2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A143 OPU-IVP AND ET IGF-I (INSULIN-LIKE GROWTH FACT CTOR I) INCREASES IN VITRO PRODUCTION, BUT DOES NOT PROTECT EMBRYOS FROM DELETERIOUS EFFECT OF HEAT STRESS IN NELORE (BOS INDICUS) AND HOLSTEIN (BOS TAUR URUS) US) BREEDS Anthony César de Souza Castilho 1 , Rafael Augusto Satrap 1 , Eduardo Montanari Razza 1 , Cintia Fernandes Silva 1 , Renato Simões 1 , Thais Nabhan 1 , Janahi Sousa Ticianelli 1 , Josmar Mazucheli 2 & Ciro Moraes Barros 1 1 UNESP, BOTUCATU, SP, BRAZIL. 2 UEM, MARINGÁ, PR, BRAZIL. To better understand the differences between taurine and zebu cattle about their resistance to heat stress (HS) we aimed to verify a) whether the addition of IGF-I (Insulin-like growth factor I) to the culture medium in the absence of fetal calf serum (FCS) would be able to maintain embryo production rates similar to those obtained in medium with FCS, b) whether the addition of IGF-I to the culture medium is more effective in decreasing the deleterious effects of HS in Holstein embryos (HOL) when compared to Nelore (NEL), and c) whether the deleterious effect of HS in embryonic development and in apoptosis rates is higher in HOL embryos when compared with the NEL. In experiment 1, NEL oocytes were matured, fertilized with NEL semen and, 10 h post insemination (10 hpi), embryos were randomly divided into four groups: FCS (FCS 5% + 0 ng/mL of IGF-I, n=165), IGF (FCS 0% + 100 ng/mL of IGF-I, n = 163); FCS + IGF (n=169) and control (0% FCS + 0 ng/mL of IGF- I, n = 168). We evaluated the rates of cleavage (Cli), morula (Mo), blastocyst (BL) and hatched blastocyst (HB). In experiments 2 and 3, HOL and NEL oocytes were matured, fertilized with semen from HOL and NEL bulls, respectively, and cultured until the BL stage. After 10 hpi embryos were randomly divided into four groups: control (cultured in absence of IGF at 39°C), HS (cultured in the absence of IGF and exposed to 41°C, for 9 h at 96 hpi, and then returning to 39°C), IGF (cultured in the presence of IGF at 39°C) and HS/IGF (IGF + HS). In experiment 2 were evaluated rates of Cli, Mo and BL and in experiment 3 we evaluated the apoptosis rate of embryos. Data were analyzed using SAS Proc GENMOD, and logistic regression and Binomial Negative-regression were performed. In experiment 1, the rate of BL was similar (P > 0.05) between groups FCS (40.6±3.4) and IGF (36.2±5.6). In experiment 2, the HS decreased BL rates, regardless of IGF treatment in Nelore (29% vs. 24%) and HOL (20% vs. 15%). The addition of IGF-I in the culture medium, regardless of HS increased (P < 0.05) the production rates of both BL in Nelore (23% vs. 29%) and HOL (15% vs. 21%). In experiment 3, HS, regardless of treatment with IGF, the rate of apoptosis was significantly increased in NEL (3.3±0.2 vs. 4.1±0.3) and especially in HOL (4.8±0,3 vs. 6.0±0.4). We conclude that: a) the addition of IGF-I to the culture medium improved embryo production in both breeds and can be used in replacement of FCS; b) for both breeds, the addition of IGF-I to the culture medium did not protect embryos from deleterious effect of HS; c) Holstein was not more sensitive the deleterious effects of HS than Nelore, in both breeds there was reduction in the blastocyst production and higher incidence of apoptosis after HS. Keywords: igf-I, heat stress, bovine. A144 OPU-IVP AND ET TREATED TED OOCYTES WITH RETINOIDS AND WITH RETINOIDS AND GROW TH FACT CTOR IMPROVE THE IN VITRO EMBRYOUS PRODUCTION AND INHIBIT THE APOPTOSIS? Ricardo de Macedo Chaves 1 , Juliana Costa Conceição 2 , Madson Atila Vidal Silva 3 , Edivaldo Rosas dos Santos Júnior 4 , Leopoldo Mayer Freitas Neto 5 , Fabíola Freitas de Paula-Lopes 6 , Paulo Bayard Dias Gonçalves 7 , Paulo Fernandes Lima 8 & Marcos Antonio Lemos de Oliveira 9 1,3 UEMA, SÃO LUIS, MA, BRAZIL. 2,4,6,9 UFRPE, RECIFE, PE, BRAZIL. 5 FACULDADE PIO DÉCIMO, ARACAJU, SE, BRAZIL. 7 UNIVERSIDADE FEDERAL DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 8 UNIVERSIDADE FEDERAL DE SANTA MARIA, SANTA MARIA, RS, BRAZIL. The objective was to evaluate in vitro the effect of adding retinol (RT) and retinoic acid (RA) in half and maturation of oocytes of both retinoids and IGF-1 in the middle of developing goat embryos to foster blastocyst production in vitro and inhibit apoptosis measured by enzyme activity of group II caspases and DNA fragmentation by TUNEL assay. Ovaries were obtained from a slaughterhouse and transported to the laboratory in warm saline at 30°C. The oocytes (n = 4320) were collected from follicles measuring 2-6 mm in diameter by aspiration with a syringe needle attached. The oocytes, tested in 10 replicates, were initially washed in medium itself, then screened and distributed in droplets of TCM 199 supplemented with RT or with RA, before finally being incubated at 39°C in a humidified atmosphere containing 5% CO2 for 24 h. Thereafter, oocytes were processed through mDM and exposed to sperm for 18 h in the same atmospheric conditions mentioned. Subsequently, presumptive zygotes were placed in droplets of KSOM medium supplemented with RT (G1, 2, 3, 7 and 8), RA (G4, 5, 6, 9 and 10), or IGF- I (G2 and 5) alone or in association with each other (G1, 3, 4, 6, 7, 8, 9 and 10), beyond the monolayer of cells of the oviduct. Finally they were incubated at 39 ° C in humid atmosphere with 5% CO2 during ten days. At the end of the 10th day of co-culture blastocysts were prepared for localization of the changes characteristic of apoptosis, using the reagent PhiPhiLux-G1D2 for determination of enzyme activity caspases group II and solutions of 4% paraformaldehyde and Phosphate -Buffered Saline + 1 mg/mL of polyvinylpyrrolidone to determine the number of blastomeres positive for apoptosis by TUNEL reaction test (DNA fragmentation). For statistical analysis, variance analysis was performed by the method of least squares. The results showed that the groups of oocytes and embryos treated only with retinoids produced fewer blastocysts (P < 0.05) than in groups of oocytes and embryos treated with retinoids associated with IGF-I. As to the blastocyst positive for apoptosis, is assessed by the activity of enzymes caspazes or DNA fragmentation, there was no difference (P > 0.05) between groups. The results suggest that the combination of retinoids with IGF-I is beneficial and can be recommended to maximize production in vitro blastocyst goats, but does not inhibit apoptosis. Keywords: retinoids, apoptosis, blastocyst. s408
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A145 OPU-IVP AND ET GENE EXPRESSION PROFILE OF IGF SYSTEM STEM MEMBERS ON IN VITRO PRODUCED BOVINE BLAST ASTOCY OCYST STS: S: COMP OMPARISON BETWEEN NELORE ( (BOS INDICUS) ) AND HOLSTEIN ( (BOS TAUR URUS US) Rafael Augusto Satrapa 1 , Anthony César de Souza Castilho 2 , Renato Simões 3 , Eduardo Montanari Razza 4 , Cintia Fernandes Silva 5 , Thais Nabhan 6 , Marcelo Pegorer 7 , Raquel Zanetti Puelker 8 , Henrique Eduardo 9 , José Buratini Junior 10 & Ciro Moraes Barros 11 1,2,3,4,5,6,8,11 DEPARTAMENTO DE FARMACOLOGIA, INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU (IBB), BOTUCATU, SP, BRAZIL. 7 DEPARTAMENTO DE REPRODUÇÃO ANIMAL - FMVZ, UNIVERSIDADE ESTADUAL PAULISTA (UNESP), BOTUCATU, SP, BRAZIL. 9 VETERINÁRIO AUTÔNOMO, BOTUCATU, SP, BRAZIL. 10 DEPARTAMENTO DE FISIOLOGIA, INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU (IBB), BOTUCATU, SP, BRAZIL. Evidences indicate that the IGF system is related to embryo quality. To better understand the differences on in vitro embryo development of Zebu and European cattle, we aim to determine the effect of heat stress on the pattern of mRNA expression of IGF-I and II, IGFR-I and II, IGFBP-2 and 4 and PAPP-A in blastocysts from Nelore (NEL) and Holstein (HOL) breeds. NEL and HOL oocytes were aspirated by OPU, matured in TCM199, fertilized with semen from NEL (n = 6) and HOL (n = 6) bulls, respectively, and cultured in SOF medium (synthetic oviduct fluid, without FCS) to the blastocyst stage. Ninety-six h post-insemination (96 hpi) embryos = 16 cells were randomly separated in two groups: control and Heat Stress (HS). Embryos from control group were continuously cultured at 39°C and embryos from HS group were exposed to 41°C for 9 h, and returned to 39°C afterwards. Embryos at 168 hpi were removed from culture medium and allocated into five pools with five embryos in each experimental group, which were subjected to RNA extraction and stocked at -80°C. The expression of target genes was performed by RT-PCR in real time with oligo-dT in reverse transcription and specific primers for the bovine species. Expression of cyclophilin-A was used as endogenous control. The means of mRNA levels of target genes were compared using T-test or Man-Whitney test whether or not data were normally distributed. The mean values of IGF-I expression were higher (P < 0.05) in blastocysts undergoing HS when compared to control group, in both NEL (22.0±11.0 vs. 0.2±0.01, respectively) and HOL (8.0±5.0 vs. 1.0±0.3), and this increase was more evident in Nelore. Moreover, relative values of gene expressions of IGFBP-2 and 4 were higher (P < 0.05) in HOL blastocysts pools undergone HS (110.0±80.0 and 6.0±1.0, respectively) when compared to NEL undergone HS (2.0±0.1 and 0.1±0.01) or not (0.2±0.01 and 0.1±0.01) and HOL control (4.0±0.1 and 0.1±0.02). The higher IGF bioavailability verified in NEL blastocysts under HS can contribute to a greater tolerance of this breed to the HS when compared to HOL. [We acknowledge FAPESP for the financial support]. Keywords: igf, gene expression, bovine. A146 OPU-IVP AND ET POTENTIAL OF THE GYR (BOS TAUR URUS US INDICUS) AND HOLSTEIN (BOS TAUR URUS US TAUR URUS) US) DONORS FOR EMBRYO PRODUCTION J oão Henr enrique Mor oreir eira V iana 1 , Ta timara a Mar aria Miy iyauchi 2 , To chimara a Apar parecida Miy iyauchi 3 , Eduar duardo do Ramos de Oliv liveir eira 4 , Jose Antonio D. Garcia 5 , Marilu Martins Gioso 6 , Carlos Antônio de Carvalho Fernandes 7 & Miller Pereira Palhão 8 1 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2,4 BIOTRAN, ALFENAS, MG, BRAZIL. 3,5,6,7,8 UNIFENAS, ALFENAS, MG, BRAZIL. N The ovarian follicle pick-up (OPU) and oocyte retrieval are critical steps for IVEP from living donors. Recently, variability in oocytes recovery has been studied in Gyr breed and it seems to be related to intrinsic potential of the donor (Oliveira et al., XXVI World Buiatrics Congress, 2010). The objective was to evaluate the potential for oocyte retrieval in two important breeds of dairy cows. Results from 245 OPU sections in Gyr (n = 132) and Holstein (n = 113) breeds were included for this purpose. Five days before OPU, the ovarian follicular wave was synchronized with auricular implant (norgestomet - Crestar ® - Intervet Schering- Plough Animal Health) and intramuscular injections of 2 mg of estradiol benzoate (2 mL of Gonadiol ® - Intervet Schering- Plough Animal Health) and 0,250 mg of D-cloprostenol (Veteglan ® - Hertape Calier Saúde Animal S.A). The OPU sections were separated in quartiles regarding the total oocytes recovered within each breed. Thereafter, the oocytes were classified according to the degree of quality and considered viable or not viable. Sex-sorted (X) semen was used for in vitro fertilization. Data from recovered oocytes (total and viable) and embryo produced in each quartile were submitted to one-way ANOVA and means between breeds were compared by “t” test (P < 0.05). The oocytes recovered (total and viable) were greater (P < 0.0003 and P < 0.0001) in OPU sections from Gyr compared to Holstein donors (34.3±9.9 vs. 25.2±7.5 oocytes and 27.8±9.5 vs. 18.4±7.0 viable, respectively, for the first quartile,). These differences were kept in OPU sections assigned in the 2nd e 3rd quartiles. However, the sections below the 3rd quartile did not change (P < 0.3 and P < 0.2) between Gyr and Holstein (5.5±2.8 vs. 4.8±2.0 oocytes and 4.0±2.2 vs. 3.5±2.0 viable, respectively). The OPU sessions with higher number of the recovered oocytes (1st quartile) confirmed the highest production of embryo, and did not differ (P < 0.6) between breeds (7.6±6.3 vs. 5.9±4.5 embryos, respectively, for Gyr vs. Holstein). However, for the other quartiles (2nd, 3rd and the remained OPU sections), the embryo production was greater in Gyr compared to Holstein donors (5.4±4.0 vs. 2.0±1.9, 3.1±2.0 vs. 1.6±1.5 and 1.6±1.3 vs. 0.9±1.2 embryos, respectively). In the 1st quartile, the conversion rate of the viable oocytes to embryo was greater (P < 0.06) in Holstein (32.1%, 154/479) than in Gyr breed (27.3%, 243/890). Previous studies have shown the importance on selecting Gyr donors using the total oocytes recovered. The results suggest that the same principles could be useful to select Holstein donors. [Support: FAPEMIG Projeto CVZ AQP 01654/09, Embrapa Projeto Rede de Inovação em Reprodução Animal (01.07.01.002)]. Keywords: dairy cattle, opu, ivep. s409
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