Effect of Mastitis on Macro-minerals of Bovine Milk and Blood Serum in Sudan

INTRODUCTION Macro-minerals in milk provide the primary mineral requirements of the neonate calf during the critical period after birth when it cannot forage for itself 1. Buruiana et al. 4 reported that milk whey from healthy cows contained more potassium and calcium, and less sodium, than that from cows with evidence of clinical mastitis. El Deeb and Hassan 7 reported significantly higher levels of ash, sodium and chloride in mastitic milk compared with healthy milk, while significantly lower levels of calcium, magnesium and inorganic phosphorus in secretions obtained from E. coli-infused glands were reported 3. However, clinically and sub-clinically mastitic milk samples had higher sodium, magnesium and calcium, and lower potassium levels than normal milk 9. Wagner and Stull 17 found differences in mean potassium and calcium contents in


INTRODUCTION
Macro-minerals in milk provide the primary mineral requirements of the neonate calf during the critical period after birth when it cannot forage for itself 1 .Buruiana et al. 4 reported that milk whey from healthy cows contained more potassium and calcium, and less sodium, than that from cows with evidence of clinical mastitis.El Deeb and Hassan 7 reported significantly higher levels of ash, sodium and chloride in mastitic milk compared with healthy milk, while significantly lower levels of calcium, magnesium and inorganic phosphorus in secretions obtained from E. coli-infused glands were reported 3 .However, clinically and subclinically mastitic milk samples had higher sodium, magnesium and calcium, and lower potassium levels than normal milk 9 .
Wagner and Stull 17 found differences in mean potassium and calcium contents in blood serum between mastitic and healthy cows.
The present study was conducted to compare the levels of the important macro-minerals (sodium, potassium, calcium, magnesium and phosphorus) in the milk and blood serum of healthy cows and cows with subclinical and clinical mastitis.

MATERIALS AND METHODS
This study was carried out on lactating Friesian cows of a herd raised under an intensive management system, on a farm belonging to the Arab Company for Agricultural Production and Processing, Dairy Unit (Dairy Land Farm).
The farm is located 30 km south of Khartoum.It was established in 1984 with a stock of Holstein-Friesian cattle originally imported from the Netherlands.The animals on the farm were housed separately as young calves, heifers, dry herds, milking herds, pregnant cows, and stud bulls.The most common breeding practice is artificial insemination.The cows are machine-milked twice a day.Screening for mastitis is carried out twice monthly.Vaccination of animals against diseases is also carried out.The farm records contain information about each individual animal, regarding production, reproduction and health.

Experimental design and procedures
According to clinical signs and California Mastitis Test (CMT) scores, the cows were grouped as clinically infected, subclinically infected and healthy cows (45 cows altogether, 15 cows in each group).In addition, the CMT result was confirmed using standard plate count agar for total bacteria 10 .Quarter milk samples were collected in sterile bottles and blood samples were collected in sterile vacutainers from the udder vein.Three weeks after the 1st collection, milk and blood were collected and analysed for all the cows in the study.

Laboratory procedures
Atomic absorption spectrophotometry.The concentration of minerals in milk and blood serum was determined with an Atomic Absorption Spectrophotometer 2380 (AAS) (Perkin Elmer, Germany).
Milk and blood serum samples were prepared according to the procedures described in the technical manual of the AAS.The procedure is based on ignition of pre-prepared ash, with concentrated nitric acid and then dilution with distilled water to 100 m .Then a series of standard metal solutions (sodium, potassium, calcium and magnesium) in parts per million (ppm) were prepared by dilution according to the procedures described in the manuals.
The AAS was calibrated and the absorption of the different concentrations of the metal standards were measured at specific wavelengths (i.e.calcium 442.7, magnesium 285.2, sodium 589.0 and potassium 766.5 nm).The standards and sample solutions were run through the AAS, their values in ppm were recorded directly and calculated according to the procedures described in the technical manuals.The data obtained were then expressed as mg/100 m .determined according to the method of Varley 16 .This method is based on the removal of protein by trichloroacetic acid with subsequent treatment of the filtrate with molybdate and reduction by methanol, giving a coloured, measurable complex.The concentrations of inorganic phosphate were then measured using a Spectrophotometer (UV-120-02; Shimadzu, Japan) at 680 nm according to the following formula:

Determination of phosphorus. Concentrations of blood serum phosphorus were
Reading of unknown Reading of standard The phosphorus content of milk samples was measured using the molybdovandate yellow colormetric method of Chapman and Pratt 6 .

Statistical analysis
The data were analysed by a randomized complete block design, using co-variance analysis and t-tests 8 .

Macro-mineral composition and content in milk
Table 1 shows that the level of sodium in the milk increased with mastitis (38.939 ± 4.599, 62.967 ± 18.516 and 90.878 ± 12.24 mg/100 m ) for healthy, subclinically and clinically mastitis-infected cows, respectively.Three weeks after the 1st milk samples were tested, sodium showed increased levels in milk from healthy cows (40.326 ± 0.99 mg/100 m ), while it decreased in subclinically and clinically mastitic milk to about 45.006 ± 8.63 and 36.783± 6.194 mg/100 m , respectively.
The reduction in potassium was more obvious in the clinically mastitic milk samples followed by subclinical samples compared with those of healthy milk (126.161± 8.38, 134.78 ± 8.87 and 160.08 ± 14.35, respectively), both during the 1st, and 3 weeks after the milk samples were tested.Potassium levels were 154.81 ± 12.47, 159.32 ± 12.16 and 142.91 ± 12.5 mg/100 m for clinically, subclinically mastitic and healthy milk, respectively.
Calcium levels in milk decreased as a result of mastitis for clinical and subclinical mastitis infection (55.61 ± 15.68 and 75.26 ± 21.84, respectively) compared with healthy milk (105.49± 14.25 mg/100 m ).Magnesium decreased in clinically and subclinically mastitic milk samples compared with those of healthy milk (7.81 ± 1.4, 9.97 ± 1.62 and 14.17 ± 1.68 mg/100 m , respectively).Phosphorus showed a decrease in the clinically mastitic milk samples, while it increased in subclinically mastitic milk samples compared with that of healthy milk samples (5.07 ± 1.04, 6.02 ± 0.94 and 5.47 ± 1.19 mg/100 m , respectively).Three weeks after the 1st collection of milk samples, the levels of phosphorus were 5.28 ± 1.001 and 4.97 ± 1.09 for clinically and subclinically mastitic milk samples, respectively, compared with healthy milk (5.89 ± 1.25 mg/100 m ).
Table 2 shows the differences in means, standard deviations and standard error of the means for healthy cow's milk and that from subclinically and clinically mastitic cows, initially and after 3 weeks.The subclinical cases showed a significant decrease in potassium (P < 0.001) and a significant increase in sodium content (P < 0.01).The clinical cases showed a significant increase in sodium (P < 0.001) and a significant decrease in potassium, magnesium (P < 0.001) and calcium (P < 0.01).
When comparing healthy cow's milk with subclinically mastitic milk, the level of sodium revealed a highly significant increase (P < 0.001) as shown in Table 3.Similarly, comparison of healthy milk with clinically mastitic milk samples revealed a significant increase in sodium (P < 0.001) and a significant decrease in calcium, magnesium (P < 0.001) and potassium (P < 0.01).Comparison between subclinically and clinically mastitic milk samples also showed a highly significant increase in calcium (P < 0.001) and sodium (P < 0.01), and a significant decrease in potassium (P < 0.05).

Blood serum macro-minerals
Blood serum mineral level (Table 4) did not change noticeably with mastitis infection, except for magnesium, which appeared higher in the subclinically mastitic cases when examined 3 weeks after the 1st collection of samples (6.14 ± 15.44 vs 2.26 ± 0.46 mg/100 m ).
Table 5 shows that, in the clinically infected blood serum samples, the levels of phosphorus and calcium decreased  significantly (P < 0.01), while in cows with subclinical mastitis, blood serum potassium levels showed a significant decrease (P < 0.05).

DISCUSSION
Comparison of means (Table 1) revealed increased levels of sodium in milk from cows with mastitis, and reduction of potassium with increased levels or scores of the disease.This finding is in agreement with those of Buriana et al. 4 , Chaiyabutr et al. 5 , Smith et al. 15 and Mahran et al. 9 , and may be due to bacterial infection of the udder, resulting in damage to the ductal secretory cells and increased permeability of the blood capillaries 12 .Thus, sodium and chloride (which are high in extra-cellular fluid) flow into the lumen of the alveolus, and to maintain osmolarity and potassium levels, decrease proportionately.
The decrease in calcium content in milk samples (Table 1) was similar to that reported by Sbodio et al. 13 and Munro et al. 11 , who found that mastitic milk contained less calcium than healthy cow's milk.This could be due to damage to the mammary gland by pathogens, often disrupting the junctional complex of the secretory epithelium which is essentially impermeable to calcium transport from milk to blood. 2 However, the present results are in contrast to those of Mahran et al. 9 , who found higher concentrations of calcium in clinically and subclinically mastitic milk than in healthy cow's milk.The reduction of magnesium levels that occurred in subclinically and clinically mastitic milk were transient, since after 3 weeks of the 1st collection, there was an increase in magnesium levels.This is similar to the findings of El Deeb and Hassan, 7 and in contrast to those of Mahran et al. 9 .The non-significant difference shown in the present data (Table 5) for magnesium levels in subclinically mastitic milk was, however, similar to the results of Singh and Ganguli 14 and Wagner and Stull 17 .Moreover, these authors concluded that the magnesium content of mastitic milk remained relatively constant with increased somatic cell counts.
The reduction in mean values for phosphorus in the clinically mastitic milk samples (Table 1) is in accordance with the results of Bogin and Ziv 3 and El Deeb and Hassan 7 .
Blood serum macro-mineral levels in milk from mastitic cows did not show noticeable changes in the present study, with the exception of magnesium (Table 4).This might be, as posited by Wagner and Stull 17 , due to homeostatic mechanisms that are sufficiently dynamic to cope with mineral losses from blood into the mastitic gland.They also considered that the magnitude of minerals passing into mastitis-diseased udder sections is so small as    to be negligible compared to the total electrolyte pool in the blood, since milk yield is reduced greatly in a mastitic gland.We conclude that the changes in macromineral levels in individual milk samples (especially increased sodium and decreased potassium) could be a good indicator of udder or quarter infection.

a
Faculty of Animal Production, Department of Dairy Production and b Faculty of Veterinary Medicine, Department of Medicine, University of Khartoum, PO Box 32, Khartoum North, 13314, Sudan.

Table 4 : Mean values of blood serum macro-minerals in healthy and subclinically and clinically mastitic cows, initially and after 3 weeks.
*R1: Initial mean values; R2: Mean values 3 weeks later.