The objective of this study was to determine risk-factors affecting increase in physical activity during estrus and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Cows were monitored continuously by 2 automated activity monitors [a collar-mounted accelerometer (HT; Heatime, SCR Engineers, Netanya, Israel) and a leg-mounted pedometer (BO; Boumatic Heat-seeker-TX, Boumatic Dairy Equipment, Madison, WI)]. When an increase in activity was detected, body condition score (BCS) and blood samples were collected, ovaries were scanned by ultrasonography, and, if the cow was eligible for breeding, artificial insemination was performed. Milk production and health-related data were recorded throughout the experimental period. Pregnancy diagnosis was performed at 42 ± 7 d of gestation. Data were analyzed using Pearson correlation, ANOVA, and logistic regression. A total of 1,099 true events of estrus from 318 lactating Holstein cows were recorded, averaging 3.46 ± 1.1 events per cow. Positive predictive value for estrus episodes detected by the HT and BO systems were 89.6 and 85.5%, respectively. Mean peak activity at estrus (PA) recorded by the HT system was 71.6 ± 20.7 index-value, and 334.3 ± 155.7% relative increase by the BO system. Compared with primiparous, multiparous cows expressed estrus with lower PA (69.3 ± 0.8 vs. 75.9 ± 1.1 index for HT; 323.9 ± 6.0 vs. 354.8 ± 8.48% for BO) and shorter duration (DU; 10.7 ± 0.2 vs. 12.0 ± 0.3 h); DU was measured by HT only. Lower BCS was associated with decreased PA measured by both systems, estrus DU, and P/AI. Peak activity was weakly correlated with milk production on the day of artificial insemination (r = -0.20); however, when categorized into quartiles, the highest-yield cows had lower PA and DU. Follicle diameter was not correlated with PA or DU, but cows with greater concentrations of estradiol had higher PA. Cows with greater PA in both systems had greater P/AI than those with lower PA (36.5 vs. 24.6% for HT, 33.5 vs. 21.4% for BO). In conclusion, measurements of estrus events captured by automated activity monitors are correlated with BCS, parity, and secondary behavior signs related to estrus. Surprisingly, estrus intensity and duration were only weakly correlated with milk production, preovulatory follicle diameter, and concentrations of estradiol at estrus. Cows that had measurements of high-intensity estrus were significantly more fertile than low-intensity estrus.

Cortisol has long been used as a marker of the stress response in animals. Cortisol can be analyzed from different media, most notably from the blood, saliva, and feces; however, the collection of cortisol from some of these media requires invasive procedures or excessive handling of the animals. Furthermore, it is not possible to capture long-term increases in circulating concentrations of cortisol from the blood, saliva, or feces. Hair cortisol has been found to be a reliable alternative for measuring chronic stress. With this emerging measure, appropriate sampling methodology must be developed and validated. The aim of this study was to determine the effects of hair color, sampling location, and processing method on cortisol concentrations in hair from lactating black and white Holstein cows (n=18). Furthermore, we aimed to measure the hair growth rates at different body locations (n=12) and test hair cortisol levels when resampled over short intervals (n=37). Both black- and white-colored hair was collected from the shoulder, top line, hip, and tail switch of Holsteins; due to breed characteristics only white hair was harvested from the tail switch. All samples were cleaned with water and isopropanol, and then ground in a ball mill or finely cut with scissors once dry. Cortisol was extracted with methanol before being measured using a commercially available ELISA kit. Concentrations of cortisol were greater in white than in black hair (7.8 ± 1.1 vs. 3.8 ± 1.1 pg/mg). When only white samples were analyzed, hair from the tail switch had more cortisol than hair from the shoulder (11.0 ± 1.2 vs. 6.2 ± 1.2 pg/mg), whereas no difference was found when compared with the hip and top line. Samples ground with a ball mill had greater concentrations of cortisol extracted than those minced with scissors (10.4 ± 1.2 vs. 4.7 ± 1.2 pg/mg). The growth rate of hair was significantly greater at the tail switch compared with the hip and shoulder (0.51 ± 0.05 vs. 0.04 ± 0.05 vs. 0.03 ± 0.05 mm/d). When hair was collected every 3 wk after calving, a tendency was detected for multiparous cows to have greater concentrations of hair cortisol and significantly greater concentrations of cortisol on d 0 and 21 after calving compared with d 42, 84, and 126. In Holsteins, the hair on the tail switch is always white, grows more rapidly than other sites, and is sensitive enough to capture changes in cortisol over intervals as short as 3 wk, making it the ideal location for measuring hair cortisol.

Hair cortisol has been used to measure chronic stress in dairy cows as it offers the advantage of being noninvasive, fast, and able to indicate levels of cortisol over long periods. The aim of this study was to determine the associations between hair cortisol with clinical disorders, reproductive status, and the development of subclinical endometritis in dairy cows. Furthermore, we aimed to determine the association between hair cortisol concentrations and blood markers associated with metabolic status and acute inflammation. In experiment 1, cows (n=64) were hair sampled every 3wk from the tail switch beginning at calving (d 0) until d 126 for cortisol analysis; blood samples were collected every 3wk from d 0 until 42 for β-hydroxybutyrate and glucose analysis. In experiment 2, cows (n=54) were chosen retrospectively by diagnosis of subclinical endometritis (END), subclinical endometritis and at least 1 clinical disease (END+CLIN), or as healthy (control) using a cytobrush and ultrasonography at 30±3d in milk. At the same time, animals were hair sampled for cortisol analysis and blood sampled for haptoglobin and ceruloplasmin analysis. Health records were recorded throughout both experimental periods. Animals with clinical disease presented higher cortisol concentrations than clinically healthy animals in experiment 1 [geometric mean (95% confidence interval); 8.8 (7.8, 9.9) vs. 10.7 (9.6, 12.0) pg/mg]; however, animals diagnosed with subclinical endometritis in experiment 2 did not differ in hair cortisol concentrations [11.7 (9.8, 14.0), 12.2 (9.3, 15.9), 10.5 (8.1, 13.6) pg/mg for control, END, and END+CLIN, respectively]. In experiment 1, an effect of sample day was noted, where d 21 had higher cortisol concentrations than d 42, 84, and 126, but not from d 0 for both parities. Within both experiments, a parity effect was present where multiparous animals consistently had higher cortisol concentrations than primiparous animals. Multiparous cows that became pregnant by 100d postpartum had lower concentrations of hair cortisol at d 42 and 84 in milk. Lastly, other biomarkers associated with metabolic status and acute inflammation, such as glucose, β-hydroxybutyrate, haptoglobin, and ceruloplasmin, were not strongly correlated with measurements of cortisol in hair. Overall, hair cortisol measurements appear to be associated with clinical disorders and have a direct association with pregnancy status; however, concentrations of hair cortisol may not be suited to differentiate situations of stress with lower magnitudes, such as the development of subclinical disease.

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.

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 ovula...