Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone.
Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus-oocytes complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries and were evaluated morphologically. Thereafter, COCs were exposed to BCB (26 μM) for 90 min at 39°C and selected based on the colour of their cytoplasm (BCB positive/BCB+ or BCB negative/BCB-). The COCs were allocated as follows: (a) IVM medium; (b) eGH group; (c) BCB-/IVM; (d) BCB+/IVM; (e) BCB-/eGH; and (f) BCB+/eGH. Then, COCs were cultured in vitro for 30 h, at 39°C in a 5%CO2 humidified air atmosphere. Cumulus-free oocytes were incubated in 10 μg/ml of bis-benzamide for 20 min at 39°C and nuclear maturation was evaluated with epifluorescence microscopy. Of the 39 COCs selected morphologically and subjected to BCB staining, 18/39 (46.2%) were classified as BCB+ and 21/39 (53.8%) as BCB- (P > 0.05). Maturation was not affected significantly by BCB classification, but the maturation rate was higher for oocytes that had been exposed to exogenous eGH versus controls (16/28, 57.1% versus 8/26, 30.8%, P < 0.05). In the present study, the BCB test was not useful for predicting competent equine oocytes prior to IVM. However, eGH enhanced equine oocyte maturation in vitro.
Federal University of Pelotas - UFPEL,School of Veterinary Medicine,Campus Capão do Leão,s/n° - Mailbox 354,Zip 96010-900,Pelotas,RS,Brasil.
Gabriel R
GR
Pereira
Animal Physiology Department,Veterinary School,Universidad Complutense de Madrid,Madrid 28040,Spain.
Pedro L
PL
Lorenzo
Animal Reproduction Department,Federal Rural University of Pernambuco,Garanhuns,PE 52296-901,Brazil.
Gustavo F
GF
Carneiro
Lethbridge Research Centre,P.O. Box 3000,5403 1Avenue S.,Lethbridge,AB T1J4B1,Canada.
Sylvie
S
Bilodeau-Goeseels
Lethbridge Research Centre,P.O. Box 3000,5403 1Avenue S.,Lethbridge,AB T1J4B1,Canada.
John P
JP
Kastelic
Department of Population Health and Reproduction,School of Veterinary Medicine,University of California,Davis,CA 95616,USA.
Alejandro
A
Esteller-Vico
Departament de Sanitat i d'Anatomia Animals,Autonomous University of Barcelona,Edifici V 08193 Bellaterra,Barcelona,Spain.
Manel
M
Lopez-Bejar
Department of Population Health and Reproduction,School of Veterinary Medicine,University of California,Davis,CA 95616,USA.
Irwin K M
IK
Liu
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D013006:Growth Hormone / Q000494:pharmacology*; D006736:Horses; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009866:Oogenesis; D010078:Oxazines / Q000032:analysis* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D010078:Oxazines; C006797:Brilliant Cresyl Blue; D013006:Growth Hormone