Artificial insemination and preservation of semen.

Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable pregnancy rates are consistently obtained with cooled semen. Conversely, techniques for cryopreservation of stallion semen will require more refinement before the procedure can be considered commercially viable on a wide scale.     

Changes in Equine Reproduction: Have They Been Good or Bad for the Horse Industry?

In the past four decades there have been tremendous changes in equine reproduction. Most breeds now allow the use of artificial insemination with fresh, cooled and frozen semen. Artificial insemination has many advantages for the breeder, in particular the control of bacteria through the use of semen extenders containing antibiotics. Deposition of sperm in small volumes onto the uterotubal junction has allowed the use of relatively low numbers of sperm. Intracytoplasmic injection of sperm into oocytes allows older, subfertile stallions to be used as breeding stallions. Advances in mare reproduction have included developing tools for hastening the onset of the breeding season. Other advances include embryo transfer, oocyte collection and transfer, and cloning. The acceptance of reproductive technology depends on the success of the technology, the attitude of the breeders/veterinarians, and the cost/benefit ratio to the industry and breed registry.     

Rectally Guided or Hysteroscopic Insemination: Is there a difference?

The increasing use of frozen semen, the high cost of a dose of semen from some stallions, and the inability of some stallions to cope with their demand has triggered an incentive to reduce the number of spermatozoa from the traditional 500 PMS to significantly lower numbers per insemination. Two techniques have been described to deposit low sperm numbers in the uterus. Although experimental results have been reported for the rectally guided deep horn insemination (RGDHI) and endoscopic deep horn insemination (EDHI) techniques, little information is available in clinical settings with respect to the results of either method. This article reports on a comparison of the rectally guided insemination and hysteroscopic techniques in a clinical setting over three breeding seasons. Semen frozen in 0.5-mL straws and concentrations of 40 to 200 million per straw was available from 88 stallions whose fertility with frozen semen ranged between 0% and 100%. Of the 2544 inseminations performed 1279 were done by RGDHI, which resulted in a 43% seasonal pregnancy rate compared with 45% for the 1265 inseminations using the EDHI technique. There was an overall, but nonsignificant, advantage for the EDHI technique over the RGDHI and semen usage was slightly lower when using the EDHI procedure. There was no difference in fertility among breeding seasons for either technique. However, fertility of some stallions was increased by increasing the sperm numbers in the inseminates. From these data and results from others, it is evident that lower sperm numbers deposited by rectally guiding a flexible pipette to the tip of the horn or by using an endoscope to deliver the semen will result in acceptable pregnancy rates with frozen semen in commercial settings.     

Advances in the Research and Application of Artificial Insemination to Equids in China: 1935–2012

China was one of the first countries to use artificial insemination (AI) in equids, and it achieved international recognition for its widespread application of AI to commercial horse breeding. This article reviews the history of equine AI in China. The technique originated from the high demand for horses to be used in agriculture, transportation, and the military. Artificial insemination was identified as an ideal tool for Chinese horse breeding to improve the productivity of native horses, especially during 1950-1970. Presently, AI is still practiced commonly in China, and it includes the use of fresh semen and transported cooled semen. The use of frozen semen has also been resumed to broaden the range of elite Sporthorse stallions and to preserve threatened or endangered native breeds. Accurate prediction of the optimum time for insemination depended mainly on transrectal palpation of the mare's ovaries. In addition to controlled methods of insemination, factors like volume and number of spermatozoa in the inseminate and timing and frequency of insemination were optimized to accomplish high fertility rates in the field. Production of hinnies and mules for agricultural labor and donkeys for meat and hide gelatin stimulated a nationwide upsurge of research into the reproductive physiology of and AI technology in donkeys. In the future, there will be further increases in the use of traditional and modern AI techniques in the breeding of Chinese equids.     

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。     

A retrospective study of artificial insemination of 251 mares using chilled and fixed time frozen-thawed semen

REASONS FOR PERFORMING STUDY: Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. HYPOTHESIS: Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. METHOD: Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 2007 northern hemisphere breeding season. Per rectum ultrasonography of the mare's reproductive tract determined the timing of insemination, and deslorelin acetate was used to induce ovulation. Chilled semen insemination was performed using a single preovulatory dose delivered into the uterine body. Frozen semen was administered as 2 doses (pre- and post ovulation) using a deep uterine insemination technique. Pregnancy was detected ultrasonographically at 15 days post insemination. Conception rates were compared using a Chi-squared test. RESULTS: Insemination with frozen semen produced a significantly (P = 0.022) higher seasonal conception rate (82.0%) than that achieved with chilled semen (69.6%). CONCLUSIONS AND POTENTIAL RELEVANCE: Insemination with frozen semen can achieve conception rates equal to those with chilled semen, enabling the mare owner a greater selection of stallions.     

Current challenges in sheep artificial insemination: A particular insight

Ovine artificial insemination (OAI) is not commonly performed because of specific problems related to semen application techniques, leading to highly variable results. The ideal methodology (frozen‐thawed semen/vaginal route) is unfeasible under field conditions due to the cervix morphology of the ewe, which prevents the process of intrauterine insemination necessary to obtain acceptable results. Currently, OAI commercial programmes use superficial cervical insemination, CAI (vaginal), with chilled semen (15°C) and intrauterine insemination, LAI (laparoscopic), with frozen‐thawed semen. The ability to improve upon these contrasting techniques may be derived from examining certain poorly studied factors such as insemination time, productive state of females and alternatives of seminal preservation, some of which we reviewed in this work. This interim solution will remain in use until AI by the vaginal route with frozen‐thawed semen is developed, but it poses new challenges in optimizing the freezing of the sperm and adapting the cervical (CAI) and/or transcervical intrauterine AI (TCAI). In this review, we address the current problems and evaluate their methodological (mechanical) and chemical (dilation) alternatives. Currently, TCAI is a methodologically complex technique with poor fertility results, so further studies are needed to improve the logistics of this procedure and the results of its application.     

Quality of Fresh,Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.     

Effects of Pentoxifylline,Caffeine, and Taurine on Post-Thaw Motility and Longevity of Equine Frozen Semen

Frozen semen provides several advantages to the breeder relative to fresh or cooled semen. However, some stallions are undesirable candidates for semen freezing due to poor post-thaw motility or longevity caused by membrane damage, osmotic stress, and oxidative stress during cryopreservation. The objective of this study was to determine the effect of post-thaw addition of pentoxifylline, caffeine, or taurine on sperm motility and longevity in equine frozen semen. Pentoxifylline, caffeine, or taurine was incorporated immediately into thawing frozen semen from nine warmblood stallions. Spermatozoa motility and longevity parameters were recorded and analyzed for each additive and for an untreated control. Of the three additives, only pentoxifylline improved total and progressive semen motility relative to that of untreated control. Pentoxifylline also increased semen curvilinear velocity, average path velocity, and straight line velocity relative to those of caffeine, taurine, or control. Semen treated with pentoxifylline also showed greater longevity relative to that of caffeine- or taurine-treated or untreated semen. Taurine improved linearity in comparison to that of semen treated with pentoxifylline, caffeine, or control but did not improve other parameters. Pentoxifylline may be useful in enhancing the quality of equine frozen semen and therefore improving its fertility. Additional studies are warranted that examine the effect of these additives on the conception rate. Pentoxifylline can be used to increase motility and longevity of equine frozen semen and theoretically increase probability of conception and overall breeding success rates.     

Influence of Semen Storage and Cryoprotectant on Post-thaw Viability and Fertility of Stallion Spermatozoa

The aim of the current study was to verify that stallion spermatozoa could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryopreservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio^® extender containing one of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5°C for 20 minutes, then frozen in nitrogen vapor. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty-three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine semen for 24 hours before freezing, while maintaining sperm viability and fertility, is possible.     

An Overview of Low Dose Insemination in the Mare

The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex‐sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex‐sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 × 10⁶ spermatozoa and is therefore, the preferred method of insemination for sex‐sorted spermatozoa. However, hysteroscopic deposition of low numbers of spermatozoa from infertile stallions does not appear to improve their fertility.     

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.     

Breeding the mare with frozen semen

Breeding with frozen semen has become more commonplace. For a successful outcome, it is important to select fertile mares and to use quality frozen semen. The method/timing of insemination will be determined primarily by the number of semen doses available per cycle. Direct insemination of semen straws is preferred as this technique results in less loss of spermatozoa in insemination equipment. Ovulatory agents are crucial to help with timing of ovulation with respect to insemination. A post breeding examination should be performed to confirm ovulation and to examine the mare for possible complications such as post mating induced endometritis. With attention to details, frozen semen can be used with very good results.     

Preliminary comparisons of a unique freezing technology to traditional cryopreservation methodology of equine spermatozoa

With recent large-breed organization acceptance, the use of frozen semen is gaining more attention in the equine industry. However, cryopreserved stallion semen is commonly associated with poor quality and decreased pregnancy rates as compared with those produced during normal mating or with cooled semen techniques. Therefore our objective was to investigate a new unique freezing technique (UFT) with the intent of improving fertility outcomes. A series of experiments tested the UFT compared with traditional liquid nitrogen methodology in combination with influence of the extenders and stallions used. In Experiment 1, post-thaw motility results of UFT variations were compared with those from liquid nitrogen methods. The averaged post-thaw motility percentages of the 4 UFT treatments were similar when compared with the traditional liquid nitrogen control group (P = .845). In Experiment 2, 2 egg-yolk–based freezing extenders, Biladyl AB intended for bovine samples and Freezing Medium Test Yolk Buffer used for human samples, were compared. A significant difference in the average post-thaw motilities was found between Biladyl AB (17%) and Freezing Medium Test Yolk Buffer (25%) (P < .002). In the third experiment, we compared variability among stallions using the UFT with the intention of creating a more consistent outcome. Post-thaw motilities and percent of original motility returns among the 4 stallions were significantly different (P < .001). In a field trial using shipped semen from a regional stallion station, the UFT demonstrated very promising results.In conclusion, the UFT may potentially be used as an alterative freezing method to replace current liquid nitrogen methodology. However, further investigation is needed to refine techniques.     

Boar Semen Studies: II. Laboratory and Fertility Results of a Method for Deep Freezing

A successful method for low temperature preservation of bull semen was modified for use with boar semen and resulted in recovery of twenty to fifty per cent motile cells immediately after thawing. Recovered cells did not survive five hours incubation at 37° C. and no pregnancies resulted following insemination of twenty-four sows and gilts with frozen semen.     

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。     

CASE STUDY: Use of Modified Phosphate-Buffered Saline as a Component of Semen Extenders Allows the Use of Transported Semen from Two Stallions

Conception rates for mares bred with transported-cooled and fresh stallion semen were collected over a 4-yr period (1998–2002) for two stallions. Both stallions stood at a commercial breeding farm. Semen from both stallions was used immediately after collection on the farm and after 24 to 48 h of cold storage when transported to locations in the U.S. and Canada. Semen for insemination of mares located on the farm was extended with a commercially available skim milk glucose extender (SKMG). Spermatozoal motility following cold storage for spermatozoa diluted in SKMG extender was unacceptable. Thus, semen from both stallions was centrifuged, and spermatozoa were resuspended in SKMG supplemented with modified PBS. In a previous study, the percentage of motile spermatozoa increased following centrifugation and reconstitution of the sperm pellet in SKMG-PBS as compared with semen dilution in SKMG (Stallion A: 15% vs 47%; Stallion B: 18% vs 43%). In the current study, 22 of 25 (88%) and 3 of 4 (75%) mares conceived with transported-cooled semen from Stallions A and B, respectively. Conception rates for mares inseminated with transported semen did not differ (P>0.05) from those inseminated on the farm with fresh semen. These data illustrate that stallion owners can modify standard cooled semen processing procedures and semen extender composition to improve post-storage spermatozoa motility and to obtain acceptable fertility.     

Semen collection techniques.

Semen collection techniques in the stallion have evolved considerably over the last 70 to 80 years and are used today primarily for artificial insemination. Semen can be collected from stallions that are otherwise unable to breed, allowing continued use of valuable animals. There are many options for collection of semen from stallions that present with ejaculatory dysfunction (see the article by McDonnell elsewhere in this issue.) Although there are many advantages to the use of artificial breeding, the collector must understand each step of the collection procedure as well as stallion preferences and proper use of an artificial vagina and mount source so that a representative semen sample is collected.     

Studies on Motility and Fertility of Cooled Stallion Spermatozoa

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.     

Artificial Insemination in Horses—More than a Century of Practice and Research

Important early studies on mammalian artificial insemination (AI) were carried out in equids, and at the end of the 19th century, the first AI programs were set up in horses. At that time, the most systematic research on equine AI was performed in Russia. After World War I, AI research shifted to cattle and sheep. This time saw major advances such as the development of artificial vaginas and phantoms for semen collection. Semen dilution counteracted the detrimental effect of seminal plasma, allowed semen storage, and increased the volume of an ejaculate for insemination of more mares. In the late 1930s, techniques for cooled semen AI as used today were in principle available. After World War II, the number of mares inseminated decreased, but with a new role of the horse as a partner in equestrian sports, new interest in equine AI was raised. In contrast to the situation in cattle, frozen semen has not replaced cooled semen AI in the horse. Recent advances in insemination of horses are the sexing of sperm, low-dose deep intrauterine insemination, and intracytoplasmic sperm injection.