pH值和含甘油稀释液平衡时间对藏獒精液冷冻保存活率的影响

试验筛选6条种公藏獒在其繁殖季节9—12月份以不同pH梯度(5.5、6.0、6.5、7.0、7.5)稀释液和不同甘油平衡时间(10、20、30、40、50、60、70、80min和90min)对藏獒精液进行冷冻,检查冷冻前后活率变化。结果表明:藏獒精子在pH为6.5的稀释液中有较高冷冻前后活率;甘油平衡时间对藏獒精液活率有较大影响,以甘油平衡时间为20min所得藏獒冷冻精液的效果较好。     

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。     

不同冷冻曲线对种公牛精液冷冻质量的影响

种公牛精液的冻后活力是评定冷冻精液品质好坏的重要指标之一,也是影响牛人工授精受胎率的重要因素.影响冻精解冻后活力的因素包括种公牛个体差异(鲜精品质、耐冻性)、稀释液(甘油浓度、稀释液成分)、生产工艺(稀释方法、平衡、冷冻方法、冷冻曲线)和解冻方法等.而冷冻速度,即精子通过对细胞产生致死性伤害的0～-60℃温度区的速度,是决定冷冻精液冻后品质好坏的关键所在.     

传统稀释液配方中添加不同保护剂对藏羊精液冷冻效果的影响

为探索在传统稀释液配方中添加保护剂对藏羊精子的冷冻保存效果,在传统稀释液配方(果糖-蔗糖-柠檬酸钠-甘油)中,由Tris(三羟甲基氨基甲烷)代替柠檬酸钠,乙二醇代替甘油,在果糖-蔗糖-Tris-乙二醇基础上添加维生素B12、维生素E、维生素C、复合维生素B等,以增强精子缓冲力,减少毒性,提高精子活力、顶体完整率及受精力。结果表明:精液冷冻保存稀释液中添加Tris比添加柠檬酸钠有更好的冷冻保存效果,能有效改善冻后精子活力和顶体完整率;精液冷冻保存稀释液中添加维生素B12对精子有良好的保护作用;用等体积乙二醇代替甘油作为防冻剂,对精子冷冻时的保护作用不亚于甘油;藏羊细管冷冻精液的制作中,用果糖-蔗糖-Tris-乙二醇-维生素B12组合稀释液配方进行2次稀释,8 min冷冻,其冷冻效果最佳,顶体完整率最高。     

几种因素对鸵鸟冷冻精液精子活力影响的研究

对每组20份精子样品,采取不同冷冻程序,分析了各单因子如:稀释液种类、稀释液中甘油浓度、稀释方法、4℃低温平衡时间、冷冻面与液氮面距离及热平衡时间对鸵鸟冷冻精液的精子解冻复苏活率的影响。结果表明:以含甘油浓度2.8%的(MEM·蛋黄)稀释液,采用两步稀释,低温平衡2.5h,冷冻面与液氮面距离1.5cm进行2min热平衡,鸵鸟精液的冷冻效果较好。     

新疆驴细管冻精的制作

试验旨在研究新疆驴细管冻精制作过程中精液冷冻液、降温平衡时间及液氮熏蒸时间对精子冻后活力的影响。研究共设计3个试验。试验1对比了INRA-Freeze、Optidyl、房氏驴精液冷冻保存液、自配马精液冷冻冷冻液等4种精液冷冻液在精子冷冻过程对精子冻后活力的影响,筛选出适合制作新疆驴细管冻精的冷冻液;试验2探索在细管冻精制作过程中平衡90、120、150 min对精子冻后活力的影响;试验3比较在细管冻精制作过程中使用液氮熏蒸5、6、7、8、9、10 min时对精子冻后活力的影响,探索液氮熏蒸的最佳时间。结果显示,使用INRA-Freeze冷冻液或房氏冷冻液在冷冻过程中对精子的损伤最小;冷冻过程中,降温平衡90、120、150 min对精子冻后活力的影响差异不显著（P>0.05）;在冷冻过程中液氮熏蒸7 min时精子冻后活力最高,显著高于熏蒸5、9、10 min的精子冻后活力（P<0.05）,与熏蒸6、8 min时精子冻后活力差异不显著（P>0.05）。研究表明,为降低成本可使用INRA-Freeze冷冻液稀释精液,于冰箱中平衡90～150 min后装入0.5 mL的细管...     

不同剂型及稀释精液平衡时间对藏獒冻精品质的影响研究

为了探讨每剂含不同前进运动精子数(≥1 000万个和≥2 000万个)和稀释精液不同平衡时间(1 h、2 h、3.5 h)对藏獒冻精品质的影响,试验比较了0.25 m L细管剂型藏獒冻精解冻后精子活力、精子顶体完整率、精子畸形率、精子37℃存活时间。结果表明:每剂含前进精子数≥2 000万个与≥1 000万个相比,藏獒冻精解冻精子活力、精子顶体完整率、精子畸形率差异不显著(P0.05);而每剂含前进运动精子≥2 000万个组的藏獒冻精解冻后37℃存活时间优于每剂含前进运动精子数≥1 000万个组,且差异显著(P0.05)。藏獒稀释精液平衡2 h组的冻精活力显著高于平衡1 h组和3.5 h组(P0.05),而平衡1 h组和3.5 h组间差异不显著(P0.05)。冻精顶体完整率以平衡2 h组最高,为(41.00±1.47)%,与平衡3.5 h组间存在显著差异(P0.05)。平衡2 h组的冻精畸形率低于平衡1 h组和3.5 h组。说明藏獒0.25 m L细管剂型以每剂含前进运动精子数≥2 000万个,稀释精液平衡时间以2 h为宜,可获得较好的冷冻效果。     

不同稀释液配方和冷冻保护剂对贵州黑山羊精液品质的影响

为探究适合贵州黑山羊冷冻精液的稀释液和冷冻保护剂,通过精液质量分析系统比较4种常用的山羊冷冻精液稀释液配方和2种冷冻保护剂对贵州黑山羊精液品质的影响。结果:不同配方中以甘油作为冷冻保护剂对贵州黑山羊精液的保护效果均优于乙二醇,且配方1的精子前进式活力最高(22.73±6.25),配方3次之(20.14±5.25)。结论:经综合评价,在贵州黑山羊冻精生产过程中可选择配方1或配方3作为冷冻精液稀释液,以甘油作为冷冻保护剂。     

提高茸鹿冻精质量关键技术

为进一步提高茸鹿冻精质量,对影响精液冷冻效果的关键因素进行了试验。结果评估得出:精液冷冻宜采用两步冷冻法,细管距液氮面2~3cm,冷冻时间5~7min;冷冻稀释液优先顺序为TriladyTrisBTrisA,Trilady稀释液从经济适用和冷冻效果上看最优;鲜精质量与冷冻效果密切相关,鲜精活力与冻存复苏率、解冻后精子活力呈正相关,R2分别为0.630 6和0.990 2;平衡时间在4℃宜平衡2~3h;平衡方法宜采用水浴中逐渐降温的方法。结果表明:通过进一步优化精液冷冻关键影响因素,可明显提高茸鹿冻精质量。     

近交五指山小型猪精液冷冻保存技术的研究

用液氮熏蒸法在氟板上制作冻精颗粒,以解冻后的精子活率、活力和质膜完整性为判定指标,比较4种冷冻稀释液及不同冷冻-解冻程序对五指山小型猪精液冷冻的效果。结果表明:①Ⅳ号冷冻稀释液冷冻解冻后精子的活率(0.610±0.036)、活力(0.427±0.025)和质膜完整性(0.503±0.015)均显著高于Ⅰ、Ⅱ和Ⅲ号冷冻稀释液(P<0.05)。②实验中精液在4℃冰箱中平衡降温2 h的精液精子活力、质膜完整性均好于在17℃平衡3 h再放入4℃冰箱中平衡2 h的解冻效果,而且精子活率差异显著(P<0.05)。③湿解法的效果优于干解法。     

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)。     

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.     

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。     

Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.     

Effects of Freezing–Thawing on DNA Integrity of Boar Spermatozoa Assessed by the Neutral Comet Assay

A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.     

Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability

Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.     

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.     

提高奶牛细管冻精精子活力的试验研究

为了提高奶牛细管冻精精子的活力,试验探索了稀释液种类、最佳熏蒸距离、最佳冷冻温度、最佳熏蒸时间、不同解冻温度及时间对精子活力的影响。结果表明:精子在由柠檬酸钠和果糖组成的稀释液中存活时间长,在三羟甲基氨基甲烷稀释液中冷冻后精子活力高于其他稀释液;牛细管冻精最佳熏蒸距离为2.5 cm,时间影响不显著(5～10 min均可);用50℃温水解冻15 s的精子活力比其他解冻温度和时间时的精子活力要好。     

红腹锦鸡精液冷冻稀释液配方的筛选

为加强濒危珍稀动物种质资源的保护,开展了濒危珍稀禽类—红腹锦鸡的人工授精研究。2005年5月26日~6月8日,对8只红腹锦鸡用手按摩其背、尾部采精12次。初测其精液品质,结果表明,采精量平均(0.114±0.016)mL(0.01~0.2 mL),每毫升精液中精子平均3.2亿个(3.0~3.3亿),活力9级以上,pH值6.5,偏酸性,淡乳白色(半透明似冲熟的藕粉),微腥。鲜精分别加11%蔗糖—卵黄稀释液(3号液);11%蔗糖—0.1%柠檬酸三钠—卵黄稀释液(5号液);5%葡萄糖—卵黄稀释液(8号液)。精子活力达8~9级。用3%柠檬酸三钠—卵黄稀释液(2号液)稀释,精子活力2级。冷冻时,用11%蔗糖溶液100 mL中加16 mL鲜卵黄,加5 mL甘油,配制的3号冷冻稀释液稀释,解冻后,精子活力达4级。优于试验中选拟的其它配方(加入5 mL甘油的5号冷冻液,解冻后精子活力为3级;加入5 mL甘油的8号冷冻液,解冻后未见活的精子)。如果冷冻稀释液中甘油改为6 mL,则解冻后精子全部死亡。