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The effect of different diets on sperm qu -

ality in the striped catfish (Pangasianodo
n hypophthalmus)

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Summary
45
The effect of different diets on the quality of sperm in striped
catfish (Pangasianodon hypophthalmus)

Wahyu Pamungkas1,2, Jadmiko Darmawan1,2, and Ikhsan Khasani1,2

30
1
Research Institute for Fish Breeding, Ministry of Marine Affairs and Fisheries,
Subang, West Java, Indonesia
2
Research Center for Fishery, National Research and Innovation Agency, Cibinong,
West Java, Indonesia

E-mail: yhoe_pamungkas@yahoo.co.id

47 27
Abstract. Sperm quality is crucial to the success of fish breeding in captivity. The present study
evaluated the effects of different diets on sperm quality in the striped catfish (Pangasianodon
hypophthalmus)
1
during a 3-month feeding trial. The thirty striped catfish were divided evenly
between the two groups (A and B). Group A was fed a commercial diet, while group B was fed
a formulated
1
diet containing essential fatty acids. Fifteen males with a bodyweight of 2.59-3.09
kg for each group were used as experimental fish. The motility and velocity of the catfish
sperm, fertilization, and hatching rate were examined to evaluate the sperm quality and its
34
association with fertilization and hatching rate. An analysis of sperm 1motility and velocity has
been performed using computer-assisted sperm analysis (CASA). The research data were
analyzed by an independent sample T-test. Group A significantly differed from group B in
35
terms of motility %, progressive motility, curvilinear velocity (VCL), straight-line velocity
1
(VSL), average path velocity (VAP), and hatching rate (HR). These findings imply that
essential fatty acid-rich diets improve the sperm quality of striped catfish.

1. Introduction
1
The striped
1
catfish, Pangasianodon hypophthalmus, is one of the most rapidly developing freshwater
species. According to the Ministry of Marine Affairs and Fisheries, Republic1
of Indonesia [1], the
national production of striped catfish in 2013 was 410.383 tons, and the increase in striped catfish
production from 2010 to 2013 reached 95.57%. Furthermore, according to the Ministry [2], total
production reached 418.002 tons in 2014 but declined by 18.8% to 339.111 tons in 2015. In 2017 there
was another decline in production by 18.67% of the total production in 2016 (392.918 tons). Ineffective
broodstock management may have contributed to the drop in fish production. Problems in10aquaculture
activities contributed to the decline in total production. Therefore, anticipating these issues is necessary
to ensure the continued availability of high-quality seeds.
The availability of good quality seeds is related to the availability of superior broodstock quality.
Improving46the quality of broodstock can be done through a genetic approach and nutrition. The nutrients
contained in broodstock diets will affect the quality of sperm and eggs produced, and in the end, will
also affect the quality of the seeds produced. According to [3], good nutrition improves not only the
quality of sperm and egg cells but also the quality and quantity of seeds. Few research has been able to
 directly link the nutrition of broodstock to spermatozoa kinetics, despite the fact that numerous studies
have linked diet to reproductive success. Sperm concentration, motility, viability, morphology,
metabolic activity, and ability to fertilize an egg are important factors that must be considered in
determining reproductive performance [4]. Meanwhile, temperature, season, stress, hormone
stimulation, and parent nutrition are factors that3
affect sperm quality [5].
In the case of broodstock nutrition, the fatty acid composition of the broodstock diet has been
discovered as a primary dietary component that impacts fish sperm quality, owing to carnivorous fish 5
being unable to manufacture specific fatty acids and hence need them to be given in their feed. In
general, both freshwater and marine species require PUFA or HUFA in their diets [3], and adding these
fatty acids to broodstock diets can result in significant
5
improvements in sperm motility indices. [6]
3
discovered that a DHA-enriched diet improved sperm quality in Senegalese sole (Solea senegalensis),
specifically
3
the sperm velocity (VCL) and percentage of progressive sperm. In European eels, diets high
in arachidonic acid (ARA) generated medium milt volumes and high sperm motility 5
[7-8], whereas diets
high in eicosapentaenoic acid (EPA) induced noteworthy milt volumes and high sperm motility. In
freshwater fish such as rainbow trout, some breeders fed a diet deficient 3
in essential fatty acids (n-3)
had lower sperm motility than breeders fed a control diet [9], while the fish fed a properly HUFA/PUFA
ratio had the highest sperm motility percentage and duration than other treatments in another rainbow
trout trial [10].
The quality of sperm is critical to the effectiveness of farmed fish spawning. In practice, 42
any
quantitative indicator that is directly connected to fertilization capacity can be used to estimate sperm 53
quality. Spermatozoa motility is the most17often used parameter in determining sperm quality [11]. The
motility and17
velocity of spermatozoa [as evaluated by computer-assisted sperm analysis (CASA)] are
frequently used to link male gamete quality to fertilization potential. Spermatozoa
17
with higher velocity
and motility have a shorter window of time 33
to reach the micropyle, which is important for fish that
spawn in highly competitive situations. Evaluation of sperm motility as well as other kinetic
characteristics such as curvilinear, straight
3
line, and average path velocities, as well as morphology, 28
is
an important aspect of determining sperm quality in many fish species [12]. This experiment was
designed to investigate into the impact of different diets on sperm quality in Striped Catfish
(Pangasianodon hypophthalmus).

2. Material and Methods 22

The research was carried out at The Research Institute for Fish Breeding (RIFB), Ministry of Marine
Affairs and Fisheries, West Java, Indonesia. The striped catfish (Pangasianodon hypophthalmus) were
acquired from the Institute's broodstock population.
18
2.1. Striped catfish rearing 1
The striped catfish males (bodyweight 2.84±0.25 kg) were used as experimental fish. The fish were
acclimated to experimental conditions
1
for 2 weeks before treatment. Thirty fish were kept in six net
cages measuring 3 m x5 m x1.5 m located in the 6,000 m2 of an earthen pond, at a stocking density of
five fish per cage for eight weeks. The fish were fed commercial and formulated feed at 3% of their
body weight (BW) twice a day during the experiment.
48
2.2. Experimental design
The fish were separated into two groups of 15 fish each (Groups A and B) and subjected to the following
experimental protocol. Commercial feed was provided to group A, while 1
a prepared diet containing
essential fatty acids was given to group B. The n-6 fatty acid source in the formulation1
feed was 2%
corn oil, while the n-3 fatty acid source was 1.5 percent fish oil (Table 1) [13]. The proximate
composition of the experimental diets is presented in Table 2. The fatty acid composition of the diets is
presented in Table 3.
 Table 1. Feed formulation of the striped catfish diet
Ingredient %
Fish meal 50.75
Soybean meal 18.61
Coconut oil 6.95
Fish oil 1.50
Corn oil 2.00
Premix 2.00
Choline chloride 0.50
Tapioca 1
5.70
CMC1 0.30
Wheat flour 11.69
1)
CMC = carboxymethyl cellulose, 2) NFE-Nitrogen Free Extract.

Table 2. Proximate composition of the striped catfish diet (%)

Diet
Proximate (%)
Commercial Formulated
Protein 35.80±0.01a 36.80±0.60b
Fat 4.90±0.16a 12.70±0.01b
a
Fiber 2.50±0.48 2.99±0.02a
a
Ash 7.80±0.05 16.40±0.02b
NFE 49.10±0.70a 31.12±0.61b
-1
Energy (Kcal kg feed) 449.780 455.624
Energy/protein 12.580 12.374
Values (means ± S.D., n=3) in the same row with different superscript letters
show significant differences (P<0.05). CMC-carboxymethyl cellulose; NFE-
nitrogen-free extract.

Table 3. The fatty acid composition of the experimental diets (% area)

Fatty acid Commercial diet Formulated diet
Σn−3 fatty acid (%) 10.10±0.02a 2.60±0.05b
Σn−6 fatty acid (%) 4.50±0.04 a
13.80±0.08b
Σn−9 fatty acid (%) 47.80±0.04 a
22.20±0.01b
ΣSaturated fatty acid (%) 53.40±0.03 a
59.30±0.02b
ΣUnsaturated fatty acid (%) 46.80±0.04 a
42.50±0.06b
n−6/n−3 ratio 0.45±0.00a 5.40±0.12b
Values (means ± S.D., n=3) in the same row with different superscript letters show
significant differences (P<0.05).

2
2.3. Sperm collection 2
During gamete maturation, fish were tested weekly for the presence of milt. Milt from
2
fish was collected
by abdominal massage into scaled-marked glass beakers whenever it was present. The milt was stripped
in a way that it did not become contaminated
2
with blood, feces, or urine. During stripping, no anesthetic
was utilized. Until further investigation, sperm samples were stored in insulated boxes (4°C) without
direct contact with ice.

2.4. Measurement of2sperm motility

Motility parameters were evaluated using CASA (CEROS II Hamilton-Thorne) connected 27
to CX41
microscope (Olympus) after 10 s of activation with a digital camera (U-TV1X- 2). The motility variables
measured included the percentage of motility and progressive motile, non-progressive motile, static,
29
curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN),
 40
and straightness (STR).
43
Measurements were done in triplicate and the average result was used in the
data analyses. Sperm with a velocity of ≥20 μm/s was defined as motile.

2.5. CASA Assessment 8

Semen was diluted and placed in ledja that had been conditioned to 37 °C. The CASA used is a Sperm
Class Analyzer (SCA) 5.2 Micro-optics, Spanish. The CASA settings were as follows: pH1 contrast
phase, 10 x 10 magnification, and a green filter on the reflector mirror. The light intensity was adjusted
to meet the standards and printed on the monitor screen. The pictures were taken in five different fields
of view, and the results were displayed in Microsoft Excel.

2.6. Fertilization and hatching rates 37

Fertilization and hatching rate values were measured to assess sperm's ability to fertilize eggs and
produce
18
offspring. Fertilization and hatching rates were calculated using the following equations:
 Fertilization rate (%) = (Number of fertilized eggs ∕Total number of eggs) × 100 [14];
 Hatching rate (%) = (Number of hatched eggs ∕Total number of fertilized eggs) × 100 [15];
18
2.7. Data analysis 52 1
The data on motility, velocity, fertilization rate, and hatching
50
rate were statistically analyzed using
Microsoft Excel 2016 and the SPSS program (ver. 25). The Independent Sample T-test was used to
examine the data.

3. Result and Discussion

44 1
The results of this study revealed that the percentages of motility and progressive motile in group A
were significantly different from those in group B, but the percentages of non-progressive motile were
not (Table 4). Group B had a higher value of the percentage of motility (96.00±1.67 %) and progressive
motile (32.10±5.90 %) than group A (77.32±5.54 % of motility; 12.35±2.63 % of progressive motile).

Table 4. Sperm motility of striped catfish fed experimental diets.

Diet
Parameter
Commercial Formulated
a
Motility (%) 77.32±5.54 96.00±1.67b
a
Progressive motile (%) 12.35±2.63 32.10±5.90b
a
Non-progressive motile (%) 64.97±4.99 63.90±4.41a
Static (%) 22.68±13.58a 4.00±4.11b
1
Mean values in the same row with different superscript letters show significant differences between the
groups (p<0.05).
36
The sperm velocity including curvilinear velocity (VCL), straight-line velocity (VSL), and average
path velocity (VAP) in group A was significantly different from group B (Table 5). The percentage of
VCL (58.87±4.33 µms¯¹), VSL (34.30±2.80 µms¯¹), and VAP (45.17±2.93 µms¯¹) in group B were
higher than in group A (36.85±4.47 µms¯¹ of VCL, 23.28±1.69 µms¯¹ 1of VSL, and 29.70±2.84 µms¯¹
of VAP). The percentage of linearity (LIN) and straightness (STR) in group A were not significantly
different from group B.

28
Table 5. Sperm velocity (VCL, VSL, VAP), linearity (Lin), and straightness (STR) of striped
catfish fed experimental diets
Diet
Parameter
Commercial Formulated
VCL (µms¯¹) 36.85±4.47a 58.87±4.33b
 VSL (µms¯¹) 23.28±1.69a 34.30±2.80b
VAP (µms¯¹) 29.70±2.84a 45.17±2.93b
Lin (%) 65.00±3.36a 58.10±1.33a
STR (%) 79.30±2.30a 75.42±1.68a
1
Mean values in the same row with different superscript letters show significant differences between the groups
(p<0.05).

Observation data for FR and HR were shown in Table 326. The hatching rate in group A was
significantly different from group B, but the percentage of FR
32
was not significantly different between
the groups. The value of the hatching rate (87.47±4.29 %) in group B was higher than in group A
(57.58±7.36 %).
Table 6. Fertilization (FR) and hatching rate (HR)
Diet
Parameter
Commercial Formulated
FR (%) 76.11±4.61a 86.66±4.77a
HR (%) 57.58±7.36a 87.47±4.29b
1
Mean values in the same row with different superscript letters show significant differences between the groups
(p<0.05).
2
Previous research on broodstock nutrition has primarily focused on female fish. However, few
studies using male broodfish show the importance of optimizing the dietary needs of both male and
female broodfish [8,10].
2
As a result, the current study investigated at how different diets high in essential
fatty acids affected sperm 38
quality and reproductive performance in striped catfish.
For viable offspring, the quality of fish sperm is just as important as the quality of female eggs.
11
Sperm morphology, density, volume, 51
motility, and fertilizing capacity, as well as seminal plasma
composition
24
and osmolality, are common parameters used to assess sperm quality in fish [15-21].
Among the24various semen quality biomarkers in fish, motility is now the most commonly used parameter
[22], and it is highly correlated with fertilization success in several fish species [23].
Sperm velocities are also reliable indicators of sperm
2
quality [24] and are important in determining
male fertility [25,19,21]. They have been linked to hatching rates and sperm motility in African catfish
[26], turbot [27], carp [28-30],39and rainbow trout [30].
In this study, striped catfish
39
sperm motility and velocity were affected by formulated diets containing
essential fatty acids. Higher sperm motility and velocity 2
values in catfish fed formulated diets indicated
a better effect (group B). According to [31] the lipid and FA profiles in broodfish diets are critical in
determining successful reproduction and early-stage development. The dietary profile of sperm
influences
2
its FA profile [33,10,32]. Lipids are the most essential substance of teleost spermatozoa
2
[33-
36]. The functionality of spermatozoa is closely related to its fatty acid [32]. Fatty acid oxidation in the
mitochondria of sperm cells produces energy, which is extremely crucial for their survival and
movement [32]. As a result, lipids and fatty acids are the primary energy sources in fish spermatozoa
and are critical to sperm viability
2
[37-38].
Apart from the above, reproductive performance is the most reliable way of determining gamete 2
quality [3,21]. Fertilization is one method for determining the quality of 49
sperm [18]. FAs content in
sperm, which is affected by broodstock dietary lipids, has been shown to affect sperm quality, regulate
its ability to fertilize eggs successfully, and increase hatching rate [38-40]. In this study, different diets
influenced the 37 value of fertilization and hatching rate in striped catfish. Fertilization and hatching rate
values describe sperm's ability 2
to fertilize eggs and produce offspring. According to the findings of the
study, dietary fatty acids in broodfish nutrition are also very important to be considered for determining
male reproductive performance.
 Good nutrition in the broodstock diet will improve the broodstock's reproductive performance,
allowing it to produce large amounts of high-quality seeds. The availability of high-quality broodstock
and seeds will actually achieve sustainable aquaculture.

4. Conclusion
The different diets affected the sperm quality of the striped catfish. Formulated diets containing essential
fatty acids, improved sperm motility and velocity of striped catfish. The high motility and velocity of
fish sperm increased fertilization and hatching rates.

Acknowledgments
1 1
The Research Institute for Fish Breeding in West Java, Indonesia, provided funding for this study. The
Agency for Marine and Fisheries Research and Human Resources, Ministry of Marine Affairs and
Fisheries, Republic of Indonesia, also contributed to the study. We also appreciate the valuable technical
assistance provided by the Research Institute for Fish Breeding's catfish commodity team.

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13 1%
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M Hajiahmadian, K Sarvi Moghanlou, N Agh, F Farrokhi Ardabili. " Sem...

14 <1%
Crossref

dergi.firat.edu.tr
15 <1%
Internet

ulspace.ul.ac.za
16 <1%
Internet

Vahid Zadmajid, Jaelen Nicole Myers, Sune Riis Sørensen, Ian Anthony...
17 <1%
Crossref

Wahyu Pamungkas, Dedi Jusadi, Muhammad Zairin, Mia Setiawati, Edd...

18 <1%
Crossref

Khrystyna Kurta, Henrik Jeuthe, Dirk Jan de Koning, Christos Palaiokos...

19 <1%
Crossref

japu.gau.ac.ir
20 <1%
Internet

frontiersin.org
21 <1%
Internet

Sources overview
 Similarity Report ID: oid:30529:121385200

download.garuda.kemdikbud.go.id
22 <1%
Internet

jifro.ir
23 <1%
Internet

link.springer.com
24 <1%
Internet

app.trdizin.gov.tr
25 <1%
Internet

journal-aquaticscience.com
26 <1%
Internet

science.gov
27 <1%
Internet

theses.cz
28 <1%
Internet

worldwidescience.org
29 <1%
Internet

Priadi Setyawan, Muhammad Hunaina Fariduddin Aththar, Imron Imron...

30 <1%
Crossref

Samuel M. Pountney, Herve Migaud, Andrew Davie. "Short term cold st...
31 <1%
Crossref

ogr.vet.chula.ac.th
32 <1%
Internet

Elisa Giaretta, Mauro Munerato, Marc Yeste, Giovanna Galeati, Marcell...

33 <1%
Crossref

Sources overview
 Similarity Report ID: oid:30529:121385200

Liu, Q.H.. "Use of computer-assisted sperm analysis (CASA) to evaluat...

34 <1%
Crossref

diamond-congress.hu
35 <1%
Internet

uel.br
36 <1%
Internet

Thea M. Edwards. "Reproductive dysgenesis in wildlife: a comparative ...

37 <1%
Crossref

Bukola Gbemisola Oguntuase, O. T Adebayo. "Sperm Quality and repro...

38 <1%
Crossref

Zoe G. Nichols, Scott Rikard, Sayyed Mohammad Hadi Alavi, William C....
39 <1%
Crossref

Koh, I.C.C.. "Cryopreservation of sperm from seven-band grouper, Epin...

40 <1%
Crossref

Priadi Setyawan, Imron Imron, Bambang Gunadi, Sander van den Burg, ...
41 <1%
Crossref

B. Tizkar, R. Kazemi, A. Alipour, A. Seidavi, G. Naseralavi, J.T. Ponce-P...

42 <1%
Crossref

Güneş Yamaner, Gökhan Tunçelli, Deniz Devrim Tosun, Özgür Çanak, D...
43 <1%
Crossref

frov.jcu.cz
44 <1%
Internet

Muzaffer Mustafa Harlioğlu, Kenan Köprücü, Ayşe Gül Harlioğlu, Serpil ...
45 <1%
Crossref

Sources overview
 Similarity Report ID: oid:30529:121385200

vdoc.pub
46 <1%
Internet

"Reproduction in Aquatic Animals", Springer Science and Business Me...

47 <1%
Crossref

Sheikhzadeh, N.. "Effect of Ergosan on semen quality of male rainbow t...

48 <1%
Crossref

buleria.unileon.es
49 <1%
Internet

cjes.guilan.ac.ir
50 <1%
Internet

orbi.ulg.ac.be
51 <1%
Internet

ALINIYA, Masoumeh, KHARA, Hossein, NOVEIRI, Shahrouz Baradaran ...

52 <1%
Publication

Emilie Henrotte. "Dietary n-3/n-6 ratio affects the biochemical composi...

53 <1%
Crossref

Sources overview