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Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Morphological evaluation of follicle deviation in tabapuã (Bos indicus) cows treated with bovine somatotropin (bST) B. Arnone 1 , C. Negrão 2 , C. Castilho 3 1 Master in Ciência Animal, UNOESTE, Presidente Prudente, SP; 2 Master in Clínica Veterinária, FMVZ, UNESP, Botucatu, SP; 3 Advisor in Ciência Animal, UNOESTE, Presidente Prudente, SP, Brasil. Introduction The selection phase of the dominant follicle (DF) is visualized by ultrasound as an increase in the dominant follicle diameter, while subordinate (SF) ceases its growth or regress. The difference in the growth rate between the two largest follicles of the wave was defined by Ginther et al. (1996) as follicular deviation. The bovine somatotropin (bST) is a hypofisarian hormone with multiple effects in growth and cellular differentiation and has shown action in the ovaries especially on follicular growth. Therefore, the objective of this study was evaluated the effect of the bST in follicular deviation in Tabapuã cows. Material and Methods There were used 16 Tabapuã cows, with 6 to 7 years of age, and body score from 2.5 to 3.0, maintained in pasture with access to mineral salt Ovarian follicular wave was synchronized with progestagen ear implants and with administration of 1mg of estradiol benzoate IM (day 0) and on day 5, coinciding with the wave emergence, they were divided in 2 groups: G-I (control, n = 8) and G-II (n = 8) treated with 500 mg bST. The bST injections were given subcutaneously in the space between the ischium and tail head. In the day 10 the implants were removed, concomitant the administration of 500 ug prostaglandin. In the day 10, the cows that presented follicles larger than 9 mm, received 300 ug of GnRH for ovulation. The females that ovulated G-I (n = 6) and G-II (n = 5) had their ovaries evaluated by real-time ultrasonography every 12 hours (Honda HS-2000 VET 5 MHz line array transrectaly transducer), during 5 days after ovulation, for evaluation the diameter of the largest dominant and future subordinate. Results and Discussion In the G-I one a cow was retreat from the analyses, due to no identifiable deviation. The moment of the visualized deviation was on average (mean ± SEM) at 57.60 ± 4.49 hours on the G-I (control) and at 50.40 ± 4.49 hours in the G-II (bST), or 2.4 and 2.1 days, respectively. In that moment the diamenter of DF and SF were 6.28 ± 0.42 and 6.26 ± 0.41 mm in the G-I and 6.08 ± 4.49 and 6.12 ± 0.39 mm in the G-II. In others studies with zebu females the deviation was observed 2.5 to 2.7 days after ovulation, with the DF measuring of 5.4 to 6.2 mm and the SF 5.3 to 5.9 mm (Sartorelli et al., 2005; Castilho et al, 2007; Gimenes et al., 2005). In Holstein females Ginther et al (1996) observed the deviation initiating 2.8 days after ovulation with the DF and SF measuring 8.5 and 7.2mm, respectively. The application of bST had not significant effect (p > 0.05) in the diameters of the DF and SF, in the moments evaluated. Kozicki et al. (2005) working with Holstein cows showed ovulatory diameter significantly larger (p < 0.05) in the treated group with bST (18.2 mm) regarding the group control (15 mm). Data of the present study indicate that bST in this dosage not alter the follicular diameter, neither the moment of the follicle deviation in cows. References Castilho C, Garcia JM, Renesto A, et al. 2007. Follicular dynamics and plasma FSH and progesterone concentrations during follicular deviation in the first post-ovulatory wave in Nelore (Bos indicus) heifers. Anim Reprod Sci, 98:189-96; Gimenes LU, SÁ Filho MF, Carvalho NA, et al. 2008. Follicle deviation and ovulatory capacity in Bos indicus heifers. Theriogenology, 69: 852-858; Ginther OJ, Wiltbank MC, Fricke PM, et al. 1996. Selection of the dominant follicle in cattle. Biol Reprod, 55:1187-1194; Kozicki LE, Segui MS, Fantini Filho JC, et al. 2005. A somatotrofina bovina (bST) e sua relação com o recrutamento folicular ovariano durante o ciclo estral de vacas. Arch Vet Sci, 10(1); Sartorelli ES, Carvalho LM, Bergfelt DR, et al. 2005. Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows. Theriogenology, 63:2382-2394. Acknowledgements: Prof. PhD. Guilherme de Paula Nogueira, Everton Reis (Intervet, Brazil) E-mail: calie@unoeste.br Anim. Reprod., v.6, n.1, p.219, Jan./Mar. 2009 219
220 Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Superovulatory treatment increases translation rate of angiogenic factors in buffalo CL L.A. Fátima 1 , D.B Campos 1 , P.S. Baruselli 2 , J Buratini Jr. 3 , P.C. Papa 1 1 Sector of Anatomy, Dept. of Surgery, 2 Dept of Animal Reproduction, FMVZ/USP, SP, Brazil, 3 Dept. of Physiology, Biosciences Institute, Universidade Estadual Paulista, Botucatu, Sao Paulo, Brazil. Introduction Biotechniques can be an effective way of improving reproduction efficiency and enhancing the production of genetically superior animals. The superovulatory treatment is a common technique aiming to spread desired genetical material, although its use in buffalos still presents limitations, mainly the low embryo recovery rate (1, 2, 3). The corpus luteum (CL) is an endocrine temporary gland that produces progesterone (P) required for the establishment and maintenance of pregnancy and regulation of reproductive cycle. CL development and function are affected by ovarian hyperestimulation (4, 5). The luteal cells of superovulated animals are described to show characteristics compatible with higher protein synthesis (6). The vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important regulators of CL function and development, and are also affected by superovulatory treatment. Several studies suggest that LH and hCG modulate expression of VEGF and both FSH and LH/hCG receptor-activated pathways are able to induce VEGF mRNA expression in granulosa cells (7, 8, 9). FSH stimulates bFGF receptors (FGFRs) expression and protein synthesis might be involved in the process (10). The aim of this work was to access gene and protein expression of VEGF and bFGF systems in cyclic CL of non-treated and superovulated water buffalos. Material and Methods Twelve water buffaloes corpora lutea (six from non-treated animals on day 6 after ovulation and six from superovulated animals in the same day of estrous cycle) were collected at the slaughterhouse, dissected, frozen immediately in liquid nitrogen for posterior protein and RNA extraction or fixed in 4% buffered formalin for posterior immunohistochemistry (IHC). Superovulatory treatment was conducted as described elsewhere (5). Protein expression of VEGF and its receptors KDR and Flt-1 as well as bFGF and its receptors FGFR1-4 was measured by western blotting (WB) and some proteins were also accessed by IHC. For the relative gene expression we used real time RT-PCR. Specifically for bFGF we also carried out an in situ hybridazation in order to localize the mRNA in CL tissue and gain a visual idea about mRNA content in superovulated CL compared to non-treated CL in the same estrous cycle phase. Results and Discussion VEGF system and bFGF protein expression accessed by WB and IHC showed an increase (p < 0.05) in superovulated CL compared to non-treated CL on day 6 after ovulation (p.o). FGFR-2 and FGFR-3 content measured by WB showed the same increase (p < 0.05) observed for the other studied proteins. On the other hand, mRNA expression from all studied genes was decreased (VEGF-A system p < 0.001, bFGF, FGFR-1 and FGFR-3, p < 0.05) or tended to decrease (FGFR-2 and FGFR-4, p < 0.1). The bFGF mRNA expression was detected by in situ hybridization in endothelial as well as luteal cells, and signal was stronger in non-treated animals, which is in agreement with real time RT-PCR data. According to Shikone et al. (10) FSH stimulates FGFRs expression through increase in protein synthesis, once cycloheximide, a protein synthesis inhibitor, could avoid the observed effect. On the other hand, hCG decreases the expression of bFGF transcript in granulosa lutein cells (11) but can also increase VEGF protein expression in CL (12). The interplay between VEGF and bFGF systems may lead to a down regulation of all transcripts once bFGF is decreased. These data indicate that the superovulatory treatment increased translation rate of angiogenic factors in CL. References (1)Drost M, Wright M, et al. 1983. Theriogenology, 20:579-584; (2) Baruselli PS, Madureira EH, et al. 2000. Theriogenology, 53:491; (3) Carvalho NAT, Baruselli PS, et al. 2002. Theriogenology, 58:1641-1650; (4) Doldi N, Bassan M, et al. 1997. Gynecol Endocrinol, 11:263-267; (5) Papa PC, Moura CEM, et al. 2007. Domest Anim Endocrinol, 33:379-389; (6) Artoni LP, Moura CEB. 2004. In: International Congress of Animal Reproduction, 2004, Porto Seguro, Brazil; (7) Ravindranath N, Little-Ihrig L, et al. 1992. Endocrinology, 131:254-260; (8) Neulen J, Yan ZS, et al. 1995. J Clin Endocrinol Metab, 80:1967-197; (9) Christenson L K, Stouffer RL. 1997. J Clin Endocrinol Metab, 82:2135-2142; (10) Shikone T, Yamoto M, Nakano. 1992. Endocrinology, 131:163-69; (11)Pham B, et al. 2006. Mol Reprod . Devel, 73:878–84; (12) Maybin, Duncan. 2004. Reproduction, 128:423-431. Acknowledgement: FAPESP for financial support. E-mail: ppapa@usp.br Anim. Reprod., v.6, n.1, p.220, Jan./Mar. 2009
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