Protocol for feeding strategy and proteomics analysis of zebrafish 
Yeast is a sustainable alternative protein source in aquaculture, rich in bioactive compounds that can act as immunostimulants to enhance fish growth and immunity. , , , Notably, the yeast Cyberlindnera jadinii has been shown to modulate inflammatory responses induced by soybean meal in fish by enhancing pathways related to wound healing and adjusting innate immunity through taurine and hypotaurine pathways. Zebrafish, Danio rerio, is commonly used in laboratory research due to its well-annotated genome, transparent embryos, small size, and short generation time. , , , To explore the impact of yeast inclusion in fish diets on immune response and health, a feeding trial was conducted using five different diets, each containing 10% of four distinct yeast strains, including autolyzed C. jadinii . This trial was performed with lab-bred zebrafish at the fish laboratory at NMBU, Norway. The fish were maintained in ZebTec Active Blue recirculating systems under controlled conditions: a temperature of 26°C–28°C, pH 7.0–7.5, and a 14/10-h light/dark cycle. Here, we present a protocol for the feeding strategy and subsequent zebrafish proteomics analysis using S-trap, iTRAQ, and 2D LC-MS/MS to characterize specifically the effects of C. jadinii . Institutional permissions This experiment was approved by the Norwegian Food Safety Authority and was performed in accordance with Norwegian regulations of June 18, 2015, No. 761 concerning the use of animals for scientific purposes. Setting up the tank Timing: variable 1. Put 20 zebrafish (3-month-old) in 3 L ZebTec Active Blue recirculating systems (Tecniplast, Italy) with the below parameters. a. Maintain temperature at 26°C–28°C. b. Maintain water pH levels between 7.0–7.5. c. Rotate photoperiod of 14/10 h light/dark cycle. Cross breeding Timing: 2–3 days 2. Put a 4-month-old male and female zebrafish (female fish have a larger underbelly, while males are slimmer. Additionally, males display more yellow in their anal fins compared to females) in a breeding tank with the below parameters. a. Maintain the same water parameters and settings as described in ‘1’. b. Breed the pair in the morning (typically 14 h of light and 10 h of darkness). c. Collect eggs in a petri dish. d. Incubate the eggs at 28°C until hatch. Feeding strategy Timing: 1 month 3. Start feeding larvae with zebrafeed (Sparos Ltd, Portugal) five days-post-fertilization (dpf). a. Feed zebrafeed 2 times a day. Note: Ensure zebrafeed size is < 100 μm. 4. Feed zebrafish with zebrafeed 14 dpf. a. Give feed 2 times a day. Note: Ensure zebrafeed size was between 100–200 μm. 5. Feed zebrafish with zebrafeed 30 dpf. a. Give feed 2 times a day. Note: Zebrafeed size was between 200–400 μm. 6. Feed zebrafish with zebrafeed and artemia two months-post-fertilization (mpf). a. Stock zebrafish of uniform size into groups of 45 in 9L recirculating tanks. b. Feed the zebrafish 2 times a day. c. Supplement the zebrafish with 0.2–0.4 mL of concentrated artemia per fish, twice a day. Note: Zebrafeed size was between 400–600 μm. 

This experiment was approved by the Norwegian Food Safety Authority and was performed in accordance with Norwegian regulations of June 18, 2015, No. 761 concerning the use of animals for scientific purposes. 

Timing: variable 1. Put 20 zebrafish (3-month-old) in 3 L ZebTec Active Blue recirculating systems (Tecniplast, Italy) with the below parameters. a. Maintain temperature at 26°C–28°C. b. Maintain water pH levels between 7.0–7.5. c. Rotate photoperiod of 14/10 h light/dark cycle. 

Timing: 2–3 days 2. Put a 4-month-old male and female zebrafish (female fish have a larger underbelly, while males are slimmer. Additionally, males display more yellow in their anal fins compared to females) in a breeding tank with the below parameters. a. Maintain the same water parameters and settings as described in ‘1’. b. Breed the pair in the morning (typically 14 h of light and 10 h of darkness). c. Collect eggs in a petri dish. d. Incubate the eggs at 28°C until hatch. 

Timing: 1 month 3. Start feeding larvae with zebrafeed (Sparos Ltd, Portugal) five days-post-fertilization (dpf). a. Feed zebrafeed 2 times a day. Note: Ensure zebrafeed size is < 100 μm. 4. Feed zebrafish with zebrafeed 14 dpf. a. Give feed 2 times a day. Note: Ensure zebrafeed size was between 100–200 μm. 5. Feed zebrafish with zebrafeed 30 dpf. a. Give feed 2 times a day. Note: Zebrafeed size was between 200–400 μm. 6. Feed zebrafish with zebrafeed and artemia two months-post-fertilization (mpf). a. Stock zebrafish of uniform size into groups of 45 in 9L recirculating tanks. b. Feed the zebrafish 2 times a day. c. Supplement the zebrafish with 0.2–0.4 mL of concentrated artemia per fish, twice a day. Note: Zebrafeed size was between 400–600 μm. 

 

 

Assembly of pelleted feed Timing: 14–16 h. The production time will depend on the volume produced and the drying capacity. This step describes the homogenization of ingredients and extrusion to form feed pellets for the feeding trial. 1. Mixing of ingredients: a. Combine and mix all dry ingredients thoroughly. b. Add the oil and mix for 10 min. c. Dissolve the gelatin in in 22°C water (ratio 1:3) and warm it to 55°C in a conventional microwave while agitating every 30–60 s. d. Add the gelatin to the mixture and mix for 10 min until homogenized. e. Add the remaining amount of water to reach approximately 30% weight of all ingredients (see ). 2. Making of feed pellet: a. Cold pellet the mash into a 0.5 mm diameter using a P35A pasta extruder (Italgi, Italy). b. Regulate the speed of the screw and knifes to obtain 0.5 mm long pellets. c. Dry the pellets at 55°C until moisture content is 8%–10% (see ). d. Store the feed in a dry place at 4°C before feeding. Analysis of chemical components in feed Timing: 10 days This step allows for chemical analysis to determine the nutritional composition of fish feed. Prior to the analysis, grind all pellets into a fine powder. 3. Dry matter, ash, crude protein, and crude lipid analysis. a. Analyze using methods described in the European Commission Regulation No 152/2009. 4. Starch chemical composition: a. Hydrolyze the starch with α-amylase and amyl glucosidase-enzymes to glucose. b. Add α-amylase (0.1%–0.5% (w/w) of starch) at temperatures between 60°C and 90°C and optimal pH between 6.0 to 7.5 and amyloglucosidase (0.1%–0.3% (w/w) of starch) at temperatures between 55°C to 65°C and optimal pH between 4.0 and 5.0. c. Use RX4041 Randox Daytona+ to determine glucose concentration using methods described by McCleary, Solah, and Gibson. , 5. Gross energy composition: a. Use PARR 6400 Automatic Isoperibol Calorimeter (Parr Instruments, Moline, Illinois, USA) according to ISO 9831. Animal feeding stuffs, animal products, and feces or urine-Determination of gross calorific value-Bomb calorimeter method. 6. Total phosphorus content: a. Combust and decompose pellet (0.5–1.0 g) in a Start D microwave digestion system (MP-AES 4200, Agilent Technologies, USA) following manufacture guidelines and between the following ranges: 15–30 min at 150°C–200°C. b. Measure phosphorus content using a microwave plasma atomic emission spectrometer (MP-AES 4200, Agilent Technologies, USA). Pilot experiment and feeding trial Timing: 28 days (4 weeks) This step describes the stocking density and amount as well as frequency of fish feeding for both the pilot experiment and feeding trial. 7. Treatment groups (pilot experiment): a. Prepare three 3.5 L tanks for three treatment groups: control high protein diet (HP), low protein (LP) and low protein diet with C. jadinii yeast ingredient (LPY). b. Stock ten 3 mpf zebrafish into each tank. c. Record the average initial body weight of the zebrafish from each group. 8. Treatment groups (feeding trial): a. Prepare five 3.5 L tanks for the five treatment groups: control diet (C) and 4 yeast containing diets (ICJ, ACJ, IWA, AWA). b. Stock twenty- 3 mpf zebrafish into each tank. c. Record the average initial body weight of the zebrafish from each group. 9. Feeding regime: a. Feed the fish at 3% body weight. b. Feed each tank three times a day at 08:00–09:00, 14:00–15:00 and 20:00–21:00. c. Record any mortality. 10. Sample collection: a. Collect 5 fish from the pilot group and 6 fish per group from the feeding trial on day 14 and day 28. b. Weigh and record the average body weight. c. Euthanize the fish using cold ice treatment. d. Collect the intestine samples for proteomic analysis. e. Freeze the samples with liquid nitrogen. f. Store at −80°C. Calculation of growth rate Timing: 2 h This step calculates the fish growth rate from the pilot study and feeding trial at days 14 and 28. The results can be used to determine if different components of the feed affect the growth rate of the fish. 11. Specific growth rate (SGR): a. Calculate the growth rate according to the following equation: S G R  = ln ( F B W ) − ln ( I B W ) d a y s × 100 Where, FBW = final body weight in g fish –1 ; IBW = initial body weight in g fish –1 . 12. Percentage body weight gain: a. Calculate the percentage of body weight gain using the equation: W e i g h t g a i n  = 100 × F i n a l w e i g h t ( g ) − I n i t i a l w e i g h t ( g ) I n i t i a l w e i g h t ( g ) 13. Feed conversion ratio (FCR): a. Calculate FCR using the equation: F C R = [ T o t a l f e e d i n t a k e ( g / D M f i s h − 1 ) ] ( F B W − I B W ) Where DM = dry matter; FBW = final body weight in g fish –1 ; IBW = initial body weight in g fish –1 . Protein isolation and quantification Timing: 70 min for step 1 4 , 24 h for incubation, 20 min for centrifugation, 10 min for washing, variable for drying This step determines the protein concentration of the intestinal samples of the zebrafish. 14. Homogenization of Frozen Intestinal Samples: a. Homogenize frozen intestine samples with 200 μL of SDS lysis buffer, 0.5 M TEAB buffer (pH 8.5), and 1× Protease Inhibitor cocktail. b. Incubate the homogenized samples at 90°C for 30 min, followed by cooling on ice for 5 min. c. Centrifuge the lysed samples at 14,000 × g for 20 min at 4°C. CRITICAL: Ensure thorough homogenization and efficient centrifugation for optimal results. 15. Protein Precipitation and Pellet Collection: a. Transfer the supernatant (containing proteins) to a new Eppendorf tube. b. Add four volumes of ice-cold acetone to the protein samples. c. Incubate the samples at −20°C for 24 h. d. Centrifuge the samples at 14,000 × g for 10 min at 4°C, collect the pellets. e. Wash the pellets twice by centrifugation at 14,000 × g at 4°C for 10 min with four volumes of ice-cold acetone. f. Dry the pellets by evaporation at 22°C to remove all liquid. CRITICAL: Handle pellets carefully to avoid loss of material. 16. Storage and Transport: a. Store the dried pellets at −80°C. 17. Resolubilization and Protein Concentration Estimation: a. Resolubilize the pellets in 200 μL of 5× SDS lysis buffer. b. Take aliquots for protein concentration estimation using Qubit 3.0 Fluorometer and Qubit Protein Assay Kit. CRITICAL: Accurate protein concentration estimation is crucial for downstream proteomics analysis. S-trap digestion and peptide quantification Timing: variable This step involves digesting 100 μg of protein from intestinal samples using the S-Trap micro column or mini (for >100 μg of protein) and utilizing Pierce quantitative colorimetric assay to determine peptide quantification. 18. Sample Preparation: a. Adjust the sample volume to equal amount of S-Trap SDS lysis buffer (5% SDS, 50 mM TEAB pH 8.5; make by diluting 2× lysis buffer 1:1 with 18.2 MΩ water). b. Add 3.5 μL of 100 mM TCEP for reduction. c. Incubate at 55°C for 10 min and cool to 22°C. d. Add 7 μL of 200 mM MMTS for alkylation. e. Incubate at 22°C for 10 min. f. Add 7 μL of 12% phosphoric acid to the solubilized, reduced, and alkylated sample. g. Add 420 μL of S-Trap binding buffer (90% MeOH, 100 mM triethylammonium bicarbonate buffer (TEAB, pH 7.1) to the acidified lysate. h. Transfer the solution into the S-Trap micro column in 200 μL increments. i. Centrifuge for 30 s-1 min at 4,000 × g . j. Keep the flow-through at −20°C. 19. Washing: a. Wash the trapped proteins by adding 150 μL S-Trap binding buffer to the column and centrifuge. b. Repeat step 19a. 4 times. 20. Trypsin Digestion: a. Add trypsin digestion buffer to each column based on the ratio (1:25 w/w) of trypsin to protein. b. Mini: Add 125 μL of digestion buffer containing trypsin to each column. Micro: Add 20 μL of digestion buffer containing trypsin to each column. c. Spin down the trypsin digestion buffer through the column at 4000 × g for 30 s, then transfer the filtrate back into the column matrix. This is to ensure that the trypsin buffer has coated the matrix and ensure sufficient buffer for incubation. d. Cap the spin column and incubate at 47°C for 1–2 h. 21. Elution: a. Mini: Sequentially add 80 μL of 50 mM TEAB, 80 μL of 0.2% aqueous formic acid, and 80 μL of 50% ACN with 0.2% FA and elute by centrifuging at 4k × g , for 30s-1 min, until all the solution has eluted. b. Micro: Sequentially add 40 μL of 50 mM TEAB, 40 μL of 0.2% aqueous formic acid, and 35 μL of 50% ACN with 0.2% FA and elute by centrifuging at 4k × g , for 30s-1 min, until all the solution has eluted. 22. Speedvac Drying: a. Dry the elutions using a Speedvac. Note: Micro <10–50 μg, Mini 100–300 μg. 23. Pierce Quantitative Colorimetric Peptide Assay: a. Prepare the standards provided by the kit and sample dilution as below in 1.5-mL tube. 24. Prepare Working Reagent (WR): a. Mix the following reagents in a 1.5-mL tube to create the Working Reagent (WR): i. 50 parts (50%) Colorimetric Peptide Assay Reagent A. ii. 48 parts Colorimetric Peptide Assay Reagent B. iii. 2 parts Colorimetric Peptide Assay Reagent C. iv. Add 180 μL of the prepared WR to each well of a 96-well microplate. Note: WR can be stored at 22°C for ≤ 30 min (keep in the dark before use). 25. Standard and Sample Addition: a. Add 20 μL of each replicate of the standard and sample into a 96-well microplate (total 3 replicates). CRITICAL: Avoid introducing air bubbles to each well. 26. Peptide Assay Working Reagent: a. Add 180 μL of the Peptide Assay Working Reagent (dye) to each well. b. Mix the plate thoroughly on a plate shaker for 30 s to 1 min. c. Cover the plate and incubate at 37°C for 15 min. 27. Absorbance Measurement: a. Measure the absorbance of the standards and samples at 480 nm. 28. Standard Curve Preparation: a. Prepare a standard curve by plotting the average Blank-corrected 480 nm measurement for each BSA standard vs. its concentration in μg/μL. b. Use the standard curve to determine the peptide concentration of each sample. iTRAQ labeling and sample clean-up Timing: 1 day This step enables the quantification of proteins using isobaric tags. 29. iTRAQ Labeling: a. Use one iTRAQ sample set containing samples from the control (C) and yeast supplemented (ACJ) diets. b. Employ the iTRAQ Reagents 8-plex kit (SCIEX, Foster City, USA) to label the digested peptides. c. Speedvac-dry the peptides and re-dissolve in 30 μL of dissolution buffer (0.5 M TEAB, pH 8.5) for labeling with iTRAQ reagent. 100 μg of peptides from each sample were used for each labeling reaction. d. Pool each sample set together according to the manufacturer’s recommendation iTRAQ Reagents - 8plex protocol . e. Lyophilize the pooled sample. 2D LC-MS/MS analysis (iTRAQ) Timing: 2 days 30. First-Dimension Separation: a. After lyophilization of the peptide samples, perform 2D LC-MS/MS. Dissolve the peptides in 20 mM ammonium formate in water, pH 10. b. Utilize high pH reversed-phase high-performance liquid chromatography unit (RP-HPLC) (1290 Infinity LC system; Agilent, Santa Clara, CA, US) equipped with a C18 column (WATERS Xbridge C18, 3.5 μm, 3.0 mm × 150 mm; Waters Milford, Massachusetts, US) for the first-dimension separation. c. Use mobile phase A (20 mM ammonium formate in water, pH 10) and mobile phase B (20 mM ammonium formate in 80% ACN, pH 10). d. Apply the following gradient elution profile: i. 0%–0% B for 10 min. ii. 0%–60% B for 60 min. iii. 60%–60% B for 5 min. iv. 60%–100% B for 1 min. v. Continued at 100% B for 5 min. vi. 100%–0% B for 1 min. vii. Subsequently, maintain at 0% B for 14 min. e. Collect the eluted fractions in 96-well, V-bottom plates at a flow rate of 0.5 mL min −1 . f. Combine eluted fractions into 10 concatenated fractions and lyophilize. 31. Second-Dimension Separation: a. Dissolve the lyophilized samples in 2% acetonitrile in water. b. For MS/MS analysis, inject 2 μL of each of the 10 fractions independently into a ProteoCol C18P (3 μm 120 Å, 300 μm × 10 mm; Trajan) trap column at 4 μL/min for 6 min. c. Elute on an C18 analytical column (Acclaim PepMap 100C18, 3 μm particle size, 75 μm i.d. × 250 mm; Thermo Scientific) using a nanoLC 425 LC system (Eksigent) in Trap-Elute configuration. d. Use mobile phase A (0.1% formic acid) and mobile phase B (0.1% formic acid in acetonitrile) with a flow rate of 300 nL min −1 . e. Apply the following gradient elution profile: i. 5%–15% B for 60 min. ii. 15%–30% B for 60 min. iii. 30%–90% B for 3 min. iv. Continued at 90% B for 20 min. v. 90%–5% B for 2 min. vi. Subsequently, maintain at 5% B for 15 min. f. Acquire MS spectra using a TripleTOF 5600+ system (AB SCIEX, Framingham, Massachusetts, US) under positive ionization mode, in the mass range of 350–1250 m/z at high sensitivity mode with an accumulation time of 250 ms per spectrum. g. Select the 30 most abundant precursors between charge range +2 to +5 per duty cycle for MS/MS analysis (accumulation time: 100 ms), in the mass range of 100–1800 m/z. h. Employ 15 s dynamic exclusion for MS/MS analysis in high sensitivity mode (>15,000) with rolling collision energy and iTRAQ reagent collision adjustment settings turned-on. Peptide and protein identification Timing: 20 h 32. Software and Database Search: a. Utilize ProteinPilot 5.0.2 software (AB SCIEX) for identification of iTRAQ-labeled proteins. b. Use the Paragon database search algorithm and the false discovery rate (FDR) cut-off functions. c. Set the search parameters as follows: i. Sample Type: iTRAQ 8plex (Peptide Labeled). ii. Cysteine Alkylation: MMTS. iii. Digestion: Trypsin. iv. Instrument: TripleTOF5600. v. Special Factors: None. vi. Species: None. vii. ID Focus: Biological Modification. viii. Database: Zebrafish reference proteome (2022_02 release, 46687). ix. Search Effort: Thorough. x. FDR Analysis: Yes. xi. Background Correction: Yes. xii. User Modified Parameter Files: Yes. 33. Protein Identification and Differential Abundance Analysis: a. Set the cut-off for protein identification at 1% FDR. b. Determine the differential abundance of proteins using paired student’s t-test. c. Define the criteria for selection of differentially abundant proteins (DAPs) as follows: i. Abundance-Increased Proteins: iTRAQ ratio ≥ 1.3 (P-value < 0.05). ii. Abundance-Decreased Proteins: iTRAQ ratio ≤ −1.3 (P-value < 0.05). Functional analysis of protein Timing: 2 days This step analyses and interprets the functions connected to the differentially abundant proteins (DAPs) via protein ratios in the LP and ACJ groups. The results will enable the classification of proteins as per Eukaryotic Orthologous Group (KOG) notation. 34. Protein classification: a. Obtain the FASTA format of protein list from the online server UniProt. b. Submit the list into eggNOG-mapper, eggNOG 4.5.1 database. c. Classify the proteins into their functional group using KOG annotation. 35. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG): a. Use ShinyGO 0.76.3 to obtain GO and KEGG pathway. Statistics Timing: variable This step explains how the data is being presented and the tests used to analyze the data. 36. Data presentation: a. Calculate the mean values and standard deviation of body weight, SGR and FCR. b. Identify and remove any ‘outside’ values. c. Represent the data of body weight, SGR and FCR as mean values with standard deviation. d. Present growth performance as diet means ± standard error of the mean. 37. Post ANOVA: a. Carry out Dunn’s test with P- value < 0.05 at 95% confidence interval to compare the mean of each group with mean of every other group. 38. Statistical analysis: a. Use GraphPad Prism version 9.5.0 for Windows. 

Timing: 14–16 h. The production time will depend on the volume produced and the drying capacity. This step describes the homogenization of ingredients and extrusion to form feed pellets for the feeding trial. 1. Mixing of ingredients: a. Combine and mix all dry ingredients thoroughly. b. Add the oil and mix for 10 min. c. Dissolve the gelatin in in 22°C water (ratio 1:3) and warm it to 55°C in a conventional microwave while agitating every 30–60 s. d. Add the gelatin to the mixture and mix for 10 min until homogenized. e. Add the remaining amount of water to reach approximately 30% weight of all ingredients (see ). 2. Making of feed pellet: a. Cold pellet the mash into a 0.5 mm diameter using a P35A pasta extruder (Italgi, Italy). b. Regulate the speed of the screw and knifes to obtain 0.5 mm long pellets. c. Dry the pellets at 55°C until moisture content is 8%–10% (see ). d. Store the feed in a dry place at 4°C before feeding. 

Timing: 10 days This step allows for chemical analysis to determine the nutritional composition of fish feed. Prior to the analysis, grind all pellets into a fine powder. 3. Dry matter, ash, crude protein, and crude lipid analysis. a. Analyze using methods described in the European Commission Regulation No 152/2009. 4. Starch chemical composition: a. Hydrolyze the starch with α-amylase and amyl glucosidase-enzymes to glucose. b. Add α-amylase (0.1%–0.5% (w/w) of starch) at temperatures between 60°C and 90°C and optimal pH between 6.0 to 7.5 and amyloglucosidase (0.1%–0.3% (w/w) of starch) at temperatures between 55°C to 65°C and optimal pH between 4.0 and 5.0. c. Use RX4041 Randox Daytona+ to determine glucose concentration using methods described by McCleary, Solah, and Gibson. , 5. Gross energy composition: a. Use PARR 6400 Automatic Isoperibol Calorimeter (Parr Instruments, Moline, Illinois, USA) according to ISO 9831. Animal feeding stuffs, animal products, and feces or urine-Determination of gross calorific value-Bomb calorimeter method. 6. Total phosphorus content: a. Combust and decompose pellet (0.5–1.0 g) in a Start D microwave digestion system (MP-AES 4200, Agilent Technologies, USA) following manufacture guidelines and between the following ranges: 15–30 min at 150°C–200°C. b. Measure phosphorus content using a microwave plasma atomic emission spectrometer (MP-AES 4200, Agilent Technologies, USA). 

Timing: 28 days (4 weeks) This step describes the stocking density and amount as well as frequency of fish feeding for both the pilot experiment and feeding trial. 7. Treatment groups (pilot experiment): a. Prepare three 3.5 L tanks for three treatment groups: control high protein diet (HP), low protein (LP) and low protein diet with C. jadinii yeast ingredient (LPY). b. Stock ten 3 mpf zebrafish into each tank. c. Record the average initial body weight of the zebrafish from each group. 8. Treatment groups (feeding trial): a. Prepare five 3.5 L tanks for the five treatment groups: control diet (C) and 4 yeast containing diets (ICJ, ACJ, IWA, AWA). b. Stock twenty- 3 mpf zebrafish into each tank. c. Record the average initial body weight of the zebrafish from each group. 9. Feeding regime: a. Feed the fish at 3% body weight. b. Feed each tank three times a day at 08:00–09:00, 14:00–15:00 and 20:00–21:00. c. Record any mortality. 10. Sample collection: a. Collect 5 fish from the pilot group and 6 fish per group from the feeding trial on day 14 and day 28. b. Weigh and record the average body weight. c. Euthanize the fish using cold ice treatment. d. Collect the intestine samples for proteomic analysis. e. Freeze the samples with liquid nitrogen. f. Store at −80°C. 

Timing: 2 h This step calculates the fish growth rate from the pilot study and feeding trial at days 14 and 28. The results can be used to determine if different components of the feed affect the growth rate of the fish. 11. Specific growth rate (SGR): a. Calculate the growth rate according to the following equation: S G R  = ln ( F B W ) − ln ( I B W ) d a y s × 100 Where, FBW = final body weight in g fish –1 ; IBW = initial body weight in g fish –1 . 12. Percentage body weight gain: a. Calculate the percentage of body weight gain using the equation: W e i g h t g a i n  = 100 × F i n a l w e i g h t ( g ) − I n i t i a l w e i g h t ( g ) I n i t i a l w e i g h t ( g ) 13. Feed conversion ratio (FCR): a. Calculate FCR using the equation: F C R = [ T o t a l f e e d i n t a k e ( g / D M f i s h − 1 ) ] ( F B W − I B W ) Where DM = dry matter; FBW = final body weight in g fish –1 ; IBW = initial body weight in g fish –1 . 

Timing: 70 min for step 1 4 , 24 h for incubation, 20 min for centrifugation, 10 min for washing, variable for drying This step determines the protein concentration of the intestinal samples of the zebrafish. 14. Homogenization of Frozen Intestinal Samples: a. Homogenize frozen intestine samples with 200 μL of SDS lysis buffer, 0.5 M TEAB buffer (pH 8.5), and 1× Protease Inhibitor cocktail. b. Incubate the homogenized samples at 90°C for 30 min, followed by cooling on ice for 5 min. c. Centrifuge the lysed samples at 14,000 × g for 20 min at 4°C. CRITICAL: Ensure thorough homogenization and efficient centrifugation for optimal results. 15. Protein Precipitation and Pellet Collection: a. Transfer the supernatant (containing proteins) to a new Eppendorf tube. b. Add four volumes of ice-cold acetone to the protein samples. c. Incubate the samples at −20°C for 24 h. d. Centrifuge the samples at 14,000 × g for 10 min at 4°C, collect the pellets. e. Wash the pellets twice by centrifugation at 14,000 × g at 4°C for 10 min with four volumes of ice-cold acetone. f. Dry the pellets by evaporation at 22°C to remove all liquid. CRITICAL: Handle pellets carefully to avoid loss of material. 16. Storage and Transport: a. Store the dried pellets at −80°C. 17. Resolubilization and Protein Concentration Estimation: a. Resolubilize the pellets in 200 μL of 5× SDS lysis buffer. b. Take aliquots for protein concentration estimation using Qubit 3.0 Fluorometer and Qubit Protein Assay Kit. CRITICAL: Accurate protein concentration estimation is crucial for downstream proteomics analysis. 

Timing: variable This step involves digesting 100 μg of protein from intestinal samples using the S-Trap micro column or mini (for >100 μg of protein) and utilizing Pierce quantitative colorimetric assay to determine peptide quantification. 18. Sample Preparation: a. Adjust the sample volume to equal amount of S-Trap SDS lysis buffer (5% SDS, 50 mM TEAB pH 8.5; make by diluting 2× lysis buffer 1:1 with 18.2 MΩ water). b. Add 3.5 μL of 100 mM TCEP for reduction. c. Incubate at 55°C for 10 min and cool to 22°C. d. Add 7 μL of 200 mM MMTS for alkylation. e. Incubate at 22°C for 10 min. f. Add 7 μL of 12% phosphoric acid to the solubilized, reduced, and alkylated sample. g. Add 420 μL of S-Trap binding buffer (90% MeOH, 100 mM triethylammonium bicarbonate buffer (TEAB, pH 7.1) to the acidified lysate. h. Transfer the solution into the S-Trap micro column in 200 μL increments. i. Centrifuge for 30 s-1 min at 4,000 × g . j. Keep the flow-through at −20°C. 19. Washing: a. Wash the trapped proteins by adding 150 μL S-Trap binding buffer to the column and centrifuge. b. Repeat step 19a. 4 times. 20. Trypsin Digestion: a. Add trypsin digestion buffer to each column based on the ratio (1:25 w/w) of trypsin to protein. b. Mini: Add 125 μL of digestion buffer containing trypsin to each column. Micro: Add 20 μL of digestion buffer containing trypsin to each column. c. Spin down the trypsin digestion buffer through the column at 4000 × g for 30 s, then transfer the filtrate back into the column matrix. This is to ensure that the trypsin buffer has coated the matrix and ensure sufficient buffer for incubation. d. Cap the spin column and incubate at 47°C for 1–2 h. 21. Elution: a. Mini: Sequentially add 80 μL of 50 mM TEAB, 80 μL of 0.2% aqueous formic acid, and 80 μL of 50% ACN with 0.2% FA and elute by centrifuging at 4k × g , for 30s-1 min, until all the solution has eluted. b. Micro: Sequentially add 40 μL of 50 mM TEAB, 40 μL of 0.2% aqueous formic acid, and 35 μL of 50% ACN with 0.2% FA and elute by centrifuging at 4k × g , for 30s-1 min, until all the solution has eluted. 22. Speedvac Drying: a. Dry the elutions using a Speedvac. Note: Micro <10–50 μg, Mini 100–300 μg. 23. Pierce Quantitative Colorimetric Peptide Assay: a. Prepare the standards provided by the kit and sample dilution as below in 1.5-mL tube. 24. Prepare Working Reagent (WR): a. Mix the following reagents in a 1.5-mL tube to create the Working Reagent (WR): i. 50 parts (50%) Colorimetric Peptide Assay Reagent A. ii. 48 parts Colorimetric Peptide Assay Reagent B. iii. 2 parts Colorimetric Peptide Assay Reagent C. iv. Add 180 μL of the prepared WR to each well of a 96-well microplate. Note: WR can be stored at 22°C for ≤ 30 min (keep in the dark before use). 25. Standard and Sample Addition: a. Add 20 μL of each replicate of the standard and sample into a 96-well microplate (total 3 replicates). CRITICAL: Avoid introducing air bubbles to each well. 26. Peptide Assay Working Reagent: a. Add 180 μL of the Peptide Assay Working Reagent (dye) to each well. b. Mix the plate thoroughly on a plate shaker for 30 s to 1 min. c. Cover the plate and incubate at 37°C for 15 min. 27. Absorbance Measurement: a. Measure the absorbance of the standards and samples at 480 nm. 28. Standard Curve Preparation: a. Prepare a standard curve by plotting the average Blank-corrected 480 nm measurement for each BSA standard vs. its concentration in μg/μL. b. Use the standard curve to determine the peptide concentration of each sample. 

Timing: 1 day This step enables the quantification of proteins using isobaric tags. 29. iTRAQ Labeling: a. Use one iTRAQ sample set containing samples from the control (C) and yeast supplemented (ACJ) diets. b. Employ the iTRAQ Reagents 8-plex kit (SCIEX, Foster City, USA) to label the digested peptides. c. Speedvac-dry the peptides and re-dissolve in 30 μL of dissolution buffer (0.5 M TEAB, pH 8.5) for labeling with iTRAQ reagent. 100 μg of peptides from each sample were used for each labeling reaction. d. Pool each sample set together according to the manufacturer’s recommendation iTRAQ Reagents - 8plex protocol . e. Lyophilize the pooled sample. 

Timing: 2 days 30. First-Dimension Separation: a. After lyophilization of the peptide samples, perform 2D LC-MS/MS. Dissolve the peptides in 20 mM ammonium formate in water, pH 10. b. Utilize high pH reversed-phase high-performance liquid chromatography unit (RP-HPLC) (1290 Infinity LC system; Agilent, Santa Clara, CA, US) equipped with a C18 column (WATERS Xbridge C18, 3.5 μm, 3.0 mm × 150 mm; Waters Milford, Massachusetts, US) for the first-dimension separation. c. Use mobile phase A (20 mM ammonium formate in water, pH 10) and mobile phase B (20 mM ammonium formate in 80% ACN, pH 10). d. Apply the following gradient elution profile: i. 0%–0% B for 10 min. ii. 0%–60% B for 60 min. iii. 60%–60% B for 5 min. iv. 60%–100% B for 1 min. v. Continued at 100% B for 5 min. vi. 100%–0% B for 1 min. vii. Subsequently, maintain at 0% B for 14 min. e. Collect the eluted fractions in 96-well, V-bottom plates at a flow rate of 0.5 mL min −1 . f. Combine eluted fractions into 10 concatenated fractions and lyophilize. 31. Second-Dimension Separation: a. Dissolve the lyophilized samples in 2% acetonitrile in water. b. For MS/MS analysis, inject 2 μL of each of the 10 fractions independently into a ProteoCol C18P (3 μm 120 Å, 300 μm × 10 mm; Trajan) trap column at 4 μL/min for 6 min. c. Elute on an C18 analytical column (Acclaim PepMap 100C18, 3 μm particle size, 75 μm i.d. × 250 mm; Thermo Scientific) using a nanoLC 425 LC system (Eksigent) in Trap-Elute configuration. d. Use mobile phase A (0.1% formic acid) and mobile phase B (0.1% formic acid in acetonitrile) with a flow rate of 300 nL min −1 . e. Apply the following gradient elution profile: i. 5%–15% B for 60 min. ii. 15%–30% B for 60 min. iii. 30%–90% B for 3 min. iv. Continued at 90% B for 20 min. v. 90%–5% B for 2 min. vi. Subsequently, maintain at 5% B for 15 min. f. Acquire MS spectra using a TripleTOF 5600+ system (AB SCIEX, Framingham, Massachusetts, US) under positive ionization mode, in the mass range of 350–1250 m/z at high sensitivity mode with an accumulation time of 250 ms per spectrum. g. Select the 30 most abundant precursors between charge range +2 to +5 per duty cycle for MS/MS analysis (accumulation time: 100 ms), in the mass range of 100–1800 m/z. h. Employ 15 s dynamic exclusion for MS/MS analysis in high sensitivity mode (>15,000) with rolling collision energy and iTRAQ reagent collision adjustment settings turned-on. 

Timing: 20 h 32. Software and Database Search: a. Utilize ProteinPilot 5.0.2 software (AB SCIEX) for identification of iTRAQ-labeled proteins. b. Use the Paragon database search algorithm and the false discovery rate (FDR) cut-off functions. c. Set the search parameters as follows: i. Sample Type: iTRAQ 8plex (Peptide Labeled). ii. Cysteine Alkylation: MMTS. iii. Digestion: Trypsin. iv. Instrument: TripleTOF5600. v. Special Factors: None. vi. Species: None. vii. ID Focus: Biological Modification. viii. Database: Zebrafish reference proteome (2022_02 release, 46687). ix. Search Effort: Thorough. x. FDR Analysis: Yes. xi. Background Correction: Yes. xii. User Modified Parameter Files: Yes. 33. Protein Identification and Differential Abundance Analysis: a. Set the cut-off for protein identification at 1% FDR. b. Determine the differential abundance of proteins using paired student’s t-test. c. Define the criteria for selection of differentially abundant proteins (DAPs) as follows: i. Abundance-Increased Proteins: iTRAQ ratio ≥ 1.3 (P-value < 0.05). ii. Abundance-Decreased Proteins: iTRAQ ratio ≤ −1.3 (P-value < 0.05). 

Timing: 2 days This step analyses and interprets the functions connected to the differentially abundant proteins (DAPs) via protein ratios in the LP and ACJ groups. The results will enable the classification of proteins as per Eukaryotic Orthologous Group (KOG) notation. 34. Protein classification: a. Obtain the FASTA format of protein list from the online server UniProt. b. Submit the list into eggNOG-mapper, eggNOG 4.5.1 database. c. Classify the proteins into their functional group using KOG annotation. 35. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG): a. Use ShinyGO 0.76.3 to obtain GO and KEGG pathway. 

Timing: variable This step explains how the data is being presented and the tests used to analyze the data. 36. Data presentation: a. Calculate the mean values and standard deviation of body weight, SGR and FCR. b. Identify and remove any ‘outside’ values. c. Represent the data of body weight, SGR and FCR as mean values with standard deviation. d. Present growth performance as diet means ± standard error of the mean. 37. Post ANOVA: a. Carry out Dunn’s test with P- value < 0.05 at 95% confidence interval to compare the mean of each group with mean of every other group. 38. Statistical analysis: a. Use GraphPad Prism version 9.5.0 for Windows. 

The anticipated outcomes of the protocol encompass a comprehensive understanding of the effects of dietary interventions on various aspects of fish physiology and molecular pathways. Through meticulous execution of the outlined methodology, researchers can expect to produce valuable data elucidating critical insights into fish growth and survival dynamics, as well as protein expression patterns in response to different dietary regimes. Specifically, the protocol aims to shed light on the impact of dietary composition on fish growth parameters and mortality rates. Researchers can anticipate generating data detailing trends in growth performance indicators, such as body weight gain, specific growth rate (SGR), and feed conversion ratio (FCR) over designated time intervals. These results will offer valuable insights into the efficacy of different dietary formulations in promoting optimal growth and health outcomes in fish populations. In our publication, the fish in the pilot study group fed the high protein diet had a significantly higher body weight at the end of 28 days when compared to the low protein diet group, but not with the low protein diet group with C. jadinii yeast. This suggests that C. jadinii poses no adverse effects on growth. In the feeding trial, all five diets yielded similar results with no significant differences in body weight gain, SGR and feed conversion ratio (FCR). In addition, the protocol characterizes the proteomic landscape of fish intestinal samples under various dietary conditions. Researchers can expect to quantify a substantial number of proteins and identify differentially abundant proteins (DAPs) in response to dietary interventions. By categorizing these proteins into higher abundance proteins (HAPs) and lower abundance proteins (LAPs), researchers can gain insights into the molecular mechanisms underlying dietary effects on protein expression. Furthermore, the protocol aims to annotate and classify the identified proteins, providing functional annotations and insights into their roles in cellular processes. Through EuKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, researchers can anticipate elucidating the functional categories and pathways associated with DAPs. This comprehensive annotation will enable researchers to unravel the biological significance of differentially expressed proteins and their implications for fish health and metabolism. In conclusion, the expected outcomes of the protocol encompass a multifaceted understanding of the interplay between dietary interventions and fish physiology at both the macroscopic and molecular levels. By generating comprehensive datasets and elucidating molecular mechanisms, the protocol aims to contribute significantly to the advancement of knowledge in aquaculture research and inform future dietary strategies for enhancing fish health and productivity. 

Regarding feed production Cold pelleting is not representative of the extruded feed use in aquaculture. However, it is still a valid and accepted approach to test the effects of novel ingredients. This is especially the case if we are testing ingredients that are sensitive to high temperature during conventional extrusion in feed manufacturing. Pellets produced through cold pelleting in a pelleting machine can have similar nutritional values, when formulated accordingly, and they can have similar technical quality regarding hardness and sinking velocities. Regarding proteomics The iTRAQ protocol enables the simultaneous quantification of multiple peptides in a single experiment, but it may not provide comprehensive coverage of the proteome. Certain proteins or peptides may be missed due to limitations in detection sensitivity or the inability to identify and quantify peptides with certain physicochemical properties. iTRAQ is more suitable for analyzing a limited number of samples (4 or 8) due to the availability of commercial multiplexing kits. For sample numbers greater than 8, they have to be divided into different groups and analyzed separately with different LC-MS runs. Only proteins commonly identified among different LC-MS runs can be compared, thus resulting in larger missing values. iTRAQ quantification can be inaccurate due to precursor ion selection issues—where ions with similar m/z values may enter the collision cell and contribute to background signals of the reporter ions—and the impurity of isotopes, as the isotopes used to label each channel are not 100% pure, leading to signal contamination in adjacent reporter channels. Variability in labeling efficiency and fragmentation during mass spectrometry can also lead to quantification errors. iTRAQ labeling requires a minimum amount of protein for successful labeling and detection. This requirement can be challenging when working with limited sample amount. 

Cold pelleting is not representative of the extruded feed use in aquaculture. However, it is still a valid and accepted approach to test the effects of novel ingredients. This is especially the case if we are testing ingredients that are sensitive to high temperature during conventional extrusion in feed manufacturing. Pellets produced through cold pelleting in a pelleting machine can have similar nutritional values, when formulated accordingly, and they can have similar technical quality regarding hardness and sinking velocities. 

The iTRAQ protocol enables the simultaneous quantification of multiple peptides in a single experiment, but it may not provide comprehensive coverage of the proteome. Certain proteins or peptides may be missed due to limitations in detection sensitivity or the inability to identify and quantify peptides with certain physicochemical properties. iTRAQ is more suitable for analyzing a limited number of samples (4 or 8) due to the availability of commercial multiplexing kits. For sample numbers greater than 8, they have to be divided into different groups and analyzed separately with different LC-MS runs. Only proteins commonly identified among different LC-MS runs can be compared, thus resulting in larger missing values. iTRAQ quantification can be inaccurate due to precursor ion selection issues—where ions with similar m/z values may enter the collision cell and contribute to background signals of the reporter ions—and the impurity of isotopes, as the isotopes used to label each channel are not 100% pure, leading to signal contamination in adjacent reporter channels. Variability in labeling efficiency and fragmentation during mass spectrometry can also lead to quantification errors. iTRAQ labeling requires a minimum amount of protein for successful labeling and detection. This requirement can be challenging when working with limited sample amount. 

Problem 1 Assembly of pelleted feed Inconsistent pellet length and/or formation of lumps with extruded pellets. The diameter of pellets is regulated by the dye at the end of the barrel of the pelleting machine. However, working with small dimensions presents some challenges to obtain a consistent pellet length. The length of the pellets is regulated by the speed of the screw and the rotation speed of the knives. Since the dye is so small, the pressure, and consequently the temperature at the end of the barrel, will increase. Elevated pressure and temperature will affect the exit speed of the pellets, leading to an uneven length of the pellets. Furthermore, the amount of water has an important influence during cold pelleting. Water contributes to the fluidity of the dough in the barrel, but most importantly, it promotes the binding capacity of the ingredients. This might be the most challenging step, since the amount of water needed depends on the water absorption capacity of the ingredients used and other factors such as temperature, processing time and even air humidity. Low levels of water in the mixture will decrease fluidity and reduce the binding capacity of the ingredients. Too much water will lead to “sticky” pellets, creating doubled size pellets, which represents a critical problem when working with small fish. Potential solution An advantage of cold pelleting is that the pellets can be re-pelleted (before drying) if the technical quality is not optimal. However, depending on the 22°C and air humidity, the moisture of the mixture can vary from batch to batch which needs to be taken into consideration. • Diameter and length of the pellet: The speed of the screw combined with the speed of the knives should be carefully tuned along the process. The temperature at the exit of the pelleting machine will increase gradually due to the pressure. Therefore, short breaks should be taken during pelleting to avoid overheating and the speed of the knife and/or the screw should be changed accordingly. • Consistency of the pellet: Keep in mind that when starting the pelleting, the end of the barrel is still at 22°C and the quality of pellets will change once the pressure and temperature increase. Therefore, before starting pelleting, one should let the mixture go through (without pelleting) until the temperature and pressure increase (the mix can be placed back to the extruder). If the strings are “too lumpy” or break too easily, it means more water should be added. If the strings are “sticky” and too flexible, it means that the water content is too high, and it will lead to the formation of clumps. If so, the strings should be air dried for some minutes before placing them back to the extruder. If in doubt, one should turn on the knife and perform a pre-test of the pellet size consistency. If most of the pellets are consistent in size, easy to separate and without dust (or have a flake-like appearance), one can start collecting the pellets. The pellets should be placed in a tray, forming a thin layer to avoid deformation of the pellets in the bottom. The pellets should be mixed a few times to facilitate the air drying and avoid the formation of clumps. Mixing is also recommended during the drying process. Problem 2 Assembly of pelleted feed There is too much dust/crumbles from the pellets. Some dust/crumbles are expected during the process of feed handling. However, large amounts of dust lead to feed waste. This situation is especially a problem when the amount of feed/ingredients are limited. Moreover, too much dust could lead to loss of feed particles during feeding thus the feed intake will be overestimated. Potential solution The dust/crumbles from pellets can have two origins during the process. • The pellets are overdried: The final water content should be between 8%–10% to reduce water activity and prevent microbial activity. However, when the pellets are too dry the chance of creating dust/crumbles increases and the quality of the pellets is reduced. The dry matter of the pellets should be measured several times during the drying, especially when the water content is close to 10%. • The water content of the mixture could be too low, leading to poor binding of the ingredients after drying: A small amount of water should be added to the mixture, but not to the point when pellets become too sticky (check ). Problem 3 Peptide and protein identification Absence of proteins by iTRAQ analysis. Potential solution • Employ other complementary techniques such as Western blotting, shotgun method or targeted mass spectrometry to verify the absence of specific proteins identified as potentially absent by iTRAQ analysis. • Optimize experimental conditions such as labeling protocols, sample preparation methods, and mass spectrometry parameters to improve quantification accuracy and minimize variability. This may involve testing different labeling reagents, optimizing digestion protocols, or adjusting collision energies during mass spectrometry analysis. • Employ sample enrichment techniques such as protein precipitation, ultrafiltration, or affinity purification to concentrate proteins from samples with low concentrations. These methods can help increase the protein content within the sample volume, meeting the minimum requirement for successful iTRAQ labeling. 

Assembly of pelleted feed Inconsistent pellet length and/or formation of lumps with extruded pellets. The diameter of pellets is regulated by the dye at the end of the barrel of the pelleting machine. However, working with small dimensions presents some challenges to obtain a consistent pellet length. The length of the pellets is regulated by the speed of the screw and the rotation speed of the knives. Since the dye is so small, the pressure, and consequently the temperature at the end of the barrel, will increase. Elevated pressure and temperature will affect the exit speed of the pellets, leading to an uneven length of the pellets. Furthermore, the amount of water has an important influence during cold pelleting. Water contributes to the fluidity of the dough in the barrel, but most importantly, it promotes the binding capacity of the ingredients. This might be the most challenging step, since the amount of water needed depends on the water absorption capacity of the ingredients used and other factors such as temperature, processing time and even air humidity. Low levels of water in the mixture will decrease fluidity and reduce the binding capacity of the ingredients. Too much water will lead to “sticky” pellets, creating doubled size pellets, which represents a critical problem when working with small fish. 

Inconsistent pellet length and/or formation of lumps with extruded pellets. The diameter of pellets is regulated by the dye at the end of the barrel of the pelleting machine. However, working with small dimensions presents some challenges to obtain a consistent pellet length. The length of the pellets is regulated by the speed of the screw and the rotation speed of the knives. Since the dye is so small, the pressure, and consequently the temperature at the end of the barrel, will increase. Elevated pressure and temperature will affect the exit speed of the pellets, leading to an uneven length of the pellets. Furthermore, the amount of water has an important influence during cold pelleting. Water contributes to the fluidity of the dough in the barrel, but most importantly, it promotes the binding capacity of the ingredients. This might be the most challenging step, since the amount of water needed depends on the water absorption capacity of the ingredients used and other factors such as temperature, processing time and even air humidity. Low levels of water in the mixture will decrease fluidity and reduce the binding capacity of the ingredients. Too much water will lead to “sticky” pellets, creating doubled size pellets, which represents a critical problem when working with small fish. 

An advantage of cold pelleting is that the pellets can be re-pelleted (before drying) if the technical quality is not optimal. However, depending on the 22°C and air humidity, the moisture of the mixture can vary from batch to batch which needs to be taken into consideration. • Diameter and length of the pellet: The speed of the screw combined with the speed of the knives should be carefully tuned along the process. The temperature at the exit of the pelleting machine will increase gradually due to the pressure. Therefore, short breaks should be taken during pelleting to avoid overheating and the speed of the knife and/or the screw should be changed accordingly. • Consistency of the pellet: Keep in mind that when starting the pelleting, the end of the barrel is still at 22°C and the quality of pellets will change once the pressure and temperature increase. Therefore, before starting pelleting, one should let the mixture go through (without pelleting) until the temperature and pressure increase (the mix can be placed back to the extruder). If the strings are “too lumpy” or break too easily, it means more water should be added. If the strings are “sticky” and too flexible, it means that the water content is too high, and it will lead to the formation of clumps. If so, the strings should be air dried for some minutes before placing them back to the extruder. If in doubt, one should turn on the knife and perform a pre-test of the pellet size consistency. If most of the pellets are consistent in size, easy to separate and without dust (or have a flake-like appearance), one can start collecting the pellets. The pellets should be placed in a tray, forming a thin layer to avoid deformation of the pellets in the bottom. The pellets should be mixed a few times to facilitate the air drying and avoid the formation of clumps. Mixing is also recommended during the drying process. 

Assembly of pelleted feed There is too much dust/crumbles from the pellets. Some dust/crumbles are expected during the process of feed handling. However, large amounts of dust lead to feed waste. This situation is especially a problem when the amount of feed/ingredients are limited. Moreover, too much dust could lead to loss of feed particles during feeding thus the feed intake will be overestimated. 

There is too much dust/crumbles from the pellets. Some dust/crumbles are expected during the process of feed handling. However, large amounts of dust lead to feed waste. This situation is especially a problem when the amount of feed/ingredients are limited. Moreover, too much dust could lead to loss of feed particles during feeding thus the feed intake will be overestimated. 

The dust/crumbles from pellets can have two origins during the process. • The pellets are overdried: The final water content should be between 8%–10% to reduce water activity and prevent microbial activity. However, when the pellets are too dry the chance of creating dust/crumbles increases and the quality of the pellets is reduced. The dry matter of the pellets should be measured several times during the drying, especially when the water content is close to 10%. • The water content of the mixture could be too low, leading to poor binding of the ingredients after drying: A small amount of water should be added to the mixture, but not to the point when pellets become too sticky (check ). 

Peptide and protein identification Absence of proteins by iTRAQ analysis. 

Absence of proteins by iTRAQ analysis. 

• Employ other complementary techniques such as Western blotting, shotgun method or targeted mass spectrometry to verify the absence of specific proteins identified as potentially absent by iTRAQ analysis. • Optimize experimental conditions such as labeling protocols, sample preparation methods, and mass spectrometry parameters to improve quantification accuracy and minimize variability. This may involve testing different labeling reagents, optimizing digestion protocols, or adjusting collision energies during mass spectrometry analysis. • Employ sample enrichment techniques such as protein precipitation, ultrafiltration, or affinity purification to concentrate proteins from samples with low concentrations. These methods can help increase the protein content within the sample volume, meeting the minimum requirement for successful iTRAQ labeling. 

Lead contact Further information and requests for resources or reagents should be directed to and will be fulfilled by the lead contact, Kathiresan Purushothaman ( purushothaman.kathiresan@nmbu.no ). Technical contact Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Kathiresan Purushothaman ( purushothaman.kathiresan@nmbu.no ). Materials availability This study did not generate new unique reagents. Data and code availability Data from mass spectrometry proteomics can be found in ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXS040431. 

Further information and requests for resources or reagents should be directed to and will be fulfilled by the lead contact, Kathiresan Purushothaman ( purushothaman.kathiresan@nmbu.no ). 

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Kathiresan Purushothaman ( purushothaman.kathiresan@nmbu.no ). 

This study did not generate new unique reagents. 

Data from mass spectrometry proteomics can be found in ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXS040431. 

This work was funded by Foods of Norway, a Centre for Research-based Innovation at the Norwegian University of Life Sciences (NMBU), which is funded by the Research Council of Norway (SFI/NFR; 237841/O30 ), CRISPR-Cas9 editing (Research Council of Norway project no. 283541), and International Travel Grant, NMBU (for K.P.). 

Conceptualization, K.P., D.K., S.D.C.R., B.M.L., L.T.M., S.V., M.O., and C.M.P.; methodology, S.D.T.R.Q., S.D.C.R., A.B.G., B.M.L., D.K., and L.Q.; writing, K.P., D.K., S.D.T.R.Q., and S.V.; funding, M.O., K.P., and C.M.P. 

The authors declare no competing interests. 

