Theriogenology 147 (2020) 102e107

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Theriogenology
journal homepage: www.theriojournal.com

The effects of laserpuncture on gonadal maturity and sperm quality of

male striped catfish (Pangasianodon hypophthalmus)
A.T. Mukti a, *, Y.G.P. Sari b, G.S.R. Agusdinata b, W.H. Satyantini a, A.S. Mubarak c,
E.M. Luqman d, Widjiati d
a
Department of Fish Health Management and Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Kampus C Unair Jl. Mulyorejo, Surabaya,
60115, Indonesia
b
Study Program of Aquaculture, Faculty of Fisheries and Marine, Universitas Airlangga, Kampus C Unair Jl. Mulyorejo, Surabaya, 60115, Indonesia
c
Department of Marine, Faculty of Fisheries and Marine, Universitas Airlangga, Kampus C Unair Jl. Mulyorejo, Surabaya, 60115, Indonesia
d
Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga, Kampus C Unair Jl. Mulyorejo, Surabaya, 60115, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Laserpuncture is one of the applicative technologies used mainly in animal and fish reproductions.
Received 23 May 2019 Laserpuncture technology has been used to improve gonadal maturity and sperm quality in fish rapidly.
Received in revised form This study aimed to determine the effects of different laserpuncture doses on gonadal maturity and
14 February 2020
sperm quality of male striped catfish. Males striped catfish (800e900 g/fish body weight) and I gonadal
Accepted 19 February 2020
maturity stage were used. Semi-conductor soft laser was used with doses of 0.2-, 0.4-, and 0.5-J, while
Available online 21 February 2020
the negative control (without the laserpuncture and the ovaprim™) and only the ovaprim™ were used
as a comparison treatment, respectively. The soft-laser was treated on reproductive acupoint every week
Keywords:
Laserpuncture
for four weeks, while the ovaprim™ was administered by intramuscular injection at dose of 0.2 mL/kg
Semi-conductor soft laser fish in final rearing period. Fish was reared in hapa at the controlled pond. Fish was fed with a com-
Gonadal maturity mercial feed containing 32% crude protein. Gonadal maturity, gonadosomatic index (GSI), hepatosomatic
Sperm quality index (HSI), and sperm quality of male striped catfish were measured in the final rearing period. The
Male striped catfish results showed that the laserpuncture on the reproductive acupoint had a highly significant effect
(P < 0.01) on the gonadal maturity, GSI, HSI, and sperm quality of male striped catfish. In terms of the
gonadal maturity, laserpuncture doses treatment of 0.4 and 0.5-J gave the most mature IV stage. While
the highest levels of GSI and HSI were found in 0.5-J of laserpuncture dose, which was 2.17% and 1.54%,
respectively. In addition, the best sperm qualities were observed in 0.5-J of laserpuncture dose, which
were 81.75% motility, 82.75% viability, and 7.0
109 cell/mL concentration. These results suggest that the
laserpuncture can accelerate a gonadal maturity and improve sperm quality in male striped catfish.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction supply of striped catfish seed, which depends on the spawning

season. Zairin [3] stated that the reproductive cycle of striped cat-
Striped catfish has been considered as one of the main cultured fish occurs naturally during the rainy season from October to April
fish in Indonesia due to several advantages, such as fast growth, every year.
easy cultivation, and high tolerance to low dissolved oxygen con- The process of gonadal maturity in striped catfish needed a long
tent in the rearing water [1]. The production of striped catfish in time and depended on the increase in gonadotropin hormone (GtH)
Indonesia was 339,060 metric tonnes (MT) in 2015 and increased and gonadal steroid. The productive period of spawning in male
rapidly in 2016 become 447,110 MT [2]. The high market demand striped catfish peaks at the age of two years or about 1.5e2.0 kg
triggers farmers to increase the amount of striped catfish produc- body weight [4]. Other constraints of a male fish are the decrease in
tion. However, one of the main problems faced by farmers was the sperm quality, such as motility and viability after spawning.
To increase production of striped catfish seed requires new
technology to improve the quality of reproduction using laser-
puncture technology. Laserpuncture technology is a stimulation
* Corresponding author.
technique on acupuncture points (acupoint) by a laser beam [5].
E-mail address: [email protected] (A.T. Mukti).

https://doi.org/10.1016/j.theriogenology.2020.02.030
0093-691X/© 2020 Elsevier Inc. All rights reserved.
 A.T. Mukti et al. / Theriogenology 147 (2020) 102e107 103

The use of the laserpuncture could reduce production costs [6]. 2.3. Experimental design
Susan [5] stated that the application of laserpuncture in the
reproductive organs could stimulate several reproductive functions This study was used the completely randomized design struc-
of male and female animals. Laserpuncture technology has been ture consisting of five treatments with four replicates. Treatments
proven to accelerate gonadal maturity, spawning processes, and to used were doses of 0.2-, 0.4-, and 0.5-J, without laserpuncture and
shorten the reproductive cycles of several aquatic species, such as ovaprim™ treatments as a negative control, and treatment of the
catfish, Clarias gariepinus [7,8] and mud crab, Scylla serrata [9]. ovaprim™ dose of 0.2 mL/kg fish body weight in male striped
Other author explained that the main advantages of laserpuncture catfish with the I gonadal maturity stage. The IV gonadal maturity
technology as a stimulation method are requiring a short time takes stage of male striped catfish as positive treatment was treated the
only 5e10 s, does not cause tissue damage, and provides a ovaprim™ dose of 0.5 mL/kg fish body weight, correctly was only
maximum response [10], depending on the type of soft-laser used. used to observe sperm quality parameters.
The low-power laser affects the biology system of humans [11] Fish were adapted and reared in a hapa sized of
and animals [12], including aquatic organisms (fish). The low- 5.0
3.0
1.5 m3 at a controlled pond and fed commercial feed
power laser around 5e30 mW improves tissue activity, such as containing 32% crude protein. Laserpuncture treatment was per-
increased production of hormones and enzymes [13]. The low- formed on reproductive acupoint every week for four weeks.
power laser gives a biological stimulus, such as changing the cell Reproductive acupoint located on the 2/3 ventral part of the body
membrane’s potential and permeability. On the other hand, it im- (governoer vessel) was measured from the anal to the pectoral fin.
proves the nerve regeneration ability located both in central and The determination of reproductive acupoint was also done using an
peripheral sides will be produced to increase the cellular activities, electro-acupuncture device tool. On the other hand, the ovaprim™
and the ability to produce hormones and enzymes [13,14]. was treated by using the intramuscular technique in the final
Several studies have shown that the laserpuncture improves the rearing stage (week 4), 8e10 h before the end rearing of fish.
vascular and endocrine systems, and various other body systems Gonadal maturity (morphology and histology), gonadosomatic in-
[15]. The low-power laser also improves the male fertility of dex (GSI) and hepatosomatic index (HSI), and sperm qualities, both
humans [16], animals [17], and aquatic organisms, such as fish [17] macroscopic and microscopic, were measured.
and sea urchins [18]. Kusuma et al. [7,8] found that the use of the
low-power laserpuncture technology on the reproductive acupoint
2.4. Morphology and histology of gonadal maturity, GSI, and HSI
precisely in 2/3 ventral parts of the body through induction once a
week is optimal for the maturation of catfish gonads. Moreover, the
At the end of the rearing process, fish was anesthetized using
low-power laser induction at the reproductive acupoint for 15 s
MS222 (Argent Laboratories, Redmond, Washington, DC) of
increases the production of the GtH [10], which is the regulator for
100 ppm [20] for 10 min. Subsequently, the fish were dissected on
produce steroid, oogenesis, and oocyte maturation [19]. On the
the abdominal part from anal to ventral. Morphological and his-
other hand, induction of laserpuncture also increases the testos-
tological gonadal maturity was observed on the shape, the length,
terone level in blood serum and the gonadosomatic index (GSI) of
the weight, the color, and the gonadal development through his-
male catfish [8].
tology preparation. Gonad sample was prepared according to Jun-
The low-power laser has a significant effect on the reproductive
queira and Carneiro [21] to measure the GSI and HSI.
performance of fish whose spawning cycle occurs throughout the
Next, the histological method was conducted by McCann [22]
season, however, does laserpuncture induction affect fish whose
using Hematoxylin-Eosin (HE) staining method according to Gen-
spawning cycle depends on the season? Therefore, this study was
ten et al. [23]. Scoring of testis cell development was conducted
aimed to determine the effect of different laserpuncture doses on
according to Çek and Yilmas [24].
gonadal maturity and sperm quality of male striped catfish.

2. Materials and methods 2.5. Sperm qualities

This study was conducted at the Fish Breeding Research Centre, The collection of sperms from the fish was conducted by using
Subang, West Java, Indonesia. Histological analysis of fish gonads the stripping method. The sperm were stored in 1.5 mL microtubes
was held at the Clinical Pathology Laboratory, Faculty of Veterinary in the refrigerator at a temperature of 4  C before the analysis of
Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. In microscopic qualities.
this study, the experimental protocols were approved by the Sci-
entific Committee, Institute of Research and Innovation, Universitas 2.5.1. Motility of spermatozoa
Airlangga (Protocol Number 886/UN3/2018). Microscopic spermatozoa motility was observed under 100

and 400
magnifications using a BH2-RFCA Olympus binocular
2.1. Animal microscope (Olympus Optical Ltd. Shinjuku-ku, Tokyo, Japan),
which was equipped with a camera. Motile and immotile of the
Male striped catfish with the average body weight of spermatozoa were calculated using a modified method by Sohouka
800e900 g/fish and the I gonadal maturity stage and never been et al. [25], as well as a progressive or active movement forward and
spawned before was used in the present study. As a precaution, non-progressive movements (such as circular, backward or silent).
male striped catfish with the IV gonadal maturity stage were also
prepared separately for positive treatment. 2.5.2. Viability of spermatozoa
The determination of spermatozoa viability was done by the
2.2. Laserpuncture staining protocol of 2% eosin yellow at sperm preparation. The
viability of the spermatozoa was observed and counted under 400

In this study, a semi-conductor soft-laser was used, which had a and 1000
magnifications using the same microscope like the one
power specification of 20 mW. The preset doses and timer set were used to observe motility of the spermatozoa. The spermatozoa
0.2, 0.4, and 0.5 with 10, 20, and 25 s, respectively. viability was counted according to Sohouka et al. [25].
104 A.T. Mukti et al. / Theriogenology 147 (2020) 102e107

2.5.3. Concentration of sperm ovaprim™ treatment (p < 0.05) as seen in Table 2. The results
The sperm concentration was calculated according to Stoss and showed that ovaprim™ treatment does not have a significant effect
Donaldson [26]. on gonadal development; the gonadal maturity only reached the II
stage (immature). It could not be stripped to collect sperms, then
2.6. Data analysis the laserpuncture-treated male fish compared with ovaprim™-
treated male fish that has the IV gonadal maturity stage (as positive
Gonadal maturity stage was statistically analyzed using Kruskal treatment) on sperm quality.
Wallis and Mann Whitney tests, while the morphology and his- This study indicated that GSI and HSI had significant differences
tology of the organ were descriptively analyzed according to Gen- between treatments (p < 0.05). The laserpuncture treatment of 0.5-
ten et al. [23]. The other data were statistically analyzed using J had the highest GSI and HSI compared to other treatments,
analysis of variant (ANOVA) with SPSS ver. 10 software and Dun- although the HSI is relatively not significantly different between
can’s multiple range test with a confidence level of 95%. treatments, except compared to the negative control (Table 1).
Similarity, the laserpuncture dose of 0.5-J had the highest effect
on testicular development. This study also showed that the laser-
3. Results
puncture of 0.5-J dose has the highest scoring of gonadal or
testicular histology in male striped catfish (Fig. 2) with testis con-
3.1. Gonadal maturity stage, GSI, and HSI
taining spermatozoa of 75e100%, as seen in Table 2.
Based on the testicular histology of male striped catfish in Fig. 2
This study showed that laserpuncture treatment affected the
shows that the seminiferous tubules are still empty and the sper-
gonadal development of male striped catfish, as seen in Fig. 1. In
matozoa are not visible (Fig. 2a and b) as one of the II gonadal
general, gonad characteristics of fish with the I gonadal maturity
maturity stage characteristics in the fish. In the III gonadal maturity
stage are testicles like threads, smaller, shorter and limited, and
stage, seminiferous tubules already containing spermatozoa
visible ends in the body cavity. The II gonadal maturity stage (Fig. 1a
(Fig. 2c), while the IV gonadal maturity stage, seminiferous tubules
and b) have more significant and more apparent the testis size and
containing more spermatozoa (Fig. 2d), even full spermatozoa
shape compared to the I gonadal maturity stage. The morphology
(Fig. 2e).
characteristics of the III gonadal maturity stage are the surface of
the testicles appear jagged, more prominent, and the whiter color
(Fig. 1c). The IV gonadal maturity stage of male striped catfish has 3.2. Sperm qualities
testis characteristics of more definite, denser, and milky white color
(Fig. 1d and e). This study showed that laserpuncture treatment produces
The IV gonadal maturity stage achieved through laserpuncture higher sperm quality, both macroscopic and microscopic, compared
treatment of 0.5-, 0.4-, and 0.2-J doses showed no significant dif- to negative and positive treatments, as seen in Tables 3 and 4. The
ference between all the joules (p > 0.05). However, there was sig- laserpuncture treatment of 0.5-J power resulted in the production
nificant difference compared to the negative control and the of the highest volume of sperms, i.e., 3.00e5.25 mL with a creamy
color (Table 3). On the other hand, the treatments showed signifi-
cant differences (p < 0.05) in microscopic sperm quality. The
laserpuncture treatment of 0.5-J power had the highest micro-
scopic sperm qualities compared to other treatments, as seen in
Table 4.

4. Discussion

The induction of laserpuncture on the reproductive acupoint in

fish has proven to accelerate the development and maturity of the
gonad including male gonad of striped catfish from I stage to IV
stage, while in the control treatments (negative control and ovap-
rim™ treatment), the maturity of the gonad developed from I stage
to only II stage during one month. This study result was consistent
with the study conducted by Matayborbir et al. [27] in the catfish
that laserpuncture exposure accelerate gonadal maturity from II
stage to IV stage of male rapidly. The condition of fish gonads at the
beginning of the study was at the initial gonadal maturity stage

Table 1
Gonadal maturity stage, GSI, and HSI of male striped catfish in the different
treatments.

Treatment Gonadal maturity stage GSI (%) HSI (%)

Negative control IIa 0.39 ± 0.15a 1.10 ± 0.07a

Ovaprim™ IIa 0.54 ± 0.29ab 1.30 ± 0.14ab
Ld of 0.2-J III to IVb 0.70 ± 0.38ab 1.32 ± 0.15ab
Ld of 0.4-J IVb 1.09 ± 0.19b 1.39 ± 0.26b
Ld of 0.5-J IVb 2.17 ± 0.68c 1.54 ± 0.17b
Fig. 1. The testicular morphology of male striped catfish in different gonadal maturity
stages; the 2nd stage in the negative control (a) and the ovaprim™ treatments (b), the Note: Negative control ¼ without the laserpuncture and the ovaprim™ treatments,
3rd stage in the laserpuncture treatment of 0.2-J dose (c), and the 4th stage in the Ld ¼ Laserpuncture dose. Different superscripts the same column show significant
laserpuncture treatments of 0.4-J (d) and 0.5-J doses (e) (bar scale ¼ 1 cm). differences (p < 0.05).
 A.T. Mukti et al. / Theriogenology 147 (2020) 102e107 105

Table 2 Table 3
Average testicular histology scoring of male striped catfish in the different Macroscopically sperm quality of male striped catfish in the different treatments.
treatments.
Treatment Spermatozoa quality parameters
Treatment Score Description
Volume (ml) pH Color Consistency
Negative control 3a Development of cells has reached spermatids
Negative control Negative Negative Negative Negative
Ovaprim™ 4b Development of cells has reached spermatids
Ovaprim™ Negative Negative Negative Negative
Ld of 0.2-J 5c Already formed spermatozoa of 25.00e49.90%
Positive 0.50 to 1.25 7 to 8 Clear White Dilute
Ld of 0.4-J 6d Already formed spermatozoa of 50.00e74.50%
Ld of 0.2-J 0.50 to 1.50 7 to 8 White milk Condensed
Ld of 0.5-J 7e Already formed spermatozoa of 75.00e100.00%
Ld of 0.4-J 0.50 to 2.00 7 to 9 White milk Condensed
Note: Negative control ¼ without the laserpuncture and the ovaprim™ treatments, Ld of 0.5-J 3.00 to 5.25 7 to 9 Creamy Condensed
Ld ¼ Laserpuncture dose. Different superscripts the same column show significant
Note: Negative control ¼ without the laserpuncture and the ovaprim™ treatments,
differences (p < 0.05).
Positive ¼ the male striped catfish with the IV gonadal maturity stage treated the
ovaprim™ (0.5 mL/kg of fish body weight), Ld ¼ Laserpuncture dose.

with a small gonadal morphology and appeared clear. Then, after

the laserpuncture treatment, the gonadal maturity reached IV stage
(fully maturated). laserpuncture exposure at reproductive acupoints in male striped
GSI has been used as one of the indicators of the development catfish increased GSI and HSI. GSI will continue to increase along
and maturity of gonad [28] in both sexes. In general, GSI increases with the maturation of the fish gonads and will reach the maximum
with the increasing gonadal maturity stage as well as a show in this value when the peak period of gonad maturity [29].
study (Table 1). Increased GSI followed the bigger size of the gonad The exposure of laserpuncture on the reproductive acupoint
(testis), as shown in Fig. 1, and increase the number of spermatozoa stimulate FSH and LH from the pituitary, which has a vital role in the
produced by testis (Fig. 2). Kusuma [7] stated that laserpuncture development and maturity of the gonad. Although FSH and LH levels
stimulates cell activations in the area governoer vessel (reproduc- were not measured in this study, a previous study [7] have reported
tive acupoint) to produce energy. The formation of energy after that laserpuncture exposure to reproductive acupoint increased FSH
laserpuncture exposure in the reproductive acupoint related to and LH levels and also accelerated LH formation which play a role in
specific proteins in cells. As the results, the development of gonads the final stage of spermatogenesis or spermatozoa production,
gradually from spermatogonia, spermatocytes, spermatids, and testicular maturation, ovulation, and spawning stimulation in cat-
spermatozoa, which are marked by the bigger testicles. The fish. It indicated that the induction of laserpuncture could increase
the performances of the activity of the hormone, which takes part in
a reproduction control system to accelerate the provision of growth,
development, and gonad maturation of fish.
Furthermore, the induction of laserpuncture on the tissue
increased gonadotropin-releasing hormone (GnRH) released by the
hypothalamus and it stimulated the anterior pituitary to secrete
FSH and LH which act on theca cells in the male gonad (testis) to
produce testosterone hormone [30]. Similarly, a study by Chang
et al. [31] showed that laserpuncture stimulated the release of
neurotransmitters. The laser beam transduced into chemical sig-
nals band being received by various ion channels, such as G-pro-
teins (GTP-binding protein)-coupled receptors subunit a and VGCC
(voltage-gated Ca2þ channels). Other possible mechanisms is
through calcium receptors, such as calcium-sensing receptor
(CaSR), located in the nervous membrane cells. Then, ligands
binding on the specific receptors triggers the release of second
messengers causing a chain reaction and bringing changes in the
cell. Electrical signals caused by depolarization of the nerve cell
membrane were propagated from cell to cell along the axon. Next,
the electrical signals were inserted from the pre-synapse mem-
brane into the post-synapse membrane and trigger the release of
neurotransmitter molecules in the synapse. The same mechanism,
the electrical signals were transmitted to the brain. intracellular
and extracellular Ca2þ ions mediated the electrical signals through
changes in spontaneous membrane potential play an essential role
in stimulating the release of GnRH from the hypothalamus and it
stimulates the pituitary to release FSH and LH. Next, FSH and LH are
then channeled into the bloodstream towards the gonads (testis),
which enable various activities. This process repeats when the
nerve cell membrane is depolarized [32].
Other study by Anglade et al. [32] explained that the release of
neurotransmitters such as gamma-aminobutyric acid (GABA) from
GABAergic neurons depends on nerve cell membrane depolariza-
Fig. 2. The testicular histology of male striped catfish in different gonadal maturity tion, action potential, calcium ions, decarboxylation of glutamate,
stages and treatments observed under a microscope at 400
magnification; the 2nd and glutamic acid decarboxylase (GAD). The anterior pituitary
stage in the negative control (a) and the ovaprim™ treatments (b), the 3rd stage in the
laserpuncture treatment of 0.2-J dose (c), and the 4th stage in the laserpuncture
directly innervates GABAergic neurons, so it has a stimulatory effect
treatments of 0.4-J (d) and 0.5-J doses (e) (Spz ¼ spermatozoa, bar scale ¼ 100 mm). on the release of LH [33].
106 A.T. Mukti et al. / Theriogenology 147 (2020) 102e107

Table 4
Microscopically sperm quality of male striped catfish in the different treatments.

Treatment Spermatozoa quality parameters

Motility (%) Viability (%) Concentration (
109 cells/mL)

Negative control Negative Negative Negative

Ovaprim™ Negative Negative Negative
Positive 58.88 ± 1.93a 58.00 ± 1.58a 4.31 ± 4.26a
Ld of 0.2-J 65.75 ± 2.32b 66.25 ± 1.75b 5.25 ± 4.56b
Ld of 0.4-J 73.00 ± 2.73c 73.00 ± 2.27c 6.06 ± 6.25c
Ld of 0.5-J 81.75 ± 1.19d 82.75 ± 1.84d 7.00 ± 5.40d

Note: Negative control ¼ without the laserpuncture and the ovaprim™ treatments, Positive ¼ the male striped catfish with the IV gonadal maturity stage treated the
ovaprim™ (0.5 mL/kg of fish body weight), Ld ¼ Laserpuncture dose. Different superscripts the same column show significant differences (p < 0.05).

Factors which determined the volume of sperm production spermatogenesis than testosterone hormone. The concurrent
include environmental conditions, such as temperature, pH, and elevation of 11-ketotestosterone and testosterone hormones level
dissolved oxygen levels. Fish which are in stress condition due to is another typical pattern in male fish [49]. Therefore, the mea-
environmental condition cannot produce large volumes of sperm. surement of 11-ketotestosterone and testosterone hormones will
The same factors have been confirmed by a study of Salisbury and be investigated for further laserpuncture studies of fish.
VanDenmark [34] where the volume of sperm production was
influenced by environmental factors, age, body size, feeding man- 5. Conclusion
agement, and sperm release frequency.
The low-power laser has significantly improved sperm quality, Laserpuncture induction of 0.5-J dose at the reproductive acu-
such as volume [35], concentration [36], motility, movement, and point increase gonadal maturity and sperm quality of male striped
viability of spermatozoa [37]. The highest motility obtained in 0.5-J catfish. Further studies on higher doses of laserpuncture and its
treatment because this dose can provide a stimulatory effect large effects on hormonal mechanisms are critical, especially in the fish.
enough to stimulate the hypothalamus neuron to release the GnRH.
GnRH stimulates pituitary neurons to release FSH and LH. FSH Declaration of competing interest
produces testosterone hormone, and LH produces an androgen-
binding protein (ABP). Testosterone hormone and ABP control No conflict of interest.
spermatogenesis and initiate spermatogenic development into
motile spermatozoa [38]. Mantayborbir et al. [27] also found that Acknowledgements
laserpuncture induction has spontaneous stimulation power and
rapidly influences the increase in the number of Leydig cells pro- This study was supported by the Universitas Airlangga through
duced. The function of Leydig cells is to produce testosterone hor- Grant Program of Faculty Priority Research. We wish to thank Prof.
mone, which binds to androgen receptors in Sertoli cells, which Dr. Raden Tatang Santanu Adikara who provided the laserpuncture
secretes ABP and helps form spermatozoa. tool, the Head of Fish Breeding Research Centre, Subang, West Java,
Several studies indicate the influence of laserpuncture on Indonesia on aid provision of striped catfish stock and facilities to
improving the sperm quality of animals including motility [35,37] this study, and Muhamad Amin, Ph.D. who helped figures and en-
and fertility [35] of spermatozoa, the adenosine triphosphate (ATP) glish editing of this revised article. Authors would like to thank and
content [39], Ca2þ concentration [40], and cell life [41]. Ca2þ appreciate the comments and corrections given by reviewers, edi-
stimulates the work of the mitochondria and the ATP synthesis in tor, and proofreader to improve this article.
the cell [42], while mitochondria and ATP play an important role in
supporting spermatozoa motility [43]. Furthermore, laserpuncture References
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