Genomic merit for reproductive traits. II: Physiological responses of Holstein heifers

Fertility traits were recently added to the evaluation of genetic merit, allowing for the selection of Holstein cattle with improved reproductive performance. In the current study, we investigated the associations among genomic merit for daughter pregnancy rate (GDPR) and heifer conception rate (GHCR) and physiological responses during proestrus and diestrus. Holstein heifers (n = 99) were classified based on GDPR [high = 3.26 ± 0.76 (1.6 to 5.3), n = 48; low = ?0.17 ± 0.75 (?1.8 to 1.0), n = 51] and GHCR [high = 2.75 ± 0.77 (1.5 to 5.5), n = 49; low = 0.06 ± 0.67 (?2.1 to 1.2), n = 50]. Heifers were fitted with an automated estrous detection device, were treated with PGF_2α for synchronization of estrus, and received either artificial insemination or embryo transfer at detected estrus. Blood was sampled at the time of PGF_2α treatment, within 24 h of the onset of estrus (d 0), and on d 7, 14, 19 ± 2, 28, and 35. Blood samples from all heifers were analyzed for concentrations of estradiol (d 0) and progesterone (on the day of PGF_2α treatment and d 0, 7, and 14). Blood samples from heifers pregnant on d 38 ± 3 were analyzed for concentrations of progesterone (d 0, 7, 14, 19 ± 2, 28, and 35), pregnancy-specific protein B (d 19 ± 2, 28, and 35), and insulin-like growth factor 1 (d 0, 7, 14, 19 ± 2, 28, and 35). Expression of mRNA for interferon-stimulated gene 15 in peripheral leukocytes isolated from blood collected on d 19 ± 2 was determined. Ovaries were scanned by ultrasound daily from d 0 to 4 or until ovulation was detected. Heifers with low GHCR tended to be less likely to be detected in estrus (78.0 vs. 91.8%). Estradiol concentration on d 0 was greater for heifers with high GDPR (4.53 ± 0.23 vs. 3.79 ± 0.23 pg/mL). The ovulatory follicle was larger for heifers with high GDPR (16.28 ± 0.33 vs. 14.55 ± 0.35 mm), whereas heifers with high GHCR tended to have smaller ovulatory follicles (15.00 ± 0.31 vs. 15.83 ± 0.37 mm). Heifers with high GDPR tended to be more likely to ovulate within 96 h of the onset of estrus (90.7 vs. 75.0%). Among heifers pregnant on d 38 ± 3, GDPR and GHCR were not associated with mRNA expression for interferon-stimulated gene 15. Heifers with high GDPR had greater concentration of pregnancy-specific protein B from d 28 to 35 (3.03 ± 0.15 vs. 2.48 ± 0.1 ng/mL). Heifers with high GHCR tended to have greater insulin-like growth factor 1 concentration from d 7 to 35 (108.0 ± 3.2 vs. 97.7 ± 4.2 ng/mL). Our results suggest that selection for Holstein cattle for GDPR may have positive effects on reproductive performance through changes in ovarian follicle development and steroidogenesis. Although selection of Holstein cattle for GHCR may negatively affect estrous expression by affecting ovarian follicle growth, selection for GHCR may improve reproductive performance by altering the somatotropic axis.     

Estrous characteristics and reproductive outcomes of Holstein heifers treated with 2 prostaglandin formulations and detected in estrus by an automated estrous detection or mounting device

Dinoprost tromethamine (DIN), a molecule similar to endogenous PGF_2α, has a half-life of approximately 9 min. Cloprostenol sodium (CLO), a synthetic analog of PGF_2α, has a half-life of approximately 3 h. We hypothesized that treatment of Holstein heifers with CLO would improve estrous detection rate, estrous characteristics, service rate, and overall reproductive performance compared with DIN. Currently in the United States, heifers are largely inseminated based on signs of estrus, which is detected visually or with the aid of mounting detection devices (MD). Automated estrous detection devices (AED) are becoming more accessible to producers, but it is not clear whether they present advantages in the reproductive management of heifers. Therefore, we hypothesized that the use of an AED would improve service and pregnancy rates compared with detection of estrus with the aid of a MD. Holstein heifers (n = 1,019) were enrolled in the experiment at 10 to 11 mo of age, when they were fitted with a Heatime HR LD System (SCR Ltd., Netanya, Israel). At 12 mo of age, we paired heifers according to estrous cycle phase and randomly assigned them to treatments in a 2 × 2 design: PGF_2α formulation (CLO vs. DIN) and estrous detection treatment (AED vs. MD). Heifers in the AED treatment were detected in estrus only by the Heatime HR LD System, whereas heifers in the MD treatment were detected in estrus only by the Kamar Heatmount Detector (Kamar Products Inc., Zionsville, IN). Treatments with the same PGF_2α formulations were repeated 14 d after the first treatment if heifers had not been detected in estrus. A sub-group of heifers had blood sampled on the day of PGF_2α treatment and within 24 h of onset of estrus to determine progesterone and estradiol concentrations. Treatment with CLO reduced the progesterone concentration within 24 h of onset of estrus compared with DIN (0.04 ± 0.01 vs. 0.11 ± 0.01 ng/mL). Among heifers in mid diestrus on the day of PGF_2α treatment, CLO reduced the interval to estrus compared with DIN (72.0 ± 2.2 vs. 82.4 ± 2.4 h). Prostaglandin F_2α formulation and estrous detection treatment did not affect pregnancy to the first service. The interval between the first and second services tended to be reduced for the AED treatment compared with the MD treatment (24.4 ± 0.5 vs. 25.7 ± 0.6 d). Prostaglandin F_2α formulation and estrous detection treatment did not affect the hazard of pregnancy. Although CLO treatment may shorten the interval to estrus in heifers at mid diestrus compared with DIN, PGF_2α formulation did not affect reproductive performance. In the current experiment, no advantages in reproductive performance were observed when estrous detection was based on an AED compared with a MD.     

Evaluation of luteolysis,follicle size,and time to ovulation in Holstein heifers treated with two different analogs and doses of prostaglandin-F2α

Objectives were to evaluate the effect of 2 analogs of PGF_2α (cloprostenol vs. dinoprost) and 2 doses (1 injection vs. 2 injections) on luteolysis, follicle diameter, hormonal concentrations, and time to ovulation in dairy heifers. Holstein heifers were fitted with automated estrus detection devices and had their estrous cycle synchronized using PGF_2α and an intravaginal insert containing progesterone. Heifers detected in estrus were blocked by weight and randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement: cloprostenol on d 7 after estrus (CLOx1; n = 45), cloprostenol on d 7 and 8 after estrus (CLOx2; n = 41), dinoprost on d 7 after estrus (DINx1; n = 43), or dinoprost on d 7 and 8 after estrus (DINx2; n = 44). Treatment with the first injection of PGF_2α was defined as experiment d 0. Area and blood flow of corpus luteum (CL) and diameter of follicles >5 mm were recorded every 12 h from d 0 to estrus and every 6 h thereafter until ovulation. Blood was sampled every 6 h from d 0 until ovulation. Heifers treated with cloprostenol had shorter interval to luteolysis (± SEM; CLOx1 = 23.5 ± 2.2, CLOx2 = 22.9 ± 2.2, DINx1 = 32.6 ± 2.7, DINx2 = 26.4 ± 2.1 h); however, time to ovulation was not affected by treatment. A smaller proportion of heifers treated with a single injection of PGF_2α underwent luteolysis compared with heifers treated with 2 injections (CLOx1 = 84.6 ± 6.2, CLOx2 = 100.0 ± 0.0, DINx1 = 59.7 ± 9.8, DINx2 = 96.3 ± 2.7%). Proportion of heifers that ovulated was smaller for DINx1 compared with other treatments (CLOx1 = 88.8 ± 5.1, CLOx2 = 100.0 ± 0.0, DINx1 = 55.2 ± 9.7, DINx2 = 94.4 ± 3.4%). Ovulatory follicle diameter was larger for DINx1 (18.2 ± 2.7 mm) compared with DINx2 (17.4 ± 2.7 mm), whereas dose did not affect the diameter of the ovulatory follicle in heifers treated with cloprostenol (CLOx1 = 17.6 ± 2.7 vs. CLOx2 = 17.8 ± 2.8 mm). Among heifers that underwent luteolysis, progesterone concentrations from 18 to 36 h after treatment were lesser in heifers treated with cloprostenol compared with those treated with dinoprost. Type of PGF_2α did not affect progesterone concentrations past 36 h from treatment; however, heifers treated with 2 PGF_2α injections had lesser progesterone concentrations and CL blood flow from 36 to 72 h after treatment compared with heifers that received a single PGF_2α injection.     

Reproductive performance of replacement dairy heifers submitted to first service with programs that favor insemination at detected estrus,timed artificial insemination,or a combination of both

Our objective was to compare the insemination dynamics and time to pregnancy for up to 100 d after the beginning of the artificial insemination period (AIP) for heifers managed with first artificial insemination (AI) service programs that relied primarily on insemination at detected estrus (AIE) after PGF_2α treatments, timed artificial insemination (TAI), or a combination of both. Holstein heifers were randomly assigned to receive first AI service with sex-selected semen after 368 ± 10 d of age with (1) AIE after synchronization of estrus with up to 3 PGF_2α treatments every 14 d starting on the first day of the AIP (PGF+AIE; n = 317). Heifers not AIE up to 9 d after the third PGF_2α received a 5-d Cosynch protocol with progesterone supplementation [GnRH + controlled internal drug release insert (CIDR)–5 d–CIDR removal and PGF_2α–3 d–GnRH and TAI] before TAI. Heifers detected in estrus from CIDR removal and PGF_2α until the day before TAI received AIE with no GnRH treatment; (2) 2 PGF_2α treatments 14 d apart with the second treatment at the beginning of the AIP (PGF+TAI; n = 334). Heifers received AIE for up to 9 d after the second PGF_2α treatment. Heifers not AIE received TAI after the 5-d Cosynch protocol and (3) TAI after the 5-d Cosynch protocol (ALL-TAI; n = 315). Heifers failing to conceive to a previous AI received a subsequent AI with conventional semen at detected estrus or TAI after the 5-d Cosynch protocol. Binomial outcomes were analyzed by logistic regression, whereas time to AI and pregnancy were analyzed with Cox's regression. The hazard of first AI up to 45 d of the AIP was greater for ALL-TAI than for PGF+AIE [hazard ratio (HR) = 1.72; 95% confidence interval (CI) =1.45 to 2.03] and PGF+TAI (HR = 1.51; 95% CI = 1.28 to 1.77), but similar for PGF+AIE and PGF+TAI (HR = 1.14; 95% CI = 0.97 to 1.33). A greater proportion of heifers received AIE in PGF+AIE (98.7%) than in PGF+TAI (78.5%). Overall, first service pregnancy per AI did not differ (PGF+AIE = 42.0%; PGF+TAI = 47.3%, ALL-TAI = 43.8%). Time to pregnancy was reduced for ALL-TAI compared with PGF+AIE (HR = 1.20, 95% CI = 1.02 to 1.42), but was similar to that of PGF+TAI (HR = 1.13, 95% CI = 0.96 to 1.33). Time to pregnancy did not differ for PGF+AIE and PGF+TAI (HR = 1.07, 95% CI = 0.91 to 1.25). Median days to pregnancy were 27, 23, and 21 for heifers in PGF+AIE, PGF+TAI, and ALL-TAI, respectively. We concluded that an ALL-TAI program for first service reduced time to pregnancy, albeit a relatively small reduction, when compared with a program that relied primarily on AIE after induction of estrus with PGF_2α treatments. The program that combined synchronization of estrus and TAI (PGF+TAI) resulted in similar time to pregnancy than the predominant TAI and predominant AIE programs.     

Effect of breeding protocols and reproductive tract score on reproductive performance of dairy heifers and economic outcome of breeding programs

The objectives of this study were to evaluate the effect of reproductive protocols and reproductive tract score on reproductive performance of dairy heifers and economic outcomes of breeding programs. Holstein heifers (n = 534), 13 ± 1 mo of age, were randomly assigned to 1 of 4 reproductive protocols. On the day of enrollment (d 0), heifers were palpated per rectum and received a score according to the maturity of their reproductive tract (1 = prepubertal; 2 = peripubertal; and 3 = puber-tal). Estrous detection-control heifers (CON, n = 146) received no treatment and were inseminated on detection of estrus for 28 d. Prostaglandin F_2α-treated heifers (PGED, n = 137) received 1 injection of PGF_2α on d 0 and were inseminated on detection of estrus; heifers not in-seminated by d 14 received a second injection of PGF_2α and were observed for estrus and artificial insemination (AI) for an additional 14 d. Heifers enrolled in the estrous detection-timed AI (EDTAI, n = 140) treatment received a controlled internal drug-release (CIDR) insert on d 0, and 7 d later, the CIDR was removed and all heifers received an injection of PGF_2α, heifers received AI on detection of estrus, and those not inseminated by 72 h after PGF_2α received an injection of GnRH concurrent with AI. Heifers in the GnRH-timed AI (GTAI, n = 111) treatment received 1 injection of GnRH on d 0, on d 6 heifers received a CIDR insert and injections of GnRH and PGF_2α, on d 13 the CIDR was removed and heifers received an injection of PGF_2α, and 48 h later all heifers received an injection of GnRH and AI. Pregnancy was diagnosed at 32 ± 3 and 62 ± 3 d after AI. Cost of reproductive protocols and their economic outcomes were calculated for a 28 d period beginning at enrollment. Heifers in the PGED treatment were inseminated at a faster rate than CON heifers. A smaller proportion of prepubertal and peripubertal heifers were inseminated within 14 d of enrollment compared with pubertal heifers. Pregnancy per AI of CON and PGED heifers was greater compared with EDTAI and GTAI heifers. Proportion of GTAI heifers pregnant at the end of the 28-d breeding program was or tended to be smaller compared with PGED and CON heifers, respectively. Heifers in the CON and PGED treatments had the smallest cost per pregnancy followed by heifers in the EDTAI and GTAI treatments, respectively. When different scenarios were evaluated, however, the mean cost per pregnancy was smallest for PGED heifers. Cost per pregnancy generated was greatest for prepubertal heifers, whereas pubertal heifers had the smallest cost per pregnancy generated. Treatment of dairy heifers with PGF_2α every 14 d until insemination and pregnancy results in the best economic outcomes, and screening heifers according to RTS may prove beneficial to identify heifers that may not be pubertal and would have compromised reproductive and economic performance in a breeding program.     

Short communication: Reproduction outcomes in dairy heifers following a 14-d progesterone insert presynchronization protocol

The objectives were to evaluate pregnancy per artificial insemination (AI), days to first AI, and proportion pregnant within 7 d of AI eligibility in dairy heifers subjected to presynchronization compared with dairy heifers not presynchronized. Thirty days before AI eligibility, Holstein heifers were assigned randomly to 1 of 3 groups: 14-d controlled internal drug release (CIDR; containing progesterone) presynchronization, PGF_2α presynchronization, or control (no presynchronization). Heifers in the 14-d CIDR presynchronization treatment (n = 119) received a CIDR on d ?30, which was removed on d ?16, followed by an injection of PGF_2α upon entry to the breeding program (d 0). Heifers in the PGF_2α presynchronization treatment (n = 118) received an injection of PGF_2α on d ?11 and d 0. Control heifers (n = 121) were not presynchronized and received an injection of PGF_2α on d 0. All heifers received tail paint on d 0 to facilitate once-daily detection of estrus (based on paint removal). Heifers detected in estrus received AI with conventional semen on the same morning as detected estrus. Generalized linear mixed models were used to assess mean treatment differences. Following PGF_2α treatment on d 0, more heifers were detected in estrus in the first 7 d after eligibility in the 14-d CIDR group (95.8%) compared with the PGF_2α (74.6%) and control (66.9%) groups. Days to first AI differed between treatments (14-d CIDR = 3.6 d vs. PGF_2α = 5.0 d vs. control = 6.8 d). Pregnancy per AI for first AI within 7 d of eligibility was 71.9% (14-d CIDR), 58.0% (PGF_2α), and 61.7% (control), and differed between 14-d CIDR and PGF_2α heifers. Presynchronization with a 14-d CIDR increased the proportion of heifers pregnant in the first 7 d of eligibility (14-d CIDR = 68.9% vs. PGF_2α = 43.2% vs. control = 41.3%). Projected days on feed (d 0 to projected calving date) were 295 (14-d CIDR), 302 (PGF_2α), and 305 (control), and were different between the 14-d CIDR and control heifers. The potential economic benefit to the producer was $15.85 per heifer presynchronized with a 14-d CIDR protocol compared with the control group. Treatment of dairy heifers with a 14-d CIDR effectively presynchronized estrus, resulting in a greater proportion detected in estrus, reduced days to first AI, and an increased proportion of heifers pregnant within the first 7 d after breeding eligibility compared with heifers presynchronized with a single PGF_2α injection and control heifers.     

Progesterone, Follicular, and Estrual Responses to Progesterone-Based Estrus and Ovulation Synchronization Protocols at Five Stages of the Estrous Cycle

The objective was to monitor changes in ovarian status in heifers exposed to a progesterone insert with or without concurrent GnRH injection. Estrus was manipulated in 283 heifers (31 breeding clusters) by administering GnRH, progesterone, and PGF_2α at 5 stages of the estrous cycle. Estrus was presynchronized with a progesterone insert (CIDR) for 7 d before PGF_2α was administered 24 h before insert removal. Successive clusters of heifers were assigned to treatments (2 heifers per treatment) on cycle d 2, 5, 10, 15, and 18. Treatments consisted of a progesterone insert (d 0) for 7 d plus: 1) PGF_2α on d 6, 24 h before insert removal (early PGF); 2) GnRH on d 0 + early PGF_2α (GnRH + early PGF); 3) PGF_2α at insert removal (late PGF); and 4) GnRH on d 0 + late PGF (GnRH + late PGF). Controls received GnRH on d 0 and PGF_2α on d 7. Ovaries were scanned by transrectal ultrasonography on d 0, 2, 7, 9, and 11 to assess follicle diameters and ovulation. Blood was collected on d 0, 2, 6, 7, 8, and 9 to quantify serum concentrations of progesterone. Insemination occurred after detected estrus or by timed artificial insemination (TAI) at 64 h after insert removal. Only 25% of 141 GnRH-treated heifers ovulated by d 2; twice as many ovulated when treatment was initiated on d 5 (46.4%) than on other cycle days (20.3%). Diameters of the largest follicle exposed to GnRH on d 0, 2, 7, or 9 did not differ regardless of whether ovulation occurred. Small treatment and stage of cycle differences in diameter of the largest follicle were detected on d 2, 7, and 9. Compared with controls, progesterone concentration was greater in all progesterone-treated heifers on d 2 and 6. Early- vs. late-PGF treatment resulted in less progesterone on d 7 and 8. Pregnancies per AI were less after TAI (44%) than after detected estrus (56%) and were less in controls than in all progesterone treatments. Heifers in which treatments were initiated on d 10 of the cycle had the most consistent (estrus vs. TAI) pregnancies per AI (65.4%) compared with other cycle days. Compared with controls, more progesterone-treated heifers ovulated by 96 h after insert removal. Application of the progesterone insert reduced variance of the interval to estrus after insert removal (or PGF_2α injection in controls) by 1.6-fold compared with controls. These results do not support the use of GnRH in a progesterone-based synchronization protocol.     

Association of estrous expression detected by an automated activity monitoring system within 40 days in milk and reproductive performance of lactating Holstein cows

The objective of this observational study was to evaluate the association of estrous expression within 40 days in milk (DIM) using a neck-mounted automated activity monitor (Heatime Pro; SCR Engineers Ltd.) with reproductive performance in lactating Holstein cows. A total of 2,077 cows (614 primiparous cows and 1,463 multiparous cows) from 5 commercial dairy farms were included in the statistical analyses. Activity data from the first 7 d after calving were excluded. An estrus event was defined as an activity change index ≥35 for more than 2 h. Cows were classified according to the number of estrus events from d 7 until d 40 postpartum into 3 categories: (1) no estrus event (Estrus0); (2) one estrus event (Estrus1), and (3) 2 or more estrus events (Estrus2). Generalized linear mixed models were used to analyze continuous and categorical data. Shared frailty models were used for time to event data. Overall, 52.7% of cows had no estrus event detected by an automated activity monitor system from d 7 until d 40 postpartum. Herd level prevalence of Estrus0 ranged from 37.5 to 58.4%. Estrous expression from d 7 until d 40 postpartum affected estrous duration and estrous intensity at first artificial insemination (AI). Cows in Estrus0 had the shortest duration (13.2 ± 0.33 h) compared with cows in Estrus1 (13.8 ± 0.36 h) and Estrus2 (14.8 ± 0.41 h). Cows in Estrus2 had a longer estrous duration at first postpartum AI compared with cows in Estrus1. Among Estrus0 cows, 46.2% had an estrus event with high intensity at first postpartum AI. Among cows in Estrus1 and Estrus2, 50.8 and 53.8% had an estrus event with high intensity at first postpartum AI, respectively. There was a significant difference between Estrus2 and Estrus0 and a tendency between Estrus0 and Estrus1. There was no difference between Estrus1 and Estrus2. For Estrus0, Estrus1, and Estrus2 cows, pregnancy per AI was 29.4, 30.9, and 37.8%, respectively. There was a significant difference between Estrus0 and Estrus2 and Estrus1 and Estrus2. There was no difference between Estrus0 and Estrus1. Estrous expression from d 7 until d 40 postpartum affected time to first AI and time to pregnancy. Compared with Estrus0 cows, cows in Estrus1 [hazard risk (HR) = 1.74] and Estrus2 (HR = 1.77) had an increased hazard of being inseminated within 100 DIM. There was no difference between Estrus1 and Estrus2. Median DIM to first AI were 70, 59, and 58 for cows in Estrus0, Estrus1, and Estrus2, respectively. Compared with Estrus0 cows, cows in Estrus1 (HR = 1.28) and Estrus2 (HR = 1.33) had an increased hazard of becoming pregnant within 200 DIM. There was no difference between Estrus1 and Estrus2. Median DIM to pregnancy were 127, 112, and 103 for Estrus0 cows, Estrus1 and Estrus2, respectively. In conclusion, cows with no estrous expression from 7 to 40 DIM had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrous activity.     

Reproductive performance and herd exit dynamics of lactating dairy cows managed for first service with the Presynch-Ovsynch or Double-Ovsynch protocol and different duration of the voluntary waiting period

The objective of this experiment was to evaluate the reproductive performance and herd exit dynamics of dairy cows managed for first service with programs varying in method of submission for insemination and voluntary waiting period (VWP) duration. Holstein cows from a commercial farm in New York were randomly allocated to receive timed artificial insemination (TAI) after the Double-Ovsynch protocol (GnRH, 7 d later PGF_2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF_2α, 56 h later GnRH, and 16 to 18 h later TAI) at 60 ± 3 d in milk (DIM) (DO60 = 458), TAI after Double-Ovsynch at 88 ± 3 DIM (DO88 = 462), or a combination of AI at detected estrus (starting at 50 ± 3 d in milk) and TAI with the Presynch-Ovsynch protocol (PGF_2α, 14 d later PGF_2α, 12 d later GnRH, 7 d later PGF_2α, 56 h later GnRH, and 16 to 18 h later TAI; PSOv = 450). Subsequent artificial insemination (AI) services were conducted at detected estrus or the Ovsynch protocol (32 ± 3 d after AI GnRH, 7 d later PGF_2α, 56 h later GnRH, and 16 to 18 h later TAI) for cows not reinseminated at detected estrus. In a subgroup of cows, cyclicity (based on progesterone concentration), uterine health (vaginal discharge and uterine cytology), and BCS were evaluated at baseline (DO60 and DO88 = 33 ± 3 DIM; PSOv = 34 ± 3 DIM), beginning of the synchronization protocol (DO60 = 33 ± 3 DIM; DO88 = 61 ± 3 DIM; PSOv = 34 ± 3 DIM), and within ?5 (PSOv) or ?10 d (DO) of the VWP end (DO60 = 50 ± 3 DIM; DO88 = 78 ± 3 DIM; PSOv = 45 ± 3 DIM). Effects of treatments were assessed with multivariable statistical methods relevant for each outcome variable. Cows in the DO88 treatment had delayed time to pregnancy during lactation (DO60 vs. DO88 hazard ratio = 1.53, 95% confidence interval = 1.32 to 1.78; PSOv vs. DO88 hazard ratio = 1.37, 95% confidence interval = 1.19 to 1.61) and, within multiparous cows, the DO88 and PSOv treatments had greater risk of leaving the herd than cows in the DO60 treatment (DO88 vs. DO60 hazard ratio = 1.49, 95% confidence interval = 1.11 to 2.00; PSOv vs. DO60 hazard ratio = 1.39, 95% confidence interval = 1.03 to 1.85). Cows in the DO88 treatment had improved uterine health, greater BCS, and reduced incidence of anovulation than cows in DO60 and PSOv; however, overall pregnancy per AI 39 ± 3 d after AI was similar for the 3 treatment groups. In summary, reproductive management strategies that led to similar average DIM to the first service (～60 d) through a combination of AI at estrus with TAI (PSOv) or all TAI (DO60) resulted in reduced time to pregnancy after calving when compared with an all TAI program (DO88) with a VWP of 88 d. Within the multiparous cow group, those that received all TAI with a VWP duration of 60 d were less likely to leave the herd than cows in the other treatments.     

Comparison of reproductive management programs for submission of Holstein heifers for first insemination with conventional or sexed semen based on expression of estrus,pregnancy outcomes,and cost per pregnancy

Our objective was to evaluate reproductive management programs for submission of Holstein heifers for first insemination with conventional or sexed semen. In experiment 1, nulliparous Holstein heifers (n = 462) were submitted to a 5-d progesterone-releasing intravaginal device (PRID)-Synch protocol [d 0, GnRH + PRID; d 5, PGF_2α − PRID; d 6, PGF_2α; d 8, GnRH + TAI] and were randomly assigned for PRID removal on d 5 or 6 of the protocol followed by timed artificial insemination (TAI) with conventional semen. Delaying PRID removal decreased early expression of estrus before scheduled TAI (0.9 vs. 12.2%), and pregnancies per AI (P/AI) did not differ between treatments. In experiment 2, nulliparous Holstein heifers (n = 736) from 3 commercial farms were randomized within farm to 1 of 3 treatments for first AI with sexed semen: (1) CIDR5 [d −6, GnRH + controlled internal drug release (CIDR); d −1, PGF_2α − CIDR; d 0, PGF_2α; d 2, GnRH + TAI]; (2) CIDR6 (d −6, GnRH + CIDR; d −1, PGF_2α; d 0, PGF_2α − CIDR; d 2, GnRH + TAI); and (3) EDAI (PGF_2α on d 0 followed by once-daily estrous detection and AI). Delaying CIDR removal decreased early expression of estrus before scheduled TAI (0.004 vs. 27.8%); however, CIDR5 heifers tended to have more P/AI at 35 (53 vs. 45 vs. 46%) and 64 (52 vs. 45 vs. 45%) days after AI than CIDR6 and EDAI heifers, respectively. Overall, CIDR5 and CIDR6 heifers had fewer days to first AI and pregnancy than EDAI heifers which resulted in less feed costs than EDAI heifers due to fewer days on feed until pregnancy. Despite greater hormonal treatment costs for CIDR5 heifers, costs per pregnancy were $16.66 less for CIDR5 than for EDAI heifers. In conclusion, delaying PRID removal by 24 h within a 5-d PRID-Synch protocol in experiment 1 suppressed early expression of estrus before TAI, and P/AI for heifers inseminated with conventional semen did not differ between treatments. By contrast, although delaying CIDR removal by 24 h within a 5-CIDR-Synch protocol in experiment 2 suppressed early expression of estrus before TAI, delaying CIDR removal by 24 h tended to decrease P/AI for heifers inseminated with sexed semen. Further, submission of heifers to a 5-d CIDR-Synch protocol for first AI tended to increase P/AI and decrease the cost per pregnancy compared with EDAI heifers.     

Impact of gonadotropin-releasing hormone administration at the time of artificial insemination on conception risk and its association with estrous expression

Cows with reduced estrous expression have compromised fertility. The aim of this study was to determine whether the administration of GnRH at the time of artificial insemination (AI) would affect ovulation rates and the fertility of animals expressing estrous behavior of lesser intensity. Cows were enrolled at the time of estrus from 3 farms (n = 2,607 estrus events; farm A: 1,507, farm B: 429, farm C: 671) and randomly assigned to receive GnRH at AI or not (control). The intensity of estrous expression, monitored through leg-mounted activity monitors, was determined using the maximum activity during estrus; estrous expression was categorized as greater or lower relative to the farm median. On farm A, cows were assessed at alert, and 24 h, 48 h, and 7 d post-alert for ovulation using ultrasonography. Pregnancy per AI was confirmed at 35 ± 7 d post-estrus for cows that were inseminated. Differences between treatments were tested using the GLIMMIX procedure of SAS. Treatment with GnRH at the time of AI increased pregnancy per AI (41.3 ± 1.6 vs. 35.7 ± 1.7%). An interaction between treatment and estrous expression on pregnancy per AI was found. Control cows with greater estrous expression had greater pregnancy per AI than those with lesser expression, whereas GnRH administration increased pregnancy per AI for cows with lesser estrous expression but not those with greater expression (GnRH, greater intensity: 43.5 ± 2.1; GnRH, lesser intensity: 37.8 ± 2.2; control, greater intensity: 42.6 ± 2.2; control, lesser intensity: 31.0 ± 2.2%). A higher proportion of cows with greater estrous expression that were administered GnRH at AI were found to ovulate by 48 h and 7 d post-estrus; however, ovulation of cows with lesser estrous expression was unaffected by GnRH administration. In conclusion, fertility of cows with reduced estrous expression may be increased using GnRH at the time of AI; however, increased ovulation rates do not seem to be the direct mechanism behind this relationship.     

Effect of transition cow health and estrous expression detected by an automated activity monitoring system within 60 days in milk on reproductive performance of lactating Holstein cows

The objective of this observational study was to evaluate the association of transition cow health and estrous expression, detected by an automated activity monitoring system (Smarttag Neck, Nedap Livestock Management), with reproductive performance in lactating Holstein cows. A total of 3,750 lactating Holstein cows (1,563 primiparous cows and 2,187 multiparous cows) from a commercial dairy farm in Slovakia calving from January 2020 until July 2021 were enrolled on an ongoing basis. Activity data were recorded from d 7 until d 60 postpartum. Within this observational period, cows were classified into 3 categories: (1) no estrus event (Estrus0), (2) 1 estrus event (Estrus1), or (3) 2 or more estrus events (Estrus2+). Transition cow health was assessed by farm personnel within the first 30 d in milk (DIM) using standard operating procedures. Generalized linear mixed models were used to analyze continuous and categorical data. Cox proportional hazard models were used for time to event data. The overall prevalence of anestrus was 20.8%. Multiparous cows had a greater risk for anestrus compared with primiparous cows [odds ratio (OR) = 1.4]. Cows with stillbirth (OR = 1.76), retained placenta (OR = 2.19), puerperal metritis (OR = 1.48), or subclinical ketosis (OR = 1.51) had a greater risk for anestrus. In addition, cows calving in summer (OR = 0.82), autumn (OR = 0.38), or winter (OR = 0.56) had a higher incidence of anestrus than cows calving in spring. Estrous expression from d 7 until d 60 postpartum was associated with estrous duration (DU) and estrous intensity at first artificial insemination (AI). Cows in Estrus0 had the shortest DU at first postpartum AI (9.4 ± 0.18 h) compared with cows in Estrus1 (10.5 ± 0.13 h) and Estrus2+ (11.4 ± 0.12 h). Cows in Estrus2+ had a longer DU at first postpartum AI compared with cows in Estrus1. For Estrus0, Estrus1, and Estrus2+ cows, pregnancy per AI at first service was 42.5%, 50.9%, and 55.4%, respectively. Estrous expression from d 7 until d 60 postpartum was associated with time to first AI and time to pregnancy. Compared with Estrus0 cows, Estrus1 [hazard ratio (HR) = 1.43] and Estrus2+ cows (HR = 1.62) had an increased hazard of being inseminated within 100 DIM. Compared with Estrus2+, Estrus1 cows had a reduced hazard of being inseminated within 100 DIM (HR = 0.89). Compared with Estrus0 cows, Estrus1 (HR = 1.24) and Estrus2+ cows (HR = 1.46) had an increased hazard of becoming pregnant within 200 DIM. Median DIM to pregnancy were 121, 96, and 92 for Estrus0, Estrus1, and Estrus2+ cows, respectively. In conclusion, cows with transition cow disorders (i.e., stillbirth, retained placenta, puerperal metritis, or subclinical ketosis) had a greater chance for anestrus compared with healthy cows. Cows in Estrus0 had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrous activity from d 7 until d 60.     

Short communication: Optimization of a timed artificial insemination program for reproductive management of heifers in Canadian dairy herds

Pregnancy per AI (P/AI) following the use of 1 of 2 timed AI (TAI) protocols and 2 different intervals between TAI and resynchronization were compared in heifers that were inseminated with either conventional or sex-sorted semen. Holstein heifers (n = 317; 527 inseminations) were submitted to a 5-d Cosynch protocol with (+) or without (–) GnRH at the time of controlled internal drug release (CIDR) insertion on d 0, CIDR removal and a single PGF_2α treatment on d 5, and TAI plus GnRH on d 8 (72 h later). Visual estrus detection (ED) was conducted on d 6 in the afternoon and d 7 in the morning and heifers observed in estrus were artificially inseminated on d 7 in the afternoon. Heifers were alternately assigned conventional or sex-sorted semen. Pregnancy was diagnosed by ultrasound 27 and 42 d after AI, and heifers diagnosed as nonpregnant were resynchronized, up to 3 times, starting on d 27 or 34 to provide an interbreeding interval of 35 or 42 d. Overall, TAI protocol had no effect on P/AI at 27 or 42 d after artificial insemination or on pregnancy loss, but P/AI following the first service tended to be higher in the –GnRH TAI group (66.3 vs. 56.8%). Pregnancy per AI at 27 d (61.9 vs. 55.5%) tended to differ between conventional and sex-sorted semen. Heifers artificially inseminated based on ED tended to have a greater P/AI (67.6 vs. 58.2%) and had decreased pregnancy loss (0.0 vs. 4.1%) than those submitted to TAI. A greater number of heifers in the –GnRH TAI protocol were artificially inseminated on ED than the +GnRH TAI protocol (21.5 vs. 13.7%). No difference in P/AI was observed between the 35- and 42-d interbreeding intervals; however, more heifers in the 42-d group were artificially inseminated based on ED than in the 35-d group (22.7 vs. 7.8%). A 5-d Cosynch+CIDR TAI protocol without the initial GnRH and with a single PGF_2α at CIDR removal is an acceptable alternative to achieve high P/AI when either conventional or sex-sorted semen is used in Holstein heifers. Breeding heifers based on detected estrus increases labor, but has the potential to increase fertility.     

Presynchronization with gonadotropin-releasing hormone does not improve fertility in Holstein heifers

Holstein dairy heifers were randomly assigned to 1 of 2 treatments to determine whether a presynchronization strategy using GnRH would improve reproductive performance after synchronization of ovulation and timed artificial insemination (TAI). Heifers (n = 82) in the first treatment (GPG) received a hormonal protocol for synchronization of ovulation and TAI (100 μg of GnRH, d 0; 25 mg of PGF_2α, d 6; 100 μg of GnRH + TAI, d 8); whereas heifers (n = 84) in the second treatment (GGPG) received GPG, but with the addition of a GnRH injection (100 μg) 7 d before initiation of the GPG protocol. The proportion of heifers receiving AI before d 8 was similar for GPG (4.9%) and GGPG (9.5%), and the proportion of heifers diagnosed pregnant 30 d after AI did not differ between treatments (45 vs. 51%, respectively). Treatment did not affect the proportion of heifers ovulating after the second GnRH injection or the proportion in which a corpus luteum regressed after treatment with PGF_2α. In conclusion, presynchronization with GnRH 7 d before initiation of synchronization of ovulation using GnRH and PGF_2α did not affect the proportion of heifers expressing estrus before TAI or improve synchronization response or fertility to the synchronization protocol.     

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AI (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AI on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF_2α and removal of the CIDR on d 5, and GnRH concurrent with timed AI on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF_2α injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF_2α did not differ between treatments and averaged 87.1%. Progesterone on the day of AI was greater for GnRH than control (0.50 ± 0.07 vs. 0.28 ± 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GnRH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF_2α and removal of the CIDR on d 5, and either GnRH 56 h after PGF_2α and AI 16 h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF_2α (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but CO S72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF_2α injection 5 d later. Moreover, extending the proestrus by delaying the final GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.     

Hot topic: Successful fixed-time insemination within 21 d after first insemination by combining chemical pregnancy diagnosis on d 18 with a rapid resynchronization program

Cattle that are not pregnant to first fixed-time artificial insemination (TAI) may be resynchronized for a second TAI if they are found nonpregnant at pregnancy diagnosis. The specific interval between first and second TAI ranges from 4 to 8 wk. The selected interval depends on the available method of pregnancy diagnosis and the efficiency of the resynchronization program. The objective of this experiment was to evaluate a pregnancy diagnosis and resynchronization system that achieved a 21-d interval between TAI. This 21-d interval approximates the natural return-to-service interval. It also enables resynchronization to be implemented within the same estrous cycle in which cattle are first inseminated. Holstein heifers were randomly assigned to a 21-d resynchronization program (21d_resynch; n = 40) or a control group in which estrus was observed for the purpose of re-insemination (control; n = 29). The 21d_resynch heifers were diagnosed for pregnancy on d 18 after a TAI (d 0) by using predetermined cut-off values for 2′-5′ oligoadenylate synthetase 1 (Oas1) gene expression in leukocytes and plasma progesterone concentration. Heifers that were not pregnant to first TAI had greater expression of Oas1 at the time of PGF_2α (d −3) than pregnant heifers, but this relationship was reversed on d 18 after TAI: the heifers that were pregnant to first TAI had almost 5-fold greater expression of Oas1 compared with nonpregnant heifers. Nonpregnant heifers in the 21d_resynch group were injected with a luteolytic dose of PGF_2α on d 19 and were injected with GnRH on d 21 and submitted to TAI. The pregnancy per AI after first insemination was similar for 21d_resynch (50.0%; pregnancy diagnosis on d 18) and control (51.7%; pregnancy diagnosis on d 27). Likewise, no difference was detected in second insemination pregnancy per AI for 21d_resynch (36.8%; nonpregnant heifers TAI on d 21) and control (35.7%; nonpregnant heifers inseminated at return to estrus or after nonpregnant diagnosis on d 27). The interval between first and second insemination was shorter for 21d_resynch compared with control (21.0 ± 0 and 27.5 ± 2.1 d). The conclusion is that a TAI resynchronization can be programmed within 21 d of previous TAI when a d 18 pregnancy test and a rapid resynchronization are used.     

Influence of energy balance and body condition on estrus and estrous cycles in Holstein heifers

The objective was to determine effects of energy balance and body condition on estrous behavior and estrous cycles in Holstein heifers. Before the experiment heifers were fed so body condition remained moderate or they became fat. During the 2 x 2 factorial experiment, moderate and fat heifers were in positive or negative energy balance. Heifers were fed individually twice daily, and energy balance was calculated daily. Heifers were observed for 30 min every 3 h, and all standing and mounting events were recorded for three consecutive estrous cycles. When progesterone in serum was less than 1 ng/ml, standing and mounting activity were profiled. Area, peak, and duration of activity profiles were examined among groups of heifers. Negative energy balance or fat body condition did not reduce peak or duration of estrous behavior and thus did not reduce detectability of estrus. Onset of diestrus was delayed in heifers that were fat and in negative energy balance relative to other heifers. We suggest that fat body condition, coincident with negative energy balance, may reduce accuracy of timing artificial insemination relative to ovulation and may consequently reduce fertility in cattle.     

Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows

Ovarian function was compared between nulliparous heifers (n = 29; 10 to 16 mo old) and lactating Holstein cows (n = 31; 55.9 +/- 3.5 d postpartum). Follicular dynamics, corpus luteum growth, and regression, and serum steroid concentrations were evaluated through ultrasonography and daily blood sampling. Most heifers (27 of 29) but only 14 of 31 cows had typical spontaneous estrous cycles after cycles were initiated. Twelve cows had atypical cycles, and 5 became anovulatory during the study. The 12 cows with atypical estrous cycles had low serum estradiol after luteolysis and failed to ovulate the dominant follicle present at luteolysis. Heifers and cows with typical cycles were compared directly. Interovulatory intervals were similar between heifers (22.0 +/- 0.4 d) and cows (22.9 +/- 0.7 d). Those animals had estrous cycles with either 2 (15 heifers; 11 cows), 3 (9 heifers; 2 cows), or 4 follicular waves (3 heifers; 1 cow). Cows ovulated later after luteolysis than heifers (5.2 +/- 0.2 vs. 4.6 +/- 0.1 d, respectively), and had more multiple ovulations (17.9 vs. 1.9%). Maximal serum estradiol concentration preceding ovulation was lower in cows than in heifers (7.9 +/- 0.8 vs. 11.3 +/- 0.6 pg/mL) even though ovulatory follicles were larger in cows (16.8 +/- 0.5 vs. 14.9 +/- 0.2 mm). Similarly, maximal serum progesterone concentration was lower for cows (5.6 +/- 0.5 vs. 7.3 +/- 0.4 ng/mL), whereas maximal volume of luteal tissue was larger for cows than heifers (11,120 +/- 678 vs. 7303 +/- 308 mm3). Thus, higher incidence of reproductive anomalies in lactating cows, such as low conception rate, ovulation failure, delayed ovulation, and multiple ovulations, may be due to lower circulating steroid concentrations in spite of larger ovulatory follicles and luteal structures.     

Factors associated with estrous expression and subsequent fertility in lactating dairy cows using automated activity monitoring

The objective of this observational study was to identify factors associated with estrous duration (DU) and intensity measured as the peak of activity (PA) change and subsequent fertility in lactating Holstein cows using a neck-mounted automated activity monitor (Heatime Pro, SCR Engineers Ltd., Netanya, Israel). Ambient temperature and relative humidity were recorded hourly to calculate the temperature-humidity index (THI). A total of 5,933 estrus events from 3,132 cows located on 8 commercial dairy farms in Germany were used for this study. Farms participated in monthly DHIA testing. Pregnancy diagnosis was performed either by transrectal palpation [farm 1: 42 ± 3 d; farm 3: 40 ± 3 d; farms 4 and 8: 38 ± 3 d; farm 5: 43 ± 3 d after artificial insemination (AI)] or transrectal ultrasonography (farms 2, 6, and 7: 30 ± 3 d after AI). Estrous intensity was categorized based on peak activity of estrus into low (35–89 index value), and high (90–100 index value) PA. Overall, 73.5% of estrus events were of high PA. The mean (± standard error of the mean) DU was 14.94 ± 0.06 h. There was a strong correlation between DU and PA (r = 0.67). In the final statistical model, only PA was associated with pregnancy per artificial insemination (P/AI), with 1.35 greater odds of pregnancy for cows with high PA compared with cows with low PA. Increased THI 1 wk before AI was associated with shorter DU, lower PA, and decreased P/AI. A small percentage of cows (4.7%) showed short interestrus intervals (i.e., more than 1 activity peak within 7 d close to the event of estrus), resulting in reduced DU, PA, and P/AI. The change of weighted rumination was associated with DU and PA, as a lower nadir was associated with a greater risk for high PA and long DU. There was no association, however, between the nadir of change of weighted rumination and P/AI. Whereas milk yield and somatic cell count from the DHIA test date before AI were negatively associated with estrous expression, neither milk yield nor somatic cell count was associated with P/AI. Surprisingly, multiparous cows expressed estrus with longer DU (13.15 ± 0.31 h) compared with primiparous cows (12.52 ± 0.32 h), whereas PA did not differ among parities. Pregnancy per AI was greater for primiparous (29.4%) than for multiparous (22.1%) cows. An estrus event with long DU or high PA was more likely later in lactation. Milk fat, milk protein, milk urea nitrogen, and lactose from the DHIA test date closest to AI had no association with estrous expression or P/AI. In conclusion, DU and PA were highly correlated, and cows with high PA were particularly associated with greater odds for pregnancy. A negative association between estrous expression and P/AI was identified for increased THI 1 wk before AI and cows with short interestrus intervals using automated activity monitor.     

Effect of estrous expression on timing and failure of ovulation of Holstein dairy cows using automated activity monitors

The aim of this study was to determine if estrous expression, as measured by an automated activity monitor (AAM), affects timing and failure of ovulation of lactating Holstein dairy cows. Cows were equipped with 2 AAM, 1 neck-mounted (AAM_C) and 1 leg-mounted (AAM_L), by 10 d postpartum and enrolled into the trial when their activity crossed the alert threshold on the AAM_C. A total of 850 episodes of estrus from 293 different cows were used for this study. When cows were enrolled, their ovaries were scanned by transrectal ultrasonography and gait and body condition scored. Ovaries of cows detected in estrus were scanned twice daily for a maximum of 3 d to determine the disappearance of the preovulatory follicle (ovulation) and the interval from estrus to ovulation was calculated. Physical activity data recorded from the AAM were used to determine estrus behavior using 2 traits: (1) peak activity and (2) duration. Peak activity was only available for the AAM_L. Peak activity was defined as the maximum activity during an estrus episode. Duration of estrus was defined as the time the activity of the cow exceeded threshold values set by the AAM software. The AAM_C correctly identified 87.8% of the estrus alerts, with 12.2% false positives. The average (±standard deviation) intervals from activity alert to ovulation were 25.8 ± 10.2 and 24.7 ± 9.3 h for the AAM_C and AAM_L, respectively. Changes in estrous expression were associated with differences in the interval from alert to ovulation. Cows with short intervals to ovulation were found to have less intense estrous expression than cows with medium and long length intervals to ovulation using the AAM_C, whereas using the AAM_L, cows with short intervals to ovulation exhibited less intense estrous expression than cows with medium but the same as those with long intervals to ovulation. Furthermore, irrespective of the AAM, estrus events with less estrous expression had increased odds of having a short interval to ovulation (below the median of 20 h) when compared with those having greater estrous expression (2.6 and 1.9 increased odds for the AAM_C and AAM_L, respectively). Ovulation failure was affected by estrous expression because estrus events with greater peak activity or longer duration had reduced ovulation failure compared with those with less estrous expression (AAM_C peak activity: 1.9 ± 1.4 vs. 9.5 ± 1.7%; AAM_L peak activity: 2.3 ± 1.4 vs. 6.2 ± 1.5%; AAM_C duration: 2.1 ± 1.4 vs. 8.9 ± 1.7%). In addition, cows with more estrous expression had greater pregnancy per artificial insemination than those with less estrous expression with both the AAM_C (42.3 ± 0.4 vs. 31.7 ± 0.4%) and the AAM_L (43.1 ± 0.4 vs. 36.3 ± 0.4%). Pregnancy per artificial insemination results were consistent even when removing cows that failed to ovulate. In conclusion, expression of estrus was highly associated with ovulation timing, ovulation failure, and fertility when using 2 different AAM. Cows with greater estrous expression have longer intervals from activity alert to ovulation, experience less ovulation failure, and have greater pregnancy per artificial insemination.