Synchronization of estrus and Ovulation in cattle

Arch. Tierz., Dummerstorf 44 (2001) Special Issue, 58-67

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Faculty of Agriculture, University of British Columbia, Vancouver, Canada , Dairy Research and technology Center, Alberta Agriculture Food and Rural Development, Edmonton, Canada, Agriculture & Agri-Food Canada Research Center, Maniloba, Canada, University of British Columbia Dairy Research Center, Agassiz, Canada.

RAJA RAJAMAHENDRAN1, DIVAKAR J. AMBROSE2, JULIE A. SMALL3

Synchronization of estrus and Ovulation in cattle Summary Estrus detection has been cited as one of the most important factors affecting the reproduetive success of artificial insemination programs. Various estrus synchronization protocols have been developed to bring a large percentage of groups of females into estrus at a predetermined time. Earlier protocols have involved Controlling estrous cycle length in cattle either by extending the life span ofthe corpus luteum by the use of progestogens or shortening the life span of the corpus luteum by the use of Prostaglandins. The reduced fertility following the earlier synchronization protocols made it necessary to understand ovarian follicular and corpus luteum dynamics in cattle. An increase in this basic understanding as well as the development of treatment regimes to manipulate ovarian follicular and corpus luteum dynamics over the last decade have resulted in development of better estrus synchronization protocols based on a) elimination of the dominant follicle and initiation of new follicular wave, b) initiation of new follicular wave, synchronization of Ovulation and timed artificial insemination. These protocols are very promising and have the potential to enhance pregnancy rates and the success of artificial insemination programs. Key Words: cattle, synchronization ofestrus and Ovulation, corpus luteum, timed artificial insemination

Zusammenfassung Titel der Arbeit: Brunst- und Ovulationssynchionisation beim Rind Die Erkennung brünstiger Rinder ist einer der bedeutendsten Faktoren ftlr die erfolgreiche Durchführung der künstlichen Besamung. Aus diesem Grund wurden verschiedene Verfahren der Brunstsynchronisation entwickelt, um einen großen Anteil brünstiger Rinder zu einer vorbestimmten Zeit zur Vertilgung zu haben. So wurde in der Vergangenheit der Zyklus mittels gestagener Substanzen verlängert oder mit Prostaglandinen verkürzt. Teilweise unbefriedigende Fruchtbarkeitsergebnisse nach der Anwendung der verschiedenen Verfahren haben zu der Erkenntnis geführt, dass die Notwendigkeit besteht, die physiologischen Prozesse der Follikeldynamik und Corpus luteum-Funktion besser zu verstehen. Das bessere Verständnis o.g. Prozesse hat in Verbindung mit neuen Möglichkeiten der Manipulation von Follikelwachstum und C.l.-Funktion zur Entwicklung besserer Verfahren der Brunstsynchronisation geführt. Die Basis der Verbesserungen besteht in der Elimination von dominanten Follikeln und in der Induktion einer neuen Follikelreifungswelle sowie in der Synchronisation von Ovulationen und in einer terminorientierten Besamung. Die Verbesserungen sind vielversprechend im Hinblick auf die Trächtigkeitsrate und den Erfolg von Besamungsprogrammen. Schlüsselwörter: Rind, Brunst- und Ovulationssynchronisation, Corpus luteum, terminorientierte Besamung

Reproduetive efficiency in dairy herds has a marked influence on profitability. For dairy cows, a calving interval of 12 to 13 months is generally considered to be economically optimal. An integral component in achieving this calving interval is the incorporation of efficient and accurate estrus detection, proper semen handling techniques, and timely artificial insemination relative to Ovulation. Estrus detection has been cited as one of the most important factors affecting the reproduetive success of artificial insemination programs (EVERETT et al., 1986). Breeding other than at the

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time of estrus (when the milk progesterone (P4) concentration is greater than 1 ng/ml may occur in 10 to 12% of all Services and may be as high as 20 to 30 % under certain management conditions (RAJAMAHENDRAN et al., 1993). Under these circumstances, not only are the costs of maintaining the cow and purchasing and holding semen wasted, but other reproduetive problems, including early embryonic death may result from wrongly timed inseminations. Thus, maximizing estrus detection can improve overall reproduetive 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 (HENRICKS et al., 1971). Therefore, various estrus synchronization protocols have been developed to bring a large percentage of a group of females into estrus at a pre-determined time. Early synchronization protocols The traditional methods of synchronizing the estrous cycle over the past 60 years have involved Controlling estrous cycle length in cattle either by a) shortening the life span ofthe corpus luteum (CL) through the use of Prostaglandin F 2 a (PGF 2a ) or estrogens; or, b) by extending the life span ofthe CL by the use of P4 or its synthetic analogues. Progesterone was the hormone originally used in attempts to synchronize the bovine estrous cycle (CHRISTIAN and CASIDA, 1948). Since the exact stage ofthe cycle is usually unknown, P 4 is administered for the length ofthe luteal phase (14 to 21 days) which is sufficient to allow the CL to regress. All animals show estrus 2 to 6 days after the termination of treatment. Numerous progestational Compounds administered by different routes (oral, ear-implant, intravaginal) have been investigated (BERARDINELLI and ALDAIR al., 1989). The fertility rates following synchronized estrus were generally far from satisfactory. The low fertility can mainly be attributed to altered follicular growth, sperm transport and abnormal embryo development (WISHART and YOUNG, 1974). Short-term progestogen treatments with the use ofa luteolytic agent (estrogens or PGF 2a ) were not completely effective in synchronizing estrus in cattle (WILTBANK and GONZALEZ-PADILLA, 1975). Following the characterization of PGF 2a as a natural luteolytic agent in cattle and the development of its potent analogues, PGF 2 a became the preferred treatment for estrus synchronization in cattle (RAJAMAHENDRAN et al., 1977). Numerous studies have shown that fertility of cattle inseminated after a single injection of PGF 2a or double injections given 12 to 14 days apart is similar or superior to that of cows inseminated after natural estrus (KASTELIC et al., 1990). However, most estrus synchronization programs involving single fixed time insemination following double injections of PGF 2a are associated with reduced fertility, because the interval from PGF 2a to Ovulation is often too variable (DAILEY et al., 1983; STEPHENS and RAJAMAHENDRAN, 1998). "ftft 1SÄKÄÄ&fe?tt\\YyfoWvMYft^\\& tot ^h^iWftU^YÖv» ^WVöWöVi ^ t \ ö N U V Ä J in times of estrus and Ovulation made it necessary to understand ovarian follicular and CL dynamics in cattle, and to develop synchronization protocols which would enable a single timed insemination to ensure high pregnancy rates. An increase in this understanding as well as development of the ability to manipulate follicular and CL

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dynamics with exogenous hormones in cattle over the past decade has made the above task possible. Ovarian follicular dynamics in cattle It has been well established, using ultrasound technology, that follicular growth occurs in a wave-like pattern during the estrous cycle in cattle (RAJAMAHENDRAN et al., 1994). A pool of follicles becomes apparent around the time of estrus followed by a period of growth, at which time a single follicle becomes dominant and continues to grow, while the remainder becomes atretic and regress. The first wave dominant follicle normally enters a static phase of no growth on about day 7-8 of the cycle, it then becomes atretic and begins to regress between days 11 to 13, to be replaced by a second wave of follicular growth. The number of waves of follicular growth during the estrous cycle appears to be related to the length of the luteal phase (TAYLOR and RAJAMAHENDRAN, 1991). If P4 concentrations in the circulation begin to decrease while the second wave follicle is in its growth phase, then the second wave dominant follicle invariably goes on to ovulate. If, on the other hand, P4 remains elevated after the second wave follicle has attained its maximum size, the dominant follicle begins to regress, to be replaced by a third wave of follicular growth. Characteristics ofa dominant follicle A dominant follicle is defined as a follicle >10mm in diameter that is recruited and selected during a follicular wave. An active dominant follicle is capable of preventing the growth of other follicles as well as the development of a new follicular wave (RAJAMAHENDRAN et al., 1994; GINTHER et al., 1996). The dominant follicle in the first wave of follicular growth has been shown to be capable of Ovulation when luteolysis is initiated on day 7 ofthe estrous cycle (KASTELIC et al., 1990). We (RAJAMAHENDRAN and TAYLOR, 1991) have shown that administration of a single Norgestomet ear implant (Synchromate-B) during the early or mid-luteal phase did not have any effect on the dominant follicle present. However, if the implant was inserted during the follicular phase, in the absence of a CL, the dominant follicle was maintained during the 9 d treatment period and ovulated after implant removal. Maintenance of the first wave dominant follicle (following induced luteolysis) and the proestrous dominant follicle, with low levels of P4 have also been reported by others (SIROIS and FORTUNE, 1990; SAVIO et al, 1993). Increasing the number of implants (one versus two) or increasing the dose of P4 causes regression of the dominant follicle maintained by a single norgestomet implant or low concentrations of P4. The regression of the dominant follicle in these studies was associated with decreased LH pulse frequency (TAYLOR and RAJAMAHENDRAN, 1994). The low fertility of cows bred at synchronized estrus following long-term P4 treatment or the use of PGF2a during the early to mid-luteal phase is mainly attributed to the abnormal development of embryos derived from ova of persistent dominant follicles (MIHM et al, 1994).

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Development ofestrus synchronization protocols based on elimination of the dominant follicle and initiation of new follicular wave Both hormonal methods and physical methods have been attempted to eliminate the dominant follicle and initiate a new follicular wave. Hormonal methods include administration of P4 (TAYLOR and RAJAMAHENDRAN, 1994) and estrogens (RAJAMAHENDRAN and WALTON, 1990; MANIKKAM and RAJAMAHENDRAN, 1997; BO et al, 1994) to induce dominant follicle regression, as well as gonadotropin releasing hormone (GnRH) and human chronic gonadotropin (hCG) to ovulate or luteinize the dominant follicle (RAJAMAHENDRAN and SIANANGAMA, 1992; SCHMITT et al, 1996; RAJAMAHENDRAN et al, 1998). Physical methods include removal by electrocautery, and aspiration of the dominant follicle (KO et al, 1991; BERGFELT, 1994). The most common methods for altering follicular tumover in conjunction with estrus synchronization have been administration of GnRH or its analogs (TWAGIRAMUNGU et al, 1995; PURSLEY et al, 1995) or estrogen administration in conjunction with progestins (BO et al, 1994). THATCHER et al. (1989) and MACMILLAN and THATCHER (1991) developed a method that synchronized both follicular development and CL regression. With this system, an injection of a GnRH agonist is followed 7 days later by an injection of PGF2a (WOLFENSON et al, 1994). The GnRH injection is aimed at luteinizing or ovulating mature follicles present during the time of treatment without considering the stage of the cycle and to initiate the recruitment and selection of a new dominant follicle 7 days later. The injection of PGF2a initiates either the regression of the spontaneous CL or the one induced by GnRH, or both. After the PGF2a injection, cows can be artificially inseminated at detected estrus. This treatment resulted in a better synchronization rate and pregnancy rate compared to those treated with two injections of PGF2a 11 days apart in dairy and beef cows (TWAGIRAMUNGU et al, 1995; PURSLEY et al, 1997). We (STEPHENS and RAJAMAHENDRAN, 1998) compared the effectiveness ofthe GnRH + PGF2a protocol and two injections of PGF2a 12 days apart in beef heifers. The pregnancy rate for the GnRH+PGF2a protocol was 40 % as opposed to 62 % in the double PGF2a group. Also heifers synchronized by the GnRH+PGF2a protocol showed poor signs of estrus and were difficult to breed. The reason for this is not clear. One problem is the premature oecurrence of estrus in animals given GnRH during the late luteal phase. This could be prevented, however, by administration of P4. Consistent results were shown in terms of dominant follicle elimination and follicular wave initiation, when estrogens were given in combination with P4, regardless of the stage of the estrous cycle (BO et al, 1994; GARCIA and SALHEDDINE, 1998). Studies were conducted to examine whether estrogen + progesterone treatment in conjunction with a controlled internal drug release device (vaginal device containing P4, CIDR-B) would minimize Variation in the interval from treatment to Ovulation. The CIDR-B device was developed for the controlled administration of P4 to cattle (MACMILLAN and PETERSON, 1993). Treatment of beef heifers with CIDR-B for 7 days, plus 100 mg of P4 and 5 mg of estradiol 17ß at the time of CIDR-B insertion and PGF2a at the time of CIDR-B removal, resulted in 75 % of the heifers ovulating between 72-82 h after CIDR-B removal, compared to 40 % of the heifers treated with two injection of PGF2a 11 days apart, and 33 % of the heifers treated with CIDR-B

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plus PGF2a (BO et al, 1994). LAGO et al. (2000) compared pregnancy rates between three synchronization protocols; CIDR-B (plus estradiol benzoate and P4 at insertion and PGF2a at removal), two injections of PGF2a (14 days apart) and natural estrus. They concluded that the CIDR-B protocol yielded the highest pregnancy rate (83 %) relative to two injections of PGF2a (64 %) and natural estrus (71 %). Development of synchronization protocols based on initiation of new follicular wave, synchronization of Ovulation and timed artificial insemination (TAI) Accurate estrus detection is required to achieve acceptable fertility in many estrus synchronization and AI regimens. However, estrus detection generally requires substantial time and labor. Therefore, timed insemination without the need for estrus detection would be of considerable benefit to dairy producers. Estrus synchronization programs involving fixed time insemination following double injections of PGF2a are associated with reduced fertility, because the interval from PGF2a to Ovulation is often too variable (DAILEY et al, 1983). It is now clear that the successful use ofestrus synchronization protocols with fixed time breeding requires not only the control ofthe CL and the initiation of a new follicular wave but also the synchronization of Ovulation. In this regard, a recently developed protocol (Ovsynch/TAI) synchronizes follicular growth, CL regression and Ovulation; pregnancy rates in lactating cows are similar to alternative regimens, without the need for estrus detection (PURSLEY et al, 1995; SCHMITT et al, 1996a). In this protocol, GnRH is injected without regard to the stage of the estrous cycle, followed by PGF2c[ 7d later, and a second injection of GnRH 48h after PGF2a. Best results are obtained when timed AI is performed about 16 h after the second injection of GnRH. However, satisfactory results may be obtained when AI is performed at the time ofthe second injection of GnRH or up to a few hours later. We (HIRAD et al, 1999) compared the effectiveness of Osynch/TAI with double injections (given 12 days apart) of PGF2a/TAI for planned breeding in dairy cows. Timed inseminations were done at 12 and 36 h after second injection of GnRH in the Ovsynch group, while in the PGF2a group, timed inseminations were done at 60 and 84 h after the second injection of PGF2a. The pregnancy rate was significantly higher for cows in the Ovsynch group (62 % Vs. 43 %). The highest pregnancy observed following single AI in the Ovsynch program was only about 40 %, when AI was performed 16-18h after the second GnRH injection in both dairy (PURSLEY et al, 1997) and beef cattle (SMALL et al, 2001). AMBROSE et al. (1999) demonstrated that Ovsynch/timed AI protocol can be successfuUy used to manage embryo transfer reeipients, in large commercial herds, in the summer months without the need for estrus detection. Ovsynch protocol was initiated in Holstein cows by a GnRH injection followed 7 d later by PGF2a and a second injection of GnRH 48 h later. Control cows (n=129) were inseminated 16 h (Day 0) after the second GnRH injection. On Day 7, a fresh (n=133) or frozen-thawed (n=142) IVF embryo was transferred to the uterine hörn on the same side as the CL, in cows assigned for timed embryo transfer. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d (14.3 %) than cows that received a frozen-thawed embryo (4.8 %) or timed insemination (4.9 %). Even though the increase in pregnancy rate following transfer of a fresh embryo was significant (P