The IGF system is related to embryo quality. We aim to determine the effect of the heat stress on the mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4, and PAPPA in in vitro production (IVP) blastocysts from Nelore and Holstein after ovum pick up (OPU) to better understand the differences between these breeds. Oocytes from four Nelore and seven Holstein were collected in six OPU sessions. Following in vitro maturation and fertilization using six Nelore or Holstein sires, embryos were divided into control (cultured at 39°C) and heat stress (HS; exposed to 41°C for 9 h). Blastocysts were submitted to RNA extraction. The IGF1 expression was higher in blastocysts under HS in both breeds, and the expression of IGFBP2 and IGFBP4 was higher in Holstein blastocysts under HS. The high PAPPA expression and the low expression of IGFBP2 and IGFBP4 are associated with a more efficient degradation of IGFBPs, which results in greater IGF bioavailability in Nelore blastocysts and may contribute to the superior HS tolerance in Nelore, when compared to Holstein.
The aim of this experiment was to evaluate the effects of cycloxigenase inhibitor drugs, i.e. flunixin meglumine (FM) and parecoxib (P), on the development of bovine embryos cultured in vitro until the blastocyst and hatched blastocyst stages. Immature oocytes were aspirated from slaughterhouse ovaries and morphologically selected for IVM (Monteiro FM et al. 2007 Anim. Reprod. 4, 51–58). Twenty hours after maturation (39°C and 5% CO2 in air), matured oocytes were transferred to fertilization media, inseminated with frozen–thawed semen, and incubated for 10 to 12 h. Presumptive zygotes (PZ) were then transferred to TCM 199 HEPES medium, vortexed to remove cumulus cells and finally to drops of IVC media (SOFaaci plus 5% BFS [Gibco] with 13 mm sodium pyruvate). Each drop of IVC medium had appropriate concentrations of FM (0.14/n = 123; 1.4/n = 122; 14/n = 117; 140/n = 44 or 1400 μg mL–1/n = 44 PZ) or P (0.09/n = 134; 0.9/n = 109; 9/n = 118; 90/n = 113 or 900 μg mL–1/n = 45 PZ), besides extra drops as control groups (CFM; n = 124 and CP; n = 149 PZ). Based on published data from bovine (FM) and human (P) administered concentrations, it was calculated the blood concentration to a bovine weighing 450 kg (FM = 14 and P = 9 μg mL–1). Both drugs were used from available commercial preparations, and in a pilot test, there were no deleterious effects of the solvent itself on the blastocyst and hatched blastocyst rates. During culture, petri dishes containing PZ/embryos were kept into plastic bags, under controlled atmosphere of 5% O2, 5% CO2 and 90% N2 at 39°C. There were 11 replicates for each treatment. In all drops (both drug concentrations and control group) the blastocyst and hatched blastocyst rates (BR and HBR, respectively) were evaluated at 144 and 192 h after fertilization, respectively. Statistical analysis was performed with ANOVA on ranks (Dunn’s test a posteriori and significance being considered when P < 0.05; BioEstat version 5.0). According to the results, FM (1400 and 140 μg mL–1) and P (900 μg mL–1) concentrations were toxic enough for a complete inhibition of in vitro bovine embryo development. There were no significant differences among the other drug concentrations and their respective control group, on the BR (27.7 ± 3.9; 29.6 ± 3.4 and 29.8% ± 4.8) and HBR (13.5 ± 4.4; 15.6 ± 3.8 and 22.1% ± 5.1), respectively to 0.14; 1.4 and 14 μg mL–1 for FM; on BR (26.0 ± 2.6; 18.2 ± 4.6; 25.8 ± 5.9 and 23.2% ± 4.8) and HBR (14.1 ± 3.3; 10.2 ± 3.3; 16.8 ± 3.8 and 12.0% ± 3.4), respectively to 0.09; 0.9; 9 and 90 μg mL–1 for P; and on BR (35.3 ± 5.2 and 36.5% ± 3.4) and HBR (26.6 ± 4.5 and 19.8% ± 3.6), respectively for CFM and CP. The results suggest that, during in vitro bovine embryo culture, there was no significant toxicity of either drug, with exception of the complete lethal concentrations of 140 and 1400 μg mL–1 (flunixin meglumine) and 900 μg mL–1 (parecoxib) on blastocyst production. Supported by FAPESP – Brazil (MFGN 06/06491-2 and 07/07705-9; EMR 07/04284-2; RAS; CFS and RALS) and CAPES – Brazil.
There is evidence that IGF system can be involved in the oocyte competence and, consequently, in the embryo development. To better understand possible differences between Bos taurus and Bos indicus in in vitro embryo development, the current work aimed to assess the expression of IGF ligands (IGF-1 and 2), types 1 and 2 IGF receptors (IGFR1 and 2), IGF-binding proteins 2 and 4 (IGFBP-2 and 4), and type A pregnancy-associated plasmatic protein (PAPP-A) mRNA in bovine immature oocytes fromBos Taurus and Bos indicus. Nellore and Holstein cows were submitted to ovum pickup, and the oocytes with grade 1, 2, and 3 were selected. To remove cumulus cells and pellucida zone, respectively, the oocytes were submitted to vortex (900 × g for 3 min) and protease treatment (Protease® from Streptomyces griseus, Sigma-Aldrich Corporation, St. Louis, MO, USA). Pools of 20 oocytes obtained from Nellore (n = 8) and Holstein (n = 4) cows were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA, USA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophlin A (CYC-A) was used as internal control. The means of mRNA levels of target genes between the breeds were compared using t-test and Mann-Whitney test when the data had normal or not normal distribution, respectively. The means values of mRNA expression of IGF-1, IGF-2, IGF receptors (1 and 2), and IGFBP 2 and 4 were greater in Holstein (0.96 ± 0.21, 0.74 ± 0.27, 1.08 ± 0.04, 1.19 ± 0.5, 1.21 ± 0.23, 0.53 ± 0.15, respectively) compared with Nellore (0.48 ± 0.10, 0.07 ± 0.02, 0.04 ± 0.03, 0.06 ± 0.02, 0.05 ± 0.01, 0.03 ± 0.15, respectively; P < 0.01). Never- theless, mRNA expression of PAPP-A was much greater in Nellore (28.10 ± 18.96) than in Holstein (1.32 ± 0.17; P < 0.05). These results suggest that high expression of PAPP-A and low expression of IGFBP-2 and 4 could allow more efficiency on the degradation of IGFBP and increase the IGF bioavailability in the oocytes from Nellore as compared with Holstein cows. Satrapa, Simões, and Castilho received a fellowship and funding from FAPESP (São Paulo, Brazil).
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