保存温度和不同种类稀释液对猪精液品质的影响

猪精液在室温保存时,含庆大霉素的稀释液保存效果优于含青、链霉素的稀释液,两种稀释液在保存72h时,精子活率、顶体完整率、GOT和LDH活性以及存活指数差异极显著(P<0.01).低温保存时,含蜂蜜和卵黄的稀释液对防精子冷刺激、维持精子活率的效果比含葡萄糖、奶粉的稀释液理想.在冷冻保存条件下,以含2%甘油的稀释液保存效果最佳.液态保存(室温、低温)的猪精液,LDH活性随精子活率下降而下降;pH值则都呈先上升后下降的变化趋势.室温保存主要是引起精子顶体脊膨大和保存后期的顶体前端膨大;低温保存主要引起顶体前部膨大和保存后期的少数顶体脱落;冷冻保存主要是造成顶体的膨大程度加大和顶体脱落.3种保存条件都造成精子线粒体透性增强.液态保存和冷冻保存精液的精清GOT活性与顶体完整率无显著相关性(P>0.05).     

猪精液稀释液室温贮存时间效果研究

为解决因水质所致的猪精液稀释液效果不稳定的问题,试验选用实验室研制的配方A和公开BTS两个配方配制成液态稀释液,过滤除菌,室温过夏天贮存后与某商业用的液态稀释液(液态B)做对照,观察3种液态稀释液室温贮存时间对精子活力和生存指数的影响。结果表明,BTS、配方A液态室温贮存396 d用于稀释猪精液,保存第3天精子活力和生存指数分别为0.85、0.75和40.80、39.60,优于液态B的0.2和24.60,仍能满足人工授精精子活力的要求。因此,BTS和配方A液态精液稀释液可以推广应用,液态室温贮存保持期可达1年。     

稀释液中添加还原型谷胱甘肽对猪精液液态保存效果的影响

为了研究还原型谷胱甘肽对猪精液液态保存效果的影响,实验通过在稀释液中分别添加不同浓度的还原型谷胱甘肽,研究了它对猪精液液态保存后的精子活力、顶体完整率、质膜完整率、DNA损伤率、体外受精卵裂率及囊胚率等影响。结果表明,向稀释液中添加1 mmol/L,5 mmol/L,10 mmol/L GSH均能显著提高精子保存质量。其中添加1 mmol/L还原型谷胱甘肽效果最好,10 mmol/L效果较差,但大部分指标仍显著高于对照组。证实了稀释液中添加还原型谷胱甘肽有助于提高液态保存猪精液的质量。     

不同浓度谷氨酰胺对常温保存猪精液质量的影响

目前,猪人工授精的精液还以液态常温保存为主,但保存时间短、质量差的问题亟需解决。本试验通过在稀释液中添加不同浓度的谷氨酰胺,检测不同保存时间的精液质量参数,来衡量谷氨酰胺对液态常温保猪存精液的影响。试验分为5组,使用改良后的Modena稀释液,分别添加不同浓度的谷氨酰胺(0 mmol/L、20 mmol/L、40 mmol/L、60 mmol/L、80 mmol/L)。试验结果表明,添加40 mmol/L谷氨酰胺的精液稀释液可以有效提高猪精液的质量并延长精子保存时间。试验结果为猪精液常温保存技术的发展提供理论依据。     

猪的人工采精和精液保存

正猪采精通常采用手握法。采精频率依公猪而定,一般每2~3天或每4~5天采集1次。采精时废弃最初射出部分液体,精子浓集部分收集后去凝胶。公猪射出的富精子部分通常在100~150 mL,在对精子的密度、活力、形态进行评估后进行稀释。1液态精液猪的输精99%采用液态精液。由于猪的精子对低温特别敏感,因此几乎所有的猪精液是采用室温稀释液。精液在室温下保存时,必须要抑制精子的活动,以保证精子能保持足够长的活力。     

公猪精液常温液态保存的研究进展

由于猪精子对于冷休克的应激反应较为敏感,使得猪精液的常温液态保存比冷冻保存得到了较快得发展。本文就猪常温精液的定义及外界影响因素、稀释液中的添加物质以及稀释粉(液)配方的设计等几个方面进行了总结,阐述了影响猪常温精液保存效果的几个主要因素,探讨了猪常温精液保存研究中的一些问题并做了展望。     

常温保存猪精液稀释液的筛选试验

为研制优良稀释液,对3种常用猪精液稀释液和1种自配猪精液稀释液进行保存效果观察试验,并对自配稀释液中所保存的精子进行了配种试验。按10级评分法评定精子的活力,根据活力变化情况,计算有效存活时间、总存活时间、生存指数,结果表明,自配稀释液保存精子的效果优于Kiew液、葡柠液和葡糖液。配种试验结果表明,用自配稀释液中保存的精液对发情母猪进行输精,其受胎率和产仔数达到了生产需求,可在生产中推广使用。     

不同稀释液对藏猪精液4℃保存效果的影响

为探讨不同精液稀释液对4℃保存藏猪精液的效果,保证优良藏猪种公猪精液的合理、高效利用,该试验选用5种常用的猪精液常温保存稀释液,并在藏猪精液4℃保存至第5天时检查藏猪精子活力、精子畸形率、精子顶体完整率、精子质膜完整率等。结果表明,在精液保存过程中,2号液对藏猪精液的保存效果最佳,精子活力在保存至第5天时仍然保持在0.54,且藏猪精子活力、精子畸形率、精子顶体完整率、精子质膜完整率均高于其余4组（P〈0.05）。该试验研究初步筛选了4℃保存藏猪精液的稀释液,为今后藏猪精液的生产应用奠定了基础。     

维生素类物质对民猪精子常温保存效果的影响

为了提高民猪精液常温保存效果,为民猪资源保护和利用提供技术保障,试验在含有民猪精液的常温稀释液中分别添加维生素C、维生素B12和水溶性维生素E,在17℃条件下保存0~6 d,测定精子的活率和质膜完整性,以评估该稀释液对民猪精液的保存效果。结果表明:与对照组比较,添加维生素类物质能够明显延长精子保存时间和提高质膜完整性,其中添加维生素E的保存效果最好,民猪精液在保存5 d后精子活率为50.8%,其质膜完整性为26.2%,仍可用于生产。说明在民猪精液的常温稀释液中添加维生素E能够延长精液保存时间,提高精子质量。     

死精子对猪精液17℃液态保存的影响

为揭示17℃液态保存过程中猪精子质量和功能持续下降的机制，本试验采集9头在役的不同品种公猪（长白、大白和杜洛克）的精液48份，使用BTS溶液等体积稀释后分为3份，其中的两份分别使用反复冻融（方法 A）和低渗处理（方法 B）方法杀死精子并离心获得稀释液A和B，将另一份精液样品离心分离精浆（Seminal Plasma,SP）和精子。分别使用不同体积比例稀释液A:精子（1:1、4:1和19:1），稀释液B:精子（1:1、4:1和19:1）,SP:精子（1:1和4:1）和BTS:精子（4:1）重悬离心后的猪精子，以SP和BTS重悬的精液及原精液样品作为对照，所有精液样品于17℃下保存3 d。结果表明：随着保存时间的延长，所有稀释液和稀释倍数处理均损伤了精子活率和活力（P<0.01）及质膜完整性（P<0.05）。稀释液A比B引起的精子脂质过氧化水平更低（P<0.05）。5倍稀释精子，稀释液B的精子活力较BTS组降低（P<0.05），稀释液B引起的精子细胞内活性氧族（ROS）水平比稀释液A更高（P<0.05），而精液总抗氧化物（TAC）水平更低（P<0.05）...     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

The reproductive performance and factors affecting on-farm application of low-dose intrauterine deposit of semen in sows

The objective of this experiment was to determine the reproductive performance and factors that affect on-farm application of low-dose intrauterine insemination (IUI) in sows. Four hundred twenty-two sows were used in a simple arrangement of four treatments to determine the effect of spermatozoa per dose (0.5 x 10(9), 1 x 10(9), or 4 x 10(9) IUI, and 4 x 10(9) with a conventional catheter) on the main effects of conception, litter size, and farrowing rate. Following weaning at approximately 18 d after parturition, estrus detection was performed daily in the presence of a mature boar. At the time of estrus detection, sows were blocked for parity (1, 2, or 3+), weaning-to-estrus interval (WEI; 3, 4, or 5 d), and assigned randomly to be serviced twice with semen from the same boar(s). Treatment services were equally divided among three technicians. Delivery of acceptable numbers of spermatozoa per dose with either device (IUI or conventional) produced similar reproductive performances; however, farrowing rate, total pigs born, and total born alive decreased (P < 0.05) when suboptimal numbers (< or = 1 x 10(9)) of spermatozoa were used with IUI. Treatment interactions with parity were not detected and were removed from the final model. Treatment interactions with WEI on farrowing rate were detected (P < 0.05), and sows with WEI of 3 d had a markedly lower (P < 0.05) farrowing rate than all other treatment groups. The results from this experiment suggest that placement of semen at the beginning of the uterine horn with conventional volumes and spermatozoa numbers produces results similar to placement of semen in the cervical cavity with a conventional AI catheter. Although there is little published evidence of reproductive performances in a commercial setting with suboptimal numbers of spermatozoa, these results suggest that insemination beyond the cervix does not offset effects of suboptimal numbers of spermatozoa.     

Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation: influence of boars and thawing diluents.

The fertilizing ability of deep frozen boar spermatozoa was assessed after artificial insemination at various intervals after human chorionic gonadotropin (HCG) induced ovulation in gilts. Boar seminal plasma and OLEP were used as thawing diluent agents. The fertilization rates were similar when insemination was performed 2 or 6 hours before the time of estimated ovulation (40 hours post-HCG administration). However, fertility rates markedly declined when insemination was performed earlier than 6 hours before expected ovulation. In inseminations performed 2 hours before ovulation, the fertility rate was higher with specimens treated with boar seminal plasma diluent than with OLEP. As the difference between the time of insemination and that of ovulation increased, the difference in fertilizing ability of spermatozoa from the 2 boars used also increased. The results show that spermatozoa which have a low resistance to freezing-thawing also have a diminished fertile life in the female genital tract.     

Fertilizing capacity of boar semen frozen in an extender supplemented with ostrich egg yolk lipoprotein fractions--a pilot study

A limited field trial was performed to evaluate the fertilizing capacity of boar spermatozoa frozen in an extender supplemented with lipoprotein fractions isolated from ostrich egg yolk (LPFo). Boar semen, diluted in an extender containing lactose with lyophilized lipoprotein fractions, glycerol and Orvus Es Paste (lactose-LPFo-G), was frozen using a controlled programmable freezer. Sperm characteristics, such as motility, plasma membrane and acrosome integrity, and mitochondrial function were monitored. Post-cervical artificial inseminations (post-CAIs) in multiparous sows (Polish Large White) were performed using the Soft & Quick catheter/cannula set. Sows were inseminated 2 to 3 times within one oestrus. Possible returns of sows to oestrus were determined from 21 to 30 days after post-CAIs. In this field trial, sows inseminated with 2 x 10(9) motile frozen-thawed spermatozoa resulted in pregnancy and farrowing rates of 75%, respectively. The average piglets born live was 10.5 +/- 0.4 (mean +/- SEM). The data of this study showed that post-CAI of boar semen frozen in LPFo-containing extender has the potential to provide acceptable fertility results. Further investigations are needed to elucidate the cause of variations in pregnancy/farrowing rate associated with frozen-thawed boar semen.     

Uterine Insemination with a Standard AI Dose in a Sow Pool System

The effect of uterine AI with a standard dose of spermatozoa on fertility of the sow was studied in a field trial. The trial involved a sow pool system with 440 sows using AI as the primary method of breeding. Sows were twice a day checked for oestrus symptoms by back pressure test in front of a boar on days 3–6 after weaning. When in standing heat, sows were randomly allocated into either a uterine insemination group (UTER, n = 157) or standard AI group (CONT, n = 169) and bred accordingly using 3 billion spermatozoa in 80 ml of extender. In both treatment groups, insemination was repeated once if the sow was still receptive 24 h later. Using pregnancy (farrowed or not) and live‐born litter size as the outcome variables, a logistic and linear regression approach, respectively, was taken to study the effect of the following factors: treatment (UTER vs CONT), AI operator, breed, satellite herd preceding weaning, parity, weaning‐to‐oestrus interval and length of lactation. Overall, live‐born litter size was 11.3 ± 2.9, repeat breeding rate 4.2% and farrowing rate 91.2%. In the UTER group, 93.6% of inseminated sows farrowed, whereas farrowing rate for the CONT group was 88.8% (p = 0.13). Intrauterine insemination with a standard AI dose did not result in a significant improvement in the live‐born litter size (11.5 ± 2.8 for the UTER and 11.1 ± 3.0 for the CONT sows, respectively, p = 0.13). However, the preceding satellite herd had a highly significant effect on the live‐born litter size (12.4 ± 2.6; 11.1 ± 2.9; 10.8 ± 2.9 and 10.9 ± 2.9 for the four satellite herds, p < 0.01). We conclude that uterine insemination did not have a significant effect on live‐born litter size and farrowing rate and we also conclude that satellite herd appears to have a major effect on fertility in a sow pool system.     

Within-herd Use of Boar Semen at 5°C, with a Note on Electronic Monitoring of Oestrus

A system was designed to allow a small swine farm in a northern latitude to use its own boars for artificial insemination (AI) conveniently. Semen was collected twice weekly for 3 day use (days 0, 1 and 2), extended in an egg yolk extender and stored at 5°C. Farm personnel were trained to manage the entire AI programme. For simplicity all semen collected was used for insemination. In the first test 47 gilts and 15 sows were inseminated with semen from four boars. One boar was subfertile with a farrowing rate of 36%. The averages for the other boars ranged from 71 to 100%. Then semen was collected from seven boars and all was used to inseminate 70 gilts and 55 sows with 3 × 10⁹ or more sperm. Overall 63% farrowed an average of 10.1 piglets per litter. Litter size for sows was 1.5 piglets larger than for gilts. There was no difference in farrowing rate when more than 3 × 10⁹ sperm were inseminated. The feasibility of initiating a complete AI programme within a small herd using herd boars was established. However, selection of the boars, use of only high quality semen, and experience with detecting oestrus was required to increase the farrowing rate. The use of various agents to protect sperm against cold shock below 15°C is worthy of further investigation. A new type of electronic probe, which measures the conductivity of cervical mucus, could be helpful if a boar is not available for conventional detection of oestrus.     

New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

The Post‐Cervical Insemination does not Impair the Reproductive Performance of Primiparous Sows

The study evaluated the reproductive performance of primiparous sows submitted to post‐cervical insemination (PCAI) compared with cervical artificial insemination (CAI). Difficulty with catheter introduction, the occurrence of bleeding or semen backflow during insemination, and volume and sperm cell backflow up to 60 min after insemination were also evaluated. Sows were homogenously distributed, according to body weight loss in lactation, lactation length, weaned piglets, weaning‐to‐oestrus interval and total born in previous farrowing, in two treatments: PCAI (n = 165) with 1.5 × 10⁹ sperm cells in 45 ml (2.4 ± 0.04 doses per sow) and CAI (n = 165) with 3 × 10⁹ sperm cells in 90 ml (2.5 ± 0.04 doses per sow). During PCAI, sows were inseminated in the absence of boars. Transabdominal real‐time ultrasonography was performed at oestrus onset, immediately before the first insemination and at 24 h after last insemination. There was no difference (P > 0.05) between treatments in farrowing rate (91.5% × 89.1%) and litter size (12.5 × 11.9 piglets born, respectively for PCAI and CAI sows). Successful passage of the intrauterine catheter in all the inseminations was possible in 86.8% (165/190) of sows initially allocated to PCAI treatment. Difficulty of introducing the catheter in at least one insemination did not affect the reproductive performance of PCAI sows (P > 0.05). Bleeding during insemination did not affect (P > 0.05) the farrowing rate in both treatments, but litter size was reduced in CAI and PCAI sows (P ≤ 0.06). Percentage of spermatozoa present in backflow within 1 h after insemination was greater in CAI than PCAI sows (P < 0.01). More than 85% of primiparous sows can be successfully post‐cervical inseminated with doses containing 1.5 × 10⁹ sperm cells in the absence of the boar during insemination without impairing the reproductive performance.     

Enhancing insemination performance in pigs through controlled release of encapsulated spermatozoa

Encapsulation of boar semen is a novel technique that allows insemination to be performed as a single intervention without the need to dilute the semen. The research reviewed in this paper shows that spermatozoa encapsulated in alginate are able to achieve the same fertility as two or three inseminations per oestrus using standard techniques and unencapsulated cells. The use of encapsulated spermatozoa is currently limited by the need for longer semen processing time and wastage of disposable material (catheters, plastic bottles, etc.). In this review, the advantages, the drawbacks and the future possibilities for artificial insemination with encapsulated spermatozoa in the sow are discussed, with the aim of applying this promising new methodology for the optimization of sow reproductive performance.     

The relationship of porcine sperm zona-binding ability to fertility

Several laboratory assays have been designed to assess the fertility potential of a semen sample before insemination, but none have been consistent and accurate predictors of fertility. To determine whether zona-binding ability may be a useful fertility predictor, we validated and used an in vitro competitive assay to measure the ability of porcine sperm to bind to the zona pellucida. The zona-binding ability of sperm from 11 boars that exhibited a broad range in average litter size and farrowing rate was determined. Sperm from each boar were compared directly with sperm from eight other boars in a systematic, pairwise fashion. Sperm from two semen samples were labeled with fluorophores at concentrations that did not affect motility or zona-binding ability. An equal number of labeled sperm from each boar was coincubated with homologous oocytes. Least squares means from analysis of variance were used to rank boars based on zona-binding ability. The competitive assay was effective in establishing a ranking of the boars (R2 = 0.62). Furthermore, there was a correlation between zona-binding ability and fertility when estimated by average litter size (r = 0.64, P < 0.05) but not when estimated by farrowing rate (r = -0.28). The explanation for this difference was that litter size and farrowing rate were poorly correlated (r = 0.14). In conclusion, a competitive zona-binding assay distinguished boars that sired either small or large litters. Competitive zona-binding ability may be useful for identifying boars with reduced fertility that produce smaller litters following insemination.