人工授精和同期发情技术在茸鹿繁殖上的应用研究

在2003年至2004年两年间,应用进口孕激素类药物CIDR对270只梅花鹿和204只马鹿进行同期发情处理,利用高产种公鹿冻精,采用人工授精技术对母鹿定时一次人工输精,梅花鹿采用特制开膣器输精95只,受胎62只,受胎率65%,直肠把握法输精175只,受胎127只,受胎率72.5%;其平均双胎率达22%。对梅花鹿采用孕激素类药物+促性腺激素的处理方法达到了发情同期化,并采用一次定时人工输精,其平均受胎率达68.75%。马鹿全部采用直肠把握法输精204只,受胎180只,受胎率88.2%。     

马鹿胚胎移植技术的试验研究

以新疆塔河马鹿为供体鹿和受体鹿 ,经过同期发情、超数排卵、人工授精、胚胎采集和胚胎移植后 ,供体鹿 3年共超排 2 7头 ,获可用胚 116枚 ,同期发情处理受体母鹿 2 2 8头 ,发情母鹿 171头 ,同期发情率 75 % ,采用人工输精后 ,进行胚胎移植 ,其中人工授精受胎率平均达 5 5 .7% ,移植 6 8头 ,受胎数 36头 ,鲜胚移植受胎率 5 2 .9%。     

天山马鹿诱导同期发情试验

采用CIDR PMSG PG方法对选用的140只天山马鹿进行同期发情处理,直肠把握人工输精。期间对马鹿的冻精制备进行了研究。结果表明:同期发情率达95%,同期率90%,情期受胎率达80%。所制备冻精的平均活力在0.4以上。     

不同剂量PMSG及输精时间对梅花鹿发情率和受胎率的影响

为了提高梅花鹿人工输精受胎率，充分发挥良种优势，研究选择辽宁地区3个鹿场分别进行同期发情试验、常规(试情)输精试验和定时输精试验，每个鹿场均选择3～8岁、健康、营养状况良好、体型大小一致、繁殖机能正常的经产母鹿90只，随机分为3组，每组30只，其中同期发情试验分为同期组Ⅰ、同期组Ⅱ、同期组Ⅲ,常规(试情)输精试验分为常规组Ⅰ、常规组Ⅱ、常规组Ⅲ,定时输精试验分为定时组Ⅰ、定时组Ⅱ和定时组Ⅲ,通过同期发情试验(CIDR+PMSG方法)探究不同剂量PMSG对母鹿发情率、受胎率和双胎率的影响，通过常规(试情)输精和定时输精试验探究不同输精时间对母鹿受胎率和双胎率的影响。结果表明：在同期发情试验中，母鹿发情率的表现为同期组Ⅲ(86.67%)>同期组Ⅱ(73.33%)>同期组Ⅰ(46.67%),且同期组Ⅱ、Ⅲ的发情率显著高于同期组Ⅰ(P<0.05);受胎率的表现为同期组Ⅱ(81.82%)>同期组Ⅰ(78.57%)>同期组Ⅲ(11.54%),且同期组Ⅰ、Ⅱ的受胎率显著高于同期组Ⅲ(P<0.05);双胎率的表现为同期组Ⅱ(16.67%)>同期组Ⅰ(9.09%...     

天山马鹿诱导同期发隋试验

采用CIDR＋PMSG＋PG方法对选用的140只天山马鹿进行同期发情处理，直肠把握人工输精。期间对马鹿的冻精制备进行了研究。结果表明：同期发情率达95％，同期率90％，情期受胎率达80％。所制备冻精的平均活力在0．4以上。     

氯前列烯醇、孕酮阴道栓单独或结合其它激素处理本地母水牛后的发情和受胎情况

本试验研究对比了PGc、CIDR、PGC +PMSG和CIDR +PGc对 2 95头本地母水牛同期发情定时输精的发情率和受胎率。同时对处理后发情的母水牛分别在第一次输精时肌注hCG、LRHA并以不注射的作对照 ,探讨hCG和LRHA对发情母水牛排卵和受胎的效果。也分析了同一处理方法对处女水牛和经产水牛同期发情率和受胎率的差异。结果表明 :采用CIDR +PGc的同期发情率和受胎率最高 (85 .13%、4 6 .0 3% ) ,PGc +PMSG、CIDR和PGc组的发情率和受胎率分别是 73.0 1%和 4 3.4 8%、78.0 2 %和 4 3.6 6 %、6 4 .18%和 4 1.6 8%。CIDR +PG组的发情率极显著高于PGc组 (P <0 .0 1) ,其它各组间无显著差异 (P >0 .0 5 )。各组母牛的受胎率无显著差异 (P >0 .0 5 )。发情的母水牛在第一次输精时肌注hCG或者LRHA3,排卵率为 89.78%和 91.38% ,极显著高于不处理母水牛 (5 9.74 % ) (P <0 .0 1) ;但受胎率与对照组无显著差异 (P >0 .0 5 )。本试验 4种同期发情处理方法在发情率上经产牛 (84 .4 7% )显著高于处女牛 (5 5 .91% ) (P <0 .0 5 ) ;但受胎率 (45 .6 1% :38.4 6 % )无显著差异 (P >0 .0 5 )。     

湖羊的同期发情

对67头湖羊分二批用氯烯前列腺醇进行同期发情处理 ，在第二次处理后53 h进行定时输精，第一批处理后进行人工授精的母羊27头，第一情 期受胎9头，受胎率33.33%，自然配种9头，受胎4头，受胎率为44.44%。第二批人工授精23 头，10头受胎，受胎率43.48%，自然交配8头，第一情期全部受胎，第一情期受胎率100%。 产羔：45头正常分娩，产羔101头，平均产羔率224.44%。     

如何提高梅花鹿同期发情后人工输精的受胎率

梅花鹿的人工授精分为小散户自然发情输精和大鹿场的同期发情处理后批量输精。大鹿场多采用同期发情后批量输精，既便于生产管理，又省时省力。但同期发情后人工授精的受胎率相对于自然发情输精受胎率较低。现就近几年从事同期发情后人工输精工作中的一些体会总结如下，供同行们参考。     

茸鹿不同人工输精方式受胎效果分析

为探明人工输精方式与受胎率关系，本试验对梅花鹿、马鹿不同输精方式进行比较研究。研究结果：马鹿、梅花鹿采取公鹿试情人工输精方式受胎率最高，受胎率分别为83．78％和62．75％；梅花鹿同期发情输精受胎率偏低（45％左右），与榆精时间（统一标准取药后56～60h输精）关系不大，主要原因是同期发情实际发情率（60％）偏低；养鹿场（户）选择人工输精方式应按“因地制宜，量力而行”的原则进行。     

多浪羊同期发情、定时输精效果探讨

运用阴道海绵栓＋PMSG法对夏季多浪羊进行同期发情处理,并用萨福克羊颗粒冻精对同期发情羊进行定时输精。结果表明：颗粒冻精输精受胎率24.52%,颗粒冻精干、湿两种解冻法对受胎率没有显著影响。     

天山马鹿诱导同期发情及人工授精试验

为解决马鹿人工授精推广存在的母鹿诱导同期发情及鉴定与马鹿采精和精液保存技术难关,采用CIDR+PMSG+PG方法对140只天山马鹿进行同期发情处理,对5头公鹿采精和保存,直肠把握人工授精。结果表明,鲜精保存72 h活率达到0.6以上,同期发情率达85%,情期受胎率达50%。     

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。     

低剂量梅花鹿XY精子马花杂交人工授精试验

采用流式细胞分离仪分离的梅花鹿X和Y型冷冻精液与常规冻精对62头3组同期化处理的马鹿进行直肠把握人工授精。结果表明,0.25 mL/支含106个有效精子的X和Y型冻精产仔率分别为43%和37%,而0.25 mL/支含107个有效精子的常规冻精产仔率为55%,X、Y型冻精与常规冻精组间差异显著(P<0.05),所产后代性别比率分别为0∶10,9∶0和5∶6,X、Y型冻精与常规冻精组间差异显著(P<0.05),X、Y型冻精与常规冻精所产后代出生及60 d时的体重差异不显著(P>0.05)。     

Fertility after different artificial insemination methods using a synthetic semen extender in sheep

The present study aimed to investigate the fertility of ewes artificially inseminated with three different methods using a synthetic semen extender, AndroMed. The three methods of artificial insemination (AI) were cervical AI with fresh-diluted or frozen-diluted semen at observed estrus, and an intrauterine AI with frozen-thawed semen. A total of 80 ewes were treated with a controlled internal drug release (CIDR) containing 0.3 g progesterone per device for 12 days. In Experiment 1 (26 Suffolk ewes), superovulation was induced with 20 mg follicle-stimulating hormone and 250 IU equine chorionic gonadotropin (eCG) two days and one day before CIDR removal, respectively, during the non-breeding season. In Experiment 2 (54 Suffolk and Suffolk crossbred ewes), an intramuscular injection of 500 IU eCG was administered one day before CIDR removal to synchronize estrus and ovulation during the breeding season. In Experiment 1, fresh-diluted or frozen-thawed semen was deposited into the cervical orifice after estrus detection, and an intrauterine AI with frozen-thawed semen was performed by laparoscopy at a fixed-time basis without estrus detection. Embryos were recovered by uterine flushing 6 days after AI, and the rates of recovered, fertilized (cleaved) ova and embryos at the morula or blastocyst stage were compared among the three AI methods. In Experiment 2, the pregnancy rates after the three AI methods were compared. In Experiment 1, the rates of recovered ova were not significantly different among the three AI methods (52.5-56.7%). The rate of fertilized ova (81.0%) by laparoscopic AI with frozen-thawed semen was significantly higher compared with cervical AI of fresh-diluted (25.5%) or frozen-thawed (3.5%) semen, but the rate of embryos at the morula or blastocyst stage (17.6%) was significantly lower than that of the cervical AI with fresh-diluted semen (69.2%). The rates of ewes yielding fertilized ova were not significantly different among the three groups (44.4, 11.1 and 62.5% for cervical AI with fresh-diluted and frozen-thawed semen and intrauterine AI with frozen-thawed semen). In Experiment 2, the pregnancy rate of ewes intrauterinally inseminated with frozen-thawed semen (72.2%) was significantly higher than those of ewes inseminated cervically with fresh-diluted (5.5%) or frozen-thawed (0.0%) semen. The present results showed that acceptable fertilization and pregnancy rates could be obtained by an intrauterine AI with frozen-thawed semen using a synthetic semen extender (AndroMed), but not sufficient by the cervical AI with either fresh or frozen semen.     

奶牛血清孕酮和总胆固醇水平与超数排卵效果的关系

为了研究血清中若干生化指标与母牛超数排卵的关系。在性周期中期第10天或11天超排处理之前颈静脉采血,用以测定血清中孕酮、总胆固醇等指标的水平,而后用FSH进行超排处理。输精后第7天左右采用非手术采卵,并通过形态学检查对胚胎进行鉴定。根据可用胚胎数将供体母牛分为A组(可用胚胎>2枚)和B组(可用胚胎≤2枚),并比较两组孕酮、总胆固醇等指标的水平。结果表明:周期中期血清孕酮水平在3.0ng/ml以上对于预选供体母牛是有效的。血清总胆固醇水平与可用胚胎数的相关系数r=0.6079(n=19;P<0.01),周期中期血清总胆固醇水平高于140mg/dl似乎可以做为预选供体牛较好的参数。     

梅花鹿电刺激采精技术研究

为提高鹿产品质量,发展优良鹿群数量,扩大优良基因在时间和地域上的利用率,2004年11月份在梅花鹿配种期对5头雄性梅花鹿进行了5次电刺激采精试验,成功5次,成功率100%,采得的精液品质优良,采精量为0.4~2.0 mL,平均采精量为1.28 mL,精子密度为5~10亿个/mL,活力为0.4~0.6,采精后雄鹿食欲、精神正常。     

纤维素酶曲添加剂对鹿茸产量及饲料消化率的影响

先后分3批次,对176头梅花鹿进行了增茸试验。结果,添喂纤维素酶曲组比对照组,鹿茸产量分别提高17.38%、26.78%和31.81%,平均每头增产129g。试验组采食量比对照组低7.48%,饲料消化率随纤维素酶曲添加水平的提高而提高。粗蛋白、酸性洗涤纤维(ADF)和中性洗涤纤维(NDF)的消化率在添喂纤维素酶曲70g/头组,分别提高9.48%(P<0.01.)、7.82%(P<0.05)和8.81%(P<0.01).试验组鹿茸氨基酸和无机元素含量都有增加的趋势。血清氨基酸除胱氨酸和组氨酸外,其它氨基酸试验组均有不同程度的提高,无机元素含量除锰外,其它元素试验组均高于对照组。     

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.     

The Construction of Cloned Sika Deer Embryos (Cervus nippon hortulorum) by Demecolcine Auxiliary Enucleation

The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 μg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.     

家养梅花鹿腹腔镜输精技术研究——CIDR+PMSG同期发情处理后输精时间是关键

2004年9月15日(第0天)给45只成年雌性东北梅花鹿阴道放置"阴道内孕酮释放装置"(CIDR),到第12天取出CIDR,同时肌肉注射孕马血清促性腺激素(PMSG),21只母鹿注射200 IU(1组),24只母鹿注射150 IU(2组)。CIDR取出后24~72 h,用带试情布的成年公鹿对处理母鹿进行持续试情。结果第1组和第2组同期发情率分别为86%(18/21)和92%(22/24)(P>0.05),取出CIDR至发情的平均时间分别为(47.54±9.33)h和(49.74±6.69)h(P>0.05)。第2组1只母鹿表现出孕期发情现象。对发情母鹿进行腹腔镜人工输精,输精时间为取出CIDR后的48~78 h。输精后第5天到11月15日,每天早晨、中午和傍晚进行试情,检查人工输精母鹿返情情况,结果第1组和第2组人工输精母鹿未返情率分别为89%(16/18)和76%(16/21)。两组母鹿产仔率分别为50%(9/18)和38%(8/21)(P>0.05)。这对同期发情梅花鹿的定时输精特别有意义。