猪精液超低温保存技术及其对养猪业未来的重要作用（续完）

上期回顾：上一期主要介绍了猪人工授精的研究历史及应用情况,介绍了超低温保存精液的潜能,另外还详细介绍了精子获能、精子蛋白的酪氨酸磷酸化、顶体反应等精子功能。     

养猪行业应向明确的保健计划方向前进

预防猪呼吸道疾病有时是一项不断摸索的工作。亚临床感染、具有相同临床症状的不同疾病、协同感染和错误的诊断可能会使疾病控制成为一项高成本的工作。完整、反复地核查可以帮助理顺混乱的信息,从而能很快找到解决和预防的方法(图1)。     

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。     

Colloid centrifugation of boar semen

Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented.     

猪精液保存的一些方法

正猪精液的保存对精液的质量维持至关重要,目前猪液态精液的保存还是主流,但在国际贸易中冻精是较受欢迎的,而近些年猪冻精发展迅速,其中的保存方法也有了一些新的尝试。1冻精的保存猪精子对低温比较敏感,单次用量大,而常温保存也能满足用量需求,随着牛等冻精的发展,对优良品种的需求、跨国运输的成本以及生物安全的内在要求,使得猪冻精得到一定发展。目前,精液冷冻保存的方法有液氮保存、干冰保     

猪冷冻精液的研究

本试验以甘油、乙二醇为抗冻剂，并添加ATP或安息香酸咖啡因，以解冻后精子活力、顶体完整率、顶体膨胀率、顶体破损率、尾部畸形率和在37℃下的存活时间几个重要评定指标为依据，筛选了几种猪颗粒冻精的冷冻稀释液配方，并优化了其冷冻程序。结果表明，与对照组相比，在冷冻液中添加0．1mg／ml的ATP或2mg／ml的安息香酸咖啡因都能显著提高解冻后精子活力(分别提高0．13和0．18)，降低顶体膨胀率(两者都将近降低了11％)，并能延长其存活时间(分别延长10．5h和11．8h)；甘油和乙二醇两种抗冻剂配合使用，将显著提高冻后精子的活力与存活时间，其较优混合量为甘油3％，乙二醇0．5％。     

猪精液冷冻保存研究进展

根据国内外的研究进展,本文简要阐述了猪精液冷冻保存的机理和操作方法,对影响猪冷冻精液质量的因素进行了分析,同时指出了目前在猪精液冷冻保存技术的理论和实践中存在的问题,并提出了该技术中需要解决的问题和展望。     

种公猪精液伪狂犬病野毒的筛查

本文基于1 100头基础母猪的规模化场进行公猪精液伪狂犬病病毒筛查,农业部规定伪狂犬病病原学检测方法是通过ELSIA方法检测猪伪狂犬病gE野毒抗体。由于抗体检测具有滞后性,导致正在发生感染的猪群g E抗体检测往往呈阴性,但此时通过病原学检测精液已经正在排毒。本文互补了ELSIA抗体检测方法与荧光定性PCR检测病原的优劣,成功实现种公猪精液伪狂犬病病毒的筛查。     

Deep-freezing of boar semen in plastic film 'cochettes'

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

Influence of boar and semen parameters on motility and acrosome integrity in liquid boar semen stored for five days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16-18 degrees C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Field data analysis of boar semen quality

This contribution provides an overview of approaches to correlate sow fertility data with boar semen quality characteristics. Large data sets of fertility data and ejaculate data are more suitable to analyse effects of semen quality characteristics on field fertility. Variation in fertility in sows is large. The effect of semen factors is relatively small and therefore impossible to find in smaller data sets. Large data sets allow for statistical corrections on both sow- and boar-related parameters. Remaining sow fertility variation can then be assigned to semen quality parameters, which is of huge interest to AI (artificial insemination) companies. Previous studies of Varkens KI Nederland to find the contribution to field fertility of (i) the number of sperm cells in an insemination dose, (ii) the sperm motility and morphological defects and (iii) the age of semen at the moment of insemination are discussed in context of the possibility to apply such knowledge to select boars on the basis of their sperm parameters for AI purposes.     

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.     

Detection of chlamydiae in boar semen and genital tracts

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.     

Assessment of storage effects in liquid preserved boar semen

Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.     

High-speed centrifugation of extender of freeze-thaw boar semen

In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and/or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison with the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.     

EGTA对猪精液液态保存的影响

猪精液液态保存体系中,钙离子浓度升高将降低精子活力,缩短精子体外保存时间。常用的金属螯合剂EDTA的溶解度较低,且容易发生沉淀,因此改进螯合剂对于改进猪精液保存液配方,提高猪精液液态保存质量十分重要。本研究中我们在精液液态保存液中分别添加不同浓度(1.5,3,6,12 mmol/L)的钙离子螯合剂EGTA,检测其对猪精子的活力、质膜完整性、顶体完整率、获能情况以及受精能力的影响。结果表明,精液保存液中添加EGTA能显著提高猪精子保存质量,其中添加3 mmol/L EGTA效果最好(P<0.05)。与对照组相比,添加3 mmol/L EGTA时,体外受精率和囊胚率显著提高(P<0.05)。因此,精液保存液中添加EGTA有助于提高猪精液液态保存的质量。