Breeding beyond 2000
Nigel Perkins, BVSc, MS, Dip ACT, FACVSc
Equine Reproduction, Massey University
This paper represents an attempt to review some of the current and recent topics of research and development in the field of equine reproduction. In addition, developments in the broader field of assisted reproductive techniques will be introduced and their relevance to the equine breeding industry discussed.
The breeding vision: one possible scenario
The year is 2040. You are sitting at your compu–TV, mousing through the current sires catalogue evaluating pedigree and performance data on stallions from around the world. Full motion images of the stallions you are interested in are flashed in intricate detail onto your wall size screen. You download data on the several stallions you are most interested in for closer comparison. You slowly work your way through data on growth and development, bony structure, veterinary reports, computerised analysis of semen quality, records of all progeny and their performance, indices reporting on progeny growth and development, mares mated to the stallion previously, and pregnancy and foaling rates. You narrow down to 2 stallions, one in New Zealand and one in New York state, USA. The USA based stallion is currently available only in doses of frozen semen. The New Zealand stallion is available in either fresh, chilled semen or frozen semen. You bring up the order screen on your world wide web interface and it asks you whether you want sexed semen. You naturally accept this offer and choose a filly foal. The order is then placed through your computer with the click of a button.
You change programs on your computer, bringing up the mare record and noting she was last in heat 2 weeks ago. You hit the on the button for the remote telemetry devices. The telemetry sensors mounted at intervals around the pasture record internal body temperature and vaginal electrical resistance at 5 minute intervals from the pea sized device permanently implanted beside her anterior vagina. The signals from all mares in the pasture are recorded simultaneously and each mare identified individually with the data automatically being collated and presented in graphical form. You note the typical appearance of the spike in core temperature and the drop in electrical resistance around the time of ovulation as it occurred during the last heat period and as you look the first of the readings for today comes in and is placed on the graph. Another button and you are in your veterinarians menu page on the local web. You select the visit option and type in the details on your mare, requesting a routine prebreeding examination for tomorrow, ovulation control regime and artificial insemination for the Thursday afternoon in 2 weeks time. The vet arrives and examines the mare using ultrasound to determine the presence or absence of any uterine infection. The mare is then given an injection of GnRH antagonist to shut down her own hormonal production for a short period of time. This also ensures that any follicles on the mares ovaries will regress over the course of the next several days. A small implant containing progesterone in a slow release form is then injected into her neck. The combination therapy is designed to inhibit the mares hormonal and ovarian activity for several days and then fade to allow the mares own hormones to increase again and stimulate her ovaries to produce follicles again in preparation for breeding. The vet speaks into her notebook computer microphone to record that the mare will need a revisit two days before the planned breed day for the next injection. You confirm the semen order and receive acknowledgement.
On the Tuesday evening, the vet arrives and injects the mare with a GnRH agonist implant designed to ensure ovulation occurs between 36 and 48 hours later. The semen shipment arrives early on Thursday morning and the vet is back to AI the mare. You check the telemetry graph with your vet. The spike and trough in the two ovulation indices are just beginning to appear and the graphic readout assures you that ovulation is 95% likely to occur during the next 12 hours. The semen shipment canister is opened and the two gel coated, semen pellets removed. Both are placed immediately into the uterus of the mare. One pellet is designed to dissolve immediately and release semen and the other more slowly to release motile spermatozoa over a 12 to 24 hour period following insemination. This ensures a continual supply of fresh sperm during the period of maximal fertility of the mare. By Thursday evening you can clearly see the graphical evidence of ovulation on the telemetry recordings and you relax confident that you have given the mare every chance of conceiving. The early blood test is done 7 days after breeding and confirms that the mare is pregnant and the follow up ultrasound done 2 weeks later shows you a tiny embryo with a just visible heart beat. A further ultrasound test at 50 days confirms that your mare is indeed carrying a filly foal. All of the things described above are either currently available or currently under research and development in some domestic animal species around the world. It is quite likely that the scenario in 40 years time will actually be more advanced than the picture painted in this paper.
The remainder of this paper will deal with several specific topics, highlighting recent developments and possible future applications.
Ultrasonography has had a huge impact on equine breeding in three main areas: determining when to breed mares, management of endometritis and pregnancy diagnosis. The potential applications of ultrasound technology are constantly expanding due to the combination of technological advances and continuing developments in skill. The following uses are either gaining momentum currently or represent potential applications for ultrasonography.
Ultrasound images are currently inspected visually for relatively simple parameters such follicle diameter, follicle shape, presence or absence of uterine oedema. Computerized image analysis is currently being performed on videotaped ultrasound images to look at detailed analysis of pixel intensity, follicle area, uterine echotexture. Digital image analysis allows appreciation of much more detail. It is very likely that future ultrasound units will contain computer hardware which will be capable of automatically evaluating several parameters. It is likely that measurements such as follicle diameter, follicle shape, follicle wall thickness, follicle fluid echotexture, uterine echotexture, uterine diameter, cervical echotexture, cervical diameter, will be used in combination to produce a score or index which will allow much more accurate prediction of ovulation within the next 12 or 24 hour period.
Ultrasonography has become an essential tool in the diagnosis and management of endometritis or infection of the uterus of the mare. This is based primarily on the detection of fluid within the lumen of the uterus and the appearance of fluid. It is highly likely that continued advances will be made for some time yet in the application of ultrasonography for this purpose. One area in particular is in the management of mares immediately after breeding. It is now known that mares that are more likely to become infected, will accumulate fluid within the uterus soon after breeding. Initially this fluid may be clear but within a short period of time, it will be followed by bacterial infection. Monthly ultrasound examinations during pregnancy are now commonly performed on individual mares considered at risk of placentitis and pregnancy loss. The measured thickness of the placenta and uterine wall for example can be used to determine whether a bacterial placental infection might be occurring. If such a problem is diagnosed and treated early it is possible to eliminate the problem and result in the pregnancy continuing and producing a live foal. In the past, such problems frequently resulted in loss of the pregnancy. A logical extension of this which was developed by researchers in the USA in the mid 1980s, is the use of ultrasonography to gather information on the normal or abnormal development of the foetus during pregnancy. This becomes particularly important in the latter stages of gestation as foaling approaches. Several parameters can be measured (placental thickness, foetal movements, foetal heart rate, foetal size, response to stimuli) and used to calculate an index termed a foetal biophysical profile. This profile can be used to identify foals which might be at risk of complications during or immediately after birth. Such foals can be managed intensively as soon as they are born. It is highly likely that individual, valuable mares will be managed in this way soon. Immediate implementation of intensive care is likely to result in a higher survivability of foals born with problems.
Foetal sexing by ultrasonography was developed in cattle in the mid 1980s and rapidly extended to the horse. Skilled clinicians from Kentucky are now performing between 600 to 1,000 fetal sexing examinations each year across the USA, in Ireland and even in Australia. The examination is based on detecting the genital tubercle which is the foetal precursor of the penis in the male and the clitoris in the female. The structure has a characteristic ultrasonic appearance and can be seen just caudal to the umbilical cord attachment in the male and just under the tail in the female. Examinations must be performed between 60-75 days of gestation although there is an alternative window of opportunity between 90-140 days. The technique requires considerable practice and skill but once familiar with it, results are very accurate. It is a great example of the potential of ultrasonography to provide us with more information.
It is possible that sending a blood sample to the laboratory may help determine the best time to breed a mare though this is less practical than the development of a simple ultrasound based method. Two possible candidates have been examined in the last few years. We are all familiar with the human pregnancy test kits sold over the counter in pharmacies around the country. These tests detect levels of a hormone in the urine which is only produced during pregnancy. Attempts have been made to produce a test which might detect an increase in the levels of Luteinizing Hormone (LH) in the urine of mares during oestrus. A large increase in LH precedes and results in ovulation or rupture of the follicle to release an egg during the mares heat period. Detecting this increase may allow mare owners to accurately predict the time of ovulation and therefore plan for breeding. However, a urine test for this particular hormone does not seem to work in the mare and this has not been successful. Recently, researchers in England looked at changes in blood levels of oestrogen, the hormone produced by large follicles which makes mares show signs of receptivity or heat. Levels of this hormone in the blood tend to rise just before ovulation and therefore this might be suitable as a predictor of ovulation, again offering use as a means of determining when to breed the mare. Again it does not seem to be reliable enough to be useful as a test in the real world. There appears to be so much variation between mares when measuring blood concentrations of hormones that it is difficult to identify a test which will work on all mares. However, it is quite possible that within any individual mare, following hormone levels may allow monitoring the change in hormone level within that mare and this may be quite useful. Is this practical? Consider the recent development of sophisticated techniques recently in the dairy cow industry which allow measurement of hormone concentrations in milk from an individual cow as the milk is flowing through a milk line from the cow to the bulk tank within the dairy shed itself. This is being tested as a possible means of providing information on whether a cow is pregnant or in heat. This sort of technology could possibly be developed to the point where scanning devices may measure hormone concentrations in blood vessels or in blood samples.
Most of our reproductive drugs have been around now for 10-20 years, including examples such as prostaglandin F2-alpha, progestagens, human chorionic gonadotropin, oestrogens. The most recent commercially applicable product has been the marketing of Ovuplant, a biodegradable implant containing a synthetic formulation of GnRH, which is currently marketed around the world for ovulation induction. A couple of other products show considerable promise for the future.
Recently researchers at Cornell University have stimulated world wide interest in the use of dopamine antagonists in mares in spring transition as a means of advancing the first ovulation of the breeding season. It appears the dopamine may play a role in the control of seasonal cyclicity in the mare and inhibiting the effect of dopamine in the brain of the mare may actually help her to begin cycling sooner than she normally would in spring. Several trials have now been performed and the results are interesting without being overwhelmingly positive. Time will tell whether this becomes the next great therapeutic breakthrough in equine seasonality.
Many researchers over the years have attempted to find a reliable, repeatable method of inducing multiple ovulations in mares. Unfortunately efforts to find a drug or regime that works well and repeatably, have not been successful to date. The most successful product so far is equine pituitary extract (EPE) which is made by obtaining pituitary glands from the heads of horses killed at slaughter houses and extracting the hormones from them. This is a very inefficient method and really only capable of producing a very small amount each year. However, researchers have been successful in producing synthetic forms of FSH and LH from other species and it is quite likely that synthetic forms of equine LH and FSH will be produced soon. Such a product could then be used to superovulate mares. Treatment could be used to increase the yield of embryos from donor mares in an embryo transfer program or simply to increase the chance of getting a problem mare pregnant. It could also be used to increase the chances of mares getting pregnant when being bred to a subfertile stallion.
Fresh and chilled semen technology
Artificial insemination(AI) using transported, chilled semen is becoming more popular around the world. It is now dominating the equine AI market. In the Swedish Warmblood industry, more mares have been bred in the last 3 years using chilled, transported semen than by either natural mating or fresh AI. In 1997, 45% of 4,220 mares were bred using chilled transported semen vs 27% by natural breeding, 24% by fresh AI and 4% by frozen semen.3
Although uniform standards and quality control measures for transported semen are more widely used than for frozen semen, there is considerable improvement yet to be experienced. The following general guidelines have been suggested for selecting a stallion to be used in a transported semen program.3
1. Stallion should be healthy, physically normal and in good body condition
2. Stallion should have large testicles and good libido
2. Good sperm production (35 billion per week)
3. Good semen quality: progressive motility >50%, >70% morphologically normal spermatozoa, concentration >100 million per ml.
4. Ejaculates free of bacterial pathogens on microbial culture of ejaculate to which antibiotic containing extender has been added.
5. Ejaculate should demonstrate at least 40% progressive sperm motility after 24 hours storage (chilled) at 4 to 6 C.
6. Each fresh semen AI dose should contain 500 million motile sperm and chilled semen AI dose should contain 1 billion motile sperm.
Stallions which fulfill these criteria can be expected to have per cycle pregnancy rates with transported semen very similar to those achieved with fresh AI or natural mating. Use of stallions who do not meet these criteria increases the risk of poor pregnancy rates with consequent damage to business relationships. Stallions which are borderline in one or more criteria may still be used in transported semen programs but it is recommended that semen from such stallions only be used if AI can be completed less than 12 hours following semen collection.
Failure to implement quality control standards is definitely having an adverse effect on results achieved with transported semen and really remains as the major area where improvements can be made immediately. Despite this, transported semen frequently produces per cycle conception rates which are similar or the same as achieved with natural mating or fresh AI.
The following areas are considered to be representative of the types of advances which can be expected in the field of transported semen during the early portion of the 21st century.
Advances in the field of frozen semen technology are likely to move utilization of frozen semen in the equine breeding industry towards the dominant breeding method as it is in the dairy industry.
At the moment, only a relatively small percentage of mares are being bred around the world with frozen semen compared to natural breeding or AI using either fresh or chilled semen. Breeders are understandably reluctant to embrace frozen semen when success rates are relatively low. At the moment average per cycle conception rates for frozen semen lie around 30-40% (range 0 to 70%). In contrast, AI with chilled, shipped semen is associated with per cycle conception rates of 60% pre cycle.5
There is much individual stallion variation in freezability. It is thought that about 25% of normal, fertile stallions have pregnancy rates using frozen semen which are comparable to those achieved with fresh or chilled semen. The remainder have reduced per cycle pregnancy rates when using frozen semen though some of these will have acceptable end of season pregnancy rates if mares are bred on repeated cycles. There does not appear to be any relationship between the fertility of a stallion using fresh semen and the fertility of that same stallion when using frozen semen.
In the dairy industry, if a bull does not freeze well, it is not likely to be a commercial success even if it is genotypically and phenotypically superior. Selection pressure has been applied over a prolonged period of time in the bovine industry which has by and large resulted in a population of bulls which produce semen able to achieve consistently high per cycle pregnancy rates using very uniform freezing techniques around the world. It is highly likely that some degree of selection pressure will be applied to stallions in the future.
There are few quality control standards which have been applied to the equine semen freezing industry. Operators freeze using many different approaches and thaw using one of several techniques. There is a wide variation in equipment standards, recording systems and general quality control. Contracts for using frozen semen vary widely and often have no in built guarantees or quality control. Widespread adoption of frozen semen will not occur until these issues are addressed and relatively uniform and clearly understood quality control and recording measures are implemented. For example operators recommend using between 200-300 million motile sperm (post thaw) per breeding dose and that a frozen semen batch only be used if it has a minimum of 30-35% progressive motility post thaw. Some individual operations are implementing good measures. Some operators currently evaluate every frozen semen batch with a Hamilton Sperm Analyser (computer assisted digitised analysis) to measure sperm concentration and progressive motility and also examine each batch post thaw with fluorescent microscopy to determine the percentage of thawed, frozen spermatozoa with intact acrosomes. Breeding doses are then based on a requirement of 300 million, motile, acrosome intact sperm in each dose. Straws are permanently marked with date of collection, stallion identification and freezing centre identification.
In contrast there appears to be reasonable agreement amongst operators about mare management and determining when to breed mares. The most common approach has been to intensively manage mares in order to inseminate with frozen semen during the window from 12 hours prior to ovulation to about 6 hours after ovulation. There is some support from Europe for once daily breeding of mares just prior to ovulation with frozen semen.
There are several areas where advances are likely to contribute to improvements in the success rate and consequently utilization rate for frozen semen.
The effect of these developments will be to produce a system which has all the benefits of frozen semen and which allows mares to be bred once daily in a manner similar to current management of mares for chilled semen. These developments will allow widespread adoption of frozen semen around the world in the equine breeding industry.
Developments in Assisted Reproductive Technologies (ART)
The following techniques may be considered as ARTs. Some or all of these are either currently being used in animal reproduction or have potential for the future.
A myriad of techniques have been tried in an attempt to reliably separate sperm based on gender and a successful technique has been described in the last decade or so, based on a cell sorting procedure called flow cytometry. Currently it appears that the technique is successful but very slow and is not capable at the moment of sorting sufficient numbers of sperm to allow animals to be inseminated. Sexed sperm have been used in IVF procedures and the first foal born from a breeding using sexed sperm was a filly called "Call Me Madam", born in Colorado on August 6, 1998. The company which sexed the sperm is XY Inc and is part owned by the Colorado State University Research Foundation. The company expects that sperm sorting for AI in the USA horse industry alone has the potential to earn in excess of $300 million annually with an equivalent potential market existing in pigs and cattle. Because the technique currently is quite slow, only a small amount of sperm can be sorted and Call Me Madam's dam was bred by placing the sexed sperm directly into her oviduct using a surgical approach through the flank. It is expected that the technique will be developed sufficiently to allow reliable sorting of sufficient numbers of sperm to allow breeding using AI and certainly for IVF procedures.
In-vitro embryo production (IVP)
IVP involves several steps which must be performed in precise order as follows:
Oocyte recovery: Unfertilized eggs (oocytes) must be recovered from mare ovaries. Oocytes may be recovered from the ovaries of horses killed for meat or other reasons, or aspirated from follicles on the ovaries of live, donor mares.
Advances in the past several years have seen IVP develop to the point where it is a commercial success in the bovine industry and there are several groups around the world which offer bovine IVP services for clients with valuable individual cows. However there appear to be problems with almost every step of the process when the technique is performed in the mare and this has delayed commercial application of the technique in horses. For example, it appears to be more difficult to actually harvest oocytes from the ovaries of mares than it is in the cow. Recently the most successful technique for oocyte aspiration in mares was reported by researchers at Louisiana State University in the USA where they aspirated oocytes from mares during early pregnancy. In addition it is more difficult to get sperm to the point where they are capable of fertilizing in a laboratory dish and also more difficult to actually have fertilization occur in a lab dish. The equine egg is also very dark in appearance and it is actually very difficult to tell if fertilization has occurred because the dark colouration of the egg prevents the changes associated with fertilization from being viewed through a microscope. Perhaps as a result of these difficulties, relatively few successes have been reported in the mare for IVP using what might be termed conventional IVF techniques. A research team led by Dr Eric Palmer of France was successful in the early 1990s in producing a live foal using oocyte aspiration, IVF and embryo transfer into the oviducts of recipient mares.
The development of ICSI techniques were heralded as a potential solution to some of these problems. ICSI involves aspirating a single spermatozoa into a very fine glass pipette (under the direction of sophisticated microscopes and manipulation equipment). The pipette is then gently pushed into the middle of an unfertilized oocyte and the single sperm cell pushed out into the cytoplasm of the oocyte, thus fertilizing the egg. This procedure allows the use of non-motile sperm, and bypasses many of the difficulties involved in trying to get spermatozoa to penetrate and fertilize an oocyte in a laboratory dish. In fact ICSI techniques have revolutionized embryo production in many species including infertile people and is also a major advance in the conservation of threatened wildlife species. The first ICSI foal was born in Colorado in 1996 ("Firecracker"). Since then two foals have been born in Australia using ICSI and researchers from Louisiana reported the birth of foals in early 1999 which were produced from ICSI management of in-vitro matured oocytes recovered from early pregnant mares.2 Still the technique is laborious and poorly successful as the following illustrative figures show:
263 ovarian follicles aspirated to recover 174 oocytes (66% recovery)
174 oocytes cultured in the lab and 86 mature to the point where fertilization can be attempted (49% maturation)
86 mature oocytes subjected to ICSI with 47 fertilized embryos resulting (55% fertilization)
47 embryos cultured in the laboratory for 48 hours and 31 of these continued to develop until the 48 hour point (66% survival)
31 embryos transferred into the oviducts of recipient mares using flank surgery and 4 mares were diagnosed pregnant using ultrasound examinations performed at about 15 days.
2 of the 4 mares subsequently delivered live foals at term with the remaining two mares undergoing pregnancy loss.2
Cloning is usually interpreted as the production of a complete individual from a single cell harvested from another embryo or individual. Cloning was reported in frogs in 1952, mice in 1977 and from early sheep and cattle embryos in the 1980s.4 Cloning achieved world wide publicity in February 1997 with the birth in Scotland of Dolly, a lamb cloned from mammary gland cells taken from an adult sheep. Dolly came from cells taken from the mammary gland of a 6 year old Finn-Dorset ewe. The process began with 277 reconstructed (cloned) eggs, of which 29 appeared to develop to a blastocyst stage and were implanted in to 13 recipients and a single lamb was born (Dolly). Dolly has had two pregnancies and given birth to apparently normal lambs.1,6
Cloning techniques are based on nuclear transfer which involves the use of at least 2 cells. The recipient cell is usually an unfertilized egg cell taken from an animal soon after ovulation. These cells are designed to begin developing after an appropriate stimulation. A researcher holds the recipient cell by fine suction under a microscope and using a very fine pipette, sucks out the chromosomes from the cell (chromosomes contain the DNA or genetic code for the cell). The donor cell (which contains the chromosomes from the adult animal) is then fused with the egg cell and this combination in some cases is followed by the egg cell beginning to divide and developing into an embryo with the genetic code from the adult animal. The resulting embryo can then be transplanted into a surrogate uterus.6
Cloning is a complex technique and remains far from completely understood. Only a small percentage of cloned embryos actually develop through to offspring and offspring appear to be at some risk of dying around the time of delivery. Several groups of researchers around the world are now trying to understand the processes which influence embryonic development and cell differentiation in order to be able to improve success rates and viability of cloned offspring. Tremendous advances are being made in this field and it is likely that cloning will soon be a commercially successful technique of replicating valuable individual animals. It has already been a spectacular success for some individuals for example a team of researchers at Ruakura in NZ have successfully cloned several calves from the last surviving Enderby Island cow ("lady") after several failed attempts to produce offspring from this same cow using embryo transfer.
1. Campbell K, McWhir J, Ritchie W, et al: Sheep cloned by nuclear transfer from a cultured cell line. Nature 385:810-813, 1997
2. Cochran R, Meintjes M, Reggio B, et al: Live foals produced from sperm injected oocytes derived from pregnant mares. Journal of Equine Veterinary Science 18:736-740, 1998
3. Darenius A. Experiences with chilled, transported equine semen. In Proceedings of the Annual Meeting of the Society for Theriogenology, Baltimore, MD, 1998 Dec, pp. 60-70
4. Griffin H. Roslin Institute Online [Web Page] 1997. Located at: http://www.ri.bbsrc.ac.uk/library/research/cloning. Accessed 1999.
5. Samper J. Why are pregnancy rates with frozen semen lower? In Proceedings of the Annual Meeting of the Scoiety for Theriogenology, Baltimore, MD, 1998 Dec, pp. 71-75
6. Wilmut I: Cloning for medicine. Scientific American 279:30-35, 1998