稀释方法对种公牛细管冻精品质的影响

在牛的冷冻精液生产中,精液稀释有一次稀释和二次稀释两种方法.一次稀释方法是将配制好的稀释液按照一定的比例直接加入到精液中,而二次稀释方法则是先用一液按精液总稀释量的一半稀释后,放在4℃的低温柜中降温,2小时后用4℃的二液在等温下稀释精液到总稀释量.为了探索两种方法对荷斯坦种公牛细管冷冻精液品质的影响,笔者进行了一次稀释法和二次稀释法的对比试验.     

低温平衡技术在种牛冷冻精液生产中的应用研究

所谓“低温平衡技术”就是在种牛冷冻精液生产过程中,创造出适合精子在超低温冷冻过程中最大程度免受低温损伤的一种保护性技术措施.它包含两个技术关键：一是精液的稀释、低温平衡：即采集的精液经检查合格后,要立即进行稀释和平衡.稀释的目的,主要是供给精子能量来源和有效保护成分,防止低温打击对精子的有害影响;提供缓冲剂以防止精液中精子在代谢过程中产生的酸性物质导致pH值过低的有害影响,维持渗透压和电解质平衡和抑制细菌的生长繁殖;平衡的目的,主要是使精液的温度平缓、均匀地降至4℃,从而尽量减小温度变化对精子的打击,缩短精液通过危险温区（0℃～15℃）的时间,提高精子的冻后活力;二是提供适宜、均衡的低温作业环境——低温平衡柜,在种牛冷冻精液生产过程中,从一次稀释后的低温平衡、二次稀释,再平衡,到分装、上架、冷冻、收集、包装等一系列操作环节,都要求在0℃～4℃的低温平衡柜中进行,使精子在冷冻生产整个过程中免受温度变化带来的损害.     

不同稀释方法对种公牛冷冻精液品质的影响

研究选用18头荷斯坦种公牛,将采集合格的精液分为三份,用一步稀释法、两步快速稀释法和两步缓慢稀释法三种不同的稀释方法进行稀释,将稀释好的精液进行细管分装、平衡、冷冻,分别比较稀释前、稀释后和冷冻后精子的活率、畸形率和顶体完整率.结果表明,不同稀释方法对精子的稀释冲击不同.三种稀释方法中两步缓慢稀释法最好,一步稀释法对精子的顶体损伤最大.     

稀释方法对绒山羊冷冻精液品质的影响

选取7只精液品质优良的雁门绒山羊种公羊,通过测定其冷冻精液解冻后的精子活率（0、4 h）、精子存活指数、顶体完整率及精清中透明质酸酶（HYD）、谷草转氨酶（GOT）、乳酸脱氢酶（LDH）酶活力,研究一步稀释法和二步稀释法对冷冻精液品质的影响。结果表明,二步稀释法解冻后的精子存活指数显著（P＜0.05）高于一步稀释法,其解冻后精清中透明质酸酶、谷草转氨酶、乳酸脱氢酶的活力均显著（P＜0.05）低于一步稀释法。表明采用二步稀释法冷冻的绒山羊精液品质好于一步稀释法。     

平衡温度对种公牛冷冻精液制作效果的影响

本试验将三头种公牛的精液稀释、平衡,封装后各分为5组,经不同的平衡温度后进行冷冻,检查牛冷冻精液解冻后的精子活力、顶体完整率、畸形率、37℃4h存活率。结果表明,牛冷冻精液在制作过程中,最佳平衡温度为4℃,在此条件下,冻精质量可以达到国家标准。     

水牛精液细管冷冻试验

对水牛细管冷冻精液的稀释液、平衡时间、始冻温度、冷冻时间、降温速度、解冻温度和时间以及输精等进行了试验。结果表明,水牛细管冷冻精液解冻后精子活率为0.4～0.6,输精196头的情期受胎牛为63.3％。     

不同稀释方法对小型宠物犬精液冷冻效果影响

为了研究Tris-果糖稀释液在不同稀释方法下对小型宠物犬精液冷冻的效果,选取常见的3个小型宠物犬品种共计6只犬进行试验,精液稀释过程分别采用一步稀释法和两步稀释法。结果表明:冷冻精液解冻后各组犬精液的同一剂型间的精子活力差异均不显著(P0.05);一步稀释法中的颗粒冻精与0.25 mL细管冻精和0.50 mL细管冻精相比,解冻后精子活力差异均显著(P0.05),而0.25 mL细管冻精与0.50 mL细管冻精的解冻后精子活力差异极显著(P0.01);采用两步稀释法的颗粒冻精、0.25mL细管冻精及0.50 mL细管冻精的解冻后精子活力组间差异均不显著(P0.05)。用Tris-果糖稀释液稀释精液时,使用两步稀释法对不同冻精剂型的精子活力影响不明显。     

冷冻精液的温度控制

冷冻精液温度控制是保证冷冻精液质量和提高母畜受胎率的技术关键。在冷冻精液生产过程中,对精液稀释、平衡温度有着严格的要求;在精液冷冻过程中必须快速通过危险温区和按照规定的精液冷冻曲线操作;在使用冷冻精液时,对冷冻精液的保存温度和解冻温度及解冻后精液温度均有特定的要求。     

细管冷冻精液生产工艺流程

细管冷冻精液的生产包括:采精、精子活率密度评定、经2次稀释、降温、、平衡、精液细管分装、冷冻、精液的予贮及入库等工艺流程如图1所示.     

稀释方法、孵育温度和时间对冷冻—解冻后猪精液质量的影响

试验以精子冻后活率、质膜完整率和顶体完整率三项指标评价了一步稀释法(Ⅰ组)和两步稀释法(Ⅱ组),以及不同保存温度和时间对解冻、稀释后的猪精液质量的影响。结果表明,采用两步稀释法对解冻后的猪细管冻精进行稀释,精子活率、质膜完整率和顶体完整率分别达到82.6%、89.4%和90.3%,极显著高于一步稀释法(P0.01)。用两步法稀释后的精液36℃保存5 min、10 min和15 min时精子活率分别为81.2%、80.8%和80.9%(P0.05),40 min时为50.1%;精子质膜完整率和顶体完整率未见显著下降(P0.05);17℃保存15 h时精子活率、质膜完整率和顶体完整率分别为81.1%、88.9%和91.4%(P0.05);60 h时分别为50.4%、61.4%和61.6%,均极显著低于初始的各项指标(P0.01)。     

Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO, and concentration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozen-thawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifugation, and after cooling down to 4°C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4°C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO.     

Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.     

Effects of liquid nitrogen vapor sensitization conditions on the quality of frozen-thawed dog spermatozoa

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.     

Cryo-scanning Electron Microscopy Discloses Differences in Dehydration of Frozen Boar Semen Stored in Large Containers

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50°C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from −5°C to −100°C, namely 2°C/min, 50°C/min and 1200°C/min, the lattermost by plunging the samples into liquid nitrogen (LN₂). The samples were thereafter fractured into LN₂ and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2°C/min and 50°C/min) but not at 1200°C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.     

Dietary Polyunsaturated Fatty Acid Supplementation Improves the Quality of Stallion Cryopreserved Semen

The objective was to assess the influence of polyunsaturated fatty acid supplementation on the quality of fresh, cooled, and frozen-thawed stallion semen. Ten stallions received their normal diet (control group) or normal diet plus 150 mL of polyunsaturated fatty acid (PUFA) linseed-based oil (PUFA group). Semen was collected every 15 days during 60 days. Stallions were reversed across the treatments after a sixty-day interval. Semen was evaluated at 2, 6, 12, and 24 hours after cooling and 24 hours after freezing. Motility (MOT), vigor, membrane viability, morphology, acrosome integrity, and osmotic tolerance test (OTT) were evaluated. In the frozen-thawed semen, sperm dynamic characteristics were analyzed by computer-assisted sperm analysis and thiobarbituric acid reactive substances (TBARs) determined. The effects of treatment, time, semen type, and their interactions were submitted to PROCMIX (SAS) and means compared by the Tukey test. There was no treatment effect on the quality of fresh and cooled semen. However, frozen-thawed semen MOT, vigor, and OTT were superior (P < .05) in control compared to PUFA group. An interactive effect of sample day by treatment was observed, such that, TBARs increased over time (P = .002) in the PUFA group after 15, 30, 45, and 60 days from the beginning of supplementation. Thus, sperm may become more susceptible to the reactive oxygen species, probably due to the incorporation of polyunsaturated fat in the cell membrane. The addition of PUFA-enriched oil may be an alternative for improving frozen-thawed semen quality by increasing its MOT and resistance to osmotic changes to which sperm cells are submitted during the freezing process.     

乌苏里貉精液冷冻技术研究

为探索乌苏里貉精液冷冻技术,通过试验,筛选出了适合于乌苏里貉精液的鲜精稀释液和冷冻保护液配方,摸索出了乌苏里貉精液的冷冻程序,包括降温、平衡和预冷冻等各技术环节以及冷冻精液的最适解冻温度,解冻后在生物显微镜下观察,精子活力均在4~5级。     

投入液氮前的平衡温度及时间对兔胚冻后存活的影响

本研究旨在了解投入液氮前的平衡温度及时间对兔胚冻后存活的影响。经0.5M蔗糖与1.5,2.0或2.5M乙二醇组成的冷冻液处理后的兔胚,在-30℃或-37℃平衡10或20分钟后即投入液氮保存或直接投入液氮保存。胚胎解冻在37℃的水浴进行。当平衡温度由-30℃下降到-37℃时,胚胎的冻后形态正常率及存活率均极显著下降(P<0.01)。在-30℃平衡20分钟与10分钟的冻后存活率无明显差异(P>0.05),但直接投入液氮时,冻后存活率则很低(8.3％)。由此表明:兔胚冻前在-30℃平衡10—20分钟是必要的。     

冷冻温度对牛精子复苏率及降温速率变化规律的研究

[目的]为了探讨温度变化对牛精子复苏率的影响,找到降温度变化规律。[方法]利用电脑程控冷冻仪、自动数字测温仪,测定精液在不同温度冷冻精子复苏率变化。[结果]表明,精液在-98℃冷冻精子复苏率最高;在-86- -118℃冷冻精子复苏率较高,精液相变适宜降温速率为35℃/min。[结论]控制精液发生相变阶段的降温速率是冻精制作成败的关键。