The immunological sperm separation method is economical compared to the existing sorting method, and it is promising for the development of new technologies by reducing sperm damage. Wholemom (WM) is a sex-regulating protein that comprises on immunoglobulin G coupled with magnetic nanoparticles (MNPs) that responds to surface proteins derived from the Y chromosome in cattle. Y sperms are restricted in motility as the WM aggregates them, and the magnet could separate the non-aggregated cells. This study was conducted to investigate the effect of WM treatment on the characteristics of bull sperm. After treating sperm with WM and incubation for 6 h, the motility parameters including total motility, progressive motility, velocity average path, velocity straight line, amplitude of lateral head displacement, and linearity were significantly higher in the WM treatment group than in the control group (p < 0.05). Sperm viability and acrosome reaction rates were similar in both groups during each incubation period (p > 0.05). In conclusion, the immunological sperm sexing procedure using a monoclonal antibody conjugated with MNPs did not affect the characteristics of bull sperm. This study suggests that compared to other techniques, the immunological method for sperm sexing could classify sperm quickly and efficiently without the use of expensive equipment.

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

The aim of this study was to investigate effect of heat stress on expression levels of plasminogen activators (PAs) related mRNAs and proteins, and changes of PAs activity in porcine endometrial explants. The endometrial explants (200 ± 50 mg) were isolated from middle part of uterine horn at follicular phase (Day 19-21) and were pre-incubated in serum-free culture medium at 38.5oC in 5% CO2 for 18 h. Then, the tissues were transferred into fresh medium and were cultured at different temperature (38.5, 39.5, 40.5 or 41.5oC) for 24 h. The expression level of urokinase-type PA (uPA), type-1 PA inhibitor (PAI-1), type-2 PAI (PAI-2), and heat shock protein-90 (HSP-90) mRNA were analysis by reverse-transcription PCR and proteins were measured by western blotting. The supernatant were used for measurement of PAs activity. In results, mRNA and protein levels of HSP-90 was higher in 41.5oC treatment groups than other treatment groups (p < 0.05). The expression of uPA, PAI-1, and PAI-2 mRNA were slightly increased by heat stress, however, there were no significant difference. Heat stress condition suppressed expression of active uPA and PAI-2 proteins (p < 0.05), whereas PAI-1 protein was increased (p < 0.01). Although PAI-1 protein was increased and active uPA was decreased, PAs activity was greatly enhanced by exposure of heat stress (p < 0.05). These results suggest that heat stress condition could change intrauterine microenvironment through regulation of PAs activity and other factors regarding with activation of PAs might be regulate by heat stress. Therefore, more studies regarding with regulatory mechanism of PAs activation are needed.

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

When sperm penetrates into the ovum, hyaluronidase plays a role of hydrolyzing the hyaluronic acid present in the membrane surrounding the oocytes. The zona pelucida of the ovum is hydrolysed to facilitate sperm entry. Therefore, the aim of this study was to investigate the effects of hyaluronidase during the in vitro maturation in porcine oocytes. The cumulus-oocyte complexes (COCs) were cultured during in vitro maturation (IVM) medium containing 0 and 0.1mg/ml hyaluronidase for 44 h. Representative images of oocytes were captured after cultured for 0 h and 22 h by using a microscope. The area was quantified using a image J software. After 44 h of IVM, nuclear maturation stage was assessed by the aceto-orcein method. In results, cumulus cells expansion was no significant difference between control and hyaluronidase treatment groups in 0 h. However, after 22 h of IVM, in 0.1mg/ml hyaluronidase group, cumulus cells diffusion was significantly reduced than control group (p<0.05). After 22 h matured COCs, the cumulus cells were normally expanded in the control group, but there was a significantly lower 0.1mg/ml hyaluronidase group than control group (p<0.05). The nuclear maturation rate was treated with 0.1mg/ml hyaluronidase, it was significantly decrease than control group (p<0.05). In conclusion, our study indicated that hyaluronidase exposure could reduce nuclear maturation in vitro by reducing the expansion of cumulus cells. According to the results, we conjectured that hyaluronidase treatment disrupted the oocyte maturation by hydrolyzing the hyaluronic acid around the oocytes and it reduces the activity of the intercellular gap junction because it weakens cumulus cell bonds and interferes with communication. However, additional studies on hyaluronidase are needed. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

In our previous study, exogenous plasminasminogen activators (PAs) influenced to fertility of boar spermatozoa via reduction of zona pellucida (ZP) resistance against protease and number of sperm binding ZP. plasminasmin (plasmin), is converted by PAs, is an important enzyme to degrade extracellular matrix and it is closely associated with fertilization process. Therefore, the aim of present study was to confirm changes of sperm penatration and ZP solubility by plasmin during in vitro fertilization (IVF). The cumulus-oocyte complasminexes (COCs) were aspirated from the antral follicles 3-6 mm in diameter and matured for 44 hours. Then, the cumulus cells were removed and denuded oocytes were co-incubated with spermatozoa for 18-20 hours in IVF medium containing 100 ng/ml plasmin. The number of sperm binding ZP and ZP solubility were measured using hoechst 33342 and 0.5% (w/v) pronase, respectively. Aceto-orcein stain was used to assess fertilization parameters. In results, sperm penetration did not affect by plasmin treatment during fertilization. Hoewever, treatment of plasmin decreased monospermic fertilization and IVF efficiency compared with control group (p<0.05). Furthermore, the number of penetrated sperm and pronucleus formation per zygote in plasmin group was significantly increased compared with control group (p<0.05). Despite of reduced monospermic fertilization by plasmin treatment, the number of sperm binding ZP was significantly higher in non-treated zygote than plasmin-treated zygote (p<0.05). Similar with previous study, ZP digestion time was reduced by plasmin treatment (p<0.05). These findings shown that plasminasmin during fertilization enhance the penetration of spermatozoa into ZP via increasing of ZP solubility and it was correspond with our previous results that fertility of spermatozoa during IVF was increased by exogenous urokinase-type PA treatment via sperm-ZP binding and increase of ZP solubility. Therefore, during the fertilization process, plasmin that is converted by PAs from oviduct epithelial cells might be closely associated with degradation of ZP proteins for penetration of sperm. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that intracellular reactive oxygen species (ROS) in mammalian oocytes were reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing 200 μM H2O2 or H2O2 with 50 μM ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress state, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II (MII) stage development was significantly reduced in H2O2 group compared to non-treated control group (61.84±1.42% and 80.00%, respectively; p<0.05) and it was slightly recovered by treatment of ALA (69.76±1.67%; p<0.05). The intracellular GSH levels was decreased in H2O2 groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, H2O2 treatment increased intracellular ROS level in oocytes and H2O2-induced ROS was decreased by treatment of ALA (p<0.05). Our findings suggested that ALA treatment under oxidative stress condition improve oocyte maturation via elevated GSH and reduced ROS levels in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

The oocyte undergoes various events during In vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, In vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, In vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and In vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

The aim of this study was to investigate change of plasminogen activators (PAs) and their inhibitors (PAIs) mRNA and protein expression level by heat stress in porcine endometrial cells. The endometrial epithelial cells were isolated from endometrial epithelium in porcine uterus and cultured in different temperature conditions (38.5 and 41.5℃) for 24 h. Expression of urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor-1 (PAI-1) and -2 (PAI-2) mRNA in epithelial cells were analyzed using reverse transcription-PCR and protein levels were measured by immunofluorescence. In result, mRNA expression of uPA, tPA, PAI-1 and PAI-2 were decreased in 41.5℃ than 38.5℃ culture condition, however, significant differences were no detected. uPA, tPA and PAI-2 protein were mainly expressed in nucleus, whereas PAI-1 was distributed in cytoplasm and nucleus. uPA and tPA protein levels were increased by heat stress treatment and significant difference was only detected in tPA level (p<0.05). In contrast, two types of PAIs protein level were decreased in 41.5℃ cultured group compared with 38.5℃ group. In present study, tPA protein expression was upregulated by heat stress in porcine endometrial cells. This result suggest that change of tPA by heat stress may be related to blood flow into uterus and intrauterine microenvironments, and could directly and indirectly influence to reproductive performance in pigs.

This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

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 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.

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 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.

This study aimed to investigate changes in the activity and mRNA expression of plasminogen activators (PAs) induced by 17β-estradiol (E₂), human chorionic gonadotropin (hCG), and interleukin-1β (IL-1β) in porcine endometrial cells. Endometrial cells were isolated from the epithelium and cultured to 80% confluence. They were then treated for 24 h with E₂ (0.2, 2, 20, and 200 ng/mL), IL-1β (0.1, 1, 10, and 100 ng/mL), and hCG (0.5, 1, 1.5 and 2 IU/mL). mRNA expressions of urokinase-type (uPA) and tissue-type (tPA) PAs were analyzed using reverse transcription PCR, and activities were measured using a PA activity assay. mRNA expressions of uPA and tPA increased with E₂ treatment; however, this was not significant. Similarly, treatment with hCG did not influence the mRNA expressions of PAs. Interestingly, treatment with 0.1 ng/mL IL-1β significantly reduced the mRNA expression of uPA, but did not affect that of tPA. Treatment with 2, 20, and 200 ng/mL E₂ increased PA activity compared with the control group; treatment with 0.1 and 1 ng/mL IL-1β significantly increased PA activity compared with the other IL-1β treatment groups, whereas treatment with 10 and 100 ng/mL IL-1β decreased. Treatment with 2 IU/mL hCG increased PA activity compared with the other treatment groups, although there were no significant differences between the hCG and control groups. In conclusion, the activity and mRNA expression of PAs were differently regulat-ed by the hormone/cytokine and its concentration in porcine endometrial cells. Therefore, understanding PA regulatory mechanisms may help to improve the reproductive potential of domestic animals.