Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm.     

不同卵黄成分、大豆卵磷脂及β-环糊精胆固醇对马精子冷冻效果的影响

本研究旨在比较添加不同浓度的卵黄成分（全卵黄（EY）、卵黄浆蛋白（EYP）、低密度脂蛋白（LDL）及水溶性卵黄蛋白（livetin））、大豆卵磷脂（SL）及β-环糊精胆固醇（CLC）对马精子冷冻效果的影响。选择4匹成年英纯血种公马进行采精,试验1：以INRA82+3.5%的混合抗冻剂作为基础冷冻液,添加0、2%、4%和8%浓度的EY、LDL、EYP或水溶性卵黄蛋白;试验2：以4%全卵黄组为对照,添加1%、2%和4% SL;试验3：在INRA82+3.5%的混合抗冻剂+4% EY基础液中添加0、0.75、1.5和3 mg/mL浓度的CLC。结果显示,EYP、LDL及水溶性卵黄蛋白3种卵黄组分均对马精子冷冻具有显著保护作用（P<0.05）,但不同卵黄组分之间及与EY组相比保护效果无显著差异（P>0.05）;添加低浓度（1%）的SL精子冻融后活力与对照组（4% EY组）相比无显著差异（P>0.05）,但添加高浓度SL组（2%和4%）冻融后精子TM、PM及RAP值均显著低于对照组（P<0.05）;添加不同浓度的CLC解冻后精子活力与未添加组无显著差异（P>0.05）。研究结果表明,卵黄中EYP、LDL和水溶性卵黄蛋白均对精子保护有效,保护效果与EY无显著差异;1% SL可获得与卵黄相似的保护效果;在含有卵黄的基础冷冻液中添加CLC未能提高精子冻后活力。     

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose-ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose-ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.     

绿原酸对猪精液冷冻保存效果的影响

为探究绿原酸对猪精液冷冻保存效果的影响,分别在TCG稀释液中添加不同浓度(15、30、50、80和100 pg/mL)的绿原酸,通过测定冷冻-解冻后精子的活率、顶体完整率、质膜完整率、DNA完整率、超氧化歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性来判定对保存效果的影响.结果表明,当添...     

黑猩猩精液冷冻和优选研究

对精液进行有效的冷冻保存和解冻优选是人工授精顺利开展的重要步骤。本研究在黑猩猩上做了尝试,分别用人工按摩采精、电刺激采精、附睾采精进行6次精液采集,所测精子活率为0.65～0.90,密度为1.69×10~（10）～6.64×10~（11）（个/L）。用自配精液冷冻液,采用CL-8800程序降温仪成功对所采集的精液进行冷冻保存。在冷冻后的10～400 d,分别对6次冻精解冻,并用人用精液优选试剂盒对其后3次进行解冻后优选。结果显示,解冻后精液活率为0.35～0.7;优选后精液活率为0.85～0.9,密度为4.5×10~（11）～5.0×10~（11）（个/L）,基本可以满足人工授精的需要。     

Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased (P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control (P < .05). A tendency (P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained <1% live acrosome-damaged spermatozoa. In the final experiment, the effects of hyaluronan supplementation post-thaw was investigated using hyaluronan concentrations of 100, 200, and 400 μg/mL. Motility parameters studied over an 8-hour period at 37°C yielded no consistent differences. In conclusion, addition of hyaluronan at a concentration of 200 μg/mL before freezing increased spermatozoal post-thaw motility. High concentration of hyaluronan (1,000 μg/mL) appeared to be detrimental to post-thaw motility. Effects of hyaluronan on fertility are beyond the scope of this study and have yet to be determined.     

高压均质鸡蛋卵黄对猪冷冻精子凋亡的影响

为探究高压均质鸡蛋卵黄对猪冷冻精子凋亡的影响,本试验采集5头健康状态良好的杜洛克公猪精液,以普通鸡蛋卵黄+Tris柠檬酸葡萄糖(TCG)冷冻基础稀释液为对照组,以添加高压均质处理的鸡蛋卵黄+Tris柠檬酸-葡萄糖(TCG)冷冻基础稀释液为试验组;精液冷冻-解冻后观察精子活力、DNA完整性、线粒体膜电位变化、细胞凋亡率、...     

哺乳动物精子冷冻工艺中防冻剂的研究进展

精子冷冻是一种简单、经济、高效的生物遗传资源保存方法,目前已经应用到许多领域,并成为治疗人类不育、牲畜繁殖和生物资源多样性保护的重要手段。在精子冷冻保存过程中的适当阶段加入防冻剂,能够有效的避免或减少精子的冷冻损伤和寒害,从而提高冷冻—解冻精子的活力。作者就防冻剂的种类、防冻剂的选择及加入与去除的方式对冷冻—解冻哺乳动物精子活力的影响作一简要综述。     

Measurement of Concentration and Viability in Stallion Sperm

Sperm concentration and sperm membrane intactness (SMI) or viability are two measures of sperm quality that provide important but different information about a stallion's reproductive capability. Sperm concentration is a measure that, by itself, informs little about the reproductive status of either the stallion or the ejaculate. Nevertheless, it is part of the product, along with semen volume, that determines total sperm number. The correct calculation of total sperm number directly affects the number of mares a stallion can breed and therefore, fertility. If either sperm concentration or semen volume is incorrectly measured, both the number of mares that a stallion can breed and the fertility of those breedings are affected. Although considerable between-stallion variation exists, sperm concentration, semen volume and total sperm number tend to be seasonal and vary with ejaculation frequency.     

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...     

Effect of Storage Time and Temperature of Equine Epididymis on the Viability,Motion Parameters,and Freezability of Epididymal Sperm

The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time.     

冷冻保存对公猪精子功能的影响

试验旨在探究公猪精液冷冻保存对其精子功能的影响。取长白猪的鲜精和优质冻精,用精子分析仪检测精子的运动能力,台盼蓝染色检测精子活率,体外受精（IVF）试验检测卵裂率与囊胚率,采用不同功能检测试剂盒检测冻精和鲜精的顶体完整率、线粒体膜通道孔（MPTP）活性、线粒体膜电位（MMP）、线粒体活性、线粒体氧化应激活性氧（ROS）以及精子DNA完整性,实时荧光定量PCR检测弱精子症相关蛋白基因SMCP、TEKT3、DNAH1、TCTE3的表达。结果表明,与猪鲜精相比,猪冻精的活率及活力均显著降低（P<0.05）,冻精的顶体完整率也明显下降（P<0.05）;冻精的卵裂率和囊胚率显著低于鲜精（P<0.05）;精子线粒体功能分析结果显示,冻精的MPTP相对荧光单位值（RFU）、线粒体膜电位荧光比率以及线粒体活性光密度（OD）值均显著低于鲜精（P<0.05）;精子线粒体ROS检测发现,冻精的RFU值显著高于鲜精（P<0.05）;精子DNA完整性检测结果显示,冻精拖尾率显著高于鲜精（P<0.05）;而弱精子症相关蛋白基因的表达与鲜精相比,差异不显著（P>0.05）。综上所述,冷冻导致猪精子活率、活力、线粒体功能、DNA完整性下降,最终使得冷冻精液精子的受精能力降低。     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.     

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）。     

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions.     

Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen

The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at +37°C, centrifuged (600 × g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 × 10⁶ spermatozoa/ml. The resulting mixture was cooled to 4°C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at +4°C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at −196°C and stored. Two straws per extender and per ejaculate were thawed in a water bath at +37°C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at +37°C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender.     

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%。     

冷冻保存对野牦牛精子酶活性的影响

研究冷冻保存（-196℃）对野牦牛精子顶体酶等酶活性的影响。应用Giemsa染色法、BAPNA底物法、改良明胶底膜法和磷酸苯二钠法分别测定不同保存年份的精子顶体完整率、顶体酶、透明质酸酶和酸性磷酸酶活性；采用酶动力学分光光度法，分别以丙酮酸钠和α-已酮酸钠为底物测定乳酸脱氢酶及其同工酶CA活性，并计算C4的相对活性。结果表明，在冷冻保存条件下，野牦牛精子有关酶活性下降；并随保存时间的延长，下降幅度增加；对保存5年以上的精液需抽样测定功能和品质后再用于生产。     

冻存液添加蜂王浆主蛋白对精子品质的影响

目的 精液冷冻保存作为人工授精不可或缺的一个技术环节,对优质畜禽种群的繁衍和保存有着至关重要的意义。目前,因精液冻存时冻存液成分、冷冻方法和外部氧化应激等因素影响,导致冻存精液品质不一。蜂王浆（RJ）已被证明能提高动物精液品质,蜂王浆主蛋白（MRJPs）作为RJ主要生物活性成分物质,具有多种生物活性和抗氧化能力。方法 为提高冻存精子品质,本研究开展了在公牛精液冻存液中添加不同浓度（0 g/25 mL、0.01 g/25 mL、0.02 g/25 mL、0.03 g/25 mL、0.04 g/25 mL）的MRJPs冻干粉,对冻存48 h后解冻精子的总活力、前进性活力、畸形率等相关参数进行了观察评估。结果 添加MRJPs呈浓度依赖性方式显著降低精子总活力、快速前进性活力、缓慢前进性活力和直行性活力（P<0.05）,而且精子畸形率和精子原地移动活力与对照组相比无显著差异（P> 0.05）。结论 精液冻存液中添加MRJPs会抑制冻存精子活力,因此,对MRJPs能以何种方式提高精液品质的研究还需要进一步开展。