Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos.

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Peritoneal fluid characteristics in normal horses after percutaneous cecal trocharization RESUMO

There is evidence that deleterious effects of heat shock (HS) on fertility are less pronounced in breeds tolerant to high temperatures, due mainly to differences in their thermoregulatory capacity. In vitro experiments have shown that Bos indicus embryos are more resistant to HS than Bos taurus. In order to better understand the differences related to HS resistance between Bos indicus and Bos taurus, the main objective of this study was to determine if tolerance to HS is caused by genetic contribution from the oocyte, spermatozoa, or both. Additionally, the influence of the time between collection of ovaries in the abattoir and oocyte aspiration in the laboratory on early embryo development was ascertained. In experiment 1, oocytes from Nellore and crossbreed Holstein cows (cHOL) were collected in a local abattoir, matured and fertilized using semen (n = 6 for each breed) from Nellore (NEL), Angus (ANG), Brahman (BRA,) and Gir (GIR) bulls. In experiment 2, oocytes from Nellore and Holstein (HOL) cows were collected in an abattoir and the oocytes were aspirated in the laboratory 4 (group 4 h) or 6.5 h (group 6.5 h) later, matured and fertilized using semen (n = 6 for each breed) from NEL, GIR, and HOL. In both experiments, 96 h post-insemination (hpi), embryos with > 16 cells were separated in 2 groups: control and HS. In the control group the embryos were cultured at 39°C, whereas in the HS group the embryos were submitted to 41°C for 12 h, and then returned to 39°C. In experiments 1 and 2 the results were analyzed by ANOVA (Proc MIXED, SAS Institute, Cary, NC, USA). In experiment 1, there was no effect of HS on blastocyst and hatched blastocyst rates in all breeds studied. The percentage of oocytes that cleaved and reached the morula stage was significantly lower (P < 0.05) in cHOL × GIR compared with the other breeds. Additionally, blastocyst rate was higher in cHOL × NEL than in cHOL × ANG and cHOL × GIR (P < 0.05). In experiment 2, cleavage, morula, and blastocyst rates in group 4 h were higher (P < 0.05) compared with group 6.5 h. The HS decreased blastocyst rates in all breeds (NEL × NEL, HOL × HOL, and HOL × GIR), and in both time intervals (4 and 6.5 h). The breed NEL × NEL had higher cleavage rate (P < 0.05) for both time intervals compared with HOL × HOL and HOL × GIR. In addition, Nellore oocytes fertilized with Nellore semen (NEL × NEL) originated higher blastocyst rates (P < 0.05) in control and HS group than the other breeds. We conclude that (a) embryos from Holstein are more susceptible to HS than embryos from crossbred Holstein; (b) the oocyte is more important than the spermatozoa for the development of thermotolerance, because the breed of the bull did not influence embryo development after HS; (c) in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the time interval between ovary collection and oocyte aspiration. Fellowships to T. Nabhan from CAPES and to R. A. Satrapa, R. A. L. Simoes, and E. M. Razza from FAPESP. Funding from FAPESP (Sao Paulo, Brazil).

Several factors affect early embryonic development in cattle, including heat stress. These factors can contribute to high early embryonic loss, probably altering gene expression. Studies using microarray-profiled genome-wide RNA expression for in vitro-produced blastocysts have compared embryos resulting in calf delivery or no pregnancy, and they have identified genes with potential roles in pregnancy and embryo competence. The aim of the present work was to compare the expression of some genes (PLAC8, HSF1, COX-2, and CDX-2) related to embryo competence and embryonic implantation between in vitro-produced embryos from the Nelore breed (Bos indicus), submitted or not submitted to heat stress. Oocytes from Nelore cows were aspirated by ovum pickup and matured for 22 h (TCM-199 with bicarbonate, supplemented with 10% FCS, 2 μL mL–1 of pyruvate, 75 μg mL–1 of amicacin, 20 μg mL–1 of FSH, and 2 IU mL–1 of hCG) at 38.5°C with 5% CO2 in air. The fertilization (Day 0) was performed with semen from Nellore bulls. After a 12-h fertilization period, in Tyrode’s lactate stock medium supplemented with 6 mg mL–1 of BSA, 2 mL mL–1 of pyruvate, 75 mg mL–1 of amicacin, 11 mg mL–1 of heparin, and 44 mL mL–1 of phenylalanine solution, presumptive zygotes were denuded and randomly divided in 2 groups: nonstressed and stressed. The culture medium was SOFaaci supplemented with sodium pyruvate (0, 2%), 5 mg mL–1 of BSA and 5% FCS. Embryo culture was performed at 38.5°C, 90% N2, 5% CO2, and 5% O2. In the stressed group, 96 h after fertilization, the embryos were subjected to heat stress of 41°C for 6 consecutive hours and then returned to a temperature of 38.5°C. On Day 7, pools of 5 blastocysts (nonstressed, n = 9; stressed, n = 7) were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the reverse transcription and bovine-specific primers in the PCR. Expression of cyclophlin A was used as an internal control. The means of mRNA levels of target genes between the groups were compared by t-test. The PLAC8 mRNA levels were higher in nonstressed blastocysts in comparison with the stressed group. The HSF1 and CDX2 mRNA was detectable only in nonstressed embryos. The COX2 mRNA levels did not differ between groups. The higher levels of PLAC8 and the CDX2 expression on nonstressed embryos indicate better competence of embryos not submitted to heat stress. Furthermore, the absence of HSF1 mRNA in the stressed embryos does not reflect the lack of biological activity of this protein. In conclusion, the data indicate that heat stress alters the gene expression pattern of in vitro-produced embryos in the Nelore breed. FAPESP (São Paulo, Brazil) is acknowledged for funding and fellowships for Castilho, Satrapa, and Razza.

In vitro-produced embryos (IVP) are known to have poor quality and be susceptible to heat and oxidative stress. In addition, they constantly undergo long-distance transport, which causes low conception rates. The aim of this study was to search for alternatives to long-distances transport of embryos produced in vitro by evaluating the use of different media and different temperatures. Bos indicus cumulus–oocyte complexes (COC; 823, quality I and II) were matured in TCM-199 bicarbonate–10% FBS (38.5°C, 5% CO2, in air) for 24 h. The fertilization was performed in TALP-IVF medium for 18 h of incubation. Presumptive zygotes were transferred to SOF medium and in vitro culture in a controlled atmosphere (5% O2, 5% CO2, 90% N2) for 7 days at 38.5°C. At Day 7, 366 embryos (quality I and II, IETS), were randomly allocated to the designated experimental groups. For Experiment 1, blastocysts were filled into straws and kept for 12 h at 36°C, using the medium as an independent variable according to the following groups: GSup, embryo support medium without FCS and amino acids (n = 115); and GHSOF, embryo culture medium SOF (HSOF) with FCS and amino acids (n = 105). Both media were buffered with HEPES. For Experiment 2, blastocysts were filled into straws with HSOF medium and sustained in transportation for 12 h, using temperature as the independent variable according to the following groups: G36, 36°C (n = 65); and G38, 38°C (n = 81). After 12 h of transport in both experiments, embryos were evaluated and classified as viable or nonviable blastocysts and were recultured in the same conditions mentioned. On Day 10, hatching rates and degeneration were evaluated. Logistic regression was used to compare the groups in each experiment. In Experiment 1, blastocyst viability after 12 h of transport was higher in the culture medium (GHSOF: 91.4%) than the support medium (GSup: 75.6%; P < 0.001). The hatching capacity in the culture medium (GHSOF: 72.4%) was higher than that in the support medium (GSup: 34.5%; P < 0.001). In Experiment 2, blastocyst viability post-transport was the same in both temperatures (G36: 92.3%, G38: 82.7%; P > 0.09). Blastocyst hatching capacity at 36°C (G36: 75.4%) was superior to that at 38°C (G38: 49%; P < 0.001). The hatching capacity immediately after 12 h of transport was minimal, so it was not possible to apply the logistic regression method in any experiment. In conclusion, the culture medium was more efficient for long-distance transport than the support medium; moreover, a temperature of 36°C for the culture medium increased embryo development compared with a temperature of 38°C during transport. Financial support was provided by CESUMAR.