牛、梅花鹿和山羊X、Y精子头部面积差异分析

用流式细胞仪分选有正常繁殖力的公牛(n=3)、公鹿(n=3)、公山羊(n=1)的精液,获得高纯度(≥91%)X和Y细管冷冻精液,对解冻后的X和Y精子进行常规染色制片,采用Motic Images Advanced 3.2软件自动测量X精子和Y精子头部面积。结果显示,公牛X精子的头部面积35.84μm2±4.12μm2,极显著高于Y精子(34.81μm2±3.72μm2)(P<0.01);梅花鹿X精子的头部面积33.29μm2±2.93μm2,极显著高于Y精子(32.90μm2±3.25μm2)(P<0.01);山羊X精子的头部面积28.53μm2±3.16μm2,也极显著高于Y精子(28.07μm2±3.19μm2)(P<0.01);不同分选纯度的公牛精液X精子之间、Y精子之间头部面积差异均不显著(P>0.05)。结论:牛、梅花鹿、山羊X精子头部面积均极显著高于Y精子。     

比利时蓝白花牛XY精子分选及冷冻精液制备

本研究利用流式细胞仪分选比利时蓝白花牛X、Y精子并制备冷冻精液。选择平均年龄4岁的健康比利时蓝白花种公牛12头，假阴道法采集精液送至实验室分选，经流式细胞仪分离、冻存、解冻后精液重分析和品质鉴定。X、Y冻精（93.4%和91.1%）性别比例显著高于常规冻精（50%）；分选后精液冻后存活率、活力和顶体完整率与常规冻精差异不显著。本研究结果显示，分选前控制公牛的精液品质（活力≥70%，畸形率≤18%）可以明显改善分选效果；分选后精子纯度和冻后活力满足低剂量人工授精要求（纯度>90%，活力>30%），精子分选对比利时蓝白花牛产业的发展具有重要意义。     

利用流式细胞仪分离奶牛精液的性别控制效果观察

目前,常见的牛性别控制方法有早期胚胎性别鉴定法和利用流式细胞仪进行X、Y精子分离法两种。流式细胞仪精子分离法是根据X、Y精子在DNA含量上的差异来分离精子。分离后的X、Y精子用于人工授精或显微授精可在授精之前就控制其性别,因而被认为是更具有实用价值的方法。此次研究通过对分离后精子的人工授精效果和性别控制效果进行试验观察,以期为该方法的进一步应用提供依据。1材料与方法1.1公牛及采精公牛为大庆田丰生物有限公司饲养的荷斯坦牛,共12头。饲以全价营养日粮,每天喂给鸡蛋1枚。采用常规的假阴道法采集精液。1.2精液的处理将采…     

高海拔地区不同品种种公牛离体精子遭受低温打击和冷冻伤害研究

精子的耐冻性在牛品种之间、个体之间均存在差异。离体精子遭受低温打击和冷冻伤害后,畸形精子数会增加,精子顶体会受到损伤。本试验对高海拔地区的荷斯坦、西门塔尔和牦牛三个品种种公牛离体精子遭受低温打击和冷冻伤害后的品质变化情况进行了研究,结果显示,荷斯坦牛、牦牛精子畸形率显著增加(P0.05),三个品种种公牛离体精子的顶体受损情况无显著差异(P0.05),但牦牛离体精子遭受低温打击和冷冻伤害后,Ⅳ型(顶体脱落)精子比例显著大于荷斯坦、西门塔尔两个品种(P0.05)。     

公牛精子分离前的保存

试验采用荷斯坦公牛精液，比较了添加抗生素、TALP稀释以及TALP稀释染色3种处理方法对常温下保存精子活力的影响。研究结果表明，新鲜精液加抗生素处理后直接保存的效果优于另外2个处理组，而分离精子经冷冻解冻后的存活率达40％，说明了该方法的可靠性。     

不同染色与冷冻方法对关中奶山羊X、Y精子的分离及应用效果研究

为探究不同Hoechst 33342的染色浓度与冷冻方法对关中奶山羊X、Y精子进行分离及应用效果的影响，本实验采集6只关中奶山羊精液，使用浓度分别为10、11、12、13μL/m L的Hoechst 33342(5 mg/m L)染色液对精液样本进行染色，以流式细胞仪分离X精子和Y精子，应用3种不同冷冻程序冷冻精液，应用体外受精与胚胎发育技术评估关中奶山羊X、Y精液的分离准确性。结果表明：12μL/m L Hoechst 33342染色条件下，奶山羊X、Y精子分离效率达到91.02%，高于10μL/m L和13μL/m L(P<0.01)，最高分选速度为4 134个/s(P<0.05)；公羊个体的精液品质越好，其精子的分离速度越快，但对分离准确率的差异不显著；使用冷冻程序2进行关中奶山羊的X、Y精子冷冻的效果较好（P<0.05）；体外受精48 h后，使用Y精子进行受精的卵细胞的卵裂率高于使用X精子（P<0.05），在胚胎培养第9天，使用Y精子进行受精的卵细胞的囊胚发育率高于使用X精子（P<0.05）。总之，当Hoechst 33342浓度为12μL/m L...     

夏季高温对种公牛精子畸形率影响的初步研究

奶牛育种是一项基础性、长期性的工作,但效益非常显著。根据美国农业部数据,育种在饲料营养、繁殖育种、疾病控制、饲养管理等主要技术因素中,对奶牛生产的贡献比例最大,达到40%。种公牛耐寒、怕热,最适宜生活的环境温度为11~18 ℃,外界气温的变化对种公牛的影响较大,特别是上海夏季高温、高湿严重,极易造成种公牛采食量下降、体温升高、精液品质和产量下降。本研究对2014年上海奶牛育种中心公牛站10 头荷斯坦种公牛精液精子畸形测定资料与上海地区气温变化情况进行分析,得出高温对种公牛冷冻精液精子畸形率影响较大,气温越高,精子畸形率越高的结论。春季和夏季公牛精子畸形率差异显著（P<0.01）,种公牛冷冻精子畸形率在2—4月最低,7—9月最差,因此采精最佳季节为10月至翌年6月。     

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.     

种公牛睾丸周径与品种、体型性状及精液产量的相关性研究

本文以来自山西省家畜冷冻精液中心的荷斯坦、夏洛来和西门塔尔3个品种的32头种公牛为材料,研究了不同品种公牛睾丸周径（SC）之间的差异以及公牛体型对睾丸周径的影响,分析了公牛睾丸周径与射精量、精子密度之间的相关。结果显示,成年之后不同品种SC差异不显著;SC与体重、胸围呈高度正相关（P〈0.01）;SC与管围呈正相关（P〈0.05）;公牛睾丸周径与平均采精量的相关系数为0.11,与平均精子密度相关性为0.21,相关系数较低。此项研究结果可为选育种公牛提供一定的参考依据。     

利用流式细胞仪建立牦牛X、Y精子分选体系的研究

旨在探索与优化牦牛精子分选条件,建立高效的牦牛X、Y精子分选技术体系。本研究制备了牦牛精液细胞悬液,采用不同量的DNA染料Hoechst33342和诱惑红共孵育精子细胞。利用流式细胞仪分选牦牛X、Y精子,通过比较分选效率、分选后精子活力及发育潜能,优化分选条件。运用RT-PCR检测分选精子的纯度,利用精子分析系统检测分选后精子的活力。将分选后的精子与体外成熟的卵母细胞进行体外受精,统计分选后精子的发育潜力,并采用SRY片段的PCR法检测早期胚胎的性别。结果显示,10 μL Hoechst33342染色40 min后的分选效果最佳,其分选产物的准确度和分选效率显著优于其他组,分选后的X、Y精子活力与20 μL Hoechest33342染色20 min组相当,显著高于其他组（P<0.05）。添加5 μmol·L^-1的诱惑红对分选的纯度无显著影响（P>0.05）,但能显著提高分选后精子的活力（P<0.05）。分选后X、Y精子分别与卵母细胞进行体外受精,受精率和囊胚形成率与未分选组差异不显著（P>0.05）,且胚胎性别比例与分选后精子纯度吻合。综上,本研究建立并优化了流式细胞仪分选牦牛X、Y精子的方法,添加诱惑红有助于改善分选后牦牛精子的活力,为后期牦牛性控精液的制备及生产奠定了基础。     

Relationships among age, body weight, scrotal circumference, semen quality and peripheral testosterone and estradiol concentrations in pubertal and postpubertal Holstein bulls

In the present study, the correlations among age, body weight, scrotal circumference (SC), semen quality and peripheral testosterone and estradiol-17beta (E(2)) concentrations were investigated in pubertal (n=5) and postpubertal (n=7) groups of Holstein bulls over a 6 week period. There were significant positive correlations (P<0.01) among age, body weight and SC in both groups, and similar significant correlations between sperm motility and SC in pubertal bulls (P<0.01) and between sperm concentration and SC in postpubertal bulls (P<0.05). The sperm motility after collection (P<0.05) and after freezing and thawing (P<0.01) of the postpubertal bulls correlated positively with the E(2) concentration. Estrogen may be important for the function of postpubertal bull testes, in which it may regulate spermatozoa motility in vivo.     

荷斯坦牛和西门塔尔牛冻精品质及其体外受精后早期胚胎发育的比较研究

为探讨荷斯坦牛和西门塔尔牛冻精的精液品质及体外受精后胚胎发育能力的差异,利用目测法、低渗膨胀法和考马斯亮蓝染色法评估了荷斯坦牛和西门塔尔牛冻精的活力、质膜完整率和顶体完整率,并比较了二者冻精体外受精后胚胎的卵裂率和囊胚率。结果表明,荷斯坦牛和西门塔尔牛冻精的活力(30.4%和27.2%)、质膜完整率(41.96%和36.22%)和顶体完整率(77.02%和73.02%)均无显著差异(P>0.05),但荷斯坦牛冻精体外受精后的卵裂率(57.5%和48.6%)和囊胚率(30.3%和23.2%)显著高于西门塔尔牛冻精(P<0.05)。提示,不同品种公牛精液体外受精后的发育能力有显著差异(P>0.05)。     

公牛精子在四种介质中的品质变化

６头荷斯坦成年公牛，每头采集３次精液，分别利用精子穿透试验和姬姆萨染色法，评定精子在发情母牛子宫颈粘液、ＶＢ１２、稀释液和解冻液等四种介质中的泳动速度、顶体完整车、畸形率以及精子有效存活时间的变化。结果表明，精子泳动速度在不同介质中的差异极显著，在宫颈粘液中显著高于其它介质，在ＶＢ１２中最低；不同介质中精子顶体完整率的差异不明显；畸形率在ＴＢ１２中显著低于解冻液；精子有效存活时间在稀释液和解冻液中显著高于ＶＢ１２中。不同公牛在四种介质中的反应亦有一定差异。     

GnRH和LHR基因多态性和牛精液品质性状的相关分析

本试验采用PCR—RFLP方法分析了GnRH基因外显子2和LHR基因内含子9在144头中国荷斯坦牛和79头河南地方肉牛品种中的多态性,利用最小二乘法分析多态位点不同基因型与精液品质性状的关系。研究结果表明,2～3岁荷斯坦牛的鲜精活力显著高于4岁以上的牛,而畸形率显著低于7岁以上的牛。对2～4岁荷斯坦牛不同精液品质性状的简单相关分析表明,畸形率与顶体完整率和冻精活力呈显著的负相关（相关系数r分别为-0．736和-0．500）。不同基因型与精液品质性状的关联分析结果表明,144头中国荷斯坦牛所研究位点不同基因型对精液品质性状没有显著影响。而河南地方肉牛GnRH基因外显子2的A883G位点GG基因型的精子密度显著低于AA和AG基因型．LHR基因G51656T位点的TT基因型精子密度显著高于GT基因型,未检测到GG基因型。并且发现随着年龄的增长,种公牛的精液品质逐渐变差。GnRH和LHR基因可作为影u向肉牛精液品质性状的候选基因。     

Morphology,morphometry and chromatin distribution in llama sperm nuclei

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 μm², perimeter 13.16 μm, length 5.12 μm, width 2.81 μm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.     

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows : Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.     

Post-thaw Survival and Longevity of Bull Spermatozoa Frozen with an Egg Yolk-based or Two Egg Yolk-free Extenders after an Equilibration Period of 18 h

The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed and Biociphos Plus as compared with the Tris-egg yolk based diluent Biladyl, for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mul aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl results in higher sperm survival and longevity than the use of Andromed or Biociphos Plus.     

Genetic and Environmental Influences on Semen Traits in A.I. Bulls

Contents: Repeatability and heritability coefficients were estimated for ejaculate volume, mass movement, individual motility, sperm concentration, first and second post-freezing control of motility, number of doses frozen, total number of spermatozoa and total number of motile spermatozoa per ejaculate in the first ten ejaculates of 175 Swiss Holstein and 673 Simmental young bulls (200 pure Simmental, 473 Simmental x Red Holstein crosses). Data were analysed using the univariate individual animal model with effects of interval between consecutive collections, age of bull at collection, season of collection, breed group of Simmental bulls according to percentage of Red Holstein genes, bull (within breed group) and permanent environment. Crossbred bulls had a better performance in all traits. Repeatability coefficients ranged from 0.28 to 0.46. They indicate that the prediction of semen quality in young bulls of the Swiss Holstein and the Simmental breed is possible to some extent. Heritabilities in Swiss Holstein bulls were generally very low and did usually not exceed 0.10, but standard errors of these estimates were high. Relatively high heritabilities (0.18–0.30) were found for Simmental bulls, Differences in heritabilities between these two breed groups were probably a consequence of the low number of Swiss Holstein bulls.     

高温对荷斯坦种公牛精液品质及精清生化指标的影响研究

以荷斯坦种公牛为试验牛研究了高温对其精液品质和精清生化指标的影响。结果表明:(1)高温可造成种公牛精液品质显著下降。使原精活力、精子密度、活精子百分数和顶体完整率分别比春季下降3·13%(P<0·05)、34·8%(P<0·01)、15·0%(P<0·01)和17·8%(P<0·01),精子畸形率比春季上升25·5%(P<0·01)。(2)夏季荷斯坦种公牛精清睾酮含量仅为4·31pg/mL,极显著低于非高温季节(P<0·01)。(3)高温环境使得荷斯坦种公牛精清钾、钙、镁处于最低水平(P<0·01),精清碱性磷酸酶(ALP)、酸性磷酸酶(ACP)处于最高水平(P<0·01),使精清钠由春季至夏季呈显著下降趋势。