Cyclicity, estrus expression and pregnancy rates in beef heifers with different reproductive tract scores following progesterone supplementation

Journal Pre-proof Cyclicity, estrus expression and pregnancy rates in beef heifers with different reproductive tract scores following progesterone supplementation

R.K. Kasimanickam, V.R. Kasimanickam, J. Oldham, M. Whitmore PII:

S0093-691X(20)30034-0

DOI:

https://doi.org/10.1016/j.theriogenology.2020.01.028

Reference:

THE 15325

To appear in:

Theriogenology

Received Date:

22 January 2019

Accepted Date:

13 January 2020

Please cite this article as: R.K. Kasimanickam, V.R. Kasimanickam, J. Oldham, M. Whitmore, Cyclicity, estrus expression and pregnancy rates in beef heifers with different reproductive tract scores following progesterone supplementation, Theriogenology (2020), https://doi.org/10.1016/j. theriogenology.2020.01.028

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Cyclicity, estrus expression and pregnancy rates in beef heifers with different reproductive tract scores following progesterone supplementation

Kasimanickam RK*, Kasimanickam VR, Oldham J, Whitmore M Department of Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164

*Corresponding author, email: [email protected]

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Abstract Two experiments were conducted to determine effects of progesterone (P4) on cyclicity, estrus expression rate (EER) and artificial insemination pregnancy rate (AIPR) in beef heifers with various reproductive tract scores (RTS; 1 to 5; 1, immature, acyclic; 5, mature, cyclic). In Experiment 1, Angus-cross heifers (n = 100, 20 per RTS category; mean (±SEM) age, 15±0.8 mo) were randomly assigned to receive a CIDR (Days -17 to -10) or no CIDR (untreated control), with weekly blood samples and ultrasonography (Days 0 to 85). Among heifers with RTS 2 to 4, median interval to cyclicity were shorter (P<0.05) for heifers in CIDR versus control. In Experiment 2, Angus-cross heifers (n=11,098) were assigned RTS, body condition score (BCS; 1 to 9; 1, emaciated; 9, obese) and temperament score (calm versus excitable). Heifers with RTS 2-5 (n=10,569) were allocated to CO-Synch (n=5,099) or CO-Synch+CIDR (n=5,470). Estrus was detected until AI (72 h after PGF2α), with pregnancy diagnosis ~70 d later. Controlling for RTS (P<0.0001), BCS (P<0.0001), temperament (P<0.0001), age (P<0.0001), treatment by RTS (P<0.01), treatment by BCS (P<0.01), and treatment by age, EER differed between CO-Synch and CO-Synch+CIDR (71.0 vs 75.9%, respectively, P<0.0001). Accounting for RTS (P<0.0001), BCS (P<0.0001), temperament (P<0.0001) and age (P<0.0001), heifers detected in estrus or not (P<0.0001), RTS by treatment (P<0.01), BCS by treatment (P<0.01), and age by treatment, AIPR differed between CO-Synch versus COSynch+CIDR (55.3 vs 61.0%, P<0.0001). In conclusion, exogenous P4 hastened cyclicity in beef heifers with RTS 2 to 4 and increased EER and AIPR. However, RTS, BCS and age influenced EER and AIPR. Among RTS 4 and 5, EER was greater for CO-Synch+CIDR vs CO-Synch. Among RTS 3 to 5, AIPR was greater for CO-Synch+CIDR versus CO-Synch. Progesterone

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status or supplementation at onset of synchronization protocols was critical to pregnancy outcomes, emphasizing heifer development for early puberty or progesterone supplementation.

Keywords: Beef heifers; Reproductive tract score; Progesterone; Cyclicity; Ovulation synchronization; Estrus expression; Pregnancy rate

1 Introduction

Estrus synchronization and artificial insemination (AI) can maximize reproductive efficiency of beef heifers and facilitate use of superior genetics [1]. Various protocols have successfully synchronized estrus or ovulation in beef heifers, facilitating AI with or without estrus detection [2-5]. Regardless, lack of adoption of AI in beef cow-calf operations limits genetic progress [6]. Labor concerns, cost and protocol complexity are major constraints stated by beef producers for not using AI, prompting development of simple, cost-effective protocols. Ages at onset of puberty and establishment of first pregnancy in beef heifers critically affect lifetime productivity [7-12]. Pubertal status before initiation of synchronization treatments, particularly protocols for fixed time AI, largely influences protocol efficiency and perhaps AI pregnancy rate (AIPR) [7]. Reproductive tract score (RTS) is a valuable tool for selecting beef heifers for breeding. Heifers are assigned a score of 1 (immature reproductive tract, acyclic) to 5 (mature reproductive tract, cyclic), based on uterine size and ovarian structures [15]. Reproductive tract scoring is an indirect measure of pubertal status and a future predictor of reproductive performance in beef heifers [13-17]. Based on RTS, pubertal heifers had higher AIPR compared to pre- or peri-pubertal heifers [7,13-17].

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In most synchronization protocols, heifers will have a range of pubertal status at protocol initiation; however, this physiological diversity offer both challenges and opportunities [7,14]. Pubertal status can be taken into account when allocating the beef heifers to cost-effective estrussynchronizing treatments [14,17]. For instance, if progesterone (P4) supplementation would benefit heifers with RTS 2 to 4 (pre- and peri-pubertal heifers) and result in acceptable AIPR, it would be economically justifiable to add a controlled internal drug release (CIDR) insert to the CO-Synch protocol for these heifers. Further, if a CO-Synch protocol would result in acceptable AIPR in heifers with RTS 5 (pubertal heifers), use of a CIDR insert could be avoided for these heifers. In a study using estrus-synchronization strategies to optimize reproductive performance [14], we assigned beef heifers to various estrus-synchronization protocols and inseminated at observed estrus. In that study, in heifers with RTS 5 after a double prostaglandin F2a (PGF2α) protocol (on Days 0 and 14), AIPR was lower compared to AIPR in heifers with RTS 2 to 4 following CIDR-PGF2α protocol (CIDR for 7 d and PGF2α at CIDR removal). Progesteronebased protocols induced cyclicity in pre- and peri-pubertal heifers [18]. The current study was designed to determine effects of ovulation synchronization strategies for a FTAI in beef heifers with various RTS. Our hypothesis was addition of progesterone supplementation to a CO-Synch fixed time AI will hasten time to cyclist, and improve estrus expression rate (EER) and AI pregnancy rate in pre- and peri-pubertal heifers. Objectives were to determine effects of P4 supplementation on cyclicity and on EER and AIPR in a CO-Synch protocol in beef heifers with various reproductive tract scores.

2 Materials and methods

2.1 Experiment 1 4

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Reproductive tracts of Angus-cross beef heifers (Angus x Simmental; Angus x Hereford; Angus x Simmental x Hereford; Mean (±SEM) age, 15±0.8 mo.) with moderate to good body condition (BCS, 5 to 7; 1, emaciated; 9, obese) during 2016 breeding season were evaluated by transrectal palpation by one clinician and assigned an RTS score (1 to 5; 1, immature, acyclic; 5, mature, cyclic; Table 1) [19]. Twenty heifers in each RTS category were selected and randomly allocated to CIDR or control groups. Ten heifers in each RTS category (n = 50) received a CIDR (1.38 g P4; Eazi-Breed™ CIDR® Cattle Insert; Zoetis Animal Health, Kalamazoo, MI, USA) insert on Day -17 that was removed on Day -10, with concurrent administration of 25 mg dinoprost (PGF2α; 5 mL, im, Lutalyse® sterile solution; Zoetis Animal Health). The remaining 10 heifers in each RTS category (n = 50) served as untreated controls. Heifers were monitored until Day 85. Once weekly (days 0, 7, 14, 21, 28, 35, 42, 49, 54, 61, 70, 77 and 85) blood samples were collected from all heifers to determine serum P4 concentrations, with concurrent transrectal ultrasonography to determine if they had a corpus luteum (CL). Heifers with serum P4 concentrations >1 ng/mL and/or presence of a CL were considered cycling. For the purpose of this experiment, heifers were intentionally not submitted for breeding until confirmation of cyclicity. Heifers with no CL and P4 < 1 ng/mL during weekly exam were considered not cyclic. Onset of cyclicity was considered as 4 d (0.5 wk) prior to weekly exam for heifers with a CL and P4 concentrations >2 ng/mL during the next weekly exam. Similarly, onset of cyclicity was considered as 7 d (1 wk) prior to weekly exam for heifers with a CL and P4 between >1 and ≤2 ng/mL (onset of cyclicity considered was 4 d earlier for heifers with CL and P4 >5 ng/mL compared to heifers with CL and P4 >1 and ≤2 ng/mL) [20-23]. After confirmation of cyclicity, heifers were grouped in pastures with bulls for breeding.

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2.1.1 Blood collection and P4 analysis

Briefly, blood samples were collected into evacuated tubes (Becton Dickinson, Franklin Lakes, NJ, USA) that were immediately placed on ice and transported to the laboratory on the same day. Upon arrival, samples were centrifuged at 1,000 × g for 15 min at 4 °C and serum removed and stored at -20 °C. Serum P4 concentrations were determined in duplicate using a coated tube labelled with 125I (Diagnostic Products Corporation, Los Angeles, CA, USA) according to manufacturer's instructions. Assay sensitivity was 0.03 ng/mL, with intra- and interassay CVs of 7.6 and 13.6%, respectively.

2.1.2 Ovarian ultrasonography

Weekly ultrasonography (Sonoscape S8, Universal imaging, Bothell, WA, USA) with 5 MHz linear-array transducer was done by one clinician.

2.2 Experiment 2

2.2.1 Heifers and treatments

Angus cross (Angus x Simmental; Angus x Hereford; Angus x Simmental x Hereford) beef heifers from 12 cow-calf operations in Washington and Oregon were included in this study. The goal of participating ranches was to have heifers calve by 2 y. Original data (from 2011 to

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2018) from paper records were entered in Excel spreadsheets. To be enrolled in the study, each heifer had to have the following information: herd (location), age (mo), treatment (CO-Synch or CO-Synch+CIDR), RTS, BCS (1 to 9; 1, emaciated; 9, obese), temperament score (0, calm, slow chute exit; walk; 1, excited, fast chute exit; jump, trot or run), estrus expression from PGF2α administration until AI (yes or no), AI time (72 h after PGF2α administration), AI sires, handlers, inseminators and pregnancy status. In total, 11,098 heifers met selection criteria. Briefly, heifers (n=11,098) were assigned RTS, BCS, and a temperament score (0, calm, slow chute exit; walk, 1, excitable, fast chute exit; jump, trot or run) when synchronization treatments were initiated. Heifers with RTS 2 to 5 (n = 10,569; mean±SEM, 15.4±1.9 mo. of age) were selected, whereas heifers with RTS 1 or diagnosed as freemartins (n=529, 4.8%) were excluded. Blocks of heifers with RTS of 2, 3, 4, or 5 were randomly and alternately assigned to either CO-Synch (n=5,099) or CO-Synch+CIDR (n=5,470) treatments (Fig. 1). On Day 0, heifers in both groups were given 100 µg of gonadorelin diacetate tetrahydrate (GnRH; 2 mL, im; Cystorelin, Merial Inc., Duluth, GA) and heifers in the CO-Synch+CIDR group were given a CIDR (1.38 g of P4; Eazi-Breed CIDR Cattle Insert; Zoetis Animal Health) vaginal insert. Seven days later, CIDRs were removed from heifers in the CO-Synch+CIDR and 25 mg of PGF2 (Lutalyse sterile solution; Zoetis Animal Health) was given to heifers in both groups. All heifers were inseminated 72 h after CIDR removal [4,5], and a second dose of GnRH given concomitantly. Times of insertion and removal of CIDR, PGF2α administration and AI were recorded for each heifer. At CIDR removal (Day 7), all heifers were fitted with estrus detection aids (Kamar® Heatmount detector patches (Kamar, Inc., Steamboat Springs, CO, USA), Estrus Alert patches (Western Point Inc., Apple Valley, MN, USA) or Chalk. Heifers were observed twice daily for

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standing estrus and or activation of estrus detection aid status until time of AI. A heifer was designated in estrus if they were observed to stand for mounting by other herd mates or if they had an activated (>90% of grey patch was red), lost (with mount marks) or partially-activated (50 to 90% of grey patch was red) heat detection aids. One clinician performed transrectal palpation of reproductive tract and assigned RTS. In addition, the same clinician assigned BCS and temperament scores. However, inseminators (n=19), AI sires (n=21) and animal handlers (n=19) differed among locations. The AI sires were selected based on sire traits and assigned to heifers to avoid inbreeding. At 2 wk after AI, heifers were exposed to natural service sires for total breeding seasons of 63 to 85 d.

2.2.2 Pregnancy diagnosis

Approximately 70 d after AI, one clinician conducted pregnancy diagnosis by transrectal ultrasonography (Aloka 500, Sysmed Lab, Inc., Chicago, IL, USA, or Sonoscape S8). Pregnancy was confirmed by visualization of viable embryo/fetus. Gestational age was estimated (to differentiate AI versus natural-service) based on size of embryo/fetus, amniotic vesicle and placentomes.

2.3 Statistical analyses

Data were analyzed commercial statistical software (SAS Version 9.4 for Windows, SAS Institute, Cary, NC, USA).

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2.3.1 Experiment 1

Differences between treatments in mean BCS, body weight, and age were analyzed using ANOVA (PROC GLM). Differences between groups in intervals from treatment to cyclicity were studied using Kaplan-Meier survival analysis (PROC LIFETEST of SAS). Kaplan-Meier survival estimates were used to calculate median days (chance of attaining cyclicity beyond that time was 50%) from treatment to cyclicity. Graphs of cumulative cyclicity risk over time were generated. In Kaplan-Meier analysis, heifers either had the event of interest (i.e. cyclicity) or were ‘censored’ when removed from the study for a reason unrelated to the event of interest, or the 85-d interval ended before the event of interest occurred. The log-rank test was used to compare overall equality of RTS survivor functions and follow-up pairwise comparisons, including within RTS categories between treatments and between RTS counterparts within treatment, using a Bonferroni-corrected log-rank test to maintain experiment-wise type-I error rate of 5%. Restricted mean survival times were obtained as the area under Kaplan-Meier survivor curves. For all analyses, P≤0.05 was considered significant.

2.3.2 Experiment 2

Differences in mean BCS and age of heifers between treatments were analyzed using ANOVA (PROC GLM). Differences between groups in mean interval from CIDR removal to time of insemination were analyzed by ANOVA, with a Bartlett test used to assess homogeneity of variance. Because variances for the mean interval were heterogeneous, log10 transformations were performed. All values are presented as non-transformed values. 9

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The EER was calculated as number of heifers that expressed estrus divided by total number of heifers. The AIPR was measured by number of heifers pregnant to AI divided by total number of heifers inseminated. PROC GLIMMIX was used to determine effects of treatments on EER and AIPR. Fixed variables, treatment (CO-Synch vs. CIDR+CO-Synch), RTS (2 to 5), BCS (3 to 8), temperament score (0 and 1), age (≤15 vs >15 mo), treatment by RTS, treatment by BCS, treatment by age and treatment by temperament score, and random variables location (n=12) nested in year (n=8) were included to determine differences between treatments in estrus expression. Mean differences, including pairwise comparisons, in estrus expression for significant fixed variables were estimated. Fixed variables included in the analysis to determine differences between treatments in AIPR were: treatment (CO-Synch vs CIDR+CO-Synch), RTS (2 to 5), BCS (3 to 7), temperament score (0 and 1), estrus expression at or before AI (yes or no), age (≤15 vs >15 mo), treatment by RTS, treatment by BCS, treatment by heifer’s age, treatment by temperament score and treatment by estrus expression at or before AI interactions. Further, location (n=12) nested in year (n=8), inseminator (n=19) nested in location (n=12), AI sire (n=21) nested in location (n=12), and animal handler (n=19) nested in location (n=12), were included as random variables. Mean differences, including pairwise comparisons, in estrus expression for significant fixed variables, were estimated. Breeding records from 12 locations across 8 y were studied. Two herds in 2016 and two other herds in 2017 did not participate due to management decisions. Although inseminators and AI sires differed among locations, all inseminators had comparable pregnancy outcomes and AI sires used had good, comparable sire fertility estimates. Inseminators and AI sires were not balanced across treatments and hence used as random variables. However, within locations, inseminators were randomly assigned to AI sires and alternatively inseminated heifers. Thus, the

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analysis included nested random effects location (year), AI sires (location) and inseminators (location) and animal handlers (location). For all analyses, P≤0.05 was considered significant. For model reduction, the P value was set at ≤0.1 for inclusion, and >0.1 for exclusion until the model contained appropriate significant main and interaction effects. Mean differences, including pairwise comparisons, in estrus expression rate and AIPR were estimated.

3 Results

3.1 Experiment 1

Mean age, body weight and body condition score are shown (Table 2). The interval (median days) from treatment to cyclicity differed between CIDR treatment and control (P<0.05, Fig. 2). Within RTS category, median days for RTS 2 to 4 differed between treatments (P<0.05, Fig. 3). Number of heifers that failed to cycle during the 85-d interval were four (one in RTS 2 and three in RTS 1) in CIDR and five (two in RTS2 and three in RTS 1) in control groups.

3.2 Experiment 2

3.2.1 Descriptive data

Mean (±SEM) age varied from 15.1±0.12 to 16.3±0.22 mo, with no differences between treatments in mean age (P>0.1) or interval from PGF2α administration to insemination (P>0.1; Table 3). Mean (±SEM) BCS varied from 5.11±0.21 to 5.60±0.19, but did not differ between 11

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treatments (P>0.1). Percentages of heifers with RTS 2, 3, 4 and 5 were 12.2 (1290), 15.7 (1658), 18.6 (1965) and 53.5 (5656). Percentages of heifers with BCS 3, 4, 5, 6, 7 and 8 were 1.3 (141), 5.9 (618), 31.5 (3328), 38.0 (4015), 20.6 (2181), 2.7% (286), respectively. Percentage of heifers with excitable temperament was 36.3% (n=3837; range,14.5 to 52.3%).

3.2.2 Effect of treatment on mean EER

Accounting for BCS (P<0.0001), temperament (P<0.0001), RTS (P<0.0001), and treatment by RTS (P<0.01), treatment by BCS (P<0.01) and heifer’s age by treatment (P<0.01) interactions, EER differed (P<0.0001) between CO-Synch and CO-Synch+CIDR groups (71.0 vs 75.9%, respectively). Mean estrus expression rate for heifers with RTS 2 (57.9 %) was lower (P<0.05) compared to RTS 3 (63.2%), 4 (64.7%) and 5 (83.2%). Mean EER for heifers with BCS 3 (48.2%) was lower (P<0.05) compared to heifers with BCS 4 (57.3%), 5 (70.7%), 6 (78.0), 7 (76.4%) or 8 (65.7%). Heifers that were >15 mo (76.3%) or calm (76.1%) had greater estrus expression rates (P<0.0001) compared to heifers ≤15 mo of age (68.8%) or excitable (68.4%). Differences in mean EER for RTS by treatment, BCS by treatment, age by treatment are shown (Table 5). There was no treatment by temperament interaction effect on estrus expression (P>0.1). The EER among locations varied from 56.8 to 74.6%. Across years, the EES for location varied from 54.3 to 71.7.

3.2.3 Effect of treatment on AIPR

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Accounting for BCS (P<0.0001), temperament (P<0.0001), RTS (P<0.0001), age (P<0.0001), heifers that expressed estrus or not (P<0.0001) and RTS by treatment (P<0.01), BCS by treatment (P<0.01) and age by treatment (P<0.01) interactions, AIPR differed (P<0.0001) between heifers in CO-Synch versus CO-Synch+CIDR groups (55.3% versus 61.0%, respectively). Mean AIPR for heifers with RTS 2 (54.3%) was lower (P<0.05) compared to RTS 3 (58.8%), 4 (59.0%) and 5 (58.8%; Table 4). Mean AIPR for heifers with BCS 3 (41.1%) was lower (P<0.05) compared to heifers with BCS 4 (51.0%), 5 (55.7%), 6 (62.4), 7 (58.4%) or 8 (52.4%; Table 4). Heifers >15 mo (61.3%) and calm (61.9%) heifers had greater AIPR (P<0.0001) compared to heifers ≤15 mo (53.2%) or excitable (51.2%) heifers, (Table 4). Heifers that expressed estrus (61.1%) had greater AIPR compared to heifers that did not (61.1 versus 50.4%, P<0.0001; Table 4). Differences in mean AIPR for RTS by treatment, BCS by treatment, age by treatment are shown (Table 5). There was no treatment by temperament or treatment by estrus expression interactions effects on AIPR (P>0.1). The AIPR among locations varied from 53.6 to 68.1%. Across years, the AIPR for location varied from 52.1 to 65.8, respectively.

4 Discussion

Median interval from the beginning of breeding season to cyclicity was shorter for beef heifers that received P4 supplementation compared to heifers that did not. Estrus expression rate and AIPR to a CO-Synch protocol were increased by P4 supplementation. However, RTS, BCS and age influenced effects of P4 supplementation on reproductive outcome, estrus expression and AIPR. The EER was greater for heifers with RTS 5 compared to heifers with an RTS of 2 to

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4. The AIPR was lower for heifers with RTS 2 compared to those with RTS ≥3. However, AIPR was not different among heifers with RTS 3 to 5. Observed differences in AIPR for heifers in the CO-Synch+CIDR group were +4.9 percentage points (pp) for heifers with RTS 5, +6.5 pp for heifers with RTS4 and +7.6 pp for heifers with RTS3 compared to heifers in the CO-Synch group. There was a trend for a difference in AIPR for heifers with lower RTS and given a CIDR (P=0.08). Among heifers with various RTS, utilization of similar ovulation synchronization protocols affected AIPR [13,14]. Perhaps P4 supplementation induced cyclic status in prepubertal heifers [13,14,17,24]. Peripheral P4 concentrations increase during initiation of puberty in the heifers [25]. Increased peripheral P4 is a prerequisite for physiologic estrous cycles to be initiated in heifers and to be resumed in postpartum suckled beef cows. In general, exogenous P4 increases LH pulse frequency during treatment [26] and stimulates LH secretion, particularly after P4 is removed [26-28]. Increased P4 decreases and/or downregulates estrogen receptors, thereby negating estradiol’s negative feedback on GnRH secretion [27]. Similar effects of P4 were reported in heifers with RTS 2 [14,26-28]. In a previous study, we reported estrus synchronization strategies to improve AIPR in beef heifers [14]. In that study, heifers were assigned to various estrus synchronization treatments and inseminated at observed estrus. The AIPR for heifers with RTS 5 inseminated at 12 h after estrus following two luteolytic doses of PGF2α (Days 0 and 14) was lower compared to AIPR for heifers with RTS 2 to 4 inseminated at 12 h after estrus following CIDR-PGF2α protocol (CIDR for 7 d and PGF2α at CIDR removal). Greater AIPR for heifers in CIDR-PGF2α group was plausibly due to the induction of cyclist in pre- and peri-pubertal heifers, explained by greater number of heifers that expressed estrus in

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that group compared to heifers in the double PGF2α group (85.1 vs 80.4%, P=0.02). This evidence illustrated that exogenous P4 induced cyclicity in pre- and peri-pubertal heifers. In a 3-y study in Florida [18], heifers were allocated to low (0.45 kg/d), medium (0.73 kg/d), or high (1.00 kg/d) growth rate diets, with or without a puberty induction protocol (CIDR inserted on d 65, removed on d 72, 100 µg GnRH on d 74, and 25 mg PGF on d 84). The Percentage of pubertal heifers was greater for medium or high versus low growth rate diets (P≤0.05), with higher puberty attainment in heifers that received puberty induction protocol (P<0.01). Although our study did not directly assess growth rates, body condition was considered as a factor and its effects on EER and AIPR were determined. Body condition scoring is a practical method for assessing energy reserves. Among heifers with good body condition (BCS 6 and 7), AIPR was greater for heifers in the CO-Synch+CIDR group compared to heifers in the CO-Synch group, with no interaction of BCS and pubertal status in the current study. In the current study, addition of CIDR resulted in 6.6 pp greater AIPR [53.3% (732/1374) for the CO-Synch v. 59.9% (942/1572) for the CO-Synch+CIDR; P<0.001] for heifers with RTS 2 and 3. Similarly, addition of CIDR resulted in 5.3 pp more in AIPR [56.1% (2089/3725) for the CO-Synch vs 61.4% (2363/3896) for the CO-Synch+CIDR; P<0.0001] for peripubertal and pubertal heifers combined. There are variable results in terms of pregnancy success in prepubertal and pubertal beef heifers following synchronization with exogenous P4. Wood-Follis et al. (2004) reported ERR, conception rate and final pregnancy rate in prepubertal and pubertal heifers following synchronization with an MGA-based protocol [1]. There was no difference between prepubertal and pubertal heifers in estrous response, conception and final pregnancy rates, 80% (56/70) and 94% (51/54), 80% (45/56) and 78% (40/51), and 97% (68/70) and 93% (50/54), respectively. In contrast, Bridges et al. (2014) used 5-d CO-Synch+CIDR, PG-6d CIDR

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and 14-d CIDR-PG protocol to synchronize beef heifers for FTAI [29]. They concluded that heifers reached puberty by initiation of synchronization had a significantly greater AIPR compared to pubertal heifers (60.7 vs 47.3%, respectively). Several studies reported greater AIPR in beef cattle expressing estrus before FTAI compared to those that did not [14,30,31]. Heifers that expressed estrus before FTAI may have had greater serum estradiol concentrations. Progestins increase ovarian follicular growth and estrogen production of by ovarian follicles [32-35]. This increased estrogen production after exogenous P4 is necessary to prepare follicular cells for luteinization and induction of a sufficient number of uterine progesterone receptors and preparation of uterus for establishment and maintenance of pregnancy [36]. Associations among plasma estradiol concentrations, ovulation, increased AIPR, and decreased pregnancy losses in beef and dairy cattle have been reported [37-39]. Increased proestrus phase estradiol initiates crucial modifications in the endometrium and oviduct [39,40]. Furthermore, estradiol concentrations during proestrus were positively correlated with: preovulatory follicle diameter; CL diameter; P4 concentration during diestrus [41]; and conception rates [42,43]. Expression of estrus promoted changes in the preimplantation endometrium, CL, and conceptus gene expression. Critical cellular pathways related to suppression of maternal immune system, attachment between conceptus and the endometrium, and CL maintenance during pregnancy were favorably active in females that expressed estrus near AI. Moreover, females with phenotypic expression of estrus had longer conceptuses, which can promote survival. These events are only possible with appropriate P4 concentrations in the previous cycle. Thus, P4 supplementation is crucial for induction of cyclicity in prepubertal heifers and is essential for pregnancy establishment and maintenance.

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Among heifers that expressed estrus, addition of CIDR did not improve AIPR for heifers with RTS 5 and 4 (P>0.1); however, addition of CIDR improved AIPR for heifers with RTS 2 and 3 (P<0.001) in the present study. Perhaps among heifers that did not receive CIDR, presence of a CL in heifers with RTS 5 and induced CL after first GnRH injection in heifers with RTS 4 (>10 mm follicle) may have contributed to comparable AIPR. It should be noted that among heifers that received CIDR, EER for heifers with RTS 4 and 5 were greater compared to those with RTS 2 and 3 in the current study. Among heifers that did not receive CIDR, EER were greater for heifers with RTS 5 compared to heifers with RTS 2 to 4. In the present study, addition of CIDR significantly improved EER in heifers with BCS 5, with a trend for improved EER for heifers with BCS 4 and 6. Similarly, addition of CIDR significantly improved AIPR in heifers with BCS 6 and 7, with a trend for improved AIPR in heifers with BCS 3. Wichtel et al. (2008) specifically examined the interaction between synchronization protocol (GnRH or CIDR based) and postpartum energy intake (Low, medium or high energy intake) on the reproductive performance of postpartum beef cattle in a FTAI program [44]. Overall, results showed that the CIDR protocol resulted in higher TAI pregnancy rate than the OVS protocol. Reducing postpartum ME intake had little effect on reproductive performance irrespective of the TAI protocol used. The authors concluded that good body condition at calving may have overcome the potential negative effect of reduced dietary intake on TAI pregnancy rate. In general, BCS categories are designated as: <5 thin; 5 to 7, moderate to good; and >7, obese. According to this categorization, there were no difference in AIPR between treatments for thin and obese classifications (P<0.1), whereas a difference in AIPR between synchronization protocols was observed for heifers with moderate to good BCS [44-46]. Gutierrez et al. (2014) reported that pregnancy was significantly affected by BCS in beef heifers

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assigned to AI and natural service or heifers assigned to natural service only [13]. Additionally, BCS at the beginning of the breeding season and BCS loss from calving to breeding affected reproductive outcomes in beef females [44,47,48]. Conversely, BCS was not associated with reproductive outcomes in dairy [16] or in beef heifers [49]. It is well known that heifers with moderate to good body condition attain puberty earlier than lighter herd-mates. It is plausible that higher percentage of heifers with lower BCS (< 5) may have had inadequate nutrient intake needed to attain puberty earlier. Excitable temperament in beef cattle is linked to poor fertility through nutritional inadequacies or stress-related responses [50-55]. Lower pregnancy rates and calving rates in beef cattle with excitable temperament compared to those with calm temperament has been reported [51-55], and these outcomes were consistent with the current study. Endocrine milieu in excitable heifers may be altered, reducing synchronization and fertilization success (including changes in ovarian follicular dynamics and follicle size) and embryonic death (reduced peripheral P4 due to small CLs) [55]. Although recommended interval from PGF2a administration to AI in beef heifers is 60±4 h, in the current study, AI was performed 72 h after PGF2α, consistent with other studies [4,5,56,57]. Peak estrous response based on a HeatWatch® Estrus Detection System was 48 to 60 h after the PGF2α treatment and optimal time to AI beef heifers is 4 to 20 h after detection of estrus [58]. Thus, acceptable AIPR can be achieved if the beef heifers are inseminated between 52 and 80 h after PGF2α. In Experiment 1, 4 days was allocated as difference in initiation of cyclicity for P4 concentrations between 1 and 2 ng/mL and >2 ng/mL. In several studies, blood P4 concentrations in heifers and non-lactating cows reached 2 and 5 ng/mL at 4 and 7 d,

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respectively, after ovulation [20-22]. It should be noted that puberty and initiation of normal estrous cycles are multifaceted events that are regulated by the hypothalamic-pituitary-ovarian axis and that age at puberty in heifers can be influenced by genetics, nutrition and body weight, and hormonal intervention. A systematic understanding of the metabolic and neuroendocrine changes that occur to initiate puberty and normal estrous cycle is needed to facilitate management of reproductive events. Development phase of CL, first 7 d after ovulation, is characterized as the interval from differentiation of the corpus hemorrhagicum to fully differentiated CL with maximum P4 secretory capacity. Regarding effect of synchronization protocol on EER and AIPR, there were differences across locations and years. There were differences among locations (P<0.05) for EER (36.8 to 74.6%) and AIPR 32.6 to 68.1%). Similarly, there were differences (P<0.05) among years for EER (59.2 to 76.2) and AIPR (52.1 to 59.3). Kasimanickam et al., (2015) reported differences in AIPR among locations from two experiments in beef heifers [5]. For example in Experiment 1 in that study, there was increased AIPR for 5-d CO-Synch+CIDR (AI at 56 h after PGF2α administration) protocol in five locations and increased AIPR for 7-d CO-Synch+CIDR (AI at 72 h after PGF2α administration) protocol in four locations. Similarly in Experiment 2, increased AIPR for the 5-d CO-Synch+CIDR (AI at 72 h after PGF2α administration) protocol in two locations and an increased AIPR for the 7-d CO-Synch+CIDR (AI at 72 h after PGF2α administration) protocol in two locations. Variability in EER and AIPR, noted in the current study, may not have been caused only by synchronization treatment per se, but there were likely other factors inherent in the AI programs in which large numbers of cattle are inseminated in a short interval [59]. When selecting a program to synchronize estrus and ovulation in cattle, however, attention should be focused on its monetary advantage compared to natural service and

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its benefit compared to other synchronization protocols in an individual herd. Utilization of protocols to synchronize estrus and ovulation as compared natural service in beef heifers provides for an advantage of enhanced pregnancy rates [60] and lifetime revenue [61]. It should be noted that it is reasonable to expect that random effects on economic benefit may vary depending on several factors, including herd size [62]. Although heifers with RTS 4 were considered pubertal in previous studies [15,16], they became pregnant 16 d later than heifers with RTS 5 (53 vs 37 d, respectively) [13]. The AI-PR was significantly different for RTS 3, 4 and 5 (48.3, 57.6 and 64.6%, respectively) [13]. In another study, AIPR for heifers with RTS <3, 3 4 and 5 were 50.4, 54.7, 58.7 and 63.1%, respectively (P<0.05) [63]. Holms et al., observed 10 percentage point difference in AIPR between RTS 4 and 5. The study observed no difference plausibly due to lower sample size [64]. Holms et al. (2016) investigated ultrasonographic reproductive tract measures as predictors of pregnancy failure and anestrus in beef heifers. The results showed that the heifers that did not have a CL and also had a largest follicle diameter less than 13 mm were at risk of being too far from puberty [65]. In addition, absence of CL predicted both anestrus and pregnancy failure for the 50-day breeding season. Events that led to puberty were declines in negative feedback of estrogen, LH surge and ovulation [49]. That these events may have not occurred or were delayed in heifers with RTS 4. Further it is plausible that Thus, it is sensible to categorize only heifers with CL as pubertal. It is arguable that the classification of heifers RTS by ultrasonography is accurate over transrectal palpation. It should be noted that the accuracy of RTS by palpation can be improved by applying more emphasis on the presence of a CL, the size of the largest follicle, and the diameter of the uterus horn as performed in the current study. Holms et al (2016) claimed transrectal ultrasonography tended to provide better prognostic

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models for reproductive failure than the current palpation model of Andersen et al [65]. However, Rosenkrans and Hardins (2003) observed that the sensitivity and specificity of palpation per rectum for diagnosis of pubertal status compared to serum progesterone levels were higher (82 and 69%, respectively) than sensitivity and specificity of ultrasonography (79 and 59%, respectively) [66]. Further, the study concluded palpation per rectum was similar to ultrasonography for classifying pubertal status in beef heifers. It should be noted that 18 and 21% (false negative rate) of the heifers palpated and ultrasounded, respectively, will be called prepubertal when they are actually pubertal. Clinicians should be aware that both ultrasonography and transrectal palpation methods may result in some misclassification but both methods are equally good in classifying heifer’s pubertal status.

4.1 Conclusion

In conclusion, exogenous progesterone hastened cyclicity in beef heifers with RTS 2 to 4 and increased EER and AIPR. However, RTS, BCS and age influenced EER and AIPR. Among RTS 4 and 5, EER was greater for CO-Synch+CIDR vs CO-Synch. Among RTS 3 to 5, AIPR was greater for CO-Synch+CIDR versus CO-Synch. Clinicians should be aware that the methodology employed to classify heifer’s pubertal status based on RTS may have some misclassification. We concluded that cyclic (progesterone) status of heifers or progesterone supplementation to acyclic heifers at commencement of synchronization protocols was critical to pregnancy outcomes, emphasizing heifer development for early puberty or P4 supplementation.

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Acknowledgment

Authors thank beef cattle producers for their participation in the study and College of Veterinary Medicine, Washington State University, Pullman, WA, Zoetis Animal Health New York, NY and Merial Inc., Duluth, GA for supporting this study. The authors also extend their gratitude to Dr. John Kastelic for editing and providing valuable suggestions to the manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

Author contributions

RK conceptualized the study; RK, VK, JO and MW collected and organized the data; RK and VK analyzed and interpreted the data; RK, VK, JO, and MW drafted the manuscript; RK and VK critically revised the manuscript.

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References

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[59] Wishart DF, Young IM, Drew SB. A comparison between the pregnancy rates of heifers inseminated once or twice after progestin treatment. Vet Rec 1977;101:230-231. [60]Rodgers JC, Bird SL, Larson JE, Dilorenzo N, Dahlen CR, Dicostanzo A, et al. An economic evaluation of estrous synchronization and timed artificial insemination in suckled beef cows. Anim Sci 2012;90:4055-4062. [61] Fench J. Evaluation of pregnancy rates following timed AI in beef heifers after synchronization of follicular waves using 14-d controlled internal drug release insert, and the lifetime productivity of beef heifers conceiving to, or sired by, AI. MS thesis. Colorado, USA: Colorado State University; 2012. p. 73-88. [62]Whittier WD, Currin JF, Schramm H, Holland S, Kasimanickam RK. Fertility in Angus cross beef cows following 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR estrus synchronization and timed artificial insemination. Theriogenology 2013;80:963-969. [63] Kasimanickam RK, Hall JB, Whittier WD. Fertility of Angus cross beef heifers after GnRH treatment on day 23 and timing of insemination in 14-day CIDR protocol. Reprod Dome Anim 2017;52:122-159. [64] Holm DE, Thompson PN, Irons PC. The value of reproductive tract scoring as a predictor of fertility and production outcomes in beef heifers. 2009;87:1934-1940. [65] Holm DE, Nielen M, Jorritsma R, Irons PC, Thompson PN. Ultrasonographic reproductive tract measures and pelvis measures as predictors of pregnancy failure and anestrus in restricted bred beef heifers. Theriogenology. 2016;85:495–501. [66] Rosenkrans KS, Hardin DK. Repeatability and accuracy of reproductive tract scoring to determine pubertal status in beef heifers. Theriogenology 2003;59:1087-1092.

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Table 1. Description of reproductive tract scores in beef heifers, based on uterine and ovarian characteristics* Uterus diameter (mm) and tone

Ovarian structures

Score

< 20, no tone

none palpable

1

20 to 25, no tone

8 mm follicles

2

20 to 25, slight tone

8 - 10 mm follicles

3

26 to 30, slight tone

>10 mm follicles, no corpus

4

luteum >30, no tone

>10 mm follicles, corpus luteum

5

Adapted from Anderson et al. 1991 [15]; *Assessed by transrectal palpation;

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1

Table 2. Mean (±SEM) characteristics, based on reproductive tract score (RTS), for beef heifers in Experiment 1. End point

Control 1RTS

N Age (mo) Body weight (kg) Body condition score2

2

1

3

2Body

1

CIDR

RTS 2

RTS 3

RTS 4

RTS 5

RTS 1

RTS 2

RTS 3

RTS 4

RTS 5

10

10

10

10

10

10

10

10

10

10

15.3±0.12

15.1±0.12

15.4±0.18

15.9±0.29

15.7±0.16

15.3±0.19

15.5±0.17

15.4±0.19

16.3±0.22

16.0 ± 0.17

320±10

323±7

324±8

319±5

325±9

320±10

322±5

317±9

326±5

324±7

5.19±0.26

5.21±0.16

5.38±0.19

5.56±0.22

5.27±0.09

5.12±0.24

5.22±0.17

5.26±0.13

5.17±0.17

5.24±0.15

See Table 1 for description of RTS; condition score (1 to 9; 1, emaciated; 9, obese);

4

32

5

Table 3. Mean (±SEM) characteristics, based on reproductive tract score (RTS)1 for beef heifers in Experiment 22. Parameter

CO-Synch 2RTS

N Age (mo)

2

CO-Synch+CIDR

RTS 3

RTS 4

RTS 5

RTS 2

RTS 3

RTS 4

RTS 5

634

740

915

2810

656

918

1050

2846

15.1±0.12

15.4±0.18

15.9±0.29

16.1±0.16

15.5±0.17

15.4±0.19

16.3±0.22

16.0 ± 0.17

Interval from PGF2α to AI (h) 72.11±0.29 71.92±0.37 72.89±0.17 71.86±0.19 72.21±0.14 72.16±0.16 71.85±0.26 72.21±0.18 Body condition score3

5.47±0.21

5.41±0.28

6

1See

7

2Refer

to Fig. 1 for synchronization protocols;

8

3Body

condition score (1 to 9; 1, emaciated; 9, obese);

5.60±0.19

5.21±0.21

5.29±0.22

5.37±0.17

5.11±0.21

5.17±0.21

Table 1 for description of RTS;

9

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10

Table 4. Effects of explanatory variables influencing estrus expression rate (EER) in Angus-

11

cross beef heifers (n=10569). Variables

Categories

No.

Mean EER (%)

P

Synchronization protocol1

CO-Synch

5099

71.0

Ref

CO-Synch+CIDR 5470

75.9

0.0001

Reproductive tract score2

Body condition score3

Age of heifers4

Temperament5

2

1290

57.9

Ref

3

1658

63.2

0.003

4

1965

64.7

0.0001

5

5656

83.2

0.0001

3

141

48.2

Ref

4

618

57.3

0.05

5

3328

70.7

0.0001

6

4015

78.0

0.0001

7

2181

76.4

0.0001

8

286

65.7

0.01

≤15

3973

68.8

Ref

>15

6596

76.3

0.0001

Calm

6762

76.1

Ref

Excitable

3837

68.4

0.0001

12

Interaction effects: Synchronization treatment by RTS - Degrees of freedom (df), 3; F value,

13

6.14, P<0.01; Synchronization treatment by BCS - df, 5; F value, 5.98, P<0.01; Synchronization

14

treatment by age of heifers - df, 1; F value, 5.16, P<0.01;

15

1Refer

16

2Reproductive

17

3Body

18

4Age

to Fig. 1 for protocols; tract scores (1 to 5; 1, acyclic, immature; 5, cyclic, mature) ranged from 2 to 5;

condition scores (1 to 9; 1, emaciated; 9, obese) ranged from 3 to 8;

(mo) ≤15 and >15;

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19

5Temperament

20

trot or run;

21

Covariance parameter estimates: Location (year), 0.03052±0.00916; Residual 0.1933±0.009135;

22

P=0.04; Fit statistics - BIC =1231.24; -2 Res log likelihood =1227.34;

score (0 or 1) - 0, calm, slow chute exit; walk; 1, excited, fast chute exit; jump,

23

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Table 5. Effects of explanatory variables influencing artificial insemination pregnancy rate

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(AIPR) in Angus-cross beef heifers (n=10569) Variables Synchronization protocol1 Reproductive tract score2

Body condition score3

Age4

Temperament5

Estrus expression6

No.

Mean AIPR (%)

P

CO-Synch

5099

55.3

Ref

CO-Synch+CIDR

5470

65.0

0.0001

2

1290

54.3

Ref

3

1658

58.8

0.02

4

1965

59.0

0.01

5

5656

58.8

0.004

3

141

41.1

Ref

4

618

51.0

0.04

5

3328

55.7

0.001

6

4015

62.4

0.0001

7

2181

58.4

0.0001

8

286

52.4

0.03

≤15

3973

53.2

-

>15

6596

61.3

0.0001

Calm

6762

61.9

-

Excitable

3837

51.2

0.0001

Yes

7769

61.1

-

No

2800

50.4

0.0001

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Interaction effects: Synchronization treatment by RTS - Degrees of freedom (df), 3; F value,

27

6.06, P<0.01; Synchronization treatment by BCS - df, 5; F value, 6.21, P<0.01; Synchronization

28

treatment by age of heifers - df, 1; F value, 5.74, P<0.01;

29

1Refer

30

2Reproductive

31

3Body

to Fig. 1 for protocols; tract scores (1 to 5; 1, acyclic, immature; 5, cyclic, mature) ranged from 2 to 5;

condition scores (1 to 9; 1, emaciated; 9, obese) ranged from 3 to 8; 36

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4Age

33

5Temperament

34

trot or run;

35

6Heifers

36

PGF2α administration to time of AI. A heifer was determined to be in estrus if the heifer was

37

visually observed to stand for mounting by other herd mates or if the heifer had an activated

38

(>90% of grey patch was red colored), lost (with mount marks) or partially-activated (50 to 90%

39

of grey patch was red colored) heat detection aids;

40

Covariance parameter estimates: Location (year), 0.02279±0.01145; A.I. Sire (Location),

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0.078925±0.03448; Inseminator (Location), 0.02319±0.04735; Animal handlers (Location),

42

0.04356±0.02528; Residual 0.2067±0.012372; P=0.16; Fit statistics - BIC =1778.18; -2 Res log

43

likelihood =1771.46;

(mo) ≤15 and >15; score (0 or 1) - 0, calm, slow chute exit; walk; 1, excited, fast chute exit; jump,

were observed twice daily for standing estrus and estrus detection aid status from

44

37

45

Table 6. Effects of treatment1 and reproductive tract score (RTS)2, body condition score (BCS)3, and age of heifers influencing mean

46

estrus expression rate (EER) and mean artificial insemination pregnancy rate (AIPR) in Angus cross beef heifers (n=10569).

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Mean EER (%) Mean AIPR (%) CO-Synch CO-Synch+CIDR CO-Synch CO-Synch+CIDR Reproductive Tract Score 5 80.9 (2272/2810)a,1 85.4 (2431/2846)a,2 56.3 (1581/2810)a,1 61.2 (1742/2846)a,2 4 58.5 (533/915)bc,1 70.3 (738/1050)a,2 55.5 (508/915)ab,1 62.0 (651/1050)a,2 3 62.1 (459/740)b,1 64.2 (589/918)b,1 54.5 (403/740)ab,1 62.1(570/918)a,2 c,1 b,1 b,1 2 56.2 (356/634) 59.6 (391/656) 51.9 (329/634) 56.7 (372/656)b,1 Body Condition Score 3 41.8 (28/67)x,1 54.2 (40/74)w,1 32.8 (22/67)x,1 48.6 (36/74)x,1 4 53.8 (169/314)x,1 60.9 (185/304)wx,1 49.7 (156/314)y,1 52.3 (159/304)x,1 y,1 y,2 y,1 5 66.2 (1064/1607) 74.9 (1289/1721) 54.6 (878/1607) 56.7 (975/1721)z,1 6 76.7 (1474/1922)z,1 79.1 (1656/2093)z,1 58.7 (1128/1922)z,1 65.9 (1379/2093)z,2 7 76.1 (798/1049)z,1 76.7 (868/1132)z,1 53.9 (565/1049)y,1 62.5 (708/1132)y,2 8 62.1 (87/140)y,1 69.2 (101/146)xy,1 51.4 (72/140)y,1 53.4 (78/146)x,1 Age of heifers (mo.) ≤15 68.4 (1282/1874)a,1 69.2 (1453/2099)a,1 53.4 (1001/1874)a,1 53.0 (1112/2099)a,1 >15 72.5 (2338/3225)b,1 80.0 (2696/3371)b,2 56.4 (1820/3225)b,1 65.9 (2223/3371)b,2 Reproductive tract score: a-cWithin a treatment, means without a common superscript differed (P<0.05); 1,2Between treatments,

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means without a common superscript differed (P<0.05);

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Body Condition score: w-zWithin a treatment, means without a common superscript differed (P<0.05); 1,2Between treatments, means

50

without a common superscript differed (P<0.05);

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Age of heifers: a,bWithin a treatment, means without a common superscript differed (P<0.05); 1,2Between treatments, means without a

52

common superscript differed (P<0.05);

Variables

38

53

1See

54

2Reproductive

55

3Body

Fig. 1 for synchronization protocols; tract score (1 to 5; 1, immature, acyclic; 5, mature, cyclic; see Table 1);

condition score (1 to 9; 1, emaciated; 9, obese);

56

39

57 58

Fig.1. Schematic presentation of ovulation synchronization protocol

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On Day 0, Angus cross heifers (n=10569) were assigned to either CO-Synch (n=5099) or CO-Synch+CIDR (n=5470)

60

protocols. Heifers were assigned a reproductive tract score (1 to 5; 1, immature, acyclic; 5, mature, cyclic), a body condition score (1

61

to 9; 1, emaciated; 9, obese), and a temperament score (0, calm, slow chute exit; walk, 1, excitable, fast chute exit; jump, trot or run)

62

when synchronization treatments were initiated. Briefly, on Day 0, all heifers in both groups were given 100 µg gonadorelin diacetate 40

63

tetrahydrate (GnRH; 2 mL, im). In addition, heifers in CO-Synch+CIDR group concomitantly received a CIDR (1.38 g progesterone).

64

Seven days later, CIDRs were removed from heifers in CO-Synch+CIDR and 25 mg of dinoprost (PGF2α; 5 mL, im) was given to all

65

heifers. Heifers in both treatments were inseminated 72 h after CIDR removal, with a second dose of GnRH administered

66

concomitantly. On day 7 (CIDR removal), all heifers were fitted with estrus detection aids and were observed for estrus twice daily for

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standing estrus and estrus detection aid status from CIDR removal to insemination. Two weeks after AI, heifers were exposed to bulls

68

(total breeding season of 63 to 85 d). Heifers were examined for pregnancy status ~70 d after AI by transrectal ultrasonography.

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Figure 2. Survival analysis for median interval between beginning of the study and cyclicity

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Median days - Chance of attaining cyclicity until that time was 50%;

72

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Median days to cyclicity

80 b b

60 b

a

a

40 b 20 0

a 0

0

RTS 5

a

a

a 0

21

RTS 4

13.5

37.5 52.5

RTS 3

CIDR treated

73

39

RTS 2

44.5

60

RTS 1

Control

74

Figure 3. Median days from start of study to cyclicity in beef heifers with various reproductive

75

tract scores.

76

Median days - Chance of attaining cyclicity until that time was 50%;

77

ab, bars without a common superscript differed (P<0.05);

78

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Author contributions

RK and VK conceptualized the study; RK, VK, JO and MW collected and organized the data; RK and VK analyzed and interpreted the data; RK, VK, JO, and MW drafted the manuscript; RK and VK critically revised the manuscript.

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Highlights 

Progesterone (P) advanced cyclicity in beef heifers with reproductive tract score (RTS) 2 to 4



CO-Synch+P improved estrus expression in heifers with RTS 4 and 5, but not for 2 and 3 compared with CO-Synch alone



CO-Synch+P protocol improved pregnancies in heifers with RTS 3, 4 and 5, but not for 1 compared with CO-Synch alone