Folliculogenesis and Morphometry of Oocyte and Follicle Growth in the Feline Ovary K Reynaud 1 , C Gicquel 1 , S Thoumire 1 , M Chebrout 1 , C Ficheux 1 , M Bestandji 1 and S Chastant-Maillard 1,2 1 UMR 1198 INRA ⁄ ENVA ⁄ CNRS FRE Biologie du De ´veloppement et Reproduction; 2 Department of Reproduction, Alfort National Veterinary College, Maisons-Alfort Cedex, France Contents This study was designed to describe, both quantitatively (morphometry) and qualitatively (histological differentiation), follicle and oocyte growth in the feline ovary. The ovaries of 43 cats were collected and processed for histology. The diameters of 832 follicle ⁄ oocyte pairs were measured, with and without zona pellucida (ZP), and a special emphasis was placed on the study of early folliculogenesis. Primordial, primary, secondary, pre-antral and early antral follicles were measured at 44.3, 86.2, 126.0, 155.6 and 223.8 lm in diameter respectively. A biphasic pattern of follicle and oocyte growth was observed. Before antrum formation, follicle (x) and oocyte ( y) size were positively and linearly correlated ( y = 0.500x + 20.01, r 2 = 0.89). Antrum formation occurred when the follicle reached 160–200 lm in diameter (when oocyte was at 102 lm). After antrum formation, a decoupling was observed, a minimal increase in oocyte size contrasting with a significant follicle development ( y = 0.001x + 114.39, r 2 = 0.01). The pre- ovulatory follicle diameter was approximately 3500 lm and the maximal oocyte diameter was 115 lm. The ZP, absent in primordial and primary follicles, appeared at the secondary stage and reached almost 6 lm at the pre-ovulatory stage. These results suggest that (i) in feline ovary, follicle and oocyte growth pattern is similar to that observed in other mammals; (ii) the antrum forms in 160–200 lm follicles, which represents 5% of the pre-ovulatory diameter and (iii) the oocyte had achieved more than 90% of its maximal growth at the stage of antrum formation. Introduction The domestic cat is the only species not considered as endangered among the 37 known felids (Pelican et al. 2006). Indeed, Felis catus suffers from overpopulation in many countries around the world (Purswell and Kolster 2006). However, cat breeders and some owners want to produce kittens from their animals. Clinicians and researchers are therefore asked simultaneously to develop biological techniques designed to limit ovarian activity and conversely to develop reproductive biotechnologies, such as artificial insemination, embryo transfer and in vitro embryo production. Both objectives demand some fundamental knowledge of reproductive physiology in cat, which is still lacking. Whereas the endocrinology of the feline oestrous cycle has been thoroughly described (Shille et al. 1979; Wildt et al. 1981; Goodrowe et al. 1989), and despite many in vitro maturation works (Goodrowe et al. 1988), follicular dynamics in this species remains largely unknown. Ovulatory diameter (3–4 mm) has been determined initially through laparotomy ⁄ laparoscopy (Wildt et al. 1981; Goodrowe et al. 1989). More recently, two studies followed terminal follicular growth (from 1 mm until ovulation) by ultrasonography (Gun- zel-Apel et al. 1998; Malandain et al. 2006). But studies encompassing evaluations of follicle diameter, follicle stage and oocyte diameter remain quite incomplete (Gosden and Telfer 1987; Barber et al. 2001; Bristol- Gould and Woodruff 2006). This study presents a complete histological and morphometric analysis of follicle growth in the cat, from the primordial through to the pre-ovulatory stage. It was conducted using evaluation of four criteria: follicle diameter, follicle stage, oocyte diameter and zona pellucida (ZP) thickness. The objective was to determine the main stages of folliculogenesis in the cats, such as antrum formation and acquisition of developmental competence of the oocyte. Materials and Methods Collection of feline ovaries Ovaries were collected from 43 cats (1.17 ± 0.09 years old; range 0.58–3.33) after routine neutering without any hormonal stimulation in the Surgery department of the Alfort veterinary college. The ovaries were stored at 38°C in phosphate buffer saline solution and selected within 1 h after collection. Only ovaries without corpora lutea but with numerous follicles measuring 1–3.5 mm in diameter were selected and fixed in formaldehyde 10% solution for 1 week. At least five of the 43 cats exhibited behavioural signs of oestrus. Histological preparation and measurements of follicle ⁄ oocyte diameters After fixation, ovaries were gradually dehydrated in alcohol solutions prior to embedding in paraffin (Parap- last; CML, Nemours, France). Both ovaries from each animal were serially sectioned at 7 lm, stained with haematoxylin–eosin–safran and mounted in Eukitt medium (CML). For each cat, primordial to pre-ovulatory follicles were observed (BX41 microscope; Olympus, Rungis, France) and measured using an ocular micrometer. Only follicles without obvious signs of atresia (regular shape, well-organized granulosa cells) were included. Follicle diameter (two perpendicular diameters, with the base- ment membrane as limit) and oocyte diameter (two perpendicular diameters, with the ZP and without ZP, with the oocyte cytoplasmic membrane as limit) were measured at the highest magnification possible (·400 magnification for follicles <300 lm, ·200 ) ·100 for Reprod Dom Anim 44, 174–179 (2009); doi: 10.1111/j.1439-0531.2007.01012.x ISSN 0936-6768 Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag