Dose Dependent Treatment with Boric Acid Induces More Changes in the Sperm Cells of Endangered Anatolian Trout Salmo Rizeensis

The aim of this study was to test the usefulness of boric acid for endangered Anatolian trout Salmo rizeensis sperm. Activation media was supplemented with boric acid (0.5, 1, 2, 3, 4 and 5 mM). Sperm motility and duration were determined in sperm samples. In addition, fertility and hatching rate were examined. Our data indicated that addition of boric acid (3 mM) to activation media was increased the percentage and duration of motile sperm, fertility and hatching rate in endangered Anatolian trout (S. rizeensis). On the other hand, the motility rate decreased with increasing the concentration of boric acid (p<0.05). In particular, a remarkable increase was observed after concentration 3 mM. Consequently, sperm quality was affected by quantitative changes different concentrations of boric acid and the best results were obtained at concentration 3 mM. Research Article


Introduction
Salmo trutta are the most important Salmonid fi sh species owing to its aquaculture potential, economic value and wide consumer demand [1]. Salmo trutta forms inhabit in the upper streams of rivers and occur in North Africa, Europe, West Asia and Anatolia [2,3]. In addition, it is an important potential species for recreational fi shery. Recently, S. t. macrostigma ecotype has been described by Turan et al. [4], as S. rizeensis [5]. In particular, populations of the species are affected by natural hybridization, the local devastation in water sources through habitat fragmentation and modifi cation, water eutrophication and contaminations, environmental instability and global warming [6][7][8][9][10]. Sperm motility is the essential functional parameter for successful fertilization in fi sh [11,12]. Sperm cells in most fi sh species immotile in seminal fl uid and require to release into the water in order to trigger motility and become metabolically active [12,13]. Therefore, characteristics of activation media are crucial in terms of initiation and progression of sperm motility [12].
Trace elements have a crucial role for the male reproductive process. Boric acid (H 3 BO 3 ) is a bioactive benefi cial element [14], and widely used in glass, ceramic, detergent, plastic, agricultural, textile, metallurgy, nuclear and medicine industries owing to its excellent characteristics and it is produced from boron ore and salt lake brine [15][16][17]. The greatest majority of boric acid is produced by Eti Maden Works (Turkey) in the world [18]. Increasing production does not meet the demand due to using as extensive [19,20]. Several studies about the nutritional benefi ts [14,19,21,22], metabolic functions [23][24][25][26], U shaped dose responses on growth of embryonic fi sh and frogs [27,28], and therapeutic applications [29][30][31], toxic effect on male reproduction system of different species (e.g. rat, rodent, dog, human) [32][33][34][35], of boron and its compounds have been published in the latest available literature. As far as the authors of this work are aware, no attempt has been made to use of boric acid in sperm activation medium of fi sh species. In this context, the aim of this study was to examine effect of activation media supplemented with boric acid (0.5 mM; 1 mM; 2 mM; 3 mM; 4 mM; 5 mM) on sperm quality and fertility of endangered trout S. rizeensis.

Materials and Methods
Six mature endangered Anatolian trout males (1388.00±0.55 g, 44.52±2.62 cm as mean±SD) were captured from Uzungöl Stream, Trabzon, Turkey for sperm collection. F spermatocrit ish were anesthetized in 50 mg/L Benzoacaine, sperm samples were collected through abdominal massage and special care was taken to prevent contamination (e.g. blood, feces or urine).

Results
Sperm parameters (mean ± SD) are presented in table 1.
Effect of boric acid on the percentage and duration of motile sperm for S. rizeensis is shown in fi gure 1. The results of the present study indicated that differences in the percentage and duration of motile sperm of S. rizeensis sperm were signifi cant among the treatments (p<0.05). Highest motility rate (80.00%) and duration of motility (60 s) were at concentration 3 mM.
After concentration 3 mM, a remarkable decrease was observed in motility rate and duration. Effect of supplementation of boric acid on fertility and hatching rate for S. rizeensis is presented in fi gure 2. The trials in this study indicated that differences in the fertility and hatching rate were signifi cant among the treatments (p<0.05). Highest fertility (92.17%) and hatching rate (82.09%) were at concentration 3 mM. In particular, a signifi cant decrease was observed in fertility and hatching rate after concentration 3 mM.
In this study, we demonstrated the usefulness of boric acid in different activation mediums for S. rizeensis sperm. Using 3 mM boric acid in activation media resulted in high sperm motility rate and duration, fertility and hatching rate of S.
rizeensis. This may be due to the fact that boric acid is involved in a number of metabolic processes and interact with critical biological substances, including pyridoxine, polysaccharides, dehydroascorbic acid, ribofl avin, and the pyridine nucleotides [38][39][40]23]. However, motility rate and duration decreased with increasing concentration of boric acid. In particular, a    In conclusion, based on these results, boric acid was used effi ciently for activation media in S. rizeensis sperm. Our study provides new insights related to use of boric acid on fi sh sperm quality. The knowledge of effects of boric acid and its mechanism of action might be helpful for both research and commercial use. Further studies would be needed to evaluation the precise mechanisms of boric acid response.

Funding
This work was funded by Karadeniz Technical University.
Authors like to express deep thanks to continuous help of Aziz KUTLUYER and Devrim ALTUNTAŞ.