JRSSEM 2021, Vol. 01, No. 5, 523 – 531
E-ISSN: 2807 - 6311, P-ISSN: 2807 - 6494
DOI : 10.36418/jrssem.v1i5.91
MOTILITY AND ABNORMALITY OF SHEEP SPERMATOZOA
THAT IS BEING FROZEN USING SOYBEAN LECITHIN
(SOYBEAN LECITHIN)
Salmin1, Marsudi2, Hendro Sukoco3, Deka Uli Fahrodi4
1Animal Husbandry Department, Faculty of Animal Husbandry and Fisheries, Universitas Tadulako
2,3,4Animal Husbandry Department, Faculty of Animal Husbandry and Fisheries, Universitas
Sulawesi Barat
e-mail: salmin.bouato@gmail.com1, marsudi@unsulbar.ac.id2, hendrosukoco@unsulbar.ac.id3,
dekaulifahrodi@unsulbar.ac.id4
*Correspondence: salmin.bouato@gmail.com
Submitted: 25 November 2021, Revised: 10 December 2021, Accepted: 20 December 2021
Abstrack. The specific purpose of this study was to determine the effect of optimal levels of
soybean lecithin in sperm extenders on motility and abnormalities of sheep spermatozoa after
undergoing the freezing process. This research was conducted experimentally in a laboratory with
a completely randomized design (CRD) with five treatments and three replications. The five
treatments tested were soybean lecithin levels in Tris (L) diluent, consisting of: L0 = 0% soybean
lecithin + 95% Tris diluent + 5% glycerol; L1 = 1% soy lecithin + 94% Tris diluent + 5% glycerol; L2
= 2% soy lecithin + 93% Tris diluent + 5% glycerol; L3 = 3% soy lecithin + 92% Tris diluent + 5%
glycerol; L4 = 4% soy lecithin + 91% Tris diluent + 5% glycerol. The independent variable in this
study was the level of soy lecithin in the Tris diluent. The dependent variable is the progressive
motility and abnormalities of post-clotting spermatozoa. Soybean lecithin levels had a significantly
different effect (P≤0.05) on the percentage of progressive motility and spermatozoa abnormalities
of post-freezing sheep. The level of 3% soya bean lecithin is the best level in maintaining the quality
of post-freezing sheep spermatozoa.
Keywords: abnormalities; spermatozoa; sheep; soybean lecithin.
Salmin, Marsudi, Hendro Sukoco, Deka Uli Fahrodi | 524
DOI : 10.36418/jrssem.v1i5.91
INTRODUCTION
The conventional method that has been
used for sperm freezing or
cryopreservation is slow freezing, but
during the slow freezing process the sperm
undergoes cold shock, freezing injury and
the formation of ice crystals (Arrebola,
González, Torres, & Abecia, 2013); (Silva,
Cajueiro, Silva, Soares, & Guerra, 2012);
(O’Hara et al., 2010) inside and outside
spermatozoa cells. Cold shock and/or
freezing injury can result in decreased
permeability of spermatozoa membranes
to water and solutions and damage the
acrosomal membrane (Jie Liu et al., 2016),
folding the middle section and tail towards
the head so that motility or sperm
movement appears to be circular or
backwards. Intracellular ice crystals can
damage the cell wall and structure of
spermatozoa while extracellular ice crystals
can increase the salt concentration which
causes cell damage or abnormalities of
spermatozoa (Forouzanfar, Abid, Hosseini,
Hajian, & Esfahani, 2013).
Motility and abnormalities have a
positive correlation with fertility, in which
the higher the number of motile
spermatozoa and the lower the number of
abnormal spermatozoa, the higher the
fertility. This correlation gradually
decreases after the freezing and thawing
processes, because during these processes
there may be damage to cell function or the
exhaustion of energy. A drastic decrease
occurs after the sperm has cooled down
and/or frozen due to cold shock, which is
characterized by the folding of the middle
section and tail towards the head so that
the sperm movement appears circular or
backwards.
The problem of sperm freezing or
cryopreservation in the form of cold shock
or freezing injury can be partially solved by
the use of a protective agent
(cryoprotective agent) in the sperm
extender (Zhou, Wu, Shi, & Zheng, 2010).
Protective materials that are popular and
universally used for freezing or
cryopreservation of sperm to date include
glycerol and lecithin (Kannaki,
Shanmugam, & Verma, 2011); (Layek,
Mohanty, Kumaresan, & Parks, 2016);
(Rickard, Pool, Druart, & de Graaf, 2019).
The main source of lecithin that has been
used for a long time is egg yolk, but the use
of this material as a source of lecithin to
prevent the effects of cold shock carries the
risk of contamination by microorganisms
that harm spermatozoa and the female
reproductive tract (Muro et al., 2016), such
as bacteria (Moran, Guzman, Ropars, &
Illmer, 2010); especially Salmonella
thyphimurium.
One of the potential protective
materials developed to protect
spermatozoa from the adverse effects of
freezing or cryopreservation is soy lecithin.
This material is a vegetable lecithin which
contains phospholipids which are very
important to protect spermatozoa
membranes in the cryopreservation
process. Naturally, the lecithin content in
soybeans is 1.48 – 3.08%. The main
components of soy lecithin are
phospholipids consisting of phosphatidyl
choline, phosphatidyl ethanolamine,
phosphatidyl inositols and glycolipids
(Saurabh, Sharma, & Gautam, 2018).
Hygienically, soybean lecithin was not
found to contain any micro-organisms that
could harm spermatozoa or female
525 | Motility and Abnormality of Sheep Spermatozoa That is Being Frozen Using Soybean
Lecithin (Soybean Lecithin)
reproductive tract (Tian et al., 2019).
Based on this description, it is deemed
necessary to conduct research on motility
and abnormality of sheep spermatozoa
being frozen using soy lecithin.
METHODS
Time and Location
This research was carried out at the
Laboratory of Animal Physiology and
Reproduction, Faculty of Animal Husbandry
and Fisheries, Tadulako University from
July to November 2021.
Experimental Livestocks
Types of experimental livestocks used in
this study are male sheeps. The number of
male sheeps as a source of sperm are three
sheeps, 2-3 years old, in good health and
have good reproductive characteristics.
Research Materials
The materials used for this research
were 10% soybean lecithin (CENTROL 3
flub, certificate number: TSC 04020, USA),
Tris (hydroxymethyl) aminomethane, citric
acid, glucose, glycerol, aquadestilata,
aquabidestilata, penicillin, streptomycin,
intermediate hypoosmotic swelling test
(HOS test), physiological NaCl, hayem's
solution, eosin Y/Negrosine, 70% alcohol
and liquid nitrogen (Zadoks et al., 2014).
Research Equipments
The equipments used in this study were
artificial vagina, thermometer, ice flask,
scale tube, test tube, measuring cup,
erlenmeyer, beaker, blender machine (Rui
Liu et al., 2021) aluminum foil, stirring rod,
micropipette (Transferpette®). dropper, lig
ht microscope (Tension), optilab camera
(Optilabpro viewer®), object glass, cover
glass, haemocytometer, Neubauer
counting chamber, pH meter (Kyte, Kleyn,
Scoggins, & Bridgen, 2013), refrigerator
(Sanken CN®), hand tallycounter
(Laboratory Dc Counter: DBC-9. K Gemini
Ind. Corp.®, USA), analytical balance, liquid
nitrogen container (Taylor Wharton),
styrofoam box (styrofoam box), tweezers,
and mini straws.
Research design
This research was conducted in a
laboratory experimental approach with a
completely randomized design with five
treatments and three replications. The five
treatments tested were the levels of soy
lecithin in the Tris (L) diluent, consisting of:
L0 = 0% soy lecithin + 95% Tris diluent +
5% glycerol
L1 = 1% soy lecithin + 94% Tris diluent +
5% glycerol
L2 = 2% soy lecithin + 93% Tris diluent +
5% glycerol
L3 = 3% soy lecithin + 92% Tris diluent +
5% glycerol
L4 = 4% soy lecithin + 91% Tris diluent +
5% glycerol
Research variable
The independent variable in this study
was the level of soy lecithin in the Tris
diluent. The dependent variable is the
progressive motility and abnormalities of
post-clotting spermatozoa.
RESULTS AND DISCUSSION
The results of observations on the
motility and abnormalities of spermatozoa
Salmin, Marsudi, Hendro Sukoco, Deka Uli Fahrodi | 526
in post-freezing sheep treated with soy
lecithin levels in the Tris extender are listed
in Table 1.
Table 1. The mean of progressive motility and abnormality of post-freezing sheep
spermatozoa from various levels of soybean lecithin in Tris extenders (%).
Lechitin Levels Soybeans (L)
Progressive Motility
Abnormality
0% (L0)
16,79±3,64a
17,03±2,84a
1% (L1)
17,55±2.52a
15,71±2,83b
2% (L2)
34,18±2,27b
14,57±2,94b
3% (L3)
43,79±3,01c
13,72±2,92b
4% (L4)
34,24±3,11d
15,62±2,90b
Note. : a,b,c,d, Different superscripts in the same column showed significant differences
(P≤0.05).
Based on the results of analysis of
diversity, the levels of soya bean lecithin in
the Tris extender showed a significantly
different effect (P≤0.05) on the motility and
abnormalities of post-freezing sheep
spermatozoa. The higher the levels of soy
lecithin in the Tris extender, the more
progressive motility percentages increased
to 3% of soy lecithin levels, then decreased
to 4% of soy lecithin levels and the
percentage of spermatozoa abnormalities
decreased to 3% soy lecithin level, then the
levels increased to 4% of soybean lecithin
(Figure 1).
Figure 1. Soybean lechitin
levels (%)
(A)
Figure 2. Soybean lechitin levels (%)
(B)
Effect of soybean lecithin levels on
progressive motility (A) and abnormalities
(B) of post-slow freezing sheep
spermatozoa.
Further test results showed that the
percentage of progressive motility (43.79%)
of post-freezing spermatozoa treated with
3% soybean lecithin (FL3) was significantly
higher (P≤0.05) and spermatozoa
abnormalities percentage (13.72%) was
significantly lower (P≤0.05) than all the
treatments that were tried. Applicatively,
the results of this study are suitable for
artificial insemination because they contain
more than 40% of progressive motile
527 | Motility and Abnormality of Sheep Spermatozoa That is Being Frozen Using Soybean
Lecithin (Soybean Lecithin)
spermatozoa according to the Indonesian
National Standard (Martin et al., 2014);
(Hoesni, 2017) and abnormal spermatozoa
was not more than 15%.
The results in this study was
presumably achieved because in the FL3
treatment, 3% of soy lecithin levels were
more effective in protecting spermatozoa
from the effects of freezing so that
progressive motility and morphological
normality of post-freezing spermatozoa
were maintained. At this level, spermatozoa
are relatively spared from the adverse
effects of cryopreservation in the form of
freezing injury (Hoesni, 2016) and freeze
kills (Kharche et al., 2013) or those that
reduce the spermatozoa’s ability to survive
(Arrebola et al., 2013). As it is known before,
the main content of soy lecithin is
phospholipids (Collares, Bongalhardo,
Deschamps, & Moreira, 2018) or egg yolk-
like phospholipids (Choudhary, Pardhi, &
Bhoyar, 2013). These phospholipids have
been identified as cryoprotective
components that protect the integrity of
spermatozoa membranes during
cryopreservation (Fermini et al., 2016). The
working mechanism of soya bean lecithin in
protecting spermatozoa membranes from
freezing injury or freeze kill is not known for
certain, but some researchers suspect that
the mechanism is similar to that of low
density lipoprotein (LDL) as found in egg
yolks. In this case there are two main
hypotheses that arise; First, as an important
component of the bio-membrane of
mammalian sperm cells, phospholipids play
an important role in regulating the
physiological function of the bio-
membrane and enter the cell to lower the
freezing point of crystals by placing
plasmalogens to reduce mechanical
damage to the bio-membrane of
spermatozoa. Some experts who agree
with this opinion believe that soy lecithin
can reduce the cholesterol/phospholipid
ratio of sperm cell membranes by seeping
into the spermatozoa membranes. In
addition, soya bean lecithin phospholipids
can replace some of the phospholipids of
sperm cell membranes to maintain their
structure and function. Second, other
experts believe that the phospholipid soy
lecithin cannot enter the sperm cell
membrane to change the bio-membrane
phospholipid concentration, but it can
integrate with the sperm cell membrane to
form a protective film. or form an interfacial
layer between the fatty acids and water
(Gallier, Gordon, & Singh, 2012) to counter
the formation of lethal intracellular ice
crystals and protect sperm cell membranes
from mechanical damage during freezing
and thawing (Zhang, Hu, Li, Jiang, & Zhang,
2009). In more detail, there are at least
three main roles carried out by the
interfacial layer, namely (a) lowering the
surface tensions on the plasma membrane,
especially those caused by the absorption
of proteins on the membrane surface (b)
forming a mechanical defense system on
the surface of the plasma membrane
through the formation of a thin layer that is
viscoelastic so as to prevent damage to the
plasma membrane structure and (c) playing
a role in controlling colloidal interactions
between fatty acids and water. This second
hypothesis is also supported by the result
of observation showing that soybean
lecithin microparticles are relatively larger
Salmin, Marsudi, Hendro Sukoco, Deka Uli Fahrodi | 528
than spermatozoa under a microscope.
Therefore, they argued that it was
impossible for soy lecithin microparticles to
enter the sperm cell membrane and this
latter opinion was the one agreed the most.
The low percentage of progressive
motility and the high percentage of sheep
spermatozoa abnormalities in other
treatments were considered to be caused
by; (1) at the concentration of 4% soybean
lecithin (FL4), the concentration of soybean
lecithin in the extender solution was
relatively higher so that the medium
became hypertonic. Visually, at this level,
the soya bean Tris-lecithin extender was
slightly thicker than the other treatments.
Hypertonic extender medium can interfere
with locomotion and harm spermatozoa. If
the movement activity of spermatozoa
after freezing continues under these
conditions, it is suspected that it will result
in the spermatozoa running out of energy
and cause damages to the morphological
structure of the spermatozoa in the form of
a severed tail or neck (Figure 2).
Spermatozoa that run out of energy in turn
will reduce motility and viability and even
death of spermatozoa. (2) at levels below
3% soya bean lecithin (L0, L1 and L2),
cryoprotectant soy lecithin is relatively too
low than the required level so that it does
not optimally protect spermatozoa from
adverse freezing effects in the form of
freezing injury or freeze kill.
Figure 2. Sperm preparation of
spermatozoa viability (A) and spermatozoa
abnormalities in the form of severed tail
and neck (B) after freezing from 4%
soybean lecithin treatment (L4)
The results of this study are different
from previous reports by researchers on pig
spermatozoa cryopreserved using a
soybean lecithin extender where the quality
of the spermatozoa they obtained was
lower at 3% soy lecithin levels but higher at
6% levels. This is thought to be caused by
species differences, in which sheep
spermatozoa may be more responsive to
cryoprotectant lecithin from soybeans at
lower levels than pig spermatozoa. Stated
that damage due to cooling and freezing
effects on spermatozoa membranes varied
between domestic species and was
influenced by several elements, namely the
cholesterol/phospholipid ratio, lipid bilayer
content, degree of chain saturation
hydrocarbons and protein/phospholipid
ratio. This opinion is supported by which
states that susceptibility to cold shock is
related to the ratio between cholesterol
and phospholipids. The lower the ratio
between cholesterol and unsaturated fatty
acids, the more vulnerable the plasma
membrane is. Spermatozoa with higher
cholesterol levels and lower levels of
unsaturated fatty acids will have a more
compact plasma membrane structure and
tend to be more resistant to cold shock.
(B
529 | Motility and Abnormality of Sheep Spermatozoa That is Being Frozen Using Soybean
Lecithin (Soybean Lecithin)
Sheep spermatozoa contain cholesterol
226 mg/1000 million cells and in pig
spermatozoa the levels may be lower, so
categorize that pig spermatozoa are very
sensitive to cold shock compared to sheep
spermatozoa.
CONCLUSIONS
Based on the the results and discussion
above, it can be concluded that the results
of the study are as follows: 1) Levels of
soybean lecithin in the Tris extender
showed a significantly different effect
(P≤0.05) on the progressive motility and
abnormality of post-freezing sheep
spermatozoa. 2) The higher the level of soy
lecithin in the Tris extender, the more the
percentage of progressive motility
increased to 3% of soy lecithin, then
decreased to 4% of soy lecithin and the
percentage of spermatozoa abnormalities
decreased to 3% of pea lecithin. soybeans,
then increased levels of 4% soy lecithin. 3)
The best progressive motility and
abnormalities of post-freezing
spermatozoa were obtained in the L3
treatment, namely 3% soybean lecithin +
92% Tris extender + 5% glycerol, with
results of 43.79% and 13.72%, respectively.
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