In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors.
This study evaluated in vitro maturation of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (5 microgram/ml) [DOSAGE ERROR CORRECTED]and butyrolactone I (12.5M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained, and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II revealed that hormonal treatment 2(incubation for 22 h - 10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h - 10 ng EGF/ml) presented higher repeatability percentages: TCM+ PVA (54.5% - 61/112); TCM+ pFF (65.0% - 63/97);NCSU23 + PVA (54.6% - 65/119), and NCSU23 + pFF (58.1% - 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/mland 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups.
Marques
Mariana Groke
MG
Department of Animal Reproduction, Faculty of Veterinary Medicine, University of São Paulo, Brazil. mgroke@usp.br
Nicacio
Alessandra Corallo
AC
de Oliveira
Viviane Purri
VP
Nascimento
Anibal Ballarotti
AB
Caetano
Heloisa Vasconcelos Amaral
HV
Mendes
Camilla Mota
CM
Mello
Marco Roberto Bourg
MR
Milazzotto
Marcella Pecora
MP
Assumpção
Mayra Elena Ortiz D'Avila
ME
Visintin
José Antônio
JA
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry*; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015571:Follicular Fluid / Q000502:physiology; D006064:Gonadotropins, Equine / Q000494:pharmacology; D008540:Meiosis / Q000187:drug effects*; D008856:Microscopy, Fluorescence / Q000662:veterinary; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development; D011142:Polyvinyl Alcohol / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
D006063:Chorionic Gonadotropin; D003470:Culture Media; D006064:Gonadotropins, Equine; D011500:Protein Synthesis Inhibitors; D004815:Epidermal Growth Factor; C082868:butyrolactone I; D011142:Polyvinyl Alcohol; D003513:Cycloheximide; D015107:4-Butyrolactone