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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 7, Num. 1, 2009, pp. 23-28
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Iranian Journal of Reproductive Medicine Vol. 7, No. 1, Winter, 2009, pp. 23-28
Semen quality characteristics, reaction
time, testis weight and seminiferous tubule diameter of buck rabbits fed neem (Azadirachta
indica A. Juss) leaf meal based diets
Ifeanyi Princewill Ogbuewu Ph.D.,
Ifeanyi Charles Okoli Ph.D., Michael Uwaezuoke Iloeje Ph.D.
Department of Animal Science and Technology,
Animal Physiology Laboratory, Federal University of Technology, Owerri,
P.M.B.1526, Owerri, Nigeria.
Corresponding Author: Ifeanyi Princewill Ogbuewu, Department of Animal Science and Technology, Animal
Physiology Laboratory, Federal University of Technology, Owerri, P.M.B.1526,
Owerri, Nigeria. E-mail: princiano2001@
yahoo.com
Received:
5 October 2008; accepted: 15 March 2009
Code Number: rm09005
Abstract
Background:
To ascertain the effects of tropical leaf meals on semen production and semen
quality.
Objective:
This study was conducted with the main objective of investigating the effect of
neem leaf meal on physiological responses of rabbit bucks fed graded levels of
neem leaf (Azadirachta indica A. Juss) meal.
Materials
and Methods: The varying levels of neem leaf meal (NLM) in the different
experimental diets were 0, 5, 10 and 15% respectively. Four groups of nine
crossbred New Zealand type rabbit bucks each, aged 7-8 months were randomly assigned
to four diets containing neem leaf meal (NLM) at 0% (control) (CD0),
5% (CD1), 10% (CD2) and 15% (CD3) respectively
for 16 weeks.
Results: The
sperm concentration values obtained were 20.15 ×106 /ml, 18.04×106
/ml, 13.65×106 /ml, 6.46 ×106 /ml for the CD0,
CD1, CD2 and CD3 groups respectively.
The results obtained indicate that sperm motility were lowest (p<0.05) in
the treatment groups than the control group. Total sperm per ejaculate was
similar (p>0.05) between the control and those on 510 %NLM dietary groups
however, the value for the 15%NLM group was significantly (p<0.05) lower
than that of the control. Abnormal sperm percentage of the bucks fed 15% NLM
was significantly (p<0.05) higher than those bucks on CD1, CD2
and CD3 groups. The seminiferous tubule diameters were significantly
smaller in the 15% NLM (203µm) than the other 3 dietary groups. All the other
variables measured including semen volume, weight of testis and reaction time
did not differ (p>0.05) among the experimental group.
Conclusion:
The results of the study indicate that the inclusion of neem leaf meal up to
15% in the ration of matured rabbit bucks could cause mild depressive effect on
the spermatogenesis, semen quality and seminiferous tubule diameter.
Key words: Rabbits, Neem, leaf, Histology, Semen.
Introduction
Reproductive inefficiency is the most
limiting constraint to efficient rabbit production in the tropics (1).
The efficiency of sperm production, libido
and quality of sperm tend to remain uniform throughout the reproductive life of
an animal but may be significantly altered by age, nutrition, environment,
health status, drugs, and chemicals (2). Among these factors nutrition is the
most prominent. The survival of an animal is dependent on the availability of
feedstuff.
The unavailability of grains and high
cost of imported feed ingredients have made the price of commercial animal feed
to go up. These problems remain the most important constraints to the expansion
of commercial livestock production in Nigeria.
The need to exploit other available but
neglected cheaper novel feed resources such as leaf meals of tropical trees
which are indigenous to our environment is urgently necessary. The neem leaf
meal, a novel feed resource has a proximate composition of 92.42% dry matter,
7.58% moisture, 20.68% crude protein, 16.60% crude fibre, 4.13% ether extract,
7.10% ash and 43.91% nitrogen free extract (3). Earlier studies on rabbits fed
neem leaf meal based diet have been focused on its feeding value. Nothing has
been reported so far on the effect of feeding the leaf meal on reproductive
performance of buck rabbits.
The good reproductive performance is
necessary for optimal production and profitability and in addition the need to reduce
feed cost is fundamental. Therefore this study attempts to investigate the
effect of graded levels of neem leaf meal on semen quality, libido and
testicular morphometry of New Zealand × chinchilla 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 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.
Preparation of neem leaf meal and
experimental diet formulation
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 its
greenish colouration. The neem leaves were processed according to the procedure
described(3, 4). 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 0%, 5.0%, 10.0% and
15.0% neem leaf meal expressed as weight of the formulated rations. It means
that in 100kg feed formulated there are 0.0kg, 5.0kg, 10kgand 15kg
of neem leaf meal for the CD0, CD1, CD2 and CD3
groups respectively. The chemical compositions of the formulated rations were
analyzed (Table I).
Table
I. Composition of experimental diets
fed to New Zealand White × Chinchilla buck rabbits.
Ingredients** |
Diets (% Neem leaf meal) |
0.0% NLM |
5.0% NLM |
10.0% NLM |
15.0% NLM
|
Spent
grain
|
55.00 |
50.00 |
45.00 |
40.00 |
Neem
leaf meal
|
- |
5.00 |
10.00 |
15.00 |
Calculated
analysis
|
Crude
protein
|
18.87 |
18.70 |
18.53 |
18.37 |
Crude
fiber
|
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 kilogram 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, NLM- neem leaf meal.
Experimental animal
and management
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 (CD1,
CD2, CD3 and CD4) of nine rabbit bucks each
and fed diets containing, 5, 10 and 15 Neem leaf meal expressed as percentage
of the total diets.
The choice of 5% to
15% inclusion levels of neem leaf meal in the present study was based on previous
study conducted by Bawa et al (5) which showed that adult rabbits can
handle up to 20% neem diet without any deleterious effect on their performance,
carcass and hematological characteristics.
The daily consumption rates of neem
leaf meal for each buck were 0.0g, 2.1g, 5.94g and 11.05g for groups fed 0%,
5%, 10% and 15% neem leaf meal respectively. The total amount of neem leaf
meal consumed by each animal during the 16 weeks feeding trial were 0.0g for buck
on 0%, 234.98g for buck on 5%, 665.06 for buck on 10% and 1237.60g for buck
on 15% neem leaf meal inclusion level.
Care was taken when placing the animals
into groups in order to balance the groups such that there were no significant
differences between them on basis of weight. 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 end of second week of the feeding trial, the general conditions of the
experimental animals and their hutches were monitored. The animals were weighed
and re-weighed at every 2 weeks interval. The feeding trial lasted for 16
weeks.
Semen
collection
The semen was collected twice a week between 9.00am and 10.00am. This was to
ensure that optimum quality semen was obtained. A matured cyclic doe was used
to tease the buck. Semen collection was done using artificial vagina that was
locally fabricated as described by Herbert (6) and Herbert and Adejumo (7). .Prior
to semen collection, the artificial vagina (AV) was warmed by allowing it to
stay for 10-15 minutes in warm water at 60oc from thermo flask
thereafter blotted dry with serviette. The AV was then lubricated with glycerol
which is a heavy organic solvent that does not contaminate semen when in
contact with it, and at the same time retains heat. Care was however, taken not
to allow the AV to get too hot as this would result in decreased efficiency of
semen collection and a possible contamination of the semen with urine.
The animals were ejaculated twice weekly for 2 months. Semen volume was read
off the collection tube and recorded in milliliters. Sperm motility was
determined on freshly collected semen placed on a warm stage at 37oc.
The samples were diluted with a physiological saline solution and observations
were made at ×400 magnification. Total spermatozoa per ejaculate were derived
by calculation.
Reaction
time (libido)
A matured cyclic doe (teaser) was introduced to the buck every 2 weeks
interval to monitor their sex drive. In this study, reaction time was
considered as an indication of libido. The time in seconds it took for the
rabbit bucks to sniff, groom and mount the female was recorded with a stop
watch, and libido scored using the scoring pattern of 1-5 (no libido - high
libido) described by Chibundu (8).
Testicular
morphometry
The animals were weighed and slaughtered at the end of the 16th
week. At slaughter, the pairs of testes were quickly milked-out, weighed and
recorded in grams after the epididymis has been trimmed off. These testicular
measurements were taken at the Animal Physiology Laboratory of Department of
Animal Science and Technology, Federal University of Technology, Owerri.
Testicular
histomorphometry
The testes were fixed in aqueous Bouin fluid. Tissue samples were taken from
the equatorial regions of the testis, washed in 50% and 70% alcohol before
being embedded in paraffin wax. After embedding, tissue samples were cut at 5µm
using a microtome. Staining was done with haematoxylin-eosin. Two slides were
prepared per testis. Histometric measurements were taken on selected slide to
generate data on seminiferous tubule diameter. This was carried out using a
Zeiss microscope fitted with an ocular micrometer according to the procedure
described by Majumdar (9). Measurements on tubules were taken twice on each testis,
the second been perpendicular to the first. The tubular diameter was calculated
as the average of the two measurements.
Statistical analysis
Statistical differences between treatment groups were determined with the
analysis of variance test (10) using the computerized statistical analysis of
SAS (11).The experimental model was completely randomized design (CRD)
experiment (Yij = µ + Ti + eij). Where
differences were observed between treatments, the means were compared using Duncans New Multiple Range Test (DNMRT).
Results
The results of buck rabbits fed Neem leaf meal based diets are shown in table II.
The treatment means were compared using Duncans New Multiple Range Test
(DNMRT) at 5% level of probability. The DNMRT = SSR × [MSE/ r] 1/2.
The abbreviation SSR, MSE and r stands for significant studentized range, error
mean square and number of replicate respectively. Whereas the square of (MSE/
r) constitute the standard error of the mean.
The body weights of the animals at pre-slaughter were found within the range of
1636.58 -1653.73g. In this study, reaction time was considered as an indication
of libido. The reaction time obtained in this study was found within the range
of 4.11-4.28 seconds. The reaction time of bucks on control diet was similar to
those on test diets.
The paired testis weights obtained in this study were found within the range of
4.414.61 g. The testis weight of bucks on controldiet was similar
to those on test diets.
From the result of present study there appeared the tendency that as the neem
leaf meal inclusion rate increased, the testicular size decreases. The
diameters of the seminiferous tubules of bucks on the control and 5.0% NLMdietswere significantly (p<0.05) larger than those on the
other two treatment diets.
The semen volume of bucks on control diet was relatively
higher when compared to those that received the test diets. The 15%NLM diet had
the least sperm concentration (6.46 ×106/ml) which was significantly
(p<0.05) lower compared to those on control diet (20.15×106/ml).
The sperm motility of bucks on control diet was significantly (p<0.05)
higher when compared to those on neem leaf meal based diets.
Table
II. Semen quality characteristics,
reaction time, testis weight and seminiferous tubule diameter of rabbit fed neem
leaf meal based diets.
Parameters |
Inclusion levels of Neem leaf meal |
S.E.M |
0% NLM |
5% NLM |
10% NLM |
15% NLM |
Body weight (g) |
1653.73 |
1648.29 |
1639.12 |
1636.58 |
2.566 |
Reaction Time (Libido) |
4.28 |
4.21 |
4.11 |
4.26 |
0.03 |
Semen volume (ml) |
0.64 |
0.56 |
0.52 |
0.51 |
0.02 |
Sperm concentration(× 106/ml) |
20.15b |
18.04b |
13.65ab |
6.46a |
2.51 |
Sperm motility (%) |
74.50a |
68.13b |
55.50c |
40.84d |
3.72 |
Total sperm / ejaculate(× 106/ml) |
14.75a |
14.44a |
10.63ab |
7.82b |
3.82 |
Abnormal spermatozoa (%) |
8.40a |
11.02a |
13.33ab |
20.96b |
2.36 |
Paired testes weight (g) |
4.61 |
4.53 |
4.46 |
4.41 |
0.02 |
STD (um) |
215a |
210a |
203ab |
194b |
6.28 |
a,b,c,d
:Means within a row with different
superscripts differs significantly (p<0.05); Neem leaf meal; STD-
seminiferous tubule diameter; S.E.M- Standard error of mean.
The bucks on 15% NLM diet had the least total sperm per ejaculate (7.82×106/ml)
while those on control group returned the highest total spermatozoa per
ejaculate (14.75×106/ml).The total sperm per ejaculate of the bucks
on control diet differed significantly (p<0.05) relative those fed 15% NLM
diet.
Discussion
The total amount of neem leaf meal consumed by each animal during the 16 weeks
feeding trial for the groups on 0%, 5%, 10% and 15% neem leaf meal treatment
was 0.00g, 234.98g, 665.06g and 1237.60g respectively.
In this study, all the semen quality parameters except abnormal sperm
percentages tend to follow a down ward trend as the inclusion rate of neem leaf
meal increases. The depression in the reproductive performance of bucks neem
leaf meal fed diets as observed in this study have been reported in male
monkey, man and albino rats (12-16). Administered aqueous neem leaf extract.
Herbert et al (4). Has reported similar results on rabbits fed Leucaena
and Gliricidia leaf meal diets. According to Herbert et al (4) feeding
20% Leucaena and Gliricidia leaf meals to rabbits had a
depressive effect on the diameter of the seminiferous tubule, sperm motility, semen
volume and sperm concentration. Rahman (17) and Rahman et al (18)
observed the disappearance of spermatozoa in the seminiferous tubules of rats
fed Leucaena leaves. He equally observed that the epithelia of the
seminiferous tubule of rats fed Leucaena leucocephala leaf meal diets
were devoid of spermatozoa.
The anti-androgenic and anti-spermatogenic properties of neem leaves had been
reported to reduce the fertilizing ability of the spermatozoa (14). The result
of the decreased sperm output was accompanied with increased proportion of
abnormal sperm and this shows that the treatment did adversely affects the
ultra structure of the spermatogenic cells during the process of
spermatogenesis or ejaculation. The percentage of abnormal sperm cell was
observed to exceed the upper limit of 20% recommended as the minimum for good
reproductive potential and fertility in either normal mating or in artificial
insemination (19,20).
This is in agreement with significant increase in abnormal sperm morphology of
rats fed neem leaves (21). Neem leaf extracts have been reported to impair spermatogenesis
and increases the number of headless spermatozoa and significantly decreased
motility of the caudal spermatozoa leading to decline in fertility index (22).
The low sperm concentration witnessed in buck fed the test diet could be
attributed to atrophy or decreased secretory activity in the lumen of the
epididymis and seminiferous that normally goes with feeding of neem leaves in
male animals (14, 23). The sperm motility was observed to decrease significantly
as the inclusion rate of neem leaf meal in the rations increases. The decreased
sperm motility as observed in this study may be opined to the earlier findings
of blockage in the energy metabolic route in animals administered Neem leaves(15, 24).
Aqueous extracts of neem leaf has been reported (13) to have adverse effects
on motility, morphology and number of spermatozoa in caudal epididymis, levels
of fructose in the seminal vesicles and on litter size. The total sperm per
ejaculate is an important semen variablerelated to fertility. It
reflects testicular volume (25, 26), and thus is a measureof total
testicular sperm output (27) which is directly related to the chances of
pregnancy aftermating.
The non significance value in reaction time (libido) observed in this study was
in line with the earlier findings (15, 24) that neem leaf extract reduces
fertility in male monkey (Macaca fascicularis) without reducing libido.
The increase in spermatozoa abnormality following inclusion of neem leaf meal
in the diet of buck rabbits as recorded in this experiment indicated that neem
leaf meal the diets could produce a suppressive effect in the maturation of
sperm cells.
The knowledge of the ability of the testes to store spermatozoa is of immense
importance in rabbit breeding program. The reduction in some of the testicular
parameters weighed in the present study with the introduction of neem leaf meal
diet is a pointer that these neem leaf meals do not promote testicular growth.
These indicate that inclusion of neem leaf meal is detrimental to the
development of spermatogenic potentials of the buck as it has been observed in
the present study. Testis size is a good indicator of the present and future
spermatozoa production of an animal (2, 28-30). The knowledge of basic
morphometric characteristics of the reproductive organs have been found to
provide valuable information in the evaluation of breeding and fertility
potential of the animals (31). The fact that the diet with 15% NLM resulted in
testes weight which was only 35% of the control confirms the poor quality of
these testes relative to those on the control diet in relation to
spermatogenesis. Larger testes without any abnormality have been reported to produce
more spermatozoa than smaller testes (20, 32-33).
Morton (34) reported that in sacrificed animals, decreased weight of the testes
indicates widespread or diffuse lost of seminiferous epithelial cells. The
testes which possess greater number of sertoli cells were heavier and produced
more spermatozoa than testes with fewer sertoli cells(35).
The small testes weight of the bucks on 15% neem leaf diets would mean that
these testes would contain fewer seminiferous tubules (the environment where
spermatogenesis takes place), fewer leydig cells, fewer sertoli cells and fewer
spermatogenic cells.
The slight decline in the size of the testis reported in this study was in
consonance with earlier findings of Anonymous (36) that oral administration of
50% ethanol extract of neem leaves caused a marked reduction in the size of the
testes and epididymis of male rats, by arresting spermatogenesis.
The reported alterations induced in the reproductive organs of Parkes (P)
strain mice by administration of aqueous neem leaf extract recovered to the
control levels after 42 days withdrawal of treatment (13). This biochemical and
histological changes in the testis after treatment with neem leaves and its
pattern of recovery revealed its possible reversible anti-androgenic property (37).
Conclusion
The sperm quality of the bucks fed neem leaf meal based diets was observed to
be of poor quality relative to those on control diet. The diameter of
seminiferous tubule and testicular size of bucks on neem leaf meal were smaller
compared to those on control diet. The 5 -15% inclusion of test diet in the
present study do not improved the sexual drive of the bucks.
The association of neem leaf meal with depressed spermatozoa production and
semen output in the bucks receiving the leaf is a source of concern and should
be given adequate attention, while recommending neem leaf meal for the diet of
breeding buck rabbits.
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