THE EFFECT OF WHITE MULBERRY, MORUS ALBA L LEAF EXTRACT ON SOME CHARACTERISTICS OF THE COOLED SEMEN OF AWASSI RAMS

Abstract

In this study, the effect of adding different levels of mulberry Morus alba L leaf extract on some characteristics of the semen of Awassi rams after different periods of preservation was studied. The study was carried out in the animal field of the Department of Animal Production, College of Agriculture, University of Baghdad, Jadiriyah. The semen samples were divided equally into four concentrations in which gear diluent was used at a dilution ratio of 1:10. The concentrations of the control group (C) were left without addition, while the three concentrations were added to the Morus alba L leaf extract in the proportions (0.5, 1, 1.5%). The results of the study showed that adding cranberry leaf extract at a concentration of 0.5% led to a significant improvement (P<0.05) on sperm motility, plasma membrane integrity, mitochondrial activity and malonaldehyde concentration during different preservation periods. It can be concluded that adding Morus alba L leaf extract to a low concentration (Tris) diluent had an effective role in improving the studied semen characteristics of the sperm of Awassi rams.

Full text

THE EFFECT OF WHITE MULBERRY, MORUS ALBA L LEAF EXTRACT ON
SOME CHARACTERISTICS OF THE COOLED SEMEN OF AWASSI RAMS
Nadia Abdul–Hadi Abdul–Ameer1, Firas A. Mahmood2 and Rabeea M. Mahmood3
1Department of Animal Production, Kufa Technical Institute, AL-Furat AL-Awssat Technical University, Iraq.
2Department of Animal Production, College of Agricultural Engineering Sciences, University of Baghdad, Iraq.
3Department of Animal Production, Collage of Agriculture, University of Karbala, Iraq.
e-mails : nadiaalneame70@gmail.com, firas.a@coagri.uobaghdad.edu.iq, rabeeaarrubaii@rocketmail.com
(Received 10 July 2021, Revised 22 September 2021, Accepted 3 October 2021)
ABSTRACT : In this study, the effect of adding different levels of mulberry Morus alba L leaf extract on some characteristics
of the semen of Awassi rams after different periods of preservation was studied. The study was carried out in the animal field of
the Department of Animal Production, College of Agriculture, University of Baghdad, Jadiriyah. The semen samples were
divided equally into four concentrations in which gear diluent was used at a dilution ratio of 1:10. The concentrations of the
control group (C) were left without addition, while the three concentrations were added to the Morus alba L leaf extract in the
proportions (0.5, 1, 1.5%). The results of the study showed that adding cranberry leaf extract at a concentration of 0.5% led to
a significant improvement (P<0.05) on sperm motility, plasma membrane integrity, mitochondrial activity and malonaldehyde
concentration during different preservation periods. It can be concluded that adding Morus alba L leaf extract to a low concentration
(Tris) diluent had an effective role in improving the studied semen characteristics of the sperm of Awassi rams.
Key words : White mulberry, Morus alba L, cooled semen, awassi rams.
How to cite : Nadia Abdul–Hadi Abdul–Ameer, Firas A. Mahmood and Rabeea M. Mahmood (2022) The effect of white mulberry,
Morus alba L leaf extract on some characteristics of the cooled semen of Awassi rams. Biochem. Cell. Arch. 22, 2683-2686.
DocID: https://connectjournals.com/03896.2022.22.2683
Biochem. Cell. Arch. Vol. 22, No. 1, pp. 2683-2686, 2022 www.connectjournals.com/bca ISSN 0972-5075
DocID: https://connectjournals.com/03896.2022.22.2683 eISSN 0976-1772
INTRODUCTION
Livestock production contributes significantly to
providing food security at the economic level of the
country due to the great importance of these products to
the consumer (Arab Organization for Agricultural
Development, 2001). Sheep constitute a large part of
this wealth, as their numbers amounted to 6.780 million
heads and constitute a major source of income for the
population of pastoral areas in Iraq (FAO, 2003). In
recent years, the use of medicinal plants has increased,
perhaps the reason for this is due to the good effect of
these plants in treating many cases, which led to the
widespread use of them in a very wide way because
they contain active ingredients with knowledge of how
they work inside the body of the organism (Health Tools,
2005). Mulberry Morus alba L is a plant its leaves rich
in effective natural antioxidants and phenolic compounds
in protecting cells from free radicals in general and fatty
acid peroxides (Andallu et al, 2014), several active
compounds have been separated from the alcoholic
extraction of Morus alba L leaves, such as quercetin
and polyphenols, phenolic acids, and flavonoids (Katsube
et al, 2006). The aim this research is to knowing the
possibility of using mulberry leaf extraction to improving
some characteristics of the semen of Awassi rams during
preservation for different periods.
MATERIALS AND METHODS
The research was proceeded in sheep and goats field
of the Department of Livestock Resources at the College
of Agriculture, Baghdad University, Jadiriyah from the
first of September 2018 to the end of November 2018. In
this study five local Awassi rams were used for studying
the influence of adding different concentrations of mulberry
Morus alba L leaf extract on some studied characteristics
of the semen of Awassi rams during cryopreservation
periods.
Experimental animals and semen collection
In this experiment, five rams were used, who were
trained for a month to collect semen using an artificial

2684 Nadia Abdul–Hadi Abdul–Ameer et al
vagina, and their ages ranged between 2.5 - 3 years and
their body weight ranged between 75-80 kg. Semen was
collected using the artificial vagina of sheep and goats
by ejaculation/ram/week. For the purpose of preparing
for ejaculation, the rams were allowed to make a false
mount (False mount) on one of the ewes to increase their
sexual desire (Badawy et al, 1975).
Semen dilution and preservation
Semen sample was diluted with Tris diluent in a
dilution ratio (10:1), the diluent was prepared as what
was mentioned by Evans and Maxwell (1990). Cranberry
leaf extract was added to gear diluent according to the
following concentrations (0, 0.5, 1, and 1.5%). After the
process of collecting the semen sample, it was placed to
a water bath at (37°C), then, it was divided into four
equal parts, and then the diluent was added to the semen
gradually inside the water bath, and the dilution ratio was
(10:1). The samples have been transferred to a beaker
contains water at (37°C). Then, the beaker was placed
in the refrigerator, and after the temperature was
stabilized at a temperature of (5°C), the readings were
taken during the times (0, 24, 48 hours) after dilution.
Studied traits
According to Chemineau et al (1991) individual
motility was estimated. While intact plasma membrane
sperm percentage was estimated according to the method
of Jeyendean et al (1984). The mitochondrial activity
was tested using 3,’3-diaminobenzidine (DAB) dye and
the percentage of mitochondrial activity was estimated
according to the method (Hrudka, 1987). The
determination of the concentration of malondidehyde in
the seminal plasma was proceeded by estimating the value
of (thiobarbutiric acid, TBA) and (Tri chloral acetic acid,
TCA) according to the method of Kumaresan et al
(2005).
Statistical analysis
Statistical Analysis System-SAS (2012) was used to
analyzing data according to a fully randomized design
(CRD) in two directions, while the differences between
the means were compared with Duncan (1955) multi-
range test.
RESULTS
The results showed that there was a significant effect
(P<0.05) in the individual motility of ram’s sperm when
kept in preservation (5°C) at time zero, as 0.5% (80%)
was superior to each of the control group, 1 and 1.5, as
their values reached (74.25, 77.25 and 77.25%)
respectively. whilst the control had the lowest value of
individual motility in 24 hours (63.5%) compared to the
other concentrations. In the same context, no significant
differences were recorded between the control group
and the concentration of 1 and 1.5% (53, 55.75 and
52.75%), respectively, at 48 hours, compared to the
concentration of 0.5% (58.75%) (Table 1). From the
results of the current study, adding Morus alba L leaf
extraction to Tris diluent produced a significant (P<0.05)
increasing in the plasma membrane integrity percentage
of Awassi ram’s sperm during zero time of preservation
(Table 2), as the concentration of Awassi rams was
superior to 0.5% (Table 2). 84.5% over the control group
(78.75%). At the time of 24 hours of preservation, no
significant differences between the control group and the
concentration of 1.5% (66.75 and 69.25%) were
observed, compared to the other concentrations. The
results of the 48 hours of preservation (5°C) for the same
trait showed a significant (P<0.05) superiority) for a
concentration of 1% (61.75%) compared to a
concentration of 0.5 and 1.5% (57.5 and 59.5%),
respectively.
The results of estimating the concentration of
malondidehyde in the seminal plasma of Awassi rams
showed (µmol/109 sperm), as it was clear from (Table 3)
that there was a significant decrease (P<0.05) for the
concentration of 0.5 and 1% (0.367 and 0.410 µmol/109
sperm), respectively on the rest of the concentrations in
the concentration of malondidehyde after cooling 50C at
Table 1 : Effect of adding different concentrations of mulberry Morus
alba L leaf extraction to Tris diluent on the individual
motility of sperm (%) of Awassi rams with different periods
of preservation.
Treatments 0 hour 24 hours 48 hours
Control L 74.250 C 63.500 B 53.000 B
0.5 80.000 A69.500 A58.750 A
177.250 B 66.250 B 55.750 B
1.5 77.250 B 64.000 B 52.750 B
Mean 77.187 65.812 55.062
SEm 0.646 0.742 0.760
Significant 0.003 0.002 0.002
Table 2 : Effect of adding different concentrations of mulberry Morus
alba L leaf extraction to Tris diluent on the integrity of the
sperm plasma membrane (%) of Awassi rams with different
preservation periods.
Treatments 0 hour 24 hours 48 hours
Control L 78.750 B 66.750 B 57.500 B
0.5 84.500 A75.00 A57.500 B
183.500A 72.500 A61.750 A
1.5 82.250 A69.250 B 59.500 AB
Mean 82.25 70.875 59.062
SEm 0.648 0.889 0.580
Significant 0.0007 0.0001 0.0087

Effect of white mulberry, M. alba L leaf extract on some characteristics of the cooled semen of Awassi rams 2685
time zero. At the time of 48 hours of preservation, the
results of the experiment exhibited a significant (P<0.05)
decrease in the concentration of malonaldehyde for
Awassi rams at a concentration of 0.5% (1.043 µmol/109
sperm) compared to the other concentrations. The results
showed that the mitochondrial activity of the sperms of
0.5% of Morus alba L leaf extract recorded the highest
percentage (53%) and was significantly (P < 0.05)
superior to the control treatment and the concentration
of 1.5% (44.66 and 42.00%), respectively, for zero hours
of preservation. While, the mitochondrial activity of
sperms after 48 hours of preservation recorded the
highest percentage of concentration 0.5 and 1% (27.66
and 27.33%), respectively incomparison with control
treatment and concentration of 1.5% (22.66 and 22.33%),
respectively (Table 4).
DISCUSSION
Recently, interest in antioxidants from natural sources
has increased due to their ability to protect biological
molecules, especially lipids, including cholesterol,
unsaturated fatty acids and proteins, from oxidative
damage (Bravo et al, 2013). The most important factors
of low sperm motility are the changes that occur in the
plasma membrane during the different preservation
periods, is the release of energy enzymes, the most
important of which is Glucose-6-phosphate
dehydrogenase, which leads to a decrease in the
production of the energy complex, adenosine triphosphate
(ATP) and an increase in the proportion of AMP / ADP).
(Manafi, 2011). In this study, a concentration of 0.5%
the highest percentage of individual motility compared to
other concentrations. The high content of flavonoids in
cranberry leaf extract may have a role in protecting the
sperm lipid membranes from oxidation by breaking the
oxidation chain reaction (Choi and Hwang, 2005 and
Shahid et al, 2012). The plasma membrane of ram sperm
is rich in polyunsaturated fatty acids, which makes the
sperm sensitive to oxidative stress (Griveau et al, 1995).
Yokozawa et al (1998) showed in a study conducted on
the effectiveness of flavonoids extracted from plants as
antioxidants, that the hydroxyl groups present in flavonoid
compounds with antioxidant activity are important in the
process of inhibiting the activity of free radicals formed
by cell self-oxidation. Thus, curbing these free radicals
leads to preventing the formation of peroxides which may
cause oxidation or destroying the free radicals formed
(Sarica et al, 2007). This was confirmed by Shahid et al
(2012) on the high percentage of effective antioxidants
in Morus alba L leaves, the most important of which are
phenols, flavonoids and ascorbic acid. Phenolic
compounds may play a role in preventing the oxidation of
unsaturated fatty acids present in sperm membranes and
energy-forming regions within the mitochondria by
interacting with free radicals (Desai et al, 2010 and
Bansal and Bilaspuri, 2011). The concentration of
Malondialdehyde in seminal plasma was reduced by the
action of the active compounds (Avdatek, 2017 and
Fayyad and Mahmood, 2019). In the current study, a
concentration of 0.5% of Morus alba L leaf extract
improved the mitochondrial activity of the sperm, and the
semen preservation steps have a key role in disrupting
the regulation of calcium ion levels due to a defect in the
permeability of the mitochondrial membrane, which
increases the calcium ion level in the cytoplasm and thus
stimulates the work of Protease enzymes, endonucleases,
phospholipases and ATPase that cause denaturation of
proteins and break down lipids in the plasma membrane
(Aman and Parks, 1994 and Aslanidi et al, 1997). From
the results of this study, it was found that lower
concentrations led to better results for the studied
characteristics of the cooled semen of Awassi rams during
a different preservation period.
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