- Email: [email protected]
Influences of culture components on the development of bovine blastocysts in defined conditions
274
Theriogenology
Embryo Culture INFLUENCES OF CULTURE COMPONENTS ON THE DEVELOPMENT OF BOVINE BLASTOCYSTS IN DEFINED CONDITIONS B.G.Brackett’, L.Keskintepe’, A.A.Simplicio”‘,
and G.C.Luvonils3
‘Department of Physiology & Pharmacology, College of Veterinary Medicine, UGA, Athens, GA 30602, USA; *Brazilian Agricultural Research Agency National Goat Research Center, PO Box D-10, Sobral, Ceara, Brazil; 31stituto di Clinical Ostetrica e Ginecologica Veterinaria, Universita‘ degli Studi di Milano, Milano, Italy. Experiments were conducted to determine influences on in vitro production (IVP) of bovine embryos of glucose (glc), citrate (c), glutathione (GSH), and growth factors, EGF and PDGF, during 9 d culture in defined conditions. Oocytes, aspirated from follicles (2-5 mm) at slaughter, with homogeneous cytoplasm and 2-3 layers of cumulus cells were washed twice in Tyrodes with 400 ug PVA/ml and 112 pg sodium pyruvate/ml and incubated in 100 ul of mTCM 199 + 10 pg oFSH and 5 pg bLH (NHPP, NIDDK, NICHD, USDA)/ml for 24 h before insemination. Then, after 6 h co-culture with heparin-capacitated sperm, ova were cultured in 50 ul drops of either c-SOF+NEA (control) (BiolReprod. 52:1410-1417) or c-SOF+NEA without glc and c (I), or without glc (II) for 5 d; then, developing embryos were transferred into c-SOF+NEA * GSH for 4 d. In another trial EGF or PDGF were added to c-SOF+NEA for the last 4 d. Culture media were renewed (50% by volume) every 24 h. Proportions of oocytes that cleaved (C) by 48 h, reached morulae (M) by 120 h, blastocysts (B) by 168 h, and expanded B (EB) by 216 h, and, of M that proceeded to B and EB were analyzed by “Sigma Stat” by means of ANOVA; differences among the treatments were analyzed by Bonferroni-t test. Removal of either glc and c, or glc, compromised results, e.g. C of 72.3 % for I, 64.8 % for II, and 86.1 % for control. Addition of GSH 5 d after beginning embryo culture was not beneficial for B development after initial culture in I, 31.3%, or II, 25.2%. Significantly more B and EB were obtained after 9 d in c-SOF+NEA (40.8 % and 34.1 %, PcO.05). Evaluation of development rates of d 5 M to B stages on d 7, 8, 9 revealed more M cultured in I followed by no GSH reached growing B stage by d 7 (27.3 %) than for other experimental groups (11.6 % for I with GSH, 14.9 % for II without GSH, and 18.9 % for II with GSH), but development was faster in cSOF+NEA (39.6 %) (PcO.05). Early inclusion of citrate was found to enhance M to B development from d 5 to d 8 and, d 9 regardless of other treatments after d 5. In another experiment, different concentrations (0.05, 0.5, 5.0 @ml) of EGF or PDGF were added to cSOF+NEA for the last 4 d of culture. The lowest concentration of either EGF or PDGF resulted in lower (PcO.05) blastocyst development (33.3 % for EGF and 46.6 % for PDGF) compared to the highest concentration (60.0 % and 80.0 %, respectively). EB development was significantly higher (PcO.05) in media containing either growth factor at 5 ng/ml; 32 of 60 M (53.3 %) reached EB with EGF, and 28 (46.6 %) of 60 M reached EB with PDGF present during the final 4 d. Results demonstrated positive influences of glc and c, absence of beneficial effect of GSH, variable patterns in M to B progression, and efficacy of EGF and PDGF in enhancing EB development. (Supported by Transgenic Products, Inc. Atlanta, GA, Atlantic Breeders Cooperative, Lancaster, PA, Shapiro Packing Co., Augusta, GA, and NHPP, NIDDK, NICHD, USDA)
Theriogenology
Embryo Culture INFLUENCES OF CULTURE COMPONENTS ON THE DEVELOPMENT OF BOVINE BLASTOCYSTS IN DEFINED CONDITIONS B.G.Brackett’, L.Keskintepe’, A.A.Simplicio”‘,
and G.C.Luvonils3
‘Department of Physiology & Pharmacology, College of Veterinary Medicine, UGA, Athens, GA 30602, USA; *Brazilian Agricultural Research Agency National Goat Research Center, PO Box D-10, Sobral, Ceara, Brazil; 31stituto di Clinical Ostetrica e Ginecologica Veterinaria, Universita‘ degli Studi di Milano, Milano, Italy. Experiments were conducted to determine influences on in vitro production (IVP) of bovine embryos of glucose (glc), citrate (c), glutathione (GSH), and growth factors, EGF and PDGF, during 9 d culture in defined conditions. Oocytes, aspirated from follicles (2-5 mm) at slaughter, with homogeneous cytoplasm and 2-3 layers of cumulus cells were washed twice in Tyrodes with 400 ug PVA/ml and 112 pg sodium pyruvate/ml and incubated in 100 ul of mTCM 199 + 10 pg oFSH and 5 pg bLH (NHPP, NIDDK, NICHD, USDA)/ml for 24 h before insemination. Then, after 6 h co-culture with heparin-capacitated sperm, ova were cultured in 50 ul drops of either c-SOF+NEA (control) (BiolReprod. 52:1410-1417) or c-SOF+NEA without glc and c (I), or without glc (II) for 5 d; then, developing embryos were transferred into c-SOF+NEA * GSH for 4 d. In another trial EGF or PDGF were added to c-SOF+NEA for the last 4 d. Culture media were renewed (50% by volume) every 24 h. Proportions of oocytes that cleaved (C) by 48 h, reached morulae (M) by 120 h, blastocysts (B) by 168 h, and expanded B (EB) by 216 h, and, of M that proceeded to B and EB were analyzed by “Sigma Stat” by means of ANOVA; differences among the treatments were analyzed by Bonferroni-t test. Removal of either glc and c, or glc, compromised results, e.g. C of 72.3 % for I, 64.8 % for II, and 86.1 % for control. Addition of GSH 5 d after beginning embryo culture was not beneficial for B development after initial culture in I, 31.3%, or II, 25.2%. Significantly more B and EB were obtained after 9 d in c-SOF+NEA (40.8 % and 34.1 %, PcO.05). Evaluation of development rates of d 5 M to B stages on d 7, 8, 9 revealed more M cultured in I followed by no GSH reached growing B stage by d 7 (27.3 %) than for other experimental groups (11.6 % for I with GSH, 14.9 % for II without GSH, and 18.9 % for II with GSH), but development was faster in cSOF+NEA (39.6 %) (PcO.05). Early inclusion of citrate was found to enhance M to B development from d 5 to d 8 and, d 9 regardless of other treatments after d 5. In another experiment, different concentrations (0.05, 0.5, 5.0 @ml) of EGF or PDGF were added to cSOF+NEA for the last 4 d of culture. The lowest concentration of either EGF or PDGF resulted in lower (PcO.05) blastocyst development (33.3 % for EGF and 46.6 % for PDGF) compared to the highest concentration (60.0 % and 80.0 %, respectively). EB development was significantly higher (PcO.05) in media containing either growth factor at 5 ng/ml; 32 of 60 M (53.3 %) reached EB with EGF, and 28 (46.6 %) of 60 M reached EB with PDGF present during the final 4 d. Results demonstrated positive influences of glc and c, absence of beneficial effect of GSH, variable patterns in M to B progression, and efficacy of EGF and PDGF in enhancing EB development. (Supported by Transgenic Products, Inc. Atlanta, GA, Atlantic Breeders Cooperative, Lancaster, PA, Shapiro Packing Co., Augusta, GA, and NHPP, NIDDK, NICHD, USDA)