Blastocyst Total Cell Number Research Articles

Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes

The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24 h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10 mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% ( P < 0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC's and DO's developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO's prevented this (31.3%, 12.7% and 29.4% respectively ( P < 0.05)) but had no effect on COC's (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4 ± 5.2, 84.7 ± 3.7 and 80.4 ± 5.8 ( P > 0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy.

Molecular Determinants of Oocyte Competence: Potential Functional Role for Maternal (Oocyte-Derived) Follistatin in Promoting Bovine Early Embryogenesis

Previous studies established a positive relationship between oocyte competence and follistatin mRNA abundance. Herein, we used the bovine model to test the hypothesis that follistatin plays a functional role in regulation of early embryogenesis. Treatment of early embryos with follistatin during in vitro culture (before embryonic genome activation) resulted in a dose-dependent decrease in time to first cleavage, increased numbers of blastocysts, and increased blastocyst total and trophectoderm cell numbers. To determine the requirement of endogenous follistatin for early embryogenesis, follistatin ablation/replacement studies were performed. Microinjection of follistatin small interfering RNA into zygotes reduced follistatin mRNA and protein and was accompanied by a reduction in number of embryos developing to eight- to 16-cell and blastocyst stages and reduced blastocyst total and trophectoderm cell numbers. Effects of follistatin ablation were rescued by culture of follistatin small interfering RNA-injected embryos in the presence of exogenous follistatin. To investigate whether follistatin regulation of early embryogenesis is potentially mediated via inhibition of endogenous activin activity, the effects of treatment of embryos with exogenous activin, SB-431542 (inhibitor of activin, TGF-beta, and nodal type I receptor signaling) and follistatin plus SB-431542 were investigated. Activin treatment mimicked positive effects of follistatin on time to first cleavage and blastocyst development, whereas negative effects of SB-431542 treatment were observed. Stimulatory effects of follistatin on embryogenesis were not blocked by SB-431542 treatment. Results support a functional role for oocyte-derived follistatin in bovine early embryogenesis and suggest that observed effects of follistatin are likely not mediated by classical inhibition of activin activity.

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.

Effect of relaxin and IGF-I on the pre-implantation development of Mongolian gerbil (Meriones unguiculatus) embryos in vitro.

Both relaxin and insulin-like growth factor (IGF) are members of the insulin super family. This study aimed to investigate the effect of relaxin and IGF-I on the pre-implantation of Mongolian gerbil of blastocyst development in vitro. Blastocysts and eight-cell stage embryos were collected from female gerbils. Eight-cell embryos and blastocysts were cultured in mM16 medium supplemented with or without relaxin or IGF-I for 24 h. Blastocysts were counted for total, inner cell mass (ICM) and trophectoderm (TE) cell numbers, and assessed apoptosis incidence. In addition, to measure incorporation of 3H-methionine, blastocysts were cultured for 3 h with relaxin or IGF-I, washed with trichloroacetic acid and measured by liquid scintiration counter. Relaxin (200 ng/ml) increased total, TE and ICM cell numbers of blastocyst (P < 0.05) when it was compared with the control. IGF-I (150 ng/ml) also has influence on total and ICM cell numbers of blastocyst when compared with control. Apoptosis incidence was relatively low, and a significant difference was not observed between each group. The effect of relaxin on incorporation of 3H-methionine was higher than the control group (P < 0.05). Relaxin increased the developmental rate from the eight-cell stage to blastocyst (P < 0.05). In conclusion, relaxin and IGF-I stimulated protein synthesis and increased cell numbers of blastocysts, promoting development of the gerbil embryo in vitro culture.

Effect of all-trans retinol on in vitro development of preimplantation buffalo embryos

Vitamin A is a well-known antioxidant and is essential for embryonic development, growth and differentiation. Oxidative stress is involved in the etiology of defective embryo development. The present study evaluated whether the presence of all-trans retinol (0, 1, 2, 5 and 10 μM) in maturation medium or embryo culture medium would enhance the developmental competence of preimplantation buffalo embryos in vitro. In experiment I, cumulus oocytes complex were matured with varying concentrations of all-trans retinol. Treatment with 5 μM all-trans retinol improved the blastocyst formation (P < 0.001) when compared with control and significant increase (P < 0.01) in total cell number was observed in 5 μM group when compared with control. Supplementation of all-trans retinol in embryo culture medium for the entire culture period under 5% O2 and 20% O2 was tested in experiments II and III, respectively. Supplementation of 10 μM all-trans retinol under 5% O2, significantly reduced blastocyst formation and cell numbers. Presence of 5 μM all-trans retinol under 20% O2 enhanced the frequency of blastocyst formation and total cell number (P < 0.001) when compared with control. DNA damage of individual embryos cultured under 20% oxygen concentration was measured by the comet assay. Supplementation of 5 μM all-trans retinol significantly reduced the comet tail (P < 0.001) when compared with control. Supplementation of all-trans retinol in embryo culture medium for first 72 h of the 8-day culture period under 5% O2 was tested in experiment IV. Addition of 5 μM all-trans retinol resulted in significant increase in blastocyst rate and total cell number (P < 0.001) when compared with control. Our results demonstrate that addition of all-trans retinol to maturation or embryo culture medium may enhance the developmental competence of buffalo embryos in vitro by enhancing blastocyst formation rate and total cell number.

152. INVOLVEMENT OF TRP53 IN SIRT1 FUNCTION DURING EMBRYO DEVELOPMENT

Sirtuin 1 (SIRT1) is an NAD+-dependant deacetylase with significant functions in cell survival and metabolism, including glucose homeostasis and mitochondrial physiology. TRP53 is a universal effector of cellular stress responses and is an important target of SIRT1. Transcriptional activity of TRP53 in the pre-implantation embryo is associated with retarded development, however examination of SIRT1 function and how it relates to TRP53 activity remains to be elucidated. We therefore assessed whether SIRT1 is involved in pre-implantation embryo development and determined whether TRP53 interacts with SIRT1 function. Zygotes were collected from superovulated female mice and cultured to the blastocyst stage in optimised conditions (F1 mouse strain, G1/G2 series sequential media, 5%CO2, 5%O2, 90%N2, group culture) or compromised conditions (C57Bl/6 strain, mHTF static media, 5%CO2 in air, individual culture). Embryo development and blastocyst cell number was assessed following exposure to a SIRT1 inhibitor (0, 1, 10, 100 or 1000µM sirtinol). In subsequent experiments, embryos were cultured in a 2x2 factorial design (±1µM sirtinol and ±30µM pifithrin-α (TRP53 inhibitor)) and embryo development and cell number determined. Sirtinol caused a dose-dependent reduction in total cell number in blastocysts during culture in both optimised and compromised conditions (p&lt;0.05), while the rate of development of zygotes was reduced for embryos in compromised but not optimised conditions (p&lt;0.05). When SIRT1 was inhibited, in the presence or absence of TRP53 inhibition, blastocyst development and cell number for embryos in optimal conditions was unchanged. However, blastocyst development (83% vs 55%, p&lt;0.05), and cell number (39 vs 54 cells, p&lt;0.05) was reduced when SIRT1 was inhibited in compromised embryos, and in the absence of TRP53, development was resolved to control levels. These results show that SIRT1 is important for embryo development, particularly under compromised conditions, and that TRP53 is a likely target for SIRT1 deacetylase activity in the mammalian embryo.

46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P &lt; 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P &lt; 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P &lt; 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P &lt; 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

237 SHORT-TERM TREATMENT OF 6-DMAP AND DEMECOLCINE IMPROVE DEVELOPMENTAL COMPETENCE OF PORCINE PARTHENOGENETIC EMBRYOS

Artificial activation is one of the essential factors for successful development of embryos derived from parthenogenetic activation or somatic cell nuclear transfer (SCNT). This study was carried out to confirm whether the short-term treatment of 6-dimethylaminopurine (6-DMAP) or demecolcine would improve developmental competence of porcine parthenogenetic activated embryos and find out the optimal activation protocol. Ovaries were collected from gilts at a local slaughterhouse and cumulus–oocyte complexes were cultured for 44 h in the tissue culture medium-199 with 10% of porcine follicular fluid. Matured oocytes were chemically activated with 10 min treatment of 0.2 mm thimerosal (Thi) followed by 30 min treatment of 8 mm dithiothrethiol (DTT). Additional treatments of 6-DMAP (2 mm) and/or demecolcine (0.4 μg mL–1) for a short term (40 min) or a long term (3 h) were conducted. Activated embryos were cultured in PZM-3. Cleavage rate, blastocyst (BL) formation rate and total cell number of BL were assessed at Day 2, 7 of culture, respectively. All data were subjected to one-way ANOVA followed by Tukey’s test using Prism version 4.0 (Graphpad Software, San Diego, CA, USA) to determine differences among experimental groups. To begin with, we evaluated that effect of short-term treatment of 6-DMAP or demecolcine. Short-term treatment of 6-DMAP improved developmental competence of activated embryos compared to long-term treatment (47.0 ± 1.0% v. 50.5 ± 1.5% for cleavage rate, 5 ± 3% v. 3 ± 1% for BL formation rate, 70 ± 13 v. 37 ± 6 for total cell numbers). Moreover, short-tem treatment of demecolcine also inhibited second polar body extrusion effectively compared with nontreated control (14 ± 2.1 v. 8.3 ± 4.1%). We confirmed that short-term treatment of 6-DMAP or demecolcine is valid. To optimize activation procedure with short-term treatment, development of embryos derived from each treatment were compared among nontreated control (C), 6-DMAP only treatment (6O), demecolcine only (DO) treatment, and 6-DMAP + demecolcine treatment (6 + D) groups. Cleavage rates of 4 groups were similar (53.5 ± 3.0, 62.1 ± 2.9, 53.6 ± 3.0 and 57.0 ± 3.0%, respectively). However, 6 + D group shows significantly greater BL formation rate (18.0 ± 2.5%) compared with C, 6O, or DO groups (9.4 ± 1.9, 14.0 ± 2.3 and 4.3 ± 1.3%, respectively). Total cell number of BL also increased in 6 + D group (52.8 ± 13.0) compared with C, 6O, or DO groups (47 ± 21.5, 31.5 ± 4.22 and 45.2 ± 13.7, respectively). In conclusion, we elucidated that short-term treatment of 6-DMAP or demecolcine improves developmental competence of embryos compared with long-term treatment. We recommend a combination of Thi/DTT with short-term treatment of both 6-DMAP and demecolcine for optimal activation procedure of porcine embryos. This study was supported by BK21, KOSEF (#M10625030005-07N250300510), HANWHA L&amp;C Corp, and Gyeonggido Veterinary Service.

225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS

Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P &lt; 0.05). The total cell number of blastocysts also increased approximately threefold (P &lt; 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P &lt; 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P &lt; 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

41 PRODUCTION OF CLONED MINIATURE PIGS USING BONE MARROW MESENCHYMAL STEM CELLS

The cloning of miniature pigs (Mini-pig) are considered to advance in genetic engineering technology and xenotransplantation. A few researches have recently been reported successfully produce cloned Mini-pigs using somatic cells, however its efficiency is still low. The present study was aimed to successfully produce cloned Mini-pigs derived from bone marrow mesenchymal stem cells (MSCs) by NT, and compared the developmental ability of cloned Mini-pigs between fetal fibroblast (FF) and differentiated MSCs. For the production of the cloned Mini-pig derived from MSCs, MSCs were isolated from a 1 month old of female Mini-pigs (T-type, PWG Micro-pig®, PWG Genetics Korea, Ltd.). MSCs were differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, FFs of Mini-pig were transferred into enucleated oocytes of domestic pigs, and 2-cell stage of 100 NT embryos were surgical transferred to the synchronized recipients. Statistical analysis was performed using one-way ANOVA, t-test, Duncan’s and Tukey’s multiple comparisons test by SPSS (SPSS, Inc., Chicago, IL, USA). The developmental potential of NT embryos derived from MSCs (differentiated and undifferentiated), the rates of blastocyst formation was significantly (P &lt; 0.05) higher than NT embryos derived from FFs, however the NT embryos derived from three different types of differentiated MSCs were significantly (P &lt; 0.05) lower than undifferentiated MSCs. In addition, total cell numbers in NT blastocysts derived from MSCs were significantly (P &lt; 0.05) higher than NT blastocysts derived from FFs, but it did not significantly (P &lt; 0.05) differ between differentiated or undifferentiated MSCs. NT embryos derived from MSCs were transferred to 5 domestic pig recipients, and 5 cloned Mini-pigs were obtained from 2 recipients (one stillbirth and 4 viable offspring). All of them were confirmed by the microsatellite analysis (8 markers) of the genomes of cloned offspring, donor MSCs and recipients. Physical and histological studies are in the process for the characterization of a cloned Mini-pig derived from MSCs as animal model. The results demonstrated that, in vitro developmental ability of NT embryos derived from undifferentiated MSCs were a higher than those from differentiated MSCs or FFs. Moreover, multipotent MSC might have a potential for the production of viable cloned Mini-pigs. Therefore, MSCs as a nuclear donor might a key to improving the production of cloned Mini-pig as animal model for xenotransplantation. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P &gt; 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P &lt; 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P &gt; 0.05). However, blastocyst rate in G2 (25.0%) was higher (P &lt; 0.05) than in G1 and G3, but was similar to G4 (P &gt; 0.05). Total cell number was similar (P &gt; 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P &lt; 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P &lt; 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P &gt; 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

Stage-dependent Effect of Leptin on Development of Porcine Embryos Derived from Parthenogenetic Activation and Transgenic Somatic Cell Nuclear Transfer

Accumulating evidence suggests that leptin may play important roles in preimplantation embryonic development, although this remain controversial, and little is known about whether leptin has a stage-dependent regulatory effect on development of porcine embryos derived by parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). The objective of this study was to investigate the effects of addition of leptin to in vitro culture (IVC) medium on development of porcine embryos derived by PA and SCNT. We found that addition of 50 ng/ml human recombinant leptin improved the rate of PA embryos reaching the blastocyst stage and increased the total cell number of blastocysts compared with the control group. The maximal blastocyst rate of SCNT embryos was achieved at 50 ng/ml, and the total cell number of blasocysts was increased significantly at 500 ng/ml leptin concentration. However, the ratio of the inner cell mass (ICM) to total cell number was not affected in any of the groups. Supplementation of leptin (50 ng/ml) from day 3, approximately the 4-8-cell stage, as in the case of the positive control, significantly increased the blatocyst rate of PA embryos compared with the negative control and inhibited cell apoptosis. There were no beneficial effects on embryonic development when 50 ng/ml leptin was added to the culture medium from day 1 to day 3 or from day 4 to day 6. These results indicate that leptin could improve the development and the quality of PA and SCNT embryos; and 50 ng/ml leptin performs its primary stimulatory effect at 4-8-cell stage and that leptin may have no effect on the maternal-zygote transition (MZT) of porcine PA and SCNT embryos.

Trichostatin A Improves Histone Acetylation in Bovine Somatic Cell Nuclear Transfer Early Embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.

Anti‐apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

Developmental potential of isolated blastomeres from early mouse embryos in the presence and absence of LIF and GM-CSF

The aim of this study was to investigate the developmental potential of isolated blastomeres in the presence and absence of leukemia inhibitory factor (LIF) and granulocyte-macrophage colony stimulating factor (GM-CSF). The blastomeres of two (1/2) and eight cells (1/8) embryos were isolated and cultured in T6 medium in the presence and absence of LIF (1,000 IU/ml) and or GM-CSF (2 ng/ml) up to 120 h. The diameter and cell number of blastocysts were measured. The developmental rates of 1/2 isolated blastomeres developed to blastocysts stages in the presence and absence of LIF and GM-CSF were 45.80, 35.10 and 48.66, 41.66, respectively. The diameter of blastocysts was higher in GM-CSF group and total cell number of blastocyst in both treated groups was higher than control (P < 0.05). No 1/8 blastomeres developed to morula and blastocyst stages. LIF and GM-CSF could improve the development of 1/2 isolated blastomeres.

Effect of Centrifugation Treatment before Vitrification on the Viability of Porcine Mature Oocytes and Zygotes Produced In Vitro

The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.

Addition of Alpha-Tocopherol to Culture Medium Improves the Quality and Cryosurvival of Nuclear-Transferred Ovine Embryos

The aim of this study was to reconstruct and cryopreserve somatic cell nuclear transferred (SCNT) ovine embryos and evaluate the effect of alpha-tocopherol on blastocyst development and subsequent cryosurvival of the SCNT embryos. The alpha-tocopherol (100 microg/ml) was added into culture medium for the SCNT embryos, the yield and total cell numbers of blastocysts were determined and the apoptosis incidences of the blastocysts were evaluated using the TUNEL assay. The blastocysts from the alpha-tocopherol and untreated groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. The results showed that there were no significant differences in blastocyst yield (26.3 vs. 22.3%) and total cell number (68.2 vs. 64.3) between the alpha-tocopherol and untreated groups. However, addition of alpha-tocopherol to the culture medium significantly decreased the apoptotic cell number (3.4 vs. 5.5) and significantly increased the cryosurvival of SCNT blastocysts (66.8 vs. 50.7%). In conclusion, addition of alpha-tocopherol to SCNT embryo culture medium was beneficial for improving embryo quality by decreasing the apoptotic blastocyst cell number and improving the tolerance of the embryos to cryopreservation.

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.