Abstract
High ambient temperature and humidity result in economic loss in pigs including growth and reproduction performances in Taiwan in subtropical area. Therefore, the strategies for thermoresistance were established and hyperthermic effects on growth performance and cardiovascular physiology were investigated exhaustively, in order to increase the economic value in pig industry.We analyzed the single nucleotide polymorphism (SNP) in 5’-flanking region of porcine heat shock protein 70.2 gene, the highly inducible form, and then analyzed the association of these SNP with growth performance in Duroc pigs. The result indicated that pigs with TT and TC genotypes at 393 nt in 5’-flanking region have thinner backfat thickness than those with CC type (P<0.05). Thus, the result suggested that the polymorphic Bsa WI site in the 5’-flanking region of porcine HSP70.2 may be used as a marker for the early selection of ultrasonic backfat thickness in Duroc pigs. However, the SNPs in structural region of porcine HSP70.2 have been not found in our further study.In order to develop the thermoresistant breeds, the strategies were established; one was to produce the transgenic pigs overexpressing HSP70.2 gene fused with GFP gene and driven by CMV promoter, another was to set up a technique for functional test to examine resistant ability to hyperthermia. First, the whole length of porcine HSP70.2 gene was amplified by RT-PCR, and then the protein sequence translated correctly by detail sequencing. Second, two transgenic pigs were produced by microinjecting pCMV-HSP70-GFP DNA into the pronucleus of fertilized eggs. Immunoblot assay revealed the varied overexpression level (6.4% and 1.4%) of HSP70-GFP in transgenic pigs. After heating at 45℃ for 3 h, the survival rate (78.1%) of the primary fibroblast cells from the highly expressing transgenic pig exceeded that from the non-transgenic pig (62.9%). This result showed that primary fibroblasts overexpressing HSP70-GFP confer cell thermotolerance. We suggested that transgenic pigs overexpressing HSP70 might improve their thermotolerance in summer season and therefore reduce the economic lost in animal production. However, it is necessary to develop a functional test in whole body to prove the ability to resistant hyperthermia.Furthermore, a technique, whole body hyperthermia (WBH), was set up to investigate the hyperthermic response in pig. Transgenic pigs expressing HSP70 were generated with a fusion marker of green fluorescent protein (GFP) following the gene. The offspring from F1 and F2 transgenic pigs (TG) were employed to monitor the cardiovascular response to heat stress by WBH. Both heart rate (HR) and mean arterial pressure (MAP) were substantially increased after WBH transgenic (TG) and non-transgenic (NTG) littermates compared with those in their shamed control groups. However, there was no significant difference in HR between the groups. Among the heated groups, there were significant different in arterial pressure parameters MAP, systolic or diastolic pressure when animal body temperatures were increased by up to 5℃ and prolonged for 1 hr after. Myocardial injury serum marker, cardiac troponin I (cTnI), was concomitantly monitored during WBH episodes. Notably, cTnI levels in heated groups were substantially enhanced when animal body temperature was increased by up to 3℃ (Δ3℃) and thereafter at rest timepoints during the experiment. Levels of cTnI for both NTG and TG animals were significantly enhanced for 1 hr after WBH. However, there was no significant difference in cTnI levels increased by WBH between the NTG and TG groups. The electrocardiogram (ECG) results were also monitored during the experiment; ST segment elevation, Q-wave changes, and ventricular premature complexes were initially occurred and consistently continued after Δ3℃. At 18 hr recover after heat stress, all animals were sacrificed and the hearts were harvested for pathological examination. Myofibril was fragmented, lysic, and myofibrostic and lymphocytes, eosinophils, and neutrophils infiltrated intermyofibrils in both HS groups. These pathological examination findings for the hearts were severe in the pigs releasing the cTnI level □0.55 ng/ml (Table 2). Above observations indicated that the dysfunction in chaperone of HSP70.2 may be insufficient expression or miss folding by GFP fused and then HSP70.2 can not protect cardiomyocytes from thermal injury.In conclusion, the backfat thickness of Duroc pigs would be affect by the SNPs in 5’-flanking region of porcine HSP70.2 gene. Nevertheless, the HSP70-GFP fusion protein could not confer the protective in cardiovascular changes in transgenic pigs from heat stress which may be caused by improper folding conformation or expressed insufficiently. Finally, a novel pig model for whole body hyperthermia was established to examine the pig’s response to hyperthermia, and it is useful for testing the thermotolerance of pigs in the future.