IN VITRO PRODUCTION OF EMBRYOS IN SWINE. L.R. Abeydeera. PIC International, Berkeley, CA ABSTRACT

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1 ELSEVIER IN VITRO PRODUCTION OF EMBRYOS IN SWINE L.R. Abeydeera PIC International, Berkeley, CA ABSTRACT In recent years, progress has been achieved in the production of pig embryos through IVM and IVF techniques. Cytoplasmic maturation of oocytes has been improved by modifications to IVM procedures. However, the historical problem of polyspennic penetration still remains a major issue to be solved. Recent studies indicate that the type of IVF medium and certain modifications to that medium can reduce polyspermy. Efforts should be directed to increase the developmental competence and quality of embryos. At present, many embryo culture (EC) media are available that can overcome the historical 4-cell block and support development of early in vivo derived embryos to the blastocyst stage. In contrast, blastocyst development of in vitro produced embryos in these culture media varies significantly. Furthermore, morphology and cell numbers in in vitro produced blastocysts are inferior to their in vivo counterparts. However,, several modifications to EC techniques have improved embryo quality and developmental competence. Testing embryo viability through surgical transfer to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although reliable in vitro systems are available for the generation of pig embryos, the problem of polyspermy and poor embryo development hamper their large-scale implementation. Further research efforts should be directed to improve oo&-te/embryo-quality and the methods to minimize polyspermy through development of novel IVM, IVF, and EC techniques by Elsevier Science Inc. Key words: pig, oocytes, IVM, IVF, embryos INTRODUCTION There is tremendous interest to produce large quantities of matured pig oocytes and embryos through IVM/IVF techniques for basic as well as biomedical research purposes. Because of their physiological similarities to humans, pigs have become increasingly important as potential xenograft donors and transgenic animals to produce specific proteins. Attempts to clone and produce transgenic pigs by pronuclear microinjection require mature oocytes and zygotes, respectively. Surgical collection of oocytes or embryos from donor animals is time consuming and expensive, and numbers are limited. Alternatively, the efficient utilization of ovaries from slaughterhouse animals to generate mature oocytes and embryos via in vitro techniques is crucial. The number and quality of oocytes that successfully enter the reproductive process will profoundly influence the ultimate efficiency of this approach. These new biotechnologies are likely to have a major impact on animal breeding in the future. Therefore, it is imperative to have a better understanding of all the steps involved in the process of in vitro embryo production. In recent years, substantial progress has been made in the development of procedures for IVM, IVF, and EC. However, further improvements are necessary to maximize embryo Theriogenology 57: , X/02/$-see front matter Elsevier Science Inc. PII: SOO93-691X(01)

2 Theriogenology production. In very early studies, although nuclear maturation was achieved, there were problems with poor male pronuclear (MPN) formation and polyspermy (20,24,65). Its becoming clear that not only nuclear maturation, but normal cytoplasmic maturation, must occur in vitro to produce viable embryos. Through the efforts of many, various modifications to the IVM system have alleviated the problem of MPN formation, but polyspermy still remains a major unresolved problem. It is not established whether polyspermy is due to inefficient culture conditions during IVM, IVF, or both. During the past decade, despite the problem of polyspermy, successful in vitro production (IVP) of pig embryos has improved dramatically. Furthermore, transfer of embryos to recipient animals has resulted in acceptable pregnancy rates and litter sizes. It is remarkable, given the present poor understanding of the mechanisms regulating oocyte maturation/fertilization and embryo development, that viable embryos can still be produced with oocytes collected from slaughterhouse-derived ovaries. Successful large-scale production of pig embryos through in vitro techniques is faced with many difficulties. These difficulties include one or more of the following: 1) inefficient oocyte maturation and fertilization techniques, 2) poor developmental capacity of in vitro produced embryos, and 3) suboptimal EC conditions. Therefore, one can conclude that optimal culture conditions are yet to be developed for reliable and efficient maturation, fertilization, and EC. Considerable research efforts in these areas would undoubtedly increase the efficiency of embryo production. This review focuses on the recent progress in pig embryo production through in vitro techniques. It is envisioned that the development of new or optimization of existing in vitro techniques will result in further advances within the discipline of embryo biotechnology. ovary Generally, ovaries of prepubertal gilts collected at a slaughterhouse are the main source of oocytes for routine IVM procedures. The ovaries are transported to the laboratory in 0.9% NaCl or PBS supplemented with antibiotics within a few hours. Transport temperature and duration from the time of ovary collection to oocyte aspiration may influence the quality of oocytes. Therefore, maintaining the viability of oocytes during this process is critical to achieve successful oocyte maturation. Walters and Graves (82) examined various ovary storage temperatures (5, 16, 25, 30, and 37 C) and durations of storage (2, 6,10, 14, and 26 h) on subsequent maturation of oocytes. At all temperatures tested, oocyte maturation was significantly decreased as the storage interval was prolonged. More than 75% of oocytes completed maturation when stored up to 5 h at 25 C. Storage for 5 h at temperatures higher or lower than 25 C compromised the ability of oocytes to complete maturation and most likely would interfere with subsequent developmental competence. Bovine oocytes recovered from ovaries stored for 8 h at 37 C produced significantly fewer embryos than ovaries stored at 25 C following IVM and IVF (91). Therefore, it seems acceptable to transport ovaries at 25 C for up to 5 h with the oocytes retrieved having the ability to complete maturation and potentially develop following fertilization. Ovarian Follicle Tvoes. Pig ovaries contain follicles of varying diameters with a size range classified as small (~3 mm), medium (3 to 6 mm), or large ( 6 mm). Generally, oocytes are aspirated from medium-sized follicles for routine IVM procedures. In the pig, the follicular antrum is fully differentiated in follicles of 0.5-mm diameter with the accompanying oocyte

3 Theriogenology reaching only three-quarters of its final size (63). These oocytes have a very limited ability to initiate nuclear WM. Oocytes reach their full size in small antral follicles of 2 to 3 mm in diameter (62). Most oocytes from these follicles initiate maturation but arrest at metaphase I (M I) of meiosis. The ability to complete the M I to M II transition is achieved in oocytes that reach their full size and nucleolar compaction (63). The ability of oocytes to resume and complete meiosis is not reached at one time point of follicular development but is acquired in a stepwise manner. Of the competent oocytes present in antral follicles, only those that have reached an adequate degree of development will respond to maturation stimuli and induce functional and morphological changes, including resumption of meiosis. A low (62%) proportion of pig oocytes isolated from <3-mm follicles will reach M II stage (L. R. Abeydeera, unpublished data). However, 82 and 94% of oocytes completed maturation when isolated from 3- to 6- and >6-mm follicles, respectively. Interestingly, only 16 and 38% of oocytes isolated from <3- and 3- to 6- mm follicles, respectively, showed cumulus expansion, but all oocytes from >6-mm follicles exhibited expansion. Yoon et al. (92) reported that more oocytes isolated from large follicles (3 to 8 mm) reached M II stage (91% vs. 58%), formed pronuclei (90% vs. 81%) and developed to blastocysts (10% vs. 2%) than those oocytes from small (~3 mm) follicles. Bovine oocytes from larger follicles have superior developmental potential as compared with small follicles (50, 67). These results suggest that full meiotic potential and subsequent developmental competence is acquired with follicular growth. However, the heterogeneity of the oocytes isolated from different sized follicles within the medium size category may also have an influence on their meiotic progression, cytoplasmic maturation, and subsequent developmental competence. Therefore, a period of pre-maturation culture may be necessary to allow those oocytes from smaller follicles to reach a comparable stage with oocytes from larger follicles. IVM Successful maturation of pig oocytes in vitro can be achieved in various culture media types (simple or complex) containing fetal calf serum (FCS) or follicular fluid (FF) and other supplements, such as gonadotropins and growth factors (16, 21, 57, 70). However, supplementation of FCS or FF introduces many unknowns and makes it difficult to identify key factors regulating normal oocyte maturation. Development of a serum-free IVM medium (7) will help to identify optimal culture conditions and will also reduce variability among laboratories. In general, the oocyte maturation process can be broadly divided into two aspects, nuclear and cy-toplasmic. Nuclear maturation is a term that refers to the resumption of meiosis and progression to the M II stage. Cytoplasmic maturation is a broader term that refers to other maturational events not directly related to meiotic processes but to other events that prepare the oocyte for fertilization and preimplantation development. In addition, relocation of cytoplasmic organelles, such as mitochondria and cortical granules (CG), also takes place during oocyte maturation (60). In general, most of the pig IVM media is supplemented with gonadotropins, specifically, LH and/or FSH, and other growth factors. Mattioli et al. (55) found that maturation to M II stage is significantly increased in the presence of LH (76%) and FSH (86%) than in their absence (35%). In the presence of gonadotropins, supplementation of epidermal growth factor (EGF) did not influence nuclear maturation (16, 21). However, MPN formation was significantly improved in

4 260 Theriogenology the presence of gonadotropins and EGF (21), indicating the beneficial effects of supplementing growth factors to improve cytoplasmic maturation. In many laboratories, oocytes selected for routine IVM procedures generally contain >3 layers of surrounding cumulus cells. These cells are considered an important element of the cumulus oocyte complex (COC) that supports nuclear and cytoplasmic maturation necessary for successful MPN formation and developmental competence. During the process of IVM, cumulus cells that surround each individual oocyte show varying degrees of expansion and may be functionally related to the nuclear or cytoplasmic maturation of the oocyte. To a certain extent, the degree of cumulus expansion could serve as an indicator of successful nuclear and cytoplasmic maturation. In most mammals, oocytes are enclosed in an expanded, mucitied mass of cumulus cells at ovulation. In mice, the predominant component in the expanded cumulus is hyaluronic acid (HA; 75) with the amount of HA synthesized being closely correlated with the degree of expansion (15). Epidermal growth factor can enhance nuclear maturation of COCs isolated from both small (<4 mm) and large (6 to 7 mm) size follicles with cumulus expansion induced only in COCs isolated from large follicles (70). It was found that EGF stimulated both HA production in COCs from large follicles and its retention within the extracellular matrix of the expanding cumulus. The results indicated that the response of COCs to EGF induced HA synthesis and cumulus expansion occurred gradually during follicular growth. The presence of cumulus cells during maturation significantly influenced nuclear maturation (M II), intracellular glutathione (GSH) content, penetration rate, MPN formation, and histone H 1 kinase activity (90). In addition, cumulus cells stabilized the distribution of CGs and their removal at various times during maturation resulted in a premature migration and partial exocytosis of CG (29). This observation may explain the lower in vitro penetrability of oocytes (90). It should be noted that the completion of nuclear maturation does not necessarily reflect normal cytoplasmic maturation, because the presence of cumulus cells appears to play a significant role in stimulating this process. In the past, many IVM media employed to culture pig oocytes resulted in successful nuclear maturation to M II stage. Although the nuclear events of maturation, germinal vesicle breakdown, and polar body extrusion seemed to occur normally, the cytoplasm of the oocyte usually failed to promote MPN formation after sperm penetration. It was concluded that cytoplasmic maturation is inadequate under certain IVM conditions. This failure may be due to inherent deficiencies in the isolated oocytes themselves and/or suboptimal IVM culture systems. Generally, the appearance of the oocyte before initiation of maturation and the meiotic stage after IVM are used to predict oocyte quality. However, it s becoming clear that these measurements are largely inadequate. IVF Successful in vitro penetration of IVM oocytes has been realized by using various types of fertilization media in conjunction with fresh or frozen-thawed spermatozoa. In many laboratories, because of various difficulties encountered in successful cryopreservation, freshly ejaculated semen is still the main source of spermatozoa for routine IVF studies. Nevertheless, large variations among boars as well as among different fractions within the same ejaculate in terms of

5 Theriogenology 261 oocyte penetration and/or polyspermy have been observed (88, 89). However, these authors found that the use of a specific sperm-rich fraction of the ejaculate can reduce the variability among different ejaculates collected from the same boar. Cryopreservation of spermatozoa offers an effective solution for long-term storage of valuable genetic material and allows production of embryos with a desirable genetic makeup through IVF in years to come. Development of successful techniques to freeze semen from a single ejaculate would minimize or eliminate the variability among trials. However, the same IVF protocol may not necessarily contain the optimal conditions for frozen semen from another boar. To realize desirable IVF parameters, it is necessary to optimize the IVF protocol for each individual batch of frozen semen. This is mainly due to the variability of spermatozoa from individual boars to withstand cryopreservation and/or their post-thaw motility. In addition, culture medium components, macromolecule type, sperm concentration, co-incubation interval, and presence or absence of caffeine have been shown to influence sperm penetration (1). Pig embryo production via IVM-IVF techniques has been hampered by two major problems, poor MPN formation and polyspermy. Various refinements to IVM culture techniques, such as addition of follicular fluid, co-culture with follicular somatic cells, limited exposure to gonadotropins, and supplementation of EGF or cysteine, significantly improved cytoplasmic maturation as evidenced by higher MPN formation after sperm penetration (1, 20). Increased MPN formation caused by cysteine supplementation is correlated with higher concentrations of intracellular GSH in matured oocytes (93). A similar effect on intracellular GSH level was also observed when oocytes were co-cultured with follicular somatic cells (4). Synthesis of GSH during oocyte maturation is known to be a prerequisite for hamster sperm nuclear chromatin decondensation and successful MPN formation (68). It is reasonable to assume that MPN formation is compromised in oocytes with lower levels of intracellular GSH. The ability of the cytoplasm to transform a penetrated sperm nucleus into a MPN is efficient in oocytes matured (56, 90) and fertilized (42, 46, 65) in the presence of cumulus cells. Intracellular GSH content seems to depend on the presence of cumulus cells during culture (23). One of the routes by which cumulus cells transport factors into the oocyte is via gap junction communications (GJC; 16, 3 1). Mori et al. (61) suggested that GJC play an important role in regulating the transport of GSH produced within cumulus cells into the oocyte and is correlated with MPN formation. This evidence strongly supports the role cumulus cells in improving cytolasmic maturation. Polyspermy Despite the significant improvements in the ability of oocytes to stimulate MPN formation following sperm penetration, a high incidence of polyspermic penetration still exists. Under in vivo conditions, fertilization occurs within a few hours after ovulation, and, in most instances, monospermic penetration ensues. Evidence from laboratory and farm animals indicates that the sperrnegg ratio at the time of initial penetration of the egg membranes is close to unity with this ratio increasing only after the establishment of a zona block (39). In comparison with the in vivo situation, in vitro matured oocytes are exposed to an excessive number of spermatozoa for a longer period of time. These conditions may predispose oocytes to penetration by more than one spermatozoa (polyspermy). It is envisioned that an ideal IVF system should result in a high penetration rate (~80%) with a low incidence of polyspenny (~10%). A tight correlation has been

6 262 Theriogenology established between the absolute number of spermatozoa per oocyte at fertilization and the degree of polyspermy (71). Theoretically, the problem of polyspermy could be overcome by reducing the number of spermatozoa within the IVF drops. However, in most cases, such adjustments are associated with low oocyte penetration rates. In mammals, sperm penetration triggers oocyte activation and subsequent CG exocytosis. Cortical granules are small, membrane-bound organelles at the periphery of the matured oocyte that undergo exocytosis in response to elevated intracellular calcium triggered by the penetrating spermatozoa (37). The CG contents released into the peri-vitelline space (PVS) are responsible for modifying the zona pellucida (ZP) and establishing the block to polyspermy. During maturation, an extensive redistribution of intracellular organelles (mitochondria and CG) occurs (17). Therefore, normal distribution of these organelles during IVM may play an important role in preventing polyspermy. Wang et al. (85) suggested that the occurrence of polyspermy in in vitro matured pig oocytes may be due to a delay in CG exocytosis. However, examination of in vitro matured and ovulated oocytes by confocal microscopy at 6 h after IVF revealed no difference in cortical granule release (84). Nevertheless, in comparison with ovulated oocytes, the incidence of polyspermy (65 vs. 28%) was higher in in vitro matured oocytes. It is possible that the delay in CG exocytosis occurs at the time of initial sperm penetration, which corresponds to the first 2 to 3 h in the IVF system used (2). Alternatively, although CG are released immediately after sperm entry, the narrow PVS in in vitro matured oocytes may interfere with the proper dispersal of CG contents and could delay the establishment of the zona block. In both of these situations, accessory sperm may gain entry before the establishment of a functional zona block. Pig oocytes matured in Whitten s medium containing a low NaCl content resulted in oocytes with a wider PVS that were, as a result, less polyspermic (26). Therefore, development of IVM media that results in matured oocytes with a wider PVS may, at least in part, resolve the problem of polyspermy. As mentioned previously, ovulated oocytes are significantly less polyspermic than in vitro matured oocytes following IVF (84). However, the in vivo counterparts of in vitro matured oocytes are the mature oocytes from pre-ovulatory follicles. Day et al. (19) compared the morphological, physical, and fertilization parameters of in vitro matured, ovulated, and preovulatory oocytes. In contrast to ovulated oocytes, in vitro matured and pre-ovulatory oocytes were similar in most of the parameters evaluated. It became clear that following ovulation, major changes occur to oocytes within the oviduct that are important in preventing polyspermy. Indeed, surgical transfer of in vitro matured oocytes to the estrus oviduct of a recipient gilt for 4 h resulted in similar morphological and physical changes as observed in ovulated oocytes (19). Furthermore, IVM oocytes exposed to the oviduct had similar penetration rates (83 vs. 87%) but were significantly less polyspermic (67 vs. 26%) than controls. At least in the pig, development of the full machinery to prevent polyspermy seems to be complete only after exposure to the oviduct. Many in vitro studies have indicated the beneficial effects of oviductal cells an&or conditioned media in reducing polyspermy (13, 43, 64, 74). In these studies, oviductal cells were obtained either from prepuberal gilts or animals showing estrus. Vatzias and Hagen (81) observed that supplementation of IVF medium with conditioned medium derived from periovulatory oviduct explant culture reduced the incidence of polyspermy as compared with conditioned medium from midluteal phase oviduct explants and the control group. Taken

7 Theriogenology 263 together, this information strongly suggests that oviductal secretions contain some factor(s) that interact with oocytes and/or spermatozoa to prevent or reduce multiple sperm entry. The oviduct, in response to ovarian hormones, synthesizes and secretes multiple proteins. This creates a microenvironment capable of supporting the events of fertilization and embryo development. A variety of proteins synthesized and secreted by the pig oviduct have been identified. Most abundant is the estrogen-dependent glycoprotein identified as porcine oviductspecific secretory glycoprotein (posp; 11). The presence of posp in the ZP and PVS of oviductal oocy-tes and embryos suggests a possible physiological role during sperm-oocyte interaction (12). Exposure of oocytes to semi-purified posp for 4 h before IVF and during fertilization significantly reduced polyspermy (61 vs. 29%) without compromising sperm penetration (74 vs. 63%; 47). In addition, the exposure of oocytes to posp also reduced sperm binding to ZP. It was not clear whether the reduction of polyspermy by posp exposure was due to its interaction with the oocyte and/or spermatozoa, as oocytes were exposed to the protein before and during fertilization. However, a recent study demonstrated that exposure of oocytes to posp alone can reduce polyspermy (57). In addition to its role in cumulus expansion, the use of HA during sperm pre-incubation or sperm-oocyte co-incubation in the presence of HA can reduce polyspermy without any significant effect on the penetration rate (77). Hyaluronic acid is present in peri- and post-ovulatory porcine oviduct fluid (80) and is known to stimulate in vitro capacitation of boar spermatozoa without inducing the acrosome reaction (73). In addition to oviductal glycoproteins, HA may also play a role in modulating sperm penetration and polyspermy. Most pig IVF media are supplemented with caffeine, a phosphodiesterase inhibitor known to increase intracellular CAMP. In a recent study, Funahashi et al. (27) examined the effect of caffeine, fertilization-promoting peptide (FPP), and adenosine on fertilization parameters of in vitro matured oocytes following IVF. In comparison with FPP (75%) and adenosine (71%), a significantly higher proportion of oocytes were penetrated when IVF medium contained caffeine (98%). Nevertheless most of the oocytes were polyspermic in the presence of caffeine (87%) when compared with FPP (25%) or adenosine (21%). In a previous study (25), these authors hypothesized that FPP and adenosine may modulate the adenyl cyclase/camp pathway in boar spermatozoa. The low incidence of polyspermy was attributed to the ability of FPP and adenosine to stimulate capacitation and inhibit spontaneous acrosome reactions (25, 27). Therefore, supplementation of IVF media with adenosine instead of caffeine would be a reasonable alternative to minimize the problem of polyspermic penetration. In addition to the components added during IVF, type of culture medium also affects the incidence of polyspermy. Martinez-Madrid et al. (54) compared two IVF media, modified Trisbuffered medium (mtbm) and modified Tyrodes medium (mtalp), for penetration rates and the incidence of polyspermy. Compared with mtbm, insemination in mtalp at 0.5 to 1 x 106/ml spermatozoa resulted in a higher penetration rate (92 to 94% vs. 61 to 77%). However, most of the oocytes fertilized in mtalp were polyspermic (86 to 89% vs. 44 to 50%). In a similar study, Kidson et al. (45) observed that, at a low sperm concentration (4 x 105/ml), both penetration (54 vs. 32%) and polyspermy (40 vs. 10%) was higher in mtalp than in mtbm. However, a lo-fold higher sperm concentration increased penetration rates to a similar

8 264 Theriogenology proportion (82 vs. 79%), but polyspermy was significantly lower in mtbm (76 vs. 26%). Therefore, it appears that proper selection of IVF medium is also a critical factor in minimizing polyspermy. EC Early attempts to culture in vivo-derived one-cell pig embryos in various culture media were consistently met with developmental arrest at the 4-cell stage (18). However, when collected at the 4-cell stage and placed into culture, embryos continued development to the blastocyst stage. In the past decade, formulation of various culture media including, modified Whitten s medium (mwm; 7), North Carolina State University 23 medium (NCSU 23; 69), Iowa State University medium (ISU; 94) and Beltsville Embryo Culture Medium 3 (BECM-3; 22), allowed >70% of in vivo-derived early embryos to reach the blastocyst stage. Although these culture media have proved to be equally competent to support development of in vivo-derived embryos to the blastocyst stage, varying degrees of success has been observed with IVM-IVF embryos in these four media (19). This indicates different sensitivities of IVM-IVF embryos to the type of culture medium (20). Highest (30%) and lowest (5%) proportions of blastocyst development were observed in NCSU 23 and mwm, respectively; the other two media (ISU and BECM) produced intermediate rates of blastocyst formation. It was concluded that a higher lactate concentration (25 mm) in mwm was detrimental. Indeed, a previous study reported a similar lactate concentration being inhibitory to the development of early stage pig embryos (18). However, reducing the lactate level in mwm did not improve blastocyst development (19). Blastocyst development in ISU (14%) was higher than mwm (5%). Comparison of medium components revealed that ISU medium contained a lower level of lactate (12.9 mm) and no glucose. It was hypothesized that the presence of glucose and a higher level of lactate in mwm may be responsible for the poor development of IVM-IVF derived embryos. Therefore, medium composition appears to have a significant influence on the developmental competence of IVM- IVF embryos. Embrvo Develonment with Modifications to IVM. In bovine, in vivo matured oocytes generate more blastocysts than in vitro matured oocytes (32, 49). Although similar comparisons are not available in pigs, in vitro matured oocytes showed lower penetration rates, asynchronous pronuclear development, and delayed and lower cleavage rates following IVF when compared with oocytes matured in vivo (48). These observations suggest that IVM conditions may not be adequate for supporting subsequent development. Efforts to stimulate the synthesis of crucial oocyte factors during IVM would influence oocyte quality and improve subsequent developmental competence and viability after transfer to recipients. Indeed, various modifications to IVM procedures have resulted in oocytes with higher developmental potential (20). Many of these modifications increased the intracellular GSH concentration in oocytes (3,4, 5) which could be attributed to their improved developmental competence. It is envisioned that following sperm penetration, some of the available intracellular GSH is utilized for sperm nuclear decondensation. Therefore, those oocytes possessing a higher GSH concentration retain more GSH than those penetrated oocytes that contained a lower GSH level. Because GSH is the major non-protein sulthydryl compound present in mammalian cells with multiple functions, including an effect on amino acid transport, DNA and protein synthesis, reduction of disultide

9 Theriogenology 265 bonds and acts as an antioxidant to protect cells from oxidative damage (58), oocytes with a higher GSH content may use its cytoplasm to counteract any detrimental effects caused by reactive oxygen species (ROS) generated during fertilization and embryo culture under the conventional 5% CO2 in air environment. Spermatozoa generate ROS and regulate their function (6). The intracellular GSH content of pig oocytes following IVM could be a potential biochemical marker to determine the effectiveness of IVM system and subsequent developmental competence of oocytes (20). In addition, higher embryonic development has been observed in oocytes isolated from the ovaries of sows as compared with prepubertal gilts (53). Therefore, the efficiency of existing in vitro embryo production could be improved by the use of oocytes derived from the ovaries of sows or cyclic gilts. Embrvo Develonment with Modifications to IVF. A significant improvement in porcine blastocyst development has been observed when GSH was supplemented during IVF (10). The higher blastocyst yields did not appear to be related to an increase GSH levels in putative zygotes. Although the exact mechanism is not known, it can be assumed that dead or dying spermatozoa generate excessive amounts of ROS under in vitro conditions, and the presence of extracellular GSH may help to reduce any detrimental effects on oocytes or zygotes. According to Grupen and Nottle (33), a IO-min sperm-oocyte co-incubation followed by transfer of oocytes with zona-bound sperm to fresh IVF medium for 5 h resulted in a higher penetration rate (57 vs. 80%) and blastocyst development (8 vs. 30%) as compared with controls. It is surprising that a shorter co-incubation period resulted in higher penetration rates. Intuitively, the presence of spermatozoa for the entire co-incubation period should result in similar or higher penetration rates when compared with the lo-min sperm-oocyte co-incubation method. Alternatively, it may be desirable to remove oocytes with zona-bound spermatozoa from suspensions of dead and dying spermatozoa as quickly as possible to avoid any oxidative insults on oocytes caused by ROS, which in turn may influence subsequent embryo development. Furthermore, in a recent study, the use of percoll separated frozen-thawed spermatozoa improved cleavage and blastocyst development following IVF (41). Because percoll treatment selects a highly motile population of spermatozoa and eliminates dead and/or less motile spermatozoa, those selected may have low ROS production during IVF. This selection process could lead to improvements in embryo development. It is worthwhile to re-examine these IVF strategies and their implications for the production of pig embryos. Embrvo Develonment with Modifications to EC. Culture of pig embryos in modified NCSU 23 without glucose but supplemented with low levels of lactate (4.5 r&i) and pyruvate (0.33 mm) for the first 72 h and followed by culture in NCSU 23 with glucose for (5.5 mm) the next 72 h improved blastocyst development compared with those cultured for 144 h in NCSU 23 with glucose (L. R. Abeydeera, unpublished data). A higher level of glucose (3 to 6 mm) during culture is detrimental to development of sheep embryos, but was beneficial at lower (1.5 mm) concentrations (78). In cattle (72) and sheep (79), embryos appear to use increased quantities of glucose from the 8-cell through blastocyst stages. In vitro derived 2- and 8-cell stage pig embryos cultured in NSCU 23 medium utilized less glucose than morula and blastocysts (30). Glucose seems to play a major role during compaction and blastocyst formation. In bovine, the generation of ROS is enhanced by the presence of higher glucose in culture medium (40). However, media with high glucose may have detrimental effects on the development of early stage IVM-IVF pig

10 266 Theriogenology embryos. Optimization of medium with appropriate energy substrates and culture in a sequential system is beneficial when the requirements of the embryo are kept in mind. An improvement in blastocyst development over controls was also observed when in vivo (70 vs. 45%) or in vitro (17 vs. 0%) derived pig embryos were cultured in Whitten s medium containing 0.4% BSA and 0.5 mg/ml HA (44, 59). The exact mechanism of HA action to improve embryo development is not known, but the promotion of embryonic viability cannot be ruled out (76). If this is the case, the use of HA to sustain embryo viability during culture should be considered. Embryo Morphology and Quality Despite improved in vitro embryo production, distinct morphological differences have been observed between in vitro and in vivo derived embryos including blastocysts (83). Well-defined blastomeres in early stage embryos and a prominent inner cell mass in blastocysts are evident in embryos recovered in vivo. According to Papaioannou and Ebert (66), the cell number of in vitro produced blastocysts is lower than their in vivo counterparts. According to Wang et al. (83), abnormal cleavage and low cell numbers in blastocysts produced in vitro are due to apparent deficiencies in actin filament distribution within the cytoplasm. Inadequate cytoplasmic maturation of in vitro matured oocytes and/or suboptimal embryo culture conditions may be responsible for poor embryo quality. Transfer of IVM-IVF zygotes to the oviduct of recipient animals (28) and culture of in vivo derived zygotes (51) for 5 to 6 d suggested that suboptimal embryo culture conditions, but not the IVM process itself, are responsible for poor embryo quality in terms of low cell numbers. Recent evidence showed that blastocyst development and cell number were increased when in vitro produced morula stage embryos were cultured in NCSU 23 under low (5%) 02 tension (52). In a previous study, no improvement in blastocyst development was observed when in vivo derived zygotes were cultured at a low (5%) 02 tension for the entire culture period when compared with conventional culture conditions (20% 02; 51). In contrast, Berthelot and Terqui (8) concluded that development of in vivo derived pig embryos cultured in modified NCSU 23 is optimal in an atmosphere of 5% 02 and 5% CO2. However, the results cannot be directly compared because the latter study significantly modified the culture medium (NCSU 23 without glucose but supplemented with lactate, pyruvate, vitamins, and higher levels of glutamine, NaHCOs, and BSA). Machaty et al. (52) reported that partial inhibition of oxidative phosphorylation at the morula stage has beneficial effects on blastocyst development and cell number. Therefore, under low 02 tension, ATP production in morula stage embryos may favor glycolysis over oxidative phosphorylation. A better understanding of how culture environment can affect embryo development as well as insights into how culture conditions could be redesigned to support optimal development is important. It is proposed that establishment of a sequential culture environment, 20% 02 up to the morula stage and 5% 02 for later stages, would generate better quality pig embryos. Embryo Transfer Although embryo development up to the blastocyst stage is possible in culture, the ultimate test of embryonic viability is to establish pregnancies and live births following transfer into

11 Theriogenology 267 recipient animals. Varying degrees of success in terms of pregnancies and live births have been achieved after transfer of in vitro produced embryos to the oviduct/uterus of recipient gilts (20, 53). It seems that only 20 to 30% of transferred embryos survive despite considerable improvements in IVP techniques. One of the major problems is the apparent asynchrony between the in vitro produced embryo and the oviduct/uterus of the recipient. Therefore, routine transfers with in vitro produced embryos are carried out in recipients that are at least 24 h behind the embryonic age with accurate estrus detection becoming a critical factor. In addition, >30 embryos are routinely deposited in the oviduct or uterus at transfer in order to increase the probability of pregnancy. In NCSU 23 containing BSA, ~10% of blastocysts hatch; the addition of 5 to 10% FCS at 4.5 to 5 d of culture increases the hatching rate to 30 to 40% (L. R. Abeydeera, unpublished data). It is possible that in vitro produced pig embryos will cleave in BSA-based media, but serum may be required for optimal blastocyst development and hatching. However, information on the ability of IVP embryos to hatch in vivo after transfer is lacking. Any problems associated with in vivo hatching of IVP embryos could negatively affect embryo survival and subsequent litter size. It is envisioned that development of embryo culture systems that stimulate in vitro hatching would most likely improve embryo survival and litter size. However, the significance of in vitro hatching as an indicator of embryo quality and viability remains uncertain. Development of Polyspermic Oocytes Penetration of oocytes by more than one spermatozoon is known to be detrimental to further development (38). However, polypronuclear oocytes (PPN) produced from IVM-IVF cleave and develop to the blastocyst stage in vitro or in vivo at a rate similar to two-pronuclear (2PN) oocytes (34). These blastocysts from PPN oocytes have fewer inner cell mass nuclei than blastocysts derived from 2PN oocytes. It should be noted that most PPN oocytes used in this study contained one female pronucleus and two male pronuclei. Blastocysts from PPN oocytes showed abnormal ploidy including haploids, triploids, and tetraploids. Interestingly, the transfer of PPN oocytes to recipients resulted in pregnancies and birth of piglets with normal ploidy (35). It seems that some of the PPN pig oocytes possess a mechanism to correct their polidy. The location of pronuclei within the cytoplasm of PPN oocytes seems to have a significant effect on determining the ploidy of the resulting embryo before the first cell division (35). In addition, a recent study suggested that lysosome activity could be the physiological mechanism for removing accessory sperm from the cytoplasm of fertilized eggs and cleaved embryos in vivo (87). To a certain degree, polyspermy seems to be a physiological condition in pigs because embryos appear to have mechanisms to remove the accessory sperm from the cytoplasm and continue development to eventually produce piglets. CONCLUSIONS Given its complexity, it is not surprising that IVM, IVF, and embryo development are related to factors such as ionic composition of media, gaseous atmosphere, and various media supplementation. It is also possible that developmental capacity is already acquired in oocytes at the time of aspiration with culture conditions having only a limited ability to change it. Refinements to IVM, IVF, and EC media have significantly improved MPN formation, reduced

12 Theriogenology polyspermy, and enhanced developmental potential of embryos. It is proposed that IVM of oocytes in the presence of gonadotropins, cysteine, thiol compounds, and growth factors along with replacing caffeine with adenosine and a short IVF interval may generate developmentally competent zygotes. In vitro matured oocytes have been successfully used to produce trangenic (14) and cloned pigs (9), indicating the practical application of in vitro techniques. Both a sequential EC medium and environment may improve the quality of blastocysts. It is envisioned that combinations of these improved systems will yield a higher proportion of viable blastocysts capable of establishing successful pregnancies and eventually reduce the number of embryos necessary per transfer. The IVP of pig embryos, in conjunction with successful non-surgical embryo transfer (36) techniques, has tremendous potential for basic research and commercial applications. Immature oocytes obtained from primodial or preantral follicles can be a valuable source of oocytes for future large-scale embryo production. To realize this, successful culture systems to stimulate oocyte growth have to be developed. In a recent study, significant embryo development was achieved after IVM-IVF of oocytes obtained following in vitro growth of preantral follicles (86). REFERENCES Abeydeera LR. In vitro fertilization and embryo development in the pig. J Reprod Fertil 2001;58(Suppl): in press. Abeydeera LR, Day BN. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biol Reprod 1997;57: Abeydeera LR, Wang WH, Cantley TC, Prather RS, Day BN. Presence of g- mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology 1998;50: Abeydeera LR, Wang WH, Cantley TC, Rieke A, Day BN. Coculture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: Relevance to intracellular glutathione. Biol Reprod 1998;58: Abeydeera LR, Wang WH, Cantley TC, Rieke A, Prather RS, Day BN. Epidermal growth factor can enhance the developmental competence of pig oocytes matured in vitro under protein-free culture conditions. Theriogenology 2000;54: Aitken RJ. Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev 1995;7: Beckmann LS, Day BN. Effects of media NaCl concentration and osmolarity on the culture of early-stage porcine embryos and the viability of embryos cultured in a selected superior medium. Theriogenology 1993;39:61 l-622. Berthelot F, Terqui M. Effects of oxygen, COz/pH and medium on the in vitro development of individually cultured porcine one- and two-cell embryos. Reprod Nutr Dev 1996;36: Betthauser J, Forsberg EJ, Jurgella G, Misica P, Golueke P, Augenstein M, Voelker G, Mallon K, Thompson S, Bishop M. Cloning pigs using in vitro systems. Theriogenology 2001;55:255 abstr.

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