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The antral follicle count (AFC) is a test in which the number of oocyte-containing follicles that are developing in both ovaries are visually counted. The count of these follicles strongly relates to the population of the growing follicle reserve on the ovaries. However, the importance of the main number of antral follicle populations (mAFC) in mono-ovulatory animal species has yet to be completely elucidated. Moreover, the investigation of the ovarian interrelationship with unilateral mAFC (main number of antral follicle populations appearing on only one side of the ovary) and bilateral mAFC (main number of antral follicle populations appearing in equivalent numbers on both sides of the ovary) and how understanding this interrelationship can offer possible indicators of ovarian response to hormonal induction have not yet been investigated in mono-ovulatory Bos indicus beef cows. The aim of this study is to investigate the different ovarian interrelationships of mAFC (unilateral and bilateral mAFC) at the time of exogenous hormonal stimulation on the total number of AFC (left and right ovaries) at the beginning of the hormonal protocol for ovarian stimulation and ovarian response at the completion of exogenous hormonal stimulation as well as their usefulness as possible biomarkers of successful hormonal stimulation in Bos indicus beef cattle. Beef cows (n = 104) with low total numbers of AFC (4.7 ± 2.4 follicles) were stimulated with a gonadotropin-releasing hormone-progesterone-prostaglandin F2α-based protocol. At the beginning of the hormonal protocol, ovarian ultrasound scans were performed to evaluate AFC from both ovaries of cows. Beef cows were divided into two groups, unilateral (n = 74) and bilateral mAFC (n = 30), according to the ovarian interrelationship. At the completion of the hormonal stimulation, ovarian ultrasound scans were performed to evaluate the dominant follicle (DF) and cows with DF > 8.5 mm in diameter emerging on their ovaries were defined as having experienced a response to hormonal stimuli. There was a difference of 19.1% between Bos indicus cows bearing unilateral mAFC that produced an increase in ovarian response (odds ratio = 2.717, p < 0.05) compared to the responsive rate of cows displaying bilateral mAFC (82.4% vs. 63.3%). In unilateral mAFC, cows bearing mAFC ipsilateral to the ovary of dominant follicle (DF) had a higher responsive rate than cows bearing mAFC contralateral to the DF ovary (50.0% vs. 32.4%, p < 0.05). In mAFC ipsilateral to the DF ovary, pregnancy rates were greatest in cows bearing mAFC and DF on the right ovary compared with cows bearing mAFC and DF on the left ovary (25.0% vs. 9.1%, p < 0.05). In primiparous and multiparous cows, unilateral mAFC occurs with a greater (p < 0.05) frequency than bilateral mAFC (69.0% and 72.0% vs. 31.0% and 28.0%, respectively). In unilateral mAFC, primiparous cows bearing mAFC ipsilateral to the DF ovary had a greater responsive rate than primiparous cows bearing mAFC contralateral to the DF ovary (55.0% vs. 20.0%, p < 0.05). In mAFC ipsilateral to the DF ovary, responsive and pregnancy rates were greatest (p < 0.05) in multiparous cows bearing mAFC and DF on the right ovary compared with multiparous cows bearing mAFC and DF on the left ovary (58.1% and 22.6% vs. 25.8% and 3.2%, respectively). Furthermore, there was a positive correlation between the mean diameter of AFC at the time of the exogenous hormonal trigger and the mean diameter of DF at the completion of hormonal synchronisation (p < 0.05). Our findings emphasise that the ovarian interrelationship with unilateral mAFC at the time of the hormonal trigger might be a promising biomarker for predicting success in ovarian response to hormonal stimulation of mono-ovulatory Bos indicus beef cows with low AFCs.