Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Influence of seminal plasma during different stages of bovine sperm cryopreservation

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 10⁶ sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (−65.2%), acrosomal membrane (−34.0%) and mitochondrial potential (−48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm?

During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO₂^-) and hydrogen peroxide (H₂O₂) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 μM CoQ10 (T3) decreased NO₂⁻ concentration (6.7 ± 2.2 μM/μg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 μM/μg protein), respectively, as well H₂O₂ concentration (1.8 ± 0.3 μM/μg protein) compared with the control (4.6 ± 0.4 μM/μg protein) and 5 μM CoQ10 treatments (4.8 ± 0.2 μM/μg protein, P < .05). In conclusion, 25 μM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO₂⁻ and H₂O₂ concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.     

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non‐permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs. 19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm.     

Effect of caspase inhibitor Z-VAD-FMK on bovine sperm cryotolerance

The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

A preliminary study on using autologous and heterologous boar sperm supernatant from freezing processes as post-thawing solution: its effect on sperm motility

The aim of this study was to evaluate the effect of post-thawing dilution with autologous and heterologous sperm supernatant on motility of frozen-thawed boar spermatozoa. During the cryopreservation, sperm supernatant (a combination of seminal plasma and semen extender, 50% v/v) or seminal plasma from nine boars (Duroc, Large White, and Landrace; three in each) was collected by centrifugation and stored frozen until use as post-thawing solution. Sperm pellets were further processed and cryopreserved using control-rate freezer and was thawed at 50°C for 12 s. After thawing, frozen thawed semen samples were diluted with seminal plasma (group A), supernatant from Landrace (group B), supernatant from Large White (group C), supernatant from Duroc (group D), and Modena™ semen extender (group E). Post-thawing motility was evaluated using a phase-contrast microscope after thawing at 1, 10, 20, and 30 min. The present results show that at 1 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (53.3%) and C (53.9%) than the other groups. At 10 min, the highest (P ≤ 0.001) progressive motility was found in groups B (65%) and C (61%). At 20 and 30 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (58.9%), C (53.5%), and D (45.6%) than groups A (3.9%) and E (20.6%). It can be stated that supernatant from the freezing processes (consisting of seminal plasma and Modena™, 50% v/v) had a beneficial effect on post-thawing progressive motility of frozen boar semen.     

Effects of cooling rates on the quality of Prochilodus lineatus (Valenciennes, 1836) sperm

This study aims to investigate the effect of different cooling rates on the semen cryopreservation of curimba (Prochilodus lineatus). Nineteen ejaculates were obtained from adults males and cryopreserved at 15°C/min (CR15), 30°C/min (CR30) (controlled temperature inside and outside straw, speed was stable during freezing) and direct freezing in liquid nitrogen vapour (~35.6°C/min) (CRNV). The straws were thawed and seminal parameters evaluated. DNA fragmentation through the comet assay was assessed. A fresh sperm sample was not frozen and used for analyses. Data were submitted to an analysis of variance (ANOVA), and means were compared by Scott–Knott test (p < 0.05) using the R Software. Mean motility percentage was 100%, and motility duration was 39.5 ± 5.7 s for the fresh sperm (subjective analysis); 58.9 ± 8.0% and 24.5 ± 5.7 s for CR15; 64.8 ± 4.8% and 26.5 ± 7.1 s for CR30; and 50.1 ± 16% and 25.7 ± 4.7 s for CRNV, respectively. Motility percentages were higher and equal between CR15 and CR30 compared to CRNV (p < 0.05). Some sperm motion kinetics, namely average path velocity (VAP) and straight line velocity (VAS), were higher for CR30 (p < 0.05), while curvilinear velocity (VCL) and velocity progression (PRO) were lower for CRNV (p < 0.05). Straightness (STR) and wobble (WOB) were the same among treatments (p > 0.05). Sperm morphology results indicated higher means for total morphological sperm alterations in CRNV. All cooling rates caused sperm DNA fragmentation, although CR30 provided a less harmful effect. This is the first report for cryopreserved P. lineatus sperm preserved under different controlled cooling rates. The cooling rate of 30°C/min is indicated for the cryopreservation of this fish sperm as it led to the lowest detrimental spermatozoa effects.     

The effect of some cryoprotectants on dromedary camel frozen‐thawed semen

The cryopreserved camel semen is often associated with poor quality and fertility. This study aimed to improve the dromedary frozen semen quality by comparing the efficiency of four cryoprotectant agents (CPAs) on sperm freezability. Semen samples were collected from seven male Maghrabi camels, diluted with Shotor diluent supplemented with glycerol (Sh‐G), dimethyl formamide (DMF, Sh‐DF), dimethyl sulfoxide (DMSO, Sh‐DS) or ethylene glycol (EG, Sh‐EG), all at 6% final concentration, and the samples were subjected to cryopreservation. The results revealed the superiority of Sh‐DF over Sh‐G and Sh‐DS in terms of post‐thaw motility (55.83 ± 2.20 vs. 47.50 ± 4.33 and 45.00 ± 2.89%, respectively), sperm membrane (49.00 ± 0.58, 39.33 ± 3.33 and 42.67 ± 1.45%, respectively) and acrosomal integrities (53.00 ± 0.58, 57.33 ± 0.88 and 52.33 ± 1.45%, respectively). Sh‐EG group showed the lowest post‐thaw motility, plasma membrane and acrosome integrities (12.50 ± 1.44, 22.67 ± 1.45 and 30.67 ± 1.45, respectively). In conclusion, the protocols of dromedary camel semen cryopreservation could be enhanced using 6% DMF as a cryoprotectant agent.     

Effect of Seminal Plasma on Post‐Thaw Quality and Functionality of Corriedale Ram Sperm Obtained by Electroejaculation and Artificial Vagina

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post‐thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.     

Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post‐thaw sperm quality in stallions

Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen‐thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold‐resistant frog, insect or plant species results in similar or better semen quality after freezing–thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose–urea extender or a Ghent combined extender (glucose–urea, trehalose‐betaine and trehalose‐proline; volume ratio of 2:1:2) in a computer‐controlled rate freezer. After freezing–thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose–urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose–urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p < .001). In conclusion, glycerol concentration in a commercial freezing extender for equine spermatozoa can be successfully reduced when urea as an additive cryoprotectant is added and the glucose concentration is elevated. However, total glycerol replacement with urea, betaine, proline and trehalose was less successful.     

Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing

This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.     

解冻稀释液中添加精浆对冻融猪精子品质的影响

【目的】 探究冷冻前添加热休克蛋白A8(heat shock protein A8, HSPA8)和解冻后添加不同浓度精浆(seminal plasma, SP)对冻融猪精子的影响。【方法】 采用手握法采集长白猪精液, 添加0.5 μg/mL HSPA8到猪精液冷冻保护剂中进行细管分装, 投入液氮中保存3周后进行解冻, 解冻后添加不同浓度精浆(0、10%、30%和50%), 对冻融后长白猪精子的运动能力、质膜完整性、顶体完整性、细胞凋亡、线粒体膜电位、鱼精蛋白缺乏及体外获能水平等进行评估。【结果】 与对照组相比(无HSPA8和精浆), 添加0.5 μg/mL HSPA8处理组(无精浆)的精子直线速度(VSL)、曲线速度(VCL)、平均路径速度(VAP)和前向性运动(STR)均显著提升(P<0.05), 精子直线性运动(LIN)和运动的摆动性(WOB)均无显著差异(P>0.05);精子质量参数中活力、质膜完整性和顶体完整性均显著升高(P<0.05), 细胞凋亡水平与线粒体膜电位均显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。之后在解冻液中添加不同浓度的精浆, 与添加0.5 μg/mL HSPA8处理组(无精浆)相比, 精浆添加量达到50%时, 精子VSL、VCL、VAP、LIN、STR和WOB均显著提升(P<0.05);精子活力、质膜完整性、顶体完整性和线粒体膜电位均显著提高(P<0.05), 细胞凋亡水平显著降低(P<0.05);精子鱼精蛋白缺失率显著降低(P<0.05);精子蛋白酪氨酸磷酸化水平显著提高(P<0.05)。【结论】 在冷冻基础液中添加0.5 μg/mL HSPA8和解冻稀释液中添加50%精浆联合使用可以有效改善冻融精子质量, 将会对猪精液的冷冻保存及商业化生产提供一定的参考。     

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.     

The Effect of Season on Spermatozoa Motility,Plasma Membrane and Acrosome Integrity in Fresh and Frozen–Thawed Semen from Xinong Saanen Bucks

The aim of this study was to evaluate whether the season of ejaculate collection influences seminal quality parameters of pre‐ and post‐freeze–thawing in Xinong Saanen bucks. Ejaculates were collected from eight bucks throughout the four seasons (spring, summer, autumn and winter) in a 12 months’ time period, identified in the Northern Hemisphere. Semen samples were evaluated by the combinations of conventional and Computer‐Assisted Sperm Analysis (CASA) when fresh and after frozen–thawed, respectively. The results clearly demonstrated that season of ejaculate collection influenced (p < 0.05) fresh semen quality. Highest semen quality was observed during autumn. On the contrary, undesirable indices (significantly lower, p < 0.05) were observed in winter as compared with the other remaining seasons. CASA has clearly shown the influences of seasonal variations on semen motility parameters. Furthermore, season of ejaculate collection was also found to influence sperm freezability. Semen characteristics after frozen–thawed followed a similar pattern with that of fresh ejaculate except in spring. The results revealed that sperm quality was higher (p < 0.01) in summer and autumn than in spring and winter. In conclusion, seasonal variation influences semen quality in Xinong Saanen bucks. In addition to summer and autumn, fresh ejaculates in spring can also be successfully used for AI. Sperm from ejaculates collected during summer and autumn are more suitable for cryopreservation. Hence, it is possible to increase the efficiency of goat breeding by manipulating the seasonal variations of semen quality for immediate AI and/or cryopreservation.     

Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen Cryopreservation Improves Sperm Viability but Results in Low Fertility in Mares

The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10⁶ spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC‐treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC‐treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC‐treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S‐MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S‐MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Orally administered Chrysin improves post‐thawed sperm quality and fertility of rooster

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post‐thawed sperm quality and fertility of Chrysin‐fed roosters were assessed in this study. Twenty 40‐week‐old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch‐0), 25 (Ch‐25), 50 (Ch‐50) or 75 (Ch‐75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post‐thawed samples) and mitochondrial activity (only in post‐thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post‐thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post‐thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post‐thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post‐thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post‐thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch‐50 and Ch‐75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post‐thawed spermatozoa was significantly improved in all Chrysin‐fed groups compared to Ch‐0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation‐induced impairment of sperm quality and fertility rate.     

Cryopreservation of Ghagus chicken semen: Effect of cryoprotectants,diluents and thawing temperature

The present study evaluated the effects of cryoprotectants, semen diluents and thawing temperature during Ghagus chicken semen cryopreservation. Four different experiments were conducted; Experiment 1—semen was cryopreserved using 6% dimethylacetamide (DMA) and 2% dimethylsulphoxide (DMSO) in Sasaki diluent (SD) and Lake and Ravie diluent (LR), Experiment 2 and 3—semen was cryopreserved using 8% ethylene glycol (EG) in SD, LRD and Red Fowl Extender (RFE), Experiment 4—semen was cryopreserved using 6% dimethylformamide (DMF) in SD, LR and Beltsville poultry semen extender (BPSE). Semen was cryopreserved in 0.5 ml French straws. Thawing was done at 5°C for 100 s in ice water in Experiments 1, 2 and 4, whereas in Experiment 3 thawing was done at 37°C for 30 s. The post-thaw sperm motility, viable sperm and acrosome-intact sperm were significantly (p < .05) lower in cryopreserved samples in all the experiments. No fertile eggs were obtained from cryopreserved samples in Experiments 1 and 2, except for 8% EG RFE treatment where the fertility was 0.83%. In Experiments 3 and 4, highest fertility was obtained in LR treatment 48.12 and 30.89%, respectively. In conclusion, using cryoprotectant EG (8%) and thawing at 37°C for 30 s, and DMF(6%) resulted in acceptable level of fertility in Ghagus chicken. Though the diluents influenced post-thaw in vitro semen parameters, the fertility was not affected. In addition, results indicated that thawing temperature may be a critical stage in the cryopreservation protocol.