Association of uterine health in the first lactation with transition cow health and reproductive performance in the second lactation of Holstein dairy cows.

The objective of this study was to evaluate the effects of puerperal metritis (PM) diagnosed and treated during the early postpartum period of the first lactation on transition cow health, milk production, reproduction, and culling of dairy cows in their second lactation. Diagnosis of PM was based on fetid watery red-brown uterine discharge and rectal temperature above 39.5°C. Two farms were enrolled in this retrospective observational cohort study (Farm A and B). In both farms, the following diseases were recorded during the first 30 DIM in lactation 1 and 2: clinical hypocalcemia (CH), retained fetal membrane (RFM), PM, hyperketonemia (KET), left displaced abomasum (LDA) and clinical mastitis (MAST). Statistical analyses were performed using SPSS for Windows separately for each farm. Linear and logistic regression models were used for continuous (e.g., milk yield) and binary (e.g., disease, pregnancy per AI, pregnancy loss) outcomes, respectively. Cox proportional hazard regression models were calculated to model the time to event outcomes for culling or death during the first 60 DIM and for pregnancy within 250 d of the second lactation. The initial models contained the following variables: year of calving, month of calving, calving ease, stillbirth, twins, days open in lactation 1, 305 d milk yield in lactation 1, PM in lactation 1, and PM in lactation 2 as explanatory variables. A total of 4,834 cows (Farm A) and 4,238 cows (Farm B) in the second lactation were considered for statistical analyses. On farm A, the incidence of PM in lactation 1 and 2 was 20.1% and 11.2%, respectively. On farm B, the incidence of PM in lactation 1 and 2 was 14.4% and 8.5%, respectively. On both farms, cows with PM in their first lactation had greater odds for RFM and PM in their second lactation, while there was no association of PM in the first lactation with any other non-uterine diseases (i.e., CH, KET, LDA, and MAST) in the second lactation. Cows with PM in lactation 2 had reduced milk yield. The reduction in milk yield in second lactation was greater for cows that already experienced PM in lactation 1. On Farm A, cows with PM in their first lactation had a greater hazard for culling within 60 DIM of the second lactation; however, the same association was not present on Farm B. Cows with PM in lactation 1 had reduced pregnancy per AI at first service in the second lactation only on farm B. Cows with PM in lactation 2 had reduced pregnancy per AI at first service in the second lactation on both farms. Pregnancy loss in lactation 2 was only associated with PM in lactation 2 but not with PM in lactation 1. On both farms, cows had a reduced hazard for pregnancy in their second lactation within 250 DIM when they experienced PM in either lactation. In conclusion, PM in the first lactation had long-lasting negative consequences (i.e., risk of uterine disease and lower reproductive performance) for cows in their next lactation.

Efficacy of an automated technology at detecting early postpartum estrus events: Can we detect resumption of cyclicity?

The objective of this observational study was to evaluate the efficacy of a neck-mounted automated activity monitor (AAM) at detecting early postpartum resumed ovarian cyclicity. A total of 192 lactating cows (primiparous = 73 and multiparous = 119) were enrolled in this study. Cows were continuously monitored by a neck-mounted AAM early postpartum (7 to 30 d in milk; DIM). Calving was classified as assisted (forced extraction of a calf) or unassisted (normal calving). Retained fetal membrane, metritis, hyperketonemia, clinical mastitis, and milk production were recorded. Cows were classified as healthy (i.e., no disease events) or sick (i.e., any disease event). Estrus events were alerted by the AAM using a proprietary algorithm set by the AAM company. Blood samples, from the coccygeal vein, were collected at 15, 18, 21, 24, 28, and 30 DIM for progesterone (P4) analysis. Resumption of cyclicity was considered when P4 concentration was ≥1 ng/mL on any collection day. Cows were considered anovular when P4 concentration was <1 ng/mL on all collection days. Cows were classified as true positive: P4 ≥ 1 ng/mL and at least one estrus alert; false positive: P4 < 1 ng/mL and at least one estrus alert; true negative: P4 < 1 ng/mL and no estrus alerts; and false negative: P4 ≥ 1 ng/mL and no estrus alerts. Statistical analyses were performed by frequency distribution and mixed effects logistic regression procedures on SAS (SAS Institute Inc.). The specificity, sensitivity, accuracy, and positive predictive value of the sensor to detect cows that had resumed cyclicity were 84.0%, 34.1%, 52.1%, and 79.2%, respectively. Out of the 192 cows, 35.9% (69/192) were anovulatory and 37.5% (72/192) had no estrus events between 7 to 30 DIM. Healthy cows were more likely to resume cyclicity in early lactation compared with cows that were sick (78.3 ± 1.9 vs. 32.8 ± 3.1%, respectively) independent of parity. In conclusion, the sensor had a high specificity for detecting anovular cows, but it had lower sensitivity, and thus was not effective at detecting cyclic cows, perhaps due to silent ovulation early postpartum.

Validation of a leg-mounted pedometer for the measurement of steps in lactating Holstein cows.

The aim of this study was to validate the pedometer AfiAct II (AfiMilk) for the measurement of steps in lactating Holstein cows housed in a freestall design by assessing its agreement with visual observation of step counts. A total of 41 primiparous (n = 12) and multiparous (n = 29) cows were enrolled in the study between August and September 2018. Steps were monitored continuously by the pedometer and visually assessed for a 24-h period using video recordings. Visually observed steps were categorized as walking and stationary steps. The total number of steps taken per cow was calculated using the sum of walking and stationary steps. Unprocessed step count data from the study day were retrieved from the AfiMilk system in time-blocks of approximately 15 min. Repeated measures correlation was used to quantify the association between the pedometer measurements and visual observation of step counts. Nonindependence among observations were accounted adjusting for interindividual (cow) variability with an analysis of covariance. Pearson correlation coefficients (r) were categorized from negligible (0.00-0.30) to very high (0.90-1.00). Bland-Altman plots were created to evaluate the bias between the pedometer and visual observations. A total of 2,261 time-blocks were used in this study with an average (mean ± standard deviation) of 55.14 ± 8.1 time-blocks per cow. A high correlation was found for the evaluation between the pedometer and observed walking steps (r = 0.74; 95% confidence interval [CI] = 0.73-0.76), stationary steps (r = 0.71; 95% CI = 0.69-0.63), and total steps (r = 0.88; 95% CI = 0.87-0.89). The results of the Bland-Altman plot suggested limited bias between the pedometer step counts and visual observation of steps, independent of the type of steps. Numerical differences and several time-block differences outside of the 95% interval of agreement suggested an overestimation of step counts by the pedometer, which increased as the number of steps increased. The pedometer measured, on average, 97.6 ± 118.5 (28%), 249.2 ± 126.2 (125%), and 297.2 ± 205.4 (196%) steps/day more than the visual observed total steps, stationary steps, and walking steps, respectively. Our findings indicate that the pedometer counts all movement in which the pedometer leg is lifted off the floor without distinguishing if there was body movement of the animal.

Association of transition cow health with pregnancy per artificial insemination and pregnancy loss in Holstein cows submitted to a Double-Ovsynch protocol for first service.

This observational study was conducted to evaluate the effect of transition cow health on pregnancy per artificial insemination (P/AI) and pregnancy loss (PL) in cows submitted to a Double-Ovsynch protocol (DO) for first service. Lactating Holstein cows (n = 15,041) from one commercial dairy farm in northern Germany between January 2015 to December 2021 were enrolled into a modified Double-Ovsynch protocol (GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 24 h later PGF2α, 32 h later GnRH, and 16 to 18 h later timed artificial insemination) for first service at 72 ± 3 d in milk. Pregnancy was diagnosed at 32 and 60 d post-AI via transrectal ultrasonography. Pregnancy loss was defined as the proportion of cows diagnosed pregnant 32 d post-artificial insemination that were diagnosed nonpregnant 60 d post-artificial insemination. Health-related events (i.e., milk fever [MF], hyperketonemia [KET], retained fetal membranes [RFM], metritis, mastitis, left displaced abomasum [LDA]) were assessed by farm personnel using standard operating procedures. Multivariable logistic regression was used for testing potential associations between transition cow health event occurrence and outcome variables, including P/AI and PL. Three separate models were built for cows in first lactation, second lactation, and ≥third lactation. Overall, 20.0% (885/4,430), 34.9% (1,391/3,989), and 53.9% (3,570/6,622) of cows had at least one transition cow health event for first, second, and ≥third lactations, respectively. The most prevalent transition cow health event for first-lactation cows was metritis (10.7%; [473/4,430]), whereas second-lactation cows suffered mostly from mastitis (16.6%; [664/3,989] and KET (16.6%; [661/3,989]), and cows with ≥third lactations were mostly affected by KET (33.2%; [2,198/6,622]). We observed a negative association between inflammatory disorders (i.e., RFM, metritis, mastitis) and P/AI in all cows irrespective of parity. Metabolic disorders (i.e., MF, KET, LDA) were negatively associated with P/AI only in multiparous cows. Irrespective of parity, only uterine diseases (i.e., RFM, metritis) were significantly associated with PL. These results show that enrolling cows into a fertility protocol, such as DO, cannot overcome the carryover effects of inflammatory and metabolic disorders on P/AI and PL and highlight the importance of optimizing transition cow health as a prerequisite for achieving high fertility in a DO protocol.

Association between genomic daughter pregnancy rate and expected milk production on the resumption of estrus behavior in Holstein cattle.

The objective of this observational study was to evaluate estrous expression at the first estrus occurring between 7 to 30 d in milk (DIM), as detected by an automated activity monitor (AAM), and its association with genomic daughter pregnancy rate (GDPR) and genomic expected milk production (GEM) in lactating dairy cows. A total of 4,119 lactations from 2,602 Holstein cows were included. Cows were enrolled as first lactation (n = 1,168), second lactation (n = 1,525) and third and greater lactation (n = 1,426). Hair samples were collected from the tail switch, and cows were genotyped using an SNP platform (Clarifide, Zoetis, São Paulo, SP, Brazil). Postpartum cows were examined daily by the farm personnel from calving until 10 DIM. Calving was classified as assisted (forced calf extraction) and unassisted (normal calving). Retained fetal membranes (RFM), hyperketonemia (KET), and left displaced abomasum (LDA) were also recorded. Mean GDPR (± SD) was -0.29 ± 1.4, and the intensity and duration of the first estrus event was 15.9 ± 13.1 x-factor (intensity unit measurement) and 11.1 ± 3.8 h, respectively. Cows that had greater GDPR had greater intensity and longer duration of estrus at the alert, independent of parity. Overall resumption of estrous expression, between 7 to 30 DIM, was 41.2% (1,695/4,119), where 58.8% (2,424/4,119) did not have an estrus event, 31.0% (1,274/4,119) of cows had one event of estrus, and 10.2% (421/4,119) of cows had 2 or more events of estrus early postpartum. Mean DIM (± SD) at first estrus event, detected by the AAM, was 19.4 ± 4.4 d. Days in milk at first event for cows with one event was 20.7 ± 1.6 d and 15.9 ± 3.1 d for cows with 2 or more events of estrus. First lactation cows were more likely to have an estrus event early postpartum when compared with second and third and greater lactation cows (45.2 ± 1.4% [530/1168] vs. 41.6 ± 1.3% [636/1525] vs. 37.2 ± 1.3% [529/1426], respectively). There was an interaction of parity and GDPR on the proportion of cows demonstrating an early postpartum estrus. There was no difference in the proportion of cows with an early postpartum estrus between those with assisted or unassisted calving, RFM, or LDA. However, cows that had KET were less likely to have an alert early postpartum when compared with cows that did not have KET. Mean genomic expected milk production (± SD) was 256.8 ± 600.1 kg. There was no interaction between GEM and parity on estrous expression (i.e., intensity and duration). There was no interaction between GEM and GDPR on the proportion of estrus early postpartum.

Use of intravaginal lactic acid bacteria prepartum as an approach for preventing uterine disease and its association with fertility of lactating dairy cows.

The objective of this study was to evaluate the effects of the use of intravaginal probiotics prepartum on the incidence risk of metritis postpartum and conception risk after first artificial insemination (AI). A total of 606 Holstein cows were enrolled 3 wk before their expected calving date from 2 farms. Cows were randomly assigned to either receive a 2-mL dose of a combination of 3 lactic acid bacteria (probiotic treatment) washed with approximately 2 mL of a sterile saline solution, into the vaginal canal twice weekly until parturition, or no intervention (control). Metritis diagnoses were carried out on 6 and 12 d postpartum. Vaginal discharge and rectal temperature were assessed, and vaginal discharge was scored on a scale from 1 to 4, where 1 = clear and 4 = fetid, purulent discharge. Metritis was defined as cows having a vaginal discharge score of 4 with or without fever (rectal temperature ≥39.5°C) on either 6 or 12 d postpartum, or both. Cows were bred after a 60-d voluntary waiting period primarily via the detection of estrus using automated activity monitors; cows not found in estrus were enrolled onto timed AI protocols to receive first breeding before 100 DIM. Pregnancy diagnosis was carried out at d 35 ± 7 post-AI on both farms. Data were analyzed via ANOVA using linear mixed regression models and survival analysis using a Cox proportional hazard model. Total incidence risk of metritis was 23.7% and 34.4% on farm A and farm B, respectively. Overall, the incidence of metritis was not different between treatment groups (control: 41.6 ± 3.8%; probiotic: 38.6 ± 4.0%); however, an interaction by farm was detected, where the probiotic treatment reduced metritis on one farm but not on the other. Conception risk after first AI was not affected by treatment. However, we detected an interaction between parity and treatment, where multiparous cows receiving the probiotic treatment were more likely to become pregnant compared with multiparous cows within the control group (hazard ratio: 1.33; 95% confidence interval: 1.10-1.60); no effect of probiotic treatment was found on the hazard of pregnancy for primiparous cows. In addition, the probiotic treatment was associated with an increased proportion of cows being detected in estrus for the first AI postpartum. In conclusion, vaginal probiotic treatment applied during the 3 wk prepartum was associated with a decreased incidence of metritis on one farm but not the other, suggesting that farm management may be a key player influencing treatment efficacy. Overall, probiotic treatment was found to have only limited effects on fertility in the current study.

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.

Association between genomic daughter pregnancy rates and reproductive parameters in Holstein dairy cattle.

The objective of this observational study was to evaluate the relationship between genomic daughter pregnancy rate (GDPR) with reproduction parameters such as pregnancy at first artificial insemination (AI), pregnancy per AI, and pregnancy losses (PL). A total of 12,949 events from 3,499 Holstein cows were included. Cows were enrolled as nulliparous (n = 1,220), primiparous (n = 1,314), or multiparous (n = 965). Cows were bred either after a timed AI protocol, timed embryo transfer (ET), or spontaneous estrus. Most lactating cows were bred following a timed AI protocol based on estradiol and progesterone, and most nulliparous were artificially inseminated following estrus detection. Hair samples were collected from the tail switch and cows were genotyped using a SNP platform (Clarifide, Zoetis). Cows that were bred by timed AI were evaluated for estrous behavior using tail chalk. Tail chalk was applied on the head of the tail 2 d before timed AI and the chalk was evaluated at AI (no estrus: 100% of chalk remaining or ≥50% of chalk remaining; Estrus: <50% of chalk remaining). Pregnancy diagnosis was performed at d 32 and 60 after AI using ultrasonography, and the presence of a heartbeat was considered a positive diagnosis. Pregnancy loss was defined as a pregnant cow on d 32 that was nonpregnant on d 60. As GDPR increased, the odds of pregnancy at first AI increased [odds ratio (OR) = 1.28, 95% CI = 1.20-1.35], the odds of pregnancy per AI increased (OR = 1.31, 95% CI = 1.25-1.36), and the odds of PL decreased (OR = 0.66, 95% CI = 0.60-0.72). Most cows that were bred on the day of the timed AI demonstrated estrus (n = 6,075; 92.9%). The odds of demonstrating estrus on the day of timed AI increased as GDPR increased (OR = 1.31, 95% CI = 1.17-1.48). There was no interaction between GDPR and parity or breeding management for pregnancy at first AI, pregnancy per AI, and PL. In conclusion, the odds of pregnancy at first AI and pregnancy per AI increased as GDPR increased. Moreover, the odds of PL increased as GDPR decreased. Greater GDPR was also associated with greater occurrence of estrus on the day of timed AI. These results suggest that selecting for higher GDPR could result in better reproductive performance, but this would need to be assessed with additional research.

Occurrence and greater intensity of estrus in recipient lactating dairy cows improve pregnancy per embryo transfer.

The aim of this study was to determine the association between occurrence and intensity of estrous expression with pregnancy success in recipient lactating dairy cows subjected to embryo transfer (ET). Two observational studies were conducted. Holstein cows were synchronized using the same timed ET protocol, based on estradiol and progesterone in both experiments. At 9 d after the end of the timed ET protocol only animals that had ovulated were implanted with a 7-d embryo [experiment 1 (Exp. 1); n = 1,401 ET events from 1,045 cows, and experiment 2 (Exp. 2); n = 1,147 ET events from 657 cows]. Embryos were produced in vivo (Exp. 1 and Exp. 2) and in vitro (only Exp. 2), then transferred to recipient cows as fresh or frozen-thawed. Pregnancy was confirmed at 29 and 58 d after the end of timed ET protocol. In Exp. 1, animals had their estrous expression monitored through a tail chalk applied on the tail head of the cows and evaluated daily for chalk removal (no estrus: 100% of chalk remaining; estrus: <50% of chalk remaining). In Exp. 2, cows were continuously monitored by a leg-mounted automated activity monitor. Estrous expression was quantified using the relative increase in physical activity at estrus in relation to the days before estrus. Estrous expression was classified as no estrus [<100% relative increase in activity (RI)], weak intensity (100-299% RI), and strong intensity (≥300% RI). Data were analyzed by analysis of variance using mixed linear regression models (GLIMMIX) in SAS (SAS Institute Inc.). A total of 65.2% (914/1,401) and 89.2% (1,019/1,142) of cows from Exp. 1 and Exp. 2, respectively, displayed estrus at the end of the ovulation synchronization protocol. In Exp. 1, cows expressing estrus before to ET had greater pregnancy per ET than those that did not [41.0 ± 2.3% (381/914) vs. 31.5 ± 2.9% (151/487), respectively]. Similarly, in Exp. 2, cows classified in the strong intensity group had greater pregnancy per ET compared with cows in the weak intensity and no estrus groups [41.3 ± 2.2% (213/571) vs. 32.7 ± 2.7% (115/353) vs. 11.3 ± 3.5% (26/218), respectively]. There was no effect of ET type on pregnancy per ET in Exp. 1. However, in Exp. 2, cows that received an in vivo-produced embryo, either fresh or frozen, had greater pregnancy per ET compared with cows that received in vitro-produced embryo. Cows receiving embryos in the early blastocyst and blastocyst stage had greater fertility compared with cows receiving embryos in the morula stage. There was an interaction between the occurrence of estrus and the stage of embryo development on pregnancy per ET, cows which displayed estrus and received a morula or early blastocyst had greater pregnancy per ET than cows that did not display estrus. In conclusion, the occurrence and the intensity of estrous expression improved pregnancy per ET in recipient lactating dairy cows and thus could be used as a tool to assist in the decision making of reproduction strategies in dairy farms.

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.

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.

Timing of artificial insemination using fresh or frozen semen after automated activity monitoring of estrus in lactating dairy cows.

The objective of this observational experiment was to determine the association between the time of artificial insemination (AI) and pregnancy per AI (P/AI) in lactating Holstein cows inseminated with either fresh or frozen semen considering different characteristics of an estrus event (i.e., onset, peak, and end) using an automated activity monitoring system. A total of 3,607 AI services based on the alert of an automated activity monitoring system (Heatime; SCR Engineers Ltd., Netanya, Israel) were evaluated from 4 commercial dairy farms in Germany. Pregnancy diagnosis was performed by transrectal palpation 38 ± 3 d after AI or by transrectal ultrasonography 30 ± 3 d after AI. Estrus intensity was categorized based on peak activity of estrus (PAE) into low (35-89 index value) and high (90-100 index value) intensity. The mean (± standard deviation) duration of an estrus event was 14.3 ± 4.6 h. The mean (± standard deviation) interval from onset of estrus (OE; moment where index value was ≥35) to AI was 16.8 ± 8.0 h, from PAE to AI was 11.9 ± 8.1 h, and from end of estrus (EE; moment where index value returned to <35) to AI was 2.5 ± 8.7 h. Primiparous cows had greater P/AI than multiparous cows, whereas first AI postpartum yielded greater P/AI compared with subsequent AI services. Type of semen was not associated with P/AI. Cows with heat stress 1 wk before AI had decreased P/AI. Cows with low estrus intensity (26.0%) were less fertile compared with cows showing high estrus intensity (32.8%). Cows with intermediate 100-d milk yield had decreased P/AI compared with cows with either low or high 100-d milk yield. There was a quadratic effect of the interval from OE to AI on P/AI. At 38 d after AI, P/AI was greatest for cows inseminated from 7 to 24 h after OE, within 18 h after PAE, or from 5 h before EE to 12 h after EE. There was no interaction between the interval from OE to AI and type of semen. There tended to be an interaction between the intervals from PAE to AI and type of semen and from EE to AI and type of semen. Cows inseminated with fresh semen within 5 h before EE had greater P/AI compared with frozen semen, whereas cows inseminated with frozen semen from 13 to 18 h after EE had greater P/AI compared with fresh semen. In conclusion, inseminating cows from 7 to 24 h after OE or 1 to 18 h after PAE yielded greatest P/AI irrespective of type of semen. In addition, high estrus intensity was positively associated with P/AI.

Technical note: Validation of an in-house bovine serum enzyme immunoassay for progesterone measurement.

Measuring circulating progesterone (P4) of dairy cows is a key component of many research studies dealing with basic and applied reproduction physiology. The gold standard in dairy cows for the measurement of P4 in serum is radioimmunoassay (RIA), but it generates radioactive waste and requires licensed facilities. The purpose of this study was to develop and validate an in-house competitive enzyme immunoassay (EIA) to measure the P4 concentration in serum of dairy cattle. The secondary objective was to validate a commercial EIA. In the present study, a competitive EIA was developed using commercially available antibodies and conjugates. Ninety-six well microtiter plates were coated with the secondary antibody and incubated overnight. Following a washing step, the wells were blocked using the primary antibody. Serum samples were prepared by first extracting P4 using petroleum ether, then diluted in working conjugate solution. Samples were pipetted into the coated and blocked plates, then the matching HRP conjugate label (P4-3-HRP, East Coast Bio, North Berwick, ME) was added. The plates were incubated for 2 h, then washed. The substrate solution was added, and the plate was incubated up to 1 h at room temperature in the dark until a blue color had developed. A stop solution was added, and the optical density measured on a microplate reader was set at 450 nm. The binding proportion was calculated by a visible spectrum absorbance reader, and the amount of P4 was calculated using a log-logit regression line. The commercial EIA was executed as suggested by the manufacturer. The validation of the in-house EIA was done by calculating the inter- and intraassay coefficients of variation (CV) and evaluating the parallelism of diluted samples. The results from the in-house and commercial EIA were also compared with the ones from the RIA graphically (scatterplots and Bland-Altman plots) and statistically, using the Spearman correlation coefficient (r) and the Cohen's kappa statistics using a threshold of 1.0 ng/mL (κ). For the in-house EIA, the intraassay CV were all <10%, but the interassay for samples with small and large P4 concentration had CV of 12.5 and 11.0%, respectively. The correlations between the results from the EIA and the RIA were strong (in-house: r = 0.90; commercial: r = 0.83). At small concentrations (<1.0 ng/mL), however, the correlation with the gold standard was weak (in-house: r = 0.27; commercial: r = 0.14). This was likely due to the lack of accuracy at small concentrations, also shown by the absence of parallelism in samples ≤0.4 ng/mL. In conclusion, results from both the in-house and commercial EIA strongly correlated with the gold standard, but less so at smaller concentrations. The in-house EIA offers good accuracy to measure P4 in samples with a concentration >0.4 ng/mL, and a perfect agreement with RIA using a threshold of 1.0 ng/mL.

Symposium review: Linking activity-sensor data and physiology to improve dairy cow fertility.

Several studies have demonstrated that the intensity of estrous expression is associated with ovulation, ovarian and uterine function, and fertility, and is dependent on social hierarchy and the housing system used. Data from recent studies involving spontaneous and induced estrus have shown that a greater relative increase and longer estrus (captured by different automated activity monitors; AAM) are both associated with improved pregnancy per artificial insemination (AI; around 10 to 14% increase) and decreased pregnancy losses. Intensity and duration of estrus were surprisingly weakly associated with preovulatory follicle diameter and concentrations of plasma estradiol at estrus, whereas ovulation failure was associated with low estrus intensity. Studies have also shown that the display of estrous behavior near AI was associated with the modification of expression of genes related to the immune system, adhesion molecules, and prostaglandin synthesis in the endometrium. Transcripts in leukocytes and in the conceptus tissue associated with maternal recognition of pregnancy as well as conceptus elongation were all associated with differences in the intensity of estrous expression. Most recently, studies from the United States and Canada have demonstrated that reproductive programs emphasizing detection of estrus using AAM can be successful and comparable to intensive timed AI protocol-based programs that incorporate GnRH and PGF2α treatments. Further, one study concluded that the administration of GnRH at AI for spontaneous estrus events greatly improved pregnancy per AI, but only for cows with reduced intensity of estrous expression, showing the potential to use AAM data as a tool in targeted reproductive programs. Quantitative information from estrus events could be used to improve estrus detection and develop decision-making strategies at the farm level. Future studies in this field should aim to better understand ovarian, conceptus, and endometrial mechanisms associated with either the expression or the intensity of estrus, and to refine the identification of phenotypes related to estrus (relative increase, absolute increase, baseline levels, duration, and repeatability within cow) to improve data usage, estrus detection, and possibly genetic selection.

Size and position of the reproductive tract impacts fertility outcomes and pregnancy losses in lactating dairy cows.

There are multiple factors that contribute to reduced fertility in lactating dairy cows. Recently, a reproductive tract size and position score (SPS) system was developed as a management tool to identify dairy cows with decreased fertility. The aim of this study was to evaluate the association between the SPS on fertility outcomes such as ovulation failure, pregnancy per artificial insemination (P/AI), concentration of pregnancy-associated glycoproteins (PAGs), and pregnancy loss in lactating dairy cows. Primiparous and multiparous lactating Holstein cows (n = 869) were enrolled at two locations. Location 1 (Loc. 1) in Minas Gerais, Brazil (n = 613) and location 2 (Loc. 2) in Agassiz, British Columbia, Canada (n = 256). At the time of AI (d 0), cows were classified as SPS (small [SPS1], medium [SPS2], or large [SPS3] sized reproductive tract) and ovulation failure was determined at 48 h and 7 d post-AI via ultrasonography (Loc. 2 only). Blood samples were collected on d 24 and 31 of gestation for quantification of PAGs and pregnancy diagnosis was performed via ultrasonography at d 31 and 60 post-AI (Loc. 1) and at d 31 ± 3 and 60 ± 3 post-AI (Loc. 2). Cows diagnosed pregnant at d 31 post-AI but not pregnant at d 60 were defined to have undergone late embryonic pregnancy loss. Parity was found to impact SPS (P < 0.01), as primiparous cows had a higher frequency of SPS1 and lower frequency of SPS3 when compared with multiparous cows (SPS1: 42.6 vs. 15.0%; SPS3: 7.0 vs. 22.0%, respectively). Cows classified as SPS3 had greater ovulation failure at 48 h (P = 0.04) and 7 d post-AI (P = 0.05). Cows classified as SPS1 had greater P/AI when compared to SPS2 and SPS3 (45.9 ± 3.3 vs. 37.4 ± 2.6 and 29.1 ± 3.5%, respectively; P = 0.004). There was no interaction between parity and SPS on P/AI. Pregnancy loss between 31 and 60 d post-AI was increased in cows classified as SPS3 compared to SPS2 and SPS1 (24.3 ± 0.05 vs. 11.6 ± 0.02 and 9.4 ± 0.02%, respectively; P = 0.04). Cows classified as SPS1 and SPS2 had greater concentrations of PAGs at 31 d post-AI when compared to SPS3 at both Loc.1 (P < 0.01) and Loc. 2 (P < 0.01). There was no interaction between SPS and pregnancy loss on PAGs at 24 and 31 d post- AI for either Loc. 1 (P = 0.75 and P = 0.76, respectively) or Loc. 2 (P = 0.61 and P = 0.81, respectively). In conclusion, cows that were classified as SPS3 had greater ovulation failure, reduced P/AI, similar concentrations of PAG on d 24, but decreased on d 31, and a greater incidence of pregnancy loss. Thus, size and position of the reproductive tract is associated with fertility and this scoring system could be used to make reproductive management decisions on dairy operations.

Short communication: Greater intensity of estrous expression is associated with improved embryo viability from superovulated Holstein heifers.

The aim of this study was to determine the association between estrous expression, measured using a breeding indicator and an automated activity monitor (AAM), and the success of embryo collection after superovulation. Holstein heifers (n = 51; 10.5 to 14.5 mo, and 325.0 ± 21.1 kg of body weight) were superovulated (n = 69 events) for the collection of embryos using a protocol based on sequential administration of FSH for follicle superstimulation and GnRH to induce ovulation. Artificial insemination (AI) was performed twice, once at the moment of GnRH administration and again 12 h later, using thawed, sexed semen. Ovaries were scanned via ultrasonography on the day of the first AI to count the total number of preovulatory follicles and 7 d later for the total number of corpora lutea present. Embryos were collected 7 d post-AI, counted, and assessed for viability. A breeding indicator (Estrotect, Rockway Inc., Spring Valley, WI) and a collar-mounted AAM (CowScout Activity Monitoring System, GEA, Dusseldorf, Germany) were used to measure standing mounts and an algorithmic estimate of estrous expression, respectively. A score for the breeding indicator was given as follows: score 1 = 100% of the indicator was intact; score 2 = 50% of the indicator was rubbed off; score 3 = greater than 50% of the indicator was rubbed off. Estrous expression detected by the AAM was quantified through the relative increase in physical activity and duration of time spent above a set threshold. Data were analyzed by ANOVA using the MIXED procedures of SAS (SAS Institute Inc., Cary, NC). The number of follicles present at AI was not affected by estrous expression. The mean (± SD) ovulatory response was 67.5 ± 26.3%. We found an effect of estrous expression as detected by the breeding indicator on the ovulatory response (42.1 ± 8.0, vs. 78.2 ± 9.0, vs. 74.0 ± 4.9%, for scores 1, 2, and 3, respectively) but not from the AAM. Heifers that had a score of 3 (versus those with scores of 1 and 2) on the breeding indicator had a greater number of embryos (4.1 ± 0.5, vs. 1.2 ± 1.0, vs. 1.8 ± 1.0 embryos), and a greater percentage of these embryos were viable (43.1 ± 0.05, vs. 35.5 ± 0.1, vs. 34.3 ± 0.1%). Similarly, heifers that showed a greater intensity of activity (as measured by the AAM) had a greater number of embryos collected (10.2 ± 1.2 vs. 6.0 ± 1.3 embryos), and a greater percentage of those embryos were viable (53.1 ± 5.0 vs. 23.4 ± 5.1%). Longer-duration estrus episodes were associated with a higher percentage of viable embryos (51.2 ± 5.2 vs. 25.3 ± 5.3%). In conclusion, stronger estrous intensity was associated with a greater number of total embryos collected and a greater percentage of viable embryos. These results suggest that monitoring the intensity of estrus could be used to predict superovulatory response as well as embryo quality in Holstein heifers.

Association of concentrations of beta-carotene in plasma on pregnancy per artificial insemination and pregnancy loss in lactating Holstein cows.

The objective of this study was to determine the association of beta-carotene concentration in plasma at the moment of artificial insemination (AI) on pregnancy/AI in lactating Holstein cows. A total of 399 events from 364 lactating Holstein cows were enrolled in the trial (143 primiparous and 221 multiparous). All cows were assigned to a timed AI protocol based on estradiol and progesterone. Blood samples were collected at the moment of AI and at 24 and 31d post-AI (samples on 31 d post-AI were collected only from cows that were diagnosed pregnant). The BCS were recorded at the time of AI. Plasma beta-carotene was quantified from blood samples taken at the time of AI using a single step denaturation and extraction into a solvent, followed by measurement using a portable spectrophotometer. Pregnancy-associated glycoproteins (PAG) were analyzed in blood samples taken at 24 and 31 d post-AI of pregnant cows. Milk production was collected for the entire experimental period. Pregnancy diagnosis was performed by ultrasound 31 and 60 d post-AI. Data was analyzed using the MIXED and GLIMMIX procedures of SAS. Cows classified as thin (<2.75) tended to have lower concentration of beta-carotene at AI when compared with those classified as Moderate (≥3.00; 3.8 ±â€¯0.1 vs. 4.3 ±â€¯0.1 µg/mL; P = 0.09). Concentration of beta-carotene were greater in multiparous compared with primiparous (P < 0.01). There was no correlation between concentration of beta-carotene and milk production (r = 0.04; P = 0.10). When plasma beta-carotene was categorized in quartiles, cows in the 1st quartile had lower pregnancy/AI and higher pregnancy losses when compared with cows that were in the 2nd, 3rd and 4th quartile (pregnancy/AI = 19.2 ±â€¯4.5, 33.7 ±â€¯4.7, 36.9 ±â€¯5.0 and 39.8 ±â€¯5.4%, respectively; P = 0.05; pregnancy losses = 41.9 ±â€¯4.8, 20.4 ±â€¯3.7, 22.1 ±â€¯4.1, and 15.7 ±â€¯4.2%, respectively; P < 0.05). There was no association between concentrations of beta-carotene at AI and PAG at 24 d post-AI (P = 0.60). Cows with greater concentrations of beta-carotene at AI were more likely to have greater concentrations of PAG at 31 d post-AI (P < 0.01). In conclusion, the concentration of beta-carotene at AI was affected by BCS and parity. Cows with higher concentrations of plasma beta-carotene at AI had greater pregnancy/AI, lower pregnancy losses and greater concentrations of PAG at d 31 post-AI, suggesting it may be associated with placental function in lactating dairy cows.

Intensity of estrus following an estradiol-progesterone-based ovulation synchronization protocol influences fertility outcomes.

The objective of this study was to examine the association between increased physical activity at the moment of timed artificial insemination (AI), detected by an automated activity monitor (AAM), and fertility outcomes. This paper also investigated factors affecting estrous expression in general. A total of 1,411 AI events from 1,040 lactating Holstein cows were recorded, averaging 1.3 ± 0.6 (±standard deviation) events per cow. Activity (measured as steps/h) was monitored continuously by a leg-mounted AAM located on the rear leg of the cow. Ovulation was synchronized by a timed AI protocol based on estradiol and progesterone. Ovarian ultrasonography was performed in all cows on d -11 (AI = d 0) and in a subset of cows on d 0 (n = 588) and d 7 (n = 819) to determine the presence of a corpus luteum and follicles. The body condition score (1 to 5 scale) was assessed on d 0 and a blood sample was collected for progesterone measurement on d 7. Using the AAM, an estrus event was determined when the relative increase (RI) in physical activity of the cow exceeded 100% of the baseline activity. The physical activity was classified as strong RI (≥300% RI), moderate RI (100-300% RI), or no estrus (<100% RI). Milk production was measured daily and averaged between d -11 and 0. Pregnancy was diagnosed at 32 and 60 d post-AI and pregnancy losses were calculated. The mean RI at estrus was 328.3 ± 132.1%. Cows with strong RI had greater pregnancy per AI than those with moderate RI and those that did not express estrus (35.1 vs. 27.3 vs. 6.2%). When including only cows that successfully ovulated after timed AI, those that displayed strong intensity RI still had greater pregnancy per AI than those with moderate intensity RI or those that did not express estrus (45.1 vs. 34.8 vs. 6.2%). Cows expressing strong RI at timed AI had greater ovulation rates compared with moderate RI and cows that did not express estrus (94.9 vs. 88.2 vs. 49.5%). Furthermore, pregnancy losses were reduced in cows with strong RI compared with cows expressing moderate RI (13.9 vs. 21.7%). Cows with a strong RI at estrus were more likely to have a corpus luteum at the beginning of the protocol and had greater concentration of progesterone 7 d post-AI. Multiparous cows expressed lower RI compared with primiparous cows. Cows with lower body condition score tended to have decreased RI at estrus. No correlation between estrous expression and pre-ovulatory follicle diameter was observed. Also, no correlation was observed between milk production at AI and RI. In conclusion, strong intensity RI of estrus events at timed AI was associated with improved ovulation rates and pregnancy per AI, and reduced pregnancy losses. These results provide further evidence that measurements of estrous expression can be used to predict fertility at the time of AI and possibly be used as a tool to assist decision making strategies of reproduction programs.

Daily lying behavior of lactating Holstein cows during an estrus synchronization protocol and its associations with fertility.

Objectives were to quantify lying behavior (LB) during an estradiol and progesterone-based synchronization protocol, to assess risk factors for ovulation, pregnancy per AI (P/AI), and degree of behavioral change at estrus, and to investigate the associations between estrus LB and walking activity. Holstein cows (43.6 ± 11.0 kg of milk/d) were fitted with leg-mounted accelerometers. Total lying time/d (L_time), bout frequency (bout_N), average lying bout duration, and relative increase in walking activity (ACT%) were evaluated for 1,411 timed artificial insemination events. The day with lowest L_time or bout_N among d -2, -1, and 0 (day of timed artificial insemination) determined the day of behavioral estrus. The variables L_time% and bout_N% represent relative ratios between lowest L_time and baseline (d -7), L_time, and lowest bout_N, and baseline (d -7) bout_N, respectively [e.g., (lowest L_time/baseline L_time) × 100]. Correlation coefficients between L_time% and bout_N% and ACT% were -0.38 and -0.31, respectively. Estrus LB change was considered large if <75% of baseline and small if ≥75% of baseline for both L_time% and bout_N%; average lying bout duration did not change with estrus. Lowest L_time% and bout_N% corresponded to, respectively, 65 ± 21% (mean ± standard deviation; 447 ± 157 min/d) and 65 ± 24% (8.5 ± 4.0 bouts/d) of baseline. The change in L_time% at estrus was smaller when cows had milk yield above average; the change in bout_N% was smaller among multiparous cows and for estrus occurring in the colder season. Likelihood of ovulation was greater when there was larger change in L_time% [odds ratio = 4.9; ovulation rate = 93 (large change) and 76% (small change)], as well as when a corpus luteum was present at start of protocol (odds ratio = 3.6; in the model with L_time%). Likelihood of pregnancy at d 32 was 1.6 times greater for estrus with large change in LB [L_time% or bout_N%; P/AI = 34% (large change in L_time%) and 26% (small change in L_time%)]. Among estrus events with ACT% ≥300% (high intensity), classification by small or large L_time% did not influence P/AI at 32 d. The magnitude of LB change at estrus and its association with fertility suggest potential application toward improved use of activity monitors (e.g., increased estrus detection, fertility prediction). The contribution of LB to accuracy of estrus detection when physical activity is known remains to be addressed. The relationship between intensity of estrus expression and fertility requires further investigations of its physiological rationale and on-farm applications.

Diagnosis of uterine and vaginal disorders by different methodologies is affected by concentration of estradiol in plasma from lactating Holstein cows.

The relationship between plasma estradiol concentration at time of examination and prevalence of uterine disorders, agreement among methods, and associations of diagnosis with pregnancy hazard and milk yield was studied in 268 Holstein cows examined at 30±3 (exam 1) and 44±3 d in milk (DIM; exam 2). Purulent vaginal discharge was sampled using 2 methods: gloved hand and Metricheck (Simcro, Hamilton, New Zealand; PVD; score ≥3). Percentage of polymorphonuclear leukocytes was determined by endometrial cytology (CYTO; exam 1: ≥18%, exam 2: ≥10%); diameter of uterine horns (UTH; >20 mm), diameter of the inner layer of the cervix (CVX; >20.5 mm), presence of fluid in the uterine lumen (FL), and ovarian structures were evaluated by ultrasonography. A blood sample was collected at each exam for estradiol analysis. Prevalence at exams 1 and 2 was, respectively, 14.2 and 18.5% (PVD), 21.4 and 10.1% (FL), and 40.6 and 50.2% (CYTO). Prevalence of PVD at exam 1 was greater among cows with estradiol ≥2 pg/mL (19.4 vs. 8.2%). Agreement of all methods with CYTO was poor, the greatest being between CYTO and FL (exam 1; kappa=0.19). Agreement between CYTO and PVD, and between CYTO and FL (exam 1; kappa=0.15 and 0.35, respectively) was higher among cows with estradiol ≥2 pg/mL. Likelihood of PVD at exam 1 was greater if cows were positive for CVX [odds ratio (OR)=3.0], FL (OR=2.6) or had estradiol ≥2 pg/mL (OR=2.7). Likelihood of CYTO increased with dystocia (OR=2.3) and FL (OR=2.5). Estradiol did not influence diagnosis at exam 2. Positive FL or CYTO at exam 1 was associated with reductions in milk yield of 59 to 180 kg by 45 DIM. Pregnancy hazard until 250 DIM was reduced by CYTO at exam 1 (hazard ratio=0.74) and by PVD (hazard ratio=0.68) at exam 2. However, FL and CYTO reduced pregnancy hazard only when estradiol was ≥2 pg/mL (exam 1), whereas PVD reduced pregnancy hazard when diagnosed at exam 2 with estradiol <2 pg/mL. Overall, agreement was poor and effects of positive diagnosis differed according to method and DIM at exam. Estradiol concentration influenced prevalence, agreement, likelihood of positive diagnosis, and its effects on days to pregnancy.