In Vitro Embryo Production
In vitro embryo production (IVEP) refers to the processes of in vitro oocyte maturation (IVM), in vitro fertilization (IVF), and the early days of in vitro embryo culture (IVC). Usually, immature oocytes are collected from the ovaries of common domestic animals, through transvaginal ultrasound-guided oocyte retrieval (OPU: Ovum Pick-Up) procedures in live animals, or after the animal’s death after slaughter.
A schematic diagram of various steps of in vitro embryo production is depicted in Fig. 24.5.24.6.1 Transvaginal Ultrasound-Guided Oocyte Retrieval (OPU: Ovum Pick-Up)
Transvaginal Ultrasound-Guided Oocyte Retrieval or OPU is an ART program that has been successfully used in cow and mare for the last three decades. Originally, the technique was introduced in human reproductive medicine. Subsequently, this minimally invasive technique became a popular method for collecting immature oocytes repeatedly from valuable donor animals of high genetic merit. A modified version of a transvaginal ovum pick-up technique, originally developed for use in human reproduction, was applied in cattle.
Animals may be sedated with an anesthetic and intestinal relaxant; however, this is not mandatory. Fecal matter is removed from the rectum and 2% lidocaine is used for epidural anesthesia for easy transrectal manipulation. Vulvar lips and adjacent areas are washed and disinfected thoroughly to lessen the chance of infection.
There are three major parts in the OPU machinery system: an ultrasonographic scanner with an appropriate transducer (probe), an aspiration pump, and a needle guidance system coupled to an oocyte collecting tube. OPU device, comprising of the transducer and the needle guidance system, is introduced into the vagina. Depending on the left or right ovary for oocyte retrieval process, the head of the ultrasound transducer is directed craniodorsally to the left or right of the cervix.
With the other hand inserted per rectum, the ovary is held against the head of the transducer so that ovary and follicles become visible on the ultrasound screen. A special biopsy line programmed within the ultrasound scanner’s software guides the precise positioning of the follicle for fruitful puncture. OPU handle can be operated with one hand from outside and the operator inserts the needle gradually until it pierces the vaginal wall and the needle is seen entering the ultrasound field. Simultaneous hand-guided movement of the needle and per rectal manipulation of the ovary helps in proper positioning of the ovary and the needle. Once the needle pierces the follicle the aspiration pump is triggered with the foot pedal. With the suction pressure, the follicular fluid and cumulus-oocyte complexes are collected into the oocyte collection medium within the embryo filter. Subsequently, the content of the embryo filter is washed and transferred into petridish. Oocytes are identified with a stereomicroscope, graded and transferred to in vitro maturation medium.24.6.2 Follicular Wave Synchronization of Donor Animals Prior to OPU
The success of OPU is governed by a plethora of factors, one of which is the quality of oocytes. In bovine, oocytes are retrieved from the ovarian follicles of at least 2 mm size on a random day of estrous cycle. But most of the follicles from which oocyte aspiration is performed are atretic in nature. IVEP with oocytes aspirated from the growth phase of the follicle results in better production of blastocysts compared to the dominance phase. Different hormonal treatment protocols have been applied for follicular wave synchronization of donor animals. Multiple FSH injections (40, 80, and 80 mg) at 24 h interval increased the number of follicles for aspirations. Another factor that determines the outcome of OPU technique is coasting, the period between the last hormonal treatment and OPU. Along with multiple FSH injections, administration of a LH injection 6 h prior to OPU procedure increases the blastocyst production.
A combination treatment of progesterone and estradiol benzoate or GnRH application on a random day of the estrous cycle synchronizes follicular wave after 2-4 days.24.6.3 Collection of Oocytes from Slaughterhouse Ovaries
Immature oocytes are obtained from ovaries collected from freshly slaughtered animals in a slaughter house. The ovaries are transported to the laboratory in a lukewarm normal saline solution (35-37 °C) containing antibiotic. Subsequently, follicles present on the ovarian surface are aspirated to obtain the oocytes. The oocytes are then searched and graded on the basis of the homogeneity of their ooplasm and the mass of cumulus cells surrounding the oocytes. Cumulus-oocyte complexes (COCs) with an unexpanded cumulus mass of >2 layers of cumulus cells, and homogeneous, evenly granular ooplasm is selected for IVM.
24.6.4 In Vitro Maturation
Usable quality oocytes are washed in maturation medium several times and cultured in a group of 15-20 oocytes per droplet of the same medium for 24 h, overlaid with mineral oil in small petridish. Maturation medium is supplemented with LH and FSH and in some cases estradiol, growth hormones, and insulin.
24.6.5 In Vitro Fertilization
Frozen straws of semen samples are thawed and processed in a fertilization medium containing compounds like caffeine, heparin that promotes capacitation and acrosomal reaction. Mature oocytes are also washed in fertilization medium, placed in a small droplet in petridish and processed semen samples containing the required amount of spermatozoa are added to it. The fertilization is carried out in a CO2 incubator for 18 h.
24.6.6 In Vitro Culture
After 18 h of sperm-oocyte co-incubation, the cumulus cells are washed off the oocytes by gentle pipetting. For culture of bovine embryos modified Charles Rosenkrans medium with amino acids (mCR2aa) containing bovine serum albumin (BSA) and fetal bovine serum (FBS) are used. After the oocytes are washed several times in IVC medium they are transferred to the original granulosa bed and cultured for
8- 9 days. Cleavage rate is observed on day 2 and subsequent stages of embryonic development are recorded at regular intervals.
24.6.7 Application
• Exploitation of female reproductive capacity (more offspring from valuable donors).
• Significant facilitation of import and export of valuable genetic material.
• Development of new breeding strategies.
• Twin production (Embryo splitting).
• Introduction of new genes into closed herds.
• Manipulation of embryos.
• Transgenesis.
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