Study On The Variation Of Lactoferrin Concentration And Its Affecting Factors In The Bovine Milk

The variation of lactoferrin concentration and affecting factors in the bovine milk were deeply researched in this study, which contained five parts as follows:Part 1: The colostrum from thirty six Holstein cows was collected in this study. The concentration of lactoferrin in the milk was detected by ELISA. Meanwhile, the relative concentration of them in the colostrums was analyzed by SDS-PAGE. From the result got in this study, we can see that the concentration of lactoferrin in colostrum has a dramatic change, especially in the 24 hours after parturition, from 1.315 mg/mL to 0.655 mg/mL.Part 2: The study animals were 198 Chinese Holstein cows randomly chosen from 4 dairy farms in the Beijing area. The cows had shown no sign of mastitis for 2 mo. Daily milk production was recorded and milk samples were taken from individual cow samples. Lactoferrin concentrations in the milk with different levels of the somatic cell count score (SCS) were examined using an enzyme-linked immunosorbent assay to determine whether milk lactorferrin concentration is influenced by parity of the cow, stage of lactation, and the somatic cell count (SCC). The lactorferrin concentration varied between 31.78 and 485.63 ug/mL in milk from normal animals. Lactoferrin was significantly associated with stage of lactation (r = 0.557), and daily milk production (r = -0.472). Nevertheless, there was no significant relationship with parity. Moreover, milk lactorferrin concentration tended to be correlated with the SCS (r = 0.375). This finding suggests that milk lactorferrin may be helpful as an indicator for intramammary infection in dairy cows.Part 3: Using the method of sequencing, we found 6 Single Nucleotide Polymorphisms (SNPs) in a 602bp region of lactorferrin promoter from 128 dairy cows, and 2 mutations of them were discovered for the first time. Least Square analysis was employed to examine the effect of 6 SNPs to the level of SCC in the milk, and the concentration of lactorferrin in the milk also was taken into the consideration. Statistical results indicated that the levels of SCS and lactorferrin in the milk were significantly correlated with the month of lactation, but with no strong relationships to the lactorferrin genotype, lactorferrin diplotypes and lactation number (P > 0.05).Part 4: The objective of this study was to evaluate the effect of implanting an Antigen Release Devices (ARD) into dairy cows during the lactation cycle to induce an immune response. Subsequently, concentrations of lactoferrin in serum and milk were measured. Forty healthy adult Chinese Holstein cows were divided into two equal groups: a test group and a control group. Animals in the test group received ARD implants, whereas the control group animals were not treated. An even spread across the two groups was maintained with animal selection based on parity, the lactation days and milk yields. The concentrations of lactoferrin in the serum and milk of all forty animals were measured using an Enzyme-Linked Immunosorbent Assay (ELISA). The results show that the implantation of an ARD did not significantly increase the concentration of lactoferrin in the serum and milk throughout the whole experiment period except on two occasions. The levels of lactoferrin in the milk and serum significantly increased on day 7 and on day 11 after implantation (P < 0.05). There was a strong correlation between milk lactoferrin and serum lactoferrin (r = 0.564, P < 0.01). Three separate ARDs were used releasing its antigen load on day 0, 14 and 28 to induce a primary, secondary and tertiary response respectively. As the significant increases in the lactoferrin levels were only observed after the first ARD release, the effects of lactoferrin appears to be associated with the early phase of the immune response.Part 5: Using mice as the model animal, we detected the change of the relative expression of lactorferrin gene in the mammary gland under immune state. One hundred and twenty KunMing White mice were divided into four groups. They were treated differently at days 4 after parturition as follows: group A - injecting isotonic Na chloride, group B– injecting Lipase + isotonic Na chloride, group C - injecting ISCOM, and group D– injecting Lipase + ISCOM. The samples, including blood, milk and mammary gland issues, were collected at days 8 and days 12 after parturition (days 4 and days 8 after injecting), respectively. The specific IgG concentration in serum and milk was determined by indirect ELISA. Meanwhile, we used the Real Time PCR to detect the relative expression of lactorferrin in the mammary gland. In the normal state, the relative expression of lactorferrin on day 1 after parturition was quite high, and on day 4, day 8 and day 12 were quite low. In addition, the relative expression of lactorferrin in the mammary gland increased significantly under immune state.