Presynchronization with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating dairy cows

The objective of this study was to compare circulating progesterone (P4) profiles and pregnancies per AI (P/AI) in lactating dairy cows bred by timed artificial insemination (TAI) following Ovsynch-56 after 2 different presynchronization protocols: Double-Ovsynch (DO) or Presynch-Ovsynch (PS). Our main hypothesis was that DO would increase fertility in primiparous cows, but not in multiparous cows. Within each herd (n = 3), lactating dairy cows (n = 1,687; 778 primiparous, 909 multiparous) were randomly assigned to DO [n = 837; GnRH-7d-PGF_2α-3d-GnRH-7d-Ovsynch-56 (GnRH-7d-PGF_2α-56h-GnRH-16hTAI)] or PS (n = 850; PGF_2α-14d-PGF_2α-12d-Ovsynch-56). In 1 herd, concentrations of P4 were determined at the first GnRH (GnRH1) of Ovsynch-56 and at d 11 after TAI (n = 739). In all herds, pregnancy was diagnosed by palpation per rectum at 39 d. In 1 herd, the incidence of late embryo loss was determined at 74 d, and data were available on P/AI at the subsequent second service. Presynchronization with DO reduced the percentage of animals with low P4 concentrations (<0.50 ng/mL) at GnRH1 of Ovsynch-56 (5.4 vs. 25.3%, DO vs. PS). A lesser percentage of both primiparous and multiparous cows treated with DO had low P4 concentrations at GnRH1 of Ovsynch-56 (3.3 vs. 19.7%, DO vs. PS primiparous; and 8.8 vs. 31.9%, DO vs. PS multiparous). Presynchronization with DO improved P/AI at the first postpartum service (46.3 vs. 38.2%, DO vs. PS). Statistically, a fertility improvement could be detected for primiparous cows treated with DO (52.5 vs. 42.3%, DO vs. PS, primiparous), but only a tendency could be detected in multiparous cows (40.3 vs. 34.3%, DO vs. PS, multiparous), consistent with our original hypothesis. Presynchronization treatment had no effect on the incidence of late embryo loss after first service (8.5 vs. 5.5%, DO vs. PS). A lower body condition score increased the percentage of cows with low P4 at GnRH1 of Ovsynch-56 and reduced fertility to the TAI. In addition, P4 concentration at d 11 after TAI was reduced by DO. The method of presynchronization at first service had no effect on P/AI at the subsequent second service (34.7 vs. 36.5%, DO vs. PS). Thus, presynchronization with DO induced cyclicity in most anovular cows and improved fertility compared with PS, suggesting that DO could be a useful reproductive management protocol for synchronizing first service in commercial dairy herds.     

Factors affecting conception rates following artificial insemination or embryo transfer in lactating Holstein cows

The objective of this study was to evaluate the factors that may affect conception rates (CR) following artificial insemination (AI) or embryo transfer (ET) in lactating Holstein cows. Estrous cycling cows producing 33.1 ± 7.2 kg of milk/d received PGF_2α injections and were assigned randomly to 1 of 2 groups (AI or ET). Cows detected in estrus (n = 387) between 48 and 96 h after the PGF_2α injection received AI (n = 227) 12 h after detection of estrus or ET (n = 160) 6 to 8 d later (1 fresh embryo, grade 1 or 2, produced from nonlactating cows). Pregnancy was diagnosed at 28 and 42 d after estrus, and embryonic loss occurred when a cow was pregnant on d 28 but not pregnant on d 42. Ovulation, conception, and embryonic loss were analyzed by a logistic model to evaluate the effects of covariates [days in milk (DIM), milk yield, body temperature (BT) at d 7 and 14 post-AI, and serum concentration of progesterone (P4) at d 7 and 14 post-AI] on the probability of success. The first analysis included all cows that were detected in estrus. The CR of AI and ET were different on d 28 (AI, 32.6% vs. ET, 49.4%) and 42 (AI, 29.1% vs. ET, 38.8%) and were negatively influenced by high BT (d 7) and DIM. The second analysis included only cows with a corpus luteum on d 7. Ovulation rate was 84.8% and was only negatively affected by DIM. Conception rates of AI and ET were different on d 28 (AI, 37.9% vs. ET, 59.4%) and 42 (AI, 33.8% vs. ET, 46.6%) and were negatively influenced by high BT (d 7). The third analysis included only ovulating cows that were 7 d postestrus. Conception rates of AI and ET were different on d 28 (AI, 37.5% vs. ET, 63.2%) and 42 (AI, 31.7% vs. ET, 51.7%) and were negatively influenced by high BT (d 7). There was a positive effect of serum concentration of P4 and a negative effect of milk production on the probability of conception for the AI group but not for the ET group. The fourth analysis was embryonic loss (AI, 10.8% vs. ET, 21.5%). The transfer of fresh embryos is an important tool to increase the probability of conception of lactating Holstein cows because it can bypass the negative effects of milk production and low P4 on the early embryo. The superiority of ET vs. AI is more evident in high-producing cows. High BT measured on d 7 had a negative effect on CR and embryonic retention.     

The use of plastic cover sheaths at the time of artificial insemination improved fertility of lactating dairy cows

An adequate and clean artificial insemination (AI) technique is recommended to improve reproductive outcomes in dairy cattle. The objective of this study was to evaluate the effectiveness of using protective plastic sheaths (PS) to minimize contamination of the AI catheter (AIC) on pregnancies per AI (PAI) in lactating dairy cattle. Lactating cows housed in freestall barns on a commercial dairy farm were presynchronized with 2 injections of PGF_2α given 14 d apart (starting at 26 ± 3 d postpartum) followed by Ovsynch (GnRH-7 d-PGF_2α-56 h-GnRH-16 h-timed-AI; TAI) 12 d later. Cows presenting signs of standing heat any time during the protocol received AI, whereas the remaining animals were subjected to TAI 16 h after second Ovsynch GnRH. At the time of AI (1 AI technician), 996 services from 773 lactating dairy cows were randomly assigned to 1 of the 2 groups; with (TRT, n = 487) or without (CON, n = 509) the use of disposable PS. In the TRT group, the AIC protected with a PS was introduced into the vagina; once in the cranial portion of the vagina adjacent to the cervical os, the PS was pulled back and only the AIC was manipulated through the cervix into the uterine body for semen deposition. In the CON group, cows were inseminated without the use of PS. Samples were taken with a sterile cotton swab from the tip of the AIC (n = 51) after AI from both treatment groups. Pregnancy diagnosis was determined by ultrasonography 39 ± 3 d after AI. Cultured swab samples revealed that the use of PS was effective in minimizing contamination of the AIC (positive bacterial growth: TRT = 61.53% vs. CON = 100%). Overall, the proportion of cows pregnant was greater for cows in TRT (42.7) compared with the CON group (36.1). For first services postpartum, PAI did not differ between CON (43.01%, n = 194) and TRT (43.8%, n = 182) groups. However, PAI for second or greater services were greater in TRT (43.8%, n = 305) than in CON cows (32.3%, n = 315). Results from this study provided evidence that the use of PS during AI improved PAI for second or greater services in lactating dairy cows. Performing a clean AI technique through the use of PS may be an effective strategy to improve reproductive outcomes in dairy cattle.     

Fertility of lactating Holstein cows submitted to a Double-Ovsynch protocol and timed artificial insemination versus artificial insemination after synchronization of estrus at a similar day in milk range

Our objective was to compare the AI submission rate and pregnancies per artificial insemination (P/AI) at first service of lactating Holstein cows submitted to a Double-Ovsynch protocol and timed artificial insemination (TAI) versus artificial insemination (AI) to a detected estrus after synchronization of estrus at a similar day in milk range. Lactating Holstein cows were randomly assigned to receive their first TAI after a Double-Ovsynch protocol (DO; n = 294) or to receive their first AI after a synchronized estrus (EST; n = 284). Pregnancy status was determined 33 ± 3 d after insemination and was reconfirmed 63 ± 3 d after insemination. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures of SAS (SAS Institute Inc., Cary, NC). By design, days in milk at first insemination did not differ between treatments (76.9 ± 0.2 vs. 76.7 ± 0.3 for DO vs. EST cows, respectively), but more DO cows were inseminated within 7 d after the end of the voluntary waiting period than EST cows (100.0 vs. 77.5%). Overall, DO cows had more P/AI than EST cows at both 33 d (49.0 vs. 38.6%) and 63 d (44.6 vs. 36.4%) after insemination, but pregnancy loss from 33 to 63 d after insemination did not differ between treatments. Primiparous cows had more P/AI than multiparous cows 33 and 63 d after insemination, but the treatment by parity interaction was not significant. Synchronization rate to the hormonal protocols was 85.3%, which did not differ between treatments; however, synchronized DO cows had more P/AI 33 d after insemination than synchronized EST cows (54.7 vs. 44.5%). In summary, submission of lactating Holstein cows to a Double-Ovsynch protocol and TAI for first insemination increased the percentage of cows inseminated within 7 d after the end of the voluntary waiting period and increased P/AI at 33 and 63 d after first insemination resulting in 64 and 58% more pregnant cows, respectively, than submission of cows for first AI after detection of estrus at a similar day in milk range. We conclude that, because the proportion of synchronized cows did not differ between treatments, DO cows had more P/AI than EST cows because of an intrinsic increase in fertility after submission to a fertility program.     

Effect of interval from timed artificial insemination to initiation of resynchronization of ovulation on fertility of lactating dairy cows

To compare 2 strategies for systematically resynchronizing ovulation, lactating Holstein cows (n = 763) at various days in milk and prior artificial insemination services were assigned randomly at timed AI (TAI) to receive the first GnRH injection of Ovsynch 26 (D26) or 33 (D33) d after TAI to resynchronize ovulation (Resynch) in cows failing to conceive. Cows in the D26 treatment received GnRH 26 d after TAI and continued Resynch only when diagnosed not pregnant by using ultrasonography 33 d after TAI, whereas D33 cows initiated Resynch only when diagnosed not pregnant 33 d after TAI. Cows were classified based on the presence or absence of a corpus luteum (CL) at the not-pregnant diagnosis, and cows without a CL received an intravaginal progesterone-releasing insert during Resynch. When analyzed as a systematic strategy, pregnancy rate per AI (PR/AI) was greater for cows assigned to the D33 than the D26 Resynch treatment (39.4 vs. 28.6%). A treatment × parity interaction was detected for PR/AI after Resynch for nonpregnant cows having a CL in which primiparous cows had a greater PR/AI than multiparous cows when Resynch was initiated 33 d after the initial TAI, and primiparous and multiparous cows when Resynch was initiated 26 d after the initial TAI. Pregnancy loss for Resynch was 6.4% between 33 and 40 d, and 2.6% between 40 and 61 d after Resynch TAI. We concluded that delaying initiation of Resynch until 33 d after TAI increased PR/AI for primiparous cows.     

Characterization of anogenital distance and its relationship to fertility in lactating Holstein cows

Anogenital distance (AGD) serves as a marker for prenatal androgenization, reproductive development, and fertility in humans and rodents. The primary objectives of this observational study in lactating dairy cows were to (1) characterize the distribution and variability of AGD, (2) determine the relationship among AGD and potential postnatal AGD determinants of age and height, and (3) evaluate the associations between AGD and pregnancy to first artificial insemination (P/AI) and cumulative pregnancy by 250 d in milk (DIM) within parity groups (first, second, and third+ parities). The secondary objective was to evaluate the association between AGD and testosterone concentrations. The AGD (mm), age (yr), and height at hip (cm) at the time of AGD determination, and aforesaid reproductive outcomes were determined in 921 Holstein cows (first, second, and third+ parity; n = 360, 256, and 305, respectively). Plasma concentrations of testosterone were determined in a subset of 93 cows. Overall, AGD had a normal distribution and high variability [mean (±standard deviation); 131.0 ± 12.2 mm], was weakly associated with cow age and height (coefficient of determination = 0.09 and 0.04, respectively), and had an inverse relationship with P/AI in first- and second-parity cows, but not in third+ parity cows. For every 1 mm increase in AGD, the odds of P/AI decreased by 3.4 and 2.4% for first- and second-parity cows, respectively. The optimal AGD threshold to predict probability of P/AI was 127.1 mm for both first- (sensitivity: 66.4; specificity: 56.6%) and second-parity cows (sensitivity: 46.0; specificity: 70.4%). Accordingly, first- and second-parity cows were categorized into either short or long AGD (≤ or >127.1 mm), and associations with reproductive outcomes were evaluated. First-parity cows with long AGD had lower P/AI (30.9 vs. 53.6%) and decreased likelihood (hazard ratio: 0.68) of pregnancy by 250 DIM than those with short AGD. Similarly, second-parity cows with long AGD had reduced P/AI (28.3 vs. 44.4%) and a tendency for decreased likelihood (hazard ratio: 0.76) of pregnancy by 250 DIM than in cows with short AGD. The association between AGD and testosterone was weak and nonsignificant. In summary, AGD in Holstein cows was normally distributed, highly variable, and weakly associated with age and height. Besides, AGD had an inverse relationship with P/AI and cumulative pregnancy by 250 DIM in first- and second-parity cows; however, such a relationship was not evident in older (third+ parity) cows.     

Characterization of luteal dynamics in lactating Holstein cows for 32 days after synchronization of ovulation and timed artificial insemination

Approximately 20 to 30% of cows diagnosed not pregnant 32 d after timed artificial insemination (TAI) lack a corpus luteum (CL), and cows submitted to a resynchronization protocol in the absence of a CL have about 10% fewer pregnancies per AI (P/AI) than cows with a CL. An understanding of luteal dynamics after synchronization of ovulation and TAI may help refine strategies for reinseminating cows failing to conceive. Lactating Holstein cows (n = 141) were synchronized for first TAI using a Double-Ovsynch protocol. Thrice weekly from 4 to 32 d after TAI, blood samples were collected for evaluation of plasma progesterone (P4) concentrations, and CL diameter was measured using transrectal ultrasonography. Pregnancy status was determined using transrectal ultrasonography 32 d after TAI. Nonsynchronized cows (n = 4) were removed from the study. For cows diagnosed pregnant 32 d after TAI (n = 57), P4 increased from 4 to 15 d and then remained constant until 32 d after TAI, whereas CL volume increased from 4 to 11 d and then remained constant until 32 d after TAI. For cows diagnosed not pregnant 32 d after TAI (n = 80), P4 profiles were evaluated using statistical cluster analysis based on the day after TAI that P4 decreased to <1 ng/mL, resulting in 5 clusters: (1) CL regression 15 d after TAI (1.3%), (2) CL regression 18 to 22 d after TAI (55.0%), (3) CL regression 25 to 27 d after TAI (17.5%), (4) CL regression 29 to 32 d after TAI (5.0%), and (5) CL maintained until 32 d after TAI (21.3%). Plasma pregnancy-associated glycoprotein (PAG) levels at 25 and 32 d after TAI differed among clusters and were below the cut-off value of the assay for the classification of cows as not pregnant for cows in clusters 2, 3, and 4, whereas more than half of the cows in cluster 5 had increased plasma PAG levels. We conclude that at least half of the nonpregnant cows that maintained their CL until 32 d after TAI were initially pregnant but underwent early pregnancy loss based on increased plasma PAG levels at 25 and 32 d after TAI.     

Dynamics of automatically generated body condition scores during early lactation and pregnancy at first artificial insemination of Holstein cows

The objective of this study was to characterize the association between body condition score (BCS) and BCS change (ΔBCS), determined by an automated camera system at multiple time points, and the subsequent pregnancy per first artificial insemination (P/AI1) of Holstein cows. A retrospective observational study was completed using data collected from 11,393 lactations in 7,928 Holstein cows calving between April 2019 and March 2021 in a commercial dairy operation located in Colorado. Cows were classified as primiparous or multiparous. Scores generated by BCS cameras at dry-off, calving, 21 days in milk (DIM), 56 DIM, and first artificial insemination were selected for the analyses and subsequently categorized as low (≤lower quartile), moderate (interquartile range), and high (≥upper quartile). Changes in BCS were calculated by periods of interest as change from dry-off to calving (multiparous cows); change from calving to 21 DIM; change from calving to 56 DIM; and change from calving to first artificial insemination and assigned into categories as large loss of BCS (top 25% of cows losing BCS); moderate loss (bottom 75% of cows losing BCS); no change (ΔBCS = 0); or gain of BCS (ΔBCS > 0). Data were examined in primiparous and multiparous cows separately using logistic regression and time-to-event analyses. Initial univariable models were followed by multivariable models that considered calving season, occurrence of disease, and milk yield up to 60 DIM as covariables. The logistic regression analyses indicated that in both parity groups the associations between BCS category and P/AI1 were more evident at 21 DIM, 56 DIM, and first artificial insemination, with lower odds of P/AI1 in cows in the low BCS category. Likewise, cows with large loss in BCS between calving and 21 DIM, calving and 56 DIM, and calving and first artificial insemination had lower odds of P/AI1 compared with other categories of ΔBCS within the same period of interest. Similarly, survival analyses evidenced that cows in the low BCS category required more time to get pregnant. In agreement, differences in the dynamics of the average daily BCS during the first 90 DIM were evident when cows were grouped by first AI outcome (pregnant vs. nonpregnant) and by their time to pregnancy category (<90 DIM; 91–150 DIM; or >150 DIM), with cows with reduced fertility showing lower BCS up to 90 DIM. Overall, low BCS and more pronounced reductions in BCS occurring closer to first artificial insemination resulted in lower odds of pregnancy per artificial insemination.     

Effects of 1 or 2 treatments with prostaglandin F2α on subclinical endometritis and fertility in lactating dairy cows inseminated by timed artificial insemination

The objectives of the current study were to investigate the efficacy of PGF_2α as a therapy to reduce the prevalence of subclinical endometritis and improve pregnancy per artificial insemination (P/AI) in cows subjected to a timed artificial insemination (AI) program. A total of 1,342 lactating Holstein dairy cows were allocated randomly at 25 ± 3 d in milk (DIM) to remain as untreated controls (control, n = 454) or to receive a single PGF_2α treatment at 39 ± 3 DIM (1PGF, n = 474) or 2 treatments with PGF_2α at 25 ± 3 and 39 ± 3 DIM (2PGF, n = 414). All cows were enrolled in the double Ovsynch program at 48 ± 3 DIM and were inseminated at 75 ± 3 DIM. A subset of 357 cows had uterine samples collected for cytological examination at 25 ± 3, 32 ± 3, and 46 ± 3 DIM to determine the percentage of polymorphonuclear leukocytes (PMNL). Subclinical endometritis was defined by the presence of ≥5% PMNL. Vaginal discharge score was evaluated at 25 ± 3 DIM and used to define the prevalence of purulent vaginal discharge. Body condition score was assessed at 25 ± 3 DIM. Pregnancy was diagnosed 32 d after AI and reconfirmed 28 d later. At 32 ± 3 DIM, the prevalence of subclinical endometritis was reduced by treatment with PGF_2α at 25 ± 3 DIM in 2PGF (control = 23.5% vs. 1PGF = 28.3% vs. 2PGF = 16.7%); however, this benefit disappeared at 46 ± 3 DIM, and 14% of the cows remained with subclinical endometritis. One or 2 treatments with PGF_2α did not influence P/AI on d 32 or 60 after timed AI, which averaged 39.9 and 35.2%. Similarly, treatment with PGF_2α had no effect on pregnancy loss between 32 and 60 d of gestation (11.9%). Cows diagnosed with both purulent vaginal discharge and subclinical endometritis had the lowest P/AI and the highest pregnancy loss compared with those diagnosed with only 1 of the 2 diseases or compared with cows having no diagnosis of uterine diseases. Interestingly, subclinical endometritis depressed P/AI and increased pregnancy loss only when it persisted until 46 DIM. On d 32 after AI, cows not diagnosed with subclinical endometritis and those that resolved subclinical endometritis by 46 DIM had greater P/AI than those that remained with subclinical endometritis at 46 DIM (45.4 and 40.0 vs. 25.0%, respectively). Similar to P/AI, cows not diagnosed with subclinical endometritis and those that resolved subclinical endometritis by 46 DIM had less pregnancy loss than those with subclinical endometritis at 46 DIM (9.6 and 13.5 vs. 43.9%, respectively). One or 2 treatments with PGF_2α before initiation of the timed AI program were unable to improve uterine health, P/AI, and maintenance of pregnancy in lactating dairy cows. Cows diagnosed with both purulent vaginal discharge and subclinical endometritis had the greatest depressions in measures of fertility at first AI, particularly when subclinical endometritis persisted in the early postpartum period.     

A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.     

Effect of pretreatment with prostaglandin F2alpha before resynchronization of ovulation on fertility of lactating dairy cows

Our objective was to assess the effect of pretreatment with PGF_2α 12 d before initiation of a protocol for resynchronization of ovulation (Resynch) using an Ovsynch protocol. Lactating Holstein cows diagnosed not pregnant 31 d after a timed artificial insemination (TAI) were randomly assigned to initiate the Resynch protocol 32 d after TAI (n = 255; RES), or receive 25 mg of PGF_2α 34 d after TAI and initiate the Resynch protocol 12 d later at 46 d after TAI (n = 272; PGF+RES). Within each treatment, a subset of cows were examined using transrectal ultrasonography to determine ovulatory response to the first GnRH injection of the Resynch protocols or a blood sample was collected to determine serum progesterone (P₄) at initiation of the Resynch protocol, or both. Overall, PGF+RES cows had more pregnancies per artificial insemination (P/AI) than RES cows 66 d after TAI (35.2 vs. 25.6%), whereas pregnancy loss from 31 to 66 d after TAI was greater for RES than PGF+RES cows (17.1 vs. 7.6%). Although P/AI was greater for cows with high (≥1.0 ng/mL) vs. low (<1.0 ng/mL) P₄ at the first GnRH injection of the Resynch protocols, treatment did not affect the proportion of cows with low P₄ at the first GnRH injection of the Resynch protocols. Overall, no effect of treatment on ovulatory response to the first GnRH injection of the Resynch protocols was detected. We conclude that pretreatment with PGF_2α 12 d before initiation of the Resynch protocol increased P/AI 66 d after TAI for cows with serum P₄ concentration >1.0 ng/mL at the first GnRH injection of the Resynch protocol and decreased pregnancy loss from 31 to 66 d after TAI. This modified resynchronization protocol may be a useful strategy for reproductive management of lactating dairy cows.     

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.     

Analysis of bull (Bos taurus) seminal vesicle fluid proteome in relation to seminal plasma proteome

The existing knowledge on the bull seminal vesicle proteome, a major seminal plasma constituent, and its relationship with seminal plasma is limited. This knowledge is prerequisite for a better understanding of seminal plasma variability, which is linked to semen quality. The objective of this study was to characterize the proteomes of seminal vesicle fluid and seminal plasma and to compare them to better understand the origin of seminal plasma proteins. We collected ejaculates and seminal vesicle fluid postmortem from 6 mature Holstein Friesian bulls. We performed the analysis and identification of proteins using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. We identified 105 proteins in bull seminal vesicle fluid and 88 proteins in seminal plasma. For both seminal vesicles and seminal plasma proteins described in our study, top biological functions were cellular movement, cell death and survival, and cellular growth and proliferation. Additionally, seminal vesicle fluid proteins were involved in protein degradation and synthesis. Seminal plasma proteins were also involved in cellular assembly and organization and cell-to-cell signaling and interactions. Proteins of both fluids were involved in the following canonical pathways: glycolysis, gluconeogenesis, liver X receptor/farnesoid X receptor, and farnesoid X receptor/retinoid X receptor activation. Additionally, seminal vesicle fluid proteins appeared to be involved in oxidative stress response mediated by nuclear factor E2-related factor 2. Our results described the bull seminal vesicle fluid proteome for the first time and allowed for significant expansion of the current knowledge on the bull seminal plasma proteome. Moreover, analysis indicated that both bull seminal vesicle fluid and seminal plasma proteomes contained interconnected protein groups related to protective functions, glycolysis, and the morphology and physiology of the spermatozoa. These proteins and their interactions could be targeted in future research.     

Associations between anogenital distance and measures of fertility in lactating North American Holstein cows: A validation study

Anogenital distance (AGD) has been defined in dairy cows as the distance from the center of the anus to the base of the clitoris. Initial reports on nulliparous Holstein heifers and first- and second-parity Holstein cows have found inverse relationships between AGD and measures of fertility. Our primary objective was to determine the relationship between AGD and measures of fertility in a larger population of North American Holstein cows to validate our previous finding that AGD is inversely related to fertility. Secondary objectives were to determine the associations between AGD and parity, and milk yield. Using digital calipers, we measured AGD in 4,709 Holstein cows [mean ± standard deviation (SD); parity 2.3 ± 1.4; days in milk (DIM) 154 ± 94; 305-d mature equivalent (ME) milk yield 13,759 ± 2,188 kg] from 18 herds in Western Canada and 1 herd in the USA. Anogenital distance (mm) was normally distributed with a mean (±SD) of 132 ± 12, ranging from 95 to 177, and a median of 133. Anogenital distance was linearly but inversely associated with pregnancy to first artificial insemination (P/AI1). For every 1-mm increase in AGD, the estimated probability of P/AI1 decreased by 0.8%. The optimum AGD cut-point that predicted probability of P/AI1 with sensitivity and specificity of 45 and 55%, respectively, was 129 mm. Consequently, data were categorized into either short (≤129) or long (>129) AGD groups across parities, and associations between AGD, parity (first, second, and third+), and fertility measures were determined. Rates of P/AI1 were greater (36 vs. 30%) in short- than in long-AGD cows; short-AGD cows required fewer AI per conception (2.3 vs. 2.4) and had fewer days open (137 vs. 142), and a greater proportion of short-AGD cows (67 vs. 64%) was pregnant by 150 DIM compared with long-AGD cows. The rates of pregnancy up to 150 (hazard ratio of 0.91) and 250 DIM (hazard ratio of 0.93) were smaller in long- than in short-AGD cows. Anogenital distance had a weak positive association with both parity (r = 0.22) and 305-d ME milk yield (r = 0.04). Results indicate an inverse relationship between AGD and measures of fertility in lactating cows, validating our earlier report. We infer that although selecting cows for short AGD is expected to have an adverse effect on milk yield, the anticipated gain in fertility will outweigh the small decline in milk yield, strengthening the potential of AGD as a novel reproductive phenotype for use in future breeding programs to improve fertility.     

Treatment with gonadotropin-releasing hormone after first timed artificial insemination improves fertility in noncycling lactating dairy cows

Lactating Holstein cows were assigned randomly to treatments to improve fertility after first postpartum timed artificial insemination (TAI). In Experiment 1, cows received no treatment (control; n = 9), a controlled internal drug releasing (CIDR) insert from 5 to 12 d after TAI (CIDR; n = 9), or 100 μg of GnRH 5 d after TAI (G5; n = 7). Although treatments did not affect circulating progesterone (P₄) concentrations from 5 to 19 d after TAI, there was a tendency for CIDR cows to have greater P₄ compared with control or G5 cows within 24 h after treatment. In 2 field trials, cows received either control (n = 223), CIDR (n = 218), or G5 (n = 227) treatments (Experiment 2), or control (n = 160), G5 (n = 159), or treatment with 100 μg of GnRH 7 d after TAI (G7; n = 163; Experiment 3). Treatment did not affect pregnancies per AI (P/AI) in Experiments 2 or 3; however, when data were combined to compare control (n = 383) and G5 (n = 386) treatments, P/AI tended to be greater for G5 (49.1%) than for control (45.8%) cows. This effect resulted from a GnRH treatment × cyclicity status interaction in which P/AI for noncycling cows receiving G5 was greater than for noncycling control cows (45.5 vs. 31.1%). In conclusion, treatment with CIDR inserts after TAI had no effect on P/AI, whereas treatment with GnRH 5 d after TAI improved P/AI for noncycling, but not for cycling cows.     

Increasing the length of an estradiol with progesterone timed artificial insemination protocol with 2 controlled internal drug release devices improves pregnancy per artificial insemination in lactating dairy cows

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day ?11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d ?4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d ?2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d ?12; 25 mg of PG and withdrawal of 1 CIDR on d ?4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d ?2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13–15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone–based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows.     

Effect of human chorionic gonadotrophin administration 2 days after insemination on progesterone concentration and pregnancy per artificial insemination in lactating dairy cows

The aim of this study was to examine the effect of a single administration of human chorionic gonadotrophin (hCG) during the establishment of the corpus luteum (CL) on progesterone (P4) concentration and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Postpartum spring-calving lactating dairy cows (n = 800; mean ± SD days in milk and parity were 78.5 ± 16.7 and 2.3 ± 0.8, respectively) on 3 farms were enrolled on the study. All cows underwent the same fixed-time AI (FTAI) protocol involving a 7-d progesterone-releasing intravaginal device with gonadotrophin-releasing hormone (GnRH) administration at device insertion, prostaglandin at device removal followed by GnRH 56 h later, and AI 16 h after the second GnRH injection. Cows were blocked on days postpartum, body condition score, and parity and randomly assigned to receive either 3,000 IU of hCG 2 d after FTAI or no further treatment (control). Blood samples were collected on d 7 and 14 postestrus by coccygeal venipuncture on a subset of 204 cows to measure serum P4 concentration, and pregnancy was diagnosed by ultrasonography approximately 30 and 70 d after FTAI. Administration of hCG caused an increase in circulating P4 concentrations compared with the control treatment on d 7 (+22.2%) and d 14 (+25.7%). The P/AI at 30 d after FTAI was affected by treatment, farm, body condition score, and calving to service interval. Overall, administration of hCG decreased P/AI (46.3% vs. 55.1% for the control). Among cows that did not become pregnant following AI, a greater proportion of control cows exhibited a short repeat interval (≤17 d) compared with cows treated with hCG (8.6% vs. 2.8%, respectively). In addition, the percentages of cows pregnant at d 21 (59.6% vs. 52.0%) and d 42 (78.3% vs. 71.9%) were greater in control than in hCG-treated cows. The overall incidence of embryo loss was 10.7% and was not affected by treatment. There was a tendency for an interaction between treatment and CL status at synchronization protocol initiation for both P4 concentration and P/AI. In conclusion, administration of hCG 2 d after FTAI increased circulating P4 concentrations. Unexpectedly, cows treated with hCG had lower fertility; however, this negative effect on fertility was manifested primarily in cows lacking a CL at the onset of the synchronization protocol.     

Characterization of Holstein heifer fertility in the United States

The overall object of this research was to characterize US Holstein (virgin) heifer fertility. This included investigation of factors influencing heifer fertility and estimation of heritability, as well as correlations with cow fertility and first-lactation milk yield. A secondary objective was to compare linear and logistic model estimates of fixed effects and linear and threshold model estimates of heritability. Data consisted of Holstein heifers, which were artificially inseminated, with their first breeding between March 2003 and August 2005. Herds were required to have at least 60 breedings across the 3 yr of data and an overall mean conception rate (CR) between 20 and 80%. After edits there were 537,938 breedings of 362,512 heifers in 2,668 herds from 41 states used for analysis. After edits, the overall mean CR for US Holstein heifers was 57%. Linear and logistic model estimates for all factors were nearly identical. Year of breeding accounted for the most variation in heifer CR, with heifer age and month of breeding being the next most important factors. Conception rate in heifers is maximal at an intermediate age of 15 to 16 mo. Heifers at 26 mo of age and older have roughly a 10% lower CR than heifers bred at younger ages. Although month of breeding affected heifer CR, effects are less than for cows. In contrast to cow fertility, heifer CR is nearly as good in the hotter summer months as in cooler months. Approximately 88% of US herds had a 40 to 70% heifer CR. Heritability estimates of heifer CR on first service were 0.5% from the linear model and 1.0% from the threshold model. Genetic correlation estimates of heifer CR on first service with cow CR on first service and with first-lactation milk yield were 0.39 and −0.19, respectively. Results indicated that selection on either the currently available US daughter pregnancy rate evaluations for cow fertility or on cow CR will also improve heifer fertility. Furthermore, heritability of heifer CR is lower than for cow CR and reporting of heifer breedings is currently less complete than for cow breedings. Thus, there are currently no immediate plans to implement a US genetic evaluation for heifer CR.     

Effect of sire fertility and timing of artificial insemination in a Presynch + Ovsynch protocol on first-service pregnancy rates

Sire fertility may influence pregnancy rate (PR) by differences in sperm survival in the female reproductive system and time required for capacitation and transport of sperm to site of fertilization. A predicted fertility index, Estimated Relative Conception Rate, was used to select 3 high-fertility artificial insemination (AI) sires (+3) and 3 average AI sires (−1). Ovulation can be predicted to occur at approximately 28 h following GnRH administration when used in an Ovsynch protocol. The objective of this study was to determine if AI at 2 times, 0 or 24 h after GnRH administration, in a Presynch + Ovsynch protocol resulted in different first-service PR when average or high-fertility sires were used. Lactating Holstein cows (n = 1,457) from 2 dairy herds located in the Piedmont region of North Carolina were utilized for 12 mo. Timing of AI did not affect first AI PR and no interaction of sire-fertility group and timing of AI was detected. First AI PR did not differ between sire-fertility groups (23.2 vs. 29.4%) for average and high-fertility groups, respectively. First-lactation cows were 53% more likely to conceive than older cows, and cows bred during April through June were 66% less likely to become pregnant compared with cows bred from October through January. No interactions were detected among parity, season, sire-fertility group, or time of AI. Using only 3 sires per group based on Estimated Relative Conception Rate estimates resulted in large variability of sire conception within groups, although group averages differed by 6 points.     

Effect of reproductive management programs that prioritized artificial insemination at detected estrus or timed artificial insemination on the economic performance of primiparous Holstein cows of different genetic merit for fertility

The objective of this randomized controlled experiment was to evaluate the effect of reproductive management programs that prioritized artificial insemination (AI) at detected estrus (AIE) or timed AI (TAI) during the first lactation on the economic performance of dairy cows of different genomically enhanced predicted transmitting ability for fertility. Lactating primiparous Holstein cows from 6 commercial farms were stratified into high, medium, and low fertility groups based on a reproduction index value calculated from multiple genomically enhanced predicted transmitting abilities to predict the number of days to achieve pregnancy. Within herd and fertility group, cows were randomly assigned either to a program that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows not AIE for all AI services or another that prioritized TAI and had an extended voluntary waiting period for first service and prioritized TAI for second and greater AI services (P-TAI; n = 1,338). Cash flow (CF) per cow accumulated for the experimental (first) and second calving interval (CIN) and cash flow per slot per 28 mo after calving in the experimental lactation were calculated. Market and rearing heifer cost values were used for estimating CF. For cows in the high fertility group, a positive effect of delayed pregnancy on milk income during the first lactation was observed (+$248 for P-TAI) but was insufficient to generate significant differences in CF between treatments mainly because of milk income compensation in the second lactation (+$125 for P-AIE) and minor reductions in reproductive cost and gains in calf value for the P-AIE treatment. In this regard, CF for 2 CIN was greater for the P-TAI treatment by $61 and $86 for market and rearing replacement heifer cost, respectively. Similarly, CF per slot was favorable to the P-TAI treatment but only by $13 and $47 for market and rearing replacement heifer cost, respectively. For cows in the low fertility group, CF was numerically in favor of the P-AIE treatment due to a pregnancy and herd exit dynamics that resulted in gains in milk income over feed cost during the first ($29) and second ($113) lactation. Differences in CF for the 2 CIN were $58 and $47 for market or rearing heifer value, respectively, and $77 and $19 for market and rearing heifer values, respectively for the slot analysis. Differences in CF between cows of different genetic merit for fertility were consistent across treatment and estimation method. Of note, cows in the low fertility group had greater CF than cows in the high fertility group in all comparisons, ranging from $198 per cow for 2 CIN to as much as $427 per slot. For the low fertility group, greater milk production contributed directly (milk income over feed cost) and indirectly (reduced culling) to increased CF. We concluded that genetic merit for fertility and CF are associated because cows of inferior genetic potential for fertility had greater CF than cows of superior genetic for fertility despite some increased costs and reduced revenues. Also, the magnitude of the CF differences observed for cows of different genetic merit for fertility managed with the P-AIE or P-TAI program may be valuable to commercial dairy farms but did not allow to conclusively support the choice of a type of reproductive management strategy for cows of different genetic merit for fertility.