11 PREGNANCY RATES AND ATP1A1 POLYMORPHISM IN THERMOTOLERANT HOLSTEIN COWS DURING SUMMER IN BRAZIL

Maximum body temperatures (BT) registered in dairy cows have been used as an indicator of heat stress (HS). However, some studies have shown that the variation in BT, which represents energy requirements for BT regulation, is more representative of HS. Cows that show less variation in BT can be considered thermotolerant. The ATP1A1 gene is critical for maintaining homeostasis through the fluctuation of plasma K+ and Na+. Thus, this experiment was designed to (1) test the correlation between the coefficient of variation (CV) in BT and pregnancy rate after artificial insemination (AI) in the summer, and (2) identify polymorphisms in the ATP1A1 gene. Seventy lactating Holstein cows kept in a freestall system were used. To measure BT, an automatic thermometer coupled to a placebo intravaginal implant was kept in the cows, for 3 days, measuring the temperature every 5 min. The temperature CV for each animal was calculated as the ratio of the standard deviation to the mean. Each animal’s daily milk production was registered on the day of implant insertion. The animals used in the experiment were AI without hormonal synchronization, 12 h after presenting oestrus. Semen from Holstein and Gir breeds were used. After 30 days, pregnancy was diagnosed using an ultrasound. Animals were re-inseminated as they showed oestrus until they became pregnant. The number of AI necessary for each animal to become pregnant was calculated. DNA was extracted from tail hair from sixty cows used in the experiment. For polymorphism detection, primers from a fragment of the ATP1A1 gene (forward 5′-AGTGCTGCGTGAAACCTG-3′, reverse 5′-GTGATGTGTGGAATGGTGC-3′) were used. PCR reactions were performed and PCR products were electrophoresed on 1% agarose gel for visualisation of the fragment sizes. Then, the PCR products were purified and sequenced. The obtained sequences were analysed and edited using the BioEdit Software (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Sequence alignment was performed using the virtual tool Clustal Omega. To exclude the effect of sire breed on pregnancy rate a logistic regression analysis using Proc Logistic (SAS for Windows, version 9.2; SAS Institute Inc., Cary, NC, USA) was performed. The correlation between temperature CV, milk production, and number of AI, and means and standard deviation were analysed by Pearson correlation coefficient using the Proc Corr of SAS. Mean milk production was 30.21 ± 7.4. Mean temperature CV was 1.32 ± 0.48. Mean AI was 1.97 ± 1.05. Results showed a positive moderate correlation between temperature CV and AI (0.33; P < 0.004). There was no correlation between milk production and CV or milk production and AI. One polymorphism, a G-A mutation, at nucleotide position 116,400,962 (exon 19) of the gene mRNA was identified in 5 animals (temperature CV: 1.04, 1.05, 1.24, 1.32, 1.41). This mutation represents an alteration from the amino acid aspartate to asparagine. In conclusion, animals that show less fluctuation in body temperature can present better pregnancy rates after AI. One novel polymorphism was identified but more studies are needed to associate it with thermoregulation.