The experimental study was undertaken in the Division of Animal Nutrition, Faculty of Veterinary Sciences & AH, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, R. S. Pura-181102. Fresh leaves of E. jambolana and P. guajava were harvested from Faculty premises. Both tree leaves were air-dried in the shed for 12-15 days. The dried leaves were micronized with a hammer mill into fine powder, weighed and kept in sterile containers for use later. The fine powder of both tree leaves were thoroughly mixed in proper ratio (50:50) on the cemented floor and suitable LMM was prepared for in-vivo trial.

The basal (starter and finisher) diets were formulated according to BIS (1992) using locally available feed ingredients to fulfil the nutrient requirements of broiler chicks. The LMM was incorporated into the diets at the rate of 0.0% (T1), 2.5 % (T2), 5.0 % (T3) and 7.5 % (T4). The T1 is control while others (T2, T3 and T4) are LMM supplemented groups. The ingredient composition of experimental broiler diets is shown in the table 1. The LMM and experimental diets were subjected chemical analysis in triplicates according to the method of AOAC (1995). Nitrogen free extract (NFE) was determined by difference between 100 and the sum of crude protein (CP), ether extract (EE), crude fibre (CF) and total ash (TA) values on dry matter (DM) basis. Calcium (Ca) was determined as per Talpatra et al. (1940) and phosphorus (P) content by spectrophotometric method (AOAC, 2000). Condensed tannins (CT) content of LMM was determined by butanol-Hcl method (Makkar, 2000). All chicks were provided fresh clean drinking water and feed ad-libitum throughout the experimental period in the morning and evening.

A total of 120 unsexed day old healthy commercial broiler chicks (Cobb-K strains) were procured from Jammu. Chicks were reared in electric brooder until 7 days of age. Chicks had free access to feed and water throughout and were maintained on a constant 24 hours light schedule. On eighth day, chicks were individually weighed, distributed into 4 treatment groups of 3 replicates with 10 chicks in deep litter pen in a completely randomized block design (CRD). Chicks in control group were fed basal diets without LMM (T₁), while other 3 treatment groups were fed the basal diet supplemented with E. jambolana and P. guajava LMM at the rate of 2.5, 5.0 and 7.0 percent in T₂, T₃ and T₄ groups, respectively.

Experimental house and brooding pens were thoroughly cleaned, disinfected, fumigated and remain closed for a period of 15 days before the arrival of chicks. All the feeders, drinkers and other utensils were thoroughly washed and disinfected. The brooding and deep litter materials were properly dried. Rice husks used as litter materials were spread on the floor of the pen and a warm temperature of 33-35 °C was maintained in the brooder with electric bulbs before the arrival of chicks and thereafter 7 days of age. On arrival, the chicks were carefully unboxed, weighed and brooded for a period of 7 days before they were randomly allocated to 4 dietary treatments. The temperature of poultry house gradually declined to 27°C by 21 days of age, after which, chicks were maintained at room temperature (25–27 °C). All the chicks were provided same care and good management practices.

Data on the feed intake, body weight gains and FCR were determined on weekly basis. The FCR was computed as feed intake divided by body weight gain. Blood samples were collected on 42 days at the time of slaughter. Two birds were randomly selected from each replicate for bleeding and slaughter. About 4 ml blood from each bird was collected in sterile centrifuge tube for serum collection and it was stored in sample vials at -20°C for estimation of biochemical constituents i.e. glucose (Teitz, 1976), total serum protein (Tietz , 1986), albumin (Doumas et al., 1972), creatinine (Newman and Price, 1999), cholesterol (Roeschlau et al., 1974), triglyceride (Cole et al., 1997) and serum enzymes viz. serum glutamic oxaloacetic transaminase (SGOT; Bergmeyer et al., 1986a), serum glutamic pyruvic transaminase (SGPT; Bergmeyer et al., 1986b), alkaline phosphatase (ALP; Bessey et al., 1946) and lactate dehydrogenase (LDH; Henry et al., 1960). The biochemical profiles were determined by using readymade diagnostic (Erba Diagnostics Mannheim GmbH) kits.

For histopathological examination, representative tissue samples of various visceral and immune organs were collected in 10% neutral buffer formalin and then processed for paraffin embedding employing alcohol as dehydrating agent and xylene as clearing agent. The sections were cut at 4-5µm thickness and stained by routine Harris haematoxylin and eosin method (Suvarna et al., 2012).

Results obtained were subjected to analysis of variance and treatment means were ranked using Duncan’s multiple range test. Periodic alterations in feed intake, body weight gain and FCR were analyzed using General linear model multivariate procedure of SPSS (16.0) software considering replicates as experimental units and the values were expressed as means ± standard error. Degree of freedom of the treatments was portioned into orthogonal polynomial, depicting linear and quadratic trends associated with increasing levels of CT containing LMM supplementation. Significance was declared at P<0.05 unless otherwise stated. Statistical procedures were done as per Snedecor and Cochran (2004).

The ingredients composition of experimental diets is presented in the table 1. Various feed ingredients viz. maize, rice polish, soybean meal, meat cum bone meal (MBM), soybean oil, di-calcium phosphate, lime stone powder, salt, mineral mixture and vitamin premix, L-lysine and DL-methionine were procured from local market for formulation of broiler starter and finisher diets as per BIS (1992). In the basal (starter and finisher) diets LMM of E. jambolana and P. guajava (50:50 ratio) was incorporated @ 0, 2.5, 5.0 and 7.5 % in T1, T2, T3 and T4 diets, respectively by replacing maize so as to maintain iso-nitrogenous diets throughout the experimental period. The proximate composition of broiler starter and finisher diets of all experimental chicks were within the range as suggested by BIS (1992).

The OM, CP, EE, CF, NFE, TA, AIA, Ca and P content on (% DM) bases of broiler starter diets were 92.55, 22.93, 4.45, 4.42, 60.75, 7.45, 2.43, 1.67 & 0.76; 92.43, 22.83, 4.40, 4.53, 60.67, 7.57, 2.61, 1.64 & 0.77; 92.11, 22.97, 4.23, 4.69, 60.22, 7.89, 2.78, 1.77 & 0.73 and 91.99, 23.07, 4.64, 4.82, 59.46, 8.01, 2.92, 1.83 & 0.70 percent, whereas, the OM, CP, EE, CF, NFE, total ash, AIA, Ca and P content on (% DM) bases of broiler finisher diets were 92.14, 20.01, 5.04, 4.63, 62.46, 7.86, 2.50, 1.73 & 0.69; 92.08, 20.13, 5.12, 4.38, 62.45, 7.92, 2.43, 1.56 & 0.67; 91.97, 19.81, 5.22, 4.72, 62.20, 8.03, 2.76, 1.83 & 0.63, and 91.88, 19.80, 5.07, 4.83, 62.18, 8.12, 2.91, 1.77 & 0.61 percent, respectively in T₁, T₂ T₃ and T₄ groups. The ME (Kcal/kg diet) contents were 2818, 2790, 2762, 2734 and 2925, 2897, 2869, 2841, for broiler starter and finisher diets, respectively in T₁, T₂ T₃ and T₄ groups. The incorporation of LMM of E. jambolana and P. guajava @ 0, 2.5, 5.0 and 7.5% by replacing maize slightly altered the nutrient composition of experimental diets but its incorporation not caused so much difference in the nutritive values of experimental (T₁, T₂, T₃ and T₄) diets.

The data of weekly feed intake, body weight gain and feed conversion ratio of all experimental chicks are presented in the table 2. The average feed intake (g) decreased significantly (P<0.05) as the level of LMM increased. Significantly (P<0.05) higher feed intake was recorded in T₁ compared to T₃ and T₄, whereas, T₂ had an intermediate value between T₁ and T₃.

The lower feed intake in T₃ and T₄ of LMM supplemented groups might be due to high CT content because CT having astringent property and at higher level it bind with glycol-proteins with some of the salivary protein and other nutrients, such complex causes a sensation in the oral cavity, which greatly reduced palatability and hence tends to depress the feed intake in broiler birds. Similar results have been reported elsewhere (Dube, 1993). Present results are contradictory to the findings of Rahman et al. (2013). They had observed that P. guajava leaf meal up to 4.5% dietary level had no detrimental effect on feed consumption.

Significantly (P<0.05) higher mean body weight gain (g) were observed in T₁ as compared to T₂, T₃ and T₄ groups, while body weight gains between T₂, T₃ and T₄ groups were showed non-significant difference. As the time period of experimental study increased, mean body weight gain of broiler chicks increased significantly (P<0.05). The maximum mean body weight gain of broiler birds was recorded at 5th week followed by 4th, 6th, 3rd, 2nd and least body weight gain was obtained at 1st week. The mean FCR among all four groups ranged from 2.08 to 2.28.

Significantly (P<0.05) better FCR was obtained in un- supplemented (T₁) group than that of LMM supplemented (T₂, T₃ and T₄) groups. The FCR when compared among weekly intervals significantly (P<0.05) better FCR was obtained in 2nd week (1.40) followed by 1st week (1.91), 3rd week (2.06), 4th week (2.50) and the worst FCR in 6th week (2.85). Similarly, El-Deek et al. (2009) study found that feeding of guava by-products, raw or treated with the higher levels (6-8 %) showed slightly reduction of broiler body weight gain. Present findings are confirmatory with the results of Shafey et al. (2013) who reported that the replacement of higher levels of Olive leaves reduced body weight gain and feed conversion efficiency in broiler chicks. But contradictory with the results of Simol et al. (2012), whose study showed that mulberry leaf powder can substitute up to 30% of commercial feed without any adverse effect on the feed intake, growth performance and FCR of broiler chicken.

The biochemical profile of broiler birds supplemented graded level of LMM is presented in table 3. Blood glucose level is an indicator of the normal physiology and well being of poultry. An increased or decreased level of blood glucose level is an indicator of stress to the birds. There were no significant differences (P>0.05) in blood glucose level among all groups with and without LMM supplementation and the glucose values were within the normal range (Kenako, 1997). However, in present study, analogous glucose level indicates normal physiological condition of all experimental birds. Total protein value was found to be lowest (P<0.000) in T₃ & T₄ than that of T₁ & T₂ but the values were statistically similar between T₁ & T₂ and T₃ & T₄. It clearly indicated that dietary incorporation LMM 2.5 % onward in broiler chicks had adversely affected the serum protein levels. However, total serum proteins were within the normal physiological range established for broiler chicks (Kenako, 1997).

^abcdefMeans with different superscript within a row and column differ significantly (P<0.05).

^abcMeans with different superscript within a row differ significantly (P<0.05).

Significantly (P<0.000) higher albumin level was recorded in T₁ as compared to T₂, T₃ and T₄. Significantly higher serum globulin level was obtained in LMM supplemented T₂ groups compared to rest of the supplemented and un- supplemented groups. The globulin levels numerically increased in T₃ and T₄ groups than that of T₁ group. Thus the elevated level of serum globulin in LMM supplemented groups clearly indicated that LMM supplementation was having immune stimulating property. The broiler chicks of T₁ group showed significantly (P<0.000) higher A:G ratio compared to rest of the supplemented groups but it was statistically similar among supplemented groups (T₂, T₃ and T₄).

Serum cholesterol and triglyceride values were significantly lower in LMM supplemented groups than that of un-supplemented group. The low cholesterol and triglyceride content observed in the birds fed with CT containing LMM would have been as a result of the hypo-cholesterolemic and hypo-lipidemic properties of CT. Upadhyay (1990) also reported a decline in blood cholesterol levels of broilers and rats fed Neem leaf meal. Present results are in accordance with previous findings (Mahmoud et al., 2013) which affirmed that dietary supplementation of dried guava leaves reduced blood cholesterol and triglyceride levels in broilers.

Creatinine content is an indicator of kidney health status. Serum creatinine levels were significantly (P<0.005) higher in T₄ as compared to T₁, T₂ and T₃ groups while statistically similar in T₁, T₂ and T₃. The higher creatinine level in T₄ might be due to higher CT level (7.5 %) which may cause kidney damage.

There was significant difference (P<0.001) between the SGPT (IU/L) levels for T₄ and those of T₁, T₂ and T₃. There were no significant differences (P>0.05) in SGPT levels among the T₁, T₂ and T₃. Comparatively higher (P<0.076, P<0.051) SGOT and LDH activities were obtained in T₄ than that of T₁, T₂ and T₃ but there were no significant difference among SGOT and LDH levels for T₁, T₂ and T₃. The ALP (IU/L) activity in T₁ group was comparatively higher than that of in T₄ group, whereas, T₂ and T₃ had an intermediate position between T₁ and T₄ which clearly indicated the higher weight gain in T₁ group. Low cholesterol in broilers was also reported by Obikaonu et al. (2012). They studied the effects of dietary inclusion of neem leaf meal at 0, 2.5, 5.0, 7.5 and 10% levels on serum biochemical indices of broiler starter and found that blood sugar was raised by the leaf meal but cholesterol, ALP, ALT and AST levels decreased with increase in leaf meal.

On histopathological examination, liver of T₁ and T₂ groups showed normal hepatocytes arranged in cord pattern with central vein and portal triad (Fig. 1). Liver of T₃ group showed mild to moderate hepatocytic degeneration (Fig. 2), however, T₄ showed mono nuclear cells (MNC’s) infiltration, fibrosis and peripheral hepatocytic degeneration (Fig. 3). Kidney of T₁ and T₂ did not show any significant microscopic changes. Nuclei of lining epithelial cells were vesicular and cytoplasm was homogenous, whereas, moderate swelling and degeneration of epithelial cells of tubules occluding the lumen was observes in T₃ group (Fig. 4).

Moreover, kidney of T₄ group showed interstitial haemorrhage, degeneration with vacuolar cytoplasm and necrosis of epithelial cells of tubules (Fig. 5). Heart of T₁ and T₂ groups showed muscle fibres of uniform thickness and minimum of intermyofibral spaces. However, in T₃ group observed thinning of myocardial fibres (Fig. 6)), while, moderate necrosis of myocardial fibre was recorded in T₄ group (Fig. 7). Intestine of T₁ and T₂ groups showed normal intestinal villi, although, in T₃ group observed mild degeneration of epithelial cells of intestinal villi (Fig. 8). However, fusion, blunting, desquamation of enterocytes and atrophy of villi with mild necrosis of enterocytes was observed in T₄ group (Fig. 9).

The observed changes and lesions in the photomicrographs of the examined vital organs (like liver, kidney heart and intestine) of the experimental chicks of higher level of LMM supplementation might be due to the higher CT level which act as anti-nutritional factors in LMM supplemented diets (5-7.5%). The higher CT level in the diet of chicks cause toxicity and could affect architectural histology of liver, kidney, heart and intestine of experimental chicks. Present findings are confirmatory with the results of previous workers (Adejumo and Ologhobo, 2015; Ewuola, 2009 and Isaac et al., 2017) who also reported lesions on the visceral organs fed toxic substance in the diet of laying hens, rabbits and albino rats, respectively.