The relationship between antral follicle count (AFC), reproductive performance, and the success of reproductive biotechnologies in cattle is a subject of ongoing research. While numerous studies have explored this relationship in various cattle breeds, many aspects of female reproductive physiology, particularly those involving subspecies differences (*Bos taurus taurus* vs. *Bos taurus indicus*) and the specific influence of antral follicle populations on fertility, remain unclear. AFC in cattle is highly variable, yet exhibits significant repeatability within individual animals, unaffected by season, follicular wave number, or lactation status. In *Bos taurus* cattle, a low AFC has been linked to negative fertility outcomes, such as smaller ovaries, fewer healthy follicles, poor reproductive performance, reduced response to superovulation, and lower progesterone and anti-Müllerian hormone concentrations. Conversely, high AFCs in *Bos indicus* and *Bos indicus*-*Bos taurus* cattle have been associated with greater in vivo and in vitro embryo production efficiency. However, previous studies on Nelore cattle (*Bos indicus*) subjected to TAI protocols have reported unexpectedly higher pregnancy rates in cows with low AFC, suggesting a complex interplay between AFC and fertility. Moreover, larger dominant follicle diameters at TAI have been associated with improved pregnancy rates, prompting further investigation into the underlying cellular and molecular mechanisms. This study aimed to test the hypothesis that a low AFC in Nelore females leads to better fertility in a TAI program than a high AFC by evaluating the impact of low/very low and high AFC on ovarian follicular dynamics and pregnancy rates and assessing the transcriptional patterns of genes important for follicular cell development.

Extensive research has explored the link between antral follicle count (AFC) and reproductive outcomes in cattle. Studies in *Bos taurus* generally demonstrate an inverse relationship between AFC and fertility. Low AFC is associated with reduced ovarian size, fewer healthy follicles and oocytes, poor reproductive performance, and lower circulating progesterone and anti-Müllerian hormone levels. Conversely, studies in *Bos indicus* have shown conflicting results, with some suggesting high AFC is linked to improved embryo production. However, recent studies in Nelore cattle (*Bos indicus*) have shown a higher pregnancy rate in cows with low AFC after timed artificial insemination (TAI), highlighting the need to clarify the complex interaction between AFC and fertility in this specific breed. The inconsistent findings across studies underscore the need for a deeper understanding of the underlying cellular and molecular mechanisms.

This study comprised two main experiments. Study I focused on ovarian follicular dynamics and pregnancy rates in Nelore cows subjected to TAI, involving 40 cows for follicular dynamics and 1428 cows for pregnancy rate analysis. Cows were categorized into very low AFC (≤15 follicles) and high AFC (≥45 follicles) groups. Ovarian follicular dynamics were assessed using ultrasonography on multiple days (days 7, 0, 5, 8, 9, 10, and 18). Progesterone concentrations were measured on day 18. Pregnancy rates were evaluated 30 days post-TAI. Study II focused on transcriptional patterns in Nelore heifers (48 total, 10 low AFC and 10 high AFC). Ovarian follicular waves were synchronized, and cumulus-oocyte complexes (COCs) were collected on day 5 via ovum pick-up (OPU). Gene expression analysis in oocytes and cumulus cells was performed using a microfluidic platform. Data were analyzed using appropriate statistical methods.

Study I revealed that cows with high AFC had significantly larger ovaries (diameter and area) than those with very low AFC. However, cows with very low AFC exhibited larger dominant follicle diameters at TAI and a trend toward higher serum progesterone concentrations on day 18. Importantly, pregnancy rates after TAI were significantly higher (P ≤ 0.05) in animals with very low and low AFC compared to those with high AFC. Study II demonstrated that the number of COCs retrieved via OPU was significantly smaller in the low AFC group compared to the high AFC group; however, the percentage of retrieved COCs was not different. Gene expression analysis revealed significant differences in the expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis, and cellular stress response in both oocytes and cumulus cells of low versus high AFC groups. Specifically, several genes associated with positive cellular functions showed higher expression in low AFC group, including genes for intercellular communication (BMP15, HAS2, EGFR, NPR2, NPR3), epigenetic regulation (DNMT3A, HDAC2, PAF1, HIFOO), cell growth (IGFBP2, GSK3A, IGF1R), and mitochondrial maintenance (TFAM). In contrast, several cell stress related genes were higher in high AFC group.

The findings of this study provide evidence that a low AFC in Nelore cows is associated with improved reproductive performance in TAI programs. The observation that cows with low AFC have larger dominant follicles at TAI, a known predictor of pregnancy success, aligns with previous research. The higher progesterone levels observed in the low AFC group are also consistent with improved fertility. The differential gene expression profiles observed between low and high AFC groups further elucidate the molecular mechanisms underlying this association. The upregulation of genes associated with intercellular communication, meiotic control, and follicular growth in the low AFC group suggests a more favorable follicular microenvironment for oocyte development and maturation. These data suggest that the impact of AFC on fertility might be modulated by the quality and development of the follicle rather than merely by the number of follicles present. This contradicts some previous literature on Bos Taurus, highlighting the subspecies differences.

This study demonstrates that low AFC in Nelore cows is associated with improved fertility in TAI programs. This is linked to larger dominant follicle diameters, a trend toward higher progesterone concentrations, and a distinct transcriptional profile in oocytes and cumulus cells that suggests improved follicle quality and oocyte competence. Future research could focus on investigating the specific interactions between the identified genes and their roles in oocyte development, as well as exploring the potential for using AFC as a selection criterion for improving reproductive efficiency in Nelore cattle.

The study primarily focused on Nelore cattle, limiting the generalizability of the findings to other cattle breeds. The sample size in Study II was relatively small; a larger sample size might reveal additional differentially expressed genes. While the study detected differences in gene expression, functional validation of these genes' roles in reproductive success is needed to further confirm their significance.