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Reproductive performance of prepubertal Bos indicus heifers after progesterone-based treatments
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Theriogenology 74 (2010) 903–911 www.theriojournal.com
Reproductive performance of prepubertal Bos indicus heifers after progesterone-based treatments I. Claro Júniora, O.G. Sá Filhoa, R.F.G. Peresa, F.H.S. Aonoa, M.L. Dayb, J.L.M. Vasconcelosa,* a
Departamento de Produção Animal, Faculdade de Medicina Veterinária e Zootecnia-UNESP, Botucatu-SP 18618-000, Brazil b Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA Received 5 January 2010; received in revised form 23 February 2010; accepted 9 April 2010
Abstract The objective was to evaluate the effects of exogenous progesterone (P4) on reproductive performance of prepubertal Bos indicus heifers. Prepubertal Nelore heifers (n ⫽ 589; 24.0 ⫾ 1.13 mo; 298.0 ⫾ 1.89 kg; body condition score of 3.2 ⫾ 0.26; mean ⫾ SEM) were randomly assigned to receive, between experimental Days ⫺12 and 0: no treatments (CIDR0; n ⫽ 113); a new intravaginal insert (CIDR) containing 1.9 g of P4 (CIDR1; n ⫽ 237); or a similar insert previously used three times, with each use occurring for 9 d (CIDR4; n ⫽ 239). An additional treatment group was pubertal heifers given 12.5 mg dinoprost tromethamine im on Day 0 (PGF; n ⫽ 346), and used as controls for evaluation of conception rates. On Day 0, transrectal palpation was done for uterine score evaluation (UtS; 1–3 scale), blood samples were taken for serum P4 concentrations, and follicle diameter (FD) was measured. The breeding season started on Day 1 and consisted of AI after detection of estrus between Days 1 and 45, and exposure to bulls between Days 46 and 90. There were effects of treatment (P ⬍ 0.05) on serum concentrations of P4 on Day 0 (0.37 ⫾ 0.16, 2.31 ⫾ 0.11, and 1.20 ⫾ 0.11 ng/mL for CIDR0, CIDR1, and CIDR4, respectively; mean ⫾ SEM), FD on Day 0 (9.45 ⫾ 0.24, 9.72 ⫾ 0.17, and 11.42 ⫾ 0.16 mm), UtS on Day 0 (1.49 ⫾ 0.06, 1.88 ⫾ 0.04, and 2.24 ⫾ 0.04), estrus detection rates at 7 d (19.5, 42.6, and 38.3%) and 45 d (52.2, 72.1, and 75.3%) of the breeding season, and on pregnancy rates at 7 d (5.3, 14.3, and 18.4%), 45 d (27.4, 39.2, and 47.7%) and 90 d (72.6, 83.5, and 83.7%) of the breeding season. Conception rate 7 d after the start of the breeding season was greater (P ⬍ 0.05) in heifers from the CIDR4 (46.8%) and PGF (43.8%) groups than in the CIDR0 (27.3%) and CIDR1 (33.7%) groups. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. Furthermore, previously used CIDR inserts were better than new inserts. © 2010 Elsevier Inc. All rights reserved. Keywords: Puberty; Progesterone; Pregnancy rates; Bos indicus; Zebu cattle
1. Introduction Compared to Bos taurus, Bos indicus heifers reach puberty older and at a higher percentage of body weight relative to mature body weight [1], resulting in a lower proportion of pubertal heifers at the beginning of breed* Corresponding Author. Tel.: ⫹55 14 3811 7185; fax: ⫹55 14 3811 7180. E-mail address: [email protected] (J.L.M. Vasconcelos). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.04.015
ing season and lesser pregnancy rates [2,3]. These characteristics contribute to reduced profitability of cowcalf enterprises using zebu cattle [4]. The key factor responsible for establishment of puberty is an increase in LH release from the anterior pituitary, in response to GnRH from the hypothalamus [5]. Day et al. [6] suggested that exposure to progesterone (P4) or other progestins reduced the hypothalamic expression of estradiol receptors, and thereby decreased the negative feedback action of estradiol on
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Fig. 1. Schematic diagram of experimental design. Between Days ⫺12 and 0, prepubertal Nelore heifers received either no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of progesterone (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used 3x (CIDR4; n ⫽ 239). An additional group of pubertal heifers (not shown in the figure; PGF; n ⫽ 346) received PGF2␣ on Day 0, and were used as controls for evaluation of conception rates. US: ultrasonographic examination (ovaries on Days ⫺19, ⫺12 and 0 and diagnosis of pregnancy on Days 75 and 120); BS: blood sampling; UtS: rectal palpation for uterine score evaluation (UtS-1: uterine horn diameter ⬍20 mm and weak uterine tone; UtS-2: uterine horn diameter ⬎20 mm and weak uterine tone; UtS-3: uterine horn diameter ⬎20 mm and strong uterine tone); BW: body weight; BCS: body condition score, on a scale from 1 (emaciated) to 5 (obese).
This study was conducted at a commercial ranch in Para, Brazil. Animals were cared for in accordance with the practices outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [16].
⫺19 of the experiment, were used. They were grazing one of six Brachiaria brizantha pastures, with ad libitum access to water and mineral supplement. Ultrasonographic examination of ovaries (Aloka SSD-500 with a 7.5 MHz linear-array transrectal transducer; Aloka, Tokyo, Japan) was performed on Days ⫺19 and ⫺12 to detect luteal tissue. Heifers with no luteal tissue detected at both examinations were considered prepubertal (n ⫽ 589) and randomly assigned to receive, within each pasture, one of the following treatments (Fig. 1): CIDR0 — heifers received no treatment (n ⫽ 113); CIDR1— between Days ⫺12 and 0, heifers received a new intravaginal insert containing 1.9 g of P4 (CIDR®—Controlled Intravaginal Drug Release, Pfizer Animal Health, São Paulo, SP, Brazil; n ⫽ 237); and CIDR4 — between Days ⫺12 and 0, heifers received a similar intravaginal insert that had previously been used three times, with each use occurring for 9 d (n ⫽ 239). Pubertal heifers received 12.5 mg of dinoprost tromethamine im (2.5 mL of Lutalyse®, Pfizer Animal Health) on Day 0, and were used exclusively as controls for evaluation of conception rates (PGF; n ⫽ 346). All heifers were observed from 0700 to 0800 h, and from 1700 to 1800 h between Days 1 and 45. Heifers detected in estrus were inseminated 10 to 14 h after detection of estrus, with AI sires (n ⫽ 9) and technicians (n ⫽ 12) randomly assigned to heifers. During the first 45 d of the breeding season, heifers that returned to estrus after the first AI were re-inseminated as described and, between Days 46 and 90, heifers were exposed to sires for natural service (one sire per 15 heifers).
2.1. Animals and treatments
2.2. Uterine score evaluation
Nelore heifers (n ⫽ 935) that were 22–26 mo of age, 280 –330 kg body weight, and had body condition score of 3.2 ⫾ 0.26 (1 ⫽ emaciated to 5 ⫽ obese [17]) on Day
On Days ⫺19, ⫺12, and 0, reproductive tracts were evaluated by transrectal palpation and uterine scores (UtS) were assigned as follows: uterine horn diameter
GnRH secretion. In that regard, in several studies with prepubertal Bos taurus heifers, exogenous progestins increased LH secretion and induced puberty [7–10]. Furthermore, exogenous P4 in non-cycling ruminants prior to induction of ovulation minimized premature luteolysis [11], which improved the likelihood of conception [12]. However, treatment with an intravaginal insert containing 1.9 g of P4 increased peripheral P4 concentrations and reduced ovarian follicular growth in Bos indicus relative to Bos taurus or crossbred heifers [13]; therefore, P4 released by the inserts may exceed subluteal concentrations and decrease the frequency of LH pulses in Bos indicus heifers. In fact, strategies to reduce peripheral P4 concentrations during P4-based synchronization protocols, such as advancing the time of prostaglandin F2␣ (PGF2␣) treatment, previously used P4 inserts, or both, improved fertility in cycling Bos indicus heifers [13–15]. The objective of this study was to evaluate the effects of treatments with exogenous P4 on rates of induced estrus, conception, and pregnancy in prepubertal Bos indicus heifers. 2. Material and methods
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⬍20 mm and weak uterine tone (UtS ⫽ 1); uterine horn diameter ⬎20 mm and weak uterine tone (UtS ⫽ 2); and uterine horn diameter ⬎20 mm and strong uterine tone (UtS ⫽ 3). All uterine tract scores, as well as body condition scores, were done by the same veterinarian. 2.3. Ultrasonography of ovarian structures and pregnancy Ovarian ultrasonography was performed on Day 0, as described above, to detect luteal tissue and to determine the diameter of the largest ovarian follicle. Diameter was calculated as the average distance for two measurements made at perpendicular angles. Heifers previously considered prepubertal that had luteal tissue on Day 0 were excluded from the study. Uterine ultrasonographic examinations were performed on Days 75 and 120 for diagnosis of pregnancy. 2.4. Blood sampling and steroid assays Blood samples were collected on Days ⫺19, ⫺12, and 0 (immediately before CIDR withdrawal) from the coccygeal vein into Vacutainer tubes (Becton Dickinson Co., Franklin Lakes, NJ, USA) for analysis of serum P4 concentrations. Blood was allowed to clot at 4 oC for 24 h and centrifuged at 1500 ⫻ g for 10 min at room temperature. Serum was removed and stored frozen (⫺20 oC) until hormonal assays were performed. Samples collected on Days ⫺19 and ⫺12 were used for the evaluation of cyclicity (in association with previous ultrasonographic findings), whereas samples collected on Day 0 were used for evaluation of the effects of treatments on progesterone concentrations of prepubertal heifers. Since some heifers with no luteal tissue detected had serum P4 concentrations ⬎1 ng/mL on Days ⫺19 and/or ⫺12, samples from those days were analyzed to determine serum cortisol concentrations, since stress may increase P4 secretion from nonluteal tissues [18,19]. Serum concentrations of P4 and cortisol were determined using a solid-phase radioimmunoassay kit containing antibody-coated tubes and 125I-labeled P4 or cortisol (Coat-a-Count; Diagnostic Products Corporation, Los Angeles, CA, USA) according to the manufacturer’s instructions. For P4 assays, the interassay CV was 5.6%, and average intra-assay CV was 5.4%; for the cortisol assay, the intra-assay CV was 1.3%. Assay sensitivities were 0.1 ng/mL for P4 and 5 ng/mL for cortisol. 2.5. Statistical analyses This experiment was analyzed as a completely randomized design. Since the main objective was to evaluate the effects of treatments with CIDR® on prepu-
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bertal heifers and fertility at first pubertal estrus was low in heifers [20], pubertal heifers (treatment PGF) were included as controls for evaluation of conception rate only, but were excluded from any other analysis. For prepubertal heifers (treatments CIDR0, CIDR1, and CIDR4), initial comparisons were performed, using PROC ANOVA and PROC GENMOD of SAS (SAS Institute Inc., Cary, NC, USA), to determine that the distribution of heifers by age, BCS, body weight, and UtS were similar among treatments. Binomially distributed data were analyzed using PROC LOGISTIC of SAS. Explanatory variables such as treatment, UtS, sire (semen), and AI technician were used in the model as classes. Explanatory variables considered covariates such as BCS, age, and body weight were previously submitted to univariate analysis; if significant, they were included in the multivariate models. In the final logistic regression models, variables were removed by backward elimination (according to the Wald’s criterion) when P ⬎ 0.2. For comparison of percentage of prepubertal heifers with serum P4 concentrations ⬎1.0 ng/mL on Days ⫺19 and ⫺12, the final model included the effect of day. For the analysis of percentage of prepubertal heifers detected in estrus during the first 7 d of the breeding season, the final model included the effects of treatment and follicular diameter within treatment. For analysis of pregnancy rates of prepubertal heifers in 7, 45, and 90 d of the breeding season, the final model included the effect of treatment. For the analysis of conception rate in 7 d of the breeding season, data from pubertal heifers were included, so four treatments were considered in the model (CIDR0, CIDR1, CIDR4, and PGF). An additional analysis was performed using only data of heifers from treatment CIDR0, to elucidate factors improving conception at first estrus in prepubertal heifers, which consisted of evaluating the effects of serum concentrations of P4 and cortisol at Day 0 on conception rate during the first 7 d of the breeding season. Continuous distributed variables, such as serum concentrations of P4 and cortisol, and follicular diameter were analyzed by PROC GLM, with final models determined by sequentially removing non-significant terms (P ⬎0.2). For the analysis of serum P4 concentrations, the final model included the effects of treatment, day, and interaction. For the analysis of follicular diameter on Day 0, the final model included the effect of treatment. For the analysis of serum concentrations of cortisol on Days ⫺19 and ⫺12, heifers were categorized as having serum P4 concentrations ⬍1.0 or ⱖ1.0 ng/mL, and the final model included the effect of categorized serum P4 concentration.
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Table 1 Reproductive end points in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of progesterone (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). Dependent variable Progesterone (ng/mL) Follicle diameter (mm) Uterine score Interval to estrus* (d) Estrus detection in 7 d (%) Conception rate in 7 d (%) Pregnancy rate in 7 d (%) Estrus detection in 45 d (%) Pregnancy rate in 45 d (%) Pregnancy rate in 90 d (%)
Treatment
P-value
CIDR0
CIDR1
CIDR4
0.37 ⫾ 0.16a 9.45 ⫾ 0.24a 1.49 ⫾ 0.06a 4.4 ⫾ 0.28a 19.5a 22/113 27.3a 6/22 5.3a 6/113 52.2a 59/113 27.4a 31/113 72.6a 82/113
2.31 ⫾ 0.11b 9.72 ⫾ 0.17a 1.88 ⫾ 0.04b 3.48 ⫾ 0.13b 42.6b 101/237 33.7a 34/101 14.3b 34/237 72.1b 171/237 39.2b 93/237 83.5b 198/237
1.20 ⫾ 0.11c 11.42 ⫾ 0.16b 2.24 ⫾ 0.04c 3.24 ⫾ 0.14b 39.3b 94/239 46.8b 44/94 18.4b 44/239 75.3b 180/239 47.7c 114/239 83.7b 200/239
⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05
The breeding season started on Day 1 and consisted of AI after estrus detection between Days 1 and 45, and exposure to bulls between Days 46 and 90. a– c Within a row, means without a common superscript differed (P ⬍ 0.05). * Considering only heifers detected in estrus during the first 7 d of breeding season.
Uterine score data were analyzed using PROC GENMOD of SAS. The model included the effects of treatment, day, follicular diameter within treatment, and interaction between treatment and day. The UtS data are reported as lsmeans ⫾ SEM. Survival analyses were performed, using the PROC LIFETEST of SAS to establish the effects of treatments on pregnancy rates throughout the first 45 d of the breeding season in prepubertal heifers. Survival analysis regressed the proportion of heifers pregnant on the days of the breeding season, and curves were compared by the Wilcoxon test. For all dependent variables, when the effect of a class with more than two levels was detected, Bonferroni (binomial distributed data) and Tukey-Kramer (continuous distributed data) tests were used for mean separation. Differences were considered significant when P ⬍ 0.05, whereas tendencies were considered when 0.1 ⬎ P ⱖ 0.05. 3. Results Serum concentrations of P4 and UtS were (mean ⫾ SEM 0.43 ⫾ 0.11 ng/mL and 1.41 ⫾ 0.04) on Day ⫺19, and were (0.64 ⫾ 0.10 ng/mL and 1.33 ⫾ 0.04) on Day ⫺12, with no significant difference among treatment groups for either end point. On Day 0, serum P4 concentrations were greater (P ⬍ 0.05) in the CIDR1 than CIDR4 and CIDR0 groups, and in the CIDR4 than CIDR0 group (P ⬍ 0.05; Table 1). The UtS was greater (P ⬍ 0.05) in the CIDR4 than CIDR1 and CIDR0 groups, and in the CIDR1 than CIDR0 groups (P ⬍ 0.05; Table 1). Heifers from the CIDR4 treatment had a greater follicular
diameter on Day 0 than in the CIDR0 and CIDR1 treatments (P ⬍ 0.05; Table 1). In heifers from treatments CIDR1 and CIDR4, the diameter of the largest follicle on Day 0 had positive relationships with UtS on Day 0 and estrus detection rate in the first 7 d of the breeding season (P ⬍ 0.05; Fig. 2 and 3); however, these relationships were not significant in the CIDR0 treatment. During the first 7 d of the breeding season, interval to estrus and variance of timing of estrus was less (P ⬍ 0.05), and the proportion of heifers detected in estrus was greater (P ⬍ 0.05) in the CIDR1 and CIDR4 than the CIDR0 treatment (Table 1; Fig. 4). Conception rate to AI during this time was similar between pubertal heifers in the PGF treatment (43.8%, 78/178) and the CIDR4 treatment, and both were greater (P ⬍ 0.05) than conception rate in the CIDR1 and CIDR0 treatments (Table 1). Pregnancy rates at 7, 45, and 90 d of the breeding season were greater (P ⬍ 0.05) in the CIDR1 and CIDR4 than in the CIDR0 treatments, and pregnancy rate in the first 45 d of the breeding season was greater (P ⬍ 0.05) in the CIDR4 than CIDR1 treatment (Table 1; Fig. 5). The percentage of heifers with no detected luteal tissue, but serum P4 concentrations ⬎1.0 ng/mL increased from Day ⫺19 to Day ⫺12 (P ⬍ 0.05): on Day ⫺19, the percentages of heifers with serum P4 concentrations ⬍1.0 ng/mL, between 1.0 and 1.5 ng/mL, and ⬎1.5 ng/mL were 91.4, 5.9, and 2.7%, respectively, whereas on Day ⫺12 these percentages were 82.6, 10.6, and 6.8%, respectively. Serum cortisol concentrations were greater in heifers with serum P4 concentrations ⱖ1.0 ng/mL than in those with ⬍1.0 ng/mL at both Day ⫺19 (25.81 ⫾ 0.25
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Fig. 2. Relationship between diameter of the largest ovarian follicle and uterine score on experimental Day 0 in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of (CIDR1; n ⫽ 237), or a similar intravaginal insert had previously been used three times (CIDR4; n ⫽ 239). The uterus was evaluated by transrectal palpation and scores were: 1 (uterine horn diameter ⬍20 mm and weak uterine tone), 2 (uterine horn diameter ⬎20 mm and weak uterine tone); or 3 (uterine horn diameter ⬎20 mm and strong uterine tone). Follicle diameter positively affected uterine score in treatments CIDR1 and CIDR4 (P ⬍ 0.05), but not in treatment CIDR0 (P ⬎ 0.1).
vs. 15.18 ⫾ 0.26 ng/mL; P ⬍ 0.01) and on Day ⫺12 (23.52 ⫾ 0.16 vs. 17.32 ⫾ 0.25 ng/mL; P ⬍ 0.01). Considering only heifers from treatment CIDR0 that were inseminated during the first 7 d of the breeding season, those that became pregnant tended to have greater circulating concentrations of P4 on Day 0 than those that did not become pregnant (0.68 ⫾ 0.17 vs. 0.33 ⫾ 0.10 ng/mL; P ⬍ 0.1); however, serum cortisol concentrations were similar among all heifers.
4. Discussion In this study, treatment for 12 d with new intravaginal inserts containing 1.9 g of P4 (CIDR1), or similar implants that had previously been used three times, with each use occurring for 9 d (CIDR4), hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. However, the CIDR4 treatment
Fig. 3. Relationship between diameter of the largest ovarian follicle on experimental Day 0 and estrus detection rates in the first 7 d of breeding season in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatments (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). The breeding season started on Day 1. Follicle diameter positively affected estrus detection rate in CIDR1 and CIDR4 treatments (P ⬍ 0.05), but not in CIDR0 treatment (P ⬎ 0.1).
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Fig. 4. Cumulative percentage of prepubertal Nelore heifers detected in estrus throughout a 45 d AI breeding season. Heifers received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239).
resulted in greater follicular diameter, UtS, conception in 7 d, and pregnancy rate in 45 d of the breeding season than the CIDR1 treatment. Furthermore, there were serum P4 concentrations ⬎1.0 ng/mL in Nelore heifers with no luteal tissue detected ultrasonographically, in association with elevated serum cortisol concentrations. Prepubertal heifers have waves of ovarian follicular development, preceded by an increase in peripheral FSH concentrations, that progress through to follicle deviation
[21–24]. However, in these heifers, their hypothalamus is sensitive to estradiol negative feedback on LH pulses [23,25]; therefore, low peripheral estradiol concentrations, perhaps derived from dominant ovarian follicle, supressed LH pulses, and limited growth and estradiol production by the follicle, thereby preventing an LH surge and ovulation [24]. The high hypothalamic sensitivity to estradiol is associated with an increased expression of estradiol receptors in the anterior and medial basal hypothalamus, which declines gradually as puberty approaches [5,25]. Prepu-
Fig. 5. Cumulative percentage of pregnancy in prepubertal Nelore heifers throughout a 45 d AI breeding season. Heifers, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). Heifers were artificially inseminated 10 to 14 h after detection of estrus; those that returned in estrus after the first service were re-inseminated.
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bertal administration of progestins effectively hastened puberty in Bos taurus heifers [26] and results from the current study, in which treatments CIDR1 and CIDR4 improved estrus detection rate in 7 d of the breeding season, indicated an acute effect of exogenous P4 on induction of puberty in Bos indicus heifers. Because P4 inhibits estradiol receptors in several tissues [27,28], it is likely that expression of hypothalamic estradiol receptors is decreased when prepubertal heifers are given exogenous P4, thereby reducing the estradiol negative feedback on LH secretion [6]. In fact, there was increased LH pulse frequency in prepubertal heifers in response to exogenous progestins [9,26]. In postpartum anestrous cows, treatment with P4 or other progestins enhanced intrafollicular and circulating concentrations of estradiol, increased release of LH pulses, and increased number of LH receptors in granulosa and theca cells in preovulatory follicles [29 – 33] that contributed to greater percentages of animals expressing estrus after P4 withdrawal [12,34]. Greater peripheral P4 concentrations were associated with lower frequency of LH pulses and, consequently, decreased peripheral estradiol concentrations and rates of dominant follicle growth and ovulation [35–39]. In agreement with a previous study [33], serum P4 concentrations on Day 0 were lower in the CIDR4 than CIDR1 treatments; the expected influence of this difference on secretion of LH provided a plausible explanation for the greater follicular diameter on Day 0 in the CIDR4 than CIDR1 treatments. Although follicle diameter was greater in CIDR4 treatment, the estrus detection rate and the mean interval between CIDR withdrawal and estrus were similar between treatments CIDR1 and CIDR4, indicating that circulating concentrations of P4 during treatment with exogenous P4 may not substantially influence the proportion of heifers induced to initiate estrous cycles. Similarly, when Anderson et al. [26] treated prepubertal heifers with either one or three implants of norgestomet, LH secretion was suppressed during treatment with the three implants, but puberty was induced similarly with one versus three implants in response to the rapid increase in LH pulses after implant withdrawal. Despite the apparent similarity between the CIDR1 and CIDR4 treatments for induction of the pubertal estrus, conception rate was greater in heifers from the CIDR4 than CIDR1 and CIDR0 treatments, and similar to that of pubertal heifers in which estrus was synchronized by administration of PGF. The enhanced conception rate in the CIDR4 heifers contributed to the increased pregnancy rate in this treatment by day 45 of the breeding season. The positive effect of CIDR4
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treatment on conception might be related to a more adequate steroidal environment and uterine development during the preovulatory period, since heifers from the CIDR0 and CIDR1 treatments had lower UtS than heifers from the CIDR4 treatment (1.49 ⫾ 0.06, 1.88 ⫾ 0.04, and 2.24 ⫾ 0.04, respectively) and studies have indicated a positive relationship between UtS and fertility in heifers during their first breeding season [40,41]. Although peripheral estradiol concentrations were not quantified in the current study, based on the greater follicular diameter and UtS for heifers in the CIDR4 treatment, we inferred that estradiol concentrations may have been elevated in these heifers. Using a 10 d treatment with a single norgestomet implant in prepubertal heifers, Anderson et al. [26] demonstrated that progestin treatment tended to increase uterine weight on the 9th d of progestin treatment (121.2 ⫾ 21 vs. 85.0 ⫾ 5.4 g) and had profound effects on uterine weight 1 d after implant withdrawal (222.3 ⫾ 30 vs. 72.7 ⫾ 10.9 g) in comparison to non-treated prepubertal heifers. It was suggested that the rapid increase of uterine weight after norgestomet implant withdrawal was related to increasing peripheral estradiol concentrations during proestrus. Estradiol causes proliferative changes in uterine tissues, reflected as growth and development of luminal and glandular epithelial cells, and luminal protein secretion [42,43]. As important as the differences detected between the CIDR1 and CIDR4 treatments, the similarity in conception rate between the CIDR4 and PGF treatments indicated that the previously used CIDR inserts resulted in induction of a more fertile pubertal estrus in Bos indicus heifers than achieved with a new CIDR, thereby increasing pregnancy rate during the breeding season. In agreement with a previous report in prepubertal Brahman-crossbred heifers [44], in the current study, serum P4 concentrations ⬎1.0 ng/mL occurred in prepubertal Nelore heifers. Cows and heifers are commonly classified as cyclic when their peripheral P4 concentrations are ⬎1.0 ng/mL in at least one of two samples collected within a 7–10 day interval [11,44 – 46], since plasma P4 concentrations ⬎1.0 ng/mL are typically restricted to cattle having a functional CL [47,48]. Although the CL is absent in prepubertal heifers, the adrenal gland is also capable of producing substantial amounts of P4, as an intermediate of cortisol synthesis, when stimulated by circulating ACTH [49]. Supporting this rationale, prepubertal heifers with serum P4 concentrations ⬎1.0 ng/mL had greater concentrations of cortisol than those with P4 concentrations ⬍1.0 ng/mL, as previously reported by Cooke and
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Arthington [44] and Cooke et al. [50], as well in studies evaluating the effects of ACTH administration to ovariectomized heifers and cows [51,52]. In a previous report, ⬎50% of prepuberal Brahmancrossbred heifers had P4 concentrations ⬎1.0 ng/mL during handling procedures [44]. The common excitable temperament in response to handling observed in Bos indicus cattle [53,54] may be a critical factor contributing to greater circulating P4 concentrations in prepubertal Bos indicus heifers, because the excitable temperament experience increases secretion and circulating concentrations of ACTH and cortisol [55]. Furthermore, that the proportion of prepubertal heifers experiencing circulating concentrations of P4 ⬎1.0 ng/mL increased from Day ⫺19 to Day ⫺12 in the present study, we inferred that multiple handlings might have stimulated adrenal secretion of P4 more than would have been expected following a single handling. Since analysis of serum P4 concentrations may result in falsely designated prepubertal heifers as having reached puberty, evaluation of pubertal status in heifers should always be associated with ovarian ultrasound examination. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Nelore heifers. Furthermore, since the CIDR4 treatment resulted in greater follicular diameter, UtS, conception in 7 d, and pregnancy rate in 45 d of breeding than treatment with CIDR1, a previously used CIDR insert is preferable to a new CIDR to induce puberty and improve reproductive performance during the breeding season in prepubertal Bos indicus heifers. References [1] Dobson H, Kamonpatana M. A review of female cattle reproduction with special reference to a comparison between buffaloes, cows and zebu. J Reprod Fertil 1986;77:1–36. [2] Patterson DJ, Corah LR, Brethour JR, Spire MF, Higgins JJ, Kiracofe GH, Stevenson JS, Simms DD. Evaluation of reproductive traits in Bos taurus and Bos indicus crossbred heifers: effects of postweaning energy manipulation. J Anim Sci 1991; 69:2349 – 61. [3] Restle J, Polli VA, De Senna DB. Efeito de grupo genético e heterose sobre a idade e peso à puberdade e sobre o desempenho reprodutivo de novilhas de corte (Effect of genetic group and heterosis on age and weight at puberty, and on reproductive performance of beef heifers). Pesquisa Agropecuária Brasileira 1999;34:701–7. [4] Eler JP, Silva JA, Ferraz JBS, Dias F, Oliveira HN, Evans JL, Golden BL. Genetic evaluation of the probability of pregnancy at 14 months for Nelore heifers. J Anim Sci 2002;80:951– 4. [5] Day ML, Imakawa K, Wolfe PL, Kittok RJ, Kinder JE. Endocrine mechanisms of puberty in heifers. Role of hypothalamo-pituitary estradiol receptors in the negative feedback of estradiol on luteinizing hormone secretion. Biol Reprod 1987;37:1054 – 65.
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[40] Anderson KJ, Lefever DG, Brinks JS, Odde KG. The use of reproductive tract scoring in beef heifers. Agri Pract 1991;2: 123– 8. [41] Ferreira MBD, Lopes BC, Dantas MS, Mourão GB, Vale Filho VR. Escore do aparelho reprodutivo pré estação de monta em novilhas Zebu aos dois anos de idade (Reproductive tract score before breeding season in two-years old Zebu heifers). Revista Brasileira de Reprodução Animal 1999;23:160 –2. [42] Murray MK. The effect of estrogen and progesterone on structural changes in the uterine glandular epithelium of the ovariectomized sheep. Biol Reprod 1992;47:408 –17. [43] Johnson ML, Redmer DA, Reynolds LP. Effects of ovarian steroids on uterine growth, morphology, and cell proliferation in ovariectomized, steroid-treated ewes. Biol Reprod 1997;57: 588 –96. [44] Cooke RF, Arthington JD. Plasma progesterone concentrations as puberty criteria for Brahman – crossbred heifers. Livest Sci 2008;123;101–5. [45] Sá Filho OG, Vilela ER, Geary TW, Vasconcelos JLM. Strategies to improve fertility in postpartum multiparous Bos indicus cows submitted to a fixed-time insemination protocol with gonadotropin-releasing hormone and prostaglandin F2␣. J Anim Sci 2009;87:2806 –14. [46] Vasconcelos JLM, Vilela ER, Sá Filho OG. Temporary weaning at two different times of the GnRH-PGF2␣-EB synchronization of ovulation protocol in post partum Nelore cows. Braz J Vet Anim Sci 2009;61:95–103. [47] Patterson DJ, Corah LR, Brethour JR. Response of prepubertal Bos taurus and Bos indicus ⫻ Bos taurus heifers to melengestrol acetate with or without gonadotropin-releasing hormone. Theriogenology 1990;33:661– 8. [48] Hoffmann B, Schuler G. The bovine placenta; a source and target of steroid hormones: observations during the second half of gestation. Domest Anim Endocrinol 2002;23:309 –20. [49] Willard S, Lay D, Friend T, Neuendorff D, Randel R. Plasma progesterone response following ACTH administration during midgestation in the pregnant Brahman heifer. Theriogenology 2005;63:1061–9. [50] Cooke RF, Austin BR, Yelich JV, Arthington JD. Effects of acclimation to handling on performance, reproductive, and physiological responses of Brahman-crossbred heifers. J Anim Sci 2009;87:3403–12. [51] Bage R, Forsberg M, Gustafsson H, Larsson B. Effect of ACTH challenge on progesterone and cortisol levels in ovariectomized repeat breeder heifers. Anim Reprod Sci 2000;63:65–76. [52] Yoshida C, Nakao T. Response of plasma cortisol and progesterone after ACTH challenge in ovariectomized lactating dairy cows. J Reprod Dev 2005;51:99 –107. [53] Fordyce GE, Dodt RM, Wythes JR. Cattle temperaments in extensive beef herds in northern Queensland. 1. Factors affecting temperament. Aust J Exp Agric 1988;28:683–7. [54] Voisinet BD, Grandin T, Tatum JD, O’Connor SF, Struthers JJ. Feedlot cattle with calm temperaments have greater average daily gains than cattle with excitable temperaments. J Anim Sci 1997;75:892– 6. [55] Curley KO, Neuendorff DA, Lewis AW, Cleere JJ, Welsh TH, Randel RD. Functional characteristics of the bovine hypothalamic–pituitary–adrenal axis vary with temperament. Horm Behav 2008;53:20 –7.
Theriogenology 74 (2010) 903–911 www.theriojournal.com
Reproductive performance of prepubertal Bos indicus heifers after progesterone-based treatments I. Claro Júniora, O.G. Sá Filhoa, R.F.G. Peresa, F.H.S. Aonoa, M.L. Dayb, J.L.M. Vasconcelosa,* a
Departamento de Produção Animal, Faculdade de Medicina Veterinária e Zootecnia-UNESP, Botucatu-SP 18618-000, Brazil b Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA Received 5 January 2010; received in revised form 23 February 2010; accepted 9 April 2010
Abstract The objective was to evaluate the effects of exogenous progesterone (P4) on reproductive performance of prepubertal Bos indicus heifers. Prepubertal Nelore heifers (n ⫽ 589; 24.0 ⫾ 1.13 mo; 298.0 ⫾ 1.89 kg; body condition score of 3.2 ⫾ 0.26; mean ⫾ SEM) were randomly assigned to receive, between experimental Days ⫺12 and 0: no treatments (CIDR0; n ⫽ 113); a new intravaginal insert (CIDR) containing 1.9 g of P4 (CIDR1; n ⫽ 237); or a similar insert previously used three times, with each use occurring for 9 d (CIDR4; n ⫽ 239). An additional treatment group was pubertal heifers given 12.5 mg dinoprost tromethamine im on Day 0 (PGF; n ⫽ 346), and used as controls for evaluation of conception rates. On Day 0, transrectal palpation was done for uterine score evaluation (UtS; 1–3 scale), blood samples were taken for serum P4 concentrations, and follicle diameter (FD) was measured. The breeding season started on Day 1 and consisted of AI after detection of estrus between Days 1 and 45, and exposure to bulls between Days 46 and 90. There were effects of treatment (P ⬍ 0.05) on serum concentrations of P4 on Day 0 (0.37 ⫾ 0.16, 2.31 ⫾ 0.11, and 1.20 ⫾ 0.11 ng/mL for CIDR0, CIDR1, and CIDR4, respectively; mean ⫾ SEM), FD on Day 0 (9.45 ⫾ 0.24, 9.72 ⫾ 0.17, and 11.42 ⫾ 0.16 mm), UtS on Day 0 (1.49 ⫾ 0.06, 1.88 ⫾ 0.04, and 2.24 ⫾ 0.04), estrus detection rates at 7 d (19.5, 42.6, and 38.3%) and 45 d (52.2, 72.1, and 75.3%) of the breeding season, and on pregnancy rates at 7 d (5.3, 14.3, and 18.4%), 45 d (27.4, 39.2, and 47.7%) and 90 d (72.6, 83.5, and 83.7%) of the breeding season. Conception rate 7 d after the start of the breeding season was greater (P ⬍ 0.05) in heifers from the CIDR4 (46.8%) and PGF (43.8%) groups than in the CIDR0 (27.3%) and CIDR1 (33.7%) groups. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. Furthermore, previously used CIDR inserts were better than new inserts. © 2010 Elsevier Inc. All rights reserved. Keywords: Puberty; Progesterone; Pregnancy rates; Bos indicus; Zebu cattle
1. Introduction Compared to Bos taurus, Bos indicus heifers reach puberty older and at a higher percentage of body weight relative to mature body weight [1], resulting in a lower proportion of pubertal heifers at the beginning of breed* Corresponding Author. Tel.: ⫹55 14 3811 7185; fax: ⫹55 14 3811 7180. E-mail address: [email protected] (J.L.M. Vasconcelos). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.04.015
ing season and lesser pregnancy rates [2,3]. These characteristics contribute to reduced profitability of cowcalf enterprises using zebu cattle [4]. The key factor responsible for establishment of puberty is an increase in LH release from the anterior pituitary, in response to GnRH from the hypothalamus [5]. Day et al. [6] suggested that exposure to progesterone (P4) or other progestins reduced the hypothalamic expression of estradiol receptors, and thereby decreased the negative feedback action of estradiol on
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Fig. 1. Schematic diagram of experimental design. Between Days ⫺12 and 0, prepubertal Nelore heifers received either no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of progesterone (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used 3x (CIDR4; n ⫽ 239). An additional group of pubertal heifers (not shown in the figure; PGF; n ⫽ 346) received PGF2␣ on Day 0, and were used as controls for evaluation of conception rates. US: ultrasonographic examination (ovaries on Days ⫺19, ⫺12 and 0 and diagnosis of pregnancy on Days 75 and 120); BS: blood sampling; UtS: rectal palpation for uterine score evaluation (UtS-1: uterine horn diameter ⬍20 mm and weak uterine tone; UtS-2: uterine horn diameter ⬎20 mm and weak uterine tone; UtS-3: uterine horn diameter ⬎20 mm and strong uterine tone); BW: body weight; BCS: body condition score, on a scale from 1 (emaciated) to 5 (obese).
This study was conducted at a commercial ranch in Para, Brazil. Animals were cared for in accordance with the practices outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [16].
⫺19 of the experiment, were used. They were grazing one of six Brachiaria brizantha pastures, with ad libitum access to water and mineral supplement. Ultrasonographic examination of ovaries (Aloka SSD-500 with a 7.5 MHz linear-array transrectal transducer; Aloka, Tokyo, Japan) was performed on Days ⫺19 and ⫺12 to detect luteal tissue. Heifers with no luteal tissue detected at both examinations were considered prepubertal (n ⫽ 589) and randomly assigned to receive, within each pasture, one of the following treatments (Fig. 1): CIDR0 — heifers received no treatment (n ⫽ 113); CIDR1— between Days ⫺12 and 0, heifers received a new intravaginal insert containing 1.9 g of P4 (CIDR®—Controlled Intravaginal Drug Release, Pfizer Animal Health, São Paulo, SP, Brazil; n ⫽ 237); and CIDR4 — between Days ⫺12 and 0, heifers received a similar intravaginal insert that had previously been used three times, with each use occurring for 9 d (n ⫽ 239). Pubertal heifers received 12.5 mg of dinoprost tromethamine im (2.5 mL of Lutalyse®, Pfizer Animal Health) on Day 0, and were used exclusively as controls for evaluation of conception rates (PGF; n ⫽ 346). All heifers were observed from 0700 to 0800 h, and from 1700 to 1800 h between Days 1 and 45. Heifers detected in estrus were inseminated 10 to 14 h after detection of estrus, with AI sires (n ⫽ 9) and technicians (n ⫽ 12) randomly assigned to heifers. During the first 45 d of the breeding season, heifers that returned to estrus after the first AI were re-inseminated as described and, between Days 46 and 90, heifers were exposed to sires for natural service (one sire per 15 heifers).
2.1. Animals and treatments
2.2. Uterine score evaluation
Nelore heifers (n ⫽ 935) that were 22–26 mo of age, 280 –330 kg body weight, and had body condition score of 3.2 ⫾ 0.26 (1 ⫽ emaciated to 5 ⫽ obese [17]) on Day
On Days ⫺19, ⫺12, and 0, reproductive tracts were evaluated by transrectal palpation and uterine scores (UtS) were assigned as follows: uterine horn diameter
GnRH secretion. In that regard, in several studies with prepubertal Bos taurus heifers, exogenous progestins increased LH secretion and induced puberty [7–10]. Furthermore, exogenous P4 in non-cycling ruminants prior to induction of ovulation minimized premature luteolysis [11], which improved the likelihood of conception [12]. However, treatment with an intravaginal insert containing 1.9 g of P4 increased peripheral P4 concentrations and reduced ovarian follicular growth in Bos indicus relative to Bos taurus or crossbred heifers [13]; therefore, P4 released by the inserts may exceed subluteal concentrations and decrease the frequency of LH pulses in Bos indicus heifers. In fact, strategies to reduce peripheral P4 concentrations during P4-based synchronization protocols, such as advancing the time of prostaglandin F2␣ (PGF2␣) treatment, previously used P4 inserts, or both, improved fertility in cycling Bos indicus heifers [13–15]. The objective of this study was to evaluate the effects of treatments with exogenous P4 on rates of induced estrus, conception, and pregnancy in prepubertal Bos indicus heifers. 2. Material and methods
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⬍20 mm and weak uterine tone (UtS ⫽ 1); uterine horn diameter ⬎20 mm and weak uterine tone (UtS ⫽ 2); and uterine horn diameter ⬎20 mm and strong uterine tone (UtS ⫽ 3). All uterine tract scores, as well as body condition scores, were done by the same veterinarian. 2.3. Ultrasonography of ovarian structures and pregnancy Ovarian ultrasonography was performed on Day 0, as described above, to detect luteal tissue and to determine the diameter of the largest ovarian follicle. Diameter was calculated as the average distance for two measurements made at perpendicular angles. Heifers previously considered prepubertal that had luteal tissue on Day 0 were excluded from the study. Uterine ultrasonographic examinations were performed on Days 75 and 120 for diagnosis of pregnancy. 2.4. Blood sampling and steroid assays Blood samples were collected on Days ⫺19, ⫺12, and 0 (immediately before CIDR withdrawal) from the coccygeal vein into Vacutainer tubes (Becton Dickinson Co., Franklin Lakes, NJ, USA) for analysis of serum P4 concentrations. Blood was allowed to clot at 4 oC for 24 h and centrifuged at 1500 ⫻ g for 10 min at room temperature. Serum was removed and stored frozen (⫺20 oC) until hormonal assays were performed. Samples collected on Days ⫺19 and ⫺12 were used for the evaluation of cyclicity (in association with previous ultrasonographic findings), whereas samples collected on Day 0 were used for evaluation of the effects of treatments on progesterone concentrations of prepubertal heifers. Since some heifers with no luteal tissue detected had serum P4 concentrations ⬎1 ng/mL on Days ⫺19 and/or ⫺12, samples from those days were analyzed to determine serum cortisol concentrations, since stress may increase P4 secretion from nonluteal tissues [18,19]. Serum concentrations of P4 and cortisol were determined using a solid-phase radioimmunoassay kit containing antibody-coated tubes and 125I-labeled P4 or cortisol (Coat-a-Count; Diagnostic Products Corporation, Los Angeles, CA, USA) according to the manufacturer’s instructions. For P4 assays, the interassay CV was 5.6%, and average intra-assay CV was 5.4%; for the cortisol assay, the intra-assay CV was 1.3%. Assay sensitivities were 0.1 ng/mL for P4 and 5 ng/mL for cortisol. 2.5. Statistical analyses This experiment was analyzed as a completely randomized design. Since the main objective was to evaluate the effects of treatments with CIDR® on prepu-
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bertal heifers and fertility at first pubertal estrus was low in heifers [20], pubertal heifers (treatment PGF) were included as controls for evaluation of conception rate only, but were excluded from any other analysis. For prepubertal heifers (treatments CIDR0, CIDR1, and CIDR4), initial comparisons were performed, using PROC ANOVA and PROC GENMOD of SAS (SAS Institute Inc., Cary, NC, USA), to determine that the distribution of heifers by age, BCS, body weight, and UtS were similar among treatments. Binomially distributed data were analyzed using PROC LOGISTIC of SAS. Explanatory variables such as treatment, UtS, sire (semen), and AI technician were used in the model as classes. Explanatory variables considered covariates such as BCS, age, and body weight were previously submitted to univariate analysis; if significant, they were included in the multivariate models. In the final logistic regression models, variables were removed by backward elimination (according to the Wald’s criterion) when P ⬎ 0.2. For comparison of percentage of prepubertal heifers with serum P4 concentrations ⬎1.0 ng/mL on Days ⫺19 and ⫺12, the final model included the effect of day. For the analysis of percentage of prepubertal heifers detected in estrus during the first 7 d of the breeding season, the final model included the effects of treatment and follicular diameter within treatment. For analysis of pregnancy rates of prepubertal heifers in 7, 45, and 90 d of the breeding season, the final model included the effect of treatment. For the analysis of conception rate in 7 d of the breeding season, data from pubertal heifers were included, so four treatments were considered in the model (CIDR0, CIDR1, CIDR4, and PGF). An additional analysis was performed using only data of heifers from treatment CIDR0, to elucidate factors improving conception at first estrus in prepubertal heifers, which consisted of evaluating the effects of serum concentrations of P4 and cortisol at Day 0 on conception rate during the first 7 d of the breeding season. Continuous distributed variables, such as serum concentrations of P4 and cortisol, and follicular diameter were analyzed by PROC GLM, with final models determined by sequentially removing non-significant terms (P ⬎0.2). For the analysis of serum P4 concentrations, the final model included the effects of treatment, day, and interaction. For the analysis of follicular diameter on Day 0, the final model included the effect of treatment. For the analysis of serum concentrations of cortisol on Days ⫺19 and ⫺12, heifers were categorized as having serum P4 concentrations ⬍1.0 or ⱖ1.0 ng/mL, and the final model included the effect of categorized serum P4 concentration.
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Table 1 Reproductive end points in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of progesterone (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). Dependent variable Progesterone (ng/mL) Follicle diameter (mm) Uterine score Interval to estrus* (d) Estrus detection in 7 d (%) Conception rate in 7 d (%) Pregnancy rate in 7 d (%) Estrus detection in 45 d (%) Pregnancy rate in 45 d (%) Pregnancy rate in 90 d (%)
Treatment
P-value
CIDR0
CIDR1
CIDR4
0.37 ⫾ 0.16a 9.45 ⫾ 0.24a 1.49 ⫾ 0.06a 4.4 ⫾ 0.28a 19.5a 22/113 27.3a 6/22 5.3a 6/113 52.2a 59/113 27.4a 31/113 72.6a 82/113
2.31 ⫾ 0.11b 9.72 ⫾ 0.17a 1.88 ⫾ 0.04b 3.48 ⫾ 0.13b 42.6b 101/237 33.7a 34/101 14.3b 34/237 72.1b 171/237 39.2b 93/237 83.5b 198/237
1.20 ⫾ 0.11c 11.42 ⫾ 0.16b 2.24 ⫾ 0.04c 3.24 ⫾ 0.14b 39.3b 94/239 46.8b 44/94 18.4b 44/239 75.3b 180/239 47.7c 114/239 83.7b 200/239
⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05 ⬍ 0.05
The breeding season started on Day 1 and consisted of AI after estrus detection between Days 1 and 45, and exposure to bulls between Days 46 and 90. a– c Within a row, means without a common superscript differed (P ⬍ 0.05). * Considering only heifers detected in estrus during the first 7 d of breeding season.
Uterine score data were analyzed using PROC GENMOD of SAS. The model included the effects of treatment, day, follicular diameter within treatment, and interaction between treatment and day. The UtS data are reported as lsmeans ⫾ SEM. Survival analyses were performed, using the PROC LIFETEST of SAS to establish the effects of treatments on pregnancy rates throughout the first 45 d of the breeding season in prepubertal heifers. Survival analysis regressed the proportion of heifers pregnant on the days of the breeding season, and curves were compared by the Wilcoxon test. For all dependent variables, when the effect of a class with more than two levels was detected, Bonferroni (binomial distributed data) and Tukey-Kramer (continuous distributed data) tests were used for mean separation. Differences were considered significant when P ⬍ 0.05, whereas tendencies were considered when 0.1 ⬎ P ⱖ 0.05. 3. Results Serum concentrations of P4 and UtS were (mean ⫾ SEM 0.43 ⫾ 0.11 ng/mL and 1.41 ⫾ 0.04) on Day ⫺19, and were (0.64 ⫾ 0.10 ng/mL and 1.33 ⫾ 0.04) on Day ⫺12, with no significant difference among treatment groups for either end point. On Day 0, serum P4 concentrations were greater (P ⬍ 0.05) in the CIDR1 than CIDR4 and CIDR0 groups, and in the CIDR4 than CIDR0 group (P ⬍ 0.05; Table 1). The UtS was greater (P ⬍ 0.05) in the CIDR4 than CIDR1 and CIDR0 groups, and in the CIDR1 than CIDR0 groups (P ⬍ 0.05; Table 1). Heifers from the CIDR4 treatment had a greater follicular
diameter on Day 0 than in the CIDR0 and CIDR1 treatments (P ⬍ 0.05; Table 1). In heifers from treatments CIDR1 and CIDR4, the diameter of the largest follicle on Day 0 had positive relationships with UtS on Day 0 and estrus detection rate in the first 7 d of the breeding season (P ⬍ 0.05; Fig. 2 and 3); however, these relationships were not significant in the CIDR0 treatment. During the first 7 d of the breeding season, interval to estrus and variance of timing of estrus was less (P ⬍ 0.05), and the proportion of heifers detected in estrus was greater (P ⬍ 0.05) in the CIDR1 and CIDR4 than the CIDR0 treatment (Table 1; Fig. 4). Conception rate to AI during this time was similar between pubertal heifers in the PGF treatment (43.8%, 78/178) and the CIDR4 treatment, and both were greater (P ⬍ 0.05) than conception rate in the CIDR1 and CIDR0 treatments (Table 1). Pregnancy rates at 7, 45, and 90 d of the breeding season were greater (P ⬍ 0.05) in the CIDR1 and CIDR4 than in the CIDR0 treatments, and pregnancy rate in the first 45 d of the breeding season was greater (P ⬍ 0.05) in the CIDR4 than CIDR1 treatment (Table 1; Fig. 5). The percentage of heifers with no detected luteal tissue, but serum P4 concentrations ⬎1.0 ng/mL increased from Day ⫺19 to Day ⫺12 (P ⬍ 0.05): on Day ⫺19, the percentages of heifers with serum P4 concentrations ⬍1.0 ng/mL, between 1.0 and 1.5 ng/mL, and ⬎1.5 ng/mL were 91.4, 5.9, and 2.7%, respectively, whereas on Day ⫺12 these percentages were 82.6, 10.6, and 6.8%, respectively. Serum cortisol concentrations were greater in heifers with serum P4 concentrations ⱖ1.0 ng/mL than in those with ⬍1.0 ng/mL at both Day ⫺19 (25.81 ⫾ 0.25
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Fig. 2. Relationship between diameter of the largest ovarian follicle and uterine score on experimental Day 0 in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert containing 1.9 g of (CIDR1; n ⫽ 237), or a similar intravaginal insert had previously been used three times (CIDR4; n ⫽ 239). The uterus was evaluated by transrectal palpation and scores were: 1 (uterine horn diameter ⬍20 mm and weak uterine tone), 2 (uterine horn diameter ⬎20 mm and weak uterine tone); or 3 (uterine horn diameter ⬎20 mm and strong uterine tone). Follicle diameter positively affected uterine score in treatments CIDR1 and CIDR4 (P ⬍ 0.05), but not in treatment CIDR0 (P ⬎ 0.1).
vs. 15.18 ⫾ 0.26 ng/mL; P ⬍ 0.01) and on Day ⫺12 (23.52 ⫾ 0.16 vs. 17.32 ⫾ 0.25 ng/mL; P ⬍ 0.01). Considering only heifers from treatment CIDR0 that were inseminated during the first 7 d of the breeding season, those that became pregnant tended to have greater circulating concentrations of P4 on Day 0 than those that did not become pregnant (0.68 ⫾ 0.17 vs. 0.33 ⫾ 0.10 ng/mL; P ⬍ 0.1); however, serum cortisol concentrations were similar among all heifers.
4. Discussion In this study, treatment for 12 d with new intravaginal inserts containing 1.9 g of P4 (CIDR1), or similar implants that had previously been used three times, with each use occurring for 9 d (CIDR4), hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. However, the CIDR4 treatment
Fig. 3. Relationship between diameter of the largest ovarian follicle on experimental Day 0 and estrus detection rates in the first 7 d of breeding season in prepubertal Nelore heifers that received, between Day ⫺12 and 0, no treatments (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). The breeding season started on Day 1. Follicle diameter positively affected estrus detection rate in CIDR1 and CIDR4 treatments (P ⬍ 0.05), but not in CIDR0 treatment (P ⬎ 0.1).
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Fig. 4. Cumulative percentage of prepubertal Nelore heifers detected in estrus throughout a 45 d AI breeding season. Heifers received, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239).
resulted in greater follicular diameter, UtS, conception in 7 d, and pregnancy rate in 45 d of the breeding season than the CIDR1 treatment. Furthermore, there were serum P4 concentrations ⬎1.0 ng/mL in Nelore heifers with no luteal tissue detected ultrasonographically, in association with elevated serum cortisol concentrations. Prepubertal heifers have waves of ovarian follicular development, preceded by an increase in peripheral FSH concentrations, that progress through to follicle deviation
[21–24]. However, in these heifers, their hypothalamus is sensitive to estradiol negative feedback on LH pulses [23,25]; therefore, low peripheral estradiol concentrations, perhaps derived from dominant ovarian follicle, supressed LH pulses, and limited growth and estradiol production by the follicle, thereby preventing an LH surge and ovulation [24]. The high hypothalamic sensitivity to estradiol is associated with an increased expression of estradiol receptors in the anterior and medial basal hypothalamus, which declines gradually as puberty approaches [5,25]. Prepu-
Fig. 5. Cumulative percentage of pregnancy in prepubertal Nelore heifers throughout a 45 d AI breeding season. Heifers, between Day ⫺12 and 0, no treatment (CIDR0; n ⫽ 113), a new intravaginal insert (CIDR1; n ⫽ 237), or a similar intravaginal insert that had previously been used three times (CIDR4; n ⫽ 239). Heifers were artificially inseminated 10 to 14 h after detection of estrus; those that returned in estrus after the first service were re-inseminated.
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bertal administration of progestins effectively hastened puberty in Bos taurus heifers [26] and results from the current study, in which treatments CIDR1 and CIDR4 improved estrus detection rate in 7 d of the breeding season, indicated an acute effect of exogenous P4 on induction of puberty in Bos indicus heifers. Because P4 inhibits estradiol receptors in several tissues [27,28], it is likely that expression of hypothalamic estradiol receptors is decreased when prepubertal heifers are given exogenous P4, thereby reducing the estradiol negative feedback on LH secretion [6]. In fact, there was increased LH pulse frequency in prepubertal heifers in response to exogenous progestins [9,26]. In postpartum anestrous cows, treatment with P4 or other progestins enhanced intrafollicular and circulating concentrations of estradiol, increased release of LH pulses, and increased number of LH receptors in granulosa and theca cells in preovulatory follicles [29 – 33] that contributed to greater percentages of animals expressing estrus after P4 withdrawal [12,34]. Greater peripheral P4 concentrations were associated with lower frequency of LH pulses and, consequently, decreased peripheral estradiol concentrations and rates of dominant follicle growth and ovulation [35–39]. In agreement with a previous study [33], serum P4 concentrations on Day 0 were lower in the CIDR4 than CIDR1 treatments; the expected influence of this difference on secretion of LH provided a plausible explanation for the greater follicular diameter on Day 0 in the CIDR4 than CIDR1 treatments. Although follicle diameter was greater in CIDR4 treatment, the estrus detection rate and the mean interval between CIDR withdrawal and estrus were similar between treatments CIDR1 and CIDR4, indicating that circulating concentrations of P4 during treatment with exogenous P4 may not substantially influence the proportion of heifers induced to initiate estrous cycles. Similarly, when Anderson et al. [26] treated prepubertal heifers with either one or three implants of norgestomet, LH secretion was suppressed during treatment with the three implants, but puberty was induced similarly with one versus three implants in response to the rapid increase in LH pulses after implant withdrawal. Despite the apparent similarity between the CIDR1 and CIDR4 treatments for induction of the pubertal estrus, conception rate was greater in heifers from the CIDR4 than CIDR1 and CIDR0 treatments, and similar to that of pubertal heifers in which estrus was synchronized by administration of PGF. The enhanced conception rate in the CIDR4 heifers contributed to the increased pregnancy rate in this treatment by day 45 of the breeding season. The positive effect of CIDR4
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treatment on conception might be related to a more adequate steroidal environment and uterine development during the preovulatory period, since heifers from the CIDR0 and CIDR1 treatments had lower UtS than heifers from the CIDR4 treatment (1.49 ⫾ 0.06, 1.88 ⫾ 0.04, and 2.24 ⫾ 0.04, respectively) and studies have indicated a positive relationship between UtS and fertility in heifers during their first breeding season [40,41]. Although peripheral estradiol concentrations were not quantified in the current study, based on the greater follicular diameter and UtS for heifers in the CIDR4 treatment, we inferred that estradiol concentrations may have been elevated in these heifers. Using a 10 d treatment with a single norgestomet implant in prepubertal heifers, Anderson et al. [26] demonstrated that progestin treatment tended to increase uterine weight on the 9th d of progestin treatment (121.2 ⫾ 21 vs. 85.0 ⫾ 5.4 g) and had profound effects on uterine weight 1 d after implant withdrawal (222.3 ⫾ 30 vs. 72.7 ⫾ 10.9 g) in comparison to non-treated prepubertal heifers. It was suggested that the rapid increase of uterine weight after norgestomet implant withdrawal was related to increasing peripheral estradiol concentrations during proestrus. Estradiol causes proliferative changes in uterine tissues, reflected as growth and development of luminal and glandular epithelial cells, and luminal protein secretion [42,43]. As important as the differences detected between the CIDR1 and CIDR4 treatments, the similarity in conception rate between the CIDR4 and PGF treatments indicated that the previously used CIDR inserts resulted in induction of a more fertile pubertal estrus in Bos indicus heifers than achieved with a new CIDR, thereby increasing pregnancy rate during the breeding season. In agreement with a previous report in prepubertal Brahman-crossbred heifers [44], in the current study, serum P4 concentrations ⬎1.0 ng/mL occurred in prepubertal Nelore heifers. Cows and heifers are commonly classified as cyclic when their peripheral P4 concentrations are ⬎1.0 ng/mL in at least one of two samples collected within a 7–10 day interval [11,44 – 46], since plasma P4 concentrations ⬎1.0 ng/mL are typically restricted to cattle having a functional CL [47,48]. Although the CL is absent in prepubertal heifers, the adrenal gland is also capable of producing substantial amounts of P4, as an intermediate of cortisol synthesis, when stimulated by circulating ACTH [49]. Supporting this rationale, prepubertal heifers with serum P4 concentrations ⬎1.0 ng/mL had greater concentrations of cortisol than those with P4 concentrations ⬍1.0 ng/mL, as previously reported by Cooke and
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Arthington [44] and Cooke et al. [50], as well in studies evaluating the effects of ACTH administration to ovariectomized heifers and cows [51,52]. In a previous report, ⬎50% of prepuberal Brahmancrossbred heifers had P4 concentrations ⬎1.0 ng/mL during handling procedures [44]. The common excitable temperament in response to handling observed in Bos indicus cattle [53,54] may be a critical factor contributing to greater circulating P4 concentrations in prepubertal Bos indicus heifers, because the excitable temperament experience increases secretion and circulating concentrations of ACTH and cortisol [55]. Furthermore, that the proportion of prepubertal heifers experiencing circulating concentrations of P4 ⬎1.0 ng/mL increased from Day ⫺19 to Day ⫺12 in the present study, we inferred that multiple handlings might have stimulated adrenal secretion of P4 more than would have been expected following a single handling. Since analysis of serum P4 concentrations may result in falsely designated prepubertal heifers as having reached puberty, evaluation of pubertal status in heifers should always be associated with ovarian ultrasound examination. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Nelore heifers. Furthermore, since the CIDR4 treatment resulted in greater follicular diameter, UtS, conception in 7 d, and pregnancy rate in 45 d of breeding than treatment with CIDR1, a previously used CIDR insert is preferable to a new CIDR to induce puberty and improve reproductive performance during the breeding season in prepubertal Bos indicus heifers. References [1] Dobson H, Kamonpatana M. A review of female cattle reproduction with special reference to a comparison between buffaloes, cows and zebu. J Reprod Fertil 1986;77:1–36. [2] Patterson DJ, Corah LR, Brethour JR, Spire MF, Higgins JJ, Kiracofe GH, Stevenson JS, Simms DD. Evaluation of reproductive traits in Bos taurus and Bos indicus crossbred heifers: effects of postweaning energy manipulation. J Anim Sci 1991; 69:2349 – 61. [3] Restle J, Polli VA, De Senna DB. Efeito de grupo genético e heterose sobre a idade e peso à puberdade e sobre o desempenho reprodutivo de novilhas de corte (Effect of genetic group and heterosis on age and weight at puberty, and on reproductive performance of beef heifers). Pesquisa Agropecuária Brasileira 1999;34:701–7. [4] Eler JP, Silva JA, Ferraz JBS, Dias F, Oliveira HN, Evans JL, Golden BL. Genetic evaluation of the probability of pregnancy at 14 months for Nelore heifers. J Anim Sci 2002;80:951– 4. [5] Day ML, Imakawa K, Wolfe PL, Kittok RJ, Kinder JE. Endocrine mechanisms of puberty in heifers. Role of hypothalamo-pituitary estradiol receptors in the negative feedback of estradiol on luteinizing hormone secretion. Biol Reprod 1987;37:1054 – 65.
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