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Animal Research International (2024) 21(2): 5518 – 5528 5518
ISSN: 1597 – 3115 ARI 2024 21(2): 5518 – 5528
www.zoo-unn.org
ANTIBACTERIAL AND ANTIOXIDATIVE EFFECTS OF
ALOE BARBADENSIS
LEAF METHANOL EXTRACTS ON QUALITY AND SPERMATOZOA
FERTILISING POTENTIAL OF EXTENDED RED SOKOTO BUCK SEMEN
1AGBAYE, Folorunso Peter and 2ALABA, Olufemi
1Department of Animal Production, Lagos State University of Science and Technology, Ikorodu,
Lagos State, Nigeria.
2Animal Physiology and Bioclimatology Unit, Department of Animal Science, University of Ibadan,
Ibadan, Oyo State, Nigeria.
Corresponding Author: Alaba, O. Animal Physiology and Bioclimatology Unit, Department of Animal
Science, Faculty of Agriculture, University of Ibadan, Ibadan, Oyo State, Nigeria. Email:
femialaba@gmail.com Phone: +234 705 463 4435
Received March 6, 2024; Revised May 19, 2024; Accepted May 21, 2024
ABSTRACT
A study was conducted to investigate the antibacterial and antioxidative effects of Aloe
barbadensis leaf methanolic extract (ABLME) on the quality and spermatozoa fertilising
potential of extended goat semen. 100 g of fresh A. barbadensis leaves were washed,
chopped, dried, ground and extracted with 95.0% methanol (ABLME). The ABLME was
added to egg yolk + sodium citrate (EYSC) diluent at 0.0, 0.5, 1.0, 1.5 and 2.0 g/L and used
to extend the goat semen. The extended semen samples were stored at 4oC and were
evaluated at 0, 24, 48 and 72 hours post storage. All treatments were replicated thrice in
a completely randomized design. Spermatozoa progressive motility (SPM), spermatozoa
liveability (SPL), normal spermatozoa (NSP), acrosome integrity (ACI), plasma membrane
integrity (PMI), secondary morphological abnormalities (SMA) and microbial load
(CFU/mL) were evaluated and analyzed using descriptive statistics and ANOVA at α0.05.
Significant reduction in microbial load was observed in samples with 2.0 g/L ABLME (6.83
± 4.80%) compared to control (27.50 ± 3.20%) at 48 hours. Reduced SMA was recorded
for 2.00 g/L ABLME (9.00 ± 0.90%) compared to control (13.67 ± 1.00%). Methanolic
extract (2.00 g/L) of A. barbadensis included in egg yolk + sodium citrate diluent gave
antibacterial activity to extended buck semen for up to 48 hours.
Keywords: Antibacterial, Anti-oxidant, Methanol Extract,
Aloe barbadensis,
Spermatozoa, Extended
semen
INTRODUCTION
Small ruminants are important genetic resources
and play a prominent role in the sustenance of
the livelihoods of impoverished families,
especially in the rural areas of tropical countries
(Adamu
et al
., 2020). The Red Sokoto goat (RSG)
or Maradi is the most predominant breed and
accounts for about 70% of Nigeria's total goat
population (Ademosun, 1994; Adamu
et al
.,
2020). Therefore, the genetic resources of this
important cold shock species need to be
exploited and this can be achieved through
assisted reproductive techniques such as artificial
insemination (AI) associated with semen
technology which allows the preserved male
genetic material to be used in females that are
isolated from the males (Morrell and Mayer,
2017; Santos and Silva, 2020). Meanwhile, the
use of semen extenders has been adjudged to be
necessary for semen preservation and storage
(Alaba
et al
., 2010). The composition of semen
extenders varies considerably depending on the
species and type of sperm preservation (i.e.,
Agbaye and Alaba 5519
Animal Research International (2024) 21(2): 5518 – 5528
cryopreserved or refrigerated). In general, the
semen extender contains nutrients (mainly
monosaccharides), cryoprotectant agents (CPAs)
as protection against, buffers, antioxidants and
antibiotics (Barbas and Mascarenhas, 2009).
Irrespective of the extender and the storage
conditions used, semen handling and
preservation negatively affect sperm quality.
Moreover, oxidative stress, which often arises
during semen storage, significantly reduces
sperm function and compromises sperm
fertilising ability by inducing oxidative damage to
proteins, lipids and nucleic acids (Ros-Santaell
and Pintus, 2021). Ordinarily, semen from a
healthy animal is expected to be free of
pathogenic contaminants, but contamination still
occurs during collection. The prepuce, foreskin
and the environment are likely sources of
contaminants. Semen processing in most cases
takes place under normal laboratory conditions
without access to sterile or contaminant-free air
resulting in possible contamination from the
laboratory atmospheric condition and equipment
hence, antibiotics used in semen extenders
inhibit microbial growth and prevent transmission
of infections to the inseminated doe. The female
genital tract is physiologically designed to destroy
infections introduced during mating but the
mechanism may be overwhelmed by a large
number of pathogens in the semen (Morrell and
Wallgren, 2011). Bacteria vary in their resistance
to antibiotics. Gram-positive organisms are
generally sensitive to penicillin because its mode
of action is to attach to the bacterial cell wall and
disintegrate the cell. Gram-negative bacteria
have a protective layer of lipids on their cell
surface and so are protected from the
attachment of penicillin molecules. Streptomycin
is more effective in inhibiting the growth or
destruction of Gram-negative bacteria (Campbell
et al
., 2009). There are many resistance factors,
such as the production of enzymes that destroy
antibiotic molecules. The best known of these is
penicillin or B-lactamase, the enzyme that
hydrolyses the B-lactam ring structure of
penicillin G rendering the antibiotic inactive. Most
strains of
Staphylococcus aureus,
a bacterium
that was generally sensitive to penicillin in the
early days of its use, have now acquired the
genes to produce these enzymes and have now
become penicillin-resistant (Campbell
et al
.,
2009). Exposure of bacteria to marginally lethal
doses of antibiotics tend to kill many cells but
permits the growth of a subset of the population
that is resistant.
Long-term use of a single drug, may,
however, favour colonization with the more
resistant bacteria. Complicating the issue is the
potential for transmission of resistance gene, R-
factor, from one bacterium to another. The
resistance gene does not have to exist in a
pathogenic cell to be passed to a drug-sensitive
pathogen. It can be contaminated with any
bacterium capable of transferring genes with a
pathogen. Fortunately, passage from the cell of
another is generally to bacteria of similar genetic
makeup. These resistance genes may become
incorporated into the chromosomal DNA or may
reside in the extrachromosomal DNA known as
plasmid DNA (Campbell
et al
., 2009). Another
factor to consider is the potential for bacteria to
mutate. Bacteria grow rapidly, sometimes
duplicating themselves in as little as 10 minutes
at the more usual generation time of 30 minutes,
a single cell can grow to one million within 10
hours and to one billion within 15 hours. if the
rate of non-lethal mutation is 10-7, 100 mutants
would be produced among these cells. Whether
resistance is natural, transferred, or because of
mutations, the presence of antibiotics that kill
much of the flora provides opportunities for
resistant cells to grow far beyond their normal
capacity: the fittest survive and rise to
prominence (Campbell
et al
., 2009).
Antibiotic resistance as well as the
emergence of new diseases and the resuscitation
of previously eradicated diseases are problems
associated with the excessive use of drugs in the
livestock industry posing a great threat to global
human health (El-Mahmood
et al.,
2008; Zaman
et al.,
2017). The majority of these antimicrobial
agents simply induce bacteriostatic antibodies.
Some are very toxic to vital organs of the body
(Tatli and Akdemir, 2005). Thus, it is critical to
tackle emerging and re-emerging infectious
diseases by focusing on the discovery of novel
medicines with a superior therapeutic profile to
combat disease outbreaks that pose a new
danger to global health security (Mohanta
et al.
,
2017).
Antibacterial and antioxidative effects of
Aloe
extract on the spermatozoa quality 5520
and fertilising potential of extended goat semen
Animal Research International (2024) 21(2): 5518 – 5528
Antibiotic-resistant microorganisms inform
the quest for new and effective antibacterial
substances to which these bacteria have not
developed resistance (El-Mahmood
et al.
, 2008).
Researchers are looking to natural chemicals
from plants as sources of antimicrobial and
antioxidative bioactive compounds to supplement
or replace the existing synthetic antibiotics,
which are becoming less effective against some
diseases (Nitta
et al.
, 2002).
Furthermore, medicinal plants are rich in
phytochemicals, some of which act as antioxidants
(Williams
et al.
, 2004). Natural antioxidants
increase the antioxidant capacity of plasma and
reduce the risk of cancer, heart disease and
stroke (Prior and Cao, 2000). Free radical
scavengers include the secondary metabolites;
phenolic and flavonoids from plants. They can be
found in the leaves, fruits, seeds and roots of the
plant. Synthetic antioxidants are commonly used
and are believed to have some detrimental
consequences including the risk of liver damage
and carcinogenesis in laboratory animals
(Lourenço
et al.,
2019). As a result, more
effective, less harmful and cost-efficient
antioxidants are required. Medicinal herbs are
preferred because they provide these desired
benefits (Namiki, 1990).
A possible alternative
to
adding antibiotics
to the semen
may
be
adding plant-based
substances
with a
known
antibacterial and antioxidative
effect on
different
bacterial genera and reactive oxygen
species.
V
arious
plant extracts
showed
some
antimicrobial and antioxidant properties
by
increasing
the
activity
of
several
antioxidative
enzymes.
Aloe vera
appears to be a more
sanitary substitute as it contains some
biologically active substances that can also act as
conventional cryoprotectants (Boudreau and
Beland, 2006). Previous studies (Gutierrez
et al
.,
2006; Aguiar
et al
., 2012; Souza
et al
., 2016)
have already shown that
A. vera
is a natural
product that can be used as a protectant in the
preservation of sperm for land animals.
A. vera
has been classified as medicinal plant with
specific bio-active ingredients such as alkaloids,
saponins, flavonoids, proteins, lipids, amino
acids, vitamins C, B (1, 2 and 6), A, E, enzymes,
organic and inorganic compounds and mineral
salts such as sodium, calcium, iron, potassium,
chloride, manganese, copper and zinc (Hamman,
2008; Darzi
et al.
, 2021). It has been used as a
natural antioxidant with a high potential to
reduce fat oxidation and oxidative stresses
(Vinson
et al
., 2005). Raw gels of
Aloe
barbadensis
Miller, 1768 (Asparagales:
Asphodelaceae) have been reported to maintain
plasma membrane integrity of spermatozoa in
extended buck semen above 60% after 48 hours
of preservation (Agbaye
et al
., 2023). Also,
pharmacological effects of
A. vera
viz: anti-
inflammatory, anti-arthritic and antimicrobial
have been documented (Newall
et al
., 1996) but
very few studies on the effects of the leaf extracts
on spermatozoa characteristics have been
reported. This study appeared to be one of the
few to document the research related to
A.
barbadensis
as an alternative to antibiotics and
antioxidants for the preservation of buck semen.
MATERIALS and METHODS
The Soxhlet extraction process was used for
methanol extracts of the leaf. Fresh
A.
barbadensis
leaves were harvested from the
Botanical Garden of the University of Ibadan, and
identification and authentication were carried out
at the Department of Botany of the same
institution, with the herbarium number UIH-
23009. The leaves were washed and cut into
pieces and 50 g was weighed into a Soxhlet reflux
apparatus containing 300 mL of absolute
methanol. The electronic hot plate was set
according to the boiling point of the solvent. The
extraction process took about 8 hours until the
refluxing solvent became clear. The extract was
concentrated by evaporating the solvent using a
rotary evaporator. This product is now referred
to as ABLME, the extracts were refrigerated (-1
°C) pending usage. The physical properties of
gels of
A. barbadensis
leaf and glycerol are
presented in Table 1.
Semen Collection: Three matured and healthy
Red Sokoto bucks aged 2 – 3 years, weighing
41.5 ± 2.0 kg were used for the experiment.
They were ejaculated weekly using the electro-
ejaculation method as described by Oyeyemi
et
al.
(2000).
Agbaye and Alaba 5519
Animal Research International (2024) 21(2): 5518 – 5528
During the collection process, the buck was
restrained in a standing position by two
attendants, the prepuce was cleansed and the
rectal probe of the ejaculator was lubricated
before inserting gently into the rectum of the
buck. The electro-ejaculator was adjusted to
supply 10 to 15 volts to create stimulation; the
process was repeated with a gradual increase in
the voltage. By regulating the voltage, the nerves
responsible for erection were stimulated,
enabling erection and ejaculation. Ejaculates
were collected into calibrated tubes using a
funnel.
Preparation of Extender and Experimental
Treatments Layout: The tris-fructose-citrate
extender (TFCE) was used for this study. The
compositions of the extender are presented in
Table 2 and the experimental layout is presented
in Table 3.
Table 2: Composition of tris-fructose-citrate
spermatozoa extender
Component (g)
Quantity
Tris Buffer
2.40
Fructose
1.00
Citrate
1.40
Penicillin
0.01
Streptomycin
0.10
Distilled Water (mL)
95.09
Semen Processing: Semen samples were
diluted in part A of the extender at 37°C (part A
is the portion without cytoprotectant). Diluted
semen and part B of the extender (the portion
with cytoprotectant) were gradually cooled to
4°C in the refrigerator before the addition of part
B extender. The addition of part B extender was
carried out in three steps at 30-minute intervals
for equilibration at this temperature
before freezing to -22oC for evaluation at
0, 24, 48 and 72 hours.
Post-Thaw Semen Quality and
Spermatozoa Fertilising Potential
Evaluation
Semen quality assessment: Spermatozoa
progressive motility (SPM), Spermatozoa
liveability (SPL), Normal spermatozoa
(NSP), pH and spermatozoa fertilising
potential assessment; plasma membrane
integrity (PMI) and acrosome integrity (ACI) were
carried out at 0, 24, 48 and 72 hours. SPM was
assessed with a phase contrast microscope at
x400 (37 °C). NSP and SPL were assessed from
two hundred spermatozoa stained with
nigrosine-eosin stain per sample reading (Rege
et al.
, 2000). The ACI percentage was estimated
from smears stained with nigrosine-eosin
examined under a phase contrast microscope at
x1000 magnification under an oil immersion
objective and bright-field (Yildiz
et al
., 2000). A
total of 200 spermatozoa in four microscopic
fields were counted. The PMI was assessed by a
hypo-osmotic swelling test (Buckett
et al
., 1997).
Table 3: Experimental layout on the effects
of
Aloe barbadensis
leaf methanolic extract
(ABLME) on the quality and spermatozoa
fertilising potentials
Treatment
(ABLME, m
g
/L)
Composition
T1
TFCE
-
A + 0.0
ABLME
T2 TFCE-B + 0.5 ABLME
T3
TFCE
-
B + 1.0
ABLME
T4
TFCE
-
B + 1.5
ABLME
T5 TFCE-B + 2.0 ABLME
TFCE = tris- fructose-citrate extender
Bacterial count: To determine the viable
bacterial count, the medium was prepared by
dissolving 23 g of nutrient Agar in 1000 mL of
distilled water and heated to boiling point. The
solution was autoclaved at 121℃ under 103.42
kPa for 20 minutes and was allowed to cool to
about 35℃. Salmonella-Shigella (SS) agar and
Eosin Methylene Blue (EMB) agar media were
used for the isolation of microorganisms and
were prepared according to the manufacturer’s
instructions. Serial dilutions were carried out to
Table 1: Comparison of physical properties of gels
of
Aloe barbadensis
leaf and glycerol
Characteristic
Aloe
barbadensis
Leaf Gel
Glycerol
†
Density (g/cm
3
)
1.20 ± 0.10
1.26
Molecular weight (g)
267.27 ± 0.57
92.09
pH
4.85 ± 0.88
6.70
-
7.50
Viscosity at 32
o
C (mPas)
6.29 ± 0.66
1.41
Solubility in water
Fairly miscible
Miscible
Boiling point (
o
C)
442.5 ± 0.45
290.00
Freezing point (
o
C)
NA
-
46.50
Melting point (
o
C)
NA
17.90
† = Source: CHEMSRC (2021),
NA = Not Available
5521
Antibacterial and antioxidative effects of
Aloe
extract on the spermatozoa quality 5520
and fertilising potential of extended goat semen
Animal Research International (2024) 21(2): 5518 – 5528
produce the bacterial load in the samples.
Distilled water (9 mL) was pipetted into a clean
test tube covered with cotton wool and foil and
was autoclaved at 121℃. 1 mL of each sample
was dispensed into the test tubes containing 9
mL of sterile distilled water and were serially
diluted. 1 mL of the last dilution factor for each
treatment with replicates was inoculated into
well-labelled empty sterile Petri dishes. Nutrient
Agar was added to each sample in the Petri-
dishes and incubated at 37℃ for 0, 24, 48 and
72 hours. Manual counting was performed with
the aid of magnification under uniform and
properly controlled artificial illumination (El-
Tayeb
et al
., 2007).
Lipid peroxidation: This test is based on a
Malondialdehyde (MDA) reaction with Thiobarbituric
acid (TBA). 2 mL of semen, 2 mL of TBA (0.7)
reagent and 1 mL of Trichloroacetic acid (TCA)
were added. The solution was thoroughly mixed
before being heated in a water bath at 80°C for
20 minutes. It was then chilled and centrifuged
for 10 minutes at 400 rpm. After centrifuging for
another 10 minutes, the absorbance of the
supernatant was evaluated at a wavelength of
540 nm. The concentration of MDA in the serum
was then calculated as follows: (Absorbance of
the test at 532 nm x Total Volume of the reaction
mixture x 1000) / (56 X 105 m-1cm-1) x (Volume
of semen x 1 cm).
Statistical Analyses: Data collected were subjected
to descriptive statistics and one-way Analysis of
variance procedure of SAS (2011) and means
were compared using Duncan’s multiple range
test of the same software.
RESULTS
Effect of ABLME on Quality and Spermatozoa
Fertilising Potential of Extended (4°C) Buck
Semen at 0 Hour: Table 4 showed that semen
samples preserved in 2.00 g/L ABLME had 84.67
± 4.57% SPL, while 0.00 g/L ABLME treatment
recorded 92.33 ± 2.67% SPL which implies about
7.66% reduction in value of SPL in the treatment
compare to the control. However, samples
preserved with other concentrations of ABLME
were statistically similar (p>0.05) to the control.
Normal spermatozoa were significantly greater
(p<0.05) in the control than in all of the ABLME-
extended samples. Semen pH in 0.00 g/L ABLME
was significantly more acidic compared to
samples preserved in ABLME extenders. There
were no statistical variations (p>0.05) in values
obtained for ACI and PMI in all treatments at
hour.
Effect of ABLME on Quality and Spermatozoa
Fertilising Potential in Extended (4°C) Buck
Semen at 24 Hours: Table 5 revealed that there
were no significant differences (p>0.05) in
results obtained for all semen samples extended
with 0.00 g/L ABLME and extenders containing
varying concentrations of ABLME for SPL and PMI
at 24 hours of storage. However, samples
extended with 0.00 g/L ABLME had significantly
higher (p<0.05) values for semen pH, NSP and
ACI compared to those extenders containing
ABLME.
Effect of ABLME on Quality and Spermatozoa
Fertilising Potential of Extended (4°C) Buck
Semen at 48 Hours: At 48 hours (Table 6), SPL
decreased significantly (p<0.05) as the concentration
of ABLME in the extenders increased. There was
an increase of about 14.34% SPL in semen
extended with 0.00 g/L ABLME with an SPL value
of 87.67 ± 1.12% compared to semen samples
extended with 2.00 g/L ABLME with an SPL value
of 73.33 ± 3.58%. The same relationship was
also seen in semen pH with samples preserved in
0.00 g/L ABLME being significantly less (p<0.05)
acidic than all samples preserved with varying
concentrations of ABLME in the extender. NSP
was also significantly greater (p<0.05) in all
semen extended with ABLME extenders
compared to being semen in 0.00 g/L ABLME
extenders. Increases of about 12.34% and
10.00% respectively were recorded for NSP in
samples extended with extenders containing 0.50
g/L with the value of 79.67 ± 2.33% NSP and
extenders containing 2.00 g/L ABLME with NSP
value of 77.33 ± 1.54% respectively compared to
that of 0.00 g/L ABLME. ACI for samples in 0.00
g/L ABLME was similar to all samples in extenders
containing varying amounts of ABLME.
552
2
Agbaye and Alaba 5519
Animal Research International (2024) 21(2): 5518 – 5528
Table 4: Effect of
Aloe barbadensis
leaf methanolic extract (ABLME) on quality, and
spermatozoa fertilising potential of extended (4°C) buck semen at 0 hour
Parameter
ABLME
(g/L)
0.00
0.50
1.00
1.50
2.00
Spermatozoa Liveability (%) 92.33 ±
2.67
c
89.00 ±
0.47
b
90.67 ±
2.27
b
91.33 ±
1.94
b
84.67 ±
4.57
a
Normal Spermatozoa (%) 92.67 ±
2.85
b
92.67 ±
2.85
b
86.33 ±
3.05
a
87.00 ±
3.05
a
84.00 ±
3.06
a
pH
6.63
±
0.24
a
6.89
±
0.01
b
6.93
±
0.01
bc
6.96
±
0.01
c
6.91
±
0.02
b
Acrosome Integrity (%)
89.33
±
6.69
85.33
±
3.08
89.33
±
3.08
84.67
±
2.75
85.33
±
2.76
Plasma Membrane Integrity (%)
85.67
±
0.00
88.33
±
0.15
84.33
±
2.00
89.00
±
0.01
90.67
±
0.03
abc = means in a row having different letter superscripts differ statistically (p<0.05)
Table 5: Effect of
Aloe barbadensis
leaf methanolic extract (ABLME) on quality, and
spermatozoa fertilising potential in extended (4°C) buck semen at 24 hours
Parameter
ABLME
(g/L)
0.00
0.50
1.00
1.50
2.00
Spermatozoa Livability (%) 85.00 ±
2.16
85.33 ±
1.99
85.33 ±
3.60
83.67 ±
4.73
83.33 ±
6.24
Normal Spermatozoa (%)
94.00
±
1.63
c
76.33
±
4.48
a
85.00
±
2.36
b
84.00
±
2.16
b
76.67
±
2.72
a
pH 6.73 ±
3.89
a
6.61 ±
3.81
b
6.57 ±
3.8
bc
6.54 ±
3.76
c
6.51 ±
0.02
c
Acrosome Integrity (%) 95.00 ±
0.47
c
79.33 ±
3.88
a
88.33 ±
1.45
b
89.00 ±
0.47
b
89.00 ±
0.58
b
Plasma Membrane Integrity (%) 70.67 ±
0.39
70.00 ±
0.58
71.00 ±
1.25
70.33 ±
1.35
75.00 ±
2.22
abc = means in a row having different letter superscripts differ statistically (p<0.05)
Table 6: Effect of
Aloe barbadensis
leaf methanolic extract (ABLME) on quality, and
spermatozoa fertilising potential of extended (4°C) buck semen at 48 hours
Parameter
ABLME
(g/L)
0.00
0.50
1.00
1.50
2.00
Spermatozoa Liveability (%) 87.67 ±
1.12
c
84.33 ±
7.12
bc
80.00 ±
3.77
b
77.67 ±
5.31
ab
73.33 ±
3.58
a
Normal Spermatozoa (%) 67.33 ±
1.18
a
79.67 ±
2.33
c
72.33 ±
5.99
b
75.67 ±
1.07
bc
77.33 ±
1.54
bc
pH 6.75 ±
0.01
c
6.65 ±
0.01
b
6.61 ±
0.01
b
6.58 ±
0.01
bc
6.54 ±
0.00
a
Acrosome Integrity (%)
76.00
±
2.90
ab
81.00
±
6.66
c
72.00
±
6.00
ab
68.00
±
3.84
a
70.33
±
2.13
b
Plasma Membrane Integrity (%)
71.00
±
1.73
c
69.33
±
1.95
bc
66.33
±
1.18
b
72.33
±
3.85
c
61.33
±
1.76
a
abc = means in a row having different letter superscripts differ statistically (p<0.05)
552
3
Antibacterial and antioxidative effects of
Aloe
extract on the spermatozoa quality 5520
and fertilising potential of extended goat semen
Animal Research International (2024) 21(2): 5518 – 5528
Effect of ABLME on Quality and Spermatozoa
Fertilising Potential of Extended (4°C) Buck
Semen at 72 Hours: In Table 7 it was observed
that above 1.00 g/L ABLME, there was a
substantial decrease in the proportion of SPL,
NSP and spermatozoa with intact acrosomes
compared to those exposed to 0.00 g/L ABLME
for 72 hours. Also, it was clear that semen
samples extended in 0.00 g/L ABLME are less
acidic than others containing different amounts
of ABLME.
Types of abnormal Spermatozoa in Buck
Semen Extended with 2.00 g/L ABLME:
Table 8 shows the classification of abnormal
spermatozoa in samples extended with 2.00 g/L
ABLME. At 0 and 72 hours, 0.20 ± 0.20 and 0.30
± 2.70% of the rudimentary tail (RDT)
respectively were the only primary abnormality
observed. Occurrence of secondary abnormalities
viz: bent tail (BNT), looped tail (LPT), coiled tail
(CLT), curved mid-piece (CMP) and detached tail
(DTT) increased gradually with duration of
storage regardless of the concentration of the
ABLME in the extender.
Effect of ABLME on Microbial Load (105 CFU/mL)
of Extended (4°C) Buck Semen: All semen
samples preserved in ABLME extenders showed
lower microbial loads compared to the control at
48 hours of storage (Table 9), In addition,
regardless of the concentration of the extract,
microbial population increased with the storage
duration. The lowest population (0.00 ± 0.00 x
105 CFU/mL) was recorded for samples extended
with 0.5 g/L ABLME after 24 hours of storage,
while the highest population (90.50 ± 7.94 x 105
CFU/mL) was obtained for samples stored with
2.0 g/L ABLME after 72 hours.
DISCUSSION
From Zero to 48 hours of storage over 70% of
life and normal spermatozoa with intact plasma
membrane integrity were recorded for all semen
samples stored with ABLME extender. Variation
in concentration shows no difference in SPL, NSP
and ACI but there were significant differences in
the values obtained for PMI and pH at 72 hours.
This result was in agreement with the findings of
Yaniz
et al.
(2008) that the slight decrease in
these parameters was due to the production of
reactive oxygen species (ROS) and the
susceptibility of the plasma membrane to
peroxidative damage caused by changes in the
pH.
Contaminant compromise of plasma
membrane integrity causes spermatozoa
progressive motility and induces a reduction in
fertility and fertility declines. The forms of
spermatozoa abnormalities observed were
similar. Morphological abnormalities and defects
are crucial because a deformed or immature
sperm cell has a reduced or no chance of
fertilizing an oocyte. RDT, BNT, CLT and CMP
were observed in samples extended with varying
concentrations of ABLME 72 hours. Different
researchers have come to different conclusions
about the most common morphological defects
and the overall percentage of morphological
abnormalities in a typical male. Tibary and
Vaughan (2006) concluded that head
abnormalities were the most common defect
(Tibary and Vaughan, 2006) why Tail deformities
were shown to be the most common in a trial at
the University of Massachusetts by Bravo
(Szymkowicz, 2012). A major factor detrimental
to the quality of semen for AI is microbial
contamination. can occur because of urinary tract
infection or during semen collection (Wu
et al
.,
2019). From this study, it is observed that the
longer the storage time, the higher the
population of microbes irrespective of the
concentration of ABLME. Between 0 to 24 hours,
the microbial load declined with time in the
control and samples extended with 0.5 g/L
ABLME but there was a significant increase in the
microbial load in samples extended with 1.0, 1.5
and 2.0 g/L ABLME, respectively. Also, a
progressive increase in the microbial loads with
time in all samples after 24 hours of storage up
to 72 hours was observed. However, all samples
preserved in extenders containing ABLME had a
lower microbial population ranging from 6.8 to
20.7 × 105 CFU/mL compared to 27.5 × 105
CFU/mL in the control containing conventional
antibiotics after 48 hours post storage.
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Table 7: Effect of
Aloe barbadensis
leaf methanolic extract (ABLME) on quality, and
spermatozoa fertilising potential of extended (4°C) buck semen at 72 hours
Parameter
ABLME
(g/L)
0.00 0.50 1.00 1.50 2.00
Spermatozoa Liveability (%) 72.67 ±
4.1
ab
75.67 ±
2.37
c
69.33 ±
5.47
bc
63.00 ±
4.04
a
67.67 ±
6.31
b
Normal Spermatozoa (%) 74.33 ±
7.02
c
70.67 ±
2.25
bc
76.67 ±
4.65
c
60.67 ±
9.68
b
65.00 ±
2.89
a
pH 6.74 ±
0.00
c
6.66 ±
0.00
b
6.64 ±
0.00
b
6.57 ±
0.00
a
6.55 ±
0.00
a
Acrosome Integrity (%) 72.00 ±
3.51
c
67.67 ±
1.56
bc
62.67 ±
3.94
b
57.67 ±
4.47
a
63.33 ±
4.9
b
Plasma Membrane Integrity
(%)
67.67
±
1.46
c
67.67
±
1.14
c
65.67
±
1.14
b
65.33
±
0.92
b
60.67
±
1.24
a
abc = Values in a row having different letter superscripts differ statistically (p<0.05)
Table 8: Types of abnormal spermatozoa in buck semen extended with 2.00 g/L
Aloe
barbadensis
leaf methanolic extract (ABLME)
Time (Hours)
Spermatozoa Abnormalities Classification (%)
RT
BT
LT
CT
CMP
DT
0
0.20
±
0.2
b
0.93
±
0.6
a
2.60
±
1.0
a
0.73
±
0.3
a
0.60
±
0.4
a
3.33
±
0.6
a
24
0.00
±
0.0
a
1.00
±
4.6
b
2.80
±
0.9
b
1.30
±
2.3
b
1.13
±
1.2
b
3.93
±
2.3
a
48
0.00
±
0.0
a
2.70
±
4.0
c
2.93
±
0.6
bc
2.60
±
0.7
c
2.13
±
1.6
c
6.27
±
1.3
b
72
0.30
±
2.7
c
5.13
±
2.6
d
3.20
±
1.1
c
3.03
±
1.7
d
2.63
±
1.2d
9.20
±
3.3
c
RDT = Rudimentary tail; BNT = Bent tail; LPT = Looped tail; CLT = Coiled tail; CMP = Curved mid-piece; DTT= Detached tail;
abc = Values in a column having different letter superscript differ statistically (p<0.05)
Table 9: Effect of
Aloe barbadensis
leaf methanolic extract (ABLME) on microbial load (105
CFU/mL) of extended (4°C) buck semen
Time (Hours)
ABLME
(g/L)
0.0
0.5
1.0
1.5
2.0
0
0.83
±
0.27
d
0.50
±
0.00
c
2.67
±
0.49
e
0.00
±
0.00
a
0.17
±
0.14
b
24
0.67
±
0.27
b
0.00
±
0.00
a
7.00
±
3.56
d
0.67
±
0.36
b
1.00
±
0.47
c
48
27.50
±
12.32
c
16.83
±
8.85
b
9.33
±
0.60
a
20.67
±
0.76
b
6.83
±
1.16
a
72
38.12
±
1.16
a
34.50
±
1.31
a
41.12
±
2.00
a
69.00
±
5.67
b
90.50
±
7.94
c
abc = Values in a row having different letter superscripts differ statistically (p<0.05)
The reduction in the microbial population in
samples extended in ABLME could be attributed
to the secondary metabolites or bioactive
ingredients in the ABLME However, the increase
in the microbial loads after 24 hours of storage
could be because of depletion in the
concentration of these metabolites with time.
This finding is consistent with the findings of
several authors, including Shihabudeen
et al.
(2010), who reported that phytoconstituents in
extender, such as alkaloids, flavonoids, tannins,
phenols, saponins and a variety of other aromatic
compounds, act as a defence mechanism against
a variety of microbes.
Streptococcus pyogenes
and
Streptococcus faecalis
are microorganisms
that have been reported to be inhibited by
A. vera
gel (Cera
et al
., 1981; Robson
et al.,
1982).
According to Saoo
et al.
(1996), lectins in a
portion of
A. vera
gel inhibit cytomegalovirus
(CMV) proliferation in cell culture by affecting
protein synthesis. Many of the medicinal effects
of
A. vera
leaf extracts have been attributed to
the polysaccharides found in the inner leaf
parenchymatous tissue (Ni
et al
., 2004). The
antibacterial activity of the extract can also be
linked to anthraquinones and saponins present in
the extract (Boateng, 2000; García-Sosa
et al.,
2006; Qun
et al.,
2023).
Conclusion: Methanol extract (2.0 g/L) of
A.
barbadensis
inclusion in egg yolk + sodium
citrate diluent gave enhanced antibacterial
activity to extend buck semen for up to 48 hours.
Since the methanol extract of
A. barbadensis
leaf
inhibits microbial growth better than
conventional antibiotics, up to 48 hours of
storage, the extract is suitable as a natural
antibacterial agent in short time buck semen
extension, not beyond 48 hours.
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and fertilising potential of extended goat semen
Animal Research International (2024) 21(2): 5518 – 5528
ACKNOWLEDGMENTS
The authors are grateful to some technical staff
of the Department of Animal Science, University
of Ibadan, Oyo State, Nigeria and the
Department of Animal Production, Lagos State
University of Science and Technology, Ikorodu,
Lagos, Nigeria for the assistance.
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