Mean Cell Number Of Blastocysts Research Articles

Effects of pre-maturational culture duration on developmental competence of bovine small-sized oocytes.

We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

Developmental Competence of Early Stage Porcine Embryos Cultured in Medium with Different Energy Substrate in vitro

To elucidate the effect of energy requirement during the early embryonic development on their developmentalability to develop to blastocyst stage, in vitro fertilized (IVF) porcine one-cell embryos were cultured in modifiedNorth Carolina State University (NCSU)-37 supplemented with different energy substrate. Result indicated that thecleavage rate of embryos in Pyr-Lac and Gluc-Pyr-Lact groups was significantly higher than in those in Gluc groupand Gluc-Rib group (P &lt; 0.05). At Day 6 of culture, the highest proportion of embryos develop to the blastocyst stagewas obtained in the presence of pyruvate-lactate only. In the medium with glucose, the addition of pyruvate-lactateor ribose slightly increased the proportion of embryos develop to the blastocyst stage, however the value were notsignificantly different form those obtained in the presence of glucose only. The mean cell number in blastocystsderived from Pyr-Lac and Gluc-Pyr-Lact groups were significantly higher than those in the Gluc group (P &lt; 0.05).These results indicated that the presence of glucose only, as energy substrate, during the first 2 days of in vitro culture(IVC) caused a decrease in development of in vitro produced (IVP) porcine embryos to the blastocyst stage and meancell number in blastocysts .Keywords: porcine embryos-energy substrate-in vitro culture

The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development

The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5-6 embryos/droplet) showed low blastocyst development compared with a control group (25-26 embryos/droplet). However, small and large numbers of embryos (5-6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25-30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1—simultaneous electrical pulses and activation group (SFA group); and Group 2—electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p<0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p>0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p>0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.

Effects of Caffeine on Maturation-Promoting Factor (MPF) Activity in Bovine Oocytes and on the Development of Somatic Cell Nuclear Transfer Embryos in White-Hanwoo

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII) oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

23 DEVELOPMENTAL POTENTIAL OF CLONED TRANSGENIC PORCINE EMBRYOS PRODUCED BY SERIAL NUCLEAR TRANSFER CAN BE IMPROVED BY TREATMENT WITH HISTONE DEACETYLASE INHIBITORS

Recently, we generated cloned transgenic pigs by nuclear transfer (NT) using fetal fibroblasts transfected with a LEA29Y gene specifically expressed in pancreatic β-cells (INS-LEA). Transfer of 216 NT embryos into 3 recipients resulted in the birth of 9 piglets. Furthermore, we examined serial NT with donor cells of the INS-LEA cloned pigs as a means of propagating the genotype of these valuable animals. Surprisingly, no piglets were obtained after transfer of 512 NT embryos into 5 recipients, which might be due to epigenetic alterations that presumably occurred during post-implantation development of the first round cloned embryos or during nuclear reprogramming in the second round of NT. In this study we tested whether in vitro development of re-cloned embryos can be improved by their treatment with histone deacetylase inhibitors (HDACi), scriptaid and suberoylanilide hydroxamic acid (SAHA). As nuclear donors, ear fibroblast cells derived from the INS-LEA cloned pig were used. Nuclear transfer was performed using in vitro-matured oocytes as previously reported (Kurome et al. 2006, Transgenic Res. 15, 229–240). After activation, reconstructed embryos were treated immediately by scriptaid (500 nM) and SAHA (10 μM) for 16 and 10 h, respectively. Development of NT embryos was assessed by cleavage and blastocyst formation during culture for 7 days. The cell number of blastocysts was also counted after fixation and staining. There was no significant difference in the cleavage rate between treated and non-treated by both HDACi, whereas treatment of NT embryos with scriptaid or SAHA significantly enhanced their development to blastocyst compared with non-treated NT embryos (22.2%, 43/194 and 22.7%, 34/150 vs 7.7%, 15/195 and 12.3%, 18/146, respectively; P &lt; 0.05). Notably, blastocyst rates obtained after treatment of re-cloned embryos with HDACi were similar to those in the first round of NT (21.2%, 33/156). Treatment of NT embryos with HDACi did not increase mean cell number of blastocysts compared with non-treated embryos. The results of our study show that in vitro developmental competence of embryos produced by serial NT can be improved by both HDACi used, scriptaid as well as SAHA, which has not been reported before in pig cloning. To determine the post-implantation developmental potential, re-cloned embryos treated with HDACi will be transferred to surrogate gilts. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung and Mukoviszidose e.V.

Effects of Inflammatory Conditions from Coculture with Activated Macrophages on Mouse Preimplantation Embryonic Development In Vitro.

Inflammatory conditions are involved in important reproductive events of mammals such as, ovulation, menstruation, implantation, labor and delivery. The inflammatory milieu in reproductive organs may have various effects on preimplantation embryonic development. The aim of this study was to evaluate the effects of inflammatory conditioning on mouse preimplantation embryonic development in vitro. To provide an inflammatory condition for preimplantation embryos, 2-cell stage embryos were cocultured with the RAW 264.7 cells, a mouse monocyte/macrophage cell line, which were activated by treatments of lipopolysaccharide (LPS) before coculture. The inflammatory response-like condition by LPS was evaluated and confirmed by RT-PCR for inflammation related genes (IL-1α, IL-6, iNOS, Cox-2, MCP-1 and TNF-α) and ELISA for secreted cytokines (IL-1α, IL-6 and MCP-1). Mouse 2-cell stage embryos were collected from oviduct and cultured in the Quinn's Advantage Blastocyst Medium (QABM) supplemented with 10% serum protein substitute (SPS) as control (CON), or directly cocultured with either intact (INT) or LPS activated (ACT) RAW 264.7 cells in the QABM without SPS. At post-hCG 120 h (72 h after culture), the blastulation rate of ACT (98.1%) was higher than that of CON (89.1%) and INT (94.5%) while there was no statistical significance between them. In addition, the hatching rate of ACT (54.5%) was higher than that of CON (47.3%) and INT (36.4%) without statistical significance. However, the mean cell number of blastocysts in ACT (112.0±6.9) was significantly higher (p<0.01) than those of CON (84.9±6.9) and INT (82.5±4.4), whereas there was no significant difference in apoptotic index among them. To examine whether embryotrophic effects of the inflammatory response-like condition on preimplantation embryos in vitro are induced by direct cell-to-cell contact, 2-cell embryos were indirectly cocultured with RAW 264.7 cells using cell culture inserts, which prevent direct contact between embryos and cocultured cells. Blastulation and hatching rates of ACT (94.1% and 47.1%) were slightly higher than that of CON (84.0% and 32.0%) and INT (90.9% and 32.7%) without statistical significance in indirect coculture system. Furthermore, the mean cell number of blastocysts in ACT (106.2±5.7) was also significantly higher (p<0.01) than those of CON (73.8±5.4) and INT (89.3±6.3) with no difference in apoptotic index. Collectively, this study demonstrates that the inflammatory response-like condition induced by activated RAW 264.7 cells could increase cell proliferation of mouse preimplantation embryos in vitro. Our results suggest that a certain inflammatory conditions could improve developmental competence of mouse preimplantation embryos in vitro by providing embryotrophic cytokines and factors. 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 (2010-0004871). (poster)

63 OOCYTE GENOME CLONING USED IN TRANSGENIC BOVINE EMBRYO PRODUCTION

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P &lt; 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) and 88.5% (108/122) respectively; (P &gt; 0.05). When the haploid activated oocytes were injected with pCX-EGFP–liposome complexes, the cleavage rate was 84.4% (54/64). These embryos showed 54.7% (35/54) of EGFP expression. In the second experiment, haploid parthenogenetic embryos (4 to 16 cells) were disaggregated and the PHB obtained were fused with a zona-free haploid male hemizygotes in order to reconstruct biparental embryos. Haploid male hemizygotes were generated by fertilization of enucleated oocytes. The haploid condition of parthenogenetic embryos and male hemizygotes was confirmed by karyotype analyzes. Reconstructed embryos were cultured in SOF medium in well of well system in 5% O2, 5% CO2, and 90% N2 at 39°C. The development of the reconstructed embryos expressing or not expressing EGFP reached 78.4% (29/37) and 61.1% (80/131) of cleavage; and 10.8% (4/37) and 8.4% (11/131) of blastocysts, respectively; (P &gt; 0.05). EGFP expression was observed in 100% of the reconstructed embryos, with 96.6% (28/29) of homogenic expression. The biparental IVF control showed 64.4% of cleavage and 22.5% of blastocysts (n = 98). The mean cell number of blastocysts were significantly different among the reconstructed biparental embryos (mean = 72.8) and the biparental IVF-embryos control (mean = 97.0). Additionally, the Oct-4 pattern expression was examined in the blastocysts, by inmunocytochemistry. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-blastocyst control. Finally, biparental reconstructed blastocysts (n = 2) were transferred in recipient cows obtaining 1 pregnancy detected at 2 months after embryo transfer. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that it is an efficient technique to generate homogeneous transgene-expressing embryos. Haploid oocyte genome replication would have high potential in livestock production, as it increases the number of embryos produced from a single oocyte, and allows the selection of favourable genetic characteristics before biparental embryo reconstruction.

267 EXPRESSION OF RELAXIN FAMILY PEPTIDE RECEPTORS RXFP1 AND RXFP2 IN PIG PREIMPLANTATION EMBRYOS AND DEVELOPMENTAL EFFECTS OF RELAXIN HORMONE

Relaxin is a polypeptide hormone secreted by male and female reproductive tissues to facilitate spermatozoa progression in the female tract and parturition. Relaxin secretions are found in the vicinity of oocytes and embryos, and exert their effects through membrane receptors, which have not yet been described in porcine embryos. Here, we determined the presence of RXFP1 and RXFP2 receptors in porcine gametes and embryo, and evaluated the developmental effects of porcine relaxin (pRLX; Yan et al. 2006 Reproduction 131, 943-950). Cumulus-oocyte complexes (COC) were aspirated from sows ovaries collected at a local abattoir. Homogeneous COC were selected for IVM (44 h) and fertilization (6 to 8 h). Presumptive zygotes were cultured in NCSU-23 + 0.4% BSA for up to 7 days. All procedures were done at 39°C, under 5% CO2 in a humidified atmosphere. Matured oocytes, BTS-diluted spermatozoa, and embryos were collected for gene expression studies. For developmental studies, COC were matured (experiment 1), or embryos cultured from the zygote stage (experiment 2) in the presence ofpRLX (0, 20, or 40 ng mL-1). In experiment 3, zygotes derived from oocytes matured in the presence of pRLX (40 ng mL-1) were cultured with pRLX (20 or 40 ng mL-1). The pRLX effects were assessed on cleaved embryos and blastocysts recorded on Days 2 and 7 postinsemination, respectively. The total cell numbers of Day-7 blastocysts were also evaluated. All data were analyzed using ANOVA. Gametes and embryos expressed RXFP1 and RXFP2 at both the mRNA and protein level. The amounts of both gene transcripts were higher in mature oocytes (metaphase II) compared with spermatozoa (P &lt; 0.05). The RXFP1/2 mRNA ratios were in favor of RXFP2 in mature oocytes (0.9×), zygotes (0.8 ×), and cleaved embryos (0.8×), and for RXFP1 in spermatozoa (1.1 ×) and blastocysts (1.1 ×). A similar pattern during embryo development was revealed at the protein level, showing a higher RXFP2 fluorescence signal in cleaved embryos and a lower signal in blastocysts compared with RXFP1 protein. In experiment 1, COC exposed to 40 ng mL-1 pRLX resulted in fewer cleaved embryos (36 ± 4%) compared with controls (42 ± 5%, P &lt; 0.05). Of the 40 ng mL-1 pRLX-derived cleaved embryos, a greater proportion developed to the blastocyst stage (38 ± 6%; P &lt; 0.05) compared with control and 20 ng mL-1 pRLX-derived cleaved embryos (26 ± 4% and 17 ± 8%, respectively). In experiment 2, however, 40 ng mL-1 pRLX induced higher cleavage but lower blastocyst rates (51 ± 5% and 20 ± 4%, respectively) compared with the control group (37 ± 4% and 32 ± 7%, respectively) (P &lt; 0.05). In experiment 3, the exposure of both oocytes and derived embryos did not affect the developmental rates (P &gt; 0.05). Nevertheless, pRLX significantly increased the mean cell number of blastocysts in all experiments (P &lt; 0.05). We concluded that pig embryos express RXFP1 and RXFP2 receptors, which may facilitate a role for pRLX during oocyte maturation and embryo development in the pig. This work was supported by the USDA-ARS Biophotonics Initiative project# 58-6402-3-0120 and the Mississippi Agricultural and Forestry Experiment Station (MAFES).

In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.

24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P &lt; 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time. Table 1. Development in vitro of bovine NT embryos produced from different donor cell types

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively). The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P &amp;lt; 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P &amp;lt; 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

Cysteamine or β-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection

The present study was carried out to investigate the effect of adding 100 μM cysteamine (Cys) or 100 μM β-mercaptoethanol (β-ME) to a defined maturation medium on in vitro maturation (IVM), and fertilization and developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The two control media for IVM culture were modified TCM199 containing 10% (v/v) porcine follicular fluid (pFF) or 0.05% (w/v) polyvinyl alcohol (PVA), and Cys or β-ME was supplemented to the PVA-control medium. There was no significant difference in the proportions of in vitro matured oocytes among the four treatment groups (94.5–98.4%). The percentages of pronuclear formation (51.0–64.2%) after ICSI were also not significantly different among the four groups. The cleavage rate (72.8%) in the oocytes treated with Cys showed no significant difference compared with those of the two control media containing pFF (72.2%) or PVA (61.5%), but was higher ( P < 0.05) than that in the oocytes treated with β-ME (56.3%). However, the rates of blastocyst formation of Cys (36.7%), β-ME (27.1%) and pFF (31.4%) were higher ( P < 0.05) than that using the control medium containing PVA (15.6%). The mean cell number of blastocysts ranged from 42 to 52 among the four groups, without significant differences. In conclusion, the addition of Cys or β-ME to a defined maturation medium enhanced blastocyst formation after ICSI, to a level similar to that achieved by adding pFF.

The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: In vitro and In vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.

350 COMPARATIVE TIME-LAPSE STUDIES ON PORCINE EMBRYOS FERTILIZED IN VITRO WITH FLOW CYTOMETRICALLY SEX SORTED SPERMATOZOA

The objective of the present time-lapse studies was to compare developmental characteristics of porcine embryos after in vitro fertilization with flow cytometrically sexed spermatozoa during an in vitro culture period. Immature oocytes were matured (n = 851) and fertilized in vitro using 50 spermatozoa of either sex per oocyte. Additionally, in vivo-matured oocytes (n = 700) were derived from hormonally stimulated (eCG/hCG) prepuberal gilts, which were slaughtered 38 h after hCG treatment. Potential zygotes were introduced into the time-lapse system (50 µL-microdrops, NCSU-23) 18 h after the onset of in vitro fertilization. The onset and duration of cell cycles and blastomere rotation as well as collapses and re-expansion of cytoplasm were investigated. At the end of the culture period (168 h), embryos were labeled with Hoechst 33342 to identify the number of cell nuclei. The time-lapse system consisted of an incubation chamber installed on an inverted phase-contrast microscope and gassed with 5% CO2 in maximally humidified air. Computer controlled positioning of the chamber allowed the capture and storage of digital images of individual embryos every 30 min over a 7-day period. Converted time values were tested by ANOVA or ANOVA on ranks. In total, 700 in vivo-matured oocytes were fertilized in vitro (Y-sperm: 342; X-sperm: 358). Cleavage rates were 45.6% for male and 40.2% for female embryos. Out of these, 45.5% developed to male and 62.5% to female blastocysts, respectively. The onset and duration of cell cycles differed significantly at the 2-cell and morula stages (P &lt; 0.01). The onset and number of rotations as well as collapses and re-expansion of cytoplasm were not different. Mean cell numbers of blastocysts were equal for both sexes (male: 35.2; female: 38.8). In parallel, 851 in vitro-matured oocytes were fertilized in vitro (Y-sperm: 431; X-sperm: 422). Cleavage rates were 45.5% for male and 49.3% for female embryos. Out of these, 54.1% developed to male and 56.7% to female blastocysts, respectively. The onset and duration of cell cycles and rotations as well as collapses were not significantly different between sexes. Mean cell numbers of blastocysts were equal for both sexes (male: 29.5; female: 27.7). Comparison between male embryos derived from in vivo (VV) or in vitro (VT)-matured oocytes showed a delay of the onset of the second cell cycle for VV (P &lt; 0.001) and at the blastocyst stage for VT (P &lt; 0.001). Accordingly, in these stages the duration of the cell cycle was shortened (P &lt; 0.001). The onset and duration of rotations as well as collapses and re-expansion of cytoplasm were not different. Similar data were obtained for the female embryos. The data suggest slight sex related differences (onset of cell cycles), but these might be masked in embryos produced from in vitro-matured oocytes due to their higher individual variability. The experiment also shows the advantages of a time-lapse system to identify dynamic cell processes. It might also be a useful tool to precisely correlate cell cycle events to activation of certain marker genes.

151 EFFECTS OF HEXOSES SUPPLEMENTED IN THE MATURATION AND PRE IMPLANTATION MEDIUM ON THE IN VITRO DEVELOPMENT OF PORCINE OOCYTES

The present study was conducted to determine the effects of hexoses on in vitro maturation, fertilization, and development of porcine oocytes. In the first experiment, porcine oocytes were matured in modified North Carolina State University (NCSU)-37 medium supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbc AMP), 10 IU/mL equine chorionic gonadotrophin, 10 IU/mL human chorionic gonadotrophin, 50 μ/mL gentamycin (Sigma-Aldrich, Tokyo, Japan), 10% (v/v) porcine follicular fluid, and various hexoses (glucose, fructose, and galactose) at various concentrations of 0 (control), 2.5, 5.5, and 10 mM. They were subsequently cultured in the maturation medium without hormones and dbcAMP for an additional 22 h. Fertilization was performed according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041); 15 oocytes were co-incubated with 1 million frozen thawed sperm/mL in fertilization medium for 5 h. Supplementation of either glucose (2.5 or 5.5 mM) or fructose (5.5 mM) in the maturation medium significantly increased the percentages of maturation to metaphase II (68.5%, 79.4%, and 70.2%, respectively) and monospermic fertilization of oocytes (55.0%, 64.5%, and 58.9%, respectively), as compared with control group (metaphase II: 52.8%; monospermic: 42.7%; P &lt; 0.05). Supplementation of galactose had no effect on the meiotic maturation and monospermic fertilization of oocytes. In the second experiment, presumptive zygotes were cultured in modified NCSU-37 supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate, and 50 μg/mL gentamycin. The cleaved embryos were collected at Day 3 after in vitro fertilization and then cultured for a further 4 days in modified NCSU-37 medium supplemented with 5.5 mM of glucose, fructose, or galactose. The percentages of blastocyst formation, calculated from cleaved embryos, were significantly higher in the glucose and fructose groups (20.4% and 18.4%, respectively) than in the galactose group (12.9%) and the non-supplemented control group (9.2%). Although fructose supplementation did not accelerate blastocyst formation, it did significantly increase the mean cell number of blastocysts (48.0 ± 2.9 vs. 38.6 ± 1.8) and reduced the index of DNA-fragmented nuclei in the blastocysts stained by the TUNEL method (7.6 ± 0.9 vs. 11.8 ± 0.9), as compared with glucose supplementation. In the present study, although all hexoses were used through the glycolysis pathway, supplementation of galactose in the maturation and embryo culture medium had no beneficial effect on development. In contrast, both glucose and fructose enhanced development with an optimal concentration of 5.5 mM. Replacement of glucose with fructose did not accelerate blastocyst formation but did enhance embryo quality in terms of increasing cell number and decreasing the number of fragmented nuclei.

Effect of the Well of the Well (WOW) System on In Vitro Culture for Porcine Embryos after Intracytoplasmic Sperm Injection

For developmental competence of porcine embryos in vitro, it is important to improve the culture environment. The present study was performed to evaluate four different culture systems for in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI); drop, well and two sizes of the well of the well (WOW) systems (500 and 1,000 microm in diameter). The cleavage rate on Day 2 and the mean cell number in blastocysts on Day 6 were not significantly different among the four treatments. However, the 1,000 microm WOW (24.6%) resulted in a significantly higher (P<0.05) blastocyst rate than those in the other culture systems (12.9, 14.8, and 7.1% for drop, well, and 500 microm WOW, respectively). The present study indicates that the microenvironment created by the 1,000 microm diameter WOW improves blastocyst production of in vitro matured porcine oocytes after ICSI, and that the effectiveness of the WOW system is dependent on the size (diameter) of the WOW.

Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos

The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO 2 incubator. While the same temperature (38.5 °C) and CO 2 concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (−300 mmHg) to create an O 2 level at 8–10% (low O 2 concentration), or in a positive air pressure (high O 2 concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O 2 concentration (Group I) or in the standard incubator with a high O 2 concentration (Group II). To assess the effect of O 2 concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O 2 concentration (Group III) or a high O 2 concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.