Cloned calves derived from somatic cell nuclear transfer embryos cultured in chemically defined medium or modified synthetic oviduct fluid

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.     

Birth weight and gestation length of Japanese black calves following transfer of embryos produced in vitro with or without co-culture.

Birth weight and gestation length of calves following the transfer of in vitro produced (IVP) embryos with or without co-culture of cumulus cells, were compared to those produced in vivo (IVD). Spermatozoa from one Japanese Black bull were used for both IVP and IVD. IVP embryos were produced using two types of culture method: 1) co-culturing with cumulus cells in TCM 199 supplemented with calf serum (IVP-Co), and 2) non-co-culturing without cumulus cells in CR1aa supplemented with BSA / calf serum (IVP-NON-Co). Both IVP and IVD embryos were transferred non-surgically to Holstein recipients on day 7+/-1 of the estrous cycle. Birth weight and gestation length of half-sib single calves were analyzed. No differences were observed in birth weight and gestation length between IVP-Co and IVP-NON-Co calves (31.0 kg and 31.8 kg, and 291.9 days and 291.0 days, respectively). However, the birth weight of the IVP-Co and IVP-NON-Co calves was significantly higher than that of the IVD calves (P<0.01). Gestation length of the IVP-Co and IVP-NON-Co calves was also significantly longer than that of the IVD calves (P<0.01).     

Estrone sulfate and progesterone profiles during late gestation in recipient cows transferred embryos produced by nuclear transfer and in vitro fertilization

The aim of present experiment was to evaluate the plasma concentrations of estrone sulfate (E(1)S) and progesterone (P(4)) during late gestation in recipient cows transferred embryos produced by nuclear transfer (NT) and in vitro fertilization (IVF). Blood samples were collected from recipients transferred embryos produced by NT (n=9) and IVF (n=13) at 160, 220, 240, 260 and 270 d of gestation and then at 5 d intervals until parturition. Plasma samples were analyzed for E(1)S and P(4) by ELISA. One NT and three IVF cows aborted between days 220 and 260 of gestation. Two NT and one IVF cow had prolonged gestation (over 290 d). One IVF cow had an overweight fetus (50 kg) after abortion (257 d). The patterns of changes in the concentrations of E(1)S during late gestation in the NT and IVF cows were almost identical. The NT and IVF cows that aborted had prolonged gestation and much higher E(1)S concentrations than the average. One NT cow aborted after 220 d of gestation and had a sudden high increase in its E(1)S concentration from 160 d to 220 d of gestation. The NT and IVF cows that had prolonged gestation also had significantly higher (P<0.05) P(4) concentrations than the average. These results raise the possibility that the E(1)S and P(4) profiles can be used to monitor some late gestational problems, such as higher birth weight, abortion and prolonged gestation.     

Glycosaminoglycans improves early development of zona-free 8-cell rat embryos to blastocysts in a chemically defined medium, but not the pregnancy rate following transfer of the blastocysts

The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.     

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.     

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed,lactating cows

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.