Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in *Bos indicus* females with a high or low antral follicle count

The study investigates how antral follicle count (AFC) relates to reproductive performance and physiology in cattle, focusing on Bos indicus (Nelore). AFC is highly variable among individuals but repeatable within an animal and is typically assessed by ultrasonography (follicles ≥3 mm). Prior work in Bos taurus has linked low AFC to smaller ovaries, fewer morphologically healthy follicles and oocytes, poorer reproductive performance, reduced superovulatory response, lower circulating progesterone and anti-Müllerian hormone, and reduced endometrial thickness. Conversely, high AFC has been associated with greater embryo production efficiency in indicus and indicus-taurus breeds. However, field studies in Nelore cows under TAI indicate higher pregnancy rates in low AFC groups and favorable follicular dynamics (larger dominant/ovulatory follicles at TAI). Given inconsistencies across subspecies and limited molecular data tied to field fertility, the authors hypothesized that low AFC in Nelore females results in better fertility in TAI programs than high AFC. Objectives: (I) evaluate effects of low/very low vs high AFC on ovarian follicular dynamics and TAI pregnancy rates; (II) assess transcriptional patterns of genes important to follicular cell development in heifers with low and high AFCs.

Background literature highlights: AFC shows high individual repeatability and does not vary with season, follicular wave number, or lactation status. In Bos taurus, low AFC correlates with smaller ovaries, fewer healthy follicles/oocytes, poorer seasonal reproductive performance, lower progesterone and AMH, reduced endometrial thickness, and reduced superovulatory response/transferable embryos. High AFC is associated with improved embryo production in indicus and indicus-taurus cattle. Yet, in Nelore subjected to TAI, cows with low AFC can have pregnancy rates up to 10% higher than high AFC cows, with larger dominant/ovulatory follicles—a known positive correlate with pregnancy. In dairy heifers, high AFC (≥25) has been linked to reduced productive life and suboptimal fertility compared to AFC ≤15. These mixed findings underscore subspecies differences and a need for molecular data (steroidogenesis, intercellular communication, meiotic control, epigenetic modulation, follicular growth, stress/apoptosis) to explain field fertility, which prior studies have largely not addressed.

Study I (cows): Ethics approved (UEL 5898201476). Conducted Aug–Nov on two commercial farms in southern Brazil (Cfa climate). Multiparous Nelore cows (48–84 months), 40–50 days postpartum, BCS 2.5–4.0, on pasture with mineral mix and water ad libitum. Two components: (a) ovarian follicular dynamics (n=40, Farm I); (b) TAI pregnancy rate (n=1,428, Farm II herds). AFC assessment by transrectal ultrasonography (7.5 MHz) counting follicles ≥3 mm. For dynamics, cows classified as very low AFC (VL-AFC ≤15; n=20) or high AFC (H-AFC ≥45; n=20); intermediate AFC excluded. Synchronization protocol (dynamics study): Day 0: ear implant with 3 mg norgestomet (Crestar) + 2 mg estradiol benzoate IM (Gonadiol). Day 8: implant removal; administer 250 µg cloprostenol (PGF2α; Ciosin), 300 IU eCG (Novormon), and 1.0 mg estradiol cypionate (ECP). Ultrasound assessments on Days −7, 0, 5, 8, 9, 10, 18. Measurements: follicle diameters (Days 5, 8, 9, 10); AFC (Days −7, 5, 18); ovary area/diameter (Day 5); CL diameter (Day 18). Dominant follicle defined as ≥8 mm on Day 10 exceeding others. Ovulation verified by disappearance of mapped dominant follicle within 12 h checks from Day 10 and CL presence 7 days later. Blood sampled via jugular after CL measurement; serum stored at −20 °C. Progesterone assayed by RIA (Immunotech RIA IM1188); sensitivity 0.1 ng/mL; intra-assay CVs 3.0% (24.240 ng/mL) and 0.1% (0.001 ng/mL). TAI pregnancy rate (field): 1,428 cows received similar hormonal management except intravaginal P4 device (DIB) used Days 0–8. Insemination at 48 h post-device removal by one technician using semen from four known-fertility bulls. Study II (heifers): Ethics approved (FMVZ/USP 8968070518). Conducted at USP Pirassununga in November. Nelore heifers (23–27 months), BCS 3.5–4.5. Day 0: 1 mg estradiol benzoate (Sincrodiol) + intravaginal P4 device (Sincrogest). Day 5: device removal and AFC by ultrasonography (≥3 mm follicles). Selected L-AFC (lowest 10; ≤29 follicles) and H-AFC (highest 10; ≥60 follicles). On Day 5, performed transvaginal OPU under epidural anesthesia; aspirated all antral follicles with 20 G needle under 80–90 mmHg. Collected COCs and granulosa cells, filtered, and selected COCs (Grades I–III). Separated cumulus cells and denuded oocytes; stored pools of 10 oocytes or cumulus from 10 COCs per animal at −80 °C. Molecular analyses: RNA extracted with TRI reagent; DNase-treated; cDNA synthesized (High Capacity cDNA kit). qPCR via Fluidigm 96.96 microfluidic platform using TaqMan assays for 95 target genes (intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis, cell stress/response). Preamplification (14 cycles), fivefold dilution, then RT-qPCR (standard protocol). Reference genes evaluated (RPL15, GAPDH, PPIA) with GeNorm; PPIA selected. Relative expression calculated by 2^−ΔΔCT. Statistics: Normality (Anderson–Darling or Shapiro–Wilk), homogeneity (Levene). Parametric data: two-tailed Student’s t-test; nonparametric: Mann–Whitney. Pregnancy rate: logistic regression with AFC group as main effect and herd, BCS, bull as covariates. Data reported as mean ± SEM or %. Significance P≤0.05 unless stated; trends noted where applicable.

Study I: Compared to VL-AFC, H-AFC cows had greater AFC (50.3±1.2 vs 13.2±0.9; P<0.0001), ovary diameter (30.0±0.1 vs 21.1±0.1 mm; P<0.0001), ovary area (73.8±0.3 vs 39.2±0.3 mm²; P<0.0001), and AFC per ovary area (7.0±0.3 vs 3.8±0.4 follicles/mm²; P<0.0001). Dominant follicle diameters were larger in VL-AFC at Day 8 (11.5±0.3 vs 9.6±0.4 mm; P=0.001), Day 9 (12.5±0.3 vs 10.7±0.4 mm; P=0.001), and Day 10 (13.6±0.3 vs 12.2±0.4 mm; P=0.008). Preovulatory follicle diameter trended larger in VL-AFC (14.6±0.3 vs 13.5±0.5 mm; P=0.06). P4 on Day 18 trended higher in VL-AFC (2.9±0.3 vs 2.1±0.3 ng/mL; P=0.06). BCS, ovulation time, and CL diameter did not differ. Pregnancy rates (TAI; n=1,428): VL-AFC (≤15): 57.9% (374/646); L-AFC (16–30): 53.1% (214/403); I-AFC (31–44): 54.9% (116/211); H-AFC (≥45): 45.2% (76/168). Pregnancy rate differed by AFC group (P=0.001), with H-AFC lowest. Herd (P=0.73), BCS (P=0.48), and bull (P=0.18) had no effect. Study II: Heifer AFC groups: L-AFC 24.0±4.7 (14–29), H-AFC 72.3±15.7 (60–107). Number of retrieved COCs lower in L-AFC (14.1±8.2) vs H-AFC (43.2±18.3; P≤0.05); retrieval percentage similar (58.8%±21.3 vs 56.6%±26.8; P>0.05). Differential gene expression: In oocytes (L-AFC vs H-AFC), 8 genes upregulated (e.g., BMP15, HAS2, EGFR, NPR3, H1FOO, IGFBP2, GAPDH, TFAM) and 3 downregulated (e.g., HSF1, PRDX3, XBP1). In cumulus cells, 21 genes upregulated (e.g., EGFR, NPR2, NPR3, DNMT3A, HDAC2, PAF1, CDK6, PA2G4, CASP9, STAT3, XBP1, HSP90AA1, HSPA5, HSPD1, SOD1; ATPL5, TFAM, PFKP; IGF1R, GSK3A) and 6 downregulated (e.g., FSHR; ARO, PTGS2; GAPDH, SREBF1, NFKB2). Patterns indicate enhanced intercellular communication, meiotic control, epigenetic modulation, cell division, stress response in L-AFC cumulus cells, with lower steroidogenesis-related genes.

Findings support the hypothesis that lower AFC in Nelore is associated with more favorable follicular dynamics during TAI (larger dominant/preovulatory follicles) and improved fertility, as evidenced by higher pregnancy rates compared to high AFC cows. Larger dominant follicles at TAI are known to correlate with estrus expression, ovulatory rate, luteal function, and pregnancy probability; the trend toward higher P4 after ovulation in VL-AFC aligns with improved luteal function, despite similar CL size, consistent with indicine-specific luteal characteristics where progesterone secretion capacity is not strictly size-dependent. Molecularly, low AFC heifers exhibited upregulation of genes in oocytes and cumulus cells involved in intercellular signaling (EGFR, BMP15, HAS2; NPR2/NPR3), meiotic control and epigenetic modulation (H1FOO; DNMT3A, HDAC2, PAF1), cell cycle (CDK6, PA2G4), metabolic maintenance and mitochondrial function (ATPL5, TFAM, PFKP), and cellular stress response (HSP90AA1, HSPA5, HSPD1, SOD1). Reduced expression of steroidogenesis-related genes (ARO, PTGS2) and FSHR in cumulus cells of L-AFC suggests distinct follicular environmental regulation. Together, these physiological and transcriptional differences provide a mechanistic basis for higher TAI fertility in low-AFC Bos indicus females and underscore subspecies differences compared to Bos taurus literature where low AFC often associates with poorer fertility.

Very low AFCs in Nelore cows were associated with larger dominant follicle diameters at TAI, a tendency toward higher progesterone concentrations post-ovulation, and higher pregnancy rates in TAI programs. Nelore heifers with low AFCs displayed distinct oocyte and cumulus cell gene expression patterns indicating enhanced intercellular communication, meiotic control, epigenetic modulation, cellular adaptation and stress response, and follicular growth. These physiological and molecular profiles likely contribute to improved reproductive outcomes in low-AFC Bos indicus under TAI.

The study notes that some differences were trends rather than statistically significant (e.g., preovulatory follicle diameter and serum progesterone, P=0.06). Gene expression analyses were limited to oocytes and cumulus cells at a single time point (Day 5), without granulosa/theca assessments, and further investigation is needed to clarify specific mechanisms. Comparisons with Bos taurus studies are limited due to subspecies differences and differing cell types analyzed. Field fertility results, while controlled for herd, BCS, and bull, reflect specific management and environmental conditions.