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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A059 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION EFFECTS OF FSH AND GROWTH DIFFERENTIATION TION FACT CTOR -9 (GDF-9) ON IN VITRO GROWTH OF BOVINE SECONDAR ONDARY FOLLICLES Regislane Pin into Rib ibeir eiro, Gisv isvani Lop opes de Vasc asconc oncelos elos, Anderson Wein einy Barbalho Silv ilva, Mar aria Juliane Passos assos, Francisc ancisco Taia Gomes Bezer erra, Katianne Freitas dos Santos, Emanuela de Lima Rebouças, Rodrigo Otávio Decaria de Salles Rossi, Ellen da Cunha de Vasconcelos & José Roberto Viana Silva UNIVERSIDADE FEDERAL DO CEARÁ, SOBRAL, CE, BRAZIL. Recently, isolation and culture of caprine secondary follicles of ~200µm has been carried out in order to produce oocytes capable of maturation (Magalhães et al., Reprod. Domest. Anim., 46:134-140, 2010). However, the effect of GDF-9 during the culture of bovine secondary follicles has not been evaluated yet. This study aimed to determine the effects of GDF-9 in the presence or absence of FSH on survival, growth and antrum formation in bovine preantral follicles cultured for 12 days. Secondary follicles were isolated and cultured, for 12 days, individually in α-MEM (control) or in α-MEM supplemented with GDF-9 (200 ng/mL), FSH (100 ng/mL from day 0 to 6, 500 ng/mL from day 6 to day 12) or GDF-9 associated with FSH. Each treatment was repeated four times, using around 24 follicles. The follicular survival was calculated according to the morphology of oocyte and granulosa cells. During culture, follicular diameter and antral cavity formation were evaluated. Data of follicular growth were compared by Kruskal-Walls test, while the percentage of viable follicles or follicles that formed an antrum were compared by chi-square test (P < 0.05). Based on the analysis, the treatments did not affect the percentage of surviving follicles after culture for 12 days. Compared to D0, during the culture of follicles with MEM alone or together with GDF-9, GDF-9+FSH or FSH there was a significant increase in follicular diameter on day 12, 8, 6 and 6, respectively. After 8 days of culture, GDF-9 (219.32 ± 15.21), GDF-9 + FSH (233.53 ± 10.57) and FSH (251.69 ± 15.49) significantly increased follicular diameter when compared to MEM (184.68 ± 9.74). Similar results were observed on day 10 (MEM: 193.78 ± 8.90; GDF-9: 232.25 ± 10.48; GDF-9 + FSH: 257.76 ± 17.18; FSH: 288.70 ± 16.09) and 12 (MEM: 204.73 ± 8.93; GDF-9: 245.50 ± 13.38; GDF-9 + FSH: 266.02 ± 17.70; FSH: 307.80 ± 19 13). On day 12, FSH (70.83%) and GDF- 9 (65%) significantly increased the rate of antrum formation when compared with control (31.81%) and GDF-9+FSH (43.82%). In conclusion, this study showed that FSH and GDF-9 promotes the growth and antrum formation of bovine secondary follicles in vitro. Keywords: gdf-9, follicles, culture. A060 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION INFLUENCE OF PROTOC OCOLS OLS FOR SUPEROVUL OVULATION ON INDUCED STRESS OF BROWN BROCKET DEER (MAZAMA GOUAZOUBIRA) Eveline Santos Zanetti & José Mauricio Barbanti Duarte FCAV - UNESP, JABOTICABAL, SP, BRAZIL. The knowledge of assisted reproduction techniques for wild animals should be useful for future in situ and ex situ conservation programs. However, stress is a limiting factor in assisted reproduction in wild animals maintained in captivity. The aims of the present study were: (1) to evaluate the influence of two different protocols designed to cause superovulation on induced stress of brown brocket deer, and (2) to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by these two different protocols. For this, six hinds were assorted into 2 groups (n = 3): the first received CIDRÒ for 8 days, followed by 0.25mg i.m. injection of OB on D-8, 700IU of eCG on D-4 and 265µg of PGF2α on D0 (Treatment 1) and the second received CIDRÒ for 7.5 days followed by 0.25mg i.m. injection of OB on D-7.5, 130mg of FSH divided into eight equal doses [beginning on D-3 and ending D-0.5] and 265µg of PGF2α on D0 (Treatment 2). The treatments were crossed over with 44-48 day intervals after CIDRÒ removal (all the animals went through all 2 Treatments). Analysis was conducted using MINITAB ® Release 14 Software (P < 0.05). The induced stress (M1 – two days before the beginning of the treatments; M2 – all the days during the treatments; M3 – the last four days of the treatments and M4 – five days after the end of the treatments) and the treatment efficacy was evaluated by observation of CL and unruptured follicles via laparoscopy 7 days after the first copulation and analyze of fecal progestin concentration. There was no significant difference for fecal cortisol concentration (ng/g feces) in four different moments between the treatments; however M1 was significantly different from M3 within each Treatment ((T1): M1 = 98.52±8.99 vs.. M3 = 170.34±22.98 and (T2): M1 = 91.40±10.93 vs. M3 = 190.54±31.82). The mean ovulation rate was higher (P = 0.05) in Treatment 1 (6.0±1.7) than in Treatment 2 (2.0±0.3), but the total ovarian stimulation (13.2±2.6 and 10.4±1.1, in 1 and 2, respectively) was not significantly different between treatments. Moreover, the mean fecal progestin concentration at the luteal phase in Treatment 1 (4293.69±769.47ng/g feces) was significantly different from Treatment 2 (1571.26±240.28 ng/g feces). Thus, regardless of the treatment, the influence of the induced stress should be considered in the superovulation protocols and, in this case, the eCG seems to promote a better gonadotrophic stimulation to overcome its effects, since Treatment 1 induced better superovulation response in the species Mazama gouazoubira,with greatest mean ovulation rates and functional corpora lutea formation. Keywords: superovulation, stress, neotropical deer. s366
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A061 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION STEADY-ST Y-STATE TE LEVELS OF VASO ASOACTIVE INTESTINAL PEPTIDE MRNA IN GOAT OVARIES AND ITS EFFECT ON THE IN VITRO DEVEL VELOPMENT OF ISOLATED PREANTRAL AL FOLLICLES Jamily Bezerra Bruno 1 Valdevane Rocha Araújo 1 , Juliana Jales de Hollanda Celestino 2 , Márcia Viviane Alves Saraiva 1 , Rebeca Magalhães Pedrosa Rocha 1 , , Ivina Rocha Brit ito 1 , Ana Bea eatr triz Graça Duar uarte 1 , Cleidson Mano anoel Gomes Silv ilva 1 , Isador sadora a Machado Teix eixeir eira Lima 1 , Mar aria Helena Tavar ares Matos 3 & José Ric icar ardo de Figueiredo 1 1 UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA, CE, BRAZIL. 2 UNIVERSIDADE DA INTEGRAÇÃO INTERNACIONAL DA LUSOFONIA AFRO-BRASILEIRA, REDENÇÃO, CE, BRAZIL. 3 UNIVERSIDADE FEDERAL DO VALE DO SÃO FRANCISCO, PETROLINA, PE, BRAZIL. The aims of this study were to evaluate the levels of vasoactive intestinal peptide (VIP) mRNA in caprine ovaries, the effects of VIP and/or follicle-stimulating hormone (FSH) on follicular development and mRNA levels of VIP, and FSH receptor (FSHR) after in vitro culture of advanced secondary follicles for 6 days. All samples were maintained in á-minimum essential medium (α-MEM+) or in α-MEM+ supplemented with FSH (100 ng/mL) and/or VIP (10 ng/mL). For expression analysis, caprine primordial, primary and secondary follicles were obtained. Cumulus-oocyte complexes (COCs), and clusters of granulosa/theca cells of small (3mm) antral goat follicles were also recovered. VIP and FSHR mRNA levels were quantified by real-time reverse transcription through the polymerase chain reaction (qRT-PCR). Glyceraldehyde-2-phosphate dehydrogenase (GAPDH) and β-actin were used as endogenous controls for fresh ovaries and for the culture follicles were used β-actin and ubiquitin (UBQ). The delta-delta-CT method was used to transform CT values into normalized relative steady-state mRNA levels. The survival, antrum formation, and follicular diameter were evaluated every 2 day. In each parameter analyzed was employed the most appropriate statistical test according to the behavior of data. (P < 0.05). qRT-PCR demonstrated that levels of VIP mRNA in secondary follicles were significantly higher than in primordial follicle. In addition, no significant difference was observed between COCs collected from small and large antral follicles. Granulosa/theca cells from large antral follicles had higher levels of VIP mRNA than small antral follicles. The addition of VIP and/or FSH to the culture medium had no effect on follicular development. However, the presence these substances significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. Conclusions included that the VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary follicles, and the expression of VIP mRNA levels after in vitro culture was reduced. Keywords: vip, fsh, expression. A062 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION STANDARD OF EMERGENCY FOLLICULAR WAVES IN LONG PROTOC OCOLS OLS (WITH OR WITHOUT CIDR REPLACEMENT CEMENT) ) AND SEASONALITY EFFECT IN SANTA A INES EWES Mar aria Emilia Franc anco Oliv liveir eira 1 , Car arla Cristina D’ama ’amato 1 , Henderson Ayr yres 2 , Luís Guilher uilherme Oliv liveir eira 1 , Pedr edro Paulo Maia Teix eixeir eira 1 , Jef eferson erson Fer erreir eira Fonsec onseca 3 & Wilter Ricardo Russiano Vicente 1 1 UNESP-FCAV, JABOTICABAL, SP, BRAZIL. 2 USP-FMVZ, SÃO PAULO, SP, BRAZIL. 3 EMBRAPA-CNPC, SOBRAL, CE, BRAZIL. N Superovulatory protocols in sheep show high variability of responses and, usually, not based on the follicular development. The present study evaluated the follicular dynamic in basis superovulation protocols in order to determine the pattern of follicular wave emergency. Season effect on variables was also studied. Seventy adult ewes were submitted to one of two synchronization protocols in three seasons (Factorial 2x3; Non-breeding: G-1CIDR, n = 12 and G-2CIDR, n=11; Transition: G-1CIDR, n = 12 and G-2CIDR, n=12; Breeding: G- 1CIDR, n = 11 and G-2CIDR, n = 12). On D0 (randomized day of estrus cycle), estrus were synchronized with a P4 device (CIDR; Pfizer- Netherlands) for 14 days. However, in G-2CIDR, the CIDR was replaced by a new one on D7. At D0 and 14, 2.5 mg of dinoprost (Lutalyse, Pfizer-Netherlands), i.m., were administered in all ewes. Ovaries ultrasonographic exam (Aquila - Pie Medical, Netherlands) was performed daily by transrectal during treatments. Follicular wave was identified as a group of follicles (2-3 mm) that grew, being that one or more reached the minimum diameter of 4.5 mm. Emergence follicular wave day was considered when the largest follicle, still with 2-3 mm, was first identified. Data were analyzed by GLIMMIX using the SAS. A total of 31.43% of ewes had corpus luteum at the beginning of protocols. Experimental ewes presented two to five follicular waves emergencies during treatments (2 waves: 4.29%; 3 waves: 34.29%; 4 waves: 52.86% and; 5 waves: 8.57% of ewes). There were no effect of treatment on emergency waves days (Wave 1: 2.05±0.42 vs.. 2.02±0.37; Wave 2: 5.69±0.42 vs.. 5.65±0.37; Wave 3: 9.77±0.42 vs.. 10.09±0.37; Wave 4: 11.85±0.39 vs.. 12.12±0.35 and; Wave 5: 12.5±0.40 vs.. 12.16±0.78 days for G- 1CIDR and G-2CIDR respectively; P > 0.05). Similarly, there were no effect of seasons on these variables (Non-breeding: 2.01±0.46, 5.11±0.47, 9.33±0.45, 12.27±0.40 and 13.50±0.40; Transition: 2.12±0.51, 5.95±0.52, 10.32±0.51, 11.61±0.50 and 11.16±0.78 and; Breeding: 1.99±0.42, 5.95±0.42, 10.14±0.42 and 12.08±0.43 days for waves 1, 2, 3, 4 and 5, respectively, P > 0.05). In Breeding season no ewes showed five waves. Although these protocols are not developed to directly induce the follicular wave emergency, there was a pattern of emergency during both treatments, regardless of season. Furthermore, these informations will be important for the definition of new protocols (i.e. superovulation or synchronization treatments); which effectively based on ovarian activity and avoid deleterious effects. Keywords: follicular dynamic, superovulation, ovine. s367
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