Table of Contents
Introduction
The intracytoplasmic sperm injection, commonly known as ICSI, is a program that is used to discover pregnancies from eggs obtained from donor mares. The process involves injecting these eggs with specific sperms from a donor and then the resultant embryo is developed in the laboratory for about seven days (Veterinary Medicine and Biomedical Services, 2017). The embryo is then transferred using a unique facility to the recipient. The process has been helpful especially to those mares that cannot get pregnant due to several factors such as cervical lacerations or uterine disease, both of which have the potential of preventing the uterus from either conceiving or holding an embryo over a given period (Chillon, Suh &Altermatt, 2010). Despite the associated benefits of producing offspring, this program is labor-intensive and expensive, necessitating the need for further research to analyze how it can be made more efficient. This paper will, therefore, give an overview of the process of intracytoplasmic sperm injection in equine and also expound on some of the associated benefits.
Overview of the Procedure
The process of injection involves fertilizing an oocyte from the desired mare and then breeding the resultant embryo in the laboratory for a duration not less than twelve hours (Veterinary Medicine and Biomedical Services, 2017). This period allows for the egg to mature by enhancing developmental changes. However, depending on the level of maturation of the oocyte immediately it has been recovered from the ovarian follicles of the mare, at times the maturation process may take up to thirty hours. While removing oocytes from the follicles, the donor is first put in sedation so that the transvaginal ultrasound-guided technique applied when the donor is still calm (Veterinary Medicine and Biomedical Services, 2017). However, care is usually taken to ensure that the animal does not fall asleep completely because the oocyte is held firm by the walls of the follicle and, therefore, to dislodge it fluid movement is critical. The transvaginal follicular aspiration process involves using a double-lined needle so as to ensure simultaneous flushing.
Oocytes can either be dislodged before ovulation from the dominant follicles or at any other time from the subordinate follicles, within a given cycle. Typically, a healthy mare contains one dominant follicle and about eight subordinates in each cycle (Chillon, Suh &Altermatt, 2010). However, this ratio is subject to the age of the mare where the old ones usually have few subordinates than the young ones. Although oocytes can be obtained from both the dominant and subordinate follicles, chances are much better when they are dislodged from the dominant ones (Chillon, Suh &Altermatt, 2010). During the removal process, numerous oocytes can be obtained but for a single donor, a period of two weeks is taken to allow the ovaries produce more oocytes and also ensure that those who will be recovered are mature enough (Veterinary Medicine and Biomedical Services, 2017). Once the oocytes are considered mature enough, they are injected with sperm from a desire stallion with the help of an ultra-powerful microscope to ensure that each of the individual sperm gets injected into the cytoplasm of only one oocyte. The already fertilized egg is then retained in the laboratory for an extra one week so that it can develop completely into a mature blastocyst (Veterinary Medicine and Biomedical Services, 2017). At this point, the resultant embryo is now suitable to be delivered to the recipient mare with the help of a special transfer facility.
Oocytes can survive in the laboratory even after they have been recovered because they are surrounded by cumulus cells that act as a source of nourishment (Chillon, Suh &Altermatt, 2010). Before they are dislodged, these complex cells are held tight on the walls of the follicles and as it develops they continue to expand such that by the time ovulation approaches they are large to the extent that their bondage to the wall is extremely weak. Therefore, minute destabilization of the fluid clears them and direct them to the oviduct (Chillon, Suh &Altermatt, 2010). This means that those oocytes that are obtained before ovulation take a little time to recover due to the loosened attachment to the follicle wall, as compared to those who are dislodged when the cumulus cells are firmly held by the wall. It is for the same reason that aspiration of follicles that are not mature leads to oocytes that will take a long time to recover (Chillon, Suh &Altermatt, 2010).
In the laboratory, oocytes can be processed in different ways. But the swim-up pull technique is the most common whereby sperm samples are placed at the bottom of the medium in use, such as a tube, the spermatozoa are then given enough time to swim before they are taken further up the tube (Chillon, Suh &Altermatt, 2010). A pipette is commonly used to stabilize the oocyte so at to ensure that injection occurs away from the polar body and in such a position that visualization will be clear. The pipette is then entered into the cytoplasm if the oocyte and part of it are aspirated with an aim if speeding up activation of the oocyte (Chillon, Suh &Altermatt, 2010). Once the oocyte has been fertilized, they are then put in the form of micro-droplets in a layer of mineral oil. They are then placed in an incubator with a higher concentration of carbon dioxide than that of oxygen (Chillon, Suh &Altermatt, 2010). Such a condition ensures that temperature, as well as the PH, are properly regulated while at the same time minimizing oxidative damage. The mineral oil, on the other hand, plays a critical role in ensuring that the medium does not get contaminated.
Expected Results
Once the follicles have been aspirated, chances of obtaining mature oocytes are typically anticipated to be above 50% (Choi, Love, Varner, Brinsko&Hinrichs, 2017). In 2013 at the Animal Teaching Hospital of Texas where most of these procedures are usually performed, every aspired follicle produced about seven oocytes and after undergoing through the laboratory incubation process over 65% of them matured (Choi, Love, Varner, Brinsko&Hinrichs, 2017). This way, there was a possibility that approximately 20% of these embryos will develop into a blastocyst if sperms are obtained from fertile stallions (Choi, Love, Varner, Brinsko&Hinrichs, 2017). Upon completion of the study, about 79% of those blastocysts that were carefully transferred into the recipients led to pregnancy (Choi, Love, Varner, Brinsko&Hinrichs, 2017). It was, therefore, realized that mares with productive complications such as cervical deterrents or oviductal anomalies do benefit immensely from this technology. Such abnormalities can result from various issues such as aberrancy in a hormonal axis which in turn deters the healthy development of the follicles as well as oocytes. Such a mare will then be infertile, but with intracytoplasmic sperm injection technique, these organisms can still be able to produce offspring (Choi, Love, Varner, Brinsko&Hinrichs, 2017).
Benefits
In comparison to the typical method whereby oocytes are transferred to the oviduct through a surgical process, giving the embryo time to develop in the laboratory has numerous advantages. For instance, the recipient mare does not go through any form of surgery that is likely to put its life in danger. This is because once the embryo is developed in the laboratory to maturity, standard facilities are used for the transfer process (Galli, Colleoni, Duchi&Lazarri, 2007). Similarly, if more oocytes are removed from the donor at a given instance, the rest do not have to be wasted but can be transferred to different recipients, thereby increasing chances of forming a foal. More also, unlike the surgical technique, intracytoplasmic sperm injection program can make use of either cold or frozen semen as well as sperms of low quality and still give the desired output. In case a frozen semen is used, only a few of the cells are necessary, and this means that the rest can still be preserved for future purposes (Galli, Colleoni, Duchi&Lazarri, 2007). When the procedure is performed on young mares that are fertile, the success rate of transfer is most often over 80%. But even if the procedure is done on aged mares, which are known to be chronically sub-fertile, the success rate is still above 40% (Galli, Colleoni, Duchi&Lazarri, 2007). Therefore, we can say that the program provides a safe alternative for mare reproductive processes as well as for stallions that are incapable of producing an offspring using conventional means.
Another major benefit of intracytoplasmic sperm injection technology is that it allows donors to b their embryo recipients. In a 2013 study conducted by Colorado State University, the researchers claimed that the main reason why it becomes necessary to have both donor and recipient mares is that is because the donor may be having reproductive problems (Choi, Love, Varner, Brinsko&Hinrichs, 2017). However, in the case where the ICSI is being used due to the stallion having poor semen, the same mare can be utilized as both the donor and the recipient. During the study, the team dislodged twelve oocytes from nine mares and then fertilized them through the ICSI technique (Choi, Love, Varner, Brinsko&Hinrichs, 2017). Eight of the embryos managed to develop to the blastocyst stage, and these were then transferred back to the donors’ uterus (Choi, Love, Varner, Brinsko&Hinrichs, 2017). A considerable success rate was noted whereby five pregnancies were recorded. Therefore, in the case that ICSI is being conducted merely because a mare has semens of poor quality, there is no need to look for a second only because the donor can still act as a recipient of its embryo after it has gone through the laboratory development process. But this approach has one major challenge whereby it is difficult to time embryo development in the laboratory and still match it with the mare’s reproductive cycle (Hinrichs, 2005).
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To critically assess how successful intracytoplasmic sperm injection since it was first put in practice, there are some factors that require consideration. The most crucial factor that determines if the program is successful in the population of mares selected for a given experiment (Hinrichs, 2017). This is because depending on the kind of population used in the process the results will be the same. In most of the cases, intracytoplasmic sperm injection approach is usually considered the last resort whereby it is only put into practice if the other conventional techniques fail to give the desired results (Hinrichs, 2017).
Conclusion
One of the significant challenges associated with aged oocytes is the loss of fertility. However, this problem can be overcome through the intracytoplasmic sperm injection program because irrespective of how poor in quality the semen is, embryo production is not affected. This technology which involves picking mature oocytes from a donor mare through the process of follicle aspiration ensures that even those oocytes which have already been harvested but are not mature enough for transfer are nurtured in the laboratory until they develop completely. Unlike the conventional surgery process, the oocyte harvesting technique is safe since it does not expose the donor to any health consequence. However, for the intracytoplasmic process to end up successfully, it is necessary that the procedure is run by a team that is substantively knowledgeable of the mare physiology. Nevertheless, further research needs to be conducted so as to have a better elucidation of the mechanism through which oocytes develop. Another shortcoming associated with intracytoplasmic sperm injection, and which requires extent study, is the aspect of aging whereby it is common that as organisms grow old, their fertility is affected yet the technique at hand does not consider such a variable.
- Chillon, L., Suh, T. &Altermatt, J. (2010). Intracytoplasmic Sperm Injection in the Horse: The Ultimate Blind Date. Acta Scientiae Veterinariae, 38(2), 559-564.
- Choi, Y., Love, C., Varner, D., Brinsko, S., & Hinrichs, K. (2017). Developmental Competence in vivo and in vitro of in vitro-matured Equine Oocytes Fertilized by Intracytoplasmic Sperm Injection with Fresh or Frozen-thawed Spermatozoa. The Journal of the Society for Reproduction and Fertility, 154(1), 455-465.
- Galli, C. Colleoni, S., Duchi, R. &Lazarri, G. (2007). Developmental Competence of Equine Oocytes and Embryos Obtained by in Vitro Procedures Ranging from in Vitro Maturation and ICSI to Embryo Culture, Cryopreservation and Somatic Cell Nuclear Transfer. Animal Reproduction Science, 98(2), 39-55.
- Hinrichs, K. (2005). Update on Equine ICSI and Cloning. Theriogenology, 64(3), 535-541.
- Veterinary Medicine and Biomedical Services. (2017). Equine Intracytoplasmic Sperm Injection Program.