Blastocyst Total Cell Number Research Articles

266 COMPARISON OF OOCYTES FROM LARGE-SIZED (≥8-mm) and MEDIUM-SIZED (3- to 7-mm) FOLLICLES FOR IN VITRO EMBRYO PRODUCTION IN PIGS

Despite recent efforts to improve culture systems, the developmental competence of in vitro-matured (IVM) porcine oocytes is still inferior compared with those that have been in vivo matured. In pigs, cumulus–oocyte complexes (COC) are usually aspirated from 3- to 7-mm follicles and matured for 42 to 44 h in vitro. In this study, we compared oocytes obtained from large-sized (≥8-mm) and medium-sized (3- to 7-mm) follicles in terms of nuclear maturation, intracellular reduced glutathione levels, gene expression, and embryo development after IVM. In the control group, COC (n = 521) were aspirated from 3- to 7-mm follicles and matured for 22 h with hormones (eCG/hCG) and subsequently matured in vitro for 20 to 22 h without hormones at 39°C, 5% CO2. In the large follicle (LF) group, COC (n = 256) were obtained from follicles larger than 7 mm and were subjected to IVM reduced for 18 h. The maturation medium was TCM-199 supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng mL–1 of epidermal growth factor, 75 µg mL–1 of kanamycin, 1 µg mL–1 of insulin, and 10% (vol/vol) porcine follicular fluid without hormones. Nuclear status and reduced glutathione content in oocytes were investigated by Hoechst 33342 staining and CellTracker Blue (CMF2HC; Invitrogen, Carlsbad, CA, USA), respectively. The abundance of messenger RNA of genes reflecting the developmental competence of oocytes was analysed in cumulus cells by real-time PCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as the reference. In addition, oocytes were subjected to parthenogenetic activation to assess in vitro embryo developmental competence. Data were analysed by Student’s t-test using SPSS version 17.0 (IBM Corporation, Armonk, NY, USA) and presented as means. All experiments were replicated at least three times. The average frequency of ovaries having ≥8-mm follicles was 2.3% (44/1953 in 11 replicates). Before IVM, the nuclear-stage oocytes from ≥8-mm follicles were as follows: germinal vesicle stage = 15.2%; metaphase I (MI) stage = 55.4%; anaphase I and telophase I (AI + TI) stages = 15.8%; and metaphase II (MII) stage = 13.6%. After 6 h of IVM, 4.2% of oocytes were at the germinal vesicle stage and frequencies of the MI, AI + TI, and MII stages were 43.6, 9.4, and 42.8%, respectively. After 18 h, IVM frequencies of the MI and MII stages were 13.0 and 87.0%, respectively. Oocytes of the LF group showed a significant (P < 0.001) increase in intracellular reduced glutathione level (1.41 v. 1.00) compared with the control (42- to 44-h matured oocytes). Cumulus cells in the LF group showed lower (P < 0.1) messenger RNA expression of COX-2 (cyclooxygenase-2) and TNFAIP6 (tumor necrosis factor, α-induced protein 6), and higher (P < 0.1) expression of PCNA (proliferating cell nuclear antigen) and Nrf2 (NF-E2-related factor 2) compared with the control. After parthenogenetic activation, the oocytes from the LF group had significantly (P < 0.05) higher blastocyst rates and total cell numbers in blastocysts than did the control group (90.1% and 73.6 v. 50.5% and 55.3, respectively). In conclusion, oocytes from preovulatory LF require only 18 h to complete maturation in vitro, and their developmental competence is higher than those obtained from MF. Although limited, oocytes from ≥8-mm follicles offer an alternative source of material for the production of transgenic pigs by somatic cell nuclear transfer. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

36 RESTORATION OF TELOMERE LENGTH IN CLONED PIG EMBRYOS DURING EARLY EMBRYOGENESIS IS NOT DEPENDENT ON TELOMERE LENGTH AND TYPE OF DONOR CELLS

Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8 ± 1.5 kb), C (24.4 ± 0.5 kb), P7 (18.7 ± 0.6 kb), and P10 (17.2 ± 0.1 kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2 ± 0.3, 17.8 ± 0.7, 18.5 ± 0.3, and 18.4 ± 0.4 kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3 ± 1.5, 23.5 ± 2.6, 20.2 ± 1.0, and 20.9 ± 1.0 kb, respectively). However, the telomere of the blastocyst was significantly longer (P < 0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2 ± 0.4, 4.0 ± 0.5, 5.1 ± 0.4, and 4.9 ± 0.4, respectively) were significantly lower (P < 0.01) than those of cloned blastocysts in the same groups (8.2 ± 1.1, 8.6 ± 0.6, 12.5 ± 2.9, and 8.3 ± 1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0 ± 5.2, 17.3 ± 2.9, 13.5 ± 2.9, and 13.1 ± 3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3 ± 2.9, 32.6 ± 3.6, 30.4 ± 3.1, and 27.4 ± 2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.

303 THE MODIFIED CULTURE SYSTEM OF PORCINE IN VITRO PRODUCTION IMPROVES COLONIZATION OF PUTATIVE EMBRYONIC STEM CELLS

The establishment of porcine embryonic stem cells (pESC) using in vitro-produced blastocysts derived from IVF, parthenogenesis (PA), and somatic cell nuclear transfer (SCNT) have a great potential. However, porcine in vitro-produced blastocysts show a lower quality than do in vivo-derived blastocysts. In this study, to improve in vitro-derived blastocyst quality, and then to establish pESC, we treated IVF embryos and PA embryos with resveratrol (RES), porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), and β-mercaptoethanol (β-ME). The control system was produced using M199 medium in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The modified group was produced using M199 with 2 µM RES in IVM and PZM5 with 10 ng mL–1 pGM-CSF, 2 µM RES, and 10 µM β-ME in IVC. Data were analysed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test. In total, 1210 PA, 612 IVF, and 5 SCNT embryos were evaluated (for SCNT, we examined only the control system). We observed that overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P < 0.05) in the modified system (54.5%) compared with the control system (43.4%) in PA and hatched blastocysts rates at Day 6 and 7 were also increased significantly. Total blastocyst cell numbers were significantly higher (P < 0.05) in the modified system (55.1) compared with the control system (45.6). Hatching rate of Day 7 IVF blastocysts was also significantly increased. After seeding porcine blastocysts, the attachment rates were higher in the modified system (32.2% in PA and 36.2% in IVF; not examined in SCNT) than the control system (19.5% in PA, 26.6% in IVF, and 40.0% in SCNT). In addition, colonization rates and cell line derivation rates were higher in the modified system than in the control system. Colonization rates of modified system were 13.1% for PA and 17.75% for IVF embryos, whereas the control system generated 2.4% for PA, 10.8% for IVF, and 20.0% for SCNT. We also investigated the correlation between blastocyst state and attachment rate. The highest attachment rate is in hatched blastocyst (78.35 ± 15.74%). Therefore, the modified system increased quality of porcine blastocysts in vitro produced, and subsequently increased attachment rates. The cell line derivation rates were 2.4% (PA), 4.2% (IVF), and 20.0% (SCNT) in control group. In modified system, they were 7.2% (PA) and 10.0% (IVF). We established 3 cell lines from PA blastocysts (1 cell line in control system and 2 cell lines in modified system) and 1 cell line from SCNT-control system. All cell lines showed alkaline phosphatase activity and expressed pluripotent markers. In conclusion, the modified system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of in vitro-produced porcine blastocysts, and subsequently increased derivation rates of putative pESC. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2012004885).

38 GENERATION OF INTERSPECIFIC CLONED BLASTOCYSTS BY ZONA PELLUCIDA-FREE NUCLEAR TRANSFER IN WILD FELIDS

Somatic cell nuclear transfer is an assisted reproductive technique that could help to preserve endangered species. Because it is difficult to obtain wild felid oocytes, interspecific cloning using domestic cat (DC) oocytes is an alternative to produce cloned embryos in these species. The aim of this study was to evaluate different cloning strategies in the DC (Felis silvestris catus) and to use the most efficient strategy to generate wild felid embryos by interspecific cloning. First, we evaluated 3 different cloning strategies: (1) enucleation of DC oocytes with zona pellucida (ZP) followed by fusion of a DC fibroblast that was injected into the perivitelline space (ZP-enclosed group), (2) the same enucleation procedure followed by intracytoplasmic injection of a DC fibroblast (ZPi group), and (3) enucleation of ZP-free oocytes followed by adhesion and fusion of a DC fibroblast (ZP-free group). After 2 h of nuclear reprogramming, the reconstructed embryos were activated with 5 µM ionomycin and 1.9 mM DMAP, and cultured in SOF. The ZP-free embryos were cultured in wells of the well system. Embryo development among treatment groups was compared by the Fisher exact test (P ≤ 0.05). The blastocyst rates were similar among the 3 groups: 11.1% (2/18), 11.1% (5/45), and 12.7% (9/71), for ZP-enclosed, ZPi, and ZP-free, respectively. However, the quantity of reconstructed embryos after the procedure was higher in the ZP-free clones because of a higher fusion rate (82.7 vs. 25.4%) and the use of a less-invasive technique than the injection. Moreover, the percentage of expanded blastocysts was also higher (0, 16.2, and 77.8% for ZP-enclosed, ZPi, and ZP-free, respectively). Parthenogenetic controls, with and without ZP, did not differ in blastocyst rates: 47.7% (42/88) and 49.4% (38/77), respectively. After this, the ZP-free strategy was chosen for the successive experiment. In experiment 2, the wild felid species selected for interspecific cloning were Bengal (a hybrid between Felis silvestris and Prionailurus bengalensis; FP group), cheetah (Acinonyx jubatus; AJ group), and tiger (Panthera tigris; PT group). The morula and blastocyst rates were higher in the FP group: 34.3% (36/105), 16.2% (16/99), and 17.5% (11/63) for morulae, and 33.3% (35/105), 1% (1/99), and 3.2% (2/63) for blastocysts of FP, AJ, and PT, respectively. Additionally, total cell number and the expression pattern of octamer-binding transcription factor 4 (Oct-4) were examined in the blastocysts by immunocytochemistry. The mean total cell number in DC, FP, AJ, and PT blastocysts was 177.9 ± 53, 229 ± 40, 53, and 41, respectively. All blastocysts expressed Oct-4 but in different proportions. The percentage of cells expressing Oct-4 in DC, FP, AJ, and PT blastocysts was 48, 66, 100, and 98%, respectively. In summary, ZP-free cloning was found to be an efficient technique in DC, with potential to be used in wild felid species. We also demonstrated that DC oocytes were able to reprogram cells of other genera. This is the first report of felid ZP-free cloning and also the first time that tiger and cheetah embryos were produced by interspecific cloning.

158 EFFECTS OF AMINO ACIDS IN EMBRYO TRANSPORT MEDIA ON PORCINE EMBRYOS

Due to the distance from the laboratory to the recipient farm, several laboratories, including ours, carry somatic cell nuclear transfer (SCNT)-derived porcine embryos to the farm using a portable incubator for a few hours. If the embryos are nourished well during the transport, viability of embryos might be increased and cloning efficiency can be improved. TALP, which is widely used as a porcine embryo transport medium, lacks amino acids (AA). Proper supply of AA in the uterus is important for the development of pre-implantation embryos because AA have functions as osmolytes, metabolic regulators, or substrates and buffers of intracellular pH. Thus, supplementation of AA could affect the embryonic viability during the transport of SCNT-derived porcine embryos. The aim of this study is to determine whether the transport medium containing AAs affects the in vitro development of parthenogenetic embryos compared to TALP. Porcine zygote medium-5 (PZM-5) was chosen as transport medium containing AA due to its similarity in constituents with TALP except for the AA. Because PZM-5 contains sodium bicarbonate as a buffer system which can not cover wide variation of pH, 10 mM HEPES was added into PZM-5 (PZM+H) as it was normally done with TALP. Porcine cumulus–oocyte complexes (COC) were collected from ovaries of slaughtered pigs and cultured for 44 h using a two-step culture protocol. After denuded, matured oocytes were activated by thimerosal for 10 min followed by dithiothreitol for 30 min. The parthenogenetic embryos were cultured in PZM-5 for 2 days, monitored for cleavage, and loaded in a straw with TALP or PZM+H, respectively. Embryos were stored in a portable incubator (MTG, Bruckberg, Germany; no CO2) at 37°C for three hours and moved to PZM-5 drop for additional 5 days culture. The development was monitored on Day 7 after activation and blastocysts (BL) were collected for total cell number counts and RNA extraction. Ten BL from the TALP group and 11 BL from the PZM+H group were stained with 10 µg mL–1 bisbenzimide (Hoechst 33342) and were visualized for cell counting under fluorescence microscopy. Messenger RNA was extracted from 7 BL of the TALP and PZM+H groups and cDNA were synthesized. Quantitative real-time PCR were done to detect expression levels of apoptosis-related genes using the cDNA. The Bax/Bcl2 ratio was investigated as expression level of apoptosis-related genes and GAPDH was used as control. Each experiment was repeated at least 3 times. Data were analyzed by paired Student’s t-test using Graphpad Prism (version 5, Graphpad Software Inc., La Jolla, CA, USA). No difference was observed between the TALP and PZM+H groups with respect to blastocyst formation rate (22.46 ± 1.47% and 23.17 ± 2.13%, respectively) and total cell number (32.9 ± 2.22 and 37.09 ± 2.18, respectively). There was no significant difference between groups in the Bax/Bcl2 ratio. The use of PZM-5 media, which contains AA, did not affect the development and apoptosis of parthenogenetic embryos. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

136 EFFECTS OF INSULIN-LIKE GROWTH FACTOR I (IGF-1) ON THE DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION BUFFALO (BUBALUS BUBALIS) EMBRYOS

The aim of this study was to explore the effects of insulin-like growth factor-I (IGF-1) on the development and apoptosis of preimplantation buffalo (Bubalus bubalis) embryos derived from IVF or somatic cell nuclear transfer (SCNT) in order to improve the quality of in vitro embryo culture (IVC). Buffalo oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% FBS) for 22–24 h, and fertilized in vitro, or enucleated and reconstructed for SCNT. Embryos were then cultured in the culture medium (CM: TCM-199 + 3% FBS) supplemented with different concentrations of IGF-1. Blastocyst development was evaluated after 7 days of culture. A total of 1566 oocytes were used in this study. The experimental data were analyzed using two-way ANOVA, P < 0.05 was considered to be statistically significant. The results showed that the cleavage rates of IVF or SCNT embryos cultured with 0, 10, 50, or 100 ng mL–1 IGF-I, were not significantly different (P > 0.05). However, the blastocyst rate of IVF embryos cultured with 50 ng mL–1 IGF-1 was significantly increased compared to the 0 ng mL–1 group (35.1 v. 23.0%; P < 0.05), but not significantly different among the 0, 10, and 100 ng mL–1 groups (23.0 v. 28.2 and 26.5%; P > 0.05). In the same line, more SCNT embryos could develop to the blastocyst stage when cultured in the CM supplemented with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (32.3 v. 20.2%; P < 0.05), but the blastocyst development decreased with 100 ng mL–1 (32.3 v. 21.4%; P < 0.05). Apoptosis and total cell number (TCN) of IVF/SCNT blastocysts were respectively detected by TUNEL or Hoechst 33342 staining. By comparison with the 0 ng mL–1 group, the TCN of IVF/SCNT blastocysts was significantly increased (IVF: 91.7 ± 6.9 v. 108.7 ± 3.9, SCNT: 76.3 ± 5.6 v. 92.8 ± 3.9; P < 0.05) and the apoptotic index was obviously decreased (IVF: 3.9 ± 0.7 v. 2.5 ± 0.7; 7.2 ± 0.5 v. 2.9 ± 0.5; P < 0.05) when the embryos were cultured in the CM with 50 ng mL–1 IGF-I. The result of RT-qPCR analysis showed that the mRNA expression level of the anti-apoptotic bcl-2 gene was distinctly enhanced, while the mRNA expression level of the pro-apoptotic bax gene was remarkably reduced in IVF/SCNT embryos cultured with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (P < 0.05). These results demonstrated that supplementing CM with 50 ng mL–1 IGF-1 could improve the developmental competence of buffalo embryos, increase the TCN of blastocysts and decrease their apoptotic index, probably by down-regulating the mRNA level of pro-apoptotic bax gene and up-regulating the mRNA level of anti-apoptotic bcl-2 gene. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus–oocyte complexes (COCs; n=4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24h with 0 or 10μM forskolin, achieving a 2×2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n=469) or kept fresh (n=546). Fresh and vitrified-warmed blastocysts were cultured for 24h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P<0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P<0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals.

Regulation of sheep oocyte maturation using cAMP modulators

Physical removal of mammalian cumulus-oocyte complexes (COCs) from ovarian follicles results in spontaneous resumption of meiosis, largely because of a decrease in cAMP concentrations, causing asynchrony between cytoplasmic and nuclear maturation and decreased oocyte developmental competence. The aim of this study was to modulate cAMP concentrations within ovine COCs to delay spontaneous nuclear maturation and improve developmental competence. Abattoir-derived sheep COCs were cultured for 2 hours (pre-IVM) in 100 μM forskolin (FSK) plus 500 μM 3-isobutyl-1-methylxanthine (IBMX). Pre-IVM (100 μM FSK and 500 μM IBMX) culture increased COC cAMP concentrations 10-fold compared with controls (P < 0.05). With regard to nuclear maturation, with FSK and IBMX and/or with FSH and cilostamide delayed completion of meiosis (metaphase II) by 3 to 4 hours compared with standard IVM (FSH-stimulated induction of meiosis). In this study, pre-IVM (with FSK and IBMX) followed by IVM (with FSH and cilostamide), increased ovine COC cAMP concentrations and delayed, but did not inhibit, completion of nuclear maturation. This did not affect embryo development rates, but increased total cell number of blastocysts compared with IVM with FSH alone (103 ± 6 vs. 66 ± 4 cells, respectively; mean ± SEM; P < 0.05). We inferred that regulation of ovine oocyte cAMP concentrations during IVM improved embryo quality compared with embryos produced by standard IVM methods.

Oxamflatin Improves Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

Abstract Aberrant epigenetic nuclear reprogramming of somatic nuclei is a major cause of low success in cloning. It has been demonstrated that treatment of histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos by alteration of epigenetic status. The aim of the present study was to investigate the effect of oxamflatin, a novel HDACi, on the developmental competence of porcine SCNT embryos. Treatment with 1 μM oxamflatin for 9 h after activation of SCNT embryos increased both in vitro and in vivo developmental competence. Treatment of SCNT embryos with 1 μM oxamflatin significantly increased blastocyst rate and total cell number in blastocysts (33.3±6.0 and 73.1±1.6, respectively) than that of controls (10.3±3.7 and 54.1±3.5, respectively) or scriptaid (16.4±4.6 and 64.4±2.1, respectively). Moreover, oxamflatin showed significant higher overall cloning efficiency from 0.9% to 3.2%, whereas scriptaid demonstrated 0% to 1.8%. In conclusion, these results indicate that oxamflatin treatment improves the developmental competence of porcine SCNT embryos.

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1–3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1–3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.

Birth of cloned calves from vitrified–warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; P<0.01). The total cell number of BNF-derived blastocysts was significantly higher (P<0.01) than that of BFF-derived blastocysts, which, in turn, was higher (P<0.01) than that of BAF-derived blastocysts. Following transfer of vitrified-warmed blastocysts to recipients, no pregnancy was obtained with fresh (n=8) or vitrified-warmed (n=18) BAF-derived blastocysts, whereas transfer of fresh BNF- (n=53) and BFF-derived (n=32) blastocysts resulted in four and three pregnancies, respectively, which aborted within 90 days of gestation. The transfer of vitrified-warmed BNF-derived blastocysts (n=39) resulted in the live birth of a calf weighing 41kg, which is now 23 months old and has no apparent abnormality, whereas the transfer of vitrified-warmed BFF-derived blastocysts (n=18) resulted in one live birth of a calf that died within 6h. These results demonstrate that cloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

Effects of lipid polarisation on survival of in vivo-derived porcine zygotes vitrified by the superfine open pulled-straw method

This study aimed to evaluate the post-warming in vitro viability of intact porcine zygotes vitrified using the superfine open pulled-straw (SOPS) method and to investigate whether cryotolerance is increased by lipid polarisation before vitrification. In vivo-derived zygotes (n=317) were either untreated before SOPS vitrification or subjected to one of the following pre-treatments: (1) centrifugation (20 min, 15000 g) or (2) equilibration in high-osmolality medium (6 min, 400 mOsm kg(-1)) followed by centrifugation. Vitrified-warmed and non-vitrified fresh zygotes were cultured in vitro for 120 h. There were no differences in the blastocyst formation rates between the vitrification groups (from 35.4±5.3% to 48.2±5.6%), but fresh zygotes exhibited higher (P<0.001) blastocyst formation rates (87.5±5.3%) than did vitrified-warmed zygotes. The total blastocyst cell number was similar among all groups (from 34.9±2.8 to 44.1±2.8). In conclusion, SOPS vitrification is a promising method for the cryopreservation of untreated in vivo-derived porcine zygotes. Neither lipid polarisation by centrifugation nor exposure to a high-osmolality medium followed by centrifugation affected the post-warming in vitro viability of zygotes. Our study also demonstrated that the donor is an important factor in determining the success of vitrification for in vivo-derived porcine zygotes.

Alanine and Glycine During the Cleavage Stages Are Detrimental to IVM/IVF Mouse Embryo Development.

Traditionally, amino acids are added to embryo culture media in a premixed formulation developed for somatic cells; MEM essential (EAA) and non-essential (NEAA) amino acids. Previous work has suggested that essential amino acids are inhibitory when present in the first step of embryo culture, and thus are generally absent. However, both EAA and NEAA are present in oviductal fluid. Our objective was to evaluate the effect of lower concentrations of EAA, as well as higher, physiological concentrations of alanine (0.5 mM, ALA) and glycine (2.0 mM, GLY), two of the most abundant NEAA in oviductal fluid, on mouse embryo development in vitro. Zygotes were produced following 18 hours in vitro maturation in a defined, in house maturation medium and 6 hours in vitro fertilization in HTF, using B6D2F1 gametes. All defined media contained 2.5 mg/mL recombinant human albumin (G-MM Recombumin, Vitrolife, Göteborg, Sweden). Embryo cleavage was recorded 22 hours post insemination, and blastocyst development examined on day 4 (96 hours) and day 5 (114 hours) of culture. In Experiment 1 (n=597), 4 treatments were examined: 1) Control (G1 48 hours, G2 66 hours; Vitrolife); 2) G1 with 0.25X EAA, ALA and GLY followed by G2; 3) G1 followed by G2 with ALA and GLY; and 4) G1 with 0.25X EAA, ALA and GLY followed by G2 with ALA and GLY. Neither embryo cleavage nor day 4 blastocyst development (control, 68.4 ± 3.8; G1+EAA+ALA+GLY/G2, 62.9 ± 4.1; G1/G2+ALA+GLY, 72.5 ± 3.6; G1+EAA+ALA+GLY/G2+ALA+GLY, 61.1 ± 4.0) was significantly affected by treatment. Inclusion of ALA, GLY and EAA in G1 decreased day 4 hatching blastocysts (control, 20.4 ± 3.3; G1+EAA+ALA+GLY/G2, 4.9 ± 1.8; G1/G2+ALA+GLY, 22.9 ± 3.4; G1+EAA+ALA+GLY/G2+ALA+GLY, 34.2 ± 3.9; p<0.01), and tended to decrease day 5 hatching blastocysts (control, 52.0 ± 4.1; G1+EAA+ALA+GLY/G2, 38.5± 4.1; G1/G2+ALA+GLY, 51.2 ± 4.1; G1+EAA+ALA+GLY/G2+ALA+GLY, 45.6 ± 4.1; p<0.07). In Experiment 2 (n=911), we further examined specific AA in step 1 of culture (0 to 48 hours) using a defined in house culture medium: 1) Control (1X NEAA, 0.5 mM alanyl-glutamine, 0.1 mM taurine); 2) 0.5x EAA; 3) ALA and GLY; 4) Methionine (0.05 mM, METH). All embryos were then cultured in defined second step medium (48 to 114 hours, also prepared in house) with ALA and GLY. When present during the first 48 hours of culture, ALA and GLY decreased day 4 hatching blastocysts (control, 39.4 ± 3.3; EAA, 48.9 ± 3.3; ALA+GLY, 32.2 ± 3.1; METH, 48.9 ± 3.3:p=0.03), although embryonic cleavage, blastocyst development (control, 72.1 ± 3.0; EAA, 76.7 ± 2.8; ALA+GLY, 66.5 ± 3.1; METH, 72.7 ± 2.9), day 5 hatching blastocysts, total blastocyst cell number (control, 94.5 ± 7.3; EAA, 83.6 ± 5.0; ALA+GLY, 79.3 ± 5.5; METH, 82.5 ± 5.4) or percentage ICM of total cells were not affected. Addition of 0.5X EAA or methionine alone to the first step of culture did not significantly improve development; neither was it inhibitory. In summary, exposure to physiological concentrations of alanine (0.5 mM) and glycine (2.0 mM) during the early cleavage stages of in vitro embryo culture slows development and inhibits the onset of hatching in mouse IVM/IVF embryos.

Development of the SCNT Embryo from Butyrolactone I Prematured Bovine Oocytes

The somatic cell nuclear transfer (SCNT) technique has been applied successfully in a range of mammalian species giving rise to offspring, however, the efficiency of development to term has remained low. Oocyte meiotic arrest is an important option to make the SCNT more flexible and increase the number of cloned embryos produced. This paper showed that the use of butyrolactone I to arrest the meiotic division for 24 h prior to in vitro maturation (IVM) provided bovine oocytes capability of supporting development of blastocysts as efficiently as non arrested oocytes. In the SCNT group, cleavage rates were not affected by prematuration when adding butyrolactone I during IVM (57.9% vs. 62.4%; control vs. 10 μmol · L−1 butyrolactone I), developmental rates to blastocyst were unaffected (22.0% vs. 20.3%; control vs. 10 μmol · L−1 butyrolactone I, p>0.05) by the addition of butyrolactone I during IVM in cloned embryos. Moreover, the total cell numbers of blastocyst were not affected by butyrolactone I (total nucleus numbers, 132±16.5 vs. 128±19.4, p>0.05). In conclusion, the SCNT embryos from butyrolactone I- prematured bovine oocytes had the same developmental potential as the non treated one. The present study provided a method for laboratories of in vitro embryos production, mainly those working on the SCNT, where prematuration could be used to increase the flexibility of the procedure.

Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.

Lacking expression of paternally-expressed gene confirms the failure of syngamy after intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis)

The objectives were to: (1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; and (2) compare the subsequent development of embryos after activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group). Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P < 0.05). However, because 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation, and IVF groups. To clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using reverse transcription polymerase chain reaction (RT-PCR). Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. Although blastocyst development after ICSI combined with chemical activation was similar to IVF oocytes, these blastocysts were generated by parthenogenesis, due to failure of male pronucleus formation.

Porcine Granulocyte-Macrophage Colony-Stimulating Factor Improves the &lt;i&gt;in vitro&lt;/i&gt; Development of Cloned Porcine Embryos

We examined the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) for the first time. We evaluated the effects of pGM-CSF on SCNT-derived blastocyst formation and investigated gene expression. A total of 522 cloned embryos in 6 replicates were treated with 10 ng/ml pGM-CSF during in vitro culture (IVC). This treatment significantly (P<0.05) increased blastocyst formation and total cell number in blastocysts compared with the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no effect on cleavage rate. The numbers of cells in the inner cell mass and trophectoderm were significantly higher in the pGM-CSF treatment group (6.0 and 43.0, respectively) compared with the control (4.4 and 31.9, respectively). Treatment with 10 ng/ml pGM-CSF significantly increased POU5F1 and Cdx2 mRNA expression in blastocysts. In addition, Bcl-2, Dnmt1 and proliferating cell nuclear antigen (PCNA) mRNA expression were upregulated in blastocysts in the pGM-CSF supplemented group compared with the control. These results suggest that pGM-CSF improves the quality and developmental viability of porcine SCNT embryos by regulating transcription factor expression.

Mycotoxin Alpha-Zearalenol Impairs the Quality of Preimplantation Porcine Embryos

Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17β-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 µM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 µM of α-ZEA groups. Only the 100 µM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 µM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding.

96 GRANULOCYTE–MACROPHAGE COLONY-STIMULATING FACTOR INCREASES DEVELOPMENTAL POTENTIAL OF PORCINE EMBRYOS IN VITRO

There have been significant improvements in the culture of porcine embryos in vitro; however, it is still suboptimal. Improvements in porcine embryo culture would benefit utilisation of porcine embryos for a variety of purposes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be expressed in the female reproductive tract and the level of its expression is high between conception and implantation. Previous studies show supplementing GM-CSF in embryo culture promotes embryonic development in human and bovine embryos. The aim of this study was to investigate the effect of GM-CSF on the culture of porcine embryos derived from somatic cell nuclear transfer (SCNT) and IVF. Different concentrations of recombinant porcine GM-CSF (0, 2, 10 ng mL–1) were introduced into Porcine Zygote Medium 3 from Day 1 to 6. Frequencies of cleaved embryos and blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell number in blastocysts from each group were counted and compared by using the Student's t-test. Differences at P &lt; 0.05 were considered significant. A total of 563 SCNT embryos from 6 different donor cell lines on 11 different days were produced for the study. Incubation of SCNT embryos with GM-CSF did not affect the frequency of cleaved embryos. Frequencies of cleaved embryos in control (0 ng mL–1), 2 ng mL–1 GM-CSF and 10 ng mL–1 GM-CSF were 64.2%, 68.1% and 65.0%, respectively. Interestingly, both concentrations of GM-CSF significantly increased the frequency of blastocyst formation as compared with the control. In 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, 30.8% and 32.3% of embryos reached blastocyst respectively, whereas only 22.4% of embryos reached blastocyst in the control group. A significant increase in total cell number in blastocysts was observed when GM-CSF was introduced into embryo culture. An average of 28.8 ± 0.9 cells was recorded in the control group, whereas 31.9 ± 1.1 and 31.8 ± 1.1 were observed in 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, respectively. Similar effects were observed when GM-CSF was introduced to the culture of IVF embryos. For IVF study, 525 embryos were generated on 10 different days and embryos cultured in the presence of GM-CSF tended to show higher blastocyst formation (P = 0.1). Frequencies of blastocyst per cleaved in the 3 groups were 55.7% (control), 65.7% (2 ng mL–1 GM-CSF) and 66.7% (10 ng mL–1 GM-CSF). In addition, culture of IVF embryos with GM-CSF significantly increased total cell number in Day 6 blastocysts. Total cell number in blastocysts in 2 ng mL–1 GM-CSF (34.2 ± 0.8) and 10 ng mL–1 GM-CSF (34.4 ± 1.2) were significantly higher compared with control (27.3 ± 1.2). Our results indicate that introducing GM-CSF into embryo culture media can increase the quality of blastocyst stage embryos. An increase in the frequency of blastocyst formation and total cell number in blastocysts suggests that GM-CSF can be used to produce better-quality embryos in vitro. Currently, effects of GM-CSF on implantation of SCNT embryos are under investigation. Further studies would elucidate the specific mechanism of GM-CSF on porcine embryos.