Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.     

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.     

新疆驴冷冻精液的研究

试验旨在探究新疆驴冷冻精液的最佳稀释保护液与处理方法。以假阴道采集新疆驴精液,并用5种不同稀释保护液稀释冷冻后,选取其中较为理想的稀释液作为对照组,再通过分别添加4%、5%、6%、8%甘油处理试验,最后再选用添加甘油后冷冻效果最优的稀释液为对照来进行离心浓缩试验。结果表明：①5号稀释液冷冻—解冻精液后,其活力、顶体完整率要显著高于其他4种稀释液（P<0.05）;②以5号稀释液为对照组,添加6%甘油(7号稀释液)冷冻后,精子的活力、顶体完整率要显著高于添加4%、5%、8%甘油组（P<0.05）;③以7号稀释液为对照组,通过离心浓缩,精液在冷冻后其活力和精子顶体完整率都显著提高（P<0.05）。结果提示,将驴精液用6%葡萄糖、2%乳糖、6%甘油为主的稀释液稀释,并经500 r/min离心浓缩10 min处理后,其冷冻效果最好。     

Freezability of Tushin Ram Semen Extended with Goat or Cow Milk Based Extenders

Cow milk is used as an extender for ram semen cryopreservation. Caseins, the major proteins of milk, appear to provide some protective effect to sperm during cryopreservation. Goat milk has unique casein structure. The aim of this study was to investigate effect of goat milk, as a main semen extender, on freezability of Tushin Ram semen. For this aim, ejaculates from four Tushin rams were collected with artificial vagina and pooled. Pooled semen was separately extended with four different extenders: TRIS based (TRIS), cow skim milk based (CSM) (10 g/100 ml), cow semi‐skim milk based (CSSM) and goat semi‐skim milk based (GSSM) extenders, containing egg yolk and glycerol. The semen was cryopreserved and stored in liquid nitrogen until examination date. After thawing (at 37°C for 1 min), sperm motility, viability, morphology, acrosome and membrane integrity (HOST) were evaluated. Although, there was not any significant differences between extenders in post‐thaw percentage of viable spermatozoa (p > 0.05), Tushin ram semen extended with GSSM or CSM extenders had significantly higher post‐thaw percentage of progressive motility (25.0% and 30.8% respectively), compared with CSSM and TRIS (7.5% and 14.1% respectively, p < 0.001). Moreover, lowest abnormality percentage of post‐thaw spermatozoa were detected in ram semen extended with GSSM (49.5%) and CSM (51.5%), compared with CSSM (65.7%) and TRIS (60.7%) (p < 0.05). Whilst the results were considered, it was concluded that goat milk based extenders may be effectively and trustfully used in cryopreservation of Tushin ram semen, instead of cow milk and Tris based extenders, as a main extender.     

Comparative quality assessment of buffalo (Bubalus bubalis) semen chilled (5°C) in egg yolk- and soya milk-based extenders

Egg yolk-Tris is most commonly used semen extender; however, its use involves hygienic risk, interference with fertility and poor microscopic examination. Therefore, replacement of egg yolk with a plant-based component with protective effects on spermatozoa would be advantageous. In present study, we observed effect of soya milk-based extenders on dilution and liquid preservation of Murrah buffalo bull semen at 5°C up to 72 h in comparison with conventional egg yolk-Tris extender (Ext.1). In experiment one, a total of 32 buffalo semen ejaculates from four animals were extended and preserved at 5°C for 72 h in soya milk-based extender (Ext.2) with different percentages (10%, 15%, 20%, 25% and 30%) of soya milk for optimization of soya milk concentration. Semen quality was assessed for individual motility, viability, membrane integrity and acrosome integrity at 0, 24, 48 and 72 h of liquid preservation. The results of experiment one indicated that 25% soya milk is an optimum concentration for buffalo bull semen extender preparation. A modified method was used to prepare another soya milk-based extender (Ext.3). In the second experiment, two soya extenders (Ext.2 and 3) with optimized concentration (25%) of soya milk were comparatively assessed with egg yolk-Tris extender (Ext.1) for semen quality parameters at 0, 24, 48 and 72 h of liquid preservation. The individual sperm motility at 0 and 24 h following dilution were found non-significant among extenders. However, after 48 h of dilution, individual motility in Ext.3 was observed significantly (p < 0.05) higher than Ext.1. After 24, 48 and 72 h of dilution sperm membrane integrity in Ext.3 was found significantly (p < 0.05) higher than Ext.1. Overall, comparative evaluation of sperm parameters obtained revealed that Ext.3 containing 25% soya milk can be used as a substitute of egg yolk-based extender for buffalo semen liquid preservation.     

Conception rates in ewes after AI with ram semen preserved in milk-egg yolk extenders supplemented with glycerol

This study aimed at comparing the effect of ram semen preserved at 5°C on two milk‐based extenders (UHT skim milk or INRA‐96^®, 5% egg yolk) supplemented with 2% glycerol, and the preservation time (24 and 48 h) on conception rates after cervical AI of ewes. In two field trials, 1198 Merino ewes were cervical AI in spontaneous oestrus. In Experiment 1, pooled semen (6 rams) was extended in UHT‐base (fresh, control) or chilled for 24 h in UHT5Y (UHT‐base 5% egg yolk), INRA5Y (INRA‐96^® 5% egg yolk), UHT5Y2G (UHT5Y 2% glycerol) or INRA5Y2G (INRA5Y 2% glycerol). In Experiment 2, AI was performed with pooled semen (7 rams) used fresh (extended in UHT‐base or UHT5Y2G, control groups) or chilled (extended in UHT5Y2G) for 24 or 48 h. Conception rate was determined by ultrasound 40 days after AI. INRA‐96^®– had similar conception as UHT‐preserved semen (56.7 vs 55.4%, p > 0.05). Addition of 2% glycerol did not modify the results (56.8 vs 55.2%, p > 0.05). Fresh semen extended in UHT‐base, and UHT5Y2G yielded similar conception rates (60 vs 64%, p > 0.05). Preservation for 24 or 48 h in UHT5Y2G gave similar results (49 vs 47%; p > 0.05). In conclusion, ram semen chilled for 24 h in UHT‐ or INRA‐96^®‐based extenders yielded similar results, and glycerol addition did not have a detrimental effect. UHT5Y2G might be used to extend ram semen for fresh AI, or to preserve it for 24 or 48 h with acceptable results.     

Comparative Study on Five Different Commercial Extenders for Boar Semen

Increasing interest in a longer preservation of diluted boar sperm raises questions in the field concerning the choice of the extender. The aim of this study was to evaluate the longevity of boar sperm extended in currently used commercial semen extenders. Three long-term extenders and two short-term extenders were compared for different semen quality parameters that can be assessed under routine laboratory conditions. Sperm morphology, motility, pH and bacteriological contamination were investigated during a 7-day period. The number of dead spermatozoa did not differ significantly among the extenders (p > 0.05). Sperm motility was not only related with storage period but most of all with pH, especially in long-term extenders. Differences between the different extenders were prominent (p < 0.05); the sperm preserved in only one long-term extender showed good motility during the whole test period. In all cases, the pH of the extended semen increased by 0.3-0.5 in the first days of storage and was significantly correlated with a decrease in motility. Bacteriological quality had no significant influence on motility or pH of the semen. In conclusion, we can state that in both short-term extenders and in only one long-term extender, sperm longevity, as evaluated by the parameters used in this study, was sufficient during the preservation period. To preserve the quality of diluted boar semen during long-term storage, the choice of the long-term extender is important. In addition, the monitoring of the pH of extended boar semen in our study emphasizes the importance of the buffering capacity of semen extenders.     

重庆板角山羊细管冷冻精液的研究

为了建立重庆板角山羊精液的细管冷冻保存方法,实验进行了不同冷冻稀释液(配方Ⅰ、Ⅱ、Ⅲ)、不同冷冻保存剂(甘油、EG)及不同离心速度(1000、1200、1400r/min)对重庆板角山羊细管精液冷冻保存效果的研究,结果表明:配方Ⅱ对重庆板角山羊精液的冻后活率显著优于配方Ⅰ和Ⅲ(P<0.05)。在配方Ⅱ中添加相同剂量(5%)的EG和甘油,精液冻后活率差异不显著(P>0.05)。以1200r/min的速度对山羊鲜精作离心处理后,冻后活率相对于对照组有所提高,但差异不显著(P>0.05)。     

Chilled storage of ram semen improves with the addition of egg yolk and glycerol to milk-based extenders

This study aimed at comparing in vitro, ultra‐heat‐treated (UHT) skim milk and INRA‐96^®‐based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5°C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (×10⁶ spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT‐based extenders yielded better (p < 0.05) SM and MI than INRA‐96^®‐based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 × 10⁶ sperm/AI dose, respectively) along the preservation time. Egg yolk–glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA‐96^® were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5°C, at least for 48 h. The combination of egg yolk–glycerol might provide extra protection in case of fluctuation of temperatures below 5°C, commonly seen under field conditions.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

The Combinatorial Effect of Different Equex STM Paste Concentrations,Cryoprotectants and the Straw‐Freezing Methods on the Post‐Thaw Boar Semen Quality

This study was to evaluate the combinatorial effect (14 treatments, A–N) of different Equex STM paste concentrations, cryoprotectants and the straw‐freezing method on the post‐thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post‐thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post‐thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw‐freezing method changes between glycerol and DMA.     

Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin‐based semen extender as a substitute for egg yolk‐based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris‐based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post‐thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early‐apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post‐thawed necrotic and late‐apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Fertility after artificial insemination using a soybean-based semen extender in sheep

The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozen-thawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility.     

Comparison of Different Extenders with Defined Protein Composition for Storage of Stallion Spermatozoa at 5°C

To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the different extenders and stored at 5 degrees C for 4 days. Total motility, membrane integrity, average path velocity (VAP), curvilinear-velocity (VCL), straight-line velocity (VSL), distance average path (DAP), distance curved line (DCL) and distance straight line (DSL) were determined by computer-assisted analysis. Total motility decreased in all extenders during storage. The parameters VAP, VCL, VSL, DAP, DCL and DSL in semen diluted in EquiPro TM at most times and in semen diluted in AndroMed-E at some times were lower than in semen diluted in EquiPro (p < 0.05). Viability on days 0 and 4 was lowest in semen diluted in AndroMed-E (p < 0.05). Velocity decreased faster when semen had been diluted in the sterilized liquid extender EquiPro TM or in AndroMed-E compared with the dry formula of EquiPro. Therefore the liquid sterilized EquiPro despite no difference in its chemical composition differs from the dry, non-sterilized EquiPro extender. Heat sterilization apparently changes effects of the extender on spermatozoa.     

Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares

Reasons for performing study: As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Aim: To compare the seminal parameters of cryopreserved donkey semen in lactose‐EDTA and lactose‐yolk extenders and the fertility rates on horse mares. Methods: Trial 1: Sperm total and progressive motility, vigour (scale 0–5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was mixed with skimmed‐milk extender, centrifuged, and then re‐suspended in lactose‐EDTA or lactose‐yolk extender. Semen was loaded into 0.5 ml straws and chilled to 5°C for 1 h, after which samples were either evaluated (chilled semen) or placed above liquid nitrogen for 20 min prior to immersion. Seminal parameters were evaluated by ANOVA and Tukey's test. Trial 2: Cryopreserved semen from 3 males was used to inseminate 53 mares at 60 oestrous cycles randomly assigned to lactose‐yolk (n = 30 cycles) or lactose‐EDTA (n = 30 cycles) extenders. Pregnancy diagnosis was performed 15 and 25 days post ovulation. The pregnancy rates were compared using Chi‐squared tests. Results: Trial 1: No significant differences were evident in any seminal parameters between extenders after either chilling or cryopreservation. Total and progressive motility were significantly (P<0.05) lower in cryopreserved semen than raw and chilled semen for both extenders. Trial 2: Pregnancy rates did not significantly differ between extenders (lactose‐EDTA extender 53.33 and 43.33%; lactose‐yolk 50.0 and 46.66% for Days 15 and 25 post ovulation, respectively). Conclusions: Cryopreservation of donkey semen using the simplified lactose‐yolk extender resulted in similar seminal parameters and fertility rates when compared to lactose‐EDTA extender. Potential relevance: Lactose‐yolk extender may be advocated as a simple, easy to prepare extender, for use in geographically isolated enterprises producing mules throughout the world.     

Fertility of ewes inseminated intrauterinally with frozen semen using extender containing bovine serum albumin

The present study was conducted to examine the fertility of ewes inseminated intrauterinally with frozen semen using semen extender containing either egg yolk or bovine serum albumin (BSA). Sixty Suffolk and cross-bred ewes were treated with controlled internal drug release (CIDR) devices during the non-breeding season (July 2006). A CIDR was inserted into the vagina for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin was administered one day before its removal. Ejaculates from a suffolk ram were diluted with a Tris-based extender containing either 15% (v/v) egg yolk or 10% (w/v) BSA, and the diluted semen was frozen in 0.25 ml straws. A fixed-time intrauterine artificial insemination (AI) was performed 43-47 h after CIDR removal, regardless of incidence of estrus. There was no significant difference in pregnancy rates at 60 days after AI between the extenders containing egg yolk (66.7%, 20/30 animals) or BSA (65.5%, 19/29 animals). Furthermore, there were no significant difference in the lambing rates (66.7% and 62.1%) and prolificacy (1.25 and 1.56) between the two semen extenders. The present study indicates that a semi-defined semen extender containing 10% BSA produces fertility after intrauterine AI that is similar to that achieved with semen extender containing egg yolk.     

Protocol Optimization for Long‐Term Liquid Storage of Goat Semen in a Chemically Defined Extender

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self‐made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid‐stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5°C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine‐transformed before being analysed with anova and Duncan’s multiple comparison test. While cooling at the rate of 0.1–0.25°C/min did not affect sperm viability parameters, doing so at the rate of 0.6°C/min from 30 to 15°C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non‐coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21‐fold diluted than when it was 11‐ or 51‐fold diluted and when extender was renewed at 48‐h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk‐containing extender was 21‐fold diluted, cooled at the rate of 0.07–0.25°C/min, stored at 5°C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro‐fertilizing potential similar to that of fresh semen was maintained for 11 days.     

Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self‐made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk‐ and yolk‐free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris–glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5°C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non‐centrifuged groups when semen was 11‐fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11‐fold diluted and stored at 5°C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.     

Cryopreservation of European Mouflon (Ovis Gmelini Musimon) Semen During the non-Breeding Season is Enhanced by the Use of Trehalose

The influence of trehalose on European mouflon spermatozoa cryopreservation during the non-breeding season was tested. Semen was frozen in two different extenders: (a) recommended Tris-based ram extender (CTR); (b) CTR extender supplemented with trehalose 0.147 mm (TRH). Sperm viability and acrosome integrity were assessed using propidium iodide and fluorescein isothiocynate labelled Pisum Sativum agglutinin. Trehalose significantly enhanced sperm viability after thawing compared with CTR extender (62.7% vs 51.8%; p < 0.05), whereas no differences were observed on acrosome integrity (42.9% vs 42.1%). Trehalose influence was also evidenced in the in vitro fertility test performed with sheep oocytes matured in vitro. Both fertilization rates (60.9% TRH vs 43.6% CTR; p < 0.05) and cleavage rates (58% TRH vs 39.8% CTR; p < 0.001) were higher for trehalose frozen semen compared with control extender frozen semen. A higher percentage of zygotes resulting from fertilization with trehalose cryopreserved semen presented the first cleavage earlier if compared with the group fertilized with control semen (48.7% vs 31.5%, respectively; p < 0.01). This result was confirmed by embryo kinetic development. Fertilization with trehalose cryopreserved semen leaded to an higher percentage of blastocysts (40.2% vs 27.8% CTR; p < 0.05), and enhanced in particular the number of blastocysts that developed on the day 6th of culture (28.6% vs 17% CTR; p < 0.05). Our data demonstrated that, during mouflon non-breeding season, trehalose extender enhances spermatozoa viability and its in vitro fertilizing capacity, allowing the production of an higher number of blastocysts.