assessing-mineral-availability-fish-feeds-using-complementary-methods

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Institute of Marine Research

Apical uptake of zinc as ionic Zn(II) or as Zn-methionine (Zn-Met) was studied in RTgutGC cell line in vitro under media compositions mirroring the gut luminal ionic concentration of freshwater (FW) and seawater (SW) acclimated salmonids. Viability of the RTgutGC cells exposed to experimental media preparations showed a time-dependent decrease in SW treated cells, with the effect being significant at 48 h (P < 0.01), but not at 12 h or 24 h. Half effective concentration of Zn exposure over 12 h (EC, in μM) was not differentially affected by media composition (FW, 59.7 ± 12.1 or SW, 83.2 ± 7.2; mean ± SE, P = 0.43). Zinc (Zn) influx in RTgutGC was not different between FW or SW treated cells, but increased significantly in the presence of methionine (2 mM, L-Met or DL-Met). An interaction effect was observed between Zn concentration and media ionic composition on the impact of Met on apical Zn uptake (L-met, P < 0.001; DL-met, P = 0.02). In the presence of Met, apical Zn uptake in SW medium was significantly lower compared to FW, but only at higher Zn concentrations (12 and 25 μM, P < 0.01). Further, Met facilitated Zn uptake was reduced in cells treated with an amino acid transport system blocker with the effect being more significant and stereospecific in SW ionic conditions. The findings of this study showed that (i) Zn speciation in the presence of Met improved apical Zn uptake in RTgutGC cells and Zn-Met species were possibly taken up through Met uptake system. (ii) The effect was differentially affected by the ionic composition of the medium. Implications and limitations of the observations towards practical Zn nutrition of salmonids are discussed.

Zinc uptake in fish intestinal epithelial model RTgutGC: Impact of media ion composition and methionine chelation.

Atlantic salmon () feeds have changed drastically in their composition from being predominantly marine-based to plant-based. This has altered the dietary supply and availability of micro-nutrients to Atlantic salmon. The impact of graded inclusion levels of a nutrient package (NP) comprising of 25 different micro-nutrients were studied in Atlantic salmon parr in freshwater (Trial 1) and post-smolts in seawater (Trial 2). In brief, the NP was included from 0 to 400%, where 100% corresponded to the recommendation by the National Research Council, 2011. Micro-nutrients, namely Zn, Mn, Se, Cu, Fe, Co, I and vitamin D3 were included in the NP with the objective of (re)evaluating the dietary need to meet the requirement of Atlantic salmon parr and post-smolt, when fed low fish meal, plant ingredient-based diets. Responses in apparent availability coefficient (AAC), whole body and vertebrae mineral concentrations, and retention were analysed. AAC of Cu, Mn, Se and Zn responded in a quadratic fashion with an increase in NP from 0 to 400% in freshwater parr; AAC could not be measured in post-smolt salmon. The whole-body concentration of Zn, Se, Co and I in Atlantic salmon parr were significantly affected by increasing NP inclusion; the same was observed for Zn, Se and Co in post-smolt Atlantic salmon. Vertebrae mineral concentration as the response criterion was non-responsive in parr; whereas, in post-smolt, Co had a linear increase, while Zn and Se showed a non-linear increase upon 0 to 400 NP inclusion. Zinc concentration and activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) in vertebrae indicated increased bone resorption in post-smolt Atlantic salmon; TRAP activity increased linearly with NP inclusion in post-smolt, but not in parr. Significant correlations between Zn and Se were observed in AAC and vertebral concentrations, indicating an interaction in intestinal uptake and vertebral deposition. Overall, Atlantic salmon parr held in freshwater were able to satisfy the requirement for the trace minerals Zn, Mn, Se, Cu, and Fe through supply from 100-150 NP, corresponding to 101-132, 47-63, 0.6-0.8, 12-16 and 150-166 mg kg , respectively; for iodine, dietary supply from 150-200 NP, corresponding to 0.7-1.6 mg kg, was required. In the seawater, Atlantic salmon post-smolt, in general, required micro-minerals and vitamin D3 levels as supplied through 150-200 NP, corresponding to 140-177, Zn; 61-67, Mn; 0.9-1, Se; 14-16, Cu; and vitamin D3, 0.06-0.09 mg kg  to fulfil the requirement, except for Cu which was satisfied at 100-150 NP, equivalent to 13-14 mg kg  diet.

Recommendations for dietary level of micro-minerals and vitamin D to Atlantic salmon () parr and post-smolt when fed low fish meal diets.

The determination of dietary mineral solubility is one of the main steps in the evaluation of their availability for a given species.

In vitro digestion method to evaluate solubility of dietary zinc, selenium and manganese in salmonid diets.

Selenium (Se) deficiency is a problem widely encountered in humans and terrestrial livestock production with increasing attention also in aquaculture. Se supports the antioxidant system, which becomes especially important during stressful conditions. In the present study, the effect of Se-supplementation in broodstock and fry diets on the performance and antioxidant metabolism of rainbow trout fry under acute hypoxia was investigated. Rainbow trout broodstock were fed plant-ingredient based diets either without any Se-supplementation (Se level: 0.3 mg/kg) or supplemented with Se supplied as sodium selenite or as hydroxy-selenomethionine (Se level: 0.6 mg/kg respectively) for 6 months prior to spawning. The progenies were subdivided into three triplicate feeding groups and fed diets with similar Se levels compared to the parental diets, resulting in a 3x3 factorial design. After 11 weeks of feeding, the fry were either sampled or subjected to a hypoxic stress challenge. One hundred fish were transferred to tanks containing water with a low oxygen level (1.7 ± 0.2 ppm) and monitored closely for 30 min. When a fish started to faint it was recorded and transferred back to normoxic water. Direct fry feeding of the hydroxy-selenomethionine supplemented diet improved the resistance towards the hypoxic stress. On the contrary, fry originating from parents fed Se-supplemented diets showed a lower stress resistance compared to fry originating from parents fed the control diet. Fry subjected to hypoxia showed elevated oxidative stress with reduced glutathione (GSH) levels and increased isoprostanes (IsoP) and phytoprostanes (PhytoP) levels produced by lipid peroxidation of polyunsaturated fatty acids (PUFA), arachidonic and α-linolenic acids respectively. Increased mRNA expression of transcription factors (nrf2, nfκb, keap1X2) and decreased mRNA expression of antioxidant enzymes (trxr, sod, gstπ) indicated a transcriptional regulation of the antioxidant response. In stressed fry, the mRNA expression of several antioxidant genes including gr, msr and gstπ was found to be higher when fed the control diet compared to the sodium selenite treatment, with a contrary effect for parental and direct Se nutrition on gpx. The long-term parental effect becomes of greater importance in stressed fry, where more than half of the genes were significantly higher expressed in the control compared to the selenite supplemented group.

Oxidative stress and antioxidant response in rainbow trout fry exposed to acute hypoxia is affected by selenium nutrition of parents and during first exogenous feeding.

Selenium is an essential micronutrient and its metabolism is closely linked to the methionine cycle and transsulfuration pathway. The present study evaluated the effect of two different selenium supplements in the diet of rainbow trout () broodstock on the one-carbon metabolism and the hepatic DNA methylation pattern in the progeny. Offspring of three parental groups of rainbow trout, fed either a control diet (NC, basal Se level: 0.3 mg/kg) or a diet supplemented with sodium selenite (SS, 0.8 mg Se/kg) or hydroxy-selenomethionine (SO, 0.7 mg Se/kg), were collected at swim-up fry stage. Our findings suggest that parental selenium nutrition impacted the methionine cycle with lower free methionine and S-adenosylmethionine (SAM) and higher methionine synthase () mRNA levels in both selenium-supplemented treatments. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) identified differentially methylated cytosines (DMCs) in offspring livers. These DMCs were related to 6535 differentially methylated genes in SS:NC, 6890 in SO:NC and 7428 in SO:SS, respectively. Genes with the highest methylation difference relate, among others, to the neuronal or signal transmitting and immune system which represent potential targets for future studies.

Parental Selenium Nutrition Affects the One-Carbon Metabolism and the Hepatic DNA Methylation Pattern of Rainbow Trout () in the Progeny.

In relation to the decrease of selenium (Se) content in aquafeeds, the impact of level and form of parental and dietary Se supplementation was investigated in rainbow trout fry using laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) bioimaging. The offspring of rainbow trout broodstock, fed either a control diet without any Se supplementation (0.3 mg Se/kg diet) or a diet supplemented with Se (0.6 mg Se/kg diet) either as sodium selenite or hydroxy-selenomethionine, were sampled at swim-up fry stage or after 11 weeks of cross-feeding. Total body Se levels were influenced by parental Se nutrition in swim-up fry and by direct Se feeding in 11-week fry with higher levels in the Se-supplemented groups compared with the control and the highest levels in the hydroxy-selenomethionine treatment. The Se retention was lower for dietary sodium selenite. Selenomethionine levels increased when Se was provided as hydroxy-selenomethionine. LA-ICP MS maps revealed yolk in swim-up fry and intestine, liver, and kidney in 11-week fed fry as tissues with high Se abundance. In swim-up fry, muscle Se was the highest abundant when parents were fed hydroxy-selenomethionine. In 11-week fed fry, muscle Se abundance was higher in the head part of fry fed both Se-supplemented diets, but only in the tail part of fry fed hydroxy-selenomethionine. Liver Se abundance was higher in fry fed sodium selenite compared with the control diet supporting the hypothesis that tissue Se distribution can be influenced by parental and dietary Se forms and levels.

Tissue localization of selenium of parental or dietary origin in rainbow trout (Oncorhynchus mykiss) fry using LA-ICP MS bioimaging.

This study evaluated how different forms of selenium (Se) supplementation into rainbow trout broodstock diets modified the one-carbon metabolism of the progeny after the beginning of exogenous feeding and followed by hypoxia challenge. The progeny of three groups of rainbow trout broodstock fed either a control diet (Se level: 0·3 µg/g) or a diet supplemented with inorganic sodium selenite (Se level: 0·6 µg/g) or organic hydroxy-selenomethionine (Se level: 0·6 µg/g) was cross-fed with diets of similar Se composition for 11 weeks. Offspring were sampled either before or after being subjected to an acute hypoxic stress (1·7 mg/l dissolved oxygen) for 30 min. In normoxic fry, parental Se supplementation allowed higher glutathione levels compared with fry originating from parents fed the control diet. Parental hydroxy-selenomethionine treatment also increased cysteine and cysteinyl-glycine concentrations in fry. Dietary Se supplementation decreased glutamate-cysteine ligase (cgl) mRNA levels. Hydroxy-selenomethionine feeding also lowered the levels of some essential free amino acids in muscle tissue. Supplementation of organic Se to parents and fry reduced betaine-homocysteine S-methyltransferase (bhmt) expression in fry. The hypoxic stress decreased whole-body homocysteine, cysteine, cysteinyl-glycine and glutathione levels. Together with the higher mRNA levels of cystathionine beta-synthase (cbs), a transsulphuration enzyme, this suggests that under hypoxia, glutathione synthesis through transsulphuration might have been impaired by depletion of a glutathione precursor. In stressed fry, S-adenosylmethionine levels were significantly decreased, but S-adenosylhomocysteine remained stable. Decreased bhmt and adenosylmethionine decarboxylase 1a (amd1a) mRNA levels in stressed fry suggest a nutritional programming by parental Se also on methionine metabolism of rainbow trout.

Antony Jesu Prabhu

Institute of Marine Research

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon