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。     

大熊猫细管冻精制备程序的建立与应用

筛选了不同的冷冻稀释液(TEST,蔗糖-卵黄-甘油液)、不同的冷冻剂型(颗粒法,细管法)、不同甘油终浓度、不同的解冻液(M199,Ham′sF10)等条件,在此基础上,建立了大熊猫细管冻精制备程序。与传统颗粒冻精制备技术相比,本试验极显著地提高了大熊猫冷冻精液质量,冷冻精液活力从35.45%提高到57.88%(P<0.01);顶体完整率从42.25%提高到78.24%(P<0.01)。从1999年至2002年共有14只雌性大熊猫在共计36只次发情配种中采用了细管冻精,较1992～1998年采用传统颗粒冻精的总受胎率提高了11.2个百分点(达到55.6%),其中经产健康适龄雌性大熊猫的受胎率提高了27.3个百分点(达到83.3%),处女大熊猫的受胎率提高了8.8个百分点(达到58.8%)。     

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。     

细管冷冻精液试验

<正> 目前世界各国盛行用塑料细管冻制牛精液,我国由于设备条件的限制,再加对细管冷冻精液的规格,型号难以统一,生产中未能推广应用。为了尽快地把细管冷冻精液技术应用到生产实践中,我们对细管冷冻精液的封装,冻制,冷冻稀释液的筛选,以及推广使用等进行了试验。试验方法1.稀释液的筛选:冷冻稀释液的好坏直接关系到冻精质量,我们在冻制细管精液时,筛选了三种稀释液配方:第一种是12％蔗糖液73cc、鲜卵黄20cc、甘油7cc;第二种是将第一种中的12％蔗糖液改为,12％蔗糖液与2.9％柠檬酸钠液按1∶1配制后提取73cc,其它成份同第一种;第三种是在第二种稀释液中以每100cc另加入果糖1克即成。     

不同稀释液配方对奶牛冷冻精液的影响

采用假阴道法采集成年种公牛精液,经精液品质检查后,合格精液用四种不同冷冻稀释液进行稀释、冷冻、以筛选出适宜的冷冻稀释液和最佳冷冻保护剂.结果表明:Ⅱ号稀释液优于其它3种稀释液(P＜0.05);甘油为最佳冷冻保护剂.优于乙二醇和DMSO冷冻效果(P＜0.05),其最佳浓度为7%,精子冷冻后活率平均可达0.45±0.575,畸形率15.44%,项体完整率57.82%;奶牛细管冻精的解冻效果以75℃水浴,2 S到6 S的解冻时间最佳.     

德国牧羊犬精液冷冻保存研究

用手握法采９条德国纯种牧羊犬（２～８岁）精液,在卵黄Ｔｒｉｓ和卵黄－葡萄糖稀释液中添加保护剂甘油和ＤＭＳＯ,液氮熏蒸制作颗粒冻精。结果表明,卵黄－Ｔｒｉｓ稀释液优于卵黄－葡萄糖稀释液。ＤＭＳＯ单独加和与甘油混合加均不利于解冻后精子活率,最佳甘油浓度为４％～６％,始冻温度－１５０℃热平衡温度－１０５℃,入氮温度－１１８℃,保持了正常的冷冻温度曲线,干解冻法解冻后活率高于湿解法（Ｐ〈０．０５）。在最优     

多浪羊细管冷冻精液制作技术的研究

为保存和利用优秀地方品种多浪羊的遗传资源,试验在多浪羊原产地进行了细管冻精制作试验.用采集的多浪羊种公羊精液,比较5种冻精保存液配方的细管冻精冷冻效果,测定解冻后精子活力.结果表明:4.83%乳糖保存液解冻后精液的活率平均为36% ,优于其它4种保存液,且差异极显著(P<0.01),解冻后精子的畸形率也均低于其它4种稀释液,差异极显著(P<0.01).表明4.83%的乳糖保存液配方是理想的绵羊细管冻精稀释液.     

牛精液采集及其处理过程对解冻后精子特性的影响

本研究中使用了4头荷斯坦公牛的32次射精。所得精液用20％卵黄—柠檬酸盐稀释(1:10),在0.5小时和3小时内冷却至5℃,再加入甘油,平衡2小时。然后将细管置于氮液面上冷冻,在-196℃液氮中贮存。将冻精分别在46、37和23℃解冻12、20和60秒。用聚乙烯做伪阴道     

不同降温方法对猪冷冻精液受胎率的影响

最近,法国对冷冻前不同降温方法对猪冷冻精液受胎率的影响作了研究。将采自9头公猪的精液分为三部分,在30℃下以800g的相对离心力离心15分钟,然后用不含甘油的卵黄葡萄糖稀释液进行第一次稀释。接着用不同方法使三组稀释液降温。第一组,先将稀释后的精液用1小时从30℃降至15℃,然后用含甘油的卵黄葡萄糖稀释液作第二次稀释,再用一小时从15℃降至     

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。     

内蒙古绒山羊精液冷冻过程中精子活力、活率及顶体变化的探讨

作者用Tris型稀释液冷冻山羊精液效果良好,解冻后精子平均活率达46.99%,最高活率为58%,顶体异常率在37%左右。     

北极狐及乌苏里貉抑制素α亚基基因克隆及生物信息学分析

试验旨在克隆北极狐及乌苏里貉抑制素α（inhibin α,INHα）亚基基因并对其进行生物信息学分析。根据GenBank中犬科INHα预测mRNA序列（登录号：XM_545660.5）设计1对引物,用RT-PCR技术从北极狐及乌苏里貉的卵巢组织中扩增出INHα亚基基因,同时将其插入到克隆载体中,进行测序及生物信息学分析。测序结果表明,北极狐及乌苏里貉的INHα亚基基因CDS序列全长为1 107 bp,编码369个氨基酸。北极狐及乌苏里貉的INHα亚基基因与犬的同源性最高,分别为97.9%与97.6%。系统进化树分析表明,北极狐及乌苏里貉与犬亲缘关系较近,同时也说明INHα亚基基因在不同物种及进化过程中具有高度保守性。对INHα亚基蛋白的高级结构预测发现,由于半胱氨酸间形成的二硫键导致其采用"蝴蝶形"或"开放手"构型,其中α-螺旋形成分子的"手腕"结构,β-折叠形成分子的"手指"结构。本研究成功克隆了北极狐及乌苏里貉的INHα亚基基因,同时进行了系统的生物信息学分析,为今后研究抑制素在卵母细胞-颗粒细胞同步发育过程中的生物学功能奠定了基础。     

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.     

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.     

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.     

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.     

Canine semen cryoconservation: Emerging data over the last 20 years

Canine semen cryoconservation was used since 1969, and this process is still nowadays in progress. This review aims to have an overview of two factors leading to a successful freezing–thawing semen. The success and efficiency of freezing process can be measured by the post-thawing sperm mobility. The first factor is the best extender used as a cryoprotectant to have a similar osmolarity and pH compared to the seminal plasma to enable sperm survival. Historically, chicken egg yolk was used since 1940, but due to microbial risks and to the presence of granules (which interfere with counting dead spermatozoa and inhibits a spermatozoal respiration), despite these disadvantages, egg yolk is considered an excellent cryoprotectant for sperm of different animal species. The low-density lipoproteins (LDL), contained in EY, when used at a concentration of 6% in a freezing medium associated with 20 mM of glutamine, show a mobility up to 54.5%, which is the best combination found. However, the sperm protection mechanism by LDL during freezing–thawing process only begins to be decrypted. But extraction protocols of LDL are not efficient for an industrial use. Therefore, egg yolk plasma is used within liquid or lyophilized state, and offering similar efficiency as the 6% LDL middle. The equilibration step, in which the diluted sperm is placed for a variable period of time at a temperature of +4°C, before freezing it. The studies show that 6 hr is the optimal duration for the canine sperm equilibration. The future of canine sperm cryopreservation is expected in liposome use and synthetic substances, which mimics LDL role.     

利用水稀释反复冻溶法、氯仿萃取法与冷乙醇沉淀法相结合进行鸡新城疫卵黄抗体提取的试验研究

本研究采用免疫学技术和物理化学方法,通过优化提取条件,改良提取工艺,建立了一种用水稀释反复冻溶法、氯仿萃取法与冷乙醇沉淀法相互结合提取鸡卵黄抗体的方法。结果显示：用5倍体积、pH5．0的稀释液稀释蛋黄,在-20℃经三次反复冻融、上清液再用同等剂量的氯仿萃取、40％的冷乙醇沉淀,提取的鸡新城疫卵黄抗体具有液体清亮,杂质含量少,抗体效价高的特点。     

Missense polymorphisms in the MC1R gene of the dog,red fox,arctic fox and Chinese raccoon dog

Coat colour variation is determined by many genes, one of which is the melanocortin receptor type 1 (MC1R) gene. In this study, we examined the whole coding sequence of this gene in four species belonging to the Canidae family (dog, red fox, arctic fox and Chinese raccoon dog). Although the comparative analysis of the obtained nucleotide sequences revealed a high conservation, which varied between 97.9 and 99.1%, we altogether identified 22 SNPs (10 in dogs, six in farmed red foxes, two in wild red foxes, three in arctic foxes and one in Chinese raccoon dog). Among them, seven appeared to be novel: one silent in the dog, three missense and one silent in the red fox, one in the 3′‐flanking region in the arctic fox and one silent in the Chinese raccoon dog. In dogs and red foxes, the SNPs segregated as 10 and four haplotypes, respectively. Taking into consideration the published reports and results of this study, the highest number of missense polymorphisms was until now found in the dog (9) and red fox (7).