SRD Outstanding Research Award Development and Application of a Chemically Defined Medium for the In Vitro Production of Porcine Embryos

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1 Journal of Reproduction and Development, Vol. 57, No. 1, 2011, E SRD Outstanding Research Award 2010 Development and Application of a Chemically Defined Medium for the In Vitro Production of Porcine Embryos Koji YOSHIOKA 1) 1) Research Team for Production Diseases, National Institute of Animal Health, Tsukuba, Ibaraki , Japan Abstract. Recent advances in systems for in vitro production (IVP) of porcine embryos, including in vitro oocyte maturation, fertilization and embryo culture, have enabled us to generate viable embryos that can develop to full term after transfer into recipients. This technology is being applied now to developments in gamete/embryo biology and agriculture, as well as in producing cloned and genetically modified pigs. Chemically defined media for IVP of embryos are useful for a precise analysis of the physical action of substances on gametogenesis and early embryogenesis, because they eliminate undefined factors present in biological materials, such as serum or serum albumin. Use of a chemically defined medium also improves the reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by eliminating protein preparations, which may be contaminated with pathogens. Therefore, it has certain advantages for research and for commercial purposes. We have recently developed a defined IVP system for porcine embryos using a single basic medium based on the composition of porcine oviductal fluid. This paper discusses the developmental ability and normality of porcine IVP embryos, and limitations and advancements in this system. Key words: Chemically defined medium, Developmental competence, In vitro production, Piglet (J. Reprod. Dev. 57: 9 16, 2011) Received: December 21, 2010 Accepted: December 21, by the Society for Reproduction and Development Correspondence: K Yoshioka ( kojiyos@affrc.go.jp) standard in vitro production (IVP) system for preimplantation embryos includes three technological steps: the in vitro maturation (IVM) of immature oocytes, in vitro fertilization (IVF) and in vitro culture (IVC) of zygotes. Successful large-scale IVP of porcine embryos will enable us to reduce the cost and time required and will be valuable for research in reproductive physiology, agriculture and biotechnology. The first successful production of piglets from in vitro-matured and in vitro-fertilized oocytes was reported by Mattioli et al. in 1989 [1], in which 2- to 4-cell embryos at 44 h after IVF were transferred into recipients. Since then, some laboratories have succeeded in producing piglets from cleaved embryos at the 2- to 4-cell stage cultured for h [2 4] and from 8-cell to morula-stage embryos cultured for 96 h [5] following IVM and IVF. Moreover, in the present decade it has been demonstrated that porcine in vitro-produced blastocysts cultured for 5 or 6 days after IVF could develop to full term [6, 7]. However, despite recent improvements in porcine IVP techniques, the developmental rate of embryos matured and fertilized in vitro to the blastocyst stage and their quality are still low compared with in vivo-derived embryos [8]. The low developmental competence of porcine in vitro-produced embryos might be caused by several factors, including a reduced incidence of male pronuclear formation, a high incidence of polyspermy and suboptimal conditions for embryo culture [9, 10]. Numerous media, such as modified Whitten s medium [11], North Carolina State University (NCSU) 23 medium [12], Beltsville embryo culture medium (BECM)-3 [13], modified synthetic oviduct fluid (msof) [6] and sequential media of NSCU37-PyrLac and NSCU37-Glu [7] are available for the successful culture of embryos to the blastocyst stage. The porcine IVC system has been improved; extending the developmental stage of embryos that can develop to full term after embryo transfer. In contrast to pigs, IVC of bovine embryos seems to have progressed toward obtaining blastocysts that could be transferred non-surgically to the uterine horn of recipients. To achieve this, co-culture with various somatic cells or medium conditioned by such cells has been applied for embryo culture [14 16]. In a co-culture system, somatic cells might provide unknown embryo growth promoting factor(s) and/or delete embryo toxic factor(s) from a basic medium [16, 17], resulting in adequate culture conditions for embryo development, relative to an in vivo environment in the oviduct or uterus. As a similar approach to mimic the in vivo environment for embryo culture, SOF [18] and human tubal fluid [19] media have been developed, which are based on the composition of sheep and human oviductal fluid, respectively. These media have proven useful for the IVC of preimplantation sheep and cattle [18] and mouse and human [19] embryos, respectively. However, the composition of inorganic elements and energy substrates in the mammalian oviduct differs among species [20 23]. A chemically defined medium on IVM of oocytes, IVF or IVC of embryos is useful for a precise analysis of the physical action of substances, such as inorganic compounds, energy substrates, hormones, cytokines and vitamins, on oocyte maturation, fertilization or early embryo development, since it eliminates the presence of undefined factors in biological materials such as serum or serum albumin. Use of a chemically defined medium also improves the

2 10 YOSHIOKA Table 1. Concentrations of inorganic and energy substrates in porcine oviductal fluid and porcine zygote medium (PZM) Component (mm) Oviducts* PZM Na Cl Ca K Mg Phosphorus Glucose Pyruvate Lactate *Adapted from Iritani et al. (1974) [20] and Nichol et al. (1992) [22]. reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by elimination of protein preparations, which risk contamination by pathogens [17]. Such a medium could also serve as a powerful tool for optimizing the IVP system and maximizing the number of embryos that survive after transfer [17] and therefore has certain advantages for research and for commercial purposes. In this paper, (1) development of chemically defined media for IVP of porcine embryos, based on the composition of pig oviductal fluid, (2) analysis of the physical action of substances and improvement of medium components using defined IVP system and (3) in vivo viability of in vitro-produced embryos after transfer to recipients were described. Defined Medium for In Vitro Embryo Culture In porcine oviductal fluid, the concentration of potassium is high [20] compared with that in the sheep [24] and cow [25] oviducts, whereas concentrations of energy substrates, such as pyruvate, lactate and glucose are low [22] in some respects compared with those in the mouse [21], rabbit [21] and human [26] oviducts. Based on this knowledge, we have developed a medium, porcine zygote medium (PZM), for IVC of porcine zygotes [27]. The inorganic and energy substrate composition of PZM was calculated according to the reported concentrations of pig oviductal fluid (Table 1) [20, 22]. This medium also contains glutamine, hypotaurine and premixed solutions of basal medium Eagle s (BME) essential amino acids and minimum essential medium (MEM) non-essential amino acids. Moreover, either bovine serum albumin (BSA) or polyvinyl alcohol (PVA) was added to PZM as macromolecular components (designated PZM-3 and PZM-4, respectively). To evaluate the efficiency of PZM for IVC of embryos, in vivoderived zygotes were cultured in PZM under different conditions and compared with in vivo development [27]. The viability of these zygotes to full term after culture was also evaluated by surgical embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 1 after artificial insemination. Culture of zygotes in PZM-3 produced better results in terms of proportion of Day 5 blastocysts, Day 7 hatching rate and numbers of inner cell mass (ICM) cells and total cells in Day 7 embryos than that in Fig. 1. Effects of culture system on the numbers of cells in porcine in vivo-derived embryos. Single-cell zygotes collected from gilts on Day 1 after artificial insemination were cultured with each medium for 4 days. *In vivo-developed embryos were collected from on Day 5 after artificial insemination. TE, trophectoderm; ICM, inner cell mass. Data are presented as mean and standard error of the mean (SEM). Different letters (a c) denote statistically significant differences (P<0.05 by Fisher s PLSD following ANOVA). Modified from Yoshioka et al. (2002) [27]. NCSU23. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO 2: 5% O 2: 90% N 2 compared with 5% CO 2 in air. The ICM and total cell numbers in Day 5 embryos cultured in PZM-3 or PZM-4 in which BSA was replaced with PVA were also greater than those of NCSU23, but less than those developed in vivo (Fig. 1). However, no difference was found in the ratio of ICM to total cell numbers among embryos developed in PZM-3, PZM-4, or in vivo. These results indicate that a medium designed to closely mimic in vivo environmental conditions in the porcine oviduct can support the normal development of porcine zygotes in vitro. When the Day 5 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Thus, porcine zygotes cultured for 4 days in a chemically defined medium showed full-term developmental potential without any severe reduction in embryo viability. The in vitro viability of porcine in vivo zygotes in the PZM appeared to be in no way inferior to that in other media described previously [11 13, 28, 29]. Moreover, PZM-3 was superior in supporting development of in vitro-matured and in vitro-fertilized [30], parthenogenetic [31] and somatic cell nuclear transferred [32, 33] porcine embryos compared with NCSU23, NCSU37 or BECM-3. In Vitro Fertilization System with Widespread Utility Various types of fertilizing media, such as tissue culture medium (TCM) 199 [34], Brackett and Oliphant medium [35], modified Tris-buffered medium [36] and Tyrode s medium [37] have been used for in vitro sperm penetration of porcine oocytes. An abnor-

3 REVIEW: DEFINED IVP SYSTEM FOR PIG EMBRYOS 11 Fig. 2. In vitro fertilization (A) and subsequent embryo development (B) of porcine oocytes matured in vitro, using various batches of boar semen cryopreserved or liquid-stored at 15 C. In total, 17 batches of frozen-thawed (#1 #12) and liquid-stored (#13 #17) spermatozoa were used in porcine IVF. *Pig breeds: L, Landrace; W, Large White; D, Duroc; H, Hampshire; and M, Meishan. Data are presented as mean and SEM. Modified from Suzuki et al. (2005) [48]. mally high incidence of polyspermic penetration in the porcine IVF system is one of the persistent problems for IVP of porcine embryos. Therefore, the low developmental rates of in vitro-produced porcine embryos might be caused not only by inadequate culture conditions but also by a high incidence of polyspermy, a genetically lethal condition in mammals [38]. Polyspermic penetration in vitro is not caused by delayed or incomplete cortical granule exocytosis [39], but more likely is caused by a delayed zona reaction and/or the simultaneous penetration by a number of viable acrosome-reacted spermatozoa [40, 41]. BSA in the fertilization medium accelerated the ability of bovine [42] and porcine [43] spermatozoa to penetrate in vitro-matured oocytes. Use of a BSAfree chemically defined medium for IVF might thus decrease the incidence of polyspermic penetration of porcine oocytes, by suppressing simultaneous sperm penetration in conditions of delayed fertilization. To develop defined conditions for IVF of porcine oocytes produced from IVM, we evaluated the effects of theophylline, adenosine and cysteine in a chemically defined medium during IVF [44]. A chemically defined medium, porcine gamete medium (PGM), was modified from PZM-4. In this experiment, PGM was used as a basal medium for IVF and PZM-4 was used for the culture of presumptive zygotes. Addition of 2.5 mm theophylline to PGM significantly increased the percentage of male pronuclear formation, compared with control (no addition). Addition of 1 μm adenosine to PGM with or without 2.5 mm theophylline significantly reduced the numbers of spermatozoa per penetrated oocyte compared with control (no addition of adenosine). Supplementation with 0.2 μm cysteine in PGM containing both 2.5 mm theophylline and 1 μm adenosine further increased the percentage of embryos developing to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as the rates of monospermy and pronuclear formation, regardless of the presence or absence of theophylline and adenosine. These results clearly demonstrate that porcine blastocysts can be produced by IVF and IVC in chemically defined media. It is known that the incidence of polyspermy in porcine IVF appears to be influenced by variation among boars, batches of semen and sources, among different fractions within the same ejaculate and among the storage and IVF protocols used in different laboratories [45 47]. The combination of multiple exogenous reagents, such as theophylline, adenosine and cysteine, to modulate the porcine IVF environment could make it possible to use semen

4 12 YOSHIOKA from unselected boars for porcine IVF by minimizing the differences between batches of semen in response to each reagent. To test this hypothesis, we evaluated fertilization in PGM supplemented with theophylline, adenosine and cysteine (PGMtac), and monitored subsequent in vitro development to the blastocyst stage in PZM-4 using frozen-thawed ejaculated (n=8), frozen-thawed epididymal (n=4) and liquid-stored ejaculated (n=5) semen, derived from a total of 17 boars [48]. For spermatozoa cryopreserved by three different methods, the numbers of spermatozoa per penetrated oocyte, blastocyst formation rate, and total numbers of cells per blastocyst differed significantly among freezing methods. When spermatozoa were liquid-stored at 15 C for 1 14 days after semen collection, the rate of sperm penetration decreased significantly as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days after collection enabled blastocyst production in vitro. Taken together, although 17 batches of semen from different boars had a wide range in potential for fertilization and subsequent embryo development (Fig. 2), porcine blastocysts (range % for blastocyst formation) could be derived from in vitro-matured oocytes that were fertilized with all batches used. This IVF system may have widespread utility both with frozen-thawed and liquid-stored spermatozoa. However, optimal IVF conditions, such as the added concentrations of theophylline, adenosine and cysteine to PGM, the concentration of spermatozoa at insemination and the co-incubation period of oocytes with spermatozoa, need to be determined for each batch of semen. In Vitro Maturation Without Porcine Follicular Fluid The standard medium for IVM of porcine oocytes is either NCSU23 or NCSU37 [12] with porcine follicular fluid (pff) supplementation [3, 4, 7]. However, follicular fluid contains numerous undefined factors and can be contaminated with viral pathogens [49 52]. Application of a fully defined IVM system eliminating follicular fluid from the medium may decrease the risk of viral contamination during IVP. In standard IVP protocols, an embryo may be exposed to three discrete media during in vitro development, because of the use of different medium for IVM, IVF and IVC. The effect of transferring the developing embryo between different culture environments is unknown, possibly resulting in reduced developmental potential, although the embryo might have the capacity to adjust to changes in osmolality, ph and/or substrate availability. An optimized IVP system using a single basic medium throughout IVM, IVF and IVC might be easier to use and maintain compared with media employed in standard IVP systems. In cattle, oocytes can be matured, fertilized and cultured in msof appropriately supplemented for specific stages of development [53]. Porcine oocyte medium (POM) is a modified PZM [54]. When POM containing pff, PGMtac4 and PZM-5 were used for IVM, IVF and IVC, respectively [54], rates of maturation, fertilization, cleavage and blastocyst formation, as well as total numbers of cells in blastocysts, were similar to those obtained using mncsu37 supplemented with pff. Thus, a single basic medium based on the Fig. 3. In vitro development of porcine in vitro-fertilized (A) and parthenogenetic (B) oocytes matured in vitro with different media. Data are presented as mean and SEM. Different letters (a, b) denote statistically significant differences (P<0.05 by Tukey s HSD following ANOVA). Modified from Yoshioka et al. (2008) [54]. composition of porcine oviductal fluid is available for IVP of porcine blastocysts as an alternative to discrete media for each phase of IVM, IVF and IVC. Addition of pff to maturation medium enhances the cytoplasmic maturation of porcine oocytes as measured by male pronucleus formation after IVF [55] and by the blastocyst formation rate [56]. On the other hand, it has been reported that pff can be replaced with PVA to produce a chemically defined medium, such as TCM199 [57] and mncsu23 [58]. The maturation rate and meiotic progression of oocytes in POM containing PVA were similar to those in POM or mncsu37 containing pff, whereas they declined in mncsu37 supplemented with PVA [54]. When oocytes were matured in POM supplemented with PVA, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation following IVF and IVC were similar to those in pff-containing POM or mncsu37. Although the percentages of embryos that developed to blastocysts after IVF did not differ between treatments with addition of PVA or pff to POM, the total number of cells in blastocysts was reduced by the use of PVA in place of pff (Fig. 3A). Because these indications of in vitro developmental competence of IVP embryos did not differ between experiments using artificially acti-

5 REVIEW: DEFINED IVP SYSTEM FOR PIG EMBRYOS 13 Table 2. Composition of defined culture media for in vitro production of porcine embryos Component (mm) basal POM PGMtac4 PZM-5 (for IVM) (for IVF) (for IVC) NaCl KCl KH 2PO MgSO 4 7H 2O NaHCO Glucose Na-pyruvate Ca-(lactate) 2 5H 2O L-glutamine Hypotaurine BME amino acids (ml/l) MEM non-essential amino acids (ml/l) Theophylline 2.5 Adenosine (μm) 1.00 L-cysteine (μm) Gentamicin (mg/ml) Polyvinyl alcohol (mg/ml) Modified from Yoshioka et al. (2008) [54]. vated oocytes (Fig. 3B), the reduced number of cells in blastocysts seems to be attributable to problems closely associated with fertilization [54]. Taken together, porcine blastocysts can be produced in this defined system, which consist of POM containing PVA (basal POM), PGMtac4 and PZM-5 for IVM, IVF and IVC, respectively (Table 2), as well as in the standard IVP system performed in IVM with pff-containing media. Improvements in the In Vitro Production System As noted above, the use of a chemically defined medium would facilitate the analysis of the physiological action of substances on gametogenesis and early embryogenesis. Addition of amino acids to culture medium promotes the development of embryos in various species [17]. Although the original PZM-4 contained 1 mm glutamine, 5 mm hypotaurine and 2% (v/v) premixed solutions of BME essential amino acids and 1% (v/v) MEM non-essential amino acids, addition of 2 mm glutamine and 5 mm hypotaurine enhanced both blastocyst yield and total number of cells in blastocysts [59, 60]. This embryo growth-promoting effect could in part be caused by the decreased accumulation of H 2O 2 and the prevention of DNA damage in Day-3 cleaved embryos, providing a suitable environment for them by protecting them from oxidative stress during IVC [60]. Addition of a macromolecular component (BSA or PVA) to PZM did not alter embryonic development, whereas refreshing the medium on Day 3 might reduce the total number of cells in Day-5 blastocysts [59]. In contrast, the timing of serum inclusion in PZM markedly affected the success rate of porcine embryo development in vitro. Supplementation with fetal bovine serum (FBS) at 20 and 48 h post-insemination reduced blastocyst formation rates, but when applied at 96 h post-insemination the rate increased compared with PVA supplementation [61]. Moreover, replacement of PVA with 10% (v/v) FBS in PZM-5 at 96 h post-insemination (Fig. 4) or later (Fig. 5) improved the subsequent development of embryos to the hatching/hatched blastocyst stage. A high incidence of polyspermy and low rates of blastocyst formation may be partly caused by incomplete maturation and poor quality of oocytes during IVM [45]. The in vivo processes of follicular development, meiotic resumption and subsequent ovulation in mammals are controlled by two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH) [62]. In maturation culture with a chemically defined POM, gonadotropins markedly enhanced the rate of nuclear maturation [63], similar to previous reports in pff- or FBS-containing IVM medium [64, 65]. Stimulation of meiotic resumption by gonadotropins occurs, at least in part, via the induction of members of the epidermal growth factor (EGF)-like growth factor family including amphiregulin, epiregulin and betacellulin, as paracrine mediators of LH signals on the surrounding cumulus cells [66]. Addition of TGF-α, one of the EGF-like growth factors, to the defined IVM medium increased the percentage of nuclear maturation in oocytes in the absence of gonadotropins, but did not improve results in the presence of gonadotropins [63]. However, porcine embryos derived from oocytes matured with TGF-α, even in the presence of gonadotropins, had a high ability for developing to the blastocyst stage. TGFα seems to improve cytoplasmic remodeling, involving intracellular organelles and lipid droplets in IVM oocytes during the completion of meiosis required for normal embryo development [63]. Furthermore, addition of a combination of dibutyryl camp with EGF and amphiregulin during the first 20 h of IVM to a chemically defined gonadotropin-free POM system improved both the incidence of meiotic resumption and progression to the metaphase II stage, to the same levels as oocytes treated with POM supplemented with gonadotropins and dibutyryl camp [67].

6 14 YOSHIOKA Fig. 4. Effects of replacing polyvinyl alcohol (PVA) with fetal bovine serum (FBS) in PZM-5 at 96 h post-insemination (hpi) after IVF on hatching rates of porcine in vitro-cultured embryos and the total number of cells in hatching and hatched blastocysts. Cleaved embryos, obtained by culture in PZM-5 (containing 3 mg/ml PVA) at 96 hpi, were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 1% (v/ v), 5% or 10% FBS until Day 8. Data are presented as mean and SEM. Different letters (a, b) denote statistically significant differences (P<0.05 by Tukey s HSD following ANOVA). Fig. 5. Effects of replacing PVA with bovine serum albumin (BSA) or FBS in PZM-5 at 130 hpi after IVF on hatching rates of porcine in vitro-produced blastocysts and the total number of cells in hatching and hatched blastocysts. Blastocysts obtained by culture in PZM-5 (containing 3 mg/ml PVA) at 130 hpi were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 3 mg/ml BSA or 10% FBS until Day 8. Data are presented as mean and SEM. Different letters (a, b) denote statistically significant differences (P<0.05 by Tukey s HSD following ANOVA). Table 3. Pregnancy results after surgical transfer of porcine blastocysts produced in vitro in PZM and its modifications References Yoshioka et al. (2003) Mito et al. (2009) Akaki et al. (2009) [44] [63] [67] Culture media In vitro maturation (basal) mncsu37 POM POM (additives) pff, gonadotropins, gonadotropins, EGF, amphiregulin, dibutyryl camp TGF-α dibutyryl camp In vitro fertilization PGMtac PGMtac PGMtac4 In vitro embryo culture PZM-4 PZM-5 PZM-5 No. of blastocysts transferred [range per recipient] [20 25] [20 22] [18 19] No. of recipients No. (%) of farrowing 4 (100) 4 (100) 3 (100) Gestation period (days) ± ± ± 1.1 No. (%) of piglets farrowed 21 (23.3) 29 (34.9) 11 (19.6) Average litter size 5.3 ± ± ± 1.8 Body weight of piglets at birth (kg) 1.35 ± ± ± 0.09 Average data are expressed as mean ± SEM. Developmental Competence of In Vitro-Produced Blastocysts Production of offspring by embryo transfer is the only method for validating the normality of in vitro-produced embryos unequivocally. Without this validation, the physiological relevance of data from oocyte maturation to early embryo development would be uncertain. Pregnancy results after surgical transfer of porcine blastocysts produced in vitro in PZM and its modifications are summarized in Table 3. Overall, transfer of blastocysts per recipient into 11 recipients resulted in pregnancy rates of 100% and efficiency of piglet production of 26.6% (61 piglets for 229 transferred blastocysts). Marchal et al. [6] reported that the transfer of 80 in vitro-produced blastocysts to four recipients (14 26 blastocysts per recipient) resulted in one pregnancy (pregnancy rate of 25%) and the birth of only two piglets (efficiency of piglet production of 2.5%). Kikuchi et al. [7] obtained an efficiency of piglet production of 13% by transferring 150 in vitro-produced blastocysts into three recipients (50 blastocysts per recipient): all recipients became pregnant and farrowed. Our results of embryo transfer are excellent compared with these reports and the gestation period and body weights of newborn piglets seem to be within normal limits. We also have succeeded in producing piglets derived from in

7 REVIEW: DEFINED IVP SYSTEM FOR PIG EMBRYOS 15 vitro-produced blastocysts after non-surgical transfer deep into the uterine horn using a flexible catheter [68, 69]. However, the farrowing rate ( %) and the efficiency of piglet production ( %) after the non-surgical transfer were both markedly inferior to those achieved by surgical transfer. Therefore, modification of non-surgical embryo transfer techniques as well as improvements in the in vitro-produced blastocyst quality will be needed before this procedure can be applied to the swine industry. Perspectives In vitro production of porcine embryos can now provide viable embryos more efficiently with less cost and time compared with the collection of in vivo-derived embryos. The swine industry greatly benefits from the technique of embryo transfer, as this facilitates the introduction of new genetic stock as replacement herd animals with minimal risk of disease transmission and cost for transportation, in comparison with the transport of live animals [70]. However, the commercial application of embryo transfer in the pig has been limited because of the need for surgical procedures for both the collection and transfer of the embryos. IVP of embryos together with non-surgical embryo transfer and embryo storage technology could alter the dissemination structure of the swine industry. Because of their physiological similarities to humans, pigs can be used as research biomodels. They are also valuable for creating genetically modified animals as potential donors of tissues and organs for human xenotransplantation. Success in IVP systems to provide large numbers of embryos can also accelerate progress in this research area. Several genetically modified pigs have been derived from embryos produced by somatic cell nuclear transfer in culture with PZM or its modifications [71 74]. For such biomedical research, application of defined IVP system for embryos could provide an advantage in biosafety aspects of protocols by elimination of undefined factors present in biological materials. Acknowledgments I wish to thank the Society for Reproduction and Development (SRD) for conferring the SRD Outstanding Research Award 2010 to this research. I am very grateful to Prof Y Takahashi (Hokkaido University) for his guidance. I would also like to express my thanks to Drs K Kikuchi (National Institute of Agribiological Sciences) and T Nagai (National Institute of Livestock and Grassland Science) for helpful comments and discussions. I also thank my colleagues at the National Institute of Animal Health for their technical support. This research was supported in part by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry from Bio-oriented Technology Research Advancement Institution (BRAIN) and Grants-in-Aid for Scientific Research C (No ) from the Japan Society for the Promotion Science (JSPS). References 1. Mattioli M, Bacci ML, Galeati G, Seren E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 1989; 31: Yoshida M, Mizoguchi Y, Ishigaki K, Kojima T, Nagai T. Birth of piglets derived from in vitro fertilization of pig oocytes matured in vitro. Theriogenology 1993; 39: Funahashi H, Kim NH, Stumpf TT, Cantley TC, Day BN. Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation porcine oocytes. Biol Reprod 1996; 54: Funahashi H, Cantley TC, Day BN. 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