Optimization of 17α-Methyl Testosterone Dose in Artemia Nauplii and Rotifer using Enrichment Technique

The recent fish production trend shows a significant contribution by aquaculture to the world fish production especially by freshwater aquaculture. As per the latest FAO report, freshwater aquaculture alone contributes around 88% of the world’s farmed fish (FAO, 2020). In order to improve the quality seed production and better fish production the sector has started using various chemical derivatives like antibiotics and hormones in the recent times (Emeka et al., 2014). In fish many of the phenotypic traits such as growth rate, colour pattern, etc. were sex based which lead to the development of mono-sex seeds, either all male or all female population. Further, the development of captive seed production systems - hatcheries - paved the way for egg or uniform yolk-sac larvae collection which opened the gate way for development of mono-sex seed production (male) using 17α-methyltestosterone through oral administration (Gale et al., 1996). In the past decades, many authors have reported that hormone is a naturally-occurring or synthetic androgens and estrogens which shown growth-promoting effect in many cultured fishes (Pelissero and Sumpter, 1992; James and Sampath, 2006). In the recent years, monosex seed production technology has become a common practice among the various animal production sectors (Bardhan et al., 2021). However, the hormone residue is creating various negative impacts to the natural ecosystem which making the hormone based sex reversal as a much challenging task in the recent times (Azizi-lalabadi and Pirsaheb. 2021). Therefore, delivering of potent hormones to the fish larvae through suitable vectors -without leaching to the environment- may reduce the negative impacts of the hormone-based mono-sex seed production.

Alternative to commercial diet, live feed - a natural diet rich of nutrients - has gained an importance in hatcheries and started playing a pivotal role in larviculture. Among the various live feed, rotifer and Artemia are widely used due to their availability, low cost, ease of culture and nutritional biochemical composition (Kolkovski et al., 1997; Sorgeloos and Lavens,1996). Additionally, these live feed act as a very good carrier of supplying essential nutrients (PUFA) and hormones (androgens or estrogens) to the target animal through enrichment process.

Therefore, the best alternative option for supplementing the hormone for monosex seed production is live feed enrichment process. In commercial feed, a particular dose is selected as optimal dose for all male production, however, it is very difficult to achieve that particular dose in live feed. Therefore, it is essential to standardize the dose of hormone in live feed through enrichment process, before recommending them as potential carrier for hormone-based mono-sex seed production in future. Keeping this in mind, the study was aimed to optimize the 17a-methyl testosterone hormone dose in Artemia nauplii and rotifer using enrichment process.

Experimental site and design
 
The present experiment was conducted at the Krishnagiri Barur-Center for sustainable Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Tamil Nadu and India and research was carried out in summer 2021. The study consisted of five treatment doses of 17α methyl testosterone namely; control (without hormone), T1 (20 mg L^-l), T2 (40 mg L^-1), T3 (60 mg L^-1) and T4 (80 mg L^-1) and each were triplicated. The doses were tested in Artemia nauplii and rotifer by following completely randomized design.
 
Decapsulation of Artemia cyst
 
The cyst of Artemia strain (Artemia salina) was procured from Tuty Enterprises, Tamil Nadu, India. The procured cysts (Dynasty TM, Salt Lake City, UT, USA) were hatched out by following standard method (Sorgeloos et al., 1986) on daily basis. First, 3 g cyst were decapsulated using sodium hypochlorite solution (at 15 mL/g cyst) for 15 min, filtered through nitex screen (50 µm) and washed with fresh water. To ensure complete removal of chlorine, the cysts were treated with 0.1% sodium thiosulphate. The washed cysts were then transferred to a conical Artemia hatching jar containing 4 L of seawater (30 ppt), with vigorous aeration and maintained at 28°C for 24 h under 1000 lux light for hatching. After 24 h, the hatched-out larvae were harvested using 50-micron nitex screen and used for enrichment study.
 
Rotifer culture
 
Pure culture of rotifer (Brachionu s calyciflorus), procured from live feed culture unit, Bharathidasan University, Tamil Nadu, India, was maintained at Krishnagiri-Barur Centre for Sustainable Aquaculture. First it was cultured in a 1000 ml flask and was periodically up-scaled to 500 litre Fibre-reinforced plastic (FRP) tanks for mass culture. The culture was maintained by feeding them with yeast @ 0.15 g/l and Chlorella vulgaris (1.5-2.5×106 cells/ml) during morning (08.00 AM) and evening (17.00 PM) times, respectively (Akter et al., 2013). Once, the culture density reached 250-450 individuals/ml, the rotifer were collected and used for enrichment process.
 
Enrichment of Artemia nauplii and rotifer

The enrichment process was carried out in plastic buckets, each of 3 L capacity and provided with aeration and each treatment were triplicated. The enrichment process of androgenic hormone, 17 α-methyl testosterones (MT), (Sigma Chemical Company, St Louis, MO, USA) in the Artemia and rotifer was carried out by following the standard method (Sorgeloos et al., 1986). The freshly hatched out Artemia nauplii and 6-8 days old rotifer were used for enrichment process. The hormone (17 α- MT) and ethanol mixture was prepared by following the method of (Stewart et al., 2001) with slight modifications. Firstly, a stock solution of hormone-ethanol mixture (20000 ppm) was prepared. This stock solution of 20000 ppm was prepared by dissolving 1000 mg of 17 α-MT in 50 ml of 70% ethanol. 1 ml of this stock solution contains 20 mg of 17 a-MT which was further used for treatment doses preparation. A pre-determined volumes of stock solution such as 1 ml, 2 ml, 3 ml and 4 ml were added to individual replicate buckets to obtain the treatment doses of T1 (20mg L^-l), T2 (40 mg L^-1), T3 (60 mg L^-1) and T4 (80 mg L^-1) , respectively. In control group, 2 ml of 70% ethanol without hormone was added. After the preparation of enrichment medium, rotifer (800 individuals/ml) and Artemia nauplii (100 individuals/ml) were introduced into the respective treatment medium and kept for different durations of enrichment.
 
Sample collection, preparation and hormone analysis
 
After enrichment, samples were collected at different time intervals. The hormone-enriched Artemia nauplii and rotifer were harvested processed by following the standard method (Sandifer and Smith 1985), Samples of 10000 Artemia nauplii and rotifer taken from each replicate on 6, 12, 18, 24, 30, 36 and 42 hrs (for rotifer up to 24 hrs only collected). Testosterone levels were measured using enzyme-linked immunosorbent assay kit (ELISA) method. Testosterone was extracted using CALBIOTECH company kits following the manufacturer’s instruction manual and ELISA reader (Model No is ER-180) was used  (href="#jestere._2014">Jesteret_al2014; Liu et al., 2013; Jiang et al., 2011).
 
Statistical analysis
 
The collected data were statistically analysed using a statistical program SPSS version 28.0. Significant difference between the mean values were tested using repeated one-way analysis of variance by keeping dose and time as independent variables (each dose vs different time intervals; each time interval vs different doses). Tukey’s test was used to rank the mean values and the statistical significance for the test was set as p>0.05.

In both live feed, the different durations enrichment found significantly higher 17 α-MT content at different doses. Similarly, Daphnia magna enriched with canola oil for different durations displayed significant difference in the lipid content between enriched and non-enriched daphnia (Fereidouni et al., 2013). In Artemia nauplii, 17 α -MT was in the range of 0.35-0.50 mg L^-1 before the commencement of experiment (0^th h). The study did not find any significant variation in 17 α-MT hormone of Artemia kept in control group at different time intervals and it was in the range of 0.35-0.49 mg L^-1. Similarly, no significant difference was found at 0^th hour in the Artemia kept in different dose groups, however, as the time increased, Artemia started accumulating 17 α-MT in its tissues at different rates (Table 1). In general, a normal Artemia has lower quantity of steroids, for example 17 α-estrodiol, than the enriched Artemia (Martin-Robichaud  et al., 1994) and it may be the reason for lower levels of 17 α-MT detection in the control group of the present study.



The increasing trend of 17 α-MT augmentation in Artemia was noticed up to 24 h. Significantly higher amount of 17 α-MT (15.33±0.09 mg L^-1) was observed in 80 mg L^-1 group at 24 hours (Table 1). Similarly, Martin-Robichaud  et al., (1994) reported that Artemia nauplii enriched in different concentrations of 17 α-estrodiol was displayed significantly higher accumulation of 17 α-estrodiol after 24 hours of enrichment. The study examined the assimilation of 17 α-MT at different time points, up to 42 hours, to know highest point of accumulation and starting point of withdrawal period. Interestingly, the study found maximum accumulation at 24 hours and, thereafter, a reduction in 17 α-MT assimilation has been noticed which may be due to higher metabolic activities and excretion activities (Navarro et al., 1991; Dhert et al., 2001). Similarly, Artemia enriched with estradiol (E2) showed increasing trend and stable up to 24 hours (Contreras-sanchez  et al., 2004). In an another study, when Artemia enriched with ascorbic acid (AA) at different durations found maximum absorption at 18 hrs and the AA content was started declining after 18 hrs of enrichment (Noshirvani et al., 2006).

In rotifer, different doses and time intervals investigation found a significant difference in hormone accumulation. 17 α-MT levels in rotifer ranged from 0.37 to 0.47 mg L^-1 at 0^th hr. The study did not find any significant differences in the 17 α-MT hormones of rotifer kept in the control group at various time intervals and it was in the range of 0.36-0.49 mg L^-1. Similarly, there was no significant difference in the 17 α-MT level among the rotifers kept in different treatment doses at 0th hour.

At 6 hours, the level of 17-MT was significantly higher in all treatment groups. Further, the dose vs time analysis revealed that rotifer accumulated statistically higher quantum of 17 MT at 6 hrs than the 12 hrs (Table 2). After 6 hrs, a decreasing trend was noticed in 17 MT accumulations and significantly higher value of 17 α-MT (17.42±0.06 mg L^-1) was recorded at 6 hrs in 60 mg L^-1. Previous studies on rotifer reported maximum accumulation of n-3 HUFA within 12 hrs of enrichment (Imada et al., 1979; Watanabe et al., 1983) and did not find any significant difference in fatty acid content when they enriched for longer duration, up to 24 hrs (Kotani et al., 2010). Similarly, rotifer kept for 8 hrs in higher concentration of enrichment medium did not produce significantly higher quantity of fatty acid (Kotani et al., 2010). At 6 hrs, there is no statistical difference in assimilation of 17 α-MT in the tissues of rotifers kept in 20 mg L^-1, 40 mg L^-1 and 80 mg L^-1 treatment groups 9 (Table 2). Generally, in rotifer, it has been suggested to increase the duration of enrichment rather than quantity of enrichment medium to obtain more efficient enrichment (Rodriguez et al., 1996; Yahyavi and Farzane, 2009). Interestingly, the present study found significantly higher assimilation of 17 α-MT in shorter enrichment period (in 6 hrs) which in turn may help to reduce cost associated with live feed production (Southgate and Lou, 1995).

The harmful residual effect of hormones, released from the hormone incorporated diets, to the natural environment is alarming. As an alternate, supplementation of hormone via live feed, using enrichment techniques, could be a viable option which improves the larval ingestion rate, survival rate, growth rate and reduces hormone residual effects. The study found higher assimilation of 17 α-MT by Artemia (at 24 hours in 80 mg L^-1) and rotifer (at 6 hours in 60 mg L^-1) which could be further explored for mono-sex seed production in a more sustainable way.

The funding support by Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, and Tamil Nadu is gratefully acknowledged. We would like to express our sincere thanks to The Vice-Chancellor, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, for his support and encouragement.