MATERIALS AND METHODS

Ethical approval: The study was conducted following the guidelines of the research policy of Chattogram Veterinary and Animal Sciences University (CVASU) and approved by the Animal Ethics Committee of CVASU, Bangladesh [Approval No. CVASU/Dir(R&E). EC/2019/94(4)].

Animal husbandry and experimental design: A total of 216 (Cobb 500) day-old broiler chicks was procured from the local renowned hatchery weighing on an average of 45.70±0.38 g each. The chicks were weighed on receipt and then randomly assigned into four dietary treatment groups i.e. D₀ (DL-Met), D₁ (0.20% L-Met), D₂ (0.25% L-Met) and D₃ (0.30% L-Met) (D₀, D₁, D₂ and D₃), where each treatment was replicated six times with nine birds per replicate in a completely randomized design (CRD). Birds were raised in battery cages for the entire trial period (day 1-33). Cages were divided into 16 pens of equal size furnished with one feeder and one drinker. All the birds had a free access to the diet, along with ad libitum fresh, clean drinking water during the entire trial period. The birds were exposed to a continuous lighting program. All the birds were vaccinated against Ranikhet (New Castle Disease) and Gumboro disease, as per the schedule mentioned in Table 3, recommended by Department of Livestock Services (DLS), Chattogram, Bangladesh from where the vaccines (lyophilized) were collected.

Diet: Ready-made broiler starter (crumble) diet was procured from the local market and used to feed the birds up to 2 weeks (day 1-14) as an adjustment period. The proximate composition and reporting values of chemical composition of ready-made starter diet are shown in Table 1. After that, finisher or test diets (mash) were prepared manually and provided the birds for the remaining trial period i.e., from day 15-33 day. Four different test diets (D₀, D₁, D₂ and D₃) were formulated as per the requirements of NRC¹⁶, shown in Table 2. All the diets were iso-caloric and iso-nitrogenous. Control diet (D₀) was formulated with the all feedstuffs without L-Met, whereas D₁, D₂ and D₃ test diets were prepared with the supplementation of L-Met at the rate of 0.20, 0.25 and 0.30%, respectively. The composition and nutritive values (calculated and analyzed in the lab) of the formulated or test diets (finisher) are shown in the Table 2.

Data and sample collection: Mortality of bird was recorded as it occurred, while body weight and feed intake were recorded weekly for the calculation of body weight gain and feed conversion ratio (FCR). Livability was calculated from mortality of birds per replicate cage. Two birds per pen were selected randomly, weighed and killed humanely on day 33 to record the relative weights of gastro-intestinal organs (liver, pancreas, bursa of fabricius, heart, small intestine, proventriculus, gizzard, spleen) for assessing the gastro-intestinal development of the birds. Tissue samples (2 or 3cm) were also collected from the ileum, by killing two birds from each replicate cage, for the measurement of intestinal morphology such as villi height, villi width, crypt depth and surface area on day 33. Feed samples were also collected prior supplying to the birds to determine the nutritive value of the feeds.

Feed sample: Collected feed samples were processed by grinding with the help of coffee grinder machine thoroughly to analyze for dry matter (DM%), moisture%, crude protein (CP%), crude fiber (CF%), ether extract (EE%) and ash using standard laboratory procedure¹⁷. Dry matter was determined by oven dry method. Crude protein was recovered by Kjeldahl process. Ether Extract was quantified by Soxhlet apparatus. Ash was measured by igniting the pre-ashing sample on a Muffle furnace at a temperature of 600°C for four to 6 h. Additionally, calcium (Ca%) and phosphorus (P%) were determined by atomic absorption and spectrophotometry, respectively. Metabolizable energy (ME) was determined indirectly on the basis of true metabolizable energy (TME) contents of the feed samples, assuming that TME was 8% higher than the ME, as it is reported that TME is 5-10% higher than ME¹⁸.

Tissue samples processing for morphometric measurement of broiler chicken: Collected tissue segments were flushed with 0.9% saline solution to remove the contents and put in the 10% neutral-buffered formalin. After that, the samples were dehydrated, cleaned and paraffin embedded. Duplicates tissue segments were processed by the paraffin sectioning at a 4 μm thickness, placed on a glass slides. The slides were stained with hematoxylin-eosin and covered with cover-slip. Histological indices were analyzed under a computer-aided light microscopic using image analyzer. Measurements of villus height and crypt depth were taken only from sections, where the plane of section ran vertically from the top of villus to the base of an adjacent crypt. The villi height (from the crypt mouth to the villi tip), crypt depth (from the base up to the region of transition between the crypt and villi), villus width (½ of the villus length) were measured. The villi surface area (the area of the villous per length unit per bird) and villi height:crypt depth ratio were also calculated.

Statistical analyses: All collected data were subjected to analyzing by one way Analysis of Variance (ANOVA) using Minitab software (Minitab, Minitab Version, 16, 2000). The significance of differences between means was tested using the Duncan’s multiple range tests (DMRT). Statistical significance was considered at p≤0.05.

RESULTS

Growth responses of broiler chicken: The results showed that the feed intake (FI) and live weight (LW) of broiler chickens up to 21 days were not influenced (p = 0.87; p = 0.57) by dietary treatments except for 33 days (Table 4). From 1-33 days, the birds of D₃ diet group consumed the highest feed (p = 0.01) compared to other groups. Live weight was also significantly (p = 0.038) greater for the broilers fed D₃ diet than the birds fed other diets up to 33 day. The feed conversion ratio (FCR) of broilers was unaffected (p = 0.133) by dietary treatment from day 1-33 (Table 4). Birds on D₃ diet group had a comparatively lower FCR value (1.56) than the other diet group, although the FCR value between treatment was statistically identical or not affected (p>0.05) throughout the trial period.