Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of α-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of α-tocopherol (0, 100, 200, 400, 600 and 800 μM) using the straw-freezing procedure and stored at −196 °C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P < 0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 μM α-tocopherol supplemented frozen-thawed groups. In α-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 °C for 3 h, the expression in 200 and 800 μM α-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P < 0.01) lower in α-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, α-tocopherol, supplemented at 200 μM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.     

Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. The aim of this study was to investigate the effects of different concentrations of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on the cryopreservation of goat spermatozoa in tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with TCG, the second aliquot was mixed with TCG and egg yolk (TCGY), third aliquot was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquots were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3 mg/ml CLC. All samples were cryopreserved in straws over liquid nitrogen vapor and sperm motion Kinetics were measured by computer-assisted semen analysis (CASA) (percent motility (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH)). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant (p < 0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thawed motility, progressive motility and recovery rate were significantly (p < 0.05) higher in 1.5 mg/ml CLC with 2.5% BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome (P > 0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation

Artificial insemination (AI) with post-thawed boar spermatozoa results in low farrowing rates and reduced litter sizes mainly due to cryoinjury or damages to spermatozoa during cryopreservation. Low viability and motility of post-thawed boar spermatozoa are highly associated with apoptosis during cryopreservation. Although glycerol is widely used a cryoprotectant (CPA) for boar spermatozoa cryopreservation, the mechanism and relationship between glycerol and apoptosis-related gene expression needs to be clarified. In this study, we treated boar spermatozoa with different concentrations of glycerol in lactose egg yolk (LEY) extender to evaluate the apoptosis-related gene expression and protease activities of caspases. These results show that: (1) low concentrations of glycerol (2% and 3%) were more suitable for boar spermatozoa cryopreservation; (2) apoptosis-related genes involved in intrinsic mitochondrial and extrinsic death receptor apoptotic signaling pathways were widely expressed in different concentrations of glycerol treated boar spermatozoa; (3) there was a significant positive correlation (r = 0.840, P = 0.037) between the percentage of Annexin V⁺/PI⁺ staining spermatozoa and caspase-6/9 protease activity. In conclusion, 2% and 3% glycerol have the best anti-apoptotic effects, and the expression of Fas/FasL and Bcl-2/Bax have a strong correlation with spermatozoa parameters.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis)

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10 mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3 h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10 mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3 h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.     

Banking North American buffalo semen

The purpose of this study was to develop a procedure to collect and preserve semen from wood bison (Bison bison athabascae) and plains bison (Bison bison bison). Semen samples from three wood and three plains bison bulls were collected by electroejaculation from June through October. In addition, sperm was collected from the cauda epididymis of seven plains bison. Semen was cryopreserved using two commercially available cryopreservation media, an egg yolk-based medium (Triladyl), and a medium free of products of animal origin (Andromed). Sperm morphology and motility were recorded on fresh and post-thawed semen samples. Total sperm motility was not different between plains and wood bison for the months of June (50%), July (69%) and October (54%). However, total sperm motility for wood bison was higher (P < 0.05) than plains bison for the months of August and September (August: 80% vs 55%; September: 73% vs 40%). Plains and wood bison did not differ in mean total and mean progressive motility (35 and 15%, respectively) of frozen-thawed sperm samples. The post-thaw motility of Triladyl-treated sperm was higher (P < 0.05) than Andromed-treated sperm (35% vs 13%, respectively). Interestingly, post-thawed epididymal spermatozoa had higher total motility (P < 0.05) than post-thawed electroejaculated sperm when cryopreserved with a medium free of products of animal origin (Andromed; 35% vs 9%, respectively). In conclusion, we used electroejaculation to collect high quality bison semen, and cryopreserved it for future needs.     

Comparing ethylene glycol with glycerol and with or without dithiothreitol and sucrose for cryopreservation of bull semen in egg-yolk containing extenders

There are few studies performed for investigating the roles of different ratio and cryoprotectants with dithiothreitol or sucrose on sperm motility characteristics and antioxidant capacities of post-thawed bull spermatozoa. The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations as cryoprotectants and dithiothreitol (D) or sucrose (S) (with/without) as antioxidants in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and diluted using both of the dithiothreitol 5 mM or sucrose 25 mM, and control (without additives) was cooled to 4 °C and frozen in 0.25-ml French straws. when compared to control, different doses cryoprotectants and antioxidants addition no significantly increased the percentages of post-thaw sperm progressive and motitilities, acrosome abnormality and plasma membrane integrity (P > 0.05). However, EG3 + S yielded the greatest percentages of the total abnormality (P < 0.05). As regard to antioxidant activities G7 and EG5 led to lowest MDA activity with or without D or S but, these results were not supported to the GPx activity (P < 0.01). The sperm motion characteristics such as VAP, VCL, ALH and BCF gave significantly different results (P < 0.05). When compared the DNA integrity, different doses cryoprotectants without antioxidants addition significantly increased the percentages of the tail intensity and tail moment (P < 0.05). There were no significant differences observed in non-return rates among all treatment groups (P > 0.05).     

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm

Plant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05).     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Effects of kinetin supplementation on the post-thaw motility,viability, and structural integrity of dog sperm

Oxidative stress is one of the major issues associated with cryopreservation because it causes a marked reduction in the post-thaw quality of semen. This study investigated the ability of kinetin to preserve the structural and functional integrity of dog sperm during cryopreservation. Pooled ejaculates were divided into 5 equal aliquots, diluted with buffer 2 supplemented with different concentrations of kinetin (0, 25, 50, 100, and 200 μM), and finally cryopreserved. The optimal concentration of kinetin was 50 μM based on the significantly improved (P < 0.05) motion characteristics and viability of post-thaw sperm samples. Moreover, kinetin-supplemented samples exhibited significantly higher (P < 0.05) sperm counts with the intact plasma membrane, normal acrosomes, mitochondria, and chromatin than control. The beneficial effects of kinetin were also reflected by the significant increase in the expression levels of anti-apoptotic (B-cell lymphoma, BCL2) and protamine-related genes (protamine 2, PRM2; protamine 3, PRM3), and decrease in the expression of pro-apoptotic (BCL2-associated X, BAX) and mitochondrial reactive oxygen species-modulating genes (ROS modulator 1, ROMO1) in kinetin-supplemented sperm samples than in control. The results demonstrated that supplementation of buffer 2 with 50 μM kinetin is ideal for reducing the magnitude of oxidative damage during semen cryoprocessing and improving the post-thaw quality of dog semen.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.     

Cryoprotectant effect of trehalose and low-density lipoprotein in extenders for frozen ram semen

This study tested trehalose and low-density lipoprotein (LDL) as cryoprotectants in extenders for frozen ram semen. In the first experiment, the extenders were Tris, with 20% egg yolk (E1-1); E1-1 with 5% glycerol (E1-2); E1-1 with 100 mM trehalose (E1-3); and E1-1 with 100 mM trehalose and 5% glycerol (E1-4). Sperm motility and membrane integrity of the E1-2, E1-3 and E1-4 extenders were greater than for E1-1 (P < 0.05), but acrosome integrity following cryopreservation did not differ. In the second experiment, the extenders were Tris, with 20% egg yolk and 100 mM trehalose (E2-1); Tris with 8% LDL and 5% glycerol (E2-2); Tris with 8% LDL and 100 mM trehalose (E2-3); and Tris with 8% LDL, 100 mM trehalose and 5% glycerol (E2-4). Sperm membrane integrity was lowest for the E2-1 extender (P < 0.05), but similar for extenders including LDL. Sperm motility post-thawing was highest for E2-2 and E2-3 extenders (P < 0.05), but acrosome integrity did not differ. Thus, extenders including trehalose and LDL as cryoprotectants recorded a post-thawing ram sperm quality similar to that achieved when using conventional cryoprotectants.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.