马精液冷冻保存技术研究进展

马精液冷冻保存是一种将种公马精液加入抗冻保护剂,再进行一系列处理,最后保存在超低温中(液氮、干冰)的技术。近年来该技术随着马冷冻精液在许多国家马匹繁殖育种中逐渐应用而得到了较好的发展。作者综述了马精液冷冻保存技术的精液稀释、离心、再稀释、降温平衡、分装剂型、降温冷冻、液氮保存的研究进展,旨在对马精液冷冻保存技术的进一步研究提供一定的参考,以期推动中国现代马业的健康可持续发展。     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

Insemination with Stallion Semen Frozen in 0,5 ml Straws

Contents: An insemination trial using frozen semen is described. The freezing procedure was slightly modified from the Hannover method. The insemination dose consisted of 7 medium straws containing approx 1 10⁹ spermatozoa. A total of 28 mares of the Norwegian Trotter breed were inseminated during the 1991 season. During oestrus the mares were examined at 12 hour intervals, and the insemination was carried out after detection of ovulation. The pregnancy rate was 43% after the first insemination, increased to 68% after second and further to 75% after the third and last insemination. The foaling rate was 61.5%.     

马、驴精液保存研究进展

马、驴精液的保存与应用技术发展速度相对较慢,文幸从马、驴精液的采集,处理,稀释,冷冻,解冻,授精等几个方面就目前国内外发展情况进行了概述.     

Methods of Concentrating Stallion Semen

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

奶牛性控冻精和常规冻精活力对比试验分析

试验观测对比奶牛性控冻精和常规冻精解冻后精子存活时间,为进一步把握输精时间,提高受胎率提供依据。试验结果表明:性控冻精刚解冻时活力较高,为0.5级以上,但活力下降快、存活时间较短,解冻后6h活力降低为零;常规冻精刚解冻时活力在0.4～0.5级之间,但存活时间较长,活力下降速度慢,解冻后8h活力降低为零。所以性控冻精解冻后,要求输精技术员尽快输精。     

Use of nutraceuticals in the stallion: Effects on semen quality and preservation

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.     

温度对奶牛冻精解冻后活力的影响

本研究对奶牛冻精解冻后在4℃,15℃,25℃温度下分别保存2 h,4 h,6 h,8 h,10 h的活力进行了测定。结果表明,4℃和15℃保存到10 h精子活力0.381±0.0170和0.379±0.0197,与初解冻接近(P0.05);25℃保存随时间延长精子活力呈下降趋势,和初解冻相比,6 h为0.349±0.0223(P0.05),8 h和10 h分别为0.302±0.0215和0.263±0.0287(P0.01)。牛冻精解冻后在4℃和15℃保存10 h以内,精子活力仍然维持在一个较高水平,符合输精要求,在此温度和时间范围内进行长距离携带,对精液质量影响小,可以达到预期的输精效果。环境温度在25℃左右时,冻精解冻后宜于4 h以内进行输精。     

奶牛性控精液与常规精液抗氧化物酶活性比较分析

通过比较常规精液和性控精液在活力、顶体完整率及精浆中四种抗氧化酶类活力的差异,对性控精液的品质进行综合评定。对用流式细胞仪分离后的性控精液和常规精液细管解冻(37.5℃,30s),分别分析精子活力和精子顶体完整率,并测定精浆中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH)、谷胱甘肽还原酶(GR)和过氧化氢酶(CAT)的活性。结果表明性控精液精子在活力显著低于常规精液,且精浆中四种抗氧化酶类的活力也都显著低于常规精液。     

马冷冻精液氧自由基的产生和清除系统

马精液中存在相对稳定的氧自由基(ROS)产生和清除系统,当精液经离心去除精清后,稳定的系统遭到破坏。马精液冷冻和解冻过程不能有效的清除ROS,将导致解冻后精子各项生理功能的下降,直接影响冻精的质量,而添加外源性的抗氧化剂则可以重新使精液的ROS产生和清除达到平衡。作者综述了马精液中精子细胞、白细胞ROS的产生系统和精液GSH-PX、CAT、SOD、VA、VE等对ROS的清除系统,旨在对人们研究马精液冷冻保存时精子中ROS的损伤机理研究提供一定的帮助。     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Heiskanen, M.-L., M. Huhtinen, A. Pirhonen and P. H. Mäenpää: Insemination results with slow-cooled stallion semen stored for approximately 40 hours. Acta vet. scand. 1994,35,257-262.– Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender

The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.     

牛冷冻精液的应用研究进展

牛人工授精技术是所有家畜中应用最为广泛的一种繁殖技术,而其巨大的发展又得益于精液冷冻保存的成功应用.本文简要介绍了牛精液冷冻技术的发展历史和精液冷冻的原理,分析了影响牛精液冷冻效果的主要因素,并对国内外现行使用的精液评定指标作了简单阐述,以期为牛高质量冷冻精液的研究和牛人工授精技术的应用提供参考.     

牛磺酸对种公猪精液品质、血清激素含量及精浆抗氧化能力的影响

本试验旨在研究牛磺酸(Tau)对种公猪性欲、精液品质、血清激素含量及精浆抗氧化能力的影响。选用24头年龄、体重相近的健康成年大约克夏种公猪,随机分为4个组,每组6个重复,每个重复1头猪。各组分别饲喂牛磺酸添加水平为0(对照组)、2、4、6 g/kg的饲粮,试验期90 d,分为1~45 d和46~90 d 2个阶段。结果表明:1)试验46~90 d时,添加6 g/kg牛磺酸显著提高了种公猪的性欲(P0.05)、采精量(P0.01)、精子活力(P0.05),4 g/kg牛磺酸改善了精子密度和精子畸形率(P0.05);2)与对照组相比,6 g/kg牛磺酸水平组极显著提高了种公猪血清促黄体素(LH)含量(P0.01),显著提高了睾酮(T)含量(P0.05);3)试验46~90 d时,6 g/kg牛磺酸显著降低了种公猪精浆丙二醛(M DA)含量(P0.05),显著提高了超氧化物歧化酶(SOD)活力(P0.05),4 g/kg牛磺酸显著提高了谷胱甘肽过氧化物酶(GSH-Px)活力(P0.05)。由此可见,饲粮中长期添加牛磺酸可以调控种公猪血清激素含量,增加精浆抗氧化能力,进而增强种公猪性欲,提高种公猪精液品质,本试验条件下牛磺酸适宜添加水平为6 g/kg。     

降温平衡对牛冷冻精液质量的影响

降温、平衡是冷冻精液生产过程中的重要环节。试验以本单位种公牛生产的精液为样本,使用低温平衡柜、测温仪、显微镜等仪器设备,研究降温、平衡对冷冻精液质量的影响。结果表明,采取一次稀释法,每盒中盛装细管精液适宜数量为100～300支;采取二次稀释法,每个烧杯中精液稀释后适宜容量为50～120mL.采取先灌装后降温、平衡,或先降温、平衡后灌装两种不同方法,对冷冻精液质量的影响差异不显著（P〉0．05）。在平衡柜温度为40℃环境下,精液从稀释后温度降至平衡温度的适宜时间为1～1．5h,平衡时间为3～4h。     

鸡精液保存技术

家禽人工授精技术在我国应用广泛,但家禽的精液冷冻保存技术虽然一直有研究机构在开发新的技术,但在实际应用中较少,主要是冷冻技术还不够成熟,解冻后的受精率不高。该文结合国内外的人工授精技术研究的情况,介绍人工授精技术及影响人工授精技术的因素,为鸡的人工授精技术研究提供新的研究思路。     

应用优选法提高牛冷冻精液活率的试验研究

在一定的初冻温度范围内，用自制冷冻箱冷冻精液。经正交试验法筛选出影响精液食冻后活率的最桂剂量、冷冻高度和初冻温度水平搭配为250支、4cm、－100℃。采用单因素对分法试验对冷冻剂量进一步评选，取得了满意的效果．该成果经生产上推广应用效果较好。