Veterinary Research Forum

Abstract Anti-oxidants are vital for protecting sperm and can mitigate the negative effects associated with cryopreservation. This study was conducted to evaluate the protective effects of adding anti-oxidants (vitamins and amino acids) to extender in sex-reversed rainbow trout (Oncorhynchus mykiss) sperm. The collected sperm was diluted at the ratio of 1 : 5 by the extenders supplemented with different anti-oxidants including 1.00 mM of ascorbic acid  and L-tryptophan, 2.00 mM of cysteine and α-tocopherol, and their combination. After dilution, the semen was aspirated into 0.50 mL straws, and the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Straws were thawed in a 30.00 ˚C water bath for 15 sec. The Sperm Class Analyzer System was used to evaluate sperm kinematics. The activity of anti-oxidant enzymes and lipid peroxidation were determined as oxidative stress indices. Our data indicated that the incorporation of anti-oxidants and amino acids increased sperm motility duration. The elevated activity of glutathione peroxidase, superoxide dismutase, and catalase in post-thaw samples indicates that the anti-oxidant system in sex-reversed rainbow trout sperm likely plays a crucial role in protecting membrane compounds from oxidation. In conclusion, the combination of 1.00 mM L-tryptophan and ascorbic acid to the extender media caused a prolonging effect in sperm motility after thawing and they have the potential to serve as effective agents for improving sperm cryosurvival.

Abstract Use of various nanoparticles and vitamins to strengthen the immune system of fish to improve growth and biochemical indice, and to mitigate the harmful effects of free radicals through enhanced antioxidant enzyme activity has become the subject of numerous studies. In this study, fish with an average weight of 55.20 ± 7.90 g, after a two-week acclimation period, were randomly distributed into 18 polyethylene 300-L tanks, with 25 fish per tank. The experiment consisted of six treatments with three replicates: Control, vitamin (Vit) C100, nano-selenium (N-Se)0.40, Vit C100 + N-Se0.10, Vit C200 + N-Se0.20, and Vit C400 + N-Se0.40. Blood samples were collected post-treatment for analysis of antioxidant enzyme activities, biochemical and hematological indices as well as post-handling stress. Results indicated significant differences in antioxidant enzyme activities among treatments. The highest catalase activity was observed in Vit C100 and Vit C100 + N-Se0.10 groups. Glutathione peroxidase activity was significantly higher in the control and Vit C200 + N-Se0.20 groups. Malondialdehyde was the highest in Vit C100 + N-Se0.10 group. Superoxide dismutase activity peaked in Vit C200 + N-Se0.20 group. Biochemical indices showed significant differences among treatments except for high-density lipoprotein. Hemoglobin and red blood cell counts were significantly different. The Vit C100 + N-Se0.10 group showed the highest serum cortisol and glucose levels post-handling stress. Overall, dietary N-Se and Vit C supplementation improved antioxidant, biochemical, hematological indices and stress resistance in rainbow trout.

Abstract Lecithotrophic larvae utilize extensive yolk reserves for early development. In this study, the effect of egg docosahexaenoic acid (DHA):eicosapentaenoic acid (EPA) ratios (i.e., 5.92, 10.08, 11.66, and 14.53) on the emerging larvae foregut development of rainbow trout was examined. Larvae samples were taken from day 22 to 36 post-fertilization. Thin whole body longitudinal sections were prepared and stained by Hematoxylin and Eosin and Alcian blue procedure. The sections were examined regarding epithelial layer thickness, intestinal fold height and mucosal layer thickness along with number of enterocytes and goblet cells. Results indicated that maximum thickness of the epithelium was observed on day 36 post fertilization in larvae hatched from eggs with DHA:EPA ratios of 14.53 and 10.08. The highest and lowest intestinal folds height were also observed in larvae hatched from eggs with DHA:EPA ratios of 10.08 and 14.53, respectively. The mucosal-submucosa layer thickness was the highest in larvae hatched from eggs with DHA:EPA ratio of 10.08. Enterocyte’s count was the highest in larvae obtained from eggs with DHA:EPA ratio of 10.08 on day 36 post-fertilization. The highest and lowest number of goblet cells were enumerated in larvae obtained from eggs with DHA:EPA ratios of 5.53 and 14.53, respectively. In conclusion, our results revealed that feeding rainbow trout broodstock with diet contained highly unsaturated fatty acids (HUFA):polyunsaturated fatty acids (PUFA) ratio of 0.28 could result in the egg with DHA:EPA ratio of 10.08 which in turn yielded larvae with better foregut development parameters compared to those larvae emerged from the eggs with increased DHA :EPA ratio.

Abstract The effects of resveratrol as an anti-oxidant in improving growth and health have been shown in several experiments. This study aimed to evaluate the effects of different dietary resveratrol inclusion levels on digestive enzymes activity and serum biochemistry of rainbow trout (Oncorhynchus mykiss). Accordingly, 225 juvenile rainbow trout with an average body weight of 10.00 ± 1.50 g were stocked in nine experimental units. The study was performed as a completely randomized design including three dietary levels of resveratrol as follows: 0.00, 400 and 800 mg kg^-1 feed. During the experiment, fish were fed based on their respective body weight using standard feeding tables at three feeding times for 8 weeks. Nine fish were randomly selected from each treatment at the end of the 4^th and 8^th weeks of the experiment. Results revealed that supplementing 800 mg kg^-1 feed resveratrol significantly increased lipase activity (31.40 ± 0.32 U mg^-1 protein) compared to the control group (29.92 ± 0.52 U mg^-1 protein) at the end of week eight. Also, at the same time, it increased serum high-density lipoprotein (123.04 ± 1.57 mg dL^-1) compared to the control group (97.055 ± 1.463 mg dL^-1). In addition, dietary supplementation of 800 mg kg^-1 feed resveratrol effectively reduced serum alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase activities along with glucose, cortisol and cholesterol. In conclusion, resveratrol can be used as a suitable food supplement to improve fish health by increasing digestive enzymes activities.

Abstract The potential of commercial astaxanthin on growth, biochemical factors, and antioxidant-related gene expression following a challenge with diazinon were studied in rainbow trout (Oncorhynchus mykiss). Fish (~ 20.70 g) were fed diets containing commercial astaxanthin (ASX) at 0.00 (CTR and ASX0), 0.50 (ASX1), 2.00 (ASX2), and 5.00 (ASX3) g kg^-1 for 60 days. Afterwards, the treated fish (ASX1, ASX2, ASX3) as well as the fish in ASX0 group were challenged with diazinon (0.11 mg L^-1) for 96 hr whereas fish in the CTR group was not challenged with diazinon. Results showed that growth pattern improved significantly with all enriched diets compared to the ASX0 group. Metabolic enzyme activities, including alanine aminotransferase and alkaline phosphatase decreased in ASX2 and ASX3 groups with respect to the ASX0 group. Serum antioxidant status also showed the same pattern with enhancement in the fish fed with the ASX2 and ASX3 supplemented diets. Feeding the fish with astaxanthin, particularly in the ASX3 group, up-regulated the expression of some antioxidant-relevant genes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and nuclear factor erythroid-2 related factor 2 (Nrf2) in the kidney and liver. Besides, the histopathological damages in kidneys and liver induced by diazinon were less pronounced in the ASX2 and ASX3 groups compared to the ASX0 group. In conclusion, commercial astaxanthin, especially at 5.00 g kg^-1, enhanced the growth performance and ameliorated the oxidative stress induced by diazinon in rainbow trout.

Abstract This study was undertaken to investigate the effects of feeding rainbow trout (Oncorhynchus mykiss) broodstocks with different ratio of plant oils to evaluate the changes in antioxidant defense status in the progenies. In the experimental diets, fish oil was replaced with different combination of plant oils including corn oil, olive oil, sunflower oil, and coconut oil, to gain different levels of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA) in the experimental diets. Fish fed eight weeks with experimental diets before reproduction. After spawning, samples were taken on days 0, 5, 10, 15, 20, 25, 30 and 35 after fertilization. The samples were homogenized, centrifuged and the supernatant was removed for determination of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activity and malondialdehyde (MDA) content. Results showed that SOD activity was significantly increased from the first sampling to day 35 in all treatment groups. The CAT activity showed a downward trend, as the highest CAT activity was observed in the eggs immediately after fertilization. The GPX activity declined until day five and then showed an increasing trend. The MDA content did not show significant changes in different groups and at different sampling times. The antioxidant enzymes activity was significantly influenced by the dietary PUFA level in the experimental groups but no change in MDA content was recorded, suggesting that the different percentages of fish oil replacement used in this study could not result in oxidative stress in early life stages of O. mykiss.

Abstract Lactococcosis disease incident caused by Lactococcus garvieae has been increased with increasing aquaculture productions and outbreaks of the disease have become a threat on farmed species. To prevent lactococcosis, inactivated vaccine has been used, however, it only provides protection when given by injection. Other than inactivated vaccine, various vaccines such as subunit vaccines can be developed. In the present study, total protein profile of 43 strains of L. garvieae isolated from fish, milk and cheese by SDS-PAGE and virulence associated immunogenic proteins of L. garvieae strains using western blot with hyper-immune rabbit sera were determined. After analyzing whole-cell lysate protein of L. garvieae strains with SDS-PAGE, protein bands were ranged between 8.00 and 140.00 kDA. Among strains, variable protein bands were ranged between 17.00 and 48.00 kDa with some variability in the staining intensity of the protein bands and formed in 6 clusters. The immunogenic protein bands were ranged between 25.00 - 75.00 kDa. Only a variable and highly immunogenic protein band was observed between 40.00 and 45.00 kDa. Most of the strain including Lgper had 44.00 kDa immunogenic protein while nonvirulent ATCC strain had 42.50 kDA immunogenic protein. Predominant immuno-reactive proteins encoded by genes can be used as a subunit vaccine.

Abstract In the present study, the antioxidant and antimicrobial effects of essential oil (EO) and extract of Pimpinella affinis on the shelf life of rainbow trout during storage at refrigerator temperature based on three indicators of microbial, chemical and sensory quality parameters were evaluated. For this purpose, fish samples were stored in zein coatings containing 1.00% EO and 2.00% extract and in combination treatment containing 1.00% of each of them in refrigerator for 12 days. All of treatments were analyzed for microbiological count (Enterobacteriaceae, psychrophilic bacteria, mesophilic bacteria and pseudomonas bacteria) and chemical including pH, thiobarbituric acid reactive substances (TBARS), total volatile base nitrogen, peroxide value (POV) and free fatty acids (FFAs) and sensory (odor, color and texture) characteristics. Results showed that the highest levels of three factors including TBARS, POV and FFAs were related to the control sample and the least amount of these parameters was observed in EO and extract combination treatments. This effect of combined treatment was also observed in the reduction of total volatile basic nitrogen and pH parameters. Total bacterial counts during storage in fish treated with the extract and EO were remained below the acceptable level and microbial spoilage was significantly decreased compared to control. According to the sensory evaluation, treatments containing EO and extract showed improvement in this index compared to control treatment during storage. Based on the results of this study, it can be concluded that the EO and extract of P. affinis as active ingredients in zein coating successfully improve the quality and shelf life of the fish in the refrigerator.

Abstract In recent years, there has been an increasing interest in using food additives from natural sources to improve taste and also extend the shelf-life of semi-preserved foodstuffs. The aim of this study was to examine the chemical and microbiological changes promoted by a local marinating process in rainbow trout fillets during chilled storage. Fish fillets were immersed in marinades and stored at 4 ˚C for 10 days and were analyzed for total volatile basic nitrogen (TVN), thiobarbitoric acid (TBA), water holding capacity (WHC), pH, mesophilic and psychrophilic bacterial count every two days. Variations in TBA and WHC were not statistically significant between marinated and control groups. The values of TVN, pH, total psychrophilic bacteria count (TPC) and total mesophilic bacteria count (TMC) in marinated samples were significantly lower than controls. The most obvious finding of this study was that traditional marinated rainbow trout fillet stored in 4 ˚C had no undesirable changes at least for eight days.

Abstract The aim of our study was to evaluate the effects of chitosan as immune stimulator on some hematological parameters and stress resistance in rainbow trout. Nine hundred rainbow trout (with initial body weight of 25 ± 0.1 g) were obtained from a local farm and acclimated to the laboratory conditions for one week. After that fish were randomly divided into four groups in three replicates. Each group received chitosan in diet at four concentrations as 0 (Control), 0.25, 0.5 and 1 percent chitosan, respectively. The trial was conducted for 8 weeks then feeding with chitosan stopped for 3 weeks later and during this time all fish were feed by control diet. The sampling was conducted to assay the hematological parameters of all groups every two weeks. In this study we assayed the resistance of fish against some environmental stresses immediately after changing the diet to the control. The results showed that using 0.25 percent chitosan in trout diets had a significant effect (P < 0.05) on hematological indices and stress resistance of rainbow trout in comparison the control group. Serum glucose level was higher in all treatment than control without any significance difference (P < 0.05). Based on the obtained results it concluded that the adding chitosan at 0.25 percent into the diet could enhance the hematological parameters and resistance against some environmental stresses in rainbow trout.