Factors affecting in VITRO PRODUCTION of bovine embryos
Department of Animal Reproduction & A.I. National Research Center, Dokki, 12622 Giza,
In vitro production (IVP) of bovine embryos takes a great attention during the last three decades. However, with the advancement of IVP procedures, variability in developmental rate and viability of the in vitro produced embryos is less than those developed in vivo. Also, the survival rate of in vitro produced embryos after freezing and thawing, and following some of the more advanced manipulation, is less than for embryos produced in vivo, indicating that the techniques used to produce embryos in vitro still require considerable improvement.
In vitro embryo development is strongly influenced by events occurring during oocyte maturation, fertilization and the subsequent development of the fertilized oocytes. So, improving the efficiency and identifying the sources of variations between IVF systems or between different laboratories are more important when routinely producing blastocysts from individuals of high genetic merits. Also, the development of specific culture regimes capable of supporting IVM/ IVF and IVC to the blastocyst stage is highly desirable.
The following review article presents description of the various factors that could affect in vitro production of bovine embryos and their ability to develop into blastocyst stage.
The ability to produce large number of embryos from donors of high genetic merit has considerable potential value in disseminating genetic improvement and shorter the progeny testing and generation interval through the national herd.
Some commercial applications of in vitro fertilization technology have included efforts to: (1) upgrade the productive and genetic performance of animals; (2) to overcome infertility of valuable high yielding animals; (3) to produce transgenic and cloned animals; (4) provide a source of sexed embryos; (5) for twin production in beef cattle; and (6) at the molecular level, the technique is used to elucidate events related to maturation, fertilization of oocytes and development of embryos, these events are difficult to study under natural conditions in living animals.
In the laboratory, embryos can be routinely produced and developed up to the blastocyst stage using three subsequent techniques: in vitro maturation (IVM) of oocytes, followed by sperm capacitation and in vitro fertilization (IVF) of matured oocytes and then in vitro culture (IVC) of the fertilized oocytes up to the blastocyst stage.
In vitro embryo development is strongly influenced by events occurring during oocyte maturation, fertilization and the subsequent development of the fertilized oocytes. So, improving the efficiency and identifying the sources of variations between IVF systems or between different laboratories are more important when routinely producing blastocysts from individuals of high genetic merits. Also, the development of specific culture regimes capable of supporting IVM/ IVF and IVC to the blastocyst stage is highly desirable.
In addition, survival rate of in vitro produced embryos after freezing and thawing, and following some of the more advanced manipulation, is less than for embryos produced in vivo, indicating that the techniques used to produce embryos in vitro still require considerable improvement.
The following review presents description of the various factors that could affect in vitro fertilization of oocytes and their ability to develop into blastocysts in cattle. These factors can be classified into:
I- Factors affecting in vitro maturation (IVM)
In vitro maturation is the most critical step in vitro embryo production. There is a constant need to emphasize the fact that effective oocyte maturation is the foundation of embryo production. Identifying these factors will improve the in vitro embryo production systems in bovine. These factors include:
1. Factors affecting oocytes yield
The recovery of large number of oocytes with high developmental competence remains an ultimate goal for the mass production of embryos in cattle. At the same time, the origin of the oocyte can play an important role in their IVF and subsequent developmental competence. Oocytes yield and quality can be affected by:
1.1 Effect of follicular size on oocyte competence
In cattle, the oocyte first acquired competence to develop into blastocysts in in vitro system at a follicular size of 2-3 mm. When follicles were pooled according to size, it was shown that large follicles (≥10 mm diameter) contain oocytes with a higher potential to become embryos (Pavlok et al., 1993; Lonergan et al., 1994). Some studies described the fate of individual oocytes according to the exact follicular size, and it confirmed an increased competence with follicle size, i.e. bovine oocyte complexes (COCs) isolated from ovaries carrying follicles of 2-5 mm in diameter showed lower rates of maturation and blastocyst formation than those from ovaries carrying follicles of > 10mm in diameter (Gandolfi et al., 1997; Kubota and Yang, 1998). Also, Blondin et al. (1997) found a significantly more oocyte developed from larger follicles (≥ 10mm) than from medium follicles. This indicate that large follicles (≥6mm diameter) provide the oocyte with a microenvironment which improves its quality (Lonergan, 1992). Dramatic changes in oocyte nuclei, especially nucleoli, are known to occur as the bovine follicle grows from 1 to 20 mm. Such changes may have a crucial effect on the developmental potential of the oocytes. It is known that a very stable form of RNA accumulates in the oocyte and that it is translated during maturation, fertilization and early embryonic development; such RNA accumulation may be influenced by the nature of the follicle growth (Sirard et al., 1992). Furthermore, Assay et al. (1992) compared the follicular environment and structure of oocytes originating from the dominant follicles (DF) with those of the two largest subordinate follicles examined a few days after ovulation. The DF is characterized by an estrogen-dominated environment, healthy cumulus cells junctionally coupled to the oocyte. Subordinate follicles are characterized by a progesterone-dominated environment, usually with degenerated cumulus cells, meiotic activation and other features of atresia.
However, other reports suggest that follicular size may not the only important criterion, since some bovine oocytes originating from large follicles failed to produce embryos, while some oocytes from medium size follicles already have this capacity (Hyttel et al., 1997).
1.2 Methods of oocytes retrieval
1.2.1 Aspiration technique
Several reports deal with comparison of the different methods used for oocyte recovery. Recovery of bovine oocytes by aspiration of vesicular follicles, using an appropriate pipette or syringe and needle, has been the method most commonly employed. The advantage of follicle aspiration is in terms of speed of operation, which may be particularly important in commercial embryo production. One of the difficulties associated with the aspiration approach lies in the fact that oocytes may only be retrieved from 30-60% of the puncture follicles (Katska, 1984). When comparing between aspiration of follicles and follicular dissection in cattle (Jiang, 1991) these work generally supports the view that significantly greater yield of oocytes in the highest-quality grades may be obtained by follicle dissection rather than aspiration. Aspiration of the oocyte can result in greater disruption of surrounding cumulus cells. There is also the possibility that aspiration does not always succeed in retrieving the highest-quality oocytes, this may be due to the cumulus oophorus being firmly attached to the stratum granulosum (Gordon, 1994).
1.2.2 Slicing of ovary
More recently, slicing procedures have been employed in cattle (Carolan et al., 1994). Those authors reported that oocyte yields average 55 per animal. This was a threefold increase on the number than recovered by aspiration. Oocyte recovery by slicing rather than aspiration can resulted in marked increase in blastocyst yield after IVM, IVF and IVC.
1.2.3 Slicing after aspiration
The slicing of ovaries after preliminary aspiration of follicles has been dealt in several reports (Takagi et al., 1992). Those authors concluded that there would be no merit, in terms of oocytes recovered or there quality in combining the two procedures.
1.8. Ovary storage: temperature and time limits
In the recovery of oocytes for in vitro maturation, the time interval between animal slaughter and oocyte recovery from the ovaries and the temperature at which the ovaries should be stored are important considerations. Cattle oocytes were recovered within 1-2h of animal slaughter; ovaries were usually stored at about 30ºC (Sekine et al., 1992). Furthermore, Pollard et al (1996) suggested that exposure of COCs to temperature below 35ºC during oocyte recovery might significantly decrease both the quantity and quality of bovine embryos produced by in vitro methods. Other reports suggested that the time from slaughter to oocyte recovery might extend up to 8 h (Solano et al., 1994).
2. Oocyte quality
Proper oocyte selection in the laboratory is crucial for successful embryo production. Presence of an intact complement of cumulus cell layers surrounding the oocyte and a homogenous appearing cytoplasm have been the best indicators of an immature oocyte ability to undergo maturation and embryonic development. Studies have evaluated the impact of cumulus morphology on subsequent development in cattle (Hazeleger and Stubbings, 1992). When immature oocytes were classified according to the number of layers of cumulus left around the oocytes following aspiration, the thicker the number of layers of cumulus cells, the better were the chances for development (Lonergan, 1992). The oocytes that are not of this type have aberrant protein synthesis (Kastrop et al., 1990) and complete meiosis at a lower frequency (De Loose et al., 1989). The role of the cumulus cells is to provide nutrients to the oocytes during its growth, to participate in the zona formation, and following the LH surge, to synthesis the matrix composed of proteins and hyaluronic acid important in oviductal transport or in sperm trapping (Bedford and Kim, 1993).
3. Culture media
The culture employed in IVM not only affect the proportion of bovine oocytes that reach metaphase II (M II) and become capable of undergoing in vitro fertilization, but can also influence subsequent embryonic development (Bavister, 1992a). In vitro maturation medium can be broadly divided into simple and complex. Simple media are usually bicarbonate-buffered systems containing physiological saline with pyruvate, lactate and glucose, and they differ in their ion concentration and in the concentrations of the energy sources. Complex media contains in addition to the basic components of simple media, amino acids, vitamins and purines.
Most IVF laboratories routinely use M-199 as the basic IVM medium in cattle (Bavister, 1992a), and there have been few reports suggesting that other media may be more appropriate. In one comparison of complex media for IVM, Hawk and Wall (1993) concluded that under their conditions, F-10 medium is superior to M-199 and B2 media. Recent comparison of IVM commercially available complex chemically defined media showed that TCM-199 was superior to RPMI-1640 (Gliedt et al., 1996). While, Wang et al. (1997) found no difference in the rate of embryo development for bovine oocytes matured in TCM-199 or CR2 media. Oocytes matured in medium leading to poor developmental competence have depressed levels of glycolysis that necessary for completion of maturation, the reduced level of glycolysis may reflect reduced activity of the pentose phosphate pathways, which plays an important role in meiotic maturation of bovine oocytes (Krisher and Bavister, 1998).
Dealing with the energy source, Hashimoto et al. (2000) showed that excessive glucose in the media used for oocyte maturation impairs the development of bovine oocytes to the blastocyst stage, possibly due to the increase of reactive oxygen species (ROS) and the decreased in the intracelluar glutathion content of bovine oocytes.
Addition of beta-mercaptoethanol to TCM-199 medium increased intracellular glutathion levels of bovine oocytes cultured individually and can improve maturation rate leading to the blastocyst stage throughout in vitro production (Mizushima and Fukui, 2001).
4. Bovine serum and other protein sources
4.1 Bovine serum albumin (BSA)
Sanbuissho and Threlfall (1988) found that FCS was superior to BSA as a protein supplement in IVM medium. It has been recognized that BSA was probably contaminated with some low molecular weight compound (Kane, 1987). For example, four lots of Fraction V BSA were tested by Bavister and McKieren (1991) for their ability to support two-cell hamster embryos in culture. Results showed that such preparations can produce highly variable effects on cultured embryos and cells ranging from highly inhibitory to highly stimulatory. Data on the amino acid content of bovine serum albumin is provided by Fallon et al. (1988). They noticed that this constituent could show wide variations; similar variability may presumably be expected with hormones, growth factors, cytokins and vitamins.
4.2 Sources of bovine serum
Bovine serum, in the form of FCS or oestrous cow serum (OCS), has been employed as the main protein source in bovine IVM studies. Lu and Gordon (1987) showed that OCS had a significant and marked effect, compared with FCS, on the percentage of secondary oocytes that were fertilized and which cleaved during culture. The same findings were reported by Schellander et al. (1990).
In the study reported by Younis et al. (1989), they suggested that pro-oestrous serum may be more effective than OCS, it was found, on analysis, it contains high levels of LH and prolactin. Recently, Boediono et al. (1994) reported that superovulated cow serum (SCS) was superior to FCS for oocyte maturation and fertilization and embryo development in vitro. Schroeder et al. (1990) reported that fetuin, a major glycoprotein constituent of FCS, can prevent hardening of the zona pellucida (ZP) during IVM by preventing the action of proteolytic enzymes originating from precociously released cortical granules.
4.3 Serum substitutes
Several commercial products are available as serum substitutes for use in in vitro cell culture. Lonergan (1992) used the Ultroser G (compounds: growth factors, adhesive factors, mineral trace elements, hormones, binding proteins and vitamins) successfully in cattle IVM without hormone supplementation at a concentrations of 1-4%. A study reported by Saeki et al., (1993) employed polyvinyl-pyrrolidone (PVP) at a 0.3% concentration as a substitutes, in the absence of hormones (Estradiol/ LH/ FSH) in the IVM medium, there was no yield of blastocyst. However, Monaghan (1993), found that PVP could effectively replaced serum in the absence of hormones.
5. Hormone supplementation of the IVM medium
Currently, most IVM protocols do employ luteinizing hormone (LH) or follicle stimulating hormone (FSH) or a combination of them. However, the effect of the gonadotropins and their relative importance on in vitro maturation and subsequent fertilization and early development is still controversial (Goto and Iritani , 1992). Zuelke and Brackett (1990) showed that the use of highly purified LH preparations of bovine origin at a certain level in their IVM medium significantly increased embryo yield after IVF/IVC. Evidence was found that LH may alter calcium distribution within the ooplasm and that the gonadotropin promotes increased glycolysis, combined with increased mitochondiral glucose oxidation, within cumulus-cell-enclosed bovine oocytes. It was also evident that LH exposure resulted in increased glutamine metabolism within the oocyte. In contrary, other reports showed no enhancement of development following addition of LH to maturation medium (Keefer et al., 1991). Reports revealed that mRNA of the LH receptors was detected exclusively in thecal cells. Absence of LH receptors in oocytes confirmed the previous results (Bevers et al., 1997).
At the same time, much of work suggested that FSH has a beneficial effect and that the presence of this gonadotropin in the in vitro maturation medium enhances expansion of the cumulus cells surrounding the oocyte, which in terms enhances sperm capacitation and the fertilization process (Eyestone and Boer, 1993). Recently, Abdoon et al. (2001) found that FSH or eCG supplementation to the IVM medium significantly increased cleavage rate and development of buffalo embryos up to the blastocyst stage when compared with negative control medium. It is concluded that cAMP dependent protein kinase activity regulating by cumulus cells following FSH stimulation plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes (Tatemoto and Terada, 1998).
Moreover, in vitro maturation of bovine cumulus oocyte complex (COCs) in serum free medium supplemented with bovine growth hormone (bGH) accelerated the progression of meiosis, induced cumulus expansion, and enhanced the cleavage rate and number of blastocyst following IVF and IVC (Izadyar et al., 1996; Kandil et al., 2000). Growth hormone can influence oocyte maturation by affecting the kinetics of the first polar body extrusion (Apa et al., 1994). Also, it causes a better cytoplasmic maturation in terms of proper distribution of cell organelles or the formation of the male decondensation factor (Izadyar et al., 1997). However, Sirotkin and Nitary (1992) recorded that GH treatment during IVM had no marked influence on the resumption of meiosis, but significantly delayed its completion in a dose related manner.
Maturation of oocytes in the presence of estradiol and FSH reduced the percentage of oocytes undergoing germinal vesicle break down (GVBD), while estradiol alone had no effect (Bevers et al., 1997). Androstenedione reduced the percentage of oocytes showing GVBD when added alone or with FSH. The presence of estradiol in the culture medium of in vitro matured human oocytes had no effect on the progression of meiosis but improved fertilization and cleavage rate suggesting that estradiol supports cytoplasmic maturational changes necessary for in vitro fertilization and early post fertilization development (Bevers et al., 1997). However, maturation of bovine oocytes in the presence of high concentrations of estradiol had a negative effect on spindle formation and first polar body extrusion (Kruip et al., 1988), and may alter protein uptake and incorporation (Pontbriand et al., 1989). Estradiol could be added at a concentration of 1µg/ml (Gordon, 1994), which is about the concentration in the follicular fluid of preovulatory follicles shortly after the LH peak.
5.3 Growth factors
The effect of growth factors on oocyte in vitro maturation has been examined in cattle by several investigators. Lonergan et al. (1996) demonstrated that the presence of epidermal growth factor (EGF) during IVM stimulated cumulus expansion and significantly increased the proportion of oocyte attaining M II. While, Takagi et al. (1991) employed EGF to cattle IVM without any evident effect on oocyte maturation.
The addition of insulin like growth factor-I (IGF-I) (Park and Lin, 1993) or transforming growth factor-α or B or IGF-2 (Jiang et al., 1991) to the IVM medium significantly improved the quality of oocyte. Nuclear maturation was not affected when denuded oocytes were cultured with EGF, indicating mediation by cumulus cells in cattle (Lorenzo et al., 1994). Stimulating activity of EGF is dependent on the cyclic AMP pathway and probably transduced by proteinase-k cytokines pathway (Coskun and Lin, 1994).
5.4 Effect of cytokins
Cytokines are small regulatory peptides or glycoproteins, with molecular weight ranging from 6000 to 60,000, which are synthesized and secreted by activated immune and mesenchymal cells (Ben-Rafael and Orvieto, 1993). Cytokines are believed to act generally in a paracrine or autocrine manner. It is possible that cytokines originating from the oocyte have a role in preparing the maternal immune or endocrine system for subsequent events in in vitro fertilization and early pregnancy (Ben-Rafael and Orvieto, 1993).
6. Effect of follicular fluid
Follicular fluid (FF) is a serum transudate modified by follicular metabolic activities, contains specific constituents such as steroids and glycoproteins synthesized by the cells of the follicle wall. Lonergan (1992) found that supplementation of the IVM medium by bovine follicular fluid (bFF) at the 10-20% level favoured subsequent embryonic development in cattle. Also, Iritani et al. (1992) recorded evidence of favourable effect from including bFF in IVM medium at 20-30% level. The implication in these various reports is that certain factors in bFF may favourably influence oocyte quality. However, Ayoub and Hunter (1993) reported that bFF from small follicles could inhibit meiosis in cattle oocytes. Follicular fluid from small and medium size follicles at estrus through mid-diestrus had more GVBD inhibition activity than at early proestrus (Romero-Arredendo and Seidel, 1996). Recently, Choi et al. (1998) reported that high concentration of bFF (10-20%) in maturation medium suppressed both resumption of meiosis, fertilization rates and embryo development.
7. Effect of maturation time
In the study of Sirard (1989) on the timing of nuclear events during IVM, the germinal vesicle (GV) was evident from 0 to 6.6 h, GVBD occurred at 6.6-8.0 h, chromatin condensation at 8-10.3 h, metaphase I at 10.3-15.4 h, anaphase I at 15.4-16.6 h, telophase I at 16.6-18.0 h and metaphase II at 18.0-24.0 h. Xu et al. (1986) found that the IVM culture period required for GVBD and abstriction of the first polar body was found to be related to the thickness and compactness of the COCs. Similarly, Spiropoulos and Long (1989) reported that partially denuded oocytes, totally denuded oocytes and oocytes with expanded cumulus cells at the start of IVM progressed to metaphase II faster than compact COCs.
Semple et al. (1993) reported that bovine oocytes achieved developmental competency within 14 h of commencing IVM; their data also suggested that early fertilization could lead to significantly higher yields of blastocyst.
Under routine IVM systems, maturation time usually 22-24 h in cattle (Monaghan et al., 1993).
II. Factors affecting in vitro fertilization (IVF)
Fertilization is a complex process, which results in the union of two gametes, the restoration of the somatic chromosome number and the start of the development of a new individual. Successful cattle IVF requires appropriate preparation of both sperm and oocyte, as well as culture conditions that are favourable to the metabolic activity of the male and female gametes (Xu and King, 1990). The first report of successful IVM and IVF of cattle oocytes was that of Iritani and Niwa (1977) in Japan, but the birth of calves was not reported until the work of Hanada et al. (1986).
1. Preparing sperm for fertilization
Fertilization of the bovine oocyte involves a sequence of events in which the sperm: (i) is motile (to reach the oocyte and move through the zona pellucida (ZP)); (ii) has the ability to undergo capacitation and express the acrosome reaction (AR); (iii) has 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; and (iv) able to fuse with the oolemma and be incorporated into the oocyte.
It is clearly important to have highly motile bull sperm available for IVF. This may be achieved by applying various procedures for isolating motile samples. There are also a number of chemical agents which may be employed to stimulate motility and AR of bull sperm and to maintain motility.
1.1 Factors affecting sperm motility and capacitation
1.1.1 Effect of bull as a source of variability in IVF
Considerable variability was exists among bulls in the ability of their sperm to become capacitated. Lambert et al. (1984) employed high ionic strength (HIS) medium to capacitate bull sperm, from five different bulls, they recorded that in vitro fertilization rates varying from 14 to 46%, they illustrated that individual variation as one of the most important factors affecting sperm preparation by the HIS method. Another authors using other capacitation medium recorded the same finding (Kroetsch and Stubbings, 1992). Moreover, Hillery et al. (1990) examined the outcome of IVF when sperm from high and low fertility bulls were employed, the yield of IVF embryos for the high fertility bulls was double than that recorded for those in the low fertility group. Individual bull variability may be related to the stage of season, age of animal, ejaculate sperm quality (Iritani et al., 1986). In this respect, seminal plasma may have been a source of variation in the sperm used in IVF as it contains: (i) decapacitation factors and variation in the level of such factors may affect ejaculated sperm (Goto et al., 1989). (ii) synthetic activity in oocytes is induced by sperm penetration (First and Parrish, 1987); and (iii) sperm may differ in the time taken for them to capacitate and this may affect subsequent embryonic development of the oocyte after IVF. Moreover, Taft et al. (1992) have recorded evidence that sperm from subfertile bulls may undergo the acrosome reaction (AR) and die prematurely. The use of pooled semen is a well-accepted method of minimizing male variability in cattle IVF work (Lu and Polge, 1992).
In contrary, Miller and Hunter (1987) failed to find evidence of significant variation among 29 AI bulls in their capability to achieve IVF. This result was confirmed by Schneider et al. (1999) who suggested that there was no predictive relationship between bull field fertility and in vitro embryo cleavage or developmental rates.
1.1.2 Use of Fresh or Frozen Semen
Various works have employed both fresh and frozen bull semen in their cattle IVF studies (Lengwinat et al., 1990). Those authors concluded that fresh semen requires a longer capacitation period than frozen semen. Meanwhile, Seaton et al. (1991) found that fresh sperm gave better penetration rates than frozen thawed sperm. Frozen-thawed bull semen is likely to deteriorate more rapidly than fresh ones (Gordon, 1994). One problem in using fresh bull sperm, they have at least passed through an initial screening before freezing.
1.1.3 Methods of sperm separation
There have been many reports characterizing Percoll density gradient, swim-up, sephadex, glass wool and other sperm separation procedures for bovine spermatozoa. The recovery rate of motile spermatozoa was higher for sperm separated by Percoll rather the swim-up method. However, swim-up procedure resulted in more ova being penetrated than did by using Percoll method. Increasing number of sperm concentrations during IVF could eliminate this problem (Parrish et al. 1995). Avery and Greve (1995) suggested that this adverse effect of Percoll is not due to Percoll particle per se, but may be ascribed to the effect of unbound PVP in the Percoll. For that reason, the presence of PVP stopped bull sperm motility.
1.2 Artificial induction of capacitation and acrosome reaction (AR)
Capacitation is a process involving the sperm in a complex series of biochemical and physiological reactions. It is believed that the initial step of capacitation involve the removal and alteration of components derived from the seminiferous tubules, epididymis, vas deferens and seminal plasma, this would permit exposure of receptors sites, allowing sperm to interact specifically with oocyte receptors. Sperm capacitation can be achieved by different methods such as:
1.2.1 Fertilization medium
Treatment of semen with a medium of high ionic strength (HIS) like Brackett and Oliphant (BO) medium (osmolarity 360 to 390 mOsm) is described for capacitation of fresh bovine semen (Brackett et al., 1982) and frozen bovine semen (Bousquet and Brackett, 1982). Sirard et al. (1986b) demonstrated that the use of the HIS method as probably being limited to certain bulls; they did not regard the procedure as suitable for general application. Recently, many authors used TALP-medium for in vitro capacitation of bovine spermatozoa (Ibrahim, 1993). In this respect, Jaakma et al. (1997) observed that a significantly higher proportion of bovine oocytes developed to blastocyst stage after insemination with spermatozoa prepared by swim-up in Fert-TALP supplemented with heparin than by centrifugation in mBO supplemented with 10mM caffeine-sodium benzoate.
1.2.2. Use of heparin and caffeine
Studies support the view that capacitation of bull sperm by heparin probably reflects the in vivo mechanism (Parrish et al., 1989). Heparin dosage and incubation period for sperm capacitation are important factors affecting bovine IVF and subsequent embryo development (Vlaenderen et al., 1991). Heparin induces changes in the calmodulin (CaM)-binding properties of sperm proteins and induces a reduction in Ca+ concentrations during capacitation (Leclerc et al., 1992). Parrish et al. (1999) found that capacitation of bovine sperm with heparin requires extracellular calcium, the maximal kinetics of heparin-induced capacitation occurs when extracellular calcium exceeds 10µMl. Changes in calcium triggre subsequent increase in cAMP, pH and tyrosine phosphorylation, that are known to be essential for capacitation (Parrish et al., 1995).
Niwa and Ohgoda (1988) reported a synergistic effect of 20-µg/ml heparin and 10nM caffeine in their capacitation treatment of frozen-thawed bull sperm. It was evident that the optimum dose of the agent was100µg/ml (Shehata, 1998). Other reports suggested that the fertilization rate may be rapidly improved by adding heparin to the IVF medium at values varied between 0.5 to 5.0µg/ml (Shamsuddin et al., 1992). Preincubation period of 15 min was found to be satisfactory.
Miller et al. (1987) reported that heparin promotes capacitation processes by mechanisms seems to depend on sperm capability for absorption of seminal proteins at time of ejaculation, which increases the ability of spermatozoa to bind heparin through its sulfate residue, a basic requirement for triggering the heparin capacitation-promoting effect.
1.2.3. Follicular fluid
Various procedures have been examined for capacitating frozen-thawed bull sperm and the subsequent use of such sperm in IVF. Sugawara et al. (1984), using frozen-thawed bull sperm preincubated in media containing bFF reported a sperm penetration rate of 56%. It is clear that bFF contains many compounds (glucose amino glycains (GAGs)) capable of capacitating bull sperm; for that reason, it has been used in bull sperm capacitation medium (Iqbal and Hunter, 1992).
1.2.4 Use of calcium ionophore (A23187)
Various authors have shown the importance of an influx of extra-cellular Ca2+ into the sperm in the capacitation process. The calcium ionophore A23187 has been employed to achieve this influx (Fukuda et al., 1988). Bird et al. (1989) showed that the ionophore treatment resulted in hyperactivation and a functional AR in bovine sperm, enabling them to penetrate zona-free hamster oocytes. Other workers compared treatment of bull sperm with A23187 and heparin, it was suggested that the simplicity of using the ionophore and the higher yield of embryos as the advantage of using that agent (Jiang et al., 1992). There are some evidences that the Ca2+ influx is the result of calcium entering a non-mitochondrial compartment as a consequence of the equilibration of the ion across the mitochondrail and plasma membrane of the sperm, and increase the respiratory activity of the sperm (Simpson and White, 1987).
Although, the ionophore-induced AR is believed by some to be similar to the normal in vivo reaction in capacitated sperm. While, Watson et al. (1991) concluded that the ionophore-induced reaction is not the same as the natural event.
More recently, Rathi et al. (2001) illustrated that Ca2+ ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR.
1.2.5 Effect of glucose in fertilization medium
In cattle, it is reported that glucose inhibits the role of heparin for inducing sperm capacitation (Tajik and Niwa, 1997). On the other hand, for cattle oocytes inseminated in chemically defined medium, glucose is required for stimulating in vitro fertilization of bovine oocyte.
2. Preparing In vitro Matured Oocytes for IVF
Removal of cumulus cells
In an effort to make the surface of the in vitro matured oocyte more accessible to sperm in IVF, researchers have attempted to remove some or all of the cumulus cell layers, either by mechanical stripping in suitably sized micropipettes (Cox et al., 1993), by the use of enzyme preparations such as hyaluornidase (Park et al., 1989) by chemical agents such as sodium citrate (Kinis et al., 1990), or by vortex (Parrish et al., 1988). Hawk et al. (1992) demonstrated that detaching most cumulus cells from oocytes before IVF increased fertilization rate. Clearly, it is undesirable that oocyte-cleaning treatments should reduce ability to be fertilized or compromise subsequent embryonic development. Younis and Brackett (1991) examined how the presence or absence of cumulus cells around the oocyte may contribute towards the variability observed in cattle IVF. They recorded that cumulus cells are necessary at the time of IVF to maximize the incidence of AR. However, Behalova and Greve (1993) have shown that sperm penetration rate was similar in denuded and cumulus-enclosed bovine oocytes, there was a significant increase in polyspermy in the denuded oocytes. While, Bottcher et al. (1990) found that removing of cumulus cells resulted in considerable increase in the incidence of defective ZPs and ooplasm abnormalities.
3. In Vitro Fertilization Culture System
It is clear that the medium employed in IVF systems must be capable of providing the secondary oocyte and the capacitated sperm with the conditions, which will permit sperm penetration to occur readily.
The basic medium for preparation of sperm droplet is IVF-TALP with heparin and caffeine. Spermatozoa are added to the droplets at a concentration of approximately 1-1.5x106 spermatozoa/ml (Madison et al., 1991). The standard conditions for co-culture of spermatozoa and oocytes are 16-22 h at 39˚C in an atmosphere of 5% CO2 in air (Fukui et al., 1990).
4. Interaction of Sperm and Oocyte
Fertilization involves activation of the oocyte by the sperm. Without this stimulus, the oocyte would be unable to form pronucleus (PN) and become a zygote. After oocyte activation the vitellus shrinks in volume, expelling fluid into the perivitteline space. At the same time, the sperm head in the vitellus swells and acquires the consistency of a gel, losing its characteristic shape, the final structure, which resembles the nucleus, it termed the male PN (Gordon, 1994).
Biochemical changes that occur as a result of sperm penetration include changes in the pattern of intracellular Ca+. It is known that such changes in Ca+ levels at fertilization are involved in the induction of cortical granules exocytosis and the resultant block to polyspermy that occur in the cow in the form of a wave (Sun et al., 1993).
5. Fertilization time
The optimal sperm/oocyte incubation time for achieving maximum fertilization rates after IVM/IVF, the highest fertilization rate and embryo yield resulted when oocytes were incubated with sperm for 24 h (Gordon, 1994).
III. Factors affecting in vitro culture of embryos
The culture of embryos in vitro requires an appropriate environment so that the early embryos can undergo several cleavage divisions to enable it to reach the blastocyst stage of development. Sheep oviducts (Gordon and Lu, 1990) or rabbit oviducts (Fukui and Ono, 1988) have been used for culture of in vitro fertilized oocytes to the morulae or blastocyst stage. However, owing to the loss of embryos in the oviduct (disappearance of the agar chips) and the impracticability of using live animals, the preferred and superior method is to use an in vitro system for embryo culture. The advantages of using the in vitro system for embryo culture are: (i) to study, in much greater detail, embryonic development, the requirements for embryonic development when maternal-embryonic transition in protein synthesis takes place; (ii) certain genes are switched on or off; and (iii) the use of very specific developmental stages for cloning and production of transgenic animals. Cleavage rate and embryo development could be affected by many factors related to:
1. Use of chemically defined culture media
Cattle embryos derived from IVM/IVF can develop in vitro to the morula stage in chemically defined, protein-free media, with no apparent advantage evident in using somatic feeder cells or serum. According to Bavister (1993), numerous laboratories using culture media without somatic cell support have obtained embryo development results equal to, and in some cases better than those reported with co-culture.
For effective blastocyst development, Pinyopummintr and Bavister (1991) found that serum factors were required, serum was beneficial in stimulating morula compaction and blastocyst formation. Moreover, Rosenkrans and First (1991) concluded that a simple medium, Charles Rosenkrans 1 (CR1), that contains essential and non-essential amino acids were beneficial to bovine embryo development in vitro in the absence of feeder cells. A comparison between CR1 medium supplemented with amino acids and BRL cell monolayer gave a similar outcome in terms of embryo yield (Moreno and Westhusin, 1993).
The effect of glucose on the development of bovine embryos has been identified. Takahashi and First (1992) showed that one-cell bovine embryos can be successfully cultured beyond the 8-16-cell block to the blastocyst stage, using a chemically defined medium without glucose but containing pyruvate, lactate, amino acids and BSA. In addition, Kim et al. (1993) found that the presence of glucose in their semi-defined IVC medium (M-199 with BSA, Lactate and pyruvate) inhibited early development, especially to the eight-cell stage; when added at 5 days after IVF, glucose improved development to the blastocyst stage. Recently, Augastin et al. (2001) noticed that glucose transport-4 (Glut4) was detected first at the blastocyst stage, and Glut-2 expression was restricted to the period of blastocyst elongation at day 14 and day 16.
Chemically defined used in many IVC systems usually contains a protein source, such as serum or BSA. Pinyopummintr and Bavister (1993) demonstrated that serum-supplemented IVC medium had a biphasic effect, inhibitory at the first cleavage stage and stimulatory at the morula/blastocyst stage, on bovine embryo development in vitro, they added that serum was most beneficial at 2 days post insemination. Elsewhere, it has been suggested that cow serum recovered from superovulated donor cows 7 days after breeding may have merit as an ingredient in IVC medium (Suzuki, 1993). The presence of FCS in SOF not only resulted in faster development and increased blastocyst production, but also enhanced overall male survival (Gutierrez-Adan et al., 1999). While, serum substitution with PVP resulted in lower blastocyst yield (Kuran et al., 2001).
2. Co-culture with Bovine Oviductal Cells
The bovine oviduct provides the microenvironment for the transport and final maturation of gametes, fertilization and early embryonic development. In setting up the bovine oviductal epithelial cell (BOEC) monolayer, researchers have attempted to provide a similar microenvironment in the laboratory. Gandolfi and Moor (1987) were among the first to demonstrate that the eight-cell developmental block did not occur if sheep early embryos were cultured on oviductal cell monolayers. Oviductal cells isolated at early and late luteal phase of the oestrous cycle were similar capable of supporting early embryonic development (Thibodeaux et al., 1992).
Some other workers have failed to achieve the same results with monolayer culture systems using IVMF oocytes as with oocytes produced and fertilized in the living animals (Van Soom and Kruif, 1992).
3. Use of preconditioned media
There have been several reports dealing with the culture of cattle embryos in vitro, using medium has been preconditioned by exposure to oviductal/ uterine or cumulus/ granulosa cells.
Medium conditioned by oviductal tissue was effective as coculture in supporting bovine embryo development from the zygote to the blastocyst stage (Eyestone et al., 1990). The supernatants prepared from cells taken around oestrus were significantly more effective than those prepared at other times (Harper et al., 1989). It was apparent that the conditioned medium contained a low-molecular-weight factor (s) acting in early cleavage and larger molecule (s) acting on blastocyst formation (Mermillod et al., 1993). The advantages of serum-free conditioned medium over coculture include eliminating the confounding presence of cells and making the search for soluble embryotrophic factors easier (Massip et al., 1993).
Although, Inzen et al. (1993) found that conditioned medium prepared from Buffalo Rat Liver cell (BRL) was as effective as coculture with BRL cells in developing cattle embryos from the two-cell to the blastocyst stage. The same findings were reported in other investigations using other media (Sikes et al., 1993).
Meanwhile, addition of high molecular weight bovine oviduct conditioned medium (BOCM) fraction to modified synthetic oviduct fluid (mSOF) medium significantly increased embryo development up to the bastocyst stage in comparison with mSOF or BOCM. (Vansteenbrugge et al., 1996).
4. Hormones and growth factors
4.1 Hormones and their effect
Hormones may be involved in the regulation of development in early embryos. Insulin has been shown to increase the rate of glucose transport in the blastocyst (Gardner and Kaye, 1991), blastocyst metabolism in vitro (Wales et al., 1985). According to Heyner et al. (1993), insulin is the only hormone, which has been shown to have a clear effect on early embryonic development. Also, Seidel et al. (1991) were able to show that insulin had a beneficial effect in IVC, possibly due to binding to IGF-I receptors.
Recently, Izadyar et al. (2000) reported that expression of GH receptors gene in preimplantation bovine embryos, presence of receptors, and the beneficial effect of GH on cleavage rate, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development. This finding is confirmed by Kolle et al. (2001), they suggested that a functional GH receptors (GHR) able to modulate carbohydrate and lipid metabolism synthesis during early preimplantation development of bovine embryos and that this GHR may be a subjected to activation by embryonic GH.
4.2 Growth factors and their effect
Studies reported by Floot et al. (1993) and Palma et al. (1993) showed no evidence that IGF-I supplementation to IVC medium improved bovine embryos. However, in mice Harvey and Kaye (1992) examined the effect of IGF-I on early embryos and found it did stimulate protein synthesis; they found that both insulin and IGF-1 stimulates mitogenesis of the inner cell mass (ICM) and morphological development via insulin and IGF-1 receptors. Moreover, Lonergan et al. (1996) showed that epidermal growth factor (EGF) is capable of significantly improving the development of two-to eight-cell cattle embryos to the morula/blastocyst stage. Studies by Floot et al. (1993) showed hat supplementation of a chemically defined medium with TGF-α did not improve IVF-derived bovine embryo development.
Larson et al. (1992) have reported a significant effect after addition of transforming growth factor-B (TGF-β) to their IVC medium when embryos were at the early balstocyst stage; they found that TGF-β mainly stimulated mitotic activity in ICM cells. Furthermore, TGF- β in CR1aa medium was also found to have a positive effect on improving bovine embryo development (Rosenkrans and First, 1991).
4.3 cytokins and their effect on embryo development in vitro
Cytokins are hormone-like polypeptides that function as intracellular signals regulating cell-cell interaction in the uterus (Tabibzadeh and Sun, 1992).
Fukui and Matsyama (1994) found that human Leukaemia inhibitory factor (hLIF) improved the development of IVMFC-derived cattle embryos when SOF medium was supplemented with BSA or PVP but not when it was supplemented with human serum. Meanwhile, the addition of LIF to SOF media has been shown to increase fourfold the number of sheep blastocyst that hatched (Fry et a., 1992), improve the viability of cultured sheep blastocyst (Fry et a., 1991).
IV. Miscellanies factors affecting IVF
1. Buffering system and osmolarity
The buffering system employed in IVM media will depend on whether the medium is exposed to air or to a carbon dioxide-enriched atmosphere. The advantage of HEPES- or phosphate-buffered media for short-term work with oocytes and embryos is that they do not require a carbon dioxide-controlled gas phase to maintain a relatively constant pH. The IVM will normally be isotonic with the natural tissue fluids in contact with the gametes. Osmolarity is generally arranged to be between 275-285mosm, which is considered to be the optimum range (Gordon, 1994).
2. Water quality
Water is a major constituent of any IVM medium. The use of ultrapure water, free from contaminants, is crucial for its preparation (Bavister, 1992a, b). The principal methods for purifying water are glass distillation, deionization, filtration, reverse osmosis, adsorption and ultrafiltration. Optimum results have been claimed using the Millipore reverse osmosis (RO) and Milli-Q (MQ) system (Fukuda et al., 1987).
3. Temperature and gas phase
In an experiment carried out by Wang (1991), oocytes were matured in a humidified atmosphere of 5% carbon dioxide in air at 36, 37, 38, 39 and 40˚C and then fertilized and cultured at 39˚C. In experiment II, oocytes were matured at 39˚C, fertilized at 36, 37, 38, 39 and 40˚C. In experiment III, oocytes were matured and fertilized at 39˚C and then cultured as early embryos at 36, 37, 38, 39 and 40˚C. It is evident from the data presented that the optimum temperature for IVM is 38-39˚C, and for IVC, the optimum temperature is 39˚C. The detrimental effect of using the 40˚C temperature is clearly demonstrated, particularly in fertilization and early development (Rivera and Hansen, 2001). It is clearly evident that during oocyte recovery, exposing of bovine oocytes to room temperature at 20˚C decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro (Azambuja et al., 1998), and induces chromosomal abnormalities, (Moor and Crosby, 1985).
A comparison was made the conventional 5% CO2 in air gas phase and one supplying 5% CO2, 5% O2 and 90% N2. Pinynopummintr and Bavister (1994) concluded that low oxygen concentration (5-10%) is detrimental for both maturation and fertilization of bovine oocytes. While, Lim et al. (1999) found that 5% oxygen, 5% CO2 and 90% N2 gas mixture provides a suitable atmosphere for early bovine embryo growth in vitro and modified bovine embryo culture medium (mBECM) + bovine serum is the optimal culture medium under this atmosphere. Most of authors have recommended that gas phase of 5% CO2 in air is a suitable incubation environments for maturation of cattle and buffalo oocytes (El-Bawab, 1994)
4. Effect of light
There are data indicating that rabbit morulae maybe affected by 24 h exposure to visible light (Schumacher and Fischer, 1988). Clearly, the general principle should always be observed of keeping embryos in darkness rather in light (Gordon, 1994).
5. Protection from oxygen toxicity
It is possible that the exposure to 20% oxygen and light during routine embryo manipulations may lead to the generation of superoxide radicals, elimination of oxygen radicals in embryos may result in significant improvements in the effectiveness of IVM/IVF and IVC systems (Rieger, 1992).
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