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.     

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.     

猪精液液态保存的研究进展

猪的人工授精主要使用液态保存精液 ,此法保存精子的存活率高、功能受损较小、授精繁殖率较高且操作简便。体外液态保存猪精液时 ,不仅要注意猪精子对温度变化敏感的特性 ,还要考虑酸碱度、离子的种类和强度、渗透压、过氧化损伤和微生物污染等对精子的影响 ,要向稀释液中添加各种保护成分 ,维持精子的存活和功能。目前主要是比较和改进各种稀释液 ,建立准确的精子质量评价方法 ,提高受精率     

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.     

果糖稀释液与葡萄糖稀释液的比较试验

用２０份荷斯坦种公牛的精液测试Ａ、Ｂ两种稀释液（简称Ａ、Ｂ）的效果。Ａ中含１０ｇ／１０００ｍｌ的果糖，Ｂ中有等摩尔浓度的葡萄糖，而没有果糖，Ａ、Ｂ中其它成分相同。通过Ａ、Ｂ液效果的对比来比较稀释液中果糖和葡萄糖的作用。实验采用配对比较的方法。分别用Ａ、Ｂ稀释同源的公牛精液，经平衡、冷冻、解冻处理，在解冻后０小时和２４小时（５℃保存）对两种稀释精液就地活力检查。结果表明：在解冻后０小时和２４小时两种稀释精液活力无显著差异。此结果建议．在冷冻精液生产中，可用廉价的葡萄糖代替稀释液中的果糖。     

不同稀释液对藏獒精液保存效果的影响

比较了8种稀释液在藏獒精液冷冻保存和低、常温保存(5 ℃,10 ℃,15 ℃,20 ℃)的效果.结果表明:2号和8号稀释液在冷冻保存时效果最好,精子活力和顶体完整率分别达到0.40和49%,与对照组相比差异显著(P<0.05).在藏獒的低温、常温保存试验中,所有稀释液在15 ℃时保存时间最长,综合考虑,7号液保存效果最好;在15℃时精子平均存活时间为103 h,显著高于对照组和其它稀释液(P<0.05).     

犬精液低温常温保存稀释液筛选试验

试验比较了 8种稀释液在 5 ,10 ,15 ,2 0℃下保存犬精液的效果。结果表明 :15℃为犬精液液态保存的较适温度 ,3号液为优等液。其中 3号液在 15℃精子的存活时间为(10 5± 10 )h,与对照液在 15℃的保存效果差异极显著 (P <0 .0 1) ;2 ,4 ,5号液是良等液 ,在 15℃精子的存活时间是 (6 4± 8)h ,与对照液在 15℃的保存效果差异显著 (P <0 .0 5 ) ;中等液为 6号液 ,在15 ,2 0℃保存时与对照液没有统计学上的差异 (P >0 .0 5 ) ,在 5 ,10℃保存时不如对照液 ;7,8号液为差等液 ,在 15℃保存时与对照液差异不显著 (P >0 .0 5 ) ,在 5 ,10 ,2 0℃时不如对照液     

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.     

不同稀释液配方对奶牛冷冻精液的影响

采用假阴道法采集成年种公牛精液,经精液品质检查后,合格精液用四种不同冷冻稀释液进行稀释、冷冻、以筛选出适宜的冷冻稀释液和最佳冷冻保护剂.结果表明:Ⅱ号稀释液优于其它3种稀释液(P＜0.05);甘油为最佳冷冻保护剂.优于乙二醇和DMSO冷冻效果(P＜0.05),其最佳浓度为7%,精子冷冻后活率平均可达0.45±0.575,畸形率15.44%,项体完整率57.82%;奶牛细管冻精的解冻效果以75℃水浴,2 S到6 S的解冻时间最佳.     

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.     

牛冷冻精液稀释液中草药配方的初步研究

通过比较以淫羊藿为主要成分的单方及复方中草药提取液对牛冷冻精液解冻后精子活率、畸形率、顶体完整率及低温保存时间的影响，以期开发牛冷冻精液中式稀释液。将三种不同配方的淫羊藿提取液分别以6．25％、12．5％、25．0％、50．0％的体积分数添加于稀释液中，制作牛颗粒冻精，利用干解冻法（40～42℃）解冻后分别检查精子活率、畸形率和顶体完整率，评定其品质。结果表明：配方三取得了较佳的冷冻效果。其提取液添加12．5％时精子冻后活率平均达到0．56，极显著高于其他各组（P〈0．01）：精子顶体完整率最高达到76．80％。畸形率仅14．61％，低温保存时间长迭138h。配方三最有利于生产牛冷冻精液。     

Effect of Antibiotic-containing Extenders on Taylorella equigenitalis Contaminated Semen

Taylorella equigenitalis is a gram-negative coccobacillus and the causative agent of a transmissible venereal disease in horses known as contagious equine metritis. Outbreaks of contagious equine metritis have been documented in various countries since 1977, with the most recent discovery in the United States in December 2008. During disease occurrences, culturing semen samples for T equigenitalis before breeding may help to prevent transmission of this disease; however, little is known about the antimicrobial activity of equine semen extenders against the organism. The purpose of this study was to investigate the infectivity levels of T equigenitalis in three equine semen extenders inoculated with known concentrations of the organism. The semen extenders used for this study included INRA 96, E-Z Mixin BF, and VMDZ. In addition, Timentin was added to INRA 96 at three different concentrations (0.5, 1.0, and 1.5 mg/mL) to investigate possible synergistic effects of antibiotic supplementation of extenders. Results were based on the visual counting of the colonies on chocolate Eugon agar plates. Both INRA 96 (with added Timentin) and VMDZ (as supplied by the manufacturer) significantly reduced the numbers of T equigenitalis isolated from semen extenders as compared with INRA 96 (as supplied by the manufacturer) or the antibiotic free E-Z Mixin BF. Our findings indicate that INRA 96 (with added Timentin) or VMDZ may significantly decrease the growth of T equigenitalis in extended semen; however, it is also important to consider the possible effects of antibiotic supplemented extenders on sperm longevity and fertility in addition to eliminating specific pathogens in semen.     

不同稀释液对Beagle犬精液保存效果的比较

通过比较4种不含卵黄的犬精液稀释保存液在低温环境中（4℃）的实际使用效果,筛选出适合的不含卵黄低温稀释保存配方.不含卵黄的配方Ⅳ （Tris 2.108 g,柠檬酸水合物1.2g,果糖1.0g,青霉素100 mg,双氢链霉素100 mg,牛血清白蛋白（BSA）4 g,加蒸馏水到100 mL）具有较好的低温稀释保存效果,并首次提出牛血清白蛋白（BSA）可作为非常时期犬精液低温稀释液中卵黄的替代物.     

山羊精液冷冻保存研究进展

精液的冷冻保存可使精液长期保存和运输,扩大精液的使用范围,使配种不受时间、地域、种畜生命的限制。充分利用优秀种公畜的遗传资源,加快育种工作进程,提高优良公畜的配种效率和种用价值。随着养羊业的迅速发展,冷冻山羊精液亟待应用于生产。山羊精液的冷冻保存是一个复杂的过程,为了获得理想的保存效果,需要平衡多种因素。从山羊精液冷冻保存剂成分、冷冻解冻的过程、冷冻过程的影响因素等方面予以综述,旨在为今后的精液冷冻保存研究工作提供一定的参考依据。     

Effect of Long-Term Storage at Different Temperatures on the Quality of Liquid Boar Semen

In this study the effect of long‐term storage of liquid boar semen at different temperatures on motility, acrosome integrity and pH was investigated. Additionally, individual variation in sperm tolerance to storage at 10°C were examined. Beltsville Thawing Solution‐diluted AI doses from 16 randomly chosen Norwegian Landrace AI boars with proven fertility were split into subsamples and stored at 25, 20, 15 and 10°C, respectively. After 0, 24, 48, 72 and 96 h of storage, sperm motility, acrosome integrity and pH were determined. After 96 h, the initial percentage of motile sperm (77.8%) was significantly reduced to 52.2, 58.8, 50.9 and 42.8% by storage at 25, 20, 15 and 10°C, respectively. After an identical period of time, the percentage of acrosome intact sperm (95.8%) at time 0 became significantly reduced to 91.3, 91.3, 81.5 and 68.3% by storage at 25, 20, 15 and 10°C, respectively. The initial pH (7.21) decreased significantly to 6.96 and 7.06 after 96 h storage at 25 and 20°C, and increased not significantly to 7.25 for storage at 15°C and significantly to 7.29 at 10°C. In conclusion, the results from this study show that, according to the variables studied, 20°C is the least harmful of the four temperatures tested for the long‐term liquid storage of boar semen. Furthermore, remarkable differences in the individual resistance of boar semen to long‐term storage at 10°C were observed.     

波尔山羊冷冻精液的研制

试验选用两种衡释保护剂配方，通过6批次460支冷冻精液的制作，获得了较为理想的乙二醇稀释保护剂。经解冻试验，角发冻后乙二醇稀释剂的精子复活力为0.39-0.40（对照组为0.29-0.30），精子存活率为49.38%-52.56%（对照组为36.25%-38.0%）。     

Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 10⁶ progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.     

Comparison of Two Semen Extenders in Terms of In Vitro Development of Bovine Embryos Following IVF

In order to conform with current EC standards with regard to antibiotic cover, the Norwegian Cattle Association is currently investigating the use of Biladyl® as an alternative to the milk-based extender which has been traditionally used in Norway. A study was carried out to investigate the effect of using semen frozen with either milk extender or Biladyl® on the outcome of in vitro fertilization and embryo culture. Semen from 6 Norwegian Red bulls was used. There was a significant difference p<0.05 in terms of cleavage rate between the 2 extenders for 1 bull, 78.2% vs 94.9% for milk and Biladyl® extenders, respectively, and for the overall total of 71.3% vs 76.1% for milk and Biladyl® extenders, respectively. There were no significant differences in terms of blastocyst yield amongst any of the bulls. In conclusion, the results suggest that Biladyl® can be used as a replacement for the traditional milk-based extender without any adverse effects on blastocyst yields following in vitro fertilization.