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Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F2α treatment affect fertility in postpuberal Nelore heifers
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Theriogenology 72 (2009) 378–385 www.theriojournal.com
Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F2a treatment affect fertility in postpuberal Nelore heifers C.C. Dias a, F.S. Wechsler a, M.L. Day b, J.L.M. Vasconcelos a,* a
Departamento de Produc¸a˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, FMVZ-UNESP, CEP 18618-000, Botucatu, SP, Brazil b Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA Received 10 September 2008; received in revised form 4 March 2009; accepted 28 March 2009
Abstract Two experiments were performed to test the hypothesis that elevated progesterone concentrations impair pregnancy rate to timed artificial insemination (TAI) in postpuberal Nelore heifers. In Experiment 1, postpuberal Nelore heifers (n = 398) received 2 mg estradiol benzoate (EB) and either a new progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) (first use) or a CIDR previously used for 9 d (second use) or for 18 d (third use) on Day 0, 12.5 mg prostaglandin F2a (PGF2a) on Day 7, 0.5 mg estradiol cypionate (ECP) and CIDR withdrawal on Day 9, and TAI on Day 11. Largest ovarian follicle diameter was determined on Day 11. The third-use CIDR treatment increased largest ovarian follicle diameter and pregnancy rate. Conception to TAI was reduced in heifers with smaller follicles in the first- and second-use CIDR treatments, but not in the third-use CIDR treatment. In Experiment 2, postpuberal Nelore heifers received the synchronization treatment described in Experiment 1 or received 12.5 mg PGF2a on Day 9 rather than Day 7. In addition, 50% of heifers received 300 IU equine chorionic gonadotropin (eCG) on Day 9. Heifers were either TAI (Experiment 2a; n = 199) or AI after detection of estrus (Experiment 2b; n = 125 of 202). In Experiment 2a, treatment with eCG increased pregnancy rate to TAI in heifers that received PGF2a on Day 9 but not on Day 7 and in heifers that received a first-use CIDR but not in heifers that received a third-use CIDR. Treatments did not influence reproductive performance in Experiment 2b. In summary, pregnancy rate to TAI in postpuberal Nelore heifers was optimized when lower concentrations of exogenous progesterone were administered, and eCG treatment was beneficial in heifers expected to have greater progesterone concentrations. # 2009 Elsevier Inc. All rights reserved. Keywords: Cattle; Pregnancy; Proestrus; Progesterone; Timed artificial insemination
1. Introduction Artificial insemination (AI) is the most effective technique available to introduce new genetics into a
* Corresponding author. Tel.: +55 14 38117185; fax: +55 14 38117180. E-mail address: [email protected] (J.L.M. Vasconcelos). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.03.006
beef cattle herd, and timed AI (TAI) protocols have been developed to overcome challenges associated with estrus detection failure. There is a need for development of TAI protocols for Bos indicus breeds because of a shorter duration of estrous behavior compared with that of Bos taurus breeds [1]. Low TAI pregnancy rate was reported in Nelore heifers in studies in which the estrous synchronization protocol was initiated with estradiol administration and
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insertion of a progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) [2,3]. Carvalho et al. [4] compared the interval from CIDR insertion and estradiol treatment to follicular wave emergence in Bos indicus (3.1 d), Bos indicus Bos taurus (3.3 d), and Bos taurus (3.2 d) heifers. Although the time of follicle emergence was similar among groups, Bos indicus heifers had the smallest dominant follicles at the end of the CIDR treatment. A lower growth rate of dominant follicles in the Bos indicus heifers appeared to be associated with greater circulating progesterone concentrations during CIDR treatment. These results suggested that high concentrations of progesterone during estrous synchronization protocol may be detrimental to follicular development in Bos indicus cattle. Because luteinizing hormone (LH) secretion and pulse frequency are negatively associated with progesterone concentration [5], perhaps endogenous gonadotropic support for follicular development may be compromised in progesterone-treated Bos indicus heifers, as described by Carvalho et al. [4]. An alternative for increasing gonadotropic support of dominant follicles before TAI is a longer interval from luteal regression to the LH surge and TAI (i.e., ‘‘proestrus’’) than that of traditional synchronization protocols. Pregnancy rate to AI has been demonstrated to be greater in cows that had a long proestrus [6–10]. Use of equine chorionic gonadotropin (eCG) is a second alternative to increase follicle development; eCG treatment increased conception rate to TAI when given at the end of a CIDR treatment in anestrous cows [11]. A third alternative may be decreasing progesterone concentrations during the synchronization protocol. The goal of the current experiments was to determine whether treatments that should increase gonadotropic support of follicular development will increase fertility in Bos indicus heifers. The overall hypothesis for these experiments was that low pregnancy rates to TAI previously reported in Nelore heifers were due to inhibition of LH secretion and in turn development of the ovulatory follicle caused by elevated systemic progesterone concentrations. The objective of the first experiment was to compare the effect of various concentrations of progesterone during a synchronization protocol on pregnancy rate to TAI by using CIDRs that were expected to deliver different concentrations of progesterone. In the second experiment, progesterone concentrations were altered by either changing the timing of luteal regression during a synchronization protocol and/or using CIDRs that would deliver different doses of progesterone. In addition, treatment with eCG was used to evaluate the effect of an exogenous gonadotropic support. The
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influence of these factors on follicular development was assessed in both TAI and AI after estrus detection. 2. Materials and methods 2.1. Experimental design, location, and heifers 2.1.1. Experiment 1 This trial was conducted between November and December 2005 in Mato Grosso do Sul, Brazil. Nelore heifers (n = 525), 24 to 26 mo of age, with body condition scores (BCSs) between 2.75 and 3.25 (1 to 5 scale [12]) were used. Heifers were maintained on two pastures (Brachiaria humidı´cola) with mineral salt ad libitum. Their ovaries were evaluated by ultrasonography for presence of a corpus luteum (CL; Aloka SSD 500 V, equipped with a 7.5-MHz linear array intrarectal transducer; Aloka, Tokyo, Japan) 7 d before the initiation of the protocol. Heifers with luteal tissue in their ovaries (n = 398) were randomly assigned to receive a new progesterone-releasing intravaginal device containing 1.9 g progesterone (CIDR; Pfizer Animal Health, Sa˜o Paulo, SP, Brazil; new CIDR; first use), a CIDR that had been used previously in a 9-d synchronization protocol (second use), or a CIDR that had been used in two previous 9-d synchronization protocols (third use). All heifers were treated with 2 mg estradiol benzoate (EB) im (Estrogin; Farmavet, Sa˜o Paulo, SP, Brazil) at CIDR insertion (Day 0 of the experiment). Blood samples for progesterone analyses were collected on Days 0 and 7, and prostaglandin F2a (PGF2a) was administered (12.5 mg, im; Lutalyse; Pfizer Animal Health) on Day 7. On Day 9, CIDRs were withdrawn, and heifers received estradiol cypionate (0.5 mg, im; estradiol cypionate (ECP); Pfizer Animal Health). Heifers were bred by TAI on Day 11 (48 h after CIDR withdrawal) by two technicians using frozenthawed semen from a single sire. Transrectal ultrasonography examinations were performed on Day 11 to assess diameter of largest ovarian follicle at TAI (defined as the average between horizontal and vertical diameters). Heifers in which the largest ovarian follicle was <6 mm were recorded as a missing observation for this variable. Ultrasonography was also performed on Day 13 to evaluate ovulation (absence of the largest ovarian follicle previously measured at TAI) and on Day 41 to detect pregnancy. Reproductive variables that were determined included ovulation, synchronization, conception, and pregnancy rates. Ovulation rate was defined as the proportion of all heifers that ovulated between Days 11 and 13, including heifers that were considered to have prematurely ovulated (presence of
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multiple small follicles that were <6 mm in diameter at TAI). Synchronized ovulation rate was defined as the proportion of all heifers that ovulated between Days 11 and 13. Conception rate was calculated as the proportion of synchronized heifers that were pregnant on Day 41, and pregnancy rate was calculated as the proportion of all heifers that were pregnant at that time. 2.1.2. Experiment 2 This experiment was conducted between November 2006 and January 2007 in Mato Grosso do Sul, Brazil. Nelore heifers (n = 650), 24 to 26 mo of age, with BCSs between 2.75 and 3.25 were observed for estrus for 21 d before initiation of the synchronization program. Heifers detected in estrus (n = 401) were used in the experiment, as they were considered as to be reproductively mature. Heifers were maintained on two pastures (Brachiaria humidı´cola) with mineral salt provided ad libitum. On Day 0, all heifers received 2 mg EB im and were randomly assigned to receive either a first-use, second-use, or third-use CIDR as described in Experiment 1, PGF2a (12.5 mg, im) given on Day 7 or Day 9, and either no eCG or 300 IU eCG im (Folligon; Intervet, Sa˜o Paulo, SP, Brazil) on Day 9. Heifers from the two pastures had their CIDRs withdrawn on Day 9 and were randomly assigned either to receive ECP (0.5 mg, im) at this time, followed by TAI on Day 11 (Experiment 2a; n = 199), or to be artificially inseminated 12 h after detection of estrus between Days 9 and 15 (Experiment 2b; n = 202). All heifers were artificially inseminated by two technicians using frozen-thawed semen from a single sire. Pregnancy diagnoses were performed by transrectal ultrasonography 30 d after TAI. 2.2. Blood samples and progesterone RIA analyses 2.2.1. Experiment 1 Blood samples for progesterone analyses were collected from a coccygeal vessel into Vacutainer tubes (Becton Dickinson Co., Franklin Lakes, NJ, USA). Blood was allowed to clot at 4 8C for 24 h and centrifuged at 1500 g for 15 min at room temperature. Serum was removed and frozen at 20 8C until assays were performed. Serum progesterone concentrations were determined using a solid-phase radioimmunoassay kit containing antibody-coated tubes and 125I-labeled progesterone (Coat-a-count; Diagnostic Products Corporation, Los Angeles, CA, USA) that had been previously validated in our laboratory [13]. The intra-assay coefficients of variation were 6.7% and 8.5% for assays 1 (Day 0) and 2 (Day 7), respectively,
and the interassay coefficient of variation was 5.4%. Sensitivity was 0.01 ng/mL. 2.3. Statistical analyses 2.3.1. Experiment 1 Continuous dependent variables (i.e., progesterone concentration on Day 7 and diameter of the largest ovarian follicle on Day 11) were analyzed by leastsquares (PROC GLM; SAS Institute Inc., Cary, NC, USA [14]) using a model that included treatment (firstuse, second-use, and third-use CIDRs), progesterone concentration on Day 0, and the interaction. Progesterone concentrations (Days 0 and 7) and largest ovarian follicle diameter were tested for normality (Kolmogorov-Smirnov test) and homogeneity of variances (Brown-Forsythe test). Progesterone concentrations were submitted to a log transformation due to heterogeneity of variance. Specific mean comparisons were made using the Tukey-Kramer test. A secondary analysis was performed to understand the factors that contribute to variation in largest ovarian follicle diameter. To accomplish this, a model that included treatment, progesterone concentration on Day 0, progesterone concentration on Day 7, and the relevant interactions, with BCS as a covariate, was used. The GLM procedure of SAS was used for this analysis. The model was reduced by sequentially removing nonsignificant (P > 0.10) terms (backwards stepwise regression), with the final model including treatment and progesterone concentration on Day 0. Dependent binomial variables (ovulation, synchronization, conception, and pregnancy rate) were analyzed by logistic regression (PROC LOGISTIC; SAS Institute Inc., Cary, NC, USA [14]) using a model that included treatment (first-use, second-use, and third-use CIDRs), progesterone concentration on Day 0, the interaction, and BCS as a covariate. For conception and pregnancy rate, AI technician and the appropriate interactions were also included in the model. Specific mean comparisons were made using the Bonferroni test. Secondary analyses were performed to understand the relationships of treatment, progesterone concentrations on Days 0 and 7, and largest ovarian follicle diameter with dependent binomial variables. For ovulation rate, the model included treatment, progesterone concentration on Day 0, progesterone concentration on Day 7, and the relevant interactions (with BCS as a covariate). For pregnancy rate, AI technician and interactions were added to the model, and largest ovarian follicle diameter was removed. For synchronization and conception rates, largest ovarian follicle diameter and interactions
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were also added. Logistic regression was used for these analyses, with the model reduced by sequentially removing non-significant (P > 0.10) terms (backwards stepwise regression). In a final analysis, heifers were categorized based on largest ovarian follicle diameter as previously described by Gimenes et al. [15]. Categories were Class 1 (follicles smaller than 8.5 mm), Class 2 (between 8.5 and 10 mm), and Class 3 (larger than 10 mm). Synchronized ovulation rate and conception rate were analyzed using the Logistic procedure of SAS, with treatment, largest ovarian follicle class, and the interaction included in the model. Specific mean comparisons were made using the least square means (LSM) procedure. 2.3.2. Experiment 2 Dependent binomial variables, pregnancy rate after TAI (Experiment 2a), percentage of heifers detected in estrus, and conception rate of these heifers (Experiment 2b) were analyzed by logistic regression (PROC LOGISTIC; SAS Institute Inc.) using a model that included CIDR uses (first, second, or third), day of PGF2a, eCG, AI technician, and the appropriate interactions. The model was reduced by sequentially removing non-significant (P > 0.10) terms (backwards stepwise regression), with the final model for Experiment 2a including CIDR uses, eCG, day of PGF2a, CIDR uses by eCG, and day of PGF2a by eCG. For Experiment 2b, the reduced model included CIDR uses, day of PGF2a, and eCG. Specific mean comparisons were made using the Bonferroni test.
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For both experiments, probability values of a 0.05 were considered significant, whereas probability values of 0.05 < a 0.1 were considered as tendencies. 3. Results 3.1. Experiment 1 3.1.1. Progesterone concentrations and largest ovarian follicle diameter On Day 0, concentration of progesterone did not differ among treatments (2.8 2.4 ng/mL). On Day 7, progesterone concentrations were lower (P < 0.05) in the third-use CIDR group than in the first-use CIDR group; with the second-use CIDR group intermediate (P > 0.1, Table 1). Concentrations of progesterone on Day 7 were not influenced by concentrations of progesterone on Day 0. Diameter of the largest ovarian follicle on Day 11 was greater (P < 0.05) for the thirduse CIDR treatment than for the first-use CIDR treatment and tended (P < 0.10) to be greater than that for the second-use CIDR treatment (Table 1). Additionally, serum progesterone concentrations on Day 0 were associated with largest ovarian follicle diameter (Fig. 1). In the secondary analysis, progesterone concentrations on Day 7 did not influence largest ovarian follicle diameter on Day 11. 3.1.2. Reproductive parameters Neither ovulation (73.6%) nor synchronized ovulation rate (68.7%) were influenced by treatment or progesterone concentration on Day 0 (Table 1). How-
Table 1 Serum progesterone concentration on Day 7, diameter of largest ovarian follicle at TAI, ovulation, synchronized rates of ovulation, conception (synchronized heifers), and pregnancy related to CIDR treatments (Experiment 1). CIDR
Serum P4 on Day 7 4
Follicle diameter at TAI,5 mm (n)
Ovulation rate,6 % (n)
Synchronized ovulation rate,7 % (n)
Conception rate,8 % (n)
Pregnancy rate,9 % (n)
First use1 Second use2 Third use 3 Total
3.0 1.9 A 2.3 1.6A,B 2.0 1.2 B 2.5 1.6
10.1 2.0A (121) 10.6 2.3A,B, a (109) 11.0 2.1B, b (106) 10.5 2.2 (336)
70.5A (103/146) 72.7A (93/128) 78.2A (97/124) 73.6 (293/398)
64.5A (78/121) 67.9A (74/109) 74.5A (79/106) 68.7 (231/336)
37.2a (29/78) 37.8a (28/74) 53.2b (42/79) 42.9 (99/231)
20.5A (30/146) 22.7A (29/128) 35.5B (44/124) 25.9 (103/398)
A,B
Values with different superscripts within a column are different (P < 0.05). Values with different superscripts within a column tended to differ (P < 0.1). 1 CIDR devices with no previous use. 2 CIDR devices with 9 d of previous use. 3 CIDR devices with 18 d of previous use. 4 Raw means SD for serum progesterone concentrations on Day 7 of the protocol. 5 Raw means SD for diameter of largest ovarian follicle at TAI. 6 Percentage of all heifers that ovulated by 48 h after TAI, including heifers that ovulated between CIDR removal and TAI, and within 48 h after TAI, reported as raw mean. 7 Percentage of heifers that ovulated within 48 h after TAI, reported as raw mean. 8 Percentage of synchronized ovulated heifers that became pregnant, reported as raw mean. 9 Percentage of all heifers pregnant after treatment with synchronization protocol, reported as raw mean. a,b
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Fig. 1. Effect (P < 0.05) of serum progesterone concentrations at the initiation of a synchronization protocol on diameter of largest ovarian follicle at TAI in postpuberal Nelore heifers. The pooled SEM value for diameter of the largest ovarian follicle at TAI was 0.22 mm.
Fig. 2. Effect (P < 0.05) of serum progesterone concentration at the initiation of a synchronization protocol on the probability of conception to TAI in postpuberal Nelore heifers.
ever, conception rate tended (P < 0.10) to be greater for the third-use treatment than those for second-use and first-use CIDR treatments. Heifers with greater progesterone concentrations on Day 0 had greater (P < 0.05) conception rate (Fig. 2). Pregnancy rate was also greater (P < 0.05) for the third-use treatment than those for the second-use and first-use CIDR treatments and in heifers with greater concentrations of progesterone on Day 0 (Fig. 3). Neither BCS nor AI technician were a significant source of variation. In the secondary analyses, progesterone on Day 7 did not affect ovulation, synchronization, conception, or pregnancy rate. Diameter of the largest ovarian follicle on
Fig. 4. Effect of largest ovarian follicle diameter at TAI (P < 0.05) on the probability of synchronized ovulation in postpuberal Nelore heifers.
Fig. 5. Effect of diameter of largest ovarian follicle at TAI (P < 0.05) on the probability of conception to TAI in postpuberal Nelore heifers.
Day 11 influenced (P < 0.05) rates of synchronization (Fig. 4) and conception (Fig. 5). When heifers were divided into largest ovarian follicle classifications (Class 1, 2, and 3), synchronized ovulation rate increased (P < 0.05) with each largest ovarian follicle class. Synchronized ovulation rate was greatest for heifers with Class 3 largest ovarian follicle (92.2%; 141 of 153), intermediate for heifers with Class 2 largest ovarian follicle (62.5%; 70 of 112), and least for heifers with Class 1 largest ovarian follicle (28.2%; 20 of 71). A treatment by largest ovarian follicle class interaction was detected (P < 0.05) for conception rate (Table 2). In the first-use and second-use CIDR treatments, conception rate was greater (P < 0.05) in heifers with Class 3 largest ovarian follicle than those for Class 1 or 2. In the third-use CIDR treatment, largest ovarian follicle class did not affect conception rate. 3.2. Experiment 2
Fig. 3. Effect (P < 0.05) of serum progesterone concentration at the initiation of a synchronization protocol on the probability of pregnancy to TAI in postpuberal Nelore heifers.
3.2.1. Experiment 2a Interactions of day of PGF2a and eCG treatment (P < 0.05) and CIDR uses and eCG treatments (P < 0.05) were detected for TAI pregnancy rate (Table 3). The eCG treatment enhanced (P < 0.05) pregnancy rate in heifers that received PGF2a on Day 9
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Table 2 Conception rate (synchronized heifers) related to the diameter of largest ovarian follicle at TAI classes within each CIDR treatment (Experiment 1). Conception rate,4 % (n)
CIDR
Class 1 1
First use Second use2 Third use 3
5
P Value Class 2
a
6
Class 3 a
9.1 (1/11) 0 (0/4)a 40.0 (2/5)a
24.0 (6/25) 21.7 (5/23)a 54.5 (12/22)a
7
52.4 (22/42)b 48.9 (23/47)b 53.8 (28/52)a
<0.05 <0.05 >0.10
Class = classification of largest ovarian follicle at TAI. a,b Values with different superscripts within a row are different (P < 0.05). 1 CIDR devices with no previous use. 2 CIDR devices with 9 d of previous use. 3 CIDR devices with 18 d of previous use. 4 Percentage of heifers that ovulated within 48 h after TAI that became pregnant, reported as raw means. 5 Heifers with follicles <8.5 mm at TAI. 6 Heifers with follicles between 8.5 and 10.0 mm at TAI. 7 Heifers with follicles >10.0 mm at TAI.
but not in those that received PGF2a on Day 7. The eCG treatment also increased pregnancy rate in heifers receiving the first-use CIDR (P < 0.05); tended to increase pregnancy rate with a second-use CIDR (P < 0.1); but had no influence on pregnancy rate in the third-use CIDR treatment. 3.2.2. Experiment 2b Overall rates of estrus (61.9%; 125 of 202), conception (64.8%; 81 of 125), and pregnancy Table 3 Pregnancy rate in postpuberal Nelore heifers at TAI protocol, with PGF2a on Days 7 or 9, with or without an eCG treatment, and with CIDR treatments of varying previous uses (Experiment 2a). Treatments
PGF2a Day 7 1 Day 9 2 CIDR First use3 Second use4 Third use 5
Pregnancy at TAI,6 % (n) Without eCG7
With eCG8
33.3 (16/48)A 26.4 (14/53)A
40.4 (21/52)A 50.0 (23/46)B
21.9 (7/32)A 24.3 (9/37)a 43.7 (14/32)A
50.0 (18/36)B 42.4 (14/33)b 41.4 (12/29)A
A,B Values for the same variable with different superscripts within a row are different (P < 0.05). a,b Values for the same variable with different superscripts within a row tended to differ (P < 0.1). 1 PGF2a (12.5 mg, im) treatment on Day 7 of synchronization protocol. 2 PGF2a (12.5 mg, im) treatment on Day 9 of synchronization protocol. 3 CIDR devices with no previous use. 4 CIDR devices with 9 d of previous use. 5 CIDR devices with 18 d of previous use. 6 Percentage of all heifers that became pregnant after treatment with synchronization protocol, reported as raw means. 7 No eCG treatment on Day 9 of synchronization protocol. 8 eCG treatment (300 IU, im) on Day 9 of synchronization protocol.
(40.1%; 81 of 202) were not affected by the CIDR, PGF2a, or eCG treatments. 4. Discussion The TAI pregnancy rate was increased when either progesterone was decreased during the synchronization protocol or eCG was administered. None of the manipulations negatively influenced fertility if the heifers were permitted to exhibit estrus and AI was performed based on detection of this physiologic end point. In the first experiment, pregnancy rate to TAI was greater in heifers treated with the third-use CIDR than with other CIDR treatments. In addition, concentrations of progesterone were different on Day 7. These differences were similar to those of previous findings in our laboratory [16]. A statistical relationship between progesterone concentration on Day 7 and pregnancy rate was not evident, suggesting that the influence of the CIDR on progesterone at this time was not a primary determinant of fertility. Because progesterone concentrations after Day 7 would be largely determined by type of CIDR (due to luteal regression), it is logical to presume that the primary effect of the type of CIDR would be emphasized from Days 7 to 9. That gonadotropic support of the follicle may have been enhanced in the third-use CIDR treatment was reinforced by the increase in follicular diameter at TAI that was detected in this treatment. Because the interval from insertion of a CIDR and estradiol treatment to follicular wave emergence was 3.1 d in Bos indicus [4], and deviation occurred 1.6 d after wave emergence, within diameter of the largest ovarian follicle of 5.9 mm at the beginning of deviation [17] or
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6.2 mm [15], and this is when dominant follicles acquire LH receptors [18], the ability of increased gonadotropic support (due to decreased progesterone) to enhance follicular maturation may have be optimized. Larger follicles at the time of AI and conception and pregnancy rates to TAI were associated with high progesterone concentrations on Day 0. In the current study, the Day 0 progesterone concentration sample was collected before the insertion of CIDR devices, and it is related to the endogenous progesterone. We inferred that heifers with greater progesterone concentrations at the outset of the protocol were in later stages of the estrous cycle, and thus, spontaneous luteal regression occurred during the protocol and contributed to this response. Follicular diameter at TAI was related to synchronization and conception rates across treatments. Heifers with larger follicles were more likely to ovulate at the appropriate time and conceive. Relationships between largest ovarian follicle diameter and synchronized ovulation heifers and conception rate were revealed when heifers were categorized into largest ovarian follicle diameter classes. Synchronized ovulation rate increased as largest ovarian follicle diameter increased, regardless of CIDR treatment. Within the third-use CIDR treatment, largest ovarian follicle classification did not influence conception rate, whereas with first-use and second-use CIDRs, conception was reduced in heifers with smaller ovarian follicles at TAI. We inferred that if progesterone concentrations during the later stages of the estrous synchronization protocol are reduced, the range of largest ovarian follicle diameters that results in acceptable fertility is increased. We speculate that when progesterone concentrations are reduced, gonadotropic stimulus is enhanced, most likely through LH concentrations, improving follicle maturity and increasing fertility. In Experiment 2a, the effect of eCG was dependent on timing of PGF2a administration (Days 7 or 9) and CIDR type. The reduced fertility in heifers with high progesterone concentrations (i.e., PGF2a on Day 9 or first-use and second-use CIDRs) could be overcome by administration of eCG at CIDR withdrawal. Strategies to reduce circulating progesterone concentration during later stages of a TAI synchronization protocol and/or increase gonadotropic stimulus improved reproductive performance in postpuberal Nelore heifers, and conversely, those that resulted in greater progesterone concentrations in the later stages of the TAI protocol negatively affected reproductive
performance. Timing of this adjustment in progesterone concentrations may be important, as a recent report in Nelore heifers demonstrated that treatment with PGF2a concurrent with CIDR insertion did not improve pregnancy rate using TAI [2]. The range of ovarian follicle diameters that resulted in acceptable fertility appeared to be extended in heifers with the lesser progesterone concentrations. We speculate that LH secretion in Nelore heifers is highly sensitive to progesterone negative feedback and that use of low concentrations of progesterone increases gonadotropic support for follicular maturation. It also suggests that synchronization protocols in postpuberal Nelore heifers should be designed to reduce concentrations of progesterone during maturation of the ovulatory follicle. In conclusion, postpuberal Nelore heifers need a period of reduced progesterone concentrations, as physiologically occurs in their estrous cycle, during the later stages of a synchronization protocol. Furthermore, gonadotropin treatment may be useful to optimize pregnancy rate to TAI in Nelore heifers treated with progesterone devices that will increase circulating progesterone to more than high physiologic concentrations. References [1] Pinheiro OL, Barros CM, Figueiredo RA, Valle ER, Encarnac¸a˜o RO, Padovani CR. Estrous behavior and estrous to ovulation interval in Nelore cattle Bos indicus with natural estrous or estrous induced with prostaglandin or norgestomet and estradiol valerate. Theriogenology 1998;49:667–81. [2] Marques MO, Sa´ Filho MF, Gimenes LU, Figueiredo TB, Soria GF, Baruselli PS. Efeito do tratamento com PGF2a na inserc¸a˜o e/ ou tratamento com eCG na remoc¸a˜o do dispositivo intravaginal de progesterona na taxa de concepc¸a˜o a` inseminac¸a˜o artificial em tempo fixo em novilhas Nelore. Acta Sci Vet 2005;1:287 [abstract]. [3] Meneghetti M, Miguel JC. Addition of eCG on a fixed timed artificial insemination protocol in the conception rate of cycling Nelore heifers. Acta Sci Vet 2008;36:638 [abstract]. [4] Carvalho JBP, Carvalho NAT, Reis EL, Nichi M, Souza AH, Baruselli PS. Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus x Bos taurus and Bos taurus heifers. Theriogenology 2008;69:167–75. [5] Kinder JE, Kojima FN, Bergfeld EG, Whermen ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996;74:1424–40. [6] Taponen J, Kulcsar M, Katila T, Katai L, Huzenicza G, Rodriguez-Martinez H. Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cycling dairy cows. Theriogenology 2002;58:1291–302. [7] Taponen J, Hjerppe P, Kopra E, Rodrı´guez-Martı´nez H, Katila T, Kindhal H. Premature prostaglandin F2a secretion causes luteal
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[13] Santos RM, Vasconcelos JLM. Concentrate intake and plasmatic progesterone concentration in Holstein cows. Braz J Vet Anim Sci 2006;58:1162–7. [14] SAS Institute Inc. SAS/STAT1 User’s Guide (Release 8.2). SAS Institute Inc., 2007. [15] Gimenes LU, Sa´ Filho MF, Carvalho NAT, Torres-Ju´nior JRS, Souza AH, Madureira EH, et al. Follicle deviation and ovulatory capacity in Bos indicus heifers. Theriogenology 2008;69:852–8. [16] Sa Filho OG, Vasconcelos JLM. Using a 27 days previously used intravaginal progesterone device (CIDR) in a synchronization of ovulation protocol in post partum Nelore cows. Acta Sci Vet 2008;36:615 [abstract]. [17] Sartorelli ES, Carvalho LM, Bergfelt DR, Ginther OJ, Barros CM. Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows. Theriogenology 2005;63:2382–94. [18] Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol Reprod 1996;55:1187–94.
Theriogenology 72 (2009) 378–385 www.theriojournal.com
Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F2a treatment affect fertility in postpuberal Nelore heifers C.C. Dias a, F.S. Wechsler a, M.L. Day b, J.L.M. Vasconcelos a,* a
Departamento de Produc¸a˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, FMVZ-UNESP, CEP 18618-000, Botucatu, SP, Brazil b Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA Received 10 September 2008; received in revised form 4 March 2009; accepted 28 March 2009
Abstract Two experiments were performed to test the hypothesis that elevated progesterone concentrations impair pregnancy rate to timed artificial insemination (TAI) in postpuberal Nelore heifers. In Experiment 1, postpuberal Nelore heifers (n = 398) received 2 mg estradiol benzoate (EB) and either a new progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) (first use) or a CIDR previously used for 9 d (second use) or for 18 d (third use) on Day 0, 12.5 mg prostaglandin F2a (PGF2a) on Day 7, 0.5 mg estradiol cypionate (ECP) and CIDR withdrawal on Day 9, and TAI on Day 11. Largest ovarian follicle diameter was determined on Day 11. The third-use CIDR treatment increased largest ovarian follicle diameter and pregnancy rate. Conception to TAI was reduced in heifers with smaller follicles in the first- and second-use CIDR treatments, but not in the third-use CIDR treatment. In Experiment 2, postpuberal Nelore heifers received the synchronization treatment described in Experiment 1 or received 12.5 mg PGF2a on Day 9 rather than Day 7. In addition, 50% of heifers received 300 IU equine chorionic gonadotropin (eCG) on Day 9. Heifers were either TAI (Experiment 2a; n = 199) or AI after detection of estrus (Experiment 2b; n = 125 of 202). In Experiment 2a, treatment with eCG increased pregnancy rate to TAI in heifers that received PGF2a on Day 9 but not on Day 7 and in heifers that received a first-use CIDR but not in heifers that received a third-use CIDR. Treatments did not influence reproductive performance in Experiment 2b. In summary, pregnancy rate to TAI in postpuberal Nelore heifers was optimized when lower concentrations of exogenous progesterone were administered, and eCG treatment was beneficial in heifers expected to have greater progesterone concentrations. # 2009 Elsevier Inc. All rights reserved. Keywords: Cattle; Pregnancy; Proestrus; Progesterone; Timed artificial insemination
1. Introduction Artificial insemination (AI) is the most effective technique available to introduce new genetics into a
* Corresponding author. Tel.: +55 14 38117185; fax: +55 14 38117180. E-mail address: [email protected] (J.L.M. Vasconcelos). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.03.006
beef cattle herd, and timed AI (TAI) protocols have been developed to overcome challenges associated with estrus detection failure. There is a need for development of TAI protocols for Bos indicus breeds because of a shorter duration of estrous behavior compared with that of Bos taurus breeds [1]. Low TAI pregnancy rate was reported in Nelore heifers in studies in which the estrous synchronization protocol was initiated with estradiol administration and
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insertion of a progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) [2,3]. Carvalho et al. [4] compared the interval from CIDR insertion and estradiol treatment to follicular wave emergence in Bos indicus (3.1 d), Bos indicus Bos taurus (3.3 d), and Bos taurus (3.2 d) heifers. Although the time of follicle emergence was similar among groups, Bos indicus heifers had the smallest dominant follicles at the end of the CIDR treatment. A lower growth rate of dominant follicles in the Bos indicus heifers appeared to be associated with greater circulating progesterone concentrations during CIDR treatment. These results suggested that high concentrations of progesterone during estrous synchronization protocol may be detrimental to follicular development in Bos indicus cattle. Because luteinizing hormone (LH) secretion and pulse frequency are negatively associated with progesterone concentration [5], perhaps endogenous gonadotropic support for follicular development may be compromised in progesterone-treated Bos indicus heifers, as described by Carvalho et al. [4]. An alternative for increasing gonadotropic support of dominant follicles before TAI is a longer interval from luteal regression to the LH surge and TAI (i.e., ‘‘proestrus’’) than that of traditional synchronization protocols. Pregnancy rate to AI has been demonstrated to be greater in cows that had a long proestrus [6–10]. Use of equine chorionic gonadotropin (eCG) is a second alternative to increase follicle development; eCG treatment increased conception rate to TAI when given at the end of a CIDR treatment in anestrous cows [11]. A third alternative may be decreasing progesterone concentrations during the synchronization protocol. The goal of the current experiments was to determine whether treatments that should increase gonadotropic support of follicular development will increase fertility in Bos indicus heifers. The overall hypothesis for these experiments was that low pregnancy rates to TAI previously reported in Nelore heifers were due to inhibition of LH secretion and in turn development of the ovulatory follicle caused by elevated systemic progesterone concentrations. The objective of the first experiment was to compare the effect of various concentrations of progesterone during a synchronization protocol on pregnancy rate to TAI by using CIDRs that were expected to deliver different concentrations of progesterone. In the second experiment, progesterone concentrations were altered by either changing the timing of luteal regression during a synchronization protocol and/or using CIDRs that would deliver different doses of progesterone. In addition, treatment with eCG was used to evaluate the effect of an exogenous gonadotropic support. The
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influence of these factors on follicular development was assessed in both TAI and AI after estrus detection. 2. Materials and methods 2.1. Experimental design, location, and heifers 2.1.1. Experiment 1 This trial was conducted between November and December 2005 in Mato Grosso do Sul, Brazil. Nelore heifers (n = 525), 24 to 26 mo of age, with body condition scores (BCSs) between 2.75 and 3.25 (1 to 5 scale [12]) were used. Heifers were maintained on two pastures (Brachiaria humidı´cola) with mineral salt ad libitum. Their ovaries were evaluated by ultrasonography for presence of a corpus luteum (CL; Aloka SSD 500 V, equipped with a 7.5-MHz linear array intrarectal transducer; Aloka, Tokyo, Japan) 7 d before the initiation of the protocol. Heifers with luteal tissue in their ovaries (n = 398) were randomly assigned to receive a new progesterone-releasing intravaginal device containing 1.9 g progesterone (CIDR; Pfizer Animal Health, Sa˜o Paulo, SP, Brazil; new CIDR; first use), a CIDR that had been used previously in a 9-d synchronization protocol (second use), or a CIDR that had been used in two previous 9-d synchronization protocols (third use). All heifers were treated with 2 mg estradiol benzoate (EB) im (Estrogin; Farmavet, Sa˜o Paulo, SP, Brazil) at CIDR insertion (Day 0 of the experiment). Blood samples for progesterone analyses were collected on Days 0 and 7, and prostaglandin F2a (PGF2a) was administered (12.5 mg, im; Lutalyse; Pfizer Animal Health) on Day 7. On Day 9, CIDRs were withdrawn, and heifers received estradiol cypionate (0.5 mg, im; estradiol cypionate (ECP); Pfizer Animal Health). Heifers were bred by TAI on Day 11 (48 h after CIDR withdrawal) by two technicians using frozenthawed semen from a single sire. Transrectal ultrasonography examinations were performed on Day 11 to assess diameter of largest ovarian follicle at TAI (defined as the average between horizontal and vertical diameters). Heifers in which the largest ovarian follicle was <6 mm were recorded as a missing observation for this variable. Ultrasonography was also performed on Day 13 to evaluate ovulation (absence of the largest ovarian follicle previously measured at TAI) and on Day 41 to detect pregnancy. Reproductive variables that were determined included ovulation, synchronization, conception, and pregnancy rates. Ovulation rate was defined as the proportion of all heifers that ovulated between Days 11 and 13, including heifers that were considered to have prematurely ovulated (presence of
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multiple small follicles that were <6 mm in diameter at TAI). Synchronized ovulation rate was defined as the proportion of all heifers that ovulated between Days 11 and 13. Conception rate was calculated as the proportion of synchronized heifers that were pregnant on Day 41, and pregnancy rate was calculated as the proportion of all heifers that were pregnant at that time. 2.1.2. Experiment 2 This experiment was conducted between November 2006 and January 2007 in Mato Grosso do Sul, Brazil. Nelore heifers (n = 650), 24 to 26 mo of age, with BCSs between 2.75 and 3.25 were observed for estrus for 21 d before initiation of the synchronization program. Heifers detected in estrus (n = 401) were used in the experiment, as they were considered as to be reproductively mature. Heifers were maintained on two pastures (Brachiaria humidı´cola) with mineral salt provided ad libitum. On Day 0, all heifers received 2 mg EB im and were randomly assigned to receive either a first-use, second-use, or third-use CIDR as described in Experiment 1, PGF2a (12.5 mg, im) given on Day 7 or Day 9, and either no eCG or 300 IU eCG im (Folligon; Intervet, Sa˜o Paulo, SP, Brazil) on Day 9. Heifers from the two pastures had their CIDRs withdrawn on Day 9 and were randomly assigned either to receive ECP (0.5 mg, im) at this time, followed by TAI on Day 11 (Experiment 2a; n = 199), or to be artificially inseminated 12 h after detection of estrus between Days 9 and 15 (Experiment 2b; n = 202). All heifers were artificially inseminated by two technicians using frozen-thawed semen from a single sire. Pregnancy diagnoses were performed by transrectal ultrasonography 30 d after TAI. 2.2. Blood samples and progesterone RIA analyses 2.2.1. Experiment 1 Blood samples for progesterone analyses were collected from a coccygeal vessel into Vacutainer tubes (Becton Dickinson Co., Franklin Lakes, NJ, USA). Blood was allowed to clot at 4 8C for 24 h and centrifuged at 1500 g for 15 min at room temperature. Serum was removed and frozen at 20 8C until assays were performed. Serum progesterone concentrations were determined using a solid-phase radioimmunoassay kit containing antibody-coated tubes and 125I-labeled progesterone (Coat-a-count; Diagnostic Products Corporation, Los Angeles, CA, USA) that had been previously validated in our laboratory [13]. The intra-assay coefficients of variation were 6.7% and 8.5% for assays 1 (Day 0) and 2 (Day 7), respectively,
and the interassay coefficient of variation was 5.4%. Sensitivity was 0.01 ng/mL. 2.3. Statistical analyses 2.3.1. Experiment 1 Continuous dependent variables (i.e., progesterone concentration on Day 7 and diameter of the largest ovarian follicle on Day 11) were analyzed by leastsquares (PROC GLM; SAS Institute Inc., Cary, NC, USA [14]) using a model that included treatment (firstuse, second-use, and third-use CIDRs), progesterone concentration on Day 0, and the interaction. Progesterone concentrations (Days 0 and 7) and largest ovarian follicle diameter were tested for normality (Kolmogorov-Smirnov test) and homogeneity of variances (Brown-Forsythe test). Progesterone concentrations were submitted to a log transformation due to heterogeneity of variance. Specific mean comparisons were made using the Tukey-Kramer test. A secondary analysis was performed to understand the factors that contribute to variation in largest ovarian follicle diameter. To accomplish this, a model that included treatment, progesterone concentration on Day 0, progesterone concentration on Day 7, and the relevant interactions, with BCS as a covariate, was used. The GLM procedure of SAS was used for this analysis. The model was reduced by sequentially removing nonsignificant (P > 0.10) terms (backwards stepwise regression), with the final model including treatment and progesterone concentration on Day 0. Dependent binomial variables (ovulation, synchronization, conception, and pregnancy rate) were analyzed by logistic regression (PROC LOGISTIC; SAS Institute Inc., Cary, NC, USA [14]) using a model that included treatment (first-use, second-use, and third-use CIDRs), progesterone concentration on Day 0, the interaction, and BCS as a covariate. For conception and pregnancy rate, AI technician and the appropriate interactions were also included in the model. Specific mean comparisons were made using the Bonferroni test. Secondary analyses were performed to understand the relationships of treatment, progesterone concentrations on Days 0 and 7, and largest ovarian follicle diameter with dependent binomial variables. For ovulation rate, the model included treatment, progesterone concentration on Day 0, progesterone concentration on Day 7, and the relevant interactions (with BCS as a covariate). For pregnancy rate, AI technician and interactions were added to the model, and largest ovarian follicle diameter was removed. For synchronization and conception rates, largest ovarian follicle diameter and interactions
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were also added. Logistic regression was used for these analyses, with the model reduced by sequentially removing non-significant (P > 0.10) terms (backwards stepwise regression). In a final analysis, heifers were categorized based on largest ovarian follicle diameter as previously described by Gimenes et al. [15]. Categories were Class 1 (follicles smaller than 8.5 mm), Class 2 (between 8.5 and 10 mm), and Class 3 (larger than 10 mm). Synchronized ovulation rate and conception rate were analyzed using the Logistic procedure of SAS, with treatment, largest ovarian follicle class, and the interaction included in the model. Specific mean comparisons were made using the least square means (LSM) procedure. 2.3.2. Experiment 2 Dependent binomial variables, pregnancy rate after TAI (Experiment 2a), percentage of heifers detected in estrus, and conception rate of these heifers (Experiment 2b) were analyzed by logistic regression (PROC LOGISTIC; SAS Institute Inc.) using a model that included CIDR uses (first, second, or third), day of PGF2a, eCG, AI technician, and the appropriate interactions. The model was reduced by sequentially removing non-significant (P > 0.10) terms (backwards stepwise regression), with the final model for Experiment 2a including CIDR uses, eCG, day of PGF2a, CIDR uses by eCG, and day of PGF2a by eCG. For Experiment 2b, the reduced model included CIDR uses, day of PGF2a, and eCG. Specific mean comparisons were made using the Bonferroni test.
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For both experiments, probability values of a 0.05 were considered significant, whereas probability values of 0.05 < a 0.1 were considered as tendencies. 3. Results 3.1. Experiment 1 3.1.1. Progesterone concentrations and largest ovarian follicle diameter On Day 0, concentration of progesterone did not differ among treatments (2.8 2.4 ng/mL). On Day 7, progesterone concentrations were lower (P < 0.05) in the third-use CIDR group than in the first-use CIDR group; with the second-use CIDR group intermediate (P > 0.1, Table 1). Concentrations of progesterone on Day 7 were not influenced by concentrations of progesterone on Day 0. Diameter of the largest ovarian follicle on Day 11 was greater (P < 0.05) for the thirduse CIDR treatment than for the first-use CIDR treatment and tended (P < 0.10) to be greater than that for the second-use CIDR treatment (Table 1). Additionally, serum progesterone concentrations on Day 0 were associated with largest ovarian follicle diameter (Fig. 1). In the secondary analysis, progesterone concentrations on Day 7 did not influence largest ovarian follicle diameter on Day 11. 3.1.2. Reproductive parameters Neither ovulation (73.6%) nor synchronized ovulation rate (68.7%) were influenced by treatment or progesterone concentration on Day 0 (Table 1). How-
Table 1 Serum progesterone concentration on Day 7, diameter of largest ovarian follicle at TAI, ovulation, synchronized rates of ovulation, conception (synchronized heifers), and pregnancy related to CIDR treatments (Experiment 1). CIDR
Serum P4 on Day 7 4
Follicle diameter at TAI,5 mm (n)
Ovulation rate,6 % (n)
Synchronized ovulation rate,7 % (n)
Conception rate,8 % (n)
Pregnancy rate,9 % (n)
First use1 Second use2 Third use 3 Total
3.0 1.9 A 2.3 1.6A,B 2.0 1.2 B 2.5 1.6
10.1 2.0A (121) 10.6 2.3A,B, a (109) 11.0 2.1B, b (106) 10.5 2.2 (336)
70.5A (103/146) 72.7A (93/128) 78.2A (97/124) 73.6 (293/398)
64.5A (78/121) 67.9A (74/109) 74.5A (79/106) 68.7 (231/336)
37.2a (29/78) 37.8a (28/74) 53.2b (42/79) 42.9 (99/231)
20.5A (30/146) 22.7A (29/128) 35.5B (44/124) 25.9 (103/398)
A,B
Values with different superscripts within a column are different (P < 0.05). Values with different superscripts within a column tended to differ (P < 0.1). 1 CIDR devices with no previous use. 2 CIDR devices with 9 d of previous use. 3 CIDR devices with 18 d of previous use. 4 Raw means SD for serum progesterone concentrations on Day 7 of the protocol. 5 Raw means SD for diameter of largest ovarian follicle at TAI. 6 Percentage of all heifers that ovulated by 48 h after TAI, including heifers that ovulated between CIDR removal and TAI, and within 48 h after TAI, reported as raw mean. 7 Percentage of heifers that ovulated within 48 h after TAI, reported as raw mean. 8 Percentage of synchronized ovulated heifers that became pregnant, reported as raw mean. 9 Percentage of all heifers pregnant after treatment with synchronization protocol, reported as raw mean. a,b
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Fig. 1. Effect (P < 0.05) of serum progesterone concentrations at the initiation of a synchronization protocol on diameter of largest ovarian follicle at TAI in postpuberal Nelore heifers. The pooled SEM value for diameter of the largest ovarian follicle at TAI was 0.22 mm.
Fig. 2. Effect (P < 0.05) of serum progesterone concentration at the initiation of a synchronization protocol on the probability of conception to TAI in postpuberal Nelore heifers.
ever, conception rate tended (P < 0.10) to be greater for the third-use treatment than those for second-use and first-use CIDR treatments. Heifers with greater progesterone concentrations on Day 0 had greater (P < 0.05) conception rate (Fig. 2). Pregnancy rate was also greater (P < 0.05) for the third-use treatment than those for the second-use and first-use CIDR treatments and in heifers with greater concentrations of progesterone on Day 0 (Fig. 3). Neither BCS nor AI technician were a significant source of variation. In the secondary analyses, progesterone on Day 7 did not affect ovulation, synchronization, conception, or pregnancy rate. Diameter of the largest ovarian follicle on
Fig. 4. Effect of largest ovarian follicle diameter at TAI (P < 0.05) on the probability of synchronized ovulation in postpuberal Nelore heifers.
Fig. 5. Effect of diameter of largest ovarian follicle at TAI (P < 0.05) on the probability of conception to TAI in postpuberal Nelore heifers.
Day 11 influenced (P < 0.05) rates of synchronization (Fig. 4) and conception (Fig. 5). When heifers were divided into largest ovarian follicle classifications (Class 1, 2, and 3), synchronized ovulation rate increased (P < 0.05) with each largest ovarian follicle class. Synchronized ovulation rate was greatest for heifers with Class 3 largest ovarian follicle (92.2%; 141 of 153), intermediate for heifers with Class 2 largest ovarian follicle (62.5%; 70 of 112), and least for heifers with Class 1 largest ovarian follicle (28.2%; 20 of 71). A treatment by largest ovarian follicle class interaction was detected (P < 0.05) for conception rate (Table 2). In the first-use and second-use CIDR treatments, conception rate was greater (P < 0.05) in heifers with Class 3 largest ovarian follicle than those for Class 1 or 2. In the third-use CIDR treatment, largest ovarian follicle class did not affect conception rate. 3.2. Experiment 2
Fig. 3. Effect (P < 0.05) of serum progesterone concentration at the initiation of a synchronization protocol on the probability of pregnancy to TAI in postpuberal Nelore heifers.
3.2.1. Experiment 2a Interactions of day of PGF2a and eCG treatment (P < 0.05) and CIDR uses and eCG treatments (P < 0.05) were detected for TAI pregnancy rate (Table 3). The eCG treatment enhanced (P < 0.05) pregnancy rate in heifers that received PGF2a on Day 9
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Table 2 Conception rate (synchronized heifers) related to the diameter of largest ovarian follicle at TAI classes within each CIDR treatment (Experiment 1). Conception rate,4 % (n)
CIDR
Class 1 1
First use Second use2 Third use 3
5
P Value Class 2
a
6
Class 3 a
9.1 (1/11) 0 (0/4)a 40.0 (2/5)a
24.0 (6/25) 21.7 (5/23)a 54.5 (12/22)a
7
52.4 (22/42)b 48.9 (23/47)b 53.8 (28/52)a
<0.05 <0.05 >0.10
Class = classification of largest ovarian follicle at TAI. a,b Values with different superscripts within a row are different (P < 0.05). 1 CIDR devices with no previous use. 2 CIDR devices with 9 d of previous use. 3 CIDR devices with 18 d of previous use. 4 Percentage of heifers that ovulated within 48 h after TAI that became pregnant, reported as raw means. 5 Heifers with follicles <8.5 mm at TAI. 6 Heifers with follicles between 8.5 and 10.0 mm at TAI. 7 Heifers with follicles >10.0 mm at TAI.
but not in those that received PGF2a on Day 7. The eCG treatment also increased pregnancy rate in heifers receiving the first-use CIDR (P < 0.05); tended to increase pregnancy rate with a second-use CIDR (P < 0.1); but had no influence on pregnancy rate in the third-use CIDR treatment. 3.2.2. Experiment 2b Overall rates of estrus (61.9%; 125 of 202), conception (64.8%; 81 of 125), and pregnancy Table 3 Pregnancy rate in postpuberal Nelore heifers at TAI protocol, with PGF2a on Days 7 or 9, with or without an eCG treatment, and with CIDR treatments of varying previous uses (Experiment 2a). Treatments
PGF2a Day 7 1 Day 9 2 CIDR First use3 Second use4 Third use 5
Pregnancy at TAI,6 % (n) Without eCG7
With eCG8
33.3 (16/48)A 26.4 (14/53)A
40.4 (21/52)A 50.0 (23/46)B
21.9 (7/32)A 24.3 (9/37)a 43.7 (14/32)A
50.0 (18/36)B 42.4 (14/33)b 41.4 (12/29)A
A,B Values for the same variable with different superscripts within a row are different (P < 0.05). a,b Values for the same variable with different superscripts within a row tended to differ (P < 0.1). 1 PGF2a (12.5 mg, im) treatment on Day 7 of synchronization protocol. 2 PGF2a (12.5 mg, im) treatment on Day 9 of synchronization protocol. 3 CIDR devices with no previous use. 4 CIDR devices with 9 d of previous use. 5 CIDR devices with 18 d of previous use. 6 Percentage of all heifers that became pregnant after treatment with synchronization protocol, reported as raw means. 7 No eCG treatment on Day 9 of synchronization protocol. 8 eCG treatment (300 IU, im) on Day 9 of synchronization protocol.
(40.1%; 81 of 202) were not affected by the CIDR, PGF2a, or eCG treatments. 4. Discussion The TAI pregnancy rate was increased when either progesterone was decreased during the synchronization protocol or eCG was administered. None of the manipulations negatively influenced fertility if the heifers were permitted to exhibit estrus and AI was performed based on detection of this physiologic end point. In the first experiment, pregnancy rate to TAI was greater in heifers treated with the third-use CIDR than with other CIDR treatments. In addition, concentrations of progesterone were different on Day 7. These differences were similar to those of previous findings in our laboratory [16]. A statistical relationship between progesterone concentration on Day 7 and pregnancy rate was not evident, suggesting that the influence of the CIDR on progesterone at this time was not a primary determinant of fertility. Because progesterone concentrations after Day 7 would be largely determined by type of CIDR (due to luteal regression), it is logical to presume that the primary effect of the type of CIDR would be emphasized from Days 7 to 9. That gonadotropic support of the follicle may have been enhanced in the third-use CIDR treatment was reinforced by the increase in follicular diameter at TAI that was detected in this treatment. Because the interval from insertion of a CIDR and estradiol treatment to follicular wave emergence was 3.1 d in Bos indicus [4], and deviation occurred 1.6 d after wave emergence, within diameter of the largest ovarian follicle of 5.9 mm at the beginning of deviation [17] or
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6.2 mm [15], and this is when dominant follicles acquire LH receptors [18], the ability of increased gonadotropic support (due to decreased progesterone) to enhance follicular maturation may have be optimized. Larger follicles at the time of AI and conception and pregnancy rates to TAI were associated with high progesterone concentrations on Day 0. In the current study, the Day 0 progesterone concentration sample was collected before the insertion of CIDR devices, and it is related to the endogenous progesterone. We inferred that heifers with greater progesterone concentrations at the outset of the protocol were in later stages of the estrous cycle, and thus, spontaneous luteal regression occurred during the protocol and contributed to this response. Follicular diameter at TAI was related to synchronization and conception rates across treatments. Heifers with larger follicles were more likely to ovulate at the appropriate time and conceive. Relationships between largest ovarian follicle diameter and synchronized ovulation heifers and conception rate were revealed when heifers were categorized into largest ovarian follicle diameter classes. Synchronized ovulation rate increased as largest ovarian follicle diameter increased, regardless of CIDR treatment. Within the third-use CIDR treatment, largest ovarian follicle classification did not influence conception rate, whereas with first-use and second-use CIDRs, conception was reduced in heifers with smaller ovarian follicles at TAI. We inferred that if progesterone concentrations during the later stages of the estrous synchronization protocol are reduced, the range of largest ovarian follicle diameters that results in acceptable fertility is increased. We speculate that when progesterone concentrations are reduced, gonadotropic stimulus is enhanced, most likely through LH concentrations, improving follicle maturity and increasing fertility. In Experiment 2a, the effect of eCG was dependent on timing of PGF2a administration (Days 7 or 9) and CIDR type. The reduced fertility in heifers with high progesterone concentrations (i.e., PGF2a on Day 9 or first-use and second-use CIDRs) could be overcome by administration of eCG at CIDR withdrawal. Strategies to reduce circulating progesterone concentration during later stages of a TAI synchronization protocol and/or increase gonadotropic stimulus improved reproductive performance in postpuberal Nelore heifers, and conversely, those that resulted in greater progesterone concentrations in the later stages of the TAI protocol negatively affected reproductive
performance. Timing of this adjustment in progesterone concentrations may be important, as a recent report in Nelore heifers demonstrated that treatment with PGF2a concurrent with CIDR insertion did not improve pregnancy rate using TAI [2]. The range of ovarian follicle diameters that resulted in acceptable fertility appeared to be extended in heifers with the lesser progesterone concentrations. We speculate that LH secretion in Nelore heifers is highly sensitive to progesterone negative feedback and that use of low concentrations of progesterone increases gonadotropic support for follicular maturation. It also suggests that synchronization protocols in postpuberal Nelore heifers should be designed to reduce concentrations of progesterone during maturation of the ovulatory follicle. In conclusion, postpuberal Nelore heifers need a period of reduced progesterone concentrations, as physiologically occurs in their estrous cycle, during the later stages of a synchronization protocol. Furthermore, gonadotropin treatment may be useful to optimize pregnancy rate to TAI in Nelore heifers treated with progesterone devices that will increase circulating progesterone to more than high physiologic concentrations. References [1] Pinheiro OL, Barros CM, Figueiredo RA, Valle ER, Encarnac¸a˜o RO, Padovani CR. Estrous behavior and estrous to ovulation interval in Nelore cattle Bos indicus with natural estrous or estrous induced with prostaglandin or norgestomet and estradiol valerate. Theriogenology 1998;49:667–81. [2] Marques MO, Sa´ Filho MF, Gimenes LU, Figueiredo TB, Soria GF, Baruselli PS. Efeito do tratamento com PGF2a na inserc¸a˜o e/ ou tratamento com eCG na remoc¸a˜o do dispositivo intravaginal de progesterona na taxa de concepc¸a˜o a` inseminac¸a˜o artificial em tempo fixo em novilhas Nelore. Acta Sci Vet 2005;1:287 [abstract]. [3] Meneghetti M, Miguel JC. Addition of eCG on a fixed timed artificial insemination protocol in the conception rate of cycling Nelore heifers. Acta Sci Vet 2008;36:638 [abstract]. [4] Carvalho JBP, Carvalho NAT, Reis EL, Nichi M, Souza AH, Baruselli PS. Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus x Bos taurus and Bos taurus heifers. Theriogenology 2008;69:167–75. [5] Kinder JE, Kojima FN, Bergfeld EG, Whermen ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996;74:1424–40. [6] Taponen J, Kulcsar M, Katila T, Katai L, Huzenicza G, Rodriguez-Martinez H. Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cycling dairy cows. Theriogenology 2002;58:1291–302. [7] Taponen J, Hjerppe P, Kopra E, Rodrı´guez-Martı´nez H, Katila T, Kindhal H. Premature prostaglandin F2a secretion causes luteal
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