散养湘西黄牛精液的采集与异地冷冻保存技术研究

通过散养湘西黄牛的精液采集与冷冻保存,有利于收集多个优良血缘,降低种公牛站内种公牛的饲养成本。2010年5月,在张家界市永定区湘西黄牛保种区,挑选年龄为2~4岁,特级种公牛10头,进行精液采集与冷冻保存。结果表明：10头未调教的公牛中,除2头（3号、7号）没有采精调教成功外,其它8头都可采精。每次采精3回,平均每次的射精量在2.33~7.66 mL、平均每次原精密度在7.18×108~1.51×109之间变化。不同个体的射精量与密度没有显著相关性。冻后细管精液在37~39℃温水中进行解冻,冻后活力在0.35~0.45之间,均能达到国家牛冷精液生产标准要求,10头公牛中有8头共生产冷冻精液3537支。因此,在保种区域现场采精,稀释处理后送至种公牛站实施湘西黄牛冻精生产的方法是可行的。     

藏獒冷冻精液保存液配方筛选试验

为了提高藏獒的繁殖力,建立完善的藏獒人工授精体系和保存优秀藏獒精子,本研究对8只藏獒进行了采精和精液的超低温冷冻保存试验,并对藏獒的精液超低温冷冻保存液进行了系统的筛选。结果表明,渗透压450 mOsm/L的配方Ⅴ（葡萄糖1.98 g、蔗糖3.42 g、柠檬酸钠0.59 g、谷氨酸钠0.75 g、三羟甲基氨基甲烷1.21 g、VB6 10 mL、VB12 10 mL、卵黄20 mL和甘油8 mL）对藏獒精液冷冻保存的效果最佳,解冻后精子的活力为0.58,畸形率为17.2%,满足输精和进行超低温冷冻保存精子的要求。     

不同浓度大豆卵磷脂替代蛋黄对东佛里生奶绵羊精液冷冻保存效果的影响

本试验皆在研究添加不同浓度大豆卵磷脂（SL）冷冻保存东佛里生奶绵羊精液的效果。我们在Tris基础稀释液中,添加18%蛋黄为对照组,添加0.5%、1%、1.5%、2%、2.5%SL设为试验组,检测冷冻精液解冻后的精子活率和顶体完整率。结果显示,添加0.5%、2.5% SL冷冻稀释液稀释的精液,解冻后精子活率和顶体完整率与其他组之间存在显著差异（P<0.05）;添加18%蛋黄和1%～2% SL冷冻稀释液稀释的精液,冷冻解冻后精子活率和顶体完整率之间无显著差异（P>0.05）;添加18%蛋黄和1.0%～1.5% SL冷冻稀释液稀释后的精液,进行人工授精后母羊的妊娠率与对照组无显著差异（P>0.05）。因此,大豆卵磷脂可以作为冷冻保护剂用于东佛里生奶绵羊精液的冷冻保存,其最佳添加浓度为1～2%（g／L）。     

五种糖类对犬精液冷冻保存效果的研究

选取8只身体健康、性欲旺盛的比格公犬,采集其精液,对鲜精进行质量检测,主要包括颜色、射精量、精子活率、活力和pH。选取精子活率达到70%以上犬精液用于后续精液冷冻试验。分别将含有葡萄糖、果糖、蔗糖、乳糖和海藻糖的精液稀释液与精液按照1∶2的比例进行稀释。经冷冻解冻后,进行活率、活力、质膜完整性和顶体完整性的精子质量检测。通过对新鲜精液进行品质检测,结果显示5只合格用犬的平均射精量为2.85 mL,密度为2.01×10~8个/mL,pH为6.56。含有果糖的冷冻稀释液中精子活率和活力最高,分别达59.90%和54.00%;其次是添加葡萄糖和乳糖的稀释液中活率较高,分别为59.21%和56.73%,且两组稀释液中精子解冻后活力均为52.00%。进一步评估精子解冻后质膜完整率,结果显示葡萄糖和果糖组显著高于其他组,分别达48.73%和49.52%;而蔗糖添加组精子质膜完整率最低,为42.21%。稀释液中添加果糖的精子解冻后顶体完整率显著高于其他组,而添加蔗糖组顶体完整率最低。在比格犬的精液冷冻保存中,添加果糖的冷冻稀释液能显著提高精子的活率和活力,从而达到提高冻精质量的效果。     

八眉猪精液品质分析研究

本试验选择生长发育良好,年龄在1.5～2.5岁的八眉猪公猪11头,按常规方法采集精液,对其精液品质进行评定分析。结果表明,11头试验猪精液颜色为乳白色或浅灰色,略带腥味,公猪平均射精量为(117.34±43.27) ml,平均精子活率为65.02%±4.26%,平均精子密度为(1.52±0.7)×10⁸个/毫升;解冻后精子活率均≥35%,精子畸形率≤20%;对精液品质特性进行相关性分析发现,精液量、精子活率、精子密度间相关性不显著(P>0.05)。经评定,其中一头公猪精子活率不达标,其余10头公猪精液符合国标要求,可以进行冻精制作及作为资源保存。     

马精液冷冻保存研究进展

从马精液冷冻程序过程中不同采精方法、稀释液添加剂、冷冻保存承载工具等方面,概述了影响精液冷冻的因素,并介绍了解冻后精液的人工授精方法。     

冷冻稀释液中添加大豆卵磷脂对梅花鹿冻融精子质量的影响

【目的】 探究在冷冻稀释液中添加大豆卵磷脂代替10%卵黄对梅花鹿精液冷冻保存效果的影响,为梅花鹿人工授精体系的完善提供参考。【方法】 采用电刺激法采集梅花鹿精液,以精液冷冻稀释液中分别添加1%、2%、3%、4%和5%大豆卵磷脂代替10%卵黄作为试验组,添加20%卵黄作为对照组,分别进行各组精液冷冻保存。5 d后,进行精液解冻,检测解冻后各组精子的活力、质膜完整率、顶体完整率、线粒体活性、存活时间,筛选合适浓度的大豆卵磷脂。选取4~5岁健康雌性梅花鹿,肌肉注射300 IU孕马血清促性腺激素（PMSG）和0.4 mg氯前列醇钠进行同期发情处理,发情后第20 h用20%卵黄组与筛选出的大豆卵磷脂组冻精进行人工输精,输精后30 d使用B超检测仪检测妊娠情况,统计妊娠率。【结果】 与对照组相比,1%大豆卵磷脂组冻融后的精子活力、向前活动力、快速前进活力、活率、质膜完整率、顶体完整率及线粒体活性均显著提高（P<0.05）;随着稀释液中大豆卵磷脂浓度的增加,其冻融后精子活力、向前活动力、快速前进活力、活率、质膜完整率、顶体完整率以及线粒体活性呈下降趋势,精子存活时间也随浓度的增加而减少。1%大豆卵磷脂组冻融精子人工授精梅花鹿的妊娠率为61.11%,高于对照组、2%和3%大豆卵磷脂组,但差异均不显著（P>0.05）。【结论】 在梅花鹿精子冷冻稀释液中添加1%大豆卵磷脂替代10%卵黄,能有效提高梅花鹿冻融精子的质量,为进一步筛选新型梅花鹿精液冷冻稀释液提供理论基础。     

上海水牛的保种冻精技术

采集上海水牛成年公牛精液进行冷冻保存,从而抢救性保护该品种。测定结果表明:上海水牛原精活率平均高于50%,活力平均高于45%;冻精解冻后精子活率平均高于45%,活力均高于40%。     

采精频率对长白种公猪精液品质的影响

为探讨采精频率对种公猪精液品质造成的影响，本试验采用计算机辅助精液分析仪检测了四川某种猪繁育场的10头长白种公猪在不同采精频率下（1、2、3、4、5次/周）的精液，分别其对精子活力、密度、精液量、精子结构变化和精子运动性能等指标的影响。结果表明：采精频率为3次/周时，精子密度最高，与采精频率为4次/周的精子密度差异不显著（P> 0.05），与采精频率为1、2和5次/周时的差异显著（P> 0.05）；采精频率为3次/周时精子活力和活率最高，与采精频率为5次/周时的活力与活率差异显著（P> 0.05）；精子总数在采精频率为3次/周时最多，精液量未呈现出规律性变化，但在采精频率为1次/周时最多，与采精频率为5次/周时的差异显著（P> 0.05）。综上所述，采精频率为3次/周时精液品质最佳；采精频率为4次/周和2次/周时精液品质次之。     

比格犬的繁殖技术Ⅰ.采精及精液冷冻

采用手握法采取比格犬精液,成功率为71.4％,平均射精量2.67mL;精液呈弱酸性,每次射出的精液分三段,精子主要在第二段,精子平均密度为2.105×10~8/mL;新鲜精液的活力为0.8～0.9;精液用两种稀释液稀释后在液氮中保存14～60d,解冻后,其孵活率分别为0.425和0.427.     

Effect of Semen Extender and Density Gradient Centrifugation on the Motility and Fertility of Turkey Spermatozoa

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.     

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis)

Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4 degrees C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean +/- SD) of motile and live sperm were 96+/-2 and 86.5+/-7.2% before freezing, and 43+/-5 and 67.4+/-3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8+/-1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

牛冷冻精液的应用研究进展

牛人工授精技术是所有家畜中应用最为广泛的一种繁殖技术,而其巨大的发展又得益于精液冷冻保存的成功应用.本文简要介绍了牛精液冷冻技术的发展历史和精液冷冻的原理,分析了影响牛精液冷冻效果的主要因素,并对国内外现行使用的精液评定指标作了简单阐述,以期为牛高质量冷冻精液的研究和牛人工授精技术的应用提供参考.     

转基因牛与非转基因牛性控精液分离及配种效果初探

本研究对转基因牛以及非转基因牛精液经流式细胞仪分离冷冻后精子活力、分离准确率进行了比较,同时对分离的性控冷冻精液进行了人工输精,对受体牛的情期受胎率进行了统计分析。结果表明,转基因牛与非转基因牛精液在冷冻解冻后活力以及分离准确率方面差异不显著(P>0.05);转基因牛与非转基因牛的性控冷冻精液的情期受胎率分别为57.4%、59.3%,二者之间差异不显著(P>0.05)。     

Stallion Sperm Selection: Past,Present, and Future Trends

Although several selection techniques are available for processing spermatozoa, only colloid centrifugation has been used to any extent in this field, starting with density gradient centrifugation and progressing more recently to single-layer centrifugation (SLC). SLC through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility and normal morphology from stallion semen. The method is easier to use and less time-consuming than density gradient centrifugation, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The potential applications of SLC in equine breeding are as follows: to improve sperm quality in artificial insemination doses for “problem” ejaculates, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.     

二甲基甲酰胺对猪精液冷冻保存效果的影响

用二甲基甲酰胺(DMF)完全替代甘油,比较不同平衡时间和不同DMF添加量对猪精液冷冻保护效果的影响。结果表明,DMF能完全替代甘油,获得较好的冷冻保护效果。最佳平衡时间为90 min,解冻后精子活力为(44.57±0.72)%,显著高于对照组和其他组(P0.05)。当DMF添加量为5%时,冻后精子活力、活率、线粒体活性、顶体完整率和质膜完整率分别为(49.91±0.39)%(、46.51±0.26)%、(47.51±0.52)%(、49.84±0.56)%、(46.30±1.61)%,均显著高于2%、3%、6%DMF添加组(P0.05),但与4%DMF添加组相比,冻后精子活力、活率和质膜完整率差异不显著(P0.05)。本试验结果表明,DMF最适添加量为5%。     

谷胱甘肽对犬精液冷冻保存效果的影响

为探讨在冷冻稀释液中添加谷胱甘肽（GSH）对犬精液冷冻保存效果的影响,采用按摩法采集5只杂种土犬的精液,离心去精清后,在冷冻液中分别加入0.5、1.0、1.5、2.0、2.5 mmol/L的GSH,制成0.25 mL的冻精进行冷冻保存,以不添加GSH的处理组作为对照组。解冻后在含有5% CO₂的空气、37 ℃、相对饱和湿度条件下孵育10 h,分别在孵育0、2、4、6、8、10 h时检查精子活力。结果显示：冻融后0 h,0.5、1.0 mmol/L GSH处理组的精子活力较高,分别为0.36和0.38,均显著（P<0.05）高于对照组和2.0、2.5 mmol/L处理组,且两者之间差异不显著（P>0.05）;1.0 mmol/L处理组的精子顶体完整率最高,为85.10%,显著（P<0.05）高于对照组,同时,其精子畸形率最低,为23.00%,显著（P<0.05）低于对照组。冻融后体外孵育2、4 h时,0.5、1.0 mmol/L处理组的精子活力均较高,其中,0.5 mmol/L处理组在体外孵育4 h时,其精子活力仍可达到0.30;孵育6 h时,1.0 mmol/L处理组精子活力最高,显著（P<0.05）高于对照组和2.0、2.5 mmol/L处理组;孵育8 h时,各GSH处理组的精子活力均显著（P<0.05）高于对照组;在孵育至10 h时,各GSH处理组的精子活力较其他孵育时间均有较大幅度的下降,未检测到对照组中有呈直线运动的精子。综上提示,在犬精液冷冻液中添加0.5~1.0 mmol/L的GSH能够显著提高冻融后的精子质量和体外存活时间。     

牛精子冷冻损伤及其改善方法

牛精液冷冻与人工授精技术相结合，在牛品种改良、保护优良种质资源中发挥着重要作用。尽管冷冻后部分牛精子活力高达60%以上，但是冷冻后精子受损现象却普遍存在，尤其是受精能力与鲜精相比显著降低。作者详细介绍了冷冻-解冻过程对牛精子造成的主要损伤，包括精子的形态完整性、活力及遗传物质的改变等；阐述了冷冻造成精子损伤的主要原因，即细胞内冰晶形成和氧化应激反应及其产生的可能机制；并且对目前常用的提高冷冻精子质量的方法，如添加冷冻保护剂、优化冷冻程序以及添加抗氧化剂等进行了详细地综述；提出了在冷冻精液研究方面值得探索的问题，以期为家畜精液冷冻保存技术的进一步优化提供理论依据。     

牛精液冷冻保护剂研究进展

牛的人工授精技术是目前应用最为广泛的动物繁殖技术之一,对牛的遗传改良做出了巨大贡献。但是,在冷冻一解冻过程中仍然有大约40％～50％的精子失去活性,限制良种公牛种用性能的发挥。论文在分析精子冷冻保存原理的基础上,阐述了渗透性、非渗透性冷冻保护剂以及低密度脂蛋白对牛精子的冷冻保护作用,以期为开发新型冷冻保护剂的研究提供一定参考,进一步提升牛冷冻精液的质量。