J. J. Barrett
J. S. Hogan 1
W. P. Weiss
K. L. Smith
The Ohio State University
Department of Animal Sciences
1 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 302 Pounden Hall, 1680 Madison Avenue, Wooster, OH 44691; 330-263-3801; e-mail: hogan.4@osu.edu
Fifteen Holstein cows were used in a trial involving intramammary challenge to determine the effects of acute clinical mastitis on the concentrations of a-tocopherol in milk, plasma, and milk and blood neutrophils. Cows were assigned to one of three experimental groups challenged by intramammary infusion of either endotoxin, Escherichia coli, or sterile phosphate-buffered saline. All quarters infused with lipo-polysaccharide or E. coli were diagnosed with clinical mastitis on days one and two after challenge. Acute inflammation caused by intramammary infusion of endotoxin of E. coli resulted in increased concentrations of a-tocopherol in milk in challenged quarters but had no effect on concentrations of a-tocopherol in plasma. Approximately 25% of the a-tocopherol in milk from glands with clinical mastitis was associated with neutrophils compared with <10% in nonmastitic glands. A shift toward sources of a-tocopherol other than synthesized milk fat occurred during acute inflammation in the mammary gland.
Nutrition has a major impact on resistance to mastitis in dairy cows. Vitamin E is an important biological free radical scavenger in mammalian cell membranes that protects phagocytes and other cells from oxidative damage. Incidence and severity of bovine mastitis were directly related to dietary intake of vitamin E. Cows fed diets that were deficient in vitamin E had increased incidence of intramammary infection (IMI), increased rates of clinical mastitis, more severe clinical signs, and infections of longer duration than did cows fed diets supplemented with vitamin E (Smith et al., 1984). Inadequate dietary intake of vitamin E in dairy cows has been associated with increases in both individual cow and bulk tank milk somatic cell count (SCC). The principal role of vitamin E in the defense system of the bovine mammary gland is to maximize neutrophil function. Vitamin E protects polyunsaturated fatty acids in the neutrophil membranes from destruction by toxic oxygen molecules that are produced to kill phagocytized bacteria (Baehner et al., 1977). Vitamin E increased intracellular kill of Escherichia coli and Staphylococcus aureus by bovine blood neutrophils (Hogan et al., 1990).
Conflicting data exist concerning the dynamics of vitamin E in milk and tissues during mastitis. A Finnish field study (Atroshi et al., 1986) found that mastitic cows had reduced concentrations of vitamin E in milk and plasma compared with healthy herdmates. In contrast, a significant increase in the concentration of a-tocopherol in milk and no change in plasma occurred when cows were challenged by intramammary infusion of E. coli (Hogan et al., 1996). The goal of the present trial was to compare concentrations of a-tocopherol in milk, plasma, and milk and blood neutrophils from cows with acute clinical mastitis with those concentrations from nonmastitic cows.
Fifteen Holstein cows from the herd at The Ohio State University's Ohio Agricultural Research and Development Center (OARDC) were assigned to five blocks of three cows. Cows were blocked by both days in milk and parity. The mean parity for the experimental cows was 2.6 and mean days in milk at challenge was 262 days. Cows were fed individually in tie stalls, and dry matter intake was recorded from one week prior to challenge until one week after challenge. Diets were balanced for all nutrients and consisted of 35.6% corn silage, 38.6% concentrate, 11.3% alfalfa hay, and 8.4% alfalfa pellets. Vitamin E was supplemented in the form of all-rac-a-tocopherol acetate at 44 IU/g of concentrate mix. Mean vitamin E supplementation per cow was 478 IU/day. One cow in each block was assigned randomly to one of three treatment groups - intramammary challenge with endotoxin, or E. coli, or a control group infused with sterile phosphate buffered saline (PBS).
Cows within a block were challenged on the same day by intramammary infusion in the right front quarter four hours after the morning milking. Concentrated endotoxin was dissolved in PBS and filter-sterilized. The challenge inoculum was 10 mg of endotoxin in 10 ml of PBS. Quarters infused with live bacteria were challenged with Escherichia coli 727. The mean number of colony-forming units in challenge inocula was 42.6 colony-forming units suspended in one ml of PBS. Control cows were infused with 10 ml of filter-sterilized PBS.
A quarter milker was used to measure milk production from the right front quarter of each cow at the morning milking on days two, one, and zero prior to challenge and on days one, two, and seven after challenge. Bulk quarter milk samples (350 to 500 ml) were used to determine SCC and concentrations of bovine serum albumin (BSA), fat, and a-tocopherol. Concentration of a-tocopherol in plasma and both milk and blood neutrophils were determined following challenge.
Challenging quarters with endotoxin or E. coli 727 resulted in acute clinical mastitis with no systemic signs on days one and two after challenge. Quarters infused with PBS did not have clinical signs of mastitis. No cows were diagnosed with systemic signs on days one, two, and seven after challenge.
Dry matter intake was not affected by treatments throughout the experimental period. Milk production was decreased in quarters challenged with endotoxin and E. coli compared to the milk production of quarters infused with PBS on days one and two after challenge. Quarter milk production returned to the pre-challenge production level by day seven after challenge in quarters infused with either endotoxin or E. coli. Milk production was not affected by intramammary infusion of PBS.
Milk SCC was greater in challenged quarters compared with quarters infused with PBS on day one, two, and seven after challenge. Mean BSA concentrations in challenged quarters were greater than concentrations in quarters infused with PBS on days one and two after challenge.
Milk-fat percentage did not differ between quarters challenged with endotoxin or E. coli and quarters infused with PBS on day one, two, or seven after challenge (Figure 1). Intramammary challenge decreased quarter milk-fat yield. Total grams of milk fat produced was lower in quarters challenged with E. coli and endotoxin compared with quarters infused with PBS on days one and two after challenge. Milk fat production was not significantly affected by infusion of PBS.
Concentrations of a-tocopherol in milk (Figure 2) were greater in challenged quarters compared with quarters infused with PBS on day two after challenge. Concentrations of a-tocopherol in milk did not differ between quarters challenged with E. coli or endotoxin and quarters infused with PBS on days one or seven after challenge. Concentrations of a-tocopherol in milk were not affected by infusion of PBS. Total a-tocopherol in milk per milking did not differ between quarters challenged with E. coli or endotoxin and quarters infused with PBS on days one, two, and seven after challenge. The mean concentrations of a-tocopherol per gram of milk fat (Figure 2) on day two after challenge was 30.9 mg/g in quarters challenged with E. coli and endotoxin compared with 21.1 mg/g from quarters infused with PBS.
Intramammary challenge had no effect on concentrations of a-tocopherol in plasma (Figure 3). The mean concentration of a-tocopherol in plasma for all cows prior to challenge was 4.07 mg/ml and 3.90 mg/ml after mammary infusions.
Intramammary challenge had no significant effect on the a-tocopherol content of neutrophils isolated from milk or blood (Figure 3). The a-tocopherol content did not differ between milk and blood neutrophils. Contribution to a-tocopherol in milk from milk neutrophils was approximately 25% in quarters challenged on days one and two. a-Tocopherol in milk from neutrophils for quarters infused with PBS was 12% on day one, 7% on day two, and 8% on day seven after challenge.
Intramammary challenge with E. coli or endo-toxin increased concentrations of a-tocopherol in milk during the acute phase of mastitis but had no effect on concentrations of a-tocopherol in plasma. Concentrations of a-tocopherol in milk were 67% higher in quarters challenged with E. coli or endotoxin compared with quarters infused with PBS on day two after challenge. In addition, concentrations of a-tocopherol in milk increased more than 80% in quarters challenged with E. coli and 76% in quarters challenged with endotoxin on day two after challenge compared with the pre-challenge concentrations of a-tocopherol in milk. These results are in agreement with an earlier trial in which acute coli-form mastitis increased the concentrations of a-tocopherol in milk 60% at 24 and 48 hours post-challenge, but had no effect on concentrations of a-tocopherol in plasma (Hogan et al., 1996). The mean concentration of a-tocopherol in milk prior to challenge in the present study was 0.69 mg/ml. This concentration was comparable to previously reported concentrations of a-tocopherol in milk from healthy cows (Atroshi et al., 1986).
The primary source of a-tocopherol in milk is the fat globule membrane. However, similar to results of an earlier trial (Hogan et al., 1996), a shift toward sources of a-tocopherol other than milk fat apparently occurred during acute inflammation in the mammary gland. Total milligrams of a-tocopherol secreted after challenge did not differ among treatments, but milk production and total grams of fat produced by challenged quarters were reduced compared with that produced by quarters infused with PBS. Consequently, quarters with clinical mastitis had an increased yield of a-tocopherol in milk per gram of milk fat.
Neutrophils in milk are a rich source of vitamin E, averaging 17.6 ng of a-tocopherol/106 cells in the present trial. Neutrophils are the principal line of defense for the mammary gland once bacteria have penetrated the teat canal. Acute inflammation in the mammary gland results in acute phase responses, including recruitment of neutrophils into the damaged tissue site and increases in milk SCC (Craven and Williams et al., 1985). Neutrophils make up more than 90% of the increase in milk SCC during the acute phase. Correlations between milk SCC and milk vitamin E concentrations were significant before and after challenge. These results suggest that the concentrations of a-tocopherol in milk and milk SCC were related in uninfected and mastitic quarters. Indeed, data from the present study suggested that a-tocopherol from milk cells can account for 10% of the concentration of a-tocopherol in milk from uninfected quarters and 25% in mastitic glands. Therefore, neutrophils normally contribute to the concentration of a-tocopherol in milk in healthy glands, and this contribution increases as milk SCC increases.
Intramammary challenge did not alter the a-tocopherol content of either milk or blood neutrophils. In addition, concentrations of a-tocopherol did not differ between milk and blood neutrophils. In contrast, glycogen is often depleted in milk neutrophils compared with blood neutrophils (Naidu and Newbould, 1973). Diapedesis into the gland has been credited for the expenditure of glycogen, thus reducing the efficiency of milk neutrophils to phagocytize and kill bacteria. Migration of neutrophils from the blood stream into milk apparently did not reduce concentration of a-tocopherol in the cells. However, a confounding factor possibly exists. Some of the a-tocopherol from the neutrophils might have been derived from milk fat. The a-tocopherol in milk fat globules phagocytized by neutrophils and the original a-tocopherol content of the neutrophil could not be distinguished. After infusing a sterile irritant into the gland, Wergin and Paape (1978) found that 68% of milk neutrophils contained fat globules, and each neutrophil averaged 2.1 fat globules per cell. Also, variation in a-tocopherol content among milk neutrophil from different cows may be due to differences in neutrophil phagocytic capabilities.
Although SCC accounted for approximately 25% of the a-tocopherol in milk from quarters with acute mastitis, the primary source of a-tocopherol apparently remained milk fat. An unknown variable was whether the major source of a-tocopherol in milk during clinical mastitis was from fat globules synthesized in the gland or from lipoproteins leaking in from the bloodstream. The decreases in milk fat and milk production indicated that inflammatory changes and tissue damage were severe enough to impair synthesis of milk components. Some of the increase in a-tocopherol might have been due to vascular leakage of blood constituents into the gland during the acute phase of mastitis, evident from the significant rise in concentrations of BSA in milk. Lipoproteins transporting a-tocopherol and leaking into the gland could be responsible for some of the increase in milk fat. If a significant amount of a-tocopherol was derived from the blood lipoproteins, then a decrease in a-tocopherol in plasma would be expected.
No significant changes in the concentrations of a-tocopherol in plasma were found. This result agrees with the results of Hogan et al. (1996), who found intramammary challenge with E. coli had no effect on concentrations of a-tocopherol in plasma, and disagrees with the results reported by Atroshi et al. (1986) that concentrations of a-tocopherol in plasma of mastitic cows were reduced compared with healthy herdmates. The mean concentration of a-tocopherol in plasma prior to challenge was 4.07 mg/ml for all cows in the present study, which is adequate a-tocopherol status according to Weiss et al. (1994). The killing ability of neutrophils was maximized when concentrations of a-tocopherol in plasma were about 3.4 ug/ml.
A shift toward sources of a-tocopherol other than synthesized milk fat occurred during acute inflammation in the mammary gland. Acute inflammation caused by intramammary infusion of endotoxin or E. coli increased concentrations of a-tocopherol in milk but had no effect on concentrations of a-tocopherol in plasma. Increases in concentrations of a-tocopherol in milk were partially due to the large decrease in milk production and the increases in SCC. Approximately 25% of the a-tocopherol in milk was from neutrophils during clinical mastitis compared with < 10% in the nonmastitic glands.
Atroshi, F., J. Tyopponen, S. Sankari, R. Kangasniemi, and J. Parantainen. 1986. Possible roles of vitamin E and glutathione metabolism in bovine mastitis. Int. J. Nutr. Res. 57:37-43.
Baehner, R. L., L. A. Boxer, J. M. Allen, and J. Davis. 1977. Auto-oxidation as a basis for altered function by polymorphonuclear leukocytes. Blood. 50:327-335.
Craven, N. and M. R. Williams. 1985. Defenses of the bovine mammary gland against infection and prospects for their enhancement. Vet. Immunol. Immunopathol. 10:71-127.
Hogan, J. S., K. L. Smith, W. P. Weiss, D. A. Todhunter, and W. L. Shockey. 1990. Relationships among vitamin E, selenium, and bovine blood neutrophils. J. Dairy Sci. 73:2372-2378.
Hogan, J. S., W. P. Weiss, K. L. Smith, L. M. Sordillo, and S. N. Williams. 1996. a-Tocopherol concentration in milk and plasma during clinical Escherichia coli mastitis. J. Dairy Sci. 79:71-75.
Naidu, T. G. and F. H. S. Newbould. 1973. Glycogen in leukocytes from bovine blood and milk. Can. J. Comp. Med. 37:47-55.
Paape, M. J., W. P. Wergin, A. J. Guidry, and R. E. Pearson. 1979. Leucocytes - second line of defense against invading pathogens. J. Dairy Sci. 62:135-153.
Smith, K. L., J. H. Harrison, D. D. Hancock, D. A. Todhunter, and H. R. Conrad. 1984. Effect of vitamin E and selenium supplementation on incidence of clinical mastitis and duration of clinical symptoms. J. Dairy Sci. 67:1293-1300.
Weiss, W. P., J. S. Hogan, and K. L. Smith. 1994. Use of a-tocopherol concentrations in blood components to assess vitamin E status of dairy cows. Agri-Practice 15:5-8.
Wergin, W. P. and M. J. Paape. 1978. Structure of polymorphonuclear leukocytes isolated from bovine blood and milk. J. Cell Biol. 75:102-112.