Theriogenology 79 (2013) 1204–1209

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Theriogenology

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The effect of equine chorionic gonadotropin on follicular size, luteal volume, circulating concentrations, and rates in anestrous beef cows treated with a novel fixed-time artificial insemination protocol

Rodrigo Dorneles Tortorella a, Rogério Ferreira b,*, Joabel Tonellotto dos Santos c, Olmiro Silveira de Andrade Neto c, Marcos Henrique Barreta d, João Francisco Oliveira c, Paulo Bayard Gonçalves c, Jairo Pereira Neves a a Programa de Pós-Graduação em Ciência Animal, Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília, UnB, Brasília, Distrito Federal, Brazil b Departamento de Zootecnia, Universidade do Estado de Santa Catarina - UDESC, Chapecó, Bandeira, Santa Catarina, Brazil c Laboratório de Biotecnologia e Reprodução Animal - BioRep, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil d Universidade Federal de Santa Catarina - Campus Universitário Curitibanos, Curitibanos, Santa Catarina, Brazil article info abstract

Article history: The objective was to determine the effects of eCG given on the day of, or 2 days before Received 31 May 2012 removal of an intravaginal progestin device, on diameter, luteal volume, Received in revised form 15 February 2013 serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time Accepted 16 February 2013 AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal Keywords: device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol Postpartum anestrus (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 mg im) eCG Follicle growth was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly ¼ ¼ assigned to receive 400 IU im eCG on Day 6 (eCG6; N 8) or Day 8 (eCG8; N 8), or to not FTAI receive eCG (control; N ¼ 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 0.2 mm) or control (8.5 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N ¼ 121) and eCG8 (N ¼ 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 857.3 mm3 and 8.1 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 505.1 mm3 and 4.5 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates. Ó 2013 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction

The success of a fixed-time artificial insemination (FTAI) * Corresponding author. Tel.: þ55 49 3322 4202; fax: þ55 49 3322 4202. program is dependent on a high ovulation rate during E-mail address: [email protected] (R. Ferreira). a short interval [1]. Therefore, the dominant follicle must

0093-691X Ó 2013 Elsevier Inc. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.theriogenology.2013.02.019 R. Dorneles Tortorella et al. / Theriogenology 79 (2013) 1204–1209 1205 acquire the capacity to ovulate in response to an ovulation cows were kept on natural pasture with free access to salt induction treatment. Bovine follicles acquire the capacity to and water. All procedures described herein were approved ovulate when they reach a diameter of approximately 10 or by the Animal Health and Welfare Committee at the 12 mm (Bos indicus and Bos taurus, respectively) [2,3]. Cows University of Santa Maria, Santa Maria, Rio Grande do Sul, under nutritional stress have low GnRH and, consequently, Brazil. low LH pulse frequency, resulting in smaller dominant In both experiments, cows received a progestin- follicles and ovarian follicular waves either without releasing IVD for 8 days and were given 2 mg im estradiol ovulation or with ovulation of a small follicle [4,5].Itis benzoate (Hubei Sanjing Biotechnology, Xianning City, noteworthy that these are major causes of prolonged China), with day of treatment defined as Day 0. Sodium interval between calving and conception, which cause cloprostenol (0.265 mg, PGF2a; Ciosin; Intervet Schering, substantial production losses in beef cattle [6]. Cotia, São Paulo, Brazil) was given im on Day 6, and gona- Progesterone (P4) is required for pregnancy mainte- dorelin (100 mg, GnRH; Profertil; Tortuga, Santo Amaro, São nance [7–9]; blood P4 concentrations during pregnancy Paulo, Brazil) was given im on Day 9, 24 hours after IVD affect embryo development and survival [10]. The size and removal (Fig. 1). The progestin IVD [18] was prepared with development of the conceptus increases under high polyurethane (5 5 10 cm, density 33 kg/m3), impreg- concentrations of endogenous P4 during early pregnancy in nated with 250 mg of medroxyprogesterone acetate cattle, whereas under low P4 concentrations, embryo (Genix-Purifarma, Anápolis, Goiás, Brazil), stored in a clean development and interferon-tau (IFN-s) production are container, and protected from light and humidity. reduced [11]. In ewes, exogenous P4 increased blastocyst In experiment 1, cows (N ¼ 24) were allocated randomly diameter by 220% on Day 9, changes from elongation to to three groups, ensuring that the groups were balanced a filamentous form on Day 12, and the concentration of according to BCS. These cows were allocated to eCG6 (N ¼ IFN-s in the uterine lumen on Day 15 of pregnancy [12].In 8) or eCG8 (N ¼ 8) treatment groups to receive 400 IU of cattle, P4 supplementation after AI has increased embryo eCG (Folligon; Intervet Schering) on Days 6 or 8, respec- survival, thereby increasing pregnancy rates [13–15]. tively, of the treatment protocol. The control group (N ¼ 8) Therefore, a successful estrus synchronization protocol was treated similarly, but did not receive eCG. Ovarian must induce ovulation of a healthy follicle which will follicular growth was assessed daily from Days 6 to 9 with result in a functional CL which will produce high P4 an Aquilla Vet ultrasound system (Pie Medical; Maastricht, concentrations. Netherlands) equipped with an 8-MHz transrectal trans- Equine chorionic gonadotropin (eCG) has been used in ducer. Ultrasonography exams were also performed on FTAI protocols in cattle to improve ovarian follicular growth Days 10 and 11 (24 and 36 hours after GnRH treatment) to and thereby produce a larger, more responsive follicle for detect ovulation (Fig. 1). Ovulation was defined as disap- ovulation [16–18], with increased pregnancy rates [17–19]. pearance of the dominant follicle, followed by formation of Equine chorionic gonadotropin is synthesized by endome- trial cups in pregnant mares and induces the formation of accessory corpora lutea. In cattle, eCG has FSH- and LH-like Control group activity [20]. EB PGF2 GnRH In FTAI protocols for beef cattle with a high incidence of postpartum anestrus, eCG has been given concurrent with MPA removal of a progestin-releasing intravaginal device (IVD), to stimulate dominant follicle growth before ovulation eCG6 group – [1,21 23]. However, a preliminary study in our laboratory EB PGF2 + eCG GnRH indicated that dominant follicle size was greater when eCG was injected 1 day before as compared with the time of IVD MPA removal (unpublished data). The present study aimed to evaluate the effects of eCG given 2 days before progestin eCG8 group

IVD withdrawal in a novel FTAI protocol for anestrous EB PGF2 eCG GnRH suckled beef cattle under nutritional stress. We hypothe- sized that administration of eCG 2 days before removing MPA the IVD would increase follicular growth, luteal volume, D0 D6 D8 D9 24 h 36 h serum P4 concentrations, and pregnancy rates. 24 h24 h 24 h 24 h 12 h Ultrasonographic exams 2. Materials and methods Fig. 1. Experimental design. Day 0 (D0): administration of medroxypro- 2.1. Cows and experimental design gesterone acetate (MPA) intravaginal device (250 mg MPA) and estradiol benzoate (EB) (2 mg im). Day 6 (D6): Administration of sodium cloprostenol (0.265 mg im). Day 8 (D8): vaginal device removal. Day 9 (D9): administration The experiments were conducted at a ranch located in of GnRH analogue (100 mg im). On D6 or D8, eCG (400 IU) was administered southern Brazil (2959’04.41”S, 5440’47.00”W) using randomly to the cows allocated to the eCG6 (N ¼ 8; experiment 1) or eCG8 anestrous multiparous suckled beef cows (Bos taurus (N ¼ 8; experiment 1) groups, respectively. The control group did not receive eCG (N ¼ 8; experiment 1). In experiment 1, cows were subjected to an crossbred; N ¼ 270), ranging from 40 to 60 days post- ovarian ultrasound exam from D6 to 36 hours after GnRH administration (Day partum, with a mean body condition score (BCS) of 2.5 on 11). In experiment 2, fixed-timed AI was done 16 hours after administration a scale from 1 to 5 (where 1 ¼ emaciated and 5 ¼ obese). All of GnRH (eCG6 group, N ¼ 121 and eCG8 group, N ¼ 125). 1206 R. Dorneles Tortorella et al. / Theriogenology 79 (2013) 1204–1209 a CL. The volume of the CL was assessed at 5, 10, and 15 the control group (P > 0.05). Cows without evidence of days postovulation using the formula V ¼ 4/3pR3, where follicle growth were removed from the statistical analysis. R ¼ (Da/2 þ Db/2)/2 and where Da and Db were the There was a day effect (P < 0.0001), and there was a tendency perpendicular diameters of the CL [24]. If a CL cavity was (P < 0.07) of an interaction between group and day (Table 1). present, its volume was subtracted from overall volume. Ovulations, regardless of group, occurred from 24 to 36 hours In experiment 2, the eCG6 (N ¼ 121) and eCG8 (N ¼ 125) after GnRH treatment. In experiment 1, ovulation ratios were protocols were compared in a larger number of cows sub- 6/7, 3/6, and 3/4 for the eCG6, eCG8, and control groups, jected to FTAI. These cows were randomly assigned to eCG6 respectively. In experiment 2, ovulation ratios were 13/20 and eCG8 treatment groups. At 16 hours after GnRH and 14/19 for the eCG6 and eCG8 groups, respectively. treatment, all cows were inseminated with semen of proven fertility. Volume of the CL was evaluated in all cows 3.2. Effects of eCG on CL volume and P4 concentration in which circulating P4 concentrations were >2 ng/mL at 10 (experiment 1) days after ovulation. However, cows with P4 concentra- tions <2 ng/mL were excluded from data analysis. Ovula- Cows without evidence of a CL and with serum P4 tion time was estimated based on the data from the first concentrations <2 ng/mL 10 days after ovulation were experiment. Pregnancy diagnoses were done 30 days after excluded from the analysis. The mean CL volumes in the FTAI using an Aquilla Vet ultrasound system (Pie Medical) eCG6 (N ¼ 6), eCG8 (N ¼ 3), and control (N ¼ 2) groups 5 equipped with a 6-MHz transrectal transducer. days after ovulation are shown (Table 2). No CL was observed in any control cows at 10 and 15 days after 2.2. Serum sampling and P4 assay ovulation using ultrasound exam, and P4 concentrations were <2 ng/mL, which provided evidence of premature In experiment 1, blood samples (N ¼ 24) for the serum luteolysis. In the control group, one cow was considered to P4 assay were obtained 5, 10, and 15 days after ovulation have ovulated, but no CL was observed 5 days later. The via coccygeal vessel puncture and were immediately mean CL volumes were larger and serum P4 concentrations refrigerated, centrifuged for 20 minutes at 1500 g, and were higher (P < 0.05) in animals in the eCG6 group than in serum stored at 20 C until assayed. Serum P4 concen- the eCG8 group at 10 and 15 days after ovulation (Table 2). trations were determined using a solid phase RIA kit with antibody-coated tubes (Coat-a-Count, Diagnostic Products Corporation, Los Angeles, CA, USA). The assay’s sensitivity 3.3. Effects of eCG on the CL volume, P4 concentration, and was 0.02 ng/mL, and the intra-assay CV was 6.96%. In pregnancy rate (experiment 2) experiment 2, serum P4 concentrations were determined in 20 cows from each group 10 days after ovulation, with The percentage of cows with serum P4 concentrations a procedure similar to that used in the first experiment. >2 ng/mL 10 days after ovulation were 40.0% (8/20) and 52.6% (10/19) in the eCG6 and eCG8 groups, respectively. 2.3. Statistical analyses Cows with serum P4 concentrations <2 ng/mL were excluded from analysis. One cow from the eCG6 group, Follicular diameter was compared using a mixed models considered an outlier, was removed from the statistical analysis with a repeated statement (PROC MIXED; SAS 9.1). analysis to avoid overestimated results; in this cow, P4 The main effects of group and day and their interactions concentrations were 9.3, 24.4, and 29.2 ng/mL at 5, 10, and were determined. Differences in follicular diameters on 15 days, respectively, after ovulation. The CL volumes at 10 a specific day were compared between groups using the days after ovulation in cows in the eCG6 group (5783.9 estimate statement. CL volume and P4 concentrations were 857.3 mm3;N¼ 8) were greater than those in the eCG8 compared using ANOVA and general linear models. Post group (3220.8 505.1 mm3;N¼ 10; P < 0.05). Moreover, hoc analyses of least squares-adjusted means were per- P4 concentrations at 10 days after ovulation were higher in formed using Student t test. Data distribution was tested for the eCG6 group (8.1 1.3 ng/mL; N ¼ 8) than in the eCG8 normality with the Shapiro–Wilk test and normalized group (4.5 0.7 ng/mL; N ¼ 10; P < 0.05). Pregnancy rate when necessary. Pregnancy and ovulation rates were analyzed using the nonparametric test used in the PROC CATMOD, and post hoc analyses performed by contrasts of Table 1 Mean SEM effects of eCG given 2 days before (Day 6; eCG6), or at the maximum likelihood estimates. The statistical analysis was time of (Day 8; eCG8) progestin device removal on dominant follicle carried out using SAS 9.1 software and significance was diameter. considered for P < 0.05. Group Dominant follicle diameter (mm)a

3. Results Day 6 Day 7 Day 8 Day 9 eCG6 (N ¼ 7) 5.9 0.4 7.2 0.5 8.5 0.4 10.0 0.4b ¼ c 3.1. Effects of eCG on follicular growth (experiment 1) and eCG8 (N 6) 6.0 0.2 6.9 0.4 7.4 0.3 8.6 0.2 Control (N¼4) 5.9 0.5 7.2 0.3 8.0 0.4 8.5 0.4c ovulation rate (experiments 1 and 2) There was a day effect on dominant follicle sizes (P < 0.001) and a tendency for an interaction between group and day (P ¼ 0.07). Development of dominant ovarian follicles was observed a Data are presented as mean standard error of the mean. in 87.5% and 75.0% of cows in the eCG6 (7/8) and eCG8 (6/8) b,c Within a column, means without a common superscript differed (P < treatment groups, respectively, and in 50% (4/8) of cows in 0.05). R. Dorneles Tortorella et al. / Theriogenology 79 (2013) 1204–1209 1207

Table 2 Corpus luteum volume (mm3) and P4 concentration (ng/mL) 5, 10, and 15 days after ovulation in a fixed-timed AI protocol with eCG administration 2 days before (eCG6) or at the time of (eCG8) progestin device removal or without eCG administration (control group).

Parameter Groups Days after ovulation

510 15 CL volume (mm3) eCG6 (N ¼ 6) 2180 266 4355 187a 4608 489a eCG8 (N ¼ 3) 1616 129 1938 1447b 2029 1685b Control (N ¼ 2)c 798 62 dd P4 concentration (ng/mL) eCG6 (N ¼ 5)d 3.1 0.4 10.0 1.8a 9.4 1.4a eCG8 (N ¼ 3) 2.1 0.5 2.6 2.0b 2.4 1.7b Control (N ¼ 2) 0.3 0.6 0.1 2.5b 0.1 2.0b

a,b Within a column, means without a common superscript differed (P < 0.05). c Control cows did not have a CL at 10 or 15 days after ovulation. d An outlier was removed from analysis (see text). was higher (P < 0.05) for cows in the eCG6 group (27.3%; Day 8, because of the greater follicle diameters in the eCG6 33/121) than in the eCG8 group (16.0%; 20/125). group (approximately 10 mm) than in the eCG8 group (approximately 8.5 mm) at the time of GnRH treatment and 4. Discussion the short interval after IVD removal. However, there was no difference in ovulation rates between the eCG6 (70.4%; 19/ The main findings of this study were that the ovulatory 27) and eCG8 (68.0%; 17/25) groups when the data from follicle size, CL volume, serum P4 concentrations, and experiments 1 and 2 were combined to increase sample pregnancy rates were higher in anestrous suckled beef size. Therefore, we inferred that size is not the only deter- cows when eCG was administered 2 days before as minant of the capacity of the dominant follicle to ovulate. compared with the day of IVD removal with this FTAI Previously, it has been reported that ovulation of a small protocol. Preovulatory follicle size is of the utmost impor- follicle results in small CL volume, low serum P4 concen- tance because it affects ovulation rate [2,3], CL volume, P4 trations, and consequently decreased pregnancy rates [25]. concentrations [25], and pregnancy rate [1,26]. Conse- Moreover, Sartori et al. [33] reported a positive correlation quently, administration of eCG concurrent with P4 removal between CL volume and P4 production at 7 days after has been included to stimulate follicle growth in several ovulation in heifers and suckling cows. However, the FTAI protocols [1,21,23]. Although ovulation is induced with correlation between P4 secretion and CL volume seems to estradiol at approximately 24 hours [23,27] or GnRH within exist only during the initial formation of the CL [29]. In view 48 hours [23,28,29] after IVD removal, to ensure the exis- of this knowledge, the FTAI protocol described herein tence of a LH-responsive follicle in most of these FTAI (eCG6 protocol) was designed to increase follicular size and protocols, administration of GnRH 24 hours after IVD hence CL size and circulating P4 concentrations after removal has been recommended to improve the synchrony ovulation. CL volumes and P4 concentrations at 10 and 15 of ovulation and to avoid expression of estrus before FTAI days after ovulation in suckled beef cattle under nutritional [17,30]. However, administration of eCG 24 hours before stress, were greater in the eCG6 group compared with administration of GnRH was expected to provide insuffi- the eCG8 treatment group, presumably because of the cient time for increased follicle growth before ovulation. larger dominant follicle which was observed just before It is well established that eCG has high sialic acid ovulation. content, resulting in a long half-life (approximately 2 days) Cows with P4 concentrations <2 ng/mL were excluded when administered in cattle; consequently, it was detected from data analysis, because it was expected that this was an in cattle serum 10 to 16 days after treatment [27,31]. indication of premature luteal regression [25]. All control Moreover, eCG stimulates estradiol production via gran- cows in experiment 1 had premature luteolysis, confirmed ulosa cells and P4 via the CL [21]. The increase in follicular via ultrasound and serum P4 concentrations at 10 and 15 estradiol production induced by eCG seems to be connected days after ovulation. In addition, premature luteal regres- to an increase in mRNA for cytochrome P450 17a-hydrox- sion was considered confirmation of the anestrous condi- ylase/17–20 lyase and LH receptors [28,32]. Considering the tion of these cows. It has been suggested that administration long half-life and the affinity for the FSH and LH receptors of a progestin to anestrous cows could reduce premature [20], eCG administration 2 days before P4 removal might be luteolysis in subsequent induced estrous cycles [34,35]. a more rational strategy to further improve dominant Previously, we reported the effectiveness of vaginal follicle size at the time of ovulation in cattle treated with implants containing medroxyprogesterone acetate in estrus a protocol designed to induce ovulation 24 hours after IVD synchronization protocols leading to a satisfactory domi- removal. nant follicle size, ovulation, and pregnancy rates [16–19]. The acquisition of ovulatory capacity is dependent, at However, premature luteolysis was still observed in some least in part, on follicular size. Full ovulatory capacity has animals, even when a progestin device was used [36]. In the been reported to be obtained when the dominant follicle present study, it was noteworthy that cows were under reaches a diameter of 10 mm in Bos indicus and 12 mm in nutritional stress, which could have affected CL lifespan Bos taurus cattle [2,3]. In the present study, a lower [37]. However, eCG, regardless of whether it was adminis- ovulation rate was expected in cows that received eCG on tered at the time of, or 2 days before progestin IVD removal, 1208 R. Dorneles Tortorella et al. / Theriogenology 79 (2013) 1204–1209 resulted in the formation of a competent CL that did not [6] Yavas Y, Walton JS. Postpartum acyclicity in suckled beef cows: – undergo premature luteolysis, even though cows were in a review. Theriogenology 2000;54:25 55. [7] Spencer TE, Johnson GA, Bazer FW, Burghardt RC. Implantation poor nutritional conditions. Mann and Lamming [38] re- mechanisms: insights from the sheep. 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