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Effect of hCG vs. GnRH at the beginning of the Ovsynch on first ovulation and conception rates in cyclic lactating dairy cows
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Theriogenology 74 (2010) 602– 607 www.theriojournal.com
Effect of hCG vs. GnRH at the beginning of the Ovsynch on first ovulation and conception rates in cyclic lactating dairy cows A. Keskina, G. Yilmazbas-Mecitoglua, A. Gumena,*, E. Karakayaa, R. Daricib, H. Okutc a
University of Uludag, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Gorukle 16059 Bursa, Turkey b TARFAS Co., Karacabey, 16190 Bursa, Turkey c University of Yuzuncu Yil, Faculty of Agriculture, Biometry of Genetics, 65080 Van, Turkey Received 28 October 2009; received in revised form 8 February 2010; accepted 13 March 2010
Abstract Ovulatory response to the first GnRH of Ovsynch is a very important factor for determining the outcome of a successful synchronization. The aim of the present study was to develop a protocol to increase the percentage of cows that ovulated in response to the first administration of Ovsynch. This study was designed to compare ovulation rates in response to GnRH or hCG at the beginning of Ovsynch and to evaluate the effects of this manipulation on pregnancy. Cows (n ⫽ 371) with corpus luteum (CL) and at least one follicle greater than 10 mm diameter size on either ovary were included in the study. Cows were divided into two groups. The Ovsynch protocol began with GnRH (10 g) in the GPG group (n ⫽ 161; GnRH-7d-PGF2␣-56h-GnRH18h-AI), whereas in the HPG group, the first GnRH of the Ovsynch was replaced with 1500 IU hCG (n ⫽ 210; hCG-7d-PGF2␣56h-GnRH-18h-AI). Ovarian ultrasonography was performed at the times of GnRH or hCG and of PGF2␣ administration, at the time of artificial insemination (AI) and seven days after AI, to determine ovulation. Maximal follicle size at the beginning of the Ovsynch did not affect on response to the first GnRH/hCG treatment. Conception rate (31 d) was 0.6 times more likely to be higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch than in those that did not respond (95% CI ⫽ 0.29 – 0.71). Conception rate was found to be different between the HPG (37.6%, 79/210) and the GPG groups (48.4%, 78/161). Thus, beginning of the Ovsynch protocol with hCG did not increase ovulation and conception rate in lactating dairy cows, suggesting that hCG is not a suitable replacement of the first GnRH of Ovsynch. However, our results do show that increasing the ovulation rate in response to the first hormonal administration of Ovsynch can have a significant effect on conception rate. © 2010 Elsevier Inc. All rights reserved. Keywords: Ovsynch; hCG; Cow; Synchronization
1. Introduction Reproductive efficiency has major impacts on profitability of dairy producers and many methodologies are being used to improve the reproductive efficiency, including the use of reproductive hormones to regulate
* Corresponding author. Tel.: ⫹90 224 2940824; fax: ⫹90 224 2941202. E-mail address: [email protected] (A. Gumen). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.009
and control the estrus cycle. The Ovsynch synchronization protocol was developed to synchronize ovulation in lactating dairy cows using GnRH and PGF2␣ [1]. This protocol synchronizes ovulation within an 8 h period from 24 –32 h after the second GnRH administration. This precise synchrony may allow for successful artificial insemination (AI) without the detection of estrus. Ovulatory response after the first GnRH administration is a critical factor for the successful synchronization of ovulation in the Ovsynch protocol [2,3].
A. Keskin et al. / Theriogenology 74 (2010) 602– 607
High ovulation rate after the first GnRH treatment increases the likelihood that cows will have a functional dominant follicle capable of ovulation during the final GnRH treatment of Ovsynch. In addition, a lower ovulation rate after the first GnRH appears to result in a lower synchronization rate after the second GnRH administration [2,3]. These positive effects of ovulatory response could increase the subsequent fertility of lactating dairy cows. In several previous studies, researchers have tried to increase ovulation response to the first GnRH and synchronization rate with presynchronization protocols such as Presynch, Double Ovsynch and so on [3– 8]. Nevertheless, these studies have yielded conflicting results. Early studies showed that response to the first GnRH in the Ovsynch protocol was 45–90% [1–3,7]. In addition, it is known that ovulation of follicles larger than 10 mm can be induced with the administration of a purified LH [9]. In cattle, a single dose of GnRH analogs elicited the release of LH for 5 h [10], a duration that is approximately half of that of the naturally occurring preovulatory surge of LH in cattle [11]. Plasma hCG was detected until 66 h after administration of hCG and it is thought to act independently of the pituitary gland [12,13]. We hypothesized that the percentage of cows that ovulated in response to the first administration of the Ovsynch protocol would increase by using hCG. This would improve conception rate because of better synchronization. Thus, the aim of the present study was to compare ovulation rate in response to GnRH or hCG at the beginning of the Ovsynch protocol and to study the effect of this manipulation on pregnancy. 2. Materials and methods 2.1. Study population This experiment was conducted on a commercial dairy herd (approximately 800 lactating dairy cows) in the South Marmara region, Bursa, Turkey, from October 2008 to July 2009. Cows were housed in free stall barns with self-catching head-locks. All cows were milked three times a day and were grouped according to their milk production. Mean milk production of the herd was 9,880 kg (305 d) per cow. Cows had free access to water and were fed complete mixed rations according to NRC recommendations [14]. Daily milk yield, reproductive health, and management records for each cow were collected from Alpro 2000 (DeLaval, Sweden). Average milk production for each cow was recorded for 7 d before and after AI. All cows had their
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body condition scored (BCS) using a 5-point (1 ⫽ thin to 5 ⫽ fat) scoring system [15]. All animal handling and procedures were approved by the Lalahan Livestock Central Research Institute Animal Care Committee. 2.2. Study design Holstein lactating dairy cows (n ⫽ 371) were randomly divided into two groups. In the first group (GPG group, n ⫽ 161), Ovsynch was started (day 0) with GnRH (Busereline acetate, 10 g, i.m., Receptal®, Intervet, Turkey). In the second group (HPG group, n ⫽ 210), Ovsynch was initiated (day 0) with hCG (1500 I.U., i.m., Chorulon®, Intervet, Turkey). Then, PGF2␣ (Cloprostenol, 500 g, i.m., Estrumate®, CEVA-DIF, Turkey) and GnRH (Busereline acetate, 10 g, i.m., Receptal®) were administered to both groups on day 7 and 9, respectively. All cows were inseminated at a fixed time 16 –18 h after the second GnRH of Ovsynch using frozen-thawed semen from bulls of proven fertility by farm veterinarians. Cows were evaluated with an ultrasound machine (Honda HS 2000 equipped with a 5.0 –7.5 MHz transducer, Honda, Japan) to determine cyclicity. Cows with corpus luteum (CL) and at least one follicle equal or larger than 10 mm size on either ovary were included in the study. Cows’ ovaries were evaluated for follicle size on the day of GnRH/hCG administration and 7 d later to determine ovulation. Ovulation in response to the first GnRH/hCG treatment was characterized by the appearance of a new CL on any ovary. Maximum follicular size was also measured at the time of AI. Ovulation after the second GnRH of Ovsynch was determined by the disappearance of the dominant follicle and the presence a new CL on the ovary 7 d after AI. Pregnancy diagnosis was performed 31 and 62 d post-insemination by ultrasonography. Pregnancy loss was calculated as the number of cows diagnosed nonpregnant at the second pregnancy check, expressed as a percentage of the number of cows diagnosed pregnant at the first pregnancy check. Conception rate was calculated as the number of cows diagnosed pregnant, divided by the number of cows receiving AI. 2.3. Statistical analysis Statistical analyses were conducted using SAS (Version 9.1; SAS Institute, 2003). Data were evaluated using PROC LOGISTIC, PROC GLM, and PROC FREQ in SAS. The model included the effects of parity, days in milk (DIM), BCS, number of services, average milk production, response to the first GnRH/hCG, and
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follicle sizes at the beginning of Ovsynch and at the time of AI. To carry out the statistical analyses, data was coded as 1 (Yes) or 0 (No) for ovulatory response to the first GnRH/hCG. Conception on day 31 and 62 after insemination was coded as 0 (not pregnant) or 1 (pregnant). Primiparous and multiparous cows were coded as 1 or 2, respectively. Number of services to each cow was coded as 0 (first service AI) or 1 (more than one service). Second ovulatory response was coded as 1 (Yes) or 0 (No). A GLM procedure was performed to compare milk production, DIM, BCS, follicle sizes at the beginning of the Ovsynch and at the time of AI between groups. Chi-square analysis using the PROC FREQ procedure was used for testing independence between parity, number of services, and the response to GnRH/hCG of Ovsynch and CR (31 and 62 d) between groups. A logistic procedure was performed to compare the effects of milk production, DIM, BCS, follicle size at the beginning of the Ovsynch and at the time of AI, parity, number of services and response to the first GnRH/hCG treatment on ovulation and on conception rates at 31 and 62 d. 3. Results Although the study began with 422 cows, 51 cows (23 cows in the GPG group and 28 cows in the HPG group) were excluded at different stages of the study due to several diseases such as mastitis, endometritis, and farm culling regime. A total of 371 cows were included in the study. The mean lactation number for cows was similar between the GPG and HPG groups (2.4 and 2.3, respectively). Average milk production did not differ between groups (40.6 ⫾ 0.6 kg/d in GPG and 41.5 ⫾ 0.5 kg/d in HPG). However, DIM was significantly different (P ⬍ 0.001) between the GPG and HPG groups (132.9 ⫾ 3.5 and 107.1 ⫾ 3.1, respectively). BCS was found to be similar between the GPG and HPG groups (2.73 ⫾ 0.02 and 2.74 ⫾ 0.01, respectively). Conception rate (at 31 and 62 d) and percentage of cows that ovulated in response to the first GnRH/hCG treatment, together with the maximal size of the ovulatory follicle on the first day of treatment, are shown in Table 1. Maximal follicle size at the beginning of Ovsynch did not differ between the GPG and HPG groups (15.8 ⫾ 0.2 mm and 15.4 ⫾ 0.2 mm, respectively), and maximal follicle size at the beginning of the Ovsynch did not affect the response to the first GnRH/ hCG treatment. Ovulation response to the first hor-
Table 1 Conception rate and percentage of cows that ovulated to first GnRH/hCG treatment and maximal size of the ovulatory follicle on day first treatment and at time of AI. GPG Maximal follicle size at the first GnRH/hCG administration Response to First GnRH/hCG % Synchronization rate % Conception rate at 30 days pregnancy % Embryonic loss % a,b
P ⬍ 0.08;
c,d
HPG
15.8 ⫾ 0.2 mm 15.4 ⫾ 0.2 mm 59.6 (96/161) 65.7 (138/210) 90.1 (145/161)a 83.8 (176/210)b 48.5 (78/161)c 37.6 (79/210)d 0.05 (4/78)
0.05 (4/79)
P ⬍ 0.04.
monal administration was similar in the GPG group (59.6%; 96/161) and in the HPG group (65.7%; 138/ 210) (Table 1). Other covariant factors, such as milk production, DIM, BCS, number of services and parity had no effect on first ovulatory response for either group. Cows that ovulated in response to the first GnRH/ hCG of Ovsynch had tendency to produce smaller (P ⬍ 0.09) follicle size at the time of AI (15.9 ⫾ 0.14 mm) than those that were nonresponsive to GnRH/hCG (16.3 ⫾ 0.21 mm). In addition, CR (31 d) was 0.64 times more likely to be higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch than in those that did not respond [95% CI ⫽ 0.29 – 0.71; actual conception rates were 49.2% (115/ 234) and 30.6% (42/137) responded and not responded cows, respectively]. This difference was also significantly higher (P ⬍ 0.03) for the conception rate at 62 d (OR ⫽ 0.45; 95% CI ⫽ 0.28 – 0.70). Conception rate was significantly different between the HPG (37.6%, 79/210) and GPG (48.4%, 78/161) groups (P ⬍ 0.04). Number of services, DIM, milk production, parity, and follicle size at the time of AI had no effect on CR. In contrast, BCS, response to the first GnRH/hCG treatment and follicle size at the beginning of Ovsynch significantly affected CR (Table 2). Conception rates at 62 d were also found to be significantly different between groups (35.7%, 75/210 in HPG and 45.9%, 74/161 in GPG). 4. Discussion We compared ovulation rate in response to GnRH or hCG in Ovsynch, and examined whether conception rate (at 31 and 62 d) was related to ovulation rate in response to the first GnRH/hCG. We selected cows according to cyclicity and follicle size at the beginning of the Ovsynch to determine the ovulatory effect of
A. Keskin et al. / Theriogenology 74 (2010) 602– 607 Table 2 Results of logistic regression model for effects of significant covariant factors on conception rate in cows. Item BCS Follicle size at the beginning of the Ovsynch Response the GnRH/hCG (No vs. Yes) 1
Odds ratio (OR)
95% (CI)1
P-values
2.56 1.08
1.05–6.22 1.01–1.16
0.03 0.02
0.52
0.32–0.87
0.01
CI ⫽ confidence interval.
GnRH and hCG. Only cyclic cows that had at least one CL and a follicle that was equal or larger than 10 mm were included in the study. Follicles smaller than 9 mm in diameter have not usually acquired ovulatory capacity in dairy cows [7,9] and ovulatory response is higher in noncyclic than in cyclic lactating dairy cows [7,16]. Therefore, the selection criteria used in our study allowed us to obtain a realistic measure of the ovulatory effect of GnRH/hCG on overall Ovsynch outcome. Ovulatory response to the first GnRH of the Ovsynch is the critical determinant for successful synchronization of ovulation in dairy cows. Early studies showed that response to the first GnRH of the Ovsynch and fertility were dependent on the stage of the estrus cycle at the beginning of the protocol [2,3]. Ovulatory response when the Ovsynch was initiated at random stages of the estrus cycle has been reported to be 45– 62% [7,16]. However, when the Ovsynch was initiated between 5–10 d of the estrus cycle, ovulatory response was increased to 85–96% [2,3]. Therefore, many researchers have developed presynchronization protocols that attempt to increase the number of cows in the optimum stage of the estrus cycle on the day of the first GnRH of the Ovsynch [3– 6]. Presynchronization protocols increased response to first GnRH of Ovsynch in previous studies [3,4]. In a recent study, Souza et al, [6] developed a new presynchronization protocol (Double Ovsynch), and they reported that ovulation rate to the first GnRH of the Ovsynch increased up to 71.8%. Nevertheless, in some other studies, the ovulatory response was negatively affected by presynchronization with PGF2␣ before the Ovsynch [7,8]. In the current study, we used hCG instead of GnRH at the beginning of the Ovsynch. Even though we were able to increase the ovulatory response up to 66% in the HGP group, the ovulatory response was found to be insignificant between the GPG and HPG groups. Despite the lack of differences in ovulatory response between the GPG and HPG groups, our results indi-
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cated that the first ovulatory response had a significant effect on pregnancy rate in lactating dairy cows. The conception rate was higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch (49.2%) than those that did not (30.6%). Increasing the first ovulatory response might have a positive effect on luteal and follicular responses to the subsequent administration of PGF2␣ and the final GnRH of the Ovsynch, and these positive effects could induce higher fertility in lactating dairy cows [2– 4,6,7]. Fertility also was associated with progesterone levels at induction of luteolysis, size, and function of preovulatory follicle. Fonseca et al [17] showed a positive relationship between concentration of progesterone at induction of luteolysis and conception rates. Conversely, lesser levels of progesterone at induction of luteolysis were associated with a decline in CR [18]. Cows that ovulated after the first GnRH of Ovsynch had increased progesterone levels at the time of PGF2␣ and were more likely to undergo luteolysis in response to the PGF2␣ of the Ovsynch [2– 4,6]. In addition, another critical factor affecting the overall outcome of Ovsynch is follicular size at the time of the second GnRH or AI, which affect ovulatory response. In our study, despite the fact that the HPG and GPG groups had similar ovulatory response to the first treatment of Ovsynch, cows in the HPG group had tendency to produce to smaller preovulatory follicle size at the time of AI than cows in GPG group (15.8 ⫾ 0.2 mm and 16.4 ⫾ 0.2 mm respectively). Another important factor is the relationship between follicle size and pregnancy. There are conflicting results among the studies addressing this issue [2,19,20]. On the one hand, Lopes et al [19] reported that larger preovulatory follicle size increased pregnancy. On the other hand, Vasconcellos et al [2] indicated that lower ovulatory follicle size produces more pregnancy in lactating dairy cows. In the present study, although preovulatory follicle size was lower in the HPG (15.8 ⫾ 0.2 mm) than the GPG group (16.4 ⫾ 0.2 mm), conception rate was lower in the HPG (37.6%) than GPG (48.4%) groups (P ⬍ 0.04). This result shows that follicle size at the time of AI could affect subsequent fertility in lactating dairy cows. Consistent with our results, some studies have indicated that fertility was greater after the ovulation of a follicle of approximately 16 mm in diameter [3,20]. Our results indicated that using of hCG replace of GnRH did not affect either ovulatory response or the overall outcome of the Ovsynch. In previous studies, treatment with hCG induced accessory corpus luteum,
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increased plasma progesterone level, and improved conception rate in lactating dairy and beef cattle [21– 24]. In early studies, hCG was used to replace the first and second GnRH of Ovsynch in lactating dairy and beef cows [22,24 –26]. However, despite the fact that usage of hCG in the Ovsynch increased cumulative conception rate in lactating dairy cows, there was no effect on conception rate in cold season [24 –26]. In the case of the beef cows, hCG was used in TAI protocol (CO-Synch) and these studies showed that hCG was not a suitable replacement for GnRH to synchronize ovulation when using the TAI protocol [21,22]. In conclusion, beginning of the Ovsynch protocol with hCG did not increase ovulation and conception rates in lactating dairy cows. Thus, hCG is not a suitable replacement for the first GnRH of Ovsynch. However, increasing ovulation rate in response to the first hormonal administration of Ovsynch can have a significant effect on conception rate.
Acknowledgements The authors thank TARFAS Co. (Bursa, TURKEY) for the use of their herd and facilities. Support was also provided by the The Scientific and Technological Research Council of Turkey (TUBITAK) grant no. TOVAG 107O227.
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[8] Navanukrav C, Redmer DA, Reynolds LP, Kirsch JD, GrazulBilska AT, Fricke PM. A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows. J Dairy Sci 2004;87:1551–7. [9] Sartori R, Fricke MP, Ferreira JCP, Ginther OJ, Wiltbank MC. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biology of Reproduction 2001;65:1403–9. [10] Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology 1990;34:81– 6. [11] Rahe CH, Owens RE, Fleeger JL, Newton HJ, Harms PG. Pattern of plasma luteinizing hormone in the cyclic cow: dependent upon the period of the cycle. Endocrinology 1980;107: 498 –503. [12] Seguin BE, Oxender WD, Britt JH. Effect of human chorionic gonadotropin and gonadotropin releasing hormone on corpus luteum function and estrus cycle duration in dairy heifers. Am J Vet Res 1977;38:1153. [13] Schmitt EJP, Diaz T, Barros CM, De la Sota RL, Drost M, Fredriksson EW, Staples CR, Thorner R, Thatcher WW. Differential response of the luteal phase and fertility in cattle following ovulation of the first-wave dominant follicle with human chorionic gonadotropin or an agonist of gonadotropinreleasing hormone. J Anim Sci 1996;74:1074 – 83. [14] National Research Council. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, D.C, 2001. [15] Ferguson JD, Galligan DT, Thomsen N. Principal descriptors of body condition score in Holstein cows. J Dairy Sci 1994;77: 2695–703. [16] Gumen A, Guenther JN, Wiltbank MC. Follicular size and response to Ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. J Dairy Sci 2003;86:3184 –94. [17] Fonseca FA, Britt JF, McDaniel BT, Wilk JC, Rakes AH. Reproductive traits of Holsteins and Jerseys. Effects of age, milk yield, and clinical abnormalities on involution of cervix and uterus, ovulation, estrus cycles, detection of estrus, conception rate, and days open. J Dairy Sci 1983;66:1128 – 47. [18] Xu Z, Burton LJ, Macmillan KL. Reproductive performance of lactating dairy cows following estrus synchronization regimens with PGF2alpha and progesterone. Theriogenology 1997;47: 687–701. [19] Lopes AS, Butler ST, Gilbert RO, Butler WR. Relationship of pre-ovulatory follicle size, estradiol concentrations and season to pregnancy outcome in dairy cows. Anim Reprod Sci 2007; 99:34 – 43. [20] Perry GA, Smith MF, Lucy MC, Green JA, Parks TE, MacNeil MD, Roberts AJ, Geary TW. Relationship between follicle size at insemination and pregnancy succes. PNAS USA 2005;102: 5268 –73. [21] Geary TW, Salverson RR, Whittier JC. Synchronization of ovulation using GnRH or hCG with the CO-Synch protocol in suckled beef cows. J Anim Sci 2001;79:2536 – 41. [22] Burns MG, Buttery BS, Dobbins CA, Martel CA, Olson KC, Lamb GC, Stevenson JS. Evaluation of human chorionic gonadotropin as a replacement for gonadotropin-releasing hormone in ovulation-synchronization protocols before fixed timed artificial insemination in beef cattle. J Anim Sci 2008; 86:2539 – 48. [23] Buttery BS, Burns MG, Stevenson JS. Ovulation and pregnancy outcomes in response to human chorionic gonadotropin before resynchronized ovulation in dairy cattle. Theriogenology 2010; 73(4):449 –59.
A. Keskin et al. / Theriogenology 74 (2010) 602– 607 [24] De Rensis F, Marconi P, Capelli T, Gatti F, Facciolongo F, Franzini S, Scaramuzzi RJ. Fertility in postpartum dairy cows in winter or summer following estrus synchronization and fixed time after the induction of an LH surge with GnRH or hCG. Theriogenology 2002;58:1675– 87. [25] De Rensis F, Bottarelli E, Battioni F, Capelli T, Techakumphu M, Garcia-Ispierto I, Lopez-Gatius F. Reproductive perfor-
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mance of dairy cows with ovarian cysts after synchronizing ovulation using GnRH or hCG during the warm or cool period of the year. Theriogenology 2008;69:481– 4. [26] De Rensis F, Valentini R, Gorrieri F, Bottarelli E, LopezGatius F. Inducing ovulation with hCG improves the fertility of dairy cows during the warm season. Theriogenology 2008; 69:1077– 82.
Theriogenology 74 (2010) 602– 607 www.theriojournal.com
Effect of hCG vs. GnRH at the beginning of the Ovsynch on first ovulation and conception rates in cyclic lactating dairy cows A. Keskina, G. Yilmazbas-Mecitoglua, A. Gumena,*, E. Karakayaa, R. Daricib, H. Okutc a
University of Uludag, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Gorukle 16059 Bursa, Turkey b TARFAS Co., Karacabey, 16190 Bursa, Turkey c University of Yuzuncu Yil, Faculty of Agriculture, Biometry of Genetics, 65080 Van, Turkey Received 28 October 2009; received in revised form 8 February 2010; accepted 13 March 2010
Abstract Ovulatory response to the first GnRH of Ovsynch is a very important factor for determining the outcome of a successful synchronization. The aim of the present study was to develop a protocol to increase the percentage of cows that ovulated in response to the first administration of Ovsynch. This study was designed to compare ovulation rates in response to GnRH or hCG at the beginning of Ovsynch and to evaluate the effects of this manipulation on pregnancy. Cows (n ⫽ 371) with corpus luteum (CL) and at least one follicle greater than 10 mm diameter size on either ovary were included in the study. Cows were divided into two groups. The Ovsynch protocol began with GnRH (10 g) in the GPG group (n ⫽ 161; GnRH-7d-PGF2␣-56h-GnRH18h-AI), whereas in the HPG group, the first GnRH of the Ovsynch was replaced with 1500 IU hCG (n ⫽ 210; hCG-7d-PGF2␣56h-GnRH-18h-AI). Ovarian ultrasonography was performed at the times of GnRH or hCG and of PGF2␣ administration, at the time of artificial insemination (AI) and seven days after AI, to determine ovulation. Maximal follicle size at the beginning of the Ovsynch did not affect on response to the first GnRH/hCG treatment. Conception rate (31 d) was 0.6 times more likely to be higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch than in those that did not respond (95% CI ⫽ 0.29 – 0.71). Conception rate was found to be different between the HPG (37.6%, 79/210) and the GPG groups (48.4%, 78/161). Thus, beginning of the Ovsynch protocol with hCG did not increase ovulation and conception rate in lactating dairy cows, suggesting that hCG is not a suitable replacement of the first GnRH of Ovsynch. However, our results do show that increasing the ovulation rate in response to the first hormonal administration of Ovsynch can have a significant effect on conception rate. © 2010 Elsevier Inc. All rights reserved. Keywords: Ovsynch; hCG; Cow; Synchronization
1. Introduction Reproductive efficiency has major impacts on profitability of dairy producers and many methodologies are being used to improve the reproductive efficiency, including the use of reproductive hormones to regulate
* Corresponding author. Tel.: ⫹90 224 2940824; fax: ⫹90 224 2941202. E-mail address: [email protected] (A. Gumen). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.009
and control the estrus cycle. The Ovsynch synchronization protocol was developed to synchronize ovulation in lactating dairy cows using GnRH and PGF2␣ [1]. This protocol synchronizes ovulation within an 8 h period from 24 –32 h after the second GnRH administration. This precise synchrony may allow for successful artificial insemination (AI) without the detection of estrus. Ovulatory response after the first GnRH administration is a critical factor for the successful synchronization of ovulation in the Ovsynch protocol [2,3].
A. Keskin et al. / Theriogenology 74 (2010) 602– 607
High ovulation rate after the first GnRH treatment increases the likelihood that cows will have a functional dominant follicle capable of ovulation during the final GnRH treatment of Ovsynch. In addition, a lower ovulation rate after the first GnRH appears to result in a lower synchronization rate after the second GnRH administration [2,3]. These positive effects of ovulatory response could increase the subsequent fertility of lactating dairy cows. In several previous studies, researchers have tried to increase ovulation response to the first GnRH and synchronization rate with presynchronization protocols such as Presynch, Double Ovsynch and so on [3– 8]. Nevertheless, these studies have yielded conflicting results. Early studies showed that response to the first GnRH in the Ovsynch protocol was 45–90% [1–3,7]. In addition, it is known that ovulation of follicles larger than 10 mm can be induced with the administration of a purified LH [9]. In cattle, a single dose of GnRH analogs elicited the release of LH for 5 h [10], a duration that is approximately half of that of the naturally occurring preovulatory surge of LH in cattle [11]. Plasma hCG was detected until 66 h after administration of hCG and it is thought to act independently of the pituitary gland [12,13]. We hypothesized that the percentage of cows that ovulated in response to the first administration of the Ovsynch protocol would increase by using hCG. This would improve conception rate because of better synchronization. Thus, the aim of the present study was to compare ovulation rate in response to GnRH or hCG at the beginning of the Ovsynch protocol and to study the effect of this manipulation on pregnancy. 2. Materials and methods 2.1. Study population This experiment was conducted on a commercial dairy herd (approximately 800 lactating dairy cows) in the South Marmara region, Bursa, Turkey, from October 2008 to July 2009. Cows were housed in free stall barns with self-catching head-locks. All cows were milked three times a day and were grouped according to their milk production. Mean milk production of the herd was 9,880 kg (305 d) per cow. Cows had free access to water and were fed complete mixed rations according to NRC recommendations [14]. Daily milk yield, reproductive health, and management records for each cow were collected from Alpro 2000 (DeLaval, Sweden). Average milk production for each cow was recorded for 7 d before and after AI. All cows had their
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body condition scored (BCS) using a 5-point (1 ⫽ thin to 5 ⫽ fat) scoring system [15]. All animal handling and procedures were approved by the Lalahan Livestock Central Research Institute Animal Care Committee. 2.2. Study design Holstein lactating dairy cows (n ⫽ 371) were randomly divided into two groups. In the first group (GPG group, n ⫽ 161), Ovsynch was started (day 0) with GnRH (Busereline acetate, 10 g, i.m., Receptal®, Intervet, Turkey). In the second group (HPG group, n ⫽ 210), Ovsynch was initiated (day 0) with hCG (1500 I.U., i.m., Chorulon®, Intervet, Turkey). Then, PGF2␣ (Cloprostenol, 500 g, i.m., Estrumate®, CEVA-DIF, Turkey) and GnRH (Busereline acetate, 10 g, i.m., Receptal®) were administered to both groups on day 7 and 9, respectively. All cows were inseminated at a fixed time 16 –18 h after the second GnRH of Ovsynch using frozen-thawed semen from bulls of proven fertility by farm veterinarians. Cows were evaluated with an ultrasound machine (Honda HS 2000 equipped with a 5.0 –7.5 MHz transducer, Honda, Japan) to determine cyclicity. Cows with corpus luteum (CL) and at least one follicle equal or larger than 10 mm size on either ovary were included in the study. Cows’ ovaries were evaluated for follicle size on the day of GnRH/hCG administration and 7 d later to determine ovulation. Ovulation in response to the first GnRH/hCG treatment was characterized by the appearance of a new CL on any ovary. Maximum follicular size was also measured at the time of AI. Ovulation after the second GnRH of Ovsynch was determined by the disappearance of the dominant follicle and the presence a new CL on the ovary 7 d after AI. Pregnancy diagnosis was performed 31 and 62 d post-insemination by ultrasonography. Pregnancy loss was calculated as the number of cows diagnosed nonpregnant at the second pregnancy check, expressed as a percentage of the number of cows diagnosed pregnant at the first pregnancy check. Conception rate was calculated as the number of cows diagnosed pregnant, divided by the number of cows receiving AI. 2.3. Statistical analysis Statistical analyses were conducted using SAS (Version 9.1; SAS Institute, 2003). Data were evaluated using PROC LOGISTIC, PROC GLM, and PROC FREQ in SAS. The model included the effects of parity, days in milk (DIM), BCS, number of services, average milk production, response to the first GnRH/hCG, and
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follicle sizes at the beginning of Ovsynch and at the time of AI. To carry out the statistical analyses, data was coded as 1 (Yes) or 0 (No) for ovulatory response to the first GnRH/hCG. Conception on day 31 and 62 after insemination was coded as 0 (not pregnant) or 1 (pregnant). Primiparous and multiparous cows were coded as 1 or 2, respectively. Number of services to each cow was coded as 0 (first service AI) or 1 (more than one service). Second ovulatory response was coded as 1 (Yes) or 0 (No). A GLM procedure was performed to compare milk production, DIM, BCS, follicle sizes at the beginning of the Ovsynch and at the time of AI between groups. Chi-square analysis using the PROC FREQ procedure was used for testing independence between parity, number of services, and the response to GnRH/hCG of Ovsynch and CR (31 and 62 d) between groups. A logistic procedure was performed to compare the effects of milk production, DIM, BCS, follicle size at the beginning of the Ovsynch and at the time of AI, parity, number of services and response to the first GnRH/hCG treatment on ovulation and on conception rates at 31 and 62 d. 3. Results Although the study began with 422 cows, 51 cows (23 cows in the GPG group and 28 cows in the HPG group) were excluded at different stages of the study due to several diseases such as mastitis, endometritis, and farm culling regime. A total of 371 cows were included in the study. The mean lactation number for cows was similar between the GPG and HPG groups (2.4 and 2.3, respectively). Average milk production did not differ between groups (40.6 ⫾ 0.6 kg/d in GPG and 41.5 ⫾ 0.5 kg/d in HPG). However, DIM was significantly different (P ⬍ 0.001) between the GPG and HPG groups (132.9 ⫾ 3.5 and 107.1 ⫾ 3.1, respectively). BCS was found to be similar between the GPG and HPG groups (2.73 ⫾ 0.02 and 2.74 ⫾ 0.01, respectively). Conception rate (at 31 and 62 d) and percentage of cows that ovulated in response to the first GnRH/hCG treatment, together with the maximal size of the ovulatory follicle on the first day of treatment, are shown in Table 1. Maximal follicle size at the beginning of Ovsynch did not differ between the GPG and HPG groups (15.8 ⫾ 0.2 mm and 15.4 ⫾ 0.2 mm, respectively), and maximal follicle size at the beginning of the Ovsynch did not affect the response to the first GnRH/ hCG treatment. Ovulation response to the first hor-
Table 1 Conception rate and percentage of cows that ovulated to first GnRH/hCG treatment and maximal size of the ovulatory follicle on day first treatment and at time of AI. GPG Maximal follicle size at the first GnRH/hCG administration Response to First GnRH/hCG % Synchronization rate % Conception rate at 30 days pregnancy % Embryonic loss % a,b
P ⬍ 0.08;
c,d
HPG
15.8 ⫾ 0.2 mm 15.4 ⫾ 0.2 mm 59.6 (96/161) 65.7 (138/210) 90.1 (145/161)a 83.8 (176/210)b 48.5 (78/161)c 37.6 (79/210)d 0.05 (4/78)
0.05 (4/79)
P ⬍ 0.04.
monal administration was similar in the GPG group (59.6%; 96/161) and in the HPG group (65.7%; 138/ 210) (Table 1). Other covariant factors, such as milk production, DIM, BCS, number of services and parity had no effect on first ovulatory response for either group. Cows that ovulated in response to the first GnRH/ hCG of Ovsynch had tendency to produce smaller (P ⬍ 0.09) follicle size at the time of AI (15.9 ⫾ 0.14 mm) than those that were nonresponsive to GnRH/hCG (16.3 ⫾ 0.21 mm). In addition, CR (31 d) was 0.64 times more likely to be higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch than in those that did not respond [95% CI ⫽ 0.29 – 0.71; actual conception rates were 49.2% (115/ 234) and 30.6% (42/137) responded and not responded cows, respectively]. This difference was also significantly higher (P ⬍ 0.03) for the conception rate at 62 d (OR ⫽ 0.45; 95% CI ⫽ 0.28 – 0.70). Conception rate was significantly different between the HPG (37.6%, 79/210) and GPG (48.4%, 78/161) groups (P ⬍ 0.04). Number of services, DIM, milk production, parity, and follicle size at the time of AI had no effect on CR. In contrast, BCS, response to the first GnRH/hCG treatment and follicle size at the beginning of Ovsynch significantly affected CR (Table 2). Conception rates at 62 d were also found to be significantly different between groups (35.7%, 75/210 in HPG and 45.9%, 74/161 in GPG). 4. Discussion We compared ovulation rate in response to GnRH or hCG in Ovsynch, and examined whether conception rate (at 31 and 62 d) was related to ovulation rate in response to the first GnRH/hCG. We selected cows according to cyclicity and follicle size at the beginning of the Ovsynch to determine the ovulatory effect of
A. Keskin et al. / Theriogenology 74 (2010) 602– 607 Table 2 Results of logistic regression model for effects of significant covariant factors on conception rate in cows. Item BCS Follicle size at the beginning of the Ovsynch Response the GnRH/hCG (No vs. Yes) 1
Odds ratio (OR)
95% (CI)1
P-values
2.56 1.08
1.05–6.22 1.01–1.16
0.03 0.02
0.52
0.32–0.87
0.01
CI ⫽ confidence interval.
GnRH and hCG. Only cyclic cows that had at least one CL and a follicle that was equal or larger than 10 mm were included in the study. Follicles smaller than 9 mm in diameter have not usually acquired ovulatory capacity in dairy cows [7,9] and ovulatory response is higher in noncyclic than in cyclic lactating dairy cows [7,16]. Therefore, the selection criteria used in our study allowed us to obtain a realistic measure of the ovulatory effect of GnRH/hCG on overall Ovsynch outcome. Ovulatory response to the first GnRH of the Ovsynch is the critical determinant for successful synchronization of ovulation in dairy cows. Early studies showed that response to the first GnRH of the Ovsynch and fertility were dependent on the stage of the estrus cycle at the beginning of the protocol [2,3]. Ovulatory response when the Ovsynch was initiated at random stages of the estrus cycle has been reported to be 45– 62% [7,16]. However, when the Ovsynch was initiated between 5–10 d of the estrus cycle, ovulatory response was increased to 85–96% [2,3]. Therefore, many researchers have developed presynchronization protocols that attempt to increase the number of cows in the optimum stage of the estrus cycle on the day of the first GnRH of the Ovsynch [3– 6]. Presynchronization protocols increased response to first GnRH of Ovsynch in previous studies [3,4]. In a recent study, Souza et al, [6] developed a new presynchronization protocol (Double Ovsynch), and they reported that ovulation rate to the first GnRH of the Ovsynch increased up to 71.8%. Nevertheless, in some other studies, the ovulatory response was negatively affected by presynchronization with PGF2␣ before the Ovsynch [7,8]. In the current study, we used hCG instead of GnRH at the beginning of the Ovsynch. Even though we were able to increase the ovulatory response up to 66% in the HGP group, the ovulatory response was found to be insignificant between the GPG and HPG groups. Despite the lack of differences in ovulatory response between the GPG and HPG groups, our results indi-
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cated that the first ovulatory response had a significant effect on pregnancy rate in lactating dairy cows. The conception rate was higher (P ⬍ 0.001) in cows that responded to the first hormonal administration of Ovsynch (49.2%) than those that did not (30.6%). Increasing the first ovulatory response might have a positive effect on luteal and follicular responses to the subsequent administration of PGF2␣ and the final GnRH of the Ovsynch, and these positive effects could induce higher fertility in lactating dairy cows [2– 4,6,7]. Fertility also was associated with progesterone levels at induction of luteolysis, size, and function of preovulatory follicle. Fonseca et al [17] showed a positive relationship between concentration of progesterone at induction of luteolysis and conception rates. Conversely, lesser levels of progesterone at induction of luteolysis were associated with a decline in CR [18]. Cows that ovulated after the first GnRH of Ovsynch had increased progesterone levels at the time of PGF2␣ and were more likely to undergo luteolysis in response to the PGF2␣ of the Ovsynch [2– 4,6]. In addition, another critical factor affecting the overall outcome of Ovsynch is follicular size at the time of the second GnRH or AI, which affect ovulatory response. In our study, despite the fact that the HPG and GPG groups had similar ovulatory response to the first treatment of Ovsynch, cows in the HPG group had tendency to produce to smaller preovulatory follicle size at the time of AI than cows in GPG group (15.8 ⫾ 0.2 mm and 16.4 ⫾ 0.2 mm respectively). Another important factor is the relationship between follicle size and pregnancy. There are conflicting results among the studies addressing this issue [2,19,20]. On the one hand, Lopes et al [19] reported that larger preovulatory follicle size increased pregnancy. On the other hand, Vasconcellos et al [2] indicated that lower ovulatory follicle size produces more pregnancy in lactating dairy cows. In the present study, although preovulatory follicle size was lower in the HPG (15.8 ⫾ 0.2 mm) than the GPG group (16.4 ⫾ 0.2 mm), conception rate was lower in the HPG (37.6%) than GPG (48.4%) groups (P ⬍ 0.04). This result shows that follicle size at the time of AI could affect subsequent fertility in lactating dairy cows. Consistent with our results, some studies have indicated that fertility was greater after the ovulation of a follicle of approximately 16 mm in diameter [3,20]. Our results indicated that using of hCG replace of GnRH did not affect either ovulatory response or the overall outcome of the Ovsynch. In previous studies, treatment with hCG induced accessory corpus luteum,
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increased plasma progesterone level, and improved conception rate in lactating dairy and beef cattle [21– 24]. In early studies, hCG was used to replace the first and second GnRH of Ovsynch in lactating dairy and beef cows [22,24 –26]. However, despite the fact that usage of hCG in the Ovsynch increased cumulative conception rate in lactating dairy cows, there was no effect on conception rate in cold season [24 –26]. In the case of the beef cows, hCG was used in TAI protocol (CO-Synch) and these studies showed that hCG was not a suitable replacement for GnRH to synchronize ovulation when using the TAI protocol [21,22]. In conclusion, beginning of the Ovsynch protocol with hCG did not increase ovulation and conception rates in lactating dairy cows. Thus, hCG is not a suitable replacement for the first GnRH of Ovsynch. However, increasing ovulation rate in response to the first hormonal administration of Ovsynch can have a significant effect on conception rate.
Acknowledgements The authors thank TARFAS Co. (Bursa, TURKEY) for the use of their herd and facilities. Support was also provided by the The Scientific and Technological Research Council of Turkey (TUBITAK) grant no. TOVAG 107O227.
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