Superovulatory response, anti-Müllerian hormone concentration and antral follicle count in Holstein cattle with short or long anogenital distance.

To understand the mechanisms underlying the inverse relationship between anogenital distance (AGD) and fertility in dairy cattle, we determined if embryo yield and quality differed between cattle of short- and long-AGD (Study 1), and whether anti-Müllerian hormone (AMH), antral follicle count (AFC) and superovulation responses differed by AGD group (Study 2). In Study 1, records of embryo yield and quality in Holstein heifers (n = 43) and cows (n = 14) from two commercial dairy herds were analyzed, retrospectively. Short- and long-AGD groups were based on the median AGD cut-points of 113 and 134 mm, for heifers and cows, respectively. The mean numbers of total (7.4 vs. 9.0; 9.4 vs.12.9), fertilized ova (5.6 vs. 6.2; 6.6 vs. 6.9), and viable embryos (4.4 vs. 4.8; 5.9 vs. 5.3) per heifer and cow did not differ between cattle of short vs. long AGD. Short-AGD cows, however, had greater proportions of fertilized ova (69.7 vs. 53.3%; P = 0.07) and viable embryos (62.1 vs. 41.1%; P = 0.03) than long-AGD cows. The odds of short-AGD cows yielding fertilized ova and viable embryos were 2.0 and 4.0 times greater, respectively, than the odds of long-AGD cows. In Study 2, lactating cows (n = 24) of a research herd had their AGD categorized as in Study 1, and AFC, AMH, and superovulation responses (i.e., no. of preovulatory follicles [≥ 10 mm] and CL) were determined. Mean AFC (27 ± 4.5 vs. 21 ± 4.0) and CL (9 ± 1.6 vs. 7 ± 1.4) per cow did not differ between short- and long-AGD groups. Serum AMH concentration (pg/mL) was lesser in short-AGD cows compared with long-AGD cows (114 ± 30.4 vs. 200 ± 26.8; P = 0.05), but the no. of preovulatory follicles was greater (15 ± 1.9 vs. 8 ± 1.7; P = 0.01) in short-AGD than in long-AGD cows. The overall proportion of follicles ≥10 mm was also greater in short-AGD cows than in long-AGD cows (56 vs 44%; P = 0.03). Preovulatory follicle number was affected by a parity x AGD group interaction (P = 0.04), with multiparous short-AGD cows accounting for the largest number of follicles. No associations were found among AGD, AFC and AMH. The associations between AFC and superovulation responses (follicles: r = 0.67, and CL: 0.58; P < 0.01) were moderate but AMH was not associated with superovulation responses. In summary, whereas AGD-associated differences in the yield and quality of embryos were not evident in heifers, the proportions of fertilized ova and viable embryos were greater in short-AGD than in long-AGD cows. In addition, the proportion of preovulatory follicles, an indicator of superovulatory response, was greater in short-AGD cows than in long-AGD cows.

Anogenital distance is associated with postpartum estrous activity, intensity of estrous expression, ovulation, and progesterone concentrations in lactating Holstein cows.

The objectives of this retrospective observational study were to determine the associations of anogenital distance (AGD) with (a) postpartum estrous activity, (b) diameter of the preovulatory follicle, (c) intensity of estrous expression, (d) postestrus ovulation, (e) corpus luteum (CL) size, and (f) concentrations of progesterone at estrus and on d 7 after estrus. Lactating Holstein cows (n = 178; 55 primiparous, 123 multiparous) were enrolled into the study during the first postpartum week. All cows were continuously monitored by a pedometer-based automated activity monitoring (AAM) system for estrus. Postpartum estrous activity was assessed using the AAM estrus alerts, in which cows with at least one true estrus alert (i.e., a relative increase in steps from each cow's baseline detected by the AAM and the presence of at least one follicle >15 mm, a CL <20 mm, or no CL detected by ultrasound) by the first 50 d in milk (DIM) were considered to have commenced estrous activity. At the estrus alert >60 DIM, ovulation was determined by ultrasound at 24 h, 48 h, and 7 d after estrus, and blood samples were collected at estrus alert and on d 7 after estrus for progesterone analysis. The AGD was measured from the center of the anus to the base of the clitoris and classified as either short- or long-AGD using 2 cut-points of 148 mm (predictive of the probability of pregnancy to first insemination; short-AGD, n = 115; long-AGD, n = 63) and 142 mm (the median AGD; short-AGD, n = 90; long-AGD, n = 88). Regardless of the cut-point used, early postpartum estrous activity by 50 DIM (67 vs. 54%), duration of estrus (11.6 vs. 9.7 h), and preovulatory follicle diameter (20 vs. 19 mm) were greater in short-AGD than in long-AGD cows. Increased peak of activity at estrus in short-AGD cows (354 vs. 258% mean relative increase) was affected by an interaction between AGD and parity in which multiparous long-AGD cows had lesser relative increase in activity than primiparous cows (217 vs. 386%, respectively). Mean progesterone concentration at estrus was lesser in short-AGD (0.47 vs. 0.61 ng/mL) than in long-AGD cows. The ovulatory response at 24 h did not differ, but at 48 h (91 vs. 78%) and on d 7 after estrus (97 vs. 84%) it was greater in short-AGD cows. Although CL diameter on d 7 after estrus did not differ, short-AGD cows had greater progesterone concentration 7 d after estrus than long-AGD cows (4.1 vs. 3.2 ng/mL, respectively). In conclusion, greater proportions of short-AGD cows commenced estrous activity by 50 DIM, had larger preovulatory follicles, exhibited greater duration of estrus, had reduced progesterone concentration at estrus, had greater ovulation rates and progesterone concentration 7 d after estrus compared with long-AGD cows, with no difference in CL size between AGD groups. Because all the differences in physiological characteristics of short-AGD cows reported herein favor improved reproductive outcomes, we infer that these are factors contributing to improved fertility reported in short-AGD cows compared with long-AGD cows.

Associations between anogenital distance and measures of fertility in lactating North American Holstein cows: A validation study.

Anogenital distance (AGD) has been defined in dairy cows as the distance from the center of the anus to the base of the clitoris. Initial reports on nulliparous Holstein heifers and first- and second-parity Holstein cows have found inverse relationships between AGD and measures of fertility. Our primary objective was to determine the relationship between AGD and measures of fertility in a larger population of North American Holstein cows to validate our previous finding that AGD is inversely related to fertility. Secondary objectives were to determine the associations between AGD and parity, and milk yield. Using digital calipers, we measured AGD in 4,709 Holstein cows [mean ± standard deviation (SD); parity 2.3 ± 1.4; days in milk (DIM) 154 ± 94; 305-d mature equivalent (ME) milk yield 13,759 ± 2,188 kg] from 18 herds in Western Canada and 1 herd in the USA. Anogenital distance (mm) was normally distributed with a mean (±SD) of 132 ± 12, ranging from 95 to 177, and a median of 133. Anogenital distance was linearly but inversely associated with pregnancy to first artificial insemination (P/AI1). For every 1-mm increase in AGD, the estimated probability of P/AI1 decreased by 0.8%. The optimum AGD cut-point that predicted probability of P/AI1 with sensitivity and specificity of 45 and 55%, respectively, was 129 mm. Consequently, data were categorized into either short (≤129) or long (>129) AGD groups across parities, and associations between AGD, parity (first, second, and third+), and fertility measures were determined. Rates of P/AI1 were greater (36 vs. 30%) in short- than in long-AGD cows; short-AGD cows required fewer AI per conception (2.3 vs. 2.4) and had fewer days open (137 vs. 142), and a greater proportion of short-AGD cows (67 vs. 64%) was pregnant by 150 DIM compared with long-AGD cows. The rates of pregnancy up to 150 (hazard ratio of 0.91) and 250 DIM (hazard ratio of 0.93) were smaller in long- than in short-AGD cows. Anogenital distance had a weak positive association with both parity (r = 0.22) and 305-d ME milk yield (r = 0.04). Results indicate an inverse relationship between AGD and measures of fertility in lactating cows, validating our earlier report. We infer that although selecting cows for short AGD is expected to have an adverse effect on milk yield, the anticipated gain in fertility will outweigh the small decline in milk yield, strengthening the potential of AGD as a novel reproductive phenotype for use in future breeding programs to improve fertility.

Repeatability of anogenital distance measurements from birth to maturity and at different physiological states in female Holstein cattle.

The inverse association between anogenital distance (AGD; the distance from the center of the anus to the base of the clitoris) and fertility, its moderate heritability, and high variability reported in dairy cattle make AGD a promising candidate for further exploration as a reproductive phenotype. In addition to heritability, repeatability (i.e., consistency in measurements taken at different time points) is important for a reproductive phenotype to be considered useful in genetic selection. Therefore, our primary objective was to determine the repeatability of AGD from birth to breeding age (≈16 mo) in Holstein heifer calves, and during different stages of the estrous cycle, gestation, and lactation in Holstein cows. We also determined the associations among AGD, height (at the hip), and body weight (BW) at birth. In calves (n = 48), we recorded BW (kg) and height (cm) at birth and measured AGD (mm) at approximately 0, 2, 6, 9, 12, and 16 mo of age. In cows, AGD was measured at different stages of the estrous cycle (proestrus, estrus, metestrus and diestrus; n = 20), gestation (30, 90, 180, and 270 d; n = 78), and lactation (30-300 d in milk in 30-d increments; n = 30). Calf height and BW at birth had a weak positive association with AGD at birth. The AGD increased linearly from birth to breeding age, but there was no association between the AGD at birth and at breeding age in heifers. Although any 2 consecutive AGD measurements were correlated, 6 mo was the earliest age at which AGD was moderately correlated (r = 0.41) with that of breeding-age heifers. The AGD was neither influenced by the different stages of estrous cycle nor lactation and remained highly repeatable (r ≥ 0.95). Although AGD measurements at 30, 90, and 180 d of gestation (126.9, 126.7, and 127.7 mm, respectively) were strongly correlated (r ≥ 0.97) with each other, AGD at 270 d of gestation (142.8 mm) differed from AGD at all earlier stages of gestation. In summary, AGD measured at birth did not reflect AGD at breeding age in heifers, but AGD measurements in cows had high repeatability at all stages of the estrous cycle, gestation, and lactation, except at 270 d of gestation. Therefore, AGD could be measured reliably at any of the aforesaid physiological states in cows due to its high repeatability, except during late gestation. The earliest gestational stage when pregnancy-associated increase in AGD occurred, however, could not be definitively established in the present study.

Relationship of anogenital distance with fertility in nulliparous Holstein heifers.

Anogenital distance (AGD), defined as the distance from the center of the anus to the base of the clitoris, in lactating dairy cows of first and second parity, has been reported to be inversely related to fertility and moderately heritable. Thus, AGD may be a useful reproductive phenotype for future genetic selection to improve fertility. The objectives of this study were to (1) characterize AGD in nulliparous dairy heifers; and (2) determine if the inverse relationship between AGD and fertility, found in lactating dairy cows, is also evident in nulliparous heifers. We measured AGD in 1,692 Holstein heifers from 16 herds in Western Canada (Alberta and British Columbia) and one herd in the United States (Washington State). Data were analyzed using MEANS, UNIVARIATE, LOGISTIC, ROC, GLIMMIX, and LIFETEST procedures of SAS (SAS Institute Inc.). Mean (±standard deviation) age at AGD measurement was 13.9 ± 1.5 mo, and AGD was normally distributed with a mean of 107.3 ± 10.5 mm, ranging from 69 to 142 mm. With every 1-mm increase in AGD, the predicted probability of pregnancy was reduced by 1.9%. Receiver operating characteristic curve analysis was used to determine the optimum threshold AGD that predicted the probability of pregnancy. Based on the optimum threshold AGD, data from heifers were categorized into short (≤110 mm) and long (>110 mm) AGD groups, and associations between AGD groups and fertility measures were determined. Heifers with short AGD required fewer services per conception (1.5 vs. 1.7) than heifers with long AGD. Consequently, heifers with short AGD conceived earlier (448.4 vs. 454.3 d) and had greater pregnancy to first AI than those with long AGD (58.3 vs. 49.6%). Moreover, heifers with long AGD had reduced hazard (hazard ratio of 0.59) for pregnancy up to 450 d of life compared with those with short AGD. In summary, AGD was normally distributed and highly variable in the population. In addition, an inverse relationship between AGD and fertility measures in nulliparous heifers was evident, confirming an earlier report of a similar relationship in lactating dairy cows. These findings strengthen the potential for AGD to be used as a fertility trait and management tool in future selection programs.

Carryover effects of pre- and postweaning planes of nutrition on reproductive tract development and estrous cycle characteristics in Holstein heifers.

The objectives of this study were to investigate the carryover effects of differing pre- and postweaning planes of nutrition on prepubertal reproductive tract development and postpubertal estrous cycle characteristics in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low or a high (5 or 10 L of whole milk/d, respectively) preweaning diet from 1 to 7 wk of age and either a low or a high (70 or 85% of concentrate dry total mixed ration, respectively) postweaning diet from 11 to 25 wk of age. Starting at wk 26 of age, heifers were subjected to weekly transrectal ultrasonography until wk 33 or until first ovulation to assess endometrial thickness and ovarian follicular count and size in the prepubertal phase. In a subset of heifers (n = 28), ovarian ultrasonography continued weekly until at least the second ovulation was confirmed; thereafter, ovarian dynamics (through ultrasonography) and blood progesterone (P4) were assessed every 2 d throughout 1 complete estrous cycle in the postpubertal phase. In the prepubertal phase, endometrial thickness (12.0 ± 0.4 vs. 10.8 ± 0.3 mm) and largest follicle size (11.8 ± 0.3 vs. 10.9 ± 0.2 mm) were greater in heifers fed the high postweaning diet than in those fed the low postweaning diet. Furthermore, the number of class 2 (6-9 mm) follicles was greater in heifers fed the high preweaning diet than in those fed the low preweaning diet (1.6 ± 0.1 vs. 1.1 ± 0.1), whereas the number of class 3 (>9 mm) follicles was greater in heifers fed the high postweaning diet than in those fed the low postweaning diet (1.2 ± 0.1 vs. 1.0 ± 0.1). In the postpubertal phase, overall corpus luteum and P4 dynamics did not differ among pre- or postweaning treatments; however, P4 at 4 d preceding luteolysis was lesser in heifers fed the high postweaning diet than in those fed the low postweaning diet (6.1 ± 0.4 vs. 7.7 ± 0.4 ng/mL). In addition, compared with heifers fed the low postweaning diet, those fed the high postweaning diet had a greater number of antral follicles (31.4 ± 2.2 vs. 21.4 ± 2.3) and tended to have more class 3 follicles (3.6 ± 0.3 vs. 2.7 ± 0.3). Results indicate positive carryover effects of increasing the preweaning plane of nutrition from 5 to 10 L of whole milk/d on prepubertal follicular growth in Holstein heifers. Furthermore, an increased postweaning plane of nutrition (85 vs. 70% of concentrate dry total mixed ration) advanced reproductive development through greater endometrial thickness and follicular growth in the prepubertal phase and increased the population of antral follicles in the postpubertal estrous cycle.