Producción Equina, Departamento de Producción Animal, Facultad de Agronomía y Veterinaria, Universidad Nacional de Río Cuarto. (5800) Río Cuarto, ARGENTINA. (llosinno@ayv.unrc.edu.ar)

Introduction

When man domesticated horses, about 5,000 years ago, he limited their foraging migrations during the seasons of the year and started to direct their reproduction. Evidence exists that certain people of Asia Minor, round 2,500 B.C., had breeding herds and castrated the males that they did not consider adequate as stallions.

Therefore reproductive biotechnology, even though empirically used, is not a new tool to man in his herd management activities.
One of the aims of biotechnology applied to reproduction is to increase reproductive efficiency, e.g. the number of descendants of an animal over a certain period of time. Small increments in reproductive efficiency have an enormous impact on production rates.
The first successful equine pregnancy with a cloned embryo is - at the time of writing this article – 60 days old and is the end product of more than 200 embryo transfers of processed ova and many early pregnancies which were lost.
This may well be the most scientifically relevant result published after the last International Symposium of Equine Reproduction, which took place recently in Colorado, USA.
Other interesting subjects discussed in this meeting were: drug induced lactation in mares that have not foaled to improve the efficiency of the production and transfer of embryos, the use of yearling mares as embryo donors and advances in molecular studies of the sperm.
Leaving aside all these interesting scientific advances, in this article we will present a summary of the main biotechnologies used in equine reproduction which can be applied to production systems with good results.

Artificial insemination (AI)
Though there are anecdotic references, the history of AI before the XVII Century is rather confused and not really reliable.
The first report of a birth by AI is attributed to the Italian monk Lázaro Spallanzzani, who in 1779, was able, by means of instruments, to impregnate a bitch and obtain a litter of puppies.
But it was not till the beginning of the 20th Century that AI came to be considered a technique with a great future in animal production.
Contrary to the acceptance of AI use in other species, especially cattle, the number of horses in AI programs has grown very slowly. There are several reasons for this, one of them is the conservative attitude of many breeders, maybe influenced by the policy of the breeders of one of the most widespread and economically important horse breeds, Purebred Racing Horse Breeders which do not allow either AI or ET (embryo transfer) to be used in breeding Race Horses.
Fear of fraud, in such valuable animals, has for many years been considered the reason for this, but the use of paternity tests by means of genetic markers before the final register of the offspring is part of the new regulations. Most pure breeds have made the use of these techniques mandatory for the final identification of registered animals.
It is in the decade of the ‘80s with the use of ultrasound and embryo transfer, that AI really increases markedly and obtains the support of the breeders of horses used in Sports, specially trotters, hurdlers and polo ponies.
The interest in the use of AI has grown over the last few years due to the risk of transmission of venereal diseases with natural reproduction and the risk this entails for stallions of great value.
Also, AI allows the use of old stallions or those that have muscular or articular problems and allows the service of a large number of mares with few mounts and therefore leads to a reduction of operational costs.
The pregnancy rates with any of the AI systems used (fresh, refrigerated or frozen semen) have recently increased sufficiently to provide commercially acceptable confidence ratios.
Artificial insemination is a technique which is expanding markedly at an international level and hundreds of thousands of mares are inseminated every year.

Artificial insemination with fresh semen
Basically in this technique the ejaculate is collected and assessed and then divided into doses that contain a minimum of 500 million sperm with progressive motility, it is then inseminated into the mares, 30-90 minutes after its collection. This system is ideal when stallions must cover 2 or more mares a day during the reproductive season and they develop muscular or articular problems or when working with a group of synchronized brood mares.
Between 5-10 mares can be inseminated with an average ejaculate and the pregnancy rates are equal to or slightly higher than (10%) those obtained with natural methods.

Artificial insemination with refrigerated semen
This system depends on the quality of the semen and its production by each stallion which varies with external temperatures, but it permits greater variation as far as management techniques.
Generally speaking semen can be refrigerated at 4-5 C during 24-48 hours, but acceptable pregnancy rates have been reported even after 80 hours using special diluents, keeping to a temperature reduction curve and maintaining the temperature.
Logistics are important when using this system since there must be synchronization between the management of the mares to be inseminated and the collection and transport of the semen. Results vary from one stallion to another, but if protocols are followed and semen refrigerated for 24 hours and used a maximum of 24 hours previous to ovulation, the pregnancy rates obtained are similar to those of natural service.
This system is currently one of the fastest growing, even allowing semen to be sent from one continent to another, especially in the cases of stallions whose semen, due to its characteristics, cannot be frozen using current methods.

AI with frozen semen
From 1957 when this method was first reported by Barker and Gandier till the ‘80s this method has had little impact on western production systems.
Currently, it has been calculated that more than 10,000 mares in Europe and 350,000 in China are inseminated using frozen semen.
Currently, there are more than 20 protocols for cryopreservation of equine semen, these use different temperature curves, diluents, containers, thawing techniques, etc.
The pregnancy rates using these different protocols are very variable and it is difficult to compare procedures.

Some of the advantages of AI with frozen semen are:
1) It is cheaper, safer, and more practical to transport a container with liquid Nitrogen and sufficient semen to inseminate hundreds of mares than to transport a stallion.
2) The costs of covering mares, transporting mares and insurance are reduced, the animals are subjected to less stress, and run less risk of contracting diseases or of suffering accidents, especially in the case of newly foaled mares, etc.
3) Stallions can continue to be used as sires even when participating in cups, shows or other events, recovering from some disease or even years after death.
4) The use of genetically inferior stallions is much reduced.
5) The semen of genetically superior stallions is preserved and more accessible.
6) Semen can be preserved in liquid Nitrogen (-196 C) during an indefinite number of years.

Many of the currently mentioned “disadvantages” of using frozen semen are, in general, due to incomplete information on the subject or not related to the technique but to mismanagement of it and the influence of regulations on its use.
Moreover, there is considerable variation between stallions as far as the capacity of their sperm to survive at below cero temperatures.
Pregnancy rates of between 0 – 70% have been reported, however for most stallions the pregnancy rate for one breeding season varies between 20-40%, though low this is considered acceptable at this point in time.

Embryo transfer (ET)
Embryo transfer is the technique by means of which an embryo is collected (by a non-surgical method such as uterine lavage) from a donor mare, either inseminated or served 6-9 days post-ovulation and this embryo is transferred (surgically or non-surgically) to the uterus of a recipient mare who has been previously hormonally synchronized.
W. Heape published the first report of a successful embryo transfer in rabbits in 1891, but it was only in 1974 that Oguri and Tsutsumi reported the first pregnancy by ET in horses.
According to available data, the first ETs in horses were carried out in Argentina during the late ‘80s. The first registers and reports in commercial centres were during the 1989 breeding season, mainly with Polo Pony mares.
Currently, there are at least 6 ET centres in operation, the number of donor mares has grown steadily and now is round 300, there are 40 stallions used in the programs and 600 receptor mares are impregnated every season.

ET is capable of increasing reproductive efficiency, but its limitations must be noted. Since it has led to excessive expectations.
Its main aims are:
1) To increase the number of foals produced each year by carefully selected mares.
2) To obtain foals from mares that are still actively competing.
3) To obtain foals from mares that cannot themselves go through a pregnancy, eg. due to a pelvis fracture.
If conditions are ideal, as far as donors, recipients, fertile stallions and trained personnel, a embryo recovery rate of 50-80% can be expected and a rate of successful transfers of 50-80%, which would give a pregnancy rate of 25-65%.

Using this technique it is possible to obtain, on average, 4 or more foals a year from a mare. However, in Argentina we have evidence of a Polo mare from which 10 pregnancies were obtained during one breeding season.

Refrigerated embryos

This system reduces the pressure on the process of synchronizing the recipient mares and allows for a greater time window for the transfer. The results obtained with embryos refrigerated for 12-30 hours in special media and containers are similar to those obtained with directly transferred embryos, therefore, in several countries this has now become a routine practice.

Frozen embryos
The birth of the first foal from a cryopreserved ( –196? C) embryo was reported in 1983. Though there have been some promising results in Argentina, this technique, in horses, has not attained the degree of development or widespread use it has in cattle.

Vitrified embryos
Vitrification is a variant of cryopreservation. In this technique high concentrations of cryoprotectors are used to avoid the formation of crystals which are damaging to the embryos. Most of the protocols use temperature curves that descend very abruptly and do not need special freezing machines, this markedly decreases the cost of this technique.
Hochi et.al were the first, in 1994, to report an equine pregnancy after transferring vitrified embryos.

Intratubaric transference or Gamete Intrafallopian Transfer (GIFT)
In this technique the oocytes of a donor mare are collected by means of intrafollicular aspiration guided by ultrasound, they are then transferred to the oviduct of the recipient mare together with a minimum dose of sperm. The follicules of the recipient mare have also been submitted to aspiration.
In 1987 McKinnon reported the first foal born using this procedure and currently it is used commercially.

Intracytomplasmic injection of sperm (IIS)
This is a variant of in vitro fertilization in which a mature oocyte receives a sperm cell injected into its cytoplasm by means of a micropipette connected to a micromanipulator.
This technique was developed in 1992 to treat male infertility problems in humans and has had such a great impact that at least 1/3 of all assisted pregnancies in humans make use of this technique.
The first report of a foal born using this procedure was in 1987, the mature oocytes used were taken from a dead mare.
In March 1998, McKinnon et al, in Australia, reported the birth of a foal using this technique but the mature oocytes from the donor mare were transported 200 km to the University lab, fertilized and maintained in vitro, then transported once more to a field clinic and transferred into the recipients oviduct.
In June 1998, researchers at the University of Louisiana reported the birth of 2 fillies, from immature oocytes collected by transvaginal aspiration of pregnant mares, matured in vitro, fertilized and surgically transferred to a recipient.

Sperm sexing
By means of a technique which detects differences in the DNA content, sperm cells that contain either X or Y chromosomes can be separated.
On the 6th of August of 1998 the first foal obtained from sexed sperm was born and at this point in time there have been many reports of fillies obtained by the use of sexed sperm.
Argentina is one of the few countries in which this technique, applied to both cattle and horses, is in full development.

Conclusions
Over the last few years reproductive efficiency in horses has greatly benefited from the use of biotechnologies such as AI and ET which are of widespread use in those systems which approved and regulated their use. A good example is Polo Pony breeding in which high performance mares, after the playing season is over, go as donors to ET centers. In this manner many foals have been obtained from good playing mares without affecting their playing.
Cryopreservation of horse semen is currently an available tool with many advantages, only limited by Breeders’ Associations’ regulations, since some forbid its use.
At this point in time only some sporting horse breeds are taking full advantage of the biotechnologies which permit the maximum use and sale of quality genetic products at a low cost, thus allowing the preservation of superior genetic qualities.