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Happy Easter Day from Juniper Online Journal of Case Studies

Wishing you an egg-ceptionally wonderful Easter. Celebrate this day with peace, love, and gratitude. Have a blessed Easter. Hope your day is blooming with love and laughter.

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Analysis of Mineral Pigments from the Gnishikadzor Area, Southeastern Armenia

Authored by: Yeghis Keheyan

Abstract

The territory of the Republic of Armenia is very rich with ores and different types of deposits, including resources of natural mineral pigments. They differ by large variation of colours and are represented by painted ores, clays, and earths, among which the most significant is the group of paints with yellow, red and brown shades (ochre). Vayots Dzor Province in South-Eastern Armenia is among the rich areas where painted earths are widely spread. Presence of red and brown ochre are very well visible in the south-western part of the province, in the gorge of the Gnishik River, which is also known as the Noravank Gorge, due to the monastic complex of Noravank located here. Red colour rocks in the area of the Noravank Gorge (Gnishikazdor) represented by the sedimentary strata of the Upper Devonian and are determined by the Famennian Stage (375-359 million years). The samples analysed were taken from the foothills of the Noravank Monastery and analysed by different techniques: Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS); FT-IR spectroscopy; XRD diffraction analysis, which allow to indicate the presence of different elements trough contrast variations (atomic number contrast), to determine spectral ranges where absorption peaks were detected, as well as to perform phase identifications. The results show that the concretion is a hard, compact mass of matter formed by the precipitation of mineral cement within the spaces between particles, and is found in sedimentary rock or soil. It is composed of a carbonate mineral such as calcite; an amorphous or microcrystalline form of silica such as chert, flint, jasper or an iron oxide or hydroxide such as goethite and hematite. Implementation of such kind of study is valuable for the future comparison of similar finds from the nearby prehistoric archaeological contexts, where inhabitants exploited red ochre as a pig.

Keywords: Mineral paints; Red ochre; Areni-1 cave; Vayots Dzor Province; Republic of Armenia

Introduction

The mountains of Armenia conceal deposits of ores. Alaverdi (Northern Armenia) and Kapan (Southern Armenia) localities are rich of copper deposits, molybdenum was found in the southeast (Dastakert deposit), in the central and southeastern areas are iron ore deposits (Hrazdan, Abovyan and Svarants deposits). Besides, there are industrial stocks of aluminium-nepheline-syenites, as well as barite with admixture of gold and silver, the deposits of lead, zinc, manganese, gold, platinum, antimony, mercury, and arsenic. There are also rare earth metals: bismuth, gallium, indium, selenium, thallium, tellurium, rhenium. Tuffs (red, orange, yellow, pink, and black), marble, travertines, limestones, are great as building and finishing materials. Semiprecious and ornamental stones are represented by agates, jaspers, amethysts, beryls, rubies, obsidians, onyxes, turquoise.

The area of the country is also rich with resources of natural mineral pigments, where 17 deposits were registered and studied. They differ by large variation of colours and are represented by painted ores, clays and earths, among which the most significant is the group of paints with yellow, red and brown shades (ochre) [1,2].

The colour shade of ochre depends on the type of the iron oxide chromophore. The red ochre contains mainly haematite (Fe2O3), while the yellowish one is rich in hydrated iron oxide goethite, FeO·OH), [3]. The presence of other minerals, such as clay minerals or some metal oxides, can also influence the colour of the ochre. The classification of ochre can be also made according to the matrix composition of kaolinite (Al2SiO5) (OH)4 and/or gypsum (CaSO4·2H2O), and/or sulphate, [4]). Green earth is a clay pigment consisting of hydrated iron, magnesium, and aluminium potassium silicates. Colour varies from a dark, greyish blue green to a dark, dull yellowish green. The colour of green earth is derived from the presence of the following minerals: glauconite or celadonite. As the yellow and red ochre, the green earth or “terreverte” has been used as a pigment all over the world since ancient times [4,5]. They have been found in artworks all over the world and in any historical period, probably due to their availability, high coloring capacities and stability to the light and to the different weather conditions.

Armenian mineral pigments were also used since the dawn of the human civilization and their exploitation by the local inhabitants continued until 1940, after which they were processed on industrial level [2,6].

Vayots Dzor Province in South-Eastern Armenia is among the rich areas where painted earths are widely spread and are represented by large deposits in Agarakadzor and Yeghegnadzor [6]. Meanwhile presence of red and brown ochre are very well visible in the south-western part of the province, in the gorge of the Gnishik River (Gnishikadzor, “dzor” in Armenian means gorge), which is also known as the Noravank Gorge, due to the monastic complex of Noravank (means New Monastery in Armenian) located here The samples analysed in this article was taken from the foothills of the Noravank Monastery, left from the road, where section of red coloured sediment is exposed during the construction of the road (Figure 1). Red colour rocks in the area of the Noravank Gorge (Gnishikazdor) are represented by the sedimentary strata of the Upper Devonian and are determined by the Famennian Stage (375-359 million years). In this area, they are exposed in the core of the so-called Gnishik anticline, spread in the basin of the middle reaches the Gnishik River. The entire stratum of Devonian deposits here is 385 m thick and is represented by ferruginous dark gray and fractured organogenic limestones, which then turn into sandy limestones with a phosphorite content. Ferruginous quartzites with large impregnations of iron oxides are also exposed in ferruginous sandy limestones, shales with carbonate nodules and rich brachiopod fauna: Productella capetatiformis Abrahamian, Plicatifera meisteri, Cyrtospirifer verneuili, Camarotoechia baitaversis, etc (Figure 2).

First evidence of exploitation of similar red coloured ochre from the area was recorded in Late Chalcolithic horizons of Areni-1 cave, located 7km north from the exposure, 2km northeast from the village of Areni, on the left bank of the Arpa River, near the point of its confluence with the tributary Gnishik and at an elevation of 1070m above the sea level. Areni-1 is a threechambered karstic cave. The excavations here began in 2007 and the major significance of this archaeological site was abundantly clear during the initial excavations when very well preserved Chalcolithic (4,300 – 3,400 BCE) and Medieval (4th –18th centuries CE) occupations were exposed. Areni-1 exhibits a transitional culture between Chalcolithic and EBA, which sheds light on the formation and the early stages of the Kura-Araxes culture in the region. Chalcolithic finds from the first gallery of the cave include numerous large storage vessels, some of which contained human skulls – of two adolescent males and a female. Grape remains and vessels typically used for wine storage, together with the results of chemical analyses of the contents, point to Chalcolithic wine production at the site. The cave had been used for different purposes since the end of the 5th millennium BCE: it was a shelter, a storeroom for food; it was used for wine production and for ritual purposes, including burial. All the data indicate clear social complexity and a ritual/productive area. Its strategic location, suitable climate of the Vayots Dzor Province compared to the surrounding mountainous area, and its numerous watercourses and highly fertile soils, make this area especially suitable for human settlement and agricultural development. Indeed, the oldest leather shoe in Eurasia and one of the oldest pieces of evidence for wine production was discovered in the Areni-1 Cave, dated to the Late Chalcolithic – and wine is still today one of the area’s main products [7-15].

Local red ochre was used by the Chalcolithic inhabitants of the cave in different purposes, i.e., for rock-paintings, in symbolic behavior (for coloring the inner parts of the ritual vessels and clay constructions, the compacted floors, as red ochre was the symbol of blood and revival as well as for decorating basketry and pottery [11,13] (Figure 2).

Experimental

The samples from Gnishikadzor or the Noravank were analysed by different techniques. Below are reported the techniques applied to characterize completely these fabulous stones.

Techniques

Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS): SEM-EDS micro-morphological and chemical investigations were carried out by a LEO 1450 VP -INCA 300 scanning electron microscope coupled with a electronic probe for X-ray microanalysis, resolution of 3,5nm with the possibility to analyze nonconductive sample by operating in novacuum conditions. The interfacing with EDS gives the possibility to have qualitative and quantitative composition of elements into area observed. For quantitative analysis this method is not sensible under 0.1% in weight. Electron beam energy is 20keV to allow the detection most of the chemical elements starting from boron. Under these experimental conditions the ancient samples have been analysed without any treatment, by using the apparatus in low vacuum. The observations in backscattered electron allow suggest the presence of different elements trough contrast variations (atomic number contrast).

FT-IR spectroscopy: The FTIR microspectra were collected with a Bruker Optic Alpha-R portable interferometer with an external reflectance head covering a circular area of about 5mm in diameter. The samples were placed directly in front of the objective and spots were selected for analysis. The recorded spectral range was 7500-375cm-1 acquired with 200 scans or more, with a resolution of 4cm-1. Spectra reported in the text, however, show only the spectral range where absorption bands were observed (4000-375cm-1). This analysis is non-destructive and non-invasive. The spectra of powdered samples were obtained using the diffuse reflectance infrared Fourier transform (DRIFT) module. In addition, very small amounts of samples were dispersed in potassium bromide (KBr, FTIR grade purity, Fluka) at different concentrations (sample/ KBr 1/100 to sample/KBr 1/1000). These were studied by collecting 200 scans or more in the same spectral range and resolution. Fourier-Transform infrared (FTIR) spectra were recorded using an Alpha FT-IR spectrometer (Bruker) equipped with the Diffuse Reflection Infrared Fourier Transform (DRIFT) module in the spectral range 7500-375cm-1 at a resolution of 2cm-1 cumulating at least 200 scans. The powdered samples were dispersed in potassium bromide (KBr), FT-IR grade of purity, Fluka) in excess. Figures reported spectral ranges where absorption peaks were detected

XRD diffraction analysis: The X-ray powder diffraction analysis has been performed in the angular range 10-90° in 2θ with a Panalytical X’Pert Pro MPD diffractometer (Cu Kα radiation, λ=1.54184 Å) equipped with X’Celerator ultrafast RTMS detector. The angular resolution (in 2θ) was 0.001°. A 0.04 rad soller slit, a 1° divergence slit, and a 20mm mask have been used on the incident beam path, while a 6.6mm anti-scatter slit and a 0.04 rad collimator have been used on the diffracted beam path. Phase identification has been performed with the Panalytical High Score Plus software.

Results and Discussion

XRD spectra (Figure 3) show prevalently the presence of ochre. From phase analysis the following chemical composition was evidenced; CaCO3, SiO2, Al2O3, Fe2O3, Al2S2(OH)4.

Different stones from the same locality have been cut off. The stone has been cut and analyzed in all parts by FTIR. It was very hard to cut it. Internal part was grey and white. The spectra obtained is reported and compared in figure 4. At 536cm-1 is hematite band and at 473cm-1 is iron oxide band. Quartz band is at 799cm-1. Infrared spectroscopy was employed to analyze the exterior red surface of the stone samples. In addition, a small stone was cut in order to examine the inner side, which appeared grey and white.

Specular reflectance produces derivative- shaped peaks in the region below 1200cm-1 because of the restrahlen effect [16]. All spectra show intense band with peaks at 1545 and 1418cm-1 assigned to the C-O stretching mode of calcite (sparitic limestone). The features at 880 and 2515cm-1 also belong to calcite and respectively assigned to O-C-O bending and combination mode. The features in the 1200-1000 cm-1 interval confidently suggest the presence of silicates, probably kaolin, characterizes by the peaks at about 3690 and 3620 cm-1 assigned to C-H stretching modes [17]. At the lower frequency range of the spectra reported in figures 5a & 5b, bands are also observed at 545 and 466cm-1, indicating the existence of iron oxides molecules in the samples [18].

The proposed assignment seems supported by the absence of mentioned features in the spectra of clear and dark points of the samples. In the last case infrared analysis shows the presence of calcium carbonate as unique component of the stone. Compares micro-FTIR (a) and DRIFT spectra (b) of a red point of the Noravank stone. Spectral differences observed can be attributed only to the different techniques employed. In fact, DRIFT spectrum confirms the components individuated in reflectance analysis suggesting only a minor content of calcium carbonate with respect to the silicates content. In addition, the DRIFT spectrum of a sample of Armenian bole is also reported (c).

SEM- EDS. The micromorphological analysis, using SEM the image detector with secondary electron resolution in non-in-air conditioning has obtained a very well-defined aspect compared to the petrographic material, with clear crystalline formations of a solid structure is observable figure 6.

Other parts of the same stone were analyzed by SEM to identify the different structures, as tested with the other techniques used figure 7 (Table 1). Various points in the area shown in figure 7 and are analyzed in EDS as reported in table 2. Several EDS analyses were carried out on several areas of the sample, the more significative are reported in table 2.

What was observed at the SEM-EDS is in line with the other types of investigations, while not providing data on the molecular formulations, but only on the composition of elements, it may be useful to consider the morphological and microstructural aspect, where, for example, it is never found its trigonal crystalline habit, but the observation of powdery material, figure 7, could be associated with its presence in conjunction with quartz and other minerals, which would explain the red color felt when handling the stone.

Hematite gave following oxide compositions; FeO 29.8%, Fe2O3 15, MgO 2,6, Al2O3 8.1, CaO 16.55, SiO2 26.7, K2O 0.55, TiO2 0.4%. The concretions with following oxide composition have been detected FeO 5.63, Fe2O3 2.8, MgO 1.55, Al2O3 11.42, CaO 33.34, SiO2 44.4.

A concretion is a hard, compact mass of matter formed by the precipitation of mineral cement within the spaces between particles and is found in sedimentary rock or soil. Concretions form within layers of sedimentary strata that have already been deposited. They usually form early in the burial history of the sediment before the rest of the sediment is hardened into rock. This concretionary cement often makes the concretion harder and more resistant to weathering than the host stratum. They are commonly composed of a carbonate mineral such as calcite; an amorphous or microcrystalline form of silica such as chert, flint, or jasper; or an iron oxide or hydroxide such as goethite and hematite. They can also be composed of other minerals that include dolomite, ankerite, siderite, pyrite, marcasite, barite, and gypsum. Although concretions often consist of a single dominant mineral, other minerals can be present depending on the environmental conditions, which created them. For example, carbonate concretions, which form in response to the reduction of sulfates by bacteria, often contain minor percentages of pyrite. Other concretions formed as a result of microbial sulfate reduction, consist a mixture of calcite, barite, and pyrite.

Conclusion

Implementation of different techniques applied to characterize completely the mineral pigment sample from Noravank is а valuable data, showing a need to conduct similar analyses for the other deposits in the Vayots Dzor Province and all Armenia. Such a study can help to create a reliable database of the mineral pigments of the country and to compare the results with the similar studies of the samples discovered from the archaeological contexts. The mineral pigments, especially, ochre, have been intensively used by prehistoric and historic populations for different purposes, especially in creation of rock-paintings, decorating the pottery and basketry, as well as in rituals. Exploitation of pigments by ancient societies will shed new light on the questions of utilization of mineral resources in the territory of Armenia and the raw-material circulation in the landscape, as well as aspects of symbolic behaviour during the complex ritual games, which took place inside the caves and other sacral spaces. This also can be significant example of benefit achieved by the combination of different scientific disciplines and tools regarding deeper study of the ancient past (Figure 8).

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Enteritis: Still a Problem in Dairy Calves

Abstract

The neonatal phase of calves is a phase that needs extra care due to newborns’ vulnerability. Enteritis - an inflammation of the intestinal mucosa, resulting mainly in diarrhea - stands out among the conditions that affect animals in this period. Enteritis are responsible for huge losses in cattle breeding, especially in the early stages of rearing. Besides the losses caused by mortality, there are also expenses with veterinarians, treatments and decreased performance of the animal throughout its productive life. The present study aimed to perform a review of diarrhea in newborn calves.

Keywords: Neonatal diarrhea; Infectious agents; Dairy cattle

Abbrevations: ETEC: E. coli enterotoxigenic; EHEC: E. coli enterohemorragic; BVDV: Bovine Viral Diarrhea Virus

Introduction

The neonatal period in cattle - that goes from birth to 28 days of age - is especially important from a health point of view, since approximately 75% of losses in young calves occur in this phase [1], and the first week of life is considered the most critical phase, with 50% of losses. Therefore, maintaining the health of calves is highly related to the hygiene of the place where they live, as they are extremely sensitive to environmental pathogens [2]. Lorenz [3] report that there are several measures to maintain calf health from birth to weaning, including the provision of good quality colostrum in adequate quantity in the first hours after birth and the need to emphasize the prevention of diseases of the gastrointestinal tract and respiratory system. Among the main conditions that cause loss in the early stages of calves development are pneumonia, malformations, central nervous system diseases, and enteritis [4]. Enteritis is clinically mainly manifested by diarrhea and stands out due to its high mortality rate [2,3,5,6], since it is commonly difficult to recover because it is almost always accompanied by malnutrition [7].

Diarrhea is a complex multifactorial disease involving animal, environmental, nutritional, and infectious agents and it is a major cause of mortality, morbidity, and economic loss in cattle worldwide [8], because the treatment of affected calves is slow and impacts on growth, weight gain to weaning and loss of genetic potential of recovered animals [9]. Due its clinical and economic importance and due the preventive measures are often neglected, it is necessary an approach on this subject, to broaden the knowledge and to promote a better conduct regarding the prevention, diagnosis and treatment of the affected animals. Therefore, the present study aimed to review diarrhea in newborn calves.

Diarrhea in Newborn Ruminants

Newborn calf diarrhea is a disease of great impact on the economic viability of cattle herds worldwide [10] (Table 1). The economic impact caused by this condition is significant, although many new intervention strategies, such as vaccine development drug development and herd management, have been developed and implemented to minimize it [2]. In this sense, the veterinarian needs to assess the status of immunoglobulins in calves, feeding, shelter, environmental disinfection, hygiene and sanitary management, to prevent neonatal deaths caused by the disease [11]. The processes involved in the pathophysiology of diarrhea are related to intestinal secretion/ hypersecretion, nutrient bad absorption and digestion, osmolarity, abnormal intestinal motility, increased hydrostatic pressure, and gastrointestinal inflammation [12-21], which may occur singly or, more commonly, by the combination of two or more factors of these mechanisms [22,23].

Secretory diarrheas occur due to abnormal stimuli to the intestinal mucosa crypts that may be caused by the action of enterotoxins and/ or the action of inflammation mediators such as prostaglandins, causing an imbalance in physiological processes, like secretion and intestinal resorption, with consequent diarrhea [24]. Diarrhea is typically profuse without blood or effort, and signs in affected calves include depression, weakness, and sometimes shock and death secondary to hypovolemia and mild acidemia [25]. The difference in osmolarity with increased concentration of solutes within the intestinal lumen, promotes greater absorption of water by the lumen, thus resulting in dehydration of the animal. Osmotic particles include poorly digested disaccharides and increased levels of D-lactate from bacterial fermentation of unabsorbed nutrients entering the colon. Reduced intestinal transit time can lead to poor digestion and malabsorption due to inadequate time for digestion and absorption of ingested food, impaired fluid resorption has a major impact on fluid balance [23].

When a calf has diarrhea, there is a huge loss of fluids and electrolytes from its body. Thus, the consequent dehydration and the appearance of metabolic acidosis are the main causes of death of these animals [26]. This happens partly because the evaluation of the animal is generally based only on clinical examination, and a more detailed approach to assessing the degree of electrolyte disturbance and acidosis through blood gas analysis is lacking or not [27]. Although this condition being common in rural properties, treatment is usually inadequate and / or insufficient, because the administration of antibiotics and anti-inflammatory drugs do not correct the hydroelectrolytic disorders and acid-base [28]. Therefore, in order for the recovering of the animal, these parameters must be measured and corrected quickly, enabling the return to homeostasis. The high frequency and persistence of calf neonatal diarrhea has attracted the interest of many researchers. The multifactorial etiology (bacteria, viruses and protozoa) influenced by nutritional and environmental factors, as well as difficulties in the precise diagnosis of the agent and the failure of treatment has required the adoption of prophylactic measures, such as cow hygiene, management and vaccination [8].

Diarrhea Infectious Agents

Diarrhea is a condition of complex multifactorial etiology, influenced by infectious, nutritional and environmental factors, as well as improper management practices. Causes include toxins, bacteria, protozoa, viruses, and management / environmental factors such as overfeeding, low temperature, poor hygiene, colostrum deprivation, and individual susceptibility of the animal [8]. Numerous infectious agents have been implicated in diarrhea of calves, such as Escherichia coli, Salmonella spp., Cryptosporidium spp., Rotavirus and coronavirus. Coinfection is commonly seen in diarrheal calves, although a single primary pathogen may be the cause in some cases. The non-infectious causes of origin are related to improper management and poor hygiene of the environment in which the animals are placed. The incidence of the disease may vary according to the geographical location of the farms, farm management practices and herd size [2]. Rotaviruses, coronaviruses and cryptosporides, the most commonly recognized enteric pathogens of calves, all produce intestinal villi atrophy, intestinal bacterial overgrowth, malabsorption, and osmotic diarrhea [25].

In general, infections caused by viruses and protozoans tend to damage the intestinal mucosa promoting alteration in intestinal absorption due to damage to intestinal cells, compromising the normal absorption of nutrients, fluids and electrolytes, without alteration in intestinal secretion [22]. Rotaviruses are the most common cause of diarrhea in newborn calves and are often involved in co-infections with other agents [11,23,25]. Clinical signs usually appear 1 to 3 days after infection lasting 5 to 9 days [23]. High environmental contamination, herds with high numbers of animals and management that favors the transmission of the agent, associated with an inexpressive immunization rate, provide favorable conditions for the spread of rotavirus in dairy herds in Brazil, justifying the prevalence and difficulty to control the infection and the spread of the virus [28]. The incidence of many etiological agents varies with the calf’s age (Table 2) and this is useful for establishing the probability of a particular agent being involved and it is generally impossible to establish a definitive field diagnosis [11].

Diarrhea may result from hypersecretion or decreased absorption. Enteropathogenic strains of E. coli are occasionally causing diarrhea in calves [29]. Enterotoxigenic E. coli, Salmonella spp, Campylobacter spp. and rotavirus cause diarrhea by secreting enterotoxins that stimulate increased intestinal secretions, while protozoa and enteric viruses cause epithelial destruction of the absorptive cell villi. Enterotoxigenic E. coli produces profuse watery diarrhea, mainly in calves older than 4 days of age and occasionally in older calves. The F5 antigen may produce a mild clinical syndrome characterized by diarrhea, dehydration and weakness in calves from 1 to 4 days of age with rapid course and may progress from healthy to decubitus and death from 6 to 12 hours [11]. Salmonella spp. is an important causative agent of diarrhea and septicemia in dairy calves and the depression caused in the animal is probably due in part to endotoxemia, not just dehydration and acidosis. Campylobacter jejuni and Campylobacter fecalis are believed to be of minor importance in calves and lambs [11].

Cryptosporidium is cited as the main agent of diarrhea in calves, not only as an opportunistic agent, but also as a primary agent. Preventive measures should be taken related to the management of cows at the time of giving birth, avoiding the agglomeration of animals and environmental contamination to reduce economic losses, and to avoid the risks to public health arising from infection [24]. The recognition of enteropathogens guides the adoption of effective prevention and control measures, besides alerting to public health reflexes, due to the zoonotic potential of several of these enteric pathogens [29,30].

Treatment

Physical examination of the diarrheal calf comprises the first step in establishing the therapeutic approach, requiring the determination of the presence of any intercurrent disease. Treatment of simple cases depends on the estimative of dehydration (Table 3), severity of acidosis, likelihood of concomitant infection, presence or absence of hypothermia and hypoglycemia [11]. The most common causes of death are dehydration and acidosis. Blood gas analysis will accurately determine the degree of metabolic acidosis [29] (Table 4). Therefore, the immediate goal in treating depressed calves is to restore them to physiological systemic status. The estimated severity of dehydration can be combined with estimates of diarrhea loss and maintenance of essential functions to manage total daily fluid requirement [11,29].

Abbreviations: pCO2, carbon dioxide pressure; pO2, oxygen pressure; HCO3-, plasma bicarbonate concentration; TCO2, total carbon dioxide in plasma; BE, base excess in the blood; StB, standard bicarbonate blood concentration; SatO2, blood oxygen saturation. Fonte: Lisbôa et al. [31]. Replacement may be administered intravenously or orally, reminding that for the latter one should be increased by 60 to 80% for partial fluid absorption [11,29]. If performed early in the disease, oral replacement can be highly effective and inexpensive. In animals with severely impaired intestinal motility, the intravenous way may be more effective in correcting hydroelectrolytic imbalances than oral administration [23]. Success of therapy is monitored based on clinical signs of calf and restoration of urination [11]. Another point to consider in chronically diarrheal calf is the need for nutritional support. When a samll quantity of milk or solid food is ingested, energyrich oral electrolytes may be used to maintain the body condition of the animal. Stop giving milk can reduce the severity of diarrhea and depression in severe diarrhea, because malabsorption exacerbates diarrhea by the osmotic effect of unabsorbed milk nutrients and also promotes bacterial proliferation and possibly poor fermentation generating organic acids. However, stop giving milk reduces weight gain [11].

Antibiotic use is frequent in the treatment of diarrhea, although few agents respond to antimicrobials, viral and parasitic agents are not directly sensitive to antibiotics. Their indiscriminate use promotes the selection of resistant strains and complicates future therapeutic efforts. However, they can attenuate clinical disease, decrease the release of pathogens to the environment and animal mortality [11,29]. Some treatment protocols include the use of anti-inflammatory drugs to help reduce the secretory effects of some agents [11]. The use of non-steroidal anti-inflammatory drugs (NSAIDs) should be restricted in dehydrated animals and administered only when the patient is sufficiently hydrated [23]. The use of probiotics, oligosaccharides and intestinal protectors is also cited, and the use of gastrointestinal motility modifiers is contraindicated, as the reduction in motility will lead to the accumulation of bacteria and pathogenic toxins [29].

Prevention

The principles of prevention are based on ensuring adequate colostral intake, specific help and nonspecific immunity, reduction of the possibility of introduction / dissemination of infectious agents [11]. Colostrum is important in preventing morbidity and mortality of diarrheal calves. Colostral antibody is responsible for the low incidence of rotavirus infections in calves under 4 days of age. Vaccination of pregnant cows is important to increase colostral immunity. Colostrum privation, lack of maternal instinct, and early separation of cow and calf are major causes of failure to transfer immunity in dairy calves [11]. Prophylactic measures include separating calves from each other with enough space to prevent contact and infection through contaminated feces and urine. All feeding facilities and equipment (buckets and bottles) must be maintained with strict hygiene conditions. There is not much difference between the patterns of disease development and the prevention of calf diarrhea according to each etiological agent. Knowledge of the causal pathogen (s) is important to accurately avaliate the current status of the affected property and to develop new interventions [2].

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[juniperpublishersoceanography]

Controlling Factors of the Population Dynamics of Two Dominant Bivalves of the Macro-benthic Community on the Sandy Tidal Flats

Authored by:  Hiroaki Tsutsumi

Abstract

The edible short-neck clam, Ruditapes philipinnarum, is one of the most dominant species in the macro-benthic community on the sandy tidal flats that face Ariake Bay in Kumamoto Prefecture, western Japan. Until the 1970s, over 40,000 tons of the clams were collected per year on the tidal flats. However, the dense patches disappeared, and the clam-harvesting fishery has suffered from extremely poor catches of less than 500 tons per year over the past three decades. We conducted environmental assessments of the sediment and did quantitative surveys of the macro-benthic community on Midori River Tidal Flats located in Kumamoto between April 2017 and April 2019 and tried to find the reasons why the clam population markedly declined. Asian mussels (Arcuatula senhousia) and short-neck clams predominated the macro-benthic community on the tidal flats. However, the former species was subject to heavy predation by ducks that visited the tidal flats during the winter, while the latter one suffered from predation by rays during the warm seasons. Although Asian mussels also affected the occurrence of short-neck clams by formation of muddy carpets on the sediment, they were accidentally destroyed due to strong wind and waves caused by a typhoon.

Keywords: Asian mussels; Muddy carpet; Population dynamics; Predation; Short-neck clams; Tidal flats

Introduction

Approximately 20,000 ha of tidal flats still remain along the coast of Ariake Bay, Kyushu, western Japan. There, innumerable blue spotted mud hoppers (Boleophthalmus pectinirostris), fiddler crabs (Uca arcuata), crabs (Macrophthalmus japonica), etc., feed on benthic diatoms thickly covered on the surface of the muddy bottom [1], and various suspension feeding clams including Ruditapes philippinarum (short-neck clams), Meretrix lusoria (Japanese hard clams) and Mactra venerformis (surf clams), etc. occur densely on the sandy bottom[2,3]. In the coast of Kumamoto Prefecture that faces Ariake Bay, most of the tidal flats are sandy, and have been used as fishery grounds for harvesting clams. Until the late 1970s, 40,000 to 65,000 tons of short-neck clams were collected on the tidal flats per year, which accounted for approximately half of the national annual catch of the clams in those days. However, it dramatically decreased in the 1980s, and declined to less than 500 tons in 1995, although the total area of the tidal flats was kept intact and the clam harvesting activities had been strictly controlled by fisherman’s associations to avoid over-fishing [4].

Nevertheless, until today, it has not shown significant recovery [5,6] (Figure 1). This dramatic decline of the clam catch indicates that the abundant primary production by microphytobenthos and phytoplankton on the tidal flats, which are available for the clam as main diets [7], is not reflected as the secondary production of the clams in the past four decades. Previous studies dealing with the occurrence of the clam on the tidal flats in Kumamoto Prefecture have found various causes of the collapse of clam harvesting fishery on the sandy tidal flats, which involve environmental disturbances due to the occurrence of extremely low salinity caused by a large amount of freshwater discharged from the river and deposition of mud transported from the upper reaches of the river during the rainy season [8], the elevation of manganese content of the sediment to intolerable levels for juveniles just after the settlement [9,10], the decrease of planktonic larvae produced during the breeding season [11], and the predation by moon snails (Glossaulax didyma) and rays (Aetobatus flagellum and Hemitrygon akajei) [12,13].

Recently, the sandy tidal flats have been thickly covered by the muddy carpets created by Asian mussels (Arcuatula (Musculista) senhousia) across the Japanese coast [14]. Those in Kumamoto Prefecture are not exceptional [15-17]. They provide new micro-habitats that enable various infaunal animals occur in them, while the sediment under them tends to fall to extremely reduced conditions as original dominant members of suspension-feeding bivalves, including short-neck clams, duck clams (Mactra quadrangularis) and razor clams (Solen strictus), etc. cannot physiologically adapt [14,15,18]. This species has invaded to the lagoons in various countries including New Zeeland [19], Australia [20,21], Europe [22-24], North America [25,26] etc., and given negative impacts on the domestic benthic ecosystem, as established dense patches with the muddy carpets [27]. The latest studies on the impact of the formation of muddy carpets by the Asian mussel on the sandy tidal flats revealed that it not only created intolerable environmental conditions for suspension-feeding clams in the sediment, but also created diet-short conditions for them by promoting the deposition of organic particles suspended in the water inside the muddy carpets [17,28].

This behavior of Asian mussels appears to be a kind of “indirect exploitative type of interference” to co-occurring other species as monopolize the food resources before the competitive species utilize them [29]. Thus, Asian mussels that create muddy carpets have potentially a strong capacity for occupying surface space on the sediment of the soft bottom just like Mediterranean mussels tend to monopolize the rock surface on the rocky shore [30,31]. The invasion process by Asian mussel on the tidal flats starts by mass settlement of planktonic larvae just after the breeding season in summer. Tsutsumi et al. [15] described the invasion process as it could establish dense patches around 5,000 ind. m-2 and 2,000 gww m-2 creating muddy carpets within several months once its mass recruitment occurred, and finally the position of the predominant species of the macro-benthic community was totally replaced with short-neck clams. None of the animals could affect the population fluctuations of the Asian mussels in this study conducted in 2008 to 2009 as long as its dense patches were mechanically destroyed by the experiment that artificially turned over the sediment with a power shovel.

In the previous studies, as predatory animals on Asian mussels in Japan, Yamamuro et al. [32] reported that several species of diving ducks that visited a lagoon for wintering favored to feed on Asian mussels and gave a marked impact on abundance during the winter. Ito [33] also found the shells of Asian mussels from the stomach contents of a specimen of dabbling duck, Anas platyrhynchos, captured beside the red algae, nori, cultivation farms set on the tidal flats. Nori has been cultivated extensively on the tidal flats and their offshore areas across the Japanese coast during the winter, and recently suffered from serious feeding damage by ducks [33-35]. In this study, we conducted the environmental assessments of the sediment and quantitative surveys of macro-benthic community on Midori River Tidal Flats, which has been one of the major sandy tidal flats that face Ariake Bay in Kumamoto Prefecture, between April 2017 and April 2019.

Here, short-neck clam-harvesting fishery activity is still carried on throughout the year, while nori cultivation farms are also set extensively during the winter. Short-neck clams originally predominated the macro-benthic community on the tidal flats, but mass-settlement of Asian mussels has recently occurred and followed the formation of muddy carpets [15]. In this paper, we report the results of the environmental assessments of the sediment and quantitative surveys of macro-benthic community on the tidal flats, analyze the mechanisms of the seasonal fluctuations of the populations of the two competitive dominant bivalves in the macro-benthic community, and discuss what factors mainly controlled their population dynamics and what we should do to re-establish dense patches of short-neck clams to recover its harvesting fishery on the tidal flats as much as actively done in the 1970s.

    Materials and Methods

The Midori River Tidal Flats is located at the river mouth of Midori River, Kumamoto, Kyushu, western Japan. It extends 5 km toward the offshore with the area of approximately 2,200 ha during the low tide in spring tide. We established a sampling site at the lower part on the tidal flats (N 32° 43′ 35.3″，E 133° 41′ 11.5″, Figure 2), where clam-harvesting fishery activity was actively carried on until the 1980s. The sampling site appears above the water when the tide level has descended to less than 40 cm.

We conducted field surveys at the sampling site during the low tide in spring tide monthly or bimonthly (15 times in total) between April 2017 and April 2019. At each sampling occasion, we collected a sediment sample up to the depth of 5 cm with a core sampler (5 cm x 5 cm x 5 cm), which was kept in a plastic bag. We also collected ten sediment samples for quantitative sampling of macro-benthic animals with a core sampler (10 cm x 10 cm x 5 cm). Each sample was sieved with a 1 mm opening mesh, and the residues retained on the mesh were kept in a plastic bag with 10 % formalin solution and a dye, Rose Bengal.

At the laboratory, the particle size composition of the sediment was determined with the sediment sample by wet-sieving method. The samples fixed with formalin solution were washed and sieved on a 1 mm opening mesh again. All of the macro-benthic animals were sorted from the residues on the mesh and identified as to its species. The total number and the wet weight of each species were confirmed. The shell lengths of the specimens of Asian mussels and short-neck clams were measured with a digital caliper.

The daily changes ratios of density (DCRD) and biomass (DCRB) of Asian mussels and short-neck clams were calculated with the following equations between two successive sampling occasions.

DCRD = (Densityi - Densityi-1)/(Di - Di-1)

DCRB = (Biomassi - Biomassi-1)/(Di- Di-1)

Densityi: Density at the sampling occasion I; Biomassi: Biomass at the sampling occasion I; Di : The number of days that passed from the survey start, 27 April 2017.

The shell length frequency distributions of the populations of Asian mussels and short-neck clams were drawn with the data of the shell length of the specimens collected at each sampling occasion, and they were treated with moving average method once by calculating the mean frequency at each shell length class every mm with shorter and larger classes to get a smoother shell length frequency distribution. In general, the shell length frequency distribution of the population is polymodal, since it is made up of a number of monomodal ones of the cohorts. It can be divided into these with a graphic method modified from Harding [36] for cohort analysis, which was computer-programmed as PROGEAN (PROgram for Generation Analysis) for a personal computer, NEC PC9801 series [37]. The shell length frequency distributions of the populations of Asian mussels and short-neck clams were divided to those of the cohorts, using PROGEAN Ver. 4.0.

    Results

Seasonal Changes in Grain Size Composition of the Sediment

Figure 3 indicates seasonal changes of the grain size composition of the sediment at the sampling site on the tidal flats between April 2017 and April 2019. The mud content of the sediment (the weight composition of fine particles of less than 63 µm in diameter) was kept above 9.8 % between April and November 2017, and the highest one, 41.3 %, was noted in May. This high mud content of the sediment was caused by the bio-deposition of fine particles suspended in the water by Asian mussels. The sediment surface was covered by muddy carpets created by this species (Figure 4a).

The mud content of the sediment rapidly decreased to 9.8 % in July, once recovered to 17.3 % in August, but decreased from 13.5 % in November 2017 to 1.0 % in January 2018 again. Since then, it fluctuated within a narrow range between 0.9 and 3.5 % until August 2018. In this period, 77.4 to 90.1 % of the sediment was made up of three components of sand (coarse sand: 500 to 1,000 µm, medium sand: 250 to 500 µm, and fine sand: 125 to 250 µm in diameter) (Figure 4b), which is the original conditions of the sediment as sandy tidal flats without the muddy carpets [4]. In October 2018, the sediment surface was temporarily covered by the muddy carpets again, and the mud content of the sediment increased to 11.5 % (Figure 4c). However, it returned to the sandy sediment with the mud content of less than 1 % by March 2019 (Figure 4 d).

Seasonal Changes of Density of Macro-Benthic Community

Figure 5a indicates seasonal fluctuations of the density of the macro-benthic community at the sampling site between April 2017 and April 2019. The community involved two exclusively dominant species, Asian mussels (A. senhousia) and short-neck clams (R. philippinarum), which occupied 48.1 % and 34.7 % of all of the specimens collected in this study, respectively. The remaining 17.2 % of the community was made up of Reticunassa festiva (snails), amphipods, polychaetes, etc. Asian mussels established dense patches of 11,290 ind. m-2 in May 2017, but the density suddenly decreased to about its half, 5,670 ind. m-2,in July. The density remained 3,670 ind. m-2 in August and 5,190 ind. m-2 in November but decreased to only 180 ind. m-2 by January 2018 and remained in low densities of 30 to 1,200 ind. m-2 until June 2018.

This species explosively increased its density from June and established extremely high-density patches of 92,140 ind. m-2 only within a couple of month and kept the dense patches of 25,610 ind. m-2 until October. However, the dense patches collapsed during the late autumn and winter again as did in the last year and returned to the extremely low density of only 40 ind. m-2 by March 2019. Short-neck clams showed more stable fluctuations of the density between 1,320 and 4,310 ind. m-2 between April 2017 and January 2018. The density increased to 39,340 ind. m-2 in May, but it also decreased to only 300 ind. m-2 by October, and the low-density patches of 200 to 410 ind. m-2 remained until April 2019. The total density of other macro-benthic animals gently fluctuated between 720 and 6,020 ind. m-2 throughout the period of this study except 17,960 ind. m-2 in April 2018.

Figure 5b indicates seasonal fluctuations of DCRD values of two exclusive dominant bivalves between May 2017 and April 2019. They further clearly show the characteristics of the seasonal fluctuations of the densities of these two species. In Asian mussels, they were characterized by an explosive increase between July and August 2018 (+3,108 ind. m-2 d-1), small scales of decreases between May and August 2017 (-96.9 ind. m-2 d-1 in July and -74.1 ind. m-2 d-1 in August) and between November 2017 and January 2018 (-84.9 ind. m-2 d-1 in January) and a collapse of dense patches between August and December 2018 (-887 ind. m-2 d-1 in October and -374 ind. m-2 d-1 in December). In short-neck clams, they were characterized by the stable fluctuations of the density between April 2017 and January 2018 (-30.3 to +27.1 ind. m-2 d-1) and followed by a rapid increase between January and May 2018 (+383 ind. m-2 d-1 in April, +112 ind. m-2 d-1 in May) and a collapse of the dense patches between May and August 2018 (-798 to -100 ind. m-2 d-1).

Seasonal Changes of Biomass of Macro-Benthic Communities

Figure 6a indicates seasonal fluctuations of the biomass of the macro-benthic community expressed by wet weight at the sampling site between April 2017 and April 2019. In biomass, Asian mussels and short-neck clams also predominated the macro-benthic community and occupied 47.1 % and 42.3 % of the total biomass of the specimens collected in this study, respectively. The remaining 10.6 % was made up of R. festiva, amphipods, polychaetes, etc. as the same manners with the density. The fluctuation patterns of the

biomass of the two dominant species roughly coincided with those of their densities. Asian mussels established dense patches of 5,186 gww m-2 in May 2017, but the biomass decreased to only 0.8 gww m-2 by July 2018. It rapidly increased to 1,406 gww m-2 by October once, following the explosive increase of the density (Figure 5a), but decreased to only 3.9 gww m-2 by March 2019 again. In contrast, the biomass of short-neck clams gradually increased from 452 gww m-2 in April 2017 to 3,572 gww m-2 in April 2018. However, it also decreased to 993 gww m-2 in May and decreased to only 5.8 gww m-2 by March 2019, although it once soon recovered to 2,274 gww m-2 in June 2018.

Figure 6b indicates seasonal fluctuations of DCRB values of the two exclusive dominant species at the sampling site between May 2017 and April 2019. The changes of DCRB values clearly revealed the occurrence of big changes of their biomass further. In Asian mussels, the fastest decline of biomass occurred between May and August 2017 (-32.2 gww m-2 d-1 in July, -74.9 gww m-2 d-1 in August), and it also suffered from the decline of the biomass during the autumn and winter (-8.2 gww m-2 d-1 in November 2018 and -10.5 gww m-2 d-1 in January 2018; -18.2 gww m-2 d-1 December 2018 and -5.4 gww m-2 d-1 in March 2019). The biomass increased between July and October 2018 (+27.0 gww m-2 d-1 in August, +8.3 gww m-2 d-1 in October 2018), following the explosive increase of the density (Figure 5a). In short-neck clams, the DCRB value had ranged between +3.0 and +21.0 gww m-2 d-1 until April 2018 except -10.1 gww m-2 d-1 in November 2017. It decreased to -56.1 gww m-2 d-1 in May 2018, once returned to positive, 42.7 gww m-2 d-1, in June, but it became negative again -48.4 gww m-2 d-1 in July and -23.1 gww m-2 d-1 in August.

Analysis of Frequency Distribution of Shell Length in the Population

The population dynamics of the two dominant bivalves, Asian mussels, and short-neck clams, in the macro-benthic community between April 2017 and April 2019 are characterized as mentioned below, based on the results of the analysis of seasonal fluctuations of the densities and biomass. We checked each of the characteristics of the population dynamics of these two species with the changes of their shell length frequency distributions (Asian mussels in Figure 7a, short-neck clams in Figure 7b) to clarify the mechanisms that caused each of the characteristic events.

Asian Mussels

Rapid decline of the biomass between 28 May and 21 August 2017

The shell length frequency distribution of the population on 21 August 2017 indicates that the population was made up of three cohorts with the shell length of 18.6±2.1 mm (mean ± S.D.)(Cohort 1), 13.4 ± 1.9 mm (Cohort 2) and 5.7 ± 1.7 mm (Cohort 3). The rapid decline of the biomass of the population (from 5,186 gww m-2 in May to 1,297 gww m-2 in August) was responsible for the marked decrease of density of Cohort 1 (from 10,819 ind. m-2 in May to 1,808 ind. m-2 in August) .

Rapid decline of the density and biomass between 8 November 2017 and 6 January 2018

The newly recruited Cohort 3 to the population in August grew up to the shell length of 9.9 ± 1.9 mm with the density of 5,054 ind. m-2 on 8 November 2017, but most members disappeared on 6 January 2018. This event indicates that Cohort 2 was subject to a strong mortality factor during this period.

Explosive population growth between 14 July and 27 October 2018

Mass recruitment by Cohort 5 to the population initiated on 14 July 2018. 42,950 ind. m-2 of the mode density of its shell length frequency distribution was recorded at the shell length class of 3 to 4 mm on 12 August, and it formed dense patches with the density of 25,610 ind. m-2 and biomass of 1,406 gww m-2 on 27 October.

Collapse of Cohort 5 between 27 October 2018 and 22 March 2019

The population was made up of only a single cohort, Cohort 5, in October 2018. As shown in the shell length frequency distribution of the population, it rapidly declined and almost disappeared by 22 March 2019, in the same manners as the rapid decline of Cohort 3 between 8 November 2017 and 6 January 2018. Thus, most members of the cohort, which were recruited to the population in the breeding season in early summer, suffered from a strong mortality factor during the late autumn and winter.

Stable fluctuations of the density and gradual increase of biomass between 27 April 2017 and 6 January 2018

The shell length frequency distribution of the population on 28 May 2017 indicates that the population was made up of four cohorts, which had the shell lengths of 29.2 ± 0.9 mm (mean ± S.D.)(Cohort 1), 21.4 ± 2.6 mm (Cohort 2), 11.8 ± 2.0 mm (Cohort 3) and 3.5 ± 1.9 mm (Cohort 4), respectively. Short-neck clams have two breeding seasons (spring and late autumn) in Kumamoto in a year (Kumamoto Prefecture Fisheries Research Center, 2006). The recruitment of a new cohort to the population was clearly recognized in the shell length frequency distribution of the population in each of two breeding seasons in 2017. Consequently, the clam population consisted of four cohorts, which had the shell lengths of 31.4 ± 2.3 mm (Cohort 3), 20.6 ± 2.2 mm (Cohort 4), 13.6 ± 2.9 mm (Cohort 5) and 3.1 ± 1.5 mm (Cohort 6) on 6 January 2018. Thus, the growth and survival processes of these cohorts could be traced along the changes of the shell length frequency distribution of the population during this period. It indicates that the clam population was not subject to strong mortality factors.

Rapid increase of density between 6 January and 17 May 2018

Cohort 6 first appeared in the shell length frequency distribution of the population on 6 January 2018, and it expanded to a mass recruitment with peak densities of around 9,100 ind. m-2 at the shell length classes of less than 4 mm on 17 May 2018. Actually, the members of this cohort have already settled on the sediment during the autumn breeding season in 2017, but they were too small to retain on the sieve with 1 mm opening mesh used for sampling. As they grew up to the shell sizes that retained on the sieve in the spring, the density rapidly increased.

Rapid decline of the biomass between 1 April and 17 May 2018

The density of the population slightly increased from 34,179 ind. m-2 to 39,340 ind. m-2 in this period (Figure 5a), while its biomass markedly decreased from 3,752 gww m-2 to 993 gww m-2 (Figure 6a) and the largest negative DCRB, -56.1 gww m-2 d-1, was noted on 17 May 2018 (Figure 6b). These contradictory changes between the density and biomass of the population were responsible for the selective elimination of Cohort 4 and Cohort 5 with the shell length of more than 17 mm from the population (see inside the frame in Figure 7b). However, this loss was compensated by the rapid increase of Cohort 6 in number.

A sign of the drastic change of the population between 17 May and 16 June 2018

The density of the population decreased to less than half in this period (15,390 ind. m-2, Figure 5a, but its biomass, nevertheless, temporarily recovered to more than double (2,274 gww m-2, Figure 6a). The fast individual growth of Cohort 6 (from 4.2±2.2 mm to 8.5±2.9 mm in shell length) due to warm conditions contributed to the increase of biomass of the population.

Collapse of the population between 16 June and 27 October 2018

The population that consisted of only Cohort 6 collapsed, and its density and biomass decreased to only 300 ind. m-2 (Figure 5a) and 61.1 gww m-2 (Figure 6a) in this period. Judging from the changes of the shell length frequency distribution of the population, the members of the population except small individuals just after the recruitment had been subject to a strong mortality factor for some reason during the warm seasons since 1 April 2018.

Scarce recruitment to the population in the spring in 2019

The population remained in the extremely low density of less than 410 ind. m-2 between 27 December 2018 and 21 April 2019, since the numbers of the recruits that should be recognized as Cohort 7 and Cohort 8 were scarce after both of the breeding seasons in the spring and autumn.

    Discussion

Factors Controlling the Population Dynamics of Asian Mussels

In this study, Asian mussels suffered from severe population decline in two different seasons (the summer in 2017, and the autumn in 2017 to the winter in 2018 and the autumn in 2018 to the winter in 2019). In the former case, the dense patches of Asian mussels with the density of 11,290 ind. m-2 and biomass of 5,186 gww m-2 on 28 May 2017 rapidly declined to less than one third of the density and about a quarter of the biomass by 21 August (Figure 5a, 6a). This species has a breeding season in July and mass recruitment of juveniles should occur to the population as it did in July to August 2018, but the number of the recruits was much restricted in 2017 (Figure 7a. In contrast, the population of short-neck clams fluctuated stably, even increasing the biomass three time larger in the same period (from 544 gww m-2 to 1,548 gww m-2). Although the habitats of these two species totally overlap on the tidal flats, the utilization in the micro-habitat level is markedly different between them. Asian mussels create muddy carpets on the sediment [38], while short-neck clams burrow the sediment [39]. Therefore, this event indicates that some strong mortality factor acted on only the benthic animals that occurred on the surface of the sediment, but it did not happen during the same season in 2018.

The seasonal fluctuations of the particle size composition of the sediment give a big hint to specify the cause of the rapid decline of Asian mussel population. The mud content of the sediment noted 41.3 % on 28 May 2017 due to the creation of muddy carpets by Asian mussels (Figure 4a), but it decreased to only 9.8 % on 25 July (Figure 3), when most of the muddy carpets disappeared and bare sandy surface appeared on the tidal flats (Figure 8a). This event indicates that a strong physical disturbance happened on the sediment, and most of the muddy carpets were washed away sometime in this period. According to the weather records at the local meteorological observatory in Kumamoto City, which is about 9 km apart from the study area, strong wind with 29.0 m·s-1 of the maximum instantaneous wind speed blew on 4 July 2017 due to a typhoon approached to the study area [40]. It is very likely that the muddy carpets with dense patches of Asian mussels were removed extensively from the tidal flats by the strong wind and waves caused by the typhoon.

In the latter case of the rapid decline of Asian mussel population during the autumn and winter, this event first reported on the present study area during the late autumn in 2014 and winter in 2015 [17]. The patches with the density of about 24,000 ind. m-2 and biomass of about 4,010 gww m-2 formed on 26 November in 2014 drastically decreased to the ones with 100 ind. m-2 and less than 10 gww m-2 by 8 March 2015, although no causes were not noted. At first, we suspected the possibility of the influence of severe winter weather. However, the weather in November and December in Kumamoto when this event starts is usually still warm, and the lowest daily mean temperature during the winter had never fallen below 0 °C except one day (-0.4 °C) in January 2011 in the past two decades [41]. It would be hard to attribute the rapid decline of the population of Asian mussels to the influence of intolerable low temperature.

In the follow-up study, we found that a large group of dabbling ducks including Anas platyrhynchos and An. acuta visited the tidal flats for feeding during low tides (Figure 8b), and the shells of Asian mussels and small juveniles of short-neck clams were found in the stomach contents of a dead individual of An. platyrhynchos) (Figure 8c). Every year, a large group of the ducks migrate from Siberia to Kumamoto for wintering [42]. It is very likely that the migratory ducks recently give a serious predation impact on the population persistence of Asian mussels and short-neck clams on the tidal flats during the late autumn and winter. In the previous studies on the feeding behaviors of the ducks on the Japanese coasts, Yamamuro et al. [32] found that three species of diving ducks (Aythya fuligula, Ay. ferina, and Ay. marila) fed mainly on bivalves including Asian mussels in estuarine lagoons that face Japan Sea, and gave a serious predation pressure on the bivalves during the winter.

On the tidal flats and near-shore areas in western Japan, nori cultivation fields are established extensively during the winter, which recently suffer from serious feeding damage by both of dabbling and diving ducks, and shells of the bivalves including Asian mussels were found with nori from their stomach contents [33-35]. Nori cultivation is popular on the tidal flats in the coast of Ariake Bay of which are the centers of the nori cultivation in Japan. All tidal flats of the bay including the present study area seem to be ones of the convenient feeding sites for the ducks during the low tide since they are able to feed on bivalves just walking on them (Figure 8b).

Factors Controlling Population Dynamics of Short-Neck Clams

The results of the population study of short-neck clams in this study indicates that it has suffered from a serious mortality factor during the spring and summer, and the first appearance of its negative impact on the population was the selective elimination of the individuals with the shell length of more than 12 mm from the population on 17 May 2018 (Figure 7b), which resulted in the rapid decline of biomass of the population (Figure 6b). During this period, we found many feeding traces marked by the rays on the sediment around the sampling site (Figure 8d). The same situations have been recently reported from various clam harvesting grounds on the tidal flats in western Japan, where the rays including A. flagellum and H. akajei gave a serious damage on the fishery activities [12,43]. Consequently, the dense patches of the clam with 34,170 ind. m-2 and 3,572 gww m-2 established on 1 April 2018 declined to ones with only 300 ind. m-2 and 61.1 gww m-2 by 17 October 2018 (Figure 5a,6a).

Such influence as depressed clam patches, however, did not occur during the warm seasons in 2017. The relatively high-density patches with 1,320 to 4,310 ind. m-2 and 452 to 1,548 gww m-2 were contrastively kept between 27 April and 8 November in 2017 (Figure 5a,6a). In the first half of this period, the clam patches were established in the muddy carpets created by Asian mussels (Figure 4a). It is likely that they provided a space refuge from the predation by the rays for the clam. In the latter half of this period, the muddy carpets were accidentally swept out from the tidal flats due to the strong wind and waves brought from the typhoon (Figure 4b). Therefore, the clam patches were not subject to a mortality factor caused by the development of reduced conditions in the muddy carpets as we had expected as a negative interference brought from Asian mussels to short-neck clams during the summer [15]. Thus, the accidental occurrence of the physical disturbance on the sediment caused by surf conditions may have worked as a key factor for the co-occurrence of Asian mussels and other macro-benthic animals including short-neck clams that favor oxidized sandy sediment.

Effective Measures to Re-establish Dense Patches of Short-Neck Clams

To re-establish dense patches of short-neck clams on the tidal flats is one of the most important issues for the sustainable development of the coastal fisheries in Japan [5]. Since this species originally predominates the macro-benthic communities on the sandy tidal flats across Japanese coast, the recovery of its population indicates to regain various functions for material circulation performed as a suspension-feeding bivalve in the ecosystem on the tidal flats [44]. However, it is apparent that the clam populations are placed under serious threats of mortality factors including predation by ducks, rays, snails etc. and there being enforced interspecific interaction with Asian mussels that have happened in the recent years.

Nevertheless, the members of the fisherman’s Associations have demonstrated by the net bag experiments that the capacity of the secondary clam production is potentially still kept intact on the tidal flats where the dense patches of the clam had disappeared. They put net bags with small pieces of oyster shell or gravels on the tidal flats to induce the settlement of planktonic larvae of the clam on the substrates inside the bags (Figure 9a). In fact, the planktonic larvae settle on the substrates, and can grow normally without any predation as they receive outside the bags (Figure 9b). Although we need to further modify this method to develop as cost-effective way, it gives as a big hint how to re-establish dense patches of short-neck clams on the tidal flats as the 1970s.

    Conclusion

Two species of bivalves, Asian mussels (A. senhousia) and short-neck clams (R. philippinarum) predominated the macro-benthic community on Midori River Tidal Flats. In the former species, innumerous planktonic larvae settled on the sediment in the breeding season of early summer, and dense patches were established, creating muddy carpets on the sediment. However, they rapidly declined due to the physical disturbance on the sediment caused by strong wind and waves when a typhoon passed through near the study area, and due to predation by the migratory dabbling ducks during the late autumn and winter. Although the creation of muddy carpets by Asian mussels expected to develop reduced conditions in the muddy carpets during the summer, and to work as a serious mortality factor to short-neck clams, the physical disturbance of the sediment caused by a typhoon swept out the muddy carpets themselves from the tidal flats. The latter species burrowed the sediment and showed a high resistance to the disturbance. However, it seriously suffered from the predation by rays during the spring and summer. Consequently, very poor macro-benthic communities were formed in both of density and biomass on the tidal flats. To recover the clam-harvesting fishery on the tidal flats, short-neck clams need to be protected not only from the development of reduced conditions in the muddy carpets during the summer as expected prior to this study but also predation by the rays.

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Obesity in Obstetrics

Mini review

The people in industrialized countries have experienced a dramatic increase in obesity in recent times. Prevalence of obesity has doubled in the last 25 years. In the United States, 17-th on the list of most obese places in the world - average BMI 28.8 Kg/m2, more than 60% of reproductive-age women are overweight and 35% are obese, representing a 70% increase in pre-pregnancy obesity. In Romania, 75th on the list- average BMI is 22.2 Kg/m2, the lowest average BMI in the European Union (9.4% obesity in 2016). [1] One of three Romanians is overweight, and one of four is obese. There are over 3.5 million obese in Romania. The highest obesity rate is recorded in Moldova, where the percentage is 23.8%. Only 10% of them see a doctor. Only one percent are included in a national obesity education program [2].

Not all ethnic groups are at equal risk. Of particular concern is the rapid increase in adolescent overweight and obesity. Concordantly, pregnancy obesity rates are also increasing. Obesity is associated with increased morbidity and 6- to 12-fold increase in mortality. Obesity is highly complex in terms of etiology and prevalence. Genetic predisposition, race, socioeconomic status, built environment (e.g., the presence of sidewalks or community design), accessibility of healthy and affordable foods, sleep habits, and geographic region all play a role. Lifestyle changes, which include consuming foods and beverages with a high glycemic index, increased food portion sizes, decreased structured physical activity, and increased screen-based sedentary behavior, have influenced the prevalence of obesity.

Antenatal Monitoring

An evaluation of dietary intake and exercise habits can provide insight into women at risk. All pregnant women without contraindications should participate in regular exercise. During prenatal visits women should be questioned and advised about their diet and exercise habits. Where available, nutritional counselling can be a helpful adjunct for women not meeting the weight gain recommendations.

The sonographer’s ability to evaluate fetal structures is largely dependent on maternal size. Approximately 15% of normally visible structures will be sub optimally seen in women with a BMI above the 90th percentile. In women with a BMI above the 97.5th percentile, only 63% of structures are well visualized. Obstetric care providers should take BMI into consideration when arranging for fetal anatomic assessment in the second trimester. Anatomic assessment at 20 to 22 weeks may be a better choice for the obese pregnant patient.

Use all available technical tools improving image quality in obesity: lower transducer emission frequencies; harmonic imaging; compound imaging; speckle reduction filters. Consider approaching the fetus through the four major abdominal areas with least subcutaneous fat: periumbilical area, suprapubic area, right and left iliac fossae. Consider using the transvaginal approach for the assessment of the central nervous system (CNS) in fetuses in vertex presentation.

Gently inform the patient and her partner that obesity will reduce the diagnostic accuracy of the scan. Consider including the BMI value among the demographic data in the report to document the presence or absence of maternal obesity. Report other cofactors of limited acoustic window, such as previous cesarean section (for the scar), twinning and myomata.

Pregnancy Complications

The risk of spontaneous abortion is increased in obese women. Lashen et al. identified an odds ratio for spontaneous abortion of 1.2 (95% CI 1.01 to 1.46) for obese women (BMI > 30 kg/m2). The authors also identified an increased risk of recurrent early miscarriages (more than 3 successive miscarriages < 12 weeks’ gestation) in the obese population, odds ratio 3.5 (95% CI 1.03to 12.01).[8] Similar risks have been identified in obese women undergoing in vitro fertilization treatment [3].

Pre-gestational diabetes is more prevalent in obese women. Therefore, testing during early in pregnancy for women with risk factors is recommended. Obese women are also at increased risk of developing gestational diabetes (GDM). Not surprisingly, obese women are also at increased risk of having a macrosomic child. Physical activity is inexpensive and can significantly reduce the risk of gestational diabetes. More relevant to the obese population, they also reported a 34% reduction in the development of gestational diabetes in women who did not participate in vigorous exercise but who did participate in brisk walking compared with those who participated in easy pace walking. Women with GDM have a 30% chance of developing type 2 diabetes later in life [4].

Intrapartum Complications and Management

Macrosomia and shoulder dystocia

The use of antenatal ultrasound to detect fetal macrosomia is associated with such obstetric interventions as labor induction and cesarean section. The rate of cesarean section is affected. Higher cesarean section is more frequent when ultrasound examination indicates a macrosomic fetus.

Fetal monitoring

The obese abdominal wall may make monitoring more difficult than in other cases, and of course, the positive predictive value of antenatal testing (e.g. cardiotocography, nonstress testing, biophysical profile assessment) is limited. There is no evidence to support the routine use of internal fetal monitoring in this population, but it may be more effective in some women. Monitoring contractions and ensuring adequate labor in obese women poses a special challenge. Obese women require more oxytocin in labor. Consider allowing longer first stage of labor before performing a cesarean for labor arrest. Although most obstetric care providers rely on manual palpation and/or external tocometry, the use of an intrauterine pressure catheter may be advantageous in some cases.

Cesarean section

The risk of cesarean section is increased in the obese parturient. The increase in cesarean section rate may be partly due to the fact that overweight and obese nulliparous women have a slower progression of the first stage of labor. When faced with lack of descent in the second stage of labor, some practitioners may opt for cesarean section rather than operative vaginal delivery because of concerns about fetal macrosomia and shoulder dystocia. This may explain the low rate of operative vaginal delivery in some series [5]. Obese women undergoing caesarean section experience more complications, including blood loss > 1000 mL, increased operative time, increased postoperative wound infection and endometritis, and need for vertical skin incision. The obese diabetic women who undergo cesarean section have an odds ratio for postoperative wound infection of 9.3 (95% CI 4.5 to 19.2), and those who require a vertical skin incision have a 12% rate of wound complication serious enough to require opening the incision [6].

For morbidly obese patients, two standard 50-cm-width operating tables secured together may be necessary. Specially constructed wider operating tables would be ideal. Weighing scales suited for obese patients are necessary not only to measure weight and evaluate weight gain during pregnancy, but also for calculating medication dosages. A wider delivery bed that is easy to move around and that may be used at all stages of delivery, including cesarean section, without the need to move the patient into another bed is most useful. Nursing care of obese patients requires ergonomic adaptation and knowledge about the special risks involved in caring for these patients. More trained nurses are necessary to care for morbidly obese patients.

The decision-to-delivery interval may be longer when an emergent or urgent cesarean section is required in obese parturient. Causes for this delay may include patient transport and bed transfer, the time to establish adequate anesthesia, and the operative time from incision to delivery. The 30-minute rule of emergency cesarean section is an arbitrary threshold rather than an evidence-based standard.

Vaginal birth after cesarean section

In the absence of contraindications, women who have had their first child by cesarean section are asked to consider vaginal birth in subsequent pregnancies. The success of vaginal birth after cesarean section is commonly quoted at 80% [7]. Obese women are less likely than their lean peers to be successful in delivering vaginally after previous cesarean section (VBAC). In women with a BMI > 29 kg/m2 the success rate is 54% to 68% [8]. The success rate is further reduced in even heavier women. Chauhan et al. found a 13% VBAC success rate in women >300 lbs (136 kg) [9].

Thromboembolism

The risk of thromboembolism is high in obese parturients. Edwards et al. reported 683 obese women (BMI > 29 kg/m2) who were matched to 660 normal weight women (BMI 19.8 to 26.0 kg/m2). The incidence of thromboembolism was 2.5% in the obese women, and only 0.6% in the controls.[29] BMI >30 plus one additional risk factor qualify for seven days of postpartum Clexane; BMI >30 plus two additional risk factors require Clexane antenatally and for 6 weeks postpartum; BMI>40 should be regarded as already having two risk factors. Clexane dosage should be calculated by weight:

Early mobilization and T.E.D. anti-embolism stockings are clinically proven to reduce the incidence of deep vein thrombosis by up to 50% and to promote increased blood flow velocity in the legs 138% of baseline by compression of the deep venous system.

Perinatal outcomes

Maternal obesity is also an established risk factor for stillbirth. The reported risk of stillbirth is 2-5 times higher in obese compared with normal-weight women. The risk of stillbirth associated with obesity increases with gestational age. Infant mortality rates increase from 2.4/1000 among normal weight women (BMI 18.5-24.9) to 5.8/1000 among women with grade 3 obesity (BMI ≥ 40.0). Maternal overweight and obesity are associated with increased risks of infant mortality due to increased mortality risk in term births and an increased prevalence of preterm births. Maternal obesity may increase the risk for intellectual disability or cognitive deficits in offspring from 1.3- to 3.6-fold. Maternal prepregnancy obesity and high gestational weight gain of > 18 kg was associated with a 3-fold increase in offspring IQ deficit (mean of 6.5 points lower) [10]. The majority of studies that have examined a link between high maternal BMI and childhood diagnosis of autism spectrum disorders have found a significant positive association. This risk may be further augmented by intrauterine growth restriction (IUGR), preterm birth, high gestational weight gain, gestational or pre-gestational diabetes, and preeclampsia [11].

Conclusion

A national information campaign is required to exploit women’s interest in having as healthy a pregnancy as possible by giving them the information they need to become fit and have a normal BMI before they consider pregnancy. Periodic health check-ups and other appointments for gynecologic care prior to pregnancy offer ideal opportunities to raise the issue of weight loss before conception. Women should be encouraged to enter pregnancy with a BMI < 30 kg/m2, and ideally < 25 kg/m2. Although obesity is not an indication for the transfer of routine obstetric care, consultation with or referral to physicians with expertise in obesity may be appropriate if the obstetrician cannot safely and effectively care for the patient because of the lack of the specialized training, experience or institutional resources.

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Omega-3 Polyunsaturated Fatty Acids, Metabolic Syndrome and Diabetes Mellitus

Authored by Victoria Serhiyenko

Abstract

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are increasingly being used to prevent cardiovascular diseases (CVD), and cardiac societies recommend the intake of 1g/day of the two ω-3 PUFAs eicosapentaenoic and docosahexaenoic acid for primary and secondary prevention of CVD. Clinical trials clearly suggest beneficial effects of ω-PUFAs consumption on lipid metabolism profile, their anti-inflammatory actions; on endothelial activation, which are likely to improve vascular function; antithrombotic and antiatherosclerotic properties. Experimental studies demonstrate direct antiarrhythmic effects, which have been challenging to document in humans. By targeting arterial stiffness and endothelial dysfunction administration of ω-3 PUFAs may prevent atherosclerosis and CVD development. A synergistic interplay showed by ω-3 PUFAs prescription suggest the potential to beneficially impact on fundamental steps involved in the development of preclinical atherosclerosis. We reviewed available evidence of the benefits of ω-PUFAs administration, especially to patients with CVD, metabolic syndrome and type 2 diabetes mellitus, including their effects on potential molecular pathways, effects on glucose and lipids metabolism parameters, thrombocyte aggregation parameters and haemostasis, endothelial function, antioxidant/anti-inflammation and antiarrhythmic properties.

Keywords: Omega-3 polyunsaturated fatty acids; Coronary heart disease, atherosclerosis; Diabetes mellitus; Glucose, lipids; Inflammation; Platelets; Haemostasis; Endothelium; Heart rate variability; Arrhythmias; Arterial stiffness

Abbrevations: ω-3 and ω-6 PUFAs: Ω-3 and ω-6 Polyunsaturated Fatty Acids; MetS: Metabolic Syndrome; T2DM: Type 2 Diabetes Mellitus; CVD: Cardiovascular Diseases; DLP: Dyslipoproteinemia; OS: Oxidative Stress

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Introduction

Numerous studies report salutary effects of ω-3 polyunsaturated fatty acids (ω-PUFAs), i.e. eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) on cardiovascular diseases (CVD) risk factors. These effects include lowering of serum triglyceride (TG) by reducing of hepatic TG production; lowering of blood pressure (BP) by improving of endothelial cell functution; decreasing of platelet aggregation by reducing of prothrombotic prostanoids; decreasing inflammation via reduction in 4-series leukotrienes (LT) production; protection from arrhythmias by modulation of electrophysiological properties of cardiac myocytes. Systematic meta analysis suggests that high doses of ω-3 PUFAs (~3g/day) produce a small, but significant decrease in systolic blood pressure (SBP) in older and hypertensive subjects [1,2]. The aim of this study was to review the latest evidence about the ω-PUFAs, metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM).

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Discussion

Ω-3 and ω-6 PUFAs are essential fatty acids, as they cannot be synthesized de novo in humans. There are limited data available regarding the exact amount of dietary ω-3 PUFAs consumed by the general population. It is reported that the total daily intake of dietary ω-3 PUFAs in the US is approximately 1.6g. Of this α-linolenic acid (α-LLA) accounts for approximately 1.4g/q.d, and only 0.1–0.2g/q.d. comes from EPA and DHA. The conversion rate from α-LLA to EPA and DHA is variable (0.2-15%). Therefore, in general, the total amount of EPA and DHA available to the body from current dietary patterns is well below the recommended amounts. EPA and DHA didn��t show a significant negative effect on glucose metabolism [3].

Several experimental studies have shown that long-chain ω-PUFAs inhibit the absorption of cholesterol in the intestine and its synthesis in the liver, lead to increased clearance of lipoproteins in the blood, prevent the development of insulin resistance (IR) in experimental diabetes, increase the level of glucose transporter 4 in skeletal muscles, have a positive effect on age related decrease of blood flow in the brain and improve glucose utilization under stress; there isn’t any influence on the development of hypertension (HT) and MetS. Ω-3 PUFAs decrease level of BP, dose-dependent prevent the development of T2DM, IR, contribute to positive changes of blood coagulation parameters; enhance endothelial cell migration and inhibits the proliferation of smooth muscle cells [4]. A meta-analysis of 18 studies found a significant effect of fish oil to lower TG concentrations and increase high-density lipoprotein cholesterol (HDL-C) in the blood; while there were no statistically significant changes in preprandial glucose, glycated hemoglobin A1c, total cholesterol, low density-lipoprotein cholesterol levels. Ω-3 PUFAs may affect the IR and glucose homeostasis by inhibition of IR in the muscle tissue >adipose tissue >>liver, inhibition of insulin secretion, which defer the development of T2DM; and on the state of lipid metabolism (in particular, reduce the concentration of TG, very low density-lipoprotein cholesterol (VLDL-C), increase of HDL-C, improve lipid profile by mixed hyperlipidaemia (HLP), slightly decrease BP, improve endothelial function, have an positive impact on the antioxidant status and inflammatory reactions [5]. Ω-3 PUFAs decrease VLDL assembly and secretion, resulting in diminished TG production, through a decreased sterol receptor element binding protein-1c activity [6,5].

The highly concentrated pharmaceutical preparation Omacor™ (Pronova Biocare, Lysaker, Norway), known as Lovaza™ (Glaxo Smith Kline, St Petersberg, FL, US) in North America is approved by the FDA as an adjunct to diet to reduce very high TG levels (≥500 mg•dL-1) in adults. Each 1-g capsule of ω-3-acid ethyl esters contains ethyl esters of EPA (0.465 g) and DHA (0.375g). Patients take a q.d. dose of 4-g or two 2-g doses (two capsules b.i.d.) [7]. Clinical trials have shown that administration of 4 g•day-1 of Lovaza™ results in a decrease in TG levels of 30-50%; does not affect the efficacy of statins [8,5]. In patients with combined HLP, co-administration of Lovaza™ with statins was a safe and effective means of lowering serum TG, despite the persistent high TG levels when the patients received statins alone [9,5].

The anti-inflammatory actions of marine ω-3 PUFAs are [10]: reduced leucocyte chemotaxis (via decreased production of some chemoattractants (e.g. leukotriene B4 down-regulated expression of receptors for chemoatttactants); reduced adhesion molecule expression and decreased leucocyte-endothelium interaction (via down-regulated expression of adhesion molecule genes [via the nuclear factor kappa B (NF-kB) (i.e. peroxisome proliferator-activated receptor-ɣ (PPAR-ɣ) etc.); decreased production of eicosanoids from arachidonic acid (AA) (via lowered membrane content of AA; inhibition of AA metabolism); decreased production of AA containing endocannabinoids (via lowered membrane content of AA); increased production of ‘weak’ eicosanoids from EPA (via increased membrane content of EPA); increased production of anti-inflammatory EPA and DHA containing endocannabinoids (via increased membrane content of EPA and DHA); increased production of pro-resolution resolvins and protectins (via increased membrane content of EPA and DHA); decreased production of inflammatory cytokines (via down-regulated expression of inflammatory cytokine genes (via NF-kB, i.e. PPAR-ɣ etc.); decreased T cell reactivity (via disruption of membrane rafts (via increased content of EPA and DHA in specific membrane regions).

Ω-3 PUFAs may decrease the risk of atherothrombosis by affecting platelet aggregation and haemostasis. The antithrombotic properties of EPA and DHA have been attributed to the incorporation into platelet phospholipids at the expense of the ω-6 PUFAs, such as AA. An important set of pathways clearly influenced by changes in the ω-3/ω-6 ratio are those for synthesis of eicosanoids. These include the cyclooxygenase (COX), lipoxygenase and cytochrome P450 epoxygenase pathways, for which EPA and DHA compete with AA as a substrate, inhibiting the production of the proaggregatory thromboxane A2 (TXA2) originating from AA. Indeed, the production of TXA2 from platelets stimulated by a variety of agonists decreased by between 60% and 80% after fatty acid supplementation both in vitro and in vivo [11,5]. The mechanism by which ω-3 PUFAs influence endothelial function is mediated by their incorporation into biological membrane phospholipids; this allows modulation of membrane composition and fluidity. The reason lies in the fact that endothelial cell membrane houses caveolae and lipid rafts where several receptors and signaling molecules crucial for cell function are concentrated [12]. Caveolae-associated receptormediated cellular signal transduction includes important pathways such as the, the nitric oxide (NO)/cyclic guanosine monophosphate signaling pathway, the nicotinamide adenine dinucleotide phosphate oxidase and tumor necrosis factor-α/ NF-kB induced COX-2 and prostaglandin E2 activation pathway. By modulating the composition of caveolae, as described for other classes of lipids ω-3 PUFAs may exert their beneficial effects, which include increased NO production and reduced production of proinflammatory mediators [13,12]. In addition to increasing NO production, ω-3 PUFAs decrease oxidative stress.

The incorporation of ω-3 PUFAs in synaptic membranes could potentially influence the autonomic control of the heart. Both nervous tissue and heart tissue have a high content of ω-3 PUFAs (especially DHA) and this may be consistent with the finding that this marine ω-3 PUFAs may modulate cardiac autonomic function as assessed by heart rate variability (HRV) [14]. Thus, ω-3 PUFAs may modulate HRV both at the level of the autonomic nervous system and the heart. Most of the data support that ω-3 PUFAs beneficially modulates cardiac autonomic control thereby possibly reducing the risk of arrhythmias. Accumulating evidence from in vivo and in vitro experiments has demonstrated that ω-3 PUFAs exert antiarrhythmic effects through modulation of myocyte electrophysiology. Ω-3 PUFAs reduce the activity of membrane Na+ channels in cardiomyocytes, thus increasing the threshold for membrane potential depolarization. EPA and DHA also modulate the activity of L-type Ca2+ channels, leading to a reduction in free cytosolic Ca2+ ion, which stabilizes myocyte electrical excitability to prevent fatal arrhythmia. EPA blocks the Na+/Ca2+ channel; however, a single amino-acid point mutation in this channel attenuated the inhibitory effect of EPA. These findings suggested that the cardioprotective effect of ω-3 PUFAs is mediated by direct interaction with membrane ion channels [15].

Ω-3 PUFAs intake has shown to reduce BP especially in HT by interacting with several mechanisms of BP regulation: reduction of stroke volume and heart rate; improvement of left ventricular (LV) diastolic filling; reduction of peripheral vascular resistances; improvement of endothelial-dependent and endothelial-independent vasodilation (stimulation of NO production; reduction of the asymmetric di-methyl-arginine; reduction of endothelin-1; relaxation of vascular smooth muscle cells; metabolic effects on perivascular adipocytes; endothelial regeneration. Mechanisms of HT-related organ damage protection: anti-inflammatory, antioxidant, and antithrombotic effects; reduction of arterial stiffness; experimental effects on LV hypertrophy and abnormal gene expression; effects on atherosclerotic plaque progression and stability [7]. Ω-3 PUFAs offer a scientifically supported means of reducing arterial stiffness and this may account for some of the purported cardioprotective effects of ω-3 PUFAs [16,17].

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Conclusion

The antiarrhythmic effects of ω-3 PUFAs, which occur by blocking various ion channels, are encouraging. So, cardiovascular benefits of ω-3 PUFAs [7,18] are: antidysrhythmic effects (reduced sudden death; possible prevention of atrial fibrillation; possible protection against pathologic ventricular arrhythmias; improvement in HRV; antiatherogenic effects (reduction in non- HDL-C levels; reduction in TG and VLDL-C levels; reduction in chylomicrons; reduction in VLDL and chylomicron remnants; increase in HDL-C levels; plaque stabilization; antithrombotic effects (decreased platelet aggregation; improved blood rheologic flow); anti-inflammatory and endothelial protective effects (reduced endothelial adhesion molecules and decreased leukocyte adhesion receptor expression; reduction in proinflammatory eicosanoids and LT’s; vasodilation); decreased SBP and diastolic BP. Thus, further research to understand the mechanism of action and confirm the beneficially effect of ω-3 PUFAs on BP profile, artery stiffness and HRV parameters in patiens with MetS, T2DM is needed.

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Expect the Unexpected with Erector Spinae Plane Block in Spine Surgery - Plan for the Worst and Hope for the Best: An Anesthesiologist Perspective-Juniper Publishers

Abstract

Spine surgery is associated with multiple postoperative complications, ranging from simple nausea and vomiting to devastating complications leading to postoperative morbidity or mortality. The postoperative neurological impairment, especially in the neurologically intact patient, is a dreadful event that makes it difficult for the surgeon to perform technically challenging or high-risk spine surgeries. Preoperative or intraoperative factors that can influence the postoperative neurological status include nature and the severity of the pathology, comorbid conditions of the patient, preexisting neurological symptoms, multiple levels involved, complex surgery or instrumentation, surgical blood loss, neurological monitoring, hemodynamic parameters, polypharmacy, and total duration of the surgery.

In addition to several known contributing factors (fixation failure, epidural hematoma, spinal cord edema, and ischemia-reperfusion injury), the role of the erector spinae plane block (ESPB) has recently been cited as a potential cause of postoperative transient paralysis after spine surgery. ESPB is considered a simple and safe regional anesthesia technique that may have an advantage in success rate and analgesic efficacy when used as an adjunct to general anesthesia in spine surgeries. Despite varied patterns of the drug spread, ESPB has been showing promising results due to consistent involvement of dorsal rami that supply all pain generators of the spine surgeries.

The potential role of ESPB in causing postoperative transient neurological complications is a diagnosis of exclusion that requires thorough clinical assessment and step-by-step evaluation using imaging modalities. Before administering ESPB in spine surgery, essential knowledge includes anatomical and technical considerations, drug distribution patterns, safe and effective volumes/types of local anesthetics, and possible associated complications. This review article describes the possible roles of all factors that lead to postoperative neurological impairment and suggests some tips and tricks for using ESPB in spine surgeries to prevent or manage such serious complications appropriately.

Keywords: Transient paraplegia; Erector spinae plane block; ESP block complications; ESP block in spine surgery; Paraplegia due to RA

Keywords: RA: Regional anesthesia; GA: General anesthesia; ESPB: Erector spinae plane block; ERAS: Enhanced recovery after surgery; LA: Local anesthetics; CT: Computed tomography; MRI: Magnetic resonance imaging; ESM: Erector spinae muscles; TP: Transverse process; SMPB: Sacral multifidus plane block; RLB: Retrolaminar block

Introduction

The occurrence of perioperative complications may be inevitable, but their prevention and management are always a shared responsibility of all team members involved. Thorough evaluation of such complications will help develop strategies to prevent and manage the same in the future. A systematic and stepwise approach is warranted before categorizing it as a surgical or anesthetic complication. Several interventions have been introduced in the surgical and anesthetic techniques to improve patient safety and satisfaction. Application of regional anesthesia (RA) alone or as an adjunct to general anesthesia (GA) is one such advance that helps reduce many polypharmacy-related side effects or complications. If a particular complication-reduction modality is inherently causing complications, it requires a comprehensive understanding of the situation and its contributing factors.

An erector spinae plane block (ESPB), a safe and simple RA technique, has shown promising results as an adjunct to multimodal analgesia in various orthopedic, general, thoracic, abdominal, obstetrics, and spine surgeries. In addition to its superior postoperative analgesic profile in spine surgeries at various levels, ESPB reduces hospitalization costs and the possible side effects of extensive anesthetic use. Since opioids have been linked to tumor recurrence [1,2], ESPB also reduces the risk of spine tumor recurrences by significantly reducing its consumption. ESPB meets all criteria suitable for enhanced recovery after surgery (ERAS) protocol [3] by facilitating early discharge and mobilization of patients. Being a novel RA technique, not many complications have been reported so far except for some anecdotal reports of bilateral quadriceps weakness, transient apathy or aphasia, minor neurological complications due to inadvertent intravascular injection of local anesthetics (LA) [4].

Recently, it has been described as a potential cause of transient paralysis after spine surgeries [5]. Therefore, it is essential to understand the differential diagnoses of postoperative neurological impairment, follow the step-by-step approach to rule them out one by one, determine the possible role of ESPB in their development, and learn the tricks for safely administering ESPB during spine surgery. This review article elaborates the essential background knowledge required before and after the administration of ESPB in spine surgeries.

Discussion

Postoperative neurological impairment after spine surgery in a neurologically intact patient is always daunting for the operating surgeon and the patient. Several common theories on neurological deterioration after decompressive spine surgeries include vascular compromise, hypotension, ischemia, direct trauma, or stretching of the neural elements. The major contributing factors of acute paralysis following spine surgery include fixation failure, epidural hematoma, spinal cord edema, and ischemia‑reperfusion injury [6].

Contributory factors

Neurons in the spinal cord are susceptible to ischemia and hypoxia. The mechanisms of spinal cord ischemia are multi-factorial and multi-channel. The pathogenesis of spinal cord lesions after spine surgeries is usually mechanical (pressure) damage via extensive hematoma or edema, resulting in pressure on the spinal cord leading to ischemic damage [7]. An altered cerebrospinal fluid flow dynamic may also cause cord compression [8]. In either case, the ultimate pathogenic cause is a secondary cellular injury due to the disruption of ionic homeostasis, development of free radicals, lipid oxidation, and degeneration of the cytoskeleton [7]. White cord syndrome, an imaging feature of spinal cord ischemia [9], is diagnosed as high intramedullary signal changes on sagittal T2 weighted MRI scans and is often seen in surgeries on the cervical spine.

The spinal infarct is one of the leading causes of paraplegia or quadriplegia in patients with preexisting vascular pathologies (thrombosis) or embolic events during surgery [10]. The anterior spinal cord has a higher risk of ischemia due to fewer anterior spinal artery feeding vessels [10] than the highly vascular posterior spinal cord due to anastomotic pial vessels. The sparing of the posterior column leads to unchanged intraoperative somatosensory evoked potentials [11]. The ischemia-reperfusion injury occurs upon restoring the blood flow to previously ischemic tissues and organs. Increased inflammatory cytokines such as TNF α and IL 1β may be considered vital indicators for evaluating decompression-associated spinal cord ischemia-reperfusion injury [12,13]. Its reported incidence is 2-5.7% following cervical and 14.5% following posterior thoracic decompression surgeries [14, 15].

Transient paralysis is one such complication that manifests itself as a temporary (up to 72 hours) loss of sensations, movements, anal reflexes, and sphincter function below the affected spinal segments [16]. It can occur after vertebroplasty, laminectomy, or thoracic decompressive procedures [17,18]. The longer duration of symptoms, multiple compression sites, and the high degree of preoperative stenosis are considered poor prognostic factors [18].

Who is the culprit?

The exact cause of the postsurgical neurological impairment is a diagnosis of exclusion requiring thorough clinical evaluation and imaging guidance to rule out each contributing factor (Table 1) in a step-by-step manner. Postoperative radiographic studies like computed tomography (CT) scan and magnetic resonance imaging (MRI) can help detect changes suggestive of misplaced implants, hematomas, edema, compressive lesions, white cord syndrome, or direct trauma to the spinal cord. Symptoms due to spinal cord edema typically occur at 48-72 hours post-surgery and may be relieved by anti-edema measures like fluid restriction [19].

The occurrence and severity of ischemia-reperfusion injury correlate with tissue ischemia time, the extent of ischemic tissue, and the oxygen requirement of the affected tissue [20]. The presence of deep tendon and superficial reflexes may rule out the possibility of hysterical paraplegia [18]. After excluding all contributing factors that may cause postoperative neurological impairment, the possible role of ESPB and LA can be considered and further evaluated. It requires an understanding of the anatomical and technical aspects, mechanism of drug spread, factors favoring neuraxial spread, and measures to avoid such incidents in the future [21].

Role of ESPB

ESPB involves depositing the local anesthetic solution between the erector spinae muscles (ESM) and the transverse process (TP) under ultrasound guidance. The ESM consists of three muscles: iliocostalis, longissimus, and spinalis. They arise from and insert into various bony components of the vertebral column [22] and form a paraspinal column that extends from the sacrum to the base of the skull. It gradually tapers upwards in the paravertebral groove on either side of the spinous processes. The retinaculum (thoracolumbar fascia in the lumbar region) that envelops this muscular column also facilitates the LA spread to several thoracic and lumbosacral levels [23]. The diverse multilayered fascial arrangement deep to the ESM may cause the inconsistent LA spread, resulting in multisegmented sensory block mainly involving dorsal rami with sometimes ventral rami.

This Para neuraxial block, when given bilaterally in spine surgery, can be advantageous in success rate and analgesic efficacy [24]. The absence of risks such as hypotension, vascular spread, or pneumothorax makes ESPB relatively safer than epidural anesthesia or paravertebral block. Bilateral ESPB offers effective perioperative analgesia without influencing the hemodynamic parameters. It significantly reduces the perioperative opioid requirements in spine surgeries at various levels (cervical, thoracic, and lumbar, and sacral) [25-32]. Its outcome depends on the volume and concentration of LA used, drug spread, and the anesthesiologist’s experience in selecting and locating the correct level of the TP.

The exact mechanism of action of the ESP block and pattern of the drug spread is still unclear. It has been suggested to anesthetize the spinal nerves by passing through the costotransverse foramen of Cruveilhier, accompanying the dorsal ramus and artery to the paravertebral space [33]. The deposited drug can spread in any direction, such as craniocaudal, anterior-posterior, and lateral-medial planes to reach the paravertebral space, neural foramina, epidural space, or sympathetic chain [34-38]. Fluoroscopic, CT, and MR imaging in living subjects have similarly confirmed the injectate tracking to the paravertebral area, intervertebral foramina, and epidural space following lumbar ESPB [39-42]. There is also a possibility of LA diffusion through the microscopic gaps in the mostly acellular architecture of interlinked collagen fibers of the fascia covering the erector spinae muscle [43].

ESPB at various spine levels

The anatomical differences at the various spine levels can cause varied drug spread and ultimately affect the outcomes of ESPB. Cervical ESPB is technically challenging due to the difficulty in identifying the tips of the cervical transverse processes due to their shorter length. It is mainly given at the C6 or C7 vertebral level. The probe needs to be kept anterolaterally rather than posteriorly to see the cervical TPs [44]. It may not be safe due to its proximity to the neuraxis (shorter transverse processes) and the possibility of bilateral phrenic nerve involvement [45-48].

Thoracic ESPB at the upper vertebral levels (T2 orT3) can be preferred in cervical spine surgery by inserting the needle from caudal-to-cranial direction to achieve the desired LA spread and avoid technical difficulties and complications associated with cervical ESPB. Thoracic ESPB can provide multilevel analgesia even with the small volumes of LA due to rigid boundaries of the thoracic paravertebral spaces that facilitate drug spread at several levels involving ventral and dorsal rami. Lower thoracic level ESPB is mainly performed for lumbar spine surgeries by inserting the needle from cranial-to-caudal direction to achieve the desired LA spread and avoid technical difficulties associated with lumbar ESPB [49,50].

The lumbar ESPB can also be technically challenging due to the increased thickness of the ESMs with their tendinous attachment to the TPs [51, 52] and increased corresponding depth of the intermuscular plane in the lumbar region. The psoas muscle is also closely adherent to the vertebral bodies and the anterior surface of the TPs. The anterior drug spread to include ventral rami may be compromised due to the lack of clear boundaries of lumbar paravertebral spaces [53]. There is a communication through the fat-filled plane between the ESM and TP with the fat-filled psoas compartment containing lumbar nerve roots and plexuses. The spread of LA to the epidural space is possible through this communication [54]. The compressed lamina and the ligaments of the lumbar spine favor LA spread more into the epidural space [55, 56]. Thus, the lumbar ESPB may result in either lumbar plexus block or epidural anesthesia. The resultant weakness in the quadriceps or lower extremity muscles depends on the LA concentration and volume used in ESPB.

Sacral ESPB is mainly described for gender reassignment surgery or perineal surgery [57-61]. Its application for lower lumbar or sacral spine surgery is yet to be determined. The sacral multifidus plane block (SMPB), one of the variants of the paraspinal block, involves the deposition of LA in the plane under the multifidus muscle and bony area between the median and intermediate crests of the sacrum. The possible mechanism of action of SMPB includes blocking the dorsal rami and medial cluneal nerves directly by LA deposition and ventral rami by anterior LA spread through dorsal and ventral sacral foramina. The SMPB may also block the pudendal nerve (S2–S4), lumbosacral plexus, and sciatic nerve via the anterior and cranial LA spread [61, 62].

The role of LA

The possible role of the LA used in ESPB in causing postoperative neurological compromise depends on its inadvertent spread into either the epidural or subarachnoid space. It can be determined based on the occurrence and recovery pattern of the neurological symptoms. Distal-to-proximal and motor-before-sensory recovery patterns are the hallmarks of the differential blockade of the LA [23]. Inadvertent spread of LA into the subarachnoid space can lead to severe hypotension and bradycardia, resulting in unstable intraoperative hemodynamics. The consequences of the epidural spread depend on the density of LA around the spinal nerves, which could be compromised in a subsequent surgical dissection affecting the potentiality of the epidural space.

The concentration of LA, which determines the mass of the drug, also affects the efficacy of any block. The deliberate use of LA in low concentrations can result in a preferred motor-sparing analgesic effect of such high-volume blocks [63, 64]. Bupivacaine and ropivacaine are the most commonly used LAs for bilateral ESPB. Both LA agents consistently display preferential blockade of C-fibres (slow pain) > A-delta fibers (fast pain) > A-beta fibers (touch/pressure) in both preclinical and clinical studies [64-66]. With the increasing concentration, these agents may result in loss of proprioception and loss of motor function. Lipid solubility and higher pKa of LA facilitate intraneural diffusion and ion channel blockade. Ropivacaine exhibits a relative motor-sparing effect due to its lower lipid solubility than bupivacaine [67]. Twenty milliliters of 0.375% ropivacaine is recommended for each side of the bilateral ESPB in adults [68, 69].

Technical aspects of ESPB

Unexpected outcomes like a neurological compromise can be correlated with possible technical errors while administrating ESPB. The first technical aspect is identifying the correct landmark under ultrasound depending on the surgical extent and the desired level of the block. It may further depend on the sonoanatomy quality and the experience of the anesthetist. Sometimes misidentifying the lamina as the tip of the TP can lead to the retrolaminar block (RLB), another variant of the paraspinal block. In RLB, the needle insertion is slightly medial, targeting the lamina of the vertebra instead of the tip of the TP. It works via diffusion of LA into the paravertebral space through the soft tissue gaps between adjacent vertebrae [70]. Both RLB and ESPB were consistently associated with the posterior spread of injectate to the back muscles and fascial layers [37].

Fluoroscopic-guided ESPB can lead to RLB due to the inability to see the tip of TP clearly like under ultrasound, resulting in deposition of the LA solution over the lamina. The proximity of the RLB to the neuraxis can lead to a high probability of epidural spread, which carries the risk of motor weakness. The second important aspect is the ergonomics associated with bilateral ESPB. Administering the bilateral ESPB by standing on only one side of the patient may result in deviation from the ideal needle trajectory on one side compared to the other. Therefore, technical considerations should focus on stabilizing the needle by one person, injecting LA by another person, and performing such bilateral blocks while standing on either side.

The third important aspect includes technical modifications such as keeping an ultrasound probe in a transverse view to help differentiate intramuscular drug spread from the effective linear drug spread between ESM and TP [71]. The fourth aspect is finding alternatives that involve dorsal rami consistently without causing drug spread to other unwanted areas. The thoracolumbar interfacial plane block is one such alternative that targets only the dorsal rami of the spinal nerve. Thus, it can provide more focused dermatomal coverage of the back required for thoracic and lumbar spine surgeries [72, 73]. However, its efficacy in spine surgeries is yet to be determined. We have suggested some tips and tricks for using ESPB in spine surgeries (Table 2), keeping all technical aspects in mind.

Conclusion

Postoperative neurological impairment following spine surgery is a serious concern for the operating surgeon and the patient. The role of ESPB in causing such complications is the diagnosis of exclusion made after a thorough evaluation of clinical symptoms and radiological studies. For that, understanding of various mechanisms involved in ESPB leading to neurological impairment is essential. It should encourage the anesthetists to take extreme precautions while administering this novel block, considering the anatomical differences at various spine levels. Surgeons should anticipate and explain the possibility of neurological deterioration while explaining the risks and benefits of the proposed surgical intervention. Intraoperatively, real-time neurophysiological monitoring is recommended as a useful tool to avoid further neurological deterioration, especially in extensive and multilevel surgeries or in high-risk and neurologically compromised patients.

After identifying or diagnosing such complications, intensive care and regular checking of spinal function are of great importance, along with simultaneous radiological workups to rule out various causative factors. Once paralysis occurs, early diagnosis and early intervention are essential in restoring spinal function. Despite the rare possibility of such complications, ESPB is still a promising option for ensuring effective perioperative analgesia in spine surgeries. It helps reduce postoperative morbidity by keeping the hemodynamic parameters stable and significantly reducing intraoperative blood loss. It can also avoid postoperative complications that lead to delay in mobility and discharge by significantly reducing the need for opioids and polypharmacy. However, further studies are needed to determine the safe concentration and volume of the LA solution used in ESPB, the exact surgery-specific vertebral level to cover desired surgical innervations, and the accurate LA deposition site to prevent spread to undesired areas.

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Class Attendance and the Performances in Physiology Board Examinations: A Study in a Caribbean Medical School-Juniper Publishers

Abstract

Objective: Many faculty members in medical schools encourage their students to attend classes regularly emphasizing that some studies have reported strong positive correlation between the performances in the National Board of Medical Examiners (NBME) examination with their class attendance. The objective of our study was to assess the association of the physiology class attendance and NBME physiology scaled score.

Methods: For this study, 93 medical students who completed two terms of medical physiology at Trinity School of Medicine (TSOM) wereselected. They had their first attempt of NBME physiology examination from summer 2014 to fall 2015. Their physiology class attendance andNBME physiology scaled score were tabulated in Microsoft Excel. The correlation of the percentage of physiology class attendance and NBME physiology scaled score was determined by a Pearson correlation coefficient and regression analysis, using the SPSS version 24.

Results: The class attendance was a significant predictor of performance in physiology NBME subject examination (R2 (91) = 0.295, p = .000). A significant positive correlation was found between the class attendance (%) and NBME physiology score (r(91) = .543, p < 0.001).

Conclusion: The class attendance (%) was significantly correlated with the NBME physiology score. The physiology class attendance appears to be related to NBME physiology scaled score. However, the impacts of other factors such as study habits, environment, cultural habits, gender difference, and personal, familial, and socioeconomic stresses need to be assessed in further studies.

Keywords: NBME; Physiology; Scaled Scores; Class Attendance; Caribbean Medical School

Abbreviations: NBME: National Board of Medical Examiners; TSOM: Trinity School of Medicine; SAT: Scholastic Assessment Test; HSGPA: High School Grade Point Average; USMLE: United States Medical Licensing Examination

Introduction

Many faculty members in medical schools encourage their students to attend classes regularly emphasizing that regular class attendance facilitates learning and enhances their performance in examinations. Class attendance appears to be a better predictor of college grades than any other known predictor of college grades - including Scholastic Assessment Test (SAT) scores, High School Grade Point Average (HSGPA), studying skills, and the amount of time spent studying [1]. The efforts to increase the class attendance rates among college students helped to achieve dramatic improvements in average grades [2].

The National Board of Medical Examiners (NBME) provides subject examinations in the basic and clinical sciences to medical schools and other institutions that educate physicians or other health professionals. The NBME subject examinations assess the educational achievement of medical students in specific subject areas. These examinations are used at virtually all allopathic medical schools and many osteopathic medical schools in the United States and Canada, and approximately 25 international schools in the Caribbean, Mexico, Europe, Middle East, and Asia. The NBME examinations are primarily used as final examinations after courses, clerkships, or other units of instruction. However, the scores achieved on NBME examinations cannot be used by examinees for credit toward the United States Medical Licensing Examination (USMLE) [3]. Some studies reported that the NBME performance has a strong positive correlation with the class attendance [4-6]. Hence, we aimed to assess the correlation of physiology class attendance (%) with the NBME physiology scaled score.

Material and Methods

The present study is an analytical comparative study. The study was conducted in Trinity School of Medicine (TSOM), St. Vincent and the Grenadines, West Indies. At Trinity, Medical Physiology is taught to students in their first and second terms each of which comprises 15 weeks. The two terms of medical physiology are designed to give the students sufficient mastery over basic physiology and its application in clinical contexts to prepare them for the physiology sections of the USMLE Step 1. The students take part in NBME physiology at the end of their second term.

The medical students who studied two terms of medical physiology and took their NBME physiology subject examination from summer 2014 to fall 2015 at TSOM were included into this study. The students who dropped their physiology course before taking the NBME physiology and continued the same next term, those who did not study their two terms of medical physiology at TSOM, those transferred from other schools to TSOM, and those with low levels of attendance due to medical leave of absence and other extenuating circumstances were excluded from the study. Ethical clearance for the study was obtained from the research ethical committee of TSOM.

Three physiology faculty members utilized sign-in sheets, which were passed out in each class period to record the class attendance. Students were informed that the class attendance was being recorded for informational purposes and that there were no consequences regarding their absences. The students’ cooperation was good. At the end of the second term, their first attempt of NBME physiology scaled score and class attendance were tabulated in the Microsoft Excel. The correlation between the class attendance and NBME physiology scaled score was determined by a Pearson correlation coefficient and regression analysis, using the SPSS version 24. A p value of 0.05 or less was considered as statistically significant.

Results

A total of 93 students were included into the study of whom 42 were female and 51 were male. The students’ physiologyclass attendance was measured by the overall attendance percent (mean = 85.1%, SD = 11.9%) (Table 1). The students’ performance was assessed by the NBME physiology scaled score (mean = 45.9, SD = 9.1) (Table 1). There was wide variation in the class attendance (%) and NBME physiology scaled score of individual students.

The correlation coefficient (adjusted r2) of our study was 0.287 (Table 2) and significance F was <0.001. A significant positive correlation was found between the class attendance (%) and NBME physiology scaled score (r(91) = .543, p < 0.001) (Table 2). The regression model significantly predicted 29.5% (R2 = 0.295) of the NBME physiology scaled score of the students (Table 2).

Simple linear regression analysis between the class attendance (%) and NBME physiology scaled score showed that the class attendance contributed 26.9% (confidence interval 0.283, 0.552) of the variation in NBME physiology scaled score (Table 3). The regression model showed that for each 1.0% increase in class attendance, the NBME physiology scaled score is expected to increase by 0.417 (Table 3). The equation obtained from the regression model is: NBME physiology scaled score = 10.423 + 0.417 x class attendance (%). Hence, according to regression model equation, a student is expected to have 94.9% of class attendance to achieve a scaled score of 50, which is the national average in physiology NBME subject examination (Figure 1).

Discussion

The principal result of our study was a significant positive correlation between the class attendance (%) and NBME physiology scaled score (r = .543, p < 0.001). The result of our study is in line with the meta-analytical review done by Crede et al. [2] who found a strong relationship between the class attendance and college grades. Hence, they suggested increasing class attendance rates among college students to achieve dramatic improvements in average grades [2]. In a study of relationship between class attendance and NBME part I examination done by Fogleman et al. [4] analysed the results of survey via a 2 X 2 chi-square for each item and scores on the NBME examination. It showed that only class attendance were significantly different between students scoring above the mean (511.2) and those below the mean on the NBME part I examination (chi-square = 4.766; p <0.05) [4].

Similarly, Hammnen et al. [7] indicated a negative correlation between grades and number of absences from class. However, their correlation was weak, indicating that other factors were involved as most of the studies, including theirs, did not have matched experimental and control groups. Hence, it is impossible to state whether regular attendance caused slightly higher scores or whether the better students attend the classes more frequently [7]. Millis et al. [8] demonstrated that a significant correlation (r = 0.203, P < 0.05) between the attendance and students’ grade averages at the end of their second year. They found increased grade averages as attendance increased. Students may assume that the self-directed study and distance learning are equivalent to the class activities. The risk of poor performance in a significant number of first-year medical students may be because of their belief that the internet- and classroom-based instructions in basic medical science courses are equivalent [8].

In a study done by Subramaniam et al. [9] in a medical college, they found a significant correlation between the attendance and the students who passed the University examinations. The number of students passing the examination was maximum (>90%) compared to those getting distinction and failing the exam after they made attendance mandatory for the medical course [9]. A moderate to strong negative correlation between absenteeism and academic achievement suggested that the class attendance is very critical for learning and important in improving the knowledge and academic achievement [10]. Bamuhair et al. [11] reported the positive effect of attendance on the academic performances with a stronger effect for lectures compared to other teaching modalities. They suggested that the lecture attendance is critical for learning even in nontraditional methods of education [11].

In a study of association between lecture attendance and grade outcomes done by Horton et al. [12] they reported the positive correlation of exam grades with the lecture attendance in male students (r = 0.29, P < 0.04) and overall (r = 0.21, P < 0.02) but not for female students considered separately (r = 0.10, NS). They also found that the overall grades were correlated positively with lecture attendance in male students (r = 0.35, P < 0.01) and overall (r = 0.31, P < 0.001) but not when female students were considered separately (r = 0.20, NS) [12].

However, Cohall et al. [13] in their study found no significant association between the improvement in attendance and improved academic performances in the examinations. Their findings suggested that the other factors are more critical to academic success [13]. Therefore, even though the present study suggests that the higher percentage of class attendance add value to academic performance in board examinations, the other factors such as quality of lectures and the study habits may also have an equal impact in academic performance of the students in board examinations.

Conclusion and Recommendation

According to our study, the physiology class attendance (%) has a strong positive correlation with the scaled score in physiology NBME subject examination. It means that the performance of students in physiology NBME subject examination improves with increase in their class attendance (%). Therefore, we recommend that the faculty members should encourage the students to attend the classes regularly by implementing incentives such as points for class attendance or by applying academic activities during the class such as discussing comprehensive questions at the end of the lecture. Implementation of class attendance policies might enhance the performance of medical students in board examinations too.

We conducted this study only in the subject of Physiology. The study that includes a larger sample size from multiple basic medical science subjects and study samples from more than one medical school might further strengthen the results. Further studies should address the quality of lectures, other ways to increase intrinsic motivation for attending lectures, whether the relationship is causal, and whether the improvement in attendance percentage can improve the NBME physiology performances. In addition, the impacts of other factors such as study habit, environment, cultural habit, gender difference, and personal, familial, and socioeconomic stresses need to be assessed in further studies.

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Exosomal Consignment in Renal Allograft Injury

Abstract

Exosomes are small mobile endocytic vesicles (30-120nm), shredded by every cell to conduct trafficking of cell generated cargo. They are found in almost all body fluids (blood, csf, saliva, urine). These include proteins, lipids, DNA, mi(cro)RNAs etc. In multicellular organisms, they are packaged into numerous vesicles mainly in exosomes to conduct their transport for various cellular activities which can be exploited clinically. Presently the survival of renal allograft is monitored by mostly invasive methods (tissue biopsy, Creatinine, GFR) where curving the injury is quite difficult. Hence potency of molecular markers like proteins and then circulating miRNAs came to picture for early detection of renal injury (Acute Kidney Injury-AKI and Chronic Kidney Disease-CKD). However, due to lack of specificity of circulating miRNAs lose their feasibility and the discovery of these exosomal cargos in cellular communication has become an efficient tool for treatment of various complicated clinical condition including renal allograft injury.

Keywords: micro RNAs,exosome, Renal Allograft Injury

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Introduction

Exosomal world: a prologue

Exosomes are membrane bound mobile vehicles that are found in almost all circulating body fluids like- blood, CSF, saliva, urine, etc. These are responsible for transport of respective cellular cargo to extracellular target sites [1]. Recent studies with exosomes have revealed that exosomal cargo delivery has many important biological, physiological and pathological significance thus, can be an effective diagnostic tool for various diseases [2]. Exosomes are small circulating units of 30-120 nm in diameter, generating from late endosomal compartments of cells by its cell membrane invagination or budding or released as shedding vesicles. Cellular cargos include proteins, lipids, DNAs, mRNAs, miRNAs, etc [1]. The exosomal cell membrane mainly constitute a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNAs).

Exsosomes were first discovered by Pan and Johnstone in 1983 [3] when they found that the release of transferrin receptors into extracellular space during sheep reticulocyte maturation was released inside a type of small vesicles. In 1989 Johnstone regarded these mammalian cargo delivering vesicles as exosomes [1-5]. Valadi et al. in 2007 first described about the composition of exosomes that apart from proteins and lipids these also contains DNAs and RNAs [6] which are recorded in ExoCarta database [7,8] . The exosomal cargo delivery requires stimulation of target cell which may be direct by receptor mediated interactions or may aid in transport from cell of origin to various bioactive molecules e.g. membrane receptors, proteins, lipids, mRNAs, miRNAs, etc [7]. When exosomes deliver its contents into the respective target sites the property and behavior of these cells changes to a great extent [8]. It is also understood from various studies done in last couple of years that miRNA composition of parent cell and exosomal components vary a lot [8] and of all the components, miRNAs have drawn the attention due to their regulatory role in gene expression as these are protected against RNAase-dependent degradation [1-8]. Thus exosomal cell-to-cell communication influence both physiological as well as pathological environment of the body. These play important roles in immune reactions, tumorigenesis and in neurodegenerative disorders [1]. e.g. in prostate cancer, ovarian cancers, lymphoma glioblastoma, etc [1].

Biogenesis

Exosomes are formed from late endosomal compartments of cells through endosomal sorting complex required for transport (ESCRT-that recognizes ubiquitylated proteins) to deliver the cargo to target cell or to fuse with lysosomes to degrade the undesired contents [1]. Earlier these exosomes were only considered to be “garbage bags” as their diversified capabilities were unknown then. But now these are the most emerging field of research. The way of formation and secretion of these vesicles from mutlivesicular bodies (MVBs) are of two types [9]:

Microvesicles, which are directly shed from cell membrane.

Exosomes, which are released by exocytosis when MVBs fuse with plasma membrane.

Exosomes can be identified by transmission microscopy as a cup-shaped morphology with negative staining [10-12]. These can be concentrated in 1.10-1.21 g/ml section of a sucrose density gradient [10-12]. Exosomes can be identified by various protein markers e.g. tetraspanin proteins- CD63, CD9, CD81, HSP70 and HSP90, etc [1, 8]. ExoQuick (a one-step precipitation procedure for exosome extraction), Immuno affinity capture, Immunobead (EpCAM), combination of EpCAM and ultracentrifugation, size exclusion chromatography and EpCAM and followed by Quantitative PCR, Microarray techniques for extraction and quantification of exosomes [1,8,13].

Exosomes formation and secretion requires enzymes and ATP. First the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed within endosomes by invagination of its cell membranes [8, 14]. Such endosomes are called MVBs. Finally, the MVBs fuse with endosomal cell membranes to release intraluminal vesicles into extracellular space which become exosomes [14].

The secretion or cell-to-cell communication of exosomes requires certain regulatory factors which were first identified by Ostrowski et al. who observed that Rab27a and Rab27b were associated with exosomal secretion [8]. It was also found that effectors of Rab27- SYTL4 and EXPH5 could also inhibit exosomal secretion in HeLa cells [15]. Also Yu et al. discovered that tumor repressor protein p53 and its downstream effector TSAP6 were required for influencing exosome secretion [16]. Another working group, Baietti et al. observed the importance of syndecan-syntenin which directly interact with ALIX protein via Leu-Tyr-Pro-X(n)-Leu motif in secrection of exosomes by endosomal budding [17]. Koumangoye et al. found that disruption of lipid rafts in exosomal membranes could inhibit its internalization in breast cell carcinoma cell line [18]. Trafficking of exosomes to target sites occurs in following mechanisms:

The transmembrane proteins of exosomes directly interact with signaling receptors of target cell membranes [19].

The exosomal fusion with plasma membrane of recipient cells to deliver the cargo into their cytosol [20].

The exosomes internalization into recipient cells have two fates[21].

in one, some exosomes are engulfed by the cell and may merge with the cell’s endosome and undergo transcytosis

in other case, engulfed exosomes fuse with endosomes and mature into lysosomes for degradation.

As per ExoCarta database records, of all the components proteins and miRNAs have been exploited for various research to correlate some application with different diseased state that could render some remedy. Due to the regulatory role of miRNAs in gene expression these are used as recent area of research as diagnostic tool [8,22]. Goldie et al. demonstrated that among small RNAs, the percentage of miRNAs is higher in exosomes than in parent cells [23]. Studies done with exosomalmiRNAs shows there are specific sorting mechanisms for miRNAs into exosomes. These are:

The neural sphingomyelinase 2 (nSMase-2)-dependent pathway by Kosaka et al. [24].

The miRNA motif and sumoylated heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent pathway by Villarroya- Beltri et al. [25].

The 3’ end of the miRNA sequence-dependent pathway by Koppers-Lalic et al. [26].

The miRNA induced silencing complex (miRISC)-related pathway and human AGO2 protein [27].

In short there are specific sequence in miRNAs as well as enzymes and proteins that guide them for their sorting into exosomes [8]. Exosomes are shed by cells during both normal as well as pathological conditions. Thus several studies have been made with exosomes in diseased states.

A brief sketch on therapeutic exosomal cargos:

Exosomal miRNA: miRNAs are the recent findings in the field of clinical research which are thought to be crucial in depicting human health and diseases. These biomarkers can also be an indicator for rejection onset of transplanted allograft. miRNAs are a class of small 18-25 nucleotide (nt) long endogenous, noncoding RNAs which play an important role in regulating gene expression [28,29]. A single miRNA has the ability to regulate expression (mostly silencing) of hundreds of mRNAs and have been known to play important role in many cellular functions that include induction of post-translational repression, mRNA degradation/silencing and transcriptional inhibition, cell development, differentiation, proliferation and functional regulation of immune response among others [28-31].

The mystery behind the functional maturation of miRNAs has been solved by research in last couple of years. Similar to mRNAs, miRNAs are also initially transcribed in the nucleus [32]. miRNAs are at first transcribed in nucleus as primary transcript by RNA polymerase II called pri-miRNA [32-35]. This pri-miRNA has a hairpin stem-loop structure (~80nt length) that contains the mature miRNAs [36]. The pri-miRNA processing include recognition of the stem loop followed by its cleavage by DROSHA (a class 2 ribonuclease III) and DGCR8 (a miRNA-processing multiprotein complex) to release pre-miRNA [32-35]. Pre-miRNA is then recognized by Exportin-5 which allows its exports to cytosol for further maturation into 19-25 nucleotide strands by RNA endonuclease III called Dicer [32- 35, 37]. Cleavage of this pre-miRNA by Dicer result in double stranded (ds) RNA molecule of which one of the single strand with more unstable 5’ base pairing is selected and transferred to an Argonaute (AGO) protein and the other strand is degraded [35,38,39]. The selected strand induces silencing of mRNAs through RNA Induced Silencing Complex(RISC) thus affecting various cellular functions like cell differentiation, proliferation as well as development and functional regulation of immune system [32-35,40]. In normal tissues, RISC remain as a low molecular weight entity with reduced regulatory activity while under stressed or replicating conditions these become high molecular weight units with intensified regulatory activity when bound to mRNA [36]. Thus mRNA silencing by miRNAs results in lower protein levels in the body [36,41].

ExosomalProteins: Proteins are the building blocks of life in all living organisms. These are amino acid chains linked by peptide bonds. They are exquisite necessity in every cellular events, may it be the formation of new cells or cell repair. Thus, can be an important biomarker in depicting biological changes. Emerging research have exploited this idea and conducted various proteomic studies. A more burning concept is ofexosomal proteins. The work done and data obtained shows that besides miRNAs another important exosomal load isexosomal proteins. TrairakPisitkun et al had worked on urinary biomarkers and found that urinary exosomal proteins can also be an efficient protein biomarker in reporting renal tubulopathies and other renal disorders [42]. Exosomes normally found in urine accounts for around 3% of the total urinary protein contents and isolation of these exosomes can result in very large enrichment of urinary proteins derived from renal tubular epithelial cells [42]. The exosomal packaging occurs when the apical membrane proteins undergo endocytosis and packaged into MVBs. These MVBs undergo encapsulation of cytosolic proteins into small vesicles. Finally outer membrane of MVBs fuse with apical plasma membrane releasing exosomes containing both membrane and cytosolic proteins [42]. The proteomics study with LC-MS/MS coupled with upstream one dimensional SDS-PAGE separation experiments had disclosed a number of proteins associated with exosome biogenesis like class E vacuolar protein sorting (VPS), ALIX, Aquaporin 1, Aquaporin 2, ESCRT, etc [43]. A total of 295 proteins of urinary exosomeswere found to be associated with renal diseases and hypertension. These have been enlisted in Urinary Exosome Protein Database [42]. In another experiment where polypeptides were considered reflect that β2- microglobulin could be an indicator of damage of renal proximal tubule cells [42,44]. The techniques used to evaluate exosomal protein change is carried out by two dimensional difference in gel electrophoresis and change proteins are identified by mass spectroscopy and validated by Western Blotting [45]. Zhou et al worked with Fetuin-A, a protein of liver as an important exosomal protein that can indicate occurrence of AKI(Acute Kidney Injury) [45].

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Early Molecular Biomarkers for Renal allograft status

Years of research with renal allograft injury for either Acute Kidney Injury (AKI) or Chronic Kidney Disease (CKD) suggest that instead of invasive detection of allograft status there are scopes for early and non-invasive detection of injury through molecular markers. The studies made at the molecular level have disclosed the fact that acute and chronic rejections to a transplanted graft at preliminary stage can be ascertained by alteration in levels as well as expressions of various molecular markers involved in signaling of graft injury. These can be measured from blood/urine samples of patients. In acute rejection the early pathological change is defined by Ischemia-Reperfusion Injury (IRI) where altered expression of various miRNAs [46] is observed 3-7 days post-injury [47]. At later stage when rejection is in progress changes in levels of miR-210,-10a and -10b as well as some proteins (like perforin, granzyme A andB mRNA, FAS Ligand, FOXP3, etc) are observed [48]. Chronic rejection in early graft injury is generally associated with Interstitial Fibrosis and Tubular Atrophy (IF/TA). Pathophysiology of IF/ TA is the deposition of Extracellular matrix (ECM) proteins and Epithelial-Mesenchymal Transition (EMT) which can be stimulated by Transforming Growth Factor beta (TGF-β)/Smad signaling cascades. Ample of literature suggest that TGF-β/ Smad signaling can cause up-regulation and down-regulation of various miRNAs (miR-21,-192 & miR-29 and -200 families under IF/TA conditions) [49,50]. Even though these biomarkers have provided fruitful information but they lack specificity and their cellular source is unknown since they circulate. So to get a much clearer picture of a particular injured cell research at molecular level have unrevealed the next generation biomarker –exosomalmiRNAs for early, specific and non-invasive detection. Moreover their cellular source is also defined so they can deliver exact status of a particular cell [1,8].

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Urinary Exosomal proteins and miRNAs in renal allograft injury as Next Gen Molecular Biomarkers

Studies done with renal diseases is pretty less and still a burning area of research that reveals the fact that urinary exosomal proteins as well as miRNAs can be a potential therapeutic tool for kidney and associated diseases [1,8].

The urinary exosomal proteins can be easily attainable by noninvasive means for diagnosis of ESRD as well as Urinary Tract Infection (UTI) [1]. In 2006 Zhou et al. reported that urinary exosomal protein- fetuin A was found to be increased in AKI (Acute Kidney Injury) patients in ICU than AKI patients in normal care [1,41,45]. In 2008, same group discovered that activating transcription factor-3 (ATF-3) was found in exosomes isolated from AKI patients than CKD patients or control [41,45,51]. Thus suggesting urinary exosomal proteins could be a diagnostic tool. Moreover, a reduced level of urinary exosomal aquaporin-1 has been observed in ischemia-reperfusion injury in rats [7]. ExosomalmiRNAs demonstrate potential diagnostic biomarker for renal fibrosis [8]. MiR-29c and CD2APmRNA [52,53] were observed in urinary exosomes of renal fibrosis patients. The findings by Stefano Gatti1 et al. showed that bone marrow derived Mesenchymal Stem Cells (MSC) Microvesicles (MV) when administered immediately after IR injury can prevent AKI and CKD in rats [8,54] through their paracrine actions. Tara K Sigdel et al have described that in AKI patients with perturbation exosomal proteins like CLCA1, PROS1, KIAA0753 were observed. In addition to that exosomal ApoM is more than soluble ApoM [55]. M.W. Welker group found that in patients with chronic Hepatitis C serum soluble exosomal CD 81, a surface protein marker is elevated [56]..

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Future Prospective and limitations

Lots of work have been done with circulating miRNAs but due to their less specificity with the exact status of injured tissues and accuracy in determining role of a miRNA and its cellular source, still more feasible molecular markers have been searched and scientists have found that the circulating vehicles of cells-circulating exosomes that carry respective cellular cargo to the target sites to conduct cell-to-cell communication can be an option. These can be more proficient in delivering the most specific information on the status of any cell, may it is normal or injured cells. The molecular composition of exosomes that has been found till date is being recorded in the ExoCarta database. By exploiting these data in different pathological diseases scientists have done lots of research with carcinomas. In renal diseases also these exosomal miRNAs are being used to find out a means for noninvasive early detection of graft rejection. The conclusion drawn from these studies that proteins like fetuin-A and activating transcription factor-3 (ATF-3) can be used as marker in acute kidney disease and miR-29c and CD2AP mRNA are identified from urinary exosomes in renal fibrosis patients.

Thus, the various convergent studies made in the field of transplantation have led to the discovery of potential therapeutic targets- non-invasive urinary exosomal miRNAs and proteins which can be used to investigate and confirm the injury of transplanted allograft at an early stage. Though the data obtained define exosomes as an appropriate marker when compared with mRNAs, still it has few limitations:

Diverse isolation procedures that can affect exosomal contents,

Exosomes containing large number of miRNAs that affect the signaling of the cell together but not itself alone and

TDifficultly in measuring the exact quantity of a particular miRNAs in a exosome when miRNA is in low concentration.

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Conclusion

The exosome cell-to-cell communication mechanisms’ experiments are still at its infant stage. There is the need for development of more sophisticated techniques to detect the exact amount of specific functional exosomal proteins and miRNAs and their proper signaling pathways. Thus more investigation are still required to exploit exosomes in clinical fields as therapeutic targets.

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Is US Patent Policy Strong Enough to Withstand the Winds of Change: A Study of the Need to Change United States Patent Policy

Author by Kent R Acheson

Abstract

The purpose of this case study was to learn how US patent policy requirements differ for the Software and Pharmaceutical Industries, specifically if United States Patent Policy adequately protects intellectual property rights [1] for two divergent industries while still encouraging research and development (R & D) investment sufficient to increase profits and innovation. Data for this study consisted of 38 witness testimonies delivered to US Congress between the years 2005 and 2010 by experts representing the two industries of interest: pharmaceutical and software. Key findings from the data analysis of these 38 testimonies revealed both within industry differences and between industry differences in patent law protection. Within industry differences showed variance based on size of the company and the degree to which they relied on their own R & D. Between industry differences reflected divergent ‘products’ with Pharmaceutical Industries needing long-term protection to recover R & D expenditures that include expenses for human trials research to proceed from patent application to market. Software industry, on the other hand, uses follow-on innovation of others to continue technological advancement by constantly improving upon existing software. The data show that these two industries use patent policy protection in different ways for different reasons. This information will enable Policymakers to develop another form of product protection in lieu of the current patent law to better meet the needs of these two industries rather than try to modify the existing law.

Introduction

Patent law was developed in parts, building on one another with a single purpose in mind of protecting all innovations in a society and this created the basis for patent laws imposed on the current and future generations. Bessen [2,3] stressed that patents may not be valuable in protecting innovation [4-6] but are used solely to diffuse new ideas in the public. Bessen and Maskin [7] had previously highlighted that little research and development (R&D) in the Software Industry is used to gain patent protections and the enforcement issue with patents is difficult, as many patents are issued for products that are not new. Levin [8] and others found much earlier that patents were rated weak at protecting the returns on innovation, far behind the protection gained through lead time and learning curve advantages.

Patent’s role in different industries

The purpose of this qualitative case study was to explore the different requirements for patent policy for the Software and Pharmaceutical Industries. All transcripts from testimonies from the spokespersons representing the two industries introduced to either House between the years 2005 to 2010 concerning the U.S. Patent Reform Bills were collected and analyzed to answer the research question in this case study. The findings could be useful in further adjusting patent policy to encourage innovation for diverse industries, or suggest the creation of another form of idea protection.

A similar problem may be in the type of intellectual property protection that a company chooses to obtain to avoid the constraints of getting a patent and extend the time frame for protection, such as copyright protection that extends protection from the 20 years for a patent to 120 years. Apple Inc. obtained a copyright protection for their popular iPhone [9], which recently lost in a suit against the Federal Government. The landmark decision helps to control copyright creep. Initially when buying an iPhone, Apple Inc. limited the service provider to AT&T and applications had to be bought from the Official Apple Store. Now, however, through a hack on the iPhone, users can choose a different service provider and load other, unofficial, applications not supported by Apple Inc., and hackers are not in violation of Copyright Law.

Policy Makers can use the findings of this study to explore new directions for the United States Patent Policy to optimize advancement of technology in the Software and Pharmaceutical Industries. Historically in the United States, there has been one patent policy. Scholars, academicians, and the United States Government still do not know the ideal amount of IPRs mainly because the objective has been to uphold one uniform policy. This study clarified if further changes were needed for patent policy through a Patent Reform Act, which enables Policy Makers to understand the needs of the Software Industry, or design another form of protection designed specifically for the Software Industry.

Crowe [10] and others stated that a case study design is most appropriate when little is known of a phenomenon in its natural context. A case study is “used to generate an in-depth, multifaceted understanding of a complex issue in its real-life context” (p. 1). The Pharmaceutical Industry has a profitable track record using the existing Patent Law to protect their R&D investments. The Software Industry is comparatively new and therefore their issues are only just now becoming obvious. Case law is outside the boundaries of this study.

The multiple dimensions of the phenomenon of the nature of protecting intellectual property rights in the Software Industry property and the Pharmaceutical Industry are worthy of study to allow all voices to be heard, including large corporations from both software and pharmaceutical companies, generic drug companies, and smaller software startups. After carefully examining all relevant transcripts, these diverse opinions can be given venue to state their needs.

Methodology and main results

The research question addressed in this study was: How do the patent policy requirements differ for the Software and Pharmaceutical Industries? From the Software and Pharmaceutical Industries’ objectives and needs for the United States Patent Policy to address, the questions spotlighted the sufficiency and effectiveness of the United States Patent Policy.

The focus of this study has two parts, they are:

1. What is the evidence United States Patent Policy adequately protects Intellectual Property Rights (IPRs) for both the Software and Pharmaceutical Industries?

2. How does the United States Patent System encourage companies to make R&D investment in the Software and the Pharmaceutical Industry?

The first research question dealt with the effectiveness of the United States Patent Policy in protecting IPRs in two industries: software and pharmaceutical. The second research question related to how companies invest in R&D with support of the United States Patent Policy. The study explored the ability of the United States Patent Policy to foster innovation with satisfactory IPR protection to encourage R&D spending focusing on two specific industries. The Software Industry experiences a sequential and complementary nature of innovations, building on previous discoveries; and may not use the patent policy in effect in the United States. If patent policy does not consider the different requirements within the Pharmaceutical Industry and is too lax then enough R&D spending will not be invested and technological advancements, including new medications, may come to the market slower or not at all.

The scope of the study is to understand how the Software and Pharmaceutical Industries use the patent system and how better to adjust the patent system to optimize technological advancement. As discussed in assumptions, because of the nature of the source of data and the possible bias that was not fully known, the assumptions may or may not have had a credible or dependable basis and may therefore have biased the findings. Qualitative designs such as the case study have inherent limitations that may threaten validity, they may lack rigor and they may not be generalizable. These limitations may be mitigated with transparency in data analysis and reporting. Crowe 5 and others explains on page 8 “seeking potential, alternative explanations, and being explicit about how interpretations and conclusions were reached, helped readers to judge the trustworthiness of the case study report.

Evidence from various sources highlight the United States Patent system does not work as intended and needs a solution to continue or increase innovative activity. The principal problem deals with innovative activity that is sequential in nature and innovative activity that involves much R&D investment to bring a product to market. Sequential inventions build on previous breakthroughs and do not require much R&D investment. Secrecy would hinder follow-on discoveries of sequential innovative products.

This study used a content analysis of witness [11] testimonies to Congress on the Software and Pharmaceutical Industries from the years 2005 to 2010, and the possibility to develop more than one patent policy to accommodate different sectors of the economy. The study concentrated on software and pharmaceutical companies, as these two industries are most at odds with each other, and have prevented the passage of the Patent Reform Act of 2005 through 2010. The Patent Reform Act of 2010 [12,13] is the result of non-passage of the 2009 Act, as was each successive year from 2005. The stance of the Software and Pharmaceutical Industries remained relatively unchanged in their requirements, but the patent reform acts changed to incorporate the majority opinion of industry. The most important recommendations of the Federal Trade Commission (FTC 11) and National Academies of Sciences (NAS) studies that were first introduced in 2005 by Senator [14] Lamar Smith were considered.

The purpose of this descriptive analysis was to examine the current United States Patent Policy and the proposed changes to United States Patent Policy, and answer the research question – How do the patent policy requirements differ for the Software and Pharmaceutical Industries? This study will help decide if the Software and Pharmaceutical Industries effectively use the U.S. Patent Policy through protecting Intellectual Property Rights (IPRs) and encouraged investment research and development (R&D). The qualitative case study was the most suitable approach to study the issues and answer the research questions because it explored real-life experiences of industries looking to patent Intellectual Property (IP).

Data and Sample Statistics

Data were collected and analysis began using the Content Analysis Guide developed for this study. The testimonies of the BSA representatives, other computer software witnesses, Computing Technology Industry Association, PhRMA representatives, other generic pharmaceutical representatives, and the Generic Pharmaceutical Association, Biotechnology Industry Association (BIO), Intellectual Property Owners Association (IPO) [15-18], and venture capital organizations were included in this study. The IPO was included because IPO members represent 30% of patent applications at the USPTO and include members from the Software and Pharmaceutical Industries, among others. The study included Venture capitalists because some members of BSA [19] and other smaller software companies began with venture capital dollars. Each data point was examined for inclusion of any reference to R&D, including duration and support for R&D, the need for patent protections [20,21], and future needs for patent policy.

The 38 documents submitted to the congressional hearings were analyzed. Documents relating to software and pharmaceutical companies reviewed were not ambiguous but very clear and straight forward following a consistent format, so that anyone conducting another study would reach the same conclusions. They all stated who authored the document, who the document represented, who presented opinion to Congress, their position on the patent reform act, and agreements and disagreements with specific points of the patent reform act. No ambiguity existed and no information required subjective judgments to interpret the information reported. The nature of the data supported the reliability of the findings.

Cisco, Hewlett-Packard, and other big high-tech companies began pushing for reform legislation to limit the number of patent infringement lawsuits and therefore the amounts paid in damages. The United States Patent and Trademark Office’s (USPTO) proceedings’ transcript from the public hearings showed the patent policy needs for BSA’s principle member and founder Microsoft. The public hearing titled Use of the Patent System to Protect Software Related Inventions took place in 1994 at the San Jose Convention Center, California, and at the Crystal Forum in Arlington, Virginia. A brief summary of Microsoft’s speech follows. Microsoft (BSA) recommended that patent protection allow an accused infringer to identify readily the activity forbidden under the claim. The success of a particular claim in meeting these objectives may depend less on the form and more on claim substance and the supporting details.

BSA represents a large base of computer software and hardware companies in the United States. Phelps (2005) from Microsoft Corporation stated that BSA does not want the patent holder to have automatically injunctive relief. Injunctive relief occurs when the courts rule an infringement occurred and automatically issue a ruling to stop the infringer from continuing operations. From the congressional hearing in 2005 on harmonization and other matters, Phelps for BSA supports publication in 18 months. Phelps [39] expressed support for establishing a post grant opposition procedure and supported third-party opportunity to alert USPTO to questionable patents during review. Phelps also supported allowing third parties the opportunity to suggest relevant prior art to examiner during review, supported a limit on damages for willful infringement to include only egregious behavior, and supported limiting damages to only the contributing, patented piece of the invention and not the market value of the whole product, as it is now.

In a congressional hearing in 2005, Simon [40] from Intel , a BSA representative, stated the patent system is difficult to maneuver because of many pieces that comprise computers and software contain “potentially hundreds of patents [that] may be relevant to a particular computer or software technology” [40]. The primary way to challenge a patent under current law is through costly litigation. Intel suggests Congress create a balanced post grant opposition enabling third parties to challenge issued patents that includes a post grant opposition of 2 years from patent grant or 1 year from receiving notice of patent infringement. Simon also encouraged Congress to create a second window to make the post grant review meaningful. Simon suggested a limit on patent application continuations and for the court not to issue a continuation on any claim broader than the broadest claim previously published or issued. BSA suggested a stay on the lower court’s decision in interlocutory appeals before final determination by the Federal Circuit Court of Appeals. Micron Technology, Inc., a non-BSA member, suggested the same patent law reforms as BSA.

In a congressional hearing in 2006, Chandler [41] of Cisco (BSA) suggested a second window triggered by receipt of an infringement complaint. During the first window, the patent issues with thousands or millions of parts making the effectiveness of the patent examination questionable. Chandler (2006) encouraged Congress to make changes to remove venue shopping, and prevent suits from worldwide damages in United States Courts like the Microsoft and AT&T case. The only patent policy need described on the BSA website dated 1994 had no updates, which is understandable because United States Patent Policy has not changed significantly for more than 50 years and the proposed changes have not made it into law. The agreement with the Patent Reform Act was from the most influential voice for the Software Industry; nevertheless, there were disagreements within the Software Industry mainly arising from smaller companies and individual inventors. Software companies wanted patent reform by Congress but differences remained among large software companies and smaller organizations. An overhaul of the patent system and other measures to promote tech development efforts are top priorities of the Business Software Alliance, Cisco, Hewlett-Packard, and other big high-tech companies . BSA members began pushing for reform legislation to limit the number of patent infringement lawsuits, and therefore, the amounts paid in damages.

In an article in PC World dated March 9, 2008, patent reform leads a list of five legislative priorities expressed by BSA in 2008. The opinion article stated that BSA members want Congress to approve the Patent Reform Act but the legislation stalled in the United States Senate because of objections from inventors, pharmaceutical companies and some small tech (computer software) firms. In addition the article proclaimed, more than 170 California businesses and organizations oppose the Patent Reform Act in its current form. They mention that research to stay competitive is both expensive and risky, but strong protections from patent policy attract the necessary investments to commercialize a new product. This is especially the case for the hundreds of smaller, venture capital-backed firms in the state, of which many spun from California’s world-class research universities and private research institutes. According to GlaxoSmithKline, California Wireless and Mi5 Networks in paragraph eight on page one of Gross [42] (2008), the Patent Reform Act “would increase costs to obtain and maintain patents, undermine patent certainty, incentivize infringement, and weaken the enforceability of patent rights and intellectual property protections.”

Dr. Myhrovold [43-45] started Dynamical Systems, a software company, in 1984 that Microsoft bought in 1986. He worked with Microsoft from 1986 to 2000 (14 years). Myhrovold retired from Microsoft in 2000 to start another company, Intellectual Ventures, which files more than 300 patents a year making it the 25th largest inventing organization in America. Dr. Myhrovold stated “[Software is] a complex topic…and it’s all about company culture and how companies use patents” (Perspectives on Patents [46,47]. Continuing Dr. Myhrovold stated “…for most tech companies patents have never been important; they have never been a way to make money” (p. 76, para. 2), and “…patents are, at best, a distraction and most tech companies have made a deliberate decision to ignore the patent system” (p. 76, para. 5). Many other non-BSA members agreed with Myhrovold.

Defensive patenting by software companies explains if a company holds enough patents then this company can steal another product company’s ideas with impunity, but the problem enters when the other entity does not create a product to attack (Myhrovold, 2006, p. 77, para 3). These are the battle lines in the patent reform debate with universities, small inventors and pharmaceutical companies whose lifeblood is the patent system on one side, and companies who decide to infringe or at least do not care about infringing on the other side. Dr. Myhrovold is a witness from the vantage point of a Microsoft senior executive in the 1990s who discussed this role with other firms in the earlier rounds of patent reform debate.

Technology companies exaggerate the problem when over the last 20 years patents have remained in last place of lawsuits for the three forms of idea protection: trademark, copyright, and patents. A study of four high-tech companies that are active in the patent reform debate paid out $3.7 billion in patent lawsuit settlement from 1993 to 2005, but those same four companies earned $1.4 trillion in revenue over the same period making the sums for infringement only 0.26% of revenues on average. The company with the highest number of lawsuits experienced sums for infringement at only 0.51% of revenues. “Patent trolls” are companies that do not market a product but only the idea for a product. Companies that do not produce a product comprise only 2% of the patent infringement lawsuits. Software companies like to blame an innocuous group of patent troll companies when they themselves perform the same litigious practices blamed on trolls. Myhrovold stated the need to embrace the trend to make the alternate resolutions more like a court trial by creating a separate Patent Court, much like the Tax Court, Bankruptcy Court, or Divorce Court to try only specific cases.

Inter Digital is a technology and software company that disagrees with BSA’s proposed changes to patent law. Inter Digital’s Bernstein summarized the differences in the Software industry on page 220 last paragraph at the 2007 congressional hearings: “…the IT industry is absolutely not united in its support for mandatory apportionment, post grant opposition, expansive USPTO rulemaking authority, and interlocutory appeals fall outside the realm of patent ‘reform’.” Bernstein continues by expressing how such an action would degrade patent rights and increase litigation for smaller innovators. The weakening of legitimate patents would protect a few corporate giants and increase the number of lawsuits Bernstein (2007), [48,49].

An article by Mc Dougall [50] and Chabrow (2006), [51,52] in InformationWeek explains the problems as they perceive them with the Patent Reform Act from other software and computer companies. Hans Hxu, founder of online gift registry Felicite.com, says the industry’s large players want the appearance of IP openness but do not practice it. “IBM patents almost everything they do, and then they sit on it, which does not encourage innovation” (Microsoft Agenda, para. 3) says Hxu, a McKinsey consultant although other critics suggest the sellers’ moves cement their advantages when they face rising [53] competition from startups. In an August 2005 essay, Harvard Law School professor and tech entrepreneur James Moore argued the collaborative patent review proposed by IBM, Microsoft, and others would result in fewer patents issued because it would give examiners more ammunition to shoot down patent applications. “If fewer patents are issued, but existing patents are not revoked, IBM and Microsoft win because they already possess vast existing portfolios,” Moore writes (Microsoft Agenda, para. 4). Some Web 2.0 companies dismiss IBM’s argument that business-method patents protect obvious ideas. “Everything is obvious after someone has done it,” says a spokesperson for online movie renter Netflix (Microsoft Agenda, para. 5), which has patents on its queue-ordering system--and is suing Blockbuster for allegedly copying the system.

Small tech companies are taking matters into their own hands, forming patent cooperatives through which they share IPRs. Search company Wink shares in Creative Commons, a group that encourages sharing of copyrights and open source licenses, but there is a line between sharing and protecting intellectual property that creates competitive advantage, says Wink’s Chief Executive Officer (CEO) Michael [54,55] Tanne. “When companies have invested in the development of technologies, they really ought to be able to protect it,” Tanne says (Microsoft Agenda, para. 6). Resolving these issues will influence developing and commercializing tech innovations. Too many lengthy and expensive legal battles will persuade IT departments to stick with familiar technology, and this is something tech vendors should consider as they take one another to court.

The largest and best known pharmaceutical companies in the Pharmaceutical Industry represented by Pharmaceuticals Researchers and Manufacturers of America (PhRMA), Biotechnology Industry Organization (BIO), and the Professional Inventors Alliance disagree with the weakening of patent protection and the long, time frame proposed for patent reexamination. High R&D characterizes these industries and the Pharmaceutical Industry realizes a shortened patent protection because patent protection begins before FDA approval. This shortens patent protection to commercialize the product to the remaining years.

On September 17, 2007, The Professional Inventors Alliance expressed through a letter to President Bush the flaws in the Patent Reform Act of 2007. The Patent Reform Act of 2007 did not pass the United States Senate because of the opposition from PhRMA, small inventors, and small tech firms . The letter from the Professional Inventors Alliance expressed that if the Patent Reform Act of 2007 passed into law it would harm the United States’ innovative character because of the inability to enforce patents and would reduce the royalties associated with a patented technology. In 1980, PHRMA’s members invested $2 billion in R&D for new medicines; although, nearly 30 years later (in 2009), PHRMA’s members invested $50.3 billion in R&D out of the $65.2 billion industry-wide total. Pharmaceutical companies rely on government-granted patents to protect their substantial investments in researching and developing new drugs. It takes 10-15 years and costs $800 million on average to bring a new medicine to market. The Pharmaceutical Research and Manufacturers of America (PhRMA) represents the country’s leading pharmaceutical research and biotechnology companies.

Without patents to protect all the inventions necessary to develop a drug for a limited time, others could simply copy the drugs immediately, offering their versions at a reduced price because they did not incur the high costs to develop the drug. This would seriously affect the pharmaceutical companies’ ability to recoup their costs and reinvest in other research projects. PhRMA stated in 2010 that “a strong patent system is crucial to our economic [56,57] competitiveness, especially in these economically trying times” (PhRMA’s website, 2001, p. 1). The companies in favor and against the Patent Reform Act of 2010 divided into the companies that have favored and opposed the previous patent reform acts, that is, computer software favoring patent reform and pharmaceutical companies and biotechnology companies opposing patent reform. Those opposing and in favor of the patent reform acts through the six years in this study have not changed their needs but, instead, Congress changed trying to create a patent policy agreeable to most patent users.

The large pharmaceutical companies also known as the name brand pharmaceutical companies and the smaller, generic pharmaceutical companies were in general agreement on most issues. Both wanted strong patent protection and both sides were against the Patent Reform Bill [58] of 2005 and 2006 as stated in the congressional hearings on patent reform. The firstinventor- to-file patent system while harmonizing with the large United States trading partners also poses some difficulties and disagreements with United States patentees. The problems lay in the grace period of 1-year and the best mode requirement in the patent application. Harmonizing with other countries’ patent systems as currently written, such as Japan and Europe, would remove the United States grace period of 1 year to file a patent application and would remove the best mode requirement when filing a patent application. The best mode requirement is the descriptive part of the patent application the inventor has to include the inventor’s idea of how best to use or combine the chemicals for complete effectiveness.

The differences between the brand name and generic pharmaceutical companies lay in eliminating the best mode factor of the patent application and the inequitable conduct defense. Brand name pharmaceutical companies say the best mode provision of the patent law is subjective, and therefore should be removed. The generic pharmaceutical companies believe the best mode provision should remain because they cannot copy the patented medication without the recipe or the “best mode” of making the drug. By removing the inequitable conduct defense, brand name pharmaceutical companies will misuse the patent system to the harm of the public and generic pharmaceutical companies. Differences exist between the brand name pharmaceuticals and the generic pharmaceuticals. One example is the issue of patent quality: Best mode. Generic pharmaceuticals want to keep the “best mode” in the patent law language because it lowers cost of medications by allowing generic companies to copy name brand drugs more easily. Ely Lilly [59,60] and PhRMA want to remove the best mode language . The Generic Pharmaceutical Association also has qualms with weakening the inequitable conduct saying that weakening this provision gives brand-name pharmaceutical companies incentive to misrepresent their inventions.

The differences between the brand name and generic pharmaceutical companies lay in eliminating the best mode factor of the patent application and the inequitable conduct defense. Brand name pharmaceutical companies say the best mode provision of the patent law is subjective, and therefore should be removed. The generic pharmaceutical companies believe the best mode provision should remain because they cannot copy the patented medication without the recipe or the “best mode” of making the drug. By removing the inequitable conduct defense, brand name pharmaceutical companies will misuse the patent system to the harm of the public and generic pharmaceutical companies. Differences exist between the brand name pharmaceuticals and the generic pharmaceuticals. One example is the issue of patent quality: Best mode. Generic pharmaceuticals want to keep the “best mode” in the patent law language because it lowers cost of medications by allowing generic companies to copy name brand drugs more easily. Ely Lilly [59,60] and PhRMA want to remove the best mode language . The Generic Pharmaceutical Association also has qualms with weakening the inequitable conduct saying that weakening this provision gives brand-name pharmaceutical companies incentive to misrepresent their inventions.

Together the Case Lawre presented the most comprehensive line of court-led patent reforms, which makes patent reform substantially different in 2010 than 2005. Patent lawyers and the law association, AIPLA [63,64], believe that legislation is not necessary and the court system will eventually find a solution for compromise for the different users of the patent system and will define patent law through successive Case Law. Larger, more market capitalized firms make more noise and are heard more clearly than smaller, less capitalized companies or individual inventors, including companies that specialize in innovation but do not concurrently produce a product, also known as patent trolls. More innovation comes from smaller firms and individual inventors than large entities. The larger software enterprises that often infringe on patents held by companies that do not produce a product (patent trolls) behave similarly to the patent trolls. IBM and Microsoft sit on patents without an accompanying product, when another company discovers something similar the patent surprises the unsuspecting company, and a licensing or royalty agreement can avoid costly litigation. IBM earned over a billion dollars in 2005 solely from license agreements and royalties. Licensing and royalty agreements are another possible direction that companies take to avoid patent infringement suits; however, their use threatens other companies to ransom licensing or royalty agreements but is cheaper and the outcome more certain than litigation.

The Pharmaceutical Industry appreciates the current patent policy and is leery of any changes that would disrupt the current manner in which they use the patent system to optimize patent protection; also the Pharmaceutical Industry like the Software Industry makes the best of the current patent policy . Although pharmaceutical firms have to wait until after drug trials and resulting FDA approval to market the medication, which includes the 20-year patent term and drug approval sometimes lasts as much as 10 years, they too have found ways to evade current patent law to extend the patent length. The Pharmaceutical Industry commonly increases the shortened patent length by adding a known chemical to the patent protected drug therapy, and adds another patent protection term of 20 years by increasing the number of patents on a drug. One specific drug therapy created by a name-brand pharmaceutical firm that a generic company was exploring to copy had patent protection by more than 200 patents spanning 40 years.

Discussion and Conclusions

The specific research questions that framed this qualitative case study were 1. What is the evidence United States Patent Policy adequately protects Intellectual Property Rights [65] (IPRs) for both the Software and Pharmaceutical Industries? 2. How does the United States Patent Policy encourage companies to make research and development (R&D) investment in both the Software Industry and in the Pharmaceutical Industry? Based on the differences on how patent policy should read, issues of effectiveness of the United States Patent Policy to both protect and encourage IPRs and R&D investment should be considered. Patent policy in the United States has remained unchanged for the last 55 years, and has been effective in protecting IPRs and encouraging R&D investment. Pharmaceutical firms have been around many years and have flourished in the current patent policy environment. Only with the creation of the personal computer have software companies entered the scene and have expressed concern for the patent policy changes to reduce the software company’s purposeful infringement. In a few words, the large software companies want to weaken patent protections and reduce their costs to defend against patent infringement lawsuits because big software companies do not care about patents or patent infringement.

Three important findings from this study are

1. The Pharmaceutical and Software Industries use patent policy differently

2. BSA explicitly states they want a strong patent policy, but, in effect, want to weaken the current patent policy, and

3. Differences exist within each industry. Congress has attempted to improve patent law 6 years without success because there is not agreement pleasing all industries, but the principle differences embodied the Software and Pharmaceutical Industries.

Firstly, pharmacy and software use patent policy differently: Pharmacy to protect R&D and Software for defensive purposes. Software Industry (BSA) does not use the patent policy as designed to protect R&D, but to defend against the threat of patent infringement lawsuits. The testimonies to Congress provided evidence to answer my research question of how the patent policy requirements differ between the Software and Pharmaceutical Industries. The testimonies to Congress were clear and straightforward. I did not have to infer the meaning or needs of the witnesses. They clearly stated their position and what they wanted in patent policy. Many people in the Pharmaceutical Industry and smaller software companies specifically stated that larger software and computer companies began calling for patent reform to limit the many patent infringement suits against them. Myhrovold shared his experience working for Microsoft in the late 90s stating that large software companies are not concerned with infringing on another’s patents and the only reason they care at all about patents is to defend against patent infringement lawsuits.

Secondly, the data from congressional testimonies clearly showed that the Software Industry (BSA) verbalized they want a strong patent policy but, instead, they want to weaken the rights of patent holders. This weakening is from: An unlimited post patent review period, placing the burden of proof for infringement on the patent holder (instead of the offender), and limiting the damage awards for infringement to only the infringing part of an innovation. The testimonies clearly stated their position and what they wanted. The previous list clearly communicated to Congress what the Software Industry (BSA) wanted in a patent policy, and refuted by other expert testimonies in the Software Industry.

All BSA representatives stated they wanted strong patent protection, and continued with the above reasons, which amount to weakening a patent holders’ legal rights to their Intellectual Property Rights (IPRs). Many testimonies contrary to BSA stated specifically the reasons BSA wants to limit a patent holders’ IPRs is to stave off patent infringement lawsuits. Myhrovold (2006) shared that patent policy did not enter into Microsoft’s and other BSA members’ culture. Patents are not how software companies protect innovation, but, rather, secrecy, and lead time or economies of scale are more effective to protect innovation in a short product lifecycle industry. Thirdly, the entire Software Industry is not united with BSA, and the entire Pharmaceutical Industry is not united with PhRMA. Differences exist between the two industries and differences exist within each industry, such as difference between larger companies and smaller companies in Software Industry and brand name pharmaceutical versus generic pharmaceutical. Each expert clearly stated what they wanted, why they wanted it, and differences within their respective industries. The witnesses to the congressional hearings succinctly stated that the BSA or PhRMA did not represent the entire industry, and the industry was not united in its desires for patent policy. Siwik [66] said in the exact words that the Pharmaceutical Industry is not united, and based on the non-BSA members’ testimonies with them vehemently disagreeing with BSA’s stance, anyone would reach the same conclusions that BSA is far from united too.

The evidence suggests the two industries use patent policy in different ways. For instance, The Software Industry does not use the patent system to protect intellectual property but rather use the patent system for defensive purposes not so much to protect innovation but to defend against infringement lawsuits. Pharmaceutical industry relies heavily on a patent protection to recover large R&D spending. The evidence was found in examples of how each industry effectively uses the patent system. Based on research of the patent system and the evidence of how each industry uses the patent system, the data would suggest agreement with many of the pharmaceutical, biotechnology, and other industries that use the patent system effectively to protect research and development dollars that the system does not need major change. Research shows the answer to the question of how the United States Patent System encourages R&D and promotes innovation; the patent system performs well according to its design. It protects ideas. The current patent policy is effective in protecting innovation and encouraging research and development spending.

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A Spin Coating of Thymol Blue Indicator on F-SnO2 Glass to fabricate a Novel Sensor Electrode in Potentiometric Acid-Base Titration

Authored by: Nasser M Abu Ghalwa

Abstract

This study deals with the investigation for preparation of conductive glass / thymol blue TB sensor electrode by spin coating of the thymol blue (TB) indicator on conductive glass formed from F-SnO2, and it’s using as indicator electrode in potentiometric acid-base titration in aqueous solution at 298K. The change of the open circuit potential with pH (E-pH) curve is linear with slope of 0.052V/dec at 298K. The standard potential of the above electrode E0, was determined with respect to the SCE as reference electrode. The recovery percentage for potentiometric acid-base titration using G/TB as indicator electrode was calculated.

Keywords: Potentiometry; Thymol blue; Sensor; Conducting glass; Titration; Indicator electrode

Introduction

Chemical sensors are devices that convert the concentration of target compounds into an analytical signal. The term analytical implies the concept of measurability. Then a chemical sensor converts the information about the presence of target compounds into a measurable quantity [1]. Chemical sensors play a big role in checking the environment we live in, contributing information on industrial production processes, quality management of food stuffs and beverages, detection and analysis of some ions and many other applications [2]. Transparent conducting oxides possess a unique combination of optical transparency and metallic conductivity in a single material. Their properties are widely exploited in a host of energy, optical, and electrical applications [3-5]. SnO2 is a wide band-gap semiconductor with a band gap of 3.6eV and was the first transparent conducting oxides to receive significant commercialization. It exhibits good transparency and can be easily n-type doped. Degenerate carrier densities can be achieved by doping with fluorine [6,7]. Potentiometric titrations are the basic chemistry laboratory technique for the quantitative analysis of substances with unknown concentrations using standard solutions of known concentration. The substance with unknown concentration and the standard solution are termed analyte and titrant respectively [8]. This method widely used in different fields such as the food industry, scientific research, and chemical, clinical and pharmaceutical laboratories. Titrimetric procedures based on a detection of the endpoint, i.e., the point at which volumetric titration is completed, are successfully employed over a wide range of concentrations and are popular because of their simplicity, speed, accuracy and good reproducibility [9]. Recently, many studies developed some types of electrodes in potentiometric acid base titration [10-13]. Thymol blue (thymolsulphonephthalein) is used as a pH indicator. A solution of thymol blue exhibits three form (red color), Neutral form (yellow color) and basic form (blue color) show Figure 1 [14].hone radiation can lead to adverse effects [10-16]. Thus, the public concern is that increasing the frequency of the radiation will also increase the effects of the radiation [14-16].

The aim of this study for preparing a spin coating of thymol blue indicator TB on conducting glass formed from F-SnO2 to prepare a new modified electrode sensor (glass / indicator electrode), for used in potentiometric acid base titration.

Experimental

Chemicals

The chemicals used in potentiometric titrations and preparation the electrode was tetraethyl orthosilicate (TEOS), Thymol blue (TB), hydrochloric acid, ammonia, Acetic acid, phosphoric acid, sodium hydroxide, sulfuric acid, citric acid and disodium phosphate. The chemicals are of analytical pure grade (Merck) Where the F-SnO2 glass from (Sigma Aldrich).

Synthesis of Materials

Preparation of Hydrolyzed TEOS

A mixture of 2. ml of absolute ethanol, 0.86ml of 0.1M of HCl were added to 2.5ml of TEOS under stirring. The obtained solution was kept under stirring at room temperature until a homogeneous clear solution was obtained. The solution was aged at least for 24 hours before used in the coating process. The hydrolyzed TEOS solution was used as a host matrix for the indicators.

Preparation of Indicators

Indicators solution (1×10-2M) thymol blue indicator (TB) were prepared using absolute ethanol as solvent.

Stock solution of indicators

The sample solution was prepared by mixing 1ml of blank hydrolyzed TEOS solution and 1ml for each indicator.

Preparation of Silica-immobilized Thin Films

Substrate Cleaning

Glasses were activated by concentrated H2SO4 for 24 hours, then washed with distilled water and ethanol. The surface was finally rubbed with cleaning paper.

Preparation of glass/TB electrodes using Spin coating method

All thin films layers prepared in this work were made by spinning three drops of the solutions onto a clean glass slide. The coating process was performed using the spin coater machine at 900rpm spinning speed for 1 min. period time. To obtain multilayers of thin films a subsequent spin coating method was performed after gradually drying of the previous layer at room temperature for 24 hours, then dried at 80oC for another 48 hours. And repeat the spin coating two or three time. Where the conducting substrate is usually conducting glass, consisting of glass coated with a thin layer of F-doped SnO2.

Sensor design of potentiometric cell

The potential of the indicator electrode relative to that of the reference electrode was measured on a digital multimeter model YDM 302C (China). Potentials were measured to ±5mv. The potential of Thymol blue, sensor indicators electrodes was measured vs. a saturated calomel electrode (SCE). The error in the measurement of the potential due to liquid- junction potentials in these electrolytes is estimated to be about 0.001V.

The solution in a beaker is stirred by means of a magnetic stirrer. The electrodes (indicator and reference) were dipped slowly into aqueous solution (acid or reductant). After the steady state potential was attained, the titration of the acid was carried out by addition of 1ml of the base to the acidic solution, waiting until the steady potential is established and then measured. The potential variation depends on the type of the base, the progress of neutralization process and on the initial concentration of the acid to be titrated. The results were reproducible to satisfactory value of ±5 mV for potential measurements. The process of addition of the titrant was repeated until the equivalence point was reached.

The E-pH relation of Thymol blue electrode:

The E-pH relation of Thymol blue electrode

According to Figure 2 the change of the open circuit potential (E) of the G/ TB indicator electrode with pH . The E-pH plot of the G/TB indicator electrode fits straight line with slope of 53.11mV at 298K. This value is close to the magnitude of the term 2.303RT/F at the corresponding temperature (59.1mV at 298 K). This value is close to the magnitude of the term 2.303RT/F (where: R gas constant, T absolute temperature and F Faraday constant) at the corresponding temperature (59.1mV at 288K). From Figure 2 the E0 value of the sensor electrode, i.e., the potential at [H+] = 1, is computed as 279.1mV relative to the saturated calomel electrode and can determination by:

This equation is applicable for the reversible behavior of working electrode. From the developed Nernst equation, we indicate that working electrodes can be used as pH-indicator. At high or low pH, the electrode indicates pH less than true value as pH glass electrode, it may be due to damage in electrode or existence of alkali metal ions in solution too.

The response time of the sensor

In general, the response time was defined as the time of sensor’s output reach to 90% of the equilibration after the measurement was started, especially to electrochemical sensors [15-17]. Figures 3a-3e show the response time of the G/TB sensor at different concentration of phosphoric acid, acetic acid, Hydrochloric acid, ammonia and NaOH respectively. Response time, in the range of (100-450) seconds was achieved, which rendered the sensor highly practical.

Effect of temperature on the response characteristics:

The importance of temperature measurement when performing pH measurements has already been mentioned in reference to slope correction. Temperature also has an effect of both pH buffers and solutions, as the hydrogen ion activity will increase with increasing pH [18].

The Thymol blue sensor response was evaluated at different temperatures, Figure 4. At lower temperatures, like 288K, the slope of the sensor was about 33.54mV/decade and the sensor would be used for pH measurements in the range from (2-11). However, when the temperature of the test solutions was adjusted to 298K, the slope significantly increased to 53.11mV/decade. By raising the temperature to 313K and 323 K the slope increased to 54.11mV/ decade and 59.75mV/decade respectively. Figure 4 shows the square of the correlation coefficient (r2) for pH measurements using the solid-state sensor, at different temperatures, as compared to pH values obtained by a conventional pH electrode (Hanna Instruments HI 1131 pH combination electrode) was found to change as the temperature increases where as r2 values for measurements at 283K, 298K, 308K, and 318K were 0.9655, 0.9386, 0.9482, 0.9876, respectively. This indicates that better results could be obtained at 298K due to easy and settable to use without heating.

The relation between conventional glass PH electrode and G/ TB indicator electrode

All potential values were converted with respect to the standard hydrogen electrode (SHE). During experiments, pH was also monitored with a commercial glass electrode that was calibrated daily using commercial standard buffer solutions (2-9) [19]. Figure 5 represents the correlation between the conventional glass PH electrode and G/Thymol blue indicator electrode, it can be easily recognized that excellent correlation between the results obtained by the solid-state pH sensor and the conventional glass pH electrode could be achieved. The slope of this relation was 0.947 and the r2 was 0.947. This indicate that G/TB indicator electrode potential values are closed to the values of conventional glass pH electrode.

Potentiometric of weak acids against NaOH

Figures 6a & 6b represent the potentiometric titration of 0.1M NaOH with different concentrations of acetic acids and phosphoric acid. The variation of G/TB electrode potential at 298K with the different volumes of standard 0.1M NaOH followed typical potentiometric titration curves. These curves show slight decrease in potential (to more negative values) with the addition of the titrant. Where Figure 6c show the potentiometric titration between the volume of 0.1M standard HCl against ammonia. The variation of the TB electrode potential at 298K with the different volumes of standard HCl followed typical potentiometric titration curves. These curves show slight increase in potential (to more positive values) with the addition of the titrant.

Location of endpoints

Figure 7a represent ΔE/ΔV against V plot for the potentiometric titrations of CH3COOH and H3PO4 with 0.1 M standard NaOH. From the plots the values of end points were determined. The obtained results and calculated values of (R%) are listed in Tables 1 & 2 for acetic acid and phosphoric acid respectively. The values of pKa for different concentration of acetic acid and phosphoric acid were calculated using the method of half neutralization as shown in Table 3. There are two jumps in the titration of phosphoric acid with NaOH using G/TB sensor. i.e two end points appear by using this electrode. The obtained values of pKa for the investigated bases are close to the previously reported values. Where Figure 7b represent ΔE/ΔV against V for the potentiometric titrations of ammonia and 0.1M standard HCl respectively. From the plots the values of end points are determined.

Finally, the values of pKb for different concentration of ammonia can be determined using the method of half neutralization. They are listed in Table 3 for the tested bases. The obtained values of pKb for the investigated bases are close to the previously reported values.

Conclusion

This study investigated the preparation of the modified electrodes of type glass/ thymol blue G/TB and their use as sensor indicator electrodes in the potentiometric acid-base titrations in aqueous solution at 298K. E-pH curve is linear with slope of 0.053.1V/decade for the G/BTB electrode at 298K. This value is close to the theoretical value 2.303RT/F (0.059V at 298K) and the recovery percentage for potentiometric acid-base titration using G/TB as indicator electrode was calculated.

i. On other hand the standard potential of the tested electrode, E0, is computed as 279mV with respect to SCE as reference electrode. Acetic acid, phosphoric acid, hydrochloric acid and ammonia were successfully potentiometric titration with NaOH as titrant in aqueous medium at 298K. Finally, this study is applied in different temperatures like 283K, 298K, 308K, and 318K were the correlation coefficient (r2) 0.9655, 0.9383, 0.9482, 0.9876, respectively.

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The Effects of Freeze-Thaw Cycles and of Storage Time on the Stability of Proakap4 Polypeptide in Raw Sperm Samples: Implications for Semen Analysis Assessment in Breeding Activities

Abstract

Evaluation of the concentrations of the sperm macromolecule called proAKAP4, has been successfully introduced as a pertinent sperm parameter to assess sperm quality and high concentrations of proAKAP4 was shown to be highly correlated with sperm motility and fertility in large mammals. The sandwich ELISA kits known as Pig 4MID® Kits allowed the artificial insemination stations to monitor sperm quality more accurately with threshold values qualifying each ejaculate and animal. Introducing new methods and procedures are always challenging and sperm frozen collections have been suggested to standardize sperm assessment in daily routine. We thus have designed an experimental study to assess proAKAP4 stability and integrity in neat frozen ejaculates. Following baseline measurements, fresh ejaculates were aliquoted and stored at -20 °C for stability experiments up to 6 months and following up to 10 freeze-thawing cycles. ProAKAP4 concentrations were assayed at each time point using the Pig 4MID® Kit and western blot. Median or mean changes from baseline concentrations were evaluated statistically. We showed that the frozen storage conditions neither modified the total proAKAP4 concentrations nor changed the degradation rates of the proAKAP4 into mature AKAP4, that should in turn ensures signaling, capacitation and motility. This sperm parameter was shown then to be robust for semen quality analysis on fresh and on frozen neat semen. Taken together, proAKAP4 polypeptide can be considered as a highly stable analyte when kept frozen in raw semen up to the semen quality analysis using the Pig 4MID® Kit

Keywords: Boar; Proakap4; 4MID®; Fertility; Stability; Precursor; Freeze-thaw cycle; Storage; Semen processing

Abbrevations: AKAP4: A-kinase Anchor Protein 4; PKA: Protein Kinase A; CASA: Computer Assisted Semen Analysis

Introduction

ProAKAP4 concentrations are considered as a new sperm parameter that have been validated by field studies for sperm analysis assessment in large mammals [1-6]. Measurement of proAKAP4 concentrations was thus reported to generate pertinent information to guide the prognosis of sow fertility and prolificity in highly competitive breeding activities [1,4]. This quantitative approach of semen assessment is based on a sandwich ELISA method that allow to compare up to 87 semen simultaneously and is commercialized under the brand name of the 4MID® Kits (4BioDx, France). The Pig 4MID® Kits provide then a reliable and valuable figure reflecting the amount of proAKAP4 in pig ejaculates, with threshold values that are allowing a follow-up of the sperm quality inside and between pig breeding centers. Structurally, proAKAP4 is a precursor protein and will have to be converted by motile and alive spermatozoa in AKAP4 (A-kinase anchor protein 4) that in turn, coordinate the main transduction signals regulating sperm motility, capacitation and fertility [1,7-11]. ProAKAP4 concentrations has been reported to be correlated with total and progressive motility in stallion, in human and in bull [2,3,6,12,13]. Clearly the proAKAP4 concentrations is a reflect of the sperm motility giving a more objective figure compared to microscopic observations of the spermatozoa that are motile only at the time of analysis. In contrast, with the Pig 4MID® Kit, the more the proAKAP4 concentration is high in the ejaculate, the more the spermatozoa will be motile and efficient to go up to the site of fecundation. They have been evidences that spermatozoa with few or without proAKAP4 will be less motile or immotile and then infertile [14-18]. Therefore, we considered as essential to determine the stability and integrity of the full-length proAKAP4 in frozen storage conditions before the critical step of the sperm quality analysis. Data concerning the effects of freezing, thawing, and long-term storage effect on sperm proAKAP4 concentrations were not yet available in the literature. In this study, we aimed then to examine the analytical stability of proAKAP4 in fresh boar semen. We then assess the variations of proAKAP4 concentrations and proAKAP4 degradation rates in following freeze-thaw cycles and in long-term storage at minus 20 °C, in a final goal to improve operating procedures for semen analysis in swine breeding centers.

Materials and Methods

Sperm Preparation

Fresh boar sperm samples (Large White strain) were obtained from a boar stud and was first checked for total volume. They were then aliquoted into 1.5-mL polypropylene cryovials for the stability experiment. For stability assessment, the sampling of each ejaculate was then composed of 5 aliquots (2 for freezethaw cycle experiment and 3 for long-term storage experiment). Following baseline measurement (T0), they were all maintained frozen until analysis. The remaining boar ejaculates were either processed for the control quality experiment or for proAKAP4 expression controls. Samples stored at -20 °C were kept in a freezer equipped with a temperature recorder.

Freeze thaw Cycles and Long-term Storage Experiments

After 24 hours, 2 frozen sperm aliquots were thawed at room temperature until completely thawed, and then mixed properly with a micropipette before analysis (freeze-thaw 1). Samples were immediately re-frozen at -20 °C. This cycle was repeated for ten consecutive time points (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10) to yield freeze- thaw processing. A group of 3 semen aliquots were stored at -20 °C for up to 1, 3 and 6 months, and then analyzed for stability at three-time intervals (T1M, T3M, T6M). As described below, the concentrations of proAKAP4 were assessed at each time point using the Pig 4MID® Kit (4BioDx, France). In parallel, proAKAP4 expressions and metabolism of the same aliquot were examined by western blotting. The results were compared with those obtained from the initial analysis of fresh samples. Median or mean changes from baseline (T0) concentrations were evaluated statistically.

Spermatozoa and Seminal Plasma Preparation from Boar Semen

A volume of 500μL of the remaining fresh semen was added in a 1.5mL Eppendorf tube and then centrifuged during 10 min at 2000rpm. The supernatant over the spermatozoa pellet was recovered with a 200μL micropipette and corresponded to the seminal plasma fraction. One volume of Tris Buffer (10 mM Tris HCl pH 6.8) with 2% SDS was added to the seminal plasma and sonicated at 22kHz (15 Watts) for 30 seconds. In parallel, 250μL of Tris Buffer with 2% SDS was added to the spermatozoa pellet, mix thoroughly with a vortex and then sonicated for 30 seconds (22 kHz, 15 Watts). Protein concentrations were determined using the Bradford’s method (BioRad, France). Then 50μL of the Tris-SDS sample was added to 1 volume of 2x concentrated NuPAGE LDS Sample Buffer (ThermoFisher, USA) and 10μL of NuPAGE Sample Reducing Agent (ThermoFisher, USA). Samples were vortexed and heated at 80 °C for 10 min.

Analysis of pig ProAKAP4 Expression and Metabolism by Western Blot

Equal protein concentrations of each sperm preparation were loaded on polyacrylamide gel (4-12% NuPage Precast Gels) and then transferred onto 0.45μm nitrocellulose membranes (G&E Healthcare, USA) using the Liquid Transfer System (Life Technologies, USA). Membranes were incubated overnight at 4 °C with the first antibody at a dilution of 1:4000 in 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (v/v) Tween 20 (TNT Buffer), either with the clone 7E10, a monoclonal antibody anti-AKAP4 (4BioDx, 4BDX-1602, France), or with the clone 6F12, a monoclonal antibody anti-proAKAP4 (4BioDx, 4BDX-1701, France). After washing 3 times in TNT (10 min), each membrane was incubated with the secondary anti-mouse antibody coupled to horseradish peroxidase at 1:50000 diluted (Vector Laboratories, Burlingame, CA USA) and revealed with the ECL™ chemioluminescence kit (G&E Healthcare, USA). Images were acquired using the Image Quant™ LAS 4000 system (G&E Healthcare, USA).

The Pig 4MID® ProAKAP4 ELISA Assays

Thawed semen samples (respectively 50, 25 and 12μL) were mix with the Pig Lysis Buffer (450, 475 and 488μL) and then proceeded for ELISA quantification using the Pig 4MID® Kit (4VDX-18K2) according to the manufacturer’s instructions (4BioDx, France). Briefly, 100μL of semen lysates was then added to each well of the antibody-coated plate. A solution with conjugated proAKAP4 antibody was then added and after appropriate washing, the complexed sandwich was incubated with a substrate solution. The resulting color intensity was proportional to the amount of proAKAP4 present in each semen sample and could be measured by spectrophotometry at 450nm. A standard curve was determined in parallel for precise concentrations of proAKAP4 in the pig semen sample. Results of proAKAP4 concentrations were always expressed in ng/mL.

Statistical Analysis

Statistical analysis was achieved using Prism 8.2 GraphPad software (GraphPad Software, USA). D’Agostino and Pearson normality tests were performed to determine if the populations were following a Gaussian distribution and Pearson correlation coefficients were determined for each proAKAP4 concentration. The threshold for statistical significance was set to be p<0.05. In normally distributed groups, results were presented as mean ± standard deviation. The significant differences from T0 value were determined by a non-parametric paired samples t-test Mann Whitney U-test. Stabilities of proAKAP4 after freeze thaw cycles and after long term storage were assessed by the percentage change from T0 for paired groups (T0-T1, T0 -T2, etc. and T0 – T1M, T0 -T3M, etc.). Bias was calculated by the formula: [(CX - C1)/C1] × 100%, with C1: the mean or median of the T0 sample; and Cx: the mean or median of the experimented sample. For non- Gaussian groups, median variations from T0 were determined by non-parametric Friedman test and Wilcoxon signed rank test

Results

ProAKAP4 Expression in Boar Raw Semen

As observed previously from other mammals [1-3] proAKAP4 was only expressed in spermatozoa preparation and not in the seminal liquid as revealed with the monoclonal antibody (clone 6F12) against proAKAP4 (Figure 1). The proAKAP4 was cleaved in AKAP4 mature protein and the prodomain was released (Figure 1A). This cleavage and metabolism of the precursor proAKAP4 can also be followed by western blotting using specific monoclonal antibodies such as the clone 7E10 which recognized the C-terminus of both proAKAP4 and AKAP4 (Figure 1B). Therefore, in this initial T0 experiment, we observed the same amount of proAKAP4 and AKAP4 in the spermatozoa preparation sample of the fresh pig ejaculate. As expected, we confirmed that proAKAP4 is a spermatozoa specific protein expressed in the flagellum of pig spermatozoa

Stability of Boar proAKAP4 during Freeze-Thaw Cycles of the Same Aliquot

The concentration of proAKAP4 was measured in the ejaculate using the Pig 4MID® Kit as T0 value for the stability experiments. The initial mean concentration of ProAKAP4 was of 50.7 ± 1.3ng / mL, reflecting a high-quality semen [1]. After semen aliquots have been frozen and thawed up to ten times, there were no statistically significant differences in proAKAP4 concentrations as quantified using the Pig 4MID® Kit from T0 to T10 (Table 1).

All concentrations were in ng /mL and indicated as a mean± SD and median (interquatile ranges). Clearly, the proAKAP4 concentrations were not modified statistically after ten freezethaw cycles and the global percentage of variations was at 9.54%. Dilutions of the neat semen (half and quarter dilution factor) had no effect on the recovery of proAKAP4 concentrations as shown graphically on Figure 2. These dilutions highlighted the robustness of the Pig 4MID® Kit to quantify accurately the amount of the proAKAP4 polypeptide in neat pig semen. We checked then the expression and metabolism of proAKAP4 by western blotting (Figure 3). None of proAKAP4 and AKAP4 expressions or metabolisms were altered by the freeze-thaw cycles. Neither the integrity of proAKAP4 or AKAP4 was shown to be altered along the 10 freeze-thawing cycles and proAKAP4 was not further converted into AKAP4 showing that proAKAP4 and AKAP4 processing were not modified by freeze thawing cycles. The proAKAP4 was therefore considered as a very stable analyte when kept frozen in raw semen until we performed the Pig 4MID® Kit analysis

Stability of the Frozen proAKAP4 Polypeptide in longterm Storage Conditions

They were no significant variation in proAKAP4 concentrations as measured with the Pig 4MID® Kit for fresh pig sperm when stored until 6 months at -20 °C (Table 2). No variations were obtained when stored at - 80 °C (data not shown). Statistical significances were evaluated as described in the Materials and methods section. Our results showed that total proAKAP4 concentrations were clearly stable up to six months of storage at -20 °C with the variation in proAKAP4 concentration always below 5%. The western-blot analysis displayed no degradation of the sample stored at -20 °C from up to 6 months highlighting the robustness of the protein when kept frozen in raw semen (data not shown).

Intra-assay and Inter-Assay of the Pig 4MID® Kit

We further assess the robustness of the Pig 4MID® Kit by evaluation of the intra-assay and inter-assay CV’s on the Pig 4MID® Kit with neat pig semen as in the design of our study. These intra-assay and inter-assay CV’s were performed with two different ejaculates of the same animal (Table 3). Inter-assay variation was assessed from 10 determinations (with 2 aliquots each day) on ten consecutive study days, and intra-assay variation was calculated from eight sequential determinations obtained from the first day of the study period

Discussion

This study examined the storage effects and repeated freezethaw cycles on pig proAKAP4 sperm protein integrity in preanalytical conditions (meaning before the 4MID® Kit procedures) to evaluate the robustness of this new parameter in daily routine of semen analysis for swine breeding activities. We clearly show that proAKAP4 polypeptide is highly stable when frozen at minus 20 °C, for a long time period (up to 6 months) and will not be altered by multiple freeze-thaw cycles in neat semen. These data are of importance as they highlighted for the first time, that specimens of one ejaculate can be aliquoted and kept at minus 20 °C until their analysis and shipped from AI stations to central laboratories without loss of proAKAP4 integrity.

The reason of this stability could be due to the localization and the inherent functionality of the proAKAP4 itself. As shown on Figure 1, the proAKAP4 is a sperm specific protein that is neither found on the membrane nor released in the seminal plasma. The proAKAP4 polypeptide is inside the spermatozoa, more precisely in the fibrous sheath of the principle piece of the flagellum [19-22] and will need to be released from the fibrous sheath to be further quantified using the 4MID® assay. ProAKAP4 has been shown to be strictly localized to the principal piece of the flagellum and not in other spermatozoa compartments [20-21], tightly anchored to the fibrous sheath, along the longitudinal columns and ribs of the sperm tail [2,3,20-21].

According to the Pig 4MID® assay procedure, the proAKAP4 has then first to be extracted from the spermatozoa. Proteins markers described in sera or in seminal fluids [1,23] are frequently reported to suffer from the shear stress induced in buffered solutions and from long-term storage conditions. In contrast of what we reported with sperm proAKAP4, proteins in buffer solution can be fragile and they may even acquire conformations susceptible to degradation during frozen and post-thawed conditions. Clearly, proAKAP4 concentrations appears to be stable as long as the polypeptide is maintained in neat semen within the spermatozoa flagellum, with the fibrous sheath bringing stability for proAKAP4 integrity. The maintenance of proAKAP4 as a fulllength precursor is then important for the aliquot processed for the initial quality assessment of the ejaculate and at further steps, for the quality control during dose processing in AI stations. High proAKAP4 concentrations in the ejaculate and then in doses, will ensure to have enough motile and functional spermatozoa populations in the hours post the artificial insemination

The total amount of proAKAP4 per spermatozoa is fully synthetized within the testis and before ejaculation. Therefore, an aliquot of the ejaculate could be frozen immediately after semen collection in boar studs as this will represent the exact picture of the long-term motility of the spermatozoa. Freezing of an aliquot of ejaculate at collection point will then facilitate the analysis of semen (related to the proAKAP4 concentration) and favors also transport of such aliquot up to external laboratories. Our results clearly showed that degradation rates of the proAKAP4 were not impacted by frozen storage conditions of the aliquot and are in favor of such collection for delocalized sperm quality assessments. Furthermore, proAKAP4 stability when stored in aliquots in sperm frozen collections, will allow to better take in account technical and logistical constraints such as i) delays in shipping frozen aliquot when in need to analyze hypofertile animal; ii) being less dependent of any power cut or voltage fluctuations of the low-cost freezers; or the use of frost-free freezer that goes through numerous defrost cycles, as may happen in small breeding centers.

In boar stud, the storage of frozen aliquoted samples could also be convenient to process all the semen in the same time to compare ejaculates of different animals at the end of collection time. The dose semen processing will then not be impacted as the 4MID® analysis will be completely run in 2 hours. The amount of proAKAP4 as a read out of sperm quality should add marketing values for AI stations by ensuring high quality semen. In swine industry, there is also a real interest to identify the best male and then to follow up the sperm production during exploitation. Boars are usually kept from 6 to 9 months in the AI stations. That will be of importance to have a stable parameter to follow animal along all his career and keeping a safe measure of the initial quality of the first ejaculate after quarantine. In this context, the storage of frozen aliquoted samples may allow likewise to identify genetical traits of interest in a particular pig strains, such as fertility, death at birth or litter size, that may be related to proAKAP4 levels of expression [1,24-27].

Finally, keeping frozen aliquots of pig semen could allow to reanalyze the same samples stored to confirm previous results or to perform additional analysis, establishing new path for boar sperm preservation investigations. Better understanding proAKAP4 stability allows now to compare ejaculates at different collection points and compared to extended semen which is being shipped and used many days later. The storage capacity of extenders should be then further explored in relation with proAKAP4 consumption and degradation rates, during several days and in chilled conditions, when spermatozoa will stay alive.

Conclusion

One of current challenge of the swine industry is to standardize the semen processing procedures within boar studs. The proAKAP4 parameter have been initially introduced to facilitate the identification of ejaculates of inferior motility and quality, that were not identified by classical sperm parameters, and that could then be withheld before their release into the field. Having a stable sperm parameter such as proAKAP4 that can be kept stable as frozen up to the analysis time should be further interesting for quality check control and to follow up this parameter evolution during all the boar career. Taken together, the proAKAP4 parameter stability present then multiple advantages in favor of harmonizing sperm quality assessment between laboratory and AI centers.

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What Indicators Could be Useful to Understand the Development of Digestive Tract, During the Early Ontogeny of Teleost Fish?

Authored by:  Cuenca-Soria C A

Abstract

Nowadays, the most of studies about the digestive physiology and structure changes during the initial ontogeny of cultured fish, have been focused to describe the indicators (associated with those changes), punctual and reclusively. Moreover, there are not enough indicators, when the digestive morphological and functional changes, are described in certain fish in particular. The present review, try to describe and compile the indicators of digestive morpho functional development, attending the feed habits, as well as the kind of ontogeny that undergo the teleost fish, which, it almost not be considered. The review tries to explain, the importance of show possible synergy between biochemistry (enzymology), histological, histochemical, molecular, mainly genomic aspects (among others); towards an integral and reliable description of the morpho functional changes in the digestive tract of fish. In this sense, the present work, try to elucidate a particular case of Mayaheros urophthalmus (a native species from the Southeast of Mexico), where it has described a pool of indicators associated to the morpho functional changes of its digestive tract. The indicators of digestive morpho functional development can be therefore, complementary mutually. Finally, it has compiled and summarized some indicators of digestive morpho functional changes, towards a better knowledge of the digestive physiology, during the initial ontogeny of teleost fish.

Keywords: Ontogeny; Indicators; Development; Morpho functional; Teleosts; Nutrition

    Introduction

At present, the optimization of the nutritional quality of live feed in teleost fish larviculture has directed research to the use of live feed with a view to strengthen the larviculture phase, the most critical in fish culture. Despite this leading to unquestionable progress in the culture of freshwater and marine fish, the high cost of establishing infrastructure for secondary cultures has seriously decreased the profitability of aquaculture. The nutritional quality of live feed does not always satisfy the nutritional requirements of larvae during culture. The production of live feed may thus face difficulties such as a variable nutritional quality and supply [1,2]. Since some decades ago, studies have tried to determine the physiological capacity of organisms to hydrolyze the ingredients present in the diet, from the point of view of their enzymatic machinery, and have progressively obtained more encouraging results, though not completely successful. Rosenlund et al. [3] established that inert diets make it possible to introduce nutrients that are not available in live feed, and to determine the capacity of organisms to digest particular ingredients. Such studies, which have revolutionized the point of view about the processes of ingestion, digestion, and assimilation of nutrients of fish farmer, during their early ontogeny.

It has increased the knowledge about the most important morphological events, in the digestive tract that take place, throughout this critical stage, as well as on the functional events (considering laboratory and field records have shown that morphology and function are closely related). The sequence of events that characterizes the most important changes, that take place, throughout the morphological and functional development of the incipient alimentary canal in fish larvae; is a clear indicator of the process that starts with fertilization and embryo development, will finally end in the morphological-structural maturation of the digestive tract, and attached glands, and their proper functioning. Such indicators, which may reflect the capacity of the fish to experiment changes, from larva to juvenile, in a relatively short time; towards a better understand the maturation process of the digestive function. It has been possible to determine the precise instant of the beginning of the change from larva to juvenile and, thus, of a change in feeding regime (from live feed to inert food).

The indicators may also represent tools for the diagnosis of the nutritional condition of cultured fish, as Gisbert et al. [4] stated. The indicators used up to now in studies on the early ontogeny of commercially important fish have been mainly morpho functional, biochemical, histochemical, immunological, and molecular. They are important because they provide a greater number of elements that will progressively make it possible to do without live feed through the design of inert food that agrees with the physiology and digestive capacity of the cultured fish and will result in a significantly more profitable fish farming activity. This review describes the morpho functional changes that take place and analyses the indicators have been already validated, as tools (and potential indicators), in order to determine the degree of development of the digestive morphology and function during the early ontogeny of teleost fish.

    Types of Ontogeny and Indicators

Ontogeny is defined as the morphological and functional changes that fishes undergo, during their early biological life. A successful development of the digestive system is essential for the survival and growth of fish larvae as it allows them to capture, ingest, digest, and absorb food [5], as do the adults of their species. Although fish larvae are morphologically capable of capturing food items in their first exogenous feeding [6,7], the digestive system requires a series of structural changes before becoming fully functional [8]. The general sequence of events during the ontogeny of teleost fish, were described by Govoni et al., [9]; who proposed that a primitive intestine is segmented into three regions: the foregut, midgut, and hindgut. The foregut forms the esophagus and stomach, the midgut forms the small and big intestines, and the hindgut forms the rectum and anus. Balon [10] described three types of ontogenies in teleost fish: indirect, transition and direct, and proposed that ontogeny is a hierarchical model of fish life history. Thus, the ontogeny had split, into the sequential periods of embryo, larva, juvenile, adult and mature (senescence) in the case of indirect ontogeny; and embryo, juvenile, adult and mature in the case of direct ontogeny (note the absence of the larval period).

In the case of transition ontogeny, the model proposed the periods of embryo, fry, juvenile, adult and mature. The fry step is a type of larval vestige according to Balon [10]. Each period, divided by natural borders and comprises a sequence of organization intervals in “jumps”. The intervals or “homeoretic” states called steps, separated by stable thresholds. The steps considered the basic units in the ontogenetic scale. Thus, the period is the longest interval and separated from the others by thresholds. The phase is the next interval by which, a stage divided by morphological units of lower ontogenetic significance, mainly for identification purposes. The step is the shortest natural interval, separated by thresholds, and the concept of stage is an instant state in ontogeny. Aspects of the ontogeny types that has been mentioned above, are discussed as follow next.

Indirect Ontogeny

The first period, the embryo, is characterized by endogenous feeding, that is to say, by the acquisition of nutrients from parental sources [11]. The time between hatching and the moment when the eyes have reached the pigmentation, is the time during which larvae feed solely on their yolk reserves [12]. This time of endogenous feeding tends to be very short and followed by a long larval stage (that depends on exogenous feeding), that leaves the larvae vulnerable and prone to a high mortality. Numerous species produce eggs and small larvae with organs and tissues that are immature at the hatching [13]. However, many fish with an indirect ontogeny produce a great number of embryos and larvae to compensate for this vulnerability [14]. The larvae may also occupy many niches (plancton, in the case of teleosts) and thus avoid competing for food. Gisbert et al. [4] stated that fish larvae generally hatch more prematurely than other vertebrates, suggesting that the space-time sequences in teleost larvae are notably different from those of the higher vertebrates.

In consequence, fish larvae go through significant morpho-structural and physiological changes during the first weeks of life, such as has been observed in flounders [15]. Many studies have been carried out, during the last two decades, in order to determinate the digestive capacity and nutritional requirements of fish larvae and juveniles [16]. Organogenesis and the changes in digestive enzyme activity, as well as the characteristics of the development of the digestive tract, have been well documented for several species, including the European seabass Dicentrarchus labrax [17], the sole Paralichthys californicus [18], the red drum Sciaenops ocellatus [19]. Morpho functional, biochemical, and molecular indicators were considered, following the digestive ontogeny in Balon model (2002), as well as the feeding habits of the species and the studies carried out to date for each case.

Carnivorous fish

At hatching, the mouth and the anus are shut, in some species [20,21]. Such characteristics were observed by Gisbert et al., [22], in the sole P. californicus, including a closed and straight tube (nondifferentiated oropharynx and anus). Between days 1 and 2 after hatching (dah), the oropharynx presents morpho histological features such as some layers of squamous cells and a few taste buds. During these days, the intestine is rudimentary and is made of simple ciliated columnar epithelium. While the yolk sac is present, the hindgut presents a 90° curve and an intestinal valve is formed to divide the intestine in two regions, the prevalvular intestine and the postvalvular intestine. Both regions present a basophilic cytoplasm and prominent eosinophilic microvilli [22]. The postvalvular intestine lacks Goblet cells [21], while the gastric glands appear (indicating digestive immaturity).

However, the activity of certain enzymes is required for larval digestion to take place immediately after hatching. Alliot et al. [23] recorded trypsin and chymotrypsin (pancreatic enzymes), using biochemical techniques, immediately after the hatching of D. labrax. García-Gasca et al. [24] detected trypsinogen gene expression in egg rRNA (75 hours after fertilization, haf) in Sphoeroides annulatus. Gisbert et al. [22] detected acidophilic zymogen grains (pancreatic enzyme precursors) in the exocrine pancreas at 1 dah, before the start of exogenous feeding, with an increase in their numbers when exogenous feeding started in P. californicus, and Yanes-Roca et al., [25], confirmed the importance of pancreatic secretions in larval development during the early ontogeny of the black snook Centropomus nigrescens. The prevalvular intestine, has been described as the main site for extracellular digestion in the digestive tract during the larval stage, as result of its alkaline pH and the presence of trypsin secreted by the exocrine pancreas [26,27]

Shortly afterwards, and coinciding with the first exogenous feeding, the alimentary canal changes and forms the oropharynx, esophagus, pre and postvalvular intestines and rectum. Several authors have observed this in other carnivorous species like Paralichthys senegalensis [28] and the yellowtail flounder Limanda ferruginea [29]. A fold formed by ciliated columnar cells starts to differentiate at 4 dahs, in the terminal region of the esophagus, from the prevalvular foregut in P. californicus [22]. The number of Goblet cells increases around 10 dahs. These cells secrete glycoproteins and mucin, the substances that make lubricating mucous membranes in the anterior oropharynx and esophagus, an organ in which no histological changes were observed, until metamorphosis, as occurs in P. californicus. At this age, one may also observe a canine tooth in the posterior region of this cavity, and the mucous membrane in the intestine is mostly rectilinear and presents several short folds. The first Goblet cells become visible in both regions of the intestine. Sphoeroides annulatus presents a marked change in the expression of trypsinogen intensity at 13 dahs [24].

The Goblet cells increase in number with the differentiation of the intestinal mucous membrane and are most abundant in the prevalvular intestine. Histologically, these cells dye a dark blue, which have a relationship, with the presence of a mixture of carboxylate and sulfated glycoproteins and of neutral glycoproteins. Folding in the prevalvular intestine increases between 19 and 23 dah and the folds occupy most of the intestinal lumen, whereas the postvalvular intestine presents few folds in a rectilinear mucous membrane [22]. The migration of the eye in P. californicus (27-30 dah) coincides with the differentiation of the gastric glands and the decline of supranuclear bodies in the postvalvular intestine [22], indicating the presence of pinocytic absorption and proteolytic digestion in the intracellular environment, that take place through a five-step process: pinocytosis, transport, accumulation, digestion, and extinction [30]. Larval digestion in fish with an indirect ontogeny is intracellular [9] and becomes extracellular when the transformation takes place.

The intracellular digestion compensates the incomplete digestion that occurs in the primitive midgut, which decreases its contribution to digestion as the alimentary canal matures, especially when a functional stomach appears, which may be the most important indicator of digestive maturation in carnivorous fish. It has been widely documented that a morpho functional indicator of the full maturity of the stomach is the presence of gastric glands in the fundal stomach, whereas a significant enzymatic indicator of stomach maturity is a high level of pepsin which, is secreted by the gastric glands. The occurrence of these three changes is an indicator of the threshold of a digestive tract in an advanced state of maturity. Govoni et al. [9] stated that the presence of vacuoles and supranuclear inclusions is an indicator of a functional alimentary canal. García-Gasca et al. [24] recorded a marked decrease in the levels of mRNA that codes for mRNA trypsinogen, starting at 28 dah in S. annulatus, which coincided with the change in diet from Artemia nauplii to a formulated micro diet. Darias et al. [31] detected a relatively constant trypsinogen expression during the first month of life of Pagrus pagrus that decreased towards the 50 dah, suggesting that trypsinogen plays a secondary role in protein digestion.

The same synchronic behaviour (a decrease in trypsin activity as an indicator of stomach differentiation) was recorded for the striped beakfish Oplegnathus fasciatus [32], although the decrease in trypsin activity was more premature (starting at a peak at 19 dah). However, the most notable change in the stomach, and the start of the juvenile stage as well, is the development of the gastric glands, as Pradhan et al. [33] pointed out. These changes may thus be useful indicators to pinpoint the process of digestive maturation.

Omnivorous fish

Few studies have been carried out on the ontogenetic dynamics of omnivorous fish. In their study on the development of digestive enzymes during the early ontogeny of Mayaheros urophthalmus, López-Ramírez et al. [34] recorded trypsin and chymotrypsin activity before hatching. This was also recorded for the rohu carp Labeo rohita, in which there is amylase, protease, lipase and alkaline phosphatase enzymatic activity at 4 dah [35]. Important digestive enzymes have been documented as present at the moment when the mouth opens [36,37]. The opening of the mouth and, thus, the beginning of exogenous feeding determine the regional differentiation of the intestine in many teleost species [38-40]. Continuing with M. urophthalmus, its larvae present a marked activity of several digestive enzymes at 13 dah, as is also the case in species like P. californicus [41].

The biochemical characterization of M. urophthalmus larvae indicates that precisely at 13 dah is the best moment to substitute live feed with artificial food, and this coincides with a visible increase in the activity of enzymes such as trypsin, chymotrypsin, aminopeptidase, carboxypeptidase, and the acid and alkaline phosphatases [34]. Aminopeptidase and alkaline phosphatase are intestinal enzymes that play a part in the digestion of small peptides and the assimilation of nutrients, respectively [42]. Their sustained increase throughout early ontogeny indicates an improvement in the intestinal digestion capacity [43]. Trypsin has been reported as the enzyme that is most responsible for alkaline proteolysis in Paralabrax maculofasciatus larvae [44], whereas in M. urophthalmus, proteolytic activity is represented by chymotrypsin. This explains why chymotrypsin has been identified as part of the enzymatic machinery of omnivorous and herbivorous fish, while trypsin has been recorded for carnivorous fish [45].

These same authors recorded the presence of trypsin, chymotrypsin, aminopeptidase and catepsin (an indicator of intracellular proteolysis and, thus, of a primitive or immature alimentary canal) before hatching (0 dah) and at 3 dahs, respectively. In the larvae of another omnivorous fish, Pagellus erythrinus (with a certain tendency towards carnivory, like M. urophthalmus), the differentiation of the digestive tract produces an oropharynx, an esophagus, a presumptive stomach and an intestine at 3 dah [46,47], morpho functional indicators that coincide with the first exogenous feeding. From these findings it follows that there is a certain functional capacity in the digestive tract at this age. The indicators that change at the beginning of exogenous feeding in omnivorous fish are an increase in trypsin activity and the start of chymotrypsin activity as an indicator of a possible beginning of mixed feeding (a mixture of endogenous and exogenous food items).

Enzymatic activity may thus be a true indicator of the digestive capacity of omnivorous fish like the common carp Cyprinus carpio Jian variety, as Yan and Qiu-Zhou [48] stated. Despite most of the studies on the early ontogeny of fish having focused on carnivorous species of the Mediterranean Sea and the Northern Atlantic, it is possible to summarize the relevant changes that may be used as indicators of the degree of maturation of a fish, on the ontogenetic scale. Yan and Qiu-Zhou [48] stated that the digestive function is correlated with the development of the intestine in fish without a stomach, such as C. carpio Jian variety. Micale et al. [46] reported on important indicators (time of mouth opening, absorption of yolk sac and formation of gastric glands, among others) for P. erythrinus larvae. Moreover, Cuenca et al., [47], revealed a relationship between the full differentiation of the stomach (histology), peaks of expression of α-amylase (molecular indicator) and peaks of acid proteases (biochemical indicator), in M. urophthalmus, at 13 dde; as synchronous events of maturation of the digestive tract.

Herbivorous fish

Hatching of herbivorous cyprinids like Ctenopharyngodon idellus produces incomplete organisms in which the differentiation of the organs, particularly of the digestive tract [9,49], continues during the post-embryonic stage [50]. Also, when growth rates are as much as 30% d-1 [51], the digestion presents an extremely pronounced enzymatic activity. Exogenous feeding starts even before the absorption of the yolk sac, and the alimentary canal is short and straight during this stage [52] and may measure 50% of the body length [50]. The short larval intestine is capable of breaking down and assimilating easily digestible elements, mainly zooplancton [53]. The intestine grows slowly during the larval stage and the time of intestinal passage is positively correlated with the relative length of the intestine [54].

The time available for the processes of digestion and absorption increases gradually, until the fish changes into an adult [52]. Thus, both the time of passage and the length of the intestine may be considered indicators of digestive maturity in cyprinids, particularly the herbivores. On another note, digestive enzyme activity is low during the first feeding stage and increases during larval development [38]. Also, the digestive enzymes in natural zooplanktonic prey survive in the digestive tract of the predators and minimally increase the enzymatic activity in the hosts [52]. Regarding trypsin activity in adults, it is significant in the foregut and midgut, though not in the hindgut where enzyme activity practically disappears [55]. In larvae, however, hydrolysis continues in the hindgut [52]. Guo-Liang et al. [56] studied the gene expression of trypsinogen and the specific activity of trypsin throughout the ontogeny of C. idellus and suggested that trypsin activity is important in larvae.

The decrease in the activity of this end protease enzyme in the hindgut throughout early ontogeny may be a good indicator of digestive development, at least for the larval stage. C. idellus does not have a stomach, which means that the whole digestive process takes place all along the intestine (mainly the foregut and midgut), which is designed to optimize the digestion and assimilation of nutrients. In agreement with this, the intestinal enzymatic machinery of C. idellus, which includes proteases, lipases, cellulases and amylases, has been analysed by Wu and Zhu [57]. Horn et al. [58] detected a marked amylase and maltase activity in Atherinops affinis (an estuarine herbivorous fish), enzymes that degrade the alpha polysaccharides present in the diet. In turn, adult herbivorous fish owe their high digestive efficiency to the great intestinal surface that is provided by a very long intestine and is available for assimilation and digestive processes.

In relation to this, Horn et al. [58] concluded that the intestine is longer and has a greater intestinal surface in A. affinis than in Atherinopsis californiensis and Leuresthes tenuis, an omnivore, and a carnivore, respectively. However, fish with no stomach and no mechanism of stomach digestion require intracellular digestion, which commonly occurs during the early ontogeny of teleost fish (until the formation of the gastric glands, indicators of the developed differentiation of a functional stomach). Liu et al. [59] detected the presence of catepsin D that plays a part in the digestion of proteins in C. idellus, an enzyme indicator of intracellular digestion like leucine alanine peptidase [27].

Transition Ontogeny

The yolk in fish with a transition ontogeny is abundant and dense during the embryo stage, providing ideal conditions for a rapid change to the juvenile stage (at least compared with fish that have an indirect ontogeny and a longer larval stage). The most representative species of this group are the fish of the Salmonidae family, which, produce big demersal eggs and well-developed fry [60]. Six hours after fertilization, salmonid eggs have small blastodiscs, narrow perivitelline spaces, and lipid globules located around the animal pole and connected to the citoplasmatic region, while the embryo and the oil droplet are located in the upper part of the yolk [60]. Dahl et al. [61] described a comparative study of the morphological characteristics of the progeny of O. mykiss obtained from the wild, from culture, and from hybrids obtained from breeding animals of both groups, in which they observed minimum differences with respect to growth.

Regarding the ontogenetic study of salmonids, Godorilov [62] described the early ontogeny of the Atlantic salmon Salmo salar, established a mathematical model to identify the stages of embryogenesis considering as the minimum unit the formation of a pair of somites, and suggested the model could be applied to other species. One of the most emblematic indicators in fish of this group is the presence of pepsin and trypsin during the hatching of O. mykiss [63]. Pavlov and Moksness [60] compared the early ontogeny of S. salar and Anarhichas lupus (a fish of direct ontogeny) and found greater technological advantages in the culture of A. lupus than in that of S. salar, considering the more complex life cycle of this last species. Rungruangsak-Torrissen et al. [64] studied the different expressions of trypsin and chymotrypsin and their effect on the growth of S. salar, and found that the first decreases in activity when growth is favoured by external (fasting, temperature and diet composition) and internal (life stage) factors, while the second increases in activity under conditions that limit growth (fasting). Thus, enzymatic activity, at least in the salmonid group, is an indicator of the way in which the internal and external factors in fish culture act and interact.

Furthermore, histological, and histochemical techniques have shown an accumulation of protein inclusion bodies, in the epithelial cells of the midgut of salmonid larvae which, results in the intracellular digestion. This event is less important in fish with a direct transition ontogeny, as they already have a functional stomach (before the absorption of the yolk sac and at the first exogenous feeding), similar to those of adult fish [9]. Thus, fry (a stage exclusive of fish with a transition ontogeny) present most of the characteristics of juveniles shortly after hatching (as they hatch also as eleuthero embryos), which makes them very developed larval vestiges. In this sense, Sarieyyüpoğlu et al. [65] recorded the start of the formation of the stomach from the mucous membrane of the esophagus in the freshwater trout O. mykiss at 2 dah. This offers advantages to fry as, at this stage in the ontogenetic scale, they present a structurally and functionally more advanced digestive tract at the time of hatching, compared with fish with an indirect ontogeny.

Direct Ontogeny

Studies on morpho functional changes in fish with a direct ontogeny are few (particularly in the case of species that are cultured). The embryonic development of this group follows the general pattern of teleost fish, though with marked differences. An internal fertilization has been showed in the Atlantic wolffish Anarhichas lupus [66]. Some species like Xenomelaniris brasiliensis (a brackish atherinid fish with an embryonic stage that lasts approximately 143 h, the moment when hatching takes place) have well pigmented eyes and the first outlines of pectoral fins even before hatching [67], these being typical indicators of fish with a direct ontogeny. On the other hand, most marine fish larvae do not have the capacity to feed immediately after hatching and depend on the yolk sac (not dense) until their eyes and mouth become functional. In contrast, at the hatching, A. lupus is an eleuthero embryo with characteristics of a premature juvenile that allow it to adapt immediately to the pelagic environment [60]. At the start of the first exogenous feeding, A lupus has a developed morphology, especially regarding the digestive system [60], that provides it with a greater probability of survival and a reduced vulnerability during early ontogeny. Hellberg and Bjerkås [68], suggested that an increase in the amount of lipids in A. lupus was associated with the maturation of epithelium cells in the intestine. Thus, the capacity to absorb lipids in the intestine of fish with a direct ontogeny may constitute a clear indicator of intestinal differentiation during ontogeny, especially during the larval stage.

    Morpho functional Indicators of Maturation

According to Ma et al. [69], there are two stages of capital importance in the process of maturation of the digestive function in fish larvae: first the functionality of pancreatic secretions and second the formation of the epithelium with the brush border membranes of the intestine. These events have described in D. labrax [70], Solea senegalensis [18] and S. ocellatus [19,71]. Notwithstanding these changes are complete in most species, physiological changes take place from the moment of the first exogenous feeding until the transformation into juveniles. Such changes include the reorganization of the muscular trunk, the differentiation of the gills, and of bone tissue; as well as the development of a functional stomach and the establishment of acid digestion [6,13,18,40,72-74].

These last changes indicate that the individual is in a condition of digestive maturation that allows it to carry out the ingestion, digestion, and assimilation of nutrients efficiently. Gisbert et al. [4] pointed out that histological indicators are a useful and precise tool that helps understand the transcendental changes that take place during the early ontogeny of teleost fish, even considering their limitations in the study of wild populations. The profile of the digestive enzymes is also an indicator of the digestibility and use of nutrients [35]. For example, it has revealed that pepsin, trypsin, and amylase activity is high in omnivore and herbivore larvae. In contrast, carnivore larvae have a high activity of pepsin, trypsin, chymotrypsin and aminopeptidase, and a very low activity of amylase, with a few exceptions like O. mykiss. The presence or absence of amylase activity is a useful indicator of the feeding habits of a species, but also of digestive maturation, as activity is high in the early larval stages of several species and decreases when morpho functional maturity is established [75]. A high amylase activity in larvae could be associated with the activity of lactase in the postnatal stages of mammals, which decreases when digestion becomes similar to that of the adults of the species.

Another important indicator of structural and functional changes during the development stages of many species is the relationship between extracellular digestion represented by enzymes of the brush border (aminopeptidase N, alkaline phosphatase, and maltase) and intracellular digestion measured by cytosolic enzymes (leucine alanine peptidase). While cytosolic activity predominates during the first days after hatching, it decreases at the same time that the enzymes of the brush border increase in activity, together with the development and establishment of the intestinal mucous membrane. According to a functional point of view, digestive activity depends on several factors, among which the hormones stand out. An important indicator is cholecystokinin (CCK), a hormone produced in the foregut and is responsible for pancreatic enzymatic secretion, the secretion of bile, and the contraction, filling and emptying of the intestine [76]. Despite there being few studies on this aspect, CCK has been detected at 1 dah in the Japanese sole Paralichthys olivaceus [77], the bluefin tuna Thunnus thynnus [78] and the sweet fish Plecoglossus altivelis [79]. However, no CCK has been detected in the intestinal tract of the Atlantic sole Hippoglossus hippoglossus [80,81], indicating that the presence of this hormone may depend on changes in the levels of expression determined by genetic and/or nutritional factors.

    Molecular Indicators of Fish Maturation

Functional genomics studies were initiated in model species, and then in those marine and freshwater which, economic importance with de goal to understand the ontogenetic changes occurring in first stages of development in order to improve the feeding schemes in these critical days. Fish physiology studies include the zebrafish Danio rerio [82], medaka Orzias latipes [83], pufferfish Fugu rubripes [84], channel catfish Ictalurus punctatus [85], Atlantic salmon Salmo salar [86], turbot S. maximus [87], European seabass D. labrax [88], between others. These studies summarize the dramatic changes, which, fish undergoes along the first days of development until reaching a definitive phenotype.

The ontogenetic changes are represented by morphological modifications, including cell differentiation and proliferation, growth, and functional maturation until reaching juvenile or adult way of life. Those process, require a high degradation degree at every modification step, which, are genetically programmed. One of main external factors influencing gene regulation is the maturation, is the nutrition, which, play a determinant role, during the first steps phases of grown and development in fish larvae [89]. In this sense, it has been the relationship between the expression of CCK, neuropeptide Y (NPY) and grown hormone (GH) versus the nutrition regime (mainly live prey) in cod larvae G morhua [90]. The former author found that the transcription of level of CCK, as well as the digestive enzymes trypsin and amylase were transcribed, according to the feeding composition, suggesting the use of these indicators of nutrition in the early phase of fish development.

More recently, the functional modifications associated to the GI-tract organogenesis were described during the methamorphosis of Atlantic halibut (Hippoglossus hippoglossus) since an anatomical, biochemical, and molecular perspectives [91]. It was observed that the multiple functions of the teleost stomach develop synchronously during methamorphosis, and it was established a correlation between the transcript abundance of ghrelin and the stomach development, as well as the establishment of the proteolytic activity. In recent years, the majority of fish ontogeny related-studies were carried out by using a small number of target genes by semi-quantitative or qPCR, and/or some studies by in situ hybridization (FISH). Subtractive hybridization and construction of cDNA libraries (SSH), where also utilized to elucidate gene that are differentially expressed and allows the comparison of two DNA population of a fraction enriched in differential distributed molecules, involved in the regulation of ontogeny and related processes. cDNA libraries construction permitted the analysis of thousands of genes or whole genome by microarrays , and to know the mechanisms related to the morphologic, structural, and functional changes in fish larvae, describing the metabolic pathways and gene individual interaction involved in the metabolism, grown, differentiation, digestion, transport, absorption, among other biological process.

The study of Sarrapoulou et al., [92] permits the insight into the molecular basis of development and stress, in aquaculture species by mean of creation of gene expression profiles for sea bream by microarray hybridization and contributes to a better understanding of the genetic background of fish physiology, which, may help to improve aquaculture practices. Functional genomics studies are also possible by the use of microarrays generated in non-model species, such as that performed by Darias et al., [88], where they used cross-species hybridization among fish of different taxonomic families. They used more than 9000 cDNA trout O. mykiis, clones originated from two pooled-tissues libraries [93] to elucidate the changes in gene expression, occurring over sea bass D. labrax larval development. They observed an overexpression of 485 genes related to organogenesis, 29 to the energetics pathways, 67 to the biosynthesis and 5 to digestion. Through this transcriptomic analysis and its validation by qPCR, has been identified several molecular markers which, indicate GI-tract maturation.

During the past several years, the information concerning fish transcriptomic are increasing because the establishment of high-through put sequencing technologies, termed next-generation sequencing (NGS) technologies which, have been exploited to analyze the dynamic transcriptome, and the resulting technology is termed RNA sequencing (RNA-seq), Qian et al., [94]. As consequence of low cost and rapid development of these technologies, the transcriptomes have been studied, in both model and non-model fish, not only for the quantification of change of in expression level, but also mapping and annotating the transcriptome with the aim to understand the biological process, such as development, adaptive evolution, host immune response, and stress response. Thus, in order to summarize, the table 1 shows some relevant indicators, that accompany the main changes of the morpho functional development in teleost fish, principally of cultured species.

    Discussion

Knowledge of the ontogenetic dynamics in teleost fish is fundamental in order to select indicators for the important events that take place in the process of digestive maturation, which may establish a reference for the design of inert food, particularly for the early life stages of commercially important species and of those with a potential for culture. It is necessary to understand the morpho functional changes, as very few studies to date have focused on relating the indicators considered in the present review to the morpho functional development of fish. Even more, little it has been done, to establish the possible synergies among the indicators that have been mentioned, which may describe more fully the relevant and particular changes that take place during the early ontogeny of teleost fish. The prevalence of one type of enzyme in the enzymatic machinery of a fish, may be a prominent indicator of digestive maturation, and thus of the composition of the feed to be provided, and of the approximate times, throughout the early ontogeny of the species under study.

Histological, biochemical, and molecular tools are widely used at present, being extremely useful and allowing a deeper and more precise knowledge of the times at which the relevant events occur throughout the early ontogeny of teleost fish. Samples taken throughout the ontogenetic series may record such changes through the determination of morphological and structural changes, enzymatic activity dynamics, the detection of the genes responsible for the molecular expression of digestive enzymes in time and, in this way, complete the information generated by other techniques. Recording the more or less exact times of enzymatic expression may turn out to be an indicator of important events throughout the histogenesis and organogenesis of the alimentary canal. Despite the high relevance of the biochemical and molecular indicators, it is necessary to first consider the structure (inherent to the morphological indicators), and then the functional aspects of fish development.

Actually, there is not enough information about the role, of abiotic and biotic factors, on the enzymatic mechanisms in teleost fish. Temperature could play a crucial role on the intestinal tract hydrogen strength (pH), of freshwater fish [95]. Furthermore, it has been studied the pH and its effect, on the activity enzymatic in teleost fish, mainly their optimal values, that differ depending on different group of digestive enzymes [96]. Moreover, the knowledge about the role of abiotic and abiotic factors, during the development of digestive tract, in the early ontogeny fish is poor. Finally, it is important to consider more biochemical, genetic, immunological, endocrinological and hematological events (among others), involving complementary disciplines like functional genomics, in order to obtain a greater number of indicators that may lead to a greater understanding of the process of digestive maturation in teleost fish, particularly of commercially valuable species and those with a potential for culture.

Finally, it is necessary to notice that not only is important to have in mind a list of molecular markers or those events involved in organogenesis to describe the changes that occur in initial ontogeny, but also the correlation with those external factors responsible for the phenotype. One of these external factors is the nutrition, because is determinant in enzyme and hormone regulation related to fundamental changes to obtain high quality juveniles. Studies on the digestive larval physiology are of the capital importance, to know their enzymatic adaptations to dietary changes [95]. The combination of physiological studies and the ecological knowledge on teleost fish, could contribute to promote the growth and survival during the larvae fish period [97].

According to the indicators of the morpho functional development above described, it has been analyzed a particular case of M. urophthalmus, a native fish from Southeast of Mexico, which is an excellent candidate for freshwater aquaculture [98]. Finally, it is recommendable, to use at least three tools of study, to interpret different kind of indicators of digestive morpho functional development, simultaneously. In this sense, several indicators could be complementary on the study of biological process, as the morpho functional development, of the digestive tract in teleost fishes.

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Influence of oligochitosans and highly molecular chitosan on Lactobacillus bulgaricus cultivation

Abstract

It was established that decrease of oligochitosans with molecular masses 7.0, 25.4, 45.3 kDa concentration in the process of Lactobacillus bulgaricus cultivation leads to fermented dairy product pH reduction and titratable acidity increase. Further increase in titratable acidity and decrease of lactic acid microorganisms’ amount was determined during the fermented dairy product storage process. Oligochitosans with molecular masses 7.0, 25.4, 45.3 kDa in concentrations interval from 0.0025 to 0.01 per cent did not exhibit prebiotic properties. Active acidity elevation and titratable acidity depression was observed at the chitosan with molecular mass 350 kDa concentration rises. Also increase of highly molecular chitosan concentration leads to elevation of lactic acid microorganisms’ total amount, which was more than three degree as many as total count of lactic acid bacteria in control sample.

Keywords: Chitosan; Oligochitosan; Lactic acidbacteria; Lactose, Lactic acid fermentation; Lactic acid

Introduction

Starters of the Lactobacillus bulgaricus species pure cultures are widely used for manufacturing of functional fermented dairy products with dietary and health-promoting properties. The prospective way of fermented milks production technological development is enrichment with chitosan [1-3]. Chitosan is a biogenic heteropolymer consists of N-acetylglucosaminaine and glucosaminresidues [2,4]. Chitosan has high molecular mass and soluble in organic acids [5,6]. Low-molecular derivatives of chitosan are represented byolygochitosans with a molecular mass from 2 to 50 kDa, which are well soluble in water. Chitosan and olygochitosans are able to interact with Lactobacillus bulgaricus cells by a different mechanism depending of their molecular mass [7-9]. Teichoic acid negatively charged molecules of lactic acidbacteria cells are capable to multi-point ion binding with positively charged high-molecular chitosan, whereas their cytoplasmic membrane proteins interact with oligochitosans [4,9]. The consequence of this process may be a change in metabolic processes in lactic acid bacteria cells. The goal of research was to study the effect of different concentrations of high-molecular chitosan and oligochitosans with varying molecular mass on lactic acid fermentation process driven by Lactobacillus bulgaricus.

Materials and Methods

Targets of research were skim milk, starter culture of lactic acid bacteria Lactobacillus bulgaricus (producer: Dairy Plant “Stavropolsky”, Russia), chitosan with a molecular mass of 350 kDa and a 95 per cent degree of deacetylation (manufacturer: “Bioprogress LLC”, Russia). Oligochitosans with molecular masses of 7.0, 25.4, 45.3 kDa and 96 per cent degree of deacetyration was prepared by the previously described technique [5]. Dry skim milk was reconstituted to a dry mass concentration of (10 ± 0.2) % by dissolving in distilled water at temperature 30 to 35 °C. Reconstituted skim milk after recombination was characterized by the following parameters: mass concentration of fat 0.15 per cent, mass concentration of protein 3.2 per cent,mass concentration of lactose 5 per cent. The solution of chitosan with molecular mass 350kDa in 2 per cent concentration lactic acid aqueous solution with mass concentration 1 per cent was added into skim milk experimental samples for preparation of mixture with final concentration of chitosan 0.0025, 0.005, 0.0075 and 0.01 per cent respectively. Similar experiments were carried out using oligohitosans with molecular masses of 7.0, 25.4, 45.3 kDa in above mentioned concentrations. The starter culture of Lactobacillus bulgaricus was inoculated in the amount of 3 per cent of the total samples volume after pasteurization of the mixture and cooling to the fermentation temperature of (43 - 45) °C. The end of the fermentation process was determined by organoleptic curd density, as well as by titratable and active acidity. Experimental and control samples were stored during 17 days at 4 ± 2 °С after completion of fermentation process. Following parameters were tested in triplicate during storage of control and experimental samples: pH by potentiometry, titratable acidity by titrimetric analysis and total count of lactic acid bacteria (CFU per gram).

Results and Discussions

The effect of highly molecular chitosan and oligohitosans with a molecular weight of 7.0, 25.4, 45.3 kDa various concentrations on fermented dairy products physical and chemical properties during the cultivation of Lactobacillus bulgaricus and long-term storage process was studied.

As shown in Tables 1 &2, decrease in the concentration of oligochitosans leads to significant decrease in pH and increase of titratable acidity after 20 hours of cultivation.

This is explained by the fact that oligohitosans with molecular masses of 7.0, 25.4, 45.3 kDa in concentrations of 0.0025 and 0.005 percent effectively interact with the proteins of the lactic acid bacteria cytoplasmic membrane. This interaction induces bacterial stress [10]. Consequently, lactose enzymatic hydrolysis and lactic acid production are accelerated resulting in titratable acidity increase. The elevation of oligohitosans concentration leads to promotion of their interaction with bacterial cells teichoic acid molecules. This type of interaction influences on lactic acid bacteria cells cytoplasmic membrane permeability and as a result inhibit rate of lactose metabolism. Highly molecular chitosan concentration variation did not lead to significant changes of pH and titratable acidity of fermented skim milk in comparison with control samples. Chitosan with a molecular mass of 350 kDa puts into effective multi-point ion binding with negatively charged teichoic acid molecules of Lactobacillus bulgaricus cells. This is due to the presence into highly molecular chitosan structure of about 1850 amino groups. Lactose assimilation and lactic acid formation rates are changed depending on highly molecular chitosan concentration.

Physical and chemical properties of fermented dairy products during long-term storage at 4 ± 2 °С were studied after the completion of the Lactobacillus bulgaricus cultivation process. It was established that optimal organoleptic attributes (taste and odor) of fermented product control sample are achieved after 5 days of storage at pH 4.2 - 4.5 and titratable acidity 70 - 140 °T. Organoleptic attributes of this product deteriorated during the further storage.

As shown in Table 3, optimal titratable acidity of fermented milks experimental samples containing oligochitosans at a concentration of 0.01 per cent persisted for up to 17 days. Further increase of titratable acidity of experimental samples containing oligochitosans at a concentration 0.0025, 0.005 and 0.0075 per cent was observed during the storage after the completion of the fermentation process.

Decrease in titratable acidity of fermented dairy product experimental samples was detected when concentration of chitosan with molecular mass 350 kDa increased in interval from 0.0025 to 0.01 percent. Therefore high-molecular chitosan concentration elevation reduces the intensity of lactic acid fermentation in experimental samples. The most powerful process of lactose homo fermentative fermentation inhibition occurred in a sample containing high-molecular chitosan in concentration of 0.01 per cent. The decrease of lactose assimilation intensity by Lactobacillus bulgaricus cells may be propelled by two reasons. The interaction between chitosan molecules and lactic acid bacteria cells cytoplasmic membrane leads to disturbance of membrane permeability for β-galactosidase enzyme, which catalases the reaction of lactose into glucose and galactose hydrolysis. At the same time structural changes in cell cytoplasmic membrane cause retardation of lactose hydrolysis products active transport into bacterial cells.

Thus, there is an inhibition of lactic acid formation in the process of fermented dairy product containing high-molecular chitosan storage, which stimulates the preservation of a large number of lactic acid bacteria. This is confirmed by the data of lactic acid microorganisms ‘quantitative accounting in control and experimental samples after 17 days of storage, as shown in Table 4.

The data presented in Table 4 indicates that oligohitosans with molecular masses of 7.0, 25.4, 45.3 kDa did not affect significantly on Lactobacillus bulgaricus grows rates during fermented dairy products storage process. Addition of highly molecular chitosan in concentrations of 0.0075 and 0.01 per cent in fermented milks increased the content of lactic acid microorganisms,which was more than three degree as many as total count of lactic acid bacteria in control sample.

Thus, tested samples ofoligohitosans with varying degrees of polymerization did not exhibit prebiotic properties and did not prolong the shelf life of fermented dairy products. High-molecular chitosan in a concentration of 0.01 per cent can be recommended as a prebiotic, prolonging the shelf life of fermented milks, manufactured with application of Lactobacillus bulgaricus starter cultures.

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