Effect Of Essential Amino Acid Concentration On The Gene Expression Of Casein α_s1 In The Mammary Gland Of Dairy Cow

This study was conducted to investigate the effect of cryoprotective solution and cryopreservation protocols on the activity of the mammary gland tissue of dairy cow, then examine the effect of Lysine, Methionine, Threonine and Phenylalanine on mRNA abundance in the culture of the cryopreserved mammary tissue.In Expt. 1, The cryoprotective solution used a 3×3 exprimental design with two factors of dimethylsulphoxide (1.0, 1.5 and 2.0mol/L) and sucrose(0.1, 0.2 and 0.3 mol/L). After cryopreservation of the mammary tissue with different cryoprotective solution, the activity of succinate dehydrogenase cryopreserved in the cryoprotective solution(contained 2.0 mol/L dimethylsulphoxide and 0.1 mol/L sucrose) was significantly higher than other cryoprotective solution (P<0.05); DNA fragmentation of tissue cryopreserved with this cryoprotective solution was lower than others cryoprotective solution (P<0.05). The cryopreservation protocols used 2×3 exprimental design with two factors of cooling rate(V1, V2) and cooling time in the -20℃for 2, 3 and 4 hours. The activity of succinate dehydrogenase cryopreserved with V2 and 4h was significantly higher than other cryopreservation protocols (P<0.05); DNA fragmentation of tissue cryopreserved with this protocols was lower than others protocols (P<0.05). The results showed that the mammary gland tissue of dairy cow can be successfully cryopreserved in the cryoprotective solution(contained 2.0 mol/L dimethylsulphoxide and 0.1 mol/L sucrose), with V2 and -20℃for 4 hours.In. Expt. 2. Four the essential amino acids (Lys, Met, Thr, Phe) at seven concentration levels each amino acid, and used in a one-way experimental design to evaluate the effects of amino acid on the mRNA abundance. Twenty-eight kinds of culture medium were prepared for the incubation of mammary tissue and contained Lysine at 120, 145, 170, 195, 220, 245, 270μg/ml; Methionine at 50, 60, 70, 80, 90, 100, 110μg/ml; Threonine at 73, 88, 103, 118, 133, 148, 163μg/ml and Phenylalanine at 75, 90, 105, 120, 135, 150, 165μg/ml, respectively. Compared with different concentration, the mRNA level was significantly increased (P<0.01) by addition of Lysine at 195 and 220μg/ml, the mRNA level was highest at 195μg/ml of Lysine. The ratio of caseinαs1 to GAPDH was significantly increased (P<0.01) by addition of Threonine at 118μg/ml and 133μg/ml. Compared with 118μg/ml, the mRNA level was greatest at 133μg/ml of Threonine. However, there were nonsignificant differences (P>0.05). The relative mRNA abundanece was significantly increased (P<0.01) by addition of Phenylalanine at 120 and 135μg/ml. The greatest mRNA level occurred when mammary tissue received 135μg/ml Phenylalanine treatment. However, there were no significant differences between 120 and 135μg/ml (P>0.05).In summary, the mammary gland tissue proved to maintain the best viability when it were cryopreserved in the cryoprotective solution (DMEM/F₁₂ contained 2.0mol/L DMSO, 0.1 mol/L Sucrose, 20% fetal bovine serum) and V2 and -20℃for 4 hours. The highest mRNA level occurred when cryopreserved mammary tissue received Lys(197μg/ml), Met(107μg/ml), Thr(121μg/ml) and Phe(126μg/ml) treatment in the culture medium, respectively.