Interest in these technical breeding solutions increased after the many breed registries began accepting foal registration from cooled and frozen sperm and embryos. Although foals have been produced using all of these technologies, these technologies are not efficient for all genetic donors or all types of gametes or embryos. For example, although cooled and frozen stallion sperm are used routinely, sperm from some stallions do not survive cooling well (and cannot be used for cooled shipped semen) and sperm from even more stallions do not survive freezing. Similarly, although small embryos (6-6.5 day embryos) survive freezing quite well; larger embryos, that can be easily flushed 7-8 days after insemination, do not survive freezing well. And equine oocytes, regardless of source, do not freeze well.
The ability to utilize frozen semen from more stallions would have a great impact on the equine industry. Improving the cryosurvival rates of stallion sperm would benefit the industry, as sperm could be used to inseminate many mares; moreover, the ability to cryopreserve sperm that have been sex-sorted could add to this impact. The ability to utilize cryopreserved embryos and oocytes would affect fewer individuals, but the genetics conserved are likely to be from elite females and therefore, could significantly affect the industry. In addition, this research would improve our understanding of cryopreservation in general, and specifically on how membrane composition, cryoprotectant interactions and cellular osmotic tolerance affect the cryosurvival rates of cells.
- Develop new techniques for preserving stallion sperm by: developing more universally tolerated cryopreservation diluents and methods to alter sperm membrane lipids
- Develop techniques to cryopreserve oocytes, ovarian tissue slices, and large embryos
- Develop techniques to cryopreserve various equine stem cell lines
Assisted Reproductive Techniques
A wide range of assisted reproductive technologies are being used in the equine industry, providing more efficient use of valuable equine genetics and providing new insight into equine reproductive physiology. CSU has been a leader in the development of assisted reproductive technologies (ARTs) – including artificial insemination, embryo transfer and oocyte procedures.
Impact on equine industry: The development of ART at CSU has resulted in profound changes in the equine industry – from methods for artificial insemination to storing and transporting embryos, semen and oocytes. Many of the procedures that were developed or advanced at CSU are commonly used by equine breeders. The program at CSU has trained numerous students, visitors and short course participants, who have helped to disseminate these technologies throughout the world.
Impact on research: The ability to collect and manipulate equine gametes has provided an opportunity to advance our understanding of gamete physiology, fertilization and early embryo development. The horse is one of few species in which advanced ARTs, such as intracytoplasmic sperm injection (ICSI), are used for clinical purposes, making the horse a potential animal model to advance and study the effects of ARTs in humans.
- Improve the efficiency and flexibility of ARTs, to reduce costs and increase use of the procedures in the equine industry through methods such as laparoscopic transfers, optimal sperm selection, and shipment of harvested ovaries and testes, oocytes and ICSI-produced embryos
- Advance our knowledge and competency in embryo culture, including effects of culture conditions on long-term animal health
- Use the unique research opportunities provided by advanced ARTs to study factors affecting oocyte viability, including maternal aging
- Study the interaction of the equine sperm and oocyte to determine what factors prevent in vitro fertilization in the horse and develop methods to produce offspring using IVF
- Increase the efficiency of procedures to increase the number of oocytes or embryos for collection per cycle
Fertilization and Pregnancy
Although thousands of foals are born every year it is noteworthy to mention that the actual mechanisms involved in fertilization remain a mystery. The interaction between the oocyte and the sperm are vaguely defined and most often related to appropriate timing and morphological normality. Listed below are areas not fully understood in normal fertilization. The horse world is currently faced with two dilemma related to reproduction. The first is an ever-growing desire to maximize reproductive efficiency and the second, a need for an effective contraception protocol. A thorough understanding of the events leading to and following the gamete interactions will potentially allow for both, a more efficient mating and/or an ability to effectively contracept the horse.
Gametogenesis: Oocyte. Current dogma suggests that the filly is born with all of the oocytes she will ever produce arrested in the ovarian stroma. Understanding oocyte production, their meiotic arrest, re-activation and maturation within the pre-ovulatory follicle are essential. These include a thorough identification of zona production and subsequent function as the failure of IVF is thought to be associated primarily with this structure.
- Improved preservation of genetic material
- Improved in vivo maturation techniques
- Diagnostics related to oocyte quality
Gametogenesis: Spermatogenesis. Spermatozoa production is an exciting area of research. We feel that the answers to many mysteries may be found in this process, not the least of which is true stem cell production as is occurring constantly in spermatogonial regeneration and spermatocyte generation. Additionally, we hope to focus on the processes that contribute to the metamorphosis of spermatocytes into spermatozoa and the associated maturational signals occurring along the epididymis. We have interesting preliminary data suggesting such things as direct action of GnRHat the site of the testis, epididymis or sperm cell itself.
- Germ cell production
- Improved sperm production
- Improved sperm quality
- Diagnostics related to sperm quality
Fertilization. We will define fertilization as the actual fusion of the equine gametes. However prior to their interaction it is imperative that the following pre-fertilization processes are adequately defined: From the mare’s side we are investigating the capacitation, the acrosome reaction, oocyte/oviductal interaction, identification and creation of the oviductal sperm reservoir. On the male side we must better define the chemical composition and role of seminal plasma as it relates to capacitation, which allows for zona binding, acrosome reaction and zona penetration. Our studies have provided great insight into the mechanisms associated with the acrosome reaction and will continue until this rate-limiting event is thoroughly defined. Oocyte activation initiated by the acrosome reacted sperm, which leads to the second resumption of meiosis, is also being studied.
- Successful IVF
- Production of transgenics
Maternal Recognition of Pregnancy. Immediately following successful fertilization it is imperative that the embryo be recognized by the dam so that 1) she ceases to ‘cycle’ and 2) the oviductal and uterine environment support the developing pregnancy. To date the oviductal signal has been identified but the mode, method or signal for communication between the embryo and uterus remains unknown. We hypothesize that this signal is initiated by the embryo and results in detectable changes in gene expression, endocrine function and metabolism of the endometrium and potentially the corpus luteum.
- Early pregnancy diagnostics
- Improved pregnancy rates
- Develop diagnostics to evaluate normal physiologic functions of gametes
- evelop diagnostics to evaluate normal physiologic functions of an embryo and developing fetus
- Develop therapeutics to address abnormalities in gametes and embryos
- Develop accurate early pregnancy diagnostics
- Develop efficient and effective contraceptives
There is a need for techniques for both contraception and sterilization of both sexes of horses.
Contraceptives that could be applied one time and inhibit reproductive activity in either mares or stallions for one year would be very useful for performance horses. Also, there is a substantial need for a sterilant for controlling wild horses and burros that inhabit federal lands and parks. Ideally, an injectable agent that induced sterility in either females or males would help mitigate the burgeoning population of horses and burros that roam the west. Such a compound could also be used to chemically sterilize domesticated mares or stallions as an alternative to surgical castration.
- Develop procedures that will interfere with fertility in both mares and stallions for up to one year.
- Develop procedures that will sterilize either mares or stallions to permanently interrupt reproductive processes.
Significant knowledge of normal reproductive physiology of the mare and stallion has been gained over the past 50 years. However, there is much more to learn and current research should build on past experiences as well as explore new diagnostic tests and procedures for enhancing reproductive efficiency.
Reproductive physiology encompasses a wide variety of reproductive functions, including neuroanatomy and endocrinology, testicular and ovarian physiology, and physiology of the male and female reproductive tracts. Moreover, a better understanding of the factors responsible for controlling the transition from anestrus to the breeding season and back to anestrus are essential to optimizing reproductive performance in the equine. The ultimate goals of the male and female reproductive systems are to provide an opportunity for male and female gametes to combine their genetic material, provide a nurturing environment for that newly created individual during embryonic and fetal development, and provide immune protection and sustenance to the newborn after birth.
Additional basic and applied research is needed for a better understanding of normal physiologic events which can be translated into enhanced success of equine breeding programs. The ultimate measure of reproductive success is the birth of live, healthy foals. Additional parameters that measure success may include ovulation rates, per cycle or per season pregnancy rates, embryo collection or transfer success rates, number of sperm per ejaculate, percentage of progressively motile sperm after cryopreservation, or other factors.
- Conduct basic research on physiologic mechanisms that control reproductive function in mares and stallions
- Develop new diagnostic tests to evaluate normal physiologic functions in mares or stallions
- Develop novel products, techniques or protocols to enhance reproductive success in mares or stallions
A wide variety of transient physiologic and permanent pathologic conditions adversely affect reproductive performance in mares and stallions. In non-pregnant mares, ovarian abnormalities such as failure of follicular development and failure of ovulation preclude mares from an opportunity to become pregnant. Persistent mating-induced endometritis and infectious endometritis create a uterine environment that is inhospitable to the developing embryo. Early embryonic death and bacterial placentitis are significant causes of pregnancy loss in mares. Testicular degeneration and epididymal dysfunction are two of the many issues that lead to reduced fertility of stallions.
Basic research is critical to understanding the pathophysiology of reproductive diseases in all domestic animals, including the horse. In many instances, the events that lead to a particular abnormality are not well understood. Advances in technology often lead to breakthrough scientific research that improves our knowledge of pathology or disease.
Applied or clinical research is needed to develop accurate and sensitive diagnostic tests for detection of the presence of a reproductive disease or abnormality. An accurate diagnosis is a key element in resolving reproductive abnormalities in the horse. Once an abnormality is detected, additional hypothesis-driven research is needed to develop and evaluate management or treatment strategies.
- Evaluate pathophysiologic mechanisms that lead to reproductive abnormalities in mares and stallions
- Develop new diagnostic tests or protocols to detect reproductive abnormalities in mares or stallions
- Develop novel therapeutic products, techniques or protocols to manage or treat reproductive abnormalities in mares or stallions