The Effects of Discarded Cocoa (Theobroma cacao) Seed Meal Based Diets on Semen Traits, Testicular Morphometry and Histomorphology of Rabbit Bucks

Abstract

A 10-weeks study was carried out to evaluate the effect of dietary intake of discarded cocoa seed meal
(DCSM) on reproductive characteristics of rabbit bucks. A total of 36 mixed-bred rabbit bucks of 20 weeks old and
having a mean weight of 1384.34 ± 283.34g were randomly assigned to 4 treatment (T) groups of 9 rabbit bucks each
replicated 3 times using a one-way analysis of variance in a completely randomized design (CRD). Water and feed
were made available ad-libitum to the bucks. Diets containing 0, 15, 30 and 45% DCSM were compounded and fed
to bucks in T1
, T2
, T3
and T4
, respectively. Prior to semen collection, rabbit bucks were trained three times per week
for semen collection with an artificial vagina (AV). At the end of the 10 weeks, semen collection and evaluation were
carried out. Thirty six (36) rabbit bucks were slaughtered and testes collected and dissected for testicular morphometry
and histomorphology evaluation. Semen colour showed milky white in all groups although T4
appeared creamy white.
There were significant (P<0.01) differences among treatment groups of rabbits in all the semen traits except in semen
volume. Semen pH showed significant (P<0.01) variations with semen of bucks in T4
having the highest value (8.67).
Sperm motility, sperm concentration, live and normal sperms in decreased with an increase in dietary DCSM whereas
non-motile, dead and abnormal sperms increased with an increase in dietary DCSM. Testicular morphometric
measurements and histomorphological evaluations were similar in all treatments (P>0.05). This investigation depicts
that DCSM inclusion above 15% affected semen quality negatively even though results on testicular morphometric
traits and histology appeared non-significant.
Keywords | Rabbit bucks, Semen traits, Sperm characters, Testicular morphometry, Histomorphology, Discarded
cocoa seed meal

Full text

Advances in Animal and Veterinary Sciences
June 2022 | Volume 10 | Issue 6 | Page 1245
INTRODUCTION
Rabbits (Oryctolagus cuniculus) have the potential to
ll the gap in human protein supply, particularly
in developing and underdeveloped countries. ey are
eective feed converters, because they can withstand
up to 30 percent crude ber compared to 10 percent in
most poultry species (Egbo et al., 2001). While rabbits
can be raised on a forage-based diet, according to Amao
and Showunmi (2016), decient and unbalanced grass
quality, as well as seasonal availability of forage, are major
constraints to successful rabbit farming. Other immense
potentials of rabbit include proverbial prolicacy, high
growth rate, short gestation period, high genetic selection
ability, relatively low cost of production, economic
utilization of space and high nutritional quality (Biobaku
and Oguntona, 1997; Hassan et al., 2012). In addition,
the consumption of rabbit meat is free from religious and
cultural biases.
Research Article
Abstract | A 10-weeks study was carried out to evaluate the eect of dietary intake of discarded cocoa seed meal
(DCSM) on reproductive characteristics of rabbit bucks. A total of 36 mixed-bred rabbit bucks of 20 weeks old and
having a mean weight of 1384.34 ± 283.34g were randomly assigned to 4 treatment (T) groups of 9 rabbit bucks each
replicated 3 times using a one-way analysis of variance in a completely randomized design (CRD). Water and feed
were made available ad-libitum to the bucks. Diets containing 0, 15, 30 and 45% DCSM were compounded and fed
to bucks in T1, T2, T3 and T4, respectively. Prior to semen collection, rabbit bucks were trained three times per week
for semen collection with an articial vagina (AV). At the end of the 10 weeks, semen collection and evaluation were
carried out. irty six (36) rabbit bucks were slaughtered and testes collected and dissected for testicular morphometry
and histomorphology evaluation. Semen colour showed milky white in all groups although T4 appeared creamy white.
ere were signicant (P<0.01) dierences among treatment groups of rabbits in all the semen traits except in semen
volume. Semen pH showed signicant (P<0.01) variations with semen of bucks in T4 having the highest value (8.67).
Sperm motility, sperm concentration, live and normal sperms in decreased with an increase in dietary DCSM whereas
non-motile, dead and abnormal sperms increased with an increase in dietary DCSM. Testicular morphometric
measurements and histomorphological evaluations were similar in all treatments (P>0.05). is investigation depicts
that DCSM inclusion above 15% aected semen quality negatively even though results on testicular morphometric
traits and histology appeared non-signicant.
Keywords | Rabbit bucks, Semen traits, Sperm characters, Testicular morphometry, Histomorphology, Discarded
cocoa seed meal
Luke Chukwudi ALi*, NNANNA ephrAim ikeh, Bright Chigozie AmAefuLe, AmArAChi LiNdA
oBiNNA, NduBuisi sAmueL mACheBe
e Eects of Discarded Cocoa (eobroma cacao) Seed Meal Based
Diets on Semen Traits, Testicular Morphometry and Histomorphology
of Rabbit Bucks
Received | January 03, 2022; Accepted | March 14, 2022; Published | May 15, 2022
*Correspondence | Ali LC, Department of Animal Science, Faculty of Agriculture, University of Nigeria, Nsukka; Email: aliluke230@gmail.com
Citation | Ali LC, Ikeh NE, Amaefule BC, Obinna AL, Machebe NS (2022). e eects of discarded cocoa (eobroma cacao) seed meal based diets on semen
traits, testicular morphometry and histomorphology of rabbit bucks. Adv. Anim. Vet. Sci. 10(6): 1245-1254.
DOI | http://dx.doi.org/10.17582/journal.aavs/2022/10.6.1245.1254
ISSN (Online) | 2307-8316
Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.
is article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.
org/licenses/by/4.0/).
Department of Animal Science, Faculty of Agriculture, University of Nigeria, Nsukka

Advances in Animal and Veterinary Sciences
June 2022 | Volume 10 | Issue 6 | Page 1246
Nutrition has a signicant impact on the reproductive
eciency of rabbit bucks (Amao and Showunmi, 2016).
Over the years, animal scientists has primarily focused
on identifying less expensive alternative feedstu that
are readily available and do not compete with the dietary
requirements of man (Egbunike, 1997). In a study of rabbit
development on tropical feed sources, it was reported that
feeding only grasses to rabbits was unsuitable due to its low
digestibility of about 10% (Cheeke and Raharjo, 1988).
Also, the availability of conventional feed ingredients of
protein and energy sources as concentrates for rabbit diets
is heavily limited. Grain production in both developed and
developing countries are primarily for human consumption,
resulting in extremely high feed prices (Ogbuewu et
al., 2008). For instance, in developing countries like
Nigeria, the key restriction to livestock development is
the inconsistency in the availability of good quality feed
in large amount throughout the year. As a result, the use
of unconventional feed resources becomes extremely
important and is promoted (Amao and Showunmi,
2016). In line with the afore-mentioned, Onifade and
Tewe (1993) noted that, tropical agricultural extraction
industries provide a large number of by-products with
untapped feeding potential. So far, certain agricultural by-
products and plants have been used in animal production
with varying degrees of success.
e rabbit buck’s reproductive system produces
spermatozoa that are deposited into the female genital
system. Spermatozoa are formed in the testes’ seminiferous
tubules and then transferred to the epididymides via the
rete testes, where they are deposited until maturity (Amao
et al., 2019). Spermatogenesis in rabbits takes 48 days
(Amann, 1982) and includes a sequence of successive
mitotic divisions, two meiotic divisions, and spermiogenesis.
However, some chemical factors like reactive oxygen species
or chemotherapeutic agents can interrupt this process,
resulting in changes in the consistency of the sperm or
the structure of the testicles (Iyama and Wilson, 2013;
Chenoweth and Lorton, 2014). Cocoa and its derivatives
(chocolate, cocoa cake or butter) are currently investigated
as possible livestock feed ingredients.
Discarded cocoa seed meal (DCSM) are agricultural
by-products that are often discarded in or around cocoa
factories or communities in Southern Nigeria, resulting in
environmental contamination as a result of inappropriate
composting (Sukha, 2003; Nwanna and Fashae, 2010).
e use of cocoa by-products as a feed option for animal
feeding has alleviated the disposal problems faced by cocoa
factories (Figueroa et al., 2019). When used as alternative
feed ingredient, feed formulation cost is reduced, and the
negative eects of climate change caused by pollution is
properly mitigated.
Investigations on the benet of the use of cocoa has
revealed that it has some therapeutic eect because of
their high contents of antioxidants primarily avonoids,
epicatechin, catechin, and procyanids (Visioli et al., 2000).
Other contents are nitrogenous compounds, minerals,
and methylxanthine (Greer et al., 2001). Even though,
methylxanthine (caeine and theobromine) are considered
as anti-nutritional factors, available report has shown that
it depends on the processing method and the quantity fed
to the animal (Rios et al., 2003). Cocoa powder has been
shown to have a variety of physiological and biological
eects in vitro, such as antioxidant, anti-inammatory,
antiviral, antibacterial, vasodilatory, anticancer, and
anti-ischemic eects, as well as improved endothelial
cell functions, which enhance cardiovascular processes
(Steinberg et al., 2003; Prochazkova et al., 2011). e
feeding of dietary antioxidants to male animals have been
reported to aid in minimizing the damaging eect of free
radicals to sperm cells and other biological functions in the
body system. us, chocolate (a cocoa product) has been
suggested for males with fertility problems (Yildirim et al.,
2014). eobromine is of primary concern in the use of
cocoa and its products as animal feed components. It has
been shown to have a detrimental eect on reproduction
in a variety of animal species, including rats (Tarka et al.,
1981), egg laying birds (Odunsi and Longe, 1995) and
rabbits (Adeyina et al., 2010).
Considering the benet of using cocoa by products/waste
as alternative feed materials, this experiment was therefore
carried out to evaluate the eect of feeding discarded cocoa
(eobroma cacao) seed meal diets on semen traits, testicular
morphometry and histomorphology of rabbit bucks.
MATERIALS AND METHODS
ethiCAL ApprovAL
is study was conducted noting the recommended research
ethics for scientic researches involving animal subjects:
code 5.4.2 and rabbit bucks were handled according the
principles of Animal Experimentation Ethics Committee
University of Nigeria, Nsukka (2013).
experimeNtAL site
is research was carried out at the Rabbit Unit of the
Animal Science Teaching and Research Farm, University
of Nigeria, Nsukka in Enugu State, South Eastern
Nigeria. Nsukka lies in the derived Savannah region and
is located at longitude 7° 21’39”E and latitude 6° 51’39”N
(Ihinegbu et al., 2019) at an elevation of 456m above sea
level (Onyenucheya and Nnamchi, 2018). e climate is
a humid tropical setting with temperature ranging from
22.9 to 27.0 % (Phil-Eze, 2012) and a relative humidity
range of 54.5 to 84.32%. In September, the peak of rainfall
is 108.55mm and January and December being the lowest

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June 2022 | Volume 10 | Issue 6 | Page 1247
with no observed rainfall (Onyenucheya and Nnamchi,
2018). e light cycle of the study area was 11 hours day
light.
mANAgemeNt of experimeNtAL ANimAL
e discarded cocoa seed meal (DCSM) used in the diet
formulation was collected in and around cocoa powder
manufacturing factories in Osun, Ogun and Oyo states
in Nigeria. e percentage and proximate composition of
the experimental ration is presented in Table 1. A total
number of thirty six (36) mixed-bred rabbit bucks of
an average weight of 1384.34 ± 283.34g were randomly
assigned to 4 treatments (T) diets according to the level
of inclusion of discarded cocoa seed meal in the diet. Each
treatment contained 9 rabbit bucks and replicated three
times with each replicates containing 3 bucks. Bucks were
held in individually washed and disinfected hutches with
a dimension of 4ft x 2ft x 2ft and a wire-screened oor
that allowed faeces and urine to pass through. Feeders and
water troughs containing feed and water, respectively were
provided to the bucks and allowed ad-libitum method of
intake. On arrival, bucks were allowed a 1-week period to
undergo acclimatization followed by an 8 weeks of feeding
of the test ingredients. e rabbit bucks were also provided
with equal supplemental forages plus the experimental
concentrates.
Table 1: Percentage (%) and proximate composition of the
experimental diets containing DCSM at various levels.
Feedstu T1T2T3T4
Maize 39.84 29.48 19.16 8.84
Wheat oal 26.56 19.66 12.77 5.89
Soya bean meal 11.84 12.74 13.63 14.51
Palm kernel cake 17.76 19.12 20.44 21.76
Cocoa seed meal 0.00 15.00 30.00 45.00
Limestone 3.00 3.00 3.00 3.00
Lysine 0.25 0.25 0.25 0.25
Methionine 0.25 0.25 0.25 0.25
Iodize salt 0.25 0.25 0.25 0.25
Vit-Min premixes 0.25 0.25 0.25 0.25
Total
proximate
100.00 100.00 100.00 100.00
Moisture 5.65 4.80 4.85 4.85
Crude protein 15.78 15.92 16.02 16.51
Crude bre 9.55 10.21 10.39 10.43
Ash 9.90 9.50 9.30 9.25
Ether extract 4.63 4.84 5.02 5.15
Nitrogen free extract 54.49 54.73 54.42 53.81
Metabolizable energy
(Kcal/kg)
2570.24 2514.75 2491.69 2490.54
semeN evALuAtioN
irty six (36) semen samples were collected after 10
weeks for evaluation from bucks in all the treatments. Prior
to semen collection, bucks were all trained for using an
articial vagina (AV). e AV used was constructed locally
with rubber rings, graduated collection tube, rubber funnel
and latex inner liner (Figure 1). e consistency of the
collected semen showed normal viscosity according to the
method of Comhaire and Vermeulen (1995). e colour
of the semen was determined by a careful visual appraisal
according to Campos et al. (2014); and the volume was
recorded via the use of calibrated tube attached as part of
the articial vagina. Sperm concentration was determined
in a counting chamber with diluting uid containing
sodium bicarbonate, formalin and distilled water in a ratio
of 1:20 using the improved Neubauer haemocytometer
slide according to the method of Ochei and Kolhatkar
(2000). e pH was measured using a specially treated
calibrated paper blot that changes colour according to the
pH of the semen (Comhaire and Vermeulen, 1995). Sperm
viability was estimated using the improved one step eosin-
nigrosin staining techniques according to Bjorndahl et al.
(2003). us, live sperms will exude the eosin-nigrosin
while dead sperm cells will not. Motility was determining
according to World Health Organization (1992) methods.
Samples from dierent treatments were dropped on a glass
slide, covered with a slip and viewed under a microscope
using x10 and x40 objectives. A minimum of 5 microscopic
elds were assessed to evaluate sperm motility. e % of
sperm motility was analyzed for actively motile and non-
motile distinguished by the movement of the sperm cells.
Sperm morphology determined by making a smear as
blood lm by placing a small drop of well mixed seminal
uid on a clean glass slide smearing it to a feather edge and
quickly xed (while still wet) in a cytological xative such
as 90% alcohol. e smear was stained with Giemsa. e
stained smear was examined under microscope using x100
objective with immersion oil. About 100 spermatozoa with
tail was counted and the percentage of abnormal forms was
noted (Ochei and Kolhatkar, 2000).
testiCuLAr morphometry of rABBit BuCks
After semen collection, bucks were slaughtered using the
procedure described by Blasco and Ouhayoun (1996).
rough the ventral region of the abdomen, the sacriced
bucks were dissected using a sharp blade to expose both
right and left testes. e organ were freed o other
connective tissues leaving behind only the testis and the
attached epididymis. Testes weight and paired testes
weight were determined using a Mettler Toledo sensitive
scale. Testes length and circumference were measured
with the use of vernier caliper and a tape. e volume of
the testes was determined using Archimedes’ principle of
water displacement with a measuring cylinder in milliliter

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June 2022 | Volume 10 | Issue 6 | Page 1248
(ml). Relative testes weight (%) and testes density (g/ml)
were calculated using the formulas described by Amao et
al. (2013).
Figure 1: e constructed articial vagina for rabbit bucks.
histomorphoLogiCAL exAmiNAtioN of rABBit
BuCks
For histomorphological test, 0.4g part of the harvested
testes were collected. e samples were xed in 10%
phosphate buered formalin for a minimum of 48 hours.
e tissues were subsequently trimmed, dehydrated in four
grades of alcohol (70%, 80%, 90% and absolute alcohol),
cleared in three grades of xylene and embedded in molten
wax. On solidifying, the blocks were sectioned, 5µm
thick with a rotary microtome, oated in water bathe and
incubated at 60°C for 30 minutes. e 5µm thick sectioned
tissues were subsequently cleared in three grades of xylene
and rehydrated in three grades of alcohol (90%, 80% and
70%). e sections were then stained with Haematoxylin
for 15 minutes. Blueing was done with ammonium
chloride. Dierentiation was done with 1% acid alcohol
before counterstaining with Eosin. Permanent mounts
were made on degreased glass slides using a mountant;
DPX. e prepared slides were examined with a Motic™
compound light microscope using x40 objective lens. e
photomicrographs were randomly taken using a Motic™
5.0 megapixels microscope camera at x400 magnications.
e method applied was according to Zahid et al. (2002)
and Ansa et al. (2017).
stAtistiCAL ANALysis
All data were analyzed by a one-way analysis of variance
(ANOVA) in a completely randomized design (CRD).
Statistically (P<0.05) dierent means among treatment
groups were separated according to the procedures of
Duncan’s New Multiple Range Test (Duncan, 1955).
RESULTS AND DISCUSSION
semeN trAits of rABBit BuCks
e results of the eect of varying dietary levels of discarded
cocoa (eobroma cacao) seed meal on semen traits of rabbit
bucks are presented in Table 2.
An evaluation of the semen appearance showed that the
inclusion of DCSM did not change the colour of the semen
as there as there were milky white except for those in T4
that appeared creamy white. Semen pH was signicantly
(P<0.01) higher for the DCSM treatment groups
compared with semen pH of those in the control group.
Higher semen pH was shown for rabbit bucks in T4 (45%
DCSM) diet and it varied from those of rabbit bucks in
T2 and T3 groups. However, semen volume of rabbit bucks
were similar (P<0.05) among treatment groups. Motility
of spermatozoa and percentage live sperm in semen
in the control, T2 and T3 were higher and did not vary
(P>0.05) among treatments. us, at 45% DCSM dietary
inclusion, the motility of sperm declined signicantly
(P<0.05) compared with others. Sperm concentration and
percentage normal sperm were higher and similar (P>0.05)
at 0 and 15% DCSM inclusion. A signicant decline in
these parameters were shown at 30% and 45% levels of
DCSM. e percentage of non-motile and dead sperms
in the semen had no signicant (P>0.05) changes as the
level of DCSM was increased from 0 to 30% inclusion
in the diet, except at 45% where it signicantly increased
(P<0.05). e percentage of abnormal sperm showed no
signicant (P>0.05) dierence at 0 and 15% DCSM,
however, the number of abnormal sperm began to rise
signicantly from 30% to 45% DCSM dietary inclusion
(P<0.05).
testiCuLAr morphometry ANd histomorphoLogy
of rABBit BuCk
e results of the eect of varying dietary levels of
discarded cocoa (eobroma cacao) seed meal on the
testicular morphometry of rabbit bucks are presented in
Table 3. e results on testicular morphometry of rabbit
bucks fed DCSM diet were not signicantly (P>0.05)
dierent among the treatment groups except in T4
with spermatogonial disruption. Figure 2 presents the
micrograph of the histomorphology of rabbit buck testes fed
varying dietary levels of discarded cocoa (eobroma cacao)
seed meal. It showed that no testicular histomorphological
dierences were observed among dierent treatments
expose to DCSM. e sections of the testes presented in
all the experimental treatments showed normal testicular
histomorphology. us, the magnied sections of the testes

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June 2022 | Volume 10 | Issue 6 | Page 1249
Table 2: Semen evaluation of rabbit bucks fed discarded cocoa seed meal based diets.
Parameters T1
(0% DCSM)
T2
(15% DCSM)
T3
(30% DCSM)
T4
(45% DCSM)
P. value
Semen appearance Milky white Milky white Milky white Creamy white
Semen pH 6.17 ± 0.17c7.00 ± 0.00b6.83 ± 0.17b8.67 ± 0.33a0.000**
Semen volume (ml) 0.83 ± 0.09 0.77 ± 0.07 1.10 ± 0.15 0.77 ± 0.12 0.190NS
Sperm motility (%) 76.67 ± 3.33a76.67 ± 3.33a70.00 ± 0.00a46.67 ± 3.33b0.000**
Sperm Con. (x106/ml) 84.67 ± 9.26a89.33 ± 5.21a57.67 ± 1.45b39.67 ± 9.35b0.003**
Non-motile sperm (%) 23.33 ± 3.33b23.33 ± 3.33b30.00 ± 3.33b53.33 ± 5.77a0.002**
Live sperm (%) 86.67 ± 3.33a86.67 ± 3.33a80.00 ± 0.00a53.33 ± 3.33b0.000**
Dead sperm (%) 13.33 ± 3.33b13.33 ± 3.33b20.00 ± 0.00b46.67 ± 3.33a0.000**
Normal sperm (%) 76.67 ± 3.33a76.67 ± 3.33a63.33 ± 3.33b36.67 ± 3.33c0.000**
Abnormal sperm (%) 23.33 ± 3.33c23.33 ± 3.33c36.67 ± 3.33b63.33 ± 3.33a0.000**
abc: Means ± SEM on the same row with dierent superscripts are signicantly dierent (P ≤ 0.05 or P ≤ 0.01); Con.: Concentration.
Table 3: Testicular morphometry of rabbit bucks fed discarded cocoa seed meal based diets.
Parameters T1
(0% DCSM)
T2
(15% DCSM)
T3
(30% DCSM)
T4
(45% DCSM)
P. Value
Live weight (kg) 2.10 ± 0.10 2.00 ± 0.20 2.05 ± 0.15 1.8 ± 0.20 0.641NS
Testicular characteristics
Right testis length (cm) 3.20 ± 0.00 3.25 ± 0.05 3.20 ± 0.10 3.10 ± 0.20 0.824NS
Left testis length (cm) 3.30 ± 0.00 3.25 ± 0.05 3.25 ± 0.15 3.10 ± 0.20 0.724NS
Right testis circumference (cm) 3.90 ± 0.30 3.95 ± 0.35 4.15 ± 0.05 3.65 ± 0.05 0.565NS
Left testis circumference (cm) 3.95 ± 0.25 4.05 ± 0.35 4.20 ± 0.00 3.65 ± 0.15 0.465NS
Right testis weight (g) 2.75 ± 0.25 2.95 ± 0.25 2.45 ± 0.05 2.10 ± 0.40 0.269NS
Left testis weight (g) 2.55 ± 0.15 2.35 ± 0.35 2.60 ± 0.10 2.10 ± 0.50 0.696NS
Paired testes weight (g) 5.25 ± 0.35 4.80 ± 0.60 5.25 ± 0.05 4.25 ± 0.85 0.578NS
Right testis volume (ml) 3.00 ± 0.00 3.00 ± 0.00 3.00 ± 0.00 2.25 ± 0.50 0.479NS
Left testis volume (ml) 2.50 ± 0.50 2.50 ± 0.50 3.50 ± 0.05 2.50 ± 0.50 0.479NS
Paired testes volume (ml) 5.50 ± 0.50 5.50 ± 0.50 6.50 ± 0.50 5.00 ± 0.00 0.242NS
Relative testes weight (%) 0.25 ± 0.01 0.24 ± 0.01 0.26 ± 0.02 0.23 ± 0.02 0.586NS
Testes density (g/ml) 0.96 ± 0.03 0.87 ± 0.03 0.81 ± 0.07 0.85 ± 0.17 0.747NS
abc: Means ± SEM on the same row with dierent superscripts are signicantly dierent (P ≤ 0.05 or P ≤ 0.01).
showed that a normal architecture were observed in
control and treated groups and a clear and well separated
seminiferous tubules surrounded by the basement
membrane were shown. e only exception is found in T4
containing 45% DCSM with some degrees of disruption
of sperm cells.
e current study was carried out to determine various
signicant eect of DCSM on the reproductive aspect
of rabbit bucks especially on semen traits, testicular
morphometry and histomorphology. Results on semen
evaluation showed that the test ingredients had various
eects on the semen quality indices. e similar milky
whitish appearance observed across T1 to T3 are indications
that DCSM up to 30% inclusion did not cause any
malfunctioning or inammation of the reproductive tract
or the accessory sex glands (Banerjee, 2010). e only
dierence was experienced in T4 with a creamy whitish
appearance which might be as a result of the dark colour
of the DCSM. Kostic (1997) stated that the alkalization
process during cocoa processing will cause a darker colour
of cocoa powder than the natural state. us, Jon (2020)
reported that dietary changes may alter the semen colour in
some cases. Semen pH and volume recorded in this work are
within the normal range according to International Rabbit
Reproduction Group (2005) and similar to values observed
by Ajuogu et al. (2018) in rabbit bucks semen. e increase
in semen pH with an increase in DCSM is in line with
the report of Li et al. (2012) that the nature of increase in
pH can be explained by the fact that the manufacturing
process of cocoa powder involves an alkalization process
which generally increases the pH of natural product from

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June 2022 | Volume 10 | Issue 6 | Page 1250
5.2 and 5.6 to between 6.8 and 8.0 depending on the
condition of alkalization. e non-signicant dierences
observed for semen volume in this research is the same as
those reported by Ojezele et al. (2016).
Figure 2: Photomicrograph of the section of the testes from
T1, T2, T3 and T4. Note: seminiferous tubules are clearly
separated and dened with thick layered spermatogenic
cells (SC); testicular interstitium (TI) are clearly separated
from the spermatogenic cells; spermatogenic cells are
clearly distributed within the relatively spacious lumen
(L); double arrow shows widen interstitial areas; arrow
shows spermatogonial disruption (H & E x 400).
e results of sperm concentration, active, live and normal
sperms showed that with an increasing concentration of
DCSM above 15%, there exist a negative eect on the
semen quality. Parameters for poor semen traits (non-
motile, dead and abnormal sperm cells) increased with an
increase in DCSM above 15% inclusion level. us, this is
an indication that DCSM should be included in a minimal
level to avoid detrimental eect on the semen quality. e
results in this study is similar with the ndings obtained
by Okunola et al. (2017). According to Munier (2018), the
addition of fermented cocoa product up to 20% level led
to a decrease in both microscopic and macroscopic semen
quality of goat. In addition, our result does not corroborate
what Collodel et al. (2014) reported, that both chocolate
(cocoa product) and propolfenol exerted a protective
eect on spermatogenesis and sperm characteristics of
rabbit bucks. Spermatogenesis is under the control of the
pituitary-hypothalamic-gonadal axis, it is possible that
bioactive constituents bound in cocoa powder might have
debilitating eects at a certain level on this axis or the
testis (Friedman et al., 1979; Minji et al., 2015). It has been
reported that theobromine in cocoa powder or the extract
showed a negative eect on the sertoli cell leading to failed
release of spermatids (Wang et al., 1992), reduction in
the prostate and seminal vesicle (Funabashi et al., 2000),
hyperplasia of the atrophied Leydig cells (Weinberger et
al., 1978), decrease in the uid volume of the seminiferous
tubules (Wang and Waller, 1994) in rats.
Results on testicular morphometry showed no signicant
(P>0.05) variations across treatment groups. Similar non-
signicant (P>0.05) observations were obtained by Amao
and Showunmi (2016) and Velasquez-Pereira et al. (1998).
Results from this research contradicts the signicant
(P<0.05) dierent results reported by Ozung et al. (2019)
on rabbit bucks reproductive tract morphometry on some
parameters after dietary cocoa husk meal intake. Amao et
al. (2013) also reported signicant (P<0.05) dierences
when rabbit bucks were fed diets containing neem
(Azadirachta Indica A. Juss) plant meal. Although the
present study observed no signicant (P>0.05) dierences
in all parameters, but are within similar range of values
reported by Abu et al. (2013); Olarotimi et al. (2015);
Abu et al. (2016); Amao et al. (2019); Ozung et al. (2019).
According to Coulter (1980), testicular circumference
is positively related to testicular weight for species like
buck and ram and this report is similar with result of this
study. Furthermore, Ahemen et al. (2016) reported that,
testis length is among the indicators for the assessment
of spermatogenesis, also Perry and Petterson (2001) and
Akpa et al. (2013) noted that, length of the testis is a
measure of the size of that testis and all these are positively
related to sperm production.
e slight non-signicant increased values observed in this
study might be as a result of cocoa polyphenols. Reactive
oxygen species (ROSs’) are important components in
reproductive mechanisms and the production of ROS is a
normal physiological process in several organs including the
testes; however, excessive production can appear deleterious
to the testis or male fertility (Akiyama, 1999; Al-Gubory
et al., 2010). us, polyphenols as a strong antioxidant
tends to ameliorate oxidative damages of ROS (Rucker,
2009). From all indications, the non-signicant (P>0.05)
dierences reported based on the eect of DCSM on the
reproductive organ of rabbit buck testis, probably may not
have been observed morphometrically at the duration of
this study. e eect of DCSM did not cause hypertrophy
or hyperplasia in the testis of rabbit bucks throughout
the duration of this study and Zar (1996) stated that
increased sizes and weights of body organs are as a result of
hypertrophy and hyperplasia. Gans et al. (1980) and Tarka
et al. (1981) reported that the eect of theobromine on the
reproductive organs of male animals were found to be both
time and dose dependent.
e photomicrograph on the testes histology obtained
were similar with the ndings of Abdul-Rahman (2015).
e anatomy and histology of the reproductive systems of
rabbits according to Herrera et al. (2014) and Zamora et al.
(2014) showed a similar morphology as the ones obtained

Advances in Animal and Veterinary Sciences
June 2022 | Volume 10 | Issue 6 | Page 1251
in this study. Uzochukwu (2016), reported a no testicular
histomorphological dierences when African catsh
(Clarias garienpinus) were fed diets containing cocoa bean
meal. In other research, non-pathological changes were
reported by Shinkut (2015) after feeding rabbit buck with
dietary supplemental garlic (Allium sativum). At 200mg/
kg of diet, it was reported that unsweetened natural cocoa
powder had no toxic eect on the testicular histology
of Sprague-Dawley rats (Asiedu-Gyekye et al., 2016).
Conversely, Oguike et al. (2019) reported a varying degree
of degeneration in the histology of rabbit bucks fed Aspilia
africana leaf meal when compared to the control group;
and similar observation was also reported in wistar rats by
Asuquo et al. (2015). is outcome probably might be as a
result of theobromine inherent in cocoa powder which is
capable of causing debilitating eect on the testis (Smit,
2011). is ideology is supported by Eteng et al. (2005);
European Food Safety Authority (2008) and Park et al.
(2015).
CONCLUSIONS AND
RECOMMENDATIONS
It was concluded from this study that feeding rabbit
bucks DCSM diet suppressed semen quality even though
results from testicular morphometry showed no signicant
(P>0.05) variations. At 45% inclusion of DCSM,
spermatogonial disruption was also reported. eobromine
inherent in DCSM might be the resulting factor leading to
these poor result. eobromine eect could be minimized
by practicing detheobromination techniques in other to
reduce its content in any sample of cocoa powder. It is
recommended that dietary inclusion of DCSM in rabbit
bucks feeding must not exceed 15% inclusion level.
ACKNOWLEDGEMENTS
e authors would like to acknowledge their aliate
university and the anonymous reviewers for their
constructive and immense contributions towards
the improvement of the manuscript. We also thank
Oguejiofor, C. Innocent and Ukwueze, O. Chidera for
their contributions during the eldwork.
NOVELTY STATEMENT
To the best of our knowledge, this study is the rst
to decipher the eects of DCSM (eobroma cacao)
based diet in rabbit bucks with interest on semen, sperm,
testicular morphometry and histomorphological traits
in a tropical environment, and therefore can be used as
alternative feedstu.
AUTHOR’S CONTRIBUTION
Ali LC: Conceptualized the work, prepared rst and nal
drafts. Ikeh NE: Statistical analysis and literature review.
Amaefule BC: Literature review and data collection.
Obinna AL: Data collection and sorting. Machebe NS:
Reviewed manuscript and supervised the eld work.
CoNfLiCt of iNterest
e authors have declared no conict of interest.
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