IVEP | Buffalopedia

IN VITRO EMBRYO PRODUCTION

Production of Buffalo Embryos in Laboratory: A Scientific Approach

Buffalo is an integral part of livestock agriculture in Asia since many centuries, because it provides draught power, milk, meat and hides to millions of people, particularly small-scale farmers. However, despite the importance of buffalo to the socioeconomy, its population has been declining, partly due to poor reproductive performance. The low reproductive efficiency in female buffalo can be attributed to delayed puberty, higher age at calving, long postpartum anoestrus period, long calving interval, lack of overt sign of heat, and low conception rate. In addition, female buffaloes have few primordial follicles and a high rate of follicular atresia. These limiting factors of reproduction also limit the embryo transfer technology in buffalo. Therefore, the emphasis has now shifted to in vitro embryo production (IVEP). IVEP has drawn the interest of innumerable researchers as it can salvage the genetic potential from infertile female and can yield large number of embryos from the ovaries of slaughtered females. This technology not only offers optimization of high-quality dams, but also allows the preservation and rapid multiplication of genetically superior characters of sire by making embryos available for animal cloning, transgenesis, cryopreservation of germplasm through embryo preservation, embryonic stem cells (ESCs) and sexing.

In vitro embryo production

It is a combination of techniques of collection of immature oocytes, in vitro maturation (IVM), fertilization (IVF) and culture (IVC).

Collection of ovaries

For genetic improvement, ovaries must be collected from genetically superior breeds. Ovaries are collected immediately after the slaughter of the animals, and kept in a thermos flask containing physiological saline (0.9% NaC1 with 400 IU/ml penicillin and 500 μm/ml streptomycin), at a temperature ranging from 32-37°C. The collected ovaries are transported to the laboratory within 4-5 h of slaughter (Fig. 1).The maintenance of temperature of the saline is important to maintain the viability of the ovaries as well as oocytes during the period of collection and transportation. Prolonging the period of ovary collection/transfer may significantly affect the viability of the oocytes for in vitro maturation. It is recommended that oocytes should be collected from the ovaries within a 6-hour period. Beyond six hours, development potential is greatly compromised.

Collection of oocytes

In the laboratory, it is necessary to wash the ovaries in fresh, sterile physiological saline (without antibiotics) to further remove any contaminants. Briefly rinsing the ovary in 70% ethyl alcohol is recommended, to eliminate surface micro organisms before processing begins. The ovaries are dried lightly with sterile paper towels before primary oocytes are recovered from 2-8 mm vesicular follicles to avoid contaminating the aspirates during oocyte retrieval. It is important to remove the big follicles (beyond 9 mm) because they contain secretions that cause jelly formation in the aspirates. This may affect the retrieval of oocytes during searching.

The methods for the collection of immature oocytes from slaughtered animal ovaries include:

1) aspiration from surface follicles using 18-20G needle attached to a 10 ml syringe containing the aspiration medium.

2) puncturing of prominent follicle

3) slicing of ovaries into small pieces.

Among these methods, aspiration is mostly used method as it is easier and faster to perform and gives higher yield of oocytes. Depending on the efficiency of aspiration and the oocyte grading system, the oocyte harvest per ovary can range from 0.46 to 3.0, with an average of only 1.5 per ovary. The limitation of collecting immature oocytes from slaughtered ovaries is that no information about pedigree of dam is obtained. This limitation is however overcome through ovum pick-up (OPU), which allows retrieval of oocytes from the best female animal without sacrificing the animal for future production. This technique has widened the application of IVEP by merging assisted reproduction with genetic improvement.

The common aspiration medium used is TCM -199 + 10% FBS and DPBS + 0.6% BSA in a 1:1 ratio. Aspiration medium can be prepared once, stored in a refrigerator and made available for one week’s activity. However, it is very important to pre-warm the aspiration medium up to 38°C before use. Alternatively, Medium-199 with Earle’s salts, L-glutamine, 25 mM HEPES, supplemented with 0.6% bovine serum albumin and trace amounts of antibiotics, can also be used.

After aspiration, the contents of the syringe, which included the aspirated oocytes, follicular fluid, granulosa cells and other debris, are slowly dispelled into a sterile centrifuge or test tube with minimum disruption of the cumulus oocyte complex (COC). Once the last ovary of a particular batch is processed, the oocytes are allowed to settle to the bottom of the tube for at least 5 minutes. The precipitate is taken using a sterilized Pasteur pipette and poured into sterile Petri dishes with grid (searching dishes) for subsequent searching of COCs. The oocytes are searched under a zoom stereo microscope at around 20X magnification. The oocytes are then shifted to 35 mm x 10 mm cell culture Petri dishes containing the washing medium (TCM-199 + 10% FBS + 0.81 mM sodium pyruvate). As much as possible, recovery work must be done in a sterile environment with a room temperature of 25°C.

Grading of oocytes

The collected oocytes are graded on the basis of their morphology as described below (Fig.2 & 3):

Grade A: COCs with an unexpanded cumulus mass having ≥4 layers of cumulus cells, and with homogenous evenly granular ooplasm.

Grade B: COCs with 2-3 layers of cumulus cells and a homogenous evenly granular ooplasm.

Grade C: Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm.

Oocytes of only Grades A and B are used for the in vitro maturation.

In vitro maturation

The success of IVM will partly depend upon quality and number of collected oocytes. The components of maturation media exhibit a marked influence upon the IVM of oocytes. The medium used for IVM varies among laboratories. The culture media employed in maturation of oocytes can be broadly divided into simple and complex. Simple media are usually a combination of various salts and bicarbonate buffered systems containing basic physiological saline with low concentration of energy source like pyruvate, lactate and glucose. The main differences between various types of simple media are their concentration of ion and the energy sources. The media are generally supplemented with serum or albumin, with trace amounts of antibiotics. Complex media contains in addition to the components of the simple media, amino acids, vitamins, purines and other substances, mainly at levels at which they are found in serum. Fixed nitrogen is present as free amino acids. The most widely used complex media for IVM of buffalo and cattle oocytes is TCM-199 with Earle’s salt, L-Glutamine and 25 mM HEPES supplemented with 10-20% serum.

In general, the collected immature oocytes of good quality are washed 3-4 times with the washing medium (TCM-199 + 10% FBS + 0.81 mM sodium pyruvate + 5% buffalo follicular fluid and 50 mg/ml gentamycin) and then with the maturation medium (washing medium + 5 µg/ml pFSH + 1 µg/ml estradiol-17β). The washed oocytes are then placed in small droplets (15-20 oocytes/droplet) of the maturation medium, covered with sterile paraffin oil, in a gelatin coated tissue culture dish and cultured for 24 h in a CO₂ incubator (5% CO₂ in air, 90-95% relative humidity) at 38.5°C.

The length of time needed for IVM is a critical factor to attain the metaphase II stage and various changes in the organization of its cytoplasm, which include alignment of cortical granules along the oolemma, rearrangement of mitochondria, continued development of the golgi store and the reduction of the Golgi compartment. The increase lipid store is probably an essential energy source to support the oocyte through maturation and early embryonic development. The expansion of cumulus cells is an indication of maturation of oocyte, reason being cumulus cells secrete hyaluronic acid (HA), a non-sulphated glycosaminoglycan bound to the cumulus cells by linker proteins. When the HA becomes hydrated, spaces between the cumulus cells become enlarged and the cells are embedded in a mucified matrix. Evidence indicates that there is no significant difference between treatments (maturation period) in terms of maturation and cleavage rates, but a significant difference in blastocyst yield has been observed in favor of the 24 h maturation period.

Assessment of cumulus expansion

After 24 h of IVM, the COCs are examined under an inverted microscope and determine the degree of cumulus expansion (Fig. 4).

Degree 0: No expansion

Degree 1: Cumulus cells non-homogeneously spread and clustered cells still observed.

Degree 2: Cumulus cells homogeneously spread and clustered cells no longer present.

Only those COCs are subjected to IVF which exhibit cumulus expansion of degrees 1 and 2.

Sperm preparation and in vitro fertilization

Mammalian fertilization is a complex event involving many different stages that must be acquired successfully to achieve fertilization. Successful IVF requires appropriate preparation of sperm and oocyte, as well as culture conditions that are favorable to the metabolic activity of the male and female gametes. The male gamete influences fertility by affecting fertilization rate and subsequent embryo development. IVF has become a valuable tool for assessing sperm functionality and for studying the success or failure of gamete interaction in most species. To enhance successful fertilization of the oocytes, sperm cells must be motile, should have the ability to undergo capacitation and express the acrosome reaction. Sperm must possess the capacity to bind to the zona pellucida and vitelline membrane by acquiring the correct binding proteins during maturation, and exposing these binding sites to the oocyte at the appropriate time. So, spermatozoa are artificially capacitated because they need to undergo biochemical and physiological changes before becoming able to fertilize oocytes. Capacitation process is the gradual removal or alteration of the protective coat from the sperm surface, especially in the region of the acrosome. The removal or alteration of this coat permits exposure of receptor sites, allowing sperm to interact with oocyte receptors. Capacitation also induces acrosome reaction that is vital for fertilization. The spermatozoa are treated with appropriate concentration of heparin to induce capacitation and subsequent acrosome reaction.

The media used for IVF suggested are BO and TALP, which contain motility enhancing substance like caffeine or theophylline. Caffeine is a cyclic nucleotide phosphodiesterase inhibitor that has been used as a motility-stimulating agent during IVF. It achieves its action by inhibiting phospodiesterase, which results in an intracellular accumulation of cAMP that activates respiration and sperm motility of spermatozoa. The bovine serum albumin is also added to fertilization medium for destabilizing cell membrane of oocytes and sperm by removing cholesterol and zinc molecules which eventually enhances capacitation and acrosome reaction. For carrying out IVF, straws of frozen‑thawed ejaculated buffalo semen are washed twice with washing BO (Brackett and Oliphant) medium (BO medium containing 10 µg/ml heparin, 137.0 µg/ml sodium pyruvate and 1.942 mg/ml caffeine sodium benzoate). The pellet is resuspended in around 0.5 ml of the washing BO medium. The spermatozoa (50 µl of the suspension) are then transferred to 50 μl droplets of capacitation and fertilization BO medium (BO medium containing 10 mg/ml fatty acid-free BSA, 10 µg/ml heparin, 137.0 µg/ml sodium pyruvate and 1.942 mg/ml caffeine sodium benzoate) in a Petri dish, covered with sterile mineral oil and placed in a CO₂ incubator (5% CO₂ in air) for 18 h at 38.5°C. The in vitro matured oocytes are washed twice with the washing BO medium and transferred to the 50 μl droplets of the capacitation and fertilization BO medium (15 to 20 oocytes per droplet) containing spermatozoa. At the end of the sperm‑oocyte incubation, the oocytes are separated from the sperm droplets and washed with the respective IVC medium (Fig. 5).

In vitro culture

At the end of sperm-oocyte incubation, IVC is carried out, where presumptive zygotes are cultured in vitro in a culture medium at 38.5°C in a CO₂ incubator for up to 9-10 days to the blastocyst stage at which these could either be transferred to suitably synchronized recipients for producing live offspring or preserved for future use. IVC is the most important step not only because of its longer duration of ~10 days compared to that of 24 h for IVM and 6-24 h for IVF but also the culture conditions and milieu during IVC have a profound influence on the outcome of embryo growth.

The media used for culture of mammalian embryos can be broadly divided into three categories.

1) Complex media like TCM-199, Ham’s F10 etc. contain a large number of components like balanced salt solutions, vitamins, amino acids, glucose, pyruvate, lactate, trace elements, purines and other metabolites. Since these media were originally designed for the culture of somatic cells, they may contain constituents which adversely affect oocyte/embryo development e.g. TCM-199 and Ham’s F10 contain hypoxanthine which can induce meiotic arrest in mice oocytes.

2) Simple media includes Chatot-Ziomek-Bavister medium, hamster embryo culture medium, simplex optimized medium, completely defined medium-Charles-Rosenkrans medium. These media were originally designed for the culture of mouse embryos but, after several modifications, keeping in view the composition of oviductal fluid, synthetic oviductal fluid was used for supporting the growth and development of embryos of diverse mammalian species.

3) Sequential media in which the embryos are cultured in a group of media sequentially. They offer the added advantage of being able to take care of the changing nutritional needs of the embryos as they grow e.g G1.2/G2.2 media.

The presumptive zygotes are subjected to IVC generally in co-culture with somatic cells like oviductal epithelial cells (OEC) (Fig. 6), 3T3 cells, BRL cells, cumulus cells or vero cells. The presence of a somatic cell monolayer in the culture medium during IVC of the developing embryo is found to be very important in enhancing its developmental potential. The provision of cell basement support provides the developing zygotes with a contented milieu, and secretes some growth factors supportive to further development in vitro, but co-culture of embryos with slaughterhouse material like OEC has the following disadvantages:

i)It is a potential source of contamination in an oocyte/embryo culture system,
ii)It could also lead to variable results in different trials since the patho-physiological status of the animal(s) from whom OEC were obtained may differ in different animals, and

iii) Co-culture with somatic cells along with the use of serum could lead to high fetal loss, complications during pregnancy and production of abnormally large calves resulting in high neonatal losses.

Evaluation of embryo development

The following morphological criteria are employed for the evaluation of buffalo embryos developed in vitro.

2‑16 cell stages	Embryos with 2, 4, 8 or 16 blastomeres are termed as 2‑cell, 4-cell, 8- cell and 16-cell stages(fig. 7)

Morula	An embryo with >32 cells in which individual blastomeres are difficult to discern from one another and the cellular mass occupy most of the perivitelline space (Fig. 8).

Compact morula	A morula in which compaction of blastomeres is clearly visible and individual blastomeres have coalesced (Fig. 9).

Early blastocysts	An embryo in which formation of blastocoel has just started. Visual differentiation between trophoblast and the inner cell mass may be possible at this stage.
Blastocyst	An embryo with a well defined blastocoel and with a pronounced differentiation of the outer trophoblast layer and the darker, more compact inner cell mass. The embryo occupie most of the perivitelline space.

Expanded blastocyss	A blastocyst which has expanded and in which zona thinning is clearly discernible.
Hatched blastocysts	A blastocyst which has partly or completely come out of the ruptures zona.

Therefore, development of an IVC system that obviates the use of somatic cell co-culture is the need of today for the IVP of buffalo embryos. So, improvements in IVP protocols with application of simple media which would increase the good quality embryo yields would be highly beneficial for all modern reproductive technologies and especially for cloning, transgenesis and ESC culture.

Contributed by Dharmendra Kumar, Taruna Anand, Pradeep Kumar and PS Yadav