Significant Differences In Cleavage Rates Research Articles

Cleavage and in vitro cultivation rates monitoring in culture media supplemented with energy sources, non-essential amino acids, and antioxidants in the buffalo embryos

AbstractThe study was designed to monitor the cleavage rate (CR) and in-vitro cultivation rate (IVC) after addition of energy sources, non-essential amino acids, and antioxidants to the Synthetic oviductal fluid (SOF) and FertiCult. After in-vitro maturation and in-vitro fertilization, presumptive zygotes were cultured in one of two culture media: FertiCult media and SOF medium, supplemented with pyruvate, glucose, and sodium lactate as energy sources, as well as 10, 20, 250, 500, and 750 mg non-essential amino acids, and antioxidants. All stages of cleavage rate (CR), and in-vitro cultivation rate (IVC) of embryonic development including morula stage (MOR) and blastocyst (BLAS) have been assessed. The findings revealed that there were no significant differences in the CR between the control and other treated groups with sources of energy when added to SOF media (P > 0.05), while there were significant differences (P < 0.05) in the IVC of embryonic development between groups (The percentages of MOR stage in the control, pyruvate, glucose and mixture of source of energy (MIX) were at 50%, 62.5%, 60%, and 63.6%, respectively). The highest percentage of the BLAS was recorded after SOF supplementation with glucose (40%). Similarly, there were no significant differences (P > 0.05) in the CR between control and FertiCult supplemented with sources of energy, while the IVC stages increased significantly (P < 0.05) in the FertiCult media supplemented with glucose, pyruvate, sodium lactate, and MIX. The percentages of the MOR stage in the control, pyruvate, glucose and mix media were at 50%, 55.6%, 55.6%, 54.5%, 57.1% respectively. The lowest percentage of the BLAS was recorded after FertiCult supplementation with pyruvate (11.1%). Replenishing the SOF maturation media with 20 mg of non-essential amino acids significantly (P < 0.05) enhanced the MOR stage (100%). There was also an improvement in the development of BLAS stage, where it reached 31.2% and 47.4% in the SOF maturation media supplemented with 10, and 750 mg non-essential amino acids, respectively. There were no significant differences (P > 0.05) in neither CR nor IVC between control and FertiCult supplemented with antioxidants. There were significant differences (P < 0.05) in the MOR stages (control, 42.9% & treated, 57.9%) and BLAS stages (control, 21.4% & treated, 42.1%) in antioxidant supplemented SOF maturation media compared to control. In conclusion, supplementation of SOF cultivation medium with energy sources, 20 mg of non-essential amino acids and antioxidant addition may improve the cleavage rate (CR) and in vitro cultivation rate (IVC) of buffalos’ embryonic development.

Effects of hepatitis B virus infection on the treatment outcomes following in vitro fertilization/intracytoplasmic sperm injection: An analysis of 21,999 first embryo transfer cycles.

To investigate the effects of hepatitis B virus (HBV) infection on the outcomes of Chinese couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the clinical data of their neonates. A total of 21,999 first embryo transfer cycles were included. They were categorized into four groups based on the couple's hepatitis B surface antigen (HBsAg) result (Group A = female HBsAg- and male HBsAg- ; Group B = female HBsAg+ and male HBsAg- ; Group C = female HBsAg- and male HBsAg+ ; Group D = female HBsAg+ and male HBsAg+ ). The fertilization rate (FR), cleavage rate (CR), implantation rate (IPR), clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate (MCR) were analysed. Multilevel logistic regression was applied to evaluate the association. The total prevalence of HBV infection was 5.74% (2526/43998). There were no statistically significant differences in CRs (98.69%, 98.76%, 98.66%, 98.72%, p > .05), IPRs (45.86%, 47.33%, 45.19%, 39.61%, p > .05), CPRs (62.84%, 65.05%, 61.80%, 56.81%, p > .05), MCRs (12.70%, 11.99%, 12.58%, 4%, p > .05) and LBRs (53.43%, 55.38%, 52.70%, 54.54%, p > .05) among the four groups. However, there were significant differences in FRs (66.25%, 66.55%, 66.32%, 61.92%, p < .05). Group D had the lowest FR. After adjusting for confounders, the multilevel logistic regression showed that HBsAg+ had no impact on the LBR, CPR or MCR. We also analysed the data of 14,465 newborns, including 8593 singletons and 2936 twins. Among the four groups, no variables reached statistical significance, including neonatal birth weight (NBW), twin ratio, gestational age, premature birth, delivery type, fetal macrosomia or low birth weight (p > .05). Our study demonstrates that, although biparental HBV infection may affect the FR, neither single-parent infection nor biparental HBV infection affects IVF/ICSI outcomes or neonatal outcomes.

Ramelteon Reduces Oxidative Stress by Maintenance of Lipid Homeostasis in Porcine Oocytes.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10−11 and 10−9 M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10−11, 10−9, and 10−7 M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10−11 and 10−9 M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10−11 M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10−11 M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10−11 M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10−11 M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10−11 M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

Influence of the sperm DNA fragmentation index on the outcome of rescue ICSI and the clinical value of rescue ICSI.

As a remedy for the failure of in vitro fertilization (IVF), rescue intracytoplasmic sperm injection (R-ICSI) has been widely carried out, but it has failed to significantly improve the fertilization rate and clinical pregnancy rate. Sperm DNA fragmentation index (DFI) was highly correlated with pregnancy outcome of artificial assisted reproduction. This study aims to investigate the effect of the sperm DFI on the outcome of R-ICSI and the clinical value of R-ICSI. This retrospective analysis was conducted among 140 infertile couples receiving R-ICSI in from January 2014 to December 2019. The subjects were assigned into a total fertilization failure (TFF)+low DFI group (R-ICSI after TFF and DFI<30%) (n=63), a TFF+high DFI group (R-ICSI after TFF and DFI≥30%) (n=16), a partial fertilization failure (PFF)+low DFI group (R-ICSI after PFF and DFI<30%) (n=52), a PFF+high DFI group (R-ICSI after PFF and DFI≥30%) (n=9). All transferred embryos were come from R-ICSI. The general clinical data [infertility duration, male age, female age, basal serum level of follicle stimulating hormone (FSH), basal serum level of luteinizing hormone (LH), antral follicle count, endometrial thickness of human chorionic gonadotropin (HCG) day, and eggs] and R-ICSI cycle outcomes (fertilization rate, normal fertilization rate, cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate and live birth rate) were analyzed. In addition, the effect of R-ICSI on the fertilization outcome of conventional IVF total fertilization failure and partial fertilization failure was explored. There was no significant difference in the general clinical data and R-ICSI cycle outcome between the TFF+low DFI group and the TFF+high DFI group (all P>0.05). There was no significant difference in the general clinical data between the PFF+low DFI group and the PFF+high DFI group (all P>0.05). The fertilization rate and normal fertilization rate in the PFF+low DFI group were significantly higher than those in the PFF+high DFI group (85.40% vs 72.41%, 71.90% vs 58.62%, respectively; both P<0.05). However, there was no significant difference in cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate, and live birth rate between the 2 groups (all P>0.05). The R-ICSI cycle of TFF: A total of 79 fresh cycles, 57 fresh transplant cycles, a total of 761 unfertilized oocytes, and 584 M II oocytes were treated with R-ICSI, the fertilization rate was 83.22%, the normal fertilization rate was 75.51%, the cleavage rate was 98.15%, the good embryo rate was 40.74%, the implantation rate was 30.56%, and the clinical pregnancy rate was 43.86%; 29 live births were obtained. The R-ICSI cycle of PFF: A total of 61 fresh cycles, 31 fresh transplant cycles, a total of 721 unfertilized oocytes, and 546 M II oocytes were treated with R-ICSI; the fertilization rate was 83.33%, the normal fertilization rate was 69.78%, the cleavage rate was 97.36%, the good embryo rate was 44.39%, the implantation rate was 25.42%, and the clinical pregnancy rate was 45.16%; 12 live births were obtained. In the case of partial fertilization failure of IVF, the sperm DFI affects the fertilization rate and normal fertilization rate of R-ICSI; whether it is a TFF of IVF or PFF of IVF, ICSI can be used as an effective remedy way.

Retrospective analysis on the effect of duration of ejaculatory abstinence on fertilization, embryo development, and pregnancy rates in patients undergoing conventional in vitro fertilization

Aim: This study aims to find out how different sexual abstinence period influences semen parameters, fertilization, embryo development, and pregnancy rates in patients undergoing conventional in vitro fertilization. Setting and designs: A retrospective analysis was conducted at a tertiary level infertility care clinic. Materials and methods: The clinical study included 532 patients between January 2017 and July 2021 who had been treated with conventional in vitro fertilization. The effect of sexual abstinence on primary outcomes such as semen volume, total sperm concentration, total motile sperm concentration, sperm morphology, and secondary outcomes such as fertilization rate, cleavage rate, top quality day 3 and day 5 embryo development rate, implantation and clinical pregnancy rate between fresh versus frozen embryo transfer on day 3 and day 5 of conventional in vitro fertilization under three groups, that is, sexual abstinence of less than or equal to 1, 2 to 5, and 6 to 7 days was evaluated. Statistical analysis used: Chi-square test was used to evaluate the qualitative variables whereas one-way analysis of variance was used to evaluate the quantitative variables. Results: Group II with 2 to 5 days of abstinence, observed a positive correlation between abstinence period and semen volume, total sperm concentration, total motile sperm concentration, and morphology. Fertilization rate was higher in groups II and III than group I (P = 0.21) with no significant difference in cleavage rate (P = 0.4) and embryo development on day 3 (P = 0.1057). The formation of AA grade blastocyst between group I and group II was 27.54 % and 23.47 %, respectively, with a P-value of 0.007154. Both fresh and frozen embryo transfers of groups II and III of abstinence period had implantation and clinical pregnancy rates of 23.39% and 43.83%, respectively. Conclusion: Our study did not find any significant difference in primary and secondary outcomes of conventional in vitro fertilization between the three groups. We recommend further prospective large randomized control studies to prove any association between duration of abstinence and pregnancy outcomes.

123 Efficiency of embryo production using ovum pickup oocytes recovered from stimulated and nonstimulated cows

Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (ovum pickup, OPU) is an essential element of modern livestock development. To increase the number of OPU oocytes retrieved, hormonal stimulation is broadly applied; however, the results are ambiguous. The aim of the present study was to compare recovery rate, quality of oocytes, and efficiency of invitro production of cattle embryos using OPU oocytes derived from hormonal treated and untreated cows. The study was performed in Simmental heifers at the age of 17 to 23 months. The heifers in the first group (n=7) were previously synchronized using prostaglandin F2α (Estrumate, MSD Animal Health) and gonadotrophin-releasing hormone (GnRH; Receptal, MSD Animal Health) and then underwent superstimulation with Stimofol (Reprobiol SPRL, Belgium) in a total amount of 0.25mg of FSH and 0.05mg of LH in a treatment of 6 injections on 3 consecutive days starting on Day −4 before OPU. The heifers of the second group (n=2) underwent OPU once a week for 5 weeks (10 OPU sessions). Groups of recovered cumulus–oocyte complexes (COCs) were matured in TCM-199 containing 10% fetal calf serum, 1mM Na-pyruvate, 50μg mL−1 gentamycin, 10μg mL−1 FSH, and 10μg mL−1 LH for 24h, fertilized in Fert-TALP (Tyrode’s-albumin-lactate-pyruvate) for 18 to 20h, and cultured in CR1aa medium for 7 days. All steps of IVP were performed at 38.5°C with 5% CO2 and 90% humidity. The cleavage and blastocysts rates were assessed on Day 2 and 7, respectively. In addition, Day-7 blastocysts were fixed, and the total cell number was determined using 4′,6-diamidino-2-phenylindole (DAPI) staining. The data were analysed by ANOVA using SigmaStat software package. As expected, the number of aspirated follicles per session in the first group of cows was higher than that in the second group: 14.6±1.2 vs. 6.8±0.5 (P&amp;lt;0.05). In total, 54 and 42 COCs were collected, which corresponds to recovery rates of 54.5±7.7 and 61.3±4.9% for stimulated and nonstimulated cows, respectively. After the quality evaluation, 36 COCs of SS heifers (67.2±1.9%) and 31 COCs of non-SS heifers (75.3±5.0%) were selected for IVP. We did not observe a significant difference in cleavage rate between two groups: 77.8% in the SS group vs. 71.0% in the non-SS group, whereas the rate of blastocyst production was higher (P&amp;lt;0.05) for SS heifers (25.0%) compared with non-SS animals (16.1%). The total number of cells in Day 7 blastocysts was significantly (P&amp;lt;0.01) higher in the first group (89.8±1.3) compared with the second group (70.6±3.2). Thus, superstimulation of heifers before OPU increases the efficiency of oocyte retrieval by ultrasound-guided transvaginal follicular aspiration as well as the developmental competence of OPU-derived bovine oocytes invitro. These studies were performed under financial support of the Russian Science Foundation (project No. 19-16-00115) and the Ministry of Science and Higher Education of Russia.

Epigenetic Alteration of Donor Cells with Histone Deacetylase Inhibitor m-Carboxycinnamic Acid Bishydroxymide Improves the In Vitro Developmental Competence of Buffalo (Bubalus bubalis) Cloned Embryos.

Epigenetic reprogramming is an indispensable process during the course of mammalian development, but aberrant in cloned embryos. The aim of this study was to examine the effect of donor cell treatment with histone deacetylase (HDAC) inhibitor m-carboxycinnamic acid bishydroxymide (CBHA) on cloned embryo development and establish its optimal concentration. Different concentrations of CBHA (2.5, 5.0, 10.0, and 20.0 μM) were used to treat buffalo adult fibroblast cells for 24 hours and effect on cell proliferation, gene expression, and histone modifications was analyzed. Based on these experiments, the best concentration was chosen to determine the effect of enhanced gene activation mark on developmental rates. Among the different concentrations, CBHA at higher concentration (20 μM) shows the sign of apoptosis and stress as indicated by proliferation rate and gene expression data. CBHA treatment significantly decreased the activity of HDACs and increased the level of gene activation mark H3K9ac and H3K4me3, but could not alter the level of H3K27ac. Based on these experiments, 5 μM CBHA was chosen for treatment of donor cells used for the production of cloned embryos. There was no significant difference in cleavage rate between the control and CBHA treatment group (98.5% ± 1.5% vs. 99.0% ± 1.0%), whereas, blastocyst rate markedly improved (46.65% ± 1.94% vs. 57.18% ± 2.68%). The level of H3K9ac and H3K27me3 did not differ significantly in cloned blastocyst produced from either control or CBHA-treated cells. Altogether, these results suggested that donor cell treatment with CBHA supports the reprogramming process and improves the cloned preimplantation development.

Suberoylanilide hydroxamic acid during in vitro culture improves development of dog-pig interspecies cloned embryos but not dog cloned embryos.

This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.

IMPROVEMENT OF BUFFALO EMBRYO DEVELOPMENT IN VITRO; EFFECT OF DIFFERENT CULTURE MEDIA

The aim of the present study to compare 4 different types of media, SOF, TCM-119, CR1aa and KSOM, for in vitro culture of buffalo embryo. This study was under taken in the Department of Artificial insemination and embryo transfer, Animal Reproduction Research Institute (ARRI), Al-Haram, Giza .The selec&#116;ed cumulus oocyte complexes(COCs) with a compact cumulus cells and evenly granulated ooplasm were in vitro matured at 38.5°C in atmosphere of 5% CO2 with high humidity .After 24h of culture, oocyte were used for in vitro fertilization . Following 18h of insemination, oocyte were randomly assigned to investigate the effect of different types of media from the zygote to day 7 post-insemination . there were no significant difference in cleavage rate among 4 media However, the morula and blastocyst stage increased significantly(P<0.05) in using SOF (26.03, 17.88% respectively) and also by using CR1aa (25.37, 16.42% respectively) compared with TCM (11.86, 5.08% respectively) and KSOM (9.62, 3.85 %respectively).There is no significant difference between SOF and CR1aa.In conclusion, the current data reveled that SOF is the best media used in embryo development in buffalo. Key words: SOF. In vitro culture media. Buffalo

Decrease in bovine in vitro embryo production efficiency during winter season in a warm-summer Mediterranean climate

Retrospective analysis of monthly embryo production from December 2011 to May 2015 and its correlation with meteorological data in our geographic zone was made. We had observed that in certain time of the year, in vitro blastocyst production decreases. Accordingly, was examined the association between blastocyst production and climatological parameters. Cleavage rates correlate positively with blastocyst rates (p<.05). Significant differences in cleavage rates between autumn and summer (79.8%; 71.5%), and between winter and autumn (71.8%; 79.8%), were found. Blastocyst production had lower efficiency in June (9±12%) and July (4.9±5.7%), which coincides with winter season. In contrast, higher embryo production was obtained in February (22.2±9.7%), March (22.9±14%) and September (25.2±6.6%), which coincides with autumn and spring season. Similarly, embryo production correlates with meteorological parameters: blastocyst production positively correlates with sunshine hours, maximum temperature and average temperature. Similarly, blastocyst production inversely correlates with total precipitation and days >1mm precipitation (p<.05). There is a significant decrease in bovine in vitro embryo production efficiency during winter season in our warm-summer Mediterranean climate zone. It remains to be investigated the direct effect of environmental factors on oocyte quality and its impact on in vitro production efficiency.

Influence of Epidermal Growth Factor with Cysteamine on in -Vitro Buffalo Embryo Development

FOR IMPROVING embryo development in buffalo, two experiments were conducted. The first one was carried out to evaluate the different concentrations (0, 5, 25, 50 ng/ml) of epidermal growth factors (EGF) on developmental competence of buffalo oocytes. The selected oocytes were cultured in the four concentrations of EGF. The embryo cleavage rate was significantly higher in oocytes exposed to 5, 25, 50 ng/ml EGF than control. There were no significant difference in four groups (0, 5, 25, 50 ng/ml EGF) in the rate of morula. But, better cleavage and blastocyst rates were observed at 5 ng/ml EGF. In the second experiment, the additive effect of 5 ng/ml EGF with 50 µM cysteamine on maturation and embryo development was studied. Oocytes were collected, matured and cultured in three groups. In the first group, media supplemented with 5 ng/ml EGF + 50 µM cysteamine combination. In the second group, media supplemented with EGF. The third group was supplemented with cysteamine. There was a significant increase in cleavage rate in combination group than EGF (P< 0.05) and cysteamine (P< 0.01) groups. But there was no significant difference in cleavage rate between EGF and cysteamine. The morula percentage was nearly similar in the three groups. But blastocyst rate was significantly (P<0.05) higher in combination group than cysteamine. Thus better cleavage and blastocyst rate were observed at combination group. It is concluded that, the addition of 5 ng/ml EGF in buffalo oocytes during in vitro culture was the best concentration for embryo developmental competence. Higher cleavage and blastocyst rate were achieved by combination of epidermal growth factor and antioxidant.

Developmental competence and cryotolerance of caprine parthenogenetic embryos cultured in chemically defined media

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 μM, Ca2+ ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.

Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

235 DEVELOPMENT OF IN VIVO-MATURED OOCYTES COLLECTED FROM JAPANESE BLACK CATTLE STIMULATED WITH DIFFERENT DURATIONS OF FOLLICULAR GROWTH

We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter &gt;8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles &gt;5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 106 sperm mL–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P &lt; 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P &lt; 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.

27 EFFECT OF A SPECIFIC INHIBITOR OF DOT1L ON PREIMPLANTATION DEVELOPMENT OF PORCINE SOMATIC CLONED EMBRYOS

EPZ004777 (EPZ), a specific inhibitor of DOT1L (a methyltransferase of H3K79), can significantly improve the generation and quality of mouse induced pluripotent stem cells [Onder et al. 2012 Nature 483(7391), 598–602), suggesting that H3K79 dimethylation (H3K79me2) is involved in controlling cell pluripotency. To date, however, it is unclear whether H3K79me2 regulates development competency of animal cloned embryos. Thus, we aimed to examine the dynamic changes of H3K79me2 in pre-implantation cloned embryos of pigs, and to explore effect of EPZ treatment of embryos on in vitro development fate in order to lay the foundation for revealing the role of H3K79me2 and mechanisms in controlling cell pluripotency. Porcine cloned embryos were treated immediately when fusion and activation stimuli were conducted, in vitro with porcine zygote medium (PZM)-3, including 0.5, 5, or 50 nM EPZ (S7353, Selleck Chemicals, Houston, TX, USA) and 1‰ DMSO (vol/vol, control group) for 24 h, respectively. Then, they were transferred into fresh PZM-3 without EPZ. We found that there was no significant difference in cleavage rate among groups, whereas the blastocyst rate of 0.5 nM EPZ group was higher than that of control group [28.97 ± 2.65% (28/96) v. 17.13 ± 2.69% (17/96)]. No obvious difference was observed for the total cell number of blastocyst among groups. We further treated the SCNT embryos with 0.5 nM EPZ for 0 (control group), 12, 24, and 36 h, respectively. No significant differences were found for cleavage rate among groups, whereas the blastocyst rates of the 12- and 24-h groups were significantly higher than that of control and 36-h groups [28.56 ± 3.51% (27/97), 28.34 ± 3.00% (25/88) v. 16.32 ± 1.93% (16/97), 17.93 ± 0.64% (18/100)]. Except for the remarkable decrease in the 36-h treatment group, no obvious difference was observed for the total cell number of blastocyst among the other 3 groups. All the above experiments were repeated at least 3 times. These results suggested that treatment of porcine SCNT embryos with 0.5 nM EPZ for 12 to 24 h could improve their development during the early stage. Then, we tested whether the EPZ favoured the in vitro development of porcine SCNT embryos by regulating H3K79me2 reprogramming. Porcine SCNT embryos were treated with 0.5 nM EPZ from the onset of electric activation and fusion stimuli was performed, and then the H3K79me2 signal (by immune-fluorescent staining) and expression of DOT1L (by RT-qPCR) at different development stages was analysed. We found that the H3K79me2 signal in control group (without EPZ treatment) decreased slowly from the time of electric stimulation to 4 hpa, and it disappeared in 8 hpa stage. In the EPZ treatment group, H3K79me2 signal started decreasing from 2 hpa, and disappeared in 8 hpa stage. The mRNA level of DOT1L in EPZ treatment group was lower than that in control group, although the difference was not significant. Taken together, treatment with EPZ at the appropriate concentration and for an appropriate time can improve the early in vitro development of pig SCNT embryos, probably by inhibiting expression of DOT1L and facilitating reprogramming of H3K79me2.Research was supported by NSFC No. 31272442.

295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P &lt; 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P &lt; 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P &lt; 0.05). Total cell numbers of BL were significantly higher (P &lt; 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

Supplementation of zinc on oocyte in vitro maturation improves preimplatation embryonic development in pigs

We investigated the effects of zinc supplementation during the IVM of porcine oocytes. Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, subsequent embryonic development, and gene expression were evaluated. Zinc concentrations in porcine plasma and follicular fluid were 0.82 and 0.84 μg/mL, respectively. Zinc was not detected in IVM medium. After treatment with various zinc concentrations (0.0, 0.4, 0.8, 1.2, and 1.6 μg/mL), no significant difference in IVM was observed among groups (85.7%, 88.7%, 90.4%, 90.3%, and 87.2%, respectively). The effects of different zinc concentrations on porcine oocyte intracellular GSH and ROS levels were examined in mature oocytes. Intracellular GSH levels were significantly higher in the 0.8-, 1.2-, and 1.6-μg/mL groups than in the control (P < 0.05). Intracellular ROS levels of oocytes matured with 0.8, 1.2, and 1.6 μg/mL were reduced significantly (P < 0.05) compared with the control and 0.4-μg/mL groups. The developmental competence of oocytes matured with different zinc concentrations was evaluated after parthenogenetic activation (PA) and in vitro fertilization (IVF). Oocytes treated with zinc during IVM showed no significant difference in cleavage rate after PA. Oocytes treated with 0.8 and 1.2 μg/mL zinc during IVM had significantly higher blastocyst formation rates after PA (41.5% and 41.1%, respectively) than the control (27.2%). IVF embryos showed similar results. The blastocyst formation rate was significantly higher (28.2%) in the 0.8-μg/mL group. TNFAIP2 and Bax were decreased in zinc-treated cumulus cells. Increased POU5F1 and decreased Bax transcript levels were observed in zinc-treated oocytes. POU5F1 and Bcl-2 transcript levels were significantly higher in zinc-treated IVF blastocysts. These results indicate that treatment with adequate zinc concentrations during IVM improved the developmental potential of porcine embryos by regulating the intracellular GSH concentration, the ROS level, and transcription factor expression.

Effect of vitrification and slow freezing on in-vitro matured prepubertal goat oocytes

The present study was conducted to study the post thaw recovery rate of morphologically normal matured oocytes subjected to vitrification and slow freezing method. Oocytes were matured in TCM-199, which comprises sodium pyruvate (0.25 mM), L- glutamine (1 mg/10 ml), gentamycin (50 μg/ml) and supplemented with 10% fetal bovine serum at 38.5°C in CO2 incubator under humidified air. After 27 h of in-vitro maturation oocytes were assigned for freezing, viz. vitrification, slow cooling and control cooling. The percentage of oocytes recovered after thawing was significantly higher for control than those for vitrification (69.67%) and slow freezing (85.00%), respectively. Moreover, the cleavage rate in vitrification, slow freezing and control were 1.14, 3.92 and 13.37%, respectively. Thus, the cleavage rate of oocytes in vitrification and slow freezing method did not differ significantly. However, there was a significant difference in cleavage rates of freezing groups and the control groups.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytesin vitro

The beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10-6 to 10-12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10-12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

Viability Selection of Bovine Oocyte Using Annexin-V Assay

Morphological characteristic is the primary reference for selecting viable cumulus-oocyte-complex (COCs). Annexin-V assay has been suggested for isolating these viable cells based on apoptosis status. This study compared fertilization rate between Annexin-V selected COCs with morphologically healthy selected COCs. Minimal binding time between Annexin-V and the COCs was determined to ensure slight disruption. Before observation with florescent microscope, eighty-two cattle’s COCs were incubated with the Annexin-V assay for 5 min, 15 min and 24 hours. Further, 157 COCs were separated into 2 groups; positively (n=76) and negatively (n=81) tagged with annexin-V-FITC and PI fluorescent signals. Once separated, control comprised of non-stained COC (n=68) and the two afore mentioned groups were matured, fertilized and cultured (IVM-IVF-IVC). After 72 hpi, cleavage rate was determined. Results shows the binding between annexin-V assay and the nonviable COC had occurred as early as 5 min with positively and negatively tagged COCs were 44 (53.7%) and 38 (46.3%) respectively. The fertilization rate of control group (66%), negatively tagged (79%) and positively (50%) tagged COCs was significantly different (?² (2) = 14.60, p < 0.05) between the groups. In conclusion, oocyte selection using annexin-V-FITC had significant differences in cleavage rate compared with oocytes assumptive as morphologically healthy. Based on COCs Annexin-V assay, COC that are assumed as morphologically healthy is a mixture of viable and nonviable oocytes.