Lipoxygenase Enzymes, Oligosaccharides (Raffinose and Stachyose) and 11sA4 and A5 Globulins of Glycinin Present in Soybean Meal Are Not Drivers of Enteritis in Juvenile Atlantic Salmon (Salmo salar)

Soybean meal has been largely investigated and commercially used in fish nutrition. However, its inclusion levels have been carefully considered due to the presence of antinutritional factors, which depending on a series of factors might induce gut inflammation damaging the mucosal integrity and causing enteritis. Several strategies including genetic engineering have been applied attempting to reduce or eliminate some of the antinutritional factors. Accordingly, we assessed the intestinal health of juvenile Atlantic salmon fed high levels of speciality soybean genotypes with reduced-to-no content amounts of lipoxygenases, altered glycinin profile and reduced levels of oligosaccharides. No major signs of enteritis, only indication of enteritis progression, was noticed in the soybean meal-based diets illustrated by mild changes in distal intestine morphology. Whereas fish, fed fishmeal control feeds, displayed normal distal intestine integrity. Speciality soybean types did not improve intestinal health of juvenile Atlantic salmon suggesting these antinutrients are not drivers of the intestinal inflammatory process in this species. No additional benefits in terms of production performance or blood biochemistry were noticed in the speciality soybean types compared to the traditional soybean.

Among the fish species, salmon appears to be one of the most sensitive to the antinutritional factors presented in SBM developing a condition known as soybean meal-induced enteropathy, which exhibits similar changes as those described above [3,4,9,17]. Some salmon species such as pink salmon Oncorhynchus gorbuscha appears to be more resistant to antinutritional factors present in dietary SBM than chinook O. tshawytscha and Atlantic salmon S. salar [18]. In the early 2000's, Buttle et al. [19], suggested the binding mechanism of soybean agglutinin (lectin) to Atlantic salmon intestinal epithelium as a primary contributor to pathological changes in this tissue. Saponins are other top candidates of key antinutritional factors present in soybean meal. A dose-response study reported increasing inflammatory process in Atlantic salmon distal intestine with greater dietary soybean saponins [20].
Several strategies have been applied attempting to reduce or eliminate some of the antinutritional factors in SBM including extrusion, fermentation, pre-processing techniques, and genetic engineering. For example, extrusion with shorter barrel retention times and higher temperatures improved utilization of SBM-rich diets (52% SBM) in salmonids [21]. Another commercial strategy is to increase the protein fraction of SBM through concentration (SPC) or isolation (SPI). A relatively small effort has been done in the genetic space focusing on selecting non-GM SBM for specific genotypes in aquafeeds. Recently, the removal of trypsin inhibitor, lectin and allergen P34/Gly m Bd 30 k from a soybean cultivar failed to alleviate inflammatory processes in Atlantic salmon [9]. Collectively, these studies suggest the challenge in identifying specific antinutritional factors present in soybean responsible for enteritis induction and highlight the complexity of interactions with compounds present in other plant ingredients largely used in aquafeed formulations. As a result, the salmon aquafeed industry has adopted ingredient inclusion limits and more processed soy protein products such as soy protein concentrate and isolate. However, from a cost-effective perspective, finding approaches to minimize or eliminate the soybean-induced enteritis in fish nutrition is worthwhile.
As part of the Australian Soybean improvement program, CSIRO has developed speciality SBM genotypes with reduced-to-no content amounts of lipoxygenases [22], altered glycinin profile [23], and reduced levels of oligosaccharides for human consumption. Accordingly, we assessed the intestinal health of juvenile Atlantic salmon fed high levels of these speciality SBM genotypes.

Formulations and Feed Manufacture
Dietary treatments are presented in Table 1. A fish meal-based diet (45%) was used as a control treatment, whereas the experimental diets contained 29% of fishmeal and 30% of SBM from three distinct genotypes of similar genetic background and matched as closely as possible for protein content: standard soybean meal (STD SBM); a soybean genotype homozygous for the gy4 allele conditioning null 11sA4 and 11sA5 globulins of Glycinin, homozygous for the l × 1, l × 2 and l × 3 alleles conditioning absence of seed lipoxygenases and homozygous for the rs2 allele conditioning near absence of seed raffinose and stachyose (TLP SBM); and a soybean genotype homozygous for the gy4 allele conditioning null 11sA4 and 11sA5 globulins of Glycinin (11sA4 null SBM).
All macro ingredients were milled to <750 µm, and well mixed with the remaining dry ingredients, and then extruded through a Baker-Perkins MPV24 twin-screw extruder. Each feed was manufactured using a 1.5 mm Ø die (~2.5 mm Ø pellets) using standard CSIRO Extrusion protocols ( Table 2). The pellets were dried at 60 • C for 12 h, after which they were vacuum infused with their specific allocation of oil. All feeds were kept in frozen storage (−20 • C) throughout the feeding trial. All uneaten feed was removed from the collection tank of all treatments 1 h following feeding, and the collected waste feed was then dried to allow calculation of apparent feed intake and feed conversion ratio.

Experimental System and Feeding Trial
Sixteen tanks with 300 L at Bribie Island Research Centre (BIRC, Queensland, Australia) were used in this experiment with~3 L/min flow of continuously aerated, recirculating freshwater at 15 • C ± 0.16 (mean ± SEM). Photoperiod was set at 12L:12D. Twenty-five salmon of 37.3 g ± 0.42 (mean ± SD) were randomly allocated to each tank. Fish were fed to apparent satiation twice daily for 56 days. This research was approved by the CSIRO Queensland Animal Ethics Committee-AEC Number: 2018-44.

Analytical Methods
Chemical analyses were carried out to confirm proximate composition of dietary treatments, and main ingredients (Tables 1 and 3) [24]. Samples were dried at 105 • C for 12 h to determine gravimetrically dry matter and followed by ashing at 550 • C for 12 h. Total nitrogen was measured by combustion (CHNS auto-analyzer, Leco Corp., St. Joseph, MI, USA) and crude protein was calculated by nitrogen conversion (%N × 6.25). Total lipid was gravimetrically determined via Folch extraction [25]. Finally, gross energy was measured via an adiabatic bomb calorimeter (Parr 6200, Par Instrument Company, Moline, IL, USA). Total amino acid (TAA) quantification was performed by mass detection following high performance reverse-phase liquid chromatography with pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl (AQC). Analyses were undertaken on a Shimadzu Nexera X2 series UHPLC (Shimadzu Corporation, Kyoto, Japan), coupled with a Shimadzu 8030 Mass Spectrometer using a modification of the Waters AccQ-tag system (Waters Corporation, Milford, MA). Bovine serum albumin (BSA) (ICN), milk powder (NIST SRM 1549a) and a well characterized aquafeed were used as reference materials. Samples or reference materials were hydrolyzed using phenolic 6N HCl at 112 • C according to the protocol for complex feed samples outlined by Waters Corp. (1996).

Production Parameters
The following production parameters were calculated for the 28 and 56 days of the feeding trial; feed conversion ratio (FCR), hepatosomatic index (HSI), and condition factor (K). No differences in production performance parameters were observed among the dietary treatments throughout the feeding trial.

Histology
Upon completion of the feeding trial, three fish per tank were randomly selected and euthanized via AQIS overdose (~70 ppm). The distal intestine, i.e., last 2 cm section of the intestine, of each fish was sampled and stored in Davidson's solution for 24 h, and then transferred to 70% ethanol until further analysis. For each fish sample, the distal intestine was divided into three sections and gradually dehydrated in ethanol, clarified in benzene and embedded in paraffin. As a result, a complete intestinal annular ring from each fish (nine per treatment) was cut into three sections (n = 36) and mounted onto individual glass slides for histological assessment.
Transversal sections of 3 µm were cut using a rotary microtome (Leica RM2245), stained with hematoxylin and eosin (H&E). The slides were blind examined after randomization, using the Zeiss Axiocam light microscope. The pictures were taken using the camera function on the Zeiss Axiocam microscope and then processed and analyzed using Zeiss Zen Light (Version 3.1) image analysis software.
To assess the degree of intestinal damage, a semiquantitative scoring system was used. In this scoring system, three parameters were quantified independently based on [10]: (1) the appearance and length of mucosal folds (MF); (2) the degree of widening of the lamina propria (LP); (3) the abundance of goblet cells (GC). For each of these parameters a score was given on a scale of 1 to 5. An increasing score value represents a greater degree of intestinal damage.

Statistical Analysis
All data were analyzed for normality and equality by Levene's tests, respectively, and then subjected to one-way ANOVA (analysis of variance, NCSS 12.0). When significant effects were identified, the post-hoc Tukey's HSD pairwise comparison test was used to determine difference among means with a significance level of 0.05.

Results
Juvenile Atlantic salmon fed the fishmeal control dietary treatment displayed normal distal intestine integrity ( Figure 1A). There were no major signs of enteritis, only an indication of enteritis progression was noticed in the SBM-based diets illustrated by mild changes in distal intestine morphology, including reduced number and length of mucosal folds, enlargement of the apical zone of mucosal folds, thickening of lamina propria, and changes in abundance of goblet cells ( Figure 1B-D). The removal of lipoxygenases, 11sA4 and A5 globulins of glycinin, and oligosaccharides from SBM failed to prevent morphological changes linked to the inflammatory process. Histology scoring demonstrated statistically higher scores of goblet cells and lamina propria in the SBM treatments than the fishmeal control (Table 4). Scoring of mucosal folds was higher in the speciality SBM groups compared to the control group.
Blood biochemistry was largely unaffected by the dietary treatments. Out of the sixteen parameters analyzed, only albumin and protein were statistically higher in TLP SBM than STD SBM (Table 5).   Survival was high across the dietary treatments (97-100%; Table 6). No differences in production performance parameters, including CV, final weight, FCR, K, and HSI were noticed between the fishmeal control group and the SBM-based groups at day 28 and day of feeding trial. Mucosal fold 1.9 ± 0.5 b 2.7 ± 0.7 ab 2.9 ± 0.6 a 3.3 ± 0.7 a 0.001 Table 5. Sixteen blood chemistry parameters of juvenile Atlantic salmon fed a fishmeal control diet (FM control) and different soybean meal types-based diets (STD SBM-standard soybean meal, C-TLP SBM-triple null soybean meal absent of seed lipoxygenases and homozygous for the rs2 allele conditioning and near absent of seed raffinose and stachyose, and D-11sA4 null SBM-soybean meal conditioning null 11sA4 and 11sA5 globulins of Glycinin) for 56 days.  Table 6. Production performance at 28 and 56 days of juvenile Atlantic salmon fed a fishmeal control diet (FM control) and different soybean meal types-based diets (STD SBM-standard soybean meal, C-TLP SBM-triple null soybean meal absent of seed lipoxygenases and homozygous for the rs2 allele conditioning and near absent of seed raffinose and stachyose, and D-11sA4 null SBM-soybean meal conditioning null 11sA4 and 11sA5 globulins of Glycinin).

Discussion
Soybean meal-induced enteritis was not observed in juvenile Atlantic salmon, despite the high dietary SBM inclusion level of 30%. Nevertheless, intestinal health impairment was characterized by mild changes in distal intestine morphology (i.e., changes in length, shape, and number of mucosal folds, changes in thickness of lamina propria and changes in number of goblet cells), indicating enteritis progression. Speciality soybean types lacking 11sA4 and A5 globulins of Glycinin, lipoxygenases and oligosaccharides did not further reduce the intestinal inflammatory process compared to STD SBM, although inflammation was mild overall. Similarly, a recent thorough study removing three proteinaceous antinutrients, namely trypsin inhibitor, lectin and the allergen P34/Gly m Bd 30 k, from soybean meal did not mitigate enteritis in Atlantic salmon [9]. The authors suggested extrusion technology inactivated these compounds during feed manufacturing. Although soybean agglutinin, a type of lectin, has been reported to bind to Atlantic salmon intestinal epithelium contributing to pathological changes in gut health, the study did not describe the feed manufacturing procedure [19]. It is unlikely that extrusion technology inactivated the compounds investigated in the present study due to their chemical compositions and exposure to lower mechanical energy during extrusion conditions compared to those reported by [9] (specific mechanical energy69-80 vs. 1872-3060 kJ/kg, respectively).
Soybean-induced enteritis in Atlantic salmon has been widely reported. However, the main drivers of enteritis and their positive and negative interactions with other compounds present in feed formulations remain unclear. A summary of seventeen studies around soybean-induced enteritis in Atlantic salmon is provided in Tables 7-9. Due to the complexity of the matter, there is a wide breadth of experimental designs with different stocking densities, fish size, duration, water temperature, salinity, feeding ration and experimental systems. For example, most literature is focused on smaller fish ranging from 41-442 g and some on larger fish 500-927 g. Similarly, soybean type and pre-processing and dietary composition varies throughout the literature. Soybeans are from different parts of the globe and are under several pre-processing conditions such as dehulled, toasted, defatted, and solvent-extracted, included at various levels 8-34% in feeds containing crude protein of 35-47%, total lipids 23-31%, and gross energy 15-24 MJ/kg. Regardless of this variation in the experimental design, most histology analyses are focused on the distal intestine describing morphological changes of standardized parameters including mucosal folds, supranuclear vacuoles, lamina propria, eosinophilic granulocytes, sub-epithelial mucosa, and connective tissue, and continue to be one of the most reliable tools to detect this histopathological condition. Other parameters such as reduced feed intake and growth commonly used as indicators of enteritis are not as reliable with contradicting findings throughout the literature. Most studies highlight the inflammatory process in the intestine of Atlantic salmon fed soybean-based feeds leading to enteritis; however, there are exceptions where only minor to mild intestinal damage were reported, including the present findings. Collectively, these studies illustrate the challenge and high complexity in tackling this issue in fish nutrition. Understandably, the aquafeed industry does not prioritize this research topic and adopt a more conservative approach of using moderate inclusion levels of plant ingredients avoiding any potential intestinal health impairments caused by antinutritional factors. It is likely extremely difficult to identify the key antinutritional factors in the major plant ingredients and their interactions with compounds from other components of the formulations having the effect. Interestingly, the intestinal health research of fish fed soybean-based feeds has gone beyond the traditional highly carnivorous species reaching a wide range of species from different feed, salinity and water temperature preferences, including for example common carp [14,15], grass carp [26], kingfish [7,12], totoaba [5,6], and largemouth bass [8]. IBW = initial body weight in g; SGR = specific growth rates in percentage of body weight; FCR = feed conversion ratio; Temp. = temperature; Ref. = references; RAS = recirculating aquaculture systems; SW = seawater; FW = freshwater; * numbers represent the following species Atlantic salmon, Chinook salmon and pink salmon.   This is not the first study where animal growth was not impaired by dietary SBM displaying equivalent performance as the fishmeal SBM-free feeds. As Krogdahl et al. [27] has demonstrated, no detrimental growth effects in feeding 20% SBM to juvenile Atlantic salmon are present; however, more aggressive inclusion levels of 40% reduced growth are evident as compared to the fishmeal control feeds. Indeed, this pattern has been described with other fish species, including California yellowtail Seriola dorsalis [7]. Removal of certain antinutritional factors from SBM also did not affect Atlantic salmon growth response compared to the standard SBM [9]. Conservative inclusion levels of standard SBM at the expense of fishmeal appears to be suitable as long as no intestinal health impairment is noticed.
High levels of dietary SBM resulted in mild intestinal inflammation indicating enteritis progression. Speciality soybean types lacking lipoxygenases, altered glycinin profile and oligosaccharides did not improve intestinal health of juvenile Atlantic salmon suggesting these antinutrients are not drivers of the intestinal inflammatory process in this species. No additional benefits in terms of production performance or blood biochemistry were noticed in the speciality soybean types compared to the traditional soybean. The present findings contribute to and summarize the growing literature in the antinutrient space, providing more insights to future research.