北极狐、乌苏里貉精液细管冷冻技术研究

为了对濒危珍稀动物种质资源的进行保护,完善精液冷冻技术方案,对北极狐37只次、乌苏里貉32只次的精液进行细管冷冻技术研究,结果表明,北极狐精液用3%柠檬酸三钠—卵黄—甘油作冷冻稀释液,乌苏里貉精液用11%蔗糖—卵黄—甘油作冷冻稀释液,在5℃冰箱内平衡1 h后,进行细管冷冻(液氮-196℃),冻溶粒子活力达4~5级。速冻法优于缓冻法。细管冻精在50℃温水中快速溶化,北极狐精液用3%柠檬酸三钠作解冻液,乌苏里貉精液用7%葡萄糖液作解冻液效果较佳。     

猪精液5mL大管冷冻方法的初步研究

以5 mL大管解冻后的精子活力、质膜完整率、顶体完整率以及人工授精结果为判定指标,筛选冷冻液Ⅰ和冷冻液Ⅱ中N-乙酰-D-氨基葡萄糖(N-Acetyl-D-Glucosamine,N-AcGA)、甘油和卵黄的适宜浓度,寻找适宜的平衡时间和冷冻高度.结果显示:(1)在冷冻液Ⅰ中添加0.1%的N-AcGA,解冻后精子的活力和顶体完整率最高,分别为50.00%和56.60%;(2)冷冻液Ⅱ中以4%的甘油浓度最好,解冻后精子的活力、弯尾率和顶体完整率可分别达52.50%、52.08%和77.67%;(3)冷冻液中的卵黄终浓度以10%效果最佳;(4)在4℃中平衡2 h,冻后顶体完整率比对照组(0 h)有显著提高(P<0.05);(5)在冷冻高度方面,距液氮面1 cm处冷冻对精子活力和顶体完整性都有明显的保护作用;(6)在人工授精方面,5 mL大管冷冻精液平均获得了超过80%的受胎率.     

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。     

金华猪精液冷冻保存技术研究

本试验用0.5 mL细管作为冷冻载体对金华猪精液进行冷冻保存研究,比较3种冷冻稀释液及不同冷冻方法、解冻程序对金华猪精液冷冻的效果,以解冻后的精子活力、畸形率和质膜完整率为判定指标。结果表明:Ⅱ、Ⅲ号冷冻稀释液处理组解冻后精子的活力、畸形率和质膜完整性都明显地高于Ⅰ号冷冻稀释液(P0.05);Ⅱ号冷冻稀释液和Ⅲ号冷冻稀释液处理组之间没有明显差异(P0.05)。采用程序冷冻仪冷冻法,使用Ⅲ号冷冻稀释液冷冻保存精子,并在60℃,8 s条件下水浴解冻精子后,精子活力和质膜完整率最高分别为42.3%和50.3%,畸形率最低为17.7%。因此,采用程序冷冻仪冷冻法,使用Ⅲ号冷冻稀释液,在60℃,8 s条件下水浴解冻方法较为适合0.5 mL细管金华猪精液冷冻保存及解冻。     

维生素B12对牛冷冻精液品质的影响

人工授精是发挥优秀公畜遗传潜力的重要现代动物生物技术,而冷冻精液正是为了提供人工授精所需精子产品的一种精子库。由卵黄、蔗糖、柠檬酸钠、甘油和抑菌物质所组成的稀释液对精子起到一定的保护作用。但是,在精子的冷冻原解冻过程中,容易发生过氧化反应,导致精液品质下降。本研究在用卵黄、蔗糖、柠檬酸钠、甘油和抑菌物质配制好的精液稀释液里面分别添加0.00%(对照)、0.25%、0.50%、0.75%、1.00%的VB12进行试验,解冻后评定精子运动特性、精子活力、顶体完整率、质膜完整性等指标。结果表明：稀释液中添加0.50%维生素B12效果最好,其精子活力、顶体完整率、质膜完整性等明显高于其他组(P<0.01)。     

维生素B12对牛冷冻精液品质的影响

人工授精是发挥优秀公畜遗传潜力的重要现代动物生物技术,而冷冻精液正是为了提供人工授精所需精子产品的一种精子库。由卵黄、蔗糖、柠檬酸钠、甘油和抑菌物质所组成的稀释液对精子起到一定的保护作用。但是,在精子的冷冻-解冻过程中,容易发生过氧化反应,导致精液品质下降。本研究在用卵黄、蔗糖、柠檬酸钠、甘油和抑菌物质配制好的精液稀释液里面分别添加0.00%(对照)、0.25%、0.50%、0.75%、1.00%的VB12进行试验,解冻后评定精子运动特性、精子活力、顶体完整率、质膜完整性等指标。结果表明:稀释液中添加0.50%维生素B12效果最好,其精子活力、顶体完整率、质膜完整性等明显高于其他组(P0.01)。     

杜泊羊细管冻精不同稀释液效果的比较

采用4种不同冷冻稀释液制作杜泊羊细管冻精,解冻后精子活力以Tris、柠檬酸、糖类、甘油和卵黄稀释液最高,达到0.408,与其他3种稀释液相比差异达极显著水平(P<0.01),柠檬酸钠、糖类稀释液为0.358;乙二醇稀释液解冻后精子活力最低,只有0.218,与其他3种稀释液相比差异极显著(P<0.01),即乙二醇的抗冷冻效果不如甘油。Tris、甘油、脱脂奶粉稀释液解冻后活力为0.337,即脱脂奶粉抗冻效果不及卵黄。     

藏獒精液冷冻技术研究

为了确定适合藏獒精液冷冻保存的方法,试验对藏獒精液的形态学指标评价、稀释液pH值、甘油浓度、甘油平衡时间及不同冷冻形态对藏獒精液冷冻及解冻后活力的影响进行研究。结果表明:三羟甲基氨基甲烷(Tris)-柠檬酸稀释液和冷冻液的pH值在6.5~7.0,4%的甘油浓度,甘油平衡时间为60 min,用液氮熏蒸法0.25 mL细管进行藏獒精液冷冻效果最佳。     

乌苏里貉精液冷冻技术研究

为探索乌苏里貉精液冷冻技术,通过试验,筛选出了适合于乌苏里貉精液的鲜精稀释液和冷冻保护液配方,摸索出了乌苏里貉精液的冷冻程序,包括降温、平衡和预冷冻等各技术环节以及冷冻精液的最适解冻温度,解冻后在生物显微镜下观察,精子活力均在4~5级。     

五种糖类对犬精液冷冻保存效果的研究

选取8只身体健康、性欲旺盛的比格公犬,采集其精液,对鲜精进行质量检测,主要包括颜色、射精量、精子活率、活力和pH。选取精子活率达到70%以上犬精液用于后续精液冷冻试验。分别将含有葡萄糖、果糖、蔗糖、乳糖和海藻糖的精液稀释液与精液按照1∶2的比例进行稀释。经冷冻解冻后,进行活率、活力、质膜完整性和顶体完整性的精子质量检测。通过对新鲜精液进行品质检测,结果显示5只合格用犬的平均射精量为2.85 mL,密度为2.01×10~8个/mL,pH为6.56。含有果糖的冷冻稀释液中精子活率和活力最高,分别达59.90%和54.00%;其次是添加葡萄糖和乳糖的稀释液中活率较高,分别为59.21%和56.73%,且两组稀释液中精子解冻后活力均为52.00%。进一步评估精子解冻后质膜完整率,结果显示葡萄糖和果糖组显著高于其他组,分别达48.73%和49.52%;而蔗糖添加组精子质膜完整率最低,为42.21%。稀释液中添加果糖的精子解冻后顶体完整率显著高于其他组,而添加蔗糖组顶体完整率最低。在比格犬的精液冷冻保存中,添加果糖的冷冻稀释液能显著提高精子的活率和活力,从而达到提高冻精质量的效果。     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.     

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.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

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.     

不同浓度大豆卵磷脂替代蛋黄对东佛里生奶绵羊精液冷冻保存效果的影响

本试验皆在研究添加不同浓度大豆卵磷脂（SL）冷冻保存东佛里生奶绵羊精液的效果。我们在Tris基础稀释液中,添加18%蛋黄为对照组,添加0.5%、1%、1.5%、2%、2.5%SL设为试验组,检测冷冻精液解冻后的精子活率和顶体完整率。结果显示,添加0.5%、2.5% SL冷冻稀释液稀释的精液,解冻后精子活率和顶体完整率与其他组之间存在显著差异（P<0.05）;添加18%蛋黄和1%～2% SL冷冻稀释液稀释的精液,冷冻解冻后精子活率和顶体完整率之间无显著差异（P>0.05）;添加18%蛋黄和1.0%～1.5% SL冷冻稀释液稀释后的精液,进行人工授精后母羊的妊娠率与对照组无显著差异（P>0.05）。因此,大豆卵磷脂可以作为冷冻保护剂用于东佛里生奶绵羊精液的冷冻保存,其最佳添加浓度为1～2%（g／L）。     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

藏獒冷冻精液保存液配方筛选试验

为了提高藏獒的繁殖力,建立完善的藏獒人工授精体系和保存优秀藏獒精子,本研究对8只藏獒进行了采精和精液的超低温冷冻保存试验,并对藏獒的精液超低温冷冻保存液进行了系统的筛选。结果表明,渗透压450 mOsm/L的配方Ⅴ（葡萄糖1.98 g、蔗糖3.42 g、柠檬酸钠0.59 g、谷氨酸钠0.75 g、三羟甲基氨基甲烷1.21 g、VB6 10 mL、VB12 10 mL、卵黄20 mL和甘油8 mL）对藏獒精液冷冻保存的效果最佳,解冻后精子的活力为0.58,畸形率为17.2%,满足输精和进行超低温冷冻保存精子的要求。     

Influence of Cool Storage before Freezing on the Quality of Frozen–Thawed Semen Samples in Dogs

The aim of this study was to determinate the semen quality of frozen–thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10⁶ spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris‐glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris‐glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post‐thaw sperm quality was assessed in 30 straws from each experimental group. After freezing–thawing, the total sperm motility (approximately 60–70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post‐thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.