公兔精液冷冻保存技术

精液的冷冻保存是一项重大的技术革新,精液通过冷冻保存,可供长期使用,进一步发挥了优秀种公兔的作用.精液冷冻保存的程序,大致分采精、精液品质检查、稀释、平衡、冷冻、解冻镜检、保子(-196℃液氮).     

牛冷冻精液的保存及使用

精液冷冻保存是将精液进行特殊处理，保存在超低温条件下以达到长期保存的目的。通常采用液氮（－１９６℃）和干冰（－７９℃）保存。其最大优点是可长期保存，使用不受时间、地域以及提高种用公牛的利用率。１稀释液配方１２％的蔗糖液７５mL、甘油５mL、卵黄液２０mL。２稀释方法将经过检查合格的精液（活力０．６以上、精子密度中等）按１∶２～５倍稀释液进行稀释，稀释后应保证每个颗粒精液中所含精子数不少于３０００～４０００万个，解冻后呈直线前进的精子数３冷冻在装有液氮的容器上置一铜纱网，距液氮面２cm左右，温度维持在－…     

猪精液超低温冷冻保存研究进展

猪人工授精(AI)技术已经广泛应用于畜牧业生产,但精液来源基本是17℃保存的新鲜猪精液,不利于精液长期保存和猪种资源保存。超低温冷冻保存技术是猪精液长期保存最有效的方法,冷冻精液可以实现不同地区、不同国家优良种猪精液间的交流,从而大大提高猪种资源的利用率,但冷冻解冻后的精液质量较差,受胎率、窝产仔数远低于鲜精,极大地限制了猪精液冷冻保存技术的广泛应用。本文综述了猪精液冷冻保存研究概况、猪精液冷冻损伤机理和猪精液冷冻保存影响因素,以期为猪精液超低温冷冻保存研究提供新的思路和方法。     

家禽精液保存技术研究进展

家禽的精液保存是人工授精技术中的关键环节,该技术水平的高低将直接影响生产过程中的经济效益。文章主要围绕短期保存过程中精液稀释液、稀释比例、保存温度及长期冷冻保存过程中冷冻保护剂、冷冻方法、平衡时间和解冻温度的研究等方面进行阐述。     

冷冻距离和冷冻保护剂种类对超低温保存新西兰兔精液品质的影响

为了探讨新西兰兔精液超低温冷冻保存的最佳方法,试验采集新西兰成年公兔精液,采用0.25 mL细管冷冻法进行超低温冷冻,以解冻后精液活率、质膜完整性、顶体完整率为指标,研究距离液氮面不同高度和冷冻保护剂种类对冷冻保存兔精液品质的影响。结果表明:冷冻精液距离液氮面3 cm,其精液活率、质膜完整性和顶体完整率明显优于1 cm和5 cm;在冷冻稀释液中添加甘油相对于其他冷冻保护剂对精液品质的保护效果好,甘油浓度为3%时相对其他浓度效果较好。说明了距液氮面3 cm冷冻,冷冻稀释液中添加3%甘油对兔精液的保存效果较好。     

猪精液冷冻技术

<正>精液冷冻保存技术(Semen freezing)是将精液特殊处理后,通过液氮(-196℃)、干冰(-79℃)或其它冷源冷冻,超低温保存,使精液达到长期保存。精液冷冻保存技术的主要原理是精子在冷冻状态下,代谢几乎停止,生命保持相对静止状态,升温后又能复苏而不失去受精能力。     

公猪精液液态低温保存试验

<正> 在实施猪的人工授精工作中,首先要解决的是精液的保存问题,近年来公猪精液冷冻保存的研究虽已取得成功,但当前猪的冷冻精液在农村中广泛应用仍有一定困难,诸如液氮罐设备、液氮来源以及精液的冷冻解冻技术要求较高、受胎率不稳定等问题一时不易解决,公猪精液的低温保存还是目前采用的主要方法,因此,研究改进公猪精液液态低温保存的方法,仍有重要的现实意义,本试验的目的,在于寻找一种经济实用的公猪精液液态保存方法,为实施猪人工授精生产服务。     

驴精液冷冻保存技术研究进展

精液冷冻技术在驴种质资源的长期保存和引进以及人工辅助繁殖技术应用等方面具有重要意义,精液冷冻保存有利于充分发挥优秀种公驴的繁殖力,降低驴养殖产业的生产成本。近年来,驴精液冷冻保护剂和冷冻方法不断优化,驴精液冷冻保存技术也取得了很大的进步。但由于驴冷冻复苏的精子活力和受胎率低,严重限制了驴冷冻精液的推广与应用。本文对驴精液冷冻保存技术的发展历程、精子冷冻损伤机制、影响驴精液冷冻保存的因素和驴冷冻精液的应用进行综述,为今后驴精液冷冻保存技术的研究提供参考。     

2种稀释液对公猪精液冷冻保存效果的影响

试验为探究2种不同冷冻稀释液对公猪精液稀释冷冻效果的影响。试验采用手握法采集同一头公猪精液,将采集的精液分为2组,进行液氮熏蒸制作细管冻精,分别使用Ⅰ号冷冻稀释液和Ⅱ号冷冻稀释液对猪精液进行冷冻保存,24 h后检测2种冷冻稀释液细管冻精的活力。结果显示,Ⅰ号冷冻稀释液冷冻解冻后的精子活力显著高于Ⅱ号冷冻稀释液(P0.05),同一种稀释液距液氮面5 cm进行熏蒸冷冻的效果较距液氮面4 cm冷冻效果好,但差异不显著(P0.05)。研究表明,利用Ⅰ号冷冻稀释液在距液氮面5 cm处进行液氮熏蒸冷冻可获得较好的公猪冷冻精液。     

绵羊精液冷冻保存技术研究综述（上）：稀释液

本文综述迄止１９９３年绵羊精液冷冻保存技术的体外和输精试验研究结果，包括精液冻前处理，降温和解冻。稀释液最早用于牛精液冷冻，但效果不太好，后来不断加以改进。已研究的稀释液有柠檬酸盐一糖类，奶类，蔗糖类，棉子糖类，三基类和其他含保护剂（甘油和卵黄）的，抗氧化剂的溶液，所试用的许多种稀释在不同情况下保存精液，均获得了较高的存活率。冻精子宫颈输精的产羔率与冷冻技术和输精操作有关，但都低于稀释的新鲜精液。     

Artificial insemination and preservation of semen.

Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable pregnancy rates are consistently obtained with cooled semen. Conversely, techniques for cryopreservation of stallion semen will require more refinement before the procedure can be considered commercially viable on a wide scale.     

Effect of seminal plasma concentration and various extenders on postthaw motility and glass wool-Sephadex filtration of cryopreserved stallion semen

OBJECTIVE: To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. SAMPLE POPULATION: 7 stallions from which we collected > or = 3 ejaculates/stallion. PROCEDURES: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or dilution rate could improve ability of such semen to survive freeze-thaw procedures. RESULTS: Use of Kenney extender as the centrifugation extender significantly improved postthaw motility and GWS filtration, compared with glucose-EDTA. Extending semen at a dilution of 1:3 was significantly better than 1:1 for both motility and GWS filtration. In addition, including seminal plasma at a concentration of 5% in the cryopreserved semen resulted in significantly higher yield of spermatozoa after GWS filtration, compared with complete removal of SP or use of seminal plasma at 25%. Lastly, semen with poor postthaw quality had significantly improved postthaw quality in regard to motility and GWS filtration when semen was frozen with seminal plasma at a concentration of 5%, compared with semen frozen with seminal plasma at a concentration of 25%. CONCLUSIONS AND CLINICAL RELEVANCE: Use of Kenney extender at a high dilution (> or = 1:3) immediately after collection of semen can improve postthaw quality of frozen stallion semen.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

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。     

Preliminary Trial: Motility Comparisons of a Unique Freezing Technology (UFT) to Liquid Nitrogen Mist Methodology for Cryopreservation of Porcine Spermatozoa

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.     

Directional Freezing of Equine Semen in Large Volumes

Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.     

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.     

冷冻方法对杜泊绵羊冷冻精液品质的影响

试验对绵羊精液分别采用液氮熏蒸冷冻法和冷冻仪冷冻法进行冷冻,通过测定精液解冻后精子活率、顶体完整率、质膜完整率和谷草转氨酶活性及乳酸脱氢酶活性来比较不同的冷冻方法对解冻后精液品质的影响;通过测定精液稀释后、平衡后、冷冻仪法冷冻后的酶活性来比较谷草转氨酶与乳酸脱氢酶在不同阶段的释放量。结果表明,采用冷冻仪法冷冻后的精子活率和质膜完整率极显著高于液氮熏蒸冷冻法（P<0.01）;冷冻仪法冷冻后的谷草转氨酶和乳酸脱氢酶活性极显著低于液氮熏蒸冷冻法（P<0.01）;精液稀释后的谷草转氨酶和乳酸脱氢酶活性极显著低于精液冷冻后的活性（P<0.01）。表明采用冷冻仪冷冻法的绵羊精液品质好于液氮熏蒸冷冻法。精子中谷草转氨酶主要在平衡阶段释放,而乳酸脱氢酶主要在冷冻阶段释放。     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

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.