The objective of this study was to investigate to influence of glutathione (GSH) on development and antioxidant enzyme activity in tetraploid porcine embryos. Tetraploid embryos were produced using parthenogenetic 2-cell embryo by electrofusion method. Tetraploid embryo development was observed every 24 hours and intracellular antioxidant enzyme activity was measured at 120 hours after electrofusion. The 4-cell to 16-cell stage tetraploid embryos was increased in 100 and 500 μM GSH-treated groups compared control group at 48 hours (P < 0.05) but cleavage rates were not significantly different among the GSH treatment groups at 48, 72, 96, and 120 hours. Blastocyst formation was significantly increased by 300 and 500 μM GSH at 120 hours in tetraploid embryos (P < 0.05). But blastocyst cell number were not significantly different among the GSH treatment groups (16.4 ± 0.8, 16.8 ± 2.6, 18.5 ± 2.8 and 17.5 ± 1.8). The intracellular antioxidant enzyme level was increased in 500 μM GSH compared to 0 and 100 μM GSH (P < 0.05). We suggest that GSH may be improve development of tetraploid embryo in pigs.

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.

The purpose of this study was to examine the effects of taurine and vitamin E on ovarian granulosa cells damaged by bromopropane (BP) in pigs. We evaluated cell viability, plasma membrane integrity (PMI) and apoptotic morphological change in porcine ovarian granulosa cells. The cells were treated with 1-BP (0, 5.0, 10, and 50 μM), 2-BP (0, 5.0, 10, and 50 mM), taurine (0, 5.0, 10, and 25 mM), and vitamin E (0, 100, 200, and 400 μM) for 24 h. 10 μM 1-BP and 50 μM 2-BP inhibited viability and PMI, and induced apoptosis in porcine ovarian granulosa cells (p < 0.05). Cell viability and PMI were increased by taurine (10 and 25 mM) and vitamin E (100 and 200 μM), and apoptosis decreased (p < 0.05). Finally, the porcine ovarian granulosa cells were co-treated with BPs (10 μM), taurine (10 mM) and/or vitamin E (200 μM). Cell viability and PMI in the co-treated cells were increased, and apoptosis was decreased. In conclusion, taurine and vitamin E can improve cell viability and inhibition of apoptosis in porcine ovarian granulosa cells damaged by bromopropane.

The aim of this study was to evaluate effect of α-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at 37.5°C for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

As a one of unsaturated fatty acid, polyunsaturated fatty acids (PUFAs) have multiple actions: as precursor of prostaglandins (PGs), steroid hormone synthesis and energy production in animal reproduction. PUFAs, which include omega-3 (n-3) and omega-6 (n-6), are derived from the diet and changed by diet, species, breed and season. The plasma membrane of spermatozoa in mammals contain various PUFAs. These composition of PUFAs regulate the membrane fluidity and cause lipid peroxidation via generation of reactive oxygen species (ROS). Induced lipid peroxidation by ROS decreased viability and motility of spermatozoa, and it is reduced by addition of antioxidant and low concentration of PUFAs. Because oocytes of animal have a high lipid components, process of oocyte maturation and embryo development are influenced by PUFAs. In in vitro study, oocyte maturation, embryo development, intracellular cAMP and MAPK activity were increased by treatment of n-3 α-linolenic acid (ALA) during maturation, whereas n-6 linoleic acid (LA) negatively influenced. Also, inhibition of fatty acid metabolism in oocyte influenced blastocyst formation of cattle. PGs are synthesized from PUFAs and various PUFAs influence PGs via regulation of PG-endoperoxide synthase (PTGS). Steroid hormone synthesis from cholesterol is regulated by expression of steroid acute regulator (StAR) protein and mRNA. Exogenous n-3 and n-6 PUFAs altered sex hormone in animal through stimulate or inhibit StAR activity. Because PUFAs altered PG and steroid hormone synthesis, follicular development was influenced by PUFAs. This effect of unsaturated fatty acid could provide information for improvement of reproductive ability in animals.

This study was to investigate effect of progesterone (P4) on prostaglandin (PG) synthases and plasminogen activators (PAs) system in bovine endometrium during estrous cycle. Endometrium tissues were collected from bovine uterus on follicular and luteal phase and were incubated with culture medium containing 0 (Control), 0.2, 2, 20 and 200 ng/ml P4 for 24 h. The PGF2α synthase (PGFS), PGE2 synthase (PGES), cyclooxygenase-2 (COX-2), urokinase PA (uPA), and PA inhibitors 1 (PAI-1) mRNA in bovine endometrium were analyzed using reverse transcription PCR and PA activity was measured using spectrophotometry. In results, COX-2 was higher at 2 ng/ml P4 group than control group in luteal phase (p<0.05), but, it did not change in follicular phase. Contrastively, PGES was significantly increased in 2 ng/ml P4 group compared to control group in follicular phase, but there were no significant differ among the treatments in luteal phase. uPA was no significant difference between P4 treatment groups and control group in both of different phase. PAI-1 was decreased in 20 ng/ml P4 group compared to control group in follicular phase (p<0.05). PA activity was decreased in 2 ng/ml P4 group compared to other groups in follicular and luteal phase (p<0.05). In conclusion, we suggest that P4 may influence to translation and post-translation process of PG production and PA activation in bovine endometrium.

The purpose of this study was to examine the effects of taurine and vitamin E on sperm characteristics damaged by bromopropane (BP) in pig. We evaluated toxicity of BP on viability, membrane integrity and mitochondrial activity of spermatozoa. 1-BP (0, 2.5, 5.0, 10, and 50 μM), 2-BP (0, 2.5, 5.0, 10, and 50 μM), taurine (0, 5.0, 10, and 25 μM) and vitamin E (0, 50, 100, and 200 μM) were treated in fresh boar semen for 6 h. 10 and 50 μM of 1-BP and 2-BP inhibited sperm viability, membrane integrity and mitochondrial activity in fresh boar semen (P<0.05). 25 μM of taurine increased sperm viability and membrane integrity (P<0.05), 100 μM of vitamin E enhanced viability and mitochondrial activity of sperm (P<0.05). Finally, 10 μM of 1-BP and 2-BP was co-treated with taurine (25 μM) and vitamin E (100 μM) in the fresh boar semen. The co-treated samples did affected viability, membrane integrity and mitochondrial activity of sperm. In conclusion, taurine and vitamin E can improve and maintain sperm quality in fresh boar semen.

This study investigated the effects of L-carnitine (LC) and nicotinic acid (NA) on sperm viability during liquid storage at 18℃ in miniature pigs. 10 μM LC and 30 mM NA, combined LC and NA (LN) were treated in fresh semen for 3, 7, and 10 days. In results, sperm survival increased in NA- and LN-treated semen on 7 and 10 days (p<0.05), mitochondrial integrity of live sperm increased in LN-treated semen on 7 days (p<0.05), but not NA-treated semen. In addition, we examined the acrosome reaction of sperm in miniature pigs. LC and NA did not influence on acrosome reaction of boar sperm. In conclusion, LC and NA effectively maintained the viability and quality of sperm during long-term storage in miniature pigs, suggesting that the combined LN may be useful for improving the semen extender for long-term liquid storage in pigs.

The objective of this study was to investigate the efficiency of nicotinic acid during in vitro fertilization (IVF) in frozen-thawed bull sperm . The ejaculated semen was diluted with Triladyl containing 20% egg-yolk and cryopreserved in liquid notrigen. The frozen sperm was thawed for 45 seconds in the 38℃ water bath. Sperm was diluted with IVF medium (Bovine-Oviduct medium; BO) containing 0, 15, 30 and 60 mM nicotinic acid (NA), which were incubated at 39℃, 5% CO2 for 0, 0.5, 1, 2 and 4h. The characteristics of frozen-thawed sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI for outer acrosomal membrane damage and Rhodamine123/PI for mitochondrial integrity using flow cytometry. And the sperm ability was analysed by Coomassie brilliant blue (CBB) staining for acrosome reaction state and Rose bengal staining for abnormality. Acrosome reaction and abnormality were analyzed using a microscope. In results, sperm viability was significantly higer in 30 mM group than 0 and 15 mM groups at 1 and 2 h (p<0.05). Outer acrosomal membrane damage was significantly lower in 30 mM group than 0 and 15 mM groups at 1, 2 and 4 h (p<0.05). And mitochondrial integrity was significantly higher in 30 mM group than 0 and 15 mM groups at 2 and 4 h (p<0.05). Also, acrosome reaction was significantly lower in 30 mM than 0 and 15 mM groups at 1 and 2 h (p<0.05) and abnormality was lower NA groups than 0 group at 1 h (p<0.05). In couclusion, we suggest that using the thawing medium containing NA for sperm dilution can be benefical for IVF in bulls

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

The oocyte undergoes various events during maturation and requires many substances for the maturation process. Various intracellular organelles are also involved in maturation of the oocyte. During the process glucose is essential for nuclear and cytoplasmic maturation, and adenosine triphosphate is needed for reorganization of the organelles and cytoskeleton. If mitochondrial function is lost, several developmental defects in meiotic chromosome segregation and maturation cause fertilization failure. The endoplasmic reticulum, a store for Ca2+, releases Ca2+ into the cytoplasm in response to various cellular signaling molecules. This event stimulates secretion of hormones, growth factors and antioxidants in oocyte during maturation. Also, oocyte nuclear maturation is stimulated by growth factors such as epidermal growth factor. This review summarizes roles of organelles with focus on the Golgi apparatus during maturation in oocyte.

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM (61.1 ± 1.5%, 64.7 ± 2.0%) of nicotinic acid than other groups (0 mM, 52.1 ± 2.3%; 20 mM, 47.8 ± 5.1%, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid (26.1 ± 1.8%, 24.9 ± 1.5%) than other groups (0 mM, 35.3 ± 0.8%; 20 mM, 36.5 ± 1.9%, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM (84.2 ± 3.6%, 88.4 ± 2.3%) of nicotinic acid than other groups (0 mM, 77.3 ± 4.4%; 20 mM, 73.3 ± 3.6%, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM (17.0 ± 1.3%) of nicotinic acid than other groups (0 mM, 9.4 ± 0.5%; 5mM, 12.6 ± 0.8%; 20 mM, 5.0 ± 1.0%, P<0.05). Moreover, total cell number was higher in 5 and 10 mM (53.6 ± 2.9%, 57.9 ± 2.8%) of nicotinic acid than other groups (0 mM, 41.0 ± 1.4%; 20 mM, 23.2 ± 2.8%, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid (0.7 ± 0.1%) than other groups (0 mM, 1.0 ± 0.1%; 10mM, 0.9 ± 0.0%; 20 mM, 1.4 ± 1.0%, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

Luteolysis is a cyclical regression of the corpus luteum in many non-primate mammalian species. Prostaglandin F2α (PGF2α) from the uterus and ovary induces functional and structural luteolysis in bovine. The action of PGF2α is mediated by PGF2α receptor located on the luteal steroidogenic and endothelial cell membranes. PGF2α plays an important role in regulating nitric oxide production in endothelial cells of the bovine corpus luteum. Nitric oxide production and nitric oxide synthase activity are stimulated and induced by PGF2α in luteal endothelial cells. Moreover, the reactive oxygen species inhibits progesterone secretion in bovine luteal cells and induces apoptosis. Thus, the interaction between PGF2α and reactive oxygen species provides important aspects in physiology of the corpus luteum forfunctional and structural luteolysis.

Objective of this study was to investigate the effect of nicotinic acid (NA) on the characteristics in fresh semen of miniature pig. We evaluated viability, acrosome reaction and mitochondrial integrity of sperm on 0, 3, 7 and 10 days during storage period with nicotinic acid. As results, the survival rate of sperm in 15 mM NA (day 3, 87.8 ± 1.2%; day 5, 84.0 ± 2.7%; day 7, 82.2 ± 0.9%) and 30 mM NA (day 3, 87.7 ± 0.3%; day 5, 84.4 ± 2.5%; day 7, 82.3 ± 0.7%) groups were higher than control and 5 mM NA groups in 3, 7 and 10 days of semen storage. The NA-treated sperm on 10 day was used day for observing acrosome integrity. The survival sperm with acrosome reaction was higher in 30 mM NA group (day 3, 2.7 ± 0.2%; day 5, 3.3 ± 0.6%; day 7, 11.4 ± 0.3%) than in the control, significantly (P<0.05). Moreover, the live sperm with mitochondrial integrity was higher in whole treatment groups of NA than control group, significantly (P<0.05). Specially, most mitochondrial integrity on 10 day of semen storage was significantly higher in 30 mM NA group (90.2 ± 1.6%) than other treatment groups (control, 81.8 ± 3.1%; 5 mM NA, 83.4 ± 3.0%; 15 mM NA, 89.1 ± 0.7%, P<0.05). In conclusion, supplement of NA in liquid semen of miniature pig can improve and maintain semen quality, such as viability, acrosome reaction, and mitochondria integrity.

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.

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased (4 ng/μl, 51.24%; 8 ng/μl, 40.88%; and 16 ng/μl; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups (4 ng/μl, 7.96%; 8 ng/μl, 6.4%; and 16 ng/μl; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups (4 ng/μl, 18.4%; 8 ng/μl, 12.5%; and 16 ng/μl; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

L-Carnitine is an antioxidant for the transport of fatty acids in mitochondria and breakdown of lipids for metabolic energy. Some studies have suggested that carnitine improves sperm motility in mammals. The objective of this study was to investigate the effect of L-carnitine on the characteristics in fresh semen of miniature pigs. The collected fresh semen was stored in modena B medium with L-carnitine (0, 1.0, 2.0, and 4.0 mg/ml) for 10 days at 18℃. The semen quality of viability, acrosome reaction and mitochondria integrity was analyzed on 0, 3, 7, and 10 day of semen storage. The percentages of live and dying sperm were not different among treatment groups with different concentrations of L-carnitine during the storage period. In acrosome reaction analysis, when the sperm stored for 7 day, the percentages of live sperm with acrosome reaction were significantly (p<0.05) lower in 1 (9.0±0.9%), 2 (7.6±0.2%) or 4mg/ml (7.9±0.8%) L-carnitine-treated groups than the control group (0 mg/ml L-carnitine) (11.12±0.2%). However, there were no difference in percentages of live sperm with acrosome reaction for 3 and 10 days of storage with each concentrations of L-carnitine. When sperm was stored for 3 and 10 days, the percentages of live sperm with mitochondria integrity were significantly higher in 2 mg/ml of L-carnitine-treated group than control group (p<0.05). In conclusion, the L-carnitine has a positive effect on acrosome reaction and mitochondria integrity in liquid state of fresh semen in miniature pigs.

The objective of this study was to investigate the changes of oxidative stress and antioxidant enzyme during in vitro development with washing culture oil in porcine embryos. During the culture, the four types of culture oil such as paraffin oil with or without washing and mineral oil with or without washing were examined. The oil was washed with PZM-3 during 7 days and collected oil only. The embryos were stained with CellTrackerTM Red, DC-FDA and Hoechst 33342 to confirm the effects of the oil. As a results, Cleavage rates and total cell number were no difference among the four oil groups. However, ≥16 cell embryos were significantly different in fore type oil treat-ment and blastocyst rate was significantly higher washing paraffin treatment than in other group(p<0.05). Also, the expression of free radical were lower in washing paraffin oil than in other groups (p<0.05). On the other hand, the expression of glutathione were not significant different among paraffin oil with or without washing and mineral oil with or without washing, however washing paraffin oil and washing mineral groups were higher than other treat-ment groups. In conclusion, the washing oil was expected with positive effects on in vitro development in porcine embryos.