Study On Thermotolerance And Heat Stress Resistant Methodology As Well As Thermotolerant Gene Identification In Buffalo

Heat stress has a detrimental effect on animal growth and development,particularly the reduction of productive performance of these species.Water buffaloes are typically adjusted to the hot/humid climates.However,they still feel great distress if exposed to direct solar radiation or working in the sunlight during hot weather,thereby affecting their physiological and production performance,such as increased respiratory rate,elevated rectal temperature,reproductive and lactation traits disorders and so on.Three mitigation strategies can be recently used for combating the adverse effects of summer heat stress in animals,such as physical modification of the environment,development of genetically thermotolerant breeds and nutritional modification.Notably,breeding for thermotolerance was the most cost-effective measure for mitigating heat stress.The traditional breeding methods for thermotolerant cultivars have progressed slowly;the emerging high-throughput sequencing technologies have provided new directions for studying heat-resistant traits.Thus,in the study we firstly explored the ability of heat tolerance in buffaloes,and secondly screened and identified candidate genes affecting thermotolerance performance using high-throughput sequencing technology,and finally evaluated the thermotolerance of sprinkled buffaloes,aiming to provide theoretical basis and technical support for improving the ability of thermotolerance in buffaloes and accelerating the breeding of high-yielding and thermotolerant breeds.1.Study on Thermotolerance and its factors in Buffaloes(1)Thermotolerance in the BuffaloesIn the study we compared the physiological,production and blood parameters of crossbred buffaloes in winter,spring,and winter.Results showed that the rectal temperature(39.24 ± 0.07°C),respiratory rate(69.74 ± 3.82breaths/min),body surface temperature(38.01 ± 0.09°C),plasma HSP70(421.28 ± 53.11pg/mL)and HSP90(3348.48±306.44 pg/mL)were significantly higher than winter(38.13 ± 0.05°C?10.49 ± 0.16breaths/min?24.51 ± 0.24°C?174.11 ± 53.13pg/mL and 947.21 ± 230.00pg/mL)and spring(38.34 ± 0.03°C?15.76 ± 0.62 breaths/min?35.44 ± 0.35°C?263.65 ± 31.36pg/mL and 1311.08 ± 222.51pg/mL);and conception rate(28.27%),diameter in ovalutory follicles(10.36 ± 0.26mm),diameter in biggest follicles(10.22 ± 0.24mm),NO.in biggest follicles(0.79 ± 0.06)and NO.in ovalutory follicles(0.71 ± 0.07)were significantly in summer compared to winter(35.48%?12.58 ± 0.32mm?12.46 ± 0.31mm?0.84 ± 0.08 and 0.90 ± 0.07)and spring(32.35%?11.77 ± 0.30mm?11.59 ± 0.29mm?0.76 ± 0.09 and 0.85 ± 0.07).In summer,a significant positively correlation between THI and RT(r = 0.81)and RR(r = 0.84)in buffaloes were found.When the THI was higher than 78,the respiratory rate and rectal temperature of buffaloes showed a significant upward trend,indicating that buffaloes was more thermotolerant than dairy cows.(2)Effect of breeds on thermotoleranceWhen THI is greater than 78,we measured the physiological and blood paremeters of pure Mediterranean(n=14)and crossbred buffaloes(n=30)in summer to compare their thermotolerance.Results showed that the rectal temperature(39.12 ± 0.09°C),respiratory rate(60.82 ± 5.45 breaths/min)and body surface temperature(37.81 ± 0.12°C)in crossbred buffaloes are significantly lower than those in Mediterranean buffaloes(39.38 ± 0.09°C?76.84 ± 4.31breaths/min and 38.23 ± 0.12°C);the plasma levels of HSP90(2938±520.73pg/mL),HSP70(375.12±64.47pg/mL)and cortisol(214.06±22.83ng/mL)in crossbred buffaloes are remarkedly higher than those of Meditereanean buffaloes(1381.61±339.00pg/mL?202.11 ± 39.45pg/mL and 145.45 ± 19.96ng/mL).Therefore,it was speculated that the crossbred buffaloes were more thermotolerant compared to purebred Mediterranean buffaloes.(3)Effect of age on thermotoleranceWhen THI was greater than 78,comparison of physiological parameters and blood parameters between calves(< 3 months old,n=15)and adult buffaloes(> 12 months old,n=30)in summer revealed that rectal temperature(39.12 ± 0.09°C),respiratory rate(60.82 ± 5.45breaths/min),and body surface temperature(37.81 ± 0.12°C)were significantly lower in adult buffaloes than in calves(39.46 ± 0.09°C,98.72 ± 7.21breaths/min,and 38.92 ± 0.12°C);plasma HSP90(2938 ± 520.73pg/mL),HSP70(375.12 ± 64.47pg/mL),and cortisol(214.06 ± 22.83ng/mL)levels were significantly higher than in calves(1098.81 ± 391.23pg/mL,172.11 ± 27.15pg/mL,and 128.25 ± 21.36ng/mL).These results indicated that adult buffaloes were more thermotolerant than that of calves.(4)Effect of milking performance on ThermotoleranceWhen the THI was greater than 78,the physiological parameters and blood parameters of high-yielding(>8~12Kg,n=16)and low-yielding(<4Kg,n=12)lactating buffaloes were compared in summer,and it was found that the rectal temperature(38.86 ± 0.07°C)and respiratory rate(56.52 ± 7.36breaths/min)were significantly lower in lowyielding buffaloes than in high-yielding buffaloes(39.21 ± 0.07°C and 84.72 ± 3.61breaths/min);the plasma HSP90(2723 ± 413.33pg/mL)and HSP70(357.14 ± 34.53pg/mL)levels were significantly higher than in high-yielding buffaloes(1278.21 ± 278.00pg/mL and 176.17 ± 29.13pg/mL).This indicates that low-yielding buffaloes were more thermotolerant than that of high-yielding buffaloes.(5)Effect of physiology on thermotolerancWhen the THI was greater than 78,the physiological parameters and blood parameters of late pregnant buffaloes(n=8)and empty buffaloes(n=17)were compared in summer,and it was found that the rectal temperature(39.03 ± 0.05°C),respiratory rate(56.62 ± 7.35breaths/min),and body surface temperature(37.91 ± 0.14°C)of empty buffaloes were significantly lower than those of late pregnant buffaloes(39.46 ± 0.05°C,106.23 ± 6.47breaths/min,and 38.77 ± 0.14°C);plasma HSP90(3458 ± 478.23pg/mL)and HSP70(359.17 ± 58.39pg/mL)levels were significantly higher than those of late pregnant buffaloes(1419.27 ± 279.00pg/mL and 237.15 ± 25.47pg/mL).These results indicate that empty buffaloes were more thermotolerant than that of late pregnant buffaloes.2.Analysis of Thermotolerant Genes in Buffaloes(1)Screening of Thermotolerance and Thermosensitive Buffaloes and CowsWhen the THI was greater than 78,the physiological parameters and blood parameters of crossed buffaloes(n=30)were measured for 5 consecutive days in summer,and obtained 4 thermotolerant and 4 thermosensitive buffaloes each.Results showed that rectal temperature and respiratory rate were 38.72 ± 0.10°C and 41.67 ± 4.43breaths/min,which were significantly lower than those in thermosensitive buffaloes(39.67 ± 0.06°C and 100.12 ± 3.48breaths/min);the plasma HSP70,HSP90 and cortisol levels were 454.26 ± 91.15pg/mL,3972.53 ± 668.49pg/mL,and 251.64 ± 10.68ng/mL in thermotolerant buffaloes,which were significantly higher than those in thermosensitive buffaloes(142.86 ± 41.35pg/mL,845.42 ± 236.96pg/mL,and 121.46 ± 24.20ng/mL),indicating that the selection of buffalo samples in the present study is feasible and can be used for further analysis.The physiological parameters,blood parameters,and milk yield changes of high-yielding Holstein cows(n=42)were measured for 7 consecutive days in summer,and three thermotolerant and thermosensitive cows were obtained.Rectal temperature and respiratory rate were 38.7 ± 0.02°C and 57 ± 1breaths/min,which were significantly lower than those in thermosensitive cows(39.7 ± 0.05°C,100 ± 6breaths/min);the plasma HSP70,HSP90,and cortisol levels in thermotolerant cows were 391 ± 59pg/mL,2747 ± 196pg/mL,and 247 ± 19ng/mL,which were significantly higher than those in thermosensitive cows(183 ± 12pg/mL,1075 ± 84pg/mL,and 144 ± 11ng/mL).(2)Transcriptome Analysis in Thermotolerant and Thermosensitive BuffaloIn the study we performed mRNA-Seq and miRNA-Seq methods to analyze the blood transcriptome of thermotolerant and thermosensitive buffaloes.Differentially expressed analysis revealed a total of 753 differential genes and 16 differential miRNAs.Applying the weighted gene co-expression network analysis(WGCNA)method,the differential genes were classified into five modules,four of which were associated with the heat stress index.In the turquoise module,six genes(IL18RAP,IL6 R,CCR1,PPBP,IL1 B,and IL1R1)associated with thermotolerance were found,and the differential miRNA bta-miR-1246 targeted the ABCC4 gene constructed an mRNA-miRNA network with hub genes,which in turn regulated the heat stress response in buffalo.The mRNA-Seq method was applied to analyze the blood transcriptome of thermotolerant and thermosensitive cows,and a total of 200 differentially expressed genes were identified.Among them,14 genes were within the protein interaction(PPI)network,and four genes related to thermotolerance(OAS2,MX2,IFIT5,and TGFB2)were enriched to immune effector processes.Integrated analysis of differential genes involved in thermotolerance in buffalo and cow,and revealed seven shared genes(BMP4,DIRAS2,CD72,DOCK5,MOV10L1,NAT14 and TSPAN15)with a consistent expression trend.In above,these results lay a foundation for further investigation of the mechanism of thermotolerance in buffaloes.(3)Analysis of Thermotolerant Gene BMP4Bovine mammary epithelial cells were used to knockdown the expression of BMP4,a thermotolerant gene shared by buffaloes and cows,by RNAi,followed by heat stress(42 ° C,3 h)treatment,and the proliferation and apoptosis rates of cells and the expression of HSP70,HSP27,Bax,and Bcl-2 genes were measured.The results showed that the apoptosis rate was significantly increased in the treatment group,but the expression levels of the genes HSP70,HSP27,BAX,and BCL-2 were not significantly different.It is tempting to speculate that upregulation of this gene may be through other pathways to enhance thermotolerance in buffaloes.3.Study on Heat stress Resistance MethologyWhen the THI was higher than 78,the effects of different spraying methods on the rectal temperature,body surface temperature and milk composition in lactating buffaloes(n=30)were compared.It was found that in view of the changes in the quality,rectal temperature and body surface temperature of buffalo milk after spraying and the water saving and pollution reduction,the scheme of spraying for 1 minute with an interval of 14 minutes was recommended in production.