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

Our objective was to compare reproductive outcomes of primiparous lactating Holstein cows of different genetic merit for fertility submitted for insemination with management programs that prioritized artificial insemination (AI) at detected estrus (AIE) or timed AI (TAI). Moreover, we aimed to determine whether subgroups of cows with different fertility potential would present a distinct response to the reproductive management strategies compared. Lactating primiparous Holstein cows (n = 6 commercial farms) were stratified into high (Hi-Fert), medium (Med-Fert), and low (Lo-Fert) genetic fertility groups (FG) based on a Reproduction Index value calculated from multiple genomic-enhanced predicted transmitting abilities. Within herd and FG, cows were randomly assigned either to a program that prioritized TAI and had an extended voluntary waiting period (P-TAI; n = 1,338) or another that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows, not AIE. Cows in P-TAI received first service by TAI at 84 ± 3 d in milk (DIM) after a Double-Ovsynch protocol, were AIE if detected in estrus after a previous AI, and received TAI after an Ovsynch-56 protocol at 35 ± 3 d after a previous AI if a corpus luteum (CL) was visualized at nonpregnancy diagnosis (NPD) 32 ± 3 d after AI. Cows with no CL visualized at NPD received TAI at 42 ± 3 d after AI after an Ovsynch-56 protocol with progesterone supplementation (P4-Ovsynch). Cows in P-AIE were eligible for AIE after a PGF2α treatment at 53 ± 3 DIM and after a previous AI. Cows not AIE by 74 ± 3 DIM or by NPD 32 ± 3 d after AI received P4-Ovsynch for TAI at 74 ± 3 DIM or 42 ± 3 d after AI. Binary data were analyzed with logistic regression, count data with Poisson regression, continuous data by ANOVA, and time to event data by Cox's proportional hazard regression. Pregnancy per AI (P/AI) to first service was greater for cows in the Hi-Fert (59.8%) than the Med-Fert (53.6%) and Lo-Fert (47.7%) groups, and for the P-TAI (58.7%) than the P-AIE (48.7%) treatment. Overall, P/AI for all second and subsequent AI combined did not differ by treatment (P-TAI = 45.2%; P-AIE = 44.5%) or FG (Hi-Fert = 46.1%; Med-Fert = 46.0%; Lo-Fert = 42.4%). The hazard of pregnancy after calving was greater for the P-AIE than the P-TAI treatment [hazard ratio (HR) = 1.27, 95% CI: 1.17 to 1.37)], and for the Hi-Fert than the Med-Fert (HR = 1.16, 95% CI: 1.05 to 1.28) and Lo-Fert (HR = 1.34, 95% CI: 1.20 to 1.49) groups. More cows in the Hi-Fert (91.2%) than the Med-Fert (88.4%) and Lo-Fert (85.8%) groups were pregnant at 200 DIM. Within FG, the hazard of pregnancy was greater for the P-AIE than the P-TAI treatment for the Hi-Fert (HR = 1.41, 95% CI: 1.22 to 1.64) and Med-Fert (HR = 1.28, 95% CI: 1.12 to 1.46) groups but not for the Lo-Fert group (HR = 1.13, 95% CI: 0.98 to 1.31). We conclude that primiparous Holstein cows of superior genetic merit for fertility had better reproductive performance than cows of inferior genetic merit for fertility, regardless of the type of reproductive management used. In addition, the effect of programs that prioritized AIE or TAI on reproductive performance for cows of superior or inferior genetic merit for fertility depended on the outcomes evaluated. Thus, programs that prioritize AIE or TAI could be used to affect certain outcomes of reproductive performance or management.

Erratum to "An automated controlled-release device for intravaginal hormone delivery" (JDS Commun. 1:15-20).

[This corrects the article DOI: 10.3168/jdsc.2020-18816.].

Evaluation and characterization of estrus alerts and behavioral parameters generated by an ear-attached accelerometer-based system for automated detection of estrus.

Our objectives were to evaluate the performance of an ear-attached automated estrus detection (AED) system (Smartbow; Zoetis) that monitored physical activity and rumination time, and to characterize AED system estrus alert features (i.e., timing and duration). Lactating Holstein cows (n = 216) commenced a protocol for the synchronization of estrus at 50 ± 3 DIM or 18 ± 3 d after artificial insemination. For 7 d after induction of luteolysis with PGF2α (d 0), we used visual observation of estrous behavior (30 min, 2 times per day) and data from an automated mounting behavior monitoring system based on a pressure-activated tail-head sensor (HeatWatch; Cowchips LLC) as a reference test (RTE) to detect behavioral estrus. Concomitantly, estrus alerts and their features were collected from the AED system. Progesterone levels confirmed luteal regression, and transrectal ultrasonography confirmed the occurrence and timing of ovulation. Performance metrics for the AED system were estimated with PROC FREQ in SAS, using the RTE or ovulation only as a reference. Performance was also estimated after the removal of cows with a discrepancy between the RTE and ovulation. Continuous outcomes with or without repeated measurements were evaluated by ANOVA using PROC MIXED in SAS. Based on the RTE, 86.6% (n = 187) of the cows presented estrus and ovulated; 1.4% (n = 3) presented estrus and did not ovulate; 6.4% (n = 14) did not present estrus but ovulated; and 5.6% (n = 12) did not present estrus or ovulation. We found no difference in the proportion of cows detected in estrus and with ovulation for the AED system (83.4%) and the RTE (86.6%). Compared with estrus events as detected by the RTE, sensitivity for the AED was 91.6% (95% CI: 87.6-95.5) and specificity was 69.2% (95% CI: 51.5-87.0). Using ovulation as reference, sensitivity was 89.6% (95% CI: 85.3-93.8) and specificity was 86.7% (95% CI 69.5-100). For all cows with agreement between the RTE and ovulation, sensitivity was 92.5% (95% CI: 88.7-96.3) and specificity was 91.7% (95% CI: 76.0-100). The mean (±SD) interval from induction of luteolysis to estrus alerts, estrus alert duration, and the onset of estrus alerts to ovulation interval were 72.2 ± 18.1, 13.5 ± 3.8, and 23.8 ± 7.1 h, respectively. We concluded that an ear-attached AED system that monitored physical activity and rumination time was effective at detecting cows in estrus and generated few false positive alerts when accounting for ovulation, cow physiological limitations, and the limitations of the RTE.

Effect of reproductive management programs for first service on replacement dairy heifer economics.

Our objective was to evaluate cash flow for dairy heifers managed for first service with programs that relied primarily on insemination at detected estrus (AIE), timed AI (TAI), or a combination of both. Holstein heifers from 2 commercial farms were randomized to receive first service with sexed semen after the beginning of the AI period (AIP) at 12 mo of age with 1 of 3 treatments: (1) PGF+AIE (n = 317): AIE after PGF2α injections every 14 d (up to 3) starting at the beginning of the AIP; heifers not AIE 9 d after the third PGF2α were enrolled in the 5d-Cosynch (5dCP) protocol; (2) ALL-TAI (n = 315): TAI after ovulation synchronization with the 5dCP protocol; and (3) PGF+TAI (n = 334): AIE after 2 PGF2α injections 14 d apart (second PGF2α at beginning of AIP). If not AIE 9 d after the second PGF2α, the 5dCP protocol was used for TAI. After first service heifers were AIE or received TAI after the 5dCP with conventional semen. Individual heifer cash flow (CF) for up to a 15-mo period (d 0 = beginning of AIP) was calculated using reproductive cost (rearing only), feed cost (rearing only), income over feed cost (lactation only), calf value, operating cost, and with or without replacement cost. A stochastic analysis with Monte Carlo simulation was used to estimate differences in CF for a range of market values for inputs and outputs. Time to pregnancy for up to 100 d after the beginning of the AIP was analyzed by Cox's proportional regression, binary data with logistic regression, and continuous outcomes by ANOVA. Time to pregnancy (hazard ratio and 95% CI) was reduced for the ALL-TAI versus the PGF+AIE treatment (1.20; 1.02-1.42), but it was similar for ALL-TAI and PGF+TAI (1.13; 0.95-1.33) and the PGF+AIE and PGF+TAI treatments (1.07; 0.91-1.25). The proportion of heifers not pregnant by 100 d did not differ (PGF+AIE = 7.0%; PGF+TAI = 6.5%; ALL-TAI = 6.8%). When including replacement cost, CF ($/slot per 15 mo) differences were $51 and $42 in favor of the PGF+TAI and ALL-TAI compared with the PGF+AIE treatment, and $9 in favor of the PGF+TAI compared with the ALL-TAI treatment but did not differ statistically. Excluding heifers that were replaced to evaluate the effect of timing of pregnancy differences only, the difference in CF between the PGF+AIE with the PGF+TAI and ALL-TAI treatment was the same (i.e., $15) and favored the programs that used more TAI, but also did not differ statistically. Stochastic simulation results were in line with those of the deterministic analysis confirming the benefit of the programs that used more TAI. We concluded that submission of heifers for first service with TAI only or TAI in combination with AIE generated numerical differences in CF of potential value to commercial dairy farms. Reduced rearing cost and increased revenue during lactation increased CF under fixed (not statistically significant) or simulated variable market conditions.

A lateral flow-based portable platform for determination of reproductive status of cattle.

Our objective was to develop and validate a tool integrating a disposable fluorescence-based lateral flow immunoassay (LFIA) coupled with a portable imaging device for estimating circulating plasma concentrations of progesterone (P4). First, we developed and optimized a competitive LFIA test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer were added on the sample pad. After 3 min, 45 µL of running buffer was added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were introduced in an LFIA portable reader wirelessly linked to a laptop to determine P4 concentration based on test-to-control-line signal (T/C ratio). In a series of experiments (n = 6), the ability of the LFIA to differentiate plasma samples with ≥1 or <1 ng/mL of P4 was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1 to 3.7 ng/mL; n = 5). The resulting linear equation was then used to determine a T/C ratio cutoff to differentiate samples with ≥1 or <1 ng/mL of P4. In addition, to evaluate the ability of the platform to assign samples to P4 concentration groups without a calibration curve for individual batches, we performed a receiver operating characteristic analysis to identify a single cutoff value for T/C ratio that could potentially be used for all batches. Overall, calibration curves showed a linear relationship between T/C ratio and P4 levels (mean coefficient of determination = 0.74; range 0.42 to 0.99). Next, plasma samples from lactating dairy cows (n = 58) were tested in triplicate to determine the ability of the LFIA system to differentiate plasma samples with ≥1 or <1 ng/mL of P4 using a RIA for P4 as reference test. Overall, the LFIA assay correctly classified 90% of the samples, with 97% sensitivity, 83% specificity, 85% positive predictive value, and 96% negative predictive value. Agreement between the tests was substantial (kappa = 0.79; 95% confidence interval 0.64 to 0.95). When using a single cutoff value for T/C ratio selected by receiver operating characteristic analysis, sensitivity and specificity to determine CL presence were 97 (95% confidence interval 82 to 99) and 79% (95% confidence interval 60 to 92), respectively. These data suggest that the developed portable LFIA system can accurately differentiate plasma samples with ≥1 or <1 ng/mL of P4.

A reproductive management program aimed at increasing reinsemination of nonpregnant dairy cows at detected estrus resulted in similar reproductive performance to a program that favored timed artificial insemination.

The objective of this experiment was to compare time to pregnancy and proportion of cows not pregnant 210 d after first service for cows managed for second and subsequent artificial insemination (AI) services with a reproductive management program that promoted reinsemination at detected estrus (AIE) or a program that promoted timed AI (TAI). After first service, lactating Holstein cows were blocked by parity and randomly assigned to d 32 Resynch (D32R; n = 464) or AIE Resynch (AIER; n = 512). To determine the effect of management strategies on time to pregnancy and cows not pregnant by the end of a 210 d at-risk period after first AI service, cows remained in AIER and D32R until pregnancy or herd exit. Cows in D32R received a GnRH treatment 32 ± 3 d after AI (first treatment intervention; FTI). Nonpregnancy diagnosis was conducted 7 d later by transrectal ultrasonography when nonpregnant cows with a corpus luteum (CL) ≥15 mm completed the Resynch protocol (PGF2α, 56 h later GnRH, and 16 to 18 h later TAI) and cows without a CL (NoCL cows) were enrolled in a PreG-Ovsynch protocol (GnRH, 7 d later GnRH, 7 d later PGF2α, 56 h later GnRH, and 16 to 18 h TAI) to receive TAI. For the AIER treatment, nonpregnant cows with a CL ≥15 mm observed by transrectal ultrasonography 32 ± 3 d after AI (i.e., FTI) received PGF2α to induce estrus. Cows not AIE within 7 d were enrolled in Resynch (GnRH, 7 d later PGF2α, 56 h later GnRH, and 16 to 18 h TAI). Cows in the NoCL group in AIER were enrolled in PreG-Ovsynch. Detection of estrus was performed based on visual observation of behavioral signs of estrus and tail-paint removal. Binomial data were analyzed with logistic regression and time to event data with Cox's proportional regression. After the FTI, a greater proportion of cows were AIE in AIER than D32R (36.0 vs. 11.9%) and more cows were AIE within 7 d of the FTI for AIER (25.0%) than D32R (4.8%). Overall pregnancy per AI at 68 ± 3 d after AI did not differ (AIER = 35.5% vs. D32R = 34.7%). The hazard of pregnancy up to 210 d after first AI for all cows enrolled (hazard ratio = 1.04, 95% CI 0.90 to 1.19) and for cows that received treatments only (D32R = 308, AIER = 349; hazard ratio = 1.00, 95% CI 0.85 to 1.19) did not differ. We conclude that a program aimed at increasing the proportion of cows reinseminated at detected estrus by treatment with PGF2α at 32 ± 3 d after AI may be an alternative strategy for dairy farms that prefer or need to inseminate more cows at detected estrus rather than by TAI.

Intravaginal instillation of prostaglandin F2α was as effective as intramuscular injection for induction of luteal regression in lactating dairy cows.

Our objectives were to test the efficacy of intravaginal (IVG) administration of PGF2α to induce corpus luteum (CL) regression, compare circulating progesterone (P4) profiles in cows receiving IVG versus intramuscular (IM) treatment with PGF2α, and evaluate reproductive outcomes. Lactating Holstein cows were synchronized using a Double-Ovsynch protocol [GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 1 d later PGF2α, 32 h later GnRH, 16 to 20 h timed artificial insemination (TAI)] to receive TAI at 67 ± 3 d in milk. Seven days after the first GnRH treatment (time 0), cows with at least 1 visible CL ≥15 mm were blocked by parity and randomly assigned to a treatment that consisted of IM injection (IM-PGF; n = 201) or IVG instillation (IVG-PGF; n = 201) of PGF2α. Cows in IM-PGF received a single 25-mg dose of PGF2α (dinoprost tromethamine) intramuscularly. Cows in IVG-PGF received two 25-mg doses of PGF2α 12 h apart delivered through a catheter in the cranial portion of the vagina. Blood samples were collected at 0, 12, 48, and 72 h after treatment. Ovulation to the first GnRH of Double-Ovsynch was determined through transrectal ultrasonography. Only cows with P4 ≥1 ng/mL (functional CL) at time 0 (IM-PGF = 169; IVG-PGF = 179) were included in the analyses. Binary and quantitative data were analyzed by logistic regression and ANOVA with repeated measures, respectively. Results are presented as least squares means. Concentrations of P4 and the proportion of cows with a new CL at time 0 did not differ. Overall, the proportion of cows with CL regression using 1 ng of P4/mL (IM-PGF = 89.0%; IVG-PGF = 86.7%) or 0.5 ng of P4/mL (IM-PGF = 82.2%; IVG-PGF = 82.1%) as the cutoff did not differ. Concentrations of P4 were affected by treatment, time, and treatment × time interaction. Cows in IVG-PGF had greater mean P4 at 12 h than cows in IM-PGF. Mean P4 did not differ at 48 or 72 h after treatment. The proportion of cows with estrus recorded within 3 d of treatment (IM-PGF = 45.4%; IVG-PGF = 48.9%), ovulation risk after treatment (IM-PGF = 88.5%; IVG-PGF = 85.1%), and pregnancies per artificial insemination after TAI (IM-PGF = 51.5%; IVG-PGF = 57.8%) did not differ. We concluded that 2 IVG doses of 25 mg of PGF2α 12 h apart were as effective as a single 25-mg IM dose of PGF2α for inducing luteal regression in lactating dairy cattle.

An automated controlled-release device for intravaginal hormone delivery.

Our objective was to develop and validate an electronically controlled hormone-delivery device for reproductive control of cattle. After development and in vitro testing of a prototype device for intravaginal (IVG) hormone release, we aimed to demonstrate the feasibility of inducing luteal regression by automated treatment with PGF2α. The IVG device comprises an outer 3D-printed plastic housing, fluid reservoirs connected to delivery pumps and tubing, a programmable circuit board, and a retention mechanism. For in vitro testing, 4 pumps were programmed to release different target volumes (0.1, 0.2, 0.5, 1.0, and 2.0 mL) in 4 replicates (n = 80). A Bland-Altman plot was constructed to assess the magnitude of disagreement between expected and delivered volumes. Observations fell within acceptable limits of agreement (1.96 standard deviations) >95% of the time, indicating overall good agreement (mean difference = -0.005 mL). To assess in vivo performance of the IVG device, lactating Holstein cows with at least 1 corpus luteum ≥15 mm in diameter were randomly allocated to 1 of 3 treatments: (1) IM-PGF (n = 6): two 25-mg intramuscular doses of PGF2α 24 h apart; (2) DEV-PGF (n = 6): four 25-mg doses of PGF2α released automatically by the IVG device at 10- or 12-h intervals; and (3) DEV-CTL (n = 4): insertion of an empty IVG device (placebo control). Blood samples were collected at 0, 12, 24, 36, 48, and 72 h after treatment. Data were analyzed by ANOVA with repeated measures. All devices (10/10) remained in situ until removed at 48 h. Progesterone (P4) concentrations from 0 to 72 h were affected by treatment, time, and their interaction. Concentrations of P4 did not differ at time 0 but differed from 24 to 72 h: cows in IM-PGF and DEV-PGF had lesser P4 than cows in DEV-CTL. Conversely, P4 did not differ for IM-PGF and DEV-PGF during the experiment. We conclude that the current IVG hormone-releasing device prototype can be programmed to automatically release PGF2α for successful induction of luteal regression in lactating dairy cows.

Reproductive performance of replacement dairy heifers submitted to first service with programs that favor insemination at detected estrus, timed artificial insemination, or a combination of both.

Our objective was to compare the insemination dynamics and time to pregnancy for up to 100 d after the beginning of the artificial insemination period (AIP) for heifers managed with first artificial insemination (AI) service programs that relied primarily on insemination at detected estrus (AIE) after PGF2α treatments, timed artificial insemination (TAI), or a combination of both. Holstein heifers were randomly assigned to receive first AI service with sex-selected semen after 368 ± 10 d of age with (1) AIE after synchronization of estrus with up to 3 PGF2α treatments every 14 d starting on the first day of the AIP (PGF+AIE; n = 317). Heifers not AIE up to 9 d after the third PGF2α received a 5-d Cosynch protocol with progesterone supplementation [GnRH + controlled internal drug release insert (CIDR)-5 d-CIDR removal and PGF2α-3 d-GnRH and TAI] before TAI. Heifers detected in estrus from CIDR removal and PGF2α until the day before TAI received AIE with no GnRH treatment; (2) 2 PGF2α treatments 14 d apart with the second treatment at the beginning of the AIP (PGF+TAI; n = 334). Heifers received AIE for up to 9 d after the second PGF2α treatment. Heifers not AIE received TAI after the 5-d Cosynch protocol and (3) TAI after the 5-d Cosynch protocol (ALL-TAI; n = 315). Heifers failing to conceive to a previous AI received a subsequent AI with conventional semen at detected estrus or TAI after the 5-d Cosynch protocol. Binomial outcomes were analyzed by logistic regression, whereas time to AI and pregnancy were analyzed with Cox's regression. The hazard of first AI up to 45 d of the AIP was greater for ALL-TAI than for PGF+AIE [hazard ratio (HR) = 1.72; 95% confidence interval (CI) =1.45 to 2.03] and PGF+TAI (HR = 1.51; 95% CI = 1.28 to 1.77), but similar for PGF+AIE and PGF+TAI (HR = 1.14; 95% CI = 0.97 to 1.33). A greater proportion of heifers received AIE in PGF+AIE (98.7%) than in PGF+TAI (78.5%). Overall, first service pregnancy per AI did not differ (PGF+AIE = 42.0%; PGF+TAI = 47.3%, ALL-TAI = 43.8%). Time to pregnancy was reduced for ALL-TAI compared with PGF+AIE (HR = 1.20, 95% CI = 1.02 to 1.42), but was similar to that of PGF+TAI (HR = 1.13, 95% CI = 0.96 to 1.33). Time to pregnancy did not differ for PGF+AIE and PGF+TAI (HR = 1.07, 95% CI = 0.91 to 1.25). Median days to pregnancy were 27, 23, and 21 for heifers in PGF+AIE, PGF+TAI, and ALL-TAI, respectively. We concluded that an ALL-TAI program for first service reduced time to pregnancy, albeit a relatively small reduction, when compared with a program that relied primarily on AIE after induction of estrus with PGF2α treatments. The program that combined synchronization of estrus and TAI (PGF+TAI) resulted in similar time to pregnancy than the predominant TAI and predominant AIE programs.

Economic performance of lactating dairy cows submitted for first service timed artificial insemination after a voluntary waiting period of 60 or 88 days.

The objective of this study was to evaluate the economic performance of dairy cows managed with a voluntary waiting period (VWP) of 60 or 88 d. A secondary objective was estimating variation in cash flow under different input pricing scenarios through stochastic Monte Carlo simulations. Lactating Holstein cows from 3 commercial farms were blocked by parity group and total milk yield in their previous lactation and then randomly assigned to a VWP of 60 (VWP60; n = 1,352) or 88 d (VWP88; n = 1,359). All cows received timed-artificial insemination (TAI) for first service after synchronization of ovulation with the Double-Ovsynch protocol. For second and greater services, cows received artificial insemination (AI) after detection of estrus or the Ovsynch protocol initiated 32 ± 3 d after AI. Two analyses were performed: (1) cash flow per cow for the calving interval of the experimental lactation and (2) cash flow per slot occupied by each cow enrolled in the experiment for an 18-mo period after calving in the experimental lactation. Extending the VWP from 60 to 88 d delayed time to pregnancy during lactation (∼20 d) and increased the risk of leaving the herd for multiparous cows (hazard ratio = 1.21). As a result, a smaller proportion of multiparous cows calved again and had a subsequent lactation (-6%). The shift in time to pregnancy combined with the herd exit dynamics resulted in longer lactation length for primiparous (22 d) but not multiparous cows. Longer lactations led to greater milk income over feed cost and a tendency for greater cash flow during the experimental lactation for primiparous but not multiparous cows in the VWP88 group. On the other hand, profitability per slot for the 18-mo period was numerically greater ($68 slot/18 mo) for primiparous cows but numerically reduced (-$85 slot/18 mo) for multiparous cows in the VWP88 treatment. For primiparous cows most of the difference in cash flow was explained by replacement cost, whereas for multiparous cows it was mostly explained by differences in replacement cost and income over feed cost. Under variable input pricing conditions generated through stochastic simulations, the longer VWP treatment always increased cash flow per 18 mo for primiparous and reduced cash flow for multiparous cows. In conclusion, extending the duration of the VWP from 60 to 88 d numerically increased profitability of primiparous cows and reduced profitability of multiparous cows. Such an effect depended mostly on the herd replacement dynamics and milk production efficiency.

Reproductive performance and herd exit dynamics of lactating dairy cows managed for first service with the Presynch-Ovsynch or Double-Ovsynch protocol and different duration of the voluntary waiting period.

The objective of this experiment was to evaluate the reproductive performance and herd exit dynamics of dairy cows managed for first service with programs varying in method of submission for insemination and voluntary waiting period (VWP) duration. Holstein cows from a commercial farm in New York were randomly allocated to receive timed artificial insemination (TAI) after the Double-Ovsynch protocol (GnRH, 7 d later PGF2α, 3 d later GnRH, 7 d later GnRH, 7 d later PGF2α, 56 h later GnRH, and 16 to 18 h later TAI) at 60 ± 3 d in milk (DIM) (DO60 = 458), TAI after Double-Ovsynch at 88 ± 3 DIM (DO88 = 462), or a combination of AI at detected estrus (starting at 50 ± 3 d in milk) and TAI with the Presynch-Ovsynch protocol (PGF2α, 14 d later PGF2α, 12 d later GnRH, 7 d later PGF2α, 56 h later GnRH, and 16 to 18 h later TAI; PSOv = 450). Subsequent artificial insemination (AI) services were conducted at detected estrus or the Ovsynch protocol (32 ± 3 d after AI GnRH, 7 d later PGF2α, 56 h later GnRH, and 16 to 18 h later TAI) for cows not reinseminated at detected estrus. In a subgroup of cows, cyclicity (based on progesterone concentration), uterine health (vaginal discharge and uterine cytology), and BCS were evaluated at baseline (DO60 and DO88 = 33 ± 3 DIM; PSOv = 34 ± 3 DIM), beginning of the synchronization protocol (DO60 = 33 ± 3 DIM; DO88 = 61 ± 3 DIM; PSOv = 34 ± 3 DIM), and within -5 (PSOv) or -10 d (DO) of the VWP end (DO60 = 50 ± 3 DIM; DO88 = 78 ± 3 DIM; PSOv = 45 ± 3 DIM). Effects of treatments were assessed with multivariable statistical methods relevant for each outcome variable. Cows in the DO88 treatment had delayed time to pregnancy during lactation (DO60 vs. DO88 hazard ratio = 1.53, 95% confidence interval = 1.32 to 1.78; PSOv vs. DO88 hazard ratio = 1.37, 95% confidence interval = 1.19 to 1.61) and, within multiparous cows, the DO88 and PSOv treatments had greater risk of leaving the herd than cows in the DO60 treatment (DO88 vs. DO60 hazard ratio = 1.49, 95% confidence interval = 1.11 to 2.00; PSOv vs. DO60 hazard ratio = 1.39, 95% confidence interval = 1.03 to 1.85). Cows in the DO88 treatment had improved uterine health, greater BCS, and reduced incidence of anovulation than cows in DO60 and PSOv; however, overall pregnancy per AI 39 ± 3 d after AI was similar for the 3 treatment groups. In summary, reproductive management strategies that led to similar average DIM to the first service (∼60 d) through a combination of AI at estrus with TAI (PSOv) or all TAI (DO60) resulted in reduced time to pregnancy after calving when compared with an all TAI program (DO88) with a VWP of 88 d. Within the multiparous cow group, those that received all TAI with a VWP duration of 60 d were less likely to leave the herd than cows in the other treatments.

A resynchronization of ovulation program based on ovarian structures present at nonpregnancy diagnosis reduced time to pregnancy in lactating dairy cows.

Our objective was to evaluate time to pregnancy after the first service postpartum and pregnancy per artificial insemination (P/AI) in dairy cows managed with 2 resynchronization of ovulation programs. After first service, lactating Holstein cows were blocked by parity (primiparous vs. multiparous) and randomly assigned to the d 32 Resynch (R32; n = 1,010) or short Resynch (SR; n = 1,000) treatments. Nonpregnancy diagnosis (NPD) was conducted 32 ± 3 d after AI by transrectal ultrasonography. Nonpregnant cows in R32 received the Ovsynch protocol: GnRH, PGF2α 7 d later, GnRH 56 h later, and timed AI (TAI) 16 to 18 h later. Cows in SR with a corpus luteum (CL) ≥15 mm and a follicle ≥10 mm at NPD received PGF2α, PGF2α 24 h later, GnRH 32 h later, and TAI 16 to 18 h later. Cows in SR without a CL ≥15 mm or a follicle ≥10 mm at NPD received a modified Ovsynch protocol with 2 PGF2α treatments and progesterone (P4) supplementation (GnRH plus CIDR, PGF2α and CIDR removal 7 d later, PGF2α 24 h later, GnRH 32 h later, and TAI 16 to 18 h later). Blood samples were collected from a subgroup of cows at the GnRH before TAI (R32 = 114; SR = 121) to measure P4 concentration. Binomial outcomes were analyzed with logistic regression and hazard of pregnancy (R32 = 485; SR = 462) with Cox's proportional regression in SAS (SAS Institute, Cary, NC). For P/AI analysis, the TAI service was the experimental unit (R32 = 720; SR = 819). Models included treatment and parity as fixed effects and farm as random effect. The hazard of pregnancy was greater for the SR treatment (hazard ratio = 1.18; 95% confidence interval: 1.01-1.37). Median time to pregnancy was 95 and 79 d for the R32 and SR treatments, respectively. At NPD, 71.3 and 71.2% of cows had a CL for the R32 and SR treatments, respectively. Treatment did not affect overall P/AI 32 ± 3 d after AI (R32 = 31.0% vs. SR = 33.9%) or for cows with a CL at NPD (R32 = 32.7% vs. SR = 32.8%). For cows with no CL at NPD, P/AI was greater for the SR treatment (36.9%) than for the R32 treatment (28.6%). Pregnancy loss from 32 to 63 d after AI was similar for all services combined (R32 = 8.3% vs. SR = 10.4%) and for cows with no CL at NPD (R32 = 13.2% vs. SR = 7.2%) but tended to be affected by treatment for cows with a CL at NPD (R32 = 6.8% vs. SR = 11.9%). Treatment affected the proportion of cows with P4 ≤0.5 ng/mL at the GnRH before TAI for all cows (R32 = 68.4% vs. SR = 81.8%), tended to have an effect among cows with a CL (R32 = 70.0% vs. SR = 81.8%), and had no effect for cows with no CL (R32 = 64.7% vs. SR = 81.8%). We concluded that the SR program reduced time to pregnancy because of a reduction of the interbreeding interval for cows with a CL at NPD and greater P/AI in cows with no CL at NPD.

Extending the duration of the voluntary waiting period from 60 to 88 days in cows that received timed artificial insemination after the Double-Ovsynch protocol affected the reproductive performance, herd exit dynamics, and lactation performance of dairy cows.

This experiment evaluated the reproductive performance, herd exit dynamics, and lactation performance of dairy cows managed with a voluntary waiting period (VWP) of 60 or 88 d. Secondary objectives were evaluating VWP effect on cyclicity status, uterine health, systemic inflammation, and body condition score (BCS) before first service. Lactating Holstein cows from 3 commercial farms in New York State cows were blocked by parity group and total milk yield in their previous lactation and then randomly assigned to VWP of 60 (VWP60; n = 1,352) or 88 (VWP88; n = 1,359) days in milk (DIM). All cows received the Double-Ovsynch protocol (GnRH-7 d-PGF2α-3 d-GnRH-7 d-GnRH-7 d-PGF2α-56 h-GnRH-16 to 20 h-timed artificial insemination; TAI) for synchronization of ovulation and TAI. For second and greater artificial insemination (AI), cows received AI after detection of estrus or the Ovsynch protocol (GnRH-7 d-PGF2α-56 h-GnRH-16 to 20 h-TAI) initiated 32 ± 3 d after AI for cows not re-inseminated at detected estrus. Cyclicity status (progesterone concentration), uterine health (vaginal discharge and uterine cytology), BCS, and systemic inflammation (haptoglobin concentration) were evaluated at baseline (33 ± 3 DIM for both treatments), beginning of the Double-Ovsynch protocol, and 10 d before TAI. Effects of treatments were assessed with multivariable statistical methods relevant for each outcome variable. Extending duration of VWP from 60 to 88 DIM increased pregnancies per AI (P/AI) to first service (VWP60 = 41%; VWP88 = 47%). Nonetheless, the greatest benefit of extending VWP on first-service P/AI was for primiparous cows (VWP60 = 46%; VWP88 = 55%), as P/AI did not differ within the multiparous cow group (VWP60 = 36%; VWP88 = 40%). Physiological status more conducive to pregnancy-characterized by improved uterine health, greater BCS, reduced systemic inflammation, and to a lesser extent more time to resume ovarian cyclicity-explained the increment in P/AI to first service. Our data also indicated that despite having greater P/AI to first service, cows with the longer VWP had delayed time to pregnancy during lactation (hazard ratio = 0.72; 95% confidence interval 0.69-0.98) and greater risk of leaving the herd, particularly for multiparous cows (hazard ratio = 1.34; 95% confidence interval 1.23-1.47). This shift in pregnancy timing led to an overall extension of the lactation length (+13 d), which resulted in greater total milk yield per lactation (+491 kg) but not greater milk yield per day of lactation. In conclusion, data from this experiment highlight the importance of considering the complex interactions between reproductive performance, herd exit dynamics, and lactation performance as well as the effects of parity at the time of defining the duration of the VWP for lactating dairy cows.

Resynchronization of ovulation protocols for dairy cows including or not including gonadotropin-releasing hormone to induce a new follicular wave: Effects on re-insemination pattern, ovarian responses, and pregnancy outcomes.

Our objectives were to evaluate the pattern of re-insemination, ovarian responses, and pregnancy per artificial insemination (P/AI) of cows submitted to different resynchronization of ovulation protocols. The base protocol started at 25 ± 3 d after artificial insemination (AI) and was as follows: GnRH, 7 and 8 d later PGF2α, GnRH 32 h after second PGF2α, and fixed timed AI (TAI) 16 to 18 h after GnRH. At 18 ± 3 d after AI, cows were randomly assigned to the G25 (n = 1,100) or NoG25 (n = 1,098) treatments. The protocol for G25 and NoG25 was the same, except that cows in NoG25 did not receive GnRH 25 ± 3 d after AI. At nonpregnancy diagnosis (NPD), 32 ± 3 d after AI, cows from G25 and NoG25 with a corpus luteum (CL) ≥15 mm in diameter and a follicle ≥10 mm completed the protocol (G25 CL = 272, NoG25 CL = 194), whereas cows from both treatments that did not meet these criteria received a modified Ovsynch protocol with P4 supplementation [controlled internal drug release insert plus GnRH, controlled internal drug release insert removal, and PGF2α 7 and 8 d later, GnRH 32 h after second PGF2α, and TAI 16 to 18 h after GnRH (G25 NoCL = 53, NoG25 NoCL = 78)]. Serum concentrations of progesterone (P4) were determined and ovarian ultrasonography was performed thrice weekly from 18 ± 3 d after AI until 1 d after TAI (G25 = 46, NoG25 = 44 cows). A greater percentage of NoG25 cows were re-inseminated at detected estrus (NoG25 = 53.5%, G25 = 44.6%), whereas more cows had a CL at NPD in G25 than NoG25 (83.7 and 71.3%). At 32 d after AI, P/AI was similar for G25 and NoG25 for inseminations at detected estrus (38.4 and 42.9%), TAI services for cows with no CL (40.4 and 36.7%), and for all services combined (39.6 and 39.0%). However, P/AI were greater for cows with a CL in G25 than NoG25 (40.6 and 32.8%) that received TAI. More cows ovulated spontaneously or in response to GnRH for the G25 than the NoG25 treatment (70 and 36%) but a similar proportion had an active follicle at NPD (G25 = 91% and NoG25 = 96%). The largest follicle diameter at NPD (G25 = 15.0 ± 0.4 mm, NoG25 = 16.5 ± 0.6 mm) and days since it reached ≥10 mm (G25 = 4.0 ± 0.3 d, NoG25 = 5.8 ± 0.6 d) were greater for the NoG25 than G25 treatment. For cows with a CL at NPD, CL regression after NPD, ovulation after TAI, and ovulatory follicle diameter did not differ. In conclusion, removing the first GnRH of a modified Resynch-25 protocol for cows with a CL at NPD and a modified Ovsynch protocol with P4 supplementation for cows without a CL at NPD resulted in a greater percentage of cows re-inseminated at detected estrus and a similar proportion of cows pregnant in spite of reduced P/AI for cows with a CL at NPD.

Intravaginal instillation of gonadotropin-releasing hormone analogues with an absorption enhancer induced a surge of luteinizing hormone in lactating dairy cows.

Our objectives were to evaluate circulating LH concentrations after intravaginal (IVG) instillation of GnRH analogs in lactating dairy cows. In 2 experiments, lactating Holstein cows (experiment 1: n = 32; experiment 2: n = 47) received the experimental treatments 48 h after the first of 2 PGF2α treatments given 12 h apart and 7 d after a modified Ovsynch protocol (GnRH at -7 d, PGF2α at -24 h, PGF2α at -56 h, GnRH at 0 h). In experiment 1, cows were stratified by parity and randomly allocated to receive the following treatments: 2 mL of saline IVG (SAL, n = 6), 100 µg of gonadorelin (Gon) i.m. (G100-IM, n = 5), and 100 (G100, n = 7), 500 (G500, n = 8), or 1,000 µg of Gon IVG (G1000, n = 7). In experiment 2, treatments were SAL (n = 8), G100-IM (n = 8), G1000 (n = 7), 1,000 µg of Gon plus 10% citric acid (CA) IVG (G1000CA, n = 8), 80 µg of buserelin IVG (B80, n = 8), and 80 µg of buserelin plus 10% CA IVG (B80CA, n = 8). In both experiments, blood was collected every 15 min from -15 min to 4 h, and every 30 min from 4 to 6 h after treatment. Data for area under the curve (AUC), mean LH concentrations, and time to maximum LH concentration were analyzed by ANOVA with (mean LH only) or without repeated measures using PROC MIXED of SAS (version 9.4, SAS Institute Inc., Cary, NC). The proportion of cows with a surge of LH was evaluated with Fisher's exact test using PROC FREQ of SAS. In both experiments, LH concentrations were affected by treatment, time, and the treatment by time interaction. In experiment 1, the AUC for LH and maximum LH concentration were greatest for the G100-IM treatment and were greater for the G1000 than for the SAL and G500 treatments. The proportion of cows with an observed surge of LH was 100 and 0% for cows that received Gon i.m. and IVG, respectively. In experiment 2, the AUC and maximum LH concentrations were greater for the G100-IM, G1000CA, and B80CA treatments than for the other IVG treatments. The proportion of cows with a surge of LH differed by treatment (SAL = 0%, G100-IM = 100%, G1000 = 14%, G1000CA = 88%, B80 = 13%, and B80CA = 100%). For the treatments with a surge of LH, time to maximum concentration of LH was the shortest for the G100-IM treatment, intermediate for the G1000CA treatment, and the longest for cows in the B80CA treatment. In conclusion, Gon (up to 1,000 µg) absorption through intact vaginal epithelium after a single IVG instillation was insufficient to elicit a surge of LH of normal magnitude. Conversely, IVG instillation of 1,000 µg of Gon and 80 µg of buserelin with the addition of citric acid as absorption enhancer resulted in a surge of LH of similar characteristics than that induced after i.m. injection of 100 µg of Gon.

Reproductive management strategies to improve the fertility of cows with a suboptimal response to resynchronization of ovulation.

The objective was to compare the reproductive performance of lactating Holstein cows managed with a strategy that included the Ovsynch protocol with exogenous progesterone (P4) supplementation or presynchronization with GnRH 7d before Ovsynch to treat cows without a corpus luteum (CL), a CL <15 mm, or cystic at the time of the PGF2α injection of Resynch (GnRH-7 d-PGF2α-56 h-GnRH-16 to 20 h-TAI). In a preliminary study, blood collection and transrectal ovarian ultrasonography were conducted (n=555) at the PGF2α of Resynch [coincident with nonpregnancy diagnosis (NPD)] to define a cutoff value for CL size that better predicted fertility after timed artificial insemination (TAI). A CL size of 15 mm was selected based on statistical differences in pregnancies per AI (P/AI) [33.2 vs. 10.3 P/AI for CL ≥15 mm (n=497) vs. no CL ≥15 mm (n=58; no CL, CL <15 mm, or cystic)]. Subsequently, in a completely randomized experiment, cows were enrolled in a management strategy that used Ovsynch with P4 supplementation [Ovsynch+P4; GnRH and controlled internal drug release device (CIDR)-7 d-PGF2α and CIDR removal-56 h-GnRH-16 to 20 h-TAI] or a PreG-Ovsynch protocol [PreG-Ovsynch; GnRH-7 d-GnRH-7 d-PGF2α-56 h-GnRH-16 to 20 h-TAI] to treat cows without a CL, a CL <15 mm, or cystic at NPD and the PGF2α of Resynch. Cows with a CL ≥15 mm at the PGF2α of Resynch completed the protocol and received TAI. Data were available from 212, 192, and 1,797 AI services after Ovsynch+P4, PreG-Ovsynch, and Resynch, respectively. At 39d after AI, P/AI tended to be greater for Ovsynch+P4 and PreG-Ovsynch combined (35.1%) than for Resynch cows (31.1%), whereas P/AI were similar for Ovsynch+P4 (34.4%) and PreG-Ovsynch (35.9%). The hazard of pregnancy for cows that received the experimental treatments at least once was similar for cows in the Ovsynch+P4 (n=124) and the PreG-Ovsynch (n=132) group (hazard ratio 1.15; 95% confidence interval: 0.87 to 1.53). Median days to pregnancy were 52 and 59 for cows in the Ovsynch+P4 and the PreG-Ovsynch groups, respectively. The presynchronizing GnRH injection of PreG-Ovsynch induced ovulation in 86.0% of the cows. At the first GnRH of Ovsynch, the proportion of cows with a CL based on ultrasound (86.6 vs. 15.0%), P4 >1 ng/mL (82.8 vs. 31.8%), a follicle ≥ 10 mm (98.0 vs. 84.4%), and P4 concentrations (3.7 vs. 1.1 ng/mL) was greater in PreG-Ovsynch than in Ovsynch+P4. Conversely, more cows ovulated in response to the first GnRH of Ovsynch in Ovsynch+P4 (71.9%) than PreG-Ovsynch (58.3%). At the PGF2α before TAI, more cows had a CL based on ultrasound (92.1 vs. 77.0%) and P4 concentrations were greater in PreG-Ovsynch than in Ovsynch+P4 (4.1 vs. 2.6 ng/mL); however, a similar proportion of cows had P4 >1 ng/mL (79.1 vs. 82.7%). We conclude that the Ovsynch+P4 and PreG-Ovsynch treatments for cows without a CL, a CL <15 mm, or cystic at the PGF2α injection of Resynch led to P/AI similar to that of cows with a CL ≥15 mm, and that both management strategies resulted in similar time to pregnancy.

Effect of extending the interval from Presynch to initiation of Ovsynch in a Presynch-Ovsynch protocol on fertility of timed artificial insemination services in lactating dairy cows.

The specific objective of this study was to determine if increasing the interval between the Presynch and Ovsynch portion of the Presynch-Ovsynch protocol (Presynch: PGF2α-14 d-PGF2α and Ovsynch: GnRH-7 d-PGF2α-56 h-GnRH-16-20 h-timed artificial insemination) from 12 to 14 d would reduce the fertility of lactating dairy cows not detected in estrus after Presynch that receive timed artificial insemination (TAI). Cows from 4 commercial dairy farms (n=3,165) were blocked by parity (primiparous vs. multiparous) and randomly assigned to a 12 (PSOv14-12; n=1,566) or 14 d (PSOv14-14; n=1,599) interval between the second PGF2α (PGF) injection of Presynch (P2) and the beginning of Ovsynch. Cows detected in estrus any time between P2 and the day of the TAI were inseminated (AIED group). From a subgroup of cows (177 and 150 in PSOv14-12 and PSOv14-14, respectively), ovarian parameters and ovulation were evaluated through determination of concentrations of progesterone (P4) in blood and transrectal ultrasonography at the time of the first GnRH (GnRH1) and the PGF injection of Ovsynch. Overall, 52.8% (n=1,671) of the cows were AIED, whereas 47.2% (n=1,494) received TAI. For cows that received TAI, pregnancies per artificial insemination 39 d after artificial insemination were similar for PSOv14-12 (36.3%) and PSOv14-14 (36.0%) but were greater for primiparous (41.5%) than multiparous cows (33.6%). Pregnancy loss from 39 to 105 d after artificial insemination was similar for PSOv14-12 (4.8%) and PSOv14-14 (8.6%), for primiparous (6.4%) and multiparous cows (7.0%), but a tendency for a treatment by parity interaction was observed. Both treatments had a similar proportion of cows with a follicle ≥ 10 mm and similar follicle size at GnRH1; however, the ovulatory response to GnRH was greater for PSOv14-12 (62.2%) than PSOv14-14 (46.4%). A greater proportion of cows with a functional corpus luteum (75.3 vs. 65.6%) and greater concentrations of P4 (3.9 vs. 3.3 ng/mL) at GnRH1 in PSOv14-14 than PSOv14-12 may have compensated for the reduction in fertility expected due to reduced ovulatory response to GnRH1. We concluded that extending the interval from Presynch to Ovsynch from 12 to 14 reduced ovulatory response to GnRH1 but did not reduce the fertility of cows that received TAI when cows were inseminated in estrus after presynchronization. Thus, farms that combine AIED and TAI during the Presynch-Ovsynch protocol may use a 14-d interval between Presynch and Ovsynch to simplify their management without reducing fertility of cows receiving TAI.