21 Invitro maturation of domestic cat oocytes: A comparison of different reproductive stages

Abstract

Domestic cat invitro embryo production (IVEP) begins with IVM of oocytes to produce mature oocytes; that is, MII. The domestic cat (Felis catus) has been used as a model to carry out assisted reproductive technology (ART) research for application in wild feline species that may be threatened or endangered. The objective of this research was to evaluate oocyte maturation of domestic cats in different reproductive stages: (1) prepubertal, (2) oestrus, (3) pregnant, and (4) anoestrus. The present study was carried out at the Universidad Autónoma Metropolitana Unidad Xochimilco in Mexico City. Unless otherwise stated, all reagents used were from Sigma-Aldrich. The domestic cat ovaries were obtained from a veterinary clinic using salpingo-oophorectomy hysterectomy (OSH). Ovaries were classified as one of the following: (1) prepubertal (female cats under 6 months of age); (2) in oestrus (one or more 2-mm mature follicles); (3) pregnant (presence of fetuses with one or more corpora lutea; and (4) anoestric (ovaries without follicular activity). The ovaries were transported (<2h) in NaCl solution (0.157M) with ampicillin (10 000 IU mL−1), streptomycin (10 000 µg mL−1) and amphotericin (25µg mL−1) to the laboratory. The cumulus–oocyte complexes (COCs) were obtained by ovary microdissection with modified Tyrode’s medium supplemented with sodium lactate (10mM), HEPES (0.50mM) and polyvinyl alcohol (0.01%). COCs were washed twice with TCM-199 medium with Earle’s salts supplemented with bovine serum albumin (BSA, 3mg mL−1), cysteine (0.1mg mL−1), HEPES (1.4mg mL−1), sodium pyruvate (0.25mg mL−1), sodium lactate (0.6mg mL−1), L-glutamine (0.15mg mL−1) and gentamicin (0.055mg mL−1). The wash medium was also used for IVM, but supplemented with human menopausal hormone (Merional® IBSA; 4.5IU mL−1). Oocyte maturation was performed with TCM-199 medium supplemented with BSA, in an atmosphere of 38.5°C, 5% CO2, 95% air, and humidity at saturation for 48h. To evaluate IVM, 300μg mL−1 of hyaluronidase was used to remove the granulosa cells for 5min at 38°C. Next, the oocytes were fixed with paraformaldehyde (4%) for 15 min; washed with a mounting solution (Imacel, invitro); then, 1.5μg mL−1 of 4’,6 diamidino-2-phenylindole dihydrochloride (DAPI) was added. The stained oocytes were evaluated under a microscope (Eclipse E600, Nikon) equipped with a fluorescence lamp and a UV filter (excitation: 330–380nm). The Student’s t-test and the Chi-squared test (χ2) were used for statistical analyses (α=P<0.05). A total of 210 ovaries were obtained from 105 female cats: prepubertal (n=38), oestrus (n=25), pregnant (n=18), and anoestrus (n=24), with a total of 1405 oocytes recovered. The meiotic maturation between the different reproductive stages after 48h of culture was similar in prepubertal (48%), oestrus (46%), pregnant (43%), and anoestrus (45%) groups and did not show a significant difference (P>0.05). This study shows that the domestic cat reproductive stage does not significantly affect the production of mature oocytes for use in ART.