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Apoptosis is the cellular mechanism of ovarian follicular atresia in mammals; the aim of this study was to examine the apoptosis‐related cyclic changes in follicular cells of different‐sized antral follicles throughout the oestrous cycle in canines. Ovaries were collected from 26 adult female dogs (1–4 years) following routine ovariohysterectomy. Antral follicles were classified as small, medium or large antral or preovulatory. The percentage of apoptotic cells was determined flow cytometrically using the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate (dUTP) DNA nick end labelling (TUNEL) assay. Apoptosis rate was quantified as the percentage of TUNEL‐positive cells on a logarithmic scale. Percentages of TUNEL‐positive cells obtained in the flow cytometric assay were compared among oestrous phases and follicular sizes using analysis of variance. Apoptotic follicles were observed in all types of canine follicles in different cycle phases and stages of development, possibly corresponding to the physiological process of the oestrous cycle. Both the oestrous phase and follicular size significantly influenced the apoptosis rate (p <.05). Apoptosis rate increased significantly (p <.05) as follicular development progressed. Apoptosis rate was the highest in large follicles during the oestrous phase (9.2%; p <.05) and the lowest in small follicles during the anestrus period (1.8%; p <.05). In conclusion, our results demonstrate significant differences in the apoptosis rate during the oestrous cycle related to follicle development in the canine ovary. Furthermore, flow cytometry using the TUNEL assay was found to be an effective method for detecting apoptosis in canine follicles. [ABSTRACT FROM AUTHOR]

Contents: Growth differentiation factor 9 (GDF‐9) and bone morphogenetic protein 15 (BMP‐15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199‐Hepes medium as (a) Control group; (b) GDF‐9 antibody (Ab); (c) BMP‐15 Ab; (d) recombinant human (rh) GDF‐9; (e) rh BMP‐15 or (f) rh BMP‐15 and GDF‐9. Data were evaluated by ANOVA. The Abs against GDF‐9 or BMP‐15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF‐9 Ab (64.4 ± 2.1%) or BMP‐15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF‐9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP‐15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF‐9 (3.7 ± 0.4%) or rhBMP‐15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF‐9 (14.7 ± 3.1) and BMP‐15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs. [ABSTRACT FROM AUTHOR]