Quantitative Proteomic Analysis Reveals Yeast Cell Wall Products Influence the Serum Proteome Composition of Broiler Chickens

With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.


Introduction
Consequent to bans on the use of antibiotic growth promoters (AGP) in animal husbandry, interest in alternative growth promoters has grown immensely. Yeast and its derivatives, such as the mannan-rich fraction (MRF) of the yeast cell wall (YCW), have emerged as viable alternatives to AGP and have been shown to impart several beneficial effects on animals and have application in the field of animal husbandry. Some noted effects of yeast-based products include modulation of the gut microbiome [1], reduced infection by pathogenic bacteria and beneficial stimulation of the host immune system [2][3][4][5][6][7][8].
Immunological responses triggered by the YCW are largely due to the action of two molecules, Mannose Oligosaccharide (MOS) and β-glucan which are highly abundant in YCW. YCW immunostimulation has been reported in a number of species including Int. J. Mol. Sci. 2022, 23, 11844 2 of 26 human [9], fish [7] and mice [10] as well as livestock such as cows [11], pigs [12,13] and broiler chickens [4] and has been shown to induce a range of immunological effects both in vivo and in vitro. These benefits include reducing the levels of pathogenic bacteria in the gut [14], enhancing growth of beneficial bacteria [15] and modulating the immune response [16]. MOS have been found to modulate the gut microbiome by acting as high affinity ligands offering competitive binding sites for mannose-specific type-1 fimbriae of pathogenic bacteria [17] and stimulating the production of mucins by goblet cells found in the digestive tract [14]. Mucins are the glycoprotein constituent of mucus [18] which are involved in the binding and clearance of bacteria from the intestine. This reduction in pathogenic bacteria can create a more favourable environment for beneficial bacteria such as Lactobacillus and Bifidobacterium spp. in the broiler intestine [19]. Thus, intestinal homeostasis depends on complex interactions between microbiota, intestinal epithelium, and host immune system [1].
The physiological effects of MOS can translate into improved performance of broilers. Several studies have suggested that birds fed MOS as a supplement to a basal diet had significantly improved weight gain when compared to those fed the basal diet alone [20,21]. A review of the different mechanisms of MOS and their subsequent effect on intestinal microflora, intestinal morphology, immunity, and performance of broilers are discussed in detail elsewhere [14]. With developments in mass spectrometry and bioinformatic technologies in the last decade [22], comparative proteomics has come to the forefront of proteomic research, including a number of reviews published highlighting its potential application in farm animal research [23][24][25]. However, despite advances in proteomic technologies, only a relatively small number of comparative proteomic studies have been conducted on avian serum in recent years [26][27][28][29][30][31].
Packialakshmi et al. revealed differential protein profiles among low abundance proteins in chicken plasma, following depletion of high abundant proteins, in an LPS challenge versus control study (n = 9 birds/group) using both LC-and MALDI-MS/MS analyses [32]. Chemokine CCLI10 and alpha 1 acid glycoprotein (A1AGP) were only detected in plasma from LPS-treated birds. Label-free proteomic analysis identified increased abundance of A1AGP, CATH-2 precursor, a heparinase precursor protein and reduced abundance of ISG12-2 in plasma following LPS-only exposure, leading the authors to propose the utility of plasma proteomic analyses to identify biomarkers of innate immunity and inflammation in chickens. Following albumin and IgG depletion from avian plasma, iTRAQ-based LC-MS/MS revealed 468 differentially abundant proteins (increased, 171; decreased, 297) in penguin sera, using an aspergillosis model system to investigate the proteomic, and inflammatory responses to Aspergillus fumigatus exposure [33]. It is notable that although 63 protein families were noted with increased abundance, and 17 individual proteins exhibited >4-fold abundance, while no significant detection of penguin complement proteins was evident. This suggests that complement does not play a role in the avian anti-Aspergillus response, as noted by [33]. Horvatić et al. used a tandem mass tag (TMT) approach linked to subsequent LC-MS/MS to quantitatively investigate the effect of LPS, and exposure time (up to 72 h), on specific protein abundance in chicken plasma (n = 12 chickens in total) [27]. Interestingly, these authors used plasma which had not previously been depleted of high abundance proteins prior to processing and LC-MS/MS. Overall, 87 significantly differentially abundant proteins were evident, of 418 total proteins detected, and identified including serum amyloid A (the major chicken acute phase protein), A1AGP and ovotransferrin in the LPS-treated cohort. Significantly elevated abundance of complement proteins was also observed by Horvatić et al. in the LPS-treated cohort of birds [27]. Using iTRAQ peptide labelling and LC-MS/MS, Shen et al. identified 239 differentially abundant proteins, when comparing laying and brooding hens (n = 6), out of a total of 1235 proteins detected in chicken serum following prior depletion of high abundance protein [34].
Although interest in, and use of, YCW-based products to improve agricultural outputs have grown enormously, to our knowledge no systematic and quantitative analysis of the serum proteome of supplemented animals has been investigated to gain an insight into  14 43 6.89 * Significant changes denote proteins which have changed in abundance with p < 0.05 in a two-sample t-test. In addition, statistically significant alterations (p < 0.05) in protein abundance are also expressed as log2fold changes in Tables 2-7, whereby log2(1) = twofold or 100% alteration in abundance, etc. ** Unique/Absent Changes denote proteins that were uniquely present in a treatment group or absent from a treatment group when compared to day-specific control. * Significant changes denote proteins which have changed in abundance with p < 0.05 in a twosample t-test. In addition, statistically significant alterations (p < 0.05) in protein abundance are also expressed as log2fold changes in Tables 2-7, whereby log2(1) = twofold or 100% alteration in abundance, etc. ** Unique/Absent Changes denote proteins that were uniquely present in a treatment group or absent from a treatment group when compared to day-specific control.
Several notable proteins showed changes in abundance in GHP1 samples. Osteocalcin (P02822) was uniquely detected in GHP1 samples on Day 21. Gastrokine-2 (A0A1D5PFM9) was uniquely present in GHP1 samples on Day 35. Transferrin receptor protein was significantly increased (p < 0.05) with a relatively large Log 2 fold change (1.7) on Day 35. Insulin-like growth factor binding protein 5 (F1ND88) was significantly decreased (p < 0.05) on Day 35. Proteins (n = 5) with involvement in carbohydrate metabolism were altered in abundance (p < 0.05) with GHP1 supplementation. These include Nphosphoglycerate kinase (F1NU17), glyceraldehyde-3-phosphate dehydrogenase (P00356) and mimecan (Q9W6H0) which were significantly increased (p < 0.05) in abundance in GHP1 samples. N-acetyl-alpha-glucosaminidase (A0A1D5NU78) was uniquely present on Day 7. Beta-hexosaminidase (F1NTQ2) was increased in abundance and approaching significance (p = 0.09) on Day 7 (Supplementary Table S1). α-enolase (A0A1D5PSH6) was significantly decreased (p < 0.05) on Day 21. α-amylase was increased in abundance and approaching significance (p = 0.08) on Day 7 (Supplementary Table S1 Table S1) and significantly decreased (p < 0.05) in abundance on Day 35 (Table 3) [8]. Proteins involved in nucleotide metabolism were significantly altered in abundance (p < 0.05) or unique to/absent from, GHP1 samples. Nucleoside diphosphate kinase (O57535) and cytidine/uridine monophosphate kinase 2 (R4GJC4) were uniquely present in GHP1 samples on Day 21 and Day 7, respectively. Adenylate kinase isoenzyme (P05081) was significantly increased (p < 0.05) on Day 21 and adenosine deaminase (Q5ZKP6) was significantly decreased (p < 0.05) on Day 21 and Day 35.      proteins involved in the immune system that were significantly increased in abundance in the serum of GHP2 supplemented broiler chickens. Highlighted lines and dots represent proteomic pathways within the immune system that contain proteins which have been increased in abundance with GHP2 supplementation. (C) Pathway map of 19 proteins involved in the immune system that were significantly increased in abundance or uniquely present in the serum of GHP3 supplemented broiler chickens. All data obtained using Reactome.org [35]. proteins involved in the immune system that were significantly increased in abundance in the serum of GHP2 supplemented broiler chickens. Highlighted lines and dots represent proteomic pathways within the immune system that contain proteins which have been increased in abundance with GHP2 supplementation. (C) Pathway map of 19 proteins involved in the immune system that were significantly increased in abundance or uniquely present in the serum of GHP3 supplemented broiler chickens. All data obtained using Reactome.org [35].
Several proteins involved in carbohydrate metabolism were also significantly increased in abundance in GHP 2 samples. Specifically, glyceraldehyde-3-phosphate dehydrogenase (P00356) and phosphoglycerate kinase (F1NU17) were significantly increased (p < 0.05) in GHP 2 samples on Day 7. Individual proteins of note also showed changes in abundance in GHP 2 samples. Osteocalcin (P02822), a marker for bone resorption, was uniquely identified on Day 21. Gastrokine-2 (A0A1D5PFM9) was uniquely present in the serum of GHP 2 treated birds on Day 35. Insulin-like growth factor II was significantly decreased (p < 0.05) in abundance on Day 35 (Table 5). Transferrin receptor protein 1 (F1NTM6) was increased in abundance and approaching significance (p = 0.06) (Supplementary Table S2) on Day 35. Proteins (n = 3) involved in vitamin A transport [36,37] were increased in abundance in GHP 2 samples. Transthyretin (P27731) was significantly increased in abundance on Day 35, Retinol binding protein 7 (E1C0M1) was uniquely present on Day 7 and Retinolbinding protein 4 (P41263) was increased in abundance and approaching significance (p = 0.08) on Day 21 (Supplementary Table S2). Several proteins involved in nucleotide metabolism were significantly altered in abundance (p < 0.05) in GHP 2 samples. These were 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (E1C8P2) which was uniquely present and adenylate kinase isoenzyme 1 (P05081) that was significantly increased (p < 0.05) on Day 7 (Table 4). Nucleoside diphosphate kinase (O57535) was uniquely present on Day 35 whereas adenosine deaminase (A0A1D5PDK4) was significantly decreased in abundance (p < 0.05) on Day 35 (Table 5). Finally, A1AGP was increased in abundance on Day 35 in samples from GHP 2 supplemented birds with a large fold change (Supplementary  Table S3). However, this change was not significant (p = 0.37).
Proteins (n = 2) involved in vitamin A transport increased in abundance in GHP3 samples. Retinol binding protein 4 (P41263) was increased in abundance and approaching significance (p = 0.06) on Day 21. Transthyretin (P27731) was significantly increased (p < 0.05) in abundance on Day 35 (Table 6). Many key proteins involved in the detoxification of ROS were significantly decreased (p < 0.05) in abundance or absent from GHP3 samples. Two catalases were absent from GHP3 samples on Day 7 and Day 21, respectively, catalase (A0A1D5PPU9) and catalase (Q5ZL24) ( Table 7). Peroxiredoxin-1 (P0CB50) was significantly decreased in abundance on Day 35. Glutathione peroxidase (F1NPJ8) was increased in abundance and approaching significance (p = 0.08 and 0.09, respectively) in GHP3 samples on Day 21 and Day 35 (Supplementary Table S5). Glutathione S-transferase (GST) (Q08392) was absent from GHP3 samples in all time points, and this protein was identified as GST alpha-class. A few proteins showed changes in abundance in GHP3 samples whereby transferrin receptor protein was significantly increased (p < 0.05) on Day 35 and gastrin releasing peptide (A0A1D5PXC4) was significantly decreased (p < 0.05) in abundance on Day 35.
GHP3-supplemented birds were supplemented an organic source of selenium in their diet instead of the inorganic source, selenium selenite, which was used for GHP1, GHP2 and control diets. To examine any potential effects of this change in selenium source, control and GHP3 samples were examined for a SeMet/SeCys substitution for methionine/cysteine using MAXQUANT [38]. Control and GHP3 samples were also searched for known selenoproteins. There was one protein identified to have SeMet/SeCys substitution in control samples (Supplementary Table S8A) which was SEC31 homolog B, COPII coat complex component (E1BXC8). The protein was detected in only one control sample with a sequence coverage of 3.6% and 1 peptide detected. Proteins (n = 4) were identified as having a SeMet/SeCys substitution in GHP3 samples (Supplementary Table S8B), coatomer subunit alpha (A0A1D5P185), golgin A4 (A0A1D5PNT3), nuclear factor related to kappaB binding protein (E1BZI6) and translocase of outer mitochondrial membrane 34 (F1P4X4). The sequence coverage of proteins identified was low, at 0.8-5.4%. Of the four proteins identified with a SeMet/SeCys substitution, three were identified in one GHP3 pooled serum sample obtained on Day 7. Using a list of previously identified selenoproteins obtained from Liu et al. [39] (Supplementary Table S7), one selenoprotein was identified in GHP3 and control samples, glutathione peroxidase (F1NPJ8) [39]. This protein was increased in abundance with p-value approaching significance on Day 21 (p = 0.08) and Day 35 (p = 0.09) (Supplementary Table S9). Selenoprotein F precursor (A0A1D5PFR6) was uniquely detected in GHP3 samples, but was not detected in GHP2, GHP1 and control samples. AIAGP was increased in abundance on Day 35 in samples from GHP3 supplemented birds with a large fold change (Supplementary Table S6), however, this was not significant (p = 0.36).

Overview
Quantitative proteomic analysis has revealed that diet supplementation with GHP1, 2 and 3 significantly alters the abundance of specific serum proteins which contribute to the immune status and health of the broiler chicken. Consistent alterations in protein abundance were seen for those involved in the complement component of the innate immune system, nutrient transport, oxidative stress, and selenium status-all of which contribute to the overall animal health and provide evidential support for the supplemental effects of GHP products.
Supplementation of broiler diet with GHP products had a detectable effect on the avian serum proteome with averages of 6.31-7.78% of proteins significantly changed in abundance (p < 0.05) or unique/absent in the serum of GHP 1-, 2-and 3-, treated birds. GHP1 samples showed the largest number of protein changes with 85 proteins significantly increased in abundance (p < 0.05) or uniquely present in comparison to control, and 63 proteins significantly decreased in abundance (p < 0.05) or absent in comparison to control. GHP3 samples showed the lowest total number of proteins changed with 65 proteins significantly increased in abundance (p < 0.05) or uniquely present in comparison to control, and 55 proteins significantly decreased in abundance (p < 0.05) or absent in comparison to control. However, sera from GHP3 supplemented birds showed the greatest number of proteins that were unique to a treatment in inter-treatment analysis and, as will be discussed, GHP3 showed greater complement activation as well as alterations to serum seleno-protein abundance which may indicate a substantial impact on broiler health status.

Individual Proteins of Note
Several individual proteins of note were seen to be significantly altered in abundance (p < 0.05) or present/absent in treatment groups with interesting links to several aspects in animal health. Gastrokine-2 is secreted by gastric mucosal cells. This protein binds gastrokine-1 and is involved in regulating homeostasis of gastric mucosa [40]. It has been documented that GHP feed supplementation can have effects on gastric mucosa [41]. Gastokine-2 was uniquely present in the serum of GHP1 and GHP2 samples on Day 35. One other protein involved in the regulation of gastric mucosa is gastrin releasing peptide (A0A1D5PXC4). Abundance of this protein was significantly reduced (p < 0.05) in GHP3 samples on Day 35. Changes in the abundance of these proteins would indicate that these GHPs may have an effect on the regulation of the gastric mucosa.
Osteocalcin (P02822), uniquely present in GHP1 and GHP2 samples on Day 21, is a marker for bone turnover and its presence in serum is an indicator of bone resorption [36]. The presence of osteocalcin, only in the serum of birds supplemented with GHP1 and GHP2, could indicate that these products affect bone formation in the early stages of life which is of critical importance in chicken husbandry. Leg problems are prevalent in broilers [37] and can lead to higher culling rates in commercial production systems which have real monetary effects on the producer [38].
In GHP1 samples, GSH-Px was also seen to be increased in abundance on Day 7, which was also accompanied by a significant increase (p < 0.05) in two other enzymes involved in the detoxification of ROS: glutaredoxin 3 (A0A1D5NW30) and peroxiredoxin 6 (F1NBV0). This increase in the abundance of proteins involved in ROS detoxification may not be due to increased oxidative stress, but rather the stimulation of the production of these proteins by feed constituents. However, elucidating the molecular mechanisms of redox homeostasis maintenance in poultry and farm animals has received limited attention and are poorly characterized. Nevertheless, determining effective means of mitigating redox stress through nutritional modulation are exciting areas of research gaining momentum [42].

Immunological Effects
As noted, GHP products can have numerous immunological effects on animals [2][3][4][5][6][7]. Across all treatment groups, there were significant (p < 0.05) increases in the level of a number of complement components in the serum of GHP-supplemented broilers harvested on Day 35, which would indicate stimulation of the complement cascade by the GHP feed constituents. Complement C4 precursor (A0A1D5P4P1), Complement C6 (B8ZX71) and Complement C7 (E1C6U2) were significantly increased (p < 0.05) in all treatments samples on Day 35. Complement C5 (E1BRS7) was increased in abundance and approaching significance in GHP2 samples and was significantly increased (p < 0.05) in abundance in GHP1 and GHP3 samples. Complement C2 was significantly increased (p < 0.05) in abundance in GHP3 samples only. Indeed, GHP3 samples showed the greatest level of complement stimulation. Increases in serum levels of complement components indicate a stimulation of the complement cascade by these GHP feed supplements.
A possible source of this complement stimulation by GHP products is through activation of the complement cascade by mannose binding protein (Q98TA4). This protein was significantly increased (p < 0.05) in abundance in GHP3 samples and increased and approaching significance in GHP2 (p = 0.2) and GHP1 (p = 0.162) samples on Day 35. Mannose binding protein, also called Mannose binding lectin (MBL) [43,44] is a C-type serum lectin [45] that binds mannose residues which results in C1-independent complement activation and binding leads to cleavage of C2 and C4 from their precursors by associated serine proteases [46]. Both C2 and C4 precursor were significantly increased in abundance (p < 0.05) in GHP3 samples. MBL may also interact directly with cell surface receptors which can initiate opsonophagocytosis [47] causing immunostimulation which could also explain the abundance rise in proteins which have been linked with the innate immune system. Interestingly, low serum concentrations of MBL has been linked to a greater susceptibility to infection [45]. There is little data available in the literature regarding stimulation of the chicken complement cascade by GHP feed supplementation. Slawinska et al. reported a downregulation of two genes influencing the complement system after in ovo administration of yeast-based prebiotics to broiler chickens followed by transcriptomic analysis [48]. Activation of the complement system by carbohydrate-based feed has been previously seen in aquaculture. Gilthead seabream were fed a diet supplemented with inulin, a branched carbohydrate comparable to β-glucan or MOS [49]. Supplementation resulted in significant increases (p < 0.05) in serum complement activity [50] which is in accordance with results found in the present study.
The increase in abundance of additional immunologically relevant proteins suggests immunostimulatory/immunomodulatory effects by these GHPs. One likely mediator of this immunostimulation is Surfactant Protein A (Q90XB2). This protein was uniquely present in GHP3 samples on Day 7, and is a C-type lectin receptor which is present in mucosal tissues and binds glycan ligands which can result in downstream immunological effects [51]. Carbohydrate recognition domains on this protein bind glycan residues resulting in innate immune stimulation [47] which could explain the increase in abundance of immune-related proteins observed. MOS-based products have been shown to elicit immunological responses without causing acute phase (fever) response [1]. Yet, the largest fold change was seen (Log 2 Fold change = 2.12) for A1AGP in LC-MS/MS analyses. This protein is in high abundance in serum and has been previously characterised as an acute phase protein [26]. Large fold changes in A1AGP were also seen between samples from GHP2 and GHP3 supplemented birds and control samples (Log 2 fold change = 3.69 and 1.86, respectively) although the p-value for these changes was not significant (p = 0.37 and 0.36, respectively). It is unlikely that the high fold changes noted for A1AGP indicate stimulation of the acute phase response because of the non-significant p-values (p < 0.05) seen in GHP2 and GHP3 as well as the lack of significant change (p < 0.05) in any other acute phase proteins (e.g., serum amyloid A). The data suggest that increases in the abundance of this protein may not represent an acute phase response but rather the natural variability of the serum level of this protein. Indeed, complementary A1AGP ELISA analysis of GHP1-treated and control samples revealed non-significant elevation in A1AGP in sera from GHP1-treated birds, in accordance with the results obtained from LC-MS/MS analysis.

Effects on Metabolism
With GHP supplementation, a number of proteins involved in carbohydrate and nucleotide metabolism were significantly altered (p < 0.05) in abundance across all treatment groups. Glyceraldehyde-3-phosphate dehydrogenase (P00356) was significantly increased (p < 0.05) in all treatment groups on Day 7. This protein is involved in glycolysis [52] and its increase could indicate a greater level of glycolysis in birds supplemented with these GHP products. Beta-hexosaminidase (F1NTQ2) was significantly increased (p < 0.05) in abundance in GHP3 samples and increased and approached significance (p = 0.08) in GHP1 samples on Day 7. This protein is involved in the release of N-acetylglucosamine and N-acetylgalactosamine from glycoproteins, which are in high concentration in the GHP [53] and its increase in abundance is likely a result of the increased glycoprotein substrate in the diet. Increase in the abundance of these proteins could indicate that the introduction of these feed supplements stimulated carbohydrate metabolism in broiler chickens. However, individual products had varying effects on the levels of carbohydrate metabolism proteins. GHP1 had a variable effect on carbohydrate metabolism. Proteins (n = 4), involved in carbohydrate metabolism were significantly increased in abundance (p < 0.05) or uniquely present in GHP1 samples. However, other proteins involved in carbohydrate metabolism showed a decrease in abundance such as α-Enolase (A0A1D5PSH6) and β-Enolase (P07322). GHP3 also had an inconsistent effect on the abundance of proteins involved in carbohydrate metabolism, with proteins both significantly increased (p < 0.05) and significantly decreased (p < 0.05) in abundance. Overall, the levels of proteins involved in carbohydrate metabolism were variable and, though significant changes (p < 0.05) were seen, no definitive effect on carbohydrate metabolism could be deduced.
Differential effects were also seen on proteins involved in nucleotide metabolism throughout all three products. Significant changes (p < 0.05) in the abundance of multiple proteins involved in this process would suggest an effect of GHP supplementation, but with proteins both increased and decreased in abundance, no common trend could be elucidated.

Vitamin A Transport
Vitamin A is of vital importance in the diet of the broiler chicken as it is not produced naturally by the animals and so must be obtained through the diet [54]. Deficiencies in broiler diets can lead to deterioration of reproductive [55] and immunological health [56,57]. Vitamin A is bound by Retinol binding protein 4 (RBP4) (P41263) which is in turn bound to Transthyretin (P27731) [58]. These proteins then transport Vitamin A throughout the animal. Retinol binding protein 7 (RBP7) (E1C0M1) is involved in the intracellular binding and transport of Vitamin A in cells [59]. RBP4 was significantly increased (p < 0.05) and RBP7 was uniquely identified in GHP1 samples on Day 7. Transthyretin was significantly increased on Day 35 in GHP2 samples and RBP7 was uniquely identified on Day 7, RBP4 was also increased in abundance and approached significance (p = 0.08) on Day 21. Transthyretin was significantly increased on Day 35 in GHP3 samples. Increases in the abundance of these proteins may indicate a greater abundance or availability of Vitamin A with the supplementation of these GHP products, however this requires further investigation. One common source of Vitamin A is β-carotene, a precursor to Vitamin A [60] that is naturally obtained through the consumption of carotenoid-producing organisms such as higher plants or photosynthetic microorganisms [61].

Effect of Organic Selenium Supplementation
Selenium is an essential element in the diet of broiler chickens and its supplementation has been seen to have beneficial effects on broiler health status and meat quality [62][63][64]. The dietary source of selenium can have an impact, as inorganic selenium is considered a pro-oxidant and organic selenium has been seen to have greater bioavailability [65][66][67]. Drip-loss has been linked to GSH-Px level [62]. GSH-Px was increased in abundance in all treatment groups with the largest fold-change noted in GHP3 samples where it was increased and approaching significance on Day 21 and Day 35. GSH-Px level has been previously shown to be influenced by selenium supplementation and a greater bioavailability of organic selenium from GHP3 would explain this increased GSH-Px level. A reduction in drip-loss and increase in serum GSH-Px has been reported previously in birds supplemented with organic selenium rather than its inorganic form [68,69]. This result was also matched in pigs with significantly higher (p < 0.01) levels of serum GSH-Px in organic selenium supplemented pigs at 0.2mg/kg and 0.3 mg/kg supplementation [70]. In this work, selenium was also supplemented to broiler diets at 0.3 mg/kg (inorganic selenium selenite for GHP1 and GHP2, Se yeast for GHP3).
The absence of the potentially pro-oxidative inorganic selenium in the diet of GHP3 supplemented birds can also be seen in the significant reduction or absence of proteins involved in the detoxification of ROS: Catalase (n = 2), A0A1D5PPU9 and Q5ZL24, which were also absent from GHP3 samples on Day 7 and Day 21, respectively. The protective effects of selenium have been seen in organs such as the liver and organic forms of selenium have been liked to reduced liver damage [66]. Glutathione S-transferase α (αGST) (Q08392) is an enzyme involved in the detoxification of reactive xenobiotics and is present in high concentrations in the cytosol of hepatocytes [71][72][73]. This enzyme conjugates glutathione to xenobiotics [74] and its presence in serum has previously been characterised as an indicative biomarker for acute hepatitis or liver damage [75]. αGST is absent from the GHP3 samples on Day 7, Day 21 and Day 35 which would indicate that this product may confer some hepatoprotective effect on the broiler chicken. This absence could be due to the reduced level of oxidative damage with an organic selenium source, the greater bioavailability of organic selenium leading to increased levels of GSH-Px which reduces oxidative damage or a dual effect of these two factors. Evidence suggests that organic selenium has a greater bioavailability than its inorganic forms [65] and this hepatoprotective effect could also be a result of the greater bioavailability of organic selenium allowing greater protective potential of enzymes that make use of the element. A greater bioavailability of organic selenium from GHP3 was not only supported by the increased levels of GSH-Px on all days, but also by the higher number of proteins detected with SeMet/SeCys substitutions and the unique presence of selenoprotein F precursor in the serum of GHP3 treated birds. An increased bioavailability might not only lead to increased benefits to the broiler chicken, but also to the producer and consumer.

Experimental Design, Sample Collection and Preservation
A total of 492 day-of-hatch male broiler chickens were used in the poultry feeding trial. Clean concrete-floor pens were used to house the birds in a medium scale trial facility on-site at Agri-Food Biosciences Institute (AFBI) (Belfast, UK). Animals were randomly split into four groups of 3 pens, with 12 pens in total (41 birds/pen; 123 birds/group) using a randomized complete block design ( Figure 4A). The pens were divided into four groups: group 1, fed a basal diet; group 2-4, fed a basal diet which included supplements GHP1, 2 and 3, respectively, at 1 kg/tonne in starter, grower and finisher diets. These supplements were mannan-rich fractions extracted from the yeast cell wall of Saccharomyces cerevisiae. Additional individual GHP details and the manufacturers recommended inclusion levels are published elsewhere (Alltech Inc., Lexington, KY, USA) [30]. Basal diets were prepared by a commercial feed mill and consisted primarily of wheat and soybean meal which met full dietary nutritional requirements [76]. Starter diets were fed from day 0 to day 10, grower diets from day 11-25 and finisher diets, day 26 to day 35. Feed and water were provided ad libitum throughout the study. Each pen was dressed in fresh litter for bedding from day zero. The temperature was initially set at 30 • C per day up to day 10 and then decreased linearly by 1 • C every second day. Birds received a lighting regimen of 16 h light and 8 h darkness until day 35. All conditions were kept uniform for all four groups. On days 7, 21 and 35, blood samples from necropsied birds were collected into 100 mL sterile sample cups (75.562.105; Sarstedt) and then transferred to BD Vacutainer ® blood collection tubes using 10 mL wide-bore serological pipettes (86.1688.010; Sarstedt). The whole blood was allowed to clot at room temperature for 30-60 min. The clot was removed by centrifugation at 4500 rpm for 10 min at 4 • C. The resulting serum supernatant was apportioned into 0.5 mL aliquots and snap frozen using liquid nitrogen. Aliquots were stored at −80 • C and thawed on ice before use.
All procedures were subject to the approval of the local Animal Welfare Ethics Review Board and subsequent approval by a Home Office Inspector. All procedures were carried out under the strict guidelines of the Animal (Scientific Procedures) Act 1986 [76].

ProteoMiner™ Serum Enrichment Evaluation
Individual and pooled avian sera were first cleared of precipitate by centrifugation (10,000× g, 10 min). Sera (200 μL) were then applied to the ProteoMiner™ Protein Enrichment kit (#1633006; BioRad) according to manufacturer's instructions. After loading and washing, 20 μL of either the provided ProteoMiner elution buffer or 0.1 M Tris-HCL, 6 M Urea and 2 M Thiourea pH 8.0 (LC-MS/MS Compatible Reagent; LCR) was applied to the column. Columns were then lightly vortexed for 5 s over 15 min. Caps were removed and columns placed in capped collection tubes and centrifuged (1000× g, 1 min). This elution step was repeated twice giving 60 μL of enriched serum sample. Enriched serum samples were stored −20 °C until LC-MS/MS analysis (Supplementary Figures S1-S3) which showed LCR gave improved protein detection.

Upgraded ProteoMiner™ Processing for Serum Enrichment
All pooled serum samples were first cleared of precipitate by centrifugation (10,000× g, 10 min). Sera (200 μL) were then applied to the ProteoMiner™ Protein Enrichment kit according to manufacturer's instructions. After washing, deionised water (200 μL) was applied to columns which were rotated end-over-end for 1 min and drained through centrifugation (1000× g, 1 min). 20 μL of LCR was applied to the column. Columns were then lightly vortexed for 5 s over 15 min. Caps were removed and columns placed in capped All procedures were subject to the approval of the local Animal Welfare Ethics Review Board and subsequent approval by a Home Office Inspector. All procedures were carried out under the strict guidelines of the Animal (Scientific Procedures) Act 1986 [76].

ProteoMiner™ Serum Enrichment Evaluation
Individual and pooled avian sera were first cleared of precipitate by centrifugation  Figures S1-S3) which showed LCR gave improved protein detection.

Upgraded ProteoMiner™ Processing for Serum Enrichment
All pooled serum samples were first cleared of precipitate by centrifugation (10,000× g, 10 min). Sera (200 µL) were then applied to the ProteoMiner™ Protein Enrichment kit according to manufacturer's instructions. After washing, deionised water (200 µL) was applied to columns which were rotated end-over-end for 1 min and drained through centrifugation (1000× g, 1 min). 20 µL of LCR was applied to the column. Columns were then lightly vortexed for 5 s over 15 min. Caps were removed and columns placed in capped collection tubes and centrifuged (1000× g, 1 min). This elution step was repeated twice giving 60 µL of enriched serum sample. Enriched serum samples were stored at −20 • C until further processing.

Protein Digestion and Q-Exactive Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Analysis
Enriched serum samples were removed from −20 • C and allowed to reach room temperature. Any precipitate was resuspended through light vortexing. Samples were tested for protein concentration by Bradford assay [77]. 10 µL of the enriched sample was then separated. Remaining samples were returned to −20 • C storage. The pH of the sample was then adjusted to between 8.5 and 9.0 using 3 to 5 volumes of Label-Free Solubilisation Buffer. The volume of diluted enriched serum sample corresponding to 5 µg protein was then removed for further processing. Protein samples (5 µg each) were then brought to 10 mM Dithiothreitol (DTT) using 0.05 M DTT and incubated at 56 • C for 30 min. Samples were then brought to 25 mM Iodoacetamide (IAA) using 0.11 M IAA. Lys-C (0.5 µL) was then added to samples at 1:100 ratio (Protease: Protein) and samples were incubated at 37 • C for 4 h. 50 mM ammonium bicarbonate (3 volumes) was then added to samples. Trypsin (0.8 µL) was then added to samples at 1:25 ratio (Protease: Protein). Protease Max (0.45 µL) was then added. Samples were incubated at 37 • C overnight. Using Sample Buffer (0.33 volumes), samples were then diluted. Peptide samples were evaporated to dryness in a SpeedVac™ (DNA 120; Thermo Scientific, Waltham, MA, USA) and stored at −20 • C prior to ZipTip treatment and transfer to Q Exactive mass spectrometer vials for injection ( Figure 4B). Peptide samples were then analysed using a Thermo Scientific™ Q Exactive connected to a Dionex Ultimate™ 2000 (RSLCnano) chromatography system. Each sample was loaded onto an EASY-Spray™ PepMap RSLC C18 column (75 µm × 500 mm) and separated by an increasing acetonitrile gradient over 120 min at a flow rate of 250 nL/min [78,79]. The mass spectrometer was operated in positive mode with MS n carried out on the 15 most abundant precursor ions at each time point. Singly charged ions were excluded from analysis.

Label-Free Quantitative (LFQ) Proteomic Analysis Using MaxQuant and Perseus
Peptide-Spectral mapping and protein peptide matching of raw files from Q-Exactive analysis of LFQ proteomic analysis were carried out using MaxQuant (version 1.5.7.0; http://maxquant.org), accessed on 9 April 2021 [80]. This utilised the Andromeda database search to match MS/MS data with the Gallus gallus 9031 reference proteome (UP000000539-Gallus gallus) from UniProt (http://uniprot.org, accessed on 9 April 2021). Search parameters included: peptide tolerance (ppm) of 20 for first search and 4.5 for main search, carbidomethylation of cysteine as a fixed modification, oxidation of methionine and acetylation of N-termini as variable modifications, maximum 2 missed cleavage sites, and a minimum 1 peptide detected per protein. The maximum protein/peptide false discovery rates were set at 1% based on a comparison to a reverse database (decoy database). The LFQ algorithm was used to generate normalised spectra intensities to infer relative protein abundance. Subsequently, protein groups were exported and processed in Perseus (version 1.5.6.0; http://coxdocs.org/doku.php?id=:perseus:start; accessed on 15 April 2021) for data filtering [81]. Proteins (i) only identified by site (ii) only identified by modification site, or (iii) identified by the decoy database were removed. Extracted LFQ intensities measured for each run were grouped according to treatment and time point. Protein abundances were log 2 transformed.

A1AGP Immunoassay
A chicken A1AGP enzyme-linked immunosorbent assay (ELISA) (ab157690) (abcam ® ) was used for complementary analysis of serum A1AGP levels accordingly to the manufacturer's instructions.

Software and Statistical Analysis
Overall, 36 pooled serum samples were prepared, extracted, and analysed by LFQ proteomics. These comprised of Control pool (Day-specific) and 3 Test pools at day 7, 21 and 35 (4 × 3 × 3 = 36). Thus, at each time point (Day 7, Day 21, and Day 35), treatment groups were compared to each day-specific control group creating three comparator groups (GHP1 vs. Control, GHP2 vs. Control and GHP3 vs. Control). A 2-sample t-test was performed to identify proteins with significant differences (p < 0.05) within each comparator group. Proteins uniquely detected in a treatment group and not detected in control, or proteins not detected in treatment and detected in control groups, were identified and tabulated. Pathway mapping was carried out using Reactome Pathway Database (https://reactome.org; accessed on 30 April 2021). Proteins were specifically analysed for features and/or functions in UniProt (http://uniprot.org).
All statistical analyses were carried out using GraphPad Prism (version 5.0; GraphPad Software Inc.) or Microsoft Excel.

Conclusions
The significant alterations in proteins involved in several key processes would suggest that these products had effects on the systemic health status of the broiler chicken that were measurable through serum quantitative proteomic analysis. In this first study of its type, the multifaceted effects of GHP supplementation including stimulation of the innate immune system, particularly the complement system, alterations in the levels of a few proteins involved in the detoxification of ROS, stimulation of metabolic processes, gastric mucosal development and transport of iron and Vitamin A, were noted. The three YCW-based products showed similarity regarding their effects, which would be expected from three related products. We recognise that the use of sample pooling may influence the precise nature of the observed changes in protein abundance. However, individual results also show that these three products have product-specific effects which may be elucidated with further study. Future investigations should ideally deploy the use of smaller pools or individual serum samples to detect alterations in discrete protein abundance.
Future animal challenge studies, using bacterial (e.g., Escherichia coli or Campylobacter jejuni) or fungal pathogens (e.g., Aspergillus fumigatus) will ultimately be required to definitively elucidate the protective effects of the GHP diets studied herein.