Determination of secretory IgA secretion and its contribution to total endogenous amino acid losses in broiler chickens fed a nitrogen-free diet or diets containing either casein or purified amino acids

Abstract

Secretory IgA (sIgA) secretion and its contributions to total endogenous amino acid (AA) losses were determined in broiler chickens. In Experiment 1, dietary treatments consisted of a nitrogen-free diet or a diet that contained 10% casein; both diets were provided to birds ad libitum. In Experiment 2, broilers received a nitrogen-free diet or diets containing 2.5, 5.0, 7.5, and 10% casein ad libitum. An additional diet was fed that contained a purified AA mixture that provided the same AA as the 10% casein diet. Another set of birds also received the 10% casein diet except birds were pair-fed to match AA intake of birds receiving the purified AA diet. In Experiment 3, broilers received ad libitum access to a nitrogen-free diet. Purified diets containing 10% casein or a purified AA mixture were also fed; the 10% casein diet was pair-fed to match AA intake of birds consuming purified AA. In each experiment, diets were fed from 18 to 21 d-of-age and sIgA secretion was measured in 6 to 11 pens of 5 broiler chickens. In Experiment 1, ileal sIgA was greater (P < 0.05) for the casein treatment compared with the nitrogen-free treatment. In Experiment 2, there was a linear increase (P < 0.05) in ileal and biliary sIgA as casein increased. Further, birds fed the 10% casein diet (pair-fed) secreted more sIgA (P < 0.05) compared with birds consuming purified AA. Proportional contributions of sIgA to total endogenous AA losses ranged from 0.5 to 5.2%. In Experiment 3, ileal sIgA was the greatest (P < 0.05) in broilers fed casein and there was no difference (P > 0.05) in ileal sIgA between the nitrogen-free and purified AA treatment. In conclusion, sIgA was a contributor to endogenous AA losses. Further, the addition of casein but not purified AA to a protein-free diet increased sIgA secretion; therefore, the presence of intact protein from casein, not the provision of AA per se, caused the increased sIgA secretion.

Introduction

The production of secretory IgA (sIgA) is needed to maintain homeostasis within the gastrointestinal tract. These antibodies are primarily produced by plasma cells which reside in the lamina propria (Murphy and Weaver, 2017). After it is produced, IgA will bind to the polymeric Ig receptor on the basolateral membrane of enterocytes and undergo transcytosis prior to being released into the intestinal lumen. In addition to the lamina propria, sIgA can enter the gastrointestinal lumen through the release of bile by the gall bladder (Fisher et al., 1979). Once released into the gastrointestinal tract, sIgA will bind to pathogens and toxins to prevent their interaction with enterocytes and reduce inflammation (Mantis et al., 2011). It has also been reported that sIgA may bind to commensal microbial species in order to increase their retention in the gastrointestinal tract (Bollinger et al., 2006; Bunker et al., 2017). Aside from IgA itself, the secretory component also has functional roles in which it will reduce the degradation of the antibodies by proteases and will provide an alternative binding site for organisms to further prevent adhesion to enterocytes (Perrier et al., 2006; Mathias and Corthésy, 2011).

Due to the importance of sIgA in maintaining intestinal homeostasis, a substantial amount of sIgA is produced and released into the intestine on a routine basis. In fact, it has been estimated that humans secrete 3 to 5 g of sIgA into the intestine daily (Van Egmond et al., 2001; Tezuka and Ohteki, 2019). Few quantitative estimates exist for poultry; however, Parsons et al. (2023) recently reported that adult White Leghorn roosters that were precision-fed purified diets excreted 5 to 30 mg of sIgA within a 24 h period. These authors also reported that sIgA accounted for up to 13% of total endogenous amino acid (AA) losses, demonstrating that although sIgA production has an important role in maintaining homeostasis, it is also a source of nutrient losses for birds.

It has been well documented that interactions between the host and microbial species within the gastrointestinal tract regulate sIgA production. Benveniste et al. (1971) demonstrated that axenic mice exhibited substantial reductions in IgA compared with mice with normal microbiota. Similar results were also reported by Moreau et al. (1978) where IgA-producing plasmacytes in the duodenum were severely reduced in axenic mice; however, these authors reported that inoculating axenic mice with select microbial species ameliorated this response. Hapfelmeier et al. (2010) further evaluated the findings of Moreau et al. (1978) and reported that inoculating germ-free mice with doses of live bacteria increased sIgA.

Although it is well known that host-microbial interactions regulate sIgA production, it is also important to consider host-diet interactions. Paula-Silva et al. (2015) reported increased sIgA in mice fed a diet containing casein compared with those fed purified AA. These authors suggested that the cause for the increased sIgA may have been due to sampling of dietary peptides from digestion by immune cells in the intestine. Parsons et al. (2023) also investigated the potential role of host-diet interactions in roosters that were precision-fed various dietary treatments. In their first experiment, the excretion of sIgA was greater in roosters fed a diet containing casein (12.5 mg / 24 h) compared with a nitrogen-free diet (6.7 mg / 24 h). It should be noted, however, that results in the study by Parsons et al. (2023) were not consistent across all 3 experiments, which was in part due to variations in sIgA secretion among individual roosters regardless of the diet that was fed. To the best of our knowledge, no work has been conducted to investigate sIgA secretion in broiler chickens in response to adding casein to a diet devoid of protein.

The objective of this study was to evaluate sIgA secretion in broiler chickens fed different levels and sources (intact versus unbound) of dietary amino acids and to determine the proportional contributions of sIgA to total endogenous AA losses.

Materials and methods

The protocols for this study were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Arkansas and University of Illinois (protocol numbers 21147, 22048, and 23006).

Diets and experimental design

Experiment 1 was conducted to determine sIgA secretion in broiler chickens fed a nitrogen-free diet or a diet that contained 10% casein. The composition of the dietary treatments is presented in Table 1. The first dietary treatment was a nitrogen-free cornstarch-dextrose-based diet, whereas treatment 2 was a similar diet except that cornstarch was replaced with dextrose and casein. A blend of cornstarch and dextrose was used in diet 1 to balance the bulk density of the diet. No cornstarch was included in treatment 2 due to concerns regarding interactions with casein which could cause issues with palatability, texture, and consistency.

Table 1.

Ingredient composition of diets 1 and 2 in Experiment 1.

Ingredients (%)	Dietary treatments
	1	2
Cornstarch	40.11	-
Dextrose	40.11	70.22
Casein	-	10.00
Soybean oil	10.00	10.00
Solka Floc1	3.00	3.00
NaHCO3	1.00	1.00
Mineral mix2	5.37	5.37
Vitamin mix3	0.20	0.20
Choline chloride (60%)	0.20	0.20
DL-alpha tocopherol acetate	0.002	0.002
Ethoxyquin	0.0125	0.0125

¹Powdered cellulose; International Fiber Corporation, Urbana, OH 43078.

²Mineral mix provided per kilogram of diet: CaCO₃, 3 g; Ca₃(PO₄)₂, 28 g; K₂HPO₄, 9 g; NaCl, 8.89 g; CuSO₄·5H₂O, 20 mg; ZnCO₃, 100 mg; MgSO₄·H₂O, 0.65 g; FeSO₄·7H₂O, 420 mg; MnSO₄·H₂O, 650 mg; H₃BO₃, 9 mg; Na₂MoO₄·2H₂O, 9 mg; KI, 40 mg; CoSO₄·7H₂O, 1 mg; Na₂SeO₃, 215 μg.

³Provided (per kilogram of diet): thiamine·HCl, 20 mg; niacin, 50 mg; riboflavin, 10 mg; d-Ca-pantothenate, 30 mg; vitamin B12, 0.04 mg; pyridoxine·HCl, 6 mg; d-biotin, 0.6 mg; folic acid, 4 mg; menadione dimethylpyridinol bisulfate, 2 mg; ascorbic acid, 250 mg; cholecalciferol, 15 mg; retinyl acetate, 1,789 μg.

Cobb 500 male broiler chickens were housed in a clean temperature and humidity-controlled room in battery cages with raised wire floors and were provided a nutritionally complete starter diet until 17 d-of-age. On d 17, feeders were removed and birds were fasted overnight. On d 18, broilers were allotted to ensure there was a similar mean BW among treatments. A completely randomized design was used and there were 6 replicate pens of 5 chickens per pen. Birds were provided ad libitum access to feed until d 21 of age. On d 21, birds were euthanized using CO₂ gas and ileal digesta were collected from Meckel’s diverticulum to the ileocecal junction via flushing. Bile was also collected from the gall bladder. Digesta were then lyophilized and ground prior to being analyzed.

Experiment 2 was conducted to further investigate the responses reported in Experiment 1. The composition of dietary treatments is presented in Table 2. Diets 1 through 5 consisted of a nitrogen-free purified dextrose-based diet that was supplemented with 2.5, 5.0, 7.5, or 10.0% casein added at the expense of dextrose. Diet 6 was a purified cornstarch-based diet that was formulated to provide the same AA as the 10% casein diet, except that all AA were provided as purified (unbound) AA. Cornstarch was used in this diet instead of dextrose to prevent the occurrence of Maillard reactions. All diets contained 0.5% TiO₂ which was used as an indigestible marker. It is important to note that after Experiment 2 was completed, it was found that there was a mislabeling of AA and no Arg was present in the purified AA mixture (the added Arg was actually Ala). The analyzed AA composition of dietary treatments is presented in Table 3.

Table 2.

Ingredient composition of diets 1 through 6 in Experiment 2.

	Dietary treatments
Ingredient, %	1	2	3	4	5	6
Dextrose	79.4	76.9	74.4	71.9	69.4	-
Cornstarch	-	-	-	-	-	69.8
Casein	-	2.5	5.0	7.5	10.0	-
Purified AA mix1	-	-	-	-	-	9.6
Soybean oil	10.0	10.0	10.0	10.0	10.0	10.0
Solka Floc2	3.0	3.0	3.0	3.0	3.0	3.0
Dicalcium phosphate	2.4	2.4	2.4	2.4	2.4	2.4
Limestone	1.3	1.3	1.3	1.3	1.3	1.3
K2CO3	1.6	1.6	1.6	1.6	1.6	1.6
NaHCO3	0.80	0.80	0.80	0.80	0.80	0.80
KCl	0.37	0.37	0.37	0.37	0.37	0.37
MgO	0.20	0.20	0.20	0.20	0.20	0.20
Mineral mix3	0.10	0.10	0.10	0.10	0.10	0.10
Vitamin mix4	0.05	0.05	0.05	0.05	0.05	0.05
Choline chloride (60%)	0.25	0.25	0.25	0.25	0.25	0.25
TiO2	0.50	0.50	0.50	0.50	0.50	0.50
Ethoxyquin	0.01	0.01	0.01	0.01	0.01	0.01
DL-alpha tocopherol acetate	0.002	0.002	0.002	0.002	0.002	0.002

¹Purified amino acid mixture provided the same amino acids as casein, except for Ala and Arg. A mislabeling of amino acids led to the mixture being devoid of Arg and the addition of twice as much Ala compared with casein.

²Powdered cellulose; International Fiber Corporation, Urbana, OH 43078.

³Provided per kg of diet: manganese, 24.4 mg; selenium, 0.04 mg; copper, 0.68 mg; iodine, 0.47 mg.

⁴Provided per kg of diet: vitamin A, 6,173 IU; vitamin D₃, 4,409 ICU; vitamin E, 44 IU; vitamin B12, 0.01 mg; menadione, 1.20 mg; riboflavin, 5.29 mg; d-panthothenic acid, 7.94 mg; thiamine, 1.23 mg; niacin, 30.86 mg; pyridoxine, 2.20 mg; folic acid, 0.71 mg; biotin, 0.07 mg.

Table 3.

Analyzed composition of diets 2 through 6 in Experiment 2 (%; as-fed basis).

Amino acids	Dietary treatments
	2	3	4	5	62
Asp1	0.16	0.31	0.48	0.62	0.62
Thr	0.09	0.18	0.28	0.36	0.37
Ser	0.11	0.22	0.34	0.44	0.52
Glu1	0.48	0.96	1.50	1.95	2.02
Pro	0.24	0.48	0.75	0.99	0.96
Gly	0.04	0.08	0.13	0.17	0.15
Ala	0.07	0.13	0.21	0.27	0.61
Cys	0.00	0.01	0.02	0.04	0.04
Val	0.14	0.28	0.44	0.58	0.52
Met	0.06	0.10	0.18	0.21	0.23
Ile	0.12	0.24	0.37	0.48	0.48
Leu	0.21	0.40	0.62	0.81	0.69
Tyr	0.08	0.14	0.24	0.34	0.39
Phe	0.11	0.22	0.34	0.45	0.47
Lys	0.18	0.34	0.54	0.70	0.75
His	0.06	0.12	0.19	0.25	0.25
Arg	0.07	0.14	0.22	0.29	0.01
Trp	0.04	0.07	0.09	0.12	0.13

¹Asp = aspartic acid + asparagine; Glu = glutamic acid + glutamine.

²A mislabeling of amino acids led to the purified AA mixture being devoid of Arg and the addition of twice as much Ala compared with dietary treatment 5.

Cobb 500 male broiler chickens were housed in a clean temperature and humidity-controlled room in battery cages with raised wire floors and were provided a nutritionally complete starter diet until 17 d-of-age. On d 17, feeders were removed and birds were fasted overnight. On d 18, broilers were allotted to ensure there was a similar mean BW among treatments. A completely randomized design was used. Dietary treatments 1 through 6 were provided ad libitum. A 7th additional treatment was included, in which diet 5 (10% casein) was pair-fed to equalize the AA intake between treatment 6 (purified AA) and 7 (10% casein). Pair-feeding for treatment 7 was not implemented until d 19 of age, as 1 d was required to establish the feed intake of birds consuming diet 6 ad libitum. Once pair-feeding commenced, birds on treatment 7 were fed 3 times per d. Treatments 1 through 5 contained 10 replicate pens of 5 chickens per pen, whereas treatments 6 and 7 that were used for pair-feeding contained 11 replicate pens of 5 chickens per pen. On d 21, birds were euthanized using CO₂ gas. Ileal digesta were then collected from Meckel’s diverticulum to the ileocecal junction via flushing and bile was collected from the gall bladder. Digesta were lyophilized and ground prior to being analyzed.

Experiment 3 was conducted to validate the findings from Experiments 1 and 2 and to correct the error in the purified AA mixture in Experiment 2 which did not contain Arg. The composition of the dietary treatments is presented in Table 4. Diet 1 was a nitrogen-free dextrose-based diet. Diet 2 was the same as diet 1, except that 10% casein was added in place of dextrose. Diet 3 was similar to diet 2, except that it was a cornstarch-based diet and all the AA were provided as purified (unbound) AA. The analyzed AA composition of dietary treatments is presented in Table 5.

Table 4.

Ingredient composition of diets 1 through 3 in Experiment 3.

	Dietary treatments
Ingredient, %	1	2	3
Dextrose	79.9	69.9	-
Cornstarch	-	-	70.3
Casein	-	10.0	-
Purified AA mix1	-	-	9.6
Soybean oil	10.0	10.0	10.0
Solka Floc2	3.0	3.0	3.0
Dicalcium phosphate	2.4	2.4	2.4
Limestone	1.3	1.3	1.3
K2CO3	1.6	1.6	1.6
NaHCO3	0.80	0.80	0.80
KCl	0.37	0.37	0.37
MgO	0.20	0.20	0.20
Mineral mix3	0.10	0.10	0.10
Vitamin mix4	0.05	0.05	0.05
Choline chloride (60%)	0.25	0.25	0.25
Ethoxyquin	0.01	0.01	0.01

¹Purified amino acid mix provided the same amino acids as casein.

²Powdered cellulose; International Fiber Corporation, Urbana, OH 43078.

³Provided per kg of diet: manganese, 24.4 mg; selenium, 0.04 mg; copper, 0.68 mg; iodine, 0.47 mg.

⁴Provided per kg of diet: vitamin A, 6,173 IU; vitamin D₃, 4,409 ICU; vitamin E, 44 IU; vitamin B12, 0.01 mg; menadione, 1.20 mg; riboflavin, 5.29 mg; d-panthothenic acid, 7.94 mg; thiamine, 1.23 mg; niacin, 30.86 mg; pyridoxine, 2.20 mg; folic acid, 0.71 mg; biotin, 0.07 mg.

Table 5.

Analyzed composition of diets 2 and 3 in Experiment 3 (%; as-fed basis).

Amino acids	Dietary treatments
	2	3
Asp1	0.65	0.63
Thr	0.38	0.37
Ser	0.45	0.47
Glu1	2.06	2.06
Pro	0.91	0.85
Gly	0.18	0.18
Ala	0.28	0.26
Cys	0.03	0.05
Val	0.61	0.60
Met	0.24	0.25
Ile	0.50	0.48
Leu	0.86	0.88
Tyr	0.38	0.36
Phe	0.48	0.44
Lys	0.74	0.72
His	0.26	0.25
Arg	0.32	0.32
Trp	0.12	0.10

¹Asp = aspartic acid + asparagine; Glu = glutamic acid + glutamine.

Ross 308 male broiler chickens were housed in a clean temperature and humidity-controlled room in battery cages with raised wire floors and were provided a nutritionally complete starter diet until 17 d-of-age. On d 17, feeders were removed and birds were fasted overnight. On d 18, broilers were allotted to ensure there was a similar mean BW among treatments. A completely randomized design was used and there were 6 replicate pens of 5 chickens per pen. During the experimental phase, broiler chickens were provided ad libitum access to diets 1 and 3. Birds receiving diet 2, however, were pair-fed to match the AA intake of birds consuming diet 3 ad libitum. Unlike Experiment 2, pair-feeding took place during the entire duration of the experimental phase. The amount of feed given to birds receiving diet 2 on d 18 was estimated based on data from Experiment 2. The amount of feed given for the remaining 2 days of the experimental period was determined based on the measured feed intake of birds consuming diet 3 ad libitum. Pair-fed birds were fed 3 times per d. On d 21, birds were euthanized and ileal digesta were collected from Meckle’s diverticulum to the ileocecal junction. Digesta were then frozen prior to being lyophilized and ground.

Chemical analyses

The CP and AA concentrations were determined at the Agricultural Experimental Station Chemical Laboratories (University of Missouri, Columbia, MO; Method 990.03 and 982.30 E [a, b, and c]; AOAC International, 2007). Titanium concentrations in experimental diets and ileal digesta were also measured using UV spectroscopy (Myers et al., 2004). Dry matter (Method 930.15; AOAC International, 2007), ash (Method 942.05; AOAC International, 2007), neutral detergent fiber (NDF; Method 2002.04; AOAC International, 2007), and acid detergent fiber (ADF; Method 973.18; AOAC International, 2007) was determined at the University of Arkansas.

sIgA analyses

The concentration of sIgA was measured using an ELISA specific for chicken IgA (Abnova, Taipei City, Taiwan; #KA2031, range 12.5 to 400 ng/ml). Recoveries were ≥ 95%, and intra and inter-assay CV were less than 5%. Approximately 0.1 g of lyophilized digesta were weighed in 50 ml centrifuge tubes and 10 ml of saline was added. Samples were then vortexed for 5 s and centrifuged at 1,400 × g for 10 min. The supernatant was decanted into 15 ml centrifuge tubes, after which it was vortexed for 5 s. Samples were then filtered using a 1.5 µm nylon syringe filter and stored frozen at −80°C. Prior to analysis, samples were thawed and dilutions were conducted for ileal digesta (1:50) and bile (1:8,000). The ELISAs were then conducted by following the manufacturer’s instructions. Briefly, 100 µl of sample was added in duplicate to a pre-coated 96-well microplate and allowed to incubate for 20 min at room temperature. After incubation, wells were aspirated, washed 4 times with wash solution, and enzyme-antibody conjugate was added. Following a 20 min incubation at room temperature in the dark, wells were once again aspirated and washed 4 times, after which 3,3′,5,5′-tetramethylbenzidine substrate solution was added. Following a 10 min incubation at room temperature in the dark, a stop solution containing 0.3 M sulfuric acid was added and absorbance was measured at 450 nm using a microplate reader. The concentration of sIgA was determined by generating a 4-parameter logistic curve using absorbance values obtained from the dilution of the standard calibrator. The sIgA concentrations were then calculated using the equations below.

$$\text{sIgA}\text{concentration}\text{in}\text{ileal}\text{digesta}\left(\mu \mathrm{g}/\mathrm{g}\right)=\left[\text{sIgA}\left(\text{ng}/\text{ml}\right)\times \text{dilution}\text{factor}\times \left(\text{saline}\left(\text{ml}\right)/\text{dry}\text{ileal}\text{digesta}\text{weight}\left(\mathrm{g}\right)\right)\right]/{10}^3$$

$$\text{sIgA}\text{concentration}\text{in}\text{bile}\left(\mu \mathrm{g}/\text{ml}\right)=\left[\text{sIgA}\left(\text{ng}/\text{ml}\right)\times \text{dilution}\text{factor}\right]/{10}^3$$

Proportional contributions of sIgA to endogenous AA losses and determination of AA digestibility

Contributions of sIgA to total endogenous AA losses were determined in Experiment 2. Due to the limited amount of sample, digesta from 2 pens of 5 chickens were pooled for AA analyses. The AA composition of sIgA was obtained from the study by Parsons et al. (2023). The calculations are described below.

Proportional contributions of sIgA to total endogenous AA losses (%) = [sIgA in ileal digesta (%) × AA in sIgA (%)] / [AA concentration in ileal digesta (%)]

$$\text{where}\text{sIgA}\text{in}\text{ileal}\text{digesta}(\%)=\left[\text{sIgA}\left(\mu \mathrm{g}/\mathrm{g}\right)/{10}^6\right]\times 100$$

Standardized ileal digestibility (SID) of AA was calculated in Experiment 2. Basal endogenous AA losses were determined from AA analysis of ileal digesta collected from broiler chickens fed diet 1 (nitrogen-free) in Experiment 2. The equation is shown below.

$$\text{SID}\text{of}\text{AA}(\%)=[(\text{AA}\text{in}\text{diet}(\%)-\text{AA}\text{in}\text{ileal}\text{digesta}(\%)+\text{endogenous}\text{AA}(\%))/\text{AA}\text{in}\text{diet}(\%)]\times 100$$

where AA in diet (%) = analyzed AA concentration in diet; AA in ileal digesta = analyzed AA concentration in ileal digesta × (Ti in diet (%) / Ti in ileal digesta (%)); endogenous AA (%) = AA in ileal digesta from birds fed a nitrogen-free diet × (Ti in diet (%) / Ti in ileal digesta (%)).

Statistical analyses

SAS software (SAS Institute, Cary, NC) was used to analyze data from all experiments. Data were analyzed using a 1-way ANOVA for a completely randomized design. Linear and quadratic contrasts, as well as single degree of freedom contrasts, were performed using PROC GLM in Experiment 2. Pairwise treatment comparisons were made using Fisher’s Least Significant Difference test (Carmer and Walker, 1985). Each pen of 5 chickens was the experimental unit for all analyses except proportional contributions of sIgA to total endogenous AA losses and SID of AA; the experimental unit for these analyses was 2 pens of 5 chickens due to limited digesta. There were 6 replicate pens per treatment in Experiments 1 and 3. In Experiment 2, dietary treatments 1 through 5 contained 10 replicate pens and treatments 6 and 7 that were used for pair-feeding contained 11 replicate pens per treatment. The significance value for all analyses was P < 0.05.

Results and discussion

One sample of casein was utilized for all 3 experiments; the nutrient composition is presented in Table 6. Casein contained 94% CP (DM basis), 0.6% ash, and no detectable NDF or ADF. Further, the sum of AA in casein was 103% (DM basis). Thus, casein was predominantly composed of moisture and AA.

Table 6.

Nutrient composition of casein in Experiments 1 through 3¹ (%).

Item, %	Casein
Moisture	9.28
CP	94.36
NDF2	ND
ADF2	ND
Ash	0.55
Amino acids, %
Asp3	6.85
Thr	4.11
Ser	4.99
Glu3	21.42
Pro	10.27
Gly	1.86
Ala	3.03
Cys	0.39
Val	6.45
Met	2.74
Ile	5.28
Leu	9.19
Tyr	5.48
Phe	5.09
Lys	7.82
His	2.93
Arg	3.62
Trp	1.27
Sum	102.78

¹Values are expressed on a DM basis, except for moisture which is expressed on an as-fed basis.

²NDF = neutral detergent fiber; ADF = acid detergent fiber. No NDF or ADF was detected in the samples.

³Asp = aspartic acid + asparagine; Glu = glutamic acid + glutamine.

Experiment 1

The growth performance and sIgA secretion of birds in Experiment 1 is presented in Table 7. As expected, broiler chickens that consumed the diet that contained 10% casein exhibited greater (P < 0.05) BW gain and gain:feed compared with birds fed the nitrogen-free diet; there was no difference (P > 0.05) in feed intake between treatments. Casein increased (P < 0.05) ileal sIgA concentration, where birds secreted 184 and 477 μg/g DM when fed the nitrogen-free and casein diets, respectively. The biliary sIgA secretion for birds fed casein was numerically higher but not different from the nitrogen-free diet (881 and 667 μg/ml, respectively; P > 0.05).

Table 7.

Growth performance and secretory IgA secretion in broiler chickens in Experiment 1^1.

Dietary treatments	Feed intake (g/chicken)	BW gain (g/chicken)	Gain:feed (g/kg)	Ileal sIgA2 (μg/g DM)	Biliary sIgA (μg/ml)
1. Nitrogen-free	241.6	−17.4	−74.7	184.1	667.1
2. Casein	278.8	61.3	219.3	476.8	881.2
Pooled SD	32.32	19.22	72.45	66.45	178.77
P-value	0.074	<0.001	<0.001	<0.001	0.065

¹Values are means of 6 pens of 5 chickens. Feed intake, BW gain, and feed efficiency were recorded from 18 to 21 d-of-age. Ileal and biliary sIgA were measured at 21 d-of-age.

²sIgA = secretory IgA.

The results from Experiment 1 were in good agreement with previous reports. Parsons et al. (2023) reported greater sIgA excretion in roosters that were precision-fed a diet that contained 10% casein compared with a nitrogen-free diet in 1 experiment. Further, a substantial difference between treatments was reported in that study, similar to what was observed herein, where roosters fed casein excreted 12.5 mg of sIgA / 24 h compared with 6.7 mg of sIgA / 24 h for birds fed a nitrogen-free diet. Similar results have been also reported in pigs. Vitari et al. (2008) observed that feeding greater levels of protein to young pigs increased sIgA excretion. Increased sIgA secretion caused by casein may have been due to interactions between dietary antigens and intestinal immune cells; however, the cause for this response could not be fully elucidated from this experiment. Thus, additional experiments were conducted herein to determine if the increased sIgA secretion caused by casein was due to the presence of intact protein or the provision of nutrients.

Experiment 2

The analyzed AA composition of diets 2 through 6 in Experiment 2 is presented in Table 3. As mentioned previously, dietary treatments 2 through 5 contained 2.5, 5.0, 7.5, and 10% casein, respectively, whereas diet 6 was formulated to provide the same AA content as diet 5 but supply AA as purified (unbound) AA. The sum of the AA for dietary treatments 2 through 5 was in good agreement with the calculated values. The AA in treatment 6, which contained a purified AA mix, was generally similar to treatment 5 (10% casein), except for Arg and Ala. As discussed previously, there was a labeling error in which the ingredient presumed to be Arg was actually Ala.

Growth performance and sIgA secretion in broiler chickens in Experiment 2 is presented in Table 8. As the concentration of casein increased, there was a linear increase (P < 0.05) in feed intake, BW gain, and gain:feed. The total feed intake was greater (P < 0.05) for birds pair-fed the 10% casein diet compared with the ad libitum-fed purified AA diet because pair-feeding was not initiated during the first 24 h of the experimental phase; the first 24 h was used to establish the feed intake of birds consuming treatment 6 ad libitum as discussed previously. The lack of Arg in treatment 6 (purified AA) combined with the higher feed intake of birds in treatment 7 (10% casein; pair-fed) is likely the reason for the greater loss in BW gain and gain:feed for treatment 6 compared with 7. The feed intake during the final 2 days of the experiment for treatments 6 and 7 once pair-feeding was initiated, however, was similar being 40.6 and 41.2 g/chicken/d, respectively.

Table 8.

Growth performance and secretory IgA secretion in broiler chickens in Experiment 2^1.

Dietary treatments	Feed intake (g/chicken)	BW gain (g/chicken)	Gain:feed (g/kg)	Ileal sIgA2 (μg/g DM)	Biliary sIgA (μg/ml)
1. Nitrogen-free	219.2c	−36.4e	−168.5d	742.3c	1495.8d
2. 2.5% casein	224.1bc	−18.5d	−85.2c	939.9bc	1689.0cd
3. 5.0% casein	224.3bc	−6.7c	−38.7c	1007.0ab	1867.2bcd
4. 7.5% casein	236.2b	11.9b	46.8b	1231.5a	2151.7b
5. 10.0% casein	251.5a	36.1a	142.8a	1240.9a	2090.0bc
6. Purified AA	133.3e	−65.3f	−497.3e	712.9c	3213.8a
7. 10% casein PF3	183.5d	−12.9cd	−71.5c	1116.2ab	2784.4a
Pooled SD	13.57	11.75	66.88	283.71	509.22
Contrasts	P-values
Linear4	<0.001	<0.001	<0.001	<0.001	<0.001
Quadratic4	0.080	0.190	0.314	0.563	0.394
1 vs. 5	<0.001	<0.001	<0.001	<0.001	0.014
1 vs. 6	<0.001	<0.001	<0.001	0.813	<0.001
5 vs. 7	<0.001	<0.001	<0.001	0.332	0.004
6 vs. 7	<0.001	<0.001	<0.001	0.002	0.052

^a-fMeans within a column with no common superscript differ (P < 0.05). Values are means of 10 pens of 5 chickens for treatments 1 through 5, and 11 pens of 5 chickens for treatments 6 and 7.

¹The BW gain, feed intake, and feed efficiency were recorded from 18 to 21 d-of-age. Ileal and biliary sIgA were measured at 21 d-of-age.

²sIgA = secretory IgA.

³PF = pair-fed. Pair-feeding was conducted during the last 48 h of the experiment for treatment 7 to match the feed intake of birds consuming treatment 6 ad libitum. The feed intake during this period for treatments 6 and 7 were 40.6 and 41.2 g/chicken/d, respectively.

⁴Linear and quadratic contrasts were performed for treatments 1 through 5.

There was a linear increase (P < 0.05) in ileal sIgA as dietary casein increased (Table 8). Further, there was no difference in ileal sIgA (P > 0.05) between the ad libitum-fed and pair-fed 10% casein treatments. This suggests that the linear response to casein may not have been due to increased supply of nutrients, rather, it may have been the result of increased concentrations of dietary antigens. Overall, the addition of 10% casein resulted in greater sIgA secretion compared with the nitrogen-free diet, being 1,241 and 742 μg/g DM, respectively. This was in good agreement with Experiment 1 and the observations by Parsons et al. (2023). The purified AA treatment which provided the same AA as the 10% casein treatment (except for Arg and Ala) resulted in lower (P < 0.05) IgA secretion compared with both the ad libitum-fed and pair-fed 10% casein treatments. Further, there was no difference (P > 0.05) in sIgA secretion between the nitrogen-free and purified AA treatments. The same results were also observed for excreta (data not shown). This is in good agreement with Paula-Silva et al. (2015) who conducted a study with mice, where it was overserved that when the AA in a casein-containing diet were replaced with all purified AA, sIgA concentrations were reduced. Data from Experiment 2 herein confirms the observation that adding casein to a protein-free diet increases sIgA secretion. Due to the lack of Arg and excess Ala in the purified AA mix in Experiment 2, however, the mechanism by which casein increased sIgA still could not be fully elucidated.

Similar to ileal sIgA, there was a linear increase (P < 0.05) in biliary sIgA as casein increased (Table 8). There was no difference (P > 0.05) in biliary sIgA concentration between treatments 6 and 7. It should be noted, however, that biliary IgA concentrations were greater (P < 0.05) in treatments 6 and 7 compared with the remaining dietary treatments. This could in part be due to analysis of only concentration of sIgA in bile and not a quantitative estimate of total sIgA within the gall bladder (i.e. total bile present multiplied by concentration of sIgA in bile). Another explanation may be the lower (P < 0.05) feed intake of birds on these treatments. Since less feed was consumed, less bile may have been released from the gall bladder into the gastrointestinal tract, subsequently allowing sIgA concentration in the bile to increase. This suggests that feed intake may be an important factor to consider when interpreting the results of sIgA concentration in bile. Lastly, the lack of difference in biliary IgA between treatments 6 and 7 may be due to the source of IgA in the bile. It has been reported in rats that biliary sIgA is primarily obtained from the blood (Fisher et al., 1979). Thus, it is possible that biliary Iga may be more indicative of systemic IgA production, while concentrations of IgA in digesta or excreta may be more representative of changes in IgA secretion by the lamina propria.

The proportional contributions of sIgA to total endogenous AA losses for dietary treatments 5 and 6 in Experiment 2 are presented in Table 9. Overall, sIgA accounted for 0.5 to 5.2% of total endogenous AA losses. The greatest proportional contributions were observed for Trp for both treatments 5 and 6, being 5.2 and 3.5%, respectively. Similar to the present study, Parsons et al. (2023) reported that sIgA contributed to greater proportions of endogenous Trp losses compared with other AA; although, they reported greater contributions ranging from 4.1 to 13.3% in precision-fed adult leghorn roosters. Further, those authors also reported that sIgA contributed to higher proportions of Val and Thr losses compared with other AA. The higher contribution to endogenous Thr losses compared with other AA was not observed herein. This discrepancy between studies may be due to differences in the age and breed of the birds, in which adult roosters in the study by Parsons et al. (2023) exhibited greater sIgA concentrations in excreta (2.6 mg/g dried excreta) compared with ileal sIgA concentrations in 21 d-old broiler chickens herein (1.0 mg/g dried ileal digesta; Table 8).

Table 9.

Proportional contributions of secretory IgA to total endogenous amino acid losses in broiler chickens at 21 d-of-age in Experiment 2¹ (%).

Amino acid	Dietary treatments		Pooled SD
	5	6		P-value
Asp2	0.96	1.71	0.461	0.032
Thr	1.25	1.69	0.442	0.161
Ser	0.95	2.39	0.563	0.004
Glu2	0.54	1.61	0.426	0.004
Pro	1.05	1.71	0.463	0.053
Gly	1.38	2.05	0.604	0.118
Ala	1.18	2.03	0.582	0.049
Cys	1.60	1.90	0.562	0.410
Val	1.23	2.54	0.757	0.025
Met	0.66	1.77	0.529	0.011
Ile	0.50	1.17	0.321	0.011
Leu	1.40	2.09	0.617	0.117
Tyr	1.76	2.11	0.638	0.412
Phe	1.10	1.45	0.452	0.261
Lys	0.64	1.15	0.323	0.039
His	0.75	1.01	0.313	0.240
Arg	1.42	2.37	0.762	0.084
Trp	5.21	3.52	1.273	0.069
Mean	1.31	1.90	0.541	0.121

¹Values are means of 5 replicates of 2 pens of 5 chickens.

²Asp = aspartic acid + asparagine; Glu = glutamic acid + glutamine.

Overall, sIgA tended to account for greater proportions of total endogenous AA losses for treatment 6 (purified AA) compared with treatment 5 (10% casein). This may be explained by the differences in AA digestibility between treatments (Table 10). The SID of AA in casein and the purified AA mixture were high, with mean AA digestibility being 92.7 and 95.9%, respectively. The SID of Cys was low for both treatments 5 and 6, being 78.5 and 75.0%, respectively. This was in good agreement with AA digestibility values for casein reported by Chung and Baker (1992) and Batal and Parsons (2003), where the digestibility of Cys in casein was lower and more variable compared with other AA. This was likely due to the low concentration of Cys in casein (0.4%, DM basis; Table 6), in which small errors in the endogenous correction would have substantial impacts on the digestibility value as indicated by the larger pooled SD for Cys compared with other AA (Table 10). Overall, the SID of AA in the purified AA mix was greater (P < 0.05) compared with casein; although, as mentioned previously, AA were generally highly digestible in both treatments. It should be noted, however, that the greater concentration of undigested AA for birds fed casein compared with purified AA likely explains the lower proportional contribution of sIgA to endogenous AA losses in the casein treatment.

Table 10.

Standardized ileal amino acid digestibility in broiler chickens at 21 d-of-age in Experiment 2¹ (%).

Amino acid	Dietary treatments		Pooled SD	P-value
	5	6
Asp2	92.0	97.2	1.17	<0.001
Thr	91.5	93.1	1.25	0.078
Ser	84.5	96.7	1.30	<0.001
Glu2	92.2	99.0	0.66	<0.001
Pro	96.9	98.5	0.40	<0.001
Gly	89.3	93.9	2.74	0.028
Ala	90.5	96.2	1.59	<0.001
Cys	78.5	75.0	6.95	0.444
Val	93.1	98.1	0.83	<0.001
Met	94.9	99.9	1.00	<0.001
Ile	90.2	97.7	0.95	<0.001
Leu	96.6	98.1	0.86	0.026
Tyr	97.4	97.2	0.86	0.733
Phe	97.5	97.9	0.91	0.454
Lys	95.7	98.7	0.94	0.001
His	96.1	96.8	0.76	0.187
Arg	94.7	-	1.75	-
Trp	97.3	93.0	1.61	<0.001
Mean	92.7	95.9	1.27	0.005

¹Values are means of 5 replicates of 2 pens of 5 chickens.

²Asp = aspartic acid + asparagine; Glu = glutamic acid + glutamine.

Experiment 3

The analyzed AA composition of diets 2 and 3 in Experiment 3 is presented in Table 5. Unlike Experiment 2, both the diet that contained 10% casein and the purified AA diet contained the same concentrations of all AA, including Arg and Ala. The growth performance and ileal sIgA secretion in Experiment 3 is presented in Table 11. The feed intake between treatment 2 (10% casein; pair-fed) and treatment 3 (purified AA; ad libitum-fed) was similar (P > 0.05); albeit, treatment 3 resulted in greater (P < 0.05) BW gain and gain:feed compared with treatment 2. The latter may be caused by slight numerical differences in feed intake and differences in AA digestibility (Table 10). Ileal sIgA in birds fed casein was 813 μg/g DM, which was greater (P < 0.05) compared with the nitrogen-free and purified AA treatment. Further, there was no difference (P > 0.05) in sIgA secretion between the nitrogen-free and purified AA treatments (383 and 422 μg/g DM, respectively).

Table 11.

Growth performance and ileal secretory IgA secretion broiler chickens in Experiment 3^1.

Dietary treatments	Feed intake (g/chicken)	BW gain (g/chicken)	Gain:feed (g/kg)	Ileal sIgA2 (μg/g DM)
1. Nitrogen-free	142.6b	−9.1c	−63.2c	382.5b
2. 10% casein PF2	148.7ab	36.6b	258.6b	813.3a
3. Purified AA	154.0a	46.8a	303.3a	421.7b
Pooled SD	6.61	3.99	24.15	169.46

^a-cMeans within a column with no common superscript differ (P < 0.05). Values are means of 6 pens of 5 chickens.

¹Feed intake, BW gain, and feed efficiency were recorded from 18 to 21 d-of-age. Ileal sIgA was measured at 21 d-of-age.

²PF = pair-fed; sIgA = secretory IgA.

The increase in sIgA secretion in Experiment 3 when casein was added to a protein-free diet and the lack of effect when the same AA were provided as purified AA is good agreement with Experiments 1 and 2 in the present study. Similar results were also reported by Paula-Silva et al. (2015) and Parsons et al. (2023) as discussed previously. No definitive conclusion from Experiment 2 could be made to explain why the addition of casein to a diet devoid of AA caused increased sIgA secretion due to the lack of Arg in the purified AA mix. In Experiment 3, however, the purified AA treatment contained the same AA content as the diet with 10% casein (Table 5). Further, the implementation of pair-feeding provided similar AA intake between broiler chickens consuming these 2 dietary treatments. Data herein demonstrates that the stimulation of sIgA secretion when casein is fed is caused by the presence of the intact protein and not due to the provision of AA in male broilers. Paula-Silva et al. (2015) hypothesized that the lower sIgA production when mice were fed purified AA compared with casein was due to host-diet interactions. Results from the present study support this hypothesis. Data from Experiment 3 indicates that as dietary proteins are digested, the peptides may be sampled by immune cells in the gastrointestinal tract, thereby increasing sIgA production. Sampling of antigens within the gastrointestinal tract occurs routinely by M-cells, dendritic cells, and macrophages (Rios et al., 2015; Murphy and Weaver, 2017). Due to the small size of single (unbound) AA, however, purified AA likely have limited capacity to interact with MHC and B-cell receptors (Blum et al., 2014; Reis et al., 2022). This would explain why there was an effect of adding intact protein but not purified (unbound) AA. Future research is warranted assessing different protein sources that vary in peptide size and antigenicity to determine if the responses reported herein are specific to the dietary protein source.

In conclusion, provision of casein to diets devoid of protein stimulates sIgA secretion in the small intestine of male broiler chickens. This effect appears to be caused by the presence of intact dietary protein and not due to the provision of nutrients per se, as there was no change in sIgA secretion when the same amino acids found in casein were fed as purified (unbound) amino acids. Furthermore, sIgA secretion in broiler chickens is a measurable contributor to endogenous amino acid losses, accounting for as much as 5% of basal endogenous amino acid losses. This study demonstrates that dietary protein can regulate sIgA secretion in the gastrointestinal tract through direct host-diet interactions. More work is needed, however, to determine if all dietary protein sources exhibit the same effect on sIgA secretion, if other components in feedstuffs other than protein affect sIgA secretion, and if the effect of the diet changes based on the length of provision of the dietary treatments.

CRediT authorship contribution statement

B.W. Parsons: Writing – original draft, Methodology, Formal analysis, Conceptualization. S.J. Rochell: Writing – review & editing, Supervision.

Disclosures

The authors declare no conflicts of interest.