COMPARISON OF LONG-TERM PROGESTERONE-BASED PROTOCOL ON REPRODUCTIVE PERFORMANCE OF PREPUBERTAL AND PUBERTAL BEEF HEIFERS

The aim of this study was to investigate estrus synchronization in prepubertal and pubertal heifers (crossbred 1⁄2 Nelore x Red Angus commercial herd) submitted to a long-term progesterone-based protocol for timed artificial insemination (TAI), as well as overall pregnancy rates. Four hundred forty-seven heifers underwent ultrasound examination of the ovaries and were assigned to one of 3 groups: (1) prepubertal heifers (PPNS; n = 114) with ovarian follicles <8 mm in diameter and no corpus luteum (CL) that were submitted to natural service (NS) by bulls for 10 days; (2) prepubertal heifers (PPTAI; n = 124) with follicles <8 mm in diameter and no CL that underwent estrus synchronization with injection of 4.0 mg estradiol benzoate (EB) and insertion of a controlledrelease intravaginal progesterone (P4) device containing 0.558 g P4 on d 0, followed by P4 device removal and injection of 150 mg prostaglandin F2α and 0.6 mg estradiol cypionate (ECP) on d 8, and TAI on d 10; and (3) pubertal heifers (PTAI; n = 209) with follicles >8 mm in diameter and/or a CL that underwent the same protocol as PPTAI heifers. Overall pregnancy rate was determined by transrectal ultrasonography on d 45 after NS or TAI. Data were analyzed using the FREQ procedure of SAS (SAS Inst. Inc., Cary, NC). Pregnancy rates were as follows: 20.1% in PPNS, 37.0% in PPTAI, and 48.5% in PTAI (P < 0.05). In conclusion, TAI after estrus synchronization with a hormonal protocol using EB and ECP in prepubertal and pubertal heifers resulted in higher pregnancy rates than prepubertal heifers exposed to natural service, and exposure of prepubertal heifers to a long-term progesterone-based protocol effectively synchronized estrus.


INTRODUCTION
Brazil has the largest commercial herd in the world-approximately 200 million animals (IBGE, 2004).Farms need to achieve good zootechnical performance and positive financial results to become profitable as quickly as possible.Low reproductive efficiency in herdsis the main cause of persistently low production rates.Therefore, animal factors, such as age, category, body condition score, reproductive health (ovaries and uterus), and calving season (Cunha, 2011), need to be taken into account when planning the reproductive strategy for each farm.
According to Soares and Araldi (2011), a critical factor in the beef industry remains low fertility of the cows in breeding herds and consequent low calving rates.Taira et al. (2010) concluded that efficiency in the calving period is a determining factor in the economic viability of production systems.In addition, the Brazilian beef cattle industry is still conducted as an extractive activity, with minimal use of inputs and biotechnology (Santos et al., 2011), which further limits economic viability.
The use of timed artificial insemination (TAI) for increasing the productivity of breeding herds is recommended by Viana et al. (2008) andTaira et al. (2010).This biotechnology has advantages over natural breeding and minimizes the variables that contribute negatively in traditional artificial insemination (AI), such as failures in the detection of estrus.Baruselli et al. (2007) report that the difficulties of estrus detection are more pronounced in Bos indicus cows, which show physiological estrus for a short period and at night.According to Moraes and Souza (2008) Hormonal protocols for TAI in lactating beef cows show decreased effectiveness when used in heifers (Sá Filho et al., 2008).Differences in reproductive physiology between Bos taurus and Bos indicus cattle may also affect responses to synchronization protocols.P4 levels during the estrous cycle are an important influencing factor.Carvalho (2004) showed that an intravaginal P4 device combined with estradiol benzoate (EB) or estradiol cypionate (ECP) (Wheaton and Lahm, 2007)  The present study investigated synchronization of estrus in prepubertal and pubertal heifers submitted to a longterm progesterone-based protocol for TAI.

MATERIAL AND METHODS
This study included 447 crossbred heifers (½ Nelore x Red Angus) from a commercial herd, aged 24-28 months, with average body condition score of 6.50 (scale of 1 = thin to 9 = obese) and weight of 325 kg.The heifers were maintained on Brachiaria brizantha, with mineral salt and water ad libitum.Before the study began, the ovaries were assessed by ultrasound examination (Tringa model, 5-MHz transducer; Pie Medical), to check for the presence of follicles greater than or less than 8 mm in diameter and corpora lutea.Three groups were assigned: (1) prepubertal (PPNS; n = 114) with follicles <8 mm in diameter and no corpus luteum (CL) served by bulls in a natural service (NS) for 10 days; (2) prepubertal (PPTAI; n = 124) with follicles <8 mm in diameter and no CL that underwent estrus synchronization with injection of 4.0 mg estradiol benzoate (EB; Cronibest ® , BE, Biogenesis-Bagó Animal Health Ltd) and insertion of a controlled-release intravaginal progesterone (P4) device (Cronipres ® , 0.558 g P4; Biogenesis-Bagó Animal Health Ltd) on d 0, followed by P4 device removal and injection of 150 mg prostaglandin F 2α (Croniben ® , Dcloprostenol; Biogenesis-Bagó Animal Health Ltd) and 0.6 mg estradiol cypionate (ECP ® estradiol cypionate; Pfizer Animal Health Laboratory) on d 8, and TAI on d 10; and (3) pubertal heifers (PTAI; n = 209) with follicles >8 mm in diameter and/or a CL that underwent the same protocol as PPTAI heifers.Overall pregnancy rate was determined by transrectal ultrasonography on d 45 after NS or TAI.All the TAI heifers were inseminated with frozen-thawed semen.Protocols to the PPTAI and PTAI groups.

Statistical analysis
Data of pregnancy rates in the groups of heifers were analyzed using Fisher's exact test and the FREQ procedure of SAS (SAS Inst.Inc., Cary, NC) at a significance level of 95% probability.

RESULTS AND DISCUSSION
The results are presented in table 1.
Pregnancy rates in beef heifers with TAI have yielded conflicting results.TAI is influenced by several factors, such as stage of the estrous cycle at the beginning of the protocol, animal category (heifers, primiparous, pluriparous), breed, body condition score, and semen quality (Gofert, 2008;Marques et al., 2008;Sá Filho et al., 2010;Soares and Araldi, 2011).
In this study, pregnancy rate (Table 1) differed significantly among groups.The animals submitted to the long-term progesterone-based protocol had significantly better pregnancy rates, 49.0% in PTAI and 37.0% in PPTAI, than the animals submitted to natural service (PPNS).The data suggest the favorable influence of the intravaginal progesterone protocol.There were also significant differences between PPTAI and PTAI heifers (P<0.05), which emphasizes the significant performance of the protocol in animals that are already cycling.Pfeifer et al.(2008) andSá Filho et al. (2010), in TAI programs in beef heifers in Brazil, have reported higher pregnancy rates in pubertal than prepubertal heifers, corroborating data of the present study.
Comparison between PPNS and PPTAI heifers suggested a marked advantage of the long-term progesterone-based protocol (P < 0.01).The pregnancy rate in PPTAI heifers (37.0%) confirmed that cyclicity of the ovaries can be induced in animals exposed to P4, even females in anestrus (Azeredo et al., 2007;Leitmann et al., 2009;Pereira, 2010).The PTAI heifers showed the best response to the study protocol (P < 0.04), corroborating reports of Sá Filho et al. (2010)

CONCLUSION
In conclusion, hormonal protocols using EB and ECP for PPTAI and PTAI resulted in better pregnancy rates than that in PPNS heifers, and exposure of prepubertal heifers to a long-term progesterone-based protocol was effective in estrus synchronization.
caused dominant follicle atresia at the start of the treatment and synchronized the emergence of a new follicular wave in Bos indicus, Bos taurus x Bos indicus, and Bos taurus cows; further, Bos indicus heifers displayed significantly lower growth rate, lower diameter of the dominant follicle, and lower rate of ovulation than Bos taurus.
Santos et al. (2011)ated the use of different P4 pessaries, noting that heifers with a smaller amount of P4 showed increased size of the dominant follicle at the time of device removal (Day 9), results also obtained bySantos et al. (2011).Azevedo et al. (2007)stated that synchronization of estrus using long-term progesterone for TAI in heifers resulted in beneficial effects on the timing of calving and on repeat conception in the next breeding season.Calving rate was significantly different between primiparous animals that received treatment in the first breeding season and those that received no treatment in the first breeding season.Application of a TAI protocol in heifers in the first breeding season resulted in higher pregnancy rate in the next breeding season.