猪深部输精技术应用研究进展

常规子宫颈人工授精(cervical artificial insemination,CAI)技术输精量大,存在浪费和不经济的问题,尤其在使用高附加值的精液产品时更为突出。近年来,一种高效利用猪精液的深部输精技术得到了研究和应用,这种技术与定时输精技术相结合,直接将精液输送至子宫体、子宫角和输卵管内,减少了每头母猪受孕所需精子数目,大幅度提高了良种公猪的利用价值,有助于实现冷冻保存精液和性控精液在养猪生产上的商业化应用。作者概述了猪人工授精中3种深部输精技术在液态保存、冷冻保存和性控精液上的研究和应用情况。无论实施哪种输精技术,公猪繁殖力、精子活力、母猪的饲养管理、输精时间和人工授精技术操作的规范性都是提高受胎率必须要考虑的因素。     

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.     

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.     

公猪繁殖性能评定

随着规模化养猪业的发展及人工授精技术的普及,公猪繁殖性能已是影响猪场经济效益的重要因素。如何准确评定公猪的繁殖能力,保证人工授精效果,进而保证母猪的繁殖性能的发挥,是养猪人工作中的重要环节。生产实践中最常用的是通过显微镜对猪精细胞水平进行检测。科研人员也在一直研究影响人工授精效果的因素,将精液的检测从细胞水平上升至分子水平,再用分子标记手段将公猪繁殖性能由检测提前至预测,最大限度地保证公猪的繁殖性能,保证优良公猪的性能得到最大程度的发挥。文章从精液品质、人工授精效果、分子水平检测、标记物等几个方面介绍公猪繁殖性能评价时需要衡量的指标,为公猪繁殖性能评定提供参考。     

Approaches towards efficient use of boar semen in the pig industry

The current cervical artificial insemination (CAI) procedure, involving deposition of excessive sperm numbers, is uneconomical for pig industry. The most obvious alternative requires uterine deposition in combination with fixed-time AI, which would reduce the number of sperm required per pregnant sow, thus allowing the best use of valuable boars and, ultimately, the commercial integration of frozen-thawed and sexed sperm. This review depicts possible best ways to implement an efficient use of liquid-stored, frozen-thawed and sexed sperm by the pig industry.     

Artificial insemination in pigs: predicting male fertility

Efficient artificial insemination (AI) is essential for future challenges in the pig industry. Knowledge on the exact relation between semen quality characteristics and fertility can have a major impact on both the genetic merit of future animals and the efficiency of AI. Variation in fertility is caused not only by farm- or sow-related parameters but also by boar- and semen-related parameters. In pig AI there is no gold standard concerning semen quality assessment. Assessing semen quality characteristics objectively and relating them to large field fertility datasets leads to an efficient production of insemination doses, which results in an efficient dissemination/descent of the breeding program required genes. Overall, this contributes to the development of semen quality assessments, which improves the prediction of porcine male fertility. Knowing which semen characteristics, and to what extent, contribute to male fertility and makes the field fertility more predictable.     

Multivariate Analyses for Determining the Association of Field Porcine Fertility With Sperm Motion Traits Analysed by Computer‐Assisted Semen Analysis and With Sperm Morphology

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate – FR and total piglets born – TB) of 14 boars was obtained from a field trial conducted during 10 week of breeding period on a commercial farm using multiparous sows (n = 948) through single‐sire mating with 2 × 10⁹ motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer‐assisted semen analysis system in raw and stored semen at 17°C for 240 h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.     

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.     

Semen alterations in porcine rubulavirus-infected boars are related to viral excretion and have implications for artificial insemination

Porcine rubulavirus (PoRV), also known as blue eye disease (BED) of swine, causes respiratory and reproductive problems in pigs at several developmental stages. To study the effect of PoRV infection on semen production, five boars were infected with 1 x 10(6) TCID(50)/ml of PoRV strain PAC-3 and evaluated for 59 days post inoculation (DPI). Infected boars developed reproductive tract pathology that included swelling of the testes and epididymides. Analysis of the semen showed that the infection had little effect on semen production in four animals, but semen from one boar showed severe alterations in sperm concentration, motility, and morphology. When motility was analyzed in BTS-diluted semen after 24, 48, or 72 h, alterations were detected in all boars. Furthermore, viral antigen was detected in semen, the seminal plasma fraction, or sperm fraction from all boars. These results showed that PoRV is excreted via semen and, therefore, artificial insemination is a potential route of dissemination.     

Fertility Results of Artificial Inseminations Performed with Liquid Boar Semen Stored in X-CellTM vs BTS Extender

The objective of the present field study was to compare the fertility results for boar semen diluted in X-cell stored up to 4-5 days before artificial insemination (AI) with semen diluted in Beltsville thawing solution (BTS) used for AI following 2-3 days of storage (where the first day being the collection day). A total number of 2601 double inseminations in Norwegian herds were included in this two-trial study. All the boars used in the study were mature cross-bred Norwegian Landrace x Duroc (LD), which were routinely used for AI in Norway. The inseminated gilts and sows were Norwegian Landrace x Yorkshire (LY). The AI doses contained 2.5 billion spermatozoa, and consisted of a mixture of semen from three, occasionally four, boars (i.e. heterospermic semen). Fertility was measured in terms of the likelihood of farrowing and subsequent litter size. The fertility of the semen in both of the extenders was satisfactory and no significant differences were found either in semen stored 4-5 days in X-cell compared with 2-3 days in BTS or in semen stored 2-3 days in X-cell compared with 2-3 days in BTS. The storage capability findings for the long-term extender X-cell could significantly simplify the practical issues of semen production and the distribution of AI doses containing 2.5 billion spermatozoa. However, in pig production systems where all semen is used within 2-3 days, the short-term extender BTS is as good as the more expensive extender X-cell.     

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.     

Porcine Field Fertility with Two Different Insemination Doses and the Effect of Sperm Morphology

In swine artificial insemination, several dose regimens are applied, ranging from 1.5 x 10(9) to 6.0 x 10(9) spermatozoa per intra-cervical insemination dose. A lower sperm dose is more profitable for artificial insemination centres and offers a more effective use of superior boars. To evaluate fertility, 50 boars were used for a total of 10 773 homospermic first inseminations at a dose of 2 billion spermatozoa. In addition, 96 boars were used at a dose of 3 billion spermatozoa for 34 789 homospermic first inseminations. Fertility was determined by a 60-day non-return rate (NR%) of first inseminations. Litter size was registered by total number of piglets born separately in primiparous and multiparous farrowings. On average, a sow was inseminated 1.5 times. A significant decrease was observed in all three fertility parameters (NR%, litter size of both primiparous and multiparous farrowings) with a dose of 2 billion spermatozoa compared with a dose of 3 billion spermatozoa. The NR% was 75.8% and 84.0% (p < 0.001), the mean litter size of primiparous farrowings 10.1 and 10.7 (p < 0.001) and the mean litter size of multiparous farrowings 11.7 and 12.1 (p < 0.001) for 2 and 3 billion spermatozoa/dose, respectively. The proportion of normal spermatozoa in the sperm morphology analysis correlated significantly with NR% in both insemination regimens: p < 0.001, r = 0.604 and p < 0.05, r = 0.223 for 2 and 3 billion spermatozoa/dose, respectively. These results confirm that quantity can at least partly compensate for poor sperm quality. When the boars with <70% normal spermatozoa in the morphology evaluation were excluded from the data there were no correlation between the sperm morphology and NR%. However, the difference between the NR% and litter size remained statistically significant (p < 0.001) in favour for the bigger insemination dose. In conclusion, a decrease in sperm dose from 3 to 2 billion spermatozoa on commercial farms will severely decrease prolificacy at least under field conditions, where a sow is inseminated an average of 1.5 times/heat, and the semen is typically used within 3 days after collection. We recommend that under commercial circumstances the homospermic semen doses contain no <3 billion spermatozoa/dose.     

Artificial insemination in swine.

This article analyses the advantages and disadvantages of artificial insemination with semen purchased from a center as well as from the herd boars on the farm. Intensive swine production could benefit greatly by adapting artificial insemination with herd boars, particularly from savings in labor and boar numbers. The techniques for semen collection, extension, and insemination are described, and sources for equipment given. Expected results of artificial insemination are quoted from experiments and international field experience.     

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.     

浅谈猪精液保存在人工授精中的发展与展望

在世界上主要的猪肉生产国中,猪人工授精(AI)技术的应用率在90%以上。尽管猪冷冻精液技术在不断进步,但由于其效率不高,在猪场实际生产中并不多见使用。在猪人工授精技术中,液态精液是目前应用最广泛的精液,与传统的牛冷冻精液不同,其独特的精子微胶囊保护膜技术,可以对猪精子进行更加完善的液态保存,因此液态精液是目前猪人工授精使用的主要方式。文章综述了猪精液保存技术在人工授精中的应用,并展望了今后的发展方向,为人工授精技术助力恢复生猪生产提供参考。     

鹅人工授精技术研究进展

中国养鹅业经过20世纪末约十年的高速发展后出现了增长缓慢的趋势,原因除了中国畜牧用地限制、疫病危害和人力成本上涨等因素外,缺乏技术创新和突破也是主要原因.鹅人工授精技术是一项先进的繁殖技术.鹅人工授精技术的完善和应用,不但可以提高优秀公鹅的配种效率,而且可以加快鹅育种的遗传进展.如果精液冷冻保存成功,鹅人工授精技术还可以用于保种和克服配种时间和空间的限制.鹅的育种落后和繁殖率低是制约养鹅业发展的瓶颈,鹅人工授精技术的应用将对养鹅业发展起到巨大推动作用.作者对近几年鹅精液收集、精液保存、精液品质评估和输精方法等方面的研究进展进行了综述.     

Genetic parameters for fertility traits in Nellore bulls

Nellore is the main cattle breed used in Brazil, being the largest commercial herd in the world. Beyond the importance of male reproductive efficiency for farm profit, the use of reproductive techniques, mainly artificial insemination, turns the evaluation of male reproductive traits even more important. Estimation of genetic parameters increases the knowledge on traits variances and allows envisaging the possibility of the inclusion of new traits as selection criterion. Genetic parameters for fifteen traits that can be classified as testicular biometry or physical and morphological semen traits were estimated for a Nellore bull population ranging from 18 to 36 months. Single-trait and bi-trait animal models were used for (co)variance components estimation. The contemporary group was considered as fixed effect and age at measurement as covariable. Scrotal circumference presented heritability of 0.47 ± 0.12. This value is similar to the heritabilities found for all testicular biometry traits (0.34–0.48). Sperm progressive motility, which has a direct effect on bull fertility, presented low heritability (0.07 ± 0.08). Major and total sperm defects presented moderate to high heritabilities (0.49 ± 0.18 and 0.39 ± 0.15, respectively), indicating that great genetic gain can be obtained through selection against sperm defects. High and positive genetic correlations were observed among testicular biometry traits, which also presented favourable genetic correlations with physical and morphological traits of the semen with magnitude ranging from high to low. Scrotal circumference presented moderate to high and favourable genetic correlations with sperm progressive motility, sperm turbulence, major sperm defects and total sperm defects. Thus, the selection for scrotal circumference results in favourable correlated genetic response for semen quality. The results show that the use of scrotal circumference as reference trait for bull fertility is appropriate, since it presents high heritability and favourable genetic correlation with semen quality.     

Intra-uterine Insemination in Farm Animals and Humans

Artificial insemination (AI) is the oldest and currently most common technique in the assisted reproduction of animals and humans. The introduction of AI in farm animals was forced by sanitary reasons and the first large-scale applications with a commercial goal were performed in cattle in the late 1930s of last century. After the Second World War, cryopreservation of semen facilitated distribution and AI was mainly performed for economic reasons, especially in dairy cattle industry. In humans however, AI was initially performed in cases of physiological and psychological sexual dysfunction, but later on also in cases of infertility caused by immunological problems. Currently, the most common indications for intra-uterine insemination (IUI) in humans are unexplained infertility and male subfertility. In these cases, IUI is considered as the treatment of the first choice, before more invasive techniques such as in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI) are used. In contrast with humans, the quantity and quality of semen produced by farm animals is much higher and permits dilution and production of several insemination doses per ejaculate. However, with the introduction of sex-sorted semen in farm animals, the same problem of low-quality semen as in humans has arisen. In cattle, pigs and horses, conventional insemination with low numbers of sex-sorted spermatozoa results in a significant decrease in fertility. To improve the fertility rates with this semen, new insemination techniques have been developed in order to deposit spermatozoa closer to the site of fertilization. In sows and mares the advantage of utero-tubal junction (UTJ) insemination has already been proven; however, in cattle it is still under investigation. In this review, the differences and similarities in the application of AI between animals and humans are discussed and as AI in farm animals is most successful in cattle, the situation in this species is elaborated the most.     

Comparative efficiency of novel laparoscopic and routine vaginal inseminations with cryopreserved semen in rabbits

Laparoscopic artificial insemination technique (LAI) is described to overcome reduced fertility problems in sheep artificial insemination (AI) programmes with frozen semen. Later on, this technology was modified for endangered non-domestic cats to deposit low quality or reduced number of sperm cells hardly obtained by electro-ejaculation into the oviduct. This technique by passes the complex structure of cervix and efficiently transfers the sperm cells to the point of fertilization. In recent years, rabbits are becoming popular transgenic animal models producing various therapeutic and commercial products, as well as being experimental animals for disease models. The worldwide transportation of frozen semen and re-establishment of transgenic lines using AI technology has become a common practice. Therefore, this study was designed to describe a laparoscopic intrauterine insemination technique, which might assist in conceiving the animals with limited number of sperm cells. The female rabbits were laparoscopically (n = 22) or vaginally (n = 13) inseminated with frozen–thawed semen samples containing approximately 10 × 10⁶ motile sperm. The laparoscopic insemination technique provided higher pregnancy rate (45.5%) than vaginal insemination technique (7.7%) (p < .05). In conclusion, the described laparoscopic AI might be a new alternative technique, thus enabling limited or low-quality frozen sperm samples to establish pregnancy in rabbits.