To use this technique in a breeding scheme, the COC could be obtained from females of high genetic merit. This could be done after accidental culling of the female or at the end of her life to ensure a last reproduction (in the same way as described for slaughterhouse oocytes) or from living female, through the Ovum Pick Up (OPU) technique.

In the bovine, OPU is mainly done by transvaginal aspiration of follicle content under ultrasonographic control. This technique has been developed in humans and has been then adapted in cattle (Pieterse et al. 1988). Aspiration parameters are as important in OPU procedure than they are during aspiration of oocytes from slaughterhouse ovaries. Increasing aspiration flow increases oocyte recovery rate but decreases oocyte quality (Bols et al. 1997). The ratio of oocyte collected from aspirated follicles is usually around 60-70 %. OPU could be performed as frequently as twice a week, with or without hormonal stimulation of follicular growth, over several months without affecting further reproductive life of the donor (Bols, 2005; van Wagtendonk-de Leeuw, 2006). It has been shown that two OPU sessions per week allow the collection of oocytes of higher quality, translating into higher rate of development to the blastocyst stage as compared with one session per week (Chaubal et al. 2006). This may be due to the appearance between weekly sessions of a dominant follicle which negatively influences the quality of oocytes in subordinate follicles. Indeed, the removal of the dominant follicles by OPU between two weekly sessions increased the blastocyst yield. An average of ten oocytes per cow and 8 per heifer may be collected at each OPU session in a twice a week protocol without stimulation, resulting in an average of 2.5 transferable embryos per cow per session and 1.5 per heifer (Galli et al. 2003). This protocol presents the advantage to avoid the repeated use of exogenous gonadotropins which could induce several reproductive pathologies and may lead to subfertility. In goat, it has been shown that LOPU could be repeated up to five time at various intervals without decrease in efficiency (Pierson et al. 2005). However, we have shown that repetition of LOPU on a weekly basis in goat under pFSH stimulation progressively decreases oocyte recovery (Baril et al., unpublished data). This decrease may be due to the repeated starvation of the donors before LOPU.

Alternatively, OPU could be done on a weekly basis after FSH priming with similar yield of total good quality oocytes. After one shot FSH stimulation (80 mg i.m. and 120 mg s.c. of pFSH), it is possible to perform two successive OPU sessions at 30 h and 126 h after FSH injection with good embryo production rate (Chaubal et al. 2006). The combined intra muscular and subcutaneous administration of FSH allows a rapid stimulation (i.m.) as well as a sustained release of the hormone (s.c.) that avoid repeated handling of animals and ensure continuous follicular growth stimulation in a twice a week stimulation protocol. In the case of stimulated protocols with multiple injections (300 mg pFSH i.m., 6 injections 12 h apart), it has been shown that a coasting period of 48 h allows to increase the rate of oocytes reaching the blastocyst stage, specially when LH (Lutropin, Vetrepharm, Canada, i.v.) was administered 6 h before OPU (Blondin et al. 2002). We did not find similar effects of this coasting period in goat LOPU (Baril et al, unpublished results). In young heifers (2-3 months of age), good IVP results (up to 3 transferable embryos per calf) could be obtained after eCG stimulation followed by oocyte collection under laparotomy (Galli et al. 2004).

Table 1. Embryo production after OPU in a twice a week unstimulated protocol (adapted from Galli et al. 2003).

In small ruminants, OPU may be done by laparotomy or, less invasively, by laparoscopy guided aspiration of ovarian follicles (Baldassarre et al. 2002; Cognie et al. 2004). In laparoscopic ovum pick-up (LOPU), aspiration conditions (size of the needle, bevel shape, aspiration flow, tubing internal diameter) should also be controlled and adapted to the species, as in OPU, to improve recovery rate and oocyte quality (Rodriguez et al. 2006). Good results can be obtained by LOPU in goat and sheep with or without ovarian stimulation by FSH or eCG (Baldassarre et al. 2003; Morton et al. 2005).

In wild ruminant species, oocyte recovery technique should be adapted to the size of the donor. Transvaginal OPU could be used in large deer species such as wapiti and red deer (Berg and Asher, 2003), whereas LOPU is recommended for smaller animals like sika deer. In sika deer, it has been shown that LOPU after oFSH stimulation of ovarian activity allows the recovery of good quality oocytes and in vitro production of viable embryos all year long, even during the non breeding season (Locatelli et al. 2006).

IN VITRO MATURATION

Nuclear maturation

In the ovary, all primary oocytes are blocked at the prophase stage of the meiotic cycle The block of meiotic progression during follicular growth is controlled by unknown factors produced by the surrounding somatic cells (Mermillod and Marchal, 1999). At this stage, the round shaped nucleus, called the germinal vesicle (GV), contains diffuse chromatin. During early folliculogenesis, the oocyte grows, undergo strong morphological modifications and stores molecules (proteins, RNAs) that will be determinant for the success of fertilization and early embryo development. The zona pellucida, a glycoprotein envelope surrounding the oocyte membrane, is synthesized by the oocyte during this period of growth. The zona pellucida has important function in regulating fertilization and protection of the embryo until hatching. The oocytes resume meiosis and progress to the metaphase II stage only if they encounter a gonadotropin surge in the preovulatory follicle. They stop their progression at this MII stage until fertilization in the oviduct, after ovulation.

Oocytes that are collected from growing follicles for IVP are blocked at the prophase stage of meiosis As soon as they are removed from the follicular inhibitory environment, meiotic resumption occurs spontaneously and progress to metaphase II (Sirard, 2001). This spontaneous meiotic resumption of the oocyte outside of the follicle is the basis of in vitro maturation.

Cytoplasmic maturation

Cytoplasmic maturation covers all morphologic and molecular events accompanying nuclear maturation after LH surge in preovulatory follicles and preparing oocyte cytoplasm to successful fertilization and embryo development. Cytoplasmic maturation includes well known morphological modifications, such as the migration of cortical granules in the cortical region of the ooplasm (Cran, 1989). These granules are stored during oocyte growth and release their enzymatic content in the perivitelline space after fertilization. These enzymes modify the structure of the zona pellucida, preventing the penetration of additional spermatozoa. Meiotic competence is acquired during early folliculogenesis, soon after the apparition of the antral cavity in the follicle (Mermillod et al. 1999). However, after this acquisition, the oocyte requires a further differentiation period during late follicular growth to reach the full competence for cytoplasmic maturation (Sirard et al. 2006). This late differentiation occurs under the meiosis inhibiting signal sent by somatic follicular cells that maintain the meiotically competent oocyte at the prophase stage.

During IVP, more than 90% of the oocytes collected from follicles larger than 3 mm in cattle are able to complete nuclear maturation. However, few of them are competent for cytoplasmic maturation. Consequently, only 30 to 40% of the oocytes reach the blastocyst stage after IVF and IVD (Figure 3). This contrast highlights the functional importance of cytoplasmic competence (Sirard et al. 2006). When oocytes harvested from larger follicles or in vivo matured oocytes are processed under the same IVF – IVD techniques, the success rate is increased (Hue et al. 1997; Lequarre et al. 2005; Marchal et al. 2002). Ovarian stimulation by gonadotropins increases the number of follicles growing to large size classes by protecting them against involution through the atresia process. Consequently, ovarian stimulation increases the number of competent oocytes collected through the OPU process.

Increasing developmental competence during late follicular growth is suspected to be related to storage in oocyte of transcripts that will be necessary for the success of early embryo development. Indeed, in ruminant species, embryo genome activity only starts after the third embryo cell cycle (8-16 Cell stage, (Memili et al. 1998). Transcription activity has been studied in growing oocytes by following ribosomal RNA synthesis (Hyttel et al. 2001). These studies allowed to establish that the oocyte is transcriptionally quiescent in primordial follicles. RNA synthesis appears in secondary follicles, peaks in tertiary follicles, at later stages of oocyte growth, and decreases thereafter to return to undetectable levels. Meiotic resumption in late preovulatory follicle is preceded by a short burst of RNA synthesis (Memili et al. 1998) which seems to be functionally important for the success or embryo development, as evidenced by -amanitin (inhibitor of RNA polymerase II) inhibition of transcription at this stage. Several maternal effect genes, such as Mater (Maternal Antigen That Embryo Require) or zygote arrest 1 (Zar1) have been identified in mouse oocytes (Tong et al. 2000; Wu et al. 2003). The storage of these maternal transcripts in oocytes is required to allow embryos to develop beyond the 2-Cell stage. Orthologues of these genes have been recently identified in oocytes from domestic species (Dalbies-Tran et al. 2005; Pennetier et al. 2006; Pennetier et al. 2004) and their expression profile has been characterized (Pennetier et al. 2006). In addition, cDNA arrays are used for a more comprehensive analysis of oocyte’s expression patterns. These studies rely on the use of both heterologous arrays (Dalbies-Tran and Mermillod, 2003) or specific arrays builded from SSH selected oocyte specific cDNA banks (Pennetier et al. 2005; Sirard et al. 2005; Vallee et al. 2006). These studies have allowed the identification of several hundreds of genes expressed in the bovine oocyte, some of them being preferentially expressed in oocyte (and sometimes testis) and not detected in other tissues. However, up to now, none of these genes as been identified as a key element of oocyte developmental competence acquisition during late folliculogenesis.

In vitro maturation

In vitro maturation is probably the most critical part of the whole process of in vitro embryo production. Ruminant oocytes are usually matured at 39°C under a 5% CO2 in a humidified atmosphere. The optimal maturation time (more than 90% of the oocytes at the metaphase II stage) is 22-24h. Due to the high lipid content of oocytes cytoplasm, it is not possible to follow the progression of their nuclear status during culture. Additionally, the presence of the cumulus cells is required during IVM for efficient cytoplasmic maturation and these cells mask the oocyte. Consequently, the only visible sign of oocyte maturation during IVM is the expansion of the cumulus cells. These cells produce hyaluronic acid which is secreted and polymerized in the extracellular matrix, leading to the increase of intercellular space.

Several culture media have been proposed for IVM (MEM, Waymouth, Ham-F12). However, the most widely used seems to be the TCM199 medium, bicarbonate buffered and containing minerals, carbon and energy sources (glucose, glutamine) as well as vitamins and amino acids. The medium is generally supplemented with high molecular weight molecules that exert a surfactant effect (BSA) and provide hormones and growth factors (fetal calf serum, serum of female in estrus, follicular fluid). These complex additives from animal sources may raise sanitary questions due to possible presence of pathogen agents, and decrease the reproducibility of experiments. They may be successfully replaced by high molecular weight polymers (Ali and Sirard, 2002) for the surfactant effect (such as polyvinyl alcohol) and by cocktails of purified or recombinant hormones and growth factors (Lonergan et al. 1996).

Hormones are usually added to the maturation medium (FSH, LH, estradiol), in addition to biological fluids. Their exact function is not clearly established, althrough the effect of FSH on cumulus expansion and metabolism has been clearly established (Sutton-McDowall et al. 2004). Estradiol effect on oocyte is exerted through intracellular receptors and seem to be detrimental to meiotic progression, inducing chromosomal abnormalities (Beker-van Woudenberg et al. 2004). Growth hormone (GH) stimulates some aspects of cytoplasmic maturation (cortical granules migration) as well as nuclear maturation, resulting in a higher blastocyst yield (Bevers and Izadyar, 2002). GH action seems to be mediated by cumulus cells (Izadyar et al. 1997). Epidermal growth factor (EGF) has been shown to stimulate nuclear and cytoplasmic maturation in a wide variety of species including mouse, pig, cattle, deer goat and sheep (Abeydeera et al. 2000; Cognie et al. 2004; Comizzoli et al. 2001b; De La Fuente et al. 1999; Guler et al. 2000; Lonergan et al. 1996). High blastocysts yields could be obtained after maturation of cattle oocytes in TCM199 only supplemented by EGF. EGF action is exerted, at least partly, directly on the oocyte and recent studies showed that EGF family of growth factors (epinephrin, amphiregullin, betacellulin) could be produced by granulosa cells under gonadotropins stimulation and could thus mediate LH signaling inside the follicle, transmitting the ovulatory signal to the oocyte (Park et al. 2004). Insulin like growth factor-I (IGF-I) has also a positive effect on cytoplasmic maturation of cattle oocytes (Bevers and Izadyar 2002). Indeed, the regulation of IGF-I and II through the controlled expression and degradation of IGF binding proteins could be one of the major processes of control of late follicular growth (Mazerbourg et al. 2003).

Another important aspect of COC environment during IVM is the antioxydative potential of the IVM medium (Ali et al. 2003). As all living cells, oocyte is sensitive to reactive oxygen species (ROS) that could induce detrimental chemical reactions (lipid or protein peroxydation). To overcome possible ROS detrimental effects during culture, antioxydative molecules (　-mercaptoethanol, cystein, vitamin A C, E), divalent cations chelators (EDTA, taurine, hypotaurine, transferrine) or ROS scavengers (superoxyde dismutase, catalase) could be added to maturation medium (Blondin et al. 1997; Ikeda et al. 2005; Kobayashi et al. 2006). However, COC are capable to produce their own protection system by secreting ROS scavenger enzymes like superoxyde dismutase and catalase (Cetica et al. 2001) or through the production of intracellular reducers molecules like glutathion (de Matos and Furnus, 2000). Stimulating these endogeneous protection systems may help oocyte to survive challenging in vitro conditions. The addition into IVM medium of cysteamine, a cell permeable precursor of cysteine, itself involved in glutathion (GSH) synthesis, has been shown to increase GSH concentration in oocytes and, in turn, to increase the resulting developmental competence after IVM in different species (Cognie et al. 2004; de Matos and Furnus, 2000; de Matos et al. 2002; Gasparrini et al. 2003; Khatir and Anouassi, 2006). Surrounding cumulus cells are also contributing to establishment of high GSH content in the oocyte (Luciano et al. 2005). Therefore, stimulating cumulus cells may also help to protect oocytes against environment ROS.

It has been established for a long time that the presence of cumulus cells is required for the success of oocyte cytoplasmic maturation and fertilization (Gilchrist et al. 2004). Therefore, maintaining the viability and functionality of cumulus cells is likely to be another important challenge for IVM success. Oocyte secreted factors (ESF) are known to drive cumulus cell differentiation during follicular growth (Li et al. 2000) and to allow cumulus expansion during oocyte maturation (Dragovic et al. 2005). The precise identification of the active factors is not fully established. However, GDF-9 and BMP-15, two members of the TGF-family of growth factors, are specifically produced by the oocyte and their effect on cumulus cells, and in turn on matured oocyte developmental competence, have been recently established (Hussein et al. 2006). Since these factors are able to protect cumulus cells against apoptosis that appear in culture (Hussein et al. 2005), their addition to IVM medium may be recommended, at least to help at maintaining cumulus functionality in culture.

IN VITRO FERTILIZATION

The techniques used for in vitro fertilization are well established in cattle as well as in small ruminants. The media used for IVF are Tyrode derived saline supplemented with BSA, lactate and pyruvate and with a capacitating agent (Parrish et al. 1986). Sperm capacitation occurs physiologically in the female genital tract. This phenomenon involves modification of spermatozoa membrane as well as changes in its mobility properties that lead to spermatozoa fertilization ability. Many protocols support sperm capacitation in vitro. In cattle, heparin is widely used for this purpose, usually without pre treatment (Parrish et al. 1989). In small ruminant, spermatozoa are usually capacitated by preliminary treatment with serum collected from females in estrus (Cognie et al. 2004). Frozen semen is generally used and a selection step is required to enrich the sperm suspension in living and motile spermatozoa. In the swim-up method, spermatozoa are deposit at the bottom of a culture medium containing tube and allowed to swim in the medium. The top fraction, containing motile sperms is then collected after a given incubation time. Alternatively, motile sperms may be purified by centrifugation in a discontinuous density gradient (Percoll). The motile fraction is collected at the bottom of the tube after centrifugation.

The sperm concentration used for IVF ranges between 0.5 and 2x10⁶ spermatozoa per ml in fertilization medium. This concentration should be optimized for each male and even for each ejaculate from a given male. The fertilization rate increases together with sperm concentration. However, after a given value, an increasing proportion of the oocytes become polyspermic. A compromise has to be established for high fertilization rate with low polyspermy rate. For better convenience, sperm and oocytes are usually co-incubated for 18-24h, although a six hours incubation time seems to be sufficient to reach the higher level of normal fertilization.

A strong male effect has been reported on the success rate of IVF and, in turn, IVF may be used as a tool to evaluate male (or ejaculate) fertility (Marquant-Le Guienne et al. 1990; Papadopoulos et al. 2005). In addition, heterologous oocytes may be used to evaluta ejaculate fertility of males from rare species (Comizzoli et al. 2001a). Different males may provide different cleavage rates. Additionally, males providing similar fertilization rates may also differ in their ability to promote the development to the blastocyst stage. These differences between males may be related to their respective ability to up regulate glucose metabolism during G1 phase at pronuclear stage (Comizzoli et al. 2003) and consequently to allow earlier onset of the S phase in the zygote, translating into earlier cleavage and higher development rate (Comizzoli et al. 2000a). Therefore, each male to be used in IVF has to be tested for optimized fertilization conditions and for its ability to promote embryo development. In the case of OPU-IVP with high genetic merit females, the bulls’ ejaculates to use should previously be tested on slaughterhouse oocytes.

IN VITRO DEVELOPMENT

In standard cell culture conditions, ruminant embryo development blocks at the 8-16 cell stage. This block was first overcome through the use of co-culture systems involving oviduct epithelial cells (Eyestone et al. 1991) or other cell lineage (Myers et al. 1994) in standard tissue culture media (TCM199). The effect of cells has not been clearly established. They may produce some growth factors positively regulating embryo development or remove toxic media components (Killian 2004). In a second step, new culture media have been designed to more exactly fit embryo requirements without the need for co-culture. One of the first media designed for this purpose was synthetic oviduct fluid or SOF (Fukui et al. 1991). The composition of SOF was established from the composition of bovine oviduct fluid. However, SOF or SOF modifications have also been successfully used in small ruminant species. More recently, sequential media have been proposed to fit the evolution of embryo requirements during early development (Gardner et al. 2000). For example, glucose seems to be toxic to early cleaving embryos, whereas morula need some glucose in the medium for further development (Donnay et al. 2002; Gardner et al. 2000).

Semi defined media represent an easy way to produce mammalian embryos in vitro. However, embryo quality (viability after cryopreservation and/or transfer to recipient) appears to be low after development in such systems (Garcia-Garcia et al. 2006). On the other hand, co-culture systems may be more complicated to manage but allow the production of higher quality embryos. Therefore, some commercial bovine IVP teams still use co-culture system or embryo development in the oviduct of transient recipient ewes. In this case, the embryos are transferred in the oviduct of synchronized ewes just after IVF and surgically collected seven days later (Galli et al. 2004). This IVP system allows the production of embryos with similar quality and cryotolerance as in vivo collected ones. Recently, as we were trying to produce deer embryos, we observed that these embryos were not able to develop beyond the morula stage in the conventional SOF development system we use in our laboratory for different domestic ruminant species. To allow sika and red deer embryos to reach the blastocyst stage, we have been obliged to develop a co culture system involving ovine oviduct epithelial cell (oOEC) monolayers in SOF medium (Table 2). This culture systems allows the production of viable embryos since we obtained kids after transfer of fresh or frozen/thawed IVP red deer embryos produced in theses conditions (Locatelli et al. 2005; Locatelli et al. 2006). This observation in deer species highlight the function of the oviduct in the regulation of early embryo development and encourage research in view to identify and use oviduct originated embryotrophic factors to increase the efficiency of IVP (Killian, 2004).

Table 2. Cleavage on Day 2 and development on Day 8 of in vitro produced red and sika deer embryos in SOF medium with or without ovine oviduct epithelial cell (oOEC) support (adapted from Locatelli et al. 2005; Locatelli et al. 2006).

Blastocyst rate was calculated from total oocyte entering into IVM.

The development usually takes place at 39°c in droplets of culture medium overlaid with oil for 7 to 9 days. A high density of embryos per volume unit (one embryo per μl) seems beneficial to development (Ferry et al. 1994). When embryos are cultured without cell support, the oxygen concentration in atmosphere should be reduced to 5% to avoid induction of oxidative stress on the embryos (Olson and Seidel, 2000; Voelkel and Hu, 1992). As during IVM, addition of antioxydant molecules may increase rate or quality of IVP embryos (Bing et al. 2003; Dalvit et al. 2005; Takahashi et al. 2002). Serum is usually added to the culture medium from the beginning or after the first cleavages (48h post insemination). The presence of serum accelerates the development of embryos but seems to negative effects on their quality (Rizos et al. 2003) translating into fetal and placental abnormalities (Large Offspring Syndrome, see below) reported during pregnancy (Thompson et al. 1995). The rate of cleavage is usually high (over 80% of the total oocytes), when appropriate bull and fertilization conditions are used. However, the rate of development to the blastocyst stage plateaus at around 40% of the total oocytes. It has been clearly established that oocyte quality (intrinsic quality of primary oocyte coupled to maturation conditions used) determine the rate of blastocyst production, whereas development environment is determinant for embryo viability (Rizos et al. 2002).

The pregnancy rates obtained after transfer of fresh IVP embryos are usually good (40 to 50% of calves born). Some problems of calf abnormality and perinatal mortality have been reported (Farin et al. 2004; Farin et al. 2006; Kruip et al. 2000). However, these problems are less frequent than those observed with cloned embryos and seem to be linked to in vitro environment and to the presence of serum in the culture medium that induces chromosomes aberrations, modifications of embryo gene expression pattern and metabolism (Lazzari et al. 2002; Lonergan et al. 2004; Rizos et al. 2003).

The main limitation to a wider use of OPU – IVP technique in breeding schemes is the low cryoresistance of the resulting embryos. Although well established techniques of embryo freezing are described in cattle, sheep and goat and provide good results of pregnancy rate after transfer of the thawed embryos, these techniques appeared sub-optimal for the cryopreservation of IVP embryos. Several alternative methods of embryo cryopreservation have been proposed to overcome the low viability of these embryos. Some vitrification procedures (high speed cooling with high concentration of cryoprotectants leading to amorphous solidification without ice crystals formation) appeared to partially solve the problem. However, the quality of the embryo which is determined by the culture condition, appears to be one of the critical factors. Similar pregnancy rates have been observed after 10% glycerol slow freezing for in vitro fertilized embryos grown in sheep oviduct than for in vivo derived embryos (Galli et al. 2004). This indicates that culture conditions still remain to be improved to better mimic the in vivo conditions.

POTENTIAL INTERESTS OF IVP

Mass embryo production

The mass production of embryos from oocytes collected in the ovaries of slaughtered females may be of interest in some breeding schemes. For example, ovaries from the same breed may be pooled at slaughterhouse and used to produce embryos of a defined breed without precise knowledge of their parental origin, except breed. These embryos produced at low cost may be used to produced valuable beef calves in dairy herds (“beef from dairy”). This allows to increase the financial value of calves in these herds and may help solving fertility problems in high producing dairy herds (Galli et al. 2003; Hansen, 2006). Similar methods could also be used to produce average genetic value low cost dairy or beef embryos to help the diffusion of genetic progress toward developing countries. A similar approach has been used with success to reconstitute genetics of herds culled for sanitary reasons due to infections like tuberculosis or to BSE suspicion (Galli et al. 2003). In addition, IVP from slaughterhouse oocytes represent the most convenient way to develop basic research on oocyte maturation and cell cycle regulation, gametes recognition and fusion and regulation of early embryo development. Most recent knowledge of gene expression patterns and gene function in these mechanisms have been made possible thanks to slaughterhouse material.

Production of embryos from selected donors

Oocyte collection by OPU coupled to IVP offers several advantages over more traditional in vivo embryo production after superovulation and artificial insemination as applied in MOET schemes (Bols, 2005; Galli et al. 2004). In 2003, the International Embryo Transfer Society (IETS, reported 106,220 transfers of IVP cattle embryos worldwide (Hansen, 2006). Indeed, the number of embryos produced through OPU-IVP technique from a given female over a given period of time is higher than the number that may be collected in vivo. Indeed, OPU could be performed twice a week for long period of time without altering the reproductive potential of the females whereas several weeks or months of rest are required between two treatments of ovarian stimulation. In addition, in small ruminants, the surgery required for embryo collection in vivo induces tissue adhesions that could impair future embryo production, whereas LOPU is less invasive and could be repeated during long periods of time (Baldassarre and Karatzas, 2004). OPU can be performed in young females (from 3 months in age through laparoscopy) as well as in old cows. In addition, OPU could be continued during the first trimester of pregnancy without risk for the fetus, and it could be also performed on animals with some pathologies of the ovary (ovarian cysts), oviduct or uterus that prevent in vivo embryo production or even compromise fertility (Galli et al. 2003). OPU-IVP also allows the use of different males for producing the embryos (different males could be used for the oocytes from one aspiration session or from different OPU sessions). Furthermore, OPU-IVP could be beneficial in the case of females which are not responding to ovarian stimulation.

Wild species

Residual population of wild species maintained in captivity often encounter reproductive disorders that impair the amplification of the herd in view of reintroduction of animals in their natural environment or for the simple maintenance of these captive residual populations (Comizzoli et al. 2000b; Pukazhenthi et al. 2006). In addition, techniques of assisted reproduction like artificial insemination or embryo transfer may help to maintain proper management of scarce genetic diversity of few remaining individuals scattered between different locations. In this view, LOPU-IVP presents several advantages over MOET, like in domestic species and, in addition, it allows to reduce stress generating manipulations of wild animals (Locatelli et al. 2006).

MAJOR LIMITATIONS AND PERSPECTIVES

There are still some limitations to the extended commercial use of IVP (Faber et al. 2003; Hansen, 2006). The low success rate of development to the blastocyst stage is increasing the cost of each embryo produced. Additionally, some females appear to be poor oocyte donors in terms on both quantity and quality (developmental potential). The embryos produced by this technique have a low viability following cryopreservation and should be transferred fresh to get an optimized pregnancy rate. This limitation implies the disposition of a large recipient herds and the synchronization of recipients at each oocyte collection to prepare fresh transfer of resulting embryos, and consequently increases the overall cost of the technique. In addition, the possibility of embryos transportation is limited and embryo storage is not recommended.

Active research in several directions may help to overcome these limitations in a near future. Manipulations of ovarian stimulation protocols have shown that increasing late intrafollicular differentiation of the oocyte allows to increase its developmental potential. For example, introducing a coasting period after FSH stimulation of ovarian activity increases the rate of oocyte development to the blastocyst stage up to 80% (Blondin et al. 2002). This late differentiation appears to be critical to oocyte competence (Lequarre et al. 2005) and several authors tried to mimic this differentiation in vitro. Since meiotic resumption appears spontaneously in vitro, after suppression of follicle induced meiotic inhibition, and rapidly blocks transcriptions suspected to support developmental competence, mimicking late oocyte differentiation in vitro requires artificial maintenance of meiotic block (Mermillod et al. 1999). Attempts to recreate in vitro a follicular-like environment by adding follicular cells together with COC, provided limited successes (Sirard, 2001; Sirard and Bilodeau, 1990). More recently, it has been established that kinase inhibitors specifically blocking the kinase activity of M-phase Promoting Factor (MPF, key regulator of G2 – M transition of the eukayotic cell cycle) were able to reversibly maintain cattle oocyte at the GV stage in culture (Lonergan et al. 2000; Mermillod et al. 2000). However, these techniques of meiotic inhibition failed to increase l competence after a 24 h pre maturation culture of the oocytes at the GV stage. Nevertheless, these approaches allowed to establish that numerous signaling pathways are involved in oocyte cell cycle control (Vigneron et al. 2003; Vigneron et al. 2004a; Vigneron et al. 2004b). The increasing knowledge of this control will allow to derive more valuable methods of oocyte meiotic inhibition in the future. In addition, future studies will benefit from the increasing knowledge of the expression of genes related to developmental competence of the oocyte (Albertini et al. 2003).

Finally, some abnormalities have been reported during pregnancies after transfer of IVP embryos that have been called large offspring syndrome or LOS (Farin et al. 2006). This translates into abnormally large offspring after longer gestation time and higher rate of perinatal mortality. In sheep, this syndrome has been clearly related to the presence of serum in embryo culture medium (van Wagtendonk-de Leeuw et al. 2000) and may result from the maintenance of gene expression alterations induced by culture environment during early development stages (Lazzari et al. 2002). These problems may be overcome by using more physiological conditions for IVD. This may be achieved by developing the embryos into the oviduct of transient recipient ewes (Galli et al. 2004). However, recent progress in the comprehension of oviduct physiology (Killian, 2004) may allow to develop in vitro culture conditions mimicking in vivo environment to increase embryo quality. In addition, the increasing knowledge of embryo gene expression pattern and its regulation will allow to use marker genes to follow more precisely the effect of modification of embryo environment (Lonergan et al. 2006).

CONCLUSIONS

In conclusion, IVP is a very promising technique that could find numerous applications in assisted reproduction of ruminant domestic and wild species. It also offers unique occasion to study early mechanisms controlling reproductive physiology and open the way to advanced biotechnology such as nuclear transfer cloning and transgenesis. The low quality of oocytes recovered from small growing follicles as well as the low resistance of IVP embryos to conventional freezing methods still limit the use of IVP in breeding schemes. Improving IVP conditions in the future will require strong research efforts in view to improve our knowledge of oocyte and early embryo physiology and gene expression and to use this knowledge to develop in vitro treatments more adapted to precise requirements of oocytes and early embryos.

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