Factors affecting in VITRO PRODUCTION of bovine embryos


Ahmed S. S. Abdoon

Department of Animal Reproduction & A.I. National Research Center, Dokki, 12622 Giza,

  Email: assabsoon@yahoo.com





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.


1.3.4 Transvaginal ultrasound-guide oocytes pick up (OPU)

            Transvaginal oocyte pick-up (OPU) is an important technique for oocyte retrieval in living previously genetically selected highly valuable donor cow. The success of OPU is measured in part by the recovery rate of oocytes, expressed as a percentage of the number of follicles punctured (Pieterse et al., 1991). This recovery rate in turn, is influence by numerous factors such as aspiration vacuum, hormonal pre-treatment of animals, puncture frequency, stage of the estrous cycle and the experience of the operator. Recovery rate declined as the aspiration pressure increased above 50mm Hg. The recovery rate of Grade 1 oocytes decreased significantly as the vacuum pressure increased with a corresponding increase in the number of denuded oocytes recovered (Ward et al., 2000). In addition, recovery rate and the recovery of oocytes considered viable for IVM/IVF procedures were both significantly higher by using a 17-g needle than a 20-g needle (Fry et al., 1997). Moreover, hormonal pretreatment of donors prior to OPU using gonadotropin significantly increased oocytes recovery rates and blastocyst production (Lonergan et al., 1994). Oocyte competence was increased when the period between p-FSH administration and OPU was extended (Sirard et al., 1999). Recently, Machatkova et al. (2000) concluded that it is possible to improve the efficiency of OPU and in vitro production of embryos by utilization of the growth phase of the first follicular wave before dominant follicle selection in cattle.


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

5.1 Gonadotropins

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.


5.2 Steroids

 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).



Abdoon, A.S.S., Kandil, O. M. Otoi, T. Suzuki, T. (2001): Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Animal Reproduction Science 65: 215-223.

Apa, R., Larnzone, A., Miceli, F., Masrrandrca, M., Caruso, A., Mancuso, S. and Canpiari, R. (1994): Growth hormone induces in vitro maturation of follicle and cumulus-enclosed rat oocytes. Molecular Reproduction and Development 106: 207-212.

Assey, R.J., Hyttel, P., Purwantara, B., Greve, T. and Schmidt, M. (1992): Oocyte structure in dominant versus subordinate follicles during the first follicular wave in cattle. Proc. 12th International Cong. Anim. Reprod. (The Hague) 1: 303-305.

Augastin, R., Pocar, P., Navarretae-Santos, A., Wrenzycki, C., Gandolfi, F., Neimann, H. and Fischer, B. (2001): Glucose transport expression is developmentally regulated in in vitro derived bovine preimplantation embryos. Molecular Reproduction and Development 60: 370-376.

Avery, B. and Greve, T. (1995): Impact of Percoll on bovine spermatozoa for in vitro insemination. Theriogenology 44: 871-878

Ayoub, M. A. and Hunter, A.G. (1993): Inhibitory effect of bovine follicular fluid on in vitro maturation of bovine oocytes. J. Dairy Science 76: 95-100.

Azambuja, R.M., Kraemer, D.C. and Westhusin, M.E. (1998): Effect of low temperature on in-vitro matured bovine oocytes. Theriogenology 49: 1155-1164.

Bavister, B.D. (1992a): Metabolism and nutrition of the preimplantation embryo. Proc. of the Nordic A.I. Virtanen Institute symposium on In vitro Culture of Domestic Animal Embryos, 5-12

Bavister, B.D. (1992b): Metabolism and nutrition of the preimplantation embryo. Proc. of the Nordic A.I. Virtanen Institute symposium on In vitro Culture of Domestic Animal Embryos, 21-27.

Bavister, B.D. (1993): Response to the use of co-culture for embryo development. Human Reproduction 8 (8): 1160-1162.

Bavister, B.D. and McKiernan, S.H. (1991): Different lots of bovine serum albumin stimulate or inhibit hamster embryo development in vitro. Serono Symposium on Preimplantation Embryo Development, Absract Vol., 52 (Abs. II-3).

Bedford, J.M. and Kim, H.H.  (1993): Cumulus oophorus as a sperm-sequestering device in vivo. J. Exp. Zoo., 265: 321-328.

Behalova, E. and Greve, T. (1993): Penetration rate of cumulus enclosed versus denuded bovine eggs fertilize in vitro. Theriogenology 39: 186.

Ben-Rafael, Z. and Orvieto, R. (1993): Cytokines-involvement in reproduction. Fertility and Sterility 58: 1093-1099.

Bevers, M.M., Dieleman, S.J., van der Hurk, R. and Izadyar, F. (1997): Regualtion of maturation in the bovine. Theriogenology 47: 13-22.

Bird, J.M., Carey, s. and Houghton, J.A. (1989): Motility and acrosomal changes in ionophpre-treated bovine spermatozoa and their relationship with in vitro penetration of zona-free hamster oocytes. Theriogenology 32: 227-242.

Blondin, P., Guilbalt, L.A. and Sirard M.A. (1997): The time interval between FSH-P administration and slaughter can influence the developmental competence of beef heifer oocytes. Theriogenology 48: 803-813.

Boediono, A., Takagi, M., Saha, S. and Suzuki, T. (1994): Influence of Day-0 and day-7 superovulated cow serum during development of bovine oocytes in vitro. Reproduction Fertility and Development 6: 261-264.

Bondioli, K.R. and Wright, R.W. (1983): In vitro fertilization of bovine oocytes by sperm capacitated in vitro. J. Animal Science 57: 1001-1005.

Bottcher, M., Blottner, S., Kruger, S. Rettig, B. and Lange, W. (1990): Effect of cumulus oophorus on the fertilization of cattle oocytes matured in vitro. Archiv Für Experimentelle Veterinar Medizin 44: 53-57.

Bousquet, D. and Brackett, B.G. (1982): Penetration of frozen-free hamster ova as a test to assess fertilizing ability of bull sperm after frozen storage. Theriogenology 17 (2): 199-213.

Brackett, B.G. Bousquet, D., Boice, M.L., Donawick, W.J., Evans, J.F. and Dressel, M.A. (1982): Normal development following in vitro fertilization in the cow. Biology of Reproduction 27: 147-158.

Carolan, C., Monaghan, P., Gallgher, M. and Gordon, I. (1994): Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro. Theriogenology 41: 1061-1068.

Choi, Y.H., Takagi, M., Kamishita, H., Wijayagunawardance, M.P.B., Acosta, T.J., Miyazawa, K. and Sato, K. (1998): Effect of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro . Theriogenology 49: 1103-1112.

Coskun, S.  and Lin, Y.C. (1994): Effects of transforming growth factors and activin-A on in vitro  porcine oocyte maturation. Molecular Reproduction and Development 38: 153-159.

Cox, J.F., Hormazabal, J. and Santa Maria, A. (1993): Effect of the cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology 40: 1259-1267.

De Loose, F., Van Vliet, C., Van Maurik, P. and Kruip, T. A. M. (1989): Morphology of immature bovine oocytes. Gamete research 24: 197-204.

El-Bawab, I. E. (1994): Meiotic maturation of Egyptian buffalo and cattle ovarian oocytes in vitro. Alex. J. Vet. Sci., 10: 79-83.

Eyestone, W.H. and Boer, H.A. de (1993): FSH enhances developmental potential of bovine oocytes matured in chemically defined medium. Theriogenology 39: 216.

Eyestone, W.H., Jones, J.M. and First, N.L. (1990): The use of oviduct-conditioned medium for culture of bovine oocytes to the blastocyst stage. Theriogenology 33: 226.

Fallon, M.N., Rammell, C.G. and Hoogenboom, J.J.L. (1988): Amino acids in bovine sera. New Zealand Veterinary J., 36: 96-98.

First, N.L. and Parrish, J.J. (1987): In vitro fertilization of ruminants. J. Reproduction and Fertility, Suppl. 34: 151-165.

Floot, M.R., Gage, T.L. and Bunch, T.D. (1993): The effect of various growth-promoting factors on preimplantation bovine embryo development in vitro. Theriogenology 39: 823-833.

Fry, R.C., Batt, P.A., Fairclough, R.J., Cameron, A.W.N. and Parr, R.A. (1991): Leukemia inhibitory factor (LIF) improves the health of cultured ovine embryos. Journal of Reproduction and Fertility, Abst. Series No. 7,, 34(Abs. 58).

Fry, R.C., Batt, P.A., Fairclough, R.J. and Parr, R.A.  (1992): Human leukemia inhibitory factor improves the viability of cultured ovine embryos. Biology of Reproduction 46: 470-474.

Fry, R.C., Niall, E.M., Simpson, T.L., Squires, T.J. and Reynolds, J. (1997): The collection of oocytes from bovine ovaries. Therogenology 47: 977-987.

Fukuda, A., Ichikawam M., Naito, K. and Toyoda, Y.  (1988): Normal development of bovine oocytes matured, fertilized and cultured with cumulus cells in vitro. Proc. 11th International Cong. Anim. Reprod. (Dublin) 3: 327-329.

Fukuda, A., Noda, Y., Tsukui, S., Matsumoto, H., Yano, J. and Mori, T. (1987): Influence of water quality on in vitro fertilization and embryo development for the mouse. J. In Vitro Fertilization and Embryo Transfer 4: 40-45.

Fukui, Y. and Matsyama, K. (1994): Development of bovine embryos matured and fertilized in vitro in media containing human leukemia inhibitory factor. Theriogenology 41: 199

Fukui, Y. and Ono, H. (1988): In vitro development to blastocyst of in vitro matured and fertilized bovine oocytes. Veterinary Record 122: 282.

Fukui, Y., Sonoyama, T., Mochoizuki, H. and Ono, H. (1990): Effect of heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro. Theriogenology 34: 575-591.

Gandolfi, F. and Moor, R.M. (1987): Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J. Reproduction and Fertility 81: 23-28.

Gandolfi, F., Luciano, A.M., Modina, S., Ponzini, A., Pocar, P., Armstrong, D.T. and Lauria, A. (1997): The in vitro developmental competence can be related to the morphology of the ovary. Theriogenology 48: 1153-1160.

Gardener, H.G. and Kaye, P.L. (1991): Insulin increases cell numbers and morphologyical development in mouse pre-implantation embryos in vitro. Reproduction Fertility and Development3: 79-91.

Gliedt, D.W., rosenkrans, C.F., Rorie, Jr. R.W. and Rakes, J.M. (1996): Effect of oocyte maturation length, sperm capacitation time and heparin on bovine embryo development. J. Dairy Science 79: 532-535.

Gordon, I. (1994): Laboratory Production of Cattle Embryo. CAB International, Wallingford, Oxon OX 108 DE, UK.

Gordon, I and Lu, K.H. (1990): Production of embryos in vitro and its impact on livestock production. Theriogenology 33: 77-87.

Goto, K. and Iritani, A. (1992): Oocyte maturation and fertilization. Animal Reproduction Science 28: 408-413.

Goto, K., Kajihara, Y., Koba, M., Kosaka, S., Nakanishi, Y. and Ogawa, K. (1989): In vitro fertilization and development of in vitro matured bovine follicular oocytes. J. Animal Science 67: 2181-2185.

Gutierrez-Adan, A., Perez, G., Granados, J., Garde, J.J, Perez-Guzman, M., Pintado, B., De La Fuente, J. (1999): Relationship between sex ratio and time of insemination according to both time on ovulation and maturational state of oocyte. Zygote 7: 37-43.

Hanada, A., Enya, Y. and Suzuki, T. (1986): Birth of calves by non-surgical transfer of in vitro fertilized embryos obtained from oocytes matured in vitro. Japanese J. Animal Reproductcion 32: 208 (Abs.)

Hashimoto, S., Minami, N, Yamada, M. and Imai, H. (2000): Excessive concentration of glucose during in vitro maturation impairs the developmental competence of bovine oocytes after in vitro fertilization: revelance to intracellular reactive oxygen species and glutathion contents. Molecular Reproduction and Development 56: 520-526.

Harper, K.M., Younis, a.I., Zuelke, K.A. and Brackett, B.G. (1989): Early bovine embryo development during culture in different estrous stage oviductal cell suspension supernatants. Biology of Reproduction 40 (Suppl. 1): 121 (Abs.222).

Harvey, M.B. and Kaye, P.L. (1992): Insulin-like growth factro-I stimulate growth of mouse embryos. Molecular Reproduction and Development 31: 195-199.

Hawk, H.W. and Wall, R.J. (1993): Experiments to improve the yield of blastocysts from in vitro produced oocytes. J. Anima Science 71 (suppl. 1), 220.

Hawk, H.W., Nel, N.D., Waterman, R.A. and Wall, R.J. (1992): Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes. Theriogenology 38: 989-998.

Hazelger, N.L. and Stubbings, R.B. (1992): Developmental potential of selected bovine oocyte cumulus complexes. Theriogenology 37: 219.

Heyner, Shah, N., Smith, Watson, A.J. and Renard, J.P. (1993): The role of growth factors in embryo production. Theriogenology 39: 151-161.

Hillery, F.L., Parrish, J.J. and First, N.L. (1990): Bull specific effect on fertilization and embryo development in vitro. Theriogenology 33: 249.

Hyttel, P., Fair, T., Callsen, H. and Greve, T. (1997): Oocyte growth, capacitation and final maturation in cattle. Theriogenology 47: 23-32.

Ibrahim, G.M.D. (1993): Bovine in vitro fertilization and embryo transfer. Ph.D.Thesis (Physiology), Fac. Vet. Med. Cairo University.

Inzen, W.G. van, Kruip, Th. A. M. and Weima, S.M. (1993): Use of conditioned medium for IVM-IVF bovine embryos in vitro culture system. Theriogenology 39: 236.

Iritani, W.G. and Niwa, K. (1977): Capacitation of bull spermatozoa and in vitro of cattle follicular oocytes matured in culture. Journal of Reproduction and Fertility 50: 119-121.

Iritani, W.G., Cheng, Z.S. and Utsumi, K. (1992): Effects of follicular factors on the IVM-IVF bovine oocytes. Proc. 12th International Congress on Animal Reproduction and Artificial Insemination (The Hague) 1, 342-344.

Iritani, A., Utsumi, K., Miyake, M. and Yamaguchi, Y. (1986): Individual variation in the in vitro fertilizing ability of bull spermatozoa. Development, Growth and Differentiation Supplementation 28: 45.

Iqbal, n. and Hunter, A.G. (1992): Comparison of various capacitating systems as to their ability to alter bovine sperm membrane proteins during capacitation. Biology of Reproduction 46 (Suppl. 1): 94 (Abs.173).

Izadyar, F., Colenbrander, B. and Bevers, M.M. (1996): In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development. Molecular Reproduction Development 45: 372-377.

Izadyar, F., Colenbrander, B. and Bevers, M.M. (1997): Growth hormone enhances fertilizability of in vitro matured bovine oocytes. Theriogenology, 47: 191 (Abst.).

Izadyar, F., Van Tol, H.T., Hage, G.W. and Bevers, M.M. (2000): Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development. Molecular Reproduction and Development 57: 247-255.

Izquierdo, D., Villamediana, P., Palomo, M.J., Mogas, T. and Paramio. (1998): Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. Theriogenology 49: 1501-1513.

Jiang, H.S. (1991): Studies in the culture of bovine embryos derived from oocytes matured and fertilized in vitro. Ph.D. Thesis, National University of Ireland, Dublin.

Jiang, H.S., Yang, X., Chang, S., Heuwieser, W. and Foote, R.H. (1991): Effect of sperm capacitation and oocyte maturation procedures on fertilization and development of bovine oocytes in vitro. Theriogenology 35: 218.

Jiang, H.S., Yang, X. and Foote, R.H. (1992): Sperm capacitation procedures for in vitro fertilization and development of bovine embryos. Theriogenology 37: 230.

Jaakma, U., Zhang, B.R., Larsson, B., Niwa, K. and Rodrihuez-Martinez, H. (1997): Effect of sperm treatments on the in vitro development of bovine oocyte in semi-defined and defined media. Theriogenology 48: 711-720.

Kandil, O.M., Abdoon, A.S.S., Otoi, T. and Suzuki, T. (2000): Effect of growth hormone and type of incubators on cleavage rate and development of in vitro fertilized bovine embryos. J. Egyptian Veterinary Medical Association 60: 43-51.

Kane, M.T. (1987): Culture media and culture of early embryos. Theriogenology 27:49-57.

Kastrop, P.M.M., Bevers, M.M., Destree, O.H. and Kruip, T.A.M. (1990): Changes in protein synthesis and phosphorylation patterns during bovine oocyte maturation in vitro. J. Reproduction and Fertility 90: 305-310.

Katska, l. (1984): Comparison of two methods for recovery of ovarian oocytes from slaughter cattle. Animal Reproduction Science 7: 461-464.

Keefer, C.L., Stice, S. and Maki-Laurila, M. (1991): Bovine embryo development in vitro: effect of in vitro maturation conditions on fertilization and blastocyst development. Theriogenology 35: 223.

Kim, J.H., Niwa, K., Lim, J.K. and Okada, K. (1993): Effect of phosphate, energy substrates, and amino acids on the development of in vitro–matured, in vitro -fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biology of Reproduction 48: 1320-1325.

Kinis, A., Vergos, E., Lonergan, P., Sharif, H., Gallgher, M. and Gordon, I. (1990): The incidence of polyspermy after zona drilling and zona cutting in bovine in vitro fertilization. J. Reproduction and Fertility, Abstract Series No. 6, Abs. 69.

Kolle, S., Stojkovic, M., Prelle, M., Waters, K., Wolf, M. and Sinowatz, F. (2001): Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogensis. Biology of Reproduction 64: 1431-1445.

Krisher, R.L. and Bavister, B.D. (1998): Response of oocytes and embryos to the culture environment. Theriogenology 49: 103-114.

Kroetsch, T.G. and Stubbings, R.B. (1992): Sire and insemination dose does affect in vitro fertilization of bovine oocytes. Theriogenology 37: 240.

Kruip, T.A.M., Van Benden, Th. H., Dieleman, S.J. and Bevers, M.M. (1988): The effect of oestradiol-17B on nuclear maturation of bovine oocytes. Proc. 11th International Congress on Animal Reproduction and Artificial Insemination (Dublin), 3: 336-338.

Kubota, C. and Yang, X. (1998): Cytoplasmic incompetence results in poor development of bovine oocytes from small follicles. Theriogenology 49: 183.

Kuran, M., Robinson, J.J., Staines, M.G. and McEvoy, T.G. (2001): Development and de novo protein synthesis activity of bovine embryos produced in vitro in different culture systems. Theriogenology55:593-606.

Lambert, R.D., Sirard, M.A., Bernard, C. and Beland,R. (1984): The fertilization performance in vitro of bovine and rabbit spermatozoa capacitated in vitro . Proc. 10th International Congress on Animal Reproduction and Artificial Insemination (illinios) 3, 292-294.

Larson, R.C., Ignotz, G.C. and Currie, W.B. (1992): Transforming growth factor-B and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle. Molecular Reproduction and Development 33: 432-435.

Leclerc, P., Sirard, M.A., Chafoules, J.G. and Naitana, S. (1992): Decrease of concentrations during heparin-induced capacitation in bovine spermatozoa. J. Reproduction and Fertility 94: 23-32.

Lengwinat, T., Blotter, S., Wesuhn, A. and Pitra, C. (1990): A comparison of the pregnancy rate, usingfresh or frozen bull semen, after in vitro maturation and fertilization of non-ovulated cattle oocytes. Monatshafte fur Veterinarmedizin 45: 345-347.

Lim, J, M., Reggio, B.C., Godke, R.A and Hansel, W. (1999): In vitro maturation and fertilization of bovine oocytes are temperature-dependent processes. Biology of Reproduction 29: 173-179.

Lonergan, P. (1992): Studies on the in vitro maturation, fertilization and culture of bovine follicular oocytes. Ph. D. Thesis, University college, Dublin. 

Lonargan, Monghan, P., Rizoz, D., Boland, M.P. and Gordon, I. (1994): Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization and culture in vitro. Molecular Reproduction and Development 37: 48-53.

Lonergan, P., Carolan, C., Van Langendonckt, A., Donnay, I., Khatir, H. and Mermillod, O. (1996): Role of epidermal growth factors in bovine oocyte maturation and preimplantation embryo development. Biology of Reproduction 54: 1420-1429.

Lu, K.H. and Gordon, I (1987): Effect of serum, hormones and cumulus cells on the in vitro maturation of f bovine oocytes. Proc. 7th Meeting of the European Embryo Transfer Association (Cambridge), 63.

Lu, K.H. and Polge, C. (1992): Summary of two years results in large scale in vitro bovine embryos production. Proc. 12th International Congress on Animal Reproduction and Artificial Insemination (The Hague) 3, 1315-13170

Machatkova, M., Jokesova, E., Horky, F. and Kreepelova, R. (2000): Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production. Theriogenology 54: 543-550.

Madison, V., Avery, B. and Greve, T.  (1991): Effect of sperm concentration on the in vitro penetration and development of bovine oocytes. Journal of Animal Science, 69 (Suppl. 1), 461.

Massip, A., Mermillod, P. Wills, C. and Dessy, F.  (1993): Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro. J. Reproduction and Fertility, 97: 65-69.

Mermillod, Vansteenbrugge, A., Wils, C., Mourmeaux, J.K., Massip, A. and Dessy, F. (1993): Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos. Biology of Reproduction 49: 582-587.

Miller, D.J. and Hunter, A.G. (1987): Individual variation for in vitro fertilization success in dairy bulls. J. Dairy Science 70: 2150-2153.

Miller, D.J., Bellin, M.E., First, N.L. and Ax, R.L (1987): Competition of follicular fluid glycosaminoglycans and desulfated heparin for heparin binding sites on bull sperm. J. Animal Science 65 (Suppl. 1): 363 (Abst).

Mizushima, S. and Fukui, Y. (2001): Freezabiliy and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium. Theriogenology 55: 1431-1445.

Monaghan, P., Carolan, C., Lonergan, P., Sharif, H., Wahid, H. and Gordon, I. (1993): The effect of maturation time on the subsequent in vitro development of bovine oocytes. Theriogenology 39: 270.

Moreno, J.F. and Westhusin, M. (1993): A comparison of two systems for culture of bovine zygotes in vitro. Biology of Reproduction 48 (Suppl. 1): 169

Moreno, J.F., Turczynski, C.J., Flores-Foxworth, G. and Kraemer, D.C. (1992): Influence of parity of donors and presence of a CL on quality and quantity of bovine oocytes from ovarian follicles aspirated post mortum. Theriogenology 39: 271 (Abst.).

Niwa, K. and Ohgoda, O. (1988): Synergistic effect of caffeine and heparin on in vitro fertilization of cattle oocytes matured in culture. Theriogenology 30: 733-741. 

O’Brien, J.K, Catt, S.L., Ireland, K.A., Maxwell, W.M.C. and Evans, G. (1997): In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep. Theriogenology 47: 1433-1443.

Palma, G.A, Clement-Sengewald, A., Kreff, M. and Brem, G. (1993): Effect of insulin-like growth factor (IGF-I) on development of in vitro produced bovine embryos in two cultured denisties. Proc. 9th Congress of the European Embryo Transfer Association (Lyon), 248.

Park, Y.S. and Lin, Y.C (1993): Effect of epidermal growth factor (EGF) and defined simple media on in vitro bovine oocyte maturation and early embryonic development. Theriogenology 39: 475-484.

Park, Y.S., Ohgoda, O. and Niwa, k. (1989): Penetration of bovine follicular oocytes by frozen-thawed spermatozoa in the presence of caffeine and heparin. J. Reproduction and Fertility 86: 577-582.

Parrish, Krogenaes, A. and Susko-Parrish, J.L. (1995): Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 44: 859-869.

Parrish, J.J., Susko-Parrish, J.L. and First, N.L.  (1989): Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH. Biology of Reproduction 41: 683-699.

Parrish, J.J., Susko-Parrish, J.L. and Graham, J.K. (1999): In vitro capacitation of bovine spermatozoa: role of intracellular calcium. Theriogenology 51: 461-472.

Parrish, J.J., Susko-Parrish, J.L., Winer, M.A.  and First, N.L. (1988): Capacitation of bovine sperm by heparin. Biology of reproduction 38: 1171-1180.

Pavlok, A., Kopecny, V., Lucas-Hahn, A. and Neimann, H. (1993): Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles. Molecular Reproduction and Development 35: 233-243.

Pieterse, M.C., Vos, P.L.A.M., Kruip, T.A.M., Wurth, Y.A., Van Benden, Th. H., Willemse, A.H. and taverne, M.A.M. (1991): Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology 35: 857-862.

Pinyopummintr, T and Bavister, B.D.  (1991): In vitro matured/ in vitro -fertilized bovine oocytes can develop into morula/blastocyst in chemically defined, protein-free culture media. Biology of Reproduction 45: 736-742. 

Pinyopummintr, T and Bavister, B.D. (1993): Effect of timing of addition and types of serum supplementation on bovine embryo development in vitro. Biology of Reproduction (Suppl. 1): 174. 

Pinyopummintr, T. and Bavister, B.D.  (1994): Effect of gaseous atmosphere on in vitro maturation and in vitro fertilization of bovine oocytes. Theriogenonogy 41: 276.

Pollard, J.W., Martino, A., Rumph, N.D., Songsasen, N., Plante, C. and Leibo, S.P.  (1996): Effect of ambient temperature during oocyte recovery on in vitro production of bovine embryos. Theriogenology 46: 849-859.

Pontbriand, D., Goff, A.K., Xu, K.P. and King, W.A. (1989): Effect of steroids on protein synthesis in mature and immature bovine oocytes. Theriogenology 31: 407-418.

Rieger, D. (1992): Relationship between energy metabolism and development of early mammalian embryos. Theriogenology 37: 75-93.

Rivera, R.M. and Hansen, P.J. (2001): Development of cocultured bovine embryos after exposure to high temperature in the physiological range. Reproduction 121: 107-115.

Romero-Arredondo, A. and Seidel, G.E. (1996): Effect of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development. Biology of Reproduction 55: 1012-1016.

Rosenkrans, C.F. and First, N.L. (1991): Culture of bovine zygotes to the blastocyst stage: effect of amino acids and vitamins. Theriogenology 35: 266.

Saeki, H., Nagao, Y., Hoshi, M. and Kainuma, H. (1993): In vitro fertilization of bovine oocytes in a protein-free medium and subsequent development to the blastocyst stage. Theriogenology 39: 302.

Sanbuissho, A. and Threlfall, W.R. (1988): Influence of serum and gonadotropins on bovine oocyte maturation in vitro. Theriogenolgy 29: 301.

Schneider, C.S., Ellington, J.E. and Wright, J.R. (1999): Relationship between bull fertility and in vitro embryo production using sperm preparation methods with and without somatic cell co-culture. Theriogenology 51: 1085-1098.

Schroeder, A., Schultz, R.M., Kopf, G.S., Taylor, F.R., Becker, R.B. and Eppig, J.J. (1990): Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2F during spontaneous mouse oocyte maturation in vitro in the absence of serum. Biology of Reproduction 43: 24-245.

Schumacher, a. and Fischer, B. (1988): Influence of visible light and room temperature on cell proliferation in preimplantation rabbit embryos. J. Reproduction and Fertility 84: 197-204.

Seaton, A.D., Catt, J.W., Rhodes, S.L., McDonald, M.F. and Welch, R.A.S. (1991): The use of unfrozen semen for in vitro fertilization of in vitro matured bovine oocytes. Proc. New Zealand Society of Animal Production  51: 67-71. 

Seidel, G.E., Nauta, W. and Olson, S.E. (1991): Effect of myoinositol, transferrin and insulin on culture of bovine embryos. J. Animal Science 69 (Suppl. 1): 403.

Sekin, Sakurada, T. and Oura, R. (1992): Optimum temperature of ovary transportation for in vitro fertilization of bovine oocytes. Veterinary Record 131: 374.

Semple, E., Loskutoff, N., Leibo, S.P.  and Betteridge, K.J. (1993): Effect of culture medium and maturation time on in vitro  development of bovine oocytes into blastocysts. Theriogenology 39: 307.

Shamsuddin, M., Larsson, Bi., Gustafsson, H. and Rodriguez- Martinez, H. (1992): Number of cells in the in vitro  pre-implantation bovine embryos obtained from different methods of in vitro  culture of IVM-IVF oocytes. Proc. Nordic A.I. Virtanen Institute symposium on the In vitro Culture of Domestic Animal Embryos. 30-31.

Shehata, S.H. (1998): In vitro buffalo sperm capacitation with special attention to acrosome reaction and oocyte penetration test. Assuit Vet. Med. J. 38: 56-72.

Sikes, D., Hernandez-Ledezma, J.J., Villanueva, C. and Roberts, R.M.  (1993): Effect of CZB versus Mediu-199 and of conditioning culture media with either bovine oviductal epithelial cells or buffalo rat liver cells on development of bovine zygotes derived by IVM-IVF procedures. Theriogenology 39: 310.

Simpson, A.M. and White, I.G. (1987): Interrelationships between motility, cAMP, respiration and calcium uptake of ram and boar sperm. Animal Reproduction Science 15: 189-207.

Sirard, M.A. (1989): Practical aspects of in vitro fertilization in cattle. J. Reproduction and Fertility, Suppl. 1 38: 127-134.

Sirard, M.A. and Blondin, P. (1996): Oocyte maturation and IVF in cattle. Animal Reproduction Science: 417-426.

Sirard, M.A., Coenen, K. and Bilodeau, S. (1992): Effect of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes. Theriogenolgy 37: 39-57.

Sirard, M.A., Lambert, R.D. and Gray, P. (1986): Enhanced motility of bovine spermatozoa treated with supernatant of bovine ovarian cell culture. Theriogenology 25: 197.

Sirard, M.A., Picard, L., Dery, M., Coenen, K. and Blondin, P. (1999): The tome interval between FSH-P administration and ovarian influences the development of cattle oocytes. Theriogenology 51: 699-708.

Sirotkin, A.V. and Nitary, J. (1992): Effect of growth hormone on bovine granulosa cells and oocytes in culture. J. Reproduction and Fertility, Abstract series No. 9, 49 (Abst. 84).

Solano, R., de Armas, R., Pupo, C.A. and Castro. F.O. (1994): Short term preservation of intrafollicular oocytes at 4°C. Theriogenology 41: 299.

Spiropoulos, J. and Long, S.E. (1989): Female meiosis in dairy cattle. J. Reproduction and Fertility, Abstract Series No.3: 48.

Sugawara, S., Hamono, K., Miyamoto, A., Kameyama, K., Horiuchi, T. and Masaki, J. (1984): In vitro fertilization and subsequent development of bovine oocytes precultured in synthetic media. Proc. 10th International Congress on Animal reproduction and Artificial Insemination (Illinois) 3, 382-384.

Sun, F.Z., Bradshaw, J.P., Galli, C. and Moor, R.M. (1993): Transient intracelluar calcium concentration (Ca2+) rises in bovine oocytes following sperm penetration and the role o IP3-induced Ca2+ release on fertilization. J. Reproduction and Fertility Abstract, Series No. 11: 66 (Abst.121).

Suzuki, T. (1993): Bovine embryo transfer and related techniques. Molecular Reproduction and development 36:236-237.

Tabibzadeh, S. and Sun, X.Z. (1992): Cytokins expression in human endometrium throughout the menstrual cycle. Human Reproduction 7: 1214-1221.

Taft, R., King, R., Anderson, S. and Killian, G. (1992): In vitro capacitation of sperm from fertile and subfertile bulls using oviductal fluid. J. Animal Science 70 (Suppl. 1) 256 (Abst. 474).

Takagi, Y., Mori, K., Takahashi, T., Sugawara, S. and Masaki, J.  (1992): Differences in development of bovine oocytes recovered by aspiration or by mincing.  J. Animal Science 70: 1923-1227.

Takagi, Y., Mori, K., Tomizawa, M., Takahashi, T., Sugawara, S. and Masaki, J. (1991): Development of bovine oocytes matured, fertilized and cultured in a sperm-free, chemically defined medium. Theriogenology 35: 1197-1207.

Takahashi, T. and First, N.L. (1992): In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 37: 963-978.

Tajik, P. and Niwa, K. (1997): Effects of caffeine and/heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development. Theriogenology 49: 771-777.

Tatemoto, H. and Terada, T. (1998): Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes. Theriogenology 49: 1007-1020.

Thibodeaux, J.K., Menezo, Y., Roussel, J.D., Hansel, W., Goodeaux, L.L., Thompson, D.L. and Godke, R.A.   (1992): Coculture of in vitro fertilized bovine embryos with oviductal epithelial cells originating from different stages of the estrous cycle. J. Dairy Science 75: 1448-1455.

Van Soom, A. and Kruif, A.de. (1992): A comparative study of in vivo and in vitro derived bovine embryos. Proc. 12th International Congress on Animal reproduction and Artificial Insemination (The Hague) 3: 1363-1365.

Vansteenbrugge, A., Van Langendoncki, A., Donnay, A., Massip, A. and Dessy, F. (1996): Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development. Theriogenology 46: 631-641.

Vlaenderen, I, Van, Soom, A. Van and Kruif, A.de. (1991): Bull specific effect on in vitro penetration, fertilization of bovine oocytes using various levels of heparin. ARTA 2: 105-106.

Wales, R.G., Khurana, N.K., Ediringhe, W.R. and Pike, I.L. (1985): Metabolism of glucose by preimlantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine. Australian Journal of Biological Science 38: 421-428.

Wang, s., Liu, Y., Holyoak, G.R. and Bunch, T.D. (1997): The effect of bovine serum albumin and fetal bovine serum on the development of pre-and post cleavage-stage bovine embryos cultured in CR2 and M199 media. Animal reproduction Science 48: 37-45.

Ward, F.A., Lonergan, P., Enright, B.P. and Boland, M.P. (2000): Factors affecting recovery and quality of oocytes for bovine embryo production in vitro using ovum pick-up technology. Theriogenology 54: 433-446.

Watson, A.J., Hogan, A., Hahnel, A., Weimer, K.E. and Schultz, G.A. (1991): Expression of growth factor ligand and receptors genes in the preimplantation bovine embryo. Molecular Reproduction and Development 31: 87-95.

Xu, K.P. and King, W.A. (1990): Effect of oviductal cells and heparin on bovine sperm capacition in vitro. Biology of Reproduction 42 (Suppl.1): 89

Xu, K.P., Greve, T., Smith,S., Liehman, P. and Hyttel, P. (1986): Chronological changes of bovine follicular oocyte matuartion in vitro . Acta Veterinaria Scandinavica 27: 505-519.

Younis, A.I and Brackett, B.G. (1991); Importance of cumulus cells and insemination intervals for development of bovine oocytes into morula and blastocysts in vitro. Theriogenology 44: 11-21.

Younis, A.I., Brackett, B.G. and Fayer-Hosken, R.A. (1989): Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Research 23: 189-201.

Zuelke, K.A. and Brackett, B.G. (1990): Luteinizing hormone-enhanced in vitro maturation of bovine oocytes with and without protein supplementation. Biology of Reproduction 43: 784-787.