提高牛细管冻精精子活力的试验研究

本文从细管冻精的3个关键技术环节研究了稀释液的种类对精子活力与存活的影响,确定了细管冻精精子在液氮冷冻度过冰晶期对其损伤最小的最佳熏蒸高度（最佳冷冻温度）和最佳熏蒸时间,并且对成品冷冻的细管冻精不同解冻温度及时间进行研究。由柠檬酸钠和果糖组成的稀释液精子存活时间长,Tris稀释液冷冻后精子活力高于其他配方;牛细管冻精最佳熏蒸距离为2cm,时间影响不显著,7~9min均可;用50℃温水15s解冻精子的活力最高。     

组织培养液M199在庆阳驴细管精液中的应用

为了研究M199对庆阳驴细管鲜精和细管冻精活力及保存时间的效果影响。在鲜精稀释液中,添加不同浓度的M199,在低温保存下,观察不同时间段精子活力;在冻精稀释液中,添加1%的M199,采用液氮熏蒸制作冻精,解冻后观察冷冻效果。结果显示,在低温保存中,稀释液中添加1%的M199,48h后精子活力仍然在0.35以上,与其他试验组和对照组差异极显著(P0.01)。在冻精稀释液中添加1%的M199,试验组与对照组冻后精子活率分别为30.83%和31.33%,两者之间差异不显著(P0.05),但试验组精子有效存活时间显著高于对照组。通过在庆阳驴精液稀释液中添加M199,均能很好的延长精子保存时间。     

不同稀释方法对小型宠物犬精液冷冻效果影响

为了研究Tris-果糖稀释液在不同稀释方法下对小型宠物犬精液冷冻的效果,选取常见的3个小型宠物犬品种共计6只犬进行试验,精液稀释过程分别采用一步稀释法和两步稀释法。结果表明:冷冻精液解冻后各组犬精液的同一剂型间的精子活力差异均不显著(P0.05);一步稀释法中的颗粒冻精与0.25 mL细管冻精和0.50 mL细管冻精相比,解冻后精子活力差异均显著(P0.05),而0.25 mL细管冻精与0.50 mL细管冻精的解冻后精子活力差异极显著(P0.01);采用两步稀释法的颗粒冻精、0.25mL细管冻精及0.50 mL细管冻精的解冻后精子活力组间差异均不显著(P0.05)。用Tris-果糖稀释液稀释精液时,使用两步稀释法对不同冻精剂型的精子活力影响不明显。     

牛细管冷冻精液最佳解冻温度的筛选试验

人工授精技术在牛繁殖改良中发挥着极其重要的作用，在实际生产中影响受胎率的因素很多，本试验通过采用不同温度、不同解冻时间对解冻后精子活力做对比试验，寻找最佳的解冻温度、解冻时间，从而提高细管冻精解冻后精子活力和受胎率。     

2种稀释液对公猪精液冷冻保存效果的影响

试验为探究2种不同冷冻稀释液对公猪精液稀释冷冻效果的影响。试验采用手握法采集同一头公猪精液,将采集的精液分为2组,进行液氮熏蒸制作细管冻精,分别使用Ⅰ号冷冻稀释液和Ⅱ号冷冻稀释液对猪精液进行冷冻保存,24 h后检测2种冷冻稀释液细管冻精的活力。结果显示,Ⅰ号冷冻稀释液冷冻解冻后的精子活力显著高于Ⅱ号冷冻稀释液(P0.05),同一种稀释液距液氮面5 cm进行熏蒸冷冻的效果较距液氮面4 cm冷冻效果好,但差异不显著(P0.05)。研究表明,利用Ⅰ号冷冻稀释液在距液氮面5 cm处进行液氮熏蒸冷冻可获得较好的公猪冷冻精液。     

牛冷冻精液在使用中应注意的几个问题

１器具消毒牛冷冻精液在解冻时应注意所有直接接触冷冻精液的器具都应是灭菌消毒过的。防止液氮罐内和解冻过程中的污染，如镊子、细管剪刀等。细管冻精从液氮中取出后先在空气中停留６秒钟，让细管口内的液氮充分挥发，以防止细管放炮。剪塑料细管封口时，应使用专用的细管剪刀，这样一来剪的口齐而且圆，输精时精液不易倒液。解冻时要用温水，３８～４０℃是适宜的解冻温度，温度低了影响精子的恢复，高于４２℃会使精子迅速死亡。经试验证明用温水解冻的冻精活力比自然解冻、手搓法解冻、冷水解冻的冻精活力高出０．１～０．２，所以冻精…     

简易冷冻装置制备鸡甘油细管冻精的优化

为优化简易装置制备鸡精液甘油细管冻精的条件,采用11%的甘油浓度、不同的熏蒸方式和熏蒸时长等条件制作细管冻精,通过测定精子活力及人工输精后的孵化效果筛选冷冻、解冻条件。结果表明:先以-15^-6℃/min的速率熏蒸1~5min后,再以-60^-25℃/min的速率熏蒸1~5min(两步均1min除外)冷冻效果最好,解冻后活力达0.8以上;以5℃的温度解冻30~60s和10℃的温度解冻15~60s的解冻效果最佳;用优化的冷冻程序、6%浓度甘油制作细管冻精,最高平均获得了77.63%的受精率和73.38%的出苗率。研究结果为鸡精液简易装置冷冻保存技术的系统化和标准化研究提供了技术支撑。     

Tris-柠檬酸添加不同植物蛋白粉冷冻奶牛精子实验研究

本文研究了Tris-柠檬酸缓冲液中添加大豆蛋白粉、花生蛋白粉、大豆和花生蛋白粉混合物,替代卵黄稀释液冷冻奶牛精子的效果。做了两方面的实验研究:(1)用3种Tris-柠檬酸蛋白粉和对照Tris-柠檬酸卵黄稀释液超低温细管冷冻奶牛精子,观察超低温冷冻奶牛精子活力的冷冻试验;(2)超低温冷冻奶牛细管冻精解冻后在4℃环境中保存的存活实验。研究结果表明,4种不同稀释液超低温冷冻奶牛精子后精子活力差异不显著(P0.05);4种不同稀释液冷冻细管冻精解冻后于4℃冷藏并连续观察,每天的精子活力变化差异显著(P0.05),第5天Tris-柠檬酸大豆和花生混合蛋白粉稀释液的活力最好,并且在显微镜下观察视野要比对照Tris柠檬酸卵黄稀释液清晰,不易滋长细菌。因此,混合大豆蛋白粉和花生蛋白粉的植物源稀释液可以替代动物源稀释液。     

犬冷冻精液技术的试验研究初报

介绍了犬微型细管(0.25 mL)冻精制作方法,优选出冷冻稀释液及稀释操作程序。制作结果:冷冻精液解冻活力达0.405±0.058,精子顶体完整率为(43.58±6.73)%,复苏率达54.6%。精子畸形率为(13.80±4.26)%;不同品种公犬鲜精和冻精品质差异不大,但个体差异显著,有的耐冻性很差;不同初始冷冻温度下,解冻效果各异,以-111~-130℃冷冻效果较好;冷冻时间10~15 min为好;稀释方法还待进一步探讨。     

牛细管冻精解冻后常温保存对受胎率的影响试验

[目的]为测试牛细管冷冻精液解冻后在常温水中的保存时间对精子活力的影响,为非定点配种寻求有效简便的冻精携带方法,观察精子在不同解冻温度下的存活时间及受胎率。[方法]选择5个解冻温区及不同时间解冻肉牛细管冻精,解冻后置于常温水(17℃~19℃)中保存,观测解冻温度和精子存活时间对受胎率的影响。[结果]50℃、60℃、70℃、解冻后精子的保存时间差异不显著,保存至72h时精子活力均低于0.35;受胎率差异不显著;随保存时间延长,受胎率呈下降趋势,在48h以内情期受胎率和总受胎率分别维持在61.8%~67.1%和91.9~95.8。[结论]70℃各时间段解冻后保存效果最佳,60℃解冻组次之,二者在精子活力≥0.35时情期受胎率和总受胎率达到63.0%、92.5%,略高于当地黄牛两项受胎率的平均值61.1%、91.7%,解冻后细管精液置于常温水中携带到较远的区域输精对精子活力、母牛受胎率无影响。     

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。     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.     

解冻温度对精子活力的影响

[目的]为了获得较为理想的解冻效果.[方法]采用不同温度,不同解冻时间,通过镜检解冻后活力的方法.[结果]发现:不同解冻温度对牛冷冻精子复苏的影响,用39.5℃温水解冻30 s精子的活力最高,冷冻精液解冻后精子活力达O.53.用40.O℃温水解冻10s、39.0℃温水解冻30 s精子的活力也较高,冷冻精液解冻后精子活力...     

不同冷冻方法对甘肃高山细毛羊精液冷冻效果的研究

为了研究甘肃高山细毛羊种公羊精液冷冻保存的效果.将液氮熏蒸距离按1.5、2.0、2.5 cm分为三个组合,每个组合用6.5、7、7.5 min三种时间进行熏蒸处理,对细毛羊种公羊冻精进行对比试验.熏蒸距离为2 cm,熏蒸时间为6.5 min、7 min和7.5 min处理组精子活率分别为24.91±3.31、42.33±3.96和32.83±2.08,与其它各组精子活率相比较好,组间差异显著（P＜0.05）.试验表明,细毛羊种公羊冻精熏蒸距离越近,熏蒸时间应减短,熏蒸距离越远,熏蒸时间应延长.最佳熏蒸距离为2 cm和熏蒸时间为7min,精子活率42.33±3.96.液氮熏蒸细管冷冻法的精子活率（42.33±3.96）,高于液氮熏蒸颗粒法（33.75±5.32）,而二者差异显著（P＜0.05）.液氮颗粒法的顶体完整率（33.75％）和细管法的顶体完整率（39.08％）差异不显著（P＞0.05）.通过调整细毛羊精液稀释液成分、pH值、渗透压,利用简易的液氮熏蒸方法可以完成甘肃高山细毛羊精液冷冻保存.     

不同季节和气候条件对种公牛精液品质的影响的研究

[目的]研究不同季节气候条件对种公牛精液品质的影响。[方法]从采精种公牛中随机抽出5头,选取气温最高（7月份）最低（1月份）和适中温度（4,9月份）进行试验,分别对不同时期所采得的精液进行检测。[结果]在最高温度月份7月份（25℃）时5头种公牛的平均精液量为5.88mL,原精活率67.2%,精液密度16.64亿/mL,冻后活率33.8%,生产冻精数143.4剂,精子畸形率23%。1月份环境温度平均在-11.2℃时,5头种公牛的精液量平均为5.7mL,原精活率66.8%,精液密度13.3亿/mL,冻后活率37.4%,生产冻精数为275.8剂,精子畸形率17.8%。在4,9月份环境温度12℃时精液量为6.6mL,原精活率73.2%,精液密度16.92亿/mL,冻后活率41.8%,生产冻精数404.4剂,精子畸形率12.6%。精液量之间差异显著（P〈0.05）,原精活率之间差异显著（P〈0.05）,精子畸形率之间差异显著（P〈0.05）。冻后活率和冻精生产数差异不显著。[结论]环境温度过高或者过低都会影响精液品质,种公牛在适宜的温度下生产的精液品质也会更加优良,种公牛在温度较高的条件下生产精液品质比在低温条件下还要差,种公牛最适宜的生产温度为12℃～16℃。     

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℃.     

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。     

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)。     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.