Blood was collected from 5 animals in each group both at the beginning and on day 43 of experimental period into sterile centrifuge tubes. Immediately after collection, the tubes were placed in an ice bath and serum was harvested subsequently by centrifuging the blood at 3500 rpm for 10 min in a centrifuge machine. The serum samples were stored at −20 °C in multiple small sterile containers for subsequent analyses. Haemoglobin content in the blood and urea and glucose in serum were determined on the day of collection.
The rumen fluid samples were collected for 2 consecutive days (on day 44 and 45). The samples were collected from 5 animals in each group using flexible stomach tube before morning feeding and 4 h post feeding. The pH of rumen fluid was determined immediately using digital pH meter (Electronics Corporation of India Limited, Hyderabad). About 15 ml of rumen fluid was preserved for enzyme estimation in sterile bottles at −20 °C. The remaining rumen fluid was strained through four layers of muslin cloth. Strained rumen fluid (0.9 ml) was mixed in tubes containing 0.1 ml of 25% metaphosphoric acid, centrifuged at 10,000g for 5 min, and supernatants were stored for determination of volatile fatty acid (VFA) concentrations at −20 °C. Saturated mercuric chloride (1 ml/100 ml of fluid) was added to remaining strained fluid and frozen for analysis of nitrogen fractions.