Elsevier

Theriogenology

Volume 59, Issue 1, 1 January 2003, Pages 151-170
Theriogenology

Embryo technologies in the horse

https://doi.org/10.1016/S0093-691X(02)01268-2Get rights and content

Abstract

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 °C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 μm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medicine, assisted reproductive techniques have been developed for the older, subfertile mare. Transfer of in vivo-matured oocytes from young, healthy mares into a recipient’s oviduct results in a 70–80% pregnancy rate compared with a 30–40% pregnancy rate when the oocytes are from older, subfertile mares. This procedure can also be used to evaluate in vitro maturation systems. In vitro production of embryos is still quite difficult in the horse. However, intracytoplasmic sperm injection (ICSI) has been used to produce several foals. Cleavage rates of 60% and blastocyst rates of 30% have been reported after ICSI of in vitro-matured oocytes. Gamete intrafallopian tube transfer (GIFT) is a possible treatment for subfertile stallions. Transfer of in vivo-matured oocytes with 200,000 sperm into the oviduct of normal mares resulted in a pregnancy rate of 55–82%. Oocyte freezing is a technique that has proven difficult in most species. However, equine oocytes vitrified in a solution of ethylene glycol, DMSO, and Ficoll and loaded onto a cryoloop resulted in three pregnancies of 26 transfers and two live foals produced. Production of a cloned horse appears to be likely, as several cloned pregnancies have recently been produced.

Introduction

The use of embryo transfer and the development of embryo technologies for the horse have increased steadily over the past two decades. The techniques of embryo collection and transfer were developed in the late 1970s, and, today, the collection and transfer of fresh and cooled embryos are permitted in most breeds. The leading countries involved in equine embryo transfer include the United States, Argentina, and Brazil. Other countries using embryo transfer to a lesser extent include Australia, Canada, Italy, Germany, and France. The unwillingness of most breed registries to allow multiple registration of foals in a given year seriously limited the widespread use of embryo transfer. However, in 2002, the largest breed registry in the United States, the American Quarter Horse Association, has approved the unlimited registration of foals from a mare during a given year using embryo transfer. This will more than double the number of embryo transfers performed each year in the United States.

The major candidates for embryo transfer include older mares with poor reproductive histories that are unable to produce a foal by conventional natural mating or artificial insemination and show mares that are competing in racing, polo, or other performance events.

Although embryo collection and transfer is a common technology used in the horse industry, other techniques such as superovulation, embryo freezing, in vitro fertilization (IVF), oocyte transfer, gamete intrafallopian tube transfer (GIFT), and oocyte freezing are still not commercially available and are emerging technologies.

Section snippets

Embryo collection and transfer

The majority of embryos collected from donor mares are from spontaneous single ovulating mares. Embryos are generally recovered 7 or 8 days after ovulation (Day 0=day of ovulation). Factors that affect embryo recovery include day of recovery, number of ovulations, age of the donor mare, and quality of semen [1]. Mean embryo recovery per cycle from single ovulating mares in commercial embryo transfer programs is approximately 50%. Mares that spontaneously double or triple ovulate during a given

Cooled, transported embryos

One of the major changes in equine embryo transfer over the past several years is the ability to store embryos at 5 °C. Cooling and storing of embryos at this temperature permit shipping of the embryos to a centralized station for transfer into recipient mares. Many breeders and practitioners are unable or unwilling to acquire, house, and manage recipient mares. Therefore, at least in the United States, there are several large recipient stations that handle shipped embryos. Procedures for

Frozen embryos

The transfer of frozen equine embryos has lagged well behind that of bovine embryos for both biological and man-made reasons. Most of the equine breed registries have not approved the registration of foals from transfer of frozen–thawed embryos. Furthermore, because superovulation is not routinely used, there are very few occasions where extra embryos are obtained such that freezing would be of benefit. Biologically, the equine embryo is unique in that it forms an acellular protein membrane

Superovulation

Currently, the majority of embryo recoveries performed are from spontaneously single ovulating mares, resulting in an embryo collection in 50% of the attempts. One of the major costs of embryo transfer is maintenance of recipient mares that are programmed but not used for embryo transfer. One method to substantially decrease the cost is to increase the number of embryos recovered per donor through induction of multiple ovulations. Other advantages of ovarian stimulation include an increase in

IVF

In several livestock species, as well as humans, in vitro-produced embryos are quite common. However, in the horse, IVF resulting in the production of embryos is not a routine procedure. This technique would be helpful for mares that have fertility problems and are unable to provide an embryo. Further, IVF could be used in a breeding program involving stallions with low sperm numbers or poor semen quality and as a laboratory test for evaluation of frozen semen. The ability to produce equine

Cloning

Somatic cell nuclear transfer (cloning) has been successfully performed in several species. Nucleated recipient oocytes are fused with somatic cells by using Sendai virus, electrofusion, or by directly injecting somatic cell nuclei into the recipient cytoplasm. Fusion of horse oocytes with adult somatic donor cells has been reported with fusion rates from 20 to 67% [58], [59]. Fusion rates of up to 82% were reported using electrofusion in combination with Sendai virus [60]. However, in all

Oocyte transfer

The older mare with a history of subfertility is often enrolled in an embryo transfer program. Unfortunately, many of these mares fail to provide embryos or pregnancies from embryo transfer. Success in an embryo transfer program is dependent on (1) the ability of the mare to ovulate, (2) the act of fertilization in the oviduct, (3) transport of the embryo into the uterus, (4) survival of the embryo until the day of recovery, and (5) pregnancy rate after transfer of the recovered embryo into a

Gamete intrafallopian tube transfer

Although oocyte transfer has proven to be a successful technique for obtaining pregnancies from subfertile mares, the procedure requires semen from fertile stallions. In contrast, GIFT can be used for stallions with low sperm numbers or perhaps in situations in which frozen semen is in limited supply and/or for use with sexed semen. During GIFT, oocytes and sperm are transferred into the oviduct. Low numbers of sperm are required for intraoviductal insemination [69]. The first successful GIFT

Oocyte freezing

A reliable cryopreservation protocol could preserve genetic material from valuable mares that die unexpectedly or those that must be euthanized. Cryopreservation of oocytes has been successful in several mammalian species including mice, cattle, humans, and horses. Recently, blastocyst formation rates after IVF of vitrified bovine oocytes have been found to be similar to those from non-vitrified oocytes [73]. Studies on cryopreservation of equine oocytes are quite limited. Survival rates of 16%

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