Zebrafish (Danio rerio) fed vitamin E-deficient diets produce embryos with increased morphologic abnormalities and mortality

Abstract

Vitamin E (α-tocopherol) is required to prevent fetal resorption in rodents. To study α-tocopherol's role in fetal development, a nonplacental model is required. Therefore, the zebrafish, an established developmental model organism, was studied by feeding the fish a defined diet with or without added α-tocopherol. Zebrafish (age, 4-6 weeks) were fed the deficient (E-), sufficient (E+) or lab diet up to 1 years. All groups showed similar growth rates. The exponential rate of α-tocopherol depletion up to ~80 day in E- zebrafish was 0.029±0.006 nmol/g, equivalent to a depletion half-life of 25±5 days. From age ~80 days, the E- fish (5±3 nmol/g) contained ~50 times less α-tocopherol than the E+ or lab diet fish (369±131 or 362±107, respectively; P<.05). E-depleted adults demonstrated decreased startle response suggesting neurologic deficits. Expression of selected oxidative stress and apoptosis genes from livers isolated from the zebrafish fed the three diets were evaluated by quantitative polymerase chain reaction and were not found to vary with vitamin E status. When E-depleted adults were spawned, they produced viable embryos with depleted α-tocopherol concentrations. The E- embryos exhibited a higher mortality (P<.05) at 24 h post-fertillization and a higher combination of malformations and mortality (P<.05) at 120 h post-fertillization than embryos from parents fed E+ or lab diets. This study documents for the first time that vitamin E is essential for normal zebrafish embryonic development.

Figure 1. Diet Vitamin E Concentrations

Measured α- and γ-tocopherol concentrations (means ± SEM, n=3 separate diet batches) of experimental diets.

Figure 2. Vitamin E Depletion Kinetics

Time course of body weights (panels A,B,C) and whole body concentrations of a- (panels D,E,F) and γ-tocopherol (panels G,H,I) from individual zebrafish fed E- (squares, n=172, panels A,D,G), E+ (triangles, n=177, panels B,E,H) or lab (circles, n=168, panels C,F, I) diets. Results are from three separate generations of fish, samples taken at noted days after initiation of dietary treatments. Body weights (A–C) were not significantly different between the diet groups, but increased over time (time effect P<0.0001; month 1< month 2< month 3–5 < month 6–10, Tukey paired comparisons, each P<0.05). (D,E,F) By month 2 (>30 days) and thereafter, the α-tocopherol concentrations of the E- zebrafish were significantly less than those of the other groups [diet × time interaction, p=0.0007; E- for month 2, month 3–5, and month 6–10, different than E+ or lab diet groups (which were not different from each other at each time interval), Tukey paired comparisons, P<0.05]. The line indicates the exponential rate of depletion. (G,H,I) Within the first month (<30 days) and thereafter, the γ-tocopherol concentrations of the E- zebrafish were significantly less than those of the other groups [diet × time interaction, P<0.0001; E- for month 1, month 2, month 3–5, and month 6–10, different than E+ or lab (which were not different from each other at each time interval), Tukey HSD P<0.05].

Figure 3. Vitamin E Deficiency Alters Startle Responses

Adult zebrafish (n=6) from each diet (E+, E- and Lab; 221 days on diet) were placed in individual 1.75 L tanks containing ~1.5 L of FW. In the baseline trials and multi-tap trials, no significant differences were observed in the average swimming velocity between the diet groups. When startled by a single tap the E+ and Lab zebrafish swam faster, while E- fish had an attenuated response (diet effect P<0.003, bars not sharing the same letter are significantly different, Tukey HSD P<0.05). When exposed to multiple taps, the fish did not show significant differences in swimming velocity.

Figure 4. α- and γ-Tocopherol Concentrations of Embryos and Adults

Adult zebrafish (n= 12 per diet) were collected for vitamin E analysis between 250 and 300 days of consuming the diets. Zebrafish from this generation were spawned at 270, 278, and 284 days. Embryos were collected for vitamin E analysis at 48 hpf in groups of 15 embryos (E- n=16, E+ n=11, lab n=12 replicates). (A) All embryo α-tocopherol concentrations (mean ± SEM: logarithmic scale) were less than those of the adult zebrafish; E- embryo α-tocopherol concentrations were less than those of E- adults; E- embryos compared with E+ or lab embryos had the lowest α-tocopherol concentrations (diet × lifestage interaction, p=0.016; bars not sharing the same letter are significantly different, Tukey HSD P<0.05). (B) Adult E+ and lab zebrafish γ-tocopherol concentrations (mean ± SEM) were greater than the adult E- zebrafish and γ-tocopherol concentrations in all of the embryos; the lab embryo γ-tocopherol concentrations were greater than those of the E+ embryos; E- embryo γ-tocopherol concentrations were not significantly different from the E- adults γ-tocopherol concentrations, while E+ and lab embryos were less than those of the adults (diet × lifestage interaction, P<0.0001, bars not sharing the same letter are significantly different, Tukey HSD P<0.05).

Figure 5. Malformations and Mortality of Zebrafish Embryos

(A) Increased mortality (mean ± SEM) was observed in the E- embryos at 24 hpf and at 120 hpf compared with the other diet groups (diet × time interaction, P<0.0001), but at 24 hpf the differences between diet groups did not reach statistical significance. Mortality increased from 24 to 120 hpf in the E- (squares, P<0.01) and the E+ embryos (triangles, P<0.01), but not in the lab diet embryos (circles). At 120 hpf, the E- embryos displayed significantly higher levels of mortality compared with the E+ and lab diet embryos (diet effect p=0.005; E- (a) > E+ (b) or lab (b), P<0.05 paired comparisons). (B) Higher levels of both malformations and mortality were observed at 120 hpf in the E- embryos compared with E+ (P<0.05, a) or lab diet embryos (P<0.001, b); E+ had greater malformations than did lab diet embryos (P<0.05, c). Embryos were analyzed in 96-well plates, one embryo per well with 48 to 120 embryos per group per spawn. Results are expressed as percentages affected per total number of embryos (n= 6 spawns per group).

Figure 6. Typical Zebrafish Morphology at 120 hpf

Representative pictures from the three diet groups are shown after 5 days (120 h). The eye and otic vesicle are indicated on all fry; malformations are illustrated on the image of the deficient fish. CF=cranial-facial malformation, BA=bent anterior-posterior axis, PE=pericardial edema, SB=swim bladder malformation, and YSE=yolk-sac edema.

Figure 7. Ascorbic Acid Concentrations in Adult Fish

Ascorbic acid concentrations (mean ± SEM) were analyzed from two generations of zebrafish (n=7 per diet). (Diet effect P<0.0035, bars not sharing the same letter are significantly different P<0.05).

Figure 8. mRNA Expression in Adult Zebrafish Liver

mRNA expression (mean ± SEM) in adult zebrafish livers from fish from each diet group (n=4 per group, on diets >200 days) was analyzed by qPCR. Genes are defined and primers shown in Table 2. Expression levels were normalized to β2M and are shown as fold change over the average of the lab diet control liver mRNA, set to 100. (Diet effect for GPX4a P=0.0011, PLA2 P=0.0027, PLA2GIV P=0.0058, AIF P=0.0222; bars not sharing the same letter are significantly different P<0.05).