The experimental procedures were carried out in accordance with Animal welfare and Ethics Censorship. The animal care protocol was reviewed and approved by Animal ethical committee of GBPUAT, Pantnagar, India (IAEC/C.V.A.Sc/VPB/388).
The study was carried out in forty healthy adult Badri cows, kept at Instructional Dairy Farm (IDF), GBPUAT, Pantnagar, Uttarakhand, India located in the foothills of Himalayas at 29.5°N latitude and 79.3°E longitudes. The animals were distributed in five groups with eight animals in each group according to lactation stages. The groups were as follow: group 1 (0-60 days), group 2 (61-120 days), group 3 (121-180 days), group 4 (181-240 days) and group 5 (to be dried off). The animals were reared under standard environmental conditions with provision of ad libitum feed and water supply.
Blood samples were collected during morning hours, from jugular vein after taking all precautionary measures in vials containing EDTA. Samples were transported immediately to the lab and were processed within 12 hours of collection. Milk samples were collected after taking all precautionary measures. All the samples were processed within 24 hours of collection. Milk samples were collected during evening milking time. Prior to milk collection, teats were washed and sterilized using 70% ethyl alcohol, then initial few streaks of milk were discarded and milk samples were collected in sterile vials. Milk samples were immediately transported to laboratory in ice box for analysis.
Determination of erythrocyte osmotic fragility
Erythrocyte osmotic fragility was determined as described by (Novozhilov et al. 2013). Sodium chloride solution (pH 7.4) was prepared at varying concentrations (0.0%, 0.1%, 0.3%, 0.5%, 0.7% and 0.9%) from 1% phosphate buffer. 5 ml of each NaCl concentration was placed in labelled test tubes serially and 0.02 ml (20 µl) of the blood sample was pipetted into each test tube. The content of the tubes was gently mixed by inverting the tubes and allowing them to stand at room temperature (24-26°C) for 30 minutes, thereafter, the tubes were centrifuged at 1500 rpm for 15 min using a centrifuge. The supernatant obtained from each tube was transferred to a clean glass cuvette and the absorbance of the supernatant was measured spectrophotometrically at a wavelength of 540 nm. The percentage haemolysis for each sample was calculated using the following Faulkner and King (1970) formula. Osmotic fragility curve of erythrocytes was determined by plotting the hemolysis percentage against various saline concentrations.
Determination of blood leucocyte indices
Blood samples were analysed for total leucocyte count (TLC) and differential leucocyte count (DLC), and absolute leucocyte count (ALC) and neutrophil: lymphocyte (N:L) ratio were calculated as per standard methods described in Jain (1986).
Determination of milk somatic cell count (SCC)
Somatic cell count was determined by the method described by Schalm et al. (1971). Milk samples were thoroughly mixed before testing. 10 µl of milk was spread over 1 cm2 marked area on a clean glass slide. Thin milk film was left at room temperature until dry. Then slide was put into methanol for 5 minutes and after which it was stained with modified Newman-Lampert stain for 3 minutes. The slide was washed in tap water for 3 times and distilled water for 2 times. Then it was dried at room temperature (Marshall, 1992). Somatic cells were counted under 100 X magnification using oil immersion power.
10 µl or 0.01 ml of milk was spread in 1 cm2, the possible number of such fields which could be counted in 1 cm2 was 4000 cells. Milk volume represented by each field was 1/100 × 1/4000 = 1/400000. Hence microscopic factor was 400000. As total number of field counted were 50, therefore, working factor was 400000/5 = 8000.
Milk SCC/ml = 8000 × no. of cells counted in 50 microscopic fields
Data were analysed using SPSS statistical software version 26.0 to determine analysis of variance between groups. Groups were subjected to one-way analysis of variance (ANOVA) and Duncan’s multiple range test. All data are expressed as mean ± standard error of the mean (SEM). Differences were considered to be statistically significant at p<0.05.