Current Reproductive Technologies Impacting Equine Embryo Production

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12–24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 μm or deflating embryos >300 μm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian’s approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5–25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.     

Live foals produced from sperm-injected oocytes derived from pregnant mares

Recently, in vitro fertilization (IVF) in the horse has met with less than anticipated results. Various problems associated with equine IVF include: (1) the inability to collect large numbers of good quality oocytes, (2) the alteration of the zona pellucida associated with in vitro maturation of equine oocytes, and (3) the improper preparation of equine sperm cells for IVF of these oocytes. Therefore, this study was conducted to achieve fertilization via sperm injection of equine oocytes and to produce live offspring from this IVF procedure. Oocytes were collected by transvaginal ultrasound-guided oocyte retrieval procedures from early pregnant mares of mixed breeds (day 14 to day 70 of pregnancy) and were matured in vitro and subjected to intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultured for 48 hours in either TCM-199 or P-1 medium (glucose and phosphate-free medium) supplemented with 15% fetal bovine serum. Cleavage rates for embryos cultured in the two culture media were different (47% vs. 63% in TCM-199 and P-1, respectively). Also, four Grade 1 embryos were surgically transferred into the oviducts of four recipient mares (one embryo/mare) at 48 hours post-ICSI, with three pregnancies (75%) developing as ultrasonically demonstrated by the presence of an embryonic vesicle in the uterine body by day 16 post-ICSI. On June 23rd one live filly was born after 328 days of gestation and subsequently, a second healthy filly was born after 319 days of gestation. To our knowledge, this is the first report of live foals resulting from in vitro fertilization (via ICSI) of in vitro matured oocytes recovered from pregnant mares using an efficient, repeatable transvaginal ultrasound-guided procedure.     

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.     

影响卵母细胞胞浆内单精子注射效率的精子相关因素

卵母细胞胞浆内单精子注射(ICSI)技术作为辅助受精的一种手段,自从出现以来,就显示出广泛的应用前景.ICSI将体外受精和胚胎的显微操作技术相结合,大大降低对精子质量的要求,在畜牧业生产实践和哺乳动物生殖生理的基础研究中都有着十分重要的意义.然而,ICSI过程中的每一个环节都可能影响到其效果,最主要和最根本的影响因素是精子和卵子本身的状态.论文就精子有关方面的因素,简要综述了精子顶体与核周鞘、细胞骨架与中心粒、DTT处理和卵母细胞激活因子(SOAF)等对ICSI的影响.     

Use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities

In some mares with lesions of the reproductive tract, embryo collection and survival rates are low, or collection of embryos is not feasible. For these mares, oocyte transfer has been proposed as a method to induce pregnancies. In this report, a method for oocyte transfer in mares and results of oocyte transfer performed over 2 breeding seasons, using mares with long histories of subfertility and various reproductive lesions, are described. Human chorionic gonadotropin or an implant containing a gonadotropin-releasing hormone analog was used to initiate follicular and oocyte maturation. Oocytes were collected by means of transvaginal ultrasound-guided follicular aspiration. Following follicular aspiration, cumulus oocyte complexes were evaluated for cumulus expansion and signs of atresia; immature oocytes were cultured in vitro to allow maturation. The recipient's ovary and uterine tube (oviduct) were exposed through a flank laparotomy with the horse standing, and the oocyte was slowly deposited within the oviduct. Oocyte transfer was attempted in 38 mares between 9 and 30 years old during 2 successive breeding seasons. All mares had a history of reproductive failure while in breeding and embryo transfer programs. Twenty pregnancies were induced. Fourteen of the pregnant mares delivered live foals. Results suggest that oocyte transfer can be a successful method for inducing pregnancy in subfertile mares in a commercial setting.     

单精注射法生产转基因小鼠的研究

精子胞质内显微受精技术（ICSI）作为辅助受精的一种手段,将体外受精研究和胚胎的显微操作技术结合起来,比以往对精子质量的要求大大降低,使其无论在畜牧业生产实践还是在哺乳动物的生殖生理基础研究中都有着十分重要的意义。本研究用小鼠精子及小鼠精子与GFP基因孵育后对小鼠卵母细胞进行ICSI,获得子代鼠。提取鼠尾基因组DNA,应用PCR、Southern blot进行整合检测。在发育至成年的11只小鼠中经PCR和Southernblot检测到3只阳性（27.3%）。结果表明,利用ICSI技术可以高效地生产转基因小鼠。     

The Development and Application of the Modern Reproductive Technologies to Horse Breeding

Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallion semen, and parallel improvements in ovulation induction and synchrony, exogenous gonadotrophic stimulation of multiple fertile ovulations and simplified, more efficient methods for non-surgical transfer of embryos to recipient mares, coupled with relaxation of breed society registration restrictions, have together contributed to a similar upsurge in the application of embryo transfer to all breeds and athletic types of horses worldwide, with the continuing and notable exception of the Thoroughbred. Although conventional in vitro fertilization remains something of an unjumped fence in equids, other modern breeding technologies like hysteroscopic low-dose insemination, fluorescence-activated sex sorting of stallion spermatozoa, between-species embryo transfer, embryo freezing and bisection, transvaginal ultrasound-guided oocyte collection, intracytoplasmic sperm injection for fertilization (ICSI), gamete intrafallopian transfer (GIFT) and now nuclear transfer (cloning), have all been applied to equids with encouraging success. Cloning, especially, holds enormous promise for the Sporthorse industry to re-create champion geldings in stallion form for breeding purposes.     

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.     

Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.     

Effect of time of oocyte collection and site of insemination on oocyte transfer in mares

The objective of the study was to compare embryo development rates after transfer of oocytes collected 22 or 33 h after hCG injection into recipients inseminated within the uterus or the oviduct. Oocytes were collected at approximately 22 or 33 h after hCG injections and incubated for approximately 16 or 1.5 h, respectively, before transfer. Intrauterine inseminations using 1 x 10(9) progressively motile sperm were done approximately 12 h before and 2 h after transfer. For intraoviductal inseminations (gamete intrafallopian transfer [GIFT]), semen was centrifuged through a Percoll gradient, and 200,000 progressively motile sperm were transferred with oocytes into the oviduct. Time of oocyte collection (22 or 33 h) after hCG injection did not affect embryo development rates (17/25, 68%, vs 12/23, 52%, respectively; P = 0.40). When results from oocyte collections at 22 and 33 h after hCG were combined, oocyte transfer with intraoviductal vs intrauterine insemination resulted in similar (P = 0.70) embryo development rates (12/22, 55%, and 17/26, 65%, respectively). However, the interaction between time of oocyte collection and site of insemination tended to be significant (P = 0.09), suggesting that GIFT using oocytes collected at 33 h after hCG may not be as effective as using oocytes collected at 22 h after hCG. Because intraoviductal insemination requires a low number of sperm, GIFT could be used in cases of male subfertility, frozen semen, or sexed sperm.     

Fertility of mares after unilateral laparoscopic tubal ligation

OBJECTIVE: To develop a technique for laparoscopic tubal (oviductal) ligation and to evaluate pregnancy rates for mares that ovulated ipsilateral or contralateral to the ligated oviduct. STUDY DESIGN: Randomized prospective clinical trial comparing pregnancy rates after unilateral laparoscopic tubal ligation. ANIMALS: Twelve mares of light horse breeds. METHODS: One oviduct in each of 6 mares was surgically ligated with a laparoscopic technique; 6 other mares served as nonligated controls. Mares with unilateral tubal ligations (UTL) were inseminated with 500 million progressively motile sperm during 1 cycle when the dominant follicle was ipsilateral to the ligation site and 1 cycle when the dominant follicle was contralateral to the ligation site. Control mares were bred during 2 cycles regardless of the side of the dominant follicle. Pregnancy examinations were performed on days 12, 14, and 16 after ovulation by transrectal ultrasonography. RESULTS: None of the mares became pregnant when ovulations occurred from the ovary adjacent to the ligated oviduct. All 6 mares became pregnant on the first cycle when an ovulation occurred from the opposite ovary. Control mares became pregnant on 10 of 12 cycles (83.3 %). CONCLUSIONS: UTL was completely effective in preventing pregnancy when ovulation occurred ipsilateral to the ligation site. The surgical procedure did not interfere with the establishment of pregnancy when ovulation occurred from the contralateral ovary. CLINICAL RELEVANCE: UTL may be a clinically useful procedure for preparing a recipient mare for gamete intrafallopian transfer. The recipient mare could be allowed to ovulate and UTL would prevent fertilization of her oocyte but would not interfere with normal corpus luteum formation. The donor oocyte could be placed into the oviduct contralateral to the UTL site.     

犬卵母细胞体外成熟及其体外受精研究进展

犬科动物胚胎对于改进犬科珍贵物种及保护濒危犬种十分重要,因此对犬科动物体外受精(IVF)以及辅助生殖(ART)的研究逐渐变得必要.通过体外受精和核移植已经成功获得能够发育的犬科动物胚胎,克隆的犬胚胎移植到受体母犬能成功产仔,但是这一技术的效率仍然很低.主要原因是犬科动物胚胎在体外发育能力很低.犬卵母细胞早期发育和其他动物不同,犬卵巢排出的卵母细胞处于生发泡(GV)期,在输卵管中恢复减数分裂.犬卵母细胞核难以观测并且不容易确定排卵时间,所以很难确定精子入卵的确切时间,并且多精受精的情况时常发生.回顾犬卵母细胞体外成熟(IVM)和体外受精取得的进展,对于提高其胚胎生产效率很有必要.     

The effect of liposomes composed of phosphatidylserine and cholesterol on fertility rates using frozen thawed equine spermatozoa

The objective of this study was to determine if the addition of liposomes composed of phosphatidylserine (PS) and cholesterol (CH) to equine sperm would improve pregnancy rates after sperm were cryopreserved. Ejaculates from four stallions, collected every other day during May and June were split, treated with PSCH liposomes or HBS (Hepes Buffered Saline) and cryopreserved. Fifty-two mares were bred over eighty estrous cycles with the frozen-thawed semen. The one cycle pregnancy rates of the mares inseminated with semen treated with liposomes (45%) were similar to mares inseminated with control semen (48%; p>0.05). Thus, at least at the levels used, PSCH liposomes added to equine sperm did not improve pregnancy rates of mares inseminated with cryopreserved semen.     

Somatic Cell Nuclear Transfer in Horses

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo -matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.     

Contagious Equine Metritis: A Review

Contagious equine metritis is a highly contagious genital infection of mares, spread venereally, and was first described in 1977. Although most contagious equine metritis outbreaks involved Thoroughbreds, infection in other breeds has also occurred. The disease has been reported in Europe, Australia and the United States. In Canada, contagious equine metritis has been designated a reportable disease under the Animal Disease and Protection Act.Contagious equine metritis is characterized by an endometritis and infertility and infected mares show no signs of systemic infection. Clinical signs have not been observed in stallions. An asymptomatic carrier state exists in both mares and stallions.Infected mares respond clinically to the topical and parenteral administration of antibacterial drugs. However, a proportion of mares remain carriers of the contagious equine metritis organism. Treatment of stallions is successful. Haemophilus equigenitalis has been proposed as the species name of the Gram-negative, microaerophilic coccobacillus.Sample collection and laboratory methods for the diagnosis of contagious equine metritis are described.     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

In Vitro Production of Equine Embryos

The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.     

Equine viral arteritis

Equine viral arteritis is reviewed with specific reference to clinical features, etiology, transmission, diagnosis, epidemiology, and current methods for the control of this disease. There is evidence of variation in pathogenicity among strains of equine arteritis virus. Virus transmission occurs primarily by the respiratory and venereal routes during the acute phase of the infection. The long-term carrier stallion appears to play a major epidemiological role in dissemination and perpetuation of the virus. Unlike the stallion, the carrier state has yet to be demonstrated in the mare or foal. A commercial modifiedlive equine arteritis virus vaccine has been shown to be safe and efficacious for stallions and mares. The disease can be controlled by identification and isolation of carrier stallions, immunization of seronegative stallions, and by restricting the breeding of equine arteritis virus-shedding stallions to equine arteritis virus vaccinated or seropositive mares.     

Physiology and endocrinology symposium: evidence that oviduct secretions influence sperm function: a retrospective view for livestock

The mammalian oviduct has long been recognized as an organ essential for successful reproduction. Bovine, ovine, porcine, and equine animal models have offered clear advantages for oviduct study related to gamete physiology, fertilization, and early embryonic development. Livestock species are amenable to surgical alteration of the reproductive tract, estrous cycle manipulation, gamete cryopreservation, and AI, as well as in vitro fertilization and embryo production. Although most reproductive technology developed for livestock was intended to benefit production animal agriculture, these techniques are a treasure trove of tools for researchers to better understand how the oviduct influences gamete function. Oviduct secretions obtained from in vitro tissue cultures or via indwelling oviduct catheters have been used for analyses to define the protein, lipid, carbohydrate, enzyme, and electrolyte compositions of the secretions during the estrous cycle or in response to hormone treatment. Oviduct secretions or components purified from them have also been used in in vitro assays to assess their ability to bind to sperm, influence sperm viability, motility, sperm capacitation, the acrosome reaction, sperm-egg binding, and egg penetration, as well as subsequent embryonic development. Compelling data have emerged which show that the composition of secretions differs during the estrous cycle and that their composition differs whether they originate from the ampullary or isthmic regions of the oviduct. These differences in composition are functionally relevant and associated with different responses by sperm. Evidence indicatess that oviduct-specific glycoproteins, glycosaminoglycans, carbohydrates, norepinepherine, catecholamines, heat-shock protein, and osteopontin are components of the oviductal milieu that have the capacity to modulate sperm function. Future research on the livestock oviduct will likely define the role that oviduct secretions have in modulating sperm function and how these modifications ultimately affect fertilization and embryo development.