Sperm pretreatment with glutathione improves IVF embryos development through increasing the viability and antioxidative capacity of sex-sorted and unsorted bull semen

The antioxidant of reduced glutathione（GSH） is the most abundant thiol in cells for the maintenance of the intracellular redox balance. The study aimed to assay the effect of sperm treatment with GSH before incubation with oocytes on the development potential of embryos obtained by in vitro fertilization（IVF）. Also the mitochondrial membrane potential（ΔΨm）, plasma membrane integrity（viability）, DNA fragmentation, reactive oxygen species（ROS） content, superoxide dismutase（SOD）, catalase（CAT） and glutathione peroxidase（GPx） activities, methane dicarboxylic aldehyde（MDA） level as indices of lipid peroxidation in sex-sorted and unsorted sperm from three bulls were investigated using flow cytometry and enzyme-labeled instrument individually. The results showed that 2 mm ol L^–1 GSH increased significantly the cleavage rate（86.68% vs. 82.78%）, 4- to 8-cell rate（82.30% vs. 73.43%） and blastocyst rate（43.15% vs. 35.24%） of IVF embryos compared with untreated group. Furthermore, addition of GSH increased significantly the ΔΨm and viability, decreased the ratio of DNA fragmentation in sex-sorted or unsorted semen（P〈0.05）, except the sex-sorted semen from bull 019. Similarly, activities of SOD, CAT and GPx were increased significantly. However, the contents of MDA were decreased significantly both in sex-sorted and unsorted semen treated with GSH（P〈0.05）. These results suggest that sperm pretreatment with GSH during IVF can maintain better the viability and fertility of sperm through reducing apoptosis and increasing the antioxidant capacity, which improves the IVF embryos development.

Sperm Quality in Holstein Bulls Friesian and Brahmans of Frozen Semen Commercially

The aim of this study was to assess sperm quality (motility, viability and acrosomal integrity) sperm from commercially frozen semen straws two breeds of bulls Bos taurus (holstein Frisian) and Bos indicus (Brahman). 9 commercial straws 0.5 ml of Holstein bull semen and 9 Brahman bull were thawed, they were kept for two hours at room temperature and motility, viability and acrosomal integrity (NAR) was assessed. The results were 30% motility, viability 40% and 30% of NAR in the Holstein breed. Brahma race for motility 40%, 50% and 40% viability was obtained NAR. In conclusion, according to the results of the variables analyzed, the Brahman breed in sperm quality was better than the Holstein breed;however, the results of both races meet minimum standards of quality sperm for use in artificial insemination (AI) field level.

Effects of Storage Temperature on the Effective Activity of Straw Frozen Semen after Thawing

The aim was to discuss the optimal storage environment and proper insemination time after thawing of 0.25 mL straw frozen semen. Straw frozen semen was thawed at 40 ℃ for 20 s, and then stored at 0 -4 ℃, 14 - 16 ℃, 25 -27 ℃ for 2, 4, 6, 8 and 10 h, respectively. The sperm motility was detected. After thawing, semen was stored at 0 - 4 ℃ and 14 - 16 ℃ for 10 h. Their sperm motilities （0.434 ±0. 016 7 and 0.423 ±0.019 6） had no significant differences （P 〉 0.05） with initial thawing motility （0.441 ± 0.030）. Sperm motility reduced as the storage time prolonged at 25 -27 ℃. Sperm motility after 6 h had signifi- cant differences with that of initial thawing motility （P 〈 O. 05 ）, and sperm motilities after 8 and 10 h showed extremely significant differences （P 〈 0.01 ）. Thus, sperm motility after thawing was still very high after stored at 0 -4 ℃ and 14 - 16 ℃ within 10 h, which met the requirements for insemination. Under this temperature and time ranges, sperm could be carried over long distances, which had small effects on sperm quality and reached the expected insemination effects. However, under the temperature of 25 - 27 ℃, semen should be used for insemination within 6 h after thawing.

Development of Annexin V Technology for Removal of Dead Spermatozoa from Bovine Spermatozoa and Its Application in Frozen Sexed Semen Production

[Objectives]This study was conducted to develop a molecular marker immunomagnetic bead sorting technology method that can specifically identify dead spermatozoa.[Methods]This study first confirmed the specific binding of Annexin V to dead bovine spermatozoa,and tried to remove dead spermatozoa in semen combining with the immunomagnetic bead technology,so as to improve the separation efficiency of target spermatozoa in the process of sex-controlled semen preparation on a flow cytometer.[Results]The spermatozoon motility,membrane integrity and mitochondrial activity after sorting and the rate of dead spermatozoa during the on-machine X/Y separation were all improved to different degrees(P<0.05),indicating that the technical process design could effectively remove some dead spermatozoa,and there was no significant effect on frozen sexed semen prepared from the separated X or Y spermatozoa(P>0.05),indicating that the technical process did not cause additional damage to the spermatozoa.[Conclusions]Combining the specificity of Annexin V with the immunomagnetic bead method could effectively remove dead spermatozoa from bovine spermatozoa,and significantly reduce the rate of dead spermatozoa in bovine permatozoa during sex-controlled separation(P<0.05).The method developed can effectively improve the production efficiency of frozen sexed semen of dairy cows,reduce the production cost,and promote the industrial application of the product.

不同抗氧化剂对牦牛冷冻精液精子活力的影响

为研究维生素C(VC)、维生素E(VE)、维生素B_(12)(VB_(12))及白藜芦醇(Res)抗氧化剂对牦牛冷冻精液精子活力的影响,在卡苏稀释液中分别添加浓度为0、200、400、600、800、1000、1200μg/mL抗氧化剂,确定抗氧化剂的单因素适宜添加量。根据单因素试验结果进行4因素3水平的响应面优化试验,确定冷冻精液中复合抗氧化剂的适宜添加量。结果表明:VC、VE、VB_(12)及Res的单因素适宜添加量分别为200、400、400、400μg/mL,精子活力最大值分别为83.28%、79.72%、78.18%、69.38%。通过响应面优化VC、VE、VB_(12)及Res的添加量分别为228.80、447.00、394.05、400.69μg/mL,精子活力理论值为82.44%。通过3次验证试验,VC、VE、VB_(12)及Res的添加量分别在230.00、450.00、400.00、400.00μg/mL时,牦牛冷冻精液精子活力平均值为83.66%,超过理论值,较好地预测实验结果。

两种氨基酸对公牛精液冷冻效果研究

[目的]试验研究L—半胱氨酸、精氨酸对牛精液冷冻效果。[方法]基础液中分别添加不同浓度的L—半胱氨酸、精氨酸进行试验,再用L_(9)(3^(4))正交表优化最佳配比,获得不同的稀释液配方,然后再进行对比试验,优选出使用效果最好的牛精液冷冻稀释液。[结果]牛精液冷冻稀释液添加0.125%、0.25%、0.375%的半胱氨酸;牛精液冷冻稀释液添加0.5%、0.75%精氨酸,牛冷冻精液解冻活力均得到提高。[结论]结果表明,半胱氨酸、精氨酸可用于牛精液冷冻保存,能改善牛精液冷冻保存效果,对提高牛冷冻精液质量效果明显。

绒山羊细管冻精保存时间对使用效果的影响

为了比较绒山羊细管冻精不同保存时间的品质变化和使用效果,笔者将辽宁绒山羊公羊的细管冻精从2005年分别保存0,5,10,18年,并分别进行抽检,选用成年母羊进行冻精输精,比较了细管冻精解冻后的活力、畸形率、冻精输配受胎率及冻精后代的产羔率、羔羊初生重和120 d断奶重。结果表明:2005年生产的辽宁绒山羊细管冻精经过0,5,10,18年的冷冻保存,解冻后的活力和畸形率没有显著变化(P>0.05),细管冻精的配种受胎效果与后代生产性能也没有显著差异(P>0.05),冻精后代的产羔率、羔羊平均初生重和120 d断奶重都没有显著差异(P>0.05)。说明细管冻精的保存时间在一定期限内对细管冻精的质量和使有效果没有不利影响。

猪精液冻融技术及其影响因素研究进展

养猪业是我国畜牧产业的重要组成部分。国内生猪养殖主要采用自繁自养模式,配种所使用的精液多采用鲜精或常温稀释保存精液,而精液冻融技术可实现猪精液的长期保存,但与牛、小鼠等物种相比较,猪精液冻融技术还有待于进一步优化改善。为进一步改进猪精液冷冻技术并实现在生产上大规模应用,本文主要综述了猪精液冻融技术发展概况、主要技术流程,影响猪精液冻融后精子活力的关键因素,包括精液的采集、冷冻前降温平衡、冷冻保护剂添加和冷冻方法等,以及精子冻融过程中的理化性质变化,包括物理结构损伤、功能性损伤、精子抗冻机制等,最后总结、展望了能够提高猪精液冻融效果的新方法。

“冻精+鲜精”混精对母猪繁殖性能的影响

为探讨“冻精+鲜精”混精使用的实用效果,该试验将长白鲜精与大白冻精设置5种混精比例,每种比例随机与配10头大白母猪,并对6个繁殖性能指标进行了统计分析。结果表明,不同混精比例的母猪受胎率、窝活仔数、窝重、仔猪初生重、妊娠期均无显著影响(P>0.05)。因此,“冻精+鲜精”混精的授精模式不会影响大白母猪的繁殖性能,研究结果对于地方猪保种的授精与非洲猪瘟威胁下中国猪繁育体系模式优化具有重要的指导意义。

牛冷冻精液精子形态活力变化研究与分析

为探讨牛常规冷冻精液解冻后不同时间内精子的形态结构及活力的变化规律,利用河南某种公牛站同一牛号、同一生产批次的300剂常规冷冻精液进行解冻,检测其解冻后不同时间内精子活力、畸形率、顶体完整率及前进运动精子数。结果表明:刚解冻时、解冻后1h精子顶体完整率下降,但差异不显著(P>0.05),解冻后3h、6h、9h的精子顶体完整率显著低于刚解冻时和解冻后1h(P<0.05)。精子畸形率在精液解冻后随着时间延长而逐渐升高,刚解冻时与解冻后1h差异不显著(P>0.05),但显著低于解冻后3h、6h(P<0.05),且解冻后3h的精子畸形率显著低于解冻后6h的畸形率(P<0.05)。精子活力在精液解冻后随着时间延长而逐渐降低,刚解冻时的精子活力最高,与解冻后1h、3h、6h、9h差异显著(P<0.05),解冻后1h精子活力显著高于6h(P<0.05),解冻后9h精子活力低于任何一个阶段的活力,且差异显著(P<0.05)。可见,人工授精时,刚解冻精液与解冻1h内精液的顶体完整率较好、精子畸形率较低,此时输精效果为佳。

不同发情阶段冻精配种对辽宁绒山羊受胎率的影响

为了提高绒山羊母羊发情鉴定的准确性、提高细管冻精配种的受胎率,开展了绒山羊不同发情阶段(不同生殖道状态)对冻配受胎率影响试验。试验选择健康经产繁殖母羊146只,在母羊发情的不同时期(4期)使用细管冻精进行子宫颈口输精,观察母羊的受胎状况并统计受胎率。结果表明:受胎率以第3期最高,达77.50%(31/40),其次为第2期,为44.00%(22/50),第1期和第4期分别为8.33%(3/36)和0(0/20)。说明辽宁绒山羊细管冻精的适宜输精期为母羊发情的第3期。

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen

The objective of the present study was to determine the effects of docosahexaenoic acid （DHA）-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk （group Ⅰ）, DHA-enriched hen egg yolk （group Ⅱ）, normal hen egg yolk with 5 mmol L^-1 of cysteine supplementation （group Ⅲ） and DHA-enriched hen egg yolk with 5 mmol L1 of cysteine supplementation （group Ⅳ）. The semen was cryopreserved using controlled rate freezer and was thawed at 50℃ for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone （group Ⅲ） improved progressive motility （P 〈 0.05）, and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk （group Ⅳ） improved both progressive motility （P 〈 0.05） and acrosome integrity （P 〈 0.01）. The use of DHA-enriched hen egg yolk alone （group Ⅱ） did not enhance any of the post-thawed semen qualities （P 〉 0.05）. In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

猪冷冻精液应用研究进展

猪冷冻精液使具有高遗传价值的公猪基因在群体中迅速传播,然而实际生产中的低繁殖率导致其推广应用受限。近20年来使用猪冷冻精液配种的繁殖成绩有了显著提高,配种分娩率达80%以上,产仔数达10头以上。显然这些重大进展不能简单地归功于冷冻保护剂的开发和冷冻程序的优化,而是解冻程序、输精方法、母猪发情排卵调控等一系列操作过程不断改进的结果。本文搜集整理了国内外20世纪80年代至今的27篇猪冷冻精液的生产及应用相关的文献,对其中母猪样本量、输精剂量、精子数量、输精方法时间以及不同处理组等技术因素进行分类,明确冻精繁殖成绩出现显著变化的时间节点,并对提高猪冷冻精液应用效果的技术因素进行分析展望,为进一步提高冷冻精液受精效率提供参考与借鉴。

Effect of Butylated Hydroxytoluene on Survival of Frozen-TIhawed Fighting Bull Spermatozoa

The aim of the present study was to evaluate effect of 3 levels of butylated hydroxytoluene （BHT） supplemented in extender for fighting bull semen. Ejaculates were obtained from 10 healthy fighting bulls by artificial vagina. Semen with motility of more than 70% was included in the experiment. Tris-fructose-glycerol-egg yolk extender containing 0, 1, 3 and 7 mM BHT was used to dilute semen to final concentration of 200 × 10^6 cells/ml. Diluted semen was frozen in 0.25 mL straws and frozen semen was thawed in a 37 ℃ water bath for 15 sec before being evaluated at 0 and 1 hour. Sperm motility at 0 and 1 hour after thawing did not differ among groups tested （ranged 47.86-53.57 and 21.79-24.29 respectively）. At 0 hour after thawing, percentage of live sperm of 3 mM BHT （37.39% ± 2.91%） was lower than those of 0 and 1 mM BHT （43.71%± 2.76% and 41.46% ±2.59% respectively, P 〈 0.05） but not different from 7 mM BHT （40.89%± 2.50%）. The effect was more significant at 1 hour after thawing and 3 mM BHT （32.07% ± 2.45%） suppressed live cells more than other concentrations （ranged 35.07%-37.46%, P 〈 0.05）. At 0 hour after thawing, percentage of membrane integrity （hypo-osmotic swelling test） was not affected by BHT concentration （ranged 23.89%-28.54%）. However, at 1 hour after thawing, percentage of membrane integrity of 3 mM BHT （19.75% ± 1.41%） was lower that of 7 mM BHT （23.29% ± 1.88%, P 〈 0.05） but not different from those of 0 and 1 mM BHT （20.32% ±1.81% and 22.07% ± 2.27% respectively）. No significant effect was found on percentage of abnormal sperm. There was no effect of BHT supplementation in extender on most of the Hamilton-thorn motility analyser parameters. It may be concluded that 3 mM BHT can be harmful to fighting bull spermatozoa and lower concentrmion can be used without detrimental effect. Further study may be needed to verify use of BHT in lower range of concentrations.

猪冷冻精液在人工授精中的研究进展

猪冷冻精液在传播优良品种、提高优秀公畜利用率和降低生产成本方面具有重要作用。随着集约化养猪模式的不断发展和人工授精技术普及率的提高,以及地方猪种质资源保存和利用的持续加强,猪冷冻精液将会得到更多应用。然而,猪精子容易受冷休克的影响,且常规人工输精模式不适用于冷冻精液,导致猪冷冻精液在生产中使用率很低。文章通过介绍目前国内外猪冷冻精液在人工授精中的应用现状,分析影响猪冷冻精液应用效果的主要因素,并提出了提高猪冷冻精液在人工授精中应用效果的相关措施,以期为猪冷冻精液生产技术进步及在人工授精中的应用提供参考。

清原马鹿新型人工输精技术的研究

试验旨在通过建立动物模型创新清原马鹿人工输精方法,有效提高冻精的利用率、确保较高受胎率。试验采用“半剂量”方法制作清原马鹿冻精,研究不同输精次数、剂量以及输精保定方式对受胎率的影响。结果显示:半支剂量组与1支剂量组解冻后的平均精子活力差异不显著(P>0.05)。当两种发情方式均采用单次输精法时,1支组的人工输精受胎率均极显著高于半支组(P<0.01);当两种发情方式均采用二次输精法时,采用“半支+半支”“1支+半支”“1支+1支”方式的人工输精受胎率均接近90%,但差异不显著(P>0.05)。机械保定组的受胎率略高于麻醉保定组,但差异不显著(P>0.05)。研究表明,试验创制的清原马鹿人工输精半支剂量的冻精细管符合人工输精的标准,二次输精法和“半支+半支”的输精剂量是清原马鹿人工输精时的最佳输精方法和输精剂量;机械保定比麻醉保定更适用于人工输精。

新鲜饲料油菜对种公牛精液质量的影响

[目的]研究新鲜饲料油菜代替胡萝卜饲喂种公牛的繁育效果,以期解决冬季种公牛青绿饲料的不足。[方法]选取20头西门塔尔、夏洛莱种公牛随机分为2组,进行为期120 d(饲喂前期和饲喂期)的试验。试验Ⅰ组和试验Ⅱ组每日饲喂相同的基础日粮,在此基础上分别额外添加2.5 kg新鲜油菜和2.5 kg新鲜胡萝卜,检测不同时期各组原精产量、密度和冻精产量。[结果]饲喂期试验Ⅰ组和试验Ⅱ组的原精产量分别比饲喂前期显著提高34.83%、54.50%。饲喂期,试验Ⅰ组的精子密度和冻精产量高于试验Ⅱ组,但差异不显著。[结论]饲料油菜可替代胡萝卜添加到基础日粮中提高种公牛精液的密度和产量。

内江猪精液品质分析及冷冻程序优化探索

【目的】围绕内江猪精液品质和冷冻精液流程优化进行研究,以更好实现对内江猪的保种和开发利用。【方法】使用同一时期采精的5头内江猪精液检测了精子的活率、活力、运动状态和生化指标(甘油三酯、过氧化氢等),分析精子活力和精浆各生化指标相关性,并根据课题组此前的研究,进行了冷冻精液流程优化。【结果】平衡12 h后内江猪精液活力指标显著下降,原地移动精子数显著上升(P<0.05)。精子活力与精浆H_(2)O_(2)和MDA含量呈负相关,而与TG含量呈正相关关系。采用温和的离心方式(2000×g,7 min)、较高的甘油浓度(终浓度8%)对冷冻精液流程优化后,精子冷冻前活率提升了10.28%,活力提升了15.75%(P<0.05)。在低温慢速(37℃,20 s)条件下,冷冻保存内江猪精液解冻后活率最高(53.18±5.68)%。【结论】根据内江猪精液特点,优化后的冷冻程序和低温慢速(37℃,20 s)的解冻程序更适合内江猪。

人工授精技术在畜牧生产中的应用历程

人工授精技术是通过人工采集雄性动物精液,经检测和处理后将新鲜或冷冻保存的精液再输入雌性动物生殖道的特定部位,以代替自然交配而繁衍后代的一种技术。人工授精技术的大规模应用可以充分发挥优秀种公畜的遗传资源,加快品种改良速度,降低养殖成本和生殖道疾病传播。人工授精技术的发展大大改变了畜牧生产的模式,但现阶段还面临着发情鉴定困难、繁殖障碍和精液冷冻与性控技术不成熟等问题。文章回顾了人工授精的发展历史与做出突出贡献的关键性人物,总结了现阶段人工授精技术应用的主要困难,以期为未来技术升级及广泛应用提供理论参考。

澳洲进口冻精对北京荷斯坦牛繁殖效果的研究

为改善北京地区荷斯坦牛的繁殖性状,引进澳洲进口冻精并观察其应用效果。在北京某牛场随机选择年龄相近身体状况良好的共363头荷斯坦母牛,采用同期发情和人工授精技术进行操作。对照组母牛使用普通冻精配种,试验组母牛使用澳洲进口冻精配种。结果显示,普通冻精配种的荷斯坦母牛受胎率为83.78%。澳洲进口冻精配种的荷斯坦母牛受胎率仅为45.24%,受胎率极显著下降(P<0.01)。与普通冻精组相比,澳洲进口冻精组母牛产犊率也下降,但差异不显著(P=0.126)。此外,分析母牛妊娠天数发现,澳洲进口冻精组母牛的妊娠天数极显著加长(P<0.01)。试验综合评估了澳洲进口冻精引入北京地区牛场后的繁殖效果,为国内牛场养殖提供参考依据。