Cryoprotective Role Research Articles

The many roles of fats in overwintering insects.

Temperate, polar and alpine insects generally do not feed over winter and hence must manage their energy stores to fuel their metabolism over winter and to meet the energetic demands of development and reproduction in the spring. In this Review, we give an overview of the accumulation, use and conservation of fat reserves in overwintering insects and discuss the ways insects modify fats to facilitate their selective consumption or conservation. Many insects are in diapause and have depressed metabolic rates over winter; together with low temperatures, this means that lipid stores are likely to be consumed predominantly in the autumn and spring, when temperatures are higher but insects remain dormant. Although there is ample evidence for a shift towards less-saturated lipids in overwintering insects, switches between the use of carbohydrate and lipid stores during winter have not been well-explored. Insects usually accumulate cryoprotectants over winter, and the resulting increase in haemolymph viscosity is likely to reduce lipid transport. For freeze-tolerant insects (which withstand internal ice), we speculate that impaired oxygen delivery limits lipid oxidation when frozen. Acetylated triacylglycerols remain liquid at low temperatures and interact with water molecules, providing intriguing possibilities for a role in cryoprotection. Similarly, antifreeze glycolipids may play an important role in structuring water and ice during overwintering. We also touch on the uncertain role of non-esterified fatty acids in insect overwintering. In conclusion, lipids are an important component of insect overwintering energetics, but there remain many uncertainties ripe for detailed exploration.

Effect of sunflower lecithin on Kalahari Red goat semen during cryopreservation

Abstract Soybean lecithin had been used as an alternative to egg yolk in domestic animal semen extender during cryopreservation due to its characteristic phospholipid content which played a major cryoprotective role. This composition of soybean lecithin informed the replacement of soybean with sunflower lecithin (SL) in the extender for the Kalahari Red (KR) buck semen cryopreservation in this study. Effect of different levels of SL on the quality of the KR buck semen during cryopreservation using slow freezing method was evaluated. Semen samples were collected from four KR bucks of between two and two and half of age using artificial vagina, evaluated for motility and then diluted in extenders containing different levels of SL (1.5%, 3.0% and 4.5%) as experimental group and 0% SL or 20% egg yolk as control. Semen parameters including motility, acrosome integrity (AcI), membrane integrity (MI), malondialdehyde (MDA) concentration, cholesterol level and seminal arginase activity were evaluated for. The results showed that motility, acrosome integrity (AI) and membrane integrity were comparable at 0%, (22.00 ± 4.58, 82.00 ± 3.51 and 96.00 ± 2.03); 1.5%, (23.00 ± 2.08, 87.00 ± 3.79 and 89.00 ± 2.08); 3.0%, (13.00 ± 2.52, 81.33 ± 0.41 and 76.67 ± 1.20) and 4.5% (11.00 ± 4.51, 85.33 ± 9.88 and 84.00 ± 8.50), respectively, after thawing. SL at 0% had the highest (P < 0.05) values for MDA, cholesterol and seminal arginase activity (1.10 ± 0.008 nmol/ml, 236.35 ± 4.08 mg/dl and 0.54 ± 3.3 E-3 units/mg protein, respectively). Our data suggest that 1.5% sunflower lecithin can be used in place of soy lecithin as a substitute for egg yolk during the cryopreservation of caprine semen.

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360–1.7) and stored at −196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05).

Physiological and Biochemical Differences in Diapausing and Nondiapausing Larvae of Eurytoma plotnikovi (Hymenoptera: Eurytomidae).

The pistachio seed wasp, Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae), is one of the main pests in various pistachio growing regions of Iran. This pest passes the winter as diapausing last instar larvae. In this study, the relationship between diapause and cold hardiness and also the physiological and biochemical characteristics the diapausing and nondiapausing larvae of E. plotnikovi were investigated. Digestive α-amylase enzyme showed a high activity (70.41 ± 2.36 µg maltose/min per mg protein) in nondiapausing larvae, but its activity vigorously decreased during the diapause period. Glycogen declined at the beginning of diapause until March. Decrease in glycogen content was proportional to increase in total simple body sugars, trehalose, myo-inositol, and sorbitol contents. Lipid accumulated from the onset of diapause in September until January reaching a high concentration of 28.74 mg/g fresh body weight, but then declined from March to end of diapause in April. The supercooling points were decreased from August (-17.68 ± 0.14°C) to January and reached to its lowest point in January (-23.14 ± 0.27°C), the coldest month of the year, then gradually increased through April (-21.38 ± 0.32°C). The survival rates at low temperature indicate that last instar larvae of E. plotnikovi are most cold tolerant in December-February when total body sugars, trehalose, myo-inositol, and sorbitol concentration is high, suggesting an alternative cryoprotective role for these compounds. The experimental data show that E. plotnikovi is freeze avoidance insect.

The cryoprotectant effect of xylooligosaccharides on denaturation of peeled shrimp (Litopenaeus vannamei) protein during frozen storage

The cryoprotective effects of xylooligosaccharides on peeled whiteleg shrimp were investigated and compared with sodium pyrophosphate treatment during frozen storage, primarily the interaction mechanisms between xylooligosaccharides and shrimp myosin. Data revealed that soaking in the xylooligosaccharides solutions prior to frozen storage markedly reduced thawing and cooking losses in frozen shrimp, which were significantly lower than that of fresh water treatment. The significant inhibitory effects on textural variables (springiness and chewiness) in oligosaccharides-treated shrimp were also found, compared to the control samples. Chemical analyses showed that these oligosaccharides maintained a higher myofibrillar protein content and Ca2+-ATPase activity in frozen shrimp. Additionally, the hematoxylin and eosin staining results indicated that the oligosaccharides significantly slowed the damage to muscle tissue structures. Our assumption was that water replacement hypothesis played a leading role in cryoprotection of frozen shrimp in this study, as shown in previous experimental results and reports. Furthermore, the homology modeling and molecular dynamics simulations confirmed that the oligosaccharides substituted water molecules around shrimp myosin surface by forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the structures in the absence of water, leading to an increase in protein stability during frozen storage. Overall, the contribution of the water-replacement activities played an important role in cryoprotective effects of this oligosaccharides on frozen shrimp. Further application of these findings might maintain better quality and extend the commercialization of refrigerated shrimp products.

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis).

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 μg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 μg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 μM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Insights into Cryoprotective Roles of Carrageenan Oligosaccharides in Peeled Whiteleg Shrimp (Litopenaeus vannamei) during Frozen Storage.

The cryoprotective effects of carrageenan oligosaccharides on peeled whiteleg shrimp were investigated and compared with sodium pyrophosphate treatment during frozen storage, primarily the interaction mechanisms between oligosaccharides and shrimp myosin. Data revealed significant profitable effects on water-holding capacity and textural variables in oligosaccharide-treated shrimp compared to the control. Chemical analyses showed that these saccharides maintained a higher myofibrillar protein content and Ca2+-ATPase activity in frozen shrimp. Additionally, the hematoxylin and eosin staining results indicated that the saccharides significantly slowed the damage to muscle tissue structures. The assumption was that water replacement hypothesis played a leading role in cryoprotection of frozen shrimp. Furthermore, the homology modeling and molecular dynamics simulations confirmed that the saccharides substituted water molecules around the shrimp myosin surface by forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the structures in the absence of water, leading to an increase in protein stability during frozen storage.

Cryoprotective roles of trehalose and alginate oligosaccharides during frozen storage of peeled shrimp (Litopenaeus vannamei).

Cryoprotective saccharides are widely accepted additives that reduce thawing loss, maintain texture, and retard protein denaturation in the frozen seafood. The present study aimed to investigate the roles of trehalose and alginate oligosaccharides on cryoprotection of frozen shrimp, primarily focusing on the interactions between myosin and saccharide molecules using a molecular dynamics (MD) simulation analysis. The results indicated that soaking in the trehalose and alginate oligosaccharides solutions markedly reduced thawing and cooking losses in frozen shrimp, with respective values decreasing to 6.02%, 8.14%, and 5.99%, 8.19% after 9weeks of storage, which were significantly lower than that of fresh water treatment (9.75% and 15.09%). Our assumption was that water replacement played a leading role in cryoprotection, as shown in previous experimental results and reports. Furthermore, homology modeling and MD simulations confirmed that trehalose and alginate oligosaccharides substituted the water molecules around the myosin surface by forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the structures in the absence of water during frozen storage. These conditions affected the flexibility of particular amino acid residues, enhanced the residue cross correlations within the two chains of myosin, and also increased the total interaction energy between myosin and water/saccharide molecules, thereby leading to an increase in protein stability. Finally, by comparing the experimental results to that of MD simulation, significant positive correlation existed between saccharides and the stabilization of myosin in shrimp muscle. The findings of the present study may help better understand the cryoprotective mechanisms of saccharides in frozen shrimp, and the two saccharides may be potentially used as alternative additives in seafood to maintain better quality during frozen storage.

Cryopreservation of goat semen: status and prospects

Artificial insemination is the main tool for the rapid dissemination of valuable germplasm to improve the genetic quality of farm animals. Freezing of spermatozoa of all the domestic species, is a challenging because of variation in size, shape and lipid composition of sperm from different species. Thus, a cryopreservation protocol for semen of one animal species, may not be necessarily ideal for other. Diluents used for semen cryopreservation of domestic animals generally contained similar ingredients. In case of goats, interactions between seminal plasma and egg yolk are deleterious to sperm, however such condition is not observed in other animal species. Therefore, a thorough understanding of sperm freezing in goat will improve the cryosurvivability and fertility of sperm. In this review, various methods for processing, freezing and thawing of buck semen and their effects on survival and fertility of spermatozoa through fertility tests or artificial insemination have been elaborated. The areas discussed in the review include the biochemical characteristics of semen, dilution rate, various diluents, osmolality, buffering capacity and pH of diluent, penetrating and non-penetrating sugars added, the role of cryoprotectants, freezing, thawing of semen and traditional and recent additives used for improving the post thaw semen quality.

Cytoskeletal Pinning Controls Phase Separation in Multicomponent Lipid Membranes

We study the effect of a minimal cytoskeletal network formed on the surface of giant unilamellar vesicles by the prokaryotic tubulin homolog, FtsZ, on phase separation in freestanding lipid membranes. FtsZ has been modified to interact with the membrane through a membrane targeting sequence from the prokaryotic protein MinD. FtsZ with the attached membrane targeting sequence efficiently forms a highly interconnected network on membranes with a concentration-dependent mesh size, much similar to the eukaryotic cytoskeletal network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that the artificial membrane-associated cytoskeleton, on the one hand, suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above the transition temperature. Our experimental observations support the ideas put forward in our previous simulation study: In particular, the picket fence effect on phase separation may explain why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.

Epifluorescence, SEM, TEM and nanoSIMS image analysis of the cold phenotype of Clostridium psychrophilum at subzero temperatures.

We have applied an image-based approach combining epifluorescence microscopy, electron microscopy and nanoscale secondary ion mass spectrometry (nanoSIMS) with stable isotope probing to examine directly the characteristic cellular features involved in the expression of the cold phenotype in the Antarctic bacterium Clostridium psychrophilum exposed to a temperature range from +5 to -15 °C under anoxic conditions. We observed dramatic morphological changes depending on temperature. At temperatures below -10 °C, cell division was inhibited and consequently filamentous growth predominated. Bacterial cells appeared surrounded by a remarkably thick cell wall and a capsule formed of long exopolysaccharide fibres. Moreover, bacteria were entirely embedded within a dense extracellular matrix, suggesting a role both in cryo-protection and in the cycling of nutrients and genetic material. Strings of extracellular DNA, transient cell membrane permeability and release of membrane vesicles were observed that suggest that evolution via transfer of genetic material may be especially active under frozen conditions. While at -5 °C, the bacterial population was metabolically healthy, at temperatures below -10 °C, most cells showed no sign of active metabolism or the metabolic flux was extremely slowed down; instead of being consumed, carbon was accumulated and stored in intracellular granules as in preparation for a long-term survival.

The protective role of antifreeze protein 3 on the structure and function of mature mouse oocytes in vitrification

Several studies have reported the oocyte damage in mice during vitrification; however, little has been known about the protective role that antifreeze protein 3 (Afp3) plays on their cellular structure and function during vitrification. In order to observe the extracellular cryo-protective role of Afp3, four groups were divided randomly. The observations were made for changes in cytoskeleton, expression of the related genes before and after vitrification, and also for changes in the in vitro developmental potential of oocytes. The outcomes were as follows: (i) microtubules, actin filaments and chromosomal integrity were more intact in the vitrification group supplemented with additional Afp3 compared to the vitrification group. In the fresh control group and the group with additional cryoprotectant containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) and sucrose, the organelles were more intact than the other two vitrification groups. (ii) Real-time PCR analysis revealed that the relative quantification of mitotic arrest deficient 2 (Mad2) and centromere protein E (Cenp-e) were significantly higher in the vitrification group with additional Afp3, the fresh control group and the one group with additional cryoprotectant, in comparison to the vitrification group. On the contrary, the expression of cold inducible RNA-binding protein (Cirbp) and kinesin-5 motor protein (Eg5) were up-regulated in the vitrification group compared to the remaining groups. (iii) The fertilization rate and the recovery rate in the fresh control group and the group with additional cryoprotectant were higher than the other two vitrification groups; furthermore, the recovery rate and the fertilization rate in the vitrification group with Afp3 were higher than the vitrification group. However, the blastocyst formation rate in all the four groups showed no statistical significance. In conclusion, Afp3 plays a positive role in the structure and function of mice oocytes in vitrification.

Cold acclimation, de-acclimation and re-acclimation of spring canola, winter canola and winter wheat: The role of carbohydrates, cold-induced stress proteins and vernalization

It is well established that a period of exposure to low temperature is required in order for temperate plants to achieve maximum freezing tolerance. During the cold acclimation process a large number of biophysical, biochemical and molecular changes occur that enable the plant to survive at below freezing temperatures. These include the alteration of carbohydrate and protein accumulation profiles, resulting large quantities of soluble sugars and cold induced stress proteins (dehydrins) thought to function in a cryoprotective role. When fully cold acclimated plants are exposed to warm temperatures (de-acclimation) a rapid turnover of these carbohydrates and proteins occurs, resulting in a plant that no longer possesses an elevated level of freezing tolerance. In certain species, re-exposure to cold acclimating temperatures (re-acclimation) results in re-accumulation of carbohydrates and proteins with a synergistic impact on freezing tolerance. In canola (Brassica napus L.), a full recovery of freezing tolerance is observed upon re-acclimation in both spring and winter cultivars. In contrast, the re-acclimation of winter wheat (Triticum aestivum L.) results in only a 39% recovery of freezing tolerance. Upon further analysis it was revealed that wheat does not accumulate carbohydrates during the re-acclimation period. While certain dehydrins also accumulate during re-acclimation, there is no clear relationship with freezing tolerance and we suggest it is the interaction of these proteins with soluble carbohydrates that is responsible for the development of freezing tolerance during re-acclimation. The role of vernalization in these processes is also discussed.

Cultivation and cryopreservation of cord tissue MSCs with Cord Blood AB Plasma

Neonatal tissues, cord and placenta, are explored as alternate sources of mesenchymal stem cells (MSCs) for their therapeutic applications. Conventionally, MSCs isolated from cord tissues are maintained and propagated in FBS containing medium for promotion of growth and survival of cells. However, for therapeutic use, FBS use is not encouraged as it is of animal origin. Thus, there is a need for replacement of FBS by equally potent and clinically acceptable cost effective sources. The current study is designed to compare the effect of cord blood plasma (CBP) with MSC qualified FBS (M FBS) during culture and cryopreservation of MSCs. MSCs were isolated from cord and placenta and propagated in either M FBS or CBP. The efficiency of the cultures was analyzed by growth curve, morphology, phenotype and functionality. The cryo-protective role of the CBP was evaluated by using it in freezing medium of MSCs. Our data showed that CBP is equivalent to M FBS for culturing placental MSCs with respect to the phenotype, proliferation rate and differentiation to various lineages. However, cord MSCs displayed slow growth rate and reduction in surface expression of CD105 marker in CBP, whereas, the other parameters were comparable. Freezing of MSCs with CBP resulted in reduction of the late apoptotic and necrotic population. Thus, CBP imparts superior protection against cryogenic insults, and appears to be a valuable substitute to M FBS for cultivation and freezing of MSCs.

Cytoskeletal Pinning Prevents Large-Scale Phase Separation in Model Membranes

During the last decades, artificial lipid bilayers have become an important tool in studies of properties of the plasma membrane of cells. One important feature of cell membranes, which has been difficult to recapitulate in the artificial bilayer systems, is the membrane-associated cytoskeleton. The cytoskeleton is believed to affect diffusion of lipid and protein molecules in the plasma membrane, and is considered to be one of the reasons for the sub-resolution size of membrane domains by preventing large-scale phase separation. Mimicking the eukaryotic actin-based cytoskeleton in vitro is inefficient and complicated, owing to the number of components involved and the nature of membrane binding of the actin-network complex. Here we describe a minimal cytoskeletal network formed by the prokaryotic tubulin homologue, FtsZ. FtsZ has been modified to interact with the membrane through a membrane targeting sequence (MTS) from MinD, another prokaryotic protein. FtsZ-MTS efficiently forms a highly interconnected network on the membrane with a concentration-dependent characteristic mesh size, much similar to the eukaryotic network underlying the plasma membrane. Using giant unilamellar vesicles formed from a quaternary lipid mixture, we demonstrate that, on the one hand, the artificial membrane-associated cytoskeleton suppresses large-scale phase separation below the phase transition temperature, and, on the other hand, preserves phase separation above transition temperature. Our experimental observations support the ideas put forward in our previous simulation study [1]: In particular, the picket-fence effect on phase separation explains why micrometer-scale membrane domains are observed in isolated, cytoskeleton-free giant plasma membrane vesicles, but not in intact cell membranes. The experimentally observed suppression of large-scale phase separation much below the transition temperatures also serves as an argument in favor of the cryoprotective role of the cytoskeleton.[1] J. Ehrig, E. P. Petrov, P. Schwille, Biophys. J. 100 (2011) 80.

Production and Characterization of the Intra- and Extracellular Carbohydrates and Polymeric Substances (EPS) of Three Sea-Ice Diatom Species, and Evidence for a Cryoprotective Role for EPS.

Diatoms and their associated extracellular polymeric substances (EPS) are major constituents of the microalgal assemblages present within sea ice. Yields and chemical composition of soluble and cell-associated polysaccharides produced by three sea-ice diatoms, Synedropsis sp., Fragilariopsis curta, and F. cylindrus, were compared. Colloidal carbohydrates (CC) contained heteropolysaccharides rich in mannose, xylose, galactose, and glucose. Synedropsis sp. CC consisted mainly of carbohydrates <8kDa size, with relatively soluble EPS, compared to high proportions of less-soluble EPS produced by both Fragilariopsis spp. F. curta colloidal EPS contained high concentrations of amino sugars (AS). Both Fragilariopsis species had high yields of hot bicarbonate (HB) soluble EPS, rich in xylose, mannose, galactose, and fucose (and AS in F. cylindrus). All species had frustule-associated EPS rich in glucose-mannose. Nutrient limitation resulted in declines in EPS yields and in glucose content of all EPS fractions. Significant similarities between EPS fractions from cultures and different components of natural EPS from Antarctic sea ice were found. Increased salinity (52) reduced growth, but increased yields of EPS in Fragilariopsis cylindrus. Ice formation was inhibited byF. cylindrus, EPS, and by enhanced EPS content (additional xanthan gum) down to -12°C, with growth rate reduced in the presence of xanthan. Differences in the production and composition of EPS between Synedropsis sp. and Fragilariopsis spp., and the association between EPS, freezing and cell survival, supports the hypothesis that EPS production is a strategy to assist polar ice diatoms to survive the cold and saline conditions present in sea ice.

Urea is not a universal cryoprotectant among hibernating anurans: Evidence from the freeze-tolerant boreal chorus frog (Pseudacris maculata)

Freeze-tolerant organisms accumulate a diversity of low molecular weight compounds to combat negative effects of ice formation. Previous studies of anuran freeze tolerance have implicated urea as a cryoprotectant in the wood frog (Lithobates sylvatica). However, a cryoprotective role for urea has been identified only for wood frogs, though urea accumulation is an evolutionarily conserved mechanism for coping with osmotic stress in amphibians. To identify whether multiple solutes are involved in freezing tolerance in the boreal chorus frog (Pseudacris maculata), we examined seasonal and freezing-induced variation in several potential cryoprotectants. We further tested for a cryoprotective role for urea by comparing survival and recovery from freezing in control and urea-loaded chorus frogs. Tissue levels of glucose, urea, and glycerol did not vary significantly among seasons for heart, liver, or leg muscle. Furthermore, no changes in urea or glycerol levels were detected with exposure to freezing temperatures in these tissues. Urea-loading increased tissue urea concentrations, but failed to enhance freezing survival or facilitate recovery from freezing in chorus frogs in this study, suggesting little role for urea as a natural cryoprotectant in this species. These data suggest that urea may not universally serve as a primary cryoprotectant among freeze-tolerant, terrestrially hibernating anurans.

Effect of Incorporation of Additives in Tris‐Based Egg Yolk Extender on Buffalo (Bubalus bubalis) Sperm Tyrosine Phosphorylation During Cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.

Skin ice nucleators and glycerol in the freezing-tolerant frog Litoria ewingii

The brown tree frog (Litoria ewingii) is the only known Southern Hemisphere vertebrate that can survive full-body freezing. Freezing challenges living organisms in many ways, with ice formation in the body producing a suite of physical and metabolic stresses which can damage cells and tissues. The present study looked at two mechanisms that address some of these stresses: cryoprotectants and ice nucleating agents (INAs). Skin secretions from L. ewingii were sampled along with microhabitat substrate and tested for the presence of INAs, which help control ice formation in the body. L. ewingii plasma was tested for seasonal and freezing-induced changes in both glucose and glycerol, which may have a cryoprotective role in freezing-tolerant frogs. Glycerol levels increased on freezing and decreased on thawing, while glucose levels did not change on freezing but increased on thawing. This suggests that glycerol may be acting as a cryoprotectant, although levels are low compared to other frogs. A clear seasonal change was seen in INA activity, with greater activity in winter than in summer. While potent INAs came from the microhabitat substrate, this work has shown for the first time that skin secretions also contain active INAs.

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37 °C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 ± 7.0; 63.4 ± 3.1%, respectively), in comparison to the controls ( P < 0.01) following the freeze–thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups ( P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls ( P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage ( P < 0.05). Malondialdehyde formation was found to be lower (1.8 ± 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze–thawing process ( P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls ( P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.