Detrimental Effects Of Fish Oil Oxidation On The Health And Its Precautions Of Juvenile Largemouth Bass Micropterus Salmoides

Five chapters were included in this thesis to investigate the in vivo detrimental effects of dietary fish oil oxidation on the healh status of largemouth bass, the in vitro cytotoxicity of malondialdehyde exposure in primary cultured hepatocytes, as well as the subsequent precautions in vivo and in vitro. Major results and conclusions were presented as follows:1. Four isonitrogenous and isolipidic diets containing fresh fish oil (peroxide value, POV:11.49meq/kg, diet FR) and three degrees of oxidized fish oil (POV:132.40,277.23and555.16meq/kg, diet OX132, OX277and OX555) were formulated to investigate the effects of dietary oxidized fish oil on growth performance, body com-position, hepatic antioxidant defense mechanism and liver histology of juvenile lar-gemouth bass. After a12-week feeding trial, a proportion of proximately3%of M. salmoide showed skull deformity or spinal malformation, and9%showed inflamma-tion and hemorrhage at the base of dorsal, pectoral and tail fin in both group OX277and OX555. Fish fed oxidized oil diets obtained significantly higher weight gain and specific growth rate due to their remarkable higher feed intakes, compared to the fresh oil receiving group. The analysis of biometric parameters and body composition indi- cated significant differences in various test diets. The activities of hepatic catalase and superoxide dismutase were significantly stimulated by oxidized oil ingestion. Hepatic glutathione peroxidase, glutathione reductase and glutathione-S-transferase activities were significantly higher, and liver glutathione content was markedly lower in group OX555than the other treatments. Oxidized oil consumption resulted in marked deple-tion of vitamin E concentration in plasma, liver and muscle tissue, increased plasma and muscle malondialdehyde content along with decreased hematocrit value. Histo-logical examinations indicated that hepatocytes with lipid vacuoles and nuclear mi-gration were shown in group OX277and OX555. The overall results in this study sug-gested that an increased oxidative stress in M. salmoide fed oxidized lipid may ac-count for their stimulated hepatic antioxidant defenses, vitamin E depletion in plasma and certain tissues, and pathological changes. The detrimental effect of lipid oxidation on fish health and the unexpectedly enhanced feed intake of oxidized feeds in M. salmoide underline the importance that cares should be taken to minimize dietary oil oxidation to the greatest extent possible.2. Six oxidized fish oil contained diets were formulated to investigate the effect of graded levels of vitamin E (VE, from α-tocopherol acetate:160,280and400mg/kg) associated with either1.2or1.8mg/kg selenium (Se, from sodium selenite) on growth, body composition and hepatic antioxidant defense mechanism of juvenile largemouth bass. Another control diet containing fresh fish oil with160mg/kg VE and1.2mg/kg Se was also prepared. Over a12-week feeding trial, about5%of M. salmoide fed diet OxSe1.2/VE160showed inflammation and haemorrhage symptoms at the base of dorsal, pectoral and tail fin. Fish in all treatments survived well (above90%). Feed intakes (88.42-89.58g/fish) of all treatments were comparable. Growth performances (weight gain and specific growth rate) and feed utilization (feed and protein efficiency ratio) were significantly impaired by dietary oil oxidation, and they didn’t benefited from neither VE nor Se supplementation. Regardless of dietary VE and Se supplemen-tation, oxidized oil ingestion resulted in markedly decreased hepatosomatic index and intraperitoneal fat ratio. Oxidized oil ingestion also induced markedly lower liver and muscle lipid contents, and these effects could be alleviated by dietary Se supplemen- tation. Dietary oil oxidation stimulated hepatic catalase activities relative to the con-trol, and supplementation of VE abrogated this effect. Hepatic reduced glutathione content in the control was markedly higher than that of treatment OxSe1.2/VE160, without any significant differences comparing with the other oxidized oil receiving groups. Hepatic glutathione peroxidase activity and liver Se concentration reflected dietary Se profile, whereas liver VE level reflected dietary VE profile. Compared with the control, fish fed diet OxSe1.2/VE160obtained markedly higher serum, liver and muscle malondialdehyde contents, which dropped significantly with increasing either VE or Se supplementation. In conclusion, the overall results in this study suggested that both VE and Se inclusion could protect largemouth bass from the oxidative dam-age challenged by dietary oil oxidation.3. The present study was designed to investigate the cytotoxicity of malondial-dehyde (MDA) in primary cultured hepatocytes from largemouth bass. MDA was prepared by acid hydrolysis of1,1,3,3-tetraethoxypropane in0.1M HCl. After at-taching to the culture plates, the hepatocytes were incubated with fresh M199medium containing50,100,250,500and1000μM MDA in addition to control (0μM) for24h. Cell counting kit-8based in vitro toxicity assay showed that a dose-dependent re-duction in cell viability was induced by MDA. An increase of lactate dehydrogenase activity was recorded at MDA exposure doses≥100and500μM in the cultured he-patocytes and culture medium, respectively. Compared with the control, catalase ac-tivity firstly increased at100μM of MDA and then decreased with≥250μM MDA, whereas superoxide dismutase activity dropped at MDA exposure dose of1000μM. The increase in cell apoptosis was associated with an increase of caspase-3/6activity since the MDA concentration was above250μM. Intracellular reactive oxygen spe-cies formation was recorded with≥100μM MDA exposure at an incubation time of12h. The reduced to oxidized glutathione ratio decreased sharply with increasing MDA dose. Cellular thiobarbituric acid reactive substances increased since the MDA exposure dose was up to100μM. The overall results in the present study suggested that MDA exposure at high level (≥250μM) would induce cell apoptosis and loss of viability to largemouth bass hepatocytes, probably by causing oxidative damage and deprivation of antioxidant stores.4. The aim of this study was to evaluate the protective role of VE pretreatment on the cytotoxicity of malondialdehyde in primary cultured largemouth bass hepatocytes. Dimethyl sulfoxide was used as a carrier to dissolve VE. After attaching to the culture plates, the hepatocytes were firstly pretreated with four levels of VE (100,250,500and1000μM, respectively) in M199medium for12h, and then were incubated with fresh M199medium containing500μM MDA for24h. Another two treatments (posi-tive control and negative control) were also prepared without VE pretreatment, but with MDA exposure for the negative control. Compared with the positive control, MDA exposure induced a loss of26.3%in cell viability and an increase in lactate de-hydrogenase activity by37.6%. When the VE levels were above250and500μM, cell viability and lactate dehydrogenase activity could be reversed by56.3-78.9%and64.3-68.6%, respectively. MDA exposure also enhanced the catalase activity by28.4%, and VE pretreatment (≥250μM) could alleviate it. In addition, as a result of MDA exposure, reduced glutathione content decreased by28.1%, while oxidized glu-tathione content increased by28.1%, whereas the ratio of reduced to oxidized gluta-thione decreased from4.53to2.14, and VE pretreatment (≥250μM) could reverse these phenomenons. After12h of MDA exposure, intracellular reactive oxygen spe-cies of the negative control increased by26.5%, whereas cellular thiobarbituric acid reactive substances increased by52%after24h of MDA incubation. When the VE level was above250μM, reactive oxygen species formation could be totally reversed, whereas cellular lipid peroxidation could be reversed by53.3-71.6%with≥500μM VE. Moreover, MDA exposure increased the activities of caspase-3(by77.4%) and caspase-6(by68.8%), which could be alleviated by VE pretreatment (≥250μM). The overall results in this study suggested that VE pretreatment (≥500μM) could reverse the deprivation of antioxidant stores and the formation of reactive oxygen species, and partially alleviate the loss in cell viability and cell apoptosis induced by MDA expo-sure.5. The present study was performed to evaluate the protective role of vitamin C pretreatment on the cytotoxicity of MDA in primary cultured largemouth bass hepa- tocytes. After attaching to the culture plates, the hepatocytes were firstly pretreated with four levels of vitamin C (250,500,1000and2000μM, respectively) in M199medium for3h, and then were incubated with fresh M199medium containing1000μM MDA for24h. Another two treatments (positive control and negative control) were also prepared without vitamin C pretreatment, but with MDA exposure for the negative control. Cell viability decreased by47.7%and lactate dehydrogenase activity increased by48.9%after24h of MDA exposure. When the vitamin C levels were above1000and500μM, cell viability and lactate dehydrogenase activity could be reversed by64.6-76.5%and54.6-79.3%, respectively. MDA exposure didn’t change the activities of catalase and superoxide dismutase, but vitamin C pretreatment (≥500μM) trended to increase the activities of these antioxidant enzymes. In addition, as a result of MDA exposure, reduced glutathione content decreased by39.9%, while oxi-dized glutathione content increased by70.8%, whereas the ratio of reduced to oxi-dized glutathione decreased from3.17to1.31. When the vitamin C levels were above500and1000μM, the changed reduced glutathione content and the ratio of reduced to oxidized glutathione could be reversed, respectively. After12h of MDA exposure, intracellular reactive oxygen species of the negative control increased by36.8%, whereas cellular thiobarbituric acid reactive substances increased by43.7%after24h of MDA incubation, and vitamin C pretreatment (≥500μM) could eliminate these phenomenons. Moreover, MDA exposure increased the activities of caspase-3(by81.1%) and caspase-6(by57.7%), which could be reversed by97.1-98.2%for cas-pase-3and51.9-59.9%for caspase-6with≥1000μM of vitamin C pretreatment. The overall results in this study suggested that vitamin C pretreatment (≥1000μM) could reverse the deprivation of antioxidant stores, reactive oxygen species formation and lipid peroxidation, and partially alleviate the loss in cell viability and cell apoptosis induced by1000μM of MDA exposure.6. The present study aimed to evaluate the protective role of sodium selenite pre-treatment on the cytotoxicity of MDA in primary cultured largemouth bass hepato-cytes. After attaching to the culture plates, the hepatocytes were firstly pretreated with four levels of Se (10,25,50and100μM, respectively) in M199medium for3h, and then were incubated with fresh M199medium containing1000μM MDA for24h. Another two treatments (positive control and negative control) were also prepared without Se pretreatment, but with MDA exposure for the negative control. Cell viabil-ity decreased by44.7%and lactate dehydrogenase activity increased by40.5%after24h of MDA exposure. With25μM Se pretreatment, cell viability and lactate dehy-drogenase activity could be reversed by33.1%and70.1%, respectively. MDA expo-sure decreased the activity of catalase by36.3%, but100μM Se pretreatment could reverse it. In addition, as a result of MDA exposure, reduced glutathione content de-creased by32.2%, while oxidized glutathione content increased by53.3%, whereas the ratio of reduced to oxidized glutathione decreased by50.9%. With low level of Se pretreatment (10-50μM), the reduced glutathione content was reversed by36.6-72.2%, but it was unchanged by100μM Se. Se pretreatment did not help to re-duce the oxidized glutathione content, but the ratio of reduced to oxidized glutathione could be reversed by28.8%with25μM selenium pretreatment. After12h of MDA exposure, intracellular reactive oxygen species of the negative control increased by32.1%, whereas cellular thiobarbituric acid reactive substances increased by26.3%after24h of MDA incubation, and25μM Se pretreatment could totally reverse the formation of reactive oxygen species, and partially reverse the lipid peroxidation by80.8%, but100μM Se pretreatment inversely enhanced intracellular lipid peroxida-tion. Moreover, MDA exposure increased the activities of caspase-3(by52.2%) and caspase-6(by52.7%), which could be reversed by68.2%for caspase-3and56.9%for caspase-6with25μM of Se pretreatment. The overall results in this study suggested that low level of Se pretreatment (10-50μM) could partially alleviate the cytotoxicity induced by MDA exposure, and the best pretreatment dose was25μM. However,100μM Se pretreatment could not alleviate but enhanced the cytotoxicity of MDA expo-sure.