Accepted Manuscript Title: Effects of triploidy induction on antioxidant defense status in rainbow trout (Oncorhynchus mykiss) during early development Author: Kaveh Taghipoor Saeed Keyvanshokooh Amir Parviz Salati Hossein Pasha-Zanoosi Samad Bahrami Babaheydari PII: DOI: Reference:
S0378-4320(16)30255-X http://dx.doi.org/doi:10.1016/j.anireprosci.2016.06.005 ANIREP 5435
To appear in:
Animal Reproduction Science
Received date: Revised date: Accepted date:
29-1-2016 7-6-2016 10-6-2016
Please cite this article as: Taghipoor, Kaveh, Keyvanshokooh, Saeed, Salati, Amir Parviz, Pasha-Zanoosi, Hossein, Babaheydari, Samad Bahrami, Effects of triploidy induction on antioxidant defense status in rainbow trout (Oncorhynchus mykiss) during early development.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2016.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Effects of triploidy induction on antioxidant defense status in rainbow trout (Oncorhynchus mykiss) during early development Kaveh Taghipoor1, Saeed Keyvanshokooh1*, Amir Parviz Salati1, Hossein PashaZanoosi2, Samad Bahrami Babaheydari1 1
Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr
University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran 2
Department of Physical Oceanography, Faculty of Marine Sciences, Khorramshahr
University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran Running head: Effect of triploidy induction on antioxidant status of trout
author: Saeed Keyvanshokooh, Department of Fisheries, Faculty of
Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran, Telephone: +98-61-53534725, Fax: +98-61-53534725, Email: [email protected]
Effect of triploidy induction on antioxidant state during development was studied.
SOD, CAT, GPx, vitamin C, and MDA were evaluated.
Heat-shock treatment changed the values of antioxidant enzymes, vitamin C and MDA.
2 ABSTRACT The objective of the present study was to examine the antioxidant status of rainbow trout (Oncorhynchus mykiss) during the early stages of development (fertilized egg, eyed egg, alevin and fry) as an effect of triploidy induction. Eggs and milt were taken from eight females and six males. After insemination, the eggs were incubated at 10 °C for 10 min. Half of the fertilized eggs were then subjected to heat-shock for 10 min submerged in a 28 °C water bath to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group (control and heat-shocked) and were incubated at 10-11 °C under the same environmental conditions in hatchery troughs until the fry stage. Triplicate samples of fertilized eggs from each experimental group were randomly selected 1.5 h post-fertilization and at the eyed egg stage of development (18 days post-fertilization, dpf). At 27 dpf, triplicate samples of alevins were chosen from each group. Based on ploidy determination experiment performed on both groups, nine diploid and nine triploid fry (76 dpf) were also selected. The triploidy induction success rate was 87.1%. Vitamin C was in lesser concentrations in fertilized eggs and eyed eggs of the heat-shock treatment group as compared with eggs of the diploid group. Alevins of the heat-shock treatment group had a lower superoxide dismutase (SOD) activity than alevins of the diploid group. Glutathione peroxidase (GPx) level was greater in fertilized eggs and alevins of the heat-shock treatment group as compared to diploids. Catalse (CAT) activity was greater in fertilized eggs, alevins and fry of the heat-shock treatment group than those of the diploid group. Malondialdehyde (MDA), as an index of lipid peroxidation, was in greater concentration in fertilized eggs of the group that was heat-shocked, but it was lesser in alevins and fry of the group in which the eggs were heat-shocked as compared to diploid counterparts. The results demonstrate
3 that heat-shock treatment leads to changes in the values of antioxidant enzymes such as SOD, CAT and GPx, and low molecular weight free-radical scavengers such as vitamin C, as well as level of lipid peroxidation.
Keywords: Rainbow trout; Triploid; Antioxidant enzymes; Lipid peroxidation; Vitamin C
1. Introduction Unwanted sexual maturation is a major problem in aquaculture since fish consume their metabolic energy for gonadal development before reaching marketable size (Taranger et al., 2010). This problem could be avoided by the use of sterile fish. Triploidy as a common chromosomal manipulation technique can be induced artificially in order to produce sterile fish. Triploid fish with three complete sets of chromosomes instead of two sets in diploid individuals can be generated by preventing the extrusion of the second polar body during the second meiotic division (Benfey, 1999). Aerobic organisms need oxygen to live, but at the same time they are also vulnerable to the effects of reactive oxygen species (ROS) as a byproduct of oxidative metabolism. The formation of ROS can cause oxidative stress including damage to DNA, inactivation of enzymes, degradation of structural proteins and peroxidation of lipids (Martinez-Alvarez et al., 2005). The antioxidant defense system can constantly suppress the production of reactive oxygen species and remove them in cells of aerobic organisms. The antioxidant systems include enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). In addition to these
4 enzymes, non-enzymatic defense system is formed by a set of low-molecular weight antioxidants such as carotenoids, vitamins A, C and E, and ubiquinol (Wilhelm Filho, 1996). The study of antioxidant enzyme expression in early life stages of fish would be imperative in understanding the origin and development of these protective mechanisms during the life history of organisms (Kalaimani et al., 2008). Levels of antioxidant enzymes and low molecular weight scavengers in eggs and larvae of Black Sea species (mollusks, crustacean and fish) have been studied (Rudneva, 1999). The activities of antioxidant enzymes in embryonic and early larval stages of various fish species have also been studied (Aceto et al., 1994; Díaz et al., 2010; Fontagné et al., 2008; Kalaimani et al., 2008; Peters and Livingstone, 1996; Peters et al., 2001). Triploidy in fish is most commonly induced through thermal or hydrostatic pressure treatment of eggs shortly after fertilization (Benfey, 1999). The current literature showed decreased survival of triploids relative to diploids in different developmental stages up to the larval phase (Fraser et al., 2012; Piferrer et al., 2009). The induction shock may be the main reason for early lowered survival in triploids (Cherfas et al., 1994). As the first developmental stages are the most sensitive to environmental stress (Rudneva, 1999), the triploidization heat-shock treatment may disturb the balance between the generation of ROS and its elimination. Although many studies indicate that triploid fish exhibit different behavioral and physiological responses when compared to their diploid counterparts (Fraser et al., 2012; Piferrer et al., 2009; Tiwary et al., 2004), there have been no investigation on effects of triploidy induction on antioxidant defense system.
5 The objective of the present work was hence to examine the antioxidant status of rainbow trout (Oncorhynchus mykiss) during the early stages of development (fertilized egg, eyed egg, alevin and fry) as an effect of triploidy induction. 2. Materials and methods 2.1. Fish and experimental conditions Artificial fertilization, heat-shock treatment procedures, and ploidy level determination were performed as described elsewhere (Salimian et al., 2016). Briefly, eight female (average weight 1600 ± 246 g) and six male (average weight 1393 ± 186 g) rainbow trout were selected from a commercial rainbow trout farm (Aligoudarz, Lorestan province, Iran) and stripped. The gametes were pooled to minimize the individual differences that may affect triploid yields. After insemination, the eggs were rinsed and incubated at 10 °C for 10 min. Half of the fertilized eggs were then subjected to heat shock for 10 min submerged in a 28 °C evenly heated, aerated water bath in order to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group (heat-shocked and control) and were incubated at 10-11°C under the same environmental conditions in hatchery troughs (about 1650 eggs per trough) until the fry stage. The first-feeding offspring were also reared under the same environmental conditions (mean water temperature of 14 ºC, pH of 7.5 and dissolved oxygen of 8.1 mg/l) and were fed on a rainbow trout commercial diet (BioMar, France) 12 times a day at the rate of 7% of their body weight. Dead eggs and fish were counted in early life stages and survival rates were obtained. The fish in the different experimental groups were weighed at the onset of first feeding and at the end of rearing period for estimation of growth. Based on weight records, percentage of body weight gain (WG
6 %) was calculated for each group as follows: WG = 100 × [final body weight (g) – initial body weight (g)] / initial body weight (g). Fish ploidy was determined by erythrocyte size measurement (Benfey et al., 1984). Briefly at the end of rearing trials, when the weight of fish reached about 2 g, blood samples were collected by cutting the caudal fin of 30 fish from each experimental group. Blood smears were fixed in 95% methanol for 3 minutes, left to air-dry and stained with 10% Giemsa solution for 15 minutes. Erythrocytes (n = 20) per slide were studied at 400X magnification to measure the size of erythrocytes using following formula. The letter "S" was used to designate the nuclear or cell area, "V" the nuclear or cell volume, "a" and "b" the small and large axis of the nucleus or cell. The formulae used for calculations, therefore, were: S = a × b × π/4; and V = [a/2] × [b/2]2 × π × 4/3. 2.2. Sample preparation and biochemical analyzes Triplicate samples of 30 fertilized eggs (FE) from each experimental group were randomly selected 1.5 h post-fertilization. At 18 days post-fertilization (dpf), triplicate samples of 30 eyed eggs (EE) were obtained. Triplicate samples of 30 alevins (A; 27 dpf) were also chosen from each group. At 76 dpf, based on ploidy determination experiment performed on both groups, nine diploid and nine triploid fry (three fish per trough) were selected. The fry were anaesthetized with clove powder (100 ppm) prior to sampling. Egg and fish samples were snap frozen in liquid nitrogen and stored at -80 °C until further analysis. Homogenates of eggs and fish were used for biochemical analysis. Eggs and fry were washed with phosphate-buffered saline (PBS), weighed, homogenized with PBS and centrifuged at 3000 g for 10 min at 4 °C. The supernatants were collected and stored at -20 °C prior to analysis.
7 Glutathione peroxidase (GPx) activity was assayed in the homogenates using the method described by Flohé and Günzler (1984). Activity of superoxide dismutase (SOD) was measured according to the method of McCord and Fridovich (1969). Catalase (CAT) activity was determined following the method of Aebi (1984). Malondialdehyde (MDA) concentarion, also known as thiobarbituric acid reactive substances (TBARS), was measured colorimetrically using the method of Buege and Aust (1978). Vitamin C was estimated by the method of Omaye et al. (1979). 2.3. Statistical analysis Statistical analyses were conducted using SPSS software version 16. Data were expressed as mean ± S.E and significance was considered as 0.05. ANOVA and Duncan post-test were used for comparisons within groups and Students't-test was utilized for comparisons between diploid and heat-shocked (triploid) groups. 3. Results As shown in Fig. 1, the survival rates of fertilized eggs and eyed eggs from the heat-shock group were significantly (P < 0.05) less than those of the diploid group. The alevin and fry survival rates in both groups were not different. Diploid fish were found to be significantly heavier than those from the heatshock treatment group at the onset and end of rearing. While the initial weight (0.087 ± 0.002 g in the heat-shock group vs. 0.096 ± 0.001 g in diploids) and final weight (2.07 ± 0.02 g in the heat-shock group vs. 2.27 ± 0.03 g in diploids) of experimental groups in our study was significantly different (P < 0.05), the WG % (2248.49 ± 72.51 in the heat-shock group vs. 2322.48 ± 48.08 in diploids) was not significantly different between experimental groups. The triploid (71.35 ± 0.72 µm3, mean ± S.E) erythrocyte nucleus volume was 1.94 times greater than that of the diploid fish (34.65 ± 1.11 µm3), while the
8 erythrocyte volume was 1.5 times greater (1132.24 ± 17.2 µm3 in triploid compared with 754.45 ± 15.6 µm3 in diploid fish; P < 0.01). Based on red blood cell analysis, the overall rate of triploidization was 87.1% and all control fish were identified as diploid. Vitamin C content decreased during development from fertilized egg phase to alevin stage, and then began to rise significantly to the end of fry stage (Fig. 2). Vitamin C was in lesser concentrations in fertilized eggs and eyed eggs of the heatshock treatment group as compared with eggs of the diploid group. SOD activity increased during development to reach the significant maximum level in alevins, and then declined until the end of the assay (Fig. 2). Alevins of the heat-shock treatment group had a lower SOD activity than alevins of the diploid group. GPx activity displayed a different trend for the diploid and the group where heat-shock treatment was imposed (Fig. 2). In diploids, GPx activity increased slowly during development and this activity was significantly higher in alevins and fry than in egg (FE and EE) stages. In heat-shock treatment group, GPx value of fertilized eggs began to decline to reach the minimum level in eyed eggs, and then began to rise in later stages of development. GPx level was greater in fertilized eggs and alevins of the heat-shock treatment group as compared to diploids. In diploids, CAT activity increased during development from the FE phase to the alevin stage before decreasing significantly until the end of the experiment (Fig. 2). In the heat-shock treatment group, CAT activity decreased significantly from FE to EE stage, and then increased to register the highest level in alevins before showing a drop in the fry stage. CAT activities were greater in fertilized eggs, alevins and fry of the heat-shock treatment group than those of the diploid group.
9 The measurement of lipid peroxidation in both experimental groups at different stages of development showed that MDA concentration increased from eyed egg phase to the alevin stage then remained constant until the end of the assay (Fig. 2). MDA was in greater concentration in fertilized eggs of the group that was heatshocked, but it was lesser in alevins and fry of the group in which the eggs were heatshocked as compared to diploid counterparts. 4. Discussion Fertilized eggs displayed significant levels of vitamin C and these levels declined during embryonic development. Similar to our results, a decrease of antioxidant vitamins during embryonic development has also been reported for rainbow trout (Fontagné et al., 2008), and for other fish species studied (Mourente et al., 1999; Palace et al., 1998; Palace and Werner, 2006). In fact, non-enzymatic antioxidants such as vitamin C are invested in eggs by mothers to ensure early antioxidant protection. The present study showed that the vitamin C values increase after exogenous feeding, between the alevin and fry stages. These data confirm that in post-larval stages, animals are able to gather nutritionally available antioxidant compounds that accumulate in their body (Nelis et al., 1988). Moreover, levels of vitamin C were lower in FE and EE of the heat-shocked group as compared with diploid group. The reduced values of vitamin C in eggs that were heat-shocked could be explained by elevated utilization of vitamin C in eggs to cope with oxidative stress arising from heat-shock treatment. Vitamin C levels were not different between diploid and heat-shock treatment group and while the initial and final weights of diploid and heatshocked fish were different, the WG % was similar in both groups. Thus,
10 comparable values of vitamin C accumulation in experimental fish body may be attributed to equal weight gain trend in both diploid and heat-shocked group. SOD and CAT are responsible for the inactivation of superoxide anion (O-2) and hydrogen peroxide (H2O2), respectively. SOD, by converting O-2 to H2O2, provides substrate for CAT (Fridovich, 1997). GPx catalyses the same reaction as CAT and also converts lipid hydroperoxides into hydroxides. In the present study, SOD and CAT displayed a similar profile, indicating that both enzymes act in chain to eliminate the superoxide anions formed. Activities of both enzymes increased during development to reach the significant maximum levels in alevins before decreasing until the end of the experiment and the highest levels of SOD and CAT activities were registered in the phase in which fish started exogenous feeding. This stage is considered as a highly energy demanding stage in the larval life-cycle, with a high requirement for oxygen (Solé et al., 2004). Changes in antioxidant enzymes have been previously reported in other fish species when their larvae shifted from endogenous feeding to exogenous feeding (Mourente et al., 1999; Peters and Livingstone, 1996; Kalaimani et al., 2008). CAT and GPx values were greater in fertilized eggs of the heat-shock treatment group compared with diploid FE. As fish species release their eggs at the metaphase stage of meiosis II, thermal shock treatments applied during meiosis II can suppress cell division and prevent the extrusion of the second polar body (Piferrer et al., 2009). Thus, increased values of CAT and GPx enzymes in heat-shock treated eggs may be explained by increased cell size in triploids due to retention of the second polar body.
11 While activities of CAT and GPx increased during development until the alevin stage in diploids, levels of both enzymes decreased significantly from FE to EE stage in the heat-shocked group. Reduced values of CAT and GPx in heat-shock treated eggs from FE to EE stage may be due to diffusion of these enzymes through the chorion as a result of egg shell damage. King et al. (2003) demonstrated that high temperature was associated with an increase in the occurrence of egg shell damage, decreased maternal investment and lowered egg survival. Therefore, observed heat-caused reduction in early survival rates during FE and EE incubation may also be related to free diffusion of antioxidant enzymes through damaged chorion of eggs. While SOD activity was lower in alevins of the group that was heatshocked, CAT and GPx values were greater in alevins of the group in which the eggs were heat-shocked as compared to diploid counterparts. As the survival rates in both experimental groups were not different from the alevin stage onwards, it may be concluded that CAT and GPx play a more effective role than SOD in protecting fish of the group in which the eggs were heatshocked. Also, the level of CAT activity was significantly higher in fry of the heat-shocked group and these results may be due to additional copies of the antioxidant enzymes genes in triploids. However, the comparable levels of other antioxidant enzymes measured in both diploid and triploid fry, and the higher value of CAT activity may also be explained by physiological responses of triploids when reared under suboptimal farming conditions. It has been suggested that triploids have a lower thermal optima than their diploid counterparts (Atkins and Benfey, 2008). While the mean temperature in this
12 study (14˚C) was within the standard optimal range for diploid rainbow trout, higher CAT value in triploid fry may imply that they have to cope with suboptimal conditions by increasing the level of CAT activity. In both diploid and heat-shocked groups, levels of MDA, as an end product of lipid peroxidation, increased between eyed egg and alevin stages. Strong development and hatching occurs within this period, and this fact may cause an increase in the consumption of environmental oxygen to meet the intense metabolic activity (Díaz et al., 2010) which may have led to higher production of ROS and increased levels of lipid peroxidation. MDA level was significantly higher in fertilized eggs of the group that was heat-shocked and the high levels of polyunsaturated fatty acids (PUFAs) present in the eggs (Mourente et al., 1999), may indicated that triploidization heat-shock treatment has increased oxidation of egg PUFAs. The higher level of MDA was also accompanied by lower level of vitamin C in heat-shocked FE indirectly suggesting a higher rate of lipid peroxidation. MDA concentrations were significantly lower in alevins and fry of the group in which the eggs were heat-shocked. These observations may be explained by a changed metabolic rate in triploids. Atkins and Benfey (2008) demonstrated that triploids of Atlantic salmon and brook trout had significantly higher metabolic rates than diploids at lower temperatures (12 °C in both species) and significantly lower metabolic rates than diploids at higher temperatures (18 and 15 °C in salmon and trout respectively). The temperature in the present study (14 °C) was higher than thermal optima for triploids and thus the lower MDA concentrations in alevins and fry of the heat-
13 shock treatment group may have been associated with their lower metabolic rate as compared to diploids. In conclusion, the aim of the present research was to study the effects of triploidization heat-shock treatment on antioxidant status in the first life phases of rainbow trout. The results demonstrate that heat-shock treatment leads to changes in the values of antioxidant enzymes such as SOD, CAT and GPx, and low molecular weight free-radical scavengers such as vitamin C, as well as level of lipid peroxidation. The results of this study may pave the way for further research on the effects of antioxidants for the management of stress during larval rearing of triploid rainbow trout.
Conflict of interest There are no known conflicts of interest
Acknowledgment This research was supported by Khorramshahr University of Marine Science and Technology (Grant No. 222945).
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Figure captions Fig. 1. Survival rates of diploid and heat-shocked rainbow trout in different stages of development. Values are means (n = 3), with standard errors represented by vertical bars. Mean values with unlike letters are significantly different (P<0.05). a
85 80 75 FE
A Stage of growth
19 Fig. 2. Levels of vitamin C, antioxidant enzymes (SOD, GPx and CAT) and lipid peroxidation (MDA) in diploid and triploid rainbow trout at different developmental stages; Each bar represents the mean value of three replicates (±SE); Differences in the same group (P<0.05; Duncan test) or between two groups (diploid and heatshocked) at the same time (t-test) are indicated by the different superscript denotations (*P<0.05, **P<0.01 and ***P<0.001) 16
Vitamin C (mg/dl)
4 2 0 FE
SOD (units/mg protein)
Stage of growth
20 18 16 14 12 10 8 6 4 2 0
A 2N B
A Stage of growth