Reproduction in Domestic Animals

Reprod Dom Anim 43 (Suppl. 2), 401–406 (2008); doi: 10.1111/j.1439-0531.2008.01191.xISSN 0936-6768Selected Aspects of Advanced Porcine Reproductive TechnologyK Kikuchi 1 , N Kashiwazaki 2 , T Nagai 3 , M Nakai 1 , T Somfai 1 , J Noguchi 1 and H Kaneko 11 Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki; 2 Laboratory of Animal Reproduction, School ofVeterinary Medicine, Azabu University, Sagamihara, Kanagawa; 3 National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, JapanContentsIn vitro fertilization (IVF) of in vitro matured (IVM) oocytesin pigs has become the most popular method of studyinggametogenesis and embryogenesis in this species. Furthermore,because of recent advances in in vitro culture (IVC) ofIVM–IVF embryos, in vitro production (IVP) of embryosnow enables us to generate viable embryos as successfully asfor in vivo-derived embryos and with less cost and in lesstime. These technologies contribute not only to developmentsin reproductive physiology and agriculture but also tothe conservation of porcine genetic resources and theproduction of cloned or genetically modified pigs. However,in IVP, there still remains the problem of abnormal ploidy,which is caused by performing procedures under nonphysiologicalconditions. In recent years, unique technologiessuch as intracytoplasmic sperm injection (ICSI) or xenograftingof gonadal tissue into immunodeficient experimentalanimals have been developed to help conserve gameteresources. These technologies combined with IVP areexpected to be useful for the conservation of gametes fromimportant genetic resources. Here, we discuss the developmentalability and normality of porcine IVP embryos andalso the utilization of ICSI and xenografting in advancingbiotechnology in pigs.IVF and the Normality of Porcine ZygotesThe in vitro developmental competence or viability ofporcine in vitro matured (IVM)–in vitro fertilized (IVF)oocytes to the blastocyst stage was first confirmed andreported by Mattioli et al. (1989). Furthermore, pigletshave been born from IVM–IVF embryos after in vitroculture (IVC) to the 2- to 4-cell stages (Mattioli et al.1989; Yoshida et al. 1993). Since then, some laboratorieshave succeeded in producing piglets from embryoscleaved at the 2- to 4-cell stages after IVM–IVF andIVC for 24–36 h (Funahashi et al. 1996, 1997). Viablepiglets were also generated after transfer of in vitroproduced (IVP) embryos at the blastocyst stage (Marchalet al. 2001; Kikuchi et al. 2002). Over several recentyears, IVC procedures have been improved, but IVMand IVF systems still have unsolved problems, including(1) imbalance of nuclear and cytoplasmic maturationand (2) polyspermy. Both these phenomena causeabnormal ploidy in IVP embryos, potentially resultingin loss of embryos after their transfer to recipients. Weneed to make every effort to achieve normality in IVPembryos after IVM and IVF.Fertilization at the immature stage, before nuclearmaturationImmature oocytes before nuclear maturation, such asthose at the metaphase-I stage, acquire fertilization oractivation potential (Kikuchi et al. 1999) and developmentalpotential, at least to the blastocyst stage (Somfaiet al. 2005). Our recent data suggest that treatment ofimmature oocytes with cytochalasin B, a drug thatinhibits actin filament polymerization, results in thefailure of extrusion of the first polar body during thefirst meiosis (Somfai et al. 2006). This failure, whichmay also be caused under physiological conditions,leads to lack of completion of meiotic maturation inoocytes (Somfai et al., unpublished). The developmentalcompetence of these immature oocytes is poor, resultingin low rates of blastocyst formation and low cellnumbers in the blastocysts. Chromosomal analysissuggests that these immature porcine oocytes fertilizedin vitro develop abnormally in terms of ploidy andespecially show increased rates of triploidy (Somfaiet al. 2005).Polyspermic fertilization of matured oocytesThe best-known problem that occurs during porcineIVF is polyspermy (Nagai 1996). Many researchershave been trying to solve this problem by differentapproaches, but so far no one has succeeded.Although it has been suggested that polyspermicoocytes have the ability to extrude the extra spermchromosomes in the early embryonic stage, beforeblastulation (Funahashi and Day 1997), the abnormalploidies are reportedly maintained until the blastocyststage (Han et al. 1999; McCauley et al. 2003; Somfaiet al. in press).Utilization of Intracytoplasmic Sperm Injectionfor PigsIntracytoplasmic sperm injection (ICSI) was first performedto study the early events of fertilization inhamsters (Uehara and Yanagimachi 1976, 1977). Fromthe 1990s, ICSI has been used for assisted reproductivetechnology in humans. Also in animals, viable offspringcan be generated by using sperm with poor motility orviability (Table 1). These successful reports confirm theooplasmic capacity for fertilization and development toterm after ICSI in both in vivo matured and IVMoocytes in mammals. If the spermatozoa have lost theirmotility, ICSI is indispensable for fertilization to producethe next generation. In pigs, the use of in vivomatured oocytes led to the first successful production ofpiglets after ICSI (Kolbe and Holtz 2000; Martin 2000);the use of ICSI in porcine IVM oocytes for successfulpiglet production was first confirmed in our laboratory(Nakai et al. 2003).Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag