鸵鸟卵黄低密度脂蛋白对猪精子冻后品质的影响

在家畜精液冷冻中,卵黄被广泛应用,且其中的低密度脂蛋白(LDL)对精子起主要保护作用。本研究利用含6%、7%、8%和9%鸵鸟卵黄LDL配制的稀释液制作猪细管冷冻精液,分析鸵鸟卵黄LDL对冷冻-解冻后猪精子质量参数的影响。结果表明:在含不同浓度鸵鸟卵黄LDL的稀释液中,8%LDL的稀释液冷冻效果最好,冻后精子活率平均可达52.13%,显著高于其他组(P<0.05);精子顶体完整率平均为58.33%,质膜完整率为72.38%,与其他处理组相比差异显著(P<0.05)。但与鸡蛋卵黄LDL和鸽子蛋卵黄LDL处理组相比,鸵鸟卵黄LDL处理组冷冻-解冻后猪精子质量参数相对较低。本研究表明,虽然鸵鸟卵黄LDL在冷冻过程中对猪精子具有一定的保护作用,但相对于鸽子蛋和鸡蛋卵黄LDL效果并不理想。     

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。     

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。     

解冻稀释液及孵育温度对冷冻-解冻后犬精液品质的影响

实验旨在比较解冻后解冻稀释液(Tris-柠檬酸-葡萄糖稀释液)的添加比例(1:0、1:1、1:2)对精液品质的影响,并比较了4种孵育温度(37、34、25、4℃)对精子寿命及精子活力的影响,探索合适的冷冻-解冻后犬精液的孵育条件。结果显示:解冻稀释液的添加与否及添加比例对解冻后孵育30 min的精子活力、活率、质膜完整率及顶体完整率均无显著影响;解冻后,在不同温度下孵育30 min,4组精子活率和质膜完整率无显著差异,而4℃孵育温度下精子活力和顶体完整率高于37℃(P0.05);解冻后孵育2 h,4℃孵育温度下的精液品质(精子活率、精子活力、质膜完整率、顶体完整率)最佳(P0.05),34℃和25℃组之间差异不显著,而37℃组的精液品质最差。结果表明,解冻稀释液对解冻后精液的孵育效果无影响,而解冻后精液的孵育温度以4℃最佳。     

不同冷冻保护液和解冻温度对猪精液冷冻效果的影响

在BF5稀释液中分别添加不同水平的牛血清白蛋白(BSA)(0.5,1,1.5 g/L)、二甲基乙酰胺(DMA)(0.5%,1%,1.5%)、甲基-β-环糊精载胆固醇(CLC)(1,2.5,5 g/L),对成年健康猪精液进行冷冻保存,于不同温度(37,50,70℃)解冻后分别检测冷冻后精子的活率、活力、质膜完整性、项体完整率、线粒体活性.结果显示,0.5 g/L BSA组冷冻后精子活力、质膜完整率、顶体完整率和线粒体活性均高于其他组,但差异不显著(P0.05).1% DMA组冷冻后精子活率和活力显著优于1.5%DMA组(P＜0.05),同时也高于对照组(3%甘油)冻后精子活率和活力,但差异不显著.不同质量浓度CLC组冷冻后精子的活率、活力、质膜完整性和线粒体活性与对照组相比差异不显著.50℃和70℃解冻组精子的活率和线粒体活性显著高于37℃解冻组的精子;50℃解冻组精子的活力和质膜完整率显著高于其他2组.因此,在猪精液冷冻稀释液中,用DMA可以代替甘油作为渗透性保护剂,且以1%DMA,50℃解冻最佳.     

海藻糖对猪精液冷冻保存效果的影响

在传统的Tris-柠檬酸-葡萄糖稀释液基础上，分别添加25％、50％、75％、100％的海藻糖，研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明，海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果，其最佳添加浓度为25％，冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高（P〈0．05），分别达到41．38％、46．34％、44．56％、43．51％和64．09％。海藻糖可以明显抑制精子获能，获能处理前精子获能率仅为3．68％，而获能处理后达到41．82％，有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2％，海藻糖只有与甘油共同作用，才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系（P〈0．01），而与获能处理前精子的获能率存在显著的负相关关系（P〈0．05）。     

不同家禽卵黄对德国牧羊犬精液冷冻的影响

为了探索不同家禽卵黄在德国牧羊犬精液冷冻中的应用效果,分别在精液冷冻稀释液中添加不同家禽(鸽、鸡、鸭、鹅和鹌鹑)和不同浓度(10%、15%、20%、25%和30%)的卵黄,并分别对冷冻前和解冻后的精子活率、顶体完整率和畸形率进行比较分析。结果:在德国牧羊犬精液冷冻稀释液中加入相同浓度的5种不同家禽卵黄,家鸽组冻后活率、顶体完整率和畸形率分别为(52.0±2.1)%、(54.3±3.7)%和(27.7±1.8)%,对德国牧羊犬精液冷冻有最佳的保护效果,其最佳浓度为20%。因此,鸽卵黄可替代鸡卵黄,作为冷冻稀释液的组成成分,用于德国牧羊犬的精液冷冻。     

大豆卵磷脂与卵黄在山羊精液冷冻过程中的联合作用研究

试验旨在探究在山羊精液冷冻保存过程中单独使用大豆卵磷脂(SL)、卵黄(EY)及其联合使用的效果。以20%EY(V/V)和2%SL(m/V)为对照,在山羊冻精基础稀释液中分别添加10%EY+2%SL、20%EY+1%SL、20%EY+2%SL,冷冻解冻后分别对精子活率、精子活力、质膜完整率、顶体完整率和线粒体活性进行检测,同时利用试剂盒检测活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量变化。结果表明:当冷冻稀释液中联合添加10%EY+2%SL,解冻后该组的精子活力、精子活率、质膜完整率、顶体完整率和线粒体活性与20%EY和2%SL对照组无显著性差异,较20%EY+1%SL和20%EY+2%SL联合添加组显著提高;与20%EY和2%SL组相比,联合添加10%EY+2%SL组精子解冻后ROS和MDA含量和SOD活性无显著性差异,但精子抗氧化能力较另外2个联合添加组显著提高。综上,山羊精液冷冻基础稀释液中添加SL可替代或者部分替代EY,EY和SL的联合添加对山羊冻精无协同作用,20%EY联合添加不同浓度的SL则会加大对冷冻精子的氧化损伤。     

褪黑素对绒山羊精液冷冻保存效果的影响

为了研究褪黑素对绒山羊精液冷冻保存效果的影响,在冷冻稀释液中外源补充不同浓度(0.125,0.250,0.500,1.000 mg/mL)的褪黑素进行冷冻试验,冷冻解冻采用计算机辅助精液分析系统(CASAS)和流式细胞仪分别检测精子活率、质膜完整率、顶体完整率以及精子细胞中活性氧(ROS)的含量。结果表明:在冷冻稀释液中添加褪黑素能够显著降低精子细胞中ROS的含量,而且在冷冻稀释液中添加浓度为0.500 mg/mL的试验组绒山羊精子活率(53.3%)、质膜完整率(44.6%)、顶体完整率(77.9%)均显著高于空白对照组(47.8%、36.3%和53.2%)(P0.05)。说明在绒山羊精液冷冻稀释液中添加0.500 mg/mL褪黑素能够显著提高绒山羊精子抗氧化能力和冷冻保存效果。     

香菇多糖对牛精液冷冻保存效果的研究

为了研究香菇多糖对牛精液冷冻保存效果的影响,在牛精液冷冻稀释液中添加不同质量浓度(1、5、25、125、625mg/L)的香菇多糖,冻融后检测牛精子活率、动力学参数以及顶体完整率、质膜完整率等。结果表明,25 mg/L组的冷冻保存效果最好,精子活率、质膜完整率、顶体完整率分别达到42.04%、43.57%和63.87%,显著高于对照组(P0.05)。而过高浓度的香菇多糖(125、625mg/L)则显著降低了冻融精子的质量。牛精液冷冻稀释液中添加香菇多糖具有显著的保护作用,其最佳添加浓度为25mg/L。     

The Cryoprotective Effect of Trehalose Supplementation on Boar Spermatozoa Quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 m m ), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 m m trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 m m , trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.     

Effects of Freezing–Thawing on DNA Integrity of Boar Spermatozoa Assessed by the Neutral Comet Assay

A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.     

Fertilizing capacity of boar semen frozen in an extender supplemented with ostrich egg yolk lipoprotein fractions--a pilot study

A limited field trial was performed to evaluate the fertilizing capacity of boar spermatozoa frozen in an extender supplemented with lipoprotein fractions isolated from ostrich egg yolk (LPFo). Boar semen, diluted in an extender containing lactose with lyophilized lipoprotein fractions, glycerol and Orvus Es Paste (lactose-LPFo-G), was frozen using a controlled programmable freezer. Sperm characteristics, such as motility, plasma membrane and acrosome integrity, and mitochondrial function were monitored. Post-cervical artificial inseminations (post-CAIs) in multiparous sows (Polish Large White) were performed using the Soft & Quick catheter/cannula set. Sows were inseminated 2 to 3 times within one oestrus. Possible returns of sows to oestrus were determined from 21 to 30 days after post-CAIs. In this field trial, sows inseminated with 2 x 10(9) motile frozen-thawed spermatozoa resulted in pregnancy and farrowing rates of 75%, respectively. The average piglets born live was 10.5 +/- 0.4 (mean +/- SEM). The data of this study showed that post-CAI of boar semen frozen in LPFo-containing extender has the potential to provide acceptable fertility results. Further investigations are needed to elucidate the cause of variations in pregnancy/farrowing rate associated with frozen-thawed boar semen.     

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.     

Breed of Boar Influences the Optimal Concentration of Gamma‐oryzanol Needed for Semen Cryopreservation

The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma‐oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A–E) according to the concentration of gamma‐oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mm , respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank (?196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma‐oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen–thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma‐oryzanol, for Duroc boar, gamma‐oryzanol at 0.16 mm (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma‐oryzanol at 0.24 mm (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma‐oryzanol needed for boar semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mm for Duroc and 0.24 mm  for Large white and Landrace.     

Comparative quality assessment of buffalo (Bubalus bubalis) semen chilled (5°C) in egg yolk- and soya milk-based extenders

Egg yolk-Tris is most commonly used semen extender; however, its use involves hygienic risk, interference with fertility and poor microscopic examination. Therefore, replacement of egg yolk with a plant-based component with protective effects on spermatozoa would be advantageous. In present study, we observed effect of soya milk-based extenders on dilution and liquid preservation of Murrah buffalo bull semen at 5°C up to 72 h in comparison with conventional egg yolk-Tris extender (Ext.1). In experiment one, a total of 32 buffalo semen ejaculates from four animals were extended and preserved at 5°C for 72 h in soya milk-based extender (Ext.2) with different percentages (10%, 15%, 20%, 25% and 30%) of soya milk for optimization of soya milk concentration. Semen quality was assessed for individual motility, viability, membrane integrity and acrosome integrity at 0, 24, 48 and 72 h of liquid preservation. The results of experiment one indicated that 25% soya milk is an optimum concentration for buffalo bull semen extender preparation. A modified method was used to prepare another soya milk-based extender (Ext.3). In the second experiment, two soya extenders (Ext.2 and 3) with optimized concentration (25%) of soya milk were comparatively assessed with egg yolk-Tris extender (Ext.1) for semen quality parameters at 0, 24, 48 and 72 h of liquid preservation. The individual sperm motility at 0 and 24 h following dilution were found non-significant among extenders. However, after 48 h of dilution, individual motility in Ext.3 was observed significantly (p < 0.05) higher than Ext.1. After 24, 48 and 72 h of dilution sperm membrane integrity in Ext.3 was found significantly (p < 0.05) higher than Ext.1. Overall, comparative evaluation of sperm parameters obtained revealed that Ext.3 containing 25% soya milk can be used as a substitute of egg yolk-based extender for buffalo semen liquid preservation.