Association of activity and subsequent fertility of dairy cows after spontaneous estrus or timed artificial insemination.

The objective of this observational study was to evaluate the association between increased physical activity at first artificial insemination (AI) and subsequent pregnancy per AI (P/AI) in lactating Holstein cows following spontaneous estrus or a timed AI (TAI) protocol. We also wanted to identify factors associated with the intensity of activity increase (PA) captured by automated activity monitors (AAM) and fertility. Two experiments were conducted, in which cows either were inseminated based on the alert of the AAM system (AAM cows) or received TAI following a 7-d Ovsynch protocol (TAI cows) if not inseminated within a farm-specific period after calving. Experiment 1 included 2,698 AI services from AAM cows and 1,042 AI services from TAI cows equipped with the Smarttag Neck (Nedap Livestock Management) from a dairy farm in Slovakia (farm 1). In the second experiment, 6,517 AI services from AAM cows and 1,226 AI services from TAI cows fitted with Heatime (Heatime Pro; SCR Engineers Ltd.) from 8 dairy farms in Germany (farms 2-9) were included. Pregnancy diagnosis was performed on a weekly basis by transrectal ultrasound (farms 1, 3, 7, 8) or by transrectal palpation (farms 2, 4-6, 9). Estrous intensity was represented by the peak value of the change in activity. In experiment 1, PA was categorized into low (x-factor 0-20) and high (x-factor 21-100) PA, and in experiment 2 into low (activity change = 35-89) and high (activity change = 90-100) PA. In TAI cows from both experiments, PA was additionally categorized into cows with no AAM alert. Data were analyzed separately for AAM and TAI cows using multinomial logistic regression models for PA in TAI cows and logistic regression models for PA in AAM cows and P/AI in both groups. In experiment 1, P/AI of AAM cows was greater for AI services performed with conventional frozen semen (57.6%) compared with sexed semen (47.2%), whereas type of semen only tended to be associated with P/AI in TAI cows (54.4% conventional frozen semen vs. 48.9% sexed semen). In experiment 2, P/AI was greater for fresh semen (AAM cows: 44.4% vs. TAI cows: 44.2%) compared with conventional frozen semen (AAM cows: 37.6% vs. TAI cows: 34.6%). In both experiments, pregnancy outcomes were associated with PA. In experiment 1, AAM cows with high PA (55.1%) had greater P/AI than cows with low PA (49.8%). Within TAI cows, cows with no alert (38.8%) had reduced P/AI compared with cows with low (54.2%) or high PA (61.8%). In experiment 2, AAM cows with high PA (45.8%) had greater P/AI compared with cows with low PA (36.4%). Timed AI cows with no alert (27.4%) had decreased P/AI compared with cows with low (41.1%) or high (50.8%) PA. The greatest risk factors for high PA were parity (experiment 1) and season of AI (except for TAI cows from experiment 1). We conclude that high PA at the time of AI is associated with greater odds of pregnancy for both AAM and TAI cows. In both experiments, about 2 thirds of AAM cows (experiment 1: 69.9% and experiment 2: 70.7%) reached high PA, whereas only approximately one-third or less of TAI cows (experiment 1: 37.3% and experiment 2: 23.6%) showed high PA. Although we observed similar results using 2 different AAM systems for the most part, risk factors for high PA might differ between farms and insemination type (i.e., AAM vs. TAI).

Effect of dose and frequency of prostaglandin F2α treatments during a 7-day Ovsynch protocol with an intravaginal progesterone releasing device on luteal regression and pregnancy outcomes in lactating Holstein cows.

Our objective was to evaluate the effect of 3 different Ovsynch protocols on progesterone (P4) and pregnancies per artificial insemination (P/AI), where all cows received a P4 releasing intravaginal device (PRID) from d 0 until d 8. We hypothesized that (1) both modified PGF2α treatments lead to decreased P4 at the second GnRH treatment (G2), resulting in greater P/AI, (2) the treatment effect is influenced by the presence of a corpus luteum (CL) at the beginning of the protocol, and (3) potential vaginal discharge caused by the PRID does not have a negative influence on fertility. Lactating Holstein cows (n = 1,056) were randomly assigned to 1 of 3 treatment groups on a weekly basis (n = 356; control: d 0, 100 µg of GnRH + PRID; d 7, 25 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the second group (n = 353) received an Ovsynch protocol with a double dose of PGF2α (DoubleDose: d 0, 100 µg of GnRH + PRID; d 7, 50 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the third group (n = 347) received an Ovsynch protocol with a second PGF2α treatment 24 h after the first one (2PGF: d 0, 100 µg of GnRH + PRID; d 7, 25 of mg dinoprost; d 8, 25 mg of dinoprost and PRID removal; d 9, 100 µg of GnRH). All cows had their ovaries scanned to determine the presence of a CL at the beginning of the Ovsynch protocol. Vaginal discharge score (VS) was evaluated at PRID removal. All cows received timed artificial insemination approximately 16 h after G2. Pregnancy diagnosis was performed via transrectal ultrasonography (d 38 ± 3 after timed artificial insemination) and rechecked on d 80 ± 7 after timed artificial insemination. Blood samples were collected on d 0, 7, and 9 of the protocol to determine P4 concentrations. Treatment affected P4 at G2. Progesterone was lower for 2PGF and DoubleDose cows compared with cows in the control group (control 0.35 ± 0.02 ng/mL; DoubleDose 0.29 ± 0.02 ng/mL; 2PGF 0.30 ± 0.02 ng/mL). Overall, P/AI did not differ among treatments. We found, however, an interaction between treatment and CL at the first GnRH treatment. Cows lacking a CL at the first GnRH treatment in the 2PGF group had greater P/AI (47.9%) compared with the same type of cows in the DoubleDose group (32.7%). We observed an effect of VS on P4 concentration at d 7. We found an increase in P4 with greater VS. Vaginal discharge score at PRID removal tended to have a positive effect on P/AI at d 38 (VS0: 36.5%; VS1: 41.3%; VS2: 49.7%). In conclusion, the addition of a second PGF treatment on d 7 and 8 of a 7-d Ovsynch protocol increased luteal regression and decreased mean P4 at G2. Cows treated with PGF2α 2 times 24 h apart showed greater P/AI, compared with cows treated with an increased dose of PGF2α.

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.

Economic impact of adding a second prostaglandin F2α treatment during an Ovsynch protocol using a meta-analytical assessment and a stochastic simulation model.

Incomplete luteal regression after treatment with a single dose of PGF2α during an Ovsynch protocol decreases fertility to timed artificial insemination (AI). An additional treatment with PGF2α 24 h after the first dose has been recommended to increase the proportion of cows with complete luteal regression and subsequent pregnancy per AI (P/AI). This is, however, associated with additional costs of labor and product. The objective was to develop a stochastic partial budget model to estimate the economic impact of an additional PGF2α treatment on d 8 during an Ovsynch protocol in primiparous and multiparous cows. A systematic review of the literature and a meta-analytical assessment was performed to evaluate the effects of adding a second PGF2α treatment during the Ovsynch protocol on P/AI in lactating dairy cows. Thirteen randomized controlled experiments from 11 published manuscripts including 9,735 cows were used. We were able to retrieve information regarding parity from 9 experiments from 7 manuscripts (2,367 primiparous cows and 5,356 multiparous cows). An additional dose of PGF2α yielded a 5.60 risk difference in P/AI [95% confidence interval (CI) = 3.69-7.52]. In primiparous cows and multiparous cows, an additional dose of PGF2α yielded a 4.24 (95% CI = 0.31-8.17) and a 5.31 risk difference in P/AI (95% CI = 2.75-7.87). Revenue was based on the associated improvement in reproductive performance because of an increase in P/AI multiplied by the value of a pregnancy (PGVAL). Median PGVAL was €252, ranging from €42 (fifth percentile) to €623 (95th percentile). Based on parity, median PGVAL was €205 (5th percentile = €43; 95th percentile = €651) and €264 (5th percentile = €88; 95th percentile = €598) for primiparous (n = 1,252) and multiparous cows (n = 3,003), respectively. Using a stochastic simulation model with 10,000 iterations, adding a second PGF2α dose on d 8 was more profitable (€7.76/cow; 5th percentile = €0.01; 95th percentile = €29.40) compared with a single PGF2α administration on d 7. In primiparous cows, adding a second PGF2α treatment was more profitable (€1.99/cow; 5th percentile = -€3.08; 95th percentile = €22.52) in 67% of all simulated iterations. In multiparous cows, adding a second PGF2α treatment was more profitable (€7.92/cow; 5th percentile = -€0.09; 95th percentile = €28.22) in 95% of all simulated iterations. In conclusion, there was a clear benefit of an additional PGF2α treatment during the Ovsynch protocol on P/AI (+5.6 percentage units). Despite additional costs for hormones and labor, an additional treatment with PGF2α on d 8 was more profitable in 95% of all scenarios because of the associated increase in fertility. It turned out, however, that adding a second PGF2α treatment was more profitable in multiparous cows compared with primiparous cows.

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.

Effect of dose and timing of prostaglandin F2α treatments during a 7-d Ovsynch protocol on progesterone concentration at the end of the protocol and pregnancy outcomes in lactating Holstein cows.

The objective of this study was to evaluate the effect of two prostaglandin F2α (PGF) treatments 24 h apart (500 µg of cloprostenol) and treatment with a double PGF dose on d 7 (1000 µg of cloprostenol) during a 7-d Ovsynch protocol on progesterone (P4) concentration and pregnancy per artificial insemination (P/AI) in lactating Holstein cows. We hypothesized that treatment leads to a decreased P4 concentration at the second GnRH treatment (G2) and an increase in P/AI compared to the traditional 7-d Ovsynch protocol. A secondary hypothesis was that the treatment effect is influenced by the presence of a corpus luteum (CL) at the first GnRH treatment (G1). Two experiments were conducted on 8 commercial dairy farms in Germany. Once a week, cows from both experiments were assigned in a consecutive manner to receive: (1) Ovsynch (control: GnRH; 7 d, PGF; 9 d, GnRH), (2) Ovsynch with a double PGF dose (GDPG: GnRH; 7 d, 2xPGF; 9 d, GnRH), or (3) Ovsynch with a second PGF treatment 24 h later (GPPG: GnRH; 7 d, PGF; 8 d, PGF; 32 h, GnRH). All cows received timed AI (TAI) approximately 16 h after G2. Pregnancy diagnosis was performed by transrectal palpation (38 ± 3 d after TAI, experiment 1) or transrectal ultrasonography (35 ± 7 d after TAI, experiment 2). Whereas farms from experiment 1 used a Presynch-Ovsynch protocol (PGF, 14 d later PGF, 12 d later GnRH, 7 d later PGF, 2 d later GnRH, and 16-18 h later TAI) to facilitate first postpartum TAI, no presynchronization protocol was used on farms from experiment 2. In experiment 1, we enrolled 1581 lactating dairy cows (60 experimental units) from 2 dairy farms. At G2, blood samples were collected from a subsample of cows (n = 491; 16 experimental units) to determine P4 concentration at G2. In experiment 2, we enrolled 1979 lactating dairy cows (252 experimental units) from 6 dairy farms. Transrectal ultrasonography was performed to determine the presence or absence of a CL at G1. In experiment 1, treatment affected P/AI (P = 0.01) and P/AI was greater for GDPG (38.2%) and GPPG (38.9%) than for control cows (29.8%). Both, GDPG and GPPG cows had decreased P4 concentration at G2 compared with control cows (P < 0.01). Whereas both treatments increased the percentage of cows with very low P4 concentration (0.00-0.09 ng/mL) at G2, only the GPPG treatment decreased the percentage of cows with high P4 concentration (≥0.6 ng/mL) at G2 compared to the control group. In experiment 2, P/AI was greater for GPPG (37.4%) than for control cows (31.0%; P = 0.03) and tended to be greater than for GDPG cows (31.8%; P = 0.05). Cows from the GDPG group had similar (P = 0.77) P/AI compared to the control group. Pregnancy per AI did not differ between cows with a CL at G1 and cows without a CL at G1 (34.1% vs. 32.6%; P = 0.50). There was no interaction between treatment and presence of a CL at G1 on P/AI (P = 0.61). Combining data from the 2 experiments but excluding cows from experiment 1 receiving presynchronization before first TAI (n = 2573; 312 experimental units), P/AI was greater for GPPG (40.3%; P < 0.01) than for control (31.8%) and GDPG cows (33.4%). Between GDPG and control cows, P/AI did not differ (P = 0.46). We conclude that overall the addition of a second PGF treatment on d 8 during a 7-d Ovsynch protocol increased P/AI compared to the traditional 7-d Ovsynch including a single PGF dose on d 7 and to a double PGF dose on d 7. Doubling the PGF dose on d 7 in a 7-d Ovsynch protocol did not affect P/AI. Use of a presynchronization protocol, however, seems to influence the effect of a dose frequency modification of PGF treatment in an Ovsynch protocol. Presynchronized cows receiving first postpartum TAI had similarly increased P/AI treated with a double PGF dose compared with treatment with a second PGF dose. Future studies need to elucidate whether the treatment effect is modified by presynchronization of the first postpartum TAI.

Machine milking-induced changes in teat canal dimensions as assessed by ultrasonography.

Changes in teat canal diameter as induced by machine milking are thought to be related to teat canal penetrability and susceptibility to new intramammary infections. The objective of this study was to describe the effects of machine milking on teat canal dimensions as assessed by ultrasonography and to evaluate postmilking changes of teat canal dimensions throughout an 8-h milking interval. Ultrasonographic images were taken of the left front and right hind teats of 80 Holstein cows milked 3 times per day. Imaging occurred before attachment of the milking unit after completion of premilking udder preparation (t-1), immediately after removal of the milking unit (t0), and then every hour after milking until 8 h had elapsed (t1 to t8). Teat canal length and teat canal diameter (at the proximal, middle, and distal regions) were measured. General linear mixed model analysis indicated differences in the relative change of teat canal length (compared with t-1) with least squares means (LSM) and 95% confidence interval (95% CI) of 11.1% (8.7-13.4%) after machine milking (t0). Teat canal length decreased throughout the 8-h milking interval but remained elevated relative to t-1. Teat canal diameter at the proximal and middle regions increased near the end of the milking interval, whereas that at the distal region did not change meaningfully during the 8 h of observation. Our findings suggest that the teat canal and its surrounding tissue is in a state of near-constant remodeling in cows milked 3 times daily. Future work is warranted to study the association between ultrasonographically assessed changes of teat canal dimensions after machine milking and teat canal penetrability to mastitis-causing pathogens.

Short communication: Effect of adding a second prostaglandin F2α injection during the Ovsynch protocol on luteal regression and fertility in lactating dairy cows: A meta-analysis.

Incomplete luteal regression after treatment with a single dose of prostaglandin F2α during an Ovsynch protocol decreases fertility to timed artificial insemination (TAI). To increase the proportion of cows with complete luteal regression and subsequently pregnancy per artificial insemination (P/AI), an additional treatment with PGF2α 24 h after the first has been recommended. A systematic review of the literature and meta-analytical assessment were performed with the objective of evaluating the effects of adding a second PGF2α treatment during the Ovsynch protocol on luteal regression and reproductive performance in lactating dairy cows. Based on the heterogeneity among the experimental treatments, a fixed or a random effects meta-analysis was conducted. Reproductive outcomes of interest were luteal regression at the end of the Ovsynch protocol, and P/AI measured 32 to 39 d after TAI. Seven randomized controlled experiments from 6 published manuscripts including 5,356 cows with the primary objective to evaluate the effect of an additional treatment with PGF2α during the Ovsynch protocol on P/AI were used. Information regarding luteal regression at the end of the Ovsynch protocol was available for 1,856 cows. Adding a second PGF2α treatment on d 8 during the Ovsynch protocol increased the relative risk (RR) of complete luteal regression at the end of the Ovsynch protocol (RR = 1.14; 95% confidence interval = 1.10 to 1.17) using a fixed effects model and the RR for pregnancy (RR = 1.14; 95% confidence interval = 1.06 to 1.22) 32 d after TAI using a fixed effects model. No heterogeneity was observed among the 6 manuscripts regarding complete luteal regression and P/AI. In summary, there was a clear benefit of an additional PGF2α treatment during the Ovsynch protocol on luteal regression (+11.6 percentage units) and on P/AI (+4.6 percentage units).

Technical note: Development and evaluation of a standard operating procedure for ultrasound-based measurements of teat canal dimensions in dairy cows.

The objectives were to (1) develop a standard operating procedure (SOP) for ultrasound-based measurements of teat canal dimensions and (2) determine the precision of ultrasound-based measurements of teat canal dimensions in dairy cows. Teat scans (n = 64) of the right hind and left front teats from 16 cows were obtained with a portable ultrasound device before and after machine milking. Teat dimensions measured were teat canal length (TCL), teat canal diameter at the proximal end of the teat canal (TCDPROX), teat canal diameter at the midpoint between the proximal and distal ends of the teat canal (TCDMID), teat canal diameter at the distal end of the teat canal (TCDDIS), teat end diameter at the midpoint between the proximal and distal ends of the teat canal (TMD), and teat canal cross-sectional area (TCLAREA). An SOP was developed, reviewed, and modified by 3 operators. Measurements were performed by the same 3 operators using an open source software program. To assess the effect of implemented modifications and the precision of ultrasound-based measurements, concordance correlation coefficients (CCC) were calculated to determine interoperator reproducibility and intraoperator repeatability. Through modifications in the SOP, interoperator CCC increased from 0.45 to 0.86 for TCL, from 0.14 to 0.66 for TCDPROX, from 0.24 to 0.66 for TCDMID, from 0.06 to 0.56 for TCDDIS, from 0.64 to 0.91 for TMD, and from 0.17 to 0.64 for TCLAREA. Intraoperator CCC over all operators were 0.91 for TCL, 0.73 for TCDPROX, 0.80 for TCDMID, 0.69 for TCDDIS, 0.94 for TMD, and 0.75 for TCLAREA. Our results indicate that measurements of teat canal dimensions can be conducted with satisfactory precision when following an SOP. Development and strict implementation of an SOP has the potential to decrease inadvertent variability of ultrasound-based measurements among operators for ultrasonographic assessment of teat dimensions.

Comparison of pregnancy outcomes using either an Ovsynch or a Cosynch protocol for the first timed AI with liquid or frozen semen in lactating dairy cows.

The objective of this study was to evaluate fertility to the first timed AI (TAI) using either liquid semen or frozen semen after an Ovsynch or a Cosynch protocol in lactating dairy cows. The hypothesis was that there is an increase in fertility to the first TAI when cows are inseminated with liquid semen compared to that when frozen semen is used in a Cosynch protocol. Lactating dairy cows (n = 1724; 540 primiparous, 1184 multiparous) from 9 commercial dairy farms were enrolled on a weekly basis to facilitate first timed AI. Two experiments were conducted. In experiment 1, all cows received GnRH, 7 d later PGF2α, and then received one of the following treatments: 1) GnRH + TAI with liquid semen 56 h after PGF2α; 2) GnRH + TAI with frozen semen 56 h after PGF2α; 3) GnRH 56 h after PGF2α + TAI with liquid semen 12-16 h after the second GnRH; 4) GnRH 56 h after PGF2α + TAI with frozen semen 12-16 h after the second GnRH. In experiment 2, all cows received GnRH, 7 d later PGF2α, and then received treatments 3 or 4 as described for experiment 1. Number of sperm per straw was 20 × 106 sperm/straw and 10 × 106 sperm/straw for frozen and liquid semen, respectively. Pregnancy diagnosis was performed by ultrasound scanning at 39 d after TAI. In experiment 1 (n = 1263), there was an interaction of semen preservation method by TAI protocol. Cows inseminated with liquid semen concurrently with the second GnRH (Cosynch-56) achieved greater pregnancy per AI (P/AI) than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P = 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P = 0.330) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P = 0.001). In experiment 2 (n = 377), there was no effect of semen preservation method (liquid semen 26.5% vs. frozen semen 25.5%; P = 0.846) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P = 0.001). The results of this study provide evidence that liquid semen achieved greater P/AI in a TAI protocol with a long time interval between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract.

Evaluation of prostaglandin F2α versus prostaglandin F2α plus gonadotropin-releasing hormone as Presynch methods preceding an Ovsynch in lactating dairy cows: A meta-analysis.

Presynchronization of cows with 2 injections of prostaglandin administered 14 d apart (Presynch-Ovsynch) is a widely adopted procedure to increase pregnancy per artificial insemination (P/AI) at first service. Recently, a presynchronization protocol including GnRH and PGF2α (Double-Ovsynch; GnRH, 7 d, PGF2α, 3 d, GnRH) followed 7 d later by an Ovsynch protocol was introduced to overcome the limitations of PGF2α-based protocols for presynchronization of anovular cows and to precisely set up cows on d 7 of the estrous cycle when the Ovsynch is initiated. A systematic review of the literature and a meta-analytical assessment was performed with the objective to compare the reproductive performance of lactating dairy cows presynchronized with these 2 protocols for the first timed AI (TAI) considering parity-specific effects. A fixed or a random effects meta-analysis was used based on the heterogeneity among the experimental groups. Reproductive outcomes of interest were P/AI measured on d 32 (28-42) and pregnancy loss between d 32 and 60 (42-74) of gestation. A total of 25 articles with 27 experimental groups from 63 herds including 21,046 cows submitted to first TAI using either a Presynch-Ovsynch or a Double-Ovsynch protocol were reviewed. Results for P/AI were then categorized by parity if available. Information was available for P/AI for 7,400 and 10,999 primiparous and multiparous cows, respectively. Information regarding pregnancy loss was available for 7,477 cows. In the random effects model for all cows, the overall proportion of P/AI was 41.7% [95% confidence interval (CI): 39.1-44.3; n = 8,213] and 46.2% (95% CI: 41.9-50.5; n = 12,833) on d 32 after TAI for Presynch-Ovsynch and Double-Ovsynch, respectively. In the random effects model for primiparous cows, the overall proportion of P/AI was 43.4% (95% CI: 36.2-47.7; n = 2,614) and 51.4% (95% CI: 47.4-55.4; n = 4,786) on d 32 after TAI for Presynch-Ovsynch and Double-Ovsynch, respectively. In the random effects model for multiparous cows, the overall proportion of P/AI was 39.2% (95% CI: 36.2-42.3; n = 3,411) and 41.4% (95% CI: 36.4-46.4; n = 7,588) on d 32 after TAI for Presynch-Ovsynch and Double-Ovsynch, respectively. The overall proportion of pregnancy loss was 11.3% (95% CI: 7.6-15.7; n = 3,247) and 11.7% (95% CI: 9.3-14.3; n = 4,230) on d 60 after AI for Presynch-Ovsynch to and Double-Ovsynch, respectively. Substantial heterogeneity existed among the experimental groups regarding P/AI and pregnancy loss. In summary, a benefit was detected for P/AI in primiparous cows presynchronized with a Double-Ovsynch protocol for the first TAI, but this benefit was not observed in multiparous cows.

Evaluation of oestrous detection in dairy cattle comparing an automated activity monitoring system to visual observation.

The objective of the study was to evaluate an automated activity monitoring (AAM) system for oestrous detection in dairy cows. Specifically, we set out to determine the oestrous detection efficiency and accuracy of the AAM system and to compare reproductive performance of cows detected either by AAM, by visual observation (VO) or by at least 1 of the 2 methods (EOM). A total of 1004 potential cow-periods from 348 cows were analysed. Oestrous detection rates (EDR) were calculated for 21 day (VWP+21), 42 day (VWP+42), and 63 day (VWP+63) after voluntary waiting period (VWP). Oestrous detection rate did not differ between AAM (42.1%) and VO (37.3%) during VWP+21 (p > 0.05) but was significantly higher in EOM (56.3%, p < 0.05). Oestrous detection by AAM achieved an efficiency (number of correctly detected oestruses/total number of cow-periods*100) of 35.6% and an accuracy (number of correctly detected oestruses/(number of true+ false oestruses)*100) of 83.8%. Visual observation resulted in an efficiency of 34.3% and accuracy of 75.1%. Pregnancy rate at 200 DIM was higher in AAM (66.8%) and EOM (68.8%) than in VO (57.1%, p < 0.05). Cows detected by AAM (p < 0.05) showed a 1.37 times greater risk to conceive than cows detected by VO as the reference (p < 0.05). The results show that an automated activity monitoring system can support oestrous detection in dairy cattle. The exclusive use of such a system, however, cannot be recommended.

MicroRNA-regulated molecular mechanism underlying bovine subclinical endometritis.

An impaired uterine environment triggered by the incidence of subclinical endometritis often compromises fertility in the bovine. The uterus is a dynamic organ with tight regulation of specific genes at the transcriptional and translational levels. Herein, we hypothesised that subclinical endometritis alters the expression of uterine microRNAs (miRNAs), which may result in the dysregulation of corresponding target genes and biological pathways. To test this hypothesis, we used a genome-wide RT(2) (Exiqon, Vedbaek, Denmark) miRNA PCR array consisting of 354 miRNA primers and analysed miRNA expression in uterine cytobrush samples taken from cows with and without subclinical endometritis. The results revealed aberrant expression of 23 miRNAs in cows with subclinical endometritis compared with healthy cows. Furthermore, we designed an in vitro endometrial cell culture model challenged by lipopolysaccharide (LPS) to validate the differential regulation of miRNAs in cytobrush samples. Interestingly, we observed similar expression miRNA patterns in cytobrush samples taken from cows with or without subclinical endometritis and in vitro cultured endometrial cells challenged by LPS. To trace signalling pathways and biological functions potentially controlled by the aberrantly expressed miRNAs, we filtered high-ranking target genes from miRBase and analysed them using ingenuity pathway analysis. The gene networks, canonical pathways and biological functions strikingly converged to signalling pathways that mediate inflammatory responses, cellular proliferation, cell movement, the cell cycle and apoptosis in the bovine endometrium. In addition, expression analysis of key genes from the gene networks confirmed their presence and the potential regulation of these genes by uterine miRNAs. Furthermore, luciferase assay data substantiated the primary information from bioinformatic prediction that generated potential target genes for the dysregulated miRNAs in subclinical endometritis. Together, these data suggest the potential regulatory role of uterine miRNAs in the development and progression of bovine subclinical endometritis.

Does oral administration of the amino acid tyrosine affect oestradiol-17ß concentration and sexual behaviour in the bitch?

The oral administration of the amino acid, tyrosine, has been for years recommended in order to improve fertility, especially to improve copulation behaviour in female dogs. However, evidence is comparatively poor. The objective of our study was to determine whether oral administration of tyrosine has an effect on oestradiol-17ß concentrations and the oestrous behaviour in the bitch. Fifty bitches were randomly allocated to one of two treatment groups in which each dog received 100mg/kg/day of either tyrosine or milk sugar orally between Day 3 and Day 9 of heat. Every two to three days, a gynaecological examination was performed and blood samples were taken to determine oestradiol-17ß and progesterone concentrations. The day of ovulation was estimated by clinical findings, and according to the specifications of the laboratory, once progesterone values exceeded 12.7nmol/l. The observed copulation behaviour was not different between the groups. No differences in volume and visual nature of vaginal discharge were observed. At the day of ovulation, mean oestradiol-17ß concentration in the treated group was 163.4pmol/l and 162.2pmol/l in the placebo group, respectively. In conclusion, feeding tyrosine to female dogs between Day 3 and Day 9 of heat did not alter visual signs of heat or copulation behaviour, and did not alter oestradiol-17ß concentration.

Investigations on the endometrial response to intrauterine administration of N-acetylcysteine in oestrous mares.

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.

Short communication: Ability of dogs to detect cows in estrus from sniffing saliva samples.

Efficient estrus detection in high-producing dairy cows is a permanent challenge for successful reproductive performance. In former studies, dogs have been trained to identify estrus-specific odor in vaginal fluid, milk, urine, and blood samples under laboratory conditions with an accuracy of more than 80%. For on-farm utilization of estrus-detection dogs it would be beneficial in terms of hygiene and safety if dogs could identify cows from the feed alley. The objective of this proof of concept study was to test if dogs can be trained to detect estrus-specific scent in saliva of cows. Saliva samples were collected from cows in estrus and diestrus. Thirteen dogs of various breeds and both sexes were trained in this study. Five dogs had no experience in scent detection, whereas 8 dogs had been formerly trained for detection of narcotics or cancer. In the training and test situation, dogs had to detect 1 positive out of 4 samples. Dog training was based on positive reinforcement and dogs were rewarded with a clicker and food for indicating saliva samples of cows in estrus. A false indication was ignored and documented in the test situation. Dogs with and without prior training were trained for 1 and 5 d, respectively. For determining the accuracy of detection, the position of the positive sample was unknown to the dog handler, to avoid hidden cues to the dog. The overall percentage of correct positive indications was 57.6% (175/304), with a range from 40 (1 dog) to 75% (3 dogs). To our knowledge, this is the first indication that dogs are able to detect estrus-specific scent in saliva of cows.

Determination of ceftiofur derivatives in serum, endometrial tissue, and lochia in puerperal dairy cows with fever or acute puerperal metritis after subcutaneous administration of ceftiofur crystalline free acid.

Acute puerperal metritis (APM) is one of the most common diseases during the puerperal period. Systemic administration of ceftiofur for 5 consecutive days has been shown to be effective for treatment of APM. The objective of this study was to determine concentrations of ceftiofur derivatives in serum, endometrial tissue, and lochia of cows with fever postpartum or APM 4 to 6d after treatment with a single subcutaneous dose of 6.6 mg of ceftiofur crystalline free acid (CCFA)/kg of estimated BW at the base of the ear. In the first experiment, samples from CCFA-treated cows with fever postpartum or APM (n=42) were taken on d 4, 5, or 6 after treatment. Concentrations of ceftiofur derivatives were quantified using an HPLC assay. Concentrations of active ceftiofur metabolite desfuroylceftiofuracetamide (DCA) were greatest at d 4 after treatment with CCFA in all samples, but they were considerably lower than the concentrations of DCA in healthy postpartum cows treated with the same dose of CCFA. The concentrations of DCA in serum, endometrial tissue, and lochia were affected by odor of vaginal discharge before treatment with CCFA. Mean concentrations of DCA could be detected above the reported minimal drug concentrations (minimum inhibitory concentrations, MIC) required to inhibit relevant pathogens such as Escherichia coli and Arcanobacterium pyogenes in serum on all days and in endometrial tissue and lochia only on d 4 in CCFA-treated cows with fetid vaginal discharge before treatment. In the second experiment, samples from CCFA-treated cows with APM (n=8) were taken on d 0 (before treatment) and d 4, 5, and 6 after treatment. Mean concentrations of DCA in serum and lochia were similar on d 4 to 6 in both laboratories. Furthermore, determined concentrations of DCA from both laboratories were correlated for serum and lochia. Mean concentrations of DCA could be detected above the reported MIC in serum and lochia only on d 4. Our 2 experiments demonstrated that in postpartum cows with fever postpartum or APM concentrations above the MIC for relevant bacteria (>0.5 µg/mL or >0.5 µg/g) of DCA could be sustained only for 4 (serum: 15/17; endometrial tissue: 2/17; lochia: 1/16) to 5d (serum: 10/13; endometrial tissue: 1/13; lochia: 2/12) after a single treatment with CCFA only in a certain proportion of cows. Overall, our data provide first pharmacological evidence that a single subcutaneous administration of 6.6g of CCFA/kg of BW might not be sufficient to efficaciously treat APM in postpartum dairy cows.

Advances in modeling of the bovine estrous cycle: synchronization with PGF2α.

Our model of the bovine estrous cycle is a set of ordinary differential equations which generates hormone profiles of successive estrous cycles with several follicular waves per cycle. It describes the growth and decay of the follicles and the corpus luteum, as well as the change of the key reproductive hormones, enzymes and processes over time. In this work we describe recent developments of this model towards the administration of prostaglandin F2α. We validate our model by showing that the simulations agree with observations from synchronization studies and with measured progesterone data after single dose administrations of synthetic prostaglandin F2α.