Use of Bovine Somatotropin in Lactating Dairy Cows Receiving Timed Artificial Insemination

Use of Bovine Somatotropin in Lactating Dairy Cows Receiving Timed Artificial Insemination F. Moreira,* C. A. Risco†, M. F. A. Pires‡, J. D. Ambrose§, M. Drost†, and W. W. Thatcher* *Department of Dairy and Poultry Sciences, University of Florida, Gainesville, 32611 †Large Animal Clinical Sciences, University of Florida, Gainesville, 32610 ‡CNPGL-EMBRAPA, Coronel Pacheco, MG 36155-000, Brazil §Alberta Agriculture, 6909-116 Street, Edmonton, Alberta T6H 4P2, Canada

ABSTRACT Objectives of the research were to examine the effect of bovine somatotropin (bST) on pregnancy rates to a timed artificial insemination protocol and to test a resynchronization system with two consecutive synchronized services. Lactating Holstein cows (n = 403) were assigned to the following treatments: bST treatment (500 mg) was initiated at 63 ± 3 d postpartum concomitantly with initiation of the timed artificial insemination protocol or bST treatment was initiated at 105 ± 3 d postpartum. At 63 ± 3 d postpartum, all cows received GnRH (100 µg), an injection of PGF2α (25 mg) 7 d later, and a GnRH injection at 48 h after PGF2α and were inseminated 16 to 20 h later. Cows were reinseminated at detected estrus or resynchronized with a GnRH injection at 20 d after insemination. At 27 d after insemination, cows were examined for pregnancy. Resynchronized cows diagnosed nonpregnant received an injection of PGF2α and were inseminated at detected estrus or received an injection of GnRH at 48 h after PGF2α and inseminated 16 to 20 h later. Cows pregnant at d 27 were reexamined for pregnancy at 45 d after insemination. First-service pregnancy rates at d 45 were increased in cows not resynchronized that initiated bST treatment at 63 ± 3 d postpartum, compared with cows initiating bST treatment at 105 ± 3 d postpartum (37.7 ± 5.8% and 22.1 ± 4.2%, respectively), but the effect of bST treatment was not observed when cows were resynchronized (25.6 ± 4.3% and 25.8 ± 5.5%, respectively). Thus, bST increased pregnancy rates to a timed artificial insemination protocol. (Key words: timed artificial insemination, bovine somatotropin, lactating cows) Abbreviation key: CL = corpus luteum, PP = days postpartum, TAI = timed artificial insemination, P4 =

Received August 13, 1999. Accepted January 5, 2000. Corresponding author: W. W. Thatcher; e-mail: THATCHER@ ANIMAL.UFL.EDU. 2000 J Dairy Sci 83:1237–1247

progesterone, RIDE = reinseminated at detected estrus, RESYNCH = resynchronized. INTRODUCTION The use of bST has been associated with a decrease in reproductive efficiency (7, 8, 49). More recently, it was observed that cows treated with bST did not express estrus as intensely as untreated controls and had a greater percentage of undetected ovulations (20). This agrees with prior results, whereby steroid-primed, ovariectomized heifers had reduced estrous expression when treated with bST (22). It was hypothesized that bST altered behavioral centers within the brain that control expression of estrus. Recently, a reproductive management system that eliminates the need for estrous detection has been developed (33, 40) and used in lactating dairy cows with promising results (2, 4, 5, 9, 32, 34). The timed artificial insemination protocol (TAI), also referred as the Ovsynch program (34), is initiated with an injection of GnRH given at random stages of the estrous cycle followed 7 d later by an injection of PGF2α. At 48 h after injection of PGF2α, a second injection of GnRH is administered and cows are inseminated at approximately 16 h after GnRH without the need for estrous detection. Such a reproductive management system, which eliminates the need for detection of estrus, provides the opportunity to evaluate the effects of bST on fertility without the management constraints of estrous detection. However, cows not conceiving to the synchronized service still need to be observed for estrous behavior to be reinseminated. A system that allows resynchronization through a timed insemination for lactating dairy cows has the potential to eliminate completely the need for estrous detection. Such a resynchronization system has been tested in which cows not conceiving to the first synchronized service were submitted to a second timed insemination within 42 to 46 d (32). It is hypothesized that such an interval may be reduced to 30 d to further decrease the interval from parturition to conception. Therefore, our objectives were to observe the effect of bST on pregnancy rates to the TAI protocol

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and also to test a resynchronization system that allows two consecutive synchronized services within a 30-d interval. MATERIALS AND METHODS The experiment was conducted on a commercial dairy in northern Florida (Trenton, FL) where lactating dairy cows were kept in free-stall facilities and milked three times daily at approximately 8-h intervals; the first milking was initiated between 0800 and 1200 h. Cows were fed a TMR that consisted of 33% forage (corn silage, alfafa hay, and bermudagrass silage) and 67% concentrate (percentage of DM; ground corn, hominy, whole cottonseed, soybean meal, and citrus pulp) containing 1.72 Mcal of NEL/kg and 16.7% CP (percentage of DM). Once a week, cows were assigned to initiate the experiment based on day postpartum (PP), starting in February and continuing through a 14-wk period until May 1996. Cows diagnosed with any evident health problems such as toxic metritis, displaced abomasum, or acute mastitis immediately before or during the experiment were not included in the analysis. Likewise, cows with a BCS of less than 2.5 on a 1 to 5 scale (11) at initiation of treatments (63 ± 3 d PP) were not included. Cows were assigned to a 2 × 2 factorial experiment with two main factors: reproductive management system and day of initiation of bST treatment. Cows received an injection of GnRH (Cystorelin, Merial Co., Athens, GA; 100 µg, i.m.) at 63 ± 3 d PP and were injected 7 d later with PGF2α (Lutalyse, PharmaciaUpjohn Co., Kalamazoo, MI; 25 mg, i.m.). At 48 h after injection of PGF2α, cows were given a second injection of GnRH (100 µg) and received their first service by timed insemination 16 to 20 h later, at 73 ± 3 d PP. Thereafter, cows not conceiving to the first synchronized service were reinseminated at detected estrus (RIDE), or were assigned to be resynchronized (RESYNCH), and received an injection of GnRH (100 µg) at 20 d after insemination. At 27 d after insemination, cows from the RIDE group which had not been reinseminated after the first synchronized service and all cows from the RESYNCH group were examined with an Aloka 500V ultrasound device (Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5-MHz linear-array transrectal transducer for the presence of a corpus luteum (CL) in the ovaries and for fetal fluid, appearance of embryo, and embryonic heart beat. Cows from the RIDE group diagnosed nonpregnant at 27 d after insemination, and which had a CL, received an injection of PGF2α (25 mg) to improve estrous detection and were reinseminated at observed estrus. Cows from the RESYNCH group diagnosed nonpregnant at ultrasonography on d 27 after insemination received Journal of Dairy Science Vol. 83, No. 6, 2000

an injection of PGF2α and a GnRH injection (100 µg) 48 h later. At 16 to 20 h after injection of GnRH, cows from the RESYNCH group were reinseminated. Thus, cows from the RESYNCH group received two synchronized services within 30 d (n = 52). Because the resynchronization treatment was a novel reproductive management system, pregnancy rates to the second synchronized service were monitored carefully. Due to low pregnancy rates following the second synchronized service, the resynchronization treatment was modified after the 5th wk of the experiment. The second injection of GnRH administered at 48 h after PGF2α injection of cows diagnosed nonpregnant at ultrasonography was terminated. Henceforth, cows from the RESYNCH group that received an injection of GnRH at 20 d after the first synchronized service, and that were diagnosed nonpregnant at ultrasonography 7 d later, were injected with PGF2α and reinseminated at detected estrus (n = 58). Only cows detected in estrus within 7 d of ultrasonography and injection of PGF2α were considered as responsive to the resynchronization treatment. For the second service, conception rate was defined as the percentage of cows pregnant out of the number of cows inseminated, whereas pregnancy rate was defined as the percentage of cows pregnant out of the total number of cows available for insemination (28). Conception and pregnancy rates to the first service are identical due to the use of timed insemination. The second main effect was initiation of bST treatment. A group of cows (bST group) received their first injection of bST (Posilac, Monsanto, St. Louis, MO; 500 mg, s.c.) at 63 ± 3 d PP when the first GnRH dose was administered. These cows were reinjected every 14 d thereafter, until 30 d before the beginning of the dry period. Alternatively, a control group of cows received their first injection of bST only at 105 ± 3 d PP and, like the bST group, were reinjected every 14 d until 30 d before the beginning of the dry period. Thus, there were four experimental groups: a RIDE-control group (RIDE group, n = 126), a RIDE-bST group (RIDE+bST group, n = 67), a RESYNCH-control group (RESYNCH group, n = 76), and a RESYNCH-bST group (RESYNCH+bST group, n = 134). To establish a practical management protocol for the staff at the dairy and to minimize assignment errors, cows were assigned to experimental groups according to the last digit of their ear tag number. This way, cows whose last ear tag digit ranged from 0 to 4 were assigned to initiate bST treatment at 63 ± 3 d PP, whereas cows whose last ear tag digit ranged from 5 to 9 initiated bST treatment at 105 ± 3 d PP. Similarly, cows with odd ear tag numbers were assigned to the RIDE group, and cows with an even ear tag number were assigned to the RESYNCH group. Although an unequal distribution of cows among

BOVINE SOMATOTROPIN AFFECTS A TAI PROTOCOL

experimental groups was anticipated, such an assignment of cows was chosen to provide a random and practical way to conduct an experiment with such a large number of cows on a commercial dairy. The loss of efficiency in detecting differences among main effects and the interaction was calculated to be only 8% (from 100 to 92%) compared with a situation in which all groups had equal numbers. Blood samples were collected from all cows prior to GnRH and PGF2α injections at d 20 after insemination and prior to ultrasonography. Blood samples were collected from the coccygeal vessel by venipuncture into heparinized tubes and kept on ice until centrifugation (3,000 × g for 30 min). Plasma was separated and stored at −20°C until assayed for progesterone (P4) by singleantibody RIA (21). Sensitivity of the P4 assay was 0.3 ng/ml, and the intrassay and interassay coefficients of variation were 11.6 and 12.6%, respectively. Cows were grouped into P4-classes according to plasma P4 concentrations of the two initial blood samples collected at the first GnRH injection at 63 ± 3 d PP, and at the injection of PGF2α administered 7 d later. Because some blood samples were missing, only 327 cows (81.1% of total experimental cows) were available for this analysis. At the first GnRH injection, cows were considered to have low plasma P4 if concentrations were equal to or lower than 1.0 ng/ml. Cows with plasma P4 concentrations greater than 1.0 ng/ml at the first GnRH injection were considered to have an active CL. However, cows in anestrus may respond to an injection of GnRH and luteinize follicles to produce plasma P4 concentrations that will not exceed 2.5 ng/ml 7 d later (T. Kassa and W. W. Thatcher, 1998, unpublished observations). Therefore, cows with low P4 concentrations at the injection of GnRH were considered to have low plasma P4 at 7 d later if concentrations were equal to or lower than 2.5 ng/ml. Thus, there were four possible classifications: cows with two consecutive low plasma P4 concentrations (LOW-LOW, ≤1.0 ng/ml and ≤ 2.5 ng/ ml, respectively; n = 24), cows with high plasma P4 concentrations at the injection of GnRH but with low plasma P4 concentrations at 7 d later (HIGH-LOW, >1.0 ng/ml and ≤1.0 ng/ml, respectively; n = 18), cows with low plasma P4 concentrations at GnRH but with high plasma P4 concentrations at 7 d later (LOW-HIGH, ≤1.0 ng/ml and >2.5 ng/ml, respectively; n = 85), and cows with high plasma P4 concentrations at both collected samples (HIGH-HIGH, >1.0 ng/ml and >1.0 ng/ml, respectively; n = 200). Similarly, cows were also classified according to their plasma P4 concentrations of samples collected prior to the second injection of GnRH, at 48 h after injection of PGF2α. Cows were considered to have undergone complete CL regression if plasma P4 concentrations were

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less than or equal to 1.0 ng/ml (n = 265). Alternatively, cows were classified as having an incomplete CL regression at 48 h after injection of PGF2α if plasma P4 concentrations were greater than 1.0 ng/ml (n = 62). Plasma P4 concentrations collected at 20 d after the first synchronized service or timed insemination were used to estimate pregnancy rates. Accuracy of pregnancy diagnosis based on two P4 samples collected at 21 and 24 d after insemination was approximately 80% (12). Cows not observed in estrus after insemination and which had plasma P4 concentrations greater than 2.0 ng/ml at 20 d after insemination, were considered pregnant, whereas cows expressing behavioral estrus or with plasma P4 concentrations equal to or lower than 2.0 ng/ml were considered nonpregnant. Plasma P4 concentrations used to categorize between cows in the proestrous phase and cows in the luteal phase (i.e., potentially pregnant) were raised to 2.0 ng/ml in an attempt to be more conservative and to avoid classifying cows in the process of luteal regression as pregnant. Cows that were diagnosed pregnant at a certain point after insemination and later diagnosed nonpregnant were considered as having lost their pregnancy. Hence, pregnancy losses were estimated between d 20 (based on plasma P4) and 27 (ultrasonography), and between d 27 and 45 (rectal palpation). Statistical Analysis Data analyses were performed by the method of least squares ANOVA using the general linear model procedures in the SAS software package (38). Two mathematical models were used: a designated basic model which included the effects of treatment, month of synchronization, parity (i.e., primiparous or multiparous), technician performing the insemination, sire, and all higher order interactions. Higher order interactions then were excluded from the models since no significant effects were observed. Differences among experimental groups were determined by preestablished orthogonal contrasts which examined the effects of bST treatment (RIDE+bST and RESYNCH+bST vs. RIDE and RESYNCH), reproductive management system (RIDE+bST and RIDE vs. RESYNCH+bST and RESYNCH), and the interaction between bST treatment and reproductive management system (RIDE+bST and RESYNCH vs. RESYNCH+bST and RIDE). The second statistical model was designated as an extended model and included all variables from the basic model plus the effects of P4-classes and CL regression status. Least squares means obtained for P4-classes were analyzed by using preestablished orthogonal contrasts to detect differences between LOW-LOW and HIGH-LOW versus LOW-HIGH and HIGH-HIGH, between LOW-LOW Journal of Dairy Science Vol. 83, No. 6, 2000

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Figure 1. Least squares means and SE for pregnancy rates to the first-service timed insemination as diagnosed by plasma progesterone (d 20), by ultrasonography (d 27), and by rectal palpation (d 45) for cows reinseminated at detected estrus and treated with bST at 63 ± 3 d postpartum (PP) (RIDE+bST; open bar), or at 105 ± 3 d PP(RIDE; dark gray bar), and for cows resynchronized and treated with bST at 63 ± 3 d PP (RESYNCH+bST; light gray bar), or at 105 ± 3 d PP (RESYNCH; black bar). An effect of bST treatment was observed at d 20 (P < 0.01), 27 (P < 0.08), and 45 (P < 0.10). An interaction was detected at d 27 (P < 0.09) and 45 (P < 0.09).

and HIGH-LOW classes, and between LOW-HIGH and HIGH-HIGH classes. The use of two different models was necessary because not all cows available for the basic model analysis (n = 403) were available for the extended model analysis (n = 327) due to missing blood samples. Body condition scores collected at 63 ± 3 d PP were included as a continuous independent variable in both basic and extended models, but were subsequently excluded because their effects were not significant. Rates of increase in cumulative pregnancy rates from the first synchronized service until 120 d PP or until 365 d PP also were analyzed with the test for homogeneity of regression (48). Comparisons among treatment response curves were tested with the same orthogonal contrasts described above. Differences were considered significant at a probability value of 0.10 or less. RESULTS Effects of Treatment Groups Reproductive responses to first synchronized service. Analysis of pregnancy rates to the first synchronized service as diagnosed by ultrasonography at 27 d after insemination detected a main effect of bST treatment (P < 0.08) and an interaction between bST treatment and resynchronization (P < 0.09; Figure 1). Cows initiating bST treatment at 63 ± 3 d PP had higher pregnancy rates than control cows that initiated bST Journal of Dairy Science Vol. 83, No. 6, 2000

treatment at 105 ± 3 d PP. An interaction that indicated that the advantage obtained by the use of bST during the TAI protocol was evident in cows not resynchronized (RIDE+bST = 46.9 ± 6.2% and RIDE = 29.6 ± 4.5%), whereas this advantage did not exist if cows were resynchronized (RESYNCH+bST = 33.8 ± 4.6% and RESYNCH = 33.4 ± 5.8%). Similarly, pregnancy rates diagnosed by rectal palpation at 45 d after insemination indicated both an effect of bST treatment (P < 0.10) and an interaction (P < 0.09) as depicted in Figure 1. Initiation of bST treatment at 63 ± 3 d PP increased pregnancy rates (RIDE+bST = 37.7 ± 5.8% and RIDE = 22.1 ± 4.2%), but such an effect was eliminated when cows were resynchronized with an injection of GnRH at 20 d after insemination (RESYNCH+bST = 25.6 ± 4.3% and RESYNCH = 25.8 ± 5.5%). Estimates of pregnancy rates based on plasma P4 concentrations collected at d 20 after insemination, immediately prior to injection of GnRH, were analyzed. Because of missing blood samples, the number of cows available for this analysis was reduced from 403 to 377 cows (RIDE+bST = 64; RIDE = 115; RESYNCH+bST = 128, RESYNCH = 70). None of the cows diagnosed nonpregnant based on plasma P4 at d 20 was diagnosed pregnant 7 d later during ultrasonography (100% accuracy). In contrast, 55.6% of the cows diagnosed pregnant at d 20 were pregnant at d 27 based on ultrasonography. Results indicated a significant effect of bST treatment (P < 0.01) which increased pregnancy rates when initiated simultaneously with the TAI protocol (Figure 1). No significant interaction was detected for the d 20 estimate of pregnancy rates. Moreover, plasma P4 concentrations at 20 d after insemination were analyzed to establish whether P4 concentrations, per se, were affected or not by administration of bST. Results indicated that cows initiating bST treatment at 63 ± 3 d PP had higher (P < 0.09) plasma P4 concentrations (RIDE+bST = 9.4 ± 0.9 ng/ml, RESYNCH+bST = 9.8 ± 0.7 ng/ml) than cows in which bST treatment was initiated at 105 ± 3 d PP (TI = 7.9 ± 0.7 ng/ml, RESYNCH = 8.8 ± 0.8 ng/ml). However, such an effect may have resulted from greater pregnancy rates of bST treated cows at 20 d after insemination. Hence, data were analyzed again, excluding cows pregnant or cows nonpregnant based on ultrasonography examination at d 27 after first service. No effects of bST treatment (P > 0.10) on plasma P4 concentrations at 20 d after timed insemination were detected for pregnant cows (RIDE+bST = 13.2 ± 1.0 ng/ml, RIDE = 13.1 ± 0.8 ng/ml, RESYNCH+bST = 13.4 ± 0.9 ng/ml, and RESYNCH = 13.4 ± 1.1 ng/ml). Concentrations of plasma P4 at 20 d after timed insemination for cows diagnosed nonpregnant at d 27, via ultrasonography, did not differ among treatment groups (RIDE+bST =

BOVINE SOMATOTROPIN AFFECTS A TAI PROTOCOL

6.1 ± 1.1 ng/ml, RIDE = 5.4 ± 0.7 ng/ml, RESYNCH+bST = 8.0 ± 0.7 ng/ml, and RESYNCH = 6.0 ± 0.9 ng/ml). Pregnancy losses. Pregnancy losses between d 20 and 27 after the first synchronized service were greater (P < 0.08) for cows resynchronized with an injection of GnRH at 20 d after insemination (RESYNCH+bST = 55.6 ± 6.0% and RESYNCH = 52.4 ± 8.1%) than for cows not resynchronized (RIDE+bST = 39.8 ± 8.2% and RIDE = 44.8 ± 6.8%). Pregnancy losses between d 27 and 45 did not differ (P > 0.10) among treatment groups (RIDE+bST = 20.8 ± 8.9%, RIDE = 27.2 ± 7.5%, RESYNCH+bST = 24.2 ± 7.7%, RESYNCH = 15.6 ± 9.6%). Reproductive responses to second service. Insemination rates to second service differed among treatment groups during the initial 5 wk of the experiment when resynchronized cows received a second timed insemination. Cows not resynchronized were considered in this analysis if estrus was detected and insemination was performed in the interval between the first synchronized service until 7 d after ultrasonography when PGF2α was injected. Second-service insemination rates for the RIDE+bST (70.9 ± 9.0%, n = 18) and RIDE (67.4 ± 6.0%, n = 42) groups were lower (P < 0.01) than the RESYNCH+bST (n = 33) and RESYNCH groups (n = 19) which had a 100% insemination rate. The interval between the first synchronized service and the second service was greater (P < 0.01) for cows in the RESYNCH+bST and RESYNCH groups that were reinseminated 30 d after the first service compared to the RIDE+bST and RIDE groups (24.0 ± 1.4 d and 20.0 ± 0.9 d, respectively). An interaction (P < 0.06) also was observed for the interval between first and second services indicating that, in cows not resynchronized, initiation of bST treatment at 63 ± 3 d PP delayed the time of the second service compared with when bST treatment was initiated at 105 ± 3 d PP (24.0 ± 1.4 d > 20.0 ± 0.9 d). Following the initial 5 wk of the experiment, cows from the resynchronized groups diagnosed nonpregnant at ultrasonography did not receive the second injection of GnRH of the TAI protocol but were, instead, inseminated at detected estrus. For the resynchronized groups, only cows detected in estrus and inseminated during the 7 d following the injection of PGF2α were considered in this analysis. Insemination rates were greater (P < 0.01) for cows not resynchronized (RIDE+bST = 79.5 ± 7.9%, n = 33; RIDE = 82.1 ± 6.1%, n = 58) in comparison to resynchronized cows (RESYNCH+bST = 49.0 ± 7.5%, n = 39; RESYNCH = 44.0 ± 10.4%, n = 19). Results also indicated that the interval between the first synchronized service and the second service was reduced (P < 0.01) for RIDE+bST (17.9 ± 1.4 d) and RIDE (20.1 ± 1.2 d) groups compared

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to RESYNCH+bST and RESYNCH groups (30.5 ± 1.5 d and 30.0 ± 2.1 d, respectively). Moreover, there were no differences detected (P > 0.10) in the interval between injection of PGF2α and insemination when the RESYNCH+bST group was compared to the RESYNCH group (3.6 ± 0.3 d and 3.2 ± 0.4 d). Conception and pregnancy rates for wk 1 to 5 did not differ compared with conception and pregnancy rates for wk 6 to 14. Therefore, data for conception and pregnancy rates to the second service were pooled. Results indicated that conception rates did not differ among RIDE+bST (24.4 ± 11.6%; n = 37), RIDE (23.0 ± 10.4%; n = 72), RESYNCH+bST (20.0 ± 10.4%; n = 50), and RESYNCH (16.7 ± 12.7%; n = 27) groups. Similarly, pregnancy rates for RIDE+bST (20.4 ± 5.5%; n = 51), RIDE (20.7 ± 3.9%; n = 100), RESYNCH+bST (18.8 ± 4.6%; n = 72), and RESYNCH (14.1 ± 6.4%; n = 38) groups did not differ. Reproductive responses at 120 and at 365 d PP. Total number of services, services per conception, days open, and pregnancy rates at 120 d PP are depicted in Table 1. Cows in which treatment with bST was initiated at 63 ± 3 d PP had fewer total services (P < 0.10), fewer services per conception (P < 0.01), and fewer days open (P < 0.05) at 120 d PP compared with cows initiating bST treatment at 105 ± 3 d PP. However, pregnancy rates at 120 d PP did not differ among treatment groups (P > 0.10). An interaction between bST treatment and reproductive management was detected (P < 0.05) when rate of accumulated pregnancy rates from the first synchronized service until 120 d PP was examined (Figure 2). A faster rate of accumulated pregnancies occurred for cows in which bST treatment was initiated at 63 ± 3 d PP, but that advantage was eliminated when cows were resynchronized. Results for the number of total services, services per conception, days open, and pregnancy rates at 365 d PP are depicted in Table 1. No differences among treatment groups were detected at 365 d PP (P > 0.10), except for days open, which were lower (P < 0.01) for cows in which bST treatment was initiated at 63 ± 3 compared with cows in which bST treatment was initiated at 105 ± 3 d PP. Analyses of the rate of accumulated pregnancy rates until 365 d PP indicated an interaction between bST treatment and reproductive management (P < 0.05; data not shown). Similarly to the results obtained by 120 d PP, a faster rate of accumulated pregnancy rates from the first service until 365 d PP was observed for cows in which bST treatment was initiated at 63 ± 3 d PP, but that advantage was not detected when cows were resynchronized. Effects of P4-Classes and CL Regression Status Reproductive responses to first synchronized service. Approximately 7.3% of the experimental cows Journal of Dairy Science Vol. 83, No. 6, 2000

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MOREIRA ET AL. Table 1. Least squares means and SE for the number of total services, services per conception, days open, and pregnancy rates at 120 d postpartum (PP) and at 365 d PP for treatment groups. RIDE+bST (n = 67) Results at 120 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%) Results at 365 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%)

RIDE (n = 126)

RESYNCH+bST (n = 134)

RESYNCH (n = 76)

1.61 1.17 75.8 47.3

± ± ± ±

0.08a 0.10c 2.4e 6.0

1.80 1.55 83.4 42.1

± ± ± ±

0.06b 0.08d 1.9f 4.3

1.61 1.35 80.7 38.9

± ± ± ±

0.06a 0.08c 1.9e 4.2

1.67 1.44 81.9 42.4

± ± ± ±

0.08b 0.10d 2.4f 5.6

3.87 3.01 130.2 77.5

± ± ± ±

0.40 0.38 12.2c 5.1

4.20 3.65 160.0 79.7

± ± ± ±

0.29 0.27 8.8d 3.7

4.04 3.19 146.1 76.6

± ± ± ±

0.28 0.27 8.7c 3.6

4.01 3.52 162.5 79.7

± ± ± ±

0.37 0.36 11.3d 4.8

Different superscripts within rows indicate significant contrasts (P < 0.10). Different superscripts within rows indicate significant contrasts (P < 0.01). e,f Different superscripts within rows indicate significant contrasts (P < 0.05). 1 Only cows conceiving were considered in these analyses. a,b c,d

were classified as LOW-LOW (24/327), 5.5% as HIGHLOW (18/327), 25.9% as LOW-HIGH (85/327), and 61.1% as HIGH-HIGH cows (200/327). Pregnancy rates determined by ultrasonography were lower (P < 0.01) for cows classified as LOW-LOW and as HIGH-LOW (8.0 ± 10.6% and 12.6 ± 11.5%, respectively) compared with LOW-HIGH and HIGH-HIGH cows (27.8 ± 6.1% and 38.4 ± 5.1%, respectively). Moreover, HIGH-HIGH cows had higher pregnancy rates than LOW-HIGH cows at d 27 after insemination (P < 0.09). Analysis of pregnancy rates by rectal palpation at d 45 after insemination indicated that reduced pregnancy rates (P < 0.01) were obtained for LOW-LOW and HIGHLOW cows (3.5 ± 9.9% and 1.9 ± 10.7%, respectively) compared with LOW-HIGH and HIGH-HIGH cows (27.1 ± 5.7% and 26.9 ± 4.7%). No differences (P > 0.10)

Figure 2. Cumulative pregnancy rates by 120 d postpartum (PP) for cows reinseminated at detected estrus and treated with bST at 63 ± 3 d PP (RIDE+bST; ——), or at 105 ± 3 d PP (RIDE; – –) in panel A, and for cows resynchronized and treated with bST at 63 ± 3 d PP (RESYNCH+bST; -----), or at 105 ± 3 d PP (RESYNCH; ..........) in panel B depicting a significant interaction between bST treatment and reproductive management system (P < 0.05). Journal of Dairy Science Vol. 83, No. 6, 2000

were observed between LOW-HIGH and HIGH-HIGH cows for pregnancy rates diagnosed by rectal palpation. Approximately 19% (62/327) of the cows were classified as having incomplete CL regression at 48 h after injection of PGF2α. Pregnancy rates were reduced for cows with incomplete CL regression compared with cows undergoing complete CL regression as determined by both ultrasonography (8.4 ± 7.3% < 35.0 ± 4.9%; P < 0.01) and rectal palpation (1.4 ± 6.8% < 28.3 ± 4.6%; P < 0.01). Pregnancy losses. Embryonic losses between d 27 and 45 after first-service timed insemination were higher (P < 0.04) for cows classified as LOW-LOW and HIGH-LOW (39.5 ± 24.0% and 77.6 ± 24.6%, respectively) compared with LOW-HIGH and HIGH-HIGH cows (12.1 ± 10.1% and 33.5 ± 6.9%, respectively). Interestingly, LOW-HIGH cows had lower (P < 0.02) embryonic losses from d 27 to 45 of pregnancy than HIGHHIGH cows (12.1 ± 10.1% < 33.5 ± 6.9%). Cows classified as having incomplete CL regression at 48 h after injection of PGF2α had greater pregnancy losses between d 27 and 45 after first-service timed insemination than did cows with complete CL regression (51.7 ± 14.6% > 29.7 ± 8.7%; P < 0.09). Reproductive responses at 120 and at 365 d PP. Results obtained at 120 d PP indicated that cows classified as LOW-LOW and HIGH-LOW had more total services (P < 0.02), more services per conception (P < 0.01), more days open (P < 0.02), and lower pregnancy rates (P < 0.02) than LOW-HIGH and HIGH-HIGH cows (Table 2). Similarly, at 365 d PP, cows classified as LOWLOW and as HIGH-LOW had more total services (P < 0.01), more services per conception (P < 0.01), and more days open (P < 0.01) than LOW-HIGH and HIGH-HIGH cows (Table 2). However, no differences in pregnancy

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BOVINE SOMATOTROPIN AFFECTS A TAI PROTOCOL Table 2. Least squares means and SE for the number of total services, services per conception, days open, and pregnancy rates at 120 d postpartum (PP) and at 365 d PP for P4 classes. LOW-LOW (n = 24) Responses at 120 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%) Responses at 365 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%)

HIGH-LOW (n = 18)

LOW-HIGH (n = 85)

HIGH-HIGH (n = 200)

2.05 1.95 89.9 18.2

± ± ± ±

0.05a 0.24c 5.9a 10.6a

1.89 2.22 92.4 25.0

± ± ± ±

0.16a 0.24c 5.8a 11.6a

1.70 1.38 79.8 41.0

± ± ± ±

0.08b 0.10d 2.4b 5.7b

1.72 1.38 81.6 39.0

± ± ± ±

0.06b 0.08d 1.9b 4.2b

5.79 5.56 208.7 69.9

± ± ± ±

0.69c 0.70c 22.6c 9.3

5.09 4.40 183.7 77.1

± ± ± ±

0.76c 0.75c 23.9c 10.2

3.79 3.43 155.3 78.3

± ± ± ±

0.37d 0.37d 11.9d 5.0

4.18 3.50 149.5 76.9

± ± ± ±

0.27d 0.27d 8.6d 3.7

Different superscripts within rows indicate significant contrasts (P < 0.02). Different superscripts within rows indicate significant contrasts (P < 0.01). 1 Only cows conceiving were considered in these analyses. a,b c,d

rates at 365 d PP were observed among P4-classes (P > 0.10; Table 2). At 120 d PP, cows with incomplete CL regression following injection of PGF2α had more total services (P < 0.01), more services per conception (P < 0.02), more days open (P < 0.01), and lower pregnancy rates (P < 0.02) than did cows with complete CL regression (Table 3). At 365 d PP, cows with incomplete regression of the CL after PGF2α injection at the first synchronized service had more total services (P < 0.07), more services per conception (P < 0.01), and more days open (P < 0.05) than did cows that had complete CL regression

Table 3. Least squares means and SE for the number of total services, services per conception, days open, and pregnancy rates at 120 d postpartum (PP) and at 365 d PP for cows with complete or incomplete CL regression. Complete CL regression (n = 265) Results at 120 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%) Results at 365 d PP Total services Services/conception1 Days open (d)1 Pregnancy rates (%)

Incomplete CL regression (n = 62)

1.66 1.56 81.7 39.2

± ± ± ±

0.06a 0.09c 2.1a 4.4c

2.02 1.90 90.1 22.6

± ± ± ±

0.10b 0.14d 3.5b 7.0d

4.24 3.64 160.1 76.3

± ± ± ±

0.29e 0.28a 9.1g 3.9

5.07 4.81 188.5 74.8

± ± ± ±

0.46f 0.47b 15.0h 6.2

a,b Different superscripts within rows indicate significant contrasts (P < 0.01). c,d Different superscripts within rows indicate significant contrasts (P < 0.02). e,f Different superscripts within rows indicate significant contrasts (P < 0.07). g,h Different superscripts within rows indicate significant contrasts (P < 0.05). 1 Only cows conceiving were considered in these analyses.

as depicted in Table 3. Pregnancy rates at 365 d PP did not differ due to CL regression status (P > 0.10; Table 3). DISCUSSION The use of bST in the present experiment resulted in increased pregnancy rates to the first synchronized service as indicated by the effects of bST at pregnancy diagnosis determined at 20, 27, and 45 d after timed insemination. Several possible explanations may account for the beneficial effects of bST on pregnancy rates, which may have occurred before or after timed artificial insemination. Injection of bST increases the production of IGF-I by the liver (31) and results in greater concentrations of IGF-I in peripheral blood and follicular fluid (27). Receptors for IGF-I have been detected in ovarian follicles (43) which indicates that IGFI may affect follicular development. Indeed, bST altered follicular dynamics in cattle (10, 25, 26), increased the number of follicles smaller than 9 mm in diameter (10, 15), and influenced the interval between follicular waves (19). Thus, evidence suggests that bST may have affected the preovulatory follicle that was synchronized during the TAI protocol prior to insemination. Such an effect may result in a healthier and more fertile oocyte. Also, altered follicular development after insemination may have affected pregnancy rates. Use of bST accelerated the emergence of a second follicular wave (19), which may result in a higher incidence of three wave cycles following insemination that are associated with increased conception rates (1). Moreover, administration of bST may have increased pregnancy rates to the first synchronized service by enhancing CL development and P4 production after insemination. Messenger RNA for bST receptors has been detected in luteal cells (23), bST treatment increased CL weight (27) and plasma P4 in peripheral blood (14, Journal of Dairy Science Vol. 83, No. 6, 2000

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24, 39), and increased plasma P4 concentrations have been associated with higher pregnancy rates (6, 45). In addition, administration of bST may attenuate PGF2α production by the endometrium during pregnancy recognition and result in increased pregnancy rates. Several reports have demonstrated that IGF-I and other growth factors modulate the expression of phospholipase A2 and cyclooxygenase-2 enzymes that lead to the production of PGF2α (3, 17, 35), and an extension of the life span of the CL was observed in bST-treated cows (24). However, bST treatment did not influence estrous cycle length nor CL regression in response to an injection of PGF2α in cows (19, 20). In the present experiment, the interval from first to second services was greater when non resynchronized cows were treated with bST during the initial 5 wk of the experiment, but no differences were observed during wk 6 to 14. The use of bST also may have affected oviductal and uterine secretions during early embryonic development because receptors for IGF-I were identified in the oviduct and uterine epithelium (42). Moreover, bST and IGF-I receptors have been identified at different stages of embryonic development (30, 42), and addition of IGFI stimulated the development of cultured bovine embryos (18, 30, 37) and growth of cultured oviductal cells (46). Thus, increased plasma concentrations of bST and IGF-I may result in increased concentrations within the oviduct and uterine endometrium to stimulate embryonic development. Hence, bST may enhance fertility through several possible mechanisms. This observation contradicts previous reports in the literature whereby bST treatment decreased reproductive efficiency (7, 8, 49). Perhaps, by eliminating the problem of increased rate of undetected estrus and ovulations upon bST treatment (20) through the use of the TAI protocol, the possible beneficial effects of bST on embryonic development and survival were detected. An interaction between bST treatment and reproductive management system on pregnancy rates observed at 27 and at 45 d (P < 0.09) after first-service timed insemination, and an effect of resynchronization treatment to increase pregnancy losses between d 20 and 27 (P < 0.07), indicated that injection of GnRH at 20 d after insemination adversely affected embryonic survival. Injection of GnRH induces an immediate increase in plasma estradiol concentrations (44), which may trigger the production of PGF2α by the endometrium and cause the demise of the CL. Moreover, there is evidence that LH receptors are found in the uterine endometrium and uterine vein (13) and that LH may stimulate PGF2α secretion (41). Hence, it is possible that an LH surge induced by an injection of GnRH may induce luteolysis. The effects of GnRH administered at 20 d Journal of Dairy Science Vol. 83, No. 6, 2000

after insemination were not expected and may indicate that resynchronization of cows prior to pregnancy diagnosis should be avoided. Despite the effects of GnRH at d 20 on first-service pregnancy rates, conception, and pregnancy rates to the second service were not affected by resynchronization treatment (P > 0.10). This was observed during the initial 5 wk of the experiment, from wk 6 to 14 of the experiment, and when all data were pooled. The interval between first and second services was increased for resynchronized cows (P < 0.01), which was expected because cows not resynchronized could be reinseminated following the first service, whereas resynchronized cows were reinseminated only after ultrasonography at 27 d after first service. Pregnancy rates by 120 and by 365 d PP were not affected by treatment (P > 0.10), indicating that neither bST nor resynchronization treatment affected subsequent fertility of experimental cows. The effect of bST treatment in reducing the number of total services and services per conception by 120 d PP, and also in decreasing the number of days open by 120 and by 365 d PP, are probably a consequence of higher pregnancy rates obtained at first service for cows in which bST treatment was initiated at 63 ± 3 d PP. Such an effect of bST on the first synchronized service was also responsible for differences in the accelerated increase in accumulative pregnancy rates until 120 (Figure 2) or 365 d PP (data not shown). The interpretation of the interaction effects observed when cumulative pregnancy rates were analyzed until 120 or 365 d PP indicated, once more, that bST increased the rate of pregnancy accumulation but that the effects of bST were eliminated when cows were resynchronized. Analysis of data following classification of cows in P4classes or according to CL regression status provided interesting insights regarding reproductive management. As expected, cows from the LOW-LOW and HIGH-LOW groups had reduced pregnancy rates to the first synchronized service compared with LOW-HIGH and HIGH-HIGH cows at 20, 27, and 45 d after insemination (P < 0.01). Cows classified as LOW-LOW were probably in anestrus at initiation of the TAI protocol and did not ovulate to the first injection of GnRH. Cows classified as HIGH-LOW were probably in estrus at the injection of PGF2α and probably ovulated prior to insemination. Such an asynchrony between ovulation and insemination seems to be related to initiation of the TAI protocol at the late luteal stages of the estrous cycle (29). Cows classified as LOW-HIGH were probably cows that were in the metestrus or proestrus stages of the estrous cycle at the initiation of the TAI protocol or, alternatively, were anestrus cows that ovulated in re-

BOVINE SOMATOTROPIN AFFECTS A TAI PROTOCOL

sponse to the injection of GnRH and formed a CL that increased plasma P4 concentrations 7 d later at the injection of PGF2α. Collectively, these cows were expected to have lower pregnancy rates than HIGH-HIGH cows. Cows initiating the TAI protocol during the metestrus or proestrus phase had lower pregnancy rates than cows that were in the early luteal phase at the first injection of GnRH (29, 47). Moreover, cows in anestrus that responded to the GnRH injection and formed a CL had a greater probability of having short estrous cycles following insemination, which would compromise embryonic survival. Indeed, cows classified as LOW-HIGH had lower pregnancy rates compared with HIGH-HIGH cows (P < 0.09) when pregnancy diagnosis was based on ultrasonography at 27 d after timed insemination. Cows with incomplete CL regression at 48 h after injection of PGF2α had lower pregnancy rates than cows with complete CL regression at 27 and at 45 d after insemination (P < 0.01). This observation agrees with prior results, which indicated that none of the cows with plasma P4 concentrations >1.0 ng/ml at 48 h after injection of PGF2α conceived to the TAI protocol (5). Low pregnancy rates experienced by cows that fail to completely regress their CL after an injection of PGF2α may be due to an incomplete maturation of the preovulatory follicle in which final development occurred in an elevated P4 environment. Such an elevated P4 environment may have reduced the pulsatile LH release between luteolysis and the LH surge which may be essential for maturation of the preovulatory follicle (36) and for prematuration of the oocyte and resumption of the ribosomal transcription in cattle oocytes (16). Also, oviductal and uterine environments exposed to an abnormal hormonal pattern may not be suitable for subsequent embryonic development and survival. In addition to a possible greater fertilization failure and embryo loss during the initial stages of embryonic development, cows with incomplete CL regression had greater pregnancy losses between ultrasonography (27 d after insemination) and rectal palpation (45 d after insemination) than cows that had complete CL regression (P < 0.09). The cause of such a greater loss is unknown but indicates that, whenever fertilization occurs in cows with incomplete CL regression, the conceptus has less chance to survive until term. Because no differences were observed for the second service among P4-classes or between cows with complete or incomplete CL regression, the differences obtained in number of total services, services per conception, in days open at 120 and 365 d PP, and also in pregnancy rates at 120 d PP likely were due to the differences obtained in pregnancy rates at the first synchronized service. Hence, these results emphasize the

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importance of enhancing fertility to the first service in lactating dairy cows because that may influence fertility parameters as late as 365 d PP. CONCLUSIONS The use of bST increased pregnancy rates to the first service TAI protocol. Continuing research should be conducted to increase our understanding on how bST affects and enhances fertility in lactating dairy cows. Resynchronization of cows with a GnRH injection at 20 d after insemination decreased embryo survival to the first service, but the same GnRH injection did not influence fertility to the second service. Administration of bST as part of a reproductive management program including TAI at approximately 63 d PP will not reduce fertility but appears to enhance first service pregnancy rate. ACKNOWLEDGMENTS Authors express their appreciation to Ron Saint-John and to Peter Gelber and his staff at Alliance Dairies (Trenton, FL) for expert management of experimental cows. Our gratitude is extended to Mario Binelli, Joan Burke, and Thais Diaz for their help during blood sample collection and ultrasonography, and to Jesse J. Johnson for conducting the radioimmunoassays. This project received partial financial support from the Agricultural Sector, Animal Agricultural Group, Monsanto Co. (St. Louis, MO) and USDA-BARD Grant 9434339-1212. Appreciation is also extended to Pharmacia-Upjohn Co. (Kalamazoo, MI) for providing Lutalyse, and to Merial Co. (Athens, GA) for the donation of Cystorelin. This is Florida Agricultural Experimental Station Journal Series No. R-07338. REFERENCES 1 Ahmad, N., E. C. Townsend, R. A. Dailey, and E. K. Inskeep. 1997. Relationships of hormonal patterns and fertility to occurrence of two or three waves of ovarian follicles, before and after breeding, in beef cows and heifers. Anim. Reprod. Sci. 49:13–28. 2 Are´ chiga, C. F., C. R. Staples, L. R. McDowell, and P. J. Hansen. 1998. Effects of timed insemination and supplemental β-carotene on reproduction and milk yield of dairy cows under heat stress. J. Dairy Sci. 81:390–402. 3 Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M. T. Corvol, and J. Masliah. 1994. Insulin-like growth factors counteract the effect of interleukin 1β on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes. FEBS Lett. 340:51–55. 4 Britt, J. S., and J. Gaska. 1998. Comparison of two estrus synchronization programs in a large, confinement-housed dairy herd. JAVMA 212:210–212. 5 Burke, J. M., R. L. de la Sota, C. A. Risco, C. R. Staples, E.J.P. Schmitt, and W. W. Thatcher. 1996. Evaluation of timed insemination using a gonadotropin-releasing hormone agonist in lactating dairy cows. J. Dairy Sci. 79:1385–1393. Journal of Dairy Science Vol. 83, No. 6, 2000

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