Pilot Study to Evaluate the Association Between the Length of the Luteal Phase and Estrous Activity Detected by Automated Activity Monitoring in Dairy Cows.

The ability of automated activity monitoring (AAM) systems to detect estrus is influenced by multiple variables. The luteal phase (LP) preceding estrus has been shown to be important for hormones release, and behavior during estrus in ruminants, but its impact on activity as measured by AAM systems has not been studied. The objective of this study was to investigate the impact of the length of the preceding LP on the intensity and duration of estrus as measured by AAM, and on the expression of estrogen receptor alpha (ERα) in the endometrium. A total of 60 cows between 46 and 53 days in milk were assigned to either a normal or a short length LP. Length of the LP was insured by the use of hormonal synchronization programs. Cows in both treatments were matched on parity, milk production, body condition score (BCS), and lameness score (assessed at enrolment). Expression of ERα receptors in the endometrium were evaluated by nuclear staining by immunohistochemistry of biopsies. Estrus was defined as the day on which the AAM system detected estrus. Cows that were not synchronized by the protocol or that were not detected in estrus by the AAM systems were excluded, which left 21 and 11 cows for analyses in the normal and short length LP, respectively. Peak activity index of estrus, duration of estrus, and expression of ERα were outcomes for multivariable linear regression models. Cows with short length LP tended to have lower peak activity at estrus, but there was no significant effect of treatment when BCS was accounted for. Cows with BCS ≤ 2.5 had less intense and shorter estrus than cows with BCS ≥ 2.75. There was no association between the length of the LP and the expression of ERα in the endometrium.

Pre-calving Intravaginal Administration of Lactic Acid Bacteria Reduces Metritis Prevalence and Regulates Blood Neutrophil Gene Expression After Calving in Dairy Cattle.

Metritis affects up to 40% of dairy cows and it is usually treated with antibiotics. In spite of their advantages, there is an increased concern about antibiotic resistance leading to the research of alternative methods. The aim of this study was to evaluate the effects of a combination of lactic acid bacteria (LAB) on the prevalence of metritis and modulation of endometrial and neutrophil inflammatory markers in dairy cows. One hundred and thirty-five cows were enrolled 3 week before calving and randomly assigned to three treatments. Treatment groups were: (1) two intravaginal doses of LAB/wk during 3 week pre-calving (vaginal, n = 45); (2) an intra-uterine dose, once 1 d after calving (uterine, n = 44); and (3) no intervention (CTRL, n = 45). Metritis was defined as body temperature > 39.5°C and purulent vaginal discharge (> 50% pus), and diagnosed 6 d after calving. Blood samples were taken at d -14, -10, -7, -4, +1, +3, +6, and +14 relative to calving for non-esterified fatty acids (NEFA) analysis. At d -10, +1, +3, and +6 neutrophils were isolated from blood for gene expression analysis by RT-qPCR. Endometrium biopsies were taken from 30 cows, 15 from CTRL and 15 from the uterine group at d +1, +3, and +6 after calving for pro-inflammatory markers analysis by NanoString®. Vaginal treatment reduced metritis prevalence (6/45) up to 58% compared with CTRL group (14/45), but there was no difference between the uterine and CTRL group. Uterine and vaginal treatments reduced blood neutrophil gene expression. Expression of pro-inflammatory markers in the endometrium did not differ between uterine and CTRL cows. Metritic cows expressed more C-X-C motif chemokine ligand 8 (CXCL8) and interleukin 1 beta (IL1B) at d 3 than healthy cows, whereas healthy cows expressed more CXCL8 at d 1 relative to calving in the endometrium. This study shows a promising potential of LAB probiotics as a preventive treatment against metritis in dairy cows.

Integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows.

The aim of this study was to compare 2 reproductive programs for the management of first postpartum artificial insemination (AI) based on activity monitors and timed AI, as well as to determine the effect of health-related factors on detection and expression of estrus. Lactating Holstein cows (n = 918) from 2 commercial farms were enrolled. Estrous cycles of all cows were presynchronized with 2 injections of PGF2α administered 2 wk apart. Treatments were (1) first insemination performed by timed AI (TAI) and (2) first insemination based upon the detection of estrus by activity monitors (ACT; Heatime, SCR Engineering, Netanya, Israel) after the presynchronization, whereas cows not inseminated by the detection of estrus were enrolled in the Ovsynch protocol. Body condition score (BCS; scale 1 to 5), hock score (scale: 1 to 4), gait score (scale: 1 to 4), and corpus luteum presence detected by ovarian ultrasonography were recorded twice during the presynchronization. On the ACT treatment, 50.5% of cows were inseminated based on detected estrus, whereas 83.2% of the cows on the TAI treatment were inseminated appropriately after the timed AI protocol. Pregnancy per AI did not differ by treatment (30.8 vs. 33.5% for ACT and TAI, respectively). Success of pregnancy was affected by parity, cyclicity, BCS, milk production, and a tendency for leg health. In addition, treatment × cyclicity and treatment × parity interactions were found to affect pregnancy success, where anovulatory cows and older cows had compromised pregnancy outcomes on the ACT treatment but not on the TAI treatment. Factors affecting pregnancy outcomes varied among farms. Hazard of pregnancy by 300 DIM was affected by farm, parity, BCS, a treatment × cyclicity interaction, and a tendency for an interaction between leg health and farm. Detection of estrus was affected by farm, parity, cyclicity, and leg health, but not BCS or milk production. Expression of estrus was compromised in anovular and older cows, and by the timing of the estrus event, but not by gait score, BCS, or milk production. Increased duration of estrus, but not intensity of estrus, improved pregnancy per AI. In conclusion, using an automated activity monitor for the detection of estrus within a Presynch-Ovsynch program resulted in similar pregnancy per AI and days open compared with a reproduction program that was strictly based on timed AI for first postpartum AI. In contrast, notable variations in reproductive outcomes were detected between farms, suggesting that the use of automated activity monitors is prone to individual farm management.

Relationship of concentrations of cortisol in hair with health, biomarkers in blood, and reproductive status in dairy cows.

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 ß-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, ß-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.

Automated and visual measurements of estrous behavior and their sources of variation in Holstein heifers. II: Standing and lying patterns.

Lying (LY) and standing (ST) behavior of Holstein heifers (n = 57) was studied from Day -7 to Day +2 relative to estrus (Day 0) using leg-mounted accelerometers. A total of 269 estrus episodes were studied. The objectives were to quantify the effects of estrus on ST and LY patterns, identify sources of variation, and provide insight into new approaches for the use of sensors in estrus detection. The sensors were used to obtain ST bout frequency (bouts per day), total daily ST time, and mean bout duration and duration of the longest bout for ST and LY on a daily basis. Estrus episodes were identified using walking activity peaks and validated by ovarian ultrasonography. Repeated-measures ANOVA was used to analyze the effects of estrus order (pubertal vs. second and greater), season, time of estrus onset, category of baseline walking activity, and number of heifers simultaneously in estrus on ST and LY variables. Overall, ST bout frequency was lower, whereas mean ST bout duration, duration of the longest ST bout, and total daily ST time were longer on Day 0 (P < 0.05). The longest ST bout increased from 232 ± 5 minutes on Day -7 to 488 ± 16 minutes on Day 0 (P < 0.05), and on Day 0, it started within -2 to 4 hours of time of estrus onset. Measurements of ST bouts varied the most on Day 0 (coefficient of variation = 21.5%-52.8%). The pubertal estrus was characterized by smaller increase in mean ST bout duration, longest ST bout duration, and total daily ST time (P < 0.05) compared to the second and greater episodes. Second and greater episodes had greater longest LY bout on Day +1, but on Day 0 it was not different from other nonestrus days (P < 0.01). The longest ST bout on Day 0 was longer for heifers with high baseline walking activity and for episodes occurring during the cold season (P < 0.05). The effect of estrus on ST variables for morning-onset episodes was observed only on Day 0. Episodes with afternoon and night onset showed the effect of estrus spread over Days -1 and 0, resulting in a smaller change between Day -7 and Day 0. The number of heifers simultaneously in estrus did not affect the studied variables (P > 0.05). Measurements of ST and LY changed during estrus and were correlated to walking activity. The large increase in duration of the longest ST bout and its occurrence in proximity of estrus onset suggest potential for automation. Incorporating measurements of ST and LY corrected for sources of variation could improve estrus detection systems.

Short communication: Factors affecting hair cortisol concentrations in lactating dairy cows.

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.