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The Journal of Reproduction and Development logoLink to The Journal of Reproduction and Development
. 2025 May 8;71(3):175–184. doi: 10.1262/jrd.2024-075

Percentage of follicle number by size over the antral follicle count and its association with subsequent reproductive performance in beef cattle

Shiori Saito KOHIGASHI 1,2, Mizuho UEMATSU 3, Go KITAHARA 2, Takeshi OSAWA 2
PMCID: PMC12151638  PMID: 40335322

Abstract

This study aimed to clarify the association between the percentage of follicle number by size over antral follicle count (AFC) and subsequent reproductive performance. A total of 306 Japanese Black cattle underwent timed artificial insemination (TAI) 41–62 days postpartum; the AFC and numbers of small, medium, and large follicles were recorded 10 days before TAI. The cross-sectional and blood flow areas of the dominant follicle (DF) on the day of TAI and the corpus luteum (CL) six days after TAI were recorded. The total number of follicles ≥ 2 mm was defined as the AFC, and the percentages of follicle number by each size defined as small (S-AFC%; 2–2.9 mm), medium (M-AFC%; 3–8.4 mm), and large (L-AFC%; ≥ 8.5 mm) follicles. The AFC and S-, M-, and L-AFC% were further grouped into low, medium, and high tertiles, and the subsequent reproductive performance compared among the groups. Plasma anti-Müllerian hormone (AMH) levels were quantified on the day of AFC measurement. No differences were observed in reproductive performance between the AFC and L-AFC% groups. The high-S-AFC% group showed a 20.6% lower conception rate, 0.58 more AI numbers, and 21.9 longer days open than those of the low-S-AFC% group (P < 0.05). The low-M-AFC% group showed an 18.0% lower conception rate after TAI and 0.54 more AI numbers than those of the high-M-AFC% group (P < 0.05). DF and CL parameters did not differ among the AFC, S-, M-, and L-AFC% groups. Plasma AMH levels in the low-AFC group were the lowest in the tertile. In conclusion, the percentage of follicles by size could be used to estimate subsequent reproductive performance.

Keywords: Antral follicle count, Japanese Black cattle, Percentage of follicle number by size, Reproductive performance

The ability to predict the fertility of each cow in the field would greatly assist in improving overall herd productivity by maintaining high-fertility cows and replacing low-fertility cows early. Ultrasound technology facilitates the noninvasive, real-time monitoring of bovine reproductive organs. This equipment enables detailed assessments of the number of follicles, whereas color Doppler techniques provide further insight into the functionality of follicles and the corpus luteum (CL) by visualizing blood flow patterns. Bovine follicles ≥ 2 mm, which can be observed via ultrasonography, are called antral follicles, and the total number of follicles observed in the right and left ovaries called the antral follicle count (AFC). AFC, along with anti-Müllerian hormone (AMH), is considered an indicator of ovarian reserve, and its association with fertility has been previously investigated. The AFC is highly repeatable in cows and can be assessed at any stage of the estrous cycle [1]. However, no consensus exists as to whether cows with a high AFC are more fertile. Holstein cows with a high AFC have a higher probability of pregnancy by 200 days postpartum (dp) and higher conception rates at the first service [2], whereas cows with a high AFC have a shorter productive herd life and lower reproductive performance [3]. Morotti et al. [4] hypothesized that one reason for this disagreement between studies may be attributable to the different grouping methods used for determining the AFC. They examined whether the relationship between AFC and conception rate after timed artificial insemination (TAI) varied according to groupings, such as the mean ± standard deviation, quartiles, and scoring. They observed that conception rates were improved in the low-AFC group for all grouping methods tested. Therefore, the reasons underlying the aforementioned disagreement among researchers remain unclear. Furthermore, the effect that the number of follicles between 2.0 and 2.9 mm in diameter has on reproductive performance has yet to be elucidated despite the existence of several reports in literature demonstrating the relationship between the number of antral follicles ≥ 2 or ≥ 3 mm and fertility [1,2,3, 5]. In addition, information on the association between the number of dominant follicles (DF) whose diameter is > 8.5 mm [6] and fertility is scarce.

Long-term administration of gonadotropin-releasing hormone (GnRH) agonists is known to suppress follicular development in cattle owing to the decreased responsiveness of gonadotropic cells in the anterior pituitary and suppression of plasma gonadotropin secretion [7]. GnRH agonist-induced inhibition of follicle development results in an increase in the number of small follicles (2–5 mm) and a decrease in the number of follicles > 5 mm [8]. Thus, the number of follicles by size represents the developmental status of follicles. However, if we only evaluate the AFC, we may end up considering cows that have ovaries with different follicular development to be of the same fertility. We hypothesized that this may be due to the inconsistent results obtained among various research groups. Therefore, evaluating the number of follicles by size, which represents the composition of each size in the AFC, is crucial. In contrast, the number of follicles of each size is influenced by the AFC, which is the total number of follicles present. On average, approximately 45 and 48% of the AFC comprise follicles with sizes of 2–3 and 4–6 mm, respectively, in Hereford crossbreeds [9]. Even if the cows have a large number of small follicles and possibly many suppressed follicles, this may simply be associated with a high AFC, and they could thus be healthy. We assumed that if the number of follicles by size is affected by the AFC, the ratio of each size is more likely to reflect the composition of the AFC rather than the number itself.

Previous studies on the relationship between the AFC and fertility vary among different cattle breeds, with some reporting higher conception rates with a high AFC in the Holstein breed [2] and others reporting better conception rates after TAI with a low AFC in the Nellore breed [10]. Additionally, the AFC was higher in Bos indicus than in B. taurus [11]. However, the relationship between the AFC and conception ability in Japanese Black cattle has yet to be elucidated.

The Doppler function of mobile ultrasound equipment has enabled the observation of blood flow distribution in bovine follicles and the CL. The atretic follicle is characterized by a lack of detectable blood flow, and the ovulatory follicle has a greater percentage of follicle wall-blood flow [12, 13]. In Holstein cows, greater blood flow to the DF and blood flow percentage in the follicle wall at 26 h after GnRH administration were previously observed in the pregnant group compared with those in the nonpregnant group [14], and a greater area of DF blood flow observed immediately before TAI [15]. Considering the CL, the blood flow area after TAI in the pregnant group was larger than that in the nonpregnant group of Japanese Black cattle [16], whereas no differences were observed in luteal size and blood flow between pregnant and nonpregnant Holstein cows [15]. Furthermore, the size of the ovulatory follicle and that of the CL that follows it affect the conception rate after AI [16, 17]. Therefore, we assumed that clarifying the relationship between follicular parameters (follicle size and blood flow area) at TAI or the luteal parameters (luteal size and blood flow area) formed after TAI and AFC, or the composition ratio of follicle number by size, would help elucidate whether the AFC and follicle number by size are associated with subsequent reproductive performance.

Our objectives were to determine whether the AFC or percentage of follicles by size could indicate subsequent reproductive performance and to clarify their association using follicular and luteal parameters in beef cattle.

Materials and Methods

Animals

This study was approved by the Ethics Committee on Animal Experimentation of the University of Miyazaki (Approval No. 2021-036) and conducted between September 2021 and March 2023. A total of 306 Japanese Black cows from the same farm in Miyazaki Prefecture were used. Fresh checks were conducted 30–47 dp, normal uterine involution confirmed, and ovulation synchronization treatment initiated within five days. The mean number ± standard deviation (SD) (range) of parity, age, and dp were 4.0 ± 2.0 (1–8), 5.3 ± 2.2 (1–9) years old, and 50.2 ± 4.5 (41–62) at TAI, respectively. Calves were weaned at 7 days of age. The body condition score (BCS) was recorded in nine grades [18], and the average BCS of the cows was 5.5 ± 0.9 (3–8).

TAI protocol

All cows, regardless of whether their ovarian cycle had resumed, underwent an ovulation synchronization protocol (CIDR-synch) at a random estrous cycle stage 31–52 dp. On day 0 of the protocol, 1 mg estradiol benzoate (Estradiol Injection “KS”; Kyoritsu Seiyaku Corporation, Tokyo, Japan) was intramuscularly administered, and an intravaginal progesterone device (CIDR 1900; Zoetis Japan, Tokyo, Japan) inserted. The CIDR was removed on day 7, and 25 mg dinoprost (Pronargon F Injection for Veterinary Use; Zoetis Japan) intramuscularly administered. After 48–50 h, 100 μg fertirelin acetate (Fertirelin Injection “Fujita”; Sasaeah Pharmaceutical Co., Ltd., Tokyo, Japan) was intramuscularly administered. Then, TAI was conducted 16–20 h after GnRH administration. Pregnancy diagnosis was conducted 42 ± 3 days after TAI via ultrasound (HS-101V; Honda Electronics Co., Ltd., Aichi, Japan) (Fig. 1).

Fig. 1.

Fig. 1.

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Experimental design. EB, PGF2α, and GnRH were intramuscularly administered. AFC, antral follicle count; AMH, anti-Müllerian hormone; EB, estradiol benzoate; PGF2α, prostaglandin F2α; GnRH, gonadotropin releasing hormone; TAI, timed artificial insemination; CIDR, intravaginal progesterone release device; US, transrectal ultrasonography; DF, dominant follicle; CL, corpus luteum.

Measurement of the AFC

On day 0, an ultrasound device (MyLab One VET; Esaote, Genova, Italy) equipped with a 10-MHz transrectal linear probe was used to record end-to-end sectional images along the long axis of both ovaries, and the AFC counted in the laboratory for follicles ≥ 2 mm. The same veterinarian (K.S.S.) performed all ovarian imaging and AFC measurements. Follicles were classified and counted as small (2.0–2.9 mm), medium (3.0–8.4 mm), and large (≥ 8.5 mm), with each classification designated as small-AFC (S-AFC), medium-AFC (M-AFC), and large-AFC (L-AFC), respectively. S-AFC, M-AFC, and L-AFC were each divided by AFC, expressed as percentiles, and defined as small-AFC% (S-AFC%), medium-AFC% (M-AFC%), and large-AFC% (L-AFC%), respectively. AFC, S-AFC%, M-AFC%, and L-AFC% were categorized into low, medium, and high tertiles (Figs. 2A–D). The average AFC, S-AFC%, M-AFC%, and L-AFC% values were 42.4 ± 18.8 (range: 6–120), 37.4 ± 15.1 (range: 0–91.8%), 59.3 ± 15.2 (range: 6.1–94.4%), and 3.32 ± 2.87% (range: 0–16.7%), respectively.

Fig. 2.

Fig. 2.

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Cow number distribution and grouping in AFC, percentage of follicle number by size, and illustration of AFC, S-AFC%, M-AFC%, and L-AFC% by group. (A) Histogram and grouping of AFC: (B) S-AFC%, (C) M-AFC%, and (D) L-AFC%. The vertical axis shows the number of cattle, and colored partial circles the percentage of follicle number by size. * The ovaries in (B) and (C) have the same AFC but with a different breakdown. AFC, antral follicle count; S-AFC%, small follicle (2.0–2.9 mm) number/AFC × 100 (%); M-AFC%, medium follicle (3.0–8.4 mm) number/AFC × 100 (%); L-AFC%, large follicle (≥ 8.5 mm) number/AFC × 100 (%).

Reproductive performance

Reproductive performance data were presented for all 306 cows regardless of their first ovulation and for 274 cows that had a CL >10 mm at the start of the TAI protocol and were considered to have their first ovulation [19]. The conception rate was calculated as follows: number of conceived cows/number of TAI cows × 100 (%). Days open referred to the number of days from the day of calving to that of AI resulting in conception and was calculated for 282 cows, excluding 24 cows wherein AI was discontinued as they were either being diverted to fattening (11 cows) or submitted for embryo transfer (13 cows). The pregnancy rate at 150 dp was calculated as the number of animals pregnant at 150 dp/total number of animals × 100 (%) (150 d-PR). AI numbers were calculated for 252 cows, excluding 30 of the 282 cows that had moved to farms and thus did not have AI records available. A total of 282 cows had conceived by March 2023.

Area and blood flow area of the dominant follicle and corpus luteum

Ovarian blood flow images and the DF diameter were recorded immediately before TAI on day 10, and those of the CL recorded on day 16, using the power Doppler method (MyLab One VET) for 231 cows that were a subset of the cows confirmed to have their first ovulation postpartum at the start of the experiment. Follicles ≥ 8.5 mm were considered the DF [6], and blood flow then observed in the largest of these follicles. For follicles, 176 cows were used for data analysis; 55 cows were not used for follicle parameter analysis for the following reasons: 17 cows did not have a DF, blood flow could not be determined owing to body movement or straining for 38 cows, and one cow had two ovulations such that the ovulatory follicle was unclear. For the CL, 219 cows were used for data analysis; 12 cows were not used for CL parameter analysis as blood flow could not be determined owing to body movement or straining. The ultrasound imaging settings used were as follows: B-mode frequency, 10 MHz; B gain, 100%; ambient light, 3; power Doppler frequency, 5.0 MHz; PRF color, 500 Hz; color map, 2; C correlation, 4; and power Doppler gain, 50%. The images were analyzed in the laboratory using Fiji software (ImageJ 1.54f; National Institutes of Health, Bethesda, MD, USA) [20]. Videos of blood flow around the DF and CL were recorded using power Doppler, and both the follicles and CL then analyzed using still images by cutting out the moment when the blood flow area was largest at the point where the follicle had the largest cross-sectional area [21]. The DF area, DF blood flow area, and perifollicular blood flow percentage were measured for the follicles, whereas the luteal and luteal blood flow areas measured for the CL. Perifollicular blood flow percentage represents the percentage of the follicle circumference covered by blood flow. The CL area was defined as the area of the CL, excluding that of the cavity, if present. All areas were measured in mm2. As a prerequisite, we initially divided the follicular and luteal parameters into three groups (low, medium, and high) and assessed whether differences in conception rate among the groups could be observed. We then investigated whether differences could be observed in mean values of the follicular/luteal parameters among the AFC and S-, M-, and L-AFC% tertile groups.

AMH assays

To measure AMH levels, 93 cows were randomly selected to ensure an approximately equal distribution of cows in the low, medium, and high AFC and S-, M-, and L-AFC% groups. Blood was collected from the jugular vein on day 0 for AMH measurements using an ethylenediamine tetraacetic acid-2K vacuum blood collection tube (Venoject II vacutainer blood collection tube: VP-DK050K; TERUMO, Tokyo, Japan) and centrifuged at 1550 × g for 15 min. Plasma was stored at −20°C until measurement. Plasma AMH from 93 cattle was quantified using a three-step sandwich immunoassay with a commercially available enzyme-linked immunosorbent assay kit (AL-114; Ansh Labs, Webster, TX, USA), according to the manufacturer’s instructions [22]. The intra- and inter-assay coefficients of variation were both 8%. The minimum concentration detected was 11 pg/ml.

Statistical analysis

EZR statistical software was used for data analysis [23]. Data are expressed as mean ± SD. The Shapiro–Wilk test was used to confirm normality. Initially, Spearman’s rank correlation coefficient was used to calculate correlations between the AFC and number of follicles of each size and between the AFC and percentage of follicles of each size to determine whether they were affected by the AFC. Correlations between S-AFC and M-AFC and between S-AFC% and M-AFC% were also assessed. The AFC and percentages of small, medium, and large follicles of each size were then divided into tertiles (low, medium, and high) to compare the conception rates after TAI and subsequent reproductive performance. Fisher’s exact probability test was used to compare the conception rates after TAI, 150d-PR, the proportion of cows without their first ovulation, the proportion of cows without DF on day 10, and the proportion of cows without CL on day 16 among the low, medium, and high groups in the AFC and S-, M-, and L-AFC% groups. This test was also used for comparisons of conception rates between cows that had their first ovulation or not and the DF/CL parameter groups. The Kruskal–Wallis test was used to compare the days open, AI number, AMH levels, DF, and CL parameters among the low, medium, and high groups in the AFC and S-, M-, and L-AFC% groups. Statistical significance was set at P < 0.05, and the significance levels corrected using the Bonferroni method.

Results

Correlation between the AFC and percentage of follicle number by size

Figure 3 shows correlations between the AFC and each of the items assessed. A strong positive correlation (P < 0.01) was observed between AFC and S-AFC or M-AFC; no correlation was observed between AFC and L-AFC (Figs. 3A1–3), and a very weak correlation observed between AFC and S-AFC% (P < 0.05). AFC and M-AFC% were not correlated; however, AFC and L-AFC% were (P < 0.01) (Figs. 3B1–3), with S-AFC and M-AFC showing a weak positive correlation and S-AFC% and M-AFC% showing a strong negative correlation (P < 0.01) (Figs. 3C1–2).

Fig. 3.

Fig. 3.

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Correlations between AFC and the number or percentage of follicle size. (A1–3) Correlations between AFC and (A1) S-AFC, (A2) M-AFC, and (A3) L-AFC. (B1–3) Correlations between AFC and (B1) S-AFC%, (B2) M-AFC%, and (B3) L-AFC%. (C1–3) Correlations between M-AFC and (C1) S-AFC, (C2) S-AFC%, and (C3) M-AFC%. The correlation coefficient r and P-values are indicated in the upper-right corner of each graph. S-, M-, and L-AFC%: S-, M-, and L-AFC were each divided by AFC and expressed as percentiles. AFC, antral follicle count; S-AFC, small follicle (2.0–2.9 mm) number; M-AFC, medium follicle (3.0–8.4 mm) number; L-AFC, large follicle (≥ 8.5 mm) number.

Comparison of reproductive performance using the AFC and percentage of follicle number by size

The conception rate after TAI did not differ between cows that had their first postpartum ovulation by day 0 and those that did not (145/274: 52.9% vs. 12/32: 37.5%; P = 0.13). The percentage of cows without their first ovulation was higher in the high AFC group than in the low-AFC group (P < 0.01) but did not differ among the S-, M-, and L-AFC% tertile groups (Table 1A).

Table 1. Percentage of cows without their first ovulation, DF, or CL by AFC and S-, M- and L-AFC% groups.

(A) Percentage of cows without their first ovulation
low medium high
AFC 4.0% a (4/100) 9.7% ab (10/103) 17.5% b (18/103)
S-AFC% 14.7% (15/102) 7.8% (8/102) 8.8% (9/102)
M-AFC% 7.9% (8/101) 8.9% (9/101) 14.4% (15/104)
L-AFC% 15.7% (16/102) 8.0% (8/100) 7.7% (8/104)
(B) Percentage of cows without DF on day 10
low medium high
AFC 8.7% (6/69) 7.1% (6/84) 6.4% (5/78)
S-AFC% 2.6% a (2/77) 3.9% ab (3/77) 15.6% b (12/77)
M-AFC% 16.0% a (12/75) 3.9% ab (3/76) 2.5%b (2/80)
L-AFC% 6.7% (5/75) 5.6% (4/72) 9.5% (8/84)
(C) Percentage of cows without CL on day 16
low medium high
AFC 3.0% (2/66) 7.4% (6/81) 5.6% (4/72)
S-AFC% 4.2% (3/72) 2.8% (2/72) 9.3% (7/75)
M-AFC% 9.6% (7/73) 2.8% (2/71) 4.0% (3/75)
L-AFC% 8.6% (6/70) 4.3% (3/69) 3.8% (3/80)
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Each category was further divided into low, medium, and high tertiles for comparison. Cows without CL on day 0 did not ovulate by the start of the examination. Cows without DF on day 10 did not undergo DF immediately prior to AI. a,b Different superscripts in the same row indicate significant differences between groups (P < 0.05). DF, dominant follicle; CL, corpus luteum; AFC, total follicle number of both the left and right ovary; S-AFC%, small follicle (2.0–2.9 mm) number/AFC × 100 (%); M-AFC%, medium follicle (3.0–8.4 mm) number/AFC × 100 (%); L-AFC%, large follicle (≥ 8.5 mm) number/AFC × 100 (%).

No differences were observed in the reproductive performance of any of the cows assessed, even for those that had their first ovulation in the AFC group (Fig. 4). The low-AFC group had lower AMH levels than those in the medium- and high-AFC groups, and the medium-AFC group had lower AMH levels than those in the high-AFC group for all cows (623.2 ± 297.6 vs. 1069.0 ± 468.7 vs. 1432.5 ± 455.8, respectively; P < 0.01), which were similar to those measured in cows that had their first ovulation (571.2 ± 253.0 vs. 1017.2 ± 465.4 vs. 1371.8 ± 466.2, respectively; P < 0.05).

Fig. 4.

Fig. 4.

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Comparison of conception rates after TAI, subsequent reproductive performance, and plasma AMH levels based on AFC, S-AFC%, M-AFC%, and L-AFC%. Each category was further divided into low, medium, and high tertiles for comparison. The conception rate was calculated as the number of conceived/TAI animals × 100 (%). Day 0 was defined as the day when the AFC and the proportion of follicles of each size were counted at 36–52 days postpartum, and when ovulation synchronization treatment was initiated. * P < 0.05. AFC, total follicle number of both the left and right ovary; S-AFC%, small follicle (2.0–2.9 mm) number/AFC × 100 (%); M-AFC%, medium follicle (3.0–8.4 mm) number/AFC × 100 (%); L-AFC%, large follicle (≥ 8.5 mm) number/AFC × 100 (%); Days open, number of days from calving to conception; 150 d-PR, number of animals pregnant at 150 days postpartum/total animals × 100 (%); Number of AI, number of AIs per conception; AMH, anti-Müllerian hormone.

The high-S-AFC% group had a lower conception rate (P < 0.05) after TAI than the low-S-AFC% group did for all cows, including those that had their first ovulation (Fig. 4). The high-S-AFC% group had longer days open than the low-S-AFC% group did (95.8 ± 56.7 vs. 73.9 ± 43.9; P < 0.01) for all cows. For cows that had their first ovulation, the high-S-AFC% group had longer days open than the medium- and low-S-AFC% groups did (94.8 ± 54.5, 78.4 ± 55.3, and 73.9 ± 44.1, respectively; P < 0.05). No differences in the 150 d-PR were observed among any of the groups. AI numbers were higher in the high-S-AFC% group than in the low-S-AFC% group for all cows (2.12 ± 1.41 vs. 1.54 ± 1.05; P < 0.01), including those that had their first ovulation (2.08 ± 1.38 vs. 1.50 ± 1.02; P < 0.01). AMH levels did not differ among the three groups for all cows, including those that had their first ovulation.

The low-M-AFC% group had a lower conception rate after TAI than the high-M-AFC% group did for all cows, including those that had their first ovulation (P < 0.05) (Fig. 4). The low-M-AFC% group had significantly longer days open than the high-M-AFC% group did for cows that had their first ovulation (92.3 ± 53.2 vs. 72.2 ± 40.3; P < 0.05); however, no difference was observed for all cows. The AI number was higher in the low-M-AFC% group than in the high-M-AFC% group for all cows (2.02 ± 1.05 vs. 1.48 ± 0.89; P < 0.01) and for those that had their first ovulation (1.97 ± 1.30 vs. 1.45 ± 0.87; P < 0.01). The 150 d-PR and AMH levels did not differ among the three groups.

No differences were observed in the conception rate after TAI, days open, 150 d-PR, or AI number of the L-AFC% group for all cows, including those that had their first ovulation (Fig. 4), whereas AMH levels were lower in the high-L-AFC% group than in the low- and medium-L-AFC% groups for all cows (P < 0.05) and for those that had their first ovulation (P < 0.01).

Comparison of conception rates between cows used for CL or DF analysis and those that were not

No differences in the conception rate was observed between cows with and without DF on day 10 (23.5% vs. 55.7%; P = 0.06). The cows that had not been used for DF parameter analysis due to body movement or straining had a conception rate similar to that of cows used for DF analysis (55.7% vs. 42.1%; P = 0.46). Cows not used for CL parameter analysis due to body movement or straining had a conception rate similar to those used for CL analysis (33.3% vs. 52.1%; P = 0.25).

Comparison of the proportion of cows without DF on day 10 and those without CL on day 16 in the AFC and S-, M-, and L-AFC% tertiles

The proportion of cows without DF on day 10 was higher (P < 0.05) in the high-S-AFC% group than in the low-S-AFC% group. The low-M-AFC% group had a higher proportion of cows without DF (P < 0.05) than that in the high-M-AFC% group. The AFC and L-AFC% groups did not differ in the proportion of cows without DF (Table 1B). In addition, no difference in the proportion of cows without CL was observed on day 16 among the AFC and S-, M-, and L-AFC% tertile groups (Table 1C).

Comparison of the follicular and luteal parameters among the low, medium, and high tertiles of the AFC and S-, M-, and L-AFC% groups

Follicular and luteal parameters among the low, medium, and high tertiles of the AFC and S-, M-, and L-AFC% groups were compared. No differences were observed in the DF area for the low, medium, and high tertiles in terms of AFC, S-AFC%, and M-AFC%. The DF area was smaller (P < 0.05) in the low-L-AFC% group than in the medium-L-AFC% group. The DF blood flow area and perifollicular blood flow percentage did not differ among the low, medium, and high tertiles of the AFC and S-, M-, and L-AFC% groups. The CL area in the low-L-AFC% group was smaller (P < 0.05) than that in the high-L-AFC% group. No differences were observed in the CL area among the AFC, S-AFC%, and M-AFC% groups. The CL blood flow area was similar among the AFC and S-, M-, and L-AFC% groups (Supplementary Table 1).

Comparison of conception rates among the low, medium, and high tertiles of the follicular and luteal parameters

The follicular and CL parameters were categorized into low, medium, and high tertiles (Fig. 5). No differences were observed in the conception rate among the groups for the follicular area, follicular blood flow area, and perifollicular blood flow percentage in the low, medium, and high groups. The conception rate in the low-CL area group was lower (P < 0.05) than that in the medium-CL area group but did not differ from that in the high-CL area group. The conception rate categorized by the CL blood-flow-area group did not differ among the groups (Fig. 5).

Fig. 5.

Fig. 5.

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Comparison of the conception rate after TAI between the follicular or CL parameter groups. Blue and yellow colors indicate the follicular and CL parameters, respectively. Numbers below the bars indicate the range of each group. TAI, timed artificial insemination; DF, dominant follicle; CL, corpus luteum.

Discussion

The present study demonstrates that the proportion of small and medium follicle counts is an appropriate indicator for assessing the composition ratio of follicular number by size in AFC (AFC breakdown). This is because the number of small- and medium-sized follicles was affected by the AFC, whereas the percentage of small- and medium-sized follicles was not. Furthermore, the percentage of large follicle counts was difficult to use as an indicator of AFC breakdown. The reason for this is that L-AFC ranged from 0–3 in all cows, regardless of the AFC; L-AFC% was divided by AFC, and L-AFC% thus depended on the AFC amount.

No relationship was observed between the AFC and conception rate or subsequent reproductive performance after TAI. In contrast, cows with S-AFC% > 42.7% or M-AFC% < 54.5% exhibited low reproductive performance. The results of our experiment suggest that it is not possible to evaluate the conception ability of a cow using AFC, and that it is necessary to consider the composition ratio of follicular number by size in AFC. Additionally, because S-AFC% and M-AFC% were negatively correlated in our study, the high-S-AFC% and low-M-AFC% groups could be considered to have similar ovarian conditions. In humans, AFC is used to diagnose polycystic ovarian syndrome (PCOS) [24]. In PCOS, AFC and blood AMH levels increase owing to the presence of many small follicles because of residual stunted follicles or ovulatory dysfunction. This characteristic ovarian sign, known as the necklace sign, results from the suppression of follicular growth [25]. Pregnancy rates have been reported to be nearly 20% lower in patients with PCOS than in healthy humans and 45% lower in spontaneous pregnancies [26]. PCOS-like cases have been reported in beef crossbreeds [27]. In contrast, AFC and AMH levels are indicators of ovarian reserve but do not reflect oocyte quality and are not necessarily related to pregnancy rates [28, 29]. Mossa et al. concluded that in cattle, although AFC and AMH levels reflect the ovarian reserve capacity in B. taurus, it is as difficult to predict fertility using these markers as it is to do so in humans [30]. During follicle growth suppression treatment in cows, the treated group had fewer medium and large follicles (>5 mm) and more small follicles (2–5 mm) than those detected in the control group, and follicle growth was limited to 2–4 mm [8]. This was similar to the ovaries observed for cows with low reproductive performance in the present study. In a model of follicular growth suppression, the long-term effect of GnRH reduced the responsiveness of the anterior pituitary gland, and follicle-stimulating hormone (FSH) levels were no longer transiently elevated [7]. Therefore, the responsiveness of the anterior pituitary gland to GnRH may have been reduced in the cows with high-S-AFC% and low-M-AFC%. In the present study, no differences were observed in the proportion of cows with CL, size of CL, or CL blood flow area on day 16 among the different AFC% groups. However, the reduced responsiveness of the anterior pituitary gland might have led to premature regression of the CL, although ovulation was induced by LH surge after exogenous GnRH administration, resulting in embryo mortality and reduced fertility. However, further research is required to confirm this finding.

In the present study, the percentage of cows without DF immediately before TAI (day 10) was higher in the high-S-AFC% and low-M-AFC% groups than in the other groups. Failure of follicular development may have resulted in the poor reproductive performance observed. In humans with PCOS, follicles fail to develop into DF, resulting in ovulatory dysfunction [31]. In contrast, the percentage of cows without CL on day 16 was similar among the low, medium, and high tertiles of the AFC and S-, M-, and L-AFC% groups. These data included cows that did not have a DF (all follicles were < 8.5 mm); however, some of them may have ovulated and formed a CL. This is because if the follicle diameter is ≥8.0 mm, ovulation could possibly occur [32, 33]. Therefore, this difference was not observed because the difference in the proportion may have become smaller when comparing the presence or absence of the CL rather than when comparing the presence or absence of the DF.

In cows with DF on day 10, no difference in the mean follicular/luteal phase parameters among the AFC and S-, M-, and L-AFC% tertile groups were observed, except for follicle size. Even when follicular growth suppression occurs, the primary issue may be the failure of follicles to develop into DF rather than the DF size after selection or the size of the CL formed after ovulation.

In this study, the low-AFC group had lower plasma AMH levels than those in the medium- and high-AFC groups. Additionally, L-AFC%, which is strongly affected by AFC, also showed different plasma AMH levels among the groups. This is consistent with previous reports stating that blood AMH levels are positively correlated with AFC [34]. Plasma AMH levels were correlated with the number of follicles <5 mm and not with the number of follicles ≥ 5 mm [35]. As our study was based on percentages rather than numbers, no notable difference in AMH levels between the S-AFC% and M-AFC% groups were observed.

No marked differences were observed in conception rates among the tertile groups (low, medium, and high) for the DF area, follicular blood flow area, and perifollicular blood flow percentage before TAI. In our study, the follicles had few areas of blood flow, and there may not have been enough differences in the area to affect the conception rate of each group. The medium-CL area group had higher conception rates than the low-CL area group did. This is consistent with previous reports of greater CL areas in a pregnant group than in a nonpregnant group [16]. As CL size correlates with blood progesterone concentration, pregnancy is more likely to be established [36].

As AFC shows less variation within the estrous cycle than between individuals and is repeatable, it can be used as an indicator of fertility at any time during the estrous cycle [1]. However, the results have been inconsistent regarding whether the number of follicles of each size changes during the estrous cycle [37, 38]. In addition, no reports have been made on changes in the percentage of follicles by size (S-AFC%, M-AFC%, and L-AFC%) during a normal estrous cycle. In future studies, daily changes in S-AFC%, M-AFC%, and L-AFC% should be observed within at least two estrous cycles to confirm the variation in proportions and repeatability among individuals during the estrous cycle. Further research is required to determine whether S-AFC% and M-AFC% can be used as indicators of fertility at any stage of the estrous cycle in cows. If these indicators can be used effectively to select cows, the productivity of an entire herd could be improved.

In conclusion, the percentage of follicle number by size, particularly of small and medium follicles, over antral follicles in postpartum beef cows is likely related to subsequent reproductive performance.

Conflict of interests

The authors have no conflicts of interest to declare.

Supplementary

Supplement Table
jrd-71-175-s001.pdf (150.3KB, pdf)

Acknowledgments

This study was supported by a grant from the Japan Association for Livestock New Technology in 2022 (Issue No. 20). We thank Dr. Iwakuma Akihiro from Zoetis for supplying the hormonal medicine. We are grateful to the students of the Laboratory of Theriogenology at the University of Miyazaki and the staff of the experimental farm.

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Supplement Table
jrd-71-175-s001.pdf (150.3KB, pdf)

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