Postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned pig embryos by increasing nuclear retention and formation of single pronucleus

The objective of this study was to investigate the effects of postactivation treatment with nocodazole on morphologic changes of donor nuclei and in vitro and in vivo development of somatic cell nucleus transfer (SCNT) embryos in pigs (Sus scrofa). Somatic cell nucleus transfer oocytes were either untreated (control) or treated with nocodazole or demecolcine after electric activation, then cultured in vitro or transferred to surrogate pigs. Treatment with nocodazole (30%) and demecolcine (29%) after electric activation improved embryo development to the blastocyst stage compared with the control (16%). The rate of oocytes that formed single clusters of chromosomes or a pronucleus 4 h after activation was higher after treatment with nocodazole (82%) and demecolcine (86%) than under the control conditions (66%), and this tendency was not altered even 12 h after activation. Pseudo-polar body extrusion was inhibited by nocodazole and demecolcine, and the rate of embryos with diploid chromosomes was higher after treatment with nocodazole (86%) and demecolcine (77%) than under control conditions (58%). Nocodazole treatment resulted in a farrowing rate of 50% with a 1.7% efficiency of piglet production, whereas controls showed a farrowing rate of 60% and a production efficiency of 3.8%. Our results demonstrate that postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned embryos likely by increasing nuclear retention and formation of single pronuclei. In vivo development could be achieved from the transfer of nocodazole-treated embryos but showed some defects compared with control.     

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.     

Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig

The objective of this study was to assess the practical usefulness of morphologically poor oocytes (MPCOCs) in relation to follicular size and oocyte diameter. Oocytes collected from medium (3–8 mm in diameter) and small (<3 mm) follicles were classified into five categories of morphologically good oocytes (MGCOCs) from medium follicles (MA, control), MPCOCs with larger and smaller diameters from medium follicles (ML and MS, respectively), and those from small follicles (SL and SS, respectively). The oocytes were examined for maturation and developmental competence after parthenogenesis and somatic cell nuclear transfer (SCNT). Nuclear maturation of ML oocytes (91%) was similar to that of control oocytes (94%), but higher than MS (80%), SL (79%), and SS (63%) oocytes. This pattern was also observed in the intracellular glutathione level, p34^cdc2 kinase activity, and gene (CDK1, PCNA, and ERK2) expression levels in in vitro‐matured oocytes. ML oocytes showed a similar proportion of blastocyst formation (20%) after SCNT to control oocytes (21%). In addition, the use of ML oocytes resulted in a 50% farrowing rate with 1.8% efficiency of piglet production after SCNT embryo transfer, while control oocytes showed a 60% farrowing rate with 2.4% production efficiency. Our results demonstrate that MPCOCs, if appropriately selected, have a comparable ability to MGCOCs in supporting not only in vitro blastocyst formation, but also development to term in vivo after SCNT. These oocytes can be used as a source for in vitro production of embryos with normal in vivo viability in pigs. Mol. Reprod. Dev. 77: 330–339, 2010. © 2009 Wiley‐Liss, Inc.     

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.     

Cotransfer of parthenogenetic embryos improves the pregnancy and implantation of nuclear transfer embryos in mouse

The majority of somatic cell nuclear transfer (SCNT) clones dies in the peri- or postimplantation period. Improvement of the full-term healthy pregnancy rates is a key issue for the economical viability and animal welfare profile of SCNT technology. In this study the effects of cotransfer of parthenogenetic or fertilized embryos on the pregnancy and implantation of SCNT mouse embryos have been investigated. SCNT embryos were produced by transferring cumulus cell nuclei into enucleated B6D2F1 mouse oocytes, whereas parthenogenetically activated (PA) and fertilized embryos were derived from ICR mice by artificial activation with strontium and in vivo fertilization, respectively. SCNT embryos were inferior in their developmental capacity to blastocyst compared to either PA or fertilized embryos. SCNT embryos were transferred alone (SCNT), or cotransferred with two to three PA (SCNT + PA) or fertilized (SCNT + Fert) embryos into the oviducts of an ICR recipient. Both pregnancy and implantation rates originating from clones in the SCNT + PA group were significantly higher than those of SCNT group (p < 0.05). The weight of placentas of clones derived from SCNT, SCNT + PA, or SCNT + Fert was in all cases significantly higher than that of fertilized controls (p < 0.001). Most of the clones derived from SCNT embryos cotransferred with PA or fertilized embryos survived to adulthood and were fertile and healthy according to histopathological observations. Our results demonstrate in mouse that cotransfer of PA embryos improves the pregnancy and implantation of SCNT embryos without compromising the overall health of the resulting clones.     

Efficient production of nuclear transferred rat embryos by modified methods of reconstruction

In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.     

In vitro development of polyspermic porcine oocytes: Relationship between early fragmentation and excessive number of penetrating spermatozoa

Embryo development during in vitro culture of polyspermic porcine oocytes was investigated in the present study. After in vitro fertilization (IVF) of in vitro matured oocytes, putative zygotes were centrifuged to visualize pronuclei. Two pronuclear (2PN) and poly-pronuclear (PPN) zygotes were selected and cultured in vitro. Their development to the blastocyst stage and total cell numbers, dead cell rates and ploidy at the blastocyst stage and morphology of resultant embryos after first cleavage were compared. A cleavage rate of PPN embryos was lower than that of 2PN (61.3% and 82.2%, respectively), however, the ability of cleaved embryos to develop to the blastocyst stage did not differ between the PPN and the 2PN groups (22.4% and 32.9%, respectively). Also there was no difference in total cell numbers and rates of dead cells between PPN and 2PN blastocysts. The majority of blastocysts in 2PN group were found to be diploid. In contrast, blastocysts in PPN group showed heterogeneous status in their ploidy including polyploidy and mixoploidy, whereas a remarkable proportion (31.3%) of them was found to be diploid. After the first cleavage (at 36 h after IVF), there was no difference in the number of nuclei/embryo between the two groups, nevertheless embryos in PPN group had significantly higher numbers of blastomeres than that of embryos in 2PN group, mainly due to an increased frequency of anuclear blastomeres. The present results indicate that correction of embryo ploidy in polyspermic embryos can occur during IVC. Nevertheless the frequency of partial fragmentation in polyspermic embryos is increased.     

Heat shock of porcine zygotes immediately after oocyte activation increases viability

We evaluated the effect of different timing variations of an applied heat shock on parthenogenetically activated (PA) porcine embryos. PA embryos were heat shocked for 9 hr at 42 degrees C from either 0-9 hr post activation (hpa; 09HS), 13-22 hpa (1322HS), or 22-31 hpa (2231HS). Analysis of 24-hr cleavage rates (P < 0.0001), day 5 cell numbers (P < 0.005), day 7 blastocyst rates (P < 0.0001), and day 7 cell numbers (P < 0.05) showed that 09HS embryos developed more successfully in vitro than did all other treated and control embryos. In vitro fertilized (IVF) embryos were exposed to similar heat treatments as described for PA embryos, and embryos derived from somatic cell nuclear transfer (SCNT) were exposed only to the control and 09HS treatments to assess the effects of the different heat treatments on the timing of first cleavage and development to blastocyst. Embryos derived from both IVF and SCNT showed higher proportions of cleaved embryos on day 1 of development when heat shocked immediately after fertilization or fusion/activation as compared to NHS controls (P < 0.05). Blastocyst rates however, showed only modest (IVF; P = 0.089) or no (SCNT; P > 0.1) improvement as compared with control embryos. In summary, exposing PA embryos to elevated temperatures immediately after oocyte activation results in dramatically enhanced developmental potential. A thorough characterization of this phenomenon may yield findings that can serve to increase the efficiency with which PA, IVF, and SCNT embryos are produced in vitro.     

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.     

Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations

This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.     

Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos

In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos. In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS). Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%). Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05). In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts. The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope. The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted. In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored. No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups. A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%). The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%). When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media. Total cell number of blastocysts were not significantly different among experimental groups. In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.     

Chromosomes in the Porcine First Polar Body Possess Competence of Second Meiotic Division within Enucleated MII Stage Oocytes

To determine whether chromosomes in the porcine first polar body (PB1) can complete the second meiotic division and subsequently undergo normal pre-implantation embryonic development, we examined the developmental competence of PB1 chromosomes injected into enucleated MII stage oocytes by nuclear transfer method (chromosome replacement group, CR group). After parthenogenetic activation (PA) or in vitro fertilization (IVF), the cleavage rate of reconstructed oocytes in the IVF group (CR-IVF group, 36.4 ± 3.2%) and PA group (CR-PA group, 50.8 ± 4.2%) were significantly lower than that of control groups in which normal MII oocytes were subjected to IVF (MII-IVF group, 75.8 ± 1.5%) and PA (MII-PA group, 86.9 ± 3.7%). Unfertilized rates was significantly higher in the CR-IVF group (48.6 ± 3.3%) than in the MII-IVF group (13.1 ± 3.4%). The blastocyst formation rate was 8.3 ± 1.9% in the CR-PA group, whereas no blastocyst formation was observed in the CR-IVF group. To produce tetraploid parthenogenetic embryos, intact MII stage oocytes injected with PB1chromosomes were electrically stimulated, treated with 7.5 μg/mL cytochalasin B for 3 h (MII oocyte + PB1 + CB group), and then cultured without cytochalasin B. The average cleavage rate of reconstructed oocytes was 72.5% (48 of 66), and the blastocyst formation rate was 18.7% (9 of 48). Chromosome analysis showed similar proportions of haploid and diploid cells in the control (normal MII oocytes) and CR groups after PA; overall, 23.6% of blastocysts were tetraploid in the MII oocyte + PB1 + CB group. These results demonstrate that chromosomes in PB1 can participate in normal pre-implantation embryonic development when injected into enucleated MII stage oocytes, and that tetraploid PA blastocysts are produced (although at a low proportion) when PB1 chromosomes are injected into intact MII stage oocytes.     

Effect of melatonin treatment on the developmental potential of parthenogenetic and somatic cell nuclear-transferred porcine oocytes in vitro

Melatonin secreted from the mammalian pineal gland is a free-radical scavenger that protects tissues from cell damage. The present study examined the effects of addition of melatonin to the culture medium on the developmental potential of parthenogenetic and somatic cell nuclear-transferred (SCNT) porcine oocytes. Supplementation of the maturation medium with melatonin did not increase the maturation rate, the proportion of oocytes that cleaved and developed into blastocysts after parthenogenetic activation, or the blastocyst cell number compared to controls. When 10-7 M melatonin was added to the culture medium, the proportion of parthenogenetic oocytes that developed to the 2-cell and 4-cell stages was significantly higher than that of controls. The potential of melatonin-treated oocytes to develop into blastocysts was high but not significantly different from that of controls. The addition of 10-7 M melatonin to the culture medium did not increase the preimplantation development of SCNT oocytes. Melatonin treatment significantly reduced the levels of reactive oxygen species in 4-cell parthenogenetic and SCNT embryos, but did not reduce the proportion of apoptotic cells in parthenogenetic and SCNT blastocysts. Although the results indicated that parthenogenetic and SCNT melatonin -treated embryos had significantly lower levels of reactive oxygen species than controls, the potential of melatonin-treated embryos to develop into blastocysts was not significantly higher than that of controls, in contrast to previous reports. The beneficial effects of melatonin on the developmental potential of oocytes might depend on the culture conditions.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.