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In vitro maturation of equine oocytes without hormones
Theriogenology
383
I N VITRO MATURATION OF EQUINE OOCYTES WITHOUT HORMONES
F.C. Landim-Alvarenga* and Y.H. Choi** *Dept. Animal Reproduction and Veterinary Radiology, Sao Paulo State University Botucatu, SP 18618.000, BRAZIL **AnimalReproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523, USA The majority of mammalian oocytes resume meiosis when cultured in vitro, as indicated by germinal vesicle breakdown (GVB), but only a variable proportion reach metaphase (MII). In mares results of in vitro maturation of ooeytes 0VM) are still poor when compared with those obtained in other domestic species. One aspect of equine IVM investigated has been variation in the sources and concentrations of LH, FSH and E2. This experiment was done to evaluate the capacity of slaughterhouse equine oocytes to undergo nuclear maturation in the absence of gonadotrophins and E2. Compact cumulusooeytes complexes collected by slicing ovaries, were matured at 38.5° C in 5% 02 in air according to the following protocols: Group t - Tissue Culture Medium 199 (TCM) with Earle's salts containing lmg/ml PVA. Group 2 - TCM 199 with Earle's salt containing 10% new born calf serum (TCMS), lnl/ml equine pituitary extract (EPE) and lpg/ml E2. Group 3 - TCMS containing 10 nl/ml EPE and l~tg/ml E2. Group 4 - TCMS containing 1~g/ml bLH, 500 ng/ml bFSH and 1~tg/ml E2. After 40 hrs of incubation, oocytes were stripped by pipetting in Modified Dulbecco's PBS with 300 IU/ml hyaluronidase and stained with 1% acetic orcein to visualize chromosomes. Table 1: Mean cumulus expansion and meiotic maturation (5 replicates). Treatment Cumulus expansion MII (%) PVA (nohormones) 2.28" 19/45 (42.2)¢ lnl EPE, E2 3.22 ,b 21/48 (43.7)c 10 nl EPE, E2 3.78 b 14/47 (29.8)° LH, FSH, E2 3.46b 15/35 (42.8)c a, b means without common superscripts differ, p<0.05, ANOVA Cumulus expansion (scored from 0, no expansion to 4, fully expanded) in Group 1 was significantly less (p<0.05, ANOVA) than in the other groups; no significant difference in nuclear maturation was observed among treatments (p>0.05, Chi-Square). These results indicate that addition of LH, FSH and/or E2, is necessary for complete cumulus expansion; however, in the doses used, these hormones did not affect nuclear maturation, which occurs spontaneously without them. This observation is in contrast to several other studies o f in vitro maturation of equine oocytes. Developmental competence in the absence ofLH, FSH and E2 still remains questionable because cytoplasmatic maturation may be incomplete without them. Possibly spontaneous maturation of equine oocytes fi'om slaughterhouse can be too rapid, resulting in asynchrony of pronuclear formation and low cleavage rates after in vitro fertilization.
383
I N VITRO MATURATION OF EQUINE OOCYTES WITHOUT HORMONES
F.C. Landim-Alvarenga* and Y.H. Choi** *Dept. Animal Reproduction and Veterinary Radiology, Sao Paulo State University Botucatu, SP 18618.000, BRAZIL **AnimalReproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523, USA The majority of mammalian oocytes resume meiosis when cultured in vitro, as indicated by germinal vesicle breakdown (GVB), but only a variable proportion reach metaphase (MII). In mares results of in vitro maturation of ooeytes 0VM) are still poor when compared with those obtained in other domestic species. One aspect of equine IVM investigated has been variation in the sources and concentrations of LH, FSH and E2. This experiment was done to evaluate the capacity of slaughterhouse equine oocytes to undergo nuclear maturation in the absence of gonadotrophins and E2. Compact cumulusooeytes complexes collected by slicing ovaries, were matured at 38.5° C in 5% 02 in air according to the following protocols: Group t - Tissue Culture Medium 199 (TCM) with Earle's salts containing lmg/ml PVA. Group 2 - TCM 199 with Earle's salt containing 10% new born calf serum (TCMS), lnl/ml equine pituitary extract (EPE) and lpg/ml E2. Group 3 - TCMS containing 10 nl/ml EPE and l~tg/ml E2. Group 4 - TCMS containing 1~g/ml bLH, 500 ng/ml bFSH and 1~tg/ml E2. After 40 hrs of incubation, oocytes were stripped by pipetting in Modified Dulbecco's PBS with 300 IU/ml hyaluronidase and stained with 1% acetic orcein to visualize chromosomes. Table 1: Mean cumulus expansion and meiotic maturation (5 replicates). Treatment Cumulus expansion MII (%) PVA (nohormones) 2.28" 19/45 (42.2)¢ lnl EPE, E2 3.22 ,b 21/48 (43.7)c 10 nl EPE, E2 3.78 b 14/47 (29.8)° LH, FSH, E2 3.46b 15/35 (42.8)c a, b means without common superscripts differ, p<0.05, ANOVA Cumulus expansion (scored from 0, no expansion to 4, fully expanded) in Group 1 was significantly less (p<0.05, ANOVA) than in the other groups; no significant difference in nuclear maturation was observed among treatments (p>0.05, Chi-Square). These results indicate that addition of LH, FSH and/or E2, is necessary for complete cumulus expansion; however, in the doses used, these hormones did not affect nuclear maturation, which occurs spontaneously without them. This observation is in contrast to several other studies o f in vitro maturation of equine oocytes. Developmental competence in the absence ofLH, FSH and E2 still remains questionable because cytoplasmatic maturation may be incomplete without them. Possibly spontaneous maturation of equine oocytes fi'om slaughterhouse can be too rapid, resulting in asynchrony of pronuclear formation and low cleavage rates after in vitro fertilization.