猪精液冷冻技术的研究

以解冻后的精子活率、活力、质膜完整率、顶体完整率和人工授精结果为判定指标 ,比较了几种冷冻稀释液、解冻液及冷冻 -解冻程序对猪精液冷冻的效果 ,并对所筛选出的最佳稀释液和冷冻程序做了改进。结果如下 :(1) 号稀释液冷冻解冻后的精子活力 (32 .4± 7.3) %、活率 (4 2 .2± 3.2 ) %、顶体完整率 (6 2 .3± 0 .8) %和弯尾率 (4 2 .6± 7.5 ) %均显著高于 、 、 号稀释液 (P<0 .0 5 )。 (2 )在 号稀释液中添加 0 .5 %、1%和 2 %的 O.E.P(氨基 -钠 -十二烷硫酸酯 )使精子活率提高 5 %、弯尾率提高 6 %、顶体完整率提高近 10 % ,与对照组相比差异显著 (P<0 .0 5 )。 (3)应用改进的稀释液 ( 液加 1% O.E.P) ,细管法比颗粒法冷冻精子活率提高近 7个百分点 ,活力提高 6个百分点 (P<0 .0 5 )。(4 )室温预平衡 4h的精子活力和活率都比对照组 (0 h)有显著提高 (P<0 .0 5 ) ,而预平衡 2、6 h的精子活力和活率与对照组差异不显著 (P>0 .0 5 )。 (5 ) 号解冻液解冻后精子活力 (4 2 .9± 2 .6 ) %显著高于 、 、 号解冻液 (P<0 .0 5 )。 (6 )用 5 0℃水浴解冻精子的活力 (4 6 .3± 2 .6 ) %显著高于 39℃ (4 2 .9± 2 .6 ) %和 70℃水浴 (4 1.1± 5 .7) %解冻的精子活力 (P<0 .0 5 )。 (7)精清和 号解冻液     

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。     

解冻稀释液及孵育温度对冷冻-解冻后犬精液品质的影响

实验旨在比较解冻后解冻稀释液(Tris-柠檬酸-葡萄糖稀释液)的添加比例(1:0、1:1、1:2)对精液品质的影响,并比较了4种孵育温度(37、34、25、4℃)对精子寿命及精子活力的影响,探索合适的冷冻-解冻后犬精液的孵育条件。结果显示:解冻稀释液的添加与否及添加比例对解冻后孵育30 min的精子活力、活率、质膜完整率及顶体完整率均无显著影响;解冻后,在不同温度下孵育30 min,4组精子活率和质膜完整率无显著差异,而4℃孵育温度下精子活力和顶体完整率高于37℃(P0.05);解冻后孵育2 h,4℃孵育温度下的精液品质(精子活率、精子活力、质膜完整率、顶体完整率)最佳(P0.05),34℃和25℃组之间差异不显著,而37℃组的精液品质最差。结果表明,解冻稀释液对解冻后精液的孵育效果无影响,而解冻后精液的孵育温度以4℃最佳。     

不同冷冻保护液和解冻温度对猪精液冷冻效果的影响

在BF5稀释液中分别添加不同水平的牛血清白蛋白(BSA)(0.5,1,1.5 g/L)、二甲基乙酰胺(DMA)(0.5%,1%,1.5%)、甲基-β-环糊精载胆固醇(CLC)(1,2.5,5 g/L),对成年健康猪精液进行冷冻保存,于不同温度(37,50,70℃)解冻后分别检测冷冻后精子的活率、活力、质膜完整性、项体完整率、线粒体活性.结果显示,0.5 g/L BSA组冷冻后精子活力、质膜完整率、顶体完整率和线粒体活性均高于其他组,但差异不显著(P0.05).1% DMA组冷冻后精子活率和活力显著优于1.5%DMA组(P＜0.05),同时也高于对照组(3%甘油)冻后精子活率和活力,但差异不显著.不同质量浓度CLC组冷冻后精子的活率、活力、质膜完整性和线粒体活性与对照组相比差异不显著.50℃和70℃解冻组精子的活率和线粒体活性显著高于37℃解冻组的精子;50℃解冻组精子的活力和质膜完整率显著高于其他2组.因此,在猪精液冷冻稀释液中,用DMA可以代替甘油作为渗透性保护剂,且以1%DMA,50℃解冻最佳.     

波尔山羊冷冻精液稀释液及稀释倍数研究

通过研究波尔山羊冷冻精液稀释液和稀释倍数,以筛选最佳稀释液配方、确定适宜的稀释倍数、提高波尔山羊精液冷冻效果。结果表明,4号稀释液解冻后精子活率高于1号、2号、3号稀释液解冻后精子的活率,差异极显著( P<0 .0 1 ) ;4号稀释液解冻后精子存活时间为61 .2 6h,长于1号、2号稀释液精子存活时间( P<0 .0 1 ) ,短于3号稀释液精子存活时间,但差异不显著( P>0 .0 5 ) ,说明4号稀释液冷冻效果最好。精液稀释3倍、4倍、5倍解冻后精子活率分别为0 .5 0 2、0 .5 0 1、0 .495 ( P>0 .0 5 ) ,均高于稀释6倍、7倍、8倍的精子活率( P<0 .0 1 ) ,表明波尔山羊冷冻精液稀释倍数以3～5倍为宜。     

羊冷冻精液的制作技术研究

通过对羊精液冷冻稀释液组成成分的研究,筛选合适的稀释液配方,研究精子平衡时间、解冻温度、稀释倍数和稀释方法等对精子活力的影响。结果如下:①解冻后的精子活率以加入Tris的一组稀释液活率最高,与其他组相比差异极显著(P<0.01)。②精液的平衡时间以平衡4～5h解冻后活率最高。③在45℃～60℃的水温条件下,取得了精子复苏率74.5%的解冻效果。④选用5种稀释比例(精液:稀释液),分别为1∶4、1∶5、1∶6、1∶6、1∶8,结果1∶8的稀释比例精液解冻后活率最高,达到了70%以上。     

新西兰兔精液冷冻保护剂的筛选

本试验的目的是分析在新西兰兔精液冷冻保存稀释液中分别添加不同浓度的海藻糖、透明质酸、维生素E(Ve)、超氧化物歧化酶(SOD)对兔精液冷冻保存效果的影响,以冻后精子活率、质膜完整性、顶体完整率等作为质量评定指标,筛选出效果较好的新西兰兔精液的冷冻保护剂种类及浓度。结果表明,精液冷冻保存稀释液中添加3种浓度的海藻糖均未提高兔精液冷冻后的精子活率、质膜完整率和顶体完整率(P0.05);添加0.5%和1%的透明质酸提高了兔精液冷冻后的精子活率(P0.05),但各组间质膜完整率和顶体完整率无显著差异;添加2 g/L Ve提高了兔精子4℃平衡后的活率、冷冻后精子活率、质膜完整率和顶体完整率(P0.05);添加4 000 IU的SOD提高了兔精子4℃平衡后的活率、冷冻后精子活率、质膜完整率和顶体完整率(P0.05)。结果证实,在新西兰兔精液冷冻保存稀释液中添加适宜浓度的Ve和SOD可提高兔精液冷冻后的精子活率、质膜完整率和顶体完整率。     

在4℃降温平衡过程中0.2g/L咖啡因与精子共孵育时间对猪颗粒冻精质量的影响

实验旨在探讨在猪精液于4℃平衡2 h过程中0.2 g/L咖啡因与精液共孵育2 h、1 h和0.5 h,对颗粒冻精解冻后精子活率、活力、质膜完整性和顶体完整性以及解冻后精子体外存活时间等指标的影响,以期进一步提高猪颗粒冻精质量。实验结果表明,在精液冷冻之前,咖啡因不同平衡时间组的精子活率和活力都呈现出升高趋势,但与对照组差异不显著;咖啡因与精液共孵育2 h、1 h和0.5 h组的精子顶体完整率和质膜完整率显著低于对照组,前3组之间差异均不显著。而颗粒冻精解冻后,咖啡因与精液共孵育2 h、1 h和0.5 h组精子活率和活力均显著高于对照组,其中共孵育1 h组显著高于其他3组(P0.05),共孵育2 h组和0.5 h组间差异不显著(P0.05);共孵育2 h组精子顶体完整率和质膜完整率显著低于对照组和共孵育0.5 h、1 h组(P0.05),但后3组之间差异不显著;共孵育2 h组精子存活时间显著低于对照组、共孵育1 h和0.5 h组(P0.05),其中共孵育1 h组精子存活时间最长,达7 d以上。总之,在精液4℃降温平衡过程中咖啡因与精液共孵育1 h对冷冻后精子质量最有利,解冻后精子活率和活力显著高于其他各组,且解冻后精子体外存活时间最长。     

小尾寒羊精液保存技术研究

为了充分发挥种公羊的生产潜力,快速扩繁优良群体,本实验以取材方便的小尾寒羊为实验动物,对其精液采用室温、低温和细管法冷冻3种保存方法进行了研究。结果表明:在低温(4±0.5)℃条件下稀释保存小尾寒羊精子,显著优于室温(23±2)℃条件下的保存效果。其中采用II液低温保存绵羊精子的时间和活率(168 h,0.35)显著优于III液(72 h,0.30)I、V液(48 h,0.35)和I液(18 h,0.42)。而采用III液冷冻保存绵羊精液,其解冻后活率(0.532±0.004)极显著高于IV液(0.470±0.003)I、液(0.445±0.004)和II液(0.432±0.012)3种冷冻稀释液(P<0.01)。4种冷冻稀释液冷冻解冻后的精子,均能在室温下6 h内保持0.35以上的活率。     

绵羊冻精稀释液加复合维生素B提高冻精解冻后的品质

将山梨醇、氯前列烯醇、复合维生素B在绵羊冷冻精液稀释液中进行添加、筛选。解冻后活率结果显示1号稀释液(41.17±4.72)%和4号稀释液(37.76±3.15)%极显著地高于对照组稀释液(22.80±4.49)%(P<0.01)。表明在绵羊冻精稀释液中添加复合维生素B可有效提高冻精解冻后活率并保护精子形态结构的完整性。 绵羊冻精稀释液加复合维生素B提高冻精解冻后的品质@哈福$甘肃农业大学动物科学技术学院!730070     

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.     

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.     

Study of Semen Cryopreservation for Banna Mini-pig Inbred Line

In this study, different concentrations of glycerol were added to semen cryoprotective extenders of Banna Mini-pig inbred line (BMI), different thawing solutions and thawing methods were studied. After thawing, the motility, the rates of plasma integrity and acrosome integrity were compared to optimize the cryopreservation method of BMI semen. The results showed that the motility, the rates of plasma integrity and acrosome integrity were significantly higher in 2% and 3% glycerol groups than those in 1%, 4% and 5% glycerol groups (P<0.05);The motility, the rates of plasma integrity and acrosome integrity were significantly higher in thawing solution Ⅰ than those in thawing solution Ⅱ, Ⅲ and Ⅳ (P<0.05); Through comparing the motility, the rates of plasma integrity and acrosome integrity, the thawing effect in 40℃ 6 s and 50℃ 6 s groups were better than that in 38℃ 30 s (P<0.05). In conclusion, the optimal semen cryoprotective method for BMI was 2% or 3% glycerol as cryoprotectant, using thawing solution Ⅰ to thaw at 40℃ 6 s or 50℃ 6 s.     

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.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

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.     

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)。     

Addition of Glutathione to an Extender for Frozen Equine Semen

The manipulation of equine semen during cryopreservation reduces sperm viability and fertility because of, among other factors, membrane lipid peroxidation that makes cells highly susceptible to free radicals and reactive oxygen species (ROS). The oxidative effect caused by the generation of ROS can be reduced by the addition of antioxidants to the seminal plasma or to the extenders used for freezing. The current study was performed to test the in vitro effect of exogenous glutathione added in five different concentrations (control, 2.5 mM, 5.0 mM, 7.5 mM, and 10 mM [treatments 1-5, respectively]) to the extender for 12 stallions. Analyzed parameters were sperm motility, viability, and acrosome and plasmatic membrane integrity. Total motility was higher in treatments 1 and 2 (P < .05); viability, progressive motility, and plasmatic membrane integrity were higher in treatment 2 (P < .001). As for acrosome membrane integrity, treatment 3 showed the best results (P < .05). The addition of 2.5 mM glutathione to the freezing extender preserves total motility and increases sperm viability, progressive motility, and plasmatic membrane integrity. Concentrations above 2.5 mM were deleterious to spermatozoa.     

不同稀释液对藏猪精液4℃保存效果的影响

为探讨不同精液稀释液对4℃保存藏猪精液的效果,保证优良藏猪种公猪精液的合理、高效利用,该试验选用5种常用的猪精液常温保存稀释液,并在藏猪精液4℃保存至第5天时检查藏猪精子活力、精子畸形率、精子顶体完整率、精子质膜完整率等。结果表明,在精液保存过程中,2号液对藏猪精液的保存效果最佳,精子活力在保存至第5天时仍然保持在0.54,且藏猪精子活力、精子畸形率、精子顶体完整率、精子质膜完整率均高于其余4组（P〈0.05）。该试验研究初步筛选了4℃保存藏猪精液的稀释液,为今后藏猪精液的生产应用奠定了基础。     

版纳微型猪近交系精子冷冻保存研究

在版纳微型猪近交系（BMI）公猪精液冷冻保存稀释液中添加不同浓度甘油,并对不同解冻液配方和解冻方法进行研究,对比解冻后的精子活力、质膜完整率和顶体完整率,优化BMI公猪精液冷冻保存方法。结果表明,解冻后,甘油浓度为2%和3%组的精子活力、质膜完整率、顶体完整率显著高于1%、4%和5%组（P<0.05）;Ⅰ号解冻液解冻后精子活力、质膜完整率、顶体完整率均显著高于Ⅱ、Ⅲ和Ⅳ号解冻液（P<0.05）;在活力、质膜完整率和顶体完整率方面,40℃ 6 s和50℃ 6 s这两种程序的解冻效果都显著优于38℃ 30 s（P<0.05）。由此可见,2%或3%的甘油作为抗冻保护剂、Ⅰ号解冻液解冻、40℃ 6 s或50℃ 6 s水浴解冻是较为理想的BMI公猪精液冷冻保存方法。