Nuclear Maturation, Histological and Ultrastructural Analysis of Oocytes in Female Domestic Cats (Felis catus)
*Maria Denise Lopes, Edilson Uiechi, Maria Dalva Cesário, Luzia A. Trinca
*Faculdade de Medicina Veterinária e Zootecnia - UNESP, Distrito de Rubião Júnior
Botucatu, São Paulo, BR
The objective of this study was to define the necessary conditions for the in vitro oocyte maturation from ovaries of domestic cats to explain the preliminary details of in vitro fertilization technique in Felidae.
This paper was divided into three studies. Study I investigated the influence of age and reproductive status of female donors on the number and quality of oocytes. Fifty-one females were submitted to ovariectomy and selected according to age and reproductive phase. The ovaries were isolated and punctured, and the aspirated oocytes were quantified and classified according to a morphological criteria. Study II evaluated follicular dynamics by histological and ultrastructural analysis of oocytes from young and adult females. Ten adult females at different phases of reproductive cycle and 5 young females aged between 3 and 6 months were used. The oocytes were collected as described in study I. After appropriate slide fixation and preparation, the material was analyzed for morphological and ultrastructural characteristics and photographed. Study III assessed the in vitro oocyte nuclear maturation from adult and young donors. Fifteen ovaries were used; 10 from adult females and 5 from young females. The oocytes collected by aspiration were quantified and classified. The oocytes classified as excellent and/or regular were grouped (10 oocytes/drop) in culture medium covered with mineral oil in disposable Petri dishes. The oocytes were incubated at 38°C and 5% CO2 for 48 hours and then washed with 0.4% hyaluronidase, fixed in methanol/acetic acid (3:1), and stained with acetic orcein.
A total of 2,159 oocytes (42.3 oocytes/female) were recovered, of which 30.99% were classified as excellent, 29.78% as regular, and 39.23% as degenerated. There was a higher oocyte recovery from young females (p=0.001); a larger number of oocytes were recovered at interestrus/anestrus when only adult donors were considered (p=0.0075). A significant association was observed between the reproductive phases and oocyte quality (p=0.002). Histological evaluation in both groups showed that morphological characteristics of the ovaries were the same. Differences were observed only in the frequency of occurrence of different stages of oogenesis. Similar results were seen using transmission and scanning electron microscopy. No ultrastructural differences were seen in oocytes of adult and young cats. The differences observed resulted from oocyte developmental phase. Evaluation of chromosomal configuration of oocytes matured in vitro 44.68% of oocytes in metaphase II in the adult female group and 25.32% in the young donor group (p<0,0001), showing that puberty influences oocyte capacity for in vitro development.
The results of this study led to the following conclusions:
1. Young cats presented a greater number of oocytes than adult cats.
2. Interestrus, anestrus and estrus were the best reproductive phases to perform the aspiration of the ovarian follicles.
3. The morphologic characteristics of the ovaries and ovarian follicles of young and adult cats were similar, just differing in the different stages of oogenesis.
4. The percentage of nuclear maturation (Metaphase II) of oocytes stimulated in vitro of young and adult cats were respectively 25,32% and 44,68%.