Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E2, 17b-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17b-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptor (EGFR). These compounds are localised in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role of gangliosides in porcine embryos is not yet clearly understood. Therefore, in this study, the relationship between ganglioside (GD1a) and EGFR activation was investigated during meiotic maturation and further development in porcine oocytes. Porcine oocytes were cultured in the NCSU-23 medium supplemented with or without EGF and GD1a for 44 h. Assessment of meiotic maturation was performed by using aceto-orcein staining method. Also, protein levels in matured oocytes were evaluated by Western blot analysis. After fertilization, presumptive zygotes were cultured in the PZM-3 medium for 6 days. All experiments were repeated more than 3 times. Data were analysed by using Student's t-test and Duncan's multiple range test. The proportion of GV arrested oocytes at 22 h was significantly different between the EGF + GD1a-treated and untreated group (41.6 ± 1.5 v. 25.0 ± 0.0%; P < 0.05). After completion of meiotic maturation (44 h), the proportion of MII was significantly different between the EGF + GD1a-treated and untreated group (89.9 ± 3.6 v. 57.4 ± 5.3%; P < 0.05). Also, expression of EGFR protein in matured porcine oocytes was increased in the presence of EGF and GD1a. After IVF, the percentage of penetrated oocytes was significantly higher in the EGF + GD1a-treated group (89.1 ± 2.3%), resulting in higher than normal pronucleus formation (2PN of 43.1 ± 5.2%). Finally, in result, differences in preimplantation developmental potential were detected between the oocytes that were matured with or without EGF and GD1a (50.4 ± 6.1 v. 27.2 ± 2.7%; P < 0.05). These results suggest that GD1a improves the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by EGFR activation.

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

The In Vitro production of porcine embryos was essential to increase of blastocyst development rate and select of high quality blastocyst in early stage. There were a lot of reports about In Vitro porcine embryo development, but there was no report about the selection of high quality embryos. Therefore, in this study, we investigated the effect of vitamin K1 (vit K1) on the development and survival rate of porcine In Vitro fertilized embryos. When vit K1 was treated for 24 hr at day 1 In Vitro culture, blastocyst development rate in the control group (35.5 ± 3.2%) was significantly lower compared to 1.0 μM, 3.0 μM, or 6.0 μM groups (14.5 ± 4.3, 0.0, or 0.0%; p<0.05). The survival rates of blastocysts at day 8 in 1.0 μM, 3.0 μM or 6.0 μM of vit K1 treated groups (22.2 ± 2.9, 0.0 or 0.0%) were significantly lower than that of the control group (31.8 ± 2.6%; p<0.05). We were added at 1.0 μM, 3.0 μM or 6.0 μM vit K1 for different durations of time at day 1 In Vitro culture. The development rate and survival rate in the group of 1.0 μM vit K1 for 6 hr was 26.5 ± 2.9% and 47.2 ± 2.8%, respectively, which were differed significantly in the group of 12 hr (p<0.05). In the group of 3.0 μM vit K1, the blastocyst development in control group was 36.4 ± 3.1% but, the survival rate 41.7 ± 3.2% in the group of 3.0 hr was significantly higher than that of the control group (p<0.05). In the group of 6.0 μM vit K1, the control group’s the blastocyst development was 32.0 ± 2.8% and the 0.5 hr supplement group’s survival rates was 42.9 ± 1.8% higher than other groups. We added vit K1 at day 1, day 2, day 4 and day 6 of In Vitro culture, on the based the results of supplemented concentration and duration. In the group of 1.0 μM 6.0 hr addition, the blastocyst development rate of day 4 and the survival rate of day 2 were the highest in each group. In the groups of 3.0 μM 3.0 hr addition or 6.0 μM 0.5 hr addition, the blastocyst development (59.5 ± 4.1% and 50.0 ± 3.6%) and survival rates (72.7 ± 5.4% and 79.2 ± 4.0%) on day 4 were significantly higher than that of control and other experiment groups (p<0.05). Meanwhile, the number of cells in blastocysts that produced by vit K1 supplementation was 53.4 ± 5.8, 49.4 ± 3.8 and 51.5 ± 4.5 respectively, which were significantly higher than that of 40.2 ± 2.3 in the control group (p<0.05). There was no difference of the number of apoptotic cells between control and experiment groups. In addition, gene expression of survival blastocyst, the Bax mRNA expression was similar between the control and the experiment groups. However, Bcl-xL mRNA expression’s in the group of 6.0 μM 0.5 hr on day 4 was highest among control and experiment groups (p<0.05). In this study suggested that the control of concentration, duration and time was effective on the survival and cell number of porcine blastocyst derived from In Vitro. We are not know what the exact reasons of the effect of vit K1 on embryo development and need to fur ther study. However, vit K1 might be using the selection of high quality porcine blastocyst

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

In conventional in vitro embryo production, oocytes are obtained from various ovaries, and donor characteristics, such as pedigree type, meat quality, or meat quantity, are not considered. The aim of this study was to examine the effects of pedigree type and meat quality and quantity of slaughterhouse donor cows on oocyte recovery, embryo development, and pregnancy rates. The ovaries of individual Korean native cows were obtained from a slaughterhouse. The ear tag numbers were obtained 24 h after slaughter, and the donor data were compared according to the pedigree type (no registration to third bloodline registration), meat quantity according to the percentage of carcass weight (more than 69%, 66 to 69%, and below 66%; Grades A, B, and C, respectively), and meat quality according to the marbling grade (marbling grade 8 to 9, 6 to 7, 4 to 5, 2 to 3, and 1; Grades 1++, 1+, 1, 2, and 3, respectively). There were 390 donors in total, and each group had 30 donors. Recipients (Holstein heifers; n = 222) were synchronized with a progesterone device, and a single blastocyst was transferred nonsurgically on Day 7 from the onset of oestrus. Data on embryo development were compared by Duncan’s multiple range tests, and pregnancy rate was analysed by chi-square test. Values of P < 0.05 were considered to indicate a significant difference. No significant difference was detected in the average number of oocytes recovered (17.5 ± 2.9 to 22.9 ± 1.2%) or blastocyst development (16.4 ± 2.6 to 19.5 ± 3.4%) from cows of different pedigrees. A higher number of oocytes were recovered from Grade A (22.9 ± 1.0) than from Grade B (19.8 ± 0.7; P < 0.05) cows. However, the meat quantity grade had no significant effect on blastocyst development (14.3 ± 1.2 to 15.0 ± 0.8%). A greater number of oocytes were recovered from the Grade 1++ (25.0 ± 1.6), 1+ (27.7 ± 2.4), and 2 (23.6 ± 1.5) groups than from the Grade 3 group (19.7 ± 1.0; P < 0.05). The developmental rate to the blastocyst stage was significantly higher in the Grades 2 and 3 groups (22.1 ± 2.2 and 19.4 ± 1.7) than in the Grade 1++, 1+, and 1 groups (9.0 ± 1.2 to 13.8 ± 2.0). Pregnancy rate was not affected by pedigree type, meat quantity, or meat quality (41.7 to 58.6%). We concluded that 1) pedigree type had no effect on oocyte recovery and embryo development; 2) meat quantity affected oocyte recovery but did not affect embryo development; 3) high meat quality (Grades 1++ and 1+) resulted in greater oocyte recovery but lower embryo development; and 4) pregnancy rate remained unaffected by pedigree type, meat quantity, and meat quality. This research was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MTT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG (2 μM) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification and slow freezing of bovine blastocysts on the survival rate and apoptosis of frozen-thawed embryos. Bovine blastocysts were vitrified and slow-freezed after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. The shrunken (Group A) and not shrunken (Group B) vitrified embryos were exposed to cryoprotectant 15% ethylene glycol-15% DMSO and 0.5 M sucrose for 1min, and the shrunken (Group C) and not shrunken (Group D) slow-freezed embryos were exposed to cryoprotectant 1.5 M ethylene glycol and 0.1 M sucrose for 5 min. Blastocysts (Group A and B) were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. Also blastocysts (Group C and D) were loaded 0.25 ml plastic straws and transferred into a controlled rate freezer and plunging into liquid nitrogen for storage. Then, after thawing, blastocysts derived from Group A and B were diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min, and blastocysts derived from Group C and D were washed three times each in Hepes buffered TCM-199 for 5min, respectively. After thawing, differences in survival and hatching rates of blastocysts derived from shrunken and not shrunken embryos were found in both vitrification (Group A vs B: 95.3±4.3 vs 81.1±7.1% for survival rate, 72.8±10.3 vs 58.9±8.3% for hatching rate P<0.05) and slow freezing (Group C vs D: 89.6±7.6 vs 53.1±12.4% for survival rate, 41.6±9.7 vs 21.4±12.9% for hatching rate, P<0.05) groups. Furthermore, we also found that the total cell numbers of blastocysts in artificial shrinkage groups were increased (Vitrification: 130.5±2.6 vs 113.7±3.5, Slow freezing: 116.7±2.4 vs 101.5±4.3, P<0.05) and the numbers of blastomeres undergoing apoptosis in artificial shrinkage groups were decreased (Vitrification: 8.5±1.5 vs 14.5±1.4, P<0.05; Slow freezing: 13.4±1.0 vs 14.3±2.6). Consistent with the results, qPCR data showed that the expression of anti-apoptotic Bcl-xl gene was greatly increased by shrunken groups, whereas the expression of pro-apoptotic Bax was decreased. Our results showed that survival and qualities in the both vitrified and slow-freezed bovine blastocysts could be improved by reducing the fluid content after artificial shrinkage. Therefore, we suggest that artificial shrinkage with pulled pasteur pipet is an effective pretreatment technique for the both vitrification and slow freezing methods of bovine blastocysts. (poster)

Oviduct fluid contains various cytokines and growth factors that enhance the embryo development during the preimplantation period. In hatched embryos, prostacyclin (PGI(2)) improves implantation, but its role during oocyte maturation and early embryo development remains contentious. Therefore, in the present study, we examined the effects of a PGI(2) analogue (iloprost) on meiotic maturation and early embryonic development in pigs, as well on the structural integrity, mitochondrial membrane potential and apoptosis in blastocysts. First, meiotic maturation in pig oocytes was examined in the presence of increasing concentrations of iloprost (1, 5 and 10 muM). After IVM, a higher proportion of iloprost-treated compared with untreated oocytes was in MII (90.0% v. 65.7%, respectively; P < 0.05). In addition, protein kinase A activity increased in iloprost-treated oocytes, indicating increased intracellular cAMP concentrations. After 22 h iloprost treatment (44 h total incubation time), western blotting demonstrated increased expression of extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (p-) ERK1/2, cAMP response element-binding protein (CREB), p-CREB and cyclo-oxygenase-2, indicating activation of the mitogen-activated protein kinase and PGI(2) pathways. In addition, the frequency of polyspermy decreased in iloprost-treated oocytes (19.9%) compared with control (35.8%), whereas the rate of blastocyst formation increased (P < 0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) showed that the number of nuclei containing fragmented DNA at the blastocyst stage decreased in the iloprost-treated group compared with control (1.2% v. 3.6%, respectively). In conclusion, iloprost appears to play a direct role in porcine oocyte maturation by enhancing blastocyst structure and survival.

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. in vitro cryopreservation ot pronuclear embryos was carried out by slow Ireezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1±2.7 and 17.0±3.1∼22.0±3.2, respectively; hence, the slow freezing group (10.2±2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8±12.6, was significantly higher than in the slow freezing group, 41.6±11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9±13.6 and 81.8+14.1, respectively, and were significantly higher than for the slow freezing group (51.8±12.6; p<0.05).

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro., there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts.

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than-on any other day, and was significantly higher than on day 0 or day I (p < 0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA ->(day-4) PZM-3+10%FBS; 3) PZM-3+0.3% BSA -> PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p < 0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at 37.8 +/- 16.1 than the other groups at 16.8 +/- 4.4 - 30.1 +/- 10.9; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.