Title Page
Contents
ABSTRACT 10
Ⅰ. Literature review 12
1.1. Introduction 12
1.2. Milk protein synthesis metabolism 15
1.3. Effect of heat stress on milk protein synthesis 16
1.4. Cell apoptosis caused by heat stress 16
1.5. L-leucine and metabolism 17
1.6. Sodium acetate and metabolism 18
Ⅱ. Effects of L-leucine and sodium acetate on milk protein synthesis in MAC-T under heat stress condition 19
2.1. Abstract 19
2.2. Introduction 21
2.3. Materials and methods 22
2.3.1. Cell culture, heat treatment and dosage selection 22
2.3.2. Cell viability assay 23
2.3.3. RNA extraction and cDNA synthesis 23
2.3.4. Quantitative real-time PCR 24
2.3.5. Protein extraction and quantification 25
2.3.6. Western blotting assay 25
2.3.7. Statistical analysis 26
2.4. Results 27
2.4.1. Identification of negative effects of MAC-T due to heat stress conditions 27
2.4.2. Effects of L-leucine and sodium acetate under heat stress condition in MAC-T 27
2.4.3. Combination effects of L-leucine and sodium acetate under heat stress condition in MAC-T 28
2.5. Discussion 46
2.6. Conclusion 51
Ⅲ. General conclusion 53
References 54
Appendix 61
Abstract (in Korean) 62
Table 2-1. Primer sequences designed for qPCR 30
Table 2-2. List of specific primary and secondary antibodies used for western blotting analysis 31
Figure 1-1. Biological changes in dairy cows due to HS 14
Figure 2-1. Relative cell viability under various heat stress condition in MAC-T for 2 hours. 32
Figure 2-2. Relative gene expression under heat stress condition at 39℃ for 2 hours in MAC-T. 33
Figure 2-3. Relative gene expression under heat stress condition at 41℃ for 2 hours in MAC-T. 34
Figure 2-4. Relative gene expression under heat stress condition at 43℃ for 2 hours in MAC-T. 35
Figure 2-5. Relative cell viability under heat stress condition at 41℃ with different levels of L-leucine or sodium acetate in MAC-T. 36
Figure 2-6. Relative heat stress-related gene expression under heat stress condition at 41℃ with different levels of L-leucine in MAC-T. 37
Figure 2-7. Relative protein synthesis-related gene expression under heat stress condition at 41℃ with different levels of L-leucine in MAC-T. 38
Figure 2-8. Relative protein expression under heat stress condition at 41℃ with different levels of L-leucine in MAC-T. 39
Figure 2-9. Relative heat stress-related gene expression under heat stress condition at 41℃ with different levels of sodium acetate in MAC-T. 40
Figure 2-10. Relative protein synthesis-related gene expression under heat stress condition at 41℃ with different levels of sodium acetate in MAC-T. 41
Figure 2-11. Relative protein expression under heat stress condition at 41℃ with different levels of sodium acetate in MAC-T. 42
Figure 2-12. Relative heat stress-related gene expression under heat stress condition at 41℃ with L-leucine and sodium acetate in MAC-T. 43
Figure 2-13. Relative protein synthesis-related gene expression under heat stress condition at 41℃ with L-leucine and sodium acetate in MAC-T. 44
Figure 2-14. Relative protein expression under heat stress condition at 41℃ with L-leucine and sodium acetate in MAC-T. 45