ASSISTED REPRODUCTIVE TECHNOLOGIES (ART): FUTURE IN SMALL RUMINANTS
Dr. Dharam Navadiya1*, Dr. Ashishkumar Patel2, Dr. Dhrumilkumar Panchal2, Dr. Nayan Raval2, Dr. Abhishek Singh Tomar1 and Dr. M. M. Islam3
1M.V.Sc. Scholar, Department of Livestock Production Management, College of Veterinary Science & A.H., Kamdhenu University, Anand, Gujarat, India
2M.V.Sc. Scholar, Department of Animal Nutrition, College of Veterinary Science & A.H., Kamdhenu University, Anand, Gujarat, India
3Associate Research Scientist and Head, Pashupalan Sanshodhan Kendra, VASREU, Kamdhenu University, Ramna Muvada, Gujarat, India
*Corresponding author: dharamdnavadiya619@gmail.com
ABSTRACT
Small ruminants, such as goats and sheep are agricultural animals with significant potential for genetic improvement in meat and milk production. Enhancing the genetic material of these species is essential to boost productivity in the small ruminant industry. Seasonal breeding of small ruminants has led to the development of assisted reproductive technologies (ART). This paper provides an overview of assisted reproductive technologies in small ruminants. ART encompasses all fertility treatments involving the handling of either eggs or embryos, including Artificial Insemination, Embryo Transfer, In vitro embryo production, Superovulation, Synchronization of Estrus, Sexed Semen, and Cryopreservation, all of which will be discussed in this article. ART is also valuable for preserving endangered species or breeds, as well as in disease eradication programs.
KEYWORD
Small ruminant, ART, Reproduction, embryo.
INTRODUCTION
Small ruminants such as goats and sheep are important agricultural animals that have the potential to be genetically improved for meat and milk production (Amiridis & Cseh, 2012). Enhancing the genetic material of these species is crucial for increasing productivity in the small ruminant industry (Paramio & Izquierdo, 2016). Sheep and goats play a significant role in global food and fiber production, especially in developing countries with harsh climates and sub-fertile lands (Amiridis & Cseh, 2012). The seasonal breeding of small ruminants has led to the development of assisted reproductive technologies (ART) to overcome reproductive restrictions and improve genetic gains. These technologies are used for out-of-season oestrus induction, enhancing reproductive performance, and genetic improvement in modern agriculture. Furthermore, ART can also contribute to the preservation of endangered species or breeds and aid in disease eradication programs (Amiridis & Cseh, 2012).
Following are the various assisted reproductive techniques which may be used in small ruminant reproduction.
Artificial Insemination (AI): Artificial insemination (AI) is a common technique used to enhance genetic progress by spreading desirable male traits (Paramio & Izquierdo, 2016). As more producers become familiar with its benefits and as research continues to improve cryopreservation techniques, AI is becoming more widely adopted (Machado et al., 1996). AI offers several advantages over natural breeding, such as reducing the number of sires individual farmers need to maintain and controlling venereal diseases (Noakes et al., 2001). However, AI is more challenging in sheep compared to cows due to difficulties in detecting oestrus without rams and in freezing ovine semen. The most widely used methods for AI include:
- intravaginal
- intracervical
- transcervical intrauterine
- laparoscopic intrauterine.
Insemination of ewes by (a) intracervical insemination and (b) laproscopic intrauterine insemination methods seen in below figure (Noakes et al., 2001).
The process of artificial insemination in goats is similar to that in sheep, but it is easier to perform intrauterine insemination via the cervix of a goat compared to an ewe (Noakes et al., 2001). In sheep, a limitation for artificial insemination is the small diameter of the cervical opening. This can be addressed by treating the cervix with PGE, along with or without oestradiol benzoate, which significantly improves the depth of cervical penetration with an insemination gun. This treatment enables transcervical access to the uterus in 80-90% of ewes and 100% of does. Another method is to mechanically dilate the cervix to deposit semen in the uterus using a transcervical applicator gun. The best results are achieved through laparoscopic insemination with uterine deposition (Machado et al., 1996). Artificial insemination (AI) is still the leading technology used on a large scale in livestock with most favourable cost benefit ratio (Galli, 2018).
Embryo Transfer: The term “embryo transfer” refers to the process of collecting embryos from a donor animal and placing them into the uterus of a recipient animal (Noakes et al., 2001). Embryo transfer techniques have been used since the late 1980s and involve methods for superovulation, embryo recovery, and transfer in various animal species. Embryo transfer (ET) improves the accuracy of screening for desirable traits. The Zona Pellucida provides natural protection that reduces or eliminates disease transmission. This protection is further enhanced through embryo washing procedures (Machado et al. 1996). However, the efficiency of Embryo Transfer (ET) is limited due to the small number of embryos obtained per donor. In small ruminants, the best results are achieved when embryos are bisected as morulae or hatched blastocysts, as the damage caused by the embryo bisection (EB) procedure is less harmful to more developed structures.
In vitro embryo production (IVEP): The in vitro production of sheep embryos (IVEP) involves specific procedures, including oocyte collection and maturation (IVM), fertilization (IVF), and culturing of the presumptive zygotes (IVC) to a particular stage. The embryos are then either frozen or transferred to synchronized recipients. For research purposes, oocytes are typically collected from slaughtered ewes after the removal of small/medium-sized antral follicles or by slicing the ovary. Immature oocytes need to undergo cytoplasmic and nuclear maturation to become fertilizable (Amiridis & Cseh 2012). Various culture media have been used for IVM. According to Amiridis & Cseh, (2012) Fresh or frozen-thawed semen can be used for in vitro fertilization of oocytes, with the latter becoming more popular due to its convenience. After co-incubation of the gametes, presumptive zygotes should be cultured until the blastocyst stage. IVF also supports the development of advanced biotechnologies such as the production of transgenic animals, nuclear transfer, gene transfer, sexing, and bioassays for sperm fertilizing ability (Machado et al., 1996). In goats, the first kid born from IVF of an ovulated oocyte was reported in 1985, and the first lamb in (Paramio & Izquierdo, 2016). The final stage of IVEP is the culture of presumptive zygotes to the blastocyst stage. Three culture systems are routinely used, including IVC in temporary recipient’s oviducts, in vitro coculture with somatic cell support and semi-defined conditions in media designed to suit embryo requirements. Currently, the most widely used medium for culturing small ruminant embryos is SOS medium with amino acids and 5% to 10% fetal calf serum or BSA (Paramio & Izquierdo, 2016). In goats and sheep, a critical factor affecting the overall efficiency of in vivo embryo production (IVEP) is the large variation in embryo response to superovulation treatments, early regression of corpora lutea in goats, and the traumatic surgical procedure of embryo recovery. Despite numerous studies on IVEP in the past 30 years, the results obtained are still low and inconsistent.
Superovulation: The MOET (Multiple Ovulation and Embryo Transfer) method is the most cost-effective way to take advantage of the genetics of female cattle and small ruminants. Unpredictable variability in the superovulatory response is the most critical factor in sheep embryo production programs (Amiridis & Cseh, 2012). The gonadotrophins used for superovulation include equine chorionic gonadotrophin (eCG), porcine or ovine follicle stimulating hormone (FSH), and human menopausal gonadotrophin (hMG). Early studies have shown that the superovulatory response using eCG is less in comparison to FSH, while the time of ovulation is not well synchronized (Amiridis & Cseh, 2012).
Synchronization of Oestrus: The male effect is a complex response involving the integration of a range of external stimuli from the buck. It is probably the most cost-effective method to induce ovulation in small ruminants. Hormonal treatments to synchronize oestrus result in a complete range of biological outcomes (Machado et al., 1996). The most common protocol for oestrus synchronization in sheep and goats is based on progestogen/progesterone treatment in the form of intravaginal implants (sponges/CIDR). Treatment with progestogens during the luteal phase accelerates follicular development but reduces the number of large follicles, increases follicular atresia rate, and supports the persistence of large estrogenic follicles. Treatment during the follicular phase reduces both the number of large follicles and ovulation rate. Prostaglandin F2 is the most potent luteolytic agent for small ruminants and can be used during the breeding season as an alternative to progestogens for oestrous synchronization. A GnRH analogue and PGF2 resulted in desirable oestrous synchronization rates, as exemplified by the acceptable conception rate (50%) subsequently to fixed-time insemination. The protocol consists of two GnRH injections given 7 days apart, while PGF2 is administered on the fifth day. Fixed-time intrauterine insemination is performed 12–14 hours after the second GnRH injection. Early studies have shown that the super ovulatory response using eCG is less in comparison to FSH, while the time of ovulation is not well synchronized (Amiridis & Cseh, 2012).
Sexed Semen: The efficiency of livestock breeding enterprises could be significantly increased if it were possible to routinely predetermine the sex of offspring. However, in the past, most claims of altering the sex ratio by separating X and Y chromosome-bearing spermatozoa have not been substantiated in practice (Noakes et al., 2001). Sperm-sexing technology using flow cytometry is crucial for accelerating genetic improvement rates and for basic research aimed at identifying potential gender-related differences in early embryo development. Maximum genetic gain can be achieved by using pre-sexed spermatozoa for in vitro embryo production, thus producing already sexed embryos for embryo transfer. Additionally, artificial insemination with pre-sexed semen would become much more commercially attractive. Spermatozoa sexing is based on differences in physical attributes of sperms that carry different sexual chromosomes. Flow cytometry can provide samples with purity ranging from 80 to 85%. Unfortunately, flow cytometry is expensive and reduces the fertility of sexed spermatozoa. Some other sexing techniques are even more damaging to sperm cells, making them of little usefulness for animal production (Machado et al., 1996). The earliest report of successful cryopreservation of mammalian embryos in 1972. Embryos have been sexed by cytological methods (Noakes et al., 2001).
Cryopreservation: Longer-term storage of semen is achieved through cryopreservation (Noakes et al., 2001). According to Machado et al., (1996) Some researchers have attempted to preserve male or female genetic information through cryopreservation of spermatozoa or oocytes (Chunrong, 2019). However, a standardized freezing procedure for mammalian oocytes has not been established because they are sensitive to cryoinjuries caused by the freezing and thawing process. Semen cryopreservation does not require raising animals, which significantly reduces costs. Additionally, this technology can contribute to the advancement of artificial insemination (AI), a significant technology in the modern livestock industry (Chunrong, 2019).
Cryopreservation methods for mammalian pre-implantation embryos have been available for about 40 years. Sheep embryos have been used in freezing experiments to gather information about the cryotolerance of embryos and to improve the efficiency of freezing and cooling procedures. The first lambs and kids from cryopreserved embryos were born in 1976 (Amiridis & Cseh, 2012). There are two major techniques used for embryo cryopreservation: controlled (traditional) slow freezing and vitrification (ultra-rapid freezing). Slow freezing was introduced first and remains the most commonly used technique. It requires a biological freezer and takes longer to be completed. On the other hand, vitrification, the ultra-rapid technique, has reduced the time and cost of the procedure.
CONCLUSION
Small ruminants, primarily raised by small farmers, are often referred to as “the poor man’s cow.” The production of sheep and goats in India, in terms of both wool and mutton quality and quantity, lags behind that of improved breeds in more developed countries. To enhance the welfare of farmers, improving the breeds of small ruminants through breeding techniques is essential. Assisted Reproductive Technologies (ART) are a promising approach, offering quick returns and addressing the growing demands of the increasing human population. We are optimistic that the younger generation, with their interest in innovation, will contribute to improving lifestyles, and this article is a valuable resource for that purpose.
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