精液质量分析系统与血球计数板法测定精子密度方法误差来源分析

正精液质量分析系统测定精子密度方法:在固定显微镜放大倍数和摄像头的情况下,摄像机的视野大小固定,以米尼图分析系统为例,20倍物镜下摄像头视野大小为563微米×563微米。此时采用厚度为20微米的精子计数板,则视野中精液体积为(0.563×0.563×0.02)毫米~3=6.34×10~(-6)(毫升),计数多个全视野精子数求平均值x,则可计算出检测样品精子密     

常见商业化猪精子计数板使用效果与测试分析

精子计数板在常规精液品质检测过程中必不可少,其搭配常用的精液检测系统如Mi ni tube AndroVision等,可快速、简便地对精液各项指标进行检测。但目前市场上精子计数板品牌众多,不同的品牌品质也有较大差距,具体表现在对精子活力、运动速度以及运动能力等精液品质相关指标的影响方面。本研究主要比对市面上常见的8款精子计数板对同一精液样品检测效果及加样后10 min各品牌精液品质变化情况,研究结果显示品牌A的精子计数板效果最好,其所测定的精子活力可达85.89%,品牌D的精子计数板效果最差,其所测定的精子活力仅51.63%,显著低于其他品牌所测结果。综上,不同品牌精子计数板品质存在差异,检测结果也存在差异。     

精子质量检测系统在鸡精子密度测定中的应用

为了明确精子质量检测系统在鸡精子密度测定中的适用性,试验采用精子质量检测系统与计数板法测定黑丝羽乌骨鸡及其杂交鸡公鸡精液精子密度,比较了精子质量检测系统在鸡精子密度测定中的重复性和准确性。结果表明:该系统的重复性和一致性较好,10倍与20倍物镜检测结果无显著差异(P0.05);该检测系统准确性较好,与计数板法测得的结果比较无显著差异(P0.05),可以在实际研究与生产中应用。     

猪卵母细胞体外受精条件的优化

猪卵母细胞体外受精(in vitro fertilization,IVF)技术为受精和胚胎早期发育机理等基础理论的研究及生产实践提供了重要的研究手段。虽然猪IVF的研究已经取得了一些进展,但其囊胚发育率仍然较低,亟需建立更为稳定、高效的体外受精方法。本试验通过比较精子浓度,精子获能处理,不同精卵共孵育培养体系以及在卵母细胞成熟过程中添加不同激素等影响猪卵母细胞体外受精胚胎发育能力的因素,以求找到最佳的猪卵母细胞体外受精体系。结果显示:在精子浓度为1×10~5~1×10~7/mL,5×10~6/mL的精子浓度显著提高体外受精效率(P<0.05);以茶碱、咖啡及咖啡因联合使用肝素作为获能物质处理精子,发现2.5 mmol/L茶碱处理组体外受精效率显著提高(P<0.05);通过比较不同的受精体系,发现使用40个卵/500μL体系显著提高体外受精效率(P<0.05);在卵母细胞成熟液中添加不同激素组合,发现添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH激素组合,体外受精效率显著提高(P<0.05)。本试验结果表明,在M199中添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH体外成熟培养卵母细胞,以5×10~6/mL精子浓度,2.5mmol/L茶碱作为获能物质处理精子,40个卵/500μL共孵育的IVF体系效果最佳,其卵裂率为(61.33±0.77)%,囊胚率为(28.33±1.08)%。本试验将为深入研究猪IVF提供理论参考。     

牛几种卵母细胞数和精子浓度对体外受精及早期胚胎发育的影响

在容量为46μl的受精液滴中,(一)滴内精子浓度固定为1.2～1.7×100/ml,每滴分别加入1、10、25、50枚体外培养成熟的卵母细胞;(二)每滴内放入10枚成熟卵母细胞,加入不同量的精子,使滴内精于浓度分别为0.64×10~6/ml、1.6×10~5/ml、3.2×10~6/ml和4.8×10~6/ml。卵母细胞的体外成熟及体外受精均按Lu等报导的方法进行。试验结果表明,当受精液滴内精子浓度为1.2～1.7×10~6/ml,活率达0.45时,每滴加50枚卵母细胞进行受精,对分裂率和胚胎发育率均无显著影响(P>0.05)。受精滴内含10枚卵母细胞,加入活率为0.45的精子,较合适的精子浓度为0.64×10~6/ml～1.6×10~6/ml。精于浓度高于1.6×10~6/ml时,对分裂率虽无影响,但对分裂球进一步发育至囊胚有一定的影响。     

获能液及精子密度对牛性控精子体外受精成功率的影响

本实验探讨了不同的精子获能添加物、精子密度、精子获能液对牛性别分离精子体外受精（IVF）的影响。结果表明：受精液中同时添加10μg/mL的肝素与5mmol／L的咖啡因,能促进牛性控精子体外获能与受精。精子密度在1．0×10^6个/mL时其囊胚发育率最高。用BO液和mTyrode’s液对牛性控精子进行获能和受精处理．其受精效果差异不显著（P〉0．05）,但BO液作用时间短对早期胚胎的发育影响较小,比较适合牛性控精子IVF。     

分光光度计和手持精子密度仪测定鸡精子密度的比较

为利用分光光度计原理测定鸡精子密度,对黑丝羽乌骨鸡及其杂交公鸡的精液,分别用吸光度法和计数板法进行精子密度的测定,得出722型分光光度计和手持精子密度仪所测定吸光度与鸡精子密度之间的回归方程。结果表明:鸡精子密度与722型分光光度计和精子密度仪吸光值之间均呈幂函数回归关系,回归方程分别为Y=19.959×X~(0.975)(R~2=0.928)、Y=10.834×X~(0.970)(R~2=0.940),与精子密度仪相关性更好。试验结果为将此回归方程植入简单且性价比高的国产手持精子密度仪,开发专用的鸡精子密度仪奠定了基础。     

国产妥曲珠利对柔嫩艾美耳球虫临床疗效试验

目的评价国产妥曲珠利对柔嫩艾美耳球虫的治疗效果,确定最佳用药剂量。方法以50×10-6mg/L、25×10-6mg/L、12.5×10-6mg/L和6.25×10-6mg/L4个剂量,饮水给药2 d进行了妥曲珠利对E.tenella的临床疗效试验。结果50×10-6mg/L和25×10-6mg/L剂量的ACI分别为190.22和187.72,12.5×10-6mg/L ACI为152.86,6.25×10-6mg/L剂量的ACI为118.50。结论妥曲珠利以25×10-6mg/L剂量饮水治疗E.tenella效果好,可以推广使用。     

多种因素对小鼠卵母细胞体外受精效果影响的分析

为了探讨不同精子获能时间,精卵孵育时间,精子密度以及颗粒细胞对小鼠卵母细胞体外受精的影响,从而达到对卵母细胞体外受精体系优化的目的。比较了精子获能时间分别为40 min、60 min、80 min试验组的受精卵卵裂率。结果表明,带颗粒细胞卵母细胞(COCs)在三个试验组中卵裂率无显著差异,不带颗粒细胞卵母细胞(NO)在精子获能时间为60 min时卵裂率最高;比较了精卵孵育时间分别为2 h、4 h、6 h、8 h试验组的受精卵卵裂率,结果显示COCs精卵孵育时间2 h试验组的效果最好,NO孵育时间为6 h试验组的效果最好;比较了精子密度分别为3×10⁵/mL,3×10⁶/mL,3×10⁷/mL试验组受精卵卵裂率,结果显示COCs和NO均为3×10⁶/mL试验组卵裂效果最好;比较COCs和NO的受精卵卵裂率,结果显示COCs与NO之间存在显著差异(P<0.05),裸卵卵裂效果显著优于颗粒细胞卵裂效果。试验结果表明,在卵母细胞体外受精过程中,精子获能时间60 min,精子密度为3×10⁶/mL,精卵孵育6 h,培养24 h后卵裂率最高。     

部分商品化非洲猪瘟病毒荧光PCR检测试剂盒的比对

为获得商品化非洲猪瘟病毒(ASFV)荧光PCR检测试剂盒的敏感性、特异性、一致性和最低检测限等试验数据,以世界动物卫生组织(OIE)推荐引物探针为金标准,以62份已灭活的ASFV阳性田间样品、32份阴性田间样品和P72基因核酸标准物质为检测对象,对农业农村部公布许可的其中17个厂家生产的商品化ASFV荧光PCR检测试剂盒开展了试验特性方面的比对研究。结果显示,17个厂家生产试剂盒的敏感性最低为77.4%,最高为96.8%,特异性最低为87.5%,最高为96.8%,均未达到100%,且有一定差别;12个厂家的试剂盒与OIE推荐引物探针检测结果一致性较好(Kappa值≥0.75),其余5个厂家的试剂盒与其一致性一般(0.4Kappa值0.75);17个厂家的试剂盒均可检测到的最低稀释度为5.9×10~3×2~(-3)拷贝/μL,但不同厂家试剂盒的最低检测限有所差别,其中5个试剂盒可检测到的最低稀释度为5.9×10~3×2~(-3)拷贝/μL,2个试剂盒为5.9×10~3×2~(-4)拷贝/μL,6个试剂盒为5.9×10~3×2~(-5)拷贝/μL,3个试剂盒为5.9×10~3×2~(-6)拷贝/μL,1个试剂盒至少为5.9×10~3×2~(-7),且大部分国产试剂盒的最低检测限比进口试剂盒低。本研究为ASFV荧光PCR检测试剂盒的筛选和比对提供了参考数据。     

Characteristics of Buck Semen with Regard to Ejaculate Numbers, Collection Intervals, Diluents and Preservation Periods

To determine the number of ejaculates which can be collected within a 20‐min period after the smallest number of days of sexual rest, and a good diluent to preserve semen for routine AI, five mature Black Bengal bucks were used in three experiments. In experiment 1, semen from the bucks were collected by using artificial vagina at homosexual mounts as many times as possible during 20 min. The ejaculate numbers 1, 3 and 4 (or 5 when the buck could produce it) were examined for important semen characteristics. The mean ejaculate volume, density, mass activity, sperm motility, sperm concentrations, total spermatozoa/ejaculate, proportion of spermatozoa with normal acrosome, midpiece and tail, and the proportion with normal head morphology varied between 267 and 342 µl, 4.1–4.5 (1–5 scale), 4.1–4.2 (1–5 scale), 77–79%, 4187 × 10⁶–5064 × 10⁶/ml, 1140 × 10⁶–1746 × 10⁶, 91–94% and 99%, respectively, depending on the collection number of the ejaculate. The difference between the ejaculates was significant only with respect to volume (p < 0.05). In experiment 2, semen was collected from the bucks successively during 20 min after 1, 2, 3 and 4 day intervals, and the first ejaculates were evaluated for the above‐mentioned semen characteristics. Semen collected after 2 or more day intervals had significantly higher volume, sperm concentration and total spermatozoa/ejaculate (p < 0.05). In experiment 3, pools of two to three ejaculates were diluted (1 : 5; semen : diluent) in splits with glucose‐citrate‐egg yolk (GCEY), Tris‐fructose‐egg yolk (TFEY) or skim milk (SM) and preserved at +4 to +7°C. Before chilling or after 0 (15 min chilling), 1, 2, 3 and 4 days of preservation, semen was evaluated for motility and proportion of normal spermatozoa with respect to acrosome, midpiece and tail. In data pooled across the bucks, the sperm motility was better in GCEY and TFEY than in SM, and the proportion of normal spermatozoa was higher in SM than in the others (p < 0.05). However, the differences in proportion of normal spermatozoa between diluents were not significant when the data were analysed separately within preservation periods. The sperm motility consistently dropped after 1 day of preservation (p < 0.01); the motility remained 50% or more up to 4 days in TFEY, 3 days in GCEY and only 2 days in SM. The proportion of spermatozoa with normal acrosome, midpiece and tail, which was generally quite high ( 90%), decreased after 3 days of preservation (p < 0.01). We conclude that Black Bengal bucks can be collected three times during 20 min, every 3 days, and that buck semen holds good motility and proportion of normal spermatozoa up to 3 days in GCEY or TFEY at 4 to 7°C.     

河北省不同地区夏季规模化羊场气载细菌的检测与分析

旨在研究河北省夏季不同地区规模化羊场气载细菌的日变化和区域性变化。选择3个区域(燕山丘陵、太行山区和平原地区)10个规模化羊场(14个舍),采用定点采样和培养计数法,对夏季各羊舍及其运动场的细菌数量进行3 d连续检测。结果:羊舍和运动场细菌数量表现为中午低、早或晚高的变化规律,早、中、晚不同地区羊舍的细菌数量分别为6.23×10^3~9.78×10^3、5.05×10^3~6.56×10^3和6.05×10^3~7.51×10^3 cfu/m^3,其中太行山区羊舍细菌在3个时间段间达显著差异(P<0.05)。从区域性分布看,太行山区羊舍细菌数量高于其他2个地区,燕山、太行和平原3个地区羊舍细菌平均数量分别达6.76×10^3、7.79×10^3和6.01×10^3 cfu/m^3;羊舍与运动场比较,虽然太行山区和平原地区羊舍细菌与运动场之间无显著性差异(P>0.05),但太行山区羊舍及其运动场的细菌数量有增加趋势(P=0.05,P=0.07),而燕山丘陵50%的羊舍细菌数量显著高于运动场(P<0.05)。另外,比较4类羊(妊娠、母带仔、育成和育肥)舍细菌得出,妊娠舍细菌平均数量最高(7.10×10^3 cfu/m^3),育肥舍或带仔母羊舍细菌数量最低(5.54×10^3 cfu/m^3,5.55×10^3 cfu/m^3)。该研究结果可为完善羊场的羊舍设计和环境调控提供可靠依据。     

2018年天津市原料奶细菌总数、体细胞数及奶牛乳房炎病原菌、耐药基因调研报告

本研究旨在调查天津市原料奶细菌总数、体细胞数及乳房炎病原菌、耐药基因,了解全市原料奶的质量状况及引起奶牛乳房炎发生的主要原因。采集天津市5家乳品加工企业奶罐车的原料奶样品,用于检测体细胞数和菌落总数;采集天津市奶牛养殖场储奶罐奶样品,用于检测体细胞数;采集奶牛场临床型乳房炎发病乳区的牛奶样品,用于检测乳房炎病原菌及耐药基因。结果显示:2018年天津市乳品加工企业原料奶体细胞数平均值为44.65万/mL,标准差42.41万/mL,变异系数94.97%,最大值为225.50万/mL,最小值为1.20万/mL,SCC≤50万/mL的样品占74.37%,50万/mL200万/mL的样品占3.13%;细菌总数平均值11.54万CFU/mL,标准差26.28万CFU/mL,变异系数227.66%,最大值190.00万CFU/mL,最小值0.095万CFU/mL,细菌总数≤10万CFU/mL的样品占74.37%,10万CFU<细菌总数≤50万CFU/mL的样品占21.25%,50万CFU<细菌总数≤100万CFU/mL的样品占1.88%,100万CFU/mL<细菌总数≤200万CFU/mL的样品占2.50%。2018年天津市奶牛养殖场原料奶体细胞数平均值为38.81万/mL,标准差36.49万/mL,变异系数94.03%,最大值为210.00万/mL,最小值为0.80万/mL,SCC≤50万/mL的样品占79.17%,50万/mL200万/mL的样品占0.83%。乳房炎病原菌检测结果显示:36个样品检出病原菌,总检出率为94.74%;共检出8种病原菌,检出率最高的是乳房链球菌,检出率为73.68%,其他病原体检出率依次为:牛支原体34.21%,牛棒状杆菌13.16%,无乳链球菌10.53%,大肠杆菌5.26%,白色念球菌5.26%,停乳链球菌2.63%,铜绿假单胞菌2.63%。14个样品检出耐药基因,总检出率为36.84%;2种耐药基因的检出率分别为β-内酰胺耐药基因CTX-M934.21%,耐甲氧西林葡萄球菌耐药基因MecA 2.63%。研究表明,2018年天津市原料奶SCC及细菌总数大部分接近欧盟标准,但仍有待进一步提高;引起天津地区临床型乳房炎的3种主要致病菌为乳房链球菌、牛支原体和牛棒状杆菌。     

Surgical intrauterine insemination with cat semen cryopreserved with Orvus ES paste or sodium lauryl sulfate

The mean post-thaw sperm motilities of feline frozen semen prepared with 1% OEP or 3 g/ml SLS as a cryoprotective agent, in addition to 7% glycerin, were 35.0 ± 2.4 and 37.0 ± 2.5%, respectively, showing no significant difference. On unilateral intrauterine insemination (UIUI) using these semen samples at a sperm number of 40 × 10(6), the conception rate was 70.0% (7/10) in the OEP group and 30% (3/10) in the SLS group, showing that the rate was higher in the OEP group, but the difference was not significant. It was suggested that sperm in frozen semen showing the above qualities were transferred to the contralateral uterine horn on UIUI.     

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.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

Semen collection and characterization in rockhopper penguins (Eudyptes chrysocome chrysocome).

Low egg fertility and hatchability is a common problem in captive populations of rockhopper penguins (Eudyptes chrysocome chrysocome). These conditions make sustaining a captive population challenging. A method for collecting and evaluating semen from rockhopper penguins was developed to assist in the evaluation of low egg fertility found in one captive population. Six adult male rockhopper penguins were conditioned to allow semen collection once a week from the start of breeding season until ejaculates no longer contained sperm. A total of 59 ejaculates was collected between 17 September and 31 December 2004. Forty-five of these samples were evaluated for volume, pH, sperm concentration, and sperm quality (motility, viability, and morphology). There was a large variation between individuals and between collections for each individual. The mean motility was 34.5% (+/- 22%). Mean volume of ejaculate was 0.23 ml (+/- .31 ml). Mean concentration was 16.9 x 10(6) sperm/ml (+/- 48.7 x 10(6) sperm/ml). Mean number of sperm per collection was 1.7 x 10(6) (+/- 4.2 x 10(6)). Mean percentage of living sperm was 82.9% (+/- 18.1%). Mean percentage of sperm with normal morphology was 82.1% (+/- 18.8%). Mean pH was 6.47 (+/- 0.49). During this season, only one of these males paired with a female. The pair produced one fertile egg, but the embryo died early in incubation. Male rockhopper penguins had low sperm concentration and low motility indicating that low male fertility may be contributing to the poor egg fertility rate. This work represents the first step in an ongoing study to improve captive breeding of rockhopper penguins.     

Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.     

The Relationship between Semen Quality and the Nonreturn Rate of Bulls

Contents
The value of routine evaluation of bull semen was analysed for 117 AI bulls placed in two studs. Data from semen analysis of a total of 1635 ejaculates was compared statistically with the nonreturn rates of the bulls. The semen parameters which were significantly correlated to nonreturn rates were the motility of the freshly collected ejaculates (p = 0.0140) and post-thaw motility (p = 0.0075). The total number of motile sperm in the inseminate ranged from 10.9 to 19.3 × 10⁶ and according to previous reports the effects of low motility should be fully 'compensated' when doses above 10 × 10⁶ sperm/dose are used for insemination. In conclusion, the motility of freshly collected semen does not appear to be 'compensation' and a low percentage of motile sperm in an ejaculate may indicate other dysfunctions of the population of motile cells. Furthermore, post-thaw motility appears to correlate significantly with nonreturn rates. The largest proportion of the variation was explained by the breed of the bull and stud (42.2% of the variation), whereas the two motility parameters explained 10% of the total variation in nonreturn rates. Objective and precise evaluation of sperm motility in combination with other semen traits are needed to improve breeding efficiency. Although microscopic evaluation of sperm motility correlates with nonreturn rates of bulls, the methods are subjectively assessed and inaccurate and therefore do not allow a satisfactory prediction of fertility.