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).     

流式细胞仪分离哺乳动物精子的研究进展

目前,分离精子性别准确率在90%以上,受精率是未分离精子的70%～80%。尽管动物分离精子还存在许多亟待解决的问题,但随着精子分离仪的改进以及与各项辅助生殖技术的结合,动物分离精子必将在实践中得到广泛的应用,并产生巨大的社会效益和经济效益。主要概述了近年来用流式细胞仪分离哺乳动物X、Y精子的研究进展、存在的问题和应用前景。     

牛XY精子分离技术的应用现状

1989年美国XY公司首次应用流式细胞仪成功将XY精子分离,并达到一定的精确度.2000年该项技术经过不断改进,开始从实验室成功进入商业化生产应用.目前XY精子的分离速度达到3 000～5 000个/s;分离精子准确度大于90%;人工授精青年母牛受孕率50%以上.至今已利用分离X精子对10 787头青年母牛进行了人工授精,受孕率达到53%.目前该项技术是世界上最先进的XY精子分离技术.如拥有一台美国XY公司的XY精子流式细胞仪,分离速度按3 000个/s计算,每天连续工作20 h,分离X 和Y 精子各2.16 亿个,每剂量200万精子,则每台仪器日产量108支.     

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.     

精子分离及其在畜牧业中的应用

概述了哺乳动物精子分离的研究和应用状况,重点阐述了流式细胞检索仪分离精子的研究和应用进展。流式细胞检索仪分离精子这种控制动物后代性别的方法,目前虽然还存在一些问题,但已经用于牛的商业化生产中,羊、猪等方面也已临近实用。随着技术的进步和设备的改进,流式细胞检索仪分离精子等精子分离技术将会得到广泛的使用,并为畜牧业生产带来巨大的效益。     

流式细胞仪分离精子研究进展

本文概述了近年来用流式细胞仪分离动物X、Y精子的研究进展、存在的问题和应用前景。流式细胞仪分离精子的研究已经取得了很大进展 ,在某些动物 (如牛 )分离的精子已开始用于人工授精。尽管该技术还存在许多亟待解决的问题 ,但随着流式细胞仪的改进和与各项辅助生殖技术 (如手术授精、体外受精、胞质内精子注射、低剂量精子人工授精 )结合 ,性别分离精子必将在实践中得到广泛的应用 ,产生巨大的社会和经济效益     

试述种公牛精子畸形的种类

精子的组织学状况是精子生成过程的反映 ,如果生成过程受到影响 ,会导致畸形精子的生成 ,当畸形率超过 18%时受精率降低 ,了解精子畸形的种类及原因对于指导生产具有重要意义。精子畸形分为原发缺陷和继发缺陷两种 ,原发缺陷是在精子发生过程中形成的 ,严重影响受精。当精子离开睾丸后 ,受到刺激而形成的缺陷为继发缺陷 ,如果数量不是很大不影响受精。但从与受胎率的关系来看 ,一些原发缺陷对受胎率的影响弱于继发缺陷。1　头部疏松　一种继发缺陷 ,发生率低 ,在公牛“排空练习”时的第一个两次或三次射精时大量出现 ,在此后的射精中迅速减…     

使用流式细胞仪分离精子进行仔猪性别控制的研究

本研究旨在探索流式细胞仪分离精子在猪性别控制中的应用。使用流式细胞仪分离猪XY精子,而后通过母猪输卵管授精生产"预知"性别的仔猪,并使用吖啶橙染色法检测粗分离对精子核酸含量的影响。结果,成功利用分离获得的猪X和Y精子对母猪进行输卵管授精,母猪怀孕率、产仔率均为100%;输Y精子母猪产仔雄性率100%(♂6/6),对照母猪产仔雄性率57.14%(♂8/14);3头输X精子母猪产母仔率91.67%(♀11/12),对照母猪产雌性仔猪40%(♀2/5);使用性别分离精子不影响母猪的怀孕率、产仔率,但窝产仔数较低;吖啶橙染色法检测结果表明,流式细胞仪粗分离对猪精子核酸含量没有显著影响(P>0.05)。本研究结果提示,使用流式细胞仪分离精子授精可以有效改变仔猪的性别比例。本研究结果为猪分离精子性别控制技术的推广应用奠定了基础。     

Three types of acrosomal aberrations of bull spermatozoa and their relation to fertility

The effect of acrosomal aberrations of the spermatozoa of Finnish Ayrshire bulls on the corrected non-return rate within 60 days of the first 500 inseminations was studied. The material consisted of sperm samples examined by the artificial insemination societies. All samples had been accepted for use in artificial insemination. One Giemsa-stained slide was studied for each of the 95 bulls concerned. Samples showing distinct acrosomal defects were studied by electron microscopy. Three different types of acrosomal aberration were found. One was obviously associated with subfertility in all 6 bulls in which it was detected.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Application of flow cytometric techniques to veterinary clinical hematology

Flow cytometry has emerged as a major new technology in veterinary clinical laboratories. Flow cytometers in current use include stand-alone instruments and cytometers incorporated into hematology analyzers. Flow cytometers offer rapid and quantitative analysis of a variety of cell types based on cell size, molecular complexity, and antigenic composition. Therefore, flow cytometry complements and extends the knowledge that can be obtained by light microscopy. Stand-alone instruments are very flexible, however, this flexibility opens the instrument to obtaining invalid or misleading results. The recent development of monoclonal antibodies specific for epitopes on blood cells of food and companion animals has greatly expanded the spectrum of tests with potential clinical application. Tests that appear to have the greatest potential for routine application include reticulocyte and reticulated platelet enumeration, detection of erythrocyte-bound immunoglobulin, immunophenotyping of leukemias and lymphomas, and bone marrow differential cell counting. This report will briefly review the technical aspects of flow cytometry and then focus on techniques with present or potential application to the veterinary clinical laboratory.     

Functional proteomic analysis of crossbred (Holstein Friesian × Sahiwal) bull spermatozoa

Male infertility is one of the prime concerns of dairy cattle production. The study was designed to find out differentially expressed proteins in categorized crossbred (Holstein Friesian × Sahiwal) bull semen to serve as potential biomarkers for male infertility. Frozen crossbred bull semen with satisfactory phenotypic records were defined as “good” and “poor” based on their fertility rates. A total of 1,547 proteins were detected in bull spermatozoa using liquid chromatography–mass spectrometer (LC‐MS/MS) analysis. Results revealed that 558 (36.1%) and 653 (42.2%) proteins were expressed to good and poor quality bull spermatozoa, respectively. A total of 336 proteins (21.7%) were reported to be unique for both good and poor quality bull semen, and among the common proteins, 224 (66.7%) and 112 (33.3%) were up‐ and downregulated in good and poor quality categorized bull semen, respectively. Gene Ontology analysis of global proteomes identified different signalling pathways, and most of them were related to cellular motility, immune systems as well as cellular metabolisms. The distinctive presence of some of the proteins may provide an insight into the molecular mechanistic role played by these proteins in crossbred bull infertility.     

不同加工贮藏方式的玉米对奶公犊牛育肥性能的影响

[目的]玉米是一种重要的能量饲料,探讨和研究不同玉米加工贮藏方法对奶公牛育肥性能的影响,以提高其利用效率,降低在奶公牛育肥中养殖应用成本.[方法]将30头奶公犊牛（平均为5月龄）随机分为3组,每组10头,分别饲喂普通粉碎玉米、蒸汽压片玉米和湿贮发酵玉米,育肥试验期为50 d.[结果]饲喂蒸汽压片玉米和湿贮发酵玉米后,日增重和日利润可达1.29kg、13.87元/d和1.24kg、13.48元/d,均高于对照组.[结论]在肉牛育肥中,对于蒸汽压片玉米和玉米籽实湿贮的加工和贮藏方法都是肉牛养殖业中值得推广的实用技术.     

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post‐thaw semen characteristics

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell^® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.     

荷斯坦公牛PRNP多态性与精子活力相关性及抗病性评估

为分析荷斯坦公牛朊蛋白基因(PRNP)多态性与精子活力的关系,并对其抗病性进行评估,选育抗病公牛,本实验以公牛精液为样品,研究脚基因中12bp、23 bp和24 bp 3个片段的插入/缺失多样性、基因mRNA转录水平及其与精子活力等指标的关系.结果表明,12bp和23 bp在群体中均得到3种基因型,24 bp只有2种基因型.不同基因型群体的mRNA转录水平存在显著差异,而基因型与精液产量和活力等指标存在相关性,抗病力和精子活力指标存在负相关.本研究中3个片段的插入-缺失多样性可以作为公牛选育的辅助标记.     

Evaluation of pentoxifylline and Basal Medium Eagle supplemented to diluent on cryopreserved goat spermatozoa

This study investigated the effect of pentoxifylline (PTX) and Basal Medium Eagle (BME) on frozen–thawed goat spermatozoa. Immediately after initial examination of ejaculated semen, samples were pooled and reexamined for quality. Then, samples were divided into eight equal aliquots and diluted with a basic tris-extender containing PTX (3, 6, 9 mM) and BME (5 mM) to reach a final concentration of 25 × 10⁹ and frozen. After 24 hr, the samples were individually thawed at 37°C for 30 s and evaluated for different characteristics. Obtained post-thaw results from Computer-Assisted Sperm Analysis indicate using of 3 and 6 mM PTX led significantly to an improvement in total motility, progressive motility and velocity characteristics of spermatozoa, except the beat/cross frequency (BCF) which indicated statistically no differences (p > .05) among control and treatments. Diluents prepared with BME (5 mM) and PTX alone (3 and 6 mM) improved significantly the membrane integrity–functionality, acrosome integrity and also hyaluronidase activity. Regarding recovery rate, the results showed significantly (p < .05) higher values for diluents containing 3 and 6 mM PTX compared to other groups. Malondialdehyde concentration exhibited also a significant difference (p < .05) in diluents supplemented with 5 mM BME, 3, 6 and 9 mM PTX, and mixture of 3 mM PTX and 5 mM BME which illustrate a similarity for active mitochondria, apoptotic-like and dead spermatozoa. Finally, the ratio of sperm chromatin dispersion stained spermatozoa presented significant differences (p < .05) among treatments in which the diluents added PTX alone demonstrated significantly lower values than control and extenders containing the mixtures of BME and PTX. In conclusion, the observation in this study indicates using of 3 and 6 mM PTX and BME alone may improve significantly (p < .05) the quality of cryopreserved goat spermatozoa.