Protocols with Different Time of Progesterone Exposure on Superestimulatory Response and Embryo Production of Locally Adapted Curraleiro Pé-Duro Cows

Transcription

1 Journal of Animal Science Advances Protocols with Different Time of Progesterone Exposure on Superestimulatory Response and Embryo Production of Locally Adapted Curraleiro Pé-Duro Cows Teixeira H. C. A., Mariante A. S., Nascimento N. V., Driessen K. and Ramos A. F. J Anim Sci Adv 2013, 3(5): DOI: /jasa Online version is available on:

2 ISSN: TEIXEIRA ET AL. Protocols with Different Time of Progesterone Exposure on Superestimulatory Response and Embryo Production of Locally Adapted Curraleiro Pé-Duro Cows 1,2 Teixeira H. C. A., 1 Mariante A. S., 1 Nascimento N. V., 1 Driessen K. and 1 Ramos A. F. Abstract 1 Embrapa Genetic Resources and Biotechnology, , Brasília-DF, Brazil. 2 Faculty of Agronomy and Veterinary Medicine, University of Brasília, , Brasília-DF, Brazil. Original Article The purpose of this study was to evaluate embryo production and embryonic quality of locally adapted Curraleiro Pé-duro cows, using protocols with different progesterone exposure. Cows were divided in three groups: Control, P24 and P36. All cows had the estrus previously synchronized and in the fifth day of the estrus cycle, the cows of the groups P24 and P36 received an intravaginal progesterone device and estradiol benzoate. Starting from the ninth day of the cycle, all cows received eight decreasing doses of FSH and two doses of D- cloprostenol together with the two last doses of FSH. Treatments P24 and P36 had the progesterone device removed 24 and 36 hours after the first application of D-cloprostenol, respectively. All cows received lecireline in the thirteenth day, with the inseminations accomplished 12 and 24 hours later. There was no difference (P > 0.05) for superestimulatory response among treatments. The number of total structures was greater (P < 0.05) in P24 than in the Control and the number of viable structures was greater (P < 0.05) in both P24 and P36 than in the Control. The use of exogenous progesterone in superovulation protocols improved embryo production and quality of locally adapted Curraleiro Pé-duro cows. Keywords: Conservation, genetic resources, germplasm, reproduction, superovulation. Corresponding author: Embrapa Genetic Resources and Biotechnology, , Brasília-DF, Brazil. heitortx@gmail.com and alexandre.floriani@embrapa.br. Received on: 12 Apr 2013 Revised on: 07 May 2013 Accepted on: 22 May 2013 Online Published on: 28 May J. Anim. Sci. Adv., 2013, 3(5):

3 PROTOCOLS WITH DIFFERENT TIME OF PROGESTERONE EXPOSURE ON Introduction In a high-production system, breeds specialized for milk and meats have been developed through extensive selection and its genetic material has been vastly widespread. In an overview, the intense selection for a small number of characteristics, and the intense use of semen from bulls with better genetic evaluation, has led to a low effective number of the population in cattle used for milk and meat, with a real risk of loss of genetic diversity (FAO, 1998; FAO, 2007). Animal genetic resources face a double challenge. At the same time that, the demand for animal products increases in developing countries, local breeds are rapidly disappearing throughout the world. From 1990 to 2005, 300 of more than 6000 breeds identified by FAO have been considered extinct (Cardellino, 2005). In livestock, increasing the genetic diversity of a particular breed is as important as the diversity between breeds, so it can be possible to exchange genetic material necessary for mating and breeding programs (Andrabi and Maxwell, 2007; Hiemstra et al., 2006). The Curraleiro Pé-duro is a genetic group that descends from cattle brought to the Americas by the Portuguese and Spanish settlers, and has a small and decreasing population size (Fioravanti, 2011). Raised extensively and extremely rustic this breed is very well adapted to harsh environments such as the plains of the semi-arid of the Brazilian Northeastern region (Bianchini et al., 2006; Mariante et al., 2003; Primo, 1992). The exceptional rusticity of the Curraleiro Pé-duro breed and its ability to survive in inhospitable regions of native pastures, are two characteristics that justify their conservation. Therefore, conservation actions of these animals should include collection and cryopreservation of semen and embryos (Egito et al., 2007; Mariante and Egito, 2002; Teixeira et al., 2011). Programs of in vivo production of cattle embryos through ovarian superstimulation, have been widely used worldwide. This technology not only increases the number of offspring obtained from a single donor (Baruselli et al., 2006) as well as can be used for mass production of embryos for cryopreservation and enrichment of germplasm banks (Hiemstra et al., 2006). The most commonly alternative used to synchronize follicular wave emergence for ovarian superstimulation in cows is the use of estradiol applied intramuscularly (im) at the time of the insertion of intravaginal progesterone/progestin device (Bó et al., 1996; Bó et al., 2006), which induces the emergence of a new follicular wave (Bó et al., 2002; Bó et al., 2006; Colazo et al., 2005). With the use of progesterone devices in ovarian superstimulation protocols, there is a concern about the time of exposure of follicles to the hormone. Progesterone plays an important role in maintaining the pulses of luteinizing hormone (LH), which is directly related with the final maturation of the follicle and oocyte (Barros and Nogueira, 2001; Bó et al., 2006). Hence, there are differences in the responses of production and embryo quality when the removal of progesterone device is done 24 or 36 hours after application of PGF 2a (Baruselli et al., 2006). Since these animals exhibit characteristics of adaptability to the dry tropical climate, efforts are needed to evaluate the superstimulatory response and embryo production of bovines of the Curraleiro Pé-duro breed in order to optimize the enrichment of germplasm banks. The purpose of this study was to evaluate the influence of different times of exposure to exogenous progesterone on superstimulatory and superovulatory responses, and embryo production and quality of cows of the Curraleiro Pé-duro breed in programs of in vivo embryo production. Materials and Methods The experiment was performed in Brasília - DF, Brazil (15º52 to 15º56 S and 48º00 to 48º02 W), with altitudes ranging from 1050 to 1250 m. The climate is the Koppen Aw, indicating dry winter and rainy summer. Twelve Curraleiro Pé-duro cows were gynecologically evaluated examination by rectal palpation and ultrasonography before the start of the experiment, in order to determine their cyclicity and absence of diseases or abnormalities in their reproductive tract. The animals were maintained on 262 J. Anim. Sci. Adv., 2013, 3(5):

4 TEIXEIRA ET AL. pasture (Brachiaria brizantha) with mineral salt and water ad libitum. The animals were divided into three groups: Control (estrus), P24 and P36, in an experimental design, in which all animals participated of all treatments (crossover). All treatments had a presynchronization of the estrous cycle, where D0 was considered the day of estrus. At D -10, a progesterone device was inserted (P4 - CIDR - Pfizer Animal Health, São Paulo-SP) and 2mg of estradiol benzoate were administered (EB - Estrogin - Farmavet, São Paulo-SP). In D -2, 150μg of D- Cloprostenol were administered (PGF2a - Veteglan - Hertape Calier Animal Health, Juatuba-MG) and at the subsequent removal of the progesterone device, at D -1, 1 mg of EB was administered. In the Control group, the superstimulatory treatment was initiated in D9 with the application of 133 mg of FSHp (FolltropinV - Bioniche Animal Health, Belleville, Ontario, Canada) in eight decreasing doses, administered each 12 hours. Along with the sixth and seventh doses of FSHp, 150μg D-Cloprostenol was administered. Twelve hours after the eighth dosis of FSHp (D13) 75mcg of Lecirelin were administered (GnRH - Gestran - Tecnopec, São Paulo-SP), and the inseminations was done 12 and 24 hours later, using semen of bulls with known fertility. Embryos were collected in the afternoon of D20 (Figure 1). In groups P24 and P36 progesterone device was inserted and 2mg of EB were administered at D5, while at D9 the superstimulatory treatment started (133mg FSHp in eight decreasing doses each 12 hours). Along with the fifth and sixth doses of FSHp a dosis of 150μg D-Cloprostenol were administered. Groups P24 and P36 had the device removed at 24 and 36 hours after the first application of PGF 2a, respectively. Twelve hours after the eighth dosis of FSHp (D13) 75mcg Lecirelin were administered and inseminations were performed 12 and 24 hours later, with embryos collection occurring in the afternoon of D20 (Figure 1). For the evaluation of the superstimulatory and superovulatory responses ultrasound examinations were made for counting follicles and corpus luteum. An ultrasound scanner B Aloka SSD 500Vet (Aloka CO., LTD. - Tokyo, Japan) where used with a transrectal linear transducer 7.5 MHz: at D5 for counting follicles at follicular wave emergence (follicles 3-5 mm); at D12 for counting superstimulated follicles (follicles 8 mm); at D15, for counting non-ovulated follicles (follicles > 8 mm) and at D20, for counting the number of corpus luteum before embryo collection (Figure 1). The embryos were collected by a non-surgical transcervical method with the aid of a Foley catheter. The uterine flush was performed with 1 liter of modified saline phosphate solution (DMPBS Flush - Nutricell, Campinas-SP, Brazil). After collection, the donors remained with ml intrauterine DMPBS for minutes for subsequent recollecting of embryos, according to a protocol by Castro Neto et al. (2005). The recovered embryos were evaluated using a stereoscopic microscope (Nikon SMZ645, Nikon, Tokyo, Japan) and classified according to the standards of the International Embryo Transfer Society (IETS, 1998), according to the stage of development (morulae, compact morulae, initial blastocyst, blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyst) and the quality (Grades 1, 2, 3, degenerated and nonfertilized ova). It was assumed that viable embryos would be those classified as grade 1, 2 and 3; freezable structures: grades 1 and 2; and unviable structures classified as degenerated or non-fertilized ova. Serum sample of which cow were collected in different moments: first application of FSH, seventh application of FSH, eighth application of FSH and on the application of GnRH, for determination of the serum progesterone level. Serum progesterone level was assessed using radioimmunoassay (RIA Siemens RIA TKPG5 kit Siemens Healthcare Diagnostics inc. London United Kingdom). The results of superstimulatory response, embryo recovery and progesterone level were assessed for normality by the Lilliefors test and homoscedasticity by Cochran test and analyzed by ANOVA and Duncan test at a significance level of 5%. The results of embryo quality were assessed by non-parametric Kruskal-Wallis test at a significance level of 5%. SAS Analysis Statistical Program v.9.2 (SAS Institute, Cary, NC, USA) was used as a tool for data analysis. 263 J. Anim. Sci. Adv., 2013, 3(5):

5 PROTOCOLS WITH DIFFERENT TIME OF PROGESTERONE EXPOSURE ON Results and Discussion No significant differences (P > 0.05) were found between treatments on the number of follicles at follicular wave emergence, of follicles after superovulation, of non-ovulated follicles and corpus luteum at the time of embryo collection (Table 1). The similarity between treatments in the number of follicles at wave emergence can be explained by the different exposures to progesterone did not have significant effect on the number of follicles. In general, Bos taurus and Bos indicus have different number of follicles (Baruselli et al., 2007), where Bos indicus recruit a larger number of follicles than Bos taurus, as described by Carvalho et al. (2008), probably because they had higher plasma IGF-I and lower concentrations of FSH (Alvarez et al., 2000; Bó et al., 2003). The number of follicles obtained with Curraleiro Pé-duro cows resemble those obtained by Bó et al. (1996) studying crossbreed Hereford versus Angus heifers (14.6 ± 2.2) and Martins (2007) with Holstein cows (13.4 ± 1.4) and are smaller than those found by Carvalho (2004) in Gir (26.5 ± 3.1) and Nellore cows (39.7 ± 4.9), suggesting that Curraleiro Péduro cows have a number of follicles at the time of wave emergence similar to Bos taurus. The number of follicles found after superovulation were similar between groups, possibly due to the use of the same dosis of FSHp (133 mg) and the similarity of the method of administration. The number of follicles after superovulation were 12.6 ± 6.2; 14.2 ± 7.4 and 15.8 ± 7.6 for Control, P24 and P36 treatments, respectively, suggesting that the superstimulatory response could be greater with a possible adjustment of the dosis used, as described by Nasser et al. (1996) and Nasser (2006) in Bos indicus (18.4 ± 3.4 and 23.0 ± 3.7, respectively) and Bó et al. (1996) in Bos taurus (18.6 ± 2.5). In general, there is a need to adapt dosages and protocols for superovulation in cows of locally adapted breeds, however, comparisons with commercial breeds may not be interesting, because these animals have undergone selection for fertility traits. Which did not occur with animals of adapted breeds? The amount of non-ovulated follicles, of corpus luteum at the time of embryo collection, and ovulation rate were similar between treatments, possibly due to the utilization of the same dosis of FSH and to the ovulation induction with GnRH analogue, which has been performed at the same time in all experimental groups. The number of corpus luteum at the time of embryo collection, obtained in this experiment with Curraleiro Pé-duro cows were 11.3 ± 5.8; 12.3 ± 5.6 and 11.9 ± 5.4, respectively for the Control, P24 and P36 treatments. Suggest that the donors of this breed have a great potential for embryo production with an adequacy of superovulation protocols. Carvalho (2004) and Bó et al. (1996), found 22.4 ± 0.5 and 16.6 ± 3.4 corpus luteum for Nelore and Angus x Hereford, respectively. The differences found may indicate that future adjustments in superovulation protocols and selection of Curraleiro Pé-duro cows for reproductive traits may increase the superovulatory response in these animals. As for embryo production, the total number of structures was higher (P < 0.05) in cows superovulated with Protocol P24 than in cows superovulated with the tradicional protocol (Control) while the number of viable embryos was higher (P < 0.05) in P24 and P36 treatments than in the Control. There were no significant differences (P > 0.05) on the recovery rate, and on the number of freezable and unviable structures between treatments (Table 2). Different studies show different responses of embryo production when using P24 and P36 protocols for Bos taurus and Bos indicus (Barros et al., 2007; Baruselli et al., 2006; Martins et al., 2005; Martins, 2007). Both P24 and P36 presented better results the control treatment, either statistically or numerically, showing that these protocols are able to produce greater quantity of embryos with higher quality in Curraleiro Pé-duro cows. Embryo recovery rate were 48.8 ± 26.6%; 60.9 ± 30.5% and 58.2 ± 26.7% for Control, P24 and P36 treatments, respectively (P > 0.05). The low recovery rate of the Control treatment may have resulted in fewer total and viable structures on the animals of this group. Furthermore, the smallest amount of viable structures presented by the Control group may be related to the concentration of progesterone, supported only by corpus luteum formed in the pre-synchronization, unlike the P J. Anim. Sci. Adv., 2013, 3(5):

6 TEIXEIRA ET AL. and P36 treatments that beyond of corpus luteum of the pre-synchronization, also had exogenous progesterone released by the device. As can be saw in Figure 2, the progesterone level of P24 and P36 were greater (P < 0.05) than Control group due to the use of progesterone device. Progesterone levels decrease in seventh and eighth application of FSH for Control and P24 groups, which not occurred in P36 group (P < 0.05). Progesterone level on the application of GnRH presented low in all groups (P > 0.05). Table 1:Average number of follicles at wave emergency, at the end of superovulation (SOV) and nonovulated, as well as number of corpus luteum at embryo collection obtained from Curraleiro Pé-duro cows submitted to traditional superstimulatory treatment (Control), and to progesterone devices removed at 24 (P24) and at 36 hours after the administration of PGF 2α (P36) Variables Control P24 P36 Follicles at wave emergency 16,3±8,8 16,1±7,7 17,2±9,5 Follicles at the end of SOV 12,6±6,2 14,2±7,4 15,8±7,6 Follicles non-ovulated 3,7±2,8 2,8±2,1 3,9±3,1 Corpus luteum at embryo collection 11,3±5,8 12,3±5,6 11,9±5,4 Table 2: Mean ± SD of total structures, viable structures, freezable structures and unviable structures of Curraleiro Pé-duro cows submitted to traditional superstimulatory treatment (Control), and to progesterone devices removed at 24 (P24) and at 36 hours after the administration of PGF 2α (P36) Variables Control P24 P36 Total estrutures 5,4±3,8 A 8,3±5,8 B 7,5±4,3 AB Viable estrutures 3,4±3,8 A 5,3±4,1 B 5,5±3,8 B Freezable estrutures 2,8±3,4 3,8±2,8 4,5±3,3 Unviable estrutures 2,1±2,5 2,9±2,7 2,0±2,4 A,B Different letters in the same variable mean statistical significance (P < 0.05) for Duncan test. Silva et al. (2002) showed the positive effect of the higher plasma progesterone level on the production and quality of embryos in Bos taurus. The increase in progesterone due to the presence of the implant can be the cause of cows superovulated with P24 and P36 have issued a better embryo production than cows superovulated with Control treatment. Moreover, Carvalho et al. (2008) when studying the effect of progesterone in a fixed-time artificial insemination (based on progesterone device) in Bos taurus, Bos indicus and Bos taurus x Bos indicus, observed that Bos indicus presented greater progesterone serum concentration than Bos taurus and crossbred animals. The authors argued that Bos indicus have lower speed to metabolize progesterone. Therefore, the more suitable protocol for Bos indicus would be P24, as these group keep higher progesterone levels for longer period, according to their metabolism, due to the anticipation in the removal of the progesterone device in these animals, allowing that pulses of LH remain frequent and do not hinder the development of follicles (Baruselli et al., 2007; Lafri et al., 2002). On the other hand, the more suitable protocol for Bos taurus would be P36, since they present a higher metabolism, and are able to metabolize progesterone more rapidly. Thus, keeping the progesterone device longer, the influence of progesterone on the modulation of LH pulses was sufficient to prevent ovulation from occurring, before the fully maturation of follicle/oocyte (Barros and Nogueira, 2001, Baruselli et al., 2006, Baruselli et al., 2007; Carvalho et al., 2008). Therefore, as observed in this experiment, it is suggested that Curraleiro Pé-duro cows keep the LH pulses appropriately for final maturation of the follicle/oocyte with the delayed withdrawal of progesterone devices, since the progesterone level maintained higher for long time and occurred a significant improvement in embryo quality on the P36 treatment. 265 J. Anim. Sci. Adv., 2013, 3(5):

7 PROTOCOLS WITH DIFFERENT TIME OF PROGESTERONE EXPOSURE ON The qualities of the embryos as a whole (nonparametric Kruskal-Wallis test) were similar (P > 0.05) for all treatments (Figure 3). However, the ratio between the number of viable and total embryos was 63.6% for the Control, 63.8% for P24 and 73.3% for P36 treatments, suggesting a possible beneficial effect of a longer exposure to progesterone on quality of the embryos produced by the Curraleiro Pé-duro cows, provided by a positive effect on the final maturation of the follicle/oocyte, like previously studied by Lafri et al. (2002) working with progesterone device based protocols, that observed a better embryo quality using long exposure of progesterone device, they discuss that the correct simulation of the luteolisys done with the device removal, do the correct modulation of the LH pulses. Fig. 1: Superestimulatory treatments to which the Curraleiro Pé-duro cows were submitted: traditional superestimulatory treatment (Control), and to progesterone devices removed at 24 (P24) and at 36 hours after the administration of PGF 2α (P36). *M, A Morning and Afternoon. 266 J. Anim. Sci. Adv., 2013, 3(5):

8 TEIXEIRA ET AL. Fig. 2: Serum progesterone level (Ng/mL) at different moments of the protocol (first, seventh and eighth application of FSH and on the application of GnRH) in Curraleiro Pé-duro cows submitted to traditional superestimulatory treatment (Control), and to progesterone devices removed at 24 (P24) and at 36 hours after the administration of PGF 2α (P36). A,B Different letters in the same moment mean statistical significance (P < 0.05) for Duncan test. Fig. 3: Distribution of the quality of embryos collected from Curraleiro Pé-duro cows submitted to traditional superestimulatory treatment (Control), and to progesterone devices removed at 24 (P24) and at 36 hours after the administration of PGF 2α (P36). Even through the Curraleiro Pé-duro cows are small frame animals, adapted to the dry tropical climate conditions (McManus et al., 2011), to heat 267 J. Anim. Sci. Adv., 2013, 3(5): stress (McManus et al., 2009) and dietary restriction, there was a similarity between treatments for all variables of superstimulatory

9 PROTOCOLS WITH DIFFERENT TIME OF PROGESTERONE EXPOSURE ON response and improvement in the quantity and quality of embryos, as it happens with commercial breeds when using P24 and P36 protocols (Carvalho, 2004; Martins, 2007; Nasser et al., 1993; Nasser, 2006). Thus, protocols developed for donors of commercial breeds may serve as a model for locally adapted breeds, requiring minimal adjustments to obtain a better response of these animals when submitted to programs of in vivo embryo production. The use of exogenous progesterone in superovulatory protocols improved embryo quality and production of Curraleiro Pé-duro cows locally adapted to the Brazilian tropical dry climate. Its suggested the use of protocols with longer exposure of progesterone to carry out the enrichment of germplasm banks with embryos of the Curraleiro Pé-duro breed. Acknowledgements To CAPES for granting a scholarship. References Alvarez P, Spicer LJ, Chase Junior CC, Payton ME, Hamilton TD, Stewart RE, Hammond AC, Olson TA, Wetteman RP (2000). Ovarian and endocrine characteristics during the estrous cycle in Angus, Brahman and Senepol cows in a subtropical environment. J. Anim. Sci., 78: Andrabi SMH, Maxwell WMC (2007). A review on reproductive biotechnologies for conservation of endangered mammalian species. Anim. Reprod. Sci., 99: Barros CM, Barcelos ACZB, Nogueira FG (2007). Superstimulatory Treatments Utilized in Bovine Embryo Transfer Protocols. Acta Scientiae Veterinariae, 35 (suppl. 3): Barros CM, Nogueira MFG (2001). Embryo Transfer in Bos indicus Cattle. Therio., 56: Baruselli PS, Filho MSF, Martins CM, Nasser LF, Nogueira MFG, Barros CM, Bó GA (2006). Superovulation and embryo transfer in Bos indicus cattle. Therio., 65: Baruselli PS, Gimenes LU, Sales JNS (2007). Reproductive physiology of Bos taurus and Bos indicus females. Brazilian J. Anim. Reprod., 31(2): Bianchini E, McManus C, Lucci CM, Fernandes MCB, Prescott E, Mariante AS, Egito AA (2006). Body traits associated with heat adaptation in naturalized Brazilian cattle breeds. Brazilian J. Agricult. Res., 41(9): Bó GA, Adams GP, Pierson RA, Mapletoft RJ (1996). Effect of progestogen plus E-17b treatment on superovulatory response in beef cattle. Therio., 45: Bó GA, Baruselli PS, Chesta PM, Martins CM (2006). The timing of ovulation and insemination schedules in superstimulated cattle. Therio., 65: Bó GA, Baruselli PS, Martínez MF (2003). Pattern and manipulation of follicular development in Bos indicus cattle. Anim. Reprod. Sci., 78: Bó GA, Baruselli PS, Moreno D, Cutaia L, Caccia M, Tribulo R, Tribulo H, Mapletoft RJ (2002). The Control of Follicular Wave Development for Self-Appointed Embryo Transfer Programs in Cattle. Therio., 57: Cardellino RA (2005). Status of the world s livestock genetic resources. Preparation of the first report on the state of the world s animal genetic resources. In: The role of biotechnology for the characterization and conservation of crop, forestry animal and fishery genetic resources, International Workshop, FAO, 1-6. Carvalho JBP (2004). Synchronization of ovulation with intravaginal with progesterone device in Bos indicus, Bos indicus x Bos taurus and Bos taurus heifers. Ph.D. Thesis, University of São Paulo, Brasil, Carvalho JBP, Carvalho NAT, Reis EL, Nichi M, Souza AH, Baruselli PS (2008). Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus Bos taurus, and Bos taurus heifers. Therio., 69: Castro Neto AS, Sanches BV, Binelli M, Seneda MM, Perri SH, Garcia JF (2005). Improvement in embryo recovery using double uterine flushing. Therio., 63: Colazo MG, Martínez MF, Small JA, Kastelic JP, Burnley CA, Ward DR, Mapletoft RJ (2005). Effect of estradiol valerate on ovarian follicle dynamics and superovulatory response in progestin-treated cattle. Therio., 63: Egito AA, Paiva SR, Albuquerque MSM, Mariante AS, Almeida LD, Castro SR, Grattapaglia D (2007). Microsatellite based genetic diversity and relationships among ten creole and commercial cattle breeds raised in Brazil. BMC Genetics, 8:83. FAO (1998). Secondary Guidelines for Development of National Farm Animal Genetic Resources Management Plans: Management of small populations at risk. FAO, 210p. FAO (2007). The State of the World s Animal Genetic Resources for Food and Agriculture. FAO, 511p. Fioravanti MCS, Juliano RS, Costa GL, Abud LJ, Cardoso VS, Carpio MG, Costa, MFO (2011). Conservación del bovino Curraleiro: cuantificación del censo y caracterización de los criadores. Anim. Gen. Resour., 48: Hiemstra SJ, Van Der Lende T, Woelders H (2006). The potential of cryopreservation and reproductive technologies for animal genetic resources conservation strategies. In: FAO. The role of biotechnology in exploring and protecting agricultural genetic resources. FAO, 187p. IETS (1998). Manual of the International Embryo Transfer 268 J. Anim. Sci. Adv., 2013, 3(5):

10 Society. 3 ed. IETS, 180p. Lafri M, Ponsart C, Nibart M, Durand M, Morel A, Jeanguyot N, Badinand F, de Mari K, Humblot P (2002). Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle. Therio., 58: Mariante AS, Egito AA (2002). Animal genetic resources in Brazil: result of five centuries of natural selection. Therio., 57(1): Mariante AS, McManus C, Mendonça JF (2003). Country report on the state of animal genetic resources: Brazil. Embrapa Genetic Resources and Biotechnology, 92p. Martins CM (2007). Different superovulation protocols with fixed time artificial insemination in Bos taurus and Bos indicus. Master Thesis, University of São Paulo, Brasil, Martins CM, Castricini ESC, Reis EL, Torres-Júnior JRS, Gimenes LU, Sá Filho MF, Baruselli PS (2005). Produção embrionária de vacas holandesas a diferentes protocolos de superovulação com inseminação artificial em tempo fixo. Acta Scientiae Veterinariae, 33 (Suppl. 1):286. McManus C, Prescott E, Paludo GR, Bianchini E, Louvandini H, Mariante AS (2009). Heat tolerance in naturalized Brazilian cattle breeds. Livestock Sci., 120: McManus C, Castanheira M, Paiva SR, Louvandini H, Fioravanti MCS, Paludo GR, Bianchini E, Corrêa PS (2011). Use of multivariate analyses for determining heat tolerance in Brazilian cattle. Trop. Ani. Health Produc., 43: Nasser LF, Adams GP, Bó GA, Mapletoft RJ (1993). Ovarian superstimulatory response relative to follicular wave emergence in heifers. Therio., 40: Nasser LFT (2006). Superovulatory response during the first follicular wave in Nelore (Bos indicus) donors. Ph.D. Thesis, University of São Paulo, Brasil, Primo AT (1992). El Ganado Bovino Iberico En Las Americas: 500 Años Después. Arch. Zootec., 41(154): Silva JC, Costa LL, Silva JR (2002). Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers. Anim. Reprod. Sci., 69:1 8. Teixeira HCA, Nascimento NV, McManus C, Egito AA, Mariante AS, Ramos AF (2011). Seasonal influence on semen traits and freezability from locally adapted Curraleiro bulls. Anim. Reprod. Sci., 125: TEIXEIRA ET AL. 269 J. Anim. Sci. Adv., 2013, 3(5):