The crosstalk between photoperiod and early mild stress on juvenile oscar (Astronotus ocellatus) after acute stress.

Early mild stress (EMS) is like preparedness and might help fish deal with stress appropriately. This study investigated how EMS and photoperiod changes can impact growth, haematology, blood biochemistry, immunological response, antioxidant system, liver enzymes, and stress response of oscar (Astronotus ocellatus; 7.29 ± 0.96 g) before and after acute confinement stress (AC stress). Ten experimental treatments included five different photoperiods 8L16D (08:16 light to dark), 12L12D (12:12 light to dark), 16L8D (16:08 light to dark), 20L4D (20:04 light to dark), and 24L0D (24:00 light to dark), and these five photoperiod schedules were conducted in an EMS condition. After 9 weeks, no significant differences were found in growth parameters, survival rate, and body composition. At the end of the experiment and after AC stress, fish farmed in 24 light hours had the lowest haematocrit, white blood cells, total protein, blood performance, lysozyme, immunoglobulin M, complement C3, superoxide dismutase, and catalase. Fish that experienced EMS had significantly higher survival rates than those farmed in normal conditions (80.67% vs 61.33%). In conclusion, considering all measured parameters, 8-h light can be suggested as an optimum photoperiod for this fish species. Under 24L0D (no EMS) conditions, there were many negative effects apparent. In addition, a positive effect of EMS was evident in terms of survival after AC stress. AC stress decreased some health parameters under 24-h light treatment, while these results were not observed in EMS-exposed fish. Therefore, the EMS schedule can be a useful tool in preventing the negative effects of stress.

Early Mild Stress along with Lipid Improves the Stress Responsiveness of Oscar (Astronotus ocellatus).

Early-life exposure to mild stressors can assist animals in coping with more stressful events in later life. This study was aimed at investigating how early stress and dietary lipid contents affect growth, hematology, blood biochemistry, immunological responses, antioxidant system, liver enzymes, and stress responses of oscar (Astronotus ocellatus) (6.8 ± 0.7 g). Six experimental treatments were HL0Stress (high-lipid diet and without stress), HL2Stresses (high-lipid diet and two-week stress), HL4Stresses (high-lipid diet and four-week stress), LL0Stress (low-lipid diet and without stress), LL2Stresses (low-lipid diet and two-week stress), and LL4Stresses (low-lipid diet and four-week stress). During the ten-week trial, fish fed high-lipid diets grew faster (46.41 ± 4.67 vs. 38.81 ± 2.81) and had a lower feed conversion ratio (2.21 vs. 2.60) than those fed low-lipid diets (P < 0.05). After acute confinement stress (AC stress), high-lipid groups had higher survival than low-lipid treatments (81.25% vs 72.92%) (P < 0.05). Fish subjected to two-time stress (2Stresses) had a higher survival rate after AC stress (90.63% vs. 62.50%), hematocrit, white blood cell, blood performance, total protein, high-density lipoproteins, cholesterol, triglyceride, alternative complement activity (ACH50), superoxide dismutase, glutathione peroxidase, and alkaline phosphatase levels than those not stressed (P < 0.05). Contrariwise, glucose, cortisol, alanine aminotransferase, and aspartate aminotransferase levels were significantly lower in the 2Stresses groups compared with 0Stress fish (P < 0.05). Collectively, these findings suggest stressing the signs of adaptation in 2Stresses fish. However, a higher number of early stress events (4Stresses) appears to exceed the threshold of manageable stress levels for this species. In conclusion, the HL2Stresses group outperformed the other treatments in terms of growth, health status, and stress responsiveness. Although fish welfare must be considered, these results suggest that early mild stress can result in a greater survival rate after fish are exposed to later acute stress.

Changes in lipid biology during ovarian development in farmed beluga sturgeon, Huso huso L.

The present study was conducted to understand key biochemical, physiological, and molecular changes associated with ovarian growth and with lipid transfer and/or accumulation into the ovary during oogenesis in captive beluga sturgeon. Plasma levels of triacylglycerides, cholesterol, phospholipid, and sex steroid hormones were determined and all were found to increase notably throughout development from the perinucleolar to the tertiary yolk stage. Using fast protein liquid chromatography, we recognized three major lipoprotein peaks in chromatograms from all samples. These peaks were characterized as containing very low-density lipoprotein (Vldl), low-density lipoprotein/high-density lipoprotein (Ldl/Hdl), and plasma proteins. While Ldl/Hdl represented the most abundant lipoprotein fraction, the relative abundance of different lipoprotein classes did not change with the stage of oogenesis. Eluted lipoproteins were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and sequenced. The peptide sequence spectra for 66-kDa, 205-kDa, 29-kDa, and 70-kDa bands matched with albumin, vitellogenin (Vtg) AB2b, immunoglobulin light-chain precursor, and immunoglobulin heavy-chain, respectively. The large amount of albumin in the plasma protein peak and the confined presence of Vtg AB2b to within Ldl/Hdl reinforce the lipoprotein classification. Lastly, transcript levels of genes encoding ovarian lipoprotein lipase (lpl), apolipoprotein E (apoe), very low-density lipoprotein receptors (vldlr), and low-density lipoprotein receptor-related protein 8-like (lrp8) were estimated using quantitative RT-PCR. The high mRNA levels of lpl, apoe, and lipoprotein receptors vldlr and lrp8 in previtellogenic females suggest that sturgeon oocytes need to be prepared to accept and traffic Vtg and lipids internally, before the start of vitellogenesis.

The effects of 11-ketotestosterone implants on transcript levels of gonadotropin receptors, and foxl2 and dmrt1 genes in the Previtellogenic ovary of cultured beluga (Huso huso).

The in vivo effect of 11-ketotestosterone (11KT) on transcript levels of the gonadotropin receptors (fshr and lhr) and sex differentiation-related genes (dmrt1 and foxl2) was examined in the ovaries of immature female beluga. For this purpose, six fish were treated with implants containing 2.5 mg 11KT and a placebo group of six females of the same age and gametogenic stage were given a blank implant. The implants were intraperitoneally inserted into 4-year-old females at the previtellogenic stage (mean body weight 5580 ± 165 g) and maintained under culture conditions for 8 weeks. Ovary samples for gene expression analysis of lhr, fshr, dmrt1 and foxl2 were collected by biopsy at 3 and 8 weeks post implantation. Diameters of oocytes increased in response to 11KT treatment, both at 3 and at 8 weeks post implantation, but no obvious changes were evident in cytology. Three weeks of 11KT treatment did not affect target gene expression, but a tendency for a time-dependent decrease of lhr and dmrt1 mRNA levels was observed in both treatment and placebo groups. By 8 weeks of treatment, however, 11KT implants provoked the upregulation of fshr and foxl2 transcript levels. Furthermore, lhr and dmrt1 transcript abundances recovered by 8 weeks of exposure in both blank- and 11KT-implanted beluga. These results suggest that 11KT, either directly or indirectly, may affect gametogenesis and regulate some key components of the reproductive axis in female beluga.

Variations of physiological and innate immunological responses in goldfish (Carassius auratus) subjected to recurrent acute stress.

This study was undertaken to investigate the influence of repeated acute stress on the physiological status and non-specific immune response of goldfish, Carassius auratus. The acute stress was a succession of a 3 min-chasing period followed by a 2 min-air exposure. The goldfish in triplicate tanks were subjected 3 times daily to this stress for one (S3) or three (S9) days. A separate group of unstressed fish was used as control for each sampling time. Blood samples were collected 12, 48 and 120 h after the last stress procedure. Variations of globulin levels, plasma anti-protease and bactericidal activities were not significant in the present study. The haematological parameters and plasma total protein and albumin strongly declined in S9 fish 12 h post-stress compared to control fish. However, plasma cortisol, glucose and lactate levels in both S3 and S9 transiently increased compared to the control fish. Similarly, plasma peroxidase activity transiently increased in both stressed groups 12 h after stress. An increase in plasma lysozyme and complement activities suggested a hormesis-like effect with one-day acute stress improving the immunological response of goldfish while an extension of the stress period to three days impaired physiology and immunity for up to 5 days. This study revealed that recurrent acute stress could immunosuppress goldfish as usually expected of chronic stress.

The Recovery Time between Early Mild Stress and Final Acute Stress Affects Survival Rate, Immunity, Health, and Physiology of Oscar (Astronotus ocellatus).

This study investigated how the time interval between the last EMS (netting) and the acute confinement stress (AC stress) at the end of the experiment can influence growth, haematology, blood biochemistry, immunological response, antioxidant system, liver enzymes, and stress response of oscar (Astronotus ocellatus; 5.7 ± 0.8 g). Nine experimental treatments were tested, as follows: Control, Stress28 (EMS in weeks two and eight), Stress27 (EMS in weeks two and seven), Stress26 (EMS in weeks two and six), Stress25 (EMS in weeks two and five), Stress24 (EMS in week two and four), Stress23 (EMS in week two and three), Stress78 (EMS in week seven and eight), and Stress67 (EMS in week six and seven). After the nine-week experimental period, while it was not significant, fish exposed to Stress78 (26.78 g) and Stress67 (30.05 g) had the lowest growth rates. After AC stress, fish exposed to Stress78 (63.33%) and Control (60.00%) showed the lowest survival rate. The Stress78 fish displayed low resilience, illustrated by values of blood performance, LDL, total protein, lysozyme, ACH50, immunoglobin, complement component 4, complement component 3, cortisol, superoxide dismutase, catalase, and alanine aminotransferase. In conclusion, gathering consecutive stress and not enough recovery time in the Stress78 group negatively affected stress responsiveness and the health of oscar.

Fish Meal Replacement and Early Mild Stress Improve Stress Responsiveness and Survival of Fish after Acute Stress.

Stress responsiveness and fish meal (FM) replacement are two of the most important concerns toward achieving sustainable aquaculture. The purpose of this study was to see how early mild stress (netting) and FM replacement with meat and bone meal (MBM) affected oscar (Astronotus ocellatus; 5.2 ± 0.9 g) growth, hematology, blood biochemistry, immune responses, antioxidant system, liver enzymes, and stress responses. Oscars were subjected to a 3 × 3 experimental design (three fish meal replacement levels: 250, 180 and 110 g/kg of FM in diets; three stress periods: 0-, 2- and 3-times early mild stress). After ten weeks of the experiment, FM levels in diets did not affect growth data, but the survival rate after the acute confinement (AC) stress was lower in 11FM treatments (47.7% compared to 67.7%) than others. Fish exposed to the 3Stress schedule had a lower growth (31.03 ± 6.50 g) and survival rate (55.5%) after the AC stress than the 2Stress group (38.92 ± 6.82 g and 70.0%). Lower survival and growth rate in the 3Stress and 11FM groups coincided with the lowest blood performance, total protein, lysozyme, complement C4, complement C3, immunoglobulin, superoxide dismutase, catalase, glutathione peroxidase, and the highest glucose, cortisol, low-density lipoprotein and aspartate aminotransferase serum levels. Altogether, this study revealed that it is possible to replace FM with MBM up to 28% (180 g/kg of FM) without negative effects on the growth and health of juvenile oscar as dietary 110 g/kg of FM impaired fish health. While fish welfare should be considered, we can conclude that mild stress (2Stress) during the farming period, but without adding excessive alternative protein sources, can improve the stress responsiveness of oscar.

11-Ketotestosterone induces oocyte growth, but does not affect oocyte cytology in pre-vitellogenic captive beluga, Huso huso L.

An effect of 11-ketotestosterone (11-KT) on growth of previtellogenic (PV) ovaries of eel, salmon and Atlantic cod has been demonstrated. The purpose of this study was to investigate the effects of 11-KT treatment (in vivo) on ovarian growth, on hormonal and biochemical changes in blood, and on ovarian mRNA levels of lipidation-related genes in captive beluga with PV oocytes. In addition, the potential involvement of lipoprotein lipase (Lpl), an important enzyme for extracellular hydrolysis of lipoprotein-associated lipids, was evaluated. Twelve beluga (4-year olds) were treated with an intraperitoneal slow-release implant of either 11-KT (2.5 mg) or a compressed matrix (control). Ovarian biopsy was done to obtain pre- (day 0: T0) and post-treatment (day 21: T21) data on histology and target gene expression. Three weeks of exposure resulted in an increase in serum 11-KT levels from 2.2 ng/mL to 83 ng/mL but did not yield significant changes in serum levels of triacylglycerides and cholesterol. Furthermore, 11-KT implantation increased oocyte diameters from 259 µm (T0) to 309 µm by T21. Regardless of the increase in oocyte size, ovaries remained in the PV stage, mostly as late perinucleolar oocytes. Meanwhile, at the molecular level, the expression of lipidation-related transcripts [lpl, apolipoprotein E (apoe), very low density lipoprotein receptors (vldlr), low-density lipoprotein receptor-related protein 8-like (lrp8)] was significantly up-regulated after three weeks. Immunostaining for Lpl by Western blotting indicated three immunoreactive bands (70, 58 and 37 kDa) in ovarian homogenates from beluga, but signal intensity was not affected by treatment. Altogether, the administration of 11-KT increased 11-KT serum levels, oocyte size, and the expression of genes associated with lipid uptake. However, this treatment did not advance ovarian development beyond the PV stage.

Effects of estradiol-17ß implantation on ovarian growth, sex steroid levels and vitellogenin proxies in previtellogenic sturgeon Huso huso.

Sexual development in female great sturgeon (Huso huso) is arrested at the previtellogenic stage for many years. The present study investigated the effects of different levels of estradiol-17ß (E2) on gonadal development, levels of sex steroids and proxies of vitellogenin in 3-year-old cultured previtellogenic great sturgeon. Fish were intraperitoneally implanted every 1.5 months over a 6-month period from January to July with capsules filled with 0, 3, 6 or 12 mg E2/kg body mass as control, low, mid and high experimental groups, respectively. Blood sampling was performed at the start of experimentation and 3 weeks after each implantation for quantification of sex steroid levels and of vitellogenin-associated variables (triacylglycerol, cholesterol, calcium, phosphorus). Gonad biopsy samples were taken at the beginning and the end of the experiment in order to determine the gonad stage and oocyte morphometrical measures were taken to evaluate treatment effects. E2 implants produced a significant elevation in serum concentrations of E2, calcium, triacylglycerol, cholesterol and phosphorus. A rapid significant decrease was observed in serum testosterone levels in a dose-independent manner, so that the highest testosterone concentrations were observed in control fish throughout the experiment. There were no significant differences in oocyte stage or morphometric end points among the treated fish. We conclude that E2 implants do not stimulate ovarian growth, and hence, E2 implants alone are insufficient to reducing the time until onset of sexual maturation in previtellogenic great sturgeon.