Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 7 (july 2022) : 843-847

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

Kartini Eriani1,*, Mustaqim Mustaqim1,2, Iwan Hasri1,3, R. Amalia1, Al Azhar4, Firman M. Nur5, Zainal A. Muchlisin6
1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia.
2Study Program of Animal Husbandry, Faculty of Agricultural and Animal Sciences, Universitas Islam Kebangsaan, Bireuen 24251, Indonesia.
3Agency for Fisheries Affair of Aceh Tengah District, Takengon, Indonesia.
4Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia.
5Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia.
6Department of Aquaculture, Faculty of Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia.
Cite article:- Eriani Kartini, Mustaqim Mustaqim, Hasri Iwan, Amalia R., Azhar Al, Nur M. Firman, Muchlisin A. Zainal (2022). Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation . Indian Journal of Animal Research. 56(7): 843-847. doi: 10.18805/IJAR.B-1379.
Background: Sperm cells are susceptible to oxidative stress during cryopreservation. Therefore, an antioxidant is necessary to protect them from damages. Fetal bovine serum (FBS) is one of potent antioxidants for fish sperm cryopreservation. Hence, the aims of this study are to examine the effect of FBS on sperm quality after a short period and to determine its optimum concentration on depik (Rasbora tawarensis).

Methods: Depik fish were obtained from the Fish hatchery of Lukup Badak, Aceh Tengah District, Indonesia. Sperms collected from the fish were diluted in Ringer extenders containing FBS concentration of 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) and 60% (P6), filled into 2 ml cryotubes and equilibrated prior immersed into liquid nitrogen for 15 days. The parameters observed were sperm motility, consistency, pH, fertilization and hatching rates and DNA fragmentation post-thawing. 

Result: The ANOVA test indicates that the application of FBS in Ringer had a significant effect on sperm motility, fertilization and hatching rates (P<0.05). The highest motility (58.33%) was recorded at FBS 60% and significantly different from those at other concentrations. The laddering analysis showed that applying FBS protected the integrity of depik sperms DNA. It is concluded that the optimum concentration of FBS on depik sperm led to a short-term cryopreservation of 60%.
Depik (Rasbora tawarensis) is one of the endemic and predominant fish with high economic value in Lake Laut Tawar, Central Aceh District, Indonesia (Muchlisin and Azizah, 2009). Presently, its population has been decreasing due to overexploitation, unfriendly fishing practices and ecological perturbation (Muchlisin, 2011; Muchlisin et al., 2011). The International Union for Conservation of Nature (IUCN) categorizes depik as a threatened fish species (CSBG, 2003; Lumbantobing, 2019). Therefore, it is crucial to save its population by prohibiting uncontrolled fishing while encouraging the Government of Aceh Tengah to develop aquaculture programs. The main obstacles faced by the program are the difficulty in locating high-quality broodstock throughout the year due to unsynchronous gonad maturation between male and female species during the rainy season in April, September and December (Muchlisin et al., 2010; Muchlisin et al., 2011b). Therefore, introducing aquaculture reproductive technology through cryopreservation program could provide high-quality germ cells as an alternative solution.
According to Eriani et al., (2008), sperm cryopreservation is a significant effort used to save the population of vulnerable and endangered animals by providing sustainable male germ cells. The success of male germ cells, however, strongly depends on the quality of sperm (Eriani et al., 2008), therefore, the best time to gather it is during spawning season (Moczarski and Koldras, 1982; Fauvel et al., 1999; 2000; Momin and Memis, 2018). Seminal plasma compositions is very important for high grade spermatozoa quality (Abishag et al., 2020) and the information about semen characteristics is helpful in selecting good quality semen for artificial fertilization thus could contribute to fishery development (Charak et al., 2020). Generally, sperm quality decreases after cryopreservation owing to temperature shock and extenders toxicity (Fauvel et al., 1999; Kasimanickam et al., 2007; Muchlisin and Azizah, 2009b; Bansal and Bilaspri, 2010). Cryoprotectant and antioxidant are the choices to overcome these potential problems. Since antioxidant can maintain the motility, viability, acrosomal integrity and plasma membrane of frozen-thawed sperms (Eriani et al., 2018), the better results are expected to be achieved by using it for the preservation of the depik sperm. Fetal bovine serum (FBS) is one of the potential antioxidants for fish sperm,this serum is extracted from a bovine fetuses blood (Maurer, 1986; Brunner et al., 2010).
The successful application of FBS in semen cryopreservation has been reported in several animals such as cattle (Reyes-Moreno, 2000), rabbit (Sarýözkan et al., 2013) and alpacas (Bravo and Valdivia, 2018). It was also applied the sperm of Sakhalin taimen fish (Kusuda et al., 2005), European eel (Garzon et al., 2008) and grouper (Yusoff et al., 2018). However, the limitation of these previous studies is that FBS was used at higher concentrations reaching 85-90%. FBS is expensive, rarely available in the local market, costly at higher concentration and not practical. In this study, the effect of FBS at low concentration on depik sperms is examined to determine its optimum value in the Ringer extender of depik spermatozoa.
Location and time
The research was conducted in June-August 2019 at Fish Hatchery of Lukup Badak, Aceh Tengah District, Indonesia.
Experimental design
The completely randomized design (CRD) consisting of six levels of treatments (FBS concentration of 10%, 20%, 30%, 40%, 50% and 60%) and three replications was used in this study.
Brood fish and sperm collection
Sixty male broodfish length ranged from 75.98-113.31 mm and body weight ranged from 2.98-7.32 g) were randomly sampled using a dedeuseun trap. The fish were kept in the plastic bag filled with oxygen then transported to the Lukup Badak Hatchery at Aceh Tengah District. The broodfish was acclimatized in a pond for 7 days before sperm collection. A total of 20 mature males fish were taken randomly from the pond, then the genital pore was wiped by the tissue to avoid contamination, after which its abdomen was gently pressured. The sperms were collected by a syringe and pooled in the beaker glass kept in an icebox (4°C), after which it was assessed for initial quality using macroscopic and microscopic evaluations. Only sperms with motility higher than 60% were used for cryopreservation.
Extender preparation
The ringer solution was mixed with six different concentrations of FBS (10%, 20%, 30%, 40%, 50% and 60%) in a total reaction volume of 10 ml. Depik sperms were diluted in the solutions at a ratio of 1:20 (Muchlisin et al., 2009a).

Cryopreservation process
Diluted sperms were filled in separate cryotubes (vol. 2 ml) and kept at 4°C for 10 minutes for equilibration. It was evaporated at a distance of 5 cm above the surface of liquid nitrogen (-60°C) for 5 minutes and immersed into the liquid nitrogen (-196°C) for 15 days.
Semen evaluation
Semen quality was measured twice. The first was on the fresh sperm including color, pH, consistency, mass movement, motility and concentration. The second was on the post-thawed semen including motility, fertility, hatching rate and DNA integrity.
Evaluation of sperm motility, fertility and hatching rates
Sperm motility was microscopically evaluated in accordance to (Muchlisin and Azizah, 2009b). Fresh or cryopreserved semen was diluted in equal volume of tap water and observed using a stereo microscope (AmScope 40X-2000-3WLED Trinocular) at 400x magnifications. The motility rate was calculated by randomly assessing at least 50 spermatozoa. The fertility and hatching rates were calculated by dividing the total numbers of fertilized and hatched eggs with total numbers of incubated sperm, respectively (Muchlisin et al., 2015; Mutmainnah et al., 2018).
DNA fragmentation analysis
Quality of spermatozoa DNA was determined by the laddering method (Zilli et al., 2003). Genomic DNA was extracted from both fresh and frozen semen using the Genomic DNA Purification Kit (Promega) and subjected to electrophoresis in 1.5% agarose gel at 135 V for 30 minutes. The DNA fragmentation and laddering were characterized by using a Uvi-Doc Machine (Yusoff et al., 2018).
Data analysis
The data of motility, fertility and hatching rates were analyzed by one-way ANOVA and followed Duncan’s multiple range tests using SPPS version 20 forsa Windows. The data of sperm viability, pH and DNA fragmentation were analyzed descriptively.
Evaluation of fresh sperm quality indicates that depik sperm was condensed and had a white-milky color. The pH ranged from 7.4-8.0, spermatozoa density was 23.58 × 109 cells ml-1, the initial motility and viability were 71.83% and 75.00%, respectively. Sperm abnormality was 11.00% (Table 1).

Table 1: Quality of fresh semen of depik fish (Rasbora tawarensis).

ANOVA test showed that FBS gave a significant effect on the sperm motility and fertility as well as egg hatching rate of the depik (P<0.05). The highest motility was found in 60% FBS (58.33% motility) and significantly different from those in other concentrations. The highest fertility and hatching rate were also recorded at 60% FBS, but the values were not significantly different with those at 50% FBS (69% vs 66.33% and 47% vs 45.67%, respectively) (Table 2).

Table 2: Average post-thawed motility and fertility of frozen depik semen and the resulted egg hatching rate.

The DNA electrophoresis results showed the presence of smears in both fresh and post-thawed sperm indicates the occurrence of DNA fragmentation. However, the application of 40% FBS showed thick and long smears, thereby, indicating a high degree of DNA fragmentation whereas the bright and short smears detected at 50% and 60% FBS applications showed low degree DNA fragmentation (Fig 1).

Fig 1: The DNA electrophoresis results of the depik (R. tawarensis) sperm.

The results showed that the addition of FBS had a positive effect on the sperms motility and fertility and egg hatching rate of depik fish. This is presumably that FBS can protect sperm during freezing due to its protein, hemoglobin, glucose, insulin, cortisol, parathyroid hormone and prostaglandin E compositions (Hayman et al., 1985; Chen et al., 1992; Garzon et al., 2008).
According to Reyes-Morino et al., (2000), protein and glucose play an essential role in protecting sperms from excessive damage during cooling, freezing and thawing. The protecting effect of glucose presence in extender for better semen quality after freezing and thawing has been proved by better post-thawing and post-freezing motilty of Turkey sperms diluted in glucose 5% containing dimethyl sulphoxide over those diluted in other extenders (tris-glucose, lactated Ringer and lactated Ringer’s glucose) (Kuzlu and Taskin, 2016).
In general, serum has multifunctional effects on sperms due to its high macromolecules and antioxidants contents (Alcay et al., 2019). It can act as an extracellular cryoprotectant for frozen spermatozoa by protecting the plasma membrane from crystallization, recrystallization or ice melting during different phases of freezing and thawing processes (Watson,1995). Besides, serum can induce capacitation (Xia and Ren, 2009) and acrosomal reactions (Hossain et al., 2007). This similar positive effect was reported by Kusuda et al., (2005) in Sakhalin taimen fish and by Yusoff et al., (2018) in groupers. However, these authors used a higher concentration of FBS (90% and 85%, respectively). Meanwhile, 50-60% was found optimal for short preservation of depik sperms without suppressing the results.
The study showed that there was a correlation between sperms motility and sperm fertility and egg hatching rate of depik as fertility and hatching rates increase proportionally to sperm motility rate. Similar results were reported by Ohta et al., (1995) in post-thawed sperm of masou salmon and turbot fish (Dreanno et al., 1998), indicating motility is an important indicator of sperm quality. Contrarily, Mutmainnah et al., (2018) reported no correlation between motility and fertility in cryopreserved sperms of seurukan fish when using glutathione as an antioxidant. The unclear relationship was also found in the sperms of brook and rainbow trout fish (Lahnsteiner et al., 2011), African catfish (Muchlisin et al., 2015) and muskellunge (Ciereszko et al., 1999). According to Ohta et al., (1995), several factors might reduce fertility including low post-thawing motility and egg stimulation. The ability of inactive sperms to fertilize egg is assumably related to hormones contained in it (Gilkey, 1981). The teleosts egg produces pheromones for example to attract sperm (Stehr and Hawkes, 1983). Gimnogamon hormone produced by egg can activate and draw spermatozoa (Yanagimachi et al., 2017). In addition, glycoprotein contained in the herring egg can activate spermatozoa (Griffin et al., 1996; Cherr et al., 2008).
The analysis of sperm DNA fragmentation after cryopreservation revealed the presence of smears in both fresh (column 2 in Fig 1) and preserved sperm (column 3, 4 and 5 in Fig 1 for 40%, 50% and 60% FBS, respectively). These results were in agreement with Zilli et al., (2003) who found DNA damage in both fresh and frozen sperms of sea bass. The long smear of DNA bands after electophoresis show fragmentation whereas clear DNA bands without smears indicate purity or integrity (Karp et al., 2012). 
The results of this study showed the addition of concentrated FBS (line 3 and 4) could maintain DNA integrity of frozen depik sperms which is similar to that of fresh sperm (line 1). This might be responsible for the high fertilization and hatching rates of the sperm. The similar findings were reported by Yusoff et al., (2018) when analyzing DNA of groupers sperm cryopreserved in a combination of polyethylene glycol 15% and FBS 85% that a slight DNA smear did not affect fertilization and hatching rates.
FBS gave a positive effect on the sperm quality of depik (Rasbora tawarensis) as shown by increased sperm motility, fertility and hatching rates as FBS concentration intensifies, with the optimum concentration at 60%.
This study was supported by Kemenristek Dikti of the Republic of Indonesia through the National Strategic Research Scheme 2019, Contract No. 91/UN11.2/PP/SP3/2019 for the Muchlizin ZA .

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