Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.15/November-2022/2.pdf
RESEARCH ARTICLE
Open Access
Effect of supplementing L-cysteine and its group analogs on frozen
semen quality of bulls: A meta-analysis
Sari Yanti Hayanti1 , Mohammad Miftakhus Sholikin1,2 , Anuraga Jayanegara2,3 , Mokhamad Fakhrul Ulum4
Marchie Astrid da Costa1 , Fitriawaty Fitriawaty1 , Surya Surya1 , Maureen Chrisye Hadiatry1 ,
Santiananda Arta Asmarasari1 , Eko Handiwirawan1 , Yenny Nur Anggraeny1 , Eni Siti Rohaeni1 ,
Salfina Nurdin Ahmad1 , Bustami Bustami1 , Aryogi Aryogi1 , Dicky Pamungkas1 , and Yenni Yusriani1
,
1. Research Center for Animal Husbandry, Research Organization for Agriculture and Food, National Research and
Innovation Agency of The Republic of Indonesia (BRIN), Cibinong Sciences Center, Cibinong, Bogor 16911, Indonesia;
2. Animal Feed and Nutrition Modelling Research Group, Faculty of Animal Science, IPB University, Bogor 16680, West
Java, Indonesia; 3. Department of Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Bogor
16680, West Java, Indonesia; 4. Division of Veterinary Reproduction, Obstetrics, and Gynaecology, Department of
Veterinary Clinic, Reproduction, and Pathology, School of Veterinary Medicine and Biomedical Sciences, IPB University,
Bogor, 16680, West Java, Indonesia.
Corresponding author: Sari Yanti Hayanti, e-mail: drh.sari.bptpjambi@gmail.com
Co-authors: MMS: mohammad.miftakhus.sholikin@gmail.com, AJ: anuraga.jayanegara@gmail.com,
MFU: ulum@apps.ipb.ac.id, MAC: marchie.astrid.da.costa@brin.go.id, FF: fitrics94@gmail.com, SS: aku.uyha@yahoo.com,
MCH: mchris0501@yahoo.co.id, SAA: santiananda19@gmail.com, EH: eh_wirawan@yahoo.com,
YNA: yennysahim@gmail.com, ESR: eni_najib@yahoo.co.id, SNA: Salfina.bptp@gmail.com, BB: bustami130@gmail.com,
AA: aryogia@gmail.com, DP: dpamungkas2000@yahoo.com, YY: yenniyusriani2009@gmail.com
Received: 10-07-2022, Accepted: 19-09-2022, Published online: 09-11-2022
doi: www.doi.org/10.14202/vetworld.2022.2517-2524 How to cite this article: Hayanti SY, Sholikin MM, Jayanegara A,
Ulum MF, da Costa MA, Fitriawaty F, Surya S, Hadiatry MC, Asmarasari SA, Handiwirawan E, Anggraeny YN, Rohaeni ES,
Ahmad SN, Bustami B, Aryogi A, Pamungkas D, and Yusriani Y (2022) Effect of supplementing L-cysteine and its group
analogs on frozen semen quality of bulls: A meta-analysis, Veterinary World, 15(11): 2517–2524.
Abstract
Background and Aim: The quality of frozen bull sperm after thawing is influenced by the primary diluent and antioxidant.
This meta-analysis was conducted to determine the effect of supplementing L-cysteine and its group analogs on the quality
of frozen bull sperm.
Materials and Methods: A total of 22 articles obtained from Google Scholar and Scopus were integrated into metadata.
The effects of adding L-cysteine and its analogs (e.g., cysteine HCl and N-acetyl-L-cysteine), both of which are known as
L-cysteine, were evaluated in this meta-analysis. The following parameters were examined: Abnormality, acrosome damage,
acrosomal integrity, DNA damage, DNA integrity, malondialdehyde (MDA) content, plasma membrane integrity, pregnancy
rate, progressive motility, sperm viability, and total motility. Data were analyzed using the mixed model methodology, with
L-cysteine dosage as a fixed effect and different studies as random effects.
Results: L-cysteine supplementation significantly increased the total motility (p < 0.05) and MDA content of semen,
following a linear pattern. Progressive motility, acrosomal integrity, and plasma membrane integrity were significantly
increased, showing a quadratic pattern (p < 0.05). Abnormality and acrosome damage were significantly decreased (p < 0.05),
following a quadratic and linear pattern, respectively. Other parameters remained unaffected by L-cysteine supplementation.
L-cysteine and cysteine HCl significantly inhibited (p = 0.001) acrosome damage in thawed frozen sperm compared with
control sperm.
Conclusion: Supplementing L-cysteine and its analog groups are recommended for freezing bull semen as it generally
improves sperm quality.
Keywords: bull cattle, cryopreservation, L-cysteine, meta-analysis, sperm.
Introduction
Artificial insemination (AI) with frozen sperm
remains the preferred technology for increasing the
number of cattle [1]. The AI allows for the propagation
and dissemination of superior sire genetics, increases
the rate of genetic improvement and production gains,
Copyright: Hayanti, et al. Open Access. This article is distributed
under the terms of the Creative Commons Attribution 4.0
International
License
(http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit
to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data
made available in this article, unless otherwise stated.
Veterinary World, EISSN: 2231-0916
allows superior sires to produce significantly more
offspring than natural service, and even enables the use
of sires that cannot reproduce naturally or are no longer alive [2]. The potential of AI has also been demonstrated in livestock [3] and wildlife conservation [4–6].
Multiple processes culminate in cryopreservation
as the final step in producing frozen bull sperm [7].
Freezing bovine sperm cells can subject them to physical and chemical stress [8]. Damaged sperm cells are
characterized by low plasma membrane integrity, acrosome and mitochondrial damage, and DNA damage,
reducing their viability and motility [9].
A success factor for AI with frozen sperm
is a small number of damaged sperm cells after
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thawing [10]. Numerous techniques, including the
addition of antioxidants to frozen semen diluents,
have been attempted to improve the quality of bull
sperm [11]. L-cysteine is found in extracellular cells
and is a member of the non-essential amino acid antioxidant group [12, 13]. It is a semi-essential amino
acid that has some analogs, including cysteine HCl,
L-cysteine, and N-acetyl-L-cysteine. L-cysteine
inhibits the production of free radicals generated by
sperm cell metabolism [14]. In studies on L-cysteine
supplementation for frozen sperm diluents, there were
differences in the breed of cattle, the macroscopic
and microscopic characteristics of fresh sperm, the
primary diluent, and the dosage of L-cysteine administered. Therefore, a systematic review is required
to determine the linearity of the effect of L-cysteine
administration on bull sperm diluent. A meta-analysis
is a powerful technique for analyzing multiple studies
using comparable variables [15].
Therefore, we conducted this meta-analysis to
determine the effect of L-cysteine supplementation on
the quality of frozen bovine sperm.
Materials and Methods
Ethical approval
This type of research does not require ethical
approval.
Study period and location
The research was conducted from March to June
2022 in Jambi Assessment Institute for Agricultural
Technology.
Metadata development
This meta-analysis was conducted according to the
methodology outlined in the Preferred Reporting Items
for Systematic Reviews and Meta-Analyses guidelines [16]. The data used in this meta-analysis were
extracted from previously published articles and entered
into a meta-data. Published articles were searched
using Google Scholar (https://scholar.google.com/) and
Scopus (https://www.scopus.com) using the search terms
“L-Cysteine,” “Cysteine HCl,” “N-Acetyl-L-Cysteine,”
“amino acid,” “antioxidant,” “bull,” “bovine,” “bull cattle,” “sperm,” “cryopreservation,” and “frozen.”
Inclusion and exclusion criteria
The primary inclusion criteria were a journal
article written in English and published by a reputable
publisher, the experimental design must have complied with the correct statistical rules, the amount of
experimental and replicated material should have met
the correct statistical standards, the study must have
explicitly used the experimental animal material for
bulls, and the number of bulls used (n) must have
met the correct statistical requirements. The inclusion
and exclusion criteria of the articles are presented in
Fugure-1.
Data extraction
Author(s) names, publication year, journal
name, breed of bulls, number of bulls, L-cysteine
Veterinary World, EISSN: 2231-0916
and its analog dosage, observed parameters, recommended extracted dosage, type of L-cysteine (e.g.,
cysteine HCl [T1], L-cysteine [T2], and N-acetylL-cysteine [T3]), units for each parameter, sampling
technique, and parameter measurement technique
were extracted from the selected articles. The search
initially retrieved approximately 55 papers describing
studies of L-cysteine supplementation for beef cattle.
However, based on their titles and abstracts, only 29
of these papers had the potential to be included.
After a comprehensive evaluation, 22 articles
were included in the metadata (Table-1). The selected
papers included 11 beef and 11 dairy cattle studies [14, 17–37]. According to the available research,
sperm is collected from sexually mature bulls.
Abnormality (%), acrosome damage (%), acrosomal
integrity (%), DNA damage (%), DNA integrity (%),
malondialdehyde (MDA) content (nmol/mL), plasma
membrane integrity (%), pregnancy rate (%), progressive motility (%), sperm viability (%), and total
motility (%) were some of the performance parameters evaluated to examine the effect of L-cysteine
supplementation. The metadata were updated with
L-cysteine dosage. No unit conversion was performed
on the data in this study because no different units
were found. Descriptive statistical analysis was conducted to determine the data distribution (Table-2).
Statistical analysis
Data analysis was conducted using the R software, version 4.2.0 (https://cran.r-project.org/), along
with supplementary packages, including “data. table,”
“DescTools,” “lme4,” “lmerTest,” “lsmeans,” “multcomp,” “multcompView,” “openxlsx,” “performance,”
“readxl,” “reshape2,” and “tidyverse” [38–40]. The
linear mixed model methodology was used for this
meta-analysis. Random effects were used in this study,
and the added dosage of L-cysteine was considered
as a fixed effect [41–43]. The following equation will
guide you through the mathematical model.
Yij = β0 + β1Dij + Si + SiDij + eij(1)
Yij = β0 + β1Dij +β2D2ij + Si + SiDij + eij(2)
Where, (1) linear mixed model of the 1st order,
(2) linear mixed model of the 2nd order, Yij was the
dependent variable, β0 was the overall intercept
across all studies (fixed effect), annotation 1 in β
was the linear regression coefficient of Y on D (fixed
effect) else was the quadratic regression coefficient
of Y on D (fixed effect), Dij was the value of the continuous predictor variable (addition of L-cysteine
dosage), Si was the random effect of study (i-th),
and SiDij was to evaluate the appropriateness of statistical models, the p-value, root mean square error
(RMSE), and R square Nakagawa were used [41, 44,
45]. If p ≤ 0.05, this indicated that the result was
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Figure-1: The selection of articles and evaluation process using the PRISMA method. PRISMA=Preferred Reporting Items
for Systematic Reviews and Meta-Analyses.
statistically significant. In addition to this, there
was a tendency for the result to be significant if the
p-value was only in the range of 0.05–0.1. To compare different types of L-cysteine, such as cysteine
HCl, L-cysteine, and N-acetyl-L-cysteine, the least
squares mean was used. Least mean squares were
also used to evaluate the effects of bull breeds on
sperm characteristics.
Results
The relationship between the addition of
L-cysteine and post-thawing frozen bovine sperm
quality is shown in Table-3. The dosage of L-cysteine
added to frozen bovine semen exerted a linear and
quadratic effect on post-thawing sperm quality. It
exerted a linear effect on total motility, sperm viability, DNA integrity, DNA damage, acrosome damage,
MDA content, and pregnancy rate and a quadratic
effect on progressive motility, abnormality, acrosomal integrity, and plasma membrane integrity. The
dosage and type of L-cysteine showed an interaction with sperm viability (p = 0.031). However, the
other parameters showed no significant interaction
between dosage × type, dosage × breed of cattle, and
type × breed of cattle.
Veterinary World, EISSN: 2231-0916
The dosage of L-cysteine exerted a positive significant effect (p < 0.05) on abnormality
(p-linear (pl) = 0.002 and p-quadratic (pq) = 0.001),
acrosome damage (p = 0.004), acrosomal integrity
(pl = 0.036 and pq = 0.024), plasma membrane integrity (p < 0.05), progressive motility (p < 0.05), MDA
content (p < 0.05), and total motility (p = 0.043).
However, the dosage of L-cysteine exerted no significant effect on DNA damage (p = 0.124), DNA integrity (p = 6.39), pregnancy rate (p = 1.73), and sperm
viability (p = 0.58). The DNA integrity, MDA content,
plasma membrane integrity, pregnancy rate, progressive motility, sperm viability, and total motility were
increased, but the abnormality, acrosome damage, and
DNA damage of frozen bull sperm cells were suppressed by the dosage of L-cysteine.
The relationship between the type of L-cysteine
and the quality of post-thawing frozen bovine semen
is presented in Table-4. L-cysteine types exerted a different effect (p = 0.001) on the acrosome damage of
post-thawing frozen semen. L-cysteine and cysteine
HCl (7.6% and 6.8%, respectively) exerted a lower
effect than the control (9.7%). However, the various
cysteine types exerted a significantly higher effect
(p = 0.003) on the total motility of post-thawing frozen
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Table-1: Studies included in the meta‑analysis.
Study
Reference
Cattle breed
Reported parameters
1
2
3
4
[17]
[18]
[19]
[20]
Sahiwal
Jersey
Friesian Holstein
Friesian Holstein
5
[21]
Bull
6
7
[22]
[23]
8
9
10
[24]
[25]
[26]
Friesian Holstein
Sahiwal×Achai and
Friesian Holstein×Jersey
Hanwoo
Hanwoo
Bull
11
[27]
Jersey
12
[28]
Jersey
13
[29]
Jersey
14
15
16
[30]
[31]
[32]
Friesian Holstein
Friesian Holstein
Friesian Holstein
17
[33]
Friesian Holstein
18
19
[34]
[14]
Friesian Holstein
Friesian Holstein
20
21
[35]
[36]
Friesian Holstein
Friesian Holstein
22
[37]
Achai and Friesian
Holstein
ACI and PMI
ACI, PGM, and TTM
PGM and TTM
ABN, ACD, MDA, PGM,
PRR, and TTM
ABN, ACD, PRR, SPV,
and TTM
ABN, PGM, and PMI
ACI, DNI, PGM, PMI,
SPV, and TTM
ACD and SPV
ACD and SPV
DND, PMI, and SPV
ABN, ACD, DND, PMI,
PRR, and SPV
ABN, ACD, PMI, SPV,
and TTM
ACD, PGM, PMI, PRR,
and TTM
DNI and PMI
DNI, PMI, SPV, and TTM
ABN, ACD, DND, PMI,
PRR, and TTM
ABN, ACD, PMI, MDA,
PRR, and TTM
ABN, ACD, and DNS
ABN, ACD, DND, MDA,
PMI, PRR, and TTM
ACD, DND, and PRR
ABN, ACD, PMI, and
TTM
ACI, PGM, PMI, and SPV
L‑cysteine
Type
Dosage (mM)
Re.
T2
T1
T2 and T3
T2
0, 0.5, 1, and 2
0, 10, 12, and 15
0, 0.5, and 1
0 and 5
2
10
1
5
T2
2 and 10
10
T2
T2
0 and 1
0, 5, 7.5, and 10
1
7.5
T2
T2
T3
10
20
10
T2
0 and 10
0, 5, 10 and 20
0, 0.01, 0.1, 1,
and 10
0 and 5
T1
0 and 5
5
T1
0 and 5
5
T2
T2
T2
0 and 10
0, 3, 5, and 7
0 and 2
10
5
2
T2
0 and 2
2
T2
T2
0 and 5
5 and 10
5
10
T2
T2
0 and 2.5
0 and 5
2.5
5
T2
0, 0.5, 1, and 1.5
1.5
5
ABN=Abnormality, ACD=Acrosome damage, ACI=Acrosomal integrity, DND=DNA damage, DNI=DNA integrity,
MDA=Malondialdehyde, PMI=Plasma membrane integrity, PRR=Pregnancy rate, PGM=Progressive motility,
Re.=Recommendation, SPV=Sperm viability, T1=Cysteine HCl, T2=L‑cysteine, T3=N‑acetyl‑L‑cysteine, TTM=Total motility
Table-2: Statistical descriptive analysis of the
meta‑analysis data.
Variable
L‑cysteine
Dosage, mM
Parameter
Abnormality, %
Acrosome damage, %
Acrosomal integrity, %
DNA damage, %
DNA integrity, %
Malondialdehyde, nmol/mL
Plasma membrane integrity, %
Pregnancy rate, %
Progressive motility, %
Sperm viability, %
Total motility, %
N Mean
SE
81 3.27 0.47
34
36
24
15
14
12
53
24
46
49
50
13.3
8.98
58.8
12.1
94.5
2.55
54.1
51.5
32.4
62.4
55.1
228
0.72
1.34
2.44
1.4
4.61
2.58
2.58
0.84
0.63
0
Max Min
20
0
23
21.5
78.2
57
100
5.96
76.8
81.5
63.9
79.3
81.4
4.8
0.89
40.2
0.84
62.6
0.7
27.8
15
9.43
40.8
38.8
N=Number of data, MAX=Maximum value of the variables,
Min=Minimum value of the variables, SE=Standard error
semen in the following increasing order: L-cysteine
(53%), N-acetate-L-cysteine (59%), and cysteine HCl
(68.5%) and, in contrast, the type of L-cysteine
exerted no different effect (p > 0.05) on abnormality
(p = 0.132), acrosomal integrity (p = 0.612), DNA
Veterinary World, EISSN: 2231-0916
damage (p = 0.39), DNA integrity (p = 0.108), MDA
content (p = 0.081), plasma membrane integrity
(p = 0.107), pregnancy rate (p = 0.63), progressive
motility (p = 0.438), and sperm viability (p = 0.057).
The effect of bull breeds on the characteristics
of frozen sperm is shown in Table-5. Compared with
Hanwoo (18.2%) and Jersey (13.4%) bulls, the acrosome damage in Friesian Holstein bull sperm was
comparatively less (4.1%). The Jersey bull showed
the maximum plasma membrane integrity among all
breeds (63.1%). The plasma membrane integrity values in the maximum-to-minimum order are as follows:
Jersey > Friesian Holstein > Achai > Sahiwal > Friesian
Holstein × Jersey > Sahiwal × Achai. The pregnancy
rate of Friesian Holstein breed was substantially
higher (p = 0.03) than that of Jersey breed. The effect
of bull breed on other sperm characteristics was not
statistically significant.
The following mathematical equation may be
derived from Table-3:
ABN =14.9 (±0.985) + 0.118D 2 (±0.0312)
−1.34D(±0.359)
(3)
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Table-3: Effect of L‑cysteine dosage (mM) on the quality of frozen bull sperm.
Parameter
N
M
β1 and β2
β0
Value
SE
0.985
Abnormality, %
34
Q
14.9
Acrosome damage, %
Acrosomal integrity, %
36
24
L
Q
9.27
56.4
DNA damage, %
DNA integrity, %
Malondialdehyde, nmol/mL
Plasma membrane integrity, %
15
14
12
53
L
L
L
Q
16.1
88.3
1.43
51.9
Pregnancy rate, %
Progressive motility, %
24
46
L
Q
49
29.4
Sperm viability, %
Total motility, %
49
50
L
L
61.5
53.5
Value
SE
Model validation
p‑value RMSE
−1.34 0.359
0.118 0.0312
1.52
−0.25 0.075
5.95
3.21
1.41
−0.279 0.112
8.5
−0.413 0.241
4.76
1.03
0.554
0.496 0.362
0.083
2.52
2.11
0.943
−0.23 0.104
4.81
0.869
0.6
4.5
1.88
0.658
−0.184 0.058
2.22
0.13
0.233
2.27
0.402
0.189
0.002
0.001
0.004
0.036
0.024
0.124
0.096
0.003
0.032
0.033
0.173
0.008
0.004
0.58
0.043
R2
Interaction
D×C
D×B
C×B
2.37
0.56
0.078
0.308
1.11
6.25
0.94
0.77
0.781
0.698
0.409
0.956
1.92 0.98
6.39
0.5
0.525 0.81
5.24 0.68
0.85
0.051
0.838
0.475
0.956
5.23
3.98
0.81
0.92 0.311
0.471
0.238
0.993
5.57
2.64
0.53
0.89
0.031
0.761
0.299
0.982
0.32
B=Breed of bulls, C=Type of L‑cysteine, D=L‑cysteine dosage (mM), L=Linear, Q=Quadratic, M=Model, N=Number of
data, RMSE=Root means square error, SE=Standard error
Table-4: The effects of various types of L‑cysteine (cysteine HCl, L‑cysteine, and N‑acetyl‑L‑cysteine) on the quality of
frozen bull sperm.
Parameter
Abnormality, %
Acrosome damage, %
Acrosomal integrity, %
DNA damage, %
DNA integrity, %
Malondialdehyde, nmol/mL
Plasma membrane integrity, %
Pregnancy rate, %
Progressive motility, %
Sperm viability, %
Total motility, %
p‑value
Control
T1
T2
0.132
0.001
0.612
0.39
0.108
0.081
0.107
0.63
0.438
0.057
0.003
14.8
9.7b
55.8
16.4
87.7
1.7
51.4
50.1
28.9
58.4
50.9a
14
6.8a
58
11.9
7.6a
60.2
13.4
96.5
3.2
53.5
52
32.2
64
53.3a
59.8
58.9
30.5
59.7
68.5b
T3
16
63.7
32.6
67.4
59ab
T1=Cysteine HCl, T2=L‑cysteine, T3=N‑acetyl‑L‑cysteine. The error rate for superscript variations on the same line is 5%
ACI = 56.4 (±5.95) − 0.279D 2 (±0.112) +
dY(ABN, ACI, PGM, and PMI)
'
=
= 0.
f (x )
(4)
3.21D(±1.41)
dD
PGM = 29.4 (±4.5) − 0.184D (±0.058)
2
+1.88D(±0.658)
PMI = 51.9 (±2.52) − 0.23D 2 (±0.104)
+2.11D(±0.943)
(5)
(6)
Where, ABN is abnormality, ACI is acrosomal integrity, D is dosage of L-cysteine (and its
analogs), PGM is progressive motility, and PMI
is plasma membrane integrity. Equations 3–6 are
quadratic equations that have been identified from
the analysis of the effect of L-cysteine dosage on
abnormality (3), acrosomal integrity (4), progressive motility (5), and plasma membrane integrity
(6). This quadratic form is useful for determining
the optimum dosage of treatment by decreasing the
quadratic function
Veterinary World, EISSN: 2231-0916
Then, the optimum value of L-cysteine dosage was
obtained and the prediction of the optimal value of
the parameters sequentially as follows: Abnormality
(D = 5.69 mM, Y = 11%); acrosomal integrity
(D = 5.76 mM, Y = 65.6%); progressive motility
(D = 5.11 mM, Y = 34.2%); and plasma membrane
integrity (D = 4.59 mM, Y = 56.8%).
Discussion
Seminal fluid plasma in sperm consists of multiple antioxidants [46] and amino acids that positively
correlate with sperm viability [47]. Frozen sperm contains lower levels of glutathione peroxidase, superoxide dismutase, reduced glutathione (GSH), and
oxidized glutathione (GSSG) than fresh sperm [48].
The change in semen temperature from freezing
to thawing affects the production of reactive oxygen
species (ROS), increasing the amount of MDA [49].
The MDA is used to determine the level of ROS in
bovine sperm [50]. According to Perumal et al. [29],
the addition of L-cysteine can inhibit the production of
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Table-5: The effect of bull breeds on the characteristics of frozen sperm.
Parameter
p‑value
Abnormality, %
Acrosome damage, %
DNA damage, %
Plasma membrane integrity, %
Pregnancy rate, %
Progressive motility, %
Sperm viability, %
Total motility, %
0.55
<0.001
0.86
0.08
0.03
0.14
0.4
0.2
B1
50.7ab
31.2
70.6
B2
13
4.1a
19.3
47.6a
54.6b
26.2
69.3
52.1
B3
B4
18.2b
52.5ab
20.4
59.6
57.2
61.2
B5
14.2
13.4b
15.2
63.1b
32.7a
57.1
60.1
63.7
B6
B7
47.7ab
46.5ab
49.6
66.8
17.6
54.9
53.6
B1=Achai, B2=Friesian Holstein, B3=Friesian Holstein × Jersey, B4=Hanwoo, B5=Jersey, B6=Sahiwal, B7=Sahiwal × Achai.
The error rate for superscript variations on the same line is 5%
lipid peroxidation, that is, MDA levels, resulting in a
low ROS concentration. This finding is consistent with
a meta-analysis that demonstrated that the administration of the antioxidant L-cysteine could inhibit MDA
production in frozen bovine sperm after thawing.
Previous studies have shown that after thawing,
the level of ROS increased in frozen bovine sperm,
diminishing their viability and motility [37, 51].
In some buffaloes and sheep, the administration of
L-cysteine inhibited the decrease in glutathione levels
in bovine sperm, similar to that in cattle [27, 52, 53].
L-cysteine functions to reduce ROS levels by boosting glutathione profiles [48]. L-cysteine is produced
by reduced L-cystine after it has traversed the cell
membrane [54]. As a precursor for protein synthesis
and GSH production, intracellular L-cysteine is essential for cellular homeostasis [12].
The addition of L-cysteine to semen exerted a significant effect (p < 0.05) in reducing the proportion of
abnormal sperm and damaged acrosomes in the postthawed frozen semen of bulls (Table-1). These findings are consistent with the previous studies [18, 23].
Low sperm count and damaged acrosomes obstruct the
enhancement of other sperm quality parameters. In terms
of motility, progressive motility, acrosomal integrity, and
plasma membrane integrity, the addition of L-cysteine
enhanced the microscopic quality of sperm. These findings are agree with studies showing that L-cysteine
administration prevents the decrease in the percentage of
total motility, progressive motility, acrosomal integrity,
and plasma membrane integrity [17, 24, 28, 33].
The optimal dosage of L-cysteine required to
suppress abnormal sperm count was 5.59 mM, assuming that the minimum abnormal sperm proportion was
11%. The optimal dosage of L-cysteine required for
acrosomal integrity is 5.67 mM, with a predicted acrosomal integrity value of 65.6%. To increase progressive
motility to 34.2%, the required dosage of L-cysteine
is 5.11 mM. Similarly, to improve the sperm plasma
membrane integrity to 56.8%, the required L-cysteine
dosage was 4.59 mM. The effectiveness of L-cysteine
dosage on these four parameters ranges from an equal
dosage of approximately 5 mM for optimizing each
parameter that determines sperm quality.
Using computer-assisted sperm analysis, it was
determined that the quality of fresh sperm obtained
Veterinary World, EISSN: 2231-0916
directly from males was superior to that of frozen
sperm after thawing [55]. The process of dilution and
freezing affects sperm quality during the production
of frozen bovine semen [56, 57]. Temperature changes
during the thawing process exert an effect on sperm
quality, including the acrosome, during the stage of
using frozen bovine semen [58, 59]. Therefore, antioxidant supplements are required to prevent organelle
damage in sperm cells [60]. This meta-analysis demonstrated that the addition of the antioxidant L-cysteine
could reduce acrosome damage.
Conclusion
The addition of L-cysteine during the process of
frozen semen production could maintain sperm quality
after thawing. Therefore, we recommend L-cysteine
as an important supplement for sperm cells. The optimal L-cysteine dosages recommended for addition to
the diluent during semen processing were 4.58–5.75
mM to increase progressive motility, acrosome integrity, and plasma membrane integrity and 5.68 mM for
reducing abnormalities.
Authors’ Contributions
SYH: Conceptual and research design. MAC, FF,
SS, and MCH: Searched the literature and collected.
SYH and SAA: Literature selection and processed
the data. MMS: Analyzed the data. SYH and MMS:
Wrote the original draft. MMS, AJ, MFU, SYH, EH,
YNA, ESR, SNA, BB, AA, DP, and YY: Supervised
the research. AJ, MFU, EH, YNA, ESR, SNA, BB,
AA, DP, and YY: Critical review of the manuscript. All
authors have read and approved the final manuscript.
Acknowledgments
The authors are thankful to Jambi Assessment
Institute for Agricultural Technology for providing the
necessary facilities for the study. The authors did not
receive any funds for this study.
Competing Interests
The authors declare that they have no competing
interests.
Publisher’s Note
Veterinary World remains neutral with regard to
jurisdictional claims in published institutional affiliation.
2522
Available at www.veterinaryworld.org/Vol.15/November-2022/2.pdf
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