Juliana Polisseni

Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis.

OBJECTIVE To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres. DESIGN Randomized study. SETTING Molecular genetics and animal reproduction laboratories. PATIENT(S) Cow ovaries obtained from slaughterhouses. INTERVENTION(S) The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development. MAIN OUTCOME MEASURE(S) Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination. RESULT(S) A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR. CONCLUSION(S) Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres.

Efeito da somatotropina na população folicular, recuperação de oócitos e produção in vitro de embriões em vacas Gir

The aim of this study was to evaluate ovarian pre-stimulation using recombinant bovine somatotropin (rbST) or rbST associated to FSH on follicular population, oocyte retrieval and in vitro embryo production in Gir cows (Bos indicus). Estrous cycle of non-lactating cycling cows with similar body and reproductive conditions were synchronized with cloprostenol followed by norgestomet ear implants, replaced every fourteen days. The animals were randomly distributed in three treatments: TI or control (punctured without pre-stimulation), TII (injection of 160 mg of rbST, before each aspiration) and TIII (injection of 160 mg of rbST followed by administration of 250 U.I. of FSH, before each aspiration). Oocytes were maturated and fertilized in vitro and presumptive zygotes were cultured in vitro for 192 hours post-fertilization. Treatments did not differ for ovarian follicular population. However, an increase in the diameter of the largest follicle and in the number of large and medium follicles with reduction in the number of small follicles was observed for TIII. Oocyte recovery did not differ between TI and TIII, but recovery rate was lower for TII. The number of grade I oocytes was higher and the number of degenerated oocytes was lower for TIII than for TI or TII. The rbST increased cleavage rate and blastocyst production, and when associated to FSH the quality of oocyte in Gir animals was improved.

280 EXPRESSION OF HSP70.1 GENE IN IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN CR2 MEDIUM SUPPLEMENTED WITH KNOCKOUT™SR

The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5°C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy® Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism® 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR® Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 ± 0.06- and 1.4 ± 0.08-fold differences relative to the PVA group without differences (P > 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions. This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vicosa, Brazil, for the real-time PCR machine.

318 EFFECT OF MATERNAL HEAT STRESS ON OOCYTE QUALITY AND IN VITRO COMPETENCE IN BOS INDICUS CATTLE

High temperatures can be harmful to the competence of cumulus–oocyte complexes and to embryo development (Al-Katanani et al. 2002 J. Dairy Sci. 85, 390–396). The aim of this study was to evaluate the effect of maternal heat stress on in vitro embryo yield. Ten multiparous nonlactating Gir (Bos indicus) cows were kept in tie stalls for an adaptive period of 28 days [pre-heat-stress period (PRE-HS)/Days -28 to -1]. Cows were subjected to 2 OPU (ovum pickup) sessions (Days -14 and -7). In the heat-stress period (HS; Days 0 to 28), cows were divided into control (C: n = 5) and heat-stressed (HS: n = 5) groups. During this period, OPU sessions were performed once a week from Days 0 to 28. The C group remained in a thermo-neutral environment, and the HS group was kept in a climatic chamber with controlled temperature and humidity (38°C and 80% during the day and 30°C and 80% during the night). In the post-heat-stress period (POST-HS; Days 28 to 147), all cows returned to thermo-neutral conditions. Then 17 OPU sessions were performed once a week from Days 35 to 147. In all periods, blood samples were collected weekly for progesterone (P4) analysis, and ovarian follicles were counted, measured, and aspirated. The COCs were evaluated and selected for the IVF procedure. Data were analyzed by ANOVA (PROC MIXED of SAS) and a chi-squared test. The luteal phase was defined as the period between 2 samples with P4 below 1.0 ng mL-1. A handling accident caused the exclusion of an HS cow after the sixth session. The C and HS groups were subjected to 125 and 107 OPU sessions, respectively. Means ± SEM for the C vs. HS groups, in the PRE-HS, HS, and POST-HS periods, respectively, were visualized follicles: 25.5 ± 2.5 vs. 28.5 ± 2.8, 24.2 ± 1.1 vs. 24.0 ± 1.9, and 15.3 ± 0.6 vs. 15.8 ± 0.8; largest follicle diameter: 12.1 ± 1.5 vs. 11.1 ± 1.7, 13.3 ± 0.8 vs. 13.0 ± 0.6, and 11.4 ± 0.4b vs. 14.0 ± 0.4a; P < 0.05; 2nd largest follicle diameter: 6.2 ± 1.3 vs. 6.0 ± 1.2, 5.9 ± 0.6 vs. 7.1 ± 0.8, and 6.3 ± 0.3b vs. 8.7 ± 0.5a; recovered COCs: 11.2 ± 2.8 vs. 14.3 ± 2.5, 9.6 ± 1.0 vs. 11.0 ± 1.3, and 8.6 ± 0.7 vs. 7.9 ± 0.6; COCs selected for IVF: 69/112 (61.6%)b vs. 108/143 (75.5%)a, 164/241 (68%) vs. 172/265 (64.9%), and 426/712 (59.8%) vs. 305/535 (75.0%); cleavage: 44/59 (74.5%) vs. 87/105 (82.9%), 72/101 (71.3%) vs. 74/121 (61.2%), and 226/317 (71.3%) vs. 159/230 (69.1%); embryos per cow/OPU: 2.1 ± 1.1y vs. 4.1 ± 1.0x, 0.4 ± 0.3 vs. 0.5 ± 0.3, and 0.9 ± 0.2x vs. 0.4 ± 0.1y; P < 0.1; and blastocyst yield: 16/59 (27.1%) vs. 33/105 (31.5%), 11/31 (35.5%) vs. 13/52 (25.0%), and 76/279 (27.2%)a vs. 25/188 (13.3%)b. In conclusion, maternal heat stress increased the percentage of short estrous cycles, decreased the P4 concentrations, and decreased the number of embryos produced by Bos indicus cows, mainly from 28 to 147 days post-heat-stress, showing long-term deleterious effects on blastocyst development.

Efeito do transporte no desenvolvimento de embriões bovinos cultivados in vitro a fresco ou reaquecidos após vitrificação

Avaliou-se a viabilidade de embrioes bovinos cultivados in vitro, a fresco ou reaquecidos apos vitrificacao, depois de transportados por 6 ou 12 horas. Oocitos obtidos de foliculos de ovarios coletados em matadouro foram maturados, fecundados e cultivados in vitro. Apos sete dias de cultivo, blastocistos com grau de qualidade I e II (segundo o manual da IETS-1998) foram selecionados, envasados em OPS (open pulled straws) e vitrificados em nitrogenio liquido. O reaquecimento foi realizado a 39oC pela passagem em solucoes de HM com concentracoes decrescentes de sacarose (0,25M - 0,15M) por cinco minutos em cada solucao. Foram avaliados tres tratamentos - V0: embrioes vitrificados, reaquecidos e cultivados in vitro (n=25); V6: embrioes vitrificados, transportados por 6 horas (simulacao em palhetas), reaquecidos e cultivados in vitro (n=29); e V12: embrioes vitrificados, transportados por 12 horas, reaquecidos e cultivados in vitro - comparados, cada um, a um tratamento controle, com embrioes a fresco-C0: embrioes a fresco cultivados in vitro (n=26); C6: embrioes a fresco cultivados in vitro apos 6 horas de transporte (n=30); e C12: embrioes a fresco cultivados in vitro apos 12 horas de transporte (n=30). Os embrioes foram co-cultivados com celulas da granulosa em microgotas de TCM 199 acrescido de SFB. Foram avaliadas as taxas de re-expansao e eclosao apos 48 horas de cultivo. A analise foi realizada pelo teste do qui-quadrado. As taxas de re-expansao entre os grupos V0, V6 e V12 nao diferiram, assim como as taxas de eclosao entre os embrioes vitrificados e os controles. As taxas de eclosao, no entanto, diferiram entre os embrioes submetidos a vitrificacao e os controles. Embrioes bovinos produzidos in vitro podem ser transportados a fresco ou vitrificados por periodos de ate 12 horas, pois possibilitam taxas de eclosao satisfatorias.