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Theriogenology 81 (2014) 446–453

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Theriogenology

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Effect of progesterone concentrations, follicle diameter,timing of artificial insemination, and ovulatory stimulus onpregnancy rate to synchronized artificial insemination inpostpubertal Nellore heifers

T. Martins a, R.F.G. Peres a, A.D.P. Rodrigues a, K.G. Pohler b, M.H.C. Pereira a,M.L. Day c, J.L.M. Vasconcelos a,*

aDepartamento de Produção Animal, Faculdade de Medicina Veterinária e Zootecnia-UNESP, Botucatu, BrasilbDepartment of Animal Sciences, University of Missouri, Columbia, Missouri, USAcDepartment of Animal Sciences, Ohio State University, Columbus, Ohio, USA

a r t i c l e i n f o

Article history:Received 26 June 2013Received in revised form 26 September 2013Accepted 16 October 2013

Keywords:ProgesteroneProestrusECPGnRHNellore heifers

* Corresponding author. Tel.: þ55 14 3880 2950.E-mail address: vasconcelos@fmvz.unesp.br (J.L.M

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.10.02

a b s t r a c t

Two experiments were designed to evaluate the effects of treatments with low versus highserum progesterone (P4) concentrations on factors associated with pregnancy success inpostpubertal Nellore heifers submitted to either conventional or fixed timed artificialinsemination (FTAI). Heifers were synchronized with a new controlled internal drugrelease device (CIDR; 1.9 g of P4 [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus2 mg of estradiol (E2) benzoate on Day 0 and 12.5 mg of prostaglandin F2a on Day 7. Inexperiment 1 (n ¼ 723), CIDR were removed on Day 7 or 9 and heifers were inseminatedafter estrus detection. In experiment 2 (n ¼ 1083), CIDR were all removed on Day 9 andFTAI was performed either 48 hours later in heifers that received E2 cypionate (ECP) onDay 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH(100 mg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P4 concen-trations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day7, 3.76 � 0.08 days vs. Day 9, 2.90 � 0.07; P < 0.01) and improved conception (Day 7, 57.1%vs. Day 9, 65.8%; P ¼ 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P ¼ 0.04). Inexperiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus(E48, 40.9%a; G54, 17.1%c; and G72, 32.0%b), but the pregnancy rate was not affected (P ¼0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3increased follicle diameter at FTAI (CIDR1, 11.07 � 0.10 vs. CIDR3, 11.61 � 0.10 mm; P <

0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception(CIDR1, 42.2 vs. CIDR3, 45.1%; P ¼ 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P¼ 0.11). In conclusion, length of treatment with P4 affected the fertility of heifers bredbased on estrus detection. When the heifers were submitted to FTAI protocol, folliclediameter at FTAI (�10.7 mm, 23.6%; 10.8–15.7 mm, 51.5%; �15.8 mm, 30.0%; P < 0.01) wasthe main factor that affected conception and pregnancy rates.

� 2014 Elsevier Inc. All rights reserved.

. Vasconcelos).

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1. Introduction

Previous reports have associated lower pregnancy ratesat fixed timed artificial insemination (FTAI) in postpubertal

T. Martins et al. / Theriogenology 81 (2014) 446–453 447

Nellore heifers and either nonlactating or lactating cyclingcows with elevated circulating concentrations of proges-terone (P4), during synchronization of ovulation protocols[1,2].

It has been suggested that high circulating concentra-tions of P4 result in reduced LH pulse frequency, whichdecreases dominant follicle development [3,4] and subse-quent follicle diameter at FTAI [2,5], compromising fertility[6–8]. Alternatives for increasing gonadotropic support ofdominant follicles before FTAI may increase fertility in beefcattle submitted to a FTAI protocol [2,5,9]. These alterna-tives include the reduction in circulating concentration ofP4 by administrating a controlled internal drug releasedevice (CIDR) previously used for 18 days [2,5], stimulationof endogenous gonadotropins release by treatment withprostaglandin (PG)F2a 2 days before CIDR removal [5,9],and administration of exogenous gonadotropin [2,5]. TheCIDR containing 1.9 g of P4 has been used successfully inestrous synchronization protocols as many as four times[9], and previous studies with postpubertal heifersdemonstrated lower serum P4 concentrations [2,5] andgreater pregnancy rates after synchronization with a CIDRpreviously used for 18 days [2] compared with the protocolwith a new CIDR.

An increased conception rate was associated withincreased preovulatory concentrations of estradiol (E2)before FTAI by increasing the length of proestrus [10,11] oradministration of exogenous E2 (estradiol cypionate [ECP])[12]. In Brazil, ECP has been used as an ovulatory stimulusconcurrent with progestin removal in synchronization ofovulation protocols [1,2,9]. Recently, Sá Filho, et al. [13]demonstrated that administration of ECP at progestinremoval increased the proportion of Nellore cows showingestrus and increased pregnancy rate compared with cowstreated with GnRH.

Therefore, reduced circulating P4 concentrations [2,5]during synchronization of ovulation protocols andincreasing preovulatory E2 before FTAI [10,11,13], couldresult in improvements in fertility. Thus, this study wasdesigned to evaluate the effects of P4 concentrations andduration of P4 treatment on follicle development duringestrous synchronization protocols, and their impacts onfertility of postpubertal Nellore heifers. In the first study(experiment 1), the effects of P4 concentrations and lengthof P4 treatment on follicle diameter, estrus distribution, andconception rate to AI based on estrus were evaluated. Toevaluate possible effects of increased preovulatory con-centrations of E2 before FTAI, the second study (experiment2) focused on comparing the impact of the ovulatorystimulus ECP or GnRH (at 54 or 72 hours) on pregnancyrates.

2. Material and methods

Experiments were conducted in commercial beef farmslocated in Mato Grosso, Brazil. Heifers were maintained onpastures (Brachiaria brizantha) with water and mineral saltad libitum. Only heifers with a CL on one of two ultraso-nography evaluations (Aloka SSD-500 with a 7.5 MHzlinear-array transrectal transducer; Aloka, Tokyo, Japan)performed within an interval of 10 days between 30 and 7

days before the initiation of protocols were included inthese studies. All animals were cared for in accordancewiththe practices outlined in the Guide for the Care and Use ofAgricultural Animals in Agricultural Research and Teaching[14].

2.1. Experiment 1

The goals of this experiment were to evaluate the effectof CIDR (new vs. previously used for 18 days) and time ofCIDR treatment (7 vs. 9 days) on follicle development andfertility of postpubertal Nellore heifers submitted to AIafter estrus detection.

Postpubertal Nellore heifers (n ¼ 723; body conditionscore [BCS] of 3.01 � 0.24 on Day 0 using a scale from 1 ¼emaciated to 5 ¼ obese [15]; 24–29 months of age) wereallocated in six pastures, and synchronized between Januaryand March 2010. Heifers were randomly assigned withineachpasture to receive either a newP4-releasing intravaginaldevice containing 1.9 g P4 (CIDR; Pfizer Animal Health, SãoPaulo, SP, Brazil, first use; CIDR1) or a CIDR that had beenpreviously inserted in two 9-days synchronization protocols(third use; CIDR3). All heifers were treated with 2 mg ofestradiol benzoate im (2.0 mL Estrogin; Farmavet, São Paulo,SP, Brazil) at CIDR insertion (Day 0) and 12.5mg imdinoprosttromethamine (PGF2a; 2.5 mL Lutalyse; Pfizer AnimalHealth) on Day 7. The CIDRs were removed on either Day 7(d7) or 9 (d9) within the CIDR1 and CIDR3 groups, resultingin treatments designated as CIDR1-d7 (n ¼ 175), CIDR1-d9(n ¼ 189), CIDR3-d7 (n ¼ 166), and CIDR3-d9 (n ¼ 193). In asubgroup of heifers (n ¼ 408) from CIDR1-d7 (n ¼ 96),CIDR1-d9 (n ¼ 87), CIDR3-d7 (n ¼ 111), and CIDR3-d9(n¼ 114), ovaries were evaluated on Days 7 and 9 by a singletechnician using ultrasonography to assess the diameter ofthe largest ovarian follicle (defined as the average betweenhorizontal and vertical diameters). In the same subgroup,blood samples were collected for subsequent P4 analyses.After CIDR removal, visual estrous detection occurred twicedaily for 1 hour (0730 and 1600 hours) during 7 days (Days8–14 in CIDR1-d7 and CIDR3-d7; Days 10–16 in CIDR1-d9and CIDR3-d9 treatments). Heifers observed in estrus wereinseminated by a single AI technician 10 to 12 hours later,with frozen–thawed commercial semen of two bulls thatwere randomly assigned to treatments. Immediately beforeinsemination, the same technician determined the diameterof the largest follicle of all heifers in estrus by transrectalultrasonography. Pregnancy diagnosis was based on detec-tion (transrectal ultrasonography) of an embryo 40days afterthe end of estrus detection period. Reproductive variablesthat were determined included estrus detection, conception,and pregnancy rates. Estrus detection rate was calculated asthe proportion of all heifers treated that were observed inestrus. Conception rate was calculated as the proportion ofheifers observed in estrus that became pregnant to AI.Pregnancy ratewas calculated as the proportion of all heifersinitially treated that became pregnant to AI.

2.2. Experiment 2

The goals of this experiment were to evaluate the effectof administration of ECP or GnRH (54 or 72 hours after CIDR

T. Martins et al. / Theriogenology 81 (2014) 446–453448

removal) on pregnancy rate of postpubertal Nellore heiferssynchronized with either first (CIDR1) or third use CIDR(CIDR3).

Postpubertal Nellore heifers, 24 to 29 months of age attwo farms (farm 1, n ¼ 570, BCS ¼ 3.08 � 0.26; and farm 2,n¼ 513, BCS¼ 2.80� 0.24)were allocated in four pastures infarm 1 and in three pastures in farm 2, and were synchro-nized between January and March 2011. Heifers wereinitially synchronized within each pasture as described inexperiment 1, except that CIDRs remained for 9 days duringthe synchronizationprotocol. OnDay 9, CIDRswere removedand heifers were randomly assigned within CIDR type toreceive one of three ovulation synchronizationmethods: 0.5mg ECP im (0.25 mL E.C.P; Pfizer Animal Health) on Day 9with FTAI 48 hours later (control group), or 100 mg GnRH im(1.0 mL Fertagyl; MSD Animal Health, São Paulo, SP, Brazil)coincidentwith FTAI at 54or 72 hours after CIDRwithdrawal.Hence, treatments were CIDR1-E48 (n ¼ 176), CIDR1-G54(n¼ 178), CIDR1-G72 (n¼ 181), CIDR3-E48 (n¼ 184), CIDR3-G54 (n ¼ 180), and CIDR3-G72 (n ¼ 184). Blood samplecollection and ovarian ultrasonographywere performed by asingle technician to determine, respectively, the serum P4concentrations and largest follicle diameter in a subset ofheifers (n¼ 250) on Day 7 and in a greater number of heifers(n¼ 532) onDay 9.Visual estrous detection in themajorityofheifers (n¼ 920), occurred twice daily for 1 hour each period(0730 and 1600 pm), from CIDR withdrawal to FTAI. Imme-diately before insemination, the same technician determinedthe diameter of the largest follicle in all heifers using ultra-sonography. Heifers were randomly assigned within treat-ments to be FTAI by experienced technicians (farm 1, n ¼ 3;farm 2, n¼ 2) using frozen–thawed commercial semen fromthree (farm 1) or two (farm 2) bulls. Blood samples for P4analyses were collected 7 days after FTAI in all heifers.Ovulationwas considered to have occurredwhenheifers hadserum P4 concentration of 1.0 ng/mL or higher 7 days afterFTAI. At FTAI, largest follicle diameter were less than 7.0 mmin 200 heifers (the capacity of follicles to respond to anovulatory stimulus is acquired in Nellore heifers around 7.0mm in diameter [16]). These heifers were presumed to haveovulated from CIDR removal to the moment of FTAI, whenserum P4 concentrations were 1.0 ng/mL or greater 7 daysafter FTAI (premature ovulation; n ¼ 142). Data for theseheifers (n ¼ 200) were included as missing observations inthe analyses of largest follicle diameter at FTAI. Reproductivevariables that were determined included ovulation,conception, and pregnancy rates. Pregnancy diagnosis wasbased on detection (transrectal ultrasonography) of an em-bryo 29 to 30 days after FTAI. The ovulation rate was definedas the proportion of all heifers that ovulated 7 days after FTAI(serum concentration of P4 � 1.0 ng/mL 7 days after FTAI),including heifers that were considered prematurely ovu-lated. The conception rate was calculated as the proportionof ovulated heifers that became pregnant to FTAI, andpregnancy rate included all heifers,whether or not theywereclassified as having ovulated.

2.3. Blood samples for P4 analyses

Blood samples for P4 analyses were collected from acoccygeal vessel intoVacutainer tubeswithout anticoagulant

(Becton Dickinson Co., Franklin Lakes, NJ, USA). Blood wasallowed to clot at 4 �C for 24 hours and centrifuged at�1500 g for 15 minutes at room temperature. Serum wasremoved and frozen at�20 �C until P4was quantified. SerumP4 concentrations were determined using a solid-phase RIAkit containing antibody-coated tubes and 125I-labeled P4(Coat-a-count; Diagnostic Products Corporation, LosAngeles, CA, USA) as previously validated in our laboratory[17]. The interassay coefficient of variation was 6.64% and7.98% and the intra-assay coefficient of variation was 3.78%and 2.93% for experiments 1 and 2, respectively.

2.4. Statistical analysis

Binominal, dependent variables were analyzed by PROCGLIMMIX of the SAS software (version 9.2, SAS InstituteInc., Cary, NC, USA) with Satterthwaite approximation todetermine the denominator degrees of freedom for tests offixed effects; with heifers treated as a random effect. Inexperiment 1, within the 2 � 2 factorial arrangement oftreatments (main effects of CIDR type and day of CIDRremoval), the effects of BCS, pasture, AI technician, AI sire,and appropriate interactions were considered in pre-liminary models. In the final models, variables wereremoved by a backward elimination (according to theWald’s criterion) when P > 0.2. The final model for theanalyses of estrus detection, conception, and pregnancyrates included only the effect of day of CIDR removal. Inexperiment 2, a similar statistical approach was taken for2� 3 factorial treatment arrangement (main effects of CIDRtype and ovulation synchronization method). The finalmodel for analyses of estrus detection, ovulation, concep-tion, and pregnancy rates included the effect of type ofCIDR and ovulation synchronization method. A secondaryanalysis that included detection of estrus as an indepen-dent variable was performed for analysis of the pregnancyrate. The general liner modeling procedure of SAS was usedto determine relationships between each individualcontinuous variable and binomial dependent variable inexperiments 1 and 2. When significant, the logisticalregression models were fit by the method of maximumlikelihood using PROC LOGISTIC of SAS to determine theintercept and slope(s) values. The logistical regressionmodels were designed using the intercept and slope valueson the following equation: Probability ¼ (elogistic equation)/(1 þ elogistic equation). In experiment 2, heifers were catego-rized in three classes based on largest follicle diameter atFTAI (class 1, <10.7 mm; class 2, between 10.8 and 15.7mm; class 3, >15.7 mm) to assess the effects on estrusdetection, conception and pregnancy rates and serum P4concentration 7 days after FTAI. These classifications weredetermined based on relationship between follicle diam-eter at FTAI and probability of conception. The minimum(<10.7 mm) and maximum (>15.7 mm) cutoff points wereestablished when the probability of conception was 10%smaller than the point of maximal probability of concep-tion (12.8 mm).

Continuous variables were analyzed by PROC MIXEDwith Satterthwaite approximation to determine the de-nominator degrees of freedom for tests of fixed effects andheifers treated as a random effect. The analyses were

Table 1Effects of type of CIDR and/or day of CIDR removal on serum P4 concentrations and follicle diameter on Days 7 and 9 in Bos indicus (Nellore) postpubertalheifers (experiment 1).

Variable Treatmentsa P valueb

CIDR1 CIDR3

Day 7 Day 9 Day 7 Day 9 CIDR Day CIDR*day

No. of heifers 96 87 111 114P4 on Day 7 (ng/mL) 4.29 � 0.18 4.53 � 0.17 2.88 � 0.19 3.14 � 0.17 <0.01Fol Day 7 (mm) 6.55 � 0.16 6.53 � 0.15 7.37 � 0.17 7.30 � 0.15 <0.01P4 Day 9 (ng/mL) 0.65 � 0.10c 3.00 � 0.10d 0.71 � 0.11c 1.77 � 0.09e <0.01 <0.01 <0.01Fol Day 9 (mm) 8.10 � 0.22 7.50 � 0.20 9.11 � 0.23 8.74 � 0.20 <0.01 0.02 0.58

Abbreviations: CIDR, controlled internal drug release device; Fol, follicle diameter; P4, progesterone.a Heifers received a new CIDR (CIDR1) or a CIDR previously used for 18 days (CIDR3) at initiation of protocol and CIDRswere removed on Day 7 or 9 in a 2�

2 factorial design.b CIDR ¼ effect of CIDR treatment (CIDR1 vs. CIDR3); Day ¼ effect of day of CIDR removal (Day 7 vs. 9); CIDR*day ¼ interaction between CIDR and day.c,d,e Values without a common superscript differed between treatments (P < 0.01).

T. Martins et al. / Theriogenology 81 (2014) 446–453 449

performed considering factorial arrangement of experi-ments and final models were determined by removingnonsignificant terms (P > 0.2) sequentially. Continuousvariables were correlated using PEARSON procedure. Inexperiment 1, the final model for the analyses of serum P4concentration and follicle diameter on Day 7 included onlythe effect of type of CIDR. Inclusion of day of CIDR removaland their interactions in the model were used for the an-alyses of serum P4 concentration and follicle diameter onDay 9. For the analyses of moment of estrus and folliclediameter at AI, the final model included effect of type ofCIDR, day of CIDR removal, and their interactions. Inexperiment 2, the analyses of serum P4 concentration andfollicle diameter on Days 7 and 9 only included effect oftype of CIDR in the final model. For the analysis of folliclediameter at FTAI, the final model included type of CIDR,ovulation synchronization method, and their interactions.A similar model was used for analysis of serum P4 con-centration 7 days after FTAI.

Results were reported as least square mean values andcomparisons were made using procedure PDIFF of SAS. Forall analyses significance was set at P � 0.05 and tendencieswere declared if P > 0.05 and �0.1.

Fig. 1. The regression of largest follicle diameter at controlled internal drugrelease device (CIDR) removal on the interval from CIDR removal to estrusfor Bos indicus (Nellore) postpubertal heifers treated with CIDR for 7 days(:, Day 7 ¼ 144; P < 0.01) or CIDR for 9 days (x, Day 9 ¼ 160; P < 0.01).

3. Results

3.1. Experiment 1

On Day 7, heifers treated with CIDR1 had greater (P <

0.01) serum P4 concentrations and smaller (P < 0.01) fol-licle diameter compared with heifers treated with CIDR3(Table 1). On Day 9, an interaction between CIDR type andday of CIDR removal was detected (P < 0.01). Serum P4concentrations were greater in the CIDR1-d9 group thanany other group (Table 1). Follicle diameter on Day 9 wasgreater (P < 0.01) in heifers synchronized with CIDR3 thanCIDR1, and greater (P¼ 0.02) in the group Day 7 than Day 9,but no interaction between CIDR type and day of CIDRremoval was detected (Table 1). Serum P4 concentrationswere negatively correlated (P < 0.01) with follicle diameteron Day 7 (r ¼ �0.20) and Day 9 (r ¼ �0.21). Across Day 7and Day 9, the interval from CIDR removal to estrus wasnegatively correlated (P < 0.01) with follicle diameter at

CIDR removal (Fig. 1). There was no effect of CIDR type (P ¼0.21) or day of CIDR removal (P ¼ 0.38) on proportion ofheifers detected in estrus (67.4% [487/723]; Table 2). Theinterval from CIDR removal to estrus was affected (P< 0.01)by CIDR (CIDR1, 3.65 � 0.08 days vs. CIDR3, 3.00 � 0.08days; Table 2) and day of CIDR removal (Day 7, 3.76 � 0.08days vs. Day 9, 2.90 � 0.07 days; Table 2; Fig. 2). At AI, theCIDR3-d9 group had increased (P < 0.05) follicle diametercompared with others groups (Table 2). There was an effectof day of CIDR removal on conception (Day 7, 57.1% [128/224] vs. Day 9, 65.8% [173/263]; P ¼ 0.05) and pregnancyrates (Day 7, 37.6% [128/341] vs. Day 9, 45.3% [173/382]; P¼0.04), but type of CIDR did not affect these variables (Table2). When compared across treatments, follicle diameter atAI was not associated with conception rate (P ¼ 0.21), andwas not detected effect of BCS or AI sire on conception rate(P > 0.10).

3.2. Experiment 2

On Day 7, heifers treated with CIDR3 had decreased (P<

0.01) serum P4 concentrations (4.73 � 0.23 vs. 6.60 � 0.28

Table 2Effects of CIDR use and/or day of CIDR removal on estrus detection, interval to estrus, largest follicle diameter at AI and probabilities of conception andpregnancy in Bos indicus (Nellore) postpubertal heifers (experiment 1).

Variable Treatmentsa P valueb

CIDR1 CIDR3

Day 7 Day 9 Day 7 Day 9 CIDR Day CIDR*day

Estrus detection, % (n) 64.0 (112/175) 66.1 (125/189) 67.5 (112/166) 71.5 (138/193) 0.21 0.38 0.79Interval from CIDR removal to estrus (day) 4.13 � 0.11 3.17 � 0.11 3.39 � 0.11 2.63 � 0.10 <0.01 <0.01 0.37Fol at AI (mm) 11.58 � 0.15c,d 11.47� 0.14c 11.91 � 0.15d 12.35 � 0.14e <0.01 0.26 0.06Conception rate, % (n) 55.4 (62/112) 65.6 (82/125) 58.9 (66/112) 65.9 (91/138) 0.66 0.05 0.72Pregnancy rate, % (n) 35.4 (62/175) 43.4 (82/189) 39.8 (66/166) 47.2 (91/193) 0.27 0.04 0.94

Abbreviations: AI, artificial insemination; CIDR, controlled internal drug release device; Fol, follicle diameter; P4, progesterone.a Heifers received a not previously used CIDR (CIDR1) or a CIDR previously used for 18 days (CIDR3) at initiation of protocol and CIDRs were removed on

Day 7 or 9 in a 2 � 2 factorial design.b CIDR ¼ effect of CIDR treatment (CIDR1 vs. CIDR3); Day ¼ effect of day of CIDR removal (Day 7 vs. 9); CIDR*day ¼ interaction between CIDR and day.c,d,e Values without a common superscript differed between treatments (P < 0.05).

T. Martins et al. / Theriogenology 81 (2014) 446–453450

ng/mL) and increased (P < 0.01) follicle diameter (7.60 �0.13 vs. 6.84 � 0.14 mm) compared with heifers treatedwith CIDR1. Similarly on Day 9, heifers treated with CIDR3had decreased (P < 0.01) serum P4 concentrations (2.85 �0.11 vs. 4.29 � 0.11 ng/mL) and increased (P < 0.01) folliclediameter (9.65 � 0.14 vs. 8.55 � 0.14 mm) compared withheifers treated with CIDR1. Serum P4 concentrations werenegatively correlated (P < 0.01) with follicle diameter onDay 7 (r ¼ �0.21) and Day 9 (r ¼ �0.16).

Estrous detection rate between Day 9 and moment ofFTAI was affected (P < 0.01) by type of CIDR (CIDR1, 24.5%[111/454] vs. CIDR3, 35.5% [166/466]) and by ovulationsynchronization method. Percentage of heifers detected inestrus before FTAI in the group E48 (40.9% [126/307]) wasgreater (P < 0.01) than G72 (32.0% [99/309]) and bothwere greater (P < 0.01) than G54 (17.1% [52/304]). Theproportion of heifers prematurely ovulated in the groupCIDR3-72 hours (40.5% [68/168]) was greater (P < 0.01)than CIDR1-72 hours (14.4% [23/160]) and both weregreater (P < 0.01) than any other group (8.5% [51/598]).The diameter of the largest follicle at FTAI was greater (P<

0.01) in heifers synchronized with CIDR3 (11.61 � 0.10

Fig. 2. Cumulative percentage of Bos indicus (Nellore) postpubertal heifersobserved in estrus throughout 7 days after controlled internal drug releasedevice (CIDR) removal. Heifers were treated with CIDR for 7 days (Day 7; n ¼224; hatched line) or CIDR for 9 days (Day 9; n ¼ 263; solid line).

mm) than CIDR1 (11.07 � 0.10 mm). Heifers observed inestrus had a greater (P < 0.01) follicle diameter on Day 9(10.34� 0.20 vs. 8.47� 0.12 mm) and at FTAI (12.52� 0.14vs. 10.74� 0.09mm) than those not in estrus, independentof treatment.

The ovulation rate was affected by type of CIDR (CIDR1,82.8% [444/535] vs. CIDR3, 88.0% [482/548]; P ¼ 0.01), andit was positively affected by follicle diameter at FTAI(Table 3; P < 0.01). Group G72 (89.8% [328/365]) hadgreater ovulation rate than those from groups G54 (83.8%[300/358]; P ¼ 0.02) and E48 (82.6% [298/360]; P < 0.01).

The conception rate was not affected by CIDR (CIDR1,42.2% [187/444] vs. CIDR3, 45.1% [217/482]; P ¼ 0.38) orsynchronization of ovulation protocol (E48, 46.9% [140/298]; G54, 42.3% [127/300]; G72, 41.7% [137/328]; P¼ 0.37).The probability of conception tended (P ¼ 0.08) to benegatively affected by serum P4 concentrations at CIDRremoval and it was positively affected (P < 0.01) by serumP4 concentrations 7 days after FTAI (Fig. 3). There was aquadratic effect (P < 0.01) of follicle diameter at FTAI onprobability of conception (Table 3; Fig. 4). A positive rela-tionship was detected (P < 0.01) between follicle diameterat FTAI and serum P4 concentrations 7 days after FTAI(Table 3). Thus, serum P4 concentrations 7 days after FTAIwas affected (P < 0.01) by type of CIDR (CIDR1, 3.60 � 0.09vs. CIDR3, 3.95 � 0.09 ng/mL) and estrus behavior (estrus,4.61 � 0.13 vs. no estrus, 3.72 � 0.09 ng/mL). The syn-chronization of ovulation protocol did not affect (P ¼ 0.69)serum P4 concentrations 7 days after FTAI.

An interaction between synchronization of ovulationprotocol and whether heifers were detected in estrus (P ¼0.02) affected the pregnancy rates. The pregnancy rates ofgroups E48 (estrus, 61.0% [75/126] vs. no estrus, 25.7% [46/181]), G54 (estrus, 47.9% [24/52] vs. no estrus, 30.3% [76/252]), G72 (estrus, 45.8% [45/99] vs. no estrus, 32.5% [68/210]) were influenced by expression of estrus, and withinheifers observed in estrus, the group E48 had greaterpregnancy rate than those from groups G54 or G72.

Pregnancy rate was greater in heifers with follicle diam-eter between 10.8 and 15.7 mm at FTAI (Table 3), indepen-dent of treatment. Pregnancy rate was not affected by CIDR(CIDR1, 34.7% [187/535] vs. CIDR3, 39.4% [217/548]; P¼ 0.11),and by synchronization of ovulation protocol (E48, 38.8%[140/360]; G54, 35.5% [127/358]; G72, 37.5% [137/365];

Table 3Association between Fol at FTAI on the probabilities of estrus, ovulation, conception, pregnancy, and serum P4 concentration 7 days after FTAI in Bos indicus(Nellore) postpubertal heifers (experiment 2).

Variable Fol at FTAI (mm)a P value

7.0–10.7 10.8–15.7 �15.8

Fol at FTAI (mm)b 9.13 � 0.06 12.56 � 0.05 16.51 � 0.25 -Estrus, % (n)c 12.4h (36/291) 38.4g (163/424) 46.7g (7/15) <0.01Ovulation rate, % (n) 75.3h,f (255/335) 96.7g (511/528) 86.7g,h,e (18/20) <0.01Conception rate, % (n) 30.8h (79/255) 53.1g,i (272/511) 28.8g,h,j (6/18) <0.01P4 7 days after FTAI (ng/mL)d 3.13 � 0.12h,j 4.03 � 0.08g 3.95 � 0.44g,h,i <0.01Pregnancy rate, % (n) 23.6h (79/335) 51.5g,e (272/528) 30.00g,h,f (6/20) <0.01

Abbreviations: AI, artificial insemination; CIDR, controlled internal drug release device; Fol, follicle diameter; FTAI, fixed timed artificial insemination; P4,progesterone.

a Based on the relationship portrayed in Figure 4.b Largest follicle diameter at FTAI.c Based on subset of heifers evaluated between CIDR withdraw and FTAI.d Progesterone concentration 7 days after FTAI in ovulated heifers.e,f Values without a common superscript differed (P < 0.05).g,h Values without a common superscript differed (P < 0.01).i,j Values without a common superscript tended to differ (P < 0.10).

T. Martins et al. / Theriogenology 81 (2014) 446–453 451

P ¼ 0.64). There was no effect of farm, BCS, AI technician, orAI sire on conception or pregnancy rates (P > 0.10).

4. Discussion

In current studies, greater circulating concentrations ofP4 reduced the follicle diameter during the synchronizationprotocol (Days 7 and 9) and decreased the likelihood ofNellore heifers conceiving to the synchronized AI. Thepossible mechanism by which higher serum P4 concen-tration during the protocol resulted in decreased folliclesize on Days 7 and 9 might be associated with a reducedfrequency of LH pulses [3,4] that negatively affect dominantfollicle development [18]. Emergence of a new follicularwave occurs 3.1 [19] to 3.3 [20] days after the onset of aprotocol that includes 2 mg of estradiol benzoate and

Fig. 3. Relationship between serum P4 concentrations (ng/mL) at timeof controlled internal drug release device (CIDR) removal (Day 9; solid line;P ¼ 0.08; n ¼ 449) or 7 days after fixed timed artificial insemination (FTAI;hatched line; P ¼ 0.01; n ¼ 926) and probability of conception in post-pubertal Bos indicus (Nellore) heifers submitted to a FTAI protocol inexperiment 2.

insertion of a P4 device. Follicular deviation occurs 2.5 [16]to 2.8 [21] days after follicle emergence coordinated byacquisition of LH receptors in the granulosa cells of thedominant follicles [22]. At this time, Bos indicus heifers havedominant follicles with diameter of 5.7 [21] to 6.2 [16] mm.The expected moment of emergence and deviation afterthe beginning of the protocol may explain the effect of P4concentration on follicle diameter on Day 7 in experiment 1(CIDR1, 6.54 vs. CIDR3, 7.33 mm) and in experiment 2(CIDR1, 6.84 vs. CIDR3, 7.60 mm). It is likely that most fol-licles had already attained deviation before Day 7 and theywere negatively affected by a greater P4 concentration[4,22].

In experiment 1, treatment with a CIDR for 9 daysresulted in greater rates of conception and pregnancycompared with a CIDR for 7 days and interval from CIDR

Fig. 4. Relationship between follicle diameter (mm) at fixed timed artificialinsemination (FTAI) and probability of conception in Bos indicus (Nellore)postpubertal heifers submitted to a FTAI protocol (experiment 2; n ¼ 784;P < 0.01). Vertical lines indicated that follicles 10.7 mm or smaller or those15.8 mm or greater in diameter at time of artificial insemination were lesslikely (P < 0.01) to support conception rate than optimally sized follicle of12.8 mm (at the predicted maximum probability of conception of 0.56).

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removal to estrus was reduced in the 9-day treatment.Recent studies in beef cattle [23,24] reported shorter in-terval from administration of PGF2a to estrus, in heifers(53.6 vs. 78.5 hours) and cows (56.7 vs. 79 hours), when theinterval between administration of estradiol benzoate andPGF2a was longer (9.5 vs. 5.5 days); however, the authorsdid not detect effects on the pregnancy rate. It has beendemonstrated that P4 plays a pivotal role, promoting oocytecompetence and establishment of uterine receptivity [25].Although we did not detect an effect of P4 concentration onthe conception rate in experiment 1, our results suggestthat duration of exposure to P4 before AI is critical to theestablishment of pregnancy and could be essential in theBos indicus cattle, owing the differences in the results foundin the present experiment and recent reports conductedwith Bos taurus [23,24].

Based on increased fertility, we chose to use the protocolwith a CIDR for 9 days in the subsequent FTAI programs toevaluate the effect of timing of AI and ovulatory stimulus onpregnancy rate. The results of experiments 1 and 2 areconsistent with prior studies [6,8] that reported increasedexpression of estrus near the moment of FTAI in cows withlarger follicles. In agreement with Sá Filho, et al. [13], thecurrent study also detected an increase in the percentage ofheifers observed in estrus after treatment with ECP. Inter-estingly, heifers treated with ECP and observed in estrushad a greater pregnancy rate compared with heifers thatwere in estrus and received GnRH at 54 or 72 hours. Souza,et al. [26] observed that dairy cows expressing estrus alongwith supplementation of E2 (1 mg of 17b estradiol) had anincreased conception rate compared with cows expressingestrus without E2 supplementation. Although the finding ofour study is confounded by moment of FTAI, it has beenreported [27] that the period for achieving acceptablepregnancy rates to AI in beef heifers seems to be approxi-mately 20 hours.

Results from these experiments confirmed studies [5,28]in which the size of the follicle at FTAI had a positive rela-tionship with P4 concentrations during the subsequentdiestrus, which in turn resulted in greater fertility. AdequateP4 concentrations during diestrus are important for fertility[5,8,28], owing to stimulation of endometrial secretions [29],embryonic development [29,30], and maternal recognition,along with establishment and maintenance of pregnancy[31].

In experiment 2, heifers with follicle diameters between10.8 and 15.7 mm at FTAI had greater pregnancy ratesowing to greater rates of ovulation and conception. Inter-estingly, this critical threshold was similar to those re-ported by Perry, et al. [8] in Bos taurus heifers and similar(>11.1 mm) to those described by Sá Filho, et al. [6] in Bosindicus cows. Perry, et al. [8] reported that pregnancy rateson CO-Synch protocol was limited by presence of smallfollicles (28%) at FTAI. We found a similar proportion (33%)of heifers with small follicles at FTAI. Causes of decreasedconception rate in heifers ovulating smaller follicles seemsto be associated with decreased preovulatory circulatingconcentrations of E2 and decreased circulating P4 concen-trations in the subsequent diestrus [7,28]. Decreasedovulation rate in heifers with smaller follicles at FTAI isconsistent with findings by Gimenes, et al. [16]. Thus, even

after addition of ECP as in the current study, ovulation ofdominant follicles with adequate status seems to be criticalfor fertility.

Based on previous findings that detected greater fertilityafter synchronization with a CIDR previously used for 18days [2] and lower serum P4 concentrations after use ofCIDR3 [2,5,9], we anticipated that in experiment 2, thepregnancy rate would be increased after synchronizationwith CIDR3. However, only a numerical increase ofapproximately 5% in the pregnancy rate was detected.Synchronizationwith CIDR3 did improve the ovulation rateand serum P4 concentrations after FTAI, consistent withfinding regarding ovulation rate, serum P4 concentrations,and largest follicle diameter at FTAI [5]. Because PGF2awasgiven to all heifers on Day 7, we suggest that the concurrentdecrease in P4 increased follicular development andfertility in both treatments, as has been noted previously[2,5,9]. This response likely minimized the differences infertility between the CIDR3 and CIDR1 and explains whywe only detected a tendency for P4 concentrations on Day 9to be related to conception rate.

Decreased conception rates were detected in heifers thathad larger follicles at FTAI (�15.8 mm), probably owing toincreased follicular age [32]. The proportion of these follicleswas small, which indicates that the E2/P4 synchronizationprogram was efficient in synchronizing follicular develop-ment. The proportion of heifers with smaller follicles (�10.7mm) seems to be a more important factor that limits preg-nancy rate in this FTAI program.

In conclusion, greater serum P4 concentrations duringfollicular development resulted in decreased folliculardiameter during the synchronization protocol and increasedinterval to estrus. Time of exposure to CIDR during the syn-chronization protocol increased the conception rate amongheifers inseminated upon estrus detection. Treatment withECP increased estrus behavior; however, fertility even afteradministration of ECP depends on follicle diameter at FTAI.Optimization of results in E2/P4 programs is linked withovulation of follicles 10.8 mm or larger.

Acknowledgments

The FAPESP (2009/06897-7) for financial support, theZoetis Animal Health (São Paulo, SP, Brazil) for donation ofLutalyse, CIDR, and E.C.P. The Agropecuária Fazenda Brasilprovided the animals used in this study.

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