不同种公牛X／Y精子分离效果及对体外受精能力的影响

本研究首先时比了3头种公牛的X/Y精子分离效果,并通过常规冻精和性控冻精的活力检测,来评价流式细胞仪分离及冷冻处理对不同种公牛精子分离效果的影响.应用体外受精试验,探讨了不同种公牛个体的常规冻精和分离冻精的受精和胚胎发育能力.试验结果表明,不同种公牛个体间除分离准确率外,精子的分离速率(4.1×103/s vs 4.5×103/s vs5.3×103/s,P<0.05)和死精率(19.3%vs 14.5%vs 11.0%,P<0.05)都存在显著差异.三头种公牛的性控冻精在体外培养过程中,精子活力的下降速率要显著快于常规冷冻精液组.不同种公牛个体的分离冻精组在4小时的精子活力也有显著差异(1号种公牛9.1%vs 2号种公牛22.1%、3号种公牛21.5%,P<0.05).此外,常规冻精和性控冻精在受精后的卵裂率和囊胚率存在显著差异(1号种公牛:64.0%vs 41.6%,26.9%vs 19.3%;2号种公牛:67.3%vs 52.3%,29.2%vs26.5%;3号种公牛:60.3%vs 43.1%,27.3%vs 29.9%).上述结果表明,不同种公牛个体间精子的分离速率、死精率、以及对分离冷冻处理的耐受性存在显著差异,造成其解冻后活力和存活时间不同程度的下降;但在体外受精试验中,3头种公牛的分离精子仍具有正常的受精能力,并能支持胚胎发育到囊胚阶段.综上结果表明,选择适宜的种公牛和降低分离及冷冻处理对精子的损伤,对于提高精子的分离效率、分离精液的品质和体外受精效率都有着重要的意义.     

不同稀释液配方对牛冻精品质的影响

选用3种稀释液配方分别对6头种公牛精液进行稀释、封装、冷冻,并检查种公牛冻精解冻后的精子活力、畸形率、顶体完整率、冻后废弃率、存活时间及镜检感光度.结果表明：3种不同稀释液配方对种公牛精液冻后活力影响差异均显著（P＜0.05）,Ⅰ稀释液生产冻精的畸形率极显著高于Ⅱ稀释液和Ⅲ稀释液（P＜0.01）,3种稀释液冻精精子顶体完整率之间差异均极显著（P＜0.01）,Ⅲ稀释液与Ⅰ稀释液和Ⅱ稀释液间对牛精液所生产冻精的冻后废弃率影响均差异显著（P＜0.05）,Ⅰ稀释液与Ⅱ稀释液和Ⅲ稀释液间对牛精液所生产冻精的存活时间影响差异也显著（P＜0.05）,Ⅲ稀释液生产的冻精精子感光度优于Ⅰ稀释液和Ⅱ稀释液,其余各项指标差异均不显著（P＞0.05）.因此,可用自配Ⅱ稀释液替代进口专用Ⅲ稀释液.     

冷冻保存对公猪精子功能的影响

试验旨在探究公猪精液冷冻保存对其精子功能的影响。取长白猪的鲜精和优质冻精,用精子分析仪检测精子的运动能力,台盼蓝染色检测精子活率,体外受精（IVF）试验检测卵裂率与囊胚率,采用不同功能检测试剂盒检测冻精和鲜精的顶体完整率、线粒体膜通道孔（MPTP）活性、线粒体膜电位（MMP）、线粒体活性、线粒体氧化应激活性氧（ROS）以及精子DNA完整性,实时荧光定量PCR检测弱精子症相关蛋白基因SMCP、TEKT3、DNAH1、TCTE3的表达。结果表明,与猪鲜精相比,猪冻精的活率及活力均显著降低（P<0.05）,冻精的顶体完整率也明显下降（P<0.05）;冻精的卵裂率和囊胚率显著低于鲜精（P<0.05）;精子线粒体功能分析结果显示,冻精的MPTP相对荧光单位值（RFU）、线粒体膜电位荧光比率以及线粒体活性光密度（OD）值均显著低于鲜精（P<0.05）;精子线粒体ROS检测发现,冻精的RFU值显著高于鲜精（P<0.05）;精子DNA完整性检测结果显示,冻精拖尾率显著高于鲜精（P<0.05）;而弱精子症相关蛋白基因的表达与鲜精相比,差异不显著（P>0.05）。综上所述,冷冻导致猪精子活率、活力、线粒体功能、DNA完整性下降,最终使得冷冻精液精子的受精能力降低。     

口蹄疫三价苗对荷斯坦种公牛冷冻精液生产的影响

为研究口蹄疫O型、亚洲Ⅰ型、A型三价灭活疫苗注射对荷斯坦种公牛冷冻精液生产的影响,本研究对2~11岁荷斯坦种公牛肌肉注射口蹄疫O型、亚洲Ⅰ型、A型三价灭活疫苗后,统计分析注射前半个月及注射后第1、2、3、4周的荷斯坦种公牛射精量、鲜精活力、鲜精密度、冻精活力、冻精精子畸形率和冻精产量的差异性。结果显示:(1)2~11岁种公牛各时间段的射精量、鲜精活力、鲜精密度、冻精活力、冻精产量无显著差异(P0.05);而注射后第1周的冻精产量、注射后第2周的畸形率显著高于注射前半个月(P0.05),其他各时间段之间无显著差异(P0.05)。(2)2岁种公牛,注射后第2周的畸形率显著高于注射前半个月(P0.05),其他各时间段之间无显著差异(P0.05);(3)3~5岁种公牛注射后第2周的畸形率显著高于注射后第1周(P0.05),其他各时间段差异不显著(P0.05)。(4)7~11岁种公牛,注射后第1周的畸形率和冻精产量显著高于注射前半个月(P0.05),其他各时间段差异不显著(P0.05)。可以得出,口蹄疫O型、亚洲Ⅰ型、A型三价灭活疫苗注射对荷斯坦种公牛射精量、鲜精活力、鲜精密度、冻精活力无显著影响,对冻精产量无不利影响,并导致畸形率升高,但对不同年龄段种公牛影响不完全相同。     

口蹄疫二价苗对荷斯坦种公牛冷冻精液生产的影响

[目的]研究口蹄疫O型、亚洲Ⅰ型二价灭活疫苗注射对荷斯坦种公牛冷冻精液生产的影响。[方法]2~9岁荷斯坦种公牛肌肉注射口蹄疫O型、亚洲Ⅰ型二价灭活疫苗后,统计分析注射前半个月、注射后第一周、注射后第二周、注射后第三周和注射后第四周的射精量、鲜精活力、鲜精密度、冻精活力、冻精精子畸形率和冻精产量的差异性。[结果](1)2~9岁种公牛各时间段的射精量、鲜精密度、冻精活力、冻精产量无显著差异(P0.05);注射后第一周的鲜精活力显著低于注射前半个月和注射后第二周(P0.05),注射后第二周的畸形率显著高于其他时间段(P0.05),其他各时间段之间无显著差异(P0.05)。(2)2岁种公牛,在各时间段之间无显著差异(P0.05);(3)5~6岁种公牛,注射后第一周的鲜精活力显著低于注射前半个月(P0.05),注射后第二周的畸形率显著高于其他各时间段(P0.05),其他各时间段差异不显著(P0.05)。(4)7~9岁种公牛,注射后第二周的鲜精密度显著低于注射后第三周(P0.05),其他各时间段差异不显著(P0.05)。[结论]口蹄疫O型、亚洲Ⅰ型二价灭活疫苗注射对荷斯坦种公牛射精量、冻精活力和冻精产量无显著影响,对鲜精活力、鲜精密度和冻精精子畸形率有一定影响,并对不同年龄段种公牛的影响有差异性。     

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.     

不同种公牛精子分离效果的比较

本研究对使用经流式细胞仪分离的4头种公牛精液的分离速率、死精率以及分离后的雌性准确率进行了比较分析,结果表明,3号种公牛的精子分离速率显著高于1号和2号种公牛(P＜0.05);1号种公牛的死精率显著高于2号和3号种公牛(P＜0.05).但是3头种公牛精液分离后的雌性准确率没有显著差异.4号种公牛的死精率达到32％,所以没有分离.同时对3头种公牛精液分离及解冻后的活率进行了比较,结果表明,3头种公牛精液经流式细胞仪分离后鲜精活率差异不显著,冷冻后0h活率差异不显著,而在冷冻4h后的精子活率差异显著(p＜0.05).     

牛冷冻精液保存时间对精液品质及其受精能力的影响

为探究不同冷冻精液保存时间对荷斯坦奶牛精液品质及其受精能力的影响,选取北京奶牛中心冻存1年(A冻精)、冻存3年(B冻精)及冻存13年(C冻精)的3批精液进行研究。利用精子分析仪分析精子运动能力,台盼蓝染色鉴定精子活率,检测精子顶体完整率、线粒体功能以及DNA完整性,利用体外受精技术检测卵裂率和囊胚率,Realtime PCR检测弱精子症相关蛋白基因表达水平。结果显示:3批精液的精子活力和直线性随冷冻时间延长逐渐下降,精子活率也显著下降;3批冷冻精液的卵裂率和囊胚率随冷冻时间延长显著下降;精子线粒体功能分析结果表明,A精液线粒体膜通道孔相对荧光单位(RFU)值和线粒体活性光密度(OD)值最高(P0.05),C精液最低(P0.05);精子DNA完整性检测结果显示,随冷冻时间延长,精子拖尾率显著增加;弱精子症相关蛋白的基因表达趋势并未显现出与体外受精结果相一致的趋势。研究证明,随着冷冻保存时间的延长,精子活力、精子线粒体功能以及精子DNA完整性逐渐下降,最终导致精子受精能力下降。     

日粮中添加胡萝卜对荷斯坦种公牛精液品质的影响

本试验选择16头荷斯坦种公牛随机分为2组，对照组饲喂基础日粮，试验组在基础日粮中添加胡萝卜，试验期为75d，观察对荷斯坦种公牛原精产量、精子密度、精子活力和制冻精数量的影响。结果表明：在荷斯坦种公牛日粮中添加胡萝卜后，试验组牛的原精产量、鲜精精子活力和制冻精数量都极显著高于对照组（P〈0．01）；精子密度与对照组间差异不显著（P〉0．05）。本试验结果验证了在荷斯坦种公牛日粮中添加胡萝卜可显著提高精液产量、精子活力和制冻精数量，提高精液品质。     

安徽不同地方牛种精液品质的分析

[目的]为研究安徽省内地方牛种的精液品质差异,[方法]试验对皖东牛、大别山牛、皖南牛3种种公牛的冬春季节所采集的精液质量进行分析与比较。[结果]结果表明:射精量方面,大别山牛射精量低于皖东牛和皖南牛,差异显著(P0.05);3种牛的精液颜色都为乳白色;精子活力方面,皖南牛的原精活力相对最高,其次是皖东牛,大别山牛最低,不同牛种间差异不显著(P0.05);皖东牛的冻精解冻后活率最低,与大别山牛和皖南牛差异显著(P0.05);皖南牛冻精解冻后精子直线运动的数量最高,与皖东牛和大别山牛差异显著(P0.05);精液细菌数方面,大别山牛的每剂冻精细菌数最高,与皖东牛和皖南牛差异显著(P0.05);畸形率方面,皖东牛的精子畸形率最低,与大别山牛和皖南牛差异显著(P0.05)。[结论]3种种公牛中,皖东牛与皖南牛的射精量高,皖东牛解冻后精子畸形率低,皖南牛与大别山牛解冻后精子活率高。     

白藜芦醇对奶牛性控冻精品质和体外受精能力的影响

本研究旨在探究不同浓度白藜芦醇处理对奶牛性控冻精品质和体外受精能力的影响。在解冻后的奶牛性控冻精中分别添加0、10~(-3)、10~(-4)、10~(-5) mol/L的白藜芦醇,各组精子在受精液中获能孵育1.5 h后,测定精子质量和体外受精能力。结果表明:添加白藜芦醇均可有效降低性控冻精中活性氧(ROS)含量、提高顶体完整活精子比例(P0.05),其中10~(-3)、10~(-4) mol/L的白藜芦醇处理对降低ROS含量最为显著;10-4 mol/L的白藜芦醇处理可显著降低丙二醛(MDA)含量并提高性控冻精的卵裂率和囊胚率(P0.05)。综上所述,白藜芦醇作为一种外源性天然抗氧化剂,通过降低性控精液中过量的ROS水平、抑制精子脂质过氧化反应、保护顶体完整性,从而提高性控精子质量和体外受精能力。     

Effect of freezing bull semen in two non‐egg yolk extenders on post‐thaw sperm quality

Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post‐thaw sperm quality was evaluated by computer‐assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56‐day non‐return rates were evaluated. Semen frozen in the liposome‐based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin‐based extender. Chromatin integrity and production of live H₂O₂+ reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome‐based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56‐day non‐return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66% ± 0.8 and 65% ± 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome‐based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.     

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 sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.     

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.     

注射口蹄疫疫苗对种公牛精液品质的影响研究

[目的]研究注射口蹄疫疫苗对种公牛精液品质的影响。[方法]随机选择6头种公牛,并采其精液进行检测,用比较方法研究。[结果]注射双价灭活疫苗前精液量6.8ml,鲜精活率70%,精液密度17.3亿/mL,冻后活率39.7%冻精数414.8剂,精子畸形率14.2%。注射双价疫苗之后精液量4.3 mL,鲜精活率6.5%,精液密度12.7亿/mL,冻后活率34.8%冻精数162.3剂,精子畸形率18%。[结论]注射疫苗后,种公牛的精液品质也随之下降,精液量、精子密度和原精活率注射后较注射前极显著降低,认为注射口蹄疫疫苗对种公牛的精液品质产生较大影响。     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Kjxstad, H., E. Ropstad and K. Andersen Berg: Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta vet. scand. 1993,34,299-303.– Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

发情母牛粘液对采精公牛交配能力及精液品质影响的研究

[目的]研究发情母牛粘液对采精种公牛交配能力以及精液品质的影响。[方法]从采精种公牛中随机抽出10头,进行2次采精试验,第一次按照正常方法采精,第二次在台牛尾根附近涂抹粘液,记录交配能力,将2次采精所采得的精液进行检测。[结果]第二次采精交配能力明显高于第一次。精液量、精液密度、活率、有效精子数、冻后活率、生产冻精数分别为：5.76 mL、19.23亿/mL、0.72、13.4、0.385、400.5,通过T检验P〈0.01,差异显著。[结论]在台牛后躯涂抹发情母牛粘液刺激公牛使交配能力和精液品质显著提高。     

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。