牛精液的贮存、解冻及品质检查

1冷冻精液的贮存和解冻方法牛的冷冻精液是以液氮做冷源进行贮存的,需要时可随时取出。为防止温度变化对精液品质的影响,取放动作要迅速,尽量减少在空气中停留的时间。从贮存容器中提取冷冻精液时,精液不应超过液氮容器的颈基部,避免因温度的回升造成精液解冻活率的下降。冷冻精液的解冻过程会影响精子的活力,要注意解冻的温度和操作方法。最初世界各国对解冻温度的要求不同,大体可分为高温40℃、室温15￣20℃和冷水10℃,当前认为40℃左右解冻效果最好。随着解冻温度的降低,精子活率有逐渐降低的趋势。颗粒精液解冻多采用将颗粒精液投入一定…     

BMY牛冷冻精液质量控制及影响因素研究

[目的]为了加大BMY牛的推广应用力度和提高人工授精受胎率,开展了BMY牛冷冻精液质量控制研究,优选出冷冻稀释液及得出冷冻精液质量控制技术程序。[方法]采用不同的初始冷冻温度、不同种类的稀释液、不同的冷冻方法对BMY牛精液冷冻及解冻效果进行试验。[结果]不同的初始冷冻温度、不同种类的稀释液、不同的冷冻方法,BMY牛精液冷冻解冻效果各异。初始冷冻温度为-121--140℃时,TRIS液为基础稀释液添加糖类和氨基酸一步法稀释BMY牛精液冷冻6-10 min BMY牛冷冻精液解冻效果较好,用程控冷冻仪冷冻BMY牛精液,同样获得良好的解冻效果,解冻活力达到0.372±0.026。BMY牛冷冻精液平均解冻活力可达0.357±0.029以上,精子解冻复苏率达50%以上。精子畸形率平均为16.8%±4.26%,BMY牛采精成功率为75.3%。[结论]全放牧条件下BMY牛电刺激采精法获得很好效果,精液质量好,更有利于种质资源的保存和利用。     

提高奶牛冷冻精液使用效果的措施

如何提高奶牛冷冻精液的使用效果，在奶牛繁殖中是人们一直追求的目标。根据多年的实际生产经验，在冷冻精液的冷冻方法、解冻液配方、解冻温度、解冻后的保存温度等方面进行了改进，取得了较好的效果。     

家禽精液保存技术研究进展

家禽的精液保存是人工授精技术中的关键环节,该技术水平的高低将直接影响生产过程中的经济效益。文章主要围绕短期保存过程中精液稀释液、稀释比例、保存温度及长期冷冻保存过程中冷冻保护剂、冷冻方法、平衡时间和解冻温度的研究等方面进行阐述。     

提高奶牛冷冻精液使用效果的措施

如何提高奶牛冷冻精液的使用效果,在奶牛繁殖中是人们一直追求的目标。作者根据多年的实际生产经验,在冷冻精液的解冻液配方、冷冻方式、解冻温度、解冻后的保存温度等方面进行了改进,取得了较好的效果。     

牛细管精液冷冻温度曲线的研究

牛冷冻精液精子复苏率的高低与原精质量、精子耐冻性、稀释液种类、稀释方法及精液冷冻和解冻速度等因素有关。其中，精液在冷冻过程中温度变化曲线是影响精子复苏率高低的重要因素之一。研究细管精液在不同条件下冷冻，精液温度变化曲线及精子复苏率变化的规律，优选出细管精液最适宜的冷冻温度曲线，可以为提高牛冷冻精液质量提供重要保障。     

猪精液冷冻和解冻影响因素的研究进展

为优化冷冻和解冻方法,提高猪精液冷冻效果,需要探讨不同冷冻效率、不同解冻温度、不同冷冻保存形式、不同冷冻方法、不同精液稀释、不同冷冻保护剂对猪精液冷冻效果的影响,从而选择最优的组合,能够减少冷冻精液与鲜精在猪场实践中的差距。     

牛精子冷冻损伤研究进展

人工授精技术是目前通过冷冻精液进行牛遗传改良而应用最为广泛的生物技术。虽然如此,由于精液产品的质量而导致人工授精的遗传影响被限制,在精液的冷冻－解冻过程中40%~50%的活精子失去完整性或功能受到损伤。作者在参阅国内外相关文献的基础上,通过阐述冷冻－解冻对精子细胞的损伤作用及机理、精浆对牛冷冻精液质量的影响、冷冻－解冻过程中精子的过氧化损伤,以及冷冻－解冻后精子相关基因与蛋白质的变化等,以期为牛高质量冷冻精液的研究提供一定参考,进一步提升冷冻精液质量。     

不同解冻方案对牛冷冻精液质量的影响

解冻是冷冻精子复苏的关键步骤,解冻方案的选择则是冷冻精子复苏的关键点。为筛选出适合目前我国规模化牧场冷冻精液的解冻方案,本研究分别采用6种解冻方案对冷冻精液进行解冻并观察其解冻活率。结果表明,随着解冻温度的升高和解冻时间的缩短,精子活率呈上升趋势,并且当解冻温度达到40℃,解冻时间为20s时,冷冻精子解冻后的活率达到最高。关于6种解冻方案对精液存活时间的影响研究结果表明,当采用38~39℃解冻30s方案时,精子存活6h的活率最好。     

鸡精液最佳冷冻时间和解冻温度的确定

以农大褐父母代蛋种鸡为材料，研究鸡精液的最佳冷冻时间和解冻温度表明，在冷冻保护液中保存3Omin的精子活力最高（0．38），冷冻精液解冻温度以30～40℃之间最好，解冻20min以内输精，方可获得较理想的受精率。     

Deep-freezing of boar semen in plastic film 'cochettes'

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

Study on Technology of Black Silkies Chicken Semen Freezing by DMA Pellet

The aim was to explore the effects of different kinds of dilution and thawing devices on the N,N-dimethylformamide (DMA) pellet frozen semen of Black Silkies. Firstly,the motility and fertility of the frozen semen thawed by different dilutions were compared;Then,the motility of the frozen semen was compared when the pellets were thawed using different tube and different number.Finally,the motility and fertility of the sperm thawed by three kinds of thawing devices (thermostat water bath,hotfunnel and hotplate ) were tested. The results showed that:①There was similar order of the sperm motility and fertility in the different dilution groups (LR > F > B > L),and there was significant difference among those groups (P < 0.05).②The motility was the best when the frozen semen was thawed with large thin-wall glass tube at 60℃.③The best temperature range of the 3 devices was different. The highest motility for thermostat water bath was 50 to 60℃ (0.51 to 0.59),and thermostat hotfunnel was 40 to 45℃ (0.42 to 0.46),while thermostat hotplate was 50 to 55℃ (0.61 to 0.63).There was no significant difference of the motility in the optimum temperature range for each device (P > 0.05).④The fertility of the different devices in their best thawing temperature was 26.91% (55℃,thermostat hotplate),23.08% (60℃, thermostat water bath), 20.93% (40℃, thermostat funnel),respectively,and there was no significant differences among those groups (P > 0.05).Therefore,the efficiency thawing condition for the Black Silkies frozen semen was the LR diluent,DMA cryoprotectant,pellet freezing,thawed in the thermostat hotplate at 54.9℃.     

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。     

黑丝羽乌骨鸡精液DMA颗粒冷冻技术研究

为研究不同稀释液与解冻装置对黑丝乌骨鸡N,N-二甲基甲酰胺（N,N-dimethylformamide,DMA）颗粒冻精解冻后精子质量的影响,试验首先采用不同稀释液对精液进行稀释并比较其解冻后精子活力与人工输精的受精率;其次,选取不同解冻管及冻精颗粒数进行恒温水浴解冻,比较其活力;最后,依据解冻后的精子活力,筛选恒温水浴、恒温漏斗、恒温板3种解冻装置各自的最佳解冻温度,并利用各自最佳解冻温度解冻后的精液进行人工输精,检测受精率。结果显示：①不同稀释液组的精子活力与受精率高低趋势一致,为LR > F > B > L组,且各组之间差异显著（P < 0.05）。②在60℃恒温水浴中,用薄壁大玻璃管解冻的精子活力最好。③不同解冻装置有各自最佳解冻活力的温度范围,恒温水浴为50~60℃（0.51~0.59）、恒温漏斗为40~45℃（0.42~0.46）、恒温板为50~55℃（0.61~0.63）,每种装置最佳温度段内的精子活力差异不显著（P > 0.05）。④3种解冻装置最佳解冻状态相比：在精子活力上,60℃恒温水浴与55℃恒温板分别显著高于40℃恒温漏斗（P < 0.05）,但60℃恒温水浴与55℃恒温板之间差异不显著（P > 0.05）;在受精率上,55℃恒温板最高（26.91%）,60℃恒温水浴次之（23.08%）、40℃恒温漏斗最低（20.93%）,三者之间差异不显著（P > 0.05）。因此,黑丝羽乌骨鸡精液应采用LR稀释液、DMA冷冻保护剂及颗粒冷冻技术,在54.9℃恒温板解冻可获得较高的受精率。     

猪精液冷冻技术试验

从稀释液和解冻液的筛选，平衡时间、平衡温度、冷冻剂型的选择等方面进行试验。试验结果：猪精液冷冻后活率为３０％－６０％；解冻后的复苏率为５１．４２％－５８．７２％；保存３０天的冻精与保存３６０天的冻精受胎率无明显差异（Ｐ＞０．０１）；冻精保存期一年以上；输精受胎率达６３．６４％（６０％－６６．６６％），平均窝产仔６．１头。     

Effects of liquid nitrogen vapor sensitization conditions on the quality of frozen-thawed dog spermatozoa

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.     

Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO, and concentration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozen-thawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifugation, and after cooling down to 4°C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4°C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO.     

冷冻方法对杜泊绵羊冷冻精液品质的影响

试验对绵羊精液分别采用液氮熏蒸冷冻法和冷冻仪冷冻法进行冷冻,通过测定精液解冻后精子活率、顶体完整率、质膜完整率和谷草转氨酶活性及乳酸脱氢酶活性来比较不同的冷冻方法对解冻后精液品质的影响;通过测定精液稀释后、平衡后、冷冻仪法冷冻后的酶活性来比较谷草转氨酶与乳酸脱氢酶在不同阶段的释放量。结果表明,采用冷冻仪法冷冻后的精子活率和质膜完整率极显著高于液氮熏蒸冷冻法（P<0.01）;冷冻仪法冷冻后的谷草转氨酶和乳酸脱氢酶活性极显著低于液氮熏蒸冷冻法（P<0.01）;精液稀释后的谷草转氨酶和乳酸脱氢酶活性极显著低于精液冷冻后的活性（P<0.01）。表明采用冷冻仪冷冻法的绵羊精液品质好于液氮熏蒸冷冻法。精子中谷草转氨酶主要在平衡阶段释放,而乳酸脱氢酶主要在冷冻阶段释放。     

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。     

Effect of addition of Orvus ES paste to frozen canine semen extender on sperm acrosomes

Using the triple-stain technique, we investigated whether sperm acrosomes in frozen canine semen were protected during freezing and thawing by addition of a surfactant, Orvus ES Paste (OEP), to the extender. Acrosomes were clearly shown to be protected by the addition of OEP to the entender when compared with those in sperm frozen without OEP addition (p<0.05).