稀释液冻干粉剂低温超长时间保存绵羊精液效果

为研究经过冷冻干燥处理的粉剂稀释液与原稀释液对绵羊精液保存的效果,采集6只盘羊与巴什拜羊杂交公羊的精液,分别用粉剂稀释液(A组)和原稀释液(B组)稀释降温后的冰水混合物长期保存,在第0、1、3、6、9和12天分别对精子活率和顶体完整率进行检测,并统计低温保存第9天的精液进行人工授精的受胎率。结果表明:粉剂稀释液与原稀释液低温长时间保存绵羊精子,在保存第9天的精子活率均能达到0.5,顶体完整率均高于65%,粉剂稀释液与原稀释液对精子活率和顶体完整率的保护能力无明显差异(P0.05);粉剂稀释液与原稀释液在低温保存第9天后进行人工授精,受胎率分别为66.9%和67.8%,两者受胎率差异不显著(P0.05)。说明稀释液冻干粉剂与液体稀释液低温保存绵羊精子具有相同效果。     

绵羊精液4℃液态保存的稀释液配方优化研究

本试验旨在通过改变绵羊精液低温保存稀释液组成成分,探讨添加脱脂奶粉替代牛奶和大豆卵磷脂替代卵黄在精液低温保存的可行性。试验一在绵羊精液稀释中,添加不同浓度的(4%、7%、10%、13%)脱脂奶粉替代鲜牛奶作为试验组,对照组添加牛奶。结果表明:10%的脱脂奶粉替代牛奶,随着绵羊精液4℃液态保存时间的延长,其精子活力、存活率、顶体完整率与牛奶组差异均不显著(P0.05),添加4%、7%和13%脱脂奶粉组的上述指标则随着精液保存时间的延长呈现显著下降趋势(P0.05)。在试验一的基础上,试验二用不同浓度(0.375%、0.75%、1.5%、3%)的大豆卵磷脂取代稀释液中的卵黄,进行绵羊精液低温保存试验。结果表明以0.375%的大豆卵磷脂取代绵羊精液低温保存稀释液中的卵黄时,对精子保存效果最好,随着保存时间的延长精子活力、存活率、顶体完整率与对照组差异均不显著(P0.05)。     

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）。     

N-乙酰半胱氨酸对绵羊精液低温保存效果的影响

为研究精液稀释液中不同浓度N-乙酰半胱氨酸(NAC)对绵羊精液低温保存效果的影响,试验用含有不同浓度(0,0.5,1.0,1.5,2.0 mmol/L)NAC的绵羊精液稀释液在4℃条件下保存绵羊精液,并对精子活率、有效存活时间以及48小时的精子畸形率和顶体完整率进行了检测。结果表明:在稀释液中添加1.0 mmol/L的NAC能显著提高精子有效存活时间、总存活时间和48小时精子的顶体完整率(P0.05)。说明添加1.0 mmol/L NAC的精液稀释液能显著改善绵羊精液的低温保存效果。     

绵羊鲜精长效保存稀释液海藻糖浓度优化试验

为了筛选获得最优海藻糖浓度的绵羊鲜精稀释液,试验用TRIS专利基础稀释液分别配制5个海藻糖浓度,0(A组)、5 mmol/dm~3(B组)、10 mmol/dm~3(C组)、15 mmol/dm~3(D组)、20 mmol/dm~3(E组),采用假阴道法采集5只公羊的精液,用稀释液分别对精液进行4倍稀释,置于30℃水浴杯中,稀释精液在2 h水浴杯降温到0℃后,冰水混合物保存,第0,24,72,144,216,288小时时对精子活率、精子低渗膨胀率和精子顶体完整率进行检测。结果表明:在TRIS精液稀释液中添加低于20 mmol/dm~3浓度的海藻糖不会提高被保存精液的精子活率,但可以显著提高被保存精子顶体完整率,优化的海藻糖添加剂量是5 mmol/dm~3。     

绵羊鲜精超长时间保存技术研究

为了研究精液稀释液对绵羊鲜精超长时间保存效果的影响,试验选择萨福克和杜泊公羊精液分别在不同季节采用专用稀释液处理并在0℃冰水混合物状态下保存,检测不同时间的鲜精顶体完整性及精子活率,同时进行人工授精,统计受胎率。结果表明:萨福克公羊精子活率在1~15 d缓慢下降;而杜泊公羊精子活率在保存第6天和第15天有一个相对快速下降的过程;5~9 d内萨福克公羊精子活率变化较为平缓,精子活率均在0.70以上;0~3 d精子顶体完整性高于6~12 d,但差异不显著(P0.05);第15天精子顶体完整性显著低于0~12 d(P0.05)。春季第9天和第12天保存的精子活率显著高于秋季(P0.05)。利用0~12 d保存的精液人工授精后,绵羊受胎率可达到70%以上,保存15 d的精液受胎率显著下降(P0.05)。说明专用稀释液能超长时间(12 d)保证精子活率,并且在春季保存效果优于秋季,受胎率得到保证。     

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...     

葡萄籽原花青素对山羊精液低温保存效果的影响

为提高精液低温保存质量,在山羊精液低温保存基础稀释液中分别添加浓度为0、10、30、50、70 mg/L葡萄籽原花青素,检测精液保存过程中各组之间的精子活率、顶体完整性、质膜完整性、线粒体活性、T-AOC、MDA等精子质量评定指标,探讨葡萄籽原花青素对山羊精液低温保存效果的影响及最适添加浓度。结果表明:一定浓度葡萄籽原花青素可提高精液保存质量,且最适添加浓度30 mg/L。在30 mg/L处理组中,精液各项指标(MDA除外)在保存期间均最高,且5 d时精子活率、顶体完整率.、线粒体活性、T-AOC显著高于其他组(P0.05),其精子活率为51.46%,顶体完整率为67.55%,质膜完整率为50.97%,线粒体活性为50.78%。     

保存温度和不同种类稀释液对猪精液品质的影响

猪精液在室温保存时,含庆大霉素的稀释液保存效果优于含青、链霉素的稀释液,两种稀释液在保存72h时,精子活率、顶体完整率、GOT和LDH活性以及存活指数差异极显著(P<0.01).低温保存时,含蜂蜜和卵黄的稀释液对防精子冷刺激、维持精子活率的效果比含葡萄糖、奶粉的稀释液理想.在冷冻保存条件下,以含2%甘油的稀释液保存效果最佳.液态保存(室温、低温)的猪精液,LDH活性随精子活率下降而下降;pH值则都呈先上升后下降的变化趋势.室温保存主要是引起精子顶体脊膨大和保存后期的顶体前端膨大;低温保存主要引起顶体前部膨大和保存后期的少数顶体脱落;冷冻保存主要是造成顶体的膨大程度加大和顶体脱落.3种保存条件都造成精子线粒体透性增强.液态保存和冷冻保存精液的精清GOT活性与顶体完整率无显著相关性(P>0.05).     

益母草碱对绵羊精液低温保存效果的影响

【目的】探究益母草碱对绵羊精液低温保存效果的影响,为实际生产中制备适宜的绵羊精液低温保存稀释液提供理论依据。【方法】采用假阴道法采集3只多浪羊公羊精液,在精液低温保存稀释液中分别添加0(对照)、20、40、60、80和100μmol/mL益母草碱,检测精液低温保存过程中精子活率、顶体完整率和质膜完整率等精液品质指标以及总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及丙二醛(MDA)含量等抗氧化指标,筛选精液保存过程中最适益母草碱浓度;添加最适浓度益母草碱后,利用实时荧光定量PCR法检测精液低温保存过程中TNF受体超家族成员6(Fas)、胱天蛋白酶3(Caspase-3)、B淋巴细胞瘤-2(Bcl-2)和B淋巴细胞瘤-2关联X蛋白(Bax)等相关凋亡基因表达量;采用子宫颈口内输精方式完成两次输精,统计母羊受胎率。【结果】与对照组相比,低温保存48 h, 40μmol/mL益母草碱组精子活率、质膜完整率、顶体完整率及T-AOC水平、SOD、GSH-Px、CAT活性均显著提高(P<0.05),MDA含量显著降低(P&l...     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

大豆卵磷脂对马精液冷冻保存的效果研究

为探索适宜浓度的大豆卵磷脂(soybean lecithin,SL)代替卵黄(egg yolk,EY)对马精液冷冻保存的效果,本试验分别以5%卵黄(V/V)和10%、20%、30%大豆卵磷脂(m/V)作为精液的冷冻保护剂冷冻解冻马精液,解冻后分别对精子细胞的运动参数、精子细胞膜的完整性、精子细胞脂质氧化物丙二醛的值和线粒体膜完整性进行检测。结果发现,精液冷冻解冻后,5%卵黄和10%、20%、30%大豆卵磷脂对精子的总运动精子数无显著影响(P>0.05),但30%大豆卵磷脂具有最大的原地摆动精子数和最小的直线前进精子数(P<0.05),其他运动参数差异不显著(P>0.05);20%大豆卵磷脂代替卵黄后具有最高的质膜完整性,同时产生最少的脂质氧化物丙二醛;线粒体膜电位经流式细胞仪检测时,30%大豆卵磷脂活精子高膜电位数最高,但与20%大豆卵磷脂之间差异不显著(P>0.05)。试验结果表明,20%大豆卵磷脂能够代替卵黄作为冷冻保护剂用于马精液的冷冻保存。     

Study on the Role of AMPK Activator in Cryopreservation of Sheep Semen

This study aimed to investigate the effects of AMPK activators metformin (Met) and acadesine (AICAR) on sheep semen cryopreservation. Firstly, Met and AICAR with different concentrations (0, 100, 200, 300, 400, 500 μmol·L^-1) were added into the frozen diluent. After freezing and thawing, the optimal concentration (400 μmol·L^-1 Met, 200 μmol·L^-1 AICAR) were selected based on sperm motility, motion performance and membrane integrity; Secondly, the collected semen was froze using different frozen diluents (control group:diluent; Met group:diluent + 400 μmol·L^-1 Met; AICAR group:diluent + 200 μmol·L^-1 AICAR). After thawing, the sperm AMPK protein expression, acrosomal enzyme activity, metabolic index, mitochondrial function and antioxidant enzyme activity were examined. The results showed that the addition of 400 μmol·L^-1 Met and 200 μmol·L^-1 AICAR could significantly improve sperm motility, motion performance and membrane integrity(P<0.05) after thawing. In 400 μmol·L^-1 Met group, the total sperm motility, acrosome integrity rate and plasma membrane integrity rate were 43.20%, 91% and 46%, respectively. Compared with the control group, the level of AMPK phosphorylation in the sperm of the Met and AICAR groups was significantly increased after thawing (P<0.05), the acrosome enzyme activity of sperm was significantly increased(P<0.05); the pyruvate level was significantly decreased (P<0.05), the lactate dehydrogenase activity, lactic acid content, and ATP content were significantly increased(P<0.05). Compared with the control group, the dilution of Met group and AICAR group was more conducive to maintain mitochondrial membrane potential (P<0.05), increase ATPase and antioxidant enzyme activity (P<0.05). The addition of appropriate concentrations of Met and AICAR could improve semen quality of cryopreservation in sheep.     

AMPK激活剂在绵羊精液冷冻保存中的作用研究

旨在研究AMPK激活剂二甲双胍（metformin,Met）和阿卡地新（acadesine,AICAR）对绵羊精液冷冻保存效果的影响。本研究首先在冷冻基础稀释液中分别添加不同浓度（0、100、200、300、400、500 μmol·L^-1）的Met和AICAR,冷冻解冻后根据精子活力、运动性能和结构完整性指标筛选出最佳的添加浓度（400 μmol·L^-1 Met、200 μmol·L^-1 AICAR）;然后分别使用不同的冷冻稀释液（对照组：稀释液;Met组：含400 μmol·L^-1 Met的稀释液;AICAR组：含200 μmol·L^-1 AICAR的稀释液）冷冻精液,解冻后检测精子中AMPK蛋白表达、顶体酶活性、代谢指标、线粒体功能以及抗氧化酶活性。结果表明,稀释液中添加400 μmol·L^-1 Met和200 μmol·L^-1AICAR均可显著提高解冻后精子活力、运动性能及精子结构完整性（P<0.05）,其中400 μmol·L^-1 Met组精子总活力达43.20%,顶体完整率为91%,质膜完整率为46%。与对照组相比,Met组和AICAR组解冻后精子中AMPK磷酸化水平显著升高（P<0.05）;顶体酶活性显著提高（P<0.05）;丙酮酸水平显著下降（P<0.05）,乳酸脱氢酶活性、乳酸以及ATP含量均显著升高（P<0.05）;与对照组相比,Met和AICAR组稀释液更有利于维持线粒体膜电位（P<0.05）,提高ATP酶（P<0.05）以及抗氧化酶的活性（P<0.05）。添加适当浓度的AMPK激活剂可以提高绵羊精液冷冻保存的效果。     

Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant

A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml^‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml^‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml^‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.     

稀释液中添加L-赖氨酸对杜泊羊鲜精液品质的影响

为了探讨添加不同水平L-赖氨酸的精液稀释液对杜泊羊鲜精液态保存下品质的影响,试验选用健康的杜泊种公羊3只,采集精液,等量分装,分别加入到含不同水平(0、0.1、0.2、0.3、0.4和0.5 g) L-赖氨酸的120 mL稀释液中,在0、6、24、30、48、54、72和78 h对精子活力、质膜完整率和顶体完整率进行检测。结果表明,添加0、0.1、0.2 g L-赖氨酸组精子活力较其他添加水平组高,添加0.4和0.5 g L-赖氨酸组精子活力下降速度快,添加0.5 g L-赖氨酸组精子活力在30 h时降为0,添加0.4 g L-赖氨酸组精子活力在48 h时降为0;添加0.1 g L-赖氨酸组精子有效存活时间和生存指数最高,与其他组间差异显著(P< 0.05);质膜完整率0.1 g组最高,贮存0~54 h间与对照组间差异不显著(P >0.05),72 h后与对照组间出现显著性差异(P< 0.05),0.4和0.5 g组最差,与其他组间差异显著(P< 0.05);顶体完整率0.1 g组最高,与对照组间差异不显著(P >0.05),0.4和0.5 g组最差,二者差异不显著(P >0.05),除贮存0和6 h外,与其他组间差异极显著(P< 0.01)。结果提示,稀释液中添加高浓度的L-赖氨酸对精液品质有抑制作用,当L-赖氨酸添加水平≥0.2 g,精子品质显著下降,添加0.1 g L-赖氨酸有助于提高杜泊羊鲜精液态保存的品质。     

Effect of L-lysine Added in Diluent on the Fresh Semen Quality of Dorper Sheep

In order to explore the effect of different L-lysine levels added in semen dilution on the fresh Dorper sheep quality stored at liquid state,3 health Dorper ram were used to collect semen which were equal-packaged and added to solution containing different levels (0,0.1,0.2,0.3,0.4 and 0.5 g) L-lysine in 120 mL diluent and at 0,6,24,30,48,54,72 and 78 h,the sperm motility,membrane integrity and acrosome integrity were detected.The results showed that the sperm motility in groups added 0,0.1 and 0.2 g L-lysine were higher than other groups,and that in 0.4 and 0.5 g L-lysine groups decreased faster than other groups;The sperm motility in 0.5 g L-lysine group reduced to 0 at 30 h,and it reduced to 0 at 48 h in the group added 0.4 g L-lysine;The effective survival time and survival index in the group added 0.1 g L-lysine was higher than other groups (P< 0.05);The membrane integrity of 0.1 g group was the highest,that had no significant difference with control group from 0 to 54 h (P >0.05),while after 72 h the two groups had significant differences (P< 0.05).The membrane integrity in 0.4 and 0.5 g groups were the worst,and that was significant different with other groups (P< 0.05);The acrosome integrity of 0.1 g group was the highest which had no significant difference with control group (P >0.05),0.4 and 0.5 g groups were the worst,the difference between them was not significant (P >0.05),while was extremely significant difference with other groups except for 0 and 6 h (P< 0.01).The results suggested that dilution added a high concentration of L-lysine could inhibit sperm quality,when the count of L-lysine was more than 0.2 g,sperm quality was significantly decreased,adding 0.1 g L-lysine could improve Dorper sheep fresh sperm quality stored at liquid state.     

猪精液4℃低温保存技术研究

为丰富猪鲜精保存技术、改善与提高精液保存质量,本研究以市场占有率高、性能稳定和可耐受4℃低温的长效稀释剂为对照,探讨本课题组研制的4℃稀释剂(低温Ⅰ号、低温Ⅱ号)对猪精液低温保存效果的影响。结果表明:低温Ⅰ号和低温Ⅱ号稀释剂对猪精液的有效保存时间和总存活时间均显著高于对照组(P<0.05)。保存至第6天时,对照组精子活力显著降低(P<0.05),保存至第10天时,低温Ⅰ号、低温Ⅱ号、对照组精子活力分别为74.6、76.0、0;保存第10天时,对照组pH显著低于2个实验组(P<0.05);保存从第5天开始,实验组质膜完整性显著高于对照组(P<0.05)。综上,本课题组研制的稀释剂对猪精液4℃低温保存效果较好,而且低温Ⅱ号对精子活力和质膜完整性保存效果更优。