154 Exposure of bulls to heat stress had deleterious effects on embryo development
Authors: Llamas Luceño N. , Van Poucke M. , Batlle Perez M. , Szymanska K. J. , Angrimani D. , Van Soom A. 
Published: December 2018

Concerns about global climate change reducing animal fertility are arising. Therefore, our objective was to determine the effects of increased environmental temperature on Holstein bulls and its effects on sperm quality and embryo development. Frozen semen samples were obtained from 6 bulls exposed to natural heat stress (HS) in August 2016, compared with a lower temperature (control) in March 2016 (temperature-humidity index of up to 74.5 and 40.6, respectively). We evaluated sperm morphology, embryo development, gene expression, inner cell mass/trophectoderm ratio (ICM/TE), and apoptosis cell ratio of Day-8 blastocysts. Sperm morphology was evaluated using eosin/nigrosin staining. Blastocysts were produced by routine in vitro methods (Wydooghe et al. 2014 Reprod. Fertil. Dev.). Cleavage rates were determined 48h after insemination, and blastocyst rates were determined on Days 7 and 8. Expression of NANOG, SOX2, POU5F1, DNMT (1, 3a, 3b), HSP (A1a, A2, A8, 10, 60, 90), HSF1, IFNT2, H19, SNRPN, IGF2, IGF2R, MEST, PHLDA2, MEG3, MEG9, PEG10 and PLAGL1 were analysed. Total RNA was extracted from Day-8 blastocysts for gene expression analysis using RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Reverse transcription and qPCR were performed with iScript (BioRad, Hercules, CA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) in a CFX Connect system (BioRad). Blastocysts were differentially stained (Wydooghe et al. 2011 Anal. Biochem.) and analysed using a Leica TCS-SP8×confocal microscope (Leica Microsystems, Wetzlar, Germany). Data analyses included a GLM procedure and paired-samples Student’s t test (P ≤ 0.05). A normal distribution was verified with a UNIVARIATE procedure and Shapiro-Wilk test. A Wilcoxon signed rank test was used to analyse qPCR data. Detached heads (P=0.006) and coiled tails (P=0.018) were significantly lower in the HS group (4.9 and 0.2%, respectively) compared with the control (5.5 and 0.5%). Moreover, proximal droplets (P=0.051) were lower in the HS group (0.7%) compared with the control (1.3%). Remarkably, cleavage and blastocyst rates at Days 7 and 8 were significantly higher in control (78.4, 19.6 and 29.5%, respectively) compared with HS (75, 14.5 and 23.2%). Early and normal blastocysts were grouped as early stage, whereas expanded, hatching and hatched blastocysts were grouped as advanced stage. There was a significant reduction in the HS group of early stage blastocysts on Day 7 and of advanced stage blastocysts on Days 7 and 8. However, in Day-8 blastocysts, there was no significant difference in gene expression for any target gene. Moreover, there were no significant differences in total number of cells or apoptosis cell ratio in blastocysts. However, the ICM/TE ratio was significantly higher (P=0.021) in control (0.7) compared with HS (0.56). Sperm samples collected in August had reduced fertility compared with those obtained in March. Although fewer sperm abnormalities were present in HS, based on decreased blastocyst rates and ICM/TE ratio in embryos produced with HS semen, we inferred that molecular mechanisms for advanced blastocyst development were affected. However, those mechanisms did not involve our target genes.
This work was funded by the European Union, Horizon 2020 Marie Sklodowska-Curie Action, REPBIOTECH 675526.