Hertape Calier Research Articles

Strategy to improve the parturition ability in crossbred recipients implanted with embryos produced in vitro

The aim of this study was to evaluate the synchronization rate, ease to parturition, calves viability and retained placenta rate for Nellore recipients treated with triancinolone (TRI) + dexametasone (DEXA) + PGF2α (d-CLO). Hundred-ninety-four crossbred recipients (½ Nellore/ ½ Caracu) were divided into two groups (TRI, n = 97; Control, n = 97) and observed from 285 to 292 days of pregnancy. On Day 285, animals from Group TRI received 1mg/60 Kg of BW/IM of triancinolone (Retardoesteróide®, Hertape Calier Saúde Animal S/A) followed of association of 25mg of dexametasone (Caliercortin®, Hertape Calier Saúde Animal S/A) and 150μg of D-cloprostenol (Veteglan®, Hertape Calier Saúde Animal S/A) on Day 292. Animals from Control received injections of saline solution in same days. TRI had higher synchronization of parturition than Control (79.4% vs. 19.5, respectively; P < 0.05), as well TRI had lower assistance of parturition. No differences were observed for assistance of parturition comparing the born weight and gender of calves. TRI had lesser placenta retained than Control (11.4% vs. 25.8%, respectively; P < 0.05). In short, this strategy of induction resulted in better conditions of assistance and quality of parturition, decreasing the placenta retained in Nellore recipients implanted with embryos produced in vitro.

192 The use of human chorionic gonadotropin seven days after synchronized estrus for the increase of luteal tissue in Morada Nova ewes

The study was performed to evaluate whether the use of hCG 7 days after synchronized oestrus in Morada Nova ewes would increase the number and size of corpora lutea. Multiparous Morada Nova ewes (n=115, mean weight of 36.6kg, body condition score 3.13 on 1 to 5 scale) were used. Oestrus was synchronized by using an intravaginal sponge impregnated with medroxyprogesterone acetate (60mg, Progespon®, Zoetis, Parsippany, NJ, USA) for 6 days and eCG (200IU, IM Novormon®, Zoetis) plus PGF2α analogue (37.5μg, D-cloprostenol, IM, Vetglan®, Hertape Calier, Spain), both administered 24h before the sponge removal. Seven days after the synchronized oestrus, hCG (300IU, IM, Vetecor®, Hertape Calier; n=57) or normal saline (control group, 1mL, IM, 0.9% NaCl, Eurofarma Laboratory SA, Sao Paulo, Brazil; n=58) was injected. B-mode ultrasound examinations of the ovaries were performed using the equipment MyLab Vet 30 Gold (Esoate, Genoa, Italy) on Day 7 (corresponding to the day of hCG or physiological solution administrations), and 6 days later (Day 13). The number of corpora lutea present and the size of luteal tissue (sum of diameter and area) were determined. Data were analysed by ANOVA with Tukey’s post hoc test (mean±s.e.m.) using SAS software (SAS Institute Inc., Cary, NC, USA). On Day 7, the number of corpora lutea was similar (P>0.05) between the hCG and control groups (1.58±0.09v. 1.57±0.08, respectively); however, the number was higher on Day 13 (P<0.05) in the hCG group (2.65±0.13) than in the control group (1.69±0.07). Similarly, there was no difference (P>0.05) between groups in diameter (16.9±0.82v. 16.0±0.71mm) and area (1.61±0.07v. 1.45±0.06cm2) for hCG and control groups, respectively, on Day 7. Differences were observed on Day 13, such that the hCG ewes had luteal tissue of larger (P<0.0001) diameters (27.13±1.25v. 18.44±0.85mm) and area (2.81±0.13v. 1.71±0.08cm2) than ewes of the control group. In conclusion, 300IU of hCG injected 7 days after synchronized oestrus is effective to increase the number of corpora lutea and in the size of luteal tissue. Despite indications of a favourable effect on luteal dynamics, the effects on progesterone production and conception rate need to be examined. This study was supported by CNPq and EMBRAPA (process no. 02.13.06.026.00.02).

82 Compensation of the Growth and Development of Individually Transferred Bovine Bisected Embryos

Embryo bisection has been used in cattle for decades to multiply embryos. Recent articles assert that each bisected hemi-embryo continues its growth and differentiation without regenerating the original number of cells, producing smaller embryos. This size difference has been verified until Day 17, when the embryo has already elongated. The objective of this research was to evaluate fetal morphometric differences between embryos and hemi-embryos during the intrauterine period and at birth. For this purpose, 6 Angus cows were superovulated using the FSH/LH protocol (Pluset®, Hertape Calier, Minas Gerais, Brazil) and all were artificially inseminated with the sire ‘Las Lilas 1931 Líder Max’. At Day 7 post-AI, 45 embryos were recovered using a non-surgical technique. In the laboratory, 30 of them were bisected under a stereoscopic magnifier using a portable micromanipulator with a microscale attached. Each hemi-embryo (N = 60) and the remaining integer embryos (N = 15, used as controls) were immediately transferred individually to a recipient. The growth of the fetuses was evaluated ultrasonographically using a Esaote Aquila Pro vet ultrasound (Esaote SpA, Genoa, Italy). Transrectal measurements were performed at 60 and 90 days, recording femur length (only at 60 days), biparietal distance, and abdominal and umbilical cord diameter. At birth, animals were weighed, measured for length and height, and the circumferences of the skull, thorax, abdomen, and metatarsus were recorded. Data were analysed by the t-test implemented in InfoStat Version 1.1 software (https://www.infostat.com.ar/). Table 1 shows the average and standard deviation of all measurements performed. No significant differences (P < 0.05) were observed between hemi-embryos and control at any stage, except for biparietal diameter on Day 90 of gestation, which was not reflected in skull circumference at birth. In conclusion, individually transferred hemi-embryos generated 60-day fetuses with almost no differences in growth and development compared with the control embryos. The compensation phenomenon should have occurred before Day 60 of gestation. Table 1.Mean and standard deviation of all measurements performed on hemi-embryos and control embryos at 60 and 90 days and at birth

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

10 TIMED ARTIFICIAL INSEMINATION PROTOCOLS TO SYNCHRONIZATION OF OVULATION IN BOS TAURUS TAURUS SUCKLING BEEF COWS

The aim of this study was to compare 3 methods for synchronization of ovulation in anestrous beef cows. The hypothesis of this study was to determine whether low doses of hCG has superior efficacy to cypionate to induce ovulation in anestrous cows and provide higher pregnancy rate in oestrus-synchronization programs. Synchronization of ovulation and conception rate to timed AI (TAI) were evaluated in anestrus Bos taurus taurus suckling beef cows 45 ± 15 days postpartum and with body condition score of 2.9 (1 to 5) maintained in a native pastured system in the south of Brazil. Females were evaluated with ultrasound on the Day 0 (D0) of the protocol (Day 0), day 8 (D8), immediately before TAI (D10), and 7 days after TAI (Day 17). All cows were synchronized with an intravaginal progesterone-releasing device (IPRD; 0.75 g of progesterone, Prociclar®, Hertape Calier Animal Health, Juatuba, Brazil) and 2 mg IM of oestradiol benzoate (EB; Benzoato HC®) on D0. On Day 8, the IPRD was removed and 150 μg of D (+) cloprostenol (Veteglan Luteolytic®), and 25 IU IM FSH/LH (Pluset®) were administered. Females of the EC (n = 84) group received 1 mg IM of oestradiol cypionate (EC; Cipionato HC®). Females on D8 of the hCG (n = 81) group received 500 IU IM of hCG (Vetecor®, Hertape Calier) at the time of TAI. The females of the EC + hCG group (n = 83) received both treatments. All cows were submitted to TAI 54 h after withdrawal of IPRD. A part of the cows (n = 102) had the ovulation evaluated every 12 h from the withdrawal of IPRD [EC (n = 34), hCG (n = 34), and hCG + EC (n = 33)]. Statistical analysis was performed using SAS PROC GLIMMIX. The dominant follicle diameter (FD) on Day 8 (8.7 ± 0.2, 8.8 ± 0.2, 8.6 ± 0.2) did not differ between treatments EC, EC + hCG, or hCG (P = 0.79). However, the FD on D10 was higher (P = 0.001) for cows treated with hCG (12.9 ± 0.3) compared with cows from the EC (11.3 ± 0.2) or EC + hCG group (11.8 ± 0.2). The interval (h) between the withdrawal of IPRD and ovulation was lower (P = 0.01) for the hCG group, (71.2 ± 1.7) compared with the groups treated with EC or EC + hCG (76.6 ± 2.18 and 74.2 ± 1.65), respectively. The ovulation rate did not differ (P = 0.61) among the EC (85.2%, 29/34), hCG (91.1%, 31/34), or EC + hCG groups (90.9%, 30/33). Corpus luteum diameter (mm) was higher (P = 0.04) on D17 for the hCG-treated group (21.4 ± 0.3) compared with others treatments (EC = 19.1 ± 0.8 or EC + hCG = 20.4 ± 0.8). However, the plasma progesterone levels on D17 were EC = 2.0 ± 0.1, hCG = 2.4 ± 0.1, and EC + hCG = 2.3 ± 0.1 ng mL–1 (P = 0.19), and the conception rate on the 28th day after TAI (EC = 43.0%; hCG = 47.0%, and EC + hCG = 48.8%; P = 0.76) was also similar. The hCG determined smallest ovulation interval, but similar rates of pregnancy were observed with both treatments.

350 EFFECT OF DIFFERENT SUB-DOSES OF EQUINE CHORIONIC GONADOTROPIN ON PREGNANCY AND TWINNING RATES OF CYCLIC ZEBU COWS SUBMITTED TO FIXED-TIME ARTIFICIAL INSEMINATION

The injection of a low dose of eCG has the potential to induce multiple ovulation and pregnancies in cattle. The present study aimed to evaluate the ovarian response, conception rate and incidence of twin pregnancies of cyclic cows receiving 1 of 2 low doses of eCG. Multiparous Nellore (Bos t. indicus) cows with plasma progesterone levels >1 ng∙mL–1 on at least one of 2 blood samples collected at 10-day intervals (Day –10 and Day 0) received an intramuscular (IM) injection of 2 mg of oestradiol benzoate (EB; Estrogin®, AUSA, São Paulo, Brazil) and a vaginal device (DIP) containing 1 g of progesterone (Primer®, Tecnopec, São Paulo, SP, Brazil) on Day 0. On Day 8, the DIP was removed and cows received an IM injection of 150 μg of cloprostenol (Veteglan®, Hertape Calier, Juatuba, MG, Brazil). At this time, the animals were randomly distributed into 3 groups. Group 1 (n = 30) received an IM injection of 2 mL of saline, whereas groups 2 (n = 41) and 3 (n = 23) received 600 IU and 900 IU of eCG (Novormon® MSD Saude Animal, São Paulo, Brazil), respectively. Twenty-four hours later (Day 9), all groups received 1 mg of EB and were submitted to fixed-time artificial insemination (FTAI) 30 h later (i.e. 54 h after DIP removal). Oestrus observation was performed daily from the time of the withdrawal of the DIP until the day of FTAI. Ovaries were examined ultrasonically at the time of FTAI, the following day and 7 days after FTAI. Pregnancy diagnosis was done by ultrasonography 30 days after FTAI and the incidence of twin or single calves was recorded at birth. Data were analysed by chi-square test. The rate of expression of oestrus was 70.0% (group 1), 82.9% (group 2), and 78.2% (group 3; P = 0.25). Cows that had 2 or more large follicles at the time of FTAI was 0% (group 1), 14.6% (group 2), and 34.8% (group 3; P < 0.05). The ovulation rate of cows in group 1 (80.0%) was higher than cows in groups 2 (48.8%) and 3 (52.2%; P < 0.05). The conception rates for groups 1, 2, and 3 were 50.0, 26.8, and 39.1%, respectively (P < 0.05). Two animals in group 3, one in group 2, and none of group 1 had twin pregnancies on Day 30 after FTAI. Only one of these cows (group 3) had a twin calving. It was concluded that the injection of 600 or 900 IU eCG, in an oestradiol/progestogen FTAI protocol does not result in an increase in the rate of twin calvings, but may negatively affect pregnancy rates of cyclic Nellore cows.Financial support was provided by FAPESP (proc. 2011/13096–0).

101 SUPPLEMENTATION WITH SUNFLOWER SEED ALTERS THE ENDOMETRIAL LIPID COMPOSITION IN BEEF COWS

Embryo death between 15 and 19 days of pregnancy is caused by the increase in the release of endometrial prostaglandin F2α (PGF2α) involved in the luteolysis process in cattle. Compounds rich in linoleic acid, such as sunflower seeds, provide lipid changes in the endometrium, and may be involved in the ability of PGF2α biosynthesis. Previous studies observed that the conception rate increased in Nelore cows supplemented with sunflower seed for 22 days from the timed AI (66.7% v. 46.3%; Peres et al. 2008, Acta Sci. Vet. 36, 639) and in crossbred heifers submitted to timed embryo transfer (55.66% v. 36.94%; Membrive et al. 2013 Acta Sci. Vet. 36, 603). We aimed to test the hypothesis that supplementation with sunflower seed promotes endometrial changes in lipid composition. Thus, we compared the composition of fatty acids in endometrial tissue in cows supplemented or not with sunflower seed. Nelore (n = 30) cows received an intravaginal device containing progesterone (1 g; DIB, Syntex Biochemistry & Pharmaceutical Industries SA, Buenos Aires, Argentina) associated with an im injection of oestradiol benzoate (2 mg; Benzoate HC, Hertape Calier Animal Health SA, Juatuba, MG, Brazil). The devices were removed after 8 days, when cows were treated im with cloprostenol sodium (2 mg; Sincrocio®, Ourofino Animal Health Ltd., Cravinhos, SP, Brazil), oestradiol cypionate (0.5 mg; ECP®, Zoetis Ltda., São Paulo, Brazil) and eCG (300 IU; Folligon®, Intervet Veterinary Ltda., Cotia, Brazil). Two days after removal of the device, females were assigned into 6 groups to receive 1.7 kg/animal/day of 40% soybean meal, 44% crude protein (CP) + 60% sunflower seed for 6 (n = 4), 14 (n = 5) and 22 days (n = 6), or 53% soybean meal, 44% CP + 47% corn for 6 (n = 4), 14 (n = 5) and 22 days (n = 6). Both diets were formulated with 72% total digestible nutrients and 24% CP. Females were slaughtered 24 h after the end of supplementation and endometrial tissue was isolated and stored at –196°C. The fatty acids in endometrial tissue were assessed by gas chromatography. Data were analysed by SAS Proc GLIMMIX (SAS Institute Inc., Cary, NC, USA). The fatty acid profile (54 compounds) was analysed and 43 fatty acids were present in the endometrial tissue. The lacking fatty acids in endometrial tissue were C4:0, C11:0, C12:1, C: 13:0, C13:0 iso, C13:0 anteiso, C14:0 iso, C15:1, C18:1 trans-16, C18:2 cis-12, trans-10, and C21:0. The fatty acids that showed a higher percentage compared with the Control group were C18:1 trans-10-trans-11-trans-12 and C10:1. The fatty acids that showed low percentage compared with the Control group were C15:0 iso, C20:5, C20:3n-3, C23:0, C24:0, and C22:5. In conclusion, supplementation with sunflower seed promotes changes in the endometrial lipid profile that may reduce the pregnancy loss in beef cows. Research supported by FAPESP, FUNDUNESP, and Santa Encarnação Farm.

167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

14 CONCEPTION RATE OF BUFFALO HEIFERS TREATED WITH OESTRADIOL CYPIONATE TO INDUCE OVULATION IN TIMED ARTIFICIAL INSEMINATION PROGRAMS

Reducing animal handling and costs in timed artificial insemination (TAI) systems are of great interest in animal production, regardless of the species. Thus, this study was carried out to investigate the effects of administering oestradiol cypionate (EC) or hCG to induce ovulation in buffalo (Bubalus bubalis) in TAI programs. The 79 Bufalipso heifers housed at Institute of Animal Science, San José de las Lajas, Cuba, were, on average, 24 months of age with a body weight of 310 to 380 kg at the beginning of the experiment. The investigation was conducted out of the breeding season (25 June through 28 July 2012). Heifers were randomly assigned to 1 of 3 treatment groups; either hCG (control group) or EC1 and EC2, in which TAI was performed at either 48 or 56 h after the EC injection, respectively. On the first day of the treatment regime (Day 0), animals received oestradiol benzoate (2 mg i.m.; Sincrodiol®, Ourofino, Brazil), followed by the insertion of an intravaginal device containing 1 g of progesterone (Primer®, Tecnopec, São Paolo, Brazil). On Day 9, an intramuscular luteolytic dose of cloprostenol (500 μg; Ciosin®, Intervet, Brazil) and eCG (500 UI; Novormon®, Intervet) were given in all heifers along with the removal of the intravaginal device. On the same day, 0.5 mg of EC (ECP®, Pfizer, Brazil) was administered intramuscularly in the EC1and EC2 groups. Animals in the hCG group received hCG (1000 IU i.m.; Vetecor®, Hertape Calier, Spain) on Day 11 and were inseminated 14 h later. Pregnancy diagnosis was performed by ultrasound 35 days after TAI. Data were analysed by a chi-squared test, with P < 0.05 taken as significant. There were no differences among treatment groups, with values of 57.7 (15/26), 60.7 (17/28), and 64% (16/25) for the EC1, EC2, and hCG groups, respectively. In conclusion, oestradiol cypionate was effective at inducing ovulation in buffalo heifers in TAI programs comparable to the control group, as evidenced by the conception rates. In addition, animals subjected to the EC protocol pass just 3 times through the corral instead of 4 when receiving hCG, which decreases the animal handling and costs.

8 DURATION OF ESTRUS AND TIME OF OVULATION IN SUPEROVULATED SANTA INÊS EWES

The aim of this study was to determine the intervals between sponge removal and the onset of estrus, sponge removal and the first detected ovulation, and onset of estrus and the first ovulation, and also to determine the duration of estrus, in superovulated Santa Inês ewes subjected to natural mating (NM) and AI. The trial was done in July and February at Cachoeiras de Macacu–Rio de Janeiro (22°27′S, 43°39′W). Fifteen non-pregnant Santa Inês ewes, age 3.4 ± 1.4 years, weighing 47.8 ± 6.3 kg, with 3.3 ± 0.4 body condition score (scale of 1 to 5), were randomly assigned to 2 groups. Both groups were superovulated using the same protocol and were mated in a crossover design by NM and laparoscopic AI. Estrus was synchronized using intravaginal sponges (60 mg of medroxyprogesterone acetate; Progespon®, Schering Plough Animal Health, São Paulo, Brazil) inserted (Day 0) and maintained for 6 days. On Day 5, the ewes received IM injections of 300 IU of eCG (Novormon®, Schering Plough Animal Health) and 0.0375 mg of cloprostenol (Prolise®, Tecnopec, São Paulo, Brazil). Twelve hours after sponge removal, 0.025 mg of gonadorelin acetate (Gestran®, Tecnopec) was administered IM. Superovulation started 60 h after sponge removal and consisted of 5 IU kg–1 of porcine FSH (pFSH; Pluset®, Hertape Calier, Minas Gerais, Brazil) IM in 6 decreasing doses (25, 25, 15, 15, 10, and 10%) at 12-h intervals. At the first pFSH dose, new sponges were inserted. At the fifth pFSH dose, 0.0375 mg of cloprostenol was administered IM and the sponges were removed. After the sponge removal, the NM group was exposed to rams twice per day for mating, until the end of estrus. In the AI group, estrus was detected using a teaser with the penis diverted. The females were permitted to be mounted twice per day until the end of estrus, and were inseminated with frozen–thawed semen 24 and 36 h after the end of superovulation. The follicular development and ovulation time were observed using real-time ultrasonography (8.0 MHz Pie Medical®, Aquila Vet, Tokyo, Japan) at 12-h intervals. For statistical analysis, a Student’s t-test was performed (5% significance level) using the BioEstat program. Results are presented as mean ± standard deviation. The time from sponge removal to onset of estrus and the duration of estrus did not differ between NM and AI groups (31.79 ± 5.94 v. 25.25 ± 10.38 h and 29.89 ± 11.54 v. 26.66 ± 8.67 h, respectively). The time from sponge removal to ovulation and from onset of estrus to the first ovulation were shorter (P < 0.001) in the NM group (32.11 ± 12.72 v. 56.48 ± 15.39 h and 8.61 ± 5.99 v. 32.25 ± 18.57 h, respectively). The time from sponge removal to ovulation in July and February was 56.07 ± 7.27 versus 56.83 ± 20.72 in the AI group and 29.54 ± 0.56 versus 33.83 ± 19.02 in the NM group, respectively, suggesting that the season of the year in a tropical region did not influence the ovulation time for each treatment. Possibly, the mechanical stimulation induced by the contact of the penis with the vagina fornix and by the accessory sex glands fluids in mating hastened the ovulation time in the NM group. The service can shorten the time of ovulation.

167 BLACK TEA DOES NOT INTERFERE IN THE NUMBER OF CORPORA LUTEA IN SUPEROVULATED RATS

The black tea originating from the Camellia sinensis plant is considered a healthy drink because it is associated at prevention of cancer, cardiovascular disease, and osteoporosis. It has been also stated that its catechins have antioxidative, antibacterial, and antitumoral activity. Despite many potential benefits of black tea consumption, the catechins present in black tea can significantly inhibit proliferation, steroidogenesis, and vascular endothelial growth factor production by swine granulosa cells. Based on these statements, the aim of this work was to verify the influence of black tea in the ovulation rate and the ovary weight in superovulated rats. This study was approved by the ethics committee of the university. Wistar rats were distributed in two groups: treated group (BT): 30 rats drinking only black tea (2.5%) ad libitum; control group (CG): 30 rats drinking water ad libitum. The experiment lasted for 3 months. Ten animals of each group were superovulated monthly with 150 UI kg–1 of eCG (Folligon®, Intervet Schering-Plough) and 150 UI kg–1 of hCG (Vetecor®, Hertape Calier) and sacrificed 48 h later. Ovaries were collected and weighted, and the corpora lutea were dissected and counted. The obtained data were submitted to the unpaired t-test with Welch’s correction, and the effect of time was assessed by ANOVA (P < 0.05). It was observed that the animal of both groups responded to the hormonal treatment because superovulation was noticed for the number of corpora lutea in both groups. The mean and standard deviation values for ovary weight (g) and corpora lutea number in both groups were as follows: first month (CG = 0.30 ± 0.10, 61.10 ± 10.94 and BT = 0.30 ± 0.07, 55.00 ± 23.03); second month (CG = 0.19 ± 0.06, 64.90 ± 27.77 and BT = 0.17 ± 0.05, 50.60 ± 19.35); and third month (CG = 0.21 ± 0.05, 62.30 ± 20.17 and BT = 0.19 ± 0.07, 48.6 ± 10.59), respectively. No difference was observed between the groups in the time points. The chronic consumption of black tea for 3 months does not interfere with ovulation aornd weight of the ovaries in superovulated rats. Financial support was provided by FAPESP 2010/20583-2 and 2010/20274-0.

Effect of hCG concentration on embryo recovery rate in a commercial equine embryo transfer program in Brazil

Injections were administered intramuscularly. Animals were split into five groups (n 1⁄4 25). G1: Acyclic recipients showing endometrial edema two days after treatment with 5.0 mg of estradiol benzoate (Estrogin , Farmavet, Sao Paulo, Brazil) were injected with 5.0 mL of P4LA300. G2: Cyclic mares showing one 35mm follicle and endometrial edema were treated with 2,000IU of hCG (Vetecor , Hertape Calier, Brazil) and 5.0 mL of P4LA300. Progesterone injections were discontinued at ET if an ovulation was detected. G3: Estrous recipients showing 25 mm follicles and endometrial edema were treated with 5.0 mL of P4LA300. G4: Diestrous recipients five to fourteen days post-ovulation, showing no endometrial edema and a visible corpus luteum were simultaneously treated with 5.0 mg of estradiol benzoate and dinoprost tromethamine (Lutalyse , Pfizer, Sao Paulo, Brazil). If considerable endometrial edema was detected two days after treatment, 5.0 mL of P4LA300 were administered. G5: Cyclic recipients whose ovulations were induced with 2.000 IU of hCG, serving as control. Progesterone-treated recipients received 5.0 mL of P4LA300 at ET, and, upon pregnancy diagnosis, were injected at fourteen-day intervals with 10.0 mL, until 120 days. Pregnancy at 15 days and loss rates at 120 days were statistically evaluated through Fisher's exact tests (P 0.05), respectively, 76.0% (19/25) and 10.5% (2/19) (G1); 68.0% (17/ 25) and 5.9% (1/17) (G2); 80.0% (20/25) and 10.0% (2/20) (G3); 60.9% (14/23) and 0% (0/14) (G4) and 60.0% (15/25) and 13.3% (2/15) (G5). In G2, 72.0% (18/25) showed a corpus luteum at ET, with a 72.2% (13/18) of pregnancy rate. Regardless of their estrous cycle phase or ovarian activity, recipients with either natural or pharmacologically induced endometrial edema, submitted to ET four days after treatment with long-acting progesterone, showed satisfactory pregnancy and embryo loss rates, allowing simple and effortless synchronization between donor and recipients.

410 ENHANCED OVARIAN RESPONSE TO LOW-DOSE TREATMENT WITH EQUINE PITUITARY EXTRACT IN MARES

The present study aimed to evaluate the ovarian response to relatively low doses of equine pituitary extract (EPE) involving 40 estrous cycles in 15 horse mares. The mares were Mangalarga Marchador and ranged in age from 5 to 24 years. Transrectal ultrasonography was used at 24-h intervals to monitor follicular development and to detect ovulations (Day 0). On Day 8, all mares received 7.5 mg i.m. of dinoprost tromethamine (Lutalyse®, Pfizer, Paulínia, Brasil) and were randomly assigned to treatment and control groups as follows: Groups 1 and 2 (n = 8 cycles/group) received 6 mg of EPE i.m. twice daily, beginning when the largest follicle was 20 to 23 mm (Group 1) or regardless of follicle size on Day 8 (Group 2); Groups 3 and 4 (n = 8 cycles/group) received 8 mg of EPE i.m. twice daily, beginning when the largest follicle was 20 to 23 mm (Group 3) or regardless of follicle size on Day 8 (Group 4); Group 5 (control; n = 8 cycles) received saline i.m. starting on Day 8. Treatments with EPE or saline continued until the largest follicle reached ≥35 mm, at which time a single dose of hCG (2500 IU i.v. Vetecor®, Hertape Calier Saúde Animal S/A, Juatuba, Brasil) was given. Groups were compared using ANOVA and mean differences among groups were located with ScottKnott test. There was an effect of group (P < 0.05) for number of follicles >30 mm, ovulations, and days of treatment as indicated in the table. In conclusion, starting EPE treatment with 8 mg (16 mg day-1) when the largest follicle reached 20 to 23 mm (Group 3) was the most effective for enhancing the number of >30 mm follicles and ovulations with minimal days of treatment. Table 1. FAPESP and JM Farm for funding the project.

405 LEVELS OF HORMONES AND NITRIC OXIDE PRESENT IN FOLLICULAR FLUID UNDER OR NOT UNDER SUPEROVULATION IN MARES

The aim of the present study was to compare the levels of 17β-estradiol, testosterone, progesterone, inibin, and nitric oxide in follicular fluid between superovulated mares (G1) using equine pituitary extract (EPE) and nonsuperovulated mares (G2) used as control. Two estrus cycles from 24 mares (G1 = 12; G2 = 12) were monitored. In G1, the first cycle was used to determine the ovulation day (Day 0), followed by the administration of PGF2 (250 μg daily of cloprostenol; Sincrocio™, Ouro Fino, São Paulo, Brazil) on the first and second day of treatment with EPE. During the second cycle, EPE (25 mg i.m.) was administered twice daily from Day 7 to the ultrasound detection of a majority of follicles at least 35 mm in diameter. At this time, 2500 IU of hCG IV (Vetecor™, Hertape Calier, Minas Gerais, Brazil) was given. The same protocol was used for G2 as was used for G1 except that EPE administration was replaced by saline injections. For follicular aspiration, the mares were sedated with xilazine (0.5 mg kg-1; Sedazine™, Fort Dodge, São Paulo, Brazil) and acepromazine (0.05 mg kg-1; Acepram 1%™, Univet, Milton, Ontario Canada); hioscin bromide (Buscofin™, Agener União, São Paulo, Brazil) was also administered to reduce intestinal motility and to make ovary manipulation by way of the rectum easier. All follicles larger than 35 mm were aspirated using a Cook® double lumen needle (12 GA) in a transvaginal aspiration probe guided by ultrasonography. The number of aspirated preovulatory follicles was 4.75 ± 2.7 and 1.0 ± 0.0 for G1 and G2, respectively. The follicular fluid collected was centrifuged at 600 × g for 10 min in order to separate the fluid from the cells. The fluid was stored at -20°C and sent to the University of California (Davis, USA) for hormonal assay. The nitric oxide dosage was determined at São Paulo State University (Botucatu, Brazil) using the luminescent reaction of Griess. The comparison between groups of hormonal levels and nitric oxide was performed using the t-test at 5% significance. The results of the present study showed that the EPE treatment (G1), under the experimental conditions, did not lead to a significant change in the mean concentrations of the evaluated hormones or of nitric oxide in the follicular fluid when compared with the levels in G2. However, differences were found among the individuals only in G1 in all hormones (P < 0.05), with exception of progesterone (P > 0.05). Table 1.Mean and standard error of the follicular fluid hormones and nitric oxide levels of groups G1 and G2 Supported by FAPESP, Brazil.