Climbing Perch Fish Sperm DNA ( Anabas testudineus ) Protection Using Cryoprotectant Agent

. Sperm cryopreservation is a sperm storage method that needs to be developed to ensure the availability of climbing perch fish ( Anabas testudineus ) sperm considering the difficulty of providing quality broodstock. This study aims to determine the effect of several types and concentrations of cryoprotectants on the DNA integrity of climbing perch fish sperm after cryopreservation for 14 days at a temperature of - 196 ℃ . Analysis of sperm DNA integrity used the gel electrophoresis method. This research consists of four types of cryoprotectants; Each type of cryoprotectant consists of two concentration levels. The cryoprotectant concentrations used were DMSO (concentrations 10 and 20%), Methanol (concentrations 10 and 20%), Glycerol (concentrations 5 and 20%), and Ethanol (concentrations 5 and 15%) as well as control treatment and fresh sperm treatment. Based on the results of the DNA integrity analysis of climbing perch fish sperm, it showed that all sperm DNA samples did not form fragments, meaning that there was no damage to climbing perch fish sperm DNA in all samples tested, both fresh sperm samples and post-cryopreserved sperm samples.


Introduction
Climbing perch fish Anabas testudineus is one of the freshwater fish that has economic value in Indonesia [1].This fish has been cultivated [2], but the technology for hatching climbing perch fish, especially sperm storage, is still in the development stage [3,4].Cryopreservation not only plays a role in fish farming activities, but also for the conservation and improvement of genetic resources [5].Cryopreservation can make an important contribution to storing genetic material over a long period of time [6].This technique has been established in several species of freshwater fish using a variety of materials (extenders and cryoprotectants) used to maintain post-thawing sperm quality [7].However, the process often triggers cell injury (cryo-injury) which is caused by mechanical damage due to the formation of ice crystals both inside the cell and outside the cell, and also by osmotic pressure or oxidative stress [8].This can affect the structure of the plasma membrane, mitochondria and chromatin [9].
One of the parameters in determining sperm quality after cryopreservation is motility [10].There are several aspects that influence sperm cell motility including mitochondrial function, plasma membrane channel activity, ATP production and flagella structure [11].For successful egg fertilization, spermatozoa need to maintain plasma membrane integrity, osmotic control, and movement capacity to reach the oocyte [5].
Apart from motility, analysis of sperm DNA integrity is also an important parameter to evaluate the success of established sperm cryopreservation protocols so as to select the best quality samples for freezing.Studies related to fish sperm DNA analysis have been carried out using several different approaches such as the gel electrophoresis method [12], the comet test (single cell gel electrophoresis) [13], TUNEL (terminal deoxynucleotidyl transferasenick-end-labelling) [14], and analysis of specific DNA sequences using RT-PCR [15,16].Sperm DNA damage has been closely linked to various indicators of reproductive health including fertilization, embryo quality, and congenital abnormalities [17,18].Thus, sperm DNA integrity must be guaranteed for successful embryo development after fertilization [19].This research was carried out as a follow-up observation to the previous publication by [20] regarding sperm DNA integrity parameters after cryopreservation with the addition of cryoprotectant using the gel electrophoresis method.Research analyzing the DNA integrity of climbing perch fish sperm after cryopreservation has never been reported before.This is important to know in order to obtain an overview of the influence of several types and concentrations of cryoprotectants on the DNA integrity of climbing perch fish sperm after cryopreservation.

Time and Site
This research was conducted from September to November 2022.The maintenance and treatment of test animals were carried out at the Fish Breeding and Rearing Laboratory of the Faculty of Marine and Fisheries, Syiah Kuala University.DNA integrity analysis was conducted at the Brackishwater Aquaculture Center (BPBAP) Ujung Batee, Banda Aceh.

Research Design
This research uses the gel electrophoresis method, which consists of four types of cryoprotectant with each type having 2 concentration levels, including: DMSO (concentration 10 and 20%), Methanol (concentration 10 and 20%), Glycerol (concentration 5 and 20%), and Ethanol (concentration 15 and 5%), which will be combined with 5% egg yolk and glucose-base and there was a control treatment which only added glucose-base and , 03010 (2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301087 5% egg yolk.Apart from that, there is also fresh sperm treatment which is used as a reference in this research.

Broodstock Maintenance and Sperm Collection
Climbing perch fish broodstock were collected from the waters of Aceh Besar, consisting of 20 male broodstock with a length of 10-14 cm and a body weight of 39-43 g.The collected broodstock were acclimated for 14 days in a broodstock pond and fed with commercial feed with a protein content of >30% at 5% of body weight.Feeding was provided twice daily at 06:00 and 18:00 WIB.Climbing perch were reared based on the guidelines of the Institutional Animal Care and Use Committee (IACUC, 2018) and have ethical approval B/034SN/XII/2022 from the animal ethic committee of the Marine and Fisheries Faculty, Syiah Kuala University.Mature male broodstock were selected, and each received an intramuscular injection of ovaprim at a dosage of 0.5 ml/kg body weight.Ovaprim was injected below the dorsal fin, with the injection needle at a 45° angle to the fish's body surface or on the dorsal fin of the fish [3].Subsequently, the fish were aerated for 7 hours.During the conditioning, the broodstock were not given any feed.After 7 hours, sperm collection was performed on the male broodstock.The sperm collection process involved cleaning the fish's genital opening using tissue, followed by gently stripping the fish's abdomen towards the genital area.The collected sperm was then transferred to a 2 ml cryotube and placed in a styrofoam container with a temperature of 4 °C [12].

Extender and Cryoprotectant Preparation
Based on previous research, the most suitable extender for climbing perch fish sperm is Glucose base [4], Therefore, this extender was used in this research, with the following combination of ingredients: NaCl 0.725 g; KCL 0.04 g; NaHCO3 0.080 g; glucose 0.20 g, mixed with aquades to a total volume of 100 ml.The previously obtained sperm was mixed with the extender at a dilution ratio of 1:60 v/v (sperm: glucose base extender).
The cryoprotectants tested were DMSO (10% and 20% concentration), Methanol (10% and 20% concentration), Glycerol (5% and 20% concentration), and Ethanol (15% and 5% concentration).The control group had 0% cryoprotectant.Each treatment was combined with 5% egg yolk.To obtain the test concentrations of 0%, 5%, 10%, 15%, and 20% cryoprotectant, 100 ml of glucose base extender was added to 1.67 ml of sperm, resulting in a sperm and glucose base extender ratio of 1:60 v/v.Then, 5 ml of egg yolk was added to this dilution.Subsequently, the dilution of sperm and egg yolk was distributed in 1.5 ml portions to 9 cryotubes.Each cryotube was then filled with the tested cryoprotectant (DMSO, methanol, glycerol, and ethanol) according to the specified test doses: 0.075 ml of cryoprotectant was added to obtain a 5% concentration, 0.15 ml to obtain a 10% concentration, 0.225 ml to obtain a 15% concentration, and 0.3 ml to obtain a 20% concentration.The 0% concentration (control) was not supplemented with cryoprotectant.

Process of Freezing and Thawing sperm samples
All cryotubes containing sperm samples were placed in an icebox at a temperature of 4 °C for 5 minutes for the initial equilibration (pre-freezing).The samples were then evaporated at a distance of 5 cm above the surface of liquid nitrogen at -79 °C for 5 minutes [21], and then transferred into liquid nitrogen (-196 ℃) and stored for 2 weeks.After two weeks, the , 03010 (2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301087 thawing process was performed.Cryotubes containing frozen sperm were taken from the container and immersed in a water bath at a temperature of 30-32 °C for 5 minutes.Subsequently, DNA integrity analysis was conducted.

DNA laddering Analysis a. Sperm DNA extraction
The sperm sample was placed in a sterile microcentrifuge tube and centrifuged to obtain a 100 μl pellet.The resulting pellet was transferred into another sterile microcentrifuge tube to which 300 μl of Cell Lysis Solution had been added, the tube containing the mixture was stirred 6 times until homogeneous, then incubated for 10 minutes at room temperature.After incubation, it was centrifuged at a speed of 13,000-16,000 rpm for 20 seconds.The supernatant layer was then discarded, then the tube containing the pellet was vortexed for 10-15 seconds until the pellet was resuspended, then 100 μl of Core Lysis Solution was added to the tube and homogenized again using a vortex to produce a lysate.35 μl of Protein Precipitation Solution (RNase Solution) was added and homogenized using a vortex for 20 seconds.Next, it was centrifuged at a speed of 13,000-16,000 rpm for 3 minutes.The supernatant was transferred into a microcentrifuge tube containing 100 μl of isopropanol, then the solution was mixed and centrifuged at a speed of 13,000-16,000 rpm at room temperature for 1 minute.The supernatant was discarded and 70% ethanol was added to the DNA sample volume at room temperature, then centrifuged again for 1 minute.The ethanol solution was discarded and the pellet was dried in air for 10-15 minutes.DNA rehydration was carried out by adding 35 μl of DNA Rehydration Solution to the tube containing the pellet, then incubating for 1 hour at 65 ℃.Finally, DNA is stored at 2-8 ℃ [22].Next, DNA concentration was measured using a Nanodrop 2000c [23].
b. Gel electrophoresis 2 g of agarose was weighed, then 1 x 100 ml of TBE buffer solution was added.Heat the agarose solution and TBE buffer until the solution becomes clear and boils.Once the solution is clear and boiling, add 2 μl of DNA dye.Next, the agarose solution is poured into the prepared gel mold and left for 30 minutes until the agarose gel hardens.Next, 1.5% agarose gel was put into the electrophoresis chamber, 1x TBE buffer solution was added.10 μl of sample and 2 μl of loading dye were mixed on parafilm paper then injected into the gel well, including 5 μl of DNA marker.Next, close the electrophoresis and turn on the electricity with a voltage of 135 V for 30 minutes.Reading of electrophoresis results was carried out using a UV transilluminator.

Data Analysis
Data on sperm DNA integrity after cryopreservation were presented descriptively.

Result
The analysis of climbing perch fish sperm DNA integrity was conducted using gel electrophoresis with four different cryoprotectants, each at two concentration levels: DMSO (10% and 20%), Methanol (10% and 20%), Glycerol (5% and 20%), and Ethanol (15% and 5%).As a comparison, testing was also performed on the control group (without the addition of cryoprotectant) and fresh sperm.The electrophoresis results showed that DNA integrity was expressed in all treatment lines and fresh sperm.The electropherogram images showed that no DNA fragments were formed in all samples of climbing perch fish sperm, indicating , 03010 (2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301087 that there was no sperm damage in the tested samples, both in fresh sperm and sperm after thawing.

Discussion
DNA is one of the cell components susceptible to cryodamage, so it is essential to maintain DNA integrity in the sperm cryopreservation process [24].The analysis of climbing perch fish sperm DNA integrity using gel electrophoresis showed that the smear pattern in the fresh sperm, control treatment, DMSO (10% and 20% concentration), Methanol (10% and 20% concentration), Glycerol (5% and 20% concentration), and Ethanol (15% and 5% concentration) all exhibited the same results, indicating the absence of DNA fragmentation in the tested sperm samples with fresh sperm samples which were used as a reference in this study.The presence of cryoprotectants in the test samples is believed to have been able to maintain the integrity of sperm DNA during the freezing process.Bhattacharya [25] added that the use of cryoprotectants can slow down the cell freezing process, and sperm cells have a longer adaptation time to extreme temperature changes, preventing cell damage due to freezing.According to [26] cryopreservation can have side effects, such as genetic changes within cells that can damage cell integrity.Therefore, the addition of cryoprotectants plays a crucial role in preserving sperm DNA integrity, reducing the risk of genetic changes and cell damage during the cryopreservation process.

Conclusion
In conclusion, no damage to climbing perch fish sperm DNA was observed in all tested samples, both in fresh sperm and sperm after cryopreservation.