我国猪冷冻精液集成技术产业化发展现状

正猪冷冻精液的研究从20世纪50年代开始,到目前为止已经取得了较大的科研进展。近几年,国内更多企业和科研机构关注猪冷冻精液技术研究与应用,实现冻精生产设备、冻精试剂零突破,猪冷冻精液商品化高效生产应用集成技术达到世界领先水平。猪冷冻精液商品化高效生产应用     

猪冷冻精液品质影响因素分析

基于猪冷冻精液品质是冻精制作流程中众多因素的合力结果。故加强各环节的操作规范性,针对影响冻精制作因素,将冻精制作流程优化,对提高冻精品质非常重要。文章就猪冻精制作流程涉及环节和影响因素逐一进行分析探讨,旨在为促进猪精液冷冻保存技术发展及冻精商业化生产提供一些实践支撑。     

异地制作保山猪冷冻精液技术

保山猪作为优良的种质资源近年来数量不断减少,冷冻精液可作为活体保护的补充方式有效保存其遗传物质。本试验保山猪精液来源于保山市保山猪繁育中心,通过公路交通远距离运送到云南省种畜繁育推广中心冷冻精液站进行细管冷冻精液的制作,对异地制作保山猪细管冻精的工艺流程和试验数据进行分析,以优化稀释液配方、筛选合适的活性剂。结果显示:公猪采精后用营养稀释液稀释、17℃恒温保存、运输、24 h内异地制作保山猪冻精,冻精质量良好,解冻活力30%以上,符合保存使用要求,为地方优良品种遗传物质保存提供可靠技术和便捷途径。     

影响猪冻精质量因素的研究进展

精液冷冻保存作为精液长期保存的最佳方法,具有保存时间长、生产成本低等优点,但由于猪精子较其他物种更容易受到低温的损伤,导致猪精液冷冻后精子活力低、受精率低,从而限制了冻精在养猪生产中的广泛使用。为优化猪精液冷冻-解冻方法,提高猪冻精质量,本文基于猪精液冷冻保存的损伤机理,从冷冻前处理、精液稀释液、冷冻和解冻程序等方面对影响猪精液冷冻质量的因素进行了详细阐述和总结。     

猪精液冷冻保存影响因素研究进展

由于猪冻精在经济生产中具有重要意义,其关键技术——猪精液冷冻保存技术已成为研究的热点。本文对猪精液冷冻原理及冷冻损伤进行了说明,并对精液稀释前处理、稀释液及添加剂、冷冻保护剂、冷冻程序与解冻、冷冻剂型等方面的研究进展进行了综述。     

科技

正我国猪冻精技术达世界水平近日,河北省廊坊市研发的"猪冷冻精液技术"的核心——《冷冻精液稀释液的制备方法》获得国家专利,在全国率先实现了冷冻精液配种与鲜精配种同等效果的繁育水平,这标志着廊坊市"猪冷冻精液技术"已达到世界先进水平。     

猪精液冷冻保存技术研究进展

猪精液冷冻保存技术对生猪产业发展有重要意义,有利于提高优秀种公猪的利用率以及实现优秀种质资源的长久保存,促进种猪场降本增效。目前中国猪精液冷冻保存技术取得了一系列进步,国内应用猪冻精配种的受胎率以及产活仔数基本达到了国外冻精配种水平。但是,由于冻融过程会对猪精子造成一定程度的损伤,精子的受精能力下降,受精窗口期缩短,国内、外应用冻精配种的生产成绩仍与常温保存精液存在不小差距。文章回顾了国内、外猪精液冷冻保存技术的发展历程,介绍了猪精液和精子的基本特点,简述了降温过程造成精子损伤的机理,并从冷冻稀释液成分这一角度分析介绍了减小冷冻损伤的机理与方法,如添加冷冻保护剂减少细胞内外冰晶形成、维持细胞内外渗透压以及稳定细胞膜结构;添加抗菌物质抑制解冻后细菌的快速繁殖;添加抗氧化物降低活性氧水平,缓解氧化应激,保护精子结构和受精能力等,最后对猪精液冷冻保存技术未来的发展方向进行展望,以期为相关研究以及实际应用提供参考。     

猪冷冻精液的生产和使用方法

正人工授精在猪的繁殖培育中应用已比较普遍。如何在生猪产业中像养牛生产一样方便快捷地使用冷冻精液,一直困扰着一线的技术和生产人员。在借鉴牛冻精生产与应用经验基础上,利用猪冷冻精液进行人工授精并成功产下仔猪后,众多科研工作者对猪冷冻精液技术进行了一系列深入的研究。目前,英国、美国、加拿大等一些国家的育种公司已经有猪冷冻精液的产品进行出售,但解冻后的精子活率以及配种后的受胎率、产仔数与常温保存猪     

猪冷冻精液的质量评价概述

猪冷冻精液的质量评价是不断提高冷冻保存技术和冻精生产应用的前提条件,是冷冻精液研究的重要内容。文章阐述了精液评价的常规分析、质膜完整性、运动参数、染色质、获能与繁殖效率六个方面,适用于开展猪冷冻精液的实验室评价和繁殖生产评价。     

猪精液冷冻技术研究进展

猪精液冷冻技术是指将猪精液作特殊处理后在超低温液氮（-196℃）状态下冷冻,以达到长期保存的目的。由于猪冷冻精液的生产难度大,冻精存活率低,受胎率低及鲜精少等诸多原因,使得该技术一直难以在生产中大面积推广,严重制约了我国猪品种的改良进程。故本文对近年来猪精液冷冻技术的研究成果作一综述,以期找到一种较好的猪精液冷冻技术方案。     

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。     

猪冷冻精液生产技术标准与规范及相关问题解读

业界普遍认为,高质量的技术标准或规范能引领和带动猪冷冻精液的商业化生产。文章基于三个猪冷冻精液相关的标准或规范逐条进行解读和探讨,使相关管理和技术人员能更好地学习和理解技术标准,加速猪冷冻精液的商业化进程;同时,标准制订者也能从管理和技术人员的反馈中及时获得标准的修订意见,从而进一步提高标准的先进性和可操作性。     

Deep Freezing of Boar Semen Packaged in Plastic Bags and Straws

For practical reasons, a large volume (i.e. 5 ml) of frozen boar semen per insemination dose is desirable, but successful freezing has not been achieved, since optimal cooling rates have not yet been established. Post-thaw motility and the acrosome intep'ty of semen from four boars frozen with a programmable freezin machine, in mini-(0.25 ml), maxi-(5 ml) plastic straws and in 10 × 5 cm PVC- or Teflon FEP-plastic bags (0.35 – 0.12 mm thick, 5 ml) was studied. The freezing of the semen was monitored using thermocouples placed in the straws and the bags. The freezing curve started from +5°C, at a rate of −3°C/min, to – 6°C, it was held for 1 min at −6°C, and was followed by further drop to −100°C at a rate of −20°C/min, with subsequent storage in LN₂. The bags had a much shorter freezing point plnteau, compared to the maxi-straws. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. The freezing procedure did not cause major acrosomal damages, significantly more normal apical ridges being present in the bags and mini-straws than in the maxi-straws. This in vitro evaluation indicates that the freezing method employed is satisfactory for freezing large volumes of boar semen into plastic bags .     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

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.     

不同类型稀释液对驴冷冻精液品质的影响

[目的]:对比分析各类型稀释液对驴冷冻精子活力、畸形率的影响,根据冻精解冻效果,筛选出适合驴精子冷冻的最佳稀释液,最适宜稀释比例,为驴冷冻精液研发提供参考。[方法]:实验共计14 d,采集6头种公驴精液,每份原精均分为3等份,分别用3种稀释液稀释,从而选取最优稀释液;按1∶0.75、1∶1.5、1∶3、1∶6不同比例混合最优稀释液与蒸馏水,每份原精均分为4等份,对应不同浓度稀释液分别进行稀释、封装、冷冻,检测冻精子活力和畸形率。[结果]:普蒂迪尔公牛稀释液、特里拉迪尔公猪稀释液、仙女座马稀释液三种稀释液中,特里拉迪尔公猪稀释液在配伍比例(稀释液与蒸馏水比值)为1∶3时,冷冻过程中能明显降低驴精子的畸形率,提升精子活力,能最大限度保存驴精子生殖功能。     

Boar Semen Studies: I. Laboratory Evaluation of Processing Phases *

A successful method for low temperature preservation of bull semen was modified for use with boar semen. Observations were made on the effects of varying cooling rate, equilibration time, freezing rate, glycerol concentration, method of glycerol addition, packaging containers, extender pH and tonicity. Observations indicate that boar semen should be cooled and frozen at a slower rate than bull semen. Within the ranges or methods examined, the other factors had little effect on recovery of motility after freezing.     

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.     

Combined effect of apigenin and ferulic acid on frozen‐thawed boar sperm quality

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high‐efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen‐thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen‐thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen‐thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen‐thawed boar sperm.     

Preservation of boar semen: An update

In the pork industry, artificial insemination and the storage of boar semen in liquid at 17°C are routinely applied to optimize the ejaculate and bring about rapid genetic changes that are reflected in the animal protein. Although the results are satisfactory, they are below what occurs with natural mating. It is currently possible to preserve boar semen with storage at 17°C and slow freezing, since to date there is only one study on vitrification, with negative results applicable only in the case of implementing an intracytoplasmic sperm injection. In both methods and due to the sensitivity of boar sperm to osmotic and temperature changes, there is a loss in the quality of the initial sample; however, slow freezing in boar semen has greater deleterious effects on the sample that are reflected in the pregnancy rates and number of live births. Therefore, only 1% of all inseminations are done with frozen semen. The aim of this review is to provide advances and results of studies conducted on the preservation of boar semen, delving more deeply into the critical points that each of the preservation techniques presents, including bacterial contamination, extender components, temperature, ice nucleation, use of additives in extenders and the main deleterious effects on sperm quality.