Revista Científica UDO Agrícola Volumen 9.
Número 4. Año 2009. Páginas: 986-991
Effect of neem (Azadirachta indica) leaf meal on serum metabolite profiles of male
rabbits
Efecto de la harina de hojas de neem (Azadirachta indica) en los perfiles metabólicos séricos de conejos machos
Ifeanyi Princewill
OGBUEWU
, Ifeanyi
Charles OKOLI and Michael Uwaezuoke ILOEJE
Animal Physiology
Laboratory, Department of Animal Science and Technology, Federal University of
Technology, P.M.B.1526, Owerri, Nigeria. E-mail: princiano2001@yahoo.com
Corresponding author
|
Received: 09/07/2009 |
First reviewing ending: 10/24/2009 |
|
First review received: 11/15/2009 |
Accepted: 11/28/2009 |
ABSTRACT
This
study was undertaken to determine the effect of neem
leaf meal (NLM) supplementation on metabolic of male rabbits. Male rabbits (36) with mean body weights of
2025 g were randomly allotted to four treatment groups (n = 9/group). Rabbits
in CD1, CD2, CD3 and CD4 groups
were fed diets containing 0% (control), 5%, 10% and 15% NLM, respectively in a
completely randomized design.. The feeding trial
lasted 16 weeks inclusive of a two week acclimatization period. At the end of
the trial, the animals were starved for 12 hours and blood samples taken from
the marginal ear vein. The serum globulin values of bucks on CD2 and
CD3 groups were significantly (p<0.05) lower than those on CD4.
The serum sodium levels of bucks on CD2 and CD4 groups
were significantly (p<0.05) different from the bucks on control group (CD1).
The bucks on CD3 and CD4 groups had significantly
(p<0.05) elevated serum urea as compared to bucks on control group. The
bucks on CD2, CD3 and CD4 groups had
significantly (p<0.05) lower serum glucose and cholesterol values relative
to control group. It may be concluded
that inclusion of NLM up to 15% in the ration of breeding male rabbits resulted
a significant reduction in serum glucose and cholesterol values.
Key words: Neem leaf meal,
serum, metabolites, male rabbits, biochemical profile
RESUMEN
El objetivo
fue determinar el efecto de la harina de hojas de neem (HHN) en los perfiles metabólicos séricos de conejos machos. Conejos con un peso corporal promedio
de 2025g se asignaron aleatoriamente
a cuatro grupos de tratamiento (n = 9/grupo). Los conejos en los grupos CD1, CD2, CD3
y CD4 se alimentaron con dietas
de HHN a 0% (control), 5%, 10% y 15%, respectivamente,
en un experimento con un diseño
completamente aleatorizado.
El ensayo de alimentación duró 16 semanas incluyendo un período de aclimatación de dos semanas. Al
final del ensayo, los
animales ayunaron durante
12 horas y se le tomaron muestras de sangre de la vena marginal de la oreja. Las muestras de sangre se transfirieron inmediatamente dentro de botellas estériles de plástico sin
anticoagulante para la prueba bioquímica
sérica. Los valores séricos de globulina de los conejos en los grupos CD2 y CD3 fueron significativamente
(p<0,05) menores que en el grupo
CD4. Los niveles séricos de
sodio de los conejos en los
grupos CD2 y CD4 fueron significativamente (p<0,05) diferentes
de los conejos en el grupo
control (CD1). Los conejos en los grupos
CD3 y CD4 tuvieron significativamente
(p <0,05) un valor sérico
de urea elevado en comparación con los conejos en el
grupo control. Los conejos
en los grupos CD2, CD3 y CD4 tuvieron
significativamente (p<0,05) menores
valores séricos de glucosa y colesterol en relación a aquellos del grupo control. Estos resultados sugieren que la inclusión hasta 15% HHN en la ración de conejos machos reproductores pudieran causar un efecto depresivo severo en los parámetros sanguíneos, especialmente los valores séricos de glucosa y colesterol.
Palabras clave: Harina de hojas de neem, conejos, pruebas bioquímicas del suero.
INTRODUCTION
Blood profiles are important
indices of the physiological state of animals (Khan and Zafar, 2005). The ability to
interpret the state of blood profiles in normal and diseased conditions is a
primary objectives of haematological and serum
biochemical studies. Research has proved that definite changes occur in the
blood throughout life (Khan et al. 1987). The serum
biochemical and haematological features have
attracted many workers to look at their indices in order to make clinical
predictions of the health status of a particular animal. The blood picture
varies with certain conditions such as stress, infections and toxicity (Khan and Zafar, 2005).
Blood constituents provide
valuable media for clinical investigations and nutritional evaluations of an
animal (Aderemi, 2004). The ingestion of numerous dietary
materials has been reported by Church et
al. (1984) to have measurable effects on blood constituents. Thus, blood provides proximate
measures for long term nutritional status of animals (Kerr et al., 1982).
Consequently, blood sampling for the assay of biochemical constituents and haematological traits are frequently employed in
nutritional studies.
With nutritional role of leaf meals in mind and its concomitant
significance to animal health. Therefore, the present study was designed with
the main objective of determining the effect of neem
leaf meal based diets on serum biochemistry of breeding male rabbits.
MATERIALS AND METHODS
Experimental location
The
study was carried out at the Rabbit Unit of the Teaching and Research Farm,
Department of Animal Science and Technology, Federal University of Technology, Owerri, Nigeria. The project site lies between latitude 4o4'
and 6o3'N and longitude 16o15' and 8o15'E. It
is about 91m above sea level with annual rainfall, temperature and humidity
ranging from 2300 -
Experimental
animals
Thirty six male rabbit bucks weighing 1025 g were procured from Shongai farm
limited, Owerri. The experiment lasted for 16 weeks
including the 14 days acclimatization period. These rabbits were randomly
separated on the basis of their weight into four treatment groups of nine
rabbits each (CD1, CD2, CD3, CD4).
All the rabbits in this study were housed individually in wooden hutch placed
in a naturally ventilated experimental room with temperature and relative
humidity of about 30oC and 70%, respectively. They were fed with
starter broiler ration (Vital feed) for the two weeks of acclimatization. Feed
and water were given ad libitum.
Processing of neem leaf meal
Fresh
matured neem leaves were harvested in and around the
Federal University of Technology, Owerri. The chopped
leaves were sun dried for about 9 hours every day for 3-4 days until they
became crispy while retaining the greenish colouration.
The sun dried leaves were later milled using electric grinding machine to
produce the neem leaf meal (NLM).
Experimental diets
The neem leaf meal (NLM) was used in the formulation of four
rabbit diets (CD1, CD2, CD3 and CD4
containing NLM at 0, 5, 10 and 15%, respectively). The chemical composition of
the experimental diets has been shown in Table 1.
|
Table
1. Ingredient composition of experimental diets fed to male rabbits. |
||||
|
Ingredients* |
Diets (%) |
|||
|
|
CD1 |
CD2 |
CD3 |
CD4 |
|
Brewer spent grain |
55.00 |
- |
- |
- |
|
Neem leaf meal |
- |
5 |
10 |
15 |
|
Calculated
nutrient composition |
||||
|
Crude protein (%) |
18.87 |
18.70 |
18.53 |
18.37 |
|
Crude fibre (%) |
10.10 |
10.78 |
11.02 |
10.27 |
|
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 |
|
Metabolizable energy (MJ/kg) |
10.42 |
10.38 |
10.33 |
10.22 |
|
*
Each diet contained white maize (35%), local fishmeal (3%), groundnut cake
(3%), bone meal (2%), oyster shell (1.5%) and common salt (0.5%). |
||||
The daily consumption of neem leaf meal was
0.0, 2.1, 5.94 and 11.05 g for CD1, CD2, CD3
and CD4 groups, respectively. The total amount of neem leaf meal consumed by each animal over the 16 weeks
feeding trial was 0.0, 234.98, 665.06 and 1237.60 g in CD1, CD2,
CD3 and CD4 groups, respectively.
Blood collection
The
blood collection was done at the end of the feeding trial. The animals were
starved for 12 hours and bled between 9.00 to 10.30 a-m. Blood was randomly
collected from the marginal ear vein of three selected rabbits per treatment
group. The rabbit was first removed from the hutch by holding it securely on
the scruff and the hind quarter supported underneath with the left hand. The
ear from which the blood was to be drawn was held upright, shaved with shaving
stick to remove the furs so as to reveal the vein more clearly. The shaved ear
was swabbed thoroughly with a clean cotton wool dipped in methylated spirit.
The blood vessel was engorged by gentle tapping of the ear after which the
hypodermic needle was inserted into the largest auricular vein and blood was
aspirated. This was rhen drained into a set of
sterile plastic bottles without anti-coagulant to harvest serum for biochemical
tests.
Serum biochemical analysis
The
serum biochemical assay was 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
and Taussky, 1985), urea nitrogen (Baker and
Silverton, 1985), serum glucose (Toro and Ackerman, 1979), sodium ions and
potassium ions by flame photometry, bicarbonate and chloride ions (Schales and Schales, 1941)
and serum enzymes (AST, ALT, ALP) by
spectrophotometric method (Rej and Hoder, 1983).
Data analysis
Data
obtained were subjected to one way analysis of variance for completely
randomized design (Steel and Torrie, 1980) using
computerized statistical analysis of SAS (2000). Treatment means were compared
using Duncan’s New Multiple Range Test (Obi, 1990).
RESULTS
Data on the effects of neem
leaf meal on serum biochemical constituents of rabbit bucks are presented in
Table 2. The serum creatinine, albumin, total
protein, HC0--3, K+, Cl-,
total bilirubin and conjugated bilirubin, alanine aminotransferase and
aspartate aminotransferase values were similar (p>0.05) among the various
treatment groups. The serum urea level of bucks on CD3 and CD4 groups
were significantly (p<0.05) different from the bucks on CD1 and
CD2 groups. Serum globulin values of bucks on CD2 and CD3
groups were significantly (p<0.05) lower than the groups on CD1
and CD4. The serum sodium value of the bucks on the control group
was significantly (p<0.05) different from the groups on CD2 and
CD4. The bucks on group CD3 and CD4 had
significantly (p<0.05) elevated serum urea values relative to the control
group. The bucks fed on various treatment groups had significantly (p<0.05)
lower serum glucose and serum cholesterol values except CD2 which
had higher (p<0.05) glucose level as compared to control bucks.
|
Table
2. Effect of graded levels of neem leaf based diets
on serum biochemical values of male rabbits |
|||||
|
Parameters |
CD1 (0% NLM) |
CD2 (5%NLM) |
CD3 (10%NLM) |
CD4 (15% NLM) |
S.E.M |
|
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 |
abc Means within a row with different
superscripts are significantly different at p<0.05;
NLM: Neem leaf meal, SEM: Standard error of the mean AST:
Aspartate aminotransferase, ALT: Alanine aminotransferase, ALP: Alkaline
Phosphatase
|
|||||
DISCUSSION
The
reduction in serum glucose value in the present study could be attributed to
the presence of bioactive compounds contained in neem
leaves which have the ability to block the energy metabolic pathway (Chattopadhyay, 1996), thus making it difficult for the
animals to meet their energy requirement (Dutta et al., 1986). The non
comparable serum urea value of bucks on control and those on CD3
and CD4 are in agreement with the findings of Kenneth and Saladin (1998) who reported that
in a state of negative nitrogen balance, muscle proteins are being broken down
and used as energy.
The
increase in serum creatinine and urea levels and the
corresponding decrease in serum glucose levels suggest that serum (urea and creatinine) and serum glucose levels were negatively
correlated in the present study. This is in support of Esonu
et al. (2001) that animals will
normally fall back on the stored energy in the muscles when there is reduction
in blood glucose level.
The
urea and creatinine concentrations in the blood were
used as kidney function test (Davis and Berdt, 1994).
The non significant differences observed in blood
proteins and creatinine in this experiment could be
compared with earlier report of protein retained in animals (Akintola and Abiola, 1999). Iyayi and Tewe (1998) and Awosanya et al.
(2000) reported the dependence of blood proteins and creatinine
on the quality and quantity of dietary proteins.
The
serum cholesterol level was found to be decreased progressively with increasing
dietary levels of neem leaf meal in rabbit bucks.
This observation probably suggests a general decrease in lipid mobilization. It
is possible that NLM has indirect inhibitory effects exerted at the levels of
HMG-CoA reductase, a key enzyme in cholesterol
biosynthesis.
The
results of serum electrolyte tended to show an improvement in the uptake of
serum sodium and chloride while serum potassium and bicarbonates decreased with
increasing levels of neem leaf meal. Serum
electrolytes are used in maintaining cellular tonicity, fluid balance, pH and
regulation of neural and muscular functions (Cheesbrough,
2000). The results of the serum electrolyte tended to show an improvement in
the uptake of sodium ions and chloride ions while bicarbonate and potassium
ions uptake decreased with increased levels of NLM diets. This suggests that,
with up to 15 % inclusion of NLM diets, the ability of the kidney in boosting
these cations and anions is not impaired.
The
serum conjugated bilirubin and serum total bilirubin values were similar among
the treatment groups. The non-elevated values of total bilirubin and conjugated
bilirubin suggest no liver damage which is usually associated with increased
serum conjugated bilirubin and total bilirubin (Cheesbrough,
2000). Serum alanine aminotransferase values obtained in this study were below
the normal range of 12 - 18 µL while the serum aspartate and serum transferase values were higher than the normal range of 9.0
- 12 µL as reported by Mitruka and Rawnsley (1977). The non significant
decrease in serum AST and alanine aminotransferase (ALT) activities of animals
on group CD3 and CD4 could indicate an improvement in
liver function due to hepatoprotective activity of neem (Chattopadhyay et al., 2000). The serum alkaline
phosphatase values were within the standard range (17 - 192 µL) reported by Mitruka and Rawnsley (1977) for
clinically healthy rabbits in the temperate climate. The observed variations in
serum ALT, serum aspartate aminotransferase (AST) and serum alkaline
phosphatase (ALP) could be attributed to environmental and sex differences.
CONCLUSION
It may be concluded
that inclusion of neem leaf meal up to 15% in the
diets of rabbits resulted in significant reductions in serum cholesterol and
glucose levels. The reduction in serum cholesterol value of the rabbit bucks
fed neem leaf meal based diets is an indication that neem leaves could reduce the deposition of cholesterol in
the skin and muscles. The reduction in serum cholesterol is a positive
development since low cholesterol meats command high market price.
LITERATURE CITED
Aderemi, F. A. 2004. Effects of
replacement of wheat bran with cassava root sieviate
supplemented or unsupplemented with enzyme on the haematology and serum biochemistry of pullet chicks. Tropical Journal of Animal Science 7: 147-153.
Akintola, S. O. and S. S. Abiola. 1999. Blood Chemistry and carcass yield of
cockerels fed melon husk diets. Tropical Journal of Animal Science 2: 39-39.
Awosanya, B.; J. K. Joseph, D. F. Apata and M. A. Ayoola. 2000.
Performance, blood chemistry and carcass quality attributes of rabbits fed raw
and processed Puereria
seed meal. Tropical Journal of Animal Science: 7: 89-96.
Baker, F. J. and R. E. Silverton. 1985. Introduction to medical
laboratory technology. 6th edition. Butterworth Ltd, London.
England. 408 p.
Boisness, R. W. and H. H. J. Taussky. 1985. Determination of creatinine
in plasma and urine. Journal of Biology and Chemistry 4: 158-581.
Chattopadhyay, R. R. 1996. Possible
mechanism of anti-hyperglycemic effects of Neem leaf
extracts. Part IV. General Pharmacology
27: 431-434.
Chattopadhyay, R. R.; R. N. Chattopadhyay and
S. K. Maitra. 2000. Effects of Neem on hepatic glycogen in rats. Indian Journal of
Pharmacology 25: 174-175.
Cheesbrough, M. 2000.
District laboratory practice in tropical countries. Part 2, Cambridge
University Press. London, England. 434 p.
Church, J. P.; J. T. Judd, C. W. Young, J. I. Kelsag
and W. W. Djum 1984. Relationship among dietary
constituent and specific serum clinical components of subjects eating
self-selected diets. Animal Journal of Clinical
Nutrition 40:
1338-1340.
Davis, M. E. and W. D. Berndt. 1994. Renal methods for toxicology. In: Hayes, A.W. (eds).
Principles and methods of toxicology, 3rd Edition. New York Raven,
USA. pp.871-894.
Dutta, P.; P. R. Bhartacharyya, O. N. Rabha, N. C.
Bordolon and J. N. Barna.
1986. Feeding deterrents for Philosamia ricini (Samia cynthia Sub rp. ricini) from Tithonia diversifolia.
Phytoparasitica 14: 77-80.
Esonu, B. O.; O. O. Emenalom, A. B. I. Udedibie, U.
Herbert, C. F. Ekpor, I. C. Okoli
and F. C. Ihukwumere. 2001. Performance and blood
chemistry of weaner pigs fed raw Mucuna
beans (Velvet bean) meal. Tropical Animal Production Investment 4: 49-54.
Ibeawuchi, I. I.; S. A. Dialoke, K. O. Ogbede, C. O. Ihejirika, E. M. Nwokeji, I. N. Chigbundu, N.
C. Adikwu and P. O. Oyibo.
2007. Influence of yam/cassava based intercropping systems with legumes in weed
suppression and disease/pest incidence reduction. Journal
of American Science. 3: 49-59.
Iyayi, E. A. and O. O. Tewe. 1998. Serum total protein, urea and creatinine levels as indices of quality cassava diets for
pigs. Tropical Veterinary 16: 57-67.
Kenneth, S. S. and M. P. Carol. 1998. Anatomy and
physiology. The unity of form and function. McGraw-Hill. Boston, Massachusetts,
USA. 1120 p.
Kerr, G. R.; E. S. Lee, E. K. Lam, R. J. Lorimor, E. Randall, R. N. Forthofer,
M. A. Davis and A. Magnettism. 1982. Relationships
between dietary and biochemical measures of nutritional status. American Journal
of Clinical Nutrition 35: 294-308.
Khan, T. A. and F. Zafar. 2005. Haematological study in response
to varying doses of estrogen in broiler chicken. International Journal of Poultry Science 10: 748-751.
Khan, K. R.; H. Mairbaurl, E. Humpler and P. Bectjen. 1987. The dose dependent
effect on corticosterone on the oxygen carrying
capacity and metabolism of RBC in human and rat. Pakistan Journal of Pharmacology 4: 23-36.
Kohn, R. A. and M. S. Allen. 1995. Enrichment of proteolytic
activity relative to nitrogen in preparations from the rumen for in vitro studies. Animal Feed Science
Technology 52: 1-14.
Mitruka, B. M. and
H. M. Rawnsley. 1977.
Clinical biochemical and haematological
reference values in normal experimental animals. Masson Publ. Co. New York,
USA. p: 102-117.
Obi, I. U. 1990. Statistical methods of detecting differences between
treatment means. 2nd edition. Snaap Press,
Enugu, Nigeria.
Peters, T.; C. T. Biomont and B. T. Doumas. 1982. Protein (total protein) in serum, urine and
cerebrospinal fluid, albumin in serum: In selected methods of clinical
chemistry, volume 9. W.R. Faulkner and S. Meites
(eds.) Washington D.C. American Association of Clinical Chemist.
Rej, R. and M. Hoder. 1983. Aspartate aminotransferase. In: Methods of enzymatic analysis. 3rd ed. H. U.
Berg Meyer and M. Grassl (eds.). Weinheim Verlag-Chemie 3: 416- 433.
SAS 2000. Institute Inc. SAS Technical Report Package 234
SAS/STAT Software. The GEMOD Procedure. Release 6.09. SAS Institutes
Inc. Cary, NC.USA.
Schales, O. and S. S. Schales.
Steel, R. G. and J.
H. Torrie. 1980. Principles and procedures of
statistics. A biometrical approach. 2nd edition. McGraw-Hill Book
Co. Inc. New York. USA. 481 p.
Toro, G. and A. Ackerman. 1979. Practical chemical chemistry. Little
Brown and Company, Boston, USA. p. 237-238.
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