| Artificial insemination techniques are conventional breeding methods of beef and dairy cattle reproduction, so semen quality has an important influence on the cattle industrial development. The main element that influences the Bull semen production is season factor, and the temperature plays a very important role on the semen quality decline, which will directly affect the economic benefits to the livestock industry. Therefore, it is of great significance to explore the regularity of high-temperature influence on the semen quality.Heat stress refers to when animals suffered from high temperature stimulus which they are unable to regulate body temperature to the normal level, resulting to all kinds of non-specific responses in their body. With the global climate greenhouse growing and highly intensive animal production development,the heat stress has become a focus of concern about animal production. Heat stress proteins are a series of high-conservative proteins that protect the body from high temperature and other adverse conditions. HSP70 is one of the most conservative and important HSPs that can be used as high-temperature state indicators of the body non-specific response. In this study, HSP70 is selected as a candidate gene to study the thermal stress state of the gene expression and the body temperature response relationship.High temperature is a disadvantageous factors for the bull reproductive traits, because of the uncontrolled temperature state in nature conditions which affect the study of heat stress to bull semen quality, we set rats as the model to establish the experimental animal model to study this issues, and investigate the gene expression of HSP70 in different temperature state, expectation for laying the foundation for the further study of the thermal stress on the semen quality of large animal.The experimental sexual maturation male Kunming mice and SD rats should be about 45 days of birth, 25 g of weight, and healthy. According to pre-experiment and related reference, they were cultivated in the light incubator to analog summer high temperature at 31℃, 34℃, 37℃, 40℃as four heat stress temperature gradient and a control (room temperature, 20℃~25℃), every temperature gradient were kept 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days separately, totally 7 treatment group (20/group). After the treatment of each experimental group, collecte semen from the epididymis organizations, first of all we carry out detection of semen quality, mainly detect the four indicators which can best reflect semen quality: percentage of the living sperms, sperm motility, sperm acrosomal integrity rate and sperm abnormality rate. Followed by total RNA and protein separation from blood and testicular tissue at the same time , then using Western blot and immunohistochemistry to detect the levels of HSP70 expression at the level of protein. Detection of the expression of HSPS in the level of RNA by fluorescence quantitative PCR;Useing Western blotting and immunohistochemistry to detect the expression of HSPs in the protein level.Comparation of the rate of acrosomal integrity and the abnormal rate comparation between the heat stress group and the control group shows that: the sperm motility, the percentage of live sperm, acrosomal integrity and abnormal sperm of control group are of the higher rate and a steady state, without significant change. The the sperm motility, the percentage of live sperm, acrosomal integrity of the 31℃, 34℃, 37℃treatment groups showed that an decreased at first for above parameters, and the rising trend and is inversely proportional to the treatmented temperature. the 40℃group showed the trend of decreasing with the increase in processing time in processing time for above parameters, but it is clear that the downward trend in the early was more obviously than later. The changing of the sperm abnomal rate just opposite to that of the the sperm motility, the percentage of live sperm and acrosomal integrity.Comprehensive analysis showed that the semen quality of control group is high and stable status and without significant change. The 31℃, 34℃, 37℃treatment group showed reduced in early stage and increased in later stage. The semen quality of 40℃treatment group showed a continuously decrease in processing time.HSP70 expression in mouse testis has a certain regularity under Heat stress, the treatment group of 31℃, 34℃, 37℃showed that the expression of HSP70 increased with processing time at first and reached to maximum amount about 5 to 7 days, and then reduced in a stabilizing trend. In 40℃group, HSP70 expression enhanced at present in pre-treatment time, and higher expression at later. The results of immunohistochemical consistent with western blot results. All the points stained in the Map were spermatogenic cells. Based on the mRNA transcription amount ofβ-actin without significant difference(p>0.05), HSP70 mRNA transcription volume in the testis tissue of rats appeared regular changes, the transcription amount of 31℃group rising rapidly with increasing of treatment time, reached the maximum at the fifth day, and followed by a descend trend; the volume of 34℃, 37℃group increased rapidly in the early time, reached the maximum at about the fifth day, and followed by a downturn and tend to a stable trend of transcription; the transcription amount of 40℃group improved rapidly with the treatment time increased, but the increasing range of early was significantly higher than the latter. Comprehensive analysis suggested that, 31℃belong to mild heat and has little effect on the animals (at an affordable range), so the volume of HSP70 mRNA transcription increased at first and reduced after shows that the body has eased the damage from outside world temperature; The temperature of 34℃, 37℃doesn't exceeded the scope of the body's ability to ease the damage, just more difficult to alleviate it, so the heat stress treatment group of 34℃, 37℃showed that the volume of HSP70 mRNA transcription increased rapidly after the first drop and tend to a stable trend; 40℃high temperature is not conducive to animal survival and exceeds the body's ability to alleviate the stress, so the volume of 40℃heat stress treatment group increased continued. In fact the transcription volume of HSP70 increasing persistently is a sign of causing serious harm to the body.The experimental results show that: under heat stress, the semen quality of rats declined first, with the expression of HSP70 increased and stability, the semen quality of rats fell back to a stable status gradually. Comprehensive analysis showed that the heat stress protein has a protective effect when the body suffered to the hot stress. But when the temperature is too high that exceed the body's own capacity, the expression of HSP70 increased while the semen quality of rats showed a gradually downward trend. In view of the rapid response and regular change of Lymphocyte HSP70 mRNA to heat stress, which could be considered as a molecular biology indicator for assessment of animal heat stress, for cattles, pigs and other large animals, because lymphocytes sampling is convenience, and application of real-time fluorescence quantitative PCR for mRNA quantification is sensitive and accurate. |
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