




































 Agricultural Science; Vol. 2, No. 2; 2020 
ISSN 2690-5396   E-ISSN 2690-4799 

https://doi.org/10.30560/as.v2n2p15 

15                             Published by IDEAS SPREAD 
 

Can Panax Ginseng Aqueous Extract Improve Chilled and 
Cryopreserved Bull Spermatozoa? 

Falah Baiee1,2, Abd Wahid Haron2, Murtadha A. AL-mudhafr 4, Innocent Damudu Peter 2,3 & Nurhusien Yimer2 
1 Department of Clinical Science, Faculty of Veterinary Medicine, University of Kufa, Najaf, Iraq 
2 Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, UPM 
Serdang, Selangor, Malaysia 
3 Faculty of Veterinary Medicine, University of Maiduguri, Maiduguri, Nigeria 
4 Department of Microbiology, Faculty of Veterinary Medicine, University of Kufa, Najaf, Iraq 
Correspondence: Falah Baiee, Department of Clinical Science, Faculty of Veterinary Medicine, University of Kufa, 
54003, Najaf, Iraq. Tel: 964-773-808-9998. E-mail: falahhali@uokufa.edu.iq  

 
Received: May 29, 2020   Accepted: June 28, 2020   Online Published: July 14, 2020 
 
The research is financed by (University Putra Malaysia, UPM researching grant; GPIPS/2016/9492800) 
 
Abstract 
This study was to evaluate the influence of Panax ginseng aqueous extract on chilled and frozen-thawed bull sperm 
quality. Samples of semen were acquired from four bulls through the use of an electro-ejaculator. Extension of the 
semen was done with tris-egg yolk diluent which was augmented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/mL 
Panax ginseng aqueous extract. Diluted chilled portions of the semen were chilled for 6 days at 5 ̊C whereas the 
frozen semen was cryopreserved in liquid nitrogen. Results revealed that in chilled and frozen-thawed semen, the 
control group, T1 and T2 recorded higher percentages in terms of sperm motility and viability in all three groups 
evaluated compared to others, while the high dose of Panax ginseng aqueous extract in T6 and T5 recorded the 
lowest percentage. Moreover, the values of sperm morphology for chilled and frozen-thawed semen were not 
significant among the groups. The results of chromatin stability of the present study showed that T2 and control 
were higher than for other groups. In conclusion, the low dosage groups (T1, T2 and T3) which were received (0.25 
mg/mL, 0.5 mg/mL and 1 mg/mL, respectively) from Panax ginseng aqueous extract were not significant as 
compared with the control group while high-dosage groups (T4, T5 and T6) which were received (2.5 mg/mL, 5 
mg/mL and 7.5 mg/mL, respectively) from Panax ginseng aqueous extract were highly decreased spermatozoa 
characteristics. 
Keywords: bull, semen cryopreservation, chilled semen, Panax ginseng, chromatin stability 
1. Introduction 
1.1 Introduce the Problem 
Bovine insemination using the artificial insemination (AI) technique is among the main difficulties in the livestock 
industry. Semen preserved in liquid or frozen form are common procedures in inseminating cows; roughly 95% of 
AI dosages are in the form of frozen semen straws (Amann and Waberski, 2014). Nevertheless, freezing-thawing 
practices of prolonged semen are accountable for a substantial decrease in motility, viability, membrane integrity 
and morphology of sperm (Amirat-Briand et al., 2010; Yimer et al., 2014; Baiee et al., 2018a) resulting in decreased 
rate of fertility of the frozen semen (Büyükleblebici et al., 2014). In actual fact, chilled semen possesses a high 
rate of fertility in comparison to frozen semen (Medeiros et al., 2002; Galarza 2019); nonetheless, chilled semen 
can be kept for a few days only (Papa et al., 2015). Consequently, the use of chilled semen in the AI technique is 
restricted (Borges-Silva et al., 2016). Therefore, one purpose of this study was to determine the ability of a natural 
material in promoting chilled seminal quality for long-time storage and usage. Many studies have been done to 
improve frozen-thawed seminal quality through addition of some additives to the extended semen quality, for 
example, anti-oxidant (Amirat-Briand et al., 2010; Khumran et al., 2015; Eidan, 2016), or amino acids (Holt et al., 
2015; Kumar et al., 2015) and unsaturated fatty acid (Kaka et al., 2015; Khoshvaght et al., 2015; Tarig et al., 2017). 
Furthermore, certain extracts of plants have been revealed as anti-oxidant additives which are used to add to semen 
or as a semen diluent (Chen et al., 1998; Baghshahi et al., 2014; Sapanidou et al., 2015), and to examine the toxic 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 16       Published by IDEAS SPREAD 
 

consequence of plant extract on sperm (Kaefer et al., 2013). Plant extracts are also used in diluting semen (Abul 
Rashid and Nurin, 2015). Based on our understanding, there are no studies that use Panax ginseng aqueous extract 
(PGe) in the tris-egg yolk diluent as enhancement for chilled and freezing semen. 
Sperm present with Chromatin deficiencies, largely due to imperfect spermatogenesis, faulty apoptosis before 
ejaculation (Baiee et al., 2018b), extreme reactive oxygen species (ROS) production in the ejaculate (Moustafa et 
al., 2004), in vitro handling (Bollwein et al., 2008), and also the stress of the freezing-thawing processes (Khalifa 
et al., 2008; Lymberopoulos and Khalifa, 2010). In fact, frozen-thawed semen processes lead to chromatin damage 
(Métayer, et al., 2002; Gadea et al., 2008; Büyükleblebici et al., 2014). However, the mechanisms accountable for 
freezing-induced chromatin damage are unidentified. Data suggests that this could be the consequence of lipid 
peroxidation (LPO) in injurious sperm chromatin (Kasimanickam et al., 2007). Moreover, sperm nuclei exposure 
to high ionic strength in the course of freezing-thawing of semen instigates deterioration of chromatin assembly 
and sequentially, makes post-thawing nuclear DNA available to the oxidative occurrence through extra- or 
intracellular ROS (Gadea et al., 2008). Additionally, perhaps, the unrestrained post-thawing influx of wandering 
calcium ions in frozen sperm (Büyükleblebici et al., 2014) may possibly encourage additional splitting of 
nucleoprotein and Chromatin through endogenous protease and nuclease stimulation (Métayer et al., 2002). 
Panax ginseng Meyer, from the family Araliaceae, is one of the most widely used traditional herbal medicines in 
Eastern and Western countries. Panax ginseng Meyer has been used in traditional medicine for over 2000 years to 
restore and enhance normal well-being, sexuality, promotion of vitality, erectile dysfunction, control of 
hypertension, and increase of resistance to stress and ageing (Attele et al., 1999; Kopalli et al., 2016). Active 
compounds in Panax ginseng include saponin (ginsenosides), acidic polysaccharides, peptides, polyacetylenes, 
alkaloids, and phenolic compounds (Attele et al., 1999). Ginsenosides of saponins are regarded as the main active 
components responsible for the pharmacological activities of Panax ginseng and approximately 40 kinds of 
saponin have been isolated from it. Most of these are protopanaxdiol and protopanaxtriol, which are aglycones of 
dammarane-type triterpenoids (Patel and Rauf 2017). Recently, physicochemical and spectroscopic data were 
acquired from four major diol-saponins: ginsenosides Rb1, Rb2, Rc, Re, Ro, and Rd (Cho et al., 2010). Thus, the 
main aim of this study was to determine and evaluate the effect of Panax ginseng aqueous extract on the quality 
of frozen-thawed bull semen. Furthermore, it was to determine the effect of Panax ginseng aqueous extract on 
chromatin stability of frozen-thawed bull semen.  
2. Method 
2.1 Animals 
This work experiment was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary 
Medicine, UPM (RO73/2015). Twenty-four ejaculates were obtained through the use of the electro-ejaculator (EE; 
Ideal® Instruments; Neogen Corporation, Lansing, Michigan; USA) from four fertile crossbred (Simmental-
Brangus) bulls from the Universiti Putra Malaysia (UPM) in Serdang, Selangor (2◦9-18.36-N; 101◦43-49.61-E) 
farm. Bulls were 4.8 ± 0.8 years old measuring 551.2 ± 41.5 kg in weight. The entire four bulls were under the 
same feeding; the animals were fed with Brachiaria decumbens grass and also commercial palm kernel cake at 3 
kg/body weight rate. Additionally, mineral blocks and water were given to the animals ad libitum. 
2.2  Chemicals 
Panax ginseng aqueous extract (GN06032014) was obtained from LJack (M)® Sdn. Bhd. Semenyih, Selangor, 
Malaysia with certification of the analysis. Tris, citric acid, sodium citrate, fructose, nigrosin, glycerol and eosin-
y were acquired from Sigma Aldrich Co. (Sigma, St. Louis, MO, USA). Penicillin-streptomycin was procured 
from Gibco® (15140122, Gibco® USA). Acridine orange stain was bought from Invitrogen® (AR7601000) 
Eugene; Oregon, USA; the straws were acquired from Minitube® (13407/0010; Slovakia). Lastly, methanol and 
glacial acetic acid were obtained from Merck (E, Merck D6100 Darmstadt, and F. R. Germany). 
2.3 Preparation of Semen Diluent 
Preparation of Tris-egg yolk diluent was as earlier described by Amirat-Briand et al. (2010) with a slight adjustment 
(Baiee et al., 2017). Tris-egg yolk diluent was divided into two portions; the first portion (P1) contained Tris: 2.24 
g, citric acid: 1.48 g, fructose: 1 g, 50000 IU penicillin-streptomycin, and 20% egg yolk in 100 mL distilled water. 
The second portion (P2) had similar constituents to the initial but with 12.8% glycerol, the double amount of 
glycerol being to create the concluding concentration of glycerol after mixing with P1 6.4%. Each portion was 
divided into seven parts in test tubes to give diverse concentrations of the extract of Panax ginseng. Furthermore, 
the diluents were weekly preparations and were kept at -20 ̊C. 
 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 17       Published by IDEAS SPREAD 
 

2.4 Semen Collection 
The collection of semen samples was done weekly very early in the morning through the use of auto mode of the 
EE. Bulls were directed to a chute. Prepuce hairs of the bulls were shaved; the preputial orifice was cleaned using 
water and dried with a clean tissue towel. Before insertion of rectal probe, rectal evacuation was done, and the EE 
device was switched on; a minimum of three individuals worked together to achieve semen sample collection. 
After sample collection, samples were conveyed to the Theriogenology and Cytogenetics lab at 37 ̊C. Computer 
assisted sperm analysis (CASA; Hamilton Thorne Bioscience; USA) was used to evaluate the concentration of 
semen and the proportion of sperm motility for fresh semen in addition to the universal sperm motility of chilled 
and cryopreserved semen. Semen samples with general sperm motility ≥ 70%, sperm viability and normal sperm 
morphology ≥ 85% and concentration of ≥ 600 ×106 sperm/mL (Kaka et al., 2015) were acknowledged to be chilled 
and cryopreserved 
2.5 Experimental Design 
The dosages of PGe were determined into six groups for all chilled and frozen-thawed experiments. Three groups 
with low concentrations were T1 0.25 mg/mL, T2 0.5 mg/mL, T3 1 mg/mL, and three groups with high 
concentrations were T4 2.5 mg/mL, T5 5 mg/mL, T6 7.5 mg/mL. Moreover, one group without addition of PGe 
was kept to be a control group. The extract was dissolved into tris-egg yolk diluent by shaking and incubation in 
water path at 37 ºC for 10 min. The successfully collected semen sample was divided into two equal aliquots, the 
1st aliquot was for the chilled semen experiment, and the 2nd aliquot was for the cryopreserved experiment. 
2.5.1 Effect of Panax Ginseng on Chilled Bull Seminal Quality 
The first aliquot of fresh semen was equally divided into seven groups which contained diverse concentrations of 
PGe which were dissolved in tris-egg yolk diluent (P1, deprived of glycerol). Extended semen groups were store 
in a refrigerator for six days at 5 ̊C. Sperm morphology, viability and motility were assessed after 1, 3 and 6 days 
for cooling. Furthermore, sperm membrane integrity was assessed at day three of chilling in the entire groups. The 
final concentration of semen in chilled experiments was about 80 × 106 sperm / mL. 
2.5.2 Effect of Panax Ginseng on Frozen-Thawed Bull Seminal Quality 
The second aliquot of the fresh semen was also equally divided into seven groups which contained tris-egg yolk 
diluent augmented with diverse concentrations of PGe (P1), they were chilled in a refrigerator at 5 ̊C for three 
hours. The second portion of diluent which contained glycerol (P2) was added to the entire treatment groups; it 
was later packaged in 0.25 mL French straws. The end concentration of glycerol in the diluent was 6.4% while the 
end concentration of spermatozoa was 20 × 106 sperm / straw. The entire groups of straws were frozen at liquid 
nitrogen for future investigation. Motility of sperm, morphology and viability values were recorded in 2, 7 and 14 
days after freezing. Sperm membrane integrity was recorded after 7 days, and sperm DNA integrity was also 
recorded after 20 days of freezing. 
2.6 Evaluation of Sperm Motility, Morphology and Viability 
Sperm viability and normal morphology proportions were investigated through the use of eosin-nigrosin stain (EN) 
as was earlier described by Felipe-Pérez et al. (2008). Briefly, a 10 μL droplet of freshly chilled or cryopreserved 
semen was added with 20 μL of EN stain; the mixture was smeared on a microscope slide and kept to dry at 45 ºC 
on a slide warmer. Subsequently the slides were viewed under a phase-contrast microscope (Nikon Japan, Eclipse 
E200) at × 400 for viability of the sperm. Sperm that were stained with pink colour were considered to be dead 
while the unstained sperm were deemed to be alive. Additionally, in the usual sperm morphology, only sperm with 
normal sperm head, midpiece and tail are referred to as normal irrespective of the colour at × 1000 with oil 
emersion, and for each test 200 sperm were counted. 
2.7 Evaluation of Sperm Membrane Reliability 
The hypo-osmotic swelling test (HOST) was done to investigate the sperm membrane reliability in chilled and 
cryopreserved sperm. Essentially, HOST was prepared as was earlier described by Revell and Mrode (1994). 
Briefly, 0.9 g fructose and 0.49 g tri-sodium citrate were dissolved in distilled water; additional distilled water was 
added to make up the volume to 100 mL. Ten microliters of chilled or cryopreserved sperm were incubated with 
90 μL of hypo-osmotic solution for 30 min at 37 ̊C. Subsequently 10 μL of incubated solution was positioned on 
a glass slide and covered using a cover slip and observed under the phase-contrast microscope at × 400. Sperm 
possessing twisted tails under hypo-osmotic conditions were considered to possess complete membranes. 
2.8 Evaluation of Sperm Chromatin Stability 
Acridine orange test (AOT) was performed in determining sperm chromatin stability in cryopreserved sperm as 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 18       Published by IDEAS SPREAD 
 

was described by Tejada et al. (1984). A metachromatic dye, acridine orange (AO) stain interpolates the DNA of 
sperm cells to release colours fluctuating between green, green yellow, yellow, orange, and red. Sperm cells with 
intact chromatin radiated green fluorescence, whereas those possessing altered chromatin radiated green yellow, 
yellow, orange, and red (Figure 1) dependent on the bulk of fragmented chromatin. Consequently, the outcomes 
were divided into five groupings based on the percentage of chromatin destroyed. A thick semen smear was made 
on a glass slide and kept to dry. Carnoy’s solution was daily prepared for the smear fixing for 3 hours at a chilled 
temperature. The slides were allowed to dry once more and stained with AO staining solution for 5 minutes; the 
slides were then washed gently using distilled water. Slides were allowed to dry and were covered using cover 
slips and observed under a fluorescence microscope (Nikon eclipse, Ti-s Japan) within 30 minutes. This is because 
this test ought to be done within 30 a minimum of three microscopic fielded positions were taken from each slide 
for each group and saved in the computer for further evaluation. 
 

Figure 1. v, w, x, y, and z. Examples of spermatozoa are from bulls stained with Acridine orange stain and 
examined under fluorescence microscope (Nikon eclipse, Ti-s Japan). Acridine orange stain emits green colour 
under blue fluorescence laser meaning to normal chromatin; but, it releases green-yellow, yellow, orange, and 

red meaning to chromatin impairment. 
v: shows a green sperm that had relatively intact chromatin 
w: green-yellow sperm that has 75% intact chromatin 
x: Yellow sperm that has 50% intact chromatin 
y: orange sperm that has 25% intact chromatin 
z: red sperm head that has relatively total chromatin impairment 
 
2.9 Statistical Analysis 
Data obtained were tested for normal distribution through the use of the Shapiro-wilk test. Two-way ANOVA was 
utilized in comparing sperm motility, viability and morphology amongst different groups and different evaluation 
times while one-way ANOVA was used in comparing sperm membrane integrity and chromatin integrity among 
groups. Two-way and one-way ANOVA were followed by the LSD test in comparing the significances among 
groups through the use of the SPSS programme version 22 for windows (SPSS Inc., Chicago, IL; the USA). 
3. Results 
The values of chilled semen for sperm motility (Table 1), morphology (Table 2) and viability (Table 3) were 
evaluated at three different times. Control, T1 and T2 groups recorded the highest percentage in terms of sperm 
motility and viability in all three evaluations as compared among groups for each time of evaluation, while the 
high dose of PGe in T5 and T6 recorded the lowest percentage. Moreover, the mean percentages of sperm 
morphology for chilled semen were not significant among groups. On the other hand, there are significant 
differences in terms of time of evaluation, since the values were significantly different for the first evaluation as 
compared to the third chilled semen evaluation. Sperm morphology values were also decreased during time of 
preservation in chilled form, but it was not significantly different among periods.   
 
 
 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 19       Published by IDEAS SPREAD 
 

Table 1. Effect of Panax ginseng aqueous extract on chilled semen motility in three different times of evaluation 

Parameters Groups 
Mean ± SEM 
1st Evaluation
1 d 

2nd Evaluation 
3 d

3rd Evaluation 
6 d 

Sperm motility  
(%) 

T1 75.69 ± 1.84aX 61.62 ± 3.27aY 28.19 ± 2.04aZ

T2 79.94 ± 1.16aX 64.87 ±  2.35aY 28.88 ± 2.02aZ

T3 66.44 ± 1.77bX 52.25 ± 2.97bX 17.13 ± 1.62bY

T4 44.19 ± 3.13cX 22.62 ± 3.49cY 3.25 ± 1.02cZ 

T5 13.13 ± 1.67cdX 3.94 ± 0.91dXY 0.70 ± 0.32cY 

T6 4.94 ± 0.71d 0.41 ± 0.11d 0.00 ± 0.00c 

Control 75.00 ± 2.06aX 59.56 ± 2.31aY 27.12 ± 2.45aZ

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24  
a, b,c, & d superscripts mean between groups were significantly different at (P˂ 0.05; within column between groups).  
x,y & z superscripts mean between times of evaluation were significantly different at (P˂ 0.01; between columns for 
the same group). 
  
Table 2. Effect of Panax ginseng aqueous extract on chilled semen normal morphology in three different times of 
evaluation 

Parameters Groups 
Mean ± SEM 

1st Evaluation
1 d

2nd Evaluation
3 d

3rd Evaluation
6 d 

Sperm 
morphology 
(%) 

T1 89.44 ± 0.66 88.47 ± 0.77 86.94 ± 1.07
T2 89.66 ± 0.79 88.12 ± 0.89 87.19 ± 0.93
T3 87.47 ± 0.83 86.59 ± 0.99 86.28 ± 0.86
T4 88.44 ± 0.97 88.03 ± 0.91 86.06 ± 1.10
T5 87.69 ± 0.93 86.75 ± 0.64 86.13 ± 1.01
T6 88.06 ± 0.96 87.03 ± 0.92 86.19 ± 1.11
Control 89.19 ± 1.00 88.25 ± 0.93 87.31 ± 1.22

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24 
 
Table 3. Effect of Panax ginseng aqueous extract on chilled semen viability in three different times of evaluation 

Parameters Groups 
Mean ± SEM 
1st Evaluation
1 d 

2nd Evaluation 
3 d

3rd Evaluation 
6 d 

Sperm viability 
 (%) 

T1 78.72 ± 1.45aX 70.41 ± 1.74aX 52.10 ± 2.21abY 

T2 80.72 ± 1.25aX 71.78 ± 1.60aX 56.47 ± 1.89aY 

T3 67.75 ± 2.39bX 60.31 ± 2.18bX 49.06 ± 2.42bY 

T4 48.56 ± 5.24cX 41.15 ± 4.58cXY 33.41 ± 3.69cY 

T5 26.03 ± 4.04cdX 17.75 ± 2.18cdXY 12.00 ± 1.79cdY 

T6 16.25 ± 2.68dX 9.22 ± 1.14dXY 4.50 ± 0.77dY 

Control 78.16 ± 1.14aX 67.22 ± 1.21aX 52.66 ± 1.35abY 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24  
a, b,c, & d superscripts mean between groups were significantly different at (P˂ 0.05; within column between groups).  
x,y & z superscripts mean between times of evaluation were significantly different at (P˂ 0.01; between columns for 
the same group). 
 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 20       Published by IDEAS SPREAD 
 

Frozen-thawed semen was also evaluated into three different times in terms of sperm motility (Table 4), 
morphology (Table 5) and viability (Table 6). Control, T1 and T2 groups were significantly higher than other 
groups in terms of sperm motility and viability values for 1st, 2nd and 3rd evaluations. The low percentage of 
sperm motility was recorded in high dose of PGe T6 = 7.5 mg/mL (0.00 ± 0.00%) in all the three different periods, 
followed by T5 = 5 mg/mL (0.31 ± 0.10, 0.25 ± 0.10, 0.28 ± 0.09, respectively). Moreover, sperm morphology 
values of frozen-thawed semen did not differ among groups which had different concentrations of PGe in all the 
three times of evaluations. On the other hand, there were no significant differences among 1st, 2nd and 3rd 
evaluations in terms of sperm motility, morphology and viability (P > 0.05). 
 
Table 4. Effect of Panax ginseng aqueous extract on frozen-thawed semen motility in three different times of 
evaluation 

Parameters Groups 
Mean ± SEM 
1st Evaluation 
2 days 

2nd Evaluation 
7 days  

3rd Evaluation 
14 days 

Sperm motility  
(%) 

T1 46.81 ± 2.98a 46.00 ± 3.15a 49.13 ± 3.05a 

T2 43.31 ± 3.37a 39.50 ± 2.24a 40.00 ± 2.42a 

T3 15.38 ± 1.05b 16.06 ± 2.09b 16.68 ± 2.05b 

T4 5.06 ± 1.07c 3.44 ± 0.88c 3.44 ± 0.65c 

T5 0.31 ± 0.10c 0.25 ± 0.10c 0.28 ± 0.09c 

T6 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 

Control 46.94 ± 2.19a 48.87 ± 3.41a 51.25 ± 2.76a 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24  
a, b & c superscripts mean between groups were significantly different at (P˂ 0.05; within column between groups). 
 
Table 5. Effect of Panax ginseng aqueous extract on frozen-thawed semen morphology in three different times of 
evaluation 

Parameters Groups 
Mean ± SEM 
1st Evaluation 
2 days 

2nd Evaluation 
7 days  

3rd Evaluation 
14 days 

Sperm 
morphology 
(%) 

T1 90.91 ± 0.67 90.11 ± 0.71 90.25 ± 0.63 
T2 90.69 ± 0.56  90.69 ± 0.59  90.59 ± 0.45 
T3 89.22 ± 0.81 89.47 ± 0.73 88.85 ± 0.70 
T4 88.91 ± 0.80 89.00 ± 0.79 89.13 ± 0.75 
T5 87.97 ± 0.80  88.25 ± 0.86 88.41 ± 0.69 
T6 88.19 ± 0.74 88.06 ± 0.88 87.69 ± 0.77 
Control 91.47 ± 0.75 91.19 ± 0.66 91.31 ± 0.54 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24 
 
 
 
 
 
 
 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 21       Published by IDEAS SPREAD 
 

Table 6. Effect of Panax ginseng aqueous extract on frozen-thawed semen viability in three different times of 
evaluation 

Parameters Groups 
Mean ± SEM
1st Evaluation
2 days 

2nd Evaluation
7 days  

3rd Evaluation 
14 days 

Sperm viability 
 (%) 

T1 65.03 ± 2.50a 65.13 ± 2.54a 65.84 ± 2.65a 

T2 58.44 ± 3.12a 57.28 ± 2.95a 57.28 ± 2.97a 

T3 31.97 ± 2.72b 32.56 ± 2.75b 33.28 ± 0.95b 

T4 13.94 ± 1.92c 13.69 ± 2.14c 13.66 ± 1.64c 

T5 6.91 ± 0.90c 6.09 ± 0.73c 6.13 ± 0.72c 

T6 3.47 ± 0.53c 3.04 ± 0.38c 3.25 ± 0.48c 

Control 66.03 ± 1.71a 66.06 ± 1.79a 66.34 ± 1.74a 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24  
a, b & c superscripts mean between groups were significantly different at (P˂ 0.05; within column between groups) 
 
Sperm membrane reliability of chilled and frozen-thawed spermatozoa was evaluated using HOST. The collected 
data of chilled semen referred to as T1 (66.03 ± 2.05%), T2 (66.84 ± 2.75%) and control (65.03 ± 1.52%) groups 
were higher significantly at (P < 0.01) as compared to T3 (53.62 ± 3.89%), T4 (36.84 ± 4.89%), T5 (14.78 ± 2.58%), 
and T6 (7.25 ± 1.39%) in chilled semen (Figure 2). Moreover, the collected data of frozen-thawed semen revealed 
that control (58.28 ± 2.13%), was higher than T1 (57.44 ± 1.96%; P = 0.73), T2 (52.56 ± 2.15%; P < 0.05), T3 
(29.31 ± 2.17%), T4 (13.12 ± 1.54%), T5 (5.97 ± 0.92%), and T6 (3.31 ± 0.54%; P < 0.01) in terms of sperm 
membrane reliability (Figure 3). There was no significant difference between T1 and T2 (P = 0.06). In fact, the 
percentage of sperm membrane reliability decreased as the dosages increased in chilled and frozen-thawed bull 
semen.  
 
 
 
 
 
 
 
   
 
 
 
 
 
 
Figure 2. Effect of Panax ginseng extract on sperm membrane integrity of chilled bull sperm (%; Mean±SEM). 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24 
a, b, c and d superscripts mean between groups were significantly different   



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 22       Published by IDEAS SPREAD 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 3. Effect of Panax ginseng extract on sperm membrane integrity of frozen-thawed bull sperm (%; 
Mean±SEM) 

T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL  
n= 24 
a, b, c, d, and e superscripts mean between groups were significantly different. 
 
Sperm chromatin integrity was evaluated using Acridine orange stain that was examined under a fluorescence 
microscope during 30 min after staining. Because the AO stain emits colour shifting from green to red, the 
calculated spermatozoa were distributed into five categories depending on the colour that emitted from them. In 
fact, only the green head of spermatozoa were considered to be entirely chromatin. The results of the present study 
showed that T2 (75.40 ± 5.27) and control (75.02 ± 5.10) groups were significantly higher than other groups except 
T1 (71.15 ± 4.93) which was not significant (Table 7). On the other hand, the chromatin impairment which situated 
under red was higher in T3 followed by T4, T6 and T5 (15.09 ± 2.33, 13.95 ± 1.26, 12.26 ± 3.51 and 11.71 ± 2.87; 
respectively). 
 
Table 7. Effect of Panax ginseng extract on chromatin stability of frozen-thawed bull sperm (%; Mean±SEM) 

Groups  Green* Green-yellow Yellow  Orange  Red  
T1 71.15 ± 4.93ab 4.14 ± 0.98 8.82 ± 1.96 12.25 ± 1.96 5.79 ± 2.22a 

T2 75.40 ± 5.27a 3.69 ± 0.99 7.43 ± 1.88 9.20 ± 2.40 5.97 ± 1.36a 

T3 70.19 ± 2.83 3,41 ± 0.94 6.05 ± 1.14 6.67 ± 2.12 15.09 ± 2.33b 

T4 64.29 ± 3.59b 4.20 ± 0.73 5.01 ± 1.58 13.75 ± 3.68 13.95 ± 1.26b 

T5 59.48 ± 2.90c 5.73 ± 1.05 11.65 ± 1.91 14.16 ± 3.34 11.71 ± 2.87b 

T6 63.53 ± 4.66c 4.08 ± 1.14 9.40 ± 1.57 12.82 ± 1.74 12.26 ± 3.51b 

Control 75.02 ± 5.10a 3.77 ± 0.91 7.85 ± 2.19 10.05 ± 4.06 5.08 ± 1.69a 

* Green: sperm had totally intact DNA                  Green-yellow: sperm had 75% intact DNA 
Yellow: sperm had 50% intact DNA                      Orange: sperm had 25% intact DNA 
 Red: sperm that had totally damaged. 
T1 = 0.25 mg/mL, T2 = 0.5 mg/mL, T3 = 1 mg/mL, T4 = 2.5 mg/mL, T5 = 5 mg/mL, T6 7.5 mg/mL, and Control = 
0.0 mg/mL.  
n= 24 
Different superscripts a, b and c in the same column demonstrates statistical different among groups at (P˂0.05) 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 23       Published by IDEAS SPREAD 
 

4. Discussion 
Studies have shown that addition of plant origin additives such as clove bud (Baghshahi et al., 2014), curcumin 
(Tvrdá et al., 2016), soy-bean lecithin (Chelucci et al., 2015), virgin coconut oil (Tarig et al., 2017), and corcin 
(Sapanidou et al., 2015) might improve some characteristics of the chilled or frozen-thawed seminal quality. Panax 
ginseng is well demonstrated in literature to remove the harmful effect of ROS, to restore and enhance normal 
wellbeing, to promote vitality, and to increase resistance to stress (Attele et al., 1999; Kopalli et al., 2016a;b). To 
our knowledge, no study has been conducted to evaluate the effect of PGe on tris-egg yolk diluent to improve the 
quality of chilled and frozen-thawed bull semen. Therefore, one purpose of this study was to determine the ability 
of natural material in promoting chilled seminal quality for long-time storage and usage. Moreover, this study was 
to determine the effect of PGe supplemented in bull semen diluent that had been stored in frozen form. In the 
present study, six doses of PGe have been distributed into six groups; three were low-dose and three were high-
dose. In fact, there are many variables which might affect the actual effect of the additive such as the quality of 
fresh semen sample, so we managed the quality of semen as mentioned in materials and methods that depended 
on previous experts of our lab (Kaka et al., 2015; Khumran et al., 2015), handling of semen sample after collection 
until dilution and also any change in freezing temperature during the freezing process which might lead to false 
results. Thus, to overcome these issues, two criteria had been used; firstly, after cryopreservation we checked the 
viability value of one straw from the control group and the frozen sample was accepted if the control group had 
50% and more viable sperms (universal standard acceptance) otherwise the sample was cancelled. The second 
criterion was we repeated the conventional parameters in terms of sperm motility, morphology and viability for 
three different times to be more precise. 
Based on our findings, in chilled form, the sperm motility (Table 1), morphology (Table 2) and viability (Table 3) 
values of T1, T2, and control groups were higher than T3, T4, T5 and T6 in all evaluation periods. These findings 
were in contrast with our hypothesis and PGe appeared to have a negative effect on chilled semen. Sperm 
membrane reliability (Figure 2) had also decreased at the same manner of sperm motility (%). Thus, these finding 
indicated that PGe had a detrimental effect on chilled semen, and the low concentration of PGe did not have a 
beneficial effect for chilled semen. In the present study the sperm motility values in groups T1, T2 and control were 
in line with Nair et al. (Nair et al., 2006) who reported (71.00 ± 3.24) at 0 h. However, our finding of the sperm 
motility (%) was higher with that study as they reported the motility value decrease to be (19.33 ± 1.95) at day 3 
of chilling in cattle bull, while in the present study the sperm motility was (64.87 ± 2.35) at day 3 in T2 group.  
Regardless of the effect of PGe, the total sperm motility, morphology, viability, and sperm membrane reliability 
were significantly decreased after six days of chilling in all groups. These findings were in agreement with previous 
studies (Murphy et al., 2013; Nair et al., 2006). 
The frozen-thawed semen showed a similar manner as in chilled semen. Since, the sperm motility (Table 4), 
morphology (Table 5) and viability (Table 6) values were significantly decreased in high doses of PGe groups as 
compared to control, T1 and T2 groups. Furthermore, PGe had a detrimental effect on sperm morphology, 
particularly sperm taillessness, dag-defect and decapitated-head (data not shown). In accordance to the time of 
evaluation of frozen-thawed semen, there were no significant differences among periods of evaluations in all 
groups of frozen-thawed semen. 
In 1998 Chen et al. (1998) were found that, the motility of human sperm was improved when incubated with 1 and 
2 mg/mL of Panax notoginseng. Our study is in agreement with a study by Gray et al. (2016) who found that, the 
total progressive and kinetic motilities of boar semen which was incubated with PGe 2 mg/mL did not differ from 
control and other groups; they attributed this to the fact that semen samples may receive insufficient amounts from 
ginsenosides Rc and Rb2 that is responsible for improving sperm motility (Chen et al., 1998). This was unlike Yun 
et al. (2016) who found that the motility of sperm treated with PGe at 2 mg/mL, was greater in comparison to the 
control group and in line with anti-oxidative enzyme groups. They concluded that, PGe exhibited an overall 
positive effect on the male reproductive system of guinea pigs. However, in the present study the quality of frozen-
thawed bull semen was diminished in dosage of 1 mg/mL and above. This contrary finding needs further 
investigations to understand the mechanism of action of PGe. 
The sperm plasma membranes are the main spot of destruction prompted through cryopreservation (Abavisani et 
al., 2013). In addition, sperm plasma membranes play an important part in the fertilization capability and sperm–
oocyte relations (Abayasekara and Wathes, 1999). Unfortunately, sperm membrane reliability were not improved 
in most treatment groups and the highest value was (58.28 ± 2.13%) in the control group (Figure 3). Based on the 
results of the present study, the high dose of PGe had a negative effect on frozen-thawed bull semen, i.e. findings 
indicated that with an increase in PGe (T3, T4, T5, and T6) resulted in poor quality of chilled and frozen-thawed 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 24       Published by IDEAS SPREAD 
 

semen. On the other hand, the low doses of PGe groups (T1, and T2) were in line with the control group in most 
parameters that were evaluated in this study. Our findings are further strengthened with Kaka et al. (2016) who 
used tris-egg yolk diluent and Abavisani et al. (2013) and Kandelousi et al. (2013) who used citrate based diluent 
supplemented with poly-unsaturated fatty acids (PUFAs). These studies found that the high dose of PUFAs could 
reduce the quality of frozen-thawed bull semen such as in terms of sperm motility, morphology, viability, and 
membrane reliability. However, findings of the present study were in contrast with Tvrdá et al. (2016) in which 
they revealed that curcumin had protective effects on the functional activities and structural integrity of 
spermatozoa even when the oxidative stress increased. The present study is also in contrast with Sapanidou et al. 
(2015) who demonstrated that addition of 1 mM of curcumin (active component of saffron plant) could improve 
the sperm motility, viability and DNA integrity of frozen-thawed bull semen.  
Sperm exposure to traumatic situations of freezing-thawing may compromise the mechanism resulting in inhibited 
fertilization or embryonic growth (Kasimanickam et al., 2007; Khalifa et al., 2008). Kasimanickam et al. (2007) 
and Lymberopoulos and Khalifa (2010) showed that rising proportions of DNA-damaged sperm was related to 
declining field fertility of frozen–thawed bull semen. Similarly, Khalifa et al. (2008) show that, a substantial 
negative association exists between the occurrence of DNA-damaged sperm in frozen-thawed semen and the 
developing capability of bovine embryos in vitro. On the other hand, one of the main objectives of the present 
study was to improve the quality of sperm chromatin stability of cryopreserved bull semen. However, PGe 
negatively affected sperm chromatin stability (Table 7). The low dosage groups (T1, T2 and T3) which were received 
(0.25 mg/mL, 0.5 mg/mL and 1 mg/mL, respectively) from PGe were not significant as compared with the control 
group while high-dosage groups (T4, T5 and T6) which were received (2.5 mg/mL, 5 mg/mL and 7.5 mg/mL, 
respectively) from PGe were highly decreased spermatozoa characteristics. 
5. Conclusion  
In conclusion, addition of Panax ginseng aqueous extract into tris-egg yolk diluent did not improve chilled and 
frozen-thawed bull semen processes in dosages that were used in this study. Furthermore, the high doses of PGe 
have a negative effect on fresh, chilled and frozen-thawed bull semen. Furthermore, this study was a good 
opportunity to investigate this medicinal plant extract’s effect directly on cell biology and function. 
Acknowledgments 
The authors wish to acknowledge the management and staff of University Putra Malaysia farm (TPU) and 
Theriogenology and Cytogenetics laboratory of the Faculty of Veterinary Medicine, UPM for their cooperation 
during this study. The authors also would like to thank the company L Jack (M)® for providing the extract of 
Panax ginseng with certification.  
Conflict of interests 
The authors declare that they have no conflict of interests. 
References 
Abavisani, A., Arshami, J., Naserian, A. A., Kandelousi, M. A. S., & Azizzadeh, M. (2013). Quality of bovine 

chilled or frozen-thawed semen after addition of omega-3 fatty acids supplementation to extender. Int J Fertil 
Steril, 7(3), 161.  

Abayasekara, D., & Wathes, D. (1999). Effects of altering dietary fatty acid composition on prostaglandin synthesis 
and fertility. Prostag Leukotr Ess, 61(5), 275-287. https://doi.org/10.1054/plef.1999.0101  

Abul Rashid, B., & Nurin Qistina, M. N. (2015). Preliminary Evaluation on the Use of Coconut Water as Semen 
Dilent for Local Kampung Cockerel. Paper presented at the Proc. 2nd ARCP & 36th MASP Ann. Conf.,1-3 
June 2015, Pordickson, Negeri Sembilan, Malaysia.  

Amann, R. P., & Waberski, D. (2014). Computer-assisted sperm analysis (CASA): capabilities and potential 
developments. Theriogenology, 81(1), 5-17. e13. https://doi.org/10.1016/j.theriogenology.2013.09.004  

Amirat-Briand, L., Bencharif, D., Vera-Munoz, O., Pineau, S., Thorin, C., Destrumelle, S., . . . Shmitt, E. (2010). 
In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: 
Preliminary results of artificial inseminations. Anim Reprod Sci, 122(3), 282-287. 
https://doi.org/10.1016/j.anireprosci.2010.09.006  

Attele, A. S., Wu, J. A., & Yuan, C. S. (1999). Ginseng pharmacology: multiple constituents and multiple actions. 
Biochem pharmacol, 58(11), 1685-1693. https://doi.org/10.1016/S0006-2952(99)00212-9  

Baghshahi, H., Riasi, A., Mahdavi, A., & Shirazi, A. (2014). Antioxidant effects of clove bud (Syzygium 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 25       Published by IDEAS SPREAD 
 

aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation. Cryobiology, 
69(3), 482-487. https://doi.org/10.1016/j.cryobiol.2014.10.009  

Baiee, F. H., Abd Wahid H., Rosnina Y., Ariff O., Yimer, N., Zaid J., Salman H., Tarig A. A., W-N. Fitri, & M. 
Umar. (2017). Kinetic Motilities of Cryopreserved Bull Spermatozoa: Owing to the Effect of Eurycoma 
longifolia Jack Aqueous Extract. American J Anim Vet Sci, 12(2). https://doi.org/10.3844/ajavsp.2017.77.84  

Baiee, F. H., Wahid, H., Rosnina, Y., Ariff, O., Yimer, N., Jeber, Z., Hammadi, S., Tarig, A., & Harighi, F. (2018a). 
Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in 
chilled and cryopreserved bull sperm. Cryobiology, 80, 43-50. https://doi.org/10.1016/j.cryobiol.2017.12.006  

Baiee, F., Wahid, H., Rosnina, Y., Ariff, O., & Yimer, N. (2018b). Sperm DNA Impairment in the Bull: Causes, 
Influences on Reproduction and Evaluations. Pertanika J Trop Agricul Sci, 41(1). 

Bollwein, H., Fuchs, I., & Koess, C. (2008). Interrelationship between plasma membrane integrity, mitochondrial 
membrane potential and DNA fragmentation in cryopreserved bovine spermatozoa. Reprod Domes Anim, 
43(2), 189-195. https://doi.org/10.1111/j.1439-0531.2007.00876.x  

Borges-Silva, J. C., Silva, M. R., Marinho, D. B., Nogueira, E., Sampaio, D. C., Oliveira, L. O. F., & Sartori, R. 
(2016). Cooled semen for fixed-time artificial insemination in beef cattle. Reprod Fert Develop, 28(7), 1004-
1008. https://doi.org/10.1071/RD14185  

Büyükleblebici, S., Tuncer, P. B., Bucak, M. N., Eken, A., Sarıözkan, S., Taşdemir, U., & Endirlik, B. Ü. (2014). 
Cryopreservation of bull sperm: Effects of extender supplemented with different cryoprotectants and 
antioxidants on sperm motility, antioxidant capacity and fertility results. Anim Reprod Sci, 150(3), 77-83. 
https://doi.org/10.1016/j.anireprosci.2014.09.006  

Chelucci, S., Pasciu, V., Succu, S., Addis, D., Leoni, G. G., Manca, M. E., … Berlinguer, F. (2015). Soybean 
lecithin–based extender preserves spermatozoa membrane integrity and fertilizing potential during goat 
semen cryopreservation. Theriogenology, 83(6), 1064-1074. 
https://doi.org/10.1016/j.theriogenology.2014.12.012  

Chen, J., Xu, M., Chen, L., Chen, Y., & Chiu, T. (1998). Effect of Panax notoginseng saponins on sperm motility 
and progression in vitro. Phytomedicine, 5(4), 289-292. https://doi.org/10.1016/S0944-7113(98)80068-8  

Cho, J.-G., Lee, M.-K., Lee, J.-W., Park, H.-J., Lee, D.-Y., Lee, Y.-H., … Baek, N.-I. (2010). Physicochemical 
Characterization and NMR Assignments of Ginsenosides Rb 1, Rb 2, R c, and Rd Isolated from Panax ginseng. 
J Ginseng Res, 34(2), 113-121. DOI:10.5142/jgr.2010.34.2.113  

Eidan, S. M. (2016). Effect on post-cryopreserved semen characteristics of Holstein bulls of adding combinations 
of vitamin C and either catalase or reduced glutathione to Tris extender. Anim Reprod Sci, 167, 1-7. 
https://doi.org/10.1016/j.anireprosci.2016.01.014  

Felipe-Pérez, Y. E., de Lourdes Juárez-Mosqueda, M., Hernández-González, E. O., & de Jesús Valencia, J. (2008). 
Viability of fresh and frozen bull sperm compared by two staining techniques. Acta Vet Bras, 2(4), 123-130. 
https://doi.org/10.21708/avb.2008.2.4.895  

Gadea, J., Gumbao, D., Canovas, S., García‐Vázquez, F. A., Grullón, L. A., & Gardón, J. C. (2008). 
Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in 
vitro fertilizing ability of frozen‐thawed bull spermatozoa. Int J Androl, 31(1), 40-49. 
https://doi.org/10.1111/j.1365-2605.2007.00756.x  

Galarza, D., de Guevara, M. L., Beltrán-Breña, P., Sánchez-Calabuig, M., López-Sebastián, A., Santiago-Moreno, 
J., & Rizos, D. (2019). 137 Assessment of fertilizing ability of Merino ram semen cold stored up to 48 h by 
heterologous IVF of bovine oocytes. Reprod Fert Develop, 31(1), 194-194. 
https://doi.org/10.1071/RDv31n1Ab137  

Gray, S. L., Lackey, B. R., & Boone, W. R. (2016). Effects of Panax ginseng, zearalenol, and estradiol on sperm 
function. J Ginseng Res, 40(3), 251-259. https://doi.org/10.1016/j.jgr.2015.08.004  

Holt, W., Del Valle, I., & Fazeli, A. (2015). Heat shock protein A8 stabilizes the bull sperm plasma membrane 
during cryopreservation: Effects of breed, protein concentration, and mode of use. Theriogenology, 84(5), 
693-701. https://doi.org/10.1016/j.theriogenology.2015.05.004  

Kaefer, C., Komninou, E. R., Campos, V. F., de Leon, P. M., Arruda, F. V. S., Nascimento, K. S., … Deschamps, 
J. C. (2013). Binding pattern and toxicological effects of lectins from genus Canavalia on bovine sperm. 
Reprod Toxicol, 38, 72-80. https://doi.org/10.1016/j.reprotox.2013.03.003  



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 26       Published by IDEAS SPREAD 
 

Kaka, A., Haron, W., Leghari, R. A., Memon, M. I., Kaka, U., Mirani, A. H., … Kalwar, Q. (2016). Effect of in-
vitro supplementation of polyunsaturated fatty acids on frozen-thawed bull sperm characteristics using 
Bioxcell® extender. Pure Appl Biol, 5(3), 399-405. http://dx.doi.org/10.19045/bspab.2016.50052  

Kaka, A., Wahid, H., Rosnina, Y., Yimer, N., Khumran, A., Behan, A. A., & Ebrahimi, M. (2015). Alpha‐Linolenic 
Acid Supplementation in Tris Extender Can Improve Frozen–Thawed Bull Semen Quality. Reprod Domes 
Anim, 50(1), 29-33. https://doi.org/10.1111/rda.12445  

Kandelousi, M. S., Arshami, J., Naserian, A., & Abavisani, A. (2013). The effects of addition of omega-3, 6, 9 fatty 
acids on the quality of bovine chilled and frozen-thawed sperm. Open Vet J, 3(1), 47.  

Kasimanickam, R., Kasimanickam, V., Thatcher, C., Nebel, R., & Cassell, B. (2007). Relationships among lipid 
peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in 
dairy bulls. Theriogenology, 67(5), 1004-1012. https://doi.org/10.1016/j.theriogenology.2006.11.013  

Khalifa, T., Rekkas, C., Lymberopoulos, A., Sioga, A., Dimitriadis, I., & Papanikolaou, T. (2008). Factors affecting 
chromatin stability of bovine spermatozoa. Anim Reprod Sci, 104(2), 143-163. 
https://doi.org/10.1016/j.anireprosci.2007.02.019  

Khoshvaght, A., Towhidi, A., Zare-shahneh, A., Noruozi, M., Zhandi, M., Davachi, N. D., & Karimi, R. (2015). 
Dietary n-3 PUFAs improve fresh and post-thaw semen quality in Holstein bulls via alteration of sperm fatty 
acid composition. Theriogenology, 85(5), 807-812. https://doi.org/10.1016/j.theriogenology.2015.10.023  

Khumran, A., Yimer, N., Rosnina, Y., Ariff, M., Wahid, H., Kaka, A., & Sarsaifi, K. (2015). Butylated 
hydroxytoluene can reduce oxidative stress and improve quality of frozen–thawed bull semen processed in 
lecithin and egg yolk based extenders. Anim Reprod Sci, 163, 128-134. 
https://doi.org/10.1016/j.anireprosci.2015.10.007  

Kopalli, S. R., Cha, K.-M., Jeong, M. S., Lee, S.-H., Sung, J. H., Seo, S. K., & Kim, S. K. (2016a). Pectinase-
treated Panax ginseng ameliorates hydrogen peroxide-induced oxidative stress in GC-2 sperm cells and 
modulates testicular gene expression in aged rats. J Ginseng Res, 40(2), 185-195. 
https://doi.org/10.1016/j.jgr.2015.08.005  

Kopalli, S. R., Won, Y. J., Hwang, S.-Y., Cha, K.-M., Kim, S. Y., Han, C. K., & Kim, S. K. (2016b). Korean red 
ginseng protects against doxorubicin-induced testicular damage: An experimental study in rats. J Funct Foods, 
20, 96-107. https://doi.org/10.1016/j.jff.2015.10.020  

Kumar, P., Kumar, D., Sikka, P., & Singh, P. (2015). Sericin supplementation improves semen freezability of 
buffalo bulls by minimizing oxidative stress during cryopreservation. Anim Reprod Sci, 152, 26-31. 
https://doi.org/10.1016/j.anireprosci.2014.11.015  

Lymberopoulos, A., & Khalifa, T. (2010). Sperm chromatin stability during in vitro manipulation of beef bull 
semen. Reprod Domes Anim, 45(2), 307-314. https://doi.org/10.1111/j.1439-0531.2008.01301.x  

Medeiros, C., Forell, F., Oliveira, A., & Rodrigues, J. (2002). Current status of sperm cryopreservation: why isn't 
it better? Theriogenology, 57(1), 327-344. https://doi.org/10.1016/S0093-691X(01)00674-4  

Métayer, S., Dacheux, F., Dacheux, J. L., & Gatti, J. L. (2002). Comparison, characterization, and identification 
of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases 
are major fluid gelatinases. Biol Reprod, 66(5), 1219-1229. https://doi.org/10.1095/biolreprod66.5.1219  

Moustafa, M. H., Sharma, R. K., Thornton, J., Mascha, E., Abdel‐Hafez, M. A., Thomas, A. J., & Agarwal, A. 
(2004). Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients 
examined for infertility. Human Reprod, 19(1), 129-138. https://doi.org/10.1093/humrep/deh024  

Murphy, C., Fahey, A., Shafat, A., & Fair, S. (2013). Reducing sperm concentration is critical to limiting the 
oxidative stress challenge in liquid bull semen. J Dairy Sci, 96(7), 4447-4454. 
https://doi.org/10.3168/jds.2012-6484  

Nair, S. J., Brar, A., Ahuja, C., Sangha, S., & Chaudhary, K. (2006). A comparative study on lipid peroxidation, 
activities of antioxidant enzymes and viability of cattle and buffalo bull spermatozoa during storage at 
refrigeration temperature. Anim Reprod Sci, 96(1), 21-29. https://doi.org/10.1016/j.anireprosci.2005.11.002  

Papa, P. M., Maziero, R. D., Guasti, P. N., Junqueira, C. R., Freitas-Dell'Aqua, C. P., Papa, F. O., … Dell'Aqua, J. 
A. (2015). Effect of glycerol on the viability and fertility of cooled bovine semen. Theriogenology, 83(1), 
107-113. https://doi.org/10.1016/j.theriogenology.2014.08.009  

Patel, S., & Rauf, A. (2017). Adaptogenic herb ginseng (Panax) as medical food: Status quo and future prospects. 



as.ideasspread.org   Agricultural Science Vol. 2, No. 2; 2020 

 27       Published by IDEAS SPREAD 
 

Biomed Pharmacother, 85, 120-127. https://doi.org/10.1016/j.biopha.2016.11.112  
Revell, S., & Mrode, R. (1994). An osmotic resistance test for bovine semen. Anim Reprod Sci, 36(1), 77-86. 

https://doi.org/10.1016/0378-4320(94)90055-8  
Sapanidou, V., Taitzoglou, I., Tsakmakidis, Ι., Kourtzelis, I., Fletouris, D., Theodoridis, A., … Tsantarliotou, M. 

(2015). Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization. Theriogenology, 84(8), 
1273-1282. https://doi.org/10.1016/j.theriogenology.2015.07.005  

Tarig, A., Wahid, H., Rosnina, Y., Yimer, N., Goh, Y., Baiee, F., Hammadi, S., & Ebrahimi, M. (2017). Effect of 
different concentrations of egg yolk and virgin coconut oil in Tris-based extenders on chilled and frozen-
thawed bull semen. Anim Reprod Sci, 182, 21-27. https://doi.org/10.1016/j.anireprosci.2017.03.024  

Tejada, R., Mitchell, J. C., Norman, A., Marik, J., & Friedman, S. (1984). A test for the practical evaluation of 
male fertility by acridine orange (AO) fluorescence. Fertil steril, 42(1), 87-91. https://doi.org/10.1016/S0015-
0282(16)47963-X  

Tvrdá, E., Tušimová, E., Kováčik, A., Paál, D., Greifová, H., Abdramanov, A., & Lukáč, N. (2016). Curcumin has 
protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress. Anim Reprod 
Sci, 172, 10-20. https://doi.org/10.1016/j.anireprosci.2016.06.008  

Yimer, N., Noraisyah, A., Rosnina, Y., Wahid, H., Sarsaifi, K., & Hafizal, A. (2014). Comparison of 
cryopreservative effect of different levels of omega-3 egg yolk in citrate extender on the quality of goat 
spermatozoa. Pakistan Vet J, 34(3), 347-350.  

Yun, S. J., Bae, G. S., Park, J. H., Song, T. H., Choi, A., Ryu, B. Y., & Chang, M. B. (2016). Antioxidant effects of 
cultured wild ginseng root extracts on the male reproductive function of boars and guinea pigs. Anim Reprod 
Sci, 170, 10. https://doi.org/10.1016/j.anireprosci.2016.04.002  

 
Copyrights 
Copyright for this article is retained by the author(s), with first publication rights granted to the journal. 
This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution 
license (http://creativecommons.org/licenses/by/4.0/). 
 

 
















<<
  /ASCII85EncodePages false
  /AllowTransparency false
  /AutoPositionEPSFiles true
  /AutoRotatePages /None
  /Binding /Left
  /CalGrayProfile (Dot Gain 20%)
  /CalRGBProfile (sRGB IEC61966-2.1)
  /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2)
  /sRGBProfile (sRGB IEC61966-2.1)
  /CannotEmbedFontPolicy /Error
  /CompatibilityLevel 1.4
  /CompressObjects /Tags
  /CompressPages true
  /ConvertImagesToIndexed true
  /PassThroughJPEGImages true
  /CreateJobTicket false
  /DefaultRenderingIntent /Default
  /DetectBlends true
  /DetectCurves 0.0000
  /ColorConversionStrategy /CMYK
  /DoThumbnails false
  /EmbedAllFonts true
  /EmbedOpenType false
  /ParseICCProfilesInComments true
  /EmbedJobOptions true
  /DSCReportingLevel 0
  /EmitDSCWarnings false
  /EndPage -1
  /ImageMemory 1048576
  /LockDistillerParams false
  /MaxSubsetPct 100
  /Optimize true
  /OPM 1
  /ParseDSCComments true
  /ParseDSCCommentsForDocInfo true
  /PreserveCopyPage true
  /PreserveDICMYKValues true
  /PreserveEPSInfo true
  /PreserveFlatness true
  /PreserveHalftoneInfo false
  /PreserveOPIComments true
  /PreserveOverprintSettings true
  /StartPage 1
  /SubsetFonts true
  /TransferFunctionInfo /Apply
  /UCRandBGInfo /Preserve
  /UsePrologue false
  /ColorSettingsFile ()
  /AlwaysEmbed [ true
  ]
  /NeverEmbed [ true
  ]
  /AntiAliasColorImages false
  /CropColorImages true
  /ColorImageMinResolution 300
  /ColorImageMinResolutionPolicy /OK
  /DownsampleColorImages true
  /ColorImageDownsampleType /Bicubic
  /ColorImageResolution 300
  /ColorImageDepth -1
  /ColorImageMinDownsampleDepth 1
  /ColorImageDownsampleThreshold 1.50000
  /EncodeColorImages true
  /ColorImageFilter /DCTEncode
  /AutoFilterColorImages true
  /ColorImageAutoFilterStrategy /JPEG
  /ColorACSImageDict <<
    /QFactor 0.15
    /HSamples [1 1 1 1] /VSamples [1 1 1 1]
  >>
  /ColorImageDict <<
    /QFactor 0.15
    /HSamples [1 1 1 1] /VSamples [1 1 1 1]
  >>
  /JPEG2000ColorACSImageDict <<
    /TileWidth 256
    /TileHeight 256
    /Quality 30
  >>
  /JPEG2000ColorImageDict <<
    /TileWidth 256
    /TileHeight 256
    /Quality 30
  >>
  /AntiAliasGrayImages false
  /CropGrayImages true
  /GrayImageMinResolution 300
  /GrayImageMinResolutionPolicy /OK
  /DownsampleGrayImages true
  /GrayImageDownsampleType /Bicubic
  /GrayImageResolution 300
  /GrayImageDepth -1
  /GrayImageMinDownsampleDepth 2
  /GrayImageDownsampleThreshold 1.50000
  /EncodeGrayImages true
  /GrayImageFilter /DCTEncode
  /AutoFilterGrayImages true
  /GrayImageAutoFilterStrategy /JPEG
  /GrayACSImageDict <<
    /QFactor 0.15
    /HSamples [1 1 1 1] /VSamples [1 1 1 1]
  >>
  /GrayImageDict <<
    /QFactor 0.15
    /HSamples [1 1 1 1] /VSamples [1 1 1 1]
  >>
  /JPEG2000GrayACSImageDict <<
    /TileWidth 256
    /TileHeight 256
    /Quality 30
  >>
  /JPEG2000GrayImageDict <<
    /TileWidth 256
    /TileHeight 256
    /Quality 30
  >>
  /AntiAliasMonoImages false
  /CropMonoImages true
  /MonoImageMinResolution 1200
  /MonoImageMinResolutionPolicy /OK
  /DownsampleMonoImages true
  /MonoImageDownsampleType /Bicubic
  /MonoImageResolution 1200
  /MonoImageDepth -1
  /MonoImageDownsampleThreshold 1.50000
  /EncodeMonoImages true
  /MonoImageFilter /CCITTFaxEncode
  /MonoImageDict <<
    /K -1
  >>
  /AllowPSXObjects false
  /CheckCompliance [
    /None
  ]
  /PDFX1aCheck false
  /PDFX3Check false
  /PDFXCompliantPDFOnly false
  /PDFXNoTrimBoxError true
  /PDFXTrimBoxToMediaBoxOffset [
    0.00000
    0.00000
    0.00000
    0.00000
  ]
  /PDFXSetBleedBoxToMediaBox true
  /PDFXBleedBoxToTrimBoxOffset [
    0.00000
    0.00000
    0.00000
    0.00000
  ]
  /PDFXOutputIntentProfile ()
  /PDFXOutputConditionIdentifier ()
  /PDFXOutputCondition ()
  /PDFXRegistryName ()
  /PDFXTrapped /False

  /CreateJDFFile false
  /Description <<
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
    /BGR <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>
    /CHS <FEFF4f7f75288fd94e9b8bbe5b9a521b5efa7684002000410064006f006200650020005000440046002065876863900275284e8e9ad88d2891cf76845370524d53705237300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c676562535f00521b5efa768400200050004400460020658768633002>
    /CHT <FEFF4f7f752890194e9b8a2d7f6e5efa7acb7684002000410064006f006200650020005000440046002065874ef69069752865bc9ad854c18cea76845370524d5370523786557406300260a853ef4ee54f7f75280020004100630072006f0062006100740020548c002000410064006f00620065002000520065006100640065007200200035002e003000204ee553ca66f49ad87248672c4f86958b555f5df25efa7acb76840020005000440046002065874ef63002>
    /CZE <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>
    /DAN <FEFF004200720075006700200069006e0064007300740069006c006c0069006e006700650072006e0065002000740069006c0020006100740020006f007000720065007400740065002000410064006f006200650020005000440046002d0064006f006b0075006d0065006e007400650072002c0020006400650072002000620065006400730074002000650067006e006500720020007300690067002000740069006c002000700072006500700072006500730073002d007500640073006b007200690076006e0069006e00670020006100660020006800f8006a0020006b00760061006c0069007400650074002e0020004400650020006f007000720065007400740065006400650020005000440046002d0064006f006b0075006d0065006e0074006500720020006b0061006e002000e50062006e00650073002000690020004100630072006f00620061007400200065006c006c006500720020004100630072006f006200610074002000520065006100640065007200200035002e00300020006f00670020006e0079006500720065002e>
    /DEU <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>
    /ESP <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>
    /ETI <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>
    /FRA <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>
    /GRE <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>
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
    /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke.  Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.)
    /HUN <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>
    /ITA <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>
    /JPN <FEFF9ad854c18cea306a30d730ea30d730ec30b951fa529b7528002000410064006f0062006500200050004400460020658766f8306e4f5c6210306b4f7f75283057307e305930023053306e8a2d5b9a30674f5c62103055308c305f0020005000440046002030d530a130a430eb306f3001004100630072006f0062006100740020304a30883073002000410064006f00620065002000520065006100640065007200200035002e003000204ee5964d3067958b304f30533068304c3067304d307e305930023053306e8a2d5b9a306b306f30d530a930f330c8306e57cb30818fbc307f304c5fc59808306730593002>
    /KOR <FEFFc7740020c124c815c7440020c0acc6a9d558c5ec0020ace0d488c9c80020c2dcd5d80020c778c1c4c5d00020ac00c7a50020c801d569d55c002000410064006f0062006500200050004400460020bb38c11cb97c0020c791c131d569b2c8b2e4002e0020c774b807ac8c0020c791c131b41c00200050004400460020bb38c11cb2940020004100630072006f0062006100740020bc0f002000410064006f00620065002000520065006100640065007200200035002e00300020c774c0c1c5d0c11c0020c5f40020c2180020c788c2b5b2c8b2e4002e>
    /LTH <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>
    /LVI <FEFF0049007a006d0061006e0074006f006a00690065007400200161006f00730020006900650073007400610074012b006a0075006d00750073002c0020006c0061006900200076006500690064006f00740075002000410064006f00620065002000500044004600200064006f006b0075006d0065006e007400750073002c0020006b006100730020006900720020012b00700061016100690020007000690065006d01130072006f00740069002000610075006700730074006100730020006b00760061006c0069007401010074006500730020007000690072006d007300690065007300700069006501610061006e006100730020006400720075006b00610069002e00200049007a0076006500690064006f006a006900650074002000500044004600200064006f006b0075006d0065006e007400750073002c0020006b006f002000760061007200200061007400760113007200740020006100720020004100630072006f00620061007400200075006e002000410064006f00620065002000520065006100640065007200200035002e0030002c0020006b0101002000610072012b00200074006f0020006a00610075006e0101006b0101006d002000760065007200730069006a0101006d002e>
    /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
    /NOR <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>
    /POL <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>
    /PTB <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>
    /RUM <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>
    /RUS <FEFF04180441043f043e043b044c04370443043904420435002004340430043d043d044b04350020043d0430044104420440043e0439043a043800200434043b044f00200441043e043704340430043d0438044f00200434043e043a0443043c0435043d0442043e0432002000410064006f006200650020005000440046002c0020043c0430043a04410438043c0430043b044c043d043e0020043f043e04340445043e0434044f04490438044500200434043b044f00200432044b0441043e043a043e043a0430044704350441044204320435043d043d043e0433043e00200434043e043f0435044704300442043d043e0433043e00200432044b0432043e04340430002e002000200421043e043704340430043d043d044b04350020005000440046002d0434043e043a0443043c0435043d0442044b0020043c043e0436043d043e0020043e0442043a0440044b043204300442044c002004410020043f043e043c043e0449044c044e0020004100630072006f00620061007400200438002000410064006f00620065002000520065006100640065007200200035002e00300020043800200431043e043b043504350020043f043e04370434043d043804450020043204350440044104380439002e>
    /SKY <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>
    /SLV <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>
    /SUO <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>
    /SVE <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>
    /TUR <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>
    /UKR <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>
    /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing.  Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
  >>
  /Namespace [
    (Adobe)
    (Common)
    (1.0)
  ]
  /OtherNamespaces [
    <<
      /AsReaderSpreads false
      /CropImagesToFrames true
      /ErrorControl /WarnAndContinue
      /FlattenerIgnoreSpreadOverrides false
      /IncludeGuidesGrids false
      /IncludeNonPrinting false
      /IncludeSlug false
      /Namespace [
        (Adobe)
        (InDesign)
        (4.0)
      ]
      /OmitPlacedBitmaps false
      /OmitPlacedEPS false
      /OmitPlacedPDF false
      /SimulateOverprint /Legacy
    >>
    <<
      /AddBleedMarks false
      /AddColorBars false
      /AddCropMarks false
      /AddPageInfo false
      /AddRegMarks false
      /ConvertColors /ConvertToCMYK
      /DestinationProfileName ()
      /DestinationProfileSelector /DocumentCMYK
      /Downsample16BitImages true
      /FlattenerPreset <<
        /PresetSelector /MediumResolution
      >>
      /FormElements false
      /GenerateStructure false
      /IncludeBookmarks false
      /IncludeHyperlinks false
      /IncludeInteractive false
      /IncludeLayers false
      /IncludeProfiles false
      /MultimediaHandling /UseObjectSettings
      /Namespace [
        (Adobe)
        (CreativeSuite)
        (2.0)
      ]
      /PDFXOutputIntentProfileSelector /DocumentCMYK
      /PreserveEditing true
      /UntaggedCMYKHandling /LeaveUntagged
      /UntaggedRGBHandling /UseDocumentProfile
      /UseDocumentBleed false
    >>
  ]
>> setdistillerparams
<<
  /HWResolution [2400 2400]
  /PageSize [612.000 792.000]
>> setpagedevice

