SHORT COMMUNICATION Pregnancy rates and peripheral progesterone levels following Ovsynch or CIDR ovulation synchronization/ timed artificial insemination protocols in postpartum dairy cows M. Aali 1, T. Pretheeban, G. Giritharan 2, and R. Rajamahendran 3 Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. Received 14 December 2007, accepted 8 May 2008. Aali, M., Pretheeban, T., Giritharan, G. and Rajamahendran, R. 2008. Pregnancy rates and peripheral progesterone levels following Ovsynch or CIDR ovulation synchronization/timed artificial insemination protocols in postpartum dairy cows. Can. J. Anim. Sci. 88: 457461. Peripheral progesterone (P 4 ) concentrations as well as pregnancy rates (PR) between cows treated with Ovsynch or CIDR ovulation synchronization/tai protocols were compared. Two hundred and twenty-seven postpartum lactating Holstein cows were randomly assigned to Ovsynch (n111) or CIDR (n116) ovulation synchronization/tai protocols. Pregnancy was diagnosed by ultrasonography at day 35 after TAI and later confirmed by rectal palpation at day 60 post TAI. Milk samples were taken from the beginning of treatment (day10) until day 35 after TAI (day 0) for P 4 determination. Pregnancy rates for Ovsynch and CIDR groups of cows were 31 and 41%, respectively. The PR of ovulation synchronized cows for Ovsynch and CIDR groups were 49 and 69%, respectively. Progesterone levels during the sampling period were similar (P0.05) between Ovsynch and CIDR treated cows. Key words: Pregnancy rate, progesterone, Ovsynch, CIDR, cows Aali, M., Pretheeban, T., Giritharan, G. et Rajamahendran, R. 2008. Taux de conception et concentration de progeste rone pe riphérique après synchronisation de l ovulation/inse mination artificielle a` temps préde termine par Ovsynch ou CIDR chez la vache laitie` re apre` s la mise bas. Can. J. Anim. Sci. 88: 457461. Les auteurs ont comparé la concentration de progeste rone pe riphe rique (P 4 ) et le taux de conception des vaches dont l ovulation avait e te synchronisée et qu on avait insémine es artificiellement selon les protocoles Ovsynch et CIDR. Pour cela, ils ont divise 227 vaches Holstein postpartum en lactation au hasard entre les protocoles Ovsynch (n111) et CIDR (n116) de synchronisation de l ovulation et d inse mination artificielle à temps préde termine. La conception a été e tablie par ultrasonographie le 35 e jour apre` s insémination et confirme ele60 e jour par toucher rectal. Des e chantillons de lait ont e té préleve sdude but du traitement (jour10) au 35 e jour suivant l inse mination (jour 0) pour le dosage de la P 4. Les groupes Ovsynch et CIDR avaient un taux de conception respectif de 31 % et de 41 %. Le taux de conception des vaches dont l ovulation avait e te synchronisée par Ovsynch et CIDR s e tablissait respectivement a` 49 % et a` 69 %. La concentration de progeste rone durant la pe riode d e chantillonnage e tait similaire (P0,05) pour les deux groupes. Mots clés: Taux de conception, progeste rone, Ovsynch, CIDR, vaches For optimum milk production and profitability, a calving interval of 12 to 13 mo has been recommended 1 Current address: Ariland Agricultural Department, Kuwait Institute for Scientific Research, PO Box 24885, Safat 13109, Kuwait. 2 Current address: Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, 513 Parnassus Avenue, Box 0556, CA, 94143-0056, USA. 3 To whom correspondence should be addressed (e-mail: raja@interchange.ubc.ca). 457 for dairy cows. An integral component in achieving this calving interval is the incorporation of efficient and accurate estrus detection, proper semen handling techniques, and timed artificial insemination (TAI) relative to ovulation. Estrus detection has been cited as one of the most important factors affecting the reproductive success of AI programs. Inseminating too early prior to Abbreviations: CL, corpus luteum; DF, dominant follicle; E 2, estradiol-17b ; EB, estradiol benzoate; GnRH, gonadotropin-releasing hormone; PGF 2a, prostaglandin F 2a ; PR, pregnancy rate; P 4, progesterone; TAI, timed artificial insemination
458 CANADIAN JOURNAL OF ANIMAL SCIENCE ovulation [when milk progesterone (P 4 ) concentration is greater than 1 ng ml 1 at observed estrus] may occur in 10 to 12% of all breedings and may be as high as 20 to 30% under certain management conditions (Rajamahendran et al. 1993). Under these circumstances, not only the costs of maintaining the cow and purchasing and holding semen wasted, but also other reproductive problems, including early embryonic mortality may result from wrongly timed inseminations (Rajamahendran et al. 1993). Thus, maximizing estrus detection can improve overall reproductive efficiency in dairy cattle. However, proper control of the time of estrus is difficult, since peak estrus activity often occurs at night and determination of the actual onset of standing estrus may be difficult without 24-h observation. To improve reproductive management and thus profitability in cattle industry, various estrus synchronization protocols have been adopted to maximize the use of AI by reducing the time and labor involved in estrus detection, and by bringing a large percentage of a group of females into estrus at a pre-determined time. Earlier protocols have involved controlling the estrous cycle length by extending the life span of the corpus luteum (CL) by the use of progestagens (Anderson and Day 1998) or shortening the life span of the CL by the use of prostaglandin F 2a (PGF 2a ) (Stephens and Rajamahendran 1998). Because maturity of the dominant follicle (DF) and thus ovulation varies with individual cows, estrus cannot be accurately synchronized with these synchronizing protocols. Animals show estrus 2 to 6d following treatment with progestagen compounds and fertility rates are far less than those following natural estrus (Rajamahendran et al. 2001). Further, a 7-d period of estrus detection is necessary following administration of PGF 2a 11 to 14 d apart (Stephens and Rajamahendran 1998). In addition, TAI, 72 to 80 h after a second PGF 2a injection resulted in a significantly lower PR than animals inseminated after natural estrus (Stevenson et al. 1989). An increase in the basic understanding of as well as the development of treatment regimes to manipulate ovarian follicular and CL dynamics over the past decade have resulted in the development of estrus/ovulation synchronization protocols, namely Ovsynch (Pursley et al. 1995) and CIDRestradiol (Martinez et al. 2000), which are based on: (a) elimination of the DF, (b) initiation of a new follicular wave, and (c) synchronization of ovulation and TAI. The Ovsynch protocol is composed of a first gonadotropin-releasing hormone (GnRH) injection for ovulation of the DF, a PGF 2a injection to cause luteal regression of existing or induced CL, and a second GnRH injection to cause ovulation of the new DF, following regression of CL. Pregnancy rates following the Ovsynch protocol have not exceeded 40% in dairy cows and heifers (Pursley et al. 1997). The CIDR protocol consists of the insertion of an intravaginal progesterone device (CIDR-B ) and injections of P 4 and E 2 to cause regression of the DF, followed by a PGF 2a injection to cause luteal regression of existing or induced CL, and a second E 2 injection following the removal of CIDR-B to synchronize ovulation (Martinez et al. 2000). Pregnancy rates ranging between 46.6 and 80% have been reported in dairy cows and heifers after treatment with CIDRestradiol-based ovulation synchronization protocols (Ryan et al. 1995b; Martinez et al. 2000). Adequate levels of P 4 are necessary for uterine function, embryonic development and pregnancy. P 4 also influences maternal recognition of the embryo. The landmark events in P 4 production after ovulation include the beginning of the luteal phase with the formation of the CL and initial rise in P 4 levels. This is followed by either the end of the luteal phase (in the case of an unsuccessful fertilization) with a decline in P 4 production or prolongation of the luteal phase (if the AI is successful and the embryo is recognized) and continued elevated P 4 production. A positive relationship between P 4 levels at the beginning of the luteal phase and PR has been observed (Ahmad et al. 1996). More data on PR are needed and information on peripheral P 4 levels following treatment with either Ovsynch or CIDR ovulation synchronization/tai protocols in lactating dairy cows is scarce. The environment, management practices adopted and the time of treatment related to breeding have also been shown to affect P 4 levels and PR following estrus/ovulation synchronization protocols (Pursely et al. 1997; Vasconcelos et al. 1999). Further, a direct comparison of these two treatment protocols has not been carried out. Therefore, the objective of the present study was to conduct a largescale study and compare in vivo P 4 production as well as PR between cows synchronized using the Ovsynch and CIDR ovulation synchronization/tai protocols. This study was conducted at the University of British Columbia Dairy Education and Research Centre, in Agassiz, BC, from September 2000 to August 2001. Two hundred and twenty-seven postpartum lactating Holstein cows between 1 and 6lactations and averaging 93926d postpartum and 4099kgd 1 milk production were randomly assigned to Ovsynch (n 111: nulliparous, 44; multiparous, 67) or CIDR (n116: nulliparous, 45; multiparous, 71) ovulation synchronization/tai protocols and treated at the same period. Ovsynch (n 111) or CIDR (n 116) ovulation synchronization/tai protocols and treated at the same period. All the cows were housed in free stall barns and fed a total mixed ration of corn and grass silage, hay and concentrates. Ovsynch treatment protocol consisted of an initial GnRH injection (100 mg of Factrel ; Fort Dodge Laboratories, Fort Dodge, IA) on day 10, followed by a PGF 2a injection (25 mg of Lutalyse ; Pharmacia Animal Health, Orangeville, ON) 7 d later (day 3), and a second injection of GnRH (100 mg) 48 h after PGF 2a injection (day1). Timed artificial insemination was carried out (day 0) 16h after the second GnRH injection (64 h after the PGF 2a injection). The CIDR treatment protocol consisted of initial injections of P 4
AALI ET AL. * PREGNANCY RATES AND PROGESTERONE IN COWS 459 (100 mg; Sigma-Aldrich, Oakville, ON) and E 2 (5 mg; Sigma-Aldrich, Oakville, ON) and vaginal insertion of controlled internal drug release device (CIDR-B ; 1.9 g progesterone, Inter-Ag, Hamilton, NZ) on day 10, followed 7 d later by 25 mg of PGF 2a injection (day3), CIDR removal on day2 and a second injection of E 2 on day 1. Timed artificial insemination was carried out (day 0) 28 h after the second injection of E 2 (76h after the PGF 2a injection). Both treatment protocols were initiated regardless of the stage of the estrous cycle of the animals. Milk samples were collected from treated cows on days10,3, 0, and 7, 14, 21, 28, 35 post TAI for P 4 determination. Measurement of P 4 was conducted using a validated commercially available solid-phase RIA kit (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA). Pregnancy was diagnosed by ultrasonography at day 35 after AI and later confirmed by rectal palpation at day 60 after TAI. Contingency tables in the chi-square analysis were used to compare: (1) the overall PR between Ovsynch and CIDR, (2) the proportion of animals which responded to treatments (ovulation synchronization rate), (3) the proportion of ovulation synchronized cows that became pregnant within treatment (Ovsynch and CIDR), (4) the proportions of cows with P 4 B1ngmL 1 on day 0,1 ng ml 1 on days 7, 14, and 21 post AI (for presumptive pregnancy rate). Least-square analysis of variance using the JMP IN statistical package (2001) was used to analyze CL function (based on P 4 levels on days 0, 7, 14, 21, 28, and 35 post AI) in the ovulationsynchronized cows (both pregnant and non-pregnant) of the Ovsynch and CIDR treatment protocols. Student t- test was performed to compare differences between means with significant difference. All handling and management of animals used in this study were in accordance with the guidelines of the Canadian Council on Animal Care (1993). The data show (Fig. 1) that ovulation in cows receiving the CIDR-based protocol tended (P 0.08) to be more synchronized than in cows receiving the Ovsynch protocol (75 vs. 61%). Similarly, the overall PR (41 vs. 31%) and PR among cows that responded to ovulation synchronization treatment (69 vs. 49%) were also better in cows receiving the CIDR-based protocol. The presumptive pregnancy rate (based on P 4 B1 ng ml 1 on day 0, and1 ngml 1 on days 7, 14 and 21 after TAI) were similar (P 0.05) between Ovsynch and CIDR groups of cows (74 vs. 64%). Embryonic mortality was estimated as the difference between presumptive pregnancy from day 21 (cows presumed to be pregnant had P 4 levelsb1 ngml 1 on the day of TAI, and P 4 levels1 ngml 1 on days 7, 14 and 21 post TAI) and actual PR obtained by ultrasound on day 35 post TAI. Cows on the CIDR-based protocol had a much smaller (P B0.05) percentage of embryonic loss (7%) than those on the Ovsynch protocol (35%). In the ovulation-synchronized cows, P 4 profiles from the time of TAI until day 35 following TAI were similar Percentage (%) 80 70 60 50 40 30 20 10 0 Ovulation synchronization rate Ovsynch Pregnancy rate among synchronized cows CIDR Overall pregnancy rate Fig. 1. Ovulation synchronization and pregnancy rates in lactating dairy cows treated with Ovsynch (black bar) or CIDR (white bar) ovulation synchronization/tai protocols. No significant differences were found among treatments for synchronization rates (P0.05) or pregnancy rates (P0.05). Numbers at the top of the bars indicate the number of animals/ group. (P 0.05) between Ovsynch and CIDR groups of cows (Fig. 2). The results of this study show that in both the Ovsynch and the CIDR ovulation synchronization/ TAI protocols, PR were substantially higher in ovulation-synchronized cows. The PR following the CIDR protocol in our study (41%) was similar to that reported in beef cows treated with estradiol benzoate (EB) (43%, Colazo et al. 2003) and lower than that of beef as well as dairy cows treated with E 2 or EB (6080%, Martinez et al. 2000). Our data also demonstrate that the CIDR ovulation synchronization/tai protocol resulted in Progesterone (ng/ml) 10 8 6 4 2 0 Ovsynch CIDR 0 7 14 21 28 35 Days post AI Fig. 2. Milk progesterone (ng ml 1 ) in ovulation synchronized cows (cows having P 4 B1.0 ng m 1 on day 0 and1.0 ng ml 1 on day 7 post AI) following the Ovsynch (n111) and CIDR (n116) ovulation synchronization/tai protocols. Milk samples were collected on days 0, 7, 14, 21, 28, and 35 post TAI. No differences were found between treatments for progesterone concentrations (P0.05).
460 CANADIAN JOURNAL OF ANIMAL SCIENCE higher PR in multiparous cows compared with the Ovsynch ovulation synchronization/tai protocols. The stage of the estrous cycle at which ovulation synchronization treatment is initiated could determine the response to treatment and therefore PR. Treatment with P 4 and E 2 together have consistently caused regression of the large follicle and stimulated the emergence of new follicular wave 35 d after treatments. Failure of cows to respond to these treatments could result in the development of persistent follicles. The persistence of a large follicle is accompanied by high levels of E 2, lower P 4 concentrations, and an increase in pulsatile release of LH. Fertility is reduced as a result of ovulation of persistent follicles and therefore ovulation of aged oocytes (Ahmad et al. 1995). Initiating CIDR P 4 E 2 treatment at late diestrus may not lead to regression of the DF; however, this could result in reduced PR following TAI as a result of ovulation of aged oocytes (Martinez et al. 2000). However, the ovulation synchronization rate to the second E 2 injection in this study was at 75% and PR in the synchronized cows was 69.9%. This finding is comparable with studies in dairy cows (Ryan et al. 1995a), dairy heifers (Ambrose et al. 2001), beef heifers and beef cows (Martinez et al. 2000), which used either E 2, EB or estradiol cypionate (ECP). PR following the Ovsynch protocol observed in our study was similar to or higher than that observed in lactating dairy cows. A direct comparison of these two ovulation synchronization protocols in lactating cows with ovarian cysts (Crane et al. 2006) also showed that both methods yield a similar PR (Ovsynch, 14.4% and CIDR, 9.5%) in treated cows. The effectiveness of the Ovsynch protocol is also influenced by the stage of follicular development at the time of treatment (Vasconcelos et al. 1999). In our study, the ovulation synchronization rate to the second GnRH injection was 61%, lower than those reported by others (Vasconcelos et al. 1999), who reported ovulation synchronization rates between 77 and 89%. The ovulation synchronization rate based on the response to the second GnRH injection varied: 92% when the animals ovulated versus 79% when animals did not ovulate after the first GnRH injection (Vasconcelos et al. 1999). In our study, 49% of the cows that responded to the second GnRH injection were pregnant, thus this pregnancy rate could be a result of 39% of the cows that did not respond to the first GnRH injection. Further, Vasconcelos et al. (1999) reported that 6% of cows ovulated before the second injection of GnRH, and these cows were in the late diestrus stage of the estrous cycle, the time when they would be expected to have normal luteal regression prior to PGF 2a treatment. Progesterone concentrations from milk sampled on days 0, 7, 14, 21, and 35 were not different between the Ovsynch and CIDR treatment protocols. Irregular estrous cycle lengths, imprecise detection of estrus and ovulation, and early embryonic mortality could all lead to higher P 4 concentration at 21 d post AI. P 4 in the CIDR cows tended (P0.1) to be higher on days 7 and 14 post AI than in the Ovsynch cows. The apparent embryonic mortality between days 21 to 35 post AI after the CIDR treatment protocol (7%) was significantly lower (P B0.05) than that found after Ovsynch treatment protocol (32%). Loss of CL maintenance around the time of implantation, the inability of the conceptus to secrete interferon-tau (IFN-t) and inhibit the uterine PGF 2a during early pregnancy could be the reasons for the early embryonic mortality. In conclusion, both the Ovsynch and the CIDR ovulation synchronization/tai protocols can be used regardless of the stage of the animal s estrous cycle, and both procedures can be effective for synchronization of ovulation and TAI programs. The stage at which the treatment protocol is initiated, the time of AI following the second injection of the treatment protocol, and the concentrations of E 2 are some of the factors that may have contributed to the PR obtained in this experiment following the Ovsynch and CIDR ovulation synchronization/tai protocols. More effort needs to be put into improving these methods to achieve maximum PR. For the Ovsynch protocol, we are currently working on: (1) supplementation of exogenous progestins during the synchronization period to minimize or prevent premature estrus or ovulation, which often occur when treatment is initiated at late diestrus, (2) presynchronization with PGF 2a 12 to 14 d apart, so that when the Ovsynch is initiated the cows will be at the diestrus stage of the estrous cycle, the time which has led to highest PR, and (3) a combination of presynchronization with PGF 2a and exogenous progestins. With regard to the CIDR protocol, our future trials will focus on: (1) reducing the dose of the second E 2 to minimize the chance of luteolysis and termination of pregnancy, and (2) initiating the CIDR protocol at times other than late diestrus, and this will be achieved by presynchronization with PGF 2a 12 to 14 d apart. This research was supported by Westgen, the Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada, and BC Investment Agriculture. Ahmad, N., Beam, S. W., Butler, W. R., Deaver, D.R., Duby, R. T., Elder, D. R., Fortune, J. E., Griel, L.C., Jones, L. S., Milvae, R. A., Pate, J. L., Revah, I., Schreiber, D. T., Townson, D. H., Tsang, P. C. W. and Inskeep, E. K. 1996. Relationship of fertility to patterns of ovarian follicular development and associated hormonal profiles in dairy cows and heifers. J. Anim. Sci. 74: 19431952. Ahmad, N., Schrick, F. N., Butcher, R. L. and Inskeep, E. K. 1995. Effect of persistent follicles on early embryonic losses in beef cows. Biol Reprod. 52: 11291135. Ambrose, D. J., Rajamahendran, R., Kastelic, J. P. and Small, J. A. 2001. Synchronization of ovulation and conception rates in Holstein heifers given an intravaginal progesterone-releasing device (CIDR), and estradiol cypionate, porcine LH or gonadotropin releasing hormone. Arch. Anim. Breed. 44: 77 79 (Special Issue).
AALI ET AL. * PREGNANCY RATES AND PROGESTERONE IN COWS 461 Anderson, L. H. and Day, M. L. 1998. Development of a progestin-based estrus synchronization program: I. Reproductive response of cows fed melengestrol acetate for 20 days with an injection of progesterone. J. Anim. Sci. 76: 12671272. Colazo, M. G., Kastelic, J. P. and Mapletoft, R. J. 2003. Effects of estradiol cypionate (ECP) on ovarian follicular dynamics, synchrony of ovulation, and fertility in CIDRbased, fixed-time AI programs in beef heifers. Theriogenology 8894: 111. Crane, M. B., Bartalome, J., Melendez, P., de Vries, A., Risco, C. and Archbald, L. F. 2006. Comparison of synchronization of ovulation with timed insemination and exogenous progesterone as therapeutic strategies for ovarian cysts in lactating dairy cows. Theriogenology 65: 1563 1574 Hanlon, D. W., Williamson, N. B., Wichtel, J. J., Sttefert, I. J., Craigie, A. L. and Pfeiffer, D. U. 1996. The effect of estradiol benzoate administration on estrous response and synchronized pregnancy rate in dairy heifers after treatment with exogenous progesterone. Theriogenology 45: 775785. Lamming, G. E., Darwash, A. O. and Black, H. L. 1989. Corpus luteum function in dairy cows and embryo mortality. J. Reprod. Fertil. 37 (Suppl.): 245252. Martinez, M. F., Kastelic, J. P., Adams, G. P., Janzen, E., McCartney, D. H. and Mapletoft, R. J. 2000. Estrus synchronization and pregnancy rates in beef cattle given CIDR-B, prostaglandin and estradiol, or GnRH. Can. Vet. J. 41: 786 790. Pursley, J. R., Mee, M. O. and Wiltbank, M. C. 1995. Synchronization of ovulation in dairy cows using PGF 2a and GnRH. Theriogenology 44: 915923. Pursley, J. R., Wiltbank, M. C., Stevenson, J. S., Ottbre, J. S., Garverick, H. A. and Anderson L. L. 1997. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J. Dairy Sci. 80: 295300. Rajamahendran, R., Burton, B. and Shelford, J. 1993. A field study on the usefulness of milk progesterone determination to confirm estrus and pregnancy of dairy cows in the Fraser Valley area of British Columbia. Can. Vet. J. 34: 349252. Rajamahendran, R., Ambrose, J. D., Small, J. A. and Dinn, N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Anim. Breed. 44: 5867 (Special Issue). Ryan, D. P., Snijders, S., Aarts, A. and O Farrell, K. J. 1995a. Effect of estradiol subsequent to induced luteolysis on development of the ovulatory follicle and interval to estrus and ovulation. Theriogenology 43: 310. Ryan, D. P., Snijders, S., Yaakub, H. and O Farrell, K. J. 1995b. An evaluation of estrus synchronization programs in reproductive management of dairy herds. J. Anim. Sci. 73: 36873695. Stephens, L. A. and Rajamahendran, R. 1998. A comparison of two estrus synchronization methods in beef heifers. Can. J. Anim. Sci. 78: 437439. Stevenson, J. S., Mee, M. O. and Stewart, R. E. 1989. Conception rates and calving intervals after prostaglandin F 2a or prebreeding in dairy cows. J. Dairy Sci. 72: 208218. Vasconcelos, J. L. M., Silcox, R. W., Rosa, G. J. M., Pursley, J. R. and Wiltbank, M. C. 1999. Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. Theriogenology 52: 10671078.