Serum biochemical evaluation and organ weight characteristics of buck rabbits fed graded levels of Neem (Azadirachta indica) leaf meals diets.

 

I.P. Ogbuewu*, Okoli, I.C., and Iloeje, M.U
Animal Physiology Laboratory, Department of Animal Science and Technology, Federal University of Technology, Owerri, P.M.B.1526, Owerri, Nigeria.

ABSTRACT


A sixteen week feeding trial was carried out to determine the effect of dietary neem leaf meal (NLM) on the organ weight characteristics and serum biochemical constituents of buck rabbits. Four treatment diets were formulated to contain the NLM at inclusion levels of 0 (control), 5%, 10% and 15% respectively. Thirty-six New Zealand white × Chinchilla buck rabbits aged 6 to 7 months were divided into 4 groups of 9 rabbits further replicated into 3 rabbits each and randomly assigned to the four dietary treatments. The lungs, skin, left kidney and pancreas weights were not significantly (p>0.05).higher relative to the control group. The serum metabolites such as creatinine, albumin, total protein, globulin, HC0-3, K+, Cl-, serum bile pigments (total bilirubin and conjugated bilirubin), and liver enzymes were statistically (p>0.05).similar for all the treatment groups. The serum sodium level was significantly (p<0.05) different among the treatment groups. The bucks fed NLM diets had significantly (p<0.05) elevated serum urea and serum creatinine level with decreased serum glucose and cholesterol levels. These results suggest that buck rabbits could tolerate 5-15% dietary levels of NLM without deleterious effects.
Key words: Neem leaf meal, buck rabbits, organ weight characteristics, serum biochemistry.

INTRODUCTION


The utilization of unconventional feed resources holds great relevance to developing countries, where the main constraint to livestock development is the scarcity and fluctuation of the quality and quantity of the year round animal feed supply. There is a serious shortage in concentrated animal feeds such as soybean, cotton seed, maize and groundnut meal, etc. in addition, the human population is increasing rapidly and arable land is decreasing , due to soil degradation, urbanization and industrialization. Production of grain in developing countries is mostly for human consumption.
The novel approaches via the utilization of tree leaves, agro- industrial by-products and aquatic sources are required to bridge the gap between supply and demand of animal feeds. As the demand for food rises, including that of animal origin, unconventional feed resources including the leaves of neem tree must play an increasingly important part in the diet of animal especially non ruminants such as poultry and rabbits in developing countries such as Nigeria.
Neem is an evergreen plant predominant in Nigeria. In Nigeria, it is known as ogwu-iba in Igbo and Dongo yaro in Hausa. Neem tree when fully grown, reaches 24m in height with a dense wide spreading crown. The crown bears large number of compound leaves each with 5-9 pairs of leaflets. Flowering is twice a year (April / May and August / September). Neem tree can yield between 20-25kg of fruits annually.
Although, numerous research efforts have been directed at the optimal utilization of Neem leaves in the feeding and medication of farm animals (Oforjindu, 2006; Esonu et al., 2006). The effects of feeding Neem leaf meal on organ weight and serum biochemical values of buck rabbits raised in the tropical environment however, have not been fully understood. Therefore, the objective of this study is to determine the effect of dietary Neem leaf meal on organ weight characteristics and serum biochemical values of buck rabbits raised in a humid tropical environment.

MATERIALS AND METHODS


Experimental location
This research was carried out in the Rabbit section, of Teaching and Research Farm of the Department of Animal Science and Technology, Federal University of Technology, Owerri, Imo state. Imo state is situated in south-eastern agro-ecological zone of Nigeria. Imo state lies between latitude 4o 4' and 6o3' N and longitude 6o15' and 8o15'E. Owerri is about 100m above sea level. The climatic data of Owerri as summarized in Ministry of Lands and Survey Atlas (1994) of Imo state is as follows: Mean annual rainfall, 2500mm; temperature range, 26.5-27.5oC and humidity range of 70-80%. Dry season duration (i.e. months with less than 65mm rainfall) is 3 months. The annual evapotranspiration is 1450mm and the soil type is essentially sandy loam with average pH of 5.5.

Processing of Neem leaf meals: Fresh matured neem leaves were harvested in and around the Federal University of Technology, Owerri, Nigeria. The leaves were chopped for effective drying. The chopped leaves were sun dried for about 9hours every day for 3-4 days until they became crispy while retaining the greenish colouration. The dry leaves were milled using a hammer mill to produce leaf meal before they were incorporated into the rations. Four experimental rations were formulated such that they contain Neem leaf meal at 0%, 5.0%, 10.0% and 15.0% dietary levels respectively. A sample of the processed leaf meal was subjected to chemical analysis to determine the proximate composition using the standard methods (A.O.A.C, 1995). The chemical compositions of the formulated rations were calculated. The chemical composition of the leaf meal and formulated diets are shown in Table 1 and 2 respectively.

Experimental animals and feeding trials: Thirty-six New Zealand white × Chinchilla rabbits buck with initial weight ranging from 950g to 1100g were randomly allocated on the weight basis to four experimental groups of six rabbit bucks each and fed diets containing the control (0%), 5, 10 and 15% Neem leaf meal respectively. The animals were housed in individual hutches measuring 1.5m × 1m × 1m with wire mesh floor with wooden frames. They were fed with starter broiler ration (vital feed) for a two week of stabilization. Feed and water were given ad libitum. Starting from the second week of the feeding trial, the general conditions of the experimental animals and their hutches were monitored. The animals were re-weighed at every 2 weeks interval. The feeding trial lasted for 16 weeks.

Blood collection: At the end of the feeding trial, three rabbits from each treatment group were bled between 9.00 - 10.30am from the marginal ear vein using 5ml syringes and needles. The 9ml of each blood samples were allowed to coagulate to produce sera for blood biochemical analysis.

Serum biochemical analysis: The biochemical assay were carried out using standard chemical procedures: Total serum protein by Golgberg refractometer method (Kohn and Allen, 1995), albumin by Bromocresol green (BCG) method (Peters et al., 1982), creatinine (Boisness et al., 1945), urea nitrogen (Baker and Silverton, 1985) , serum glucose (Toro and Ackerman, 1975), Sodium ions and Potassium ions by flame photometry, bicarbonate ions and chloride ions (Schales and Schales, 1941), Serum enzymes by spectrophotometric method ( Rej and Hoder, 1985).

Data analysis: Statistical differences between treatment groups were determined with the analysis of variance test (Steel and Torrie, 1980) using the computerized statistical analysis of SAS (2000). Where differences were observed between treatments, the means were compared using Duncan’s New Multiple Range Test (DNMRT).

 

Table 1.0 Proximate composition of Neem leaf meals (dry matter basis).

Nutrient

Percentage dry matter

Dry matter

92.42

Crude protein

20.68

Crude fibre

16.60

Ash

7.10

Ether extracts

4.13

Nitrogen free extracts

43.91

 

 

Table 2.  The composition of experimental diets fed to New Zealand White × Chinchilla rabbits.

Ingredients**

Diets (% Neem leaf meal)

0.0(T0)

5.0(T1)

10.0(T2)

15.0(T3)

Spent grain

55.00

50.00

45.00

40.00

Neem leaf meal

0.00

5.00

10.00

15.00

Calculated analysis

Crude protein

18.87

18.70

18.53

18.37

Crude fibre

10.1

10.78

11.02

11.27

Ether extract

5.97

5.95

5.93

5.91

Calcium

1.41

1.39

1.38

1.36

Phosphorus

0.66

0.62

0.58

0.53

ME (MJ/kg)

10.42

10.38

10.33

10.22

**Each diet contained 35% maize, 3% local fish meal and groundnut cake each, 2% bone meal, 1% oyster shell, 0.50% vitamin./mineral  premix*, 0.5% common salt, *contributed the following to each kilogramme of diet: vit.A 500 IU; Vit. D2 1500 IU; Vit. E 3 IU; Vit. K 2mg; Riboflavin 3mg; Panthothenic acid 6mg; Niacin 15mg; Vit B12  0.8mg; Choline, 3mg; Folic acid 4mg; Mn 8mg; Zn 0.5mg;  Iodine 1.0mg; Co 1.2mg.

 

 

Results

Data on organ weights as percentage of body weight are shown in Table 3. The internal organ weight of the control was in the other hand were similar to the treatment groups, with the exception of pancreas weight at 5% which differed significantly (p<0.05). The bucks fed 5% NLM had the highest liver, pancreas, lungs and kidney weights, although this differed not significantly (p>0.05).

 

Table 3 Effect of graded levels of Neem (Azadirachta indica) leaf meal on organ weight characteristics of buck rabbits.

Parameters

                             Inclusion levels of Neem leaf meal

S.E.M

T0 (0% NLM)

T1 (5% NLM)

T2 (10% NLM)

T3 (15% NLM)

Body weight (g)

 

 

 

 

 

Dressed weight (%)

 

 

 

 

 

Head  (% of live weight)

9.54a

9.62a

7.86b

8.12ab

5.638

Skin  (% of live weight)

9.39a

10.47a

6.39b

9.49a

0.044

Heart  (% of live weight)

0.18

0.18

0.22

0.21

0.005

Lungs  (% of live weight)

0.60a

0.65a

0.42ab

0.39ab

1.032

Left kidney (% of live weight)

0.38

0.38

0.32

0.39

0.005

Right kidney (% of live weight)

0.37

0.31

0.31

0.38

0.001

Paired kidney (% of live weight)

0.75

0.69

0.63

0.77

0.016

Pancreas (% of live weight)

0.03b

0.06a

0.04ab

0.05ab

0.003

Liver (% of live weight)

2.58ab

2.72ab

2.21b

2.94a

0.077

 

a, b,c Means within a row with different superscripts differs significantly (p<0.05); NLM-Neem leaf meal.

 

The effects of different levels of dietary NLM on serum biochemical constituents of rabbit bucks is shown in Table 4. The total serum protein, albumin and globulin values of the rabbits on 5 and 10% were observed to be lower than that of the control with the exception of the serum albumin value of 10%, while the 15% NLM had a higher value relative to control. However, these serum proteins proved not to be significantly different (p>0.05). The serum urea and creatinine levels in this study appeared to follow a similar trend. There was a significant (p<0.05) difference between the urea values at 15% NLM relative to the bucks on control diet. The serum glucose and cholesterol values of the treatment groups were significantly (p<0.05) different from the bucks on control diet. The serum electrolytes, liver enzymes and serum bile pigments of the treatment groups were not significantly different from the control diets except chloride value at 15%Neem leaf meal diet which differed significantly (p<0.05).

 

Table 4.  Serum biochemical characteristics of buck rabbits fed graded levels of Neem (Azadirachta indica) leaf meal.

Parameters

                              Inclusion levels of Neem leaf meal (NLM)

S.E.M

T0 (0% NLM)

T1 (5% NLM)

T2 (10% NLM)

T3 (15% NLM)

Total protein (g/dl)

6.10

3.00

3.20

6.90

0.50

Globulin (g/dl)

4.70a

2.10b

1.50b

5.10a

0.38

Albumin (g/dl)

1.40aB

0.90B

1.70a

1.80a

2.10

Urea (mg/dl)

46.50b

41.00b

57.20a

64.80a

2.67

Creatinine (mg/dl)

0.80

0.70

1.20

1.20

0.07

Cholesterol (mg/dl)

174.60a

115.20b

95.40c

56.50d

12.31

Glucose (mg/dl)

63.50b

75.80a

48.30c

18.00d

6.24

Sodium (mmol/l)

198.60b

155.50c

203.40b

269.20a

11.73

Potassium (mmol/l)

4.40ab

5.30a

3.10b

3.53a

0.24

Chloride (mmol/l)

117.10b

112.00b

119.20b

134.50a

2.42

Bicarbonate (mmol/l)

26.40ab

33.00a

19.60b

20.20b

1.57

Total bilirubin (mg/dl)

0.40

0.40

0.30

0.40

0.01

Conj. bilirubin (mg/dl)

0.30

0.20

0.20

0.20

0.01

ALT (µ/l)

10.00

11.00

9.00

7.00

0.42

AST  (µ/l)

15.00

17.00

13.00

11.00

0.65

ALP (µ/l)

117.90b

97.70c

130.90a

105.10bc

13.67

a,b,c Mean within a row with different superscripts differs significantly (P<0.05), AST- Aspartate Transferase, ALT- Alanine Transferase, ALP- Alkaline Phosphatase

 

DISCUSSION


The result of the body weight of the groups showed that higher dietary inclusion levels of test diets resulted in decreased weight gain. This could be probably due to the effect of incomplete elimination of toxic factors (Opender et al., 2004, Esonu et al., 2001). Other workers (Opender et al.,2004; Amaefule and Obioha, 2001; Iheukwumere and Okoli, 2002), have also reported the effects of nutrient imbalance and poor metabolism (Fanimo et al.,1995) on monogastric animals fed high levels of unconventional feed ingredients.
The decreased, dressed weight percentage whole carcass weight obtained as the dietary levels of NLM diets increases indicate depletion of peripheral glucose level (Chattopadhyay et al., 2000). In addition, the non-significant increase in some organ weight as observed in this of study as the inclusion levels of NLM diets increases indicate a higher physiological activity of this vital organ. This finding, were in slight disagreement with earlier results of Esonu et al. (2006) and Amaefule and Obioha (2001) on birds fed NLM and pigeon (Cabanas cajan)seeds respectively. The relative increase in the values of liver, as the NLM inclusion increases was as a result of hepatoprotective nature of the test ingredient (Chattopadhyay et al., 2000).These slight differences may reflect the ability of the ability of rabbits to adequately handle and tolerate anti- nutritional factors at these levels of NLM diets. This capacity may be mediated through coprophagy and caecal fermentation. This can equally be attributed to species differences or the nature of the active ingredients contained in this test materials.
The reduction in the serum glucose level in the present study could be attributed to the presence of bioactive compounds contained in Neem leaves which has the ability to block the energy metabolic pathway, thus making it difficult for the animals to meet their energy requirement. This was in line with Kenneth and Saladin (1998) who reported that in a state of negative nitrogen balance, the body protein (mainly muscles and liver proteins) are being broken down and used as energy. The peripheral utilization of glucose and gycogenolytic effect due to epinephrine action has been reported to block by NLM completely in diabetic rats (Chattopadhyay, 1996; Bopanna et al., 1997). The result of this study was in support of Khallare and Shrivastav (2003) who reported that the administration of aqueous neem leaf extract in human, led to the blockage of the biochemical energy pathway.
The increase in serum creatinine and serum urea level and corresponding decrease in serum glucose levels suggest that serum (urea and creatinine) and serum glucose level were negatively correlated. This is in support of Esonu et al. (2001) that animal will normally falls back to the stored energy in the muscles (phosphocreatine) when there is a reduction in blood glucose level. The presence of increasing urea and creatinine concentration in the blood is used in the evaluation of the effects of chemicals on the kidney (Davis and Berdt, 1994). The non comparable serum urea value of the control diet and the groups on 15% NLM diet is an indication of poor quality of amino acid profile of the NLM at 15% inclusion rate.
The non significance differences observed in blood proteins and creatinine in this experiment could be viewed with earlier report of protein retained in animals (Akintola and Abiola, 1999). Iyayi and Tewe (1998) and Awosanya et al. (1999) reported the dependence of blood proteins and creatinine on the quality and quantity of dietary proteins. The phosphagens (Arginine phosphate and creatine phosphate) acts as special reservoir of chemical energy for rapid phosphorylation of adenosine diphosphate (ADP) to reconstitute adenosine triphosphate (ATP) during catabolism of the muscle tissues (Allen et al., 1999).
Serum cholesterol levels were observed to decrease progressively with increasing dietary levels of NLM in this present study. This fall in serum cholesterol level of rabbits fed NLM diets probably suggest a general decrease in lipid mobilization. This could be that NLM have indirect inhibitory effects exerted at the levels of HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. This suggests that Neem leaf meal diets were capable of reducing serum cholesterol, thereby helping to reduce the deposition of cholesterol in the skin and muscles. This equally implies that NLM should be used to produce animal product with reduced cholesterol content. The fall in serum cholesterol level of rabbits fed NLM diets probably suggest a general decrease in lipid mobilization. The reduction in the cholesterol level of rabbit fed NLM diets are in agreement with the earlier findings of (Upadhyay, 1990; Oforjindu, 2006) that Neem leaf meal in the diets of broiler birds and rats resulted to a decrease in the cholesterol and liver lipid levels. Chemical analysis revealed that Neem leaf extract contains the following compounds: Quercetin-3-O-β-D-glucoside, Myricetin-3-O-rutinoside, Kaempferol-3-O-β-D-glucoside, Quercetin-3-O-glucose and L-rhamnoside (Chattopadhyay, 1996). It is presumed that these compounds either partially or wholly may be responsible for anti- hyperlipidermic activity of Neem leaves.
Increased urea and creatinine have been used as important indices for evaluation of the effect of chemicals on the kidney (Davis and Berdt, 1994). The results of serum electrolyte tended to show an improvement in the up take of sodium and chloride ions while potassium and bicarbonates ions decreases with increasing levels of NLM. This reflects that Neem leaf meal up to 15% could still maintain the integrity of the kidney in boosting these cations and anions. The concentration of the other electrolyte in the rations and the intestinal environment of the animal probably also influence these values.
The serum total bilirubin and conjugated bilirubin investigated in this study, however, were within the standard range of 0.3-0.4mg/100ml and 0.2-0.4mg/100ml recommended (Mitruka and Rawnsley, 1977) for serum total bilirubin and conjugated bilirubin respectively. The long duration of Neem leaf meal treatment seem not to impose stress on the animals. The non-elevated values of total bilirubin and conjugated bilirubin suggest no liver damage which is usually associated with increased conjugated bilirubin and total bilirubin (Cheesbrough, 2000).
The progressive decrease in AST and ALT levels beyond 5% inclusion and 10% for ALP value suggest the hepato-protective activity of Neem leaf meal (Anonymous, 2007). The ALT values obtained in this study were below the normal range of 12-18 µL as recommended by (Mitruka and Rawnsley, 1977), while the AST values were higher than the normal range of 9.0-12 µL (Mitruka and Rawnsley, 1977). The ALP value were within the standard range (17-192 µL) recommended by the same author for clinically healthy rabbits in the temperate climate. This may be due to environmental differences.

CONCLUSION
We conclude that up to 15% inclusion of NLM diets result to decreased weight gain as evidenced in relatively poor weight gain by buck rabbits receiving increasing levels of neem leaf meal. Neem leaf meals did not improve many of the physiological and biochemical parameters measured. Further detailed research pathophysiology of bucks fed ad libitum of the same dietary levels might be of help in determining the actual differences in structural, functional and biochemical values of rabbit bucks fed Neem leaf meal.

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First Published September 2008

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