Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 microg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1-10 microg/ml compared to the control (P<0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 microg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.     

A novel approach for human sperm cryopreservation with AFPIII

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 μg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 μg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 μg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.     

Shedding off specific lipid constituents from sperm cell membrane during cryopreservation

Membrane damage is one of the main reasons for reduced motility and fertility of sperm cells during cryopreservation. Using a model system of sperm cryopreservation developed in our laboratory, we have investigated the detailed changes due to cryopreservation in the plasma membrane lipid composition of the goat epididymal sperm cells. Total lipid and its components, i.e., neutral lipids, glycolipids and phospholipids decreased significantly after cryopreservation. Among neutral lipids sterols, steryl esters and 1-O-alkyl-2,3-diacyl glycerols decreased appreciably, while among phospholipids, major loss was observed for phosphatidyl choline and phosphatidyl ethanolamine. Unsaturated fatty acids bound to the phospholipids diminished while the percentage of saturated acids increased. The cholesterol:phospholipid ratio enhanced and the amount of hydrocarbon, which was unusually high, increased further on cryopreservation. The data indicates that profound increase of the hydrophobicity of the cell membrane is one of the major mechanisms by which spermatozoa acquire potential to resist or combat stress factors like cryodamage. The results are compatible with the view that for survival against cryodamage, sperm cells modulate the structure of their outer membrane by shedding off preferentially some hydrophilic lipid constituents of the cell membrane.     

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.     

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing

In the present study, the applicability of proton NMR spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to the analysis of the lipid composition of human spermatozoa and seminal fluids as well as changes after cryopreservation of human spermatozoa was investigated. Whereas NMR spectra primarily indicated a high content of double bonds within the spermatozoa but no marked differences upon cryopreservation, MS detected intense peaks which could be assigned to phosphatidylcholines containing one docosahexaenoic and one palmitic or stearic acid residue (m/z=806 and 834). In contrast, the seminal plasma contained more saturated fatty acids and especially more sphingomyelin (SM). A freezing/thawing cycle markedly influences the lipid composition of spermatozoa. There was a diminution of phosphatidylcholines (16:0, 22:6 and 18:0, 22:6) and SM (16:0) and the appearance of lysophosphatidylcholines (16:0 and 18:0) and ceramide (16:0). These data demonstrate the release or activation of both phospholipase A(2) and sphingomyelinase in human spermatozoa due to the freezing/thawing cycle. These results were finally confirmed by experiments on the action of phospholipases on lipids containing docosahexaenoic acid.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

Effects of cryopreservation procedures on sperm membranes

Empirical approaches to semen cryopreservation have resulted in the production of young in a broad range of species. However, acceptable levels of fertility in most domestic animal species has not been achieved. In this review, an attempt has been made to describe the complexity of the sperm plasma membrane and the many steps in a cryopreservation procedure where membrane perturbations can occur. Improvement in sperm cryopreservation procedures will require a careful consideration of the complexity of the sperm plasma membrane, the interaction of its components and the influence of cooling, freezing and thawing on these interactions.     

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Spermatozoa were damaged significantly by the cryopreservation cycle in all experiments. Spermatozoa with the best fresh quality had accumulated the largest quantity of lipids upon thawing. A general decrease in the proportion of polyunsaturated fatty acids was observed after thawing. The yolks enriched in n-3 fatty acids failed to improve the quality of sperm following cryopreservation. The proportion of DHA was significantly higher in spermatozoan phospholipids from thawed cells that had been in contact with n-3 yolks. A significant reduction in cholesterol was observed in spermatozoa after the cryopreservation cycle, which correlated with an increased number of acrosome-reacted cells and changes in the parameters of motility. The addition of 3% fish oil to the daily boar ration significantly increased the content of DHA (from 33 to 45% of the total fatty acids) in the spermatozoa. Ejaculate concentrations were significantly increased in the experimental group. DHA-enriched semen did not show improved freezability, at least not as assessed by in vitro parameters.     

Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing

Diluents containing sonicated liposomes of purified phosphatidylserine (PS), phosphatidylcholine (PC) with varying fatty acyl chain lengths and double bonds and cholesterol (CH) alone or in combination, or egg yolk lecithin were evaluated for protection of bull sperm during cold shock produced by rapid cooling from 25 to 0 degrees C and during freezing and thawing. Bull semen was washed twice and diluted to 50 X 10(6) sperm/ml in diluents containing no lipid, 0.5 or 5 mM sonicated lipid or 20% egg yolk and plunged into ice water to cold shock the sperm. Sperm so treated were frozen using conventional methods. The percentage of progressively motile sperm (MS) was estimated prior to cooling, after cold shock, and after freezing and thawing. Lipids with fatty acyl chains of less than 12 carbons were toxic to sperm cells. Phosphatidylserine alone or in combination with PC or CH, but not PC or CH alone, protected sperm from cold shock as well as did egg yolk lecithin liposomes or egg yolk. Liposomes of PS/PC or PS/CH were not better than PS in protecting sperm from cold shock. Lipid concentrations of 0.5 mM were more effective than liposomes at 5 mM in protecting sperm during freezing and thawing. During freezing, PS alone or in combination with PC partially protected sperm, but only PS/CH was as effective as egg yolk in protecting sperm from freeze-thaw damage. It is concluded that defined diluents, particularly those containing PS, may be useful in studies of cryobiology of spermatozoa.     

Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation

The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma membrane disorders (mean ± SEM 9.48 ± 0.65 and 1.62 ± 0.23% in raw and frozen-thawed sperm, respectively). However, both membrane peroxidation and protein phosphorylation were increased (P < 0.05) compared to raw semen (1.74 and 5.20-fold, respectively). There was a correlation (r = 0.73; P < 0.05) between the increase in lipid peroxidation and tyrosine phosphorylation. Seminal plasma, regardless of origin, reduced (P > 0.05) tyrosine phosphorylation present on the surface of cryopreserved sperm; however, lipid peroxidation was not significantly reduced. In conclusion, we inferred that emergence of phosphorylated proteins on the surface of cryopreserved sperm was due to increased lipid peroxidation that occurred during the freezing/thawing process; however, reduced tyrosine phosphorylation that occurred after addition of seminal plasma was triggered by other mechanisms, apparently independent from the reduction in membrane peroxidation.     

The surface coat of chylomicrons: lipid chemistry

Chylomicrons from the thoracic duct lymph of dogs fed corn oil were isolated by centrifugation and disrupted by either freezing and thawing or rotary evaporation and rehydration. A pellet, representing the surface coat, was isolated by centrifugation. Pellets isolated by freezing and thawing contained a higher percentage of saturated triglycerides than pellets isolated by rotary evaporation; the presence of saturated triglyceride in the pellet was probably an artifact of the preparation of the surface coat material at low temperature. Exchange of free cholesterol between surface and core lipid of chylomicrons was complete within 1 hr. The percentage of cholesterol in pellets of surface material isolated by freezing and thawing was about twice that found for pellets after rotary evaporation at 25-40 degrees C. Cholesteryl ester was not present in the surface lipid and that present in the core lipid did not exchange with serum lipoprotein cholesteryl ester. For phosphatidyl choline, the percentage of linoleic acid in lymph chylomicrons was markedly higher than that in clear lymph or plasma, while the percentage of arachidonic acid was lower. Sphingomyelin of lymph chylomicrons was characterized by very high levels of 16:0 and relatively small percentages of very long-chain fatty acids as compared with clear lymph or plasma. The data are consistent with the view that in lymph chylomicrons: (a) cholesteryl esters are dissolved in a core of triglycerides which contain fatty acids derived primarily from dietary fatty acids, (b) free cholesterol is partitioned between core and surface and is freely exchangeable between the two, (c) the phospholipid fractions are present on the surface and are intracellular in origin.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Sea bass sperm freezability is influenced by motility variables and membrane lipid composition but not by membrane integrity and lipid peroxidation

Cryopreserved sperm quality depends on the characteristics of fresh sperm. Thus, it is necessary to establish a group of variables to predict the cryopreservation potential of the fresh samples with the aim of optimizing resources. Motility, viability, lipid peroxidation and lipid profile of European sea bass (Dicentrarchus labrax) sperm were determined before and after cryopreservation to establish which variables more accurately predict the sperm cryopreservation potential in this species. Cryopreservation compromised sperm quality, expressed as a reduction of motility (46.5 ± 2.0% to 35.3 ± 2.5%; P<0.01) and viability (91.3 ± 0.7% to 69.9 ± 1.6%; P<0.01), and produced an increase in lipid peroxidation (2.4 ± 0.4 to 4.0 ± 0.4 μmoles MDA/mill spz; P<0.01). Also, significant changes were observed in the lipid composition before and after freezing, resulting in a reduction in the cholesterol/phospholipids ratio (1.4 ± 0.1 to 1.1 ± 0.0; P<0.01), phosphatidylcholine (47.7 ± 0.8% to 44.2 ± 0.8%; P<0.01) and oleic acid (8.7 ± 0.2% to 8.3 ± 0.2%; P<0.05) in cryopreserved sperm, as well as an increase in lysophosphatidylcholine (4.4 ± 0.3% to 4.8 ± 0.3%; P<0.01) and C24:1n9 fatty acid (0.5 ± 0.1% to 0.6 ± 0.1%; P<0.05). Motility, velocity, cholesterol/phospholipids ratio, monounsaturated fatty acids and the n3/n6 ratio were positively correlated (P<0.05) before and after freezing, whereas, viability and lipid peroxidation were not correlated. Motility and the cholesterol/phospholipids (CHO/PL) ratio were negatively correlated (P<0.05) with each other and the CHO/PL ratio was positively correlated (P<0.05) with lipid peroxidation. Therefore, the results demonstrated that motility and plasma membrane lipid composition (CHO/PL) were the most desirable variables determined in fresh samples to predict cryo-resistance in European sea bass sperm, taking into account the effect of both on cryopreserved sperm quality.     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

Hyperglycemia-induced changes in hepatic membrane fatty acid composition correlate with increased caspase-3 activities and reduced chick embryo viability

Fertile chicken eggs were injected with various concentrations of either d-glucose or l-glucose during the first three days of embryonic development. The exogenous glucose concentrations ranged from 0 to 18.58 micromol/kg egg. At 18 days of development (theoretical stage 44), brains, livers, and blood from chorio-allantoic vessels were isolated from living embryos. Exogenous d-glucose and l-glucose caused increased plasma d-glucose levels, increased plasma alanine aminotransferase (ALT) activities, and decreased embryo viability. Embryo viability was monitored by a reduction in the percentage of living embryos at theoretical stage 44, reduced embryo masses, reduced brain masses, and reduced liver masses. When compared to controls, embryonic exposure to either exogenous d-glucose or l-glucose caused increased caspase-3 activities and increased lipid hydroperoxide (LPO) levels in both brain and liver tissues. Because lipid hydroperoxides are lipid peroxidation intermediates that result in the attack of any unsaturated neutral lipid or unsaturated phospholipid, the effect of exogenous glucose on hepatic membrane fatty acid composition was studied. Exogenous glucose (either d-glucose or l-glucose) promoted reduced levels of several unsaturated, long-chain fatty acids and increased levels of saturated, short-chain fatty acids within hepatic membranes. Exogenous-glucose induced decreases in the ratios of unsaturated/saturated fatty acids and long-chain/short-chain fatty acids within hepatic membranes which strongly correlated with glucose-induced increases in plasma ALT activities and moderately correlated to hepatic LPO levels. These observations are consistent with the hypothesis that embryonic hyperglycemia promotes hepatic membrane lipid peroxidation and hepatic cell death.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.