This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% eggyolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide(H2O2) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitromatured pig oocytes and treated for 4 h after electric activation with 0.5 μM latrunculin A (LA), 10.4 μM cytochalasins B (CB), and 4.9 μM cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8～88.6%), blastocyst formation (30.7～ 32.4%), and mean cell number of blastocysts (33.5～33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5～78.0%) and mean blastocyst cell number (33.6～35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5～85.7%) and blastocyst cell number (41.1～43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.