The aim of the present study was to assess the effects of L-carnitine, an enhancer of lipid metabolism and mitochondrial activity, during in vitro maturation (IVM) on nuclear maturation and in vitro fertilisation of porcine follicular oocytes and subsequent embryo development. Mitochondrial functions, intracellular lipid content and reactive oxygen species (ROS) levels in oocytes were also investigated. L-carnitine supplementation in 0.6-5mgmL(-1) concentration during IVM significantly improved (P<0.05) the rates of metaphase-II (MII) stage oocytes compared with the control; however, fertilisation rates and monospermy were not improved. Although supplementation of IVM medium with L-carnitine significantly increased oocyte cleavage (P<0.05), further development to the blastocyst stage was not improved. The density of active mitochondria was significantly higher and the density of lipid droplets was significantly lower (P<0.05) in L-carnitine-treated oocytes compared with the control. Furthermore, the ROS levels in L-carnitine-treated oocytes were significantly lower than those in the control. In conclusion, enhancing mitochondrial functions by L-carnitine improved oocyte maturation and cleavage underlining the importance of lipid metabolism for nuclear and cytoplasmic maturation of porcine oocytes.

Abstract.The following selection markers for in vitro-produced porcine embryos were investigated: the timing, pattern and evenness of the first cleavage and the timing of the second cleavage. The embryos that cleaved by 30 h postinsemination (hpi) developed to blastocysts at a significantly higher rate (60.9%) and with a significantly higher cell number (33.6 cells) than those of embryos cleaved by 36 hpi (26.4% and 23.6 cells, respectively, P<0.05). Blastocyst proportions derived from 2-and 3-cell embryos cleaved by 30 hpi (68.2 and 65.3%, respectively) were significantly higher than those of 4-and >4-cell embryos (46.3 and 42.6%, respectively, P<0.05). The cell number per blastocyst generated from 2-cell embryos was significantly greater (37.3 cells) than those from 3-, 4-and >4-cell embryos (23.6-27.8 cells, P<0.05). Among embryos cleaved by 30 hpi, the blastocysts derived from evenly cleaved embryos (40.6 cells) were of significantly better quality than those derived from unevenly cleaved embryos (33.2 cells, P<0.05), although their blastocyst rates did not differ. The evenly cleaved embryos that underwent subsequent cleavage within 18 h had significantly higher blastocyst rates (72.7-81.0%) and quality (36.2-40.9 cells) than those without subsequent cleavage (48.3% and 22.5 cells, respectively, P<0.05) during the same period. In conclusion, the timing, pattern and evenness of the first cleavage and the timing of the second cleavage affected the developmental competence and quality of in vitroproduced porcine embryos. Key words: Cell division, Early stage, Embryo morphology, In vitro fertilization, Pig (J. Reprod. Dev. 56: [593][594][595][596][597][598][599][600] 2010) he developmental competence of porcine embryos produced in vitro after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) for a short period has been confirmed [1,2]. Birth of piglets has been accomplished from IVM-IVF embryos after IVC to the 2-to 4-cell stages [3][4][5] or 8-cell to morula stage [6]. Although transfer of in vitro-produced (IVP) blastocysts has also led to pregnancies and live births [7][8][9], utilization of early IVP embryos for transfer remains preferable because the viability of IVP porcine embryos is decreased by IVC after IVF [10]. High oxygen tension, imperfect culture media and polyspermy are thought to reduce the developmental competence of IVP porcine embryos [11]. Thus, large numbers of early-stage IVP embryos should be transferred to recipients to ensure multiple pregnancies.For the success of embryo transfer (ET) programs, selection of high-quality embryos for ET and elimination of those with low developmental competence at early stages are key factors [12,13]. To achieve this goal, transfer of cleavage stage embryos chosen by an effective embryo selection system is necessary. The timing of the first cleavage and cleavage evenness are among the most frequently used morphological criteria for choosing good quality embryos for ET. Studies in cattle [14], humans [15], mice [16] and pigs [13,17] have...

The pig is considered to be a suitable source of cells and organs for xenotransplants, as well as a transgenic animal to produce specific proteins, given the biological similarities it shares with human beings. However, the in vitro embryo production system in pigs is inefficient compared with those in other mammals, such as cattle or mice. Although numerous modifications have been applied to improve the efficiency of in vitro embryo production systems in pigs, not much progress has been made to overcome the problem of polyspermy, and low developmental ability due to insufficient cytoplasmic abilities of in vitro matured oocytes and improper culture conditions for the in vitro produced embryos. Recent achievements, such as the establishment of chemically defined medium and utilization of 'zona hardening' technique, have gained some success. However, further research for the reduction of polyspermy and detrimental effects of the culture systems in pigs is still needed.

We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22 hr, last 22 hr, or entire 44 hr duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P < 0.05) and obvious induction of cumulus expansion were observed over the whole time span (44 hr) in the IVM group. When cumulus cells were removed at 22 hr and denuded oocytes were further cultured, pLIF showed no effect on maturation rate. Oocytes matured in pLIF-supplemented medium showed a tendency for more rapid blastocyst development (21.1% vs. 16.2%, P = 0.0715). Examination of transcripts and proteins of the LIF signaling pathway in COCs revealed that LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) are present in both cumulus cells and oocytes. The amount of phosphorylated STAT3 (p-STAT3) markedly increased in both cumulus cells and oocytes cultured in pLIF-supplemented media, although oocyte p-STAT3 disappeared after 44 hr of IVM. These results suggest that the LIF/STAT3 pathway is functional during IVM of porcine oocytes, and supplementing pLIF in the IVM medium can improve oocyte maturation by activating this pathway.

Abstract. Pigs are one of the most important domesticated animals in Vietnam. They are the main source of meat for the Vietnamese. According to FAO statistics, Vietnam is among the top 5 countries raising pigs in the world, with nearly 27 million hogs. This review article introduces the distribution, morphology, growth potential, meat-producing ability and reproductive efficiency of six Vietnamese indigenous pig breeds: I, Mong Cai, Muong Khuong, Soc, Meo and Co. The collected data showed that these Vietnamese pigs are less effective in comparison with Western pigs in terms of reproductive and meat-producing ability as well as weight growth. However, these Vietnamese indigenous breeds have some special characteristics, such as very early sexual maturity, and good adaptability to harsh raising conditions or poor feeding. Moreover, recent genetic research has shown that Vietnamese pigs are genetically diverse. Thus, conservation of these pig breeds using assisted reproductive techniques is urgent and important.

Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus-oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.

In this study, we evaluated the effect of different concentrations of cysteine in in vitro maturation (IVM) medium during IVM under low oxygen tension (5% O(2)) of porcine oocytes on the intracellular content of glutathione (GSH) and subsequent in vitro fertilization (IVF) and development. Cumulus oocyte complexes (COCs) were collected from ovaries obtained at a local slaughterhouse, cultured in IVM medium supplemented with 0 (control), 0.05, 0.1, 0.2 or 0.6 mM cysteine for 44-46 h, fertilized in vitro and subsequently cultured for 6 days in total. The GSH content of the IVM oocytes exposed to 0, 0.05, 0.1, 0.2 or 0.6 mM cysteine increased significantly (P<0.05) as the concentration of cysteine increased (12.2, 14.0, 15.1, 16.4 and 16.4 pmol/oocyte, respectively). However, the rates of oocyte maturation, sperm penetration, male pronuclear formation, monospermy and even cleavage on Day 2 (the day of IVF was defined as Day 0) and blastocyst formation on Day 6 did not differ among the groups. Moreover, the cell numbers of blastomeres in blastocysts were uniform among the groups. These results indicate that supplementation with 0.05-0.6 mM cysteine during IVM under 5% O(2) tension significantly increased the intracellular GSH contents of IVM oocytes; however, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation and subsequent embryonic development to the blastocyst stage.