Emerging evidence from in vivo as well as
in vitro studies indicates that botanicals play noteworthy roles in the
treatment, prevention and management of diseases. Use of natural compounds in
botanicals has been proposed as potential alternative to conventional
therapeutic options. Therefore this study aimed to evaluate the effect of
African walnut powder (Tetracarpidiumconophorum)
on semen characteristics of rabbit bucks.
Methods
The semen characteristic of bucks placed on
varying levels (0%, 1.5%, 2.0% and 2.5%) of African walnut powder as
supplement was examined.A total
number of 16 matured bucks were allotted into four treatments. African walnut
was obtained from Osun state, Nigeria and processed
into powder. The experimental diets were administered after 2 weeks of
acclimatization. Progressive motility, acrosome integrity and morphology,
semen volume, live dead ratio, semen pH and reaction time were evaluated
after semen collection through the use of artificial vagina. Hormones like
testosterone, cortisol, thyroxin and triiodo – thyronine were also assayed. Data obtained were subjected
to one-way analysis of variance.
Results
Significant differences were observed in
semen volume across the treatment (p<0.05), semen volume ranged between
0.16ml (2.0%AWNP) and 0.38ml (2.0%AWNP) across the treatment. Result shows
that progressive motility ranged from 73.30% (1.5% and 2.0%AWNP) and 83.30%
(2.5%AWNP). Acrosome integrity of the animals placed on 2.0% and 2.5%AWNP had
mean value of 90%, while 0.0% and 1.5% had 83.33% and 86.66% respectively.
Morphology was observed to be statistically uniform across the treatments
(p>0.05). Live dead ratio had higher mean value (90%) than control
animals, (80.0%) though the difference is negligible statistically (p>0.05).
Reaction time across the treatments showed no significant difference
(p>0.05). Triiodo-thyronine showed higher mean
value of 17.02pg/ml at 2.0%AWNP while animal on 2.5%AWNP had the least value
of 12.98pg/ml. Thyroxine and cortisol had similar
trend of increased value with increased value of AWNP. Testosterone showed
significant difference across the treatment (p<0.05) with 2.0%AWNP having
the highest value (42.5pg/ml) while the least value was obtained from
1.5%AWNP.
Conclusions
To
improve bucks semen characteristic and fertility, 2.0% level of AWNP is
recommended based on the result of progressive motility, morphology, acrosome
integrity and testosterone which had an increase in value more than the
control which was lower compare to other percentage levels of AWNP.
List of Abbreviations
AWNP:African
Walnut Powder
PKC:Palm Kernel Cake
ANOVA:
Analysis of Variance
INTRODUCTION
Rabbits are induced breeder but have an annual cycle of reproduction
which is shown by change in conception rate, acceptance of male and ovulation
rate and in male by significant seasonal change in testicular size (Habbebet al.,
1992).The most obvious limitation to
rabbit production in hot climate area is their susceptibility to heat stress
that evoke a series of drastic changes in their biological function which in
turn end with impairment of production and reproduction. Such detrimental
effect is punctuated during summer which are reflected in limiting the breeding
season of rabbit to be normal from September to May each year,
(Maraiet al., 2002a, and Maraiet al., 2002b). Abd
El-Azim and EL- Kamash
(2015) reported the spermatozoa behavior during transit along the female
reproductive tract (Castellini, 2008). Variations in the seminal characteristic contribute to the
large variability in semen quality trait. (Alvarino, 2000), it is important to evaluate
seminal inclusion to improve semen output. Oxidative stress results from
increased production of free radicals and reactive oxygen species and a
decrease in antioxidant defenses. (Trevasan, et al., 2001; William, et al.,
2002).Tetracarpidiumconophorum
like many other plants in Africa and other part of the world has been proven to
have decorative, nutritive, medicinal, agricultural and industrial value over
the years. Phytochemical analysis of Africa walnut
indicates that it contains bioactive compounds such as oxalates, phytates, tannins, saponins and
alkaloids, which partly show the use of the seed, leaves and roots in
herbal medicine (Ojobor et al., 2015 and Ayolola et al., 2011). The presence of tannin, support its
anti-inflammatory property. As a rich source of alkaloids, couple with the
presence of the essential vitamins and minerals, T.conophorum can be seen as a potential
source of useful food and drugs. High content of ascorbic acid also indicates
that the plant can also be used to prevent or at least minimize the formation
of carcinogenic substance from dietary materials (Ojoboret al., 2015). Studies have shown that the nuts are rich in protein,
carbohydrate, fat and oil, vitamin and minerals (Ojobo et al., 2015, Onawumi et al., 2013 and Ihemeje et al., 2010).
MATERIALS AND METHODS
Experimental Site
The
study was carried out at the rabbit unit of Department of Animal Production,
The Oke-Ogun Polytechnic Saki (TOPS). The pen was
adequately and thoroughly cleaned by removing the cobweb, rabbit waste and the
cage was thoroughly disinfected with the use of formalin 2 days before the
arrival of the experimental animals.
Experimental Animal
and Management
16
matured male rabbit (bucks) were obtained from a reputable farm in Saki, Oyo
state, Nigeria and they were weighed individually and distributed randomly
(Completely randomized design was used) into four experimental treatments with
4 bucks to each of the treatment. They were reared in cage system under similar
environmental condition. The animals were acclimatized for two weeks; they were
given prophylactic treatment which includes deworming and antibiotics
administration. The bucks were weighed at the beginning of the study to obtain
their initial body weight and they were individually weighed into the nearest
gram at weekly intervals
Experimental diet
The
experiment comprised of four treatments viz, 0% AWNP,
1.5% AWNP, 2.0% AWNP and 2.5% AWNP as supplement in the bucks’ diet.
Table 1: Gross
formulation for the experiment
Ingredient
0% AWNP
1.5% AWNP
2.0% AWNP
2.5% AWNP
Maize
20.00
20.00
20.00
20.00
Cornbran
50.50
50.50
50.50
50.50
Soya
meal
20.00
20.00
20.00
20.00
PKC
05.00
05.00
05.00
05.00
Premix
00.25
00.25
00.25
00.25
Bone
meal
03.00
03.00
03.00
03.00
Salt
00.25
00.25
00.25
00.25
Fish
meal
01.00
01.00
01.00
01.00
Walnut
powder
0.00
1.00
1.30
1.65
Total(Kg)
100
100
100
100
Calculated
nutrient
Crude
protein
17.00
17.00
17.00
17.00
Metabolizable
energy(ME)(kcal/kg)
2502.42
2505.42
2505.42
2505.42
Crude
fiber (%)
11.36
11.36
11.36
11.36
Table: 2 Proximate composition of African walnut powder
Parameter
%
Moisture
31.40
Ash
6.01
Crude
fiber
8.66
Crude
protein
28.85
Carbohydrate
Energy
21.30
234.57kcal
(Nneromet al.,
2013)
Sources and
Processing of African Walnut Powder
T. conophorum (African walnut) was obtained from Osun state Nigeria. The walnuts were sliced into smaller
size and it was dried. The dried nut was then milled using pestle and mortal to
obtain AWNP powder. The AWNP was kept in airtight container till the period of
usage.
Materials Used in
Semen Collection and Semen Processing
An
artificial vagina was made for the collection of the semen from the buck
without causing any injury to the animal. Ejaculate were
submerged into 370C water bath before evaluation. The semen samples
were examined for consistency, color, spermatozoa motility, volume, mass
activity, morphology and concentration. The ejaculate volume was determined by
collecting semen into a graduated tube. Color was evaluated by visual observation.
The consistency was scored as 1= watery cloudy, 2=milky, 3=thin creamy,
4=creamy and 5=creamy-grany (Shamsudeenet al., 1994). To evaluate mass
activity (wave motion) a drop of (2ul) of undiluted semen was placed on a
pre-warmed slide at 370C, without a cover slip and examined under
phase contrast microscope (100X magnification) (Nikon, Eclipes,
E200, Japan). The mass activity was scored 0 = no motility, 1 = weak
spermatozoa with weak movement (<20%), 2 = some motile spermatozoa (20 -
40%) without wave movement, 3 = slow wave movement (40 - 60%) with motile
spermatozoa, 4 = rapid wave movement without whirl pool (60 - 80%) with motile
spermatozoa and 5 = very rapid wave movement with clear whirlpools (>80%)
with motile spermatozoa (Avudiet al., 2004). The spermatozoa motility was estimated subjectively
by preparing a wet mount of diluted semen (normal saline) by placing a 5ul drop
of fresh semen under coverslip with magnification of 200X using a phase
contrast microscope (Nikon, Eclipse, E200, Japan). The spermatozoa quality
assay (motility, viability, acrosome integrity, morphology, and pH) were
performed. A drop of semen sample was placed on a pre-warmed glass slide and a
cover slip. Progressive motility was assessed with phase contrast microscope at
X 400 at 370C. The motility was examined at least with 200
spermatozoa selected randomly from a minimum of four microscopic fields. Eosin-nigrosin stain was used to examine the morphologically
normal spermatozoa (Evans and Maxwell, 1997). Spermatozoa viability was
evaluated using eosin-nigrosin stain (Evan and
Maxwell, 1987). The acrosome integrity (normal apical ridges) was determine
from semen smear with nigrosin-eosin examined under
phase contrast microscope 1000x magnification under oil immersion (Yildizet al.,
2000). A total of 200 spermatozoa were counted in a least four microscopic
field.
Statistical Analysis
All
data collected were subjected to analysis of variance (ANOVA) of completely
randomized design using SAS (1999) package and the means were separated using
Duncan multiple range test of the same software at 5% level of significance.
RESULTS AND
DISCUSSION
Semen volume of the
experimental bucks ranged from 0.16 - 0.38ml and this is lower than the semen
value reported by Iwuji and Herbert (2012) who
reported the range of 0.60 - 0.78ml, the disparity might result from breed
differences. This result shows that African walnut powder did not significantly
(p>0.05) influence mass activity of the spermatozoa of rabbit buck and is in
agreement with the report of Ajayiet al., (2009) who fed rabbits with
blood sun-flower meal. However, the mass activity value that ranged from 76.66
– 85.0% is higher than the value (66 – 77%) obtained by Ajayiet al,.
(2009). Similarly, the result of the study is much higher than those reported
by Abu and Uchende (2010) who study anti
spermatogenesis effect of aqueous and ethanolic
extract of Hymenocardiaacida
stem bark on sperm activities of laboratory rodent and obtain value of 23 –
28%. The present investigation shows significant difference (p<0.05) in the
progressive motility of bucks treated with different levels of AWNP which
ranges from 73.33 – 83.33%. In farm animals, diverse studies reveal a good
correlation between several sperm quality parameters (progressive motility and
velocity) according to the straight path, damage acrosome and apoptotic cells
and scores of penetration test (Richardson et
al; 2011; Martins-Rodriguez et al
2012). Animals treated with 0.0% supplementary levels of walnut powder recorded
the lowest mean value of percentage live spermatozoa (80%). Result of this
experiment also demonstrated that 1.5%, 2.0%, 2.5% supplementary level of
walnut powder to rabbit buck achieve the maximum percentage of live spermatozoa
(90%).Table 3 showed significant
difference (p<0.05) in the acrosome integrity of the bucks across the
treatments with values ranging from 83.33 (0%AWNP) to 90% (2.0 and 2.5%AWNP).
This is similar to the work of Roca et al
(2005) who registered similar value for acrosome integrity (90.0%) in hybrid
male rabbit. However, Lavaraet al (2005) observed lower acrosome integrity (80.2%). This means
that AWNP has significant effect on the acrosome integrity of rabbit bucks on
2.0% and 2.5% which has the highest values. Reaction time (seconds) as an index
for libido (sexual drive) showed no significant difference (p>0.05) among
the experimental animals. Reaction time value ranged from 50.33(0.0%AWNP) to
172 (2.5%AWNP). This is not in agreement with the studies by Iwuji and Herbert (2012) who reported the range of 11.00 -
27.75s in animals treated with Garcina kola. No
significant difference (p>0.05) was observed in the morphology of bucks fed
supplementary walnut powder. Morphometric parameters of rabbit spermatozoa
could be important in predicting buck fertility since they remain fairly
constant with time. The pH obtained in table 3 tends to maintain an upward
trend with the level of African walnut powder across the treatment. According
to Meachem (2002) semen has a high buffering capacity
much higher ability than that of most other fluid in the body but the buffering
ability tends to decrease shortly after ejaculation as a result of loss of
carbon dioxide by sperm cell. The comparable semen pH value in all treatment
groups was an indication that African walnut powder supplementation in buck
diet up to 2.5% did not affect the semen acid/ alkaline equilibrium. The
recommended pH of rabbit semen is 5.40 – 9.60 (Peters and Sally 1976). pH result from this study (7.30 – 8.00) falls within the
recommended range, this reveals that administration of AWNP does not alter the
semen pH negatively.Triiodothyronine
and thyroxine ranges from 12.98 – 17.02 and 23.19 –
25.47 respectively. Table 4 shows that there is significant difference
(p<0.05) among rabbit buck fed supplementary walnut powder. Triiodothyronine is a thyroid hormone that plays vital
roles in reproduction, body’s metabolic rate, heart and digestive functions,
muscle control, brain development and function, and the maintenance of bones (Maria
et al., 2021).
Thyroxine is the main hormone
secreted into the blood stream by thyroid gland. It is the inactive form and
most of it is converted to an active form called triiodothyronine
by organs such as the liver and kidney. The thyroid hormone plays vital roles
in regulating and digestive function, muscle control, brain development and
maintenance of bone. Thyroxine values obtained from
this study were lower than the result of who reported Chieriatoet al., (1997) 32.4- 38.0 ng/ml.
Table 4 shows significant
different (p<0.05) in testosterone of rabbit buck fed different levels of
supplementary walnut powder where the highest recorded values was observed in
buck group placed on 2.0% ANWP (42.59). Sajjadet al (2007) reported that the level of
blood serum testosterone is correlated with scrotal circumference and semen
volume correlated with scrotal circumferences. Testosterone is thought to
regulate sex drive (libido), bone mass, fat distribution, and muscle mass and
strength, also the production of red blood cell and sperm cells. A small amount
of circulatory testosterone is converted to estradiol, a form of estrogen. As
anima aged, they often make less testosterone, and though they produce less
estradiol as well. Thus changes often attributed to testosterone deficiency
might be partly or entirely due to accompanying decline in estradiol.
Testosterone is produced by the interstitial cell of the testis and necessary
for the completion of spermatogenesis. The cortisol ranges from 65.33 (0.0%
AWNP) – 67.50 (2.5% AWNP). These shows that AWNP has effect on cortisol hormone
on rabbit buck. Cortisol is also called stress hormone because of its
connection to the stress response; however, cortisol is much more than just a
hormone release during stress. Cortisol is one of the steroid hormones and is
made in the adrenal gland. Most cells within the body have cortisol receptors.
Secretion is controlled by the hypothalamus, the pituitary gland and the
adrenal gland a combination of glands often refers to as HPA axis.
Table 3: Semen
characteristic of rabbit bucks fed AWNP
Parameters
0.0% AWNP
1.5% AWNP
2.0% AWNP
2.5% AWNP
SEM
Volume
(ml)
0.33ab
0.38a
0.16b
0.30ab
0.03
Mass
activity (%)
80.00
80.00
76.66
85.00
2.40
Progressive
motility (%)
80.00ab
73.33b
73.33b
83.33a
1.44
Live:dead (%)
80.00ab
90.00a
90.00a
90.00a
0.00
Acrosome
integrity (%)
83.33b
86.66ab
90.00a
90.00a
1.07
Reaction
time (seconds)
50.33
95.00
101.33
172.00
40.14
Morphology
(%)
81.66
80.00
83.33
80.00
3.29
pH
8.00a
7.30c
7.50b
7.50b
0.00
Semen
color
a,b,c,d mean having
different superscript letters in the same row differ significantly (p<0.05)
Table 4: Hormonal
profile of rabbit bucks fed AWNP
Parameters
0.0%
1.5%
2.0%
2.5%
AWNP
AWNP
AWNP
AWNP
SEM
Triiodothyronine (pg/ml)
15.77ab
16.03ab
17.02a
12.98b
0.62
Thyroxine (pg/ml)
23.60b
23.55b
23.19b
25.47a
0.31
Testosterone
(pg/ml)
39.95c
39.54c
42.59a
41.03b
0.12
Cortisol
(pg/ml)
65.33b
66.42ab
66.49ab
67.50a
0.29
a,b,c,d mean having
different superscript letters in the same row differ significantly (p<0.05)
CONCLUSION
To
improve bucks semen characteristic and fertility, 2.0% level of AWNP is
recommended based on the result of progressive motility, morphology, acrosome
integrity and testosterone which had an increase in value more than the control
which was lower compare to other percentage levels of AWNP.
Hence AWNP could be supplemented into the
diet of buck at 2% level to improve fertility and productivity of bucks.
Disclosure of
Conflict of Interest:
The
authors declare no conflicts of Interest regarding the publication of this
paper.
Competing interests:
The
authors declare no competing of Interest regarding the publication of this
paper.
Author’s Contribution:
Amao, Emmanuel Ayodele:
Conceived and designed the experiment and manuscript preparation.
Amao, OyetounDunmola: Made available animals and feed for the animals
throughout the period of experiment.
Tijani, HaulatTemitope: Management of the animals and data collection.
Agbaye, Folorunso
Peter: Statistical analysis and interpretation of the results.
Afolabi, Ojo
Samuel: Laboratory analysis of blood samples.
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Cite this Article: Amao, EA; Amao, OD; Tijani,
HT; Agbaye, FP; Afolabi,
OS (2024). Semen Characteristics of Rabbit Bucks Fed
Supplementary Walnut Powder Tetracarpidiumconophorum.Greener Journal of Agricultural Sciences, 14(1): 1-7.
