J.S. Hogan , W.P. Weiss, and K.L. Smith
Department of Animal Sciences
Eighteen cows were challenged by intramammary infusion with Escherichia coli to determine the effects of acute clinical mastitis on alpha-Tocopherol concentrations in plasma and milk. Cows were fed diets supplemented with 1000 International Units (IU) of vitamin E/day from calving through the experimental period. Geometric mean days in milk at challenge was 33 days. Each mammary quarter was diagnosed with an intermammary infection (IMI) and clinical mastitis at 24 and 48 hours after challenge. The alpha-Tocopherol concentrations in milk from challenged quarters were approximately 60% greater by 24 and 48 hours after challenge compared with concentrations prechallenge and 168 hours postchallenge. Plasma alpha-Tocopherol concentrations did not change after intramammary challenge. alpha-Tocopherol in plasma and milk was correlated at 48 and 168 hours postchallenge but not at prechallenge or 24 hours postchallenge. Milk alpha-Tocopherol and SCC were correlated positively across all sample periods. Milk fat and milk alpha-Tocopherol concentrations were correlated at each sample period except 24 hours postchallenge. Increases in milk alpha-Tocopherol during clinical mastitis were not correlated to milk production, dry matter intake, or bovine serum albumin concentration in milk. Changes in milk alpha-Tocopherol concentration during clinical mastitis were similar to the dynamics of milk SCC.
The incidence and severity of bovine mastitis have been related to dietary intake of vitamin E in many dairy herds (Weiss et al., 1990). Previous trials have shown that cows fed diets deficient in vitamin E had an increased incidence of IMI, increased rates of clinical mastitis, more severe clinical signs, and infections of longer duration than cows fed diets sufficient in vitamin E (Smith et al., 1984; 1993). A principal role of alpha-Tocopherol in maintaining host defense appears to involve leucocytes (Boxer, 1986; Hogan et al., 1993). Dietary and parenteral supplementation with vitamin E enhanced bovine phagocytic cell function (Hogan et al., 1990; 1992; 1994; Politis et al., 1995).
Research investigating the relationships between mammary health and vitamin E has consistently demonstrated the prophylactic effects of dietary vitamin E. However, limited data exist concerning the dynamics of alpha-Tocopherol in tissues during mastitis. Atroshi et al. (1986) reported cows with naturally occurring clinical mastitis had depressed alpha-Tocopherol concentrations in plasma and milk compared with those of healthy herdmates. These results suggested that either the mastitic cows were more susceptible because of inadequate alpha-Tocopherol status prior to the disease or that the disease caused a depression of alpha-Tocopherol concentrations in plasma and milk. Decreases in alpha-Tocopherol concentrations in blood during other infectious diseases and periods of stress have been reported (Chan et al., 1989). The purpose of the current trial was to determine changes in milk and plasma alpha-Tocopherol concentrations during acute mastitis following intramammary challenge with Escherichia coli.
Experimental Cows. Eighteen cows in the Ohio Agricultural Research and Development Center dairy herd were assigned to six blocks of three. Geometric mean parity was 3.2 (range: 2 to 7) and geometric mean days in milk at challenge was 33 (range 20 to 61 days). Cows were housed in tie-stalls and individually fed a diet containing 1000 IU/day of supplemental alpha-Tocopherol (provided as all-rac--tocopheryl acetate) from parturition through the experimental period. Escherichia coli 727, originally isolated from a naturally occurring IMI, was used as the intramammary challenge strain. Intramammary challenge cultures were prepared as detailed by Hogan et al. (1995). The geometric mean for challenge inoculum was 58 colony-forming units (range: 44 to 90 colony- forming units) suspended in 1 ml of saline. Cows within a block were challenged on the same day. Infusions were in either the right or left front mammary quarter 4 hours after morning milking. Only uninfected quarters were infused.
Quarter Foremilk Samples. Quarter foremilk samples were collected 3 days prior to bacterial challenge, immediately prior to challenge, and 24, 48, and 168 hours postchallenge. Sample collection and microbiological procedures were as previously described (Smith et al., 1984). All Gram-negative isolates were identified by the API-20E system (Analytab Products, Plainview, NY).
The colony-forming units per milliliter and SCC were determined in quarter foremilk samples during the postchallenge period. Colony-forming units were determined by appropriate 10-fold dilutions of sample. The initial inocula were duplicate 1 ml pour plates of undiluted milk in McConkey agar. Dilutions were plated on the surface of McConkey agar plates. All dilutions were in duplicate. The SCC per milliliter of milk were determined by Fossomatic milk cell counter (Type 15600; Foss Electric, Hillerød, Denmark). Samples from clinical quarters were diluted 1:10 and 1:50 (milk:saline) for counting. Data were expressed as log10 colony-forming units per milli-liter of milk and log10 SCC per milliliter of milk.
Clinical status of all quarters was recorded at the time quarter foremilk samples were obtained. Clinical status was recorded on a five-point scale: 1 = normal milk and normal quarter, 2 = normal quarter but milk was questionable, 3 = normal quarter but abnormal milk, 4 = a swollen quarter and abnormal milk, and 5 = swollen quarter, abnormal milk, and systemic signs of infection. Rectal temperatures were measured immediately prior to challenge and when quarter foremilk samples were collected postchallenge.
Milk Production and Dry Matter Intake. Milk production was measured electronically at each milking. Daily dry matter intake was recorded for each cow from 7 days prior to challenge through 7 days after challenge. Postchallenge daily milk production and dry matter intake were expressed as percentages of means for the 7 days prior to challenge [(b/a X 100), where a = mean value for the 7 days prior to challenge, and b = daily value postchallenge].
Plasma alpha-Tocopherol. Peripheral blood was collected immediately prior to challenge and 24, 48, and 168 hours postchallenge for alpha-Tocopherol analysis. Plasma was stored frozen and protected from light until analyzed. Plasma alpha-Tocopherol was determined by HPLC equipped with a reverse phase column and a fluorescence detector (Weiss et al., 1992).
Milk alpha-Tocopherol, Bovine Serum Albumin, and Fat. Mammary secretions from challenged quarters were collected immediately prior to challenge and 24, 48, and 168 hours postchallenge for alpha-Tocopherol, bovine serum albumin, and fat analyses. Bulk quarter milk samples were collected approximately 4 hours after morning milking. Mammary secretion samples were frozen, saponified, and alpha-Tocopherol extracted with petroleum ether (Indyk, 1988) for alpha-Tocopherol analysis. Milk alpha-Tocopherol was quantified as described for plasma alpha-Tocopherol. Concentrations of bovine serum albumin in whey from challenged quarters were determined by electroimmunodiffusion (Schanbacher and Smith, 1974). Milk fat was measured using the Roese-Gottlieb method (AOAC, 1980).
Milk samples from each infused quarter were bacteriologically positive for E. coli and had signs of clinical mastitis (clinical score 3 or 4) at 24 and 48 hours after challenge. Thirteen of 18 (72.2%) quarters were bacteriologically negative at 168 hours after challenge. Clinical signs were present in only 2 (11.1%) quarters at 168 hours postchallenge. None of the challenged cows were diagnosed with systemic signs (clinical score 5). Bacterial counts, bovine serum albumin, and SCC each were significantly greater at 24, 48, and 168 hours after challenge than prior to challenge (Figures 1 and 2). Milk production was reduced at 24 and 48 hours postchallenge compared with prechallenge and 168 hours after E. coli infusion (Figure 1). Milk fat percentage was approximately 50% lower at 24 hours after challenge than at prechallenge (Figure 2). Milk fat percentage did not differ among samples collected at 0, 48, and 168 hours after challenge. Intramammary challenge had no effect on dry matter intake (Figure 1). In summary, each challenged quarter developed acute mastitis with limited systemic signs.
Atroshi et al. (1986) reported cows with naturally occurring clinical mastitis had depressed plasma and milk alpha-Tocopherol concentrations compared with healthy herd mates. In contrast, acute coliform mastitis altered milk alpha-Tocopherol concentrations but had no effect on plasma alpha-Tocopherol in the present study (Figure 2). Milk alpha-Tocopherol increased approximately 60% at 24 and 48 hours after challenge compared with prechallenge concentrations. Mean concentration of milk alpha-Tocopherol (0.57 g/ml) before challenge was comparable with values reported by Atroshi et al. (1986) and Hidiroglou (1989) for milk collected from cows without clinical mastitis. The primary source of alpha-Tocopherol in milk is fat globule membranes (Broersma et al., 1991). Therefore, alpha-Tocopherol and fat concentrations are correlated in milk from uninfected quarters (Weiss et al., 1994). Milk fat and alpha-Tocopherol concentrations were correlated in the present study, except at 24 hours after challenge. Correlation coefficients between alpha-Tocopherol and milk fat were 0.73, 0.27, 0.74, and 0.71 at 0, 24, 48, and 168 hours postchallenge, respectively. During the acute phase of mastitis, milk fat concentration decreased as milk alpha-Tocopherol concentration increased. At 48 hours after challenge, milk fat concentrations returned to prechallenge levels, but milk alpha-Tocopherol remained elevated. Changes in milk alpha-Tocopherol concentrations also appeared to be independent of milk synthesis at 24 and 48 hours after challenge. Milk production and alpha-Tocopherol were not correlated. The severity of experimentally induced IMI was sufficient to impair milk synthesis as indicated by decreases in both total milk production and fat concentration. Therefore, the increased concentration in milk alpha-Tocopherol apparently resulted from factors other than local milk fat synthesis.
Mean plasma alpha-Tocopherol was 2.3 g/ml at time of challenge and did not change following intramammary infusion of E. coli. Plasma alpha-Tocopherol is transported as lipoproteins, and no specific alpha-Tocopherol transport protein has been described (Drevon, 1991). The uptake of alpha-Tocopherol in peripheral tissues might occur during catabolism of lipoproteins by specific lipases or by nonspecific transport into cells (Drevon, 1991). Plasma alpha-Tocopherol concentrations did not change during clinical mastitis and were not correlated with milk alpha-Tocopherol concentrations at 24 hours postchallenge. Correlation coefficients between plasma and milk alpha-Tocopherol concentrations were 0.45, 0.26, 0.34, and 0.47 at 0, 24, 48, and 168 hours postchallenge, respectively. Blood constituents in milk increased during clinical mastitis. Concentration of bovine serum albumin in milk increased during clinical mastitis because of vascular leakage into the gland (Schanbacher and Smith, 1974). Concentrations of bovine serum albumin in milk increased approximately 50-fold at 24 hours after challenge but were not correlated with milk alpha-Tocopherol concentrations. Although bovine serum albumin concentrations in milk were not associated with milk alpha-Tocopherol, SCC were correlated with the increase in milk alpha-Tocopherol. The cell population that increases the most dramatically during acute mammary inflammation is neutrophils. Neutrophils are a rich source of alpha-Tocopherol, averaging 5 ng alpha-Tocopherol/106 cells (Weiss et al., 1992). The neutrophil influx appeared to account partially for the increase in milk alpha-Tocopherol.
Neutrophil alpha-Tocopherol inhibits autoxidation of polyunsaturated fatty acids by free radicals in cellular membranes (Baehner et al., 1977; Boxer, 1986). Cellular alpha-Tocopherol is localized in membranes in close proximity to the mixed function oxidase enzymes that initiate the production of free radicals. The importance of neutrophils in host defense against bovine IMI is well documented (Craven and Williams, 1985). Incidence and severity of clinical signs associated with IMI depend on responsiveness of neutrophils (Craven and Williams, 1985). The concentration of alpha-Tocopherol in mammary gland secretion appeared to be increased during clinical mastitis by shifting the primary source from that associated with milk fat to neutrophil alpha-Tocopherol.
Figure 1. Changes in milk bovine serum albumin
(BSA) (top), bacterial counts (2nd from top), dry matter intake (DMI) (3rd from top), and milk
production (bottom) following intramammary challenge with Escherichia
coli (n = 18). Asterisk indicates that means differ from
prechallenge mean (P < .05).
Figure 2. Changes in SCC (top), milk fat percentage
(2nd from top), plasma alpha-Tocopherol (3rd from top), and milk alpha-Tocopherol (bottom) following
intramammary challenge with Escherichia coli (n = 18).
Asterisk indicates that means differ from prechallenge mean (P
< .05).
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