RESUMO
Siamese fishing fish (Betta splendens) or betta are usually subjected to a special method of transportation for global trade, where they are individually conveyed in plastic bags containing just enough water to cover their bodies. This study aimed to investigate the effects of transportation on their stress response by measuring hematological values, stress hormone levels, glucose levels, and stress-related gene expression. Betta fish (average body weight 1.91 ± 0.42 g; n = 30) were exposed to simulated transport in a water volume of 40 mL for 12, 24, and 48 h. Baseline levels (pre-transport) were measured prior to the experiment. The control group was transported using water without adding clove oil. Two treatment groups were transported using water with the addition of 1 and 3 mg/L concentrations of clove oil, respectively. The results revealed that transportation can be a factor that affects water quality. The pH and dissolved oxygen levels were significantly lower than baseline, while nitrite and total ammonia concentrations significantly increased. Correlating to the stress responses, significantly increasing total red blood cell counts, plasma cortisol levels, and up-regulating the expression of stress-related genes, including HSP70, GR, MR, and HIF-1α. The addition of 1 mg/L clove oil was found to reduce stress during the transport simulation, as evidenced by a reduction in these stress parameters. Conversely, increasing the concentration of clove oil to 3 mg/L significantly increased plasma cortisol after 12 h of simulated transport, and up-regulated GR, MR, and HIF-1α expression. This study revealed that the transport process can stimulates stress in betta fish but adding a concentration of 1 mg/L clove oil to the transport water could mitigate this stress response and promote animal welfare during their transportation.
RESUMO
17α, 20ß-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20ß-hydroxysteroid dehydrogenase (CR/20ß-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17ß-hydroxysteroid dehydrogenase, type 12-like (17ß-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17ß-HSD12 (presumably orthologous to salmon 17ß-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17α-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20ß-HSD or 17ß-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 17OHP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17ß-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17ß. CR/20ß-HSD showed strong 17ß-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 µg/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 20ß-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of 17OHP to DHP by ovarian follicles incubated with SPE and HCG peaked at 16 h, subsequent with increased follicular hsd17b12 mRNA levels, which were significantly higher than those in control incubations. However, the levels of CR/20bhsd mRNA remained low and did not differ among time points. The present study strongly suggests that 17ß-HSD12, and not CR/20ß-HSD, is the 20ß-HSD responsible for DHP production by ovarian follicles during oocyte maturation in Nile tilapia.
