t m P p a o ( a 2 d P © Available online at www.sciencedirect.com Theriogenology 78 (2012) 510–516 0 h Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol J.N.S. Salesa,f,*, J.B.P. Carvalhob, G.A. Crepaldic, R.S. Ciprianod, J.O. Jacominia, J.R.G. Maioc, J.C. Souzae, G.P. Nogueirad, P.S. Barusellia a Department of Animal Reproduction, FMVZ-USP, São Paulo-SP, Brazil b APTA-Vale do Paraíba Regional Center, Pindamonhangaba, SP, Brazil c Ouro Fino Saúde Animal, Ribeirão Preto, SP, Brazil d Department of Support, Animal Production and Health - Unesp, Araçatuba, SP, Brazil e Department of Animal Science, UFLA, Lavras, MG, Brazil f Center of Veterinary Science, UFPB, Areia, PB, Brazil Received 17 December 2011; received in revised form 26 February 2012; accepted 26 February 2012 Abstract The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used o evaluate the effect of EB (n � 5) and EC (n � 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P � 0.001), agnitude (20.5 and 9.4 ng/mL; P � 0.005), duration (8.6 and 16.5 h; P � 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; � 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n � 60) and 3 (pregnancy er AI; P/AI; n � 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were ssigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal r 1 mg of EC im at P4 device removal. There was no difference (P � 0.05) between EB and EC treatments on follicular responses maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; nd ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P � 0.22) between the cows treated with EB (57.5%; 77/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 evice removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar /AI in suckled Bos indicus beef cows submitted to TAI. 2012 Elsevier Inc. All rights reserved. Keywords: Bovine; Ovarian response; Estradiol; Tai; LH; Synchronization www.theriojournal.com * Corresponding author. Tel.: 55 11 3091 7674. E-mail address: znlogan@yahoo.com.br (J.N.S. Sales). 093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. ttp://dx.doi.org/10.1016/j.theriogenology.2012.02.031 1. Introduction It is currently possible to efficiently synchronize ovarian follicular growth and induce ovulation of a dominant follicle at a known moment, allowing insem- ination of beef zebu females on a predetermined day and hour, without the need to detect estrus [1]. Timed mailto:znlogan@yahoo.com.br www.theriojournal.com http://dx.doi.org/10.1016/j.theriogenology.2012.02.031 w o i c s B c P c S 2 t w a c c u w s i s 2 2 R C s a s t t 2 g s c ( 511J.N.S. Sales et al. / Theriogenology 78 (2012) 510–516 artificial insemination (TAI) may be employed either in cyclic or anestrous cows and started independent of estrous cycle phase [2]. Recently, many ovulation synchronization protocols have been studied in zebu cows [3–5]. These protocols are based on synchronizing emergence of the wave of follicular growth by simultaneous administration of progesterone and estradiol [6,7]. At the end of a protocol, it is necessary to reduce circulating progesterone concentrations by removal of the progesterone device (exogenous source) and administration of prostaglandin to promote CL regression (endogenous source), so that, ovulation may occur. Lastly, it is necessary to promote the final growth of the follicle and to induce a synchronized ovulation, which allows insemination at a pre- determined moment [2]. Adequate results have been achieved by TAI (�50%) ith protocols employing estradiol benzoate (EB) as the vulation inducing agent in Bos taurus [8,9] and in Bos ndicus [1,4] beef cows. However, it is necessary to handle ows at least four times to perform TAI (Day 0, EB � P4 device insertion; Day 8, PGF � P4 device removal; Day 9, EB; and Day 10, TAI [10]). It is known that estradiol esters are capable of inducing ovulation in beef cows, however, their pharmacokinetics differ. In some studies, EB had a shorter half-life and induced an earlier LH peak compared to estradiol cypionate (EC) [11,12]. Thus, based on the distinct pharmacokinetics of estradiol esters and aiming to reduce TAI-related cow handling, three studies were conducted to evaluate the effect of EB and EC on induction of ovulation of suckled beef cows submitted to TAI. The objective of Ex- periment 1 was to evaluate the circulating LH-release profile after giving EB and EC administration to ovariectomized Bos indicus heifers. Experiment 2 aimed to evaluate the effect of EC (treated at P4 device removal) or EB (treated 24 h post P4 device removal) on ovarian responses of Nelore cows (Bos indicus) submitted to a TAI protocol. Experiment 3 aimed to evaluate the effect of EC and EB, as ovulation inducers, on the pregnancy rate of Nelore cows (Bos indicus) submitted to TAI. The hypothesis was that EC administration (at device removal) decreases cow handling without affecting fertility of beef cows submitted to TAI programs, compared to EB given 24 h after P4 device removal. 2. Materials and methods 2.1. Experiment 1: circulating LH profiles in ovariectomized Nelore heifers 2.1.1. Animals and handling Experiment 1 was conducted on the research farm of the University of the State of Sao Paulo (UNESP) in Araçatuba, SP, Brazil. Nelore heifers, 36.0 � 0.5 mo p old, weighing 427.8 � 9.3 kg had their ovaries re- moved (via laparotomy) to eliminate endogenous estra- diol. Heifers were maintained on Brachiaria brizantha pastures and given mineralized-salt and free access to water. 2.1.2. Experimental design Ovariectomized heifers (n � 10) were synchronized using an estradiol/progesterone-based TAI protocol 1. Heifers received an intravaginal device containing 1 g of P4 (Sincrogest, Ourofino Agronegocio, Sao Paulo, Brazil) plus 2 mg of estradiol benzoate im (EB; Sin- crodiol, Ourofino Agronegocio, Sao Paulo, Brazil). Eight days later, the progesterone device was removed and heifers received 500 �g PGF2� im (sodium clopro- tenol; Sincrocio, Ourofino Agronegocio, Sao Paulo, razil). Concurrently, EC-Group heifers (n � 5) re- eived 1 mg of estradiol cypionate im (ECP, Pfizer, Sao aulo, SP, Brazil) and EB-Group heifers (n � 5) re- eived 1 mg EB im (Sincrodiol, Ourofino Agronegocio, ao Paulo, SP, Brazil). .1.3. Blood sampling and LH assay Jugular veins were cannulated 1 day before onset of reatments with the two estradiol esters. Blood samples ere collected in evacuated tubes every 3 h after initi- tion of treatments (total of 72 h). Within 4 h after ollection, samples were refrigerated (4 °C) and then entrifuged (3000g for 15 min) and stored at �20 °C ntil assayed for LH. Plasma concentrations of LH ere determined by double antibody radioimmunoas- ay, as previously described [13]. The intraassay and nterassay CVs were 10.3 and 13.1%, respectively. As- ay sensitivity was 0.02 ng/mL. .2. Experiment 2: ovarian responses in Nelore cows .2.1. Animals and handling procedures The second experiment was conducted at the State esearch Farm (APTA - Pindamonhagaba Regional enter), in Pindamonhagaba, Sao Paulo, Brazil. Sixty uckled multiparous Nelore cows scoring 2.8 � 0.4 on (1-thin to 5-obese) body condition score (BCS; [14]) cale. All cows were maintained on Brachiaria brizan- ha pastures and given mineralized-salt and free access o water. .2.2. Experimental design All cows were synchronized using an estradiol/pro- esterone-based TAI protocol 1. Hormonal treatments tarted between 30 and 60 days postpartum. Cows re- eived an intravaginal device containing 1 g of P4 Sincrogest, Ourofino Agronegocio, Sao Paulo, Brazil) lus an administration of 2 mg of estradiol benzoate im o B i d s e 5 s m p t d i e 2 ( 2 c u s p u b f 2 m s T i 4 f h A w c s i d 2 A L a t d c T i s t f t A l v 512 J.N.S. Sales et al. / Theriogenology 78 (2012) 510–516 (EB; Sincrodiol, Ourofino Agronegocio). Eight days later, the device was removed and 500 �g PGF2� (clo- prostenol sodium; Sincrocio, Ourofino Agronegocio) and 300 IU of equine chorionic gonadotropin (eCG; Folligon, Intervet-Schering-Plough, Boxmeer, Nether- lands) administered im. At P4-device removal, EC- Group cows (n � 30) received 1 mg of estradiol cypi- nate im (ECP, Pfizer Animal Health, Sao Paulo, SP, razil) and EB-Group cows (n � 30) received 1 mg EB m (Sincrodiol, Ourofino Agronegocio) 24 h after P4- evice removal (Fig. 1). At the beginning of the estrous ynchronization procedures (day 0), ultrasonographic xaminations were performed (USA; Chison 600 VET, MHz linear transducer, China) to evaluate ovarian tatus (presence of CL and ovarian follicles �8 or �8 m [1]). Moreover, ultrasonographic evaluations were erformed every 12 h from P4-device removal to de- ection of ovulation. The moment of ovulation was efined as the time of disappearance of a previously dentified dominant follicle (DF) from one ultrasound xamination to the next, minus 6 h. .3. Experiment 3: pregnancy per AI in Nelore cows Bos indicus) .3.1. Animals and management The experiment was conducted on two commer- ial farms located in northwestern Brazil, from Jan- ary to March 2009. A total of 953 multiparous uckled Nelore (Bos indicus) cows, 30 to 60 days ostpartum and average BCS of 2.79 � 0.41, were sed [14]. All cows were maintained on Brachiaria rizantha pastures and given mineralized-salt and Fig. 1. Experimental design (P4, progesterone; EB, estradiol benzoate AI). ree access to water. l .3.2. Experimental design Postpartum suckled cows were allocated into treat- ent groups according to calving date. All cows were ynchronized using an estradiol/progesterone-based AI protocol and treatments similar to those described n Experiment 2. In this experiment, TAI was performed 8 to 52 h after P4 device removal. Frozen semen straws rom single ejaculates of each of the three sires were omogeneously distributed between experimental groups. ll inseminations were performed by the same technician ho had no previous knowledge of cow treatment allo- ation. Pregnancy examinations were conducted by tran- rectal ultrasonography (Chison D600VET, Chison Med- cal Imaging, Co, China) 30 days after AI. The experimental esign is shown (Fig. 1). .4. Statistical analysis Statistical analyses were performed using Statistical nalysis System for Windows [15]. In Experiment 1, H surge was defined as an increase in LH � 2 SD bove the overall within-cow mean of LH concentra- ions. The magnitude of the LH surge (MLH) was efined as the difference between the maximum con- entrations observed during the surge and baseline. iming of LH surge (TLH) was defined as the moment n which the LH surge occurred. Duration of the LH urge (DLH) was determined as the time interval from he onset of the LH surge (baseline concentration be- ore the increase in LH concentration induced by es- radiol) to its first return to baseline concentrations. rea under the curve (AUC) of LH release was calcu- ated by the trapezoid method. In Experiment 2, the ariables evaluated were diameter of the ovulatory fol- stradiol cypionate; eCG, equine chorionic gonadotropin; TAI, Timed ; EC, e icle, time from P4-device removal to ovulation, and L G b T E o p N D M M I O D 513J.N.S. Sales et al. / Theriogenology 78 (2012) 510–516 ovulation rate. Continuous data were tested for normal- ity of the residues and analyzed by the UNIVARIATE procedure (transformed when necessary) and subjected to Bartlett’s test to assess homogeneity of variances. The GLM procedure with Tukey adjustment was used to determine significant differences among groups. All values are expressed as mean � SEM. In Experiment 3, the variables initially included in the models were treat- ment (EB and EC), farm and BCS at the first day of the synchronization protocol, and their interactions. Data were analyzed by a multivariate logistic regression us- ing the LOGISTIC procedure of SAS. Variables were removed by backward elimination, based on the Wald statistics criterion when P � 0.20. Variables included in the final model for analysis of P/AI were treatment, BCS and farm. Adjusted odds ratio (AOR) and 95% confidence interval (CI) were generated during the lo- gistic regression. Results are presented as proportions and AOR. The P/AI was analyzed using the GLIMMIX procedure of SAS. 3. Results 3.1. Experiment 1: circulating LH profile in ovariectomized Nelore heifers The LH release profile was different between EC- and EB-Group heifers (Fig. 2). The TLH, MLH, DLH and AUC for the EC and EB treatments are summarized (Table 1). Onset of the LH peak differed (P � 0.001) between EC and EB groups, such that in the latter LH peaked 30.9 h earlier than in the former. Additionally, LH peak magnitude was greater (P � 0.005) for EB heifers. Moreover, heifers receiving EB as the ovula- Fig. 2. Mean � SEM effect of treatment with EC or EB on circulating H concentrations in Nelore cows. D tion inducing agent had shorter (P � 0.001) peak du- ration and smaller (P � 0.01) area under the curve for LH concentrations. Finally, LH peak dispersion was smaller (P � 0.01) in Group-EB compared to that of EC-Group heifers. 3.2. Experiment 2: ovarian responses in Nelore cows Of the 60 cows initially used in this experiment, four (6.7%) did not respond (absence of a DF �6 mm at P4-device removal) to the TAI protocol and were there- fore removed from the study. Follicular response data are shown (Table 2). Maximum diameters of the DF (P � 0.12), and of the ovulatory follicle (P � 0.11) and interval from P4-device removal to ovulation did not differ between cows that received EB 24 h after P4- device removal and those receiving EC as described. Ovulation rate after the TAI protocol was similar be- tween the EB (77.8%; 21/27) and the EC groups Table 1 Plasma LH profile in ovariectomized Nelore heifers treated with EC or benzoate (EB). EB EC P No. animals 5 5 Magnitude of LH surge (ng/mL) 20.5 � 1.9 9.4 � 2.2 0.001 Time of LH surge (h) 19.6 � 1.2 50.5 � 3.6 0.005 Duration of LH surge (h) 8.6 � 0.2 16.5 � 1.0 0.001 Area under LH curve (ng/mL/72 h) 158.6 � 26.1 339.4 � 36.4 0.01 Protocol: day 0–1 intravaginal device of P4; 2 mg of EB; Day 8 - progesterone device was removed; 500 �g PGF2�; treatments (EC roup —1 mg of estradiol cypionate or EB Group —1 mg estradiol enzoate). able 2 ffects of EB - estradiol benzoate or EC - estradiol cypionate, as vulation inducers in suckled Nelore cows submitted to rogesterone-based TAI protocols, on ovarian responses. EB EC P o. animals 27 29 iameter of DF on Day 8 (mm) 10.2 � 0.3 9.9 � 0.3 0.76 aximum diameter of the DF (mm) 13.4 � 0.4 14.2 � 0.3 0.12 aximum diameter of the OF (mm) 13.1 � 0.4 13.9 � 0.4 0.11 nterval P4 device removal to ovulation (h) 70.2 � 1.8 68.5 � 1.9 0.53 vulation rate (%) 77.8 (21/27) 82.8 (24/29) 0.82 ouble ovulation 4.8 (1/21) 4.2 (1/24) 0.89 F, dominant follicle; OF, ovulatory follicle. 4 a 3 ( a f e f B w 4 e g s 514 J.N.S. Sales et al. / Theriogenology 78 (2012) 510–516 (82.8%, 24/29). Similarly, there was no difference in double ovulation rates (EB � 4.8%; 1/21 and EC � .2%; 1/24). Moreover, the variability of ovulation was lso similar (P � 0.59) between treatments (Fig. 3). .3. Experiment 3: pregnancy per AI in Nelore cows Bos indicus) There was no interaction between ovulation inducer nd explanatory variables, such as BCS (P � 0.96) and arm (P � 0.21) on P/AI. There was no significant ffect of ovulation inducer (EB and EC; P � 0.22) and arm (P � 0.15) on P/AI (Table 3). However, increased CS on the first day of the synchronization protocol as associated with increases in P/AI (Table 3). . Discussion To our knowledge, this is the first report studying the ffects of distinct ovulation inducers in estradiol and pro- esterone based protocols on LH profiles, follicular re- ponses and fertility of Bos indicus females submitted to TAI. In the present study, despite differences between the LH releases characteristics of the two ovulation inducers, ovarian response and fertility of Bos indicus cows were similar. These results were reached because of the 24 h earlier EC administration used in the ovulation synchro- nization protocol. The experimental hypothesis was con- firmed, since fertility between groups was similar. The main advantage of EC is the reduced need to handle these animals to perform the TAI. LH peaks occurred 19.6 and 50.5 h after the admin- Fig. 3. Distribution of the time of ovulation (h) after P4 device removal in Nelore cows (Bos indicus) submitted to TAI according to EC treatment at P4 device removal (handled three times; n � 29) or EB 24 h after (handled four times; n � 27). istration of EB and EC, respectively. These results were consistent with previous studies in which the adminis- tration of EB to beef cows (Bos taurus) or EC to crossbred beef heifers promoted LH surges at approx- imately 20.4 [12] or 54.6 h [11] after treatment, respec- tively. This is result of the distinct pharmacokinetics of the estradiol esters used. The EC is formed by esteri- fication of estradiol by cyclepentano propionic acid, resulting in low solubility in water and consequent slower release from the administration site and pro- longed biological activity compared to estradiol benzo- ate [16]. In addition to LH peak anticipation, differ- ences in the pharmacokinetics of the estradiol esters are responsible for the greater LH release and shorter LH peak duration of animals treated with estradiol benzo- ate. The recognition of these differences is important to determine the appropriate time to administer the ovu- lation inducer in TAI protocols in which estradiol and progesterone are used. Despite the greater LH peak synchronization of animals receiving EB as the ovula- tion inducer, ovulation synchronization between the two experimental groups (EB and EC) was similar. Presently, progesterone and estradiol based TAI pro- tocols are highly efficient in synchronizing ovulation in Bos indicus cows [17,18]. In the present study, �80% of the cows responded to the synchronization protocol and in only 6.7% a DF was not detected at P4-device removal. Other authors reported that 4.7% [19] and 6.3% [20] of females synchronized with estradiol and progesterone based protocols did not exhibit a new wave of follicular growth. Another important characteristic of TAI protocols is the ovulatory follicle diameter (OFD), because it is directly related to CL size during the subsequent di- estrus [21]. The CL diameter is highly correlated to the progesterone it synthesizes [22] which is essential for Table 3 Risk factors for P/AI following two estradiol/progesterone-based synchronization protocols in suckled Bos indicus cows. Variable P/AI, % (n/n) Adjusted OR (95% CI)a P Ovulation inducer EB 57.5 (277/482) Referentb 0.22 EC 61.8 (291/471) 1.18 (0.91–1.53) Farm A 58.7 (229/390) Referentb 0.15 B 60.2 (339/563) 1.24 (0.93–1.65) BCS category Low (2.0–2.5) 54.9 (218/397) Referentb 0.01 Moderate to high (�2.5) 63.0 (350/556) 1.39 (1.07–1.81) a OR, odds ratio; CI, confidence interval. b Referent, reference group for adjusted risk ratio. s a d a r c c t p m s t r n a t b a w f a c n f c A ( A h m B i O G C R [ [ 515J.N.S. Sales et al. / Theriogenology 78 (2012) 510–516 post-fertilization embryonic development [23]. In the present study, OFD did not differ between ovulation inducers or between treatments and diameter of the ovulatory follicle was �13 mm at TAI. Satisfactory pregnancy rates were observed in cows with OF diam- eters �13 mm at TAI [24]. Besides their efficiency in synchronizing the LH peak, both estradiol esters induced high ovulation rates (�80%), approximately 70 h after P4-device removal. Ovulation synchronization allows cows to be insemi- nated at a predetermined time point, thereby avoiding the need for estrus detection [1]. Ovulation synchroni- zation is important in situations in which the flexibility in relation to the time of AI is low, as in cows insem- inated with sex-sorted semen [18]. The moment of ovulation in relation to the P4- device removal was similar between estradiol esters because of the 24-h delay on the EB administration, since the LH peak in this group occurred 30 h earlier than in EC group. This delay allowed a similar final growth of the ovulatory follicle between cows receiving either EB or EC. In a previous study, EB administration at progesterone device removal resulted in smaller ovu- latory follicle diameters and lower pregnancy rates in cows inseminated 54 h after P4-device removal [17]. Fertility was not influenced by estradiol esters in this tudy. Similar results were observed by Meneghetti, et l. [25] in which no pregnancy rate differences were etected between cows treated with EB (50.8%) 24 h fter P4-device removal or EC (51.9%) at P4-device emoval. The estradiol esters studied had similar effi- acy in inducing ovulation and fertility for TAI in beef ows. Body condition scores lower than 2.5 (1–5 scale) led o lower TAI pregnancy rates in beef cows [2]. In the resent study, the risk for P/AI was higher in cows of oderate to high BCS in both treatments. In various tudies with beef cows, it was reported that low BCS at he beginning of TAI synchronization protocols may educe fertility [4,25,26]. Thus, management tech- iques aiming at maintain or improving BCS before nd, mainly, after parturition are important to guarantee he future fertility of cattle herds. In conclusion, despite pharmacologic differences etween the two ovulation inducing agents studied (EB nd EC), both were effective in inducing an LH peak hich resulted in synchronized ovulations, adequate ertility in suckled Bos indicus beef cows submitted to n estradiol/progesterone-based synchronization proto- ol. However, the use of EC allows for reduction on the umber times cows must be handled without reduced ertility. This is also a desirable characteristic for in- reasing the adoption and spread of TAI. cknowledgments The authors are thankful to DIMAFE Agropecuária Jacareuna Farm - são Felix do Araguaia, MT, Brazil), PTA-Vale do Paraíba Regional Center (Pindamon- angaba, SP, Brazil) and Department of Support, Ani- al Production and Health - UNESP (Araçatuba, SP, razil) for allowing the use of their animals and facil- ties for this study. 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Theriogenology 2009;72:179–89. 26] Sá Filho MF, Santos JE, Ferreira RM, Sales JN, Baruselli PS. Importance of estrus on pregnancy per insemination in suck- led Bos indicus cows submitted to estradiol/progesterone- based timed insemination protocols. Theriogenology 2011; 76:455– 63. Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulat ... 1. Introduction 2. Materials and methods 2.1. Experiment 1: circulating LH profiles in ovariectomized Nelore heifers 2.1.1. Animals and handling 2.1.2. Experimental design 2.1.3. Blood sampling and LH assay 2.2. Experiment 2: ovarian responses in Nelore cows 2.2.1. Animals and handling procedures 2.2.2. Experimental design 2.3. Experiment 3: pregnancy per AI in Nelore cows (Bos indicus) 2.3.1. Animals and management 2.3.2. Experimental design 2.4. Statistical analysis 3. Results 3.1. Experiment 1: circulating LH profile in ovariectomized Nelore heifers 3.2. Experiment 2: ovarian responses in Nelore cows 3.3. Experiment 3: pregnancy per AI in Nelore cows (Bos indicus) 4. Discussion Acknowledgments References