Influence of epidermal growth factor and insulin-like growth factor 1 on nuclear maturation and fertilization of buffalo cumulus oocyte complexes in serum free media and their subsequent development in vitro.
The in vitro maturation, fertilization and development of Indian water buffalo (Bubalus sp.) cumulus oocyte complexes (COCs) to blastocysts were studied during culture, either in serum free tissue culture medium 199 (TCM 199) or Waymouth MB (WM). Based on different supplements added to these media, the experimental groups included: (a) no supplement (control); (b) hormones (FSH, LH and oestradiol) (c) Epidermal growth factor (EGF); (d) IGF-1; and (e) EGF + IGF-1. Experiments were conducted to note three end points: (1) nuclear maturation 24 h after culture (eight replicates); (2) fertilization 24 h after insemination (10 replicates); (3) development to blastocysts (nine replicates). The oocytes were cultured in groups of up to five per drop. Using a two-way (5 x 2) factorial model with interactions, the results were compared using generalized linear models with binomial errors and the logit link function. In experiment 1, the proportion of oocytes reaching metaphase II was higher for all the supplement treatments than the control treatment (t = 3.68, p < 0.0001). The proportion of oocytes reaching metaphase II was 74.7, 63.2, 64.7 and 81% with hormone (chi2 = 17.23, p < 0.0001), EGF (chi2 = 7.07, p = 0.007), IGF-1 (chi2 = 19.21, p = 0.002) and EGF + IGF-1 (chi2 = 33.04, p < 0.0001) supplementation, respectively, compared to 46.6% in the control (no supplement) group. Media did not have an effect on outcome. In experiment 2, the proportion of oocytes fertilized was significantly higher with hormones (31.0%, chi2 = 12.5, p = 0.0004), IGF-1 (35.7%, chi2 = 20.53, p < 0.0001), and the EGF + IGF-1 combination (49.7%, chi2 = 51.35, p < 0.0001) compared to control (16.2%). No significant effect of media was seen. In experiment 3, the proportion of oocytes that cleaved at 48 h after culturing was significantly higher for all supplement treatments compared to control. IGF-1 supplementation was the only treatment that did not produce a significantly higher rate of progression to blastocysts compared to the control. Once again, media had no effect on outcome. It was concluded that maturation, fertilization and development of buffalo oocytes were enhanced by all supplements tested. Enhancement was maximal with the combination of EGF+IGF-1. In contrast, no significant differences were found between the two types of media used.
Purohit
G N
GN
Department of Animal Reproduction Gynaecology and Obstetrics, College of Veterinary and Animal Science, Rajasthan Agricultural University, Bikaner, Rajasthan 334001, India. gnpvog@yahoo.co.in
Brady
M S
MS
Sharma
S S
SS
D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000502:physiology; D002020:Buffaloes / Q000196:embryology / Q000502:physiology*; D016895:Culture Media, Serum-Free; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004958:Estradiol / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005640:Follicle Stimulating Hormone / Q000502:physiology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D016014:Linear Models; D007986:Luteinizing Hormone / Q000502:physiology; D008297:Male; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology; D011247:Pregnancy
D016895:Culture Media, Serum-Free; D004958:Estradiol; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone