Reproductive efficiency of a new modified boar semen extender for liquid storage

In the present study the Authors developed a new modified boar semen extender for short-term liquid storage, based on the use of amikacin sulphate and fructose rather than gentamicin and glucose. The new extender (ME-S) was evaluated and compared in vitro to commercial ones (CRONOS^™, TRIXcell^™) and to a modified extender designated for long term storage (ME-L) for progressive motility. Progressive motility was not different (P>0.05) among extenders until 120 h of storage, as differences among extenders became significant (P<0.05) at 144 and 166 h. Motility data across time were better for ME-S than TRIXcell^™ (P<0.05). No differences were observed about the morphology and membrane integrity (ORT) among the new extender (ME-S) and the commercial ones. Following the results of the in vitro comparison, an artificial insemination field trial was performed for reproductive efficacy. In this trial ME-L was not used because it was not completely reliable yet. A total of 1011 sows were bred: 506 with ME-S and 505 with a commercial one (CRONOS^™). The pregnancy rate for ME-S was 93.68% (474 pregnant sows), as the commercial extender resulted in 452 pregnancies (89.5%). The statistical comparison was significant (P<0.05) and the number of live piglets born showed an increase of 52.     

猪精液常温保存的试验

设计了4个猪精液稀释液配方,以台湾生产的猪精液常温保存稀释粉为对照,筛选出保存效果较好的配方,再进行不同因素对精液保存的影响试验。结果显示,以含有Tris和半胱胺酸的配方4,并在稀释液中添加庆大霉素(100 IU/mL)和林肯霉素(200 μg/mL)作抗生素,在24-30℃,pH为6．4,稀释倍数为1:1时保存效果最佳。     

抗生素对猪精液常温保存效果的影响

采集9头公猪的精液（其中长白4头、大白4头、杜洛克1头）,对精液进行稀释后,分别加入9种抗生素,在18℃保存,测定精子保存时间和精子存活指数.结果表明：磺胺抗菌效果最好,其有效保存时间为4.38 d,精子存活指数为3.14.恩诺沙星、阿奇霉素、林肯霉素的抗菌效果次之,庆大霉素、青链霉素的抗菌效果再次之,新霉素、卡那霉素的抗菌效果最差.     

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.     

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。     

Assessment of sperm quality traits in relation to fertility in boar semen

Background

Several studies have been published where sperm plasma membrane integrity correlated to fertility. In this study we describe a simple fluorometer-based assay where we monitored the fluorescence intensity of artificially membrane-ruptured spermatozoa with a fixed time staining with fluorescent DNA dyes.

Methods

Membrane-impermeant fluorescent dyes Hoechst 33258 (H258) and propidium iodide (PI) were used to measure the fluorescence of the nucleus in artificially membrane ruptured spermatozoa and membrane-permeant dye Hoechst 33342 (H342) was used to measure fluorescence of intact spermatozoa. The concentration of spermatozoa in insemination doses varied from 31.2 × 10⁶/ml to 50 × 10⁶/ml and the average value was 35 × 10⁶/ml. Each boar was represented by three consecutive ejaculates, collected at weekly intervals. Nonreturn rate within 60 days of first insemination (NR %) and litter size (total number of piglets born) of multiparous farrowings were used as fertility measures.

Results

Sperm fluorescence intensity of H258 and H342, but not the fluorescence intensity of PI-stained spermatozoa correlated significantly with the litter size of multiparous farrowings, values being r = - 0.68 (P < 0.01) for H258, r = - 0.69 (P < 0.01) for H342 and r = - 0.38, (P = 0.11) for PI.

Conclusions

The increase in fluorescence values of membrane-ruptured H258 and unruptured H342-stained spermatozoa in boar AI doses can be associated with smaller litter size after AI. This finding indicates that the fluorescence properties of the sperm nucleus could be used to select for AI doses with greater fertilizing potential.     

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     

猪精液带毒与精液净化

在养猪生产中人工授精是加速遗传改良的最好方法。公猪精液带毒也是人工授精经常面临的问题。正常饲养或健康的公猪精液中可能检测到细菌、衣原体和病毒等多种病原,因为产生精子的睾丸是机体免疫豁免区。公猪精液中一旦带毒,特别是病原微生物,除了影响精液质量、造成公猪不育外,在配种过程中还会引起母猪的感染,甚至再通过母猪垂直感染仔猪造成疾病的传播,给猪场带来巨大的经济损失。     

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.