Growth and response to Escherichia coli lipopolysaccharide challenge in Lohmann LSL-Lite pullets when fed a source of omega-3 fatty acids and yeast bioactives from hatch through to 16 wk of age

Abstract

Growth and response to Escherichia coli lipopolysaccharide (LPS) challenge in Lohmann LSL-Lite pullets when fed a source of omega-3 fatty acids (n-3 FA) and yeast bioactives (YB) from hatch through to 16 wk of age (woa) were investigated. Co-extruded full fat flaxseed and pulse mixture (FFF; 1:1 wt/wt) supplied n-3 FA and YB were yeast cell walls processed with β-1,3-glucan hydrolase. A total of 1,064-day-old pullets were placed in cages (19 birds/cage) and allocated to 7 diets (n = 8). The iso-caloric and iso-nitrogenous diets were control, control + 1, 3, or 5% FFF and + 0.025, 0.05, or 0.1% YB. The birds had ad libitum access to feed and water. Body weight (BW), feed intake, and lymphoid organs weight were recorded. At 15 woa, 2 pairs of pullets/cage received intravenous injection of either 1 mL of sterile saline without or with 8 mg LPS/kg BW. Injected pullets were bled, monitored for BW and cloaca temperature at time points within 168 h. Birds fed 1% FFF were heavier (P < 0.04) than birds fed other diets at 16 woa with FFF and YB exhibiting non-linear responses. Control birds had heavier (P = 0.02) thymus at 4 woa. Spleen weight increased quadratically (P < 0.05) in response to FFF at 8 and YB at 16 woa. The LPS increased cloaca temperature and altered concentration of several plasma metabolites (P < 0.05). The interaction (P < 0.05) between LPS and diet was such that control birds exhibited lower creatine kinase (CK) upon challenge with LPS relative to birds fed other diets. The LPS birds fed 1% FFF and 0.05% YB showed higher plasma albumin than non-LPS cohorts. Non-LPS birds fed control, 1 and 3% FFF had higher plasma K than LPS cohorts. In general, FFF and YB exhibited linear and quadratic effects (P < 0.05) on select plasma metabolites. In conclusion, dietary provision of n-3 FA and YB influenced pullet BW at sexual maturity, development of lymphoid organs and modulated some plasma metabolites in response to LPS.

INTRODUCTION

The digestive, immune, and the integument (skin and feathers) systems undergo rapid growth and development in the first 6 wk of age (woa) of modern pullet life (Silva et al., 2015). Pullets that have higher levels of stress during this growth spurt are more likely to have poor uniformity, which can negatively affect lifetime egg production (Leeson and Summers, 2005). For example, flocks with poor uniformity, the birds on the low end of the body weight scale will not come into production until 5 to 10 wk relative to other birds (Leeson and Summers, 2005). Rearing period is also critical for optimal development of structural bones pivotal for mitigating inevitable structural bones loss in the course of laying cycle (Akbari Moghaddam Kakhki et al., 2018,2020). Indeed, it is during the growth spurt that, the cortical (outer structural shell) and trabecular (inner for strength and support) bone are formed (Fleming et al., 2006). It has been estimated that 95% of the skeleton is developed by 12 woa (ISA, 2011; Hanlon et al., 2022). Collectively, any lag in growth during this period will affect the optimal body weight (BW) and composition at sexual maturity, delay the onset of egg production, impair egg production, and increase risks of osteoporosis, feather pecking, and poor eggshell quality (Leeson and Summers, 2005).

In practice, pullets are exposed to a variety of stressors associated with routine rearing practices, including vaccination, handling, heat, and feed change. Whether these stressors occur singly or simultaneously, they have the potential to stimulate stress response, impair immunity and negatively affect bird growth and well-being (Widowski and Torrey, 2018). Healthy birds with functioning immune systems are remarkably resilient to stressors and in fighting off diseases (Olsen et al., 2012; Linden, 2015). However, the immune system of growing pullet is uniquely fragile due to aforementioned production stressors and transient immunosuppression caused by routine vaccination regimens in early phases of pullet life (Butcher and Mojtaba, 2008; Kapczynski et al., 2013). In conjunction with restriction on use of antibiotics, changes in housing environment could increase the risk of proliferation of pathogenic and zoonotic bacteria in the gut. A Swiss survey 12 years after the ban of cages showed a significant decrease in the incidence of viral diseases but a marked increase in bacterial diseases (Kaufmann-Bart and Hoop, 2009). According to a survey in Canada, cumulative mortality rate of 1.8 ± 1.7% with a range of 0.01 to 8.3% was reported for pullets. The reasons for mortality included birds getting trapped in housing equipment (28.2%), leg injuries (24.4%), disease (21.8%), and smothering (15.4%) (Van Staaveren et al., 2019).

Immunomodulatory feedstuffs can alleviate stress and growth perturbations in domestic animals (Yang et al., 2008). There are numerous feedstuffs that have been shown to have immunomodulatory effects in young birds such as functional fatty acids (omega-3 polyunsaturated fatty acids, n-3 FA), yeast metabolites and probiotics (Lu et al., 2019; Neijat et al., 2019; Rubio, 2019). The effects of n-3 FA on the immune system are centered on their ability to alter cytokine production and secondarily to diminish eicosanoid biosynthesis (Anderson and Fritsche, 2002). The health benefits of n-3 FA are largely due long chained eicosapentaenoic acid (20:5 n−3, EPA) and docosahexaenoic acid (22:6 n−3, DHA) (Cherian, 2015). Both EPA and DHA can be supplemented in the diet by using fish and microalgae sources; but albeit with lower efficiency they could also be synthesized de novo from dietary α-linolenic acid (18:3 n−3, ALA) (Cherian, 2015). In general, although underlying mechanisms are poorly understood yeast and yeast metabolites have been shown to result in improved growth performance, resilience against enteric and systemic pathogens and consistent reductions in mortality of broiler chickens (Hooge, 2004; Roberts et al., 2015). These affects have been associated with modulation of immune system (Leung et al., 2018; Leung et al., 2019; Lu et al., 2019), mitigation of environmental stressors (Nelson et al., 2020), increase antibody titers production after vaccination (Cortés-Coronado et al., 2017) and interactions with gut constituents particularly reduction of enteric pathogens in various animal models (Price et al., 2010; Kiarie et al., 2012; Feye et al., 2016; Kiarie et al., 2019a,b).

There is little consideration on the utility of immunomodulating dietary components such as n-3 FA and yeast in feeding program for pullets. It is plausible such feedstuffs holds great promise in optimal pullet rearing particularly in high-stress housing environments. However, there is dearth of the data on the impact of feeding these functional feedstuffs on pullet development and overall health. Therefore, the objective of the present study was to investigate growth, body weight uniformity, lymphoid organ development and response to Escherichia coli lipopolysaccharide (LPS) challenge in egg-type Lohmann LSL-Lite pullets when fed a source of n-3 FA and YB from hatch through to 16 woa.

MATERIALS AND METHODS

The experimental protocol (#3634) was approved by the University of Guelph Animal Care Committee, and birds were cared for in accordance with the Canadian Council on Animal Care guidelines throughout the experiment (CCAC, 2009).

Birds and Housing

A total of 1,064-day-old Lohmann LSL-Lite pullets were procured from a commercial hatchery (Archer poultry farm Ltd., Brighton, Canada) and placed in 56 of conventional cages (L × W × H; 76 × 71 × 46 cm, Ford Dickinson Inc., Mitchell, Canada) based on body weight (BW) (19 pullets/pen). The cages were in one environmentally controlled room at the Arkell poultry research station, University of Guelph (Guelph, Canada). The cages were equipped with 2 nipple drinkers and trough feeder as described (Khanal et al., 2020). The room temperature was set at 35°C for the first 3 d, then gradually decreased to 20°C by 5 woa and held to the end. The relative humidity was 50% to 60% throughout the experiment. Light (30 Lux) was provided continuously for the first 2 D, then gradually decreased according to the lighting program for Lohmann LSL-Lite to 10L:14D at the intensity of 5 Lux. The light schedule was provided with a 20 min step up (dawn period) with full intensity at 05:30 h and a 20 min step down (dusk period) with light fully off at 19:30 h. Birds were vaccinated for Marek, and Newcastle disease (ND) at hatchery via neck inject. Birds were spray vaccinated for infectious bronchitis virus (IBV) at 3 woa, first booster for IBV and ND at 6 woa, fowl laryngotracheitis virus via wing web and avian encephalomyelitis and fowl pox via eye-drop at 8 woa. The second and third boosters for IBV and ND were via spray at 11 woa, and thigh injection at 15 woa, respectively.

Experimental Diets

Seven diets were prepared (1) control, mainly corn and soybean meal based; (2–4) control plus 1 or 3 or 5% of co-extruded full fat flaxseed and pulse mixture (FFF, 1:1 wt/wt) as a source of n3-FA (linPRO, O&T Farms Ltd., Regina, Canada), and (5–7) control plus 0.025 or 0.05 or 0.1% of yeast bioactives (YB, Maxi-Nutrio, CBS Bio Platforms Inc., Calgary, Canada). The YB are yeast cell walls originated from Saccharomyces cerevisiae processed with β-1,3-glucan hydrolase (Patterson et al., 2023). The doses of FFF and YB were based on our previous studies in broilers and pullet breeders (Lu et al., 2019; Akbari Moghaddam Kakhki et al., 2020; Thanabalan and Kiarie, 2021). All diets were formulated to meet or exceed the breeder specifications (Lohmann, 2016): starter, d 0 to 3 woa (Table 1); grower, 4 to 8 woa (Table 2), and developer, 9 to 16 woa (Table 3).

Table 1.

Composition of experimental diets in starter phase, as fed basis.¹

Item	Control	Flaxseed inclusion, %2			Yeast bioactives inclusion, %3
		1	3	5	0.025	0.05	0.10
Corn	43.5	43.2	42.5	41.8	43.5	43.5	43.4
Soybean meal	29.2	28.7	27.8	26.8	29.2	29.2	29.2
Wheat	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Barley	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Corn germ meal, 60%	3.00	3.00	3.00	3.00	3.00	3.00	3.00
Wheat middlings	2.50	2.50	2.50	2.50	2.50	2.50	2.50
Soy oil	2.49	2.29	1.89	1.49	2.49	2.49	2.49
Monocalcium phosphate	2.15	2.15	2.14	2.14	2.15	2.15	2.15
Limestone	0.86	0.86	0.87	0.88	0.86	0.86	0.86
Vitamins and trace minerals premix4	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Salt	0.29	0.29	0.29	0.29	0.29	0.29	0.29
Sodium bicarbonate	0.21	0.21	0.21	0.21	0.21	0.21	0.21
L-Lysine HCl	0.15	0.16	0.16	0.17	0.15	0.15	0.15
DL-Methionine, 99%	0.10	0.10	0.11	0.11	0.10	0.10	0.10
L-Threonine, 98%	0.04	0.04	0.05	0.05	0.04	0.04	0.04
Flaxseed	-	1.00	3.00	5.00	-	-	-
Yeast bioactives	-	-	-	-	0.025	0.05	0.10
Calculated provisions
AME, kcal/kg	2,800	2,800	2,800	2,800	2,800	2,800	2,800
Crude protein, %	20.0	20.0	20.0	20.0	20.0	20.0	20.0
SID Lys, %	0.98	0.98	0.98	0.98	0.98	0.98	0.98
SID Met + Cys, %	0.68	0.68	0.68	0.68	0.68	0.68	0.68
SID Thr, %	0.65	0.65	0.65	0.65	0.65	0.65	0.65
Calcium, %	1.05	1.05	1.05	1.05	1.05	1.05	1.05
Available phosphorus, %	0.48	0.48	0.48	0.48	0.48	0.48	0.48
Chloride, %	0.23	0.23	0.23	0.23	0.23	0.23	0.23
Sodium, %	0.18	0.18	0.18	0.18	0.18	0.18	0.18
Analyzed provisions
Dry matter, %	87.0	87.3	87.6	87.8	87.6	87.7	87.4
Crude protein, %	20.1	19.8	20.9	20.7	20.6	21.2	20.0
Gross energy, kcal/kg	3,993	3,987	3,959	4,021	4,096	4,055	4,088
Crude fat, %	5.60	5.91	3.74	3.83	4.10	4.45	4.44
Calcium, %	0.87	0.86	0.85	0.86	0.82	0.86	0.86
Phosphorus, %	0.66	0.68	0.68	0.65	0.64	0.68	0.66
Fatty acids, % of total fat
C18:3	5.26	7.33	10.6	14.5	-	-	-
C20:5	0.11	0.10	0.09	0.09	-	-	-
C22:6	0.02	0.01	0.01	0.01	-	-	-
∑ n-3	5.26	7.33	10.6	14.5	-	-	-
∑ n-6	47.2	51.2	50.6	48.0	-	-	-
∑ n-6: ∑ n-3 ratio	8.98	6.98	4.76	3.31	-	-	-

¹Starter: 0 to 3 wk of age (woa).

²Co-extruded full-fat flaxseed and pulse mixture (1:1 wt/wt), as a source of a-linolenic acid, O & T Farms Ltd., Saskatoon, Saskatchewan, Canada.

³Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives (CBS Bio Platforms Inc., Calgary, Canada).

⁴Provided in kg of diet: vitamin A (retinol), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E, 100 mg; vitamin K3 (menadione), 5.0 mg; vitamin B1 (thiamin), 4.0 mg; vitamin B2 (riboflavin), 10.0 mg; vitamin B3 (niacin), 50.0 mg; vitamin B5 (pantothenic acid), 20.0 mg; vitamin B6 (pyridoxine), 4.0 mg; vitamin B9 (folic acid), 2.0 mg; vitamin B12 (cyanocobalamin), 30.0 mg; biotin, 200 mcg; choline, 400.0 mg; Mg, 110 mg; Zn, 80 mg; Fe, 40.0 mg; Cu, 10.0 mg; I, 1 mg, Se, 0.31 mg.

Table 2.

Composition of experimental diets in grower phase, as fed basis.¹

Item	Control	Flaxseed inclusion, %2			Yeast bioactives inclusion, %3
		1	3	5	0.025	0.05	0.10
Corn	42.8	42.5	41.8	41.1	42.8	42.8	42.7
Soybean meal	21.3	20.8	19.9	19.9	21.3	21.3	21.3
Wheat	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Barley	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Corn germ meal, 60%	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Wheat middlings	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Soy oil	1.88	1.68	1.28	0.88	1.88	1.88	1.88
Monocalcium phosphate	2.03	2.03	2.02	2.02	2.03	2.03	2.03
Limestone fine	0.84	0.85	0.85	0.86	0.84	0.84	0.84
Vitamins and trace minerals premix4	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Salt	0.29	0.29	0.29	0.29	0.29	0.29	0.29
Sodium bicarbonate	0.16	0.17	0.17	0.17	0.16	0.16	0.16
L-Lysine HCl	0.14	0.14	0.15	0.15	0.14	0.14	0.14
DL-Methionine, 99%	0.04	0.04	0.04	0.04	0.04	0.04	0.04
L-Threonine, 98%	0.03	0.03	0.03	0.04	0.03	0.03	0.03
Flaxseed	-	1.00	3.00	5.00	-	-	-
Yeast bioactives	-	-	-	-	0.025	0.05	0.10
Calculated provisions
AME, kcal/kg	2,800	2,800	2,800	2,800	2,800	2,800	2,800
Crude protein, %	18.5	18.5	18.5	18.5	18.5	18.5	18.5
SID Lys, %	0.82	0.82	0.82	0.82	0.82	0.82	0.82
SID Met + Cys, %	0.60	0.60	0.60	0.60	0.60	0.60	0.60
SID Thr, %	0.57	0.57	0.57	0.57	0.57	0.57	0.57
Calcium, %	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Available phosphorus, %	0.45	0.45	0.45	0.45	0.45	0.45	0.45
Chloride, %	0.23	0.23	0.23	0.23	0.23	0.23	0.23
Sodium, %	0.17	0.17	0.17	0.17	0.17	0.17	0.17
Analyzed provisions
Dry matter, %	87.8	87.8	88.1	88.3	87.9	87.7	87.9
Crude protein, %	19.7	19.6	19.2	18.6	20.4	19.5	19.5
Gross energy, kcal/kg	4,011	3,923	4,045	4,025	3,996	4,036	4,000
Crude fat, %	3.00	3.59	3.88	3.81	2.19	3.21	3.39
Calcium, %	0.77	0.81	0.84	0.82	0.80	0.78	0.75
Phosphorus, %	0.62	0.68	0.64	0.62	0.61	0.65	0.62
Fatty acids, % of total fat
C18:3	5.66	7.88	11.8	15.9	-	-	-
C20:5	0.16	0.14	0.13	0.12	-	-	-
C22:6	0.01	0.02	0.02	0.02	-	-	-
∑ n-3	5.66	7.88	11.8	15.9	-	-	-
∑ n-6	55.4	54.3	50.7	47.3	-	-	-
∑ n-6: ∑ n-3 ratio	9.79	6.89	4.28	2.97	-	-	-

¹Grower: 4 to 8 wk of age (woa).

²Co-extruded full-fat flaxseed and pulse mixture (1:1 wt/wt), as a source of a-linolenic acid, O & T Farms Ltd.

³Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives (CBS Bio Platforms Inc.).

⁴Provided in kg of diet: vitamin A (retinol), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E, 100 mg; vitamin K3 (menadione), 5.0 mg; vitamin B1 (thiamin), 4.0 mg; vitamin B2 (riboflavin), 10.0 mg; vitamin B3 (niacin), 50.0 mg; vitamin B5 (pantothenic acid), 20.0 mg; vitamin B6 (pyridoxine), 4.0 mg; vitamin B9 (folic acid), 2.0 mg; vitamin B12 (cyanocobalamin), 30.0 mg; biotin, 200 mcg; choline, 400.0 mg; Mg, 110 mg; Zn, 80 mg; Fe, 40.0 mg; Cu, 10.0 mg; I, 1 mg, Se, 0.31 mg.

Table 3.

Composition of experimental diets in developer phase, as fed basis.¹

Item	Control	Flaxseed inclusion, %2			Yeast bioactives inclusion, %3
		1	3	5	0.025	0.05	0.10
Corn	47.0	45.6	42.3	40.2	47.0	47.0	46.9
Soybean meal	6.77	6.12	4.83	3.53	6.77	6.77	6.77
Wheat	10.0	10.0	10.0	10.0	10.0	10.0	10.0
Barley	15.0	15.0	15.0	15.0	15.0	15.0	15.0
Corn germ meal, 60%	7.00	7.00	7.00	7.00	7.00	7.00	7.00
Wheat middlings	10.1	11.1	13.1	15.1	10.1	10.1	10.1
Soy oil	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Monocalcium phosphate	1.69	1.69	1.69	1.69	1.69	1.69	1.69
Limestone fine	0.79	0.80	0.82	0.84	0.79	0.79	0.79
Vitamins and trace minerals premix4	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Salt	0.27	0.27	0.27	0.26	0.27	0.27	0.27
Sodium bicarbonate	0.16	0.16	0.16	0.17	0.16	0.16	0.16
L-Lysine HCL	0.17	0.18	0.19	0.20	0.17	0.17	0.17
DL-Methionine, 99%	0.02	0.02	0.02	0.03	0.02	0.02	0.02
L-Tryptophan, 98%	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Flaxseed	-	1.00	3.00	5.00	-	-	-
Yeast bioactives	-	-	-	-	0.025	0.05	0.10
Calculated provisions
AME, kcal/kg	2,800	2,800	2,800	2,800	2,800	2,800	2,800
Crude protein, %	15.0	15.0	15.0	15.0	15.0	15.0	15.0
SID Lys, %	0.57	0.57	0.57	0.57	0.57	0.57	0.57
SID Met + Cys, %	0.52	0.52	0.52	0.52	0.52	0.52	0.52
SID Thr, %	0.42	0.42	0.41	0.40	0.42	0.42	0.42
Calcium, %	0.90	0.90	0.90	0.90	0.90	0.90	0.90
Available phosphorus, %	0.37	0.37	0.37	0.37	0.37	0.37	0.37
Chloride, %	0.23	0.23	0.23	0.23	0.23	0.23	0.23
Sodium, %	0.16	0.16	0.16	0.16	0.16	0.16	0.16
Calculated provisions
Dry matter, %	88.8	87.8	87.5	87.9	87.8	87.6	88.0
Crude protein, %	16.2	15.8	16.5	16.1	15.8	15.8	15.3
Gross energy, kcal/kg	4,040	4,028	3,970	3,986	3,996	3,952	4,021
Crude fat, %	2.84	3.11	3.79	3.91	3.00	3.10	3.05
Calcium, %	0.75	0.68	0.81	0.70	0.72	0.72	0.79
Phosphorus, %	0.58	0.57	0.50	0.57	0.53	0.51	0.56
Fatty acids, % of total fat
C18:3	3.83	6.71	11.7	16.4	-	-	-
C20:5	0.21	0.15	0.13	0.10	-	-	-
C22:6	0.03	0.02	0.02	0.02	-	-	-
∑ n-3	3.83	6.71	11.7	16.4	-	-	-
∑ n-6	54.0	54.2	50.4	47.3	-	-	-
∑ n-6: ∑ n-3 ratio	14.1	8.08	4.30	2.88	-	-	-

¹Developer: 9 to 16 wk of age (woa).

²Co-extruded full-fat flaxseed and pulse mixture (1:1 wt/wt), as a source of a-linolenic acid, O & T Farms Ltd.

³Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives (CBS Bio Platforms Inc.).

⁴Provided in kg of diet: vitamin A (retinol), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E, 100 mg; vitamin K3 (menadione), 5.0 mg; vitamin B1 (thiamin), 4.0 mg; vitamin B2 (riboflavin), 10.0 mg; vitamin B3 (niacin), 50.0 mg; vitamin B5 (pantothenic acid), 20.0 mg; vitamin B6 (pyridoxine), 4.0 mg; vitamin B9 (folic acid), 2.0 mg; vitamin B12 (cyanocobalamin), 30.0 mg; biotin, 200 mcg; choline, 400.0 mg; Mg, 110 mg; Zn, 80 mg; Fe, 40.0 mg; Cu, 10.0 mg; I, 1 mg, Se, 0.31 mg.

Experimental Procedures and Samples Collection

The diets were allocated based on BW in a completely randomized design to give 8 replicate cages per diet. Birds had free access of water and feed for 16 wk experimental period. The BW and feed intake on cage bases was recorded every 2 wk. For BW uniformity, 6 birds per cage were wing tagged at placement and were individually weighed every 2 wk. At 4, 8, 12, and 16 woa, one bird per cage was randomly selected, weighed, euthanized, and subsequently dissected for liver, thymus, bursa, and spleen weight. At the beginning of 15 woa, 4 pullets per cage were randomly selected, marked, and weighed for LPS challenge. In each cage, 2 of these pullets were subcutaneously injected with 1 mL of LPS (8 mg/kg of BW, L4130, Sigma-Aldrich, St. Louis, MO) suspended in saline and the other 2 equal volume of sterile saline. The BW of the 4 pullets was monitored at 24, 72 and 168 h post-challenge. Cloaca temperature was measured at 3, 6, 9, 12, 24 h postchallenge. At 6 and 12 h postchallenge, blood samples were taken from alternate LPS or saline pullet per cage at each sampling point (i.e. blood was only drawn once for pullet that received either LPS or saline). Blood was drawn from brachial wing vein using a 25-gauge, 1 inch needle using 10 mL syringe and immediately transferred in tubes coated with lithium heparin (Becton Dickinson & Co, Franklin Lakes, NJ). The samples were placed on ice, transported to the laboratory, and immediately centrifuged at 2,000 × g for 10 min at 4°C to recover plasma, which was immediately stored at −20°C until used for analyses.

Sample Processing and Analyses

Samples of diet were ground in a coffee grinder, thoroughly mixed to be analyzed for dry matter (DM), crude protein (CP), crude fat, calcium, and phosphorous in a commercial laboratory (SGS Canada Inc., Guelph, Canada). Gross energy was measured using a bomb calorimeter (IKA Calorimeter System C 6000; IKA Works, Wilmington, NC). Fatty acids concentration in control and FFF diet samples were analyzed in a commercial lab (Activation Laboratories, Ancaster, Canada) according to O’Fallon et al. (2007). Plasma samples were submitted to Animal Health Laboratory (AHL) (University of Guelph) for analyses of select panel of avian biochemistry profile with the methods described by Greenacre et al. (2008).

Calculations and Statistical Analyses

The BW uniformity was calculated as coefficient of variation (CV, %) by dividing individual BW standard deviation by mean BW. Organ weights were expressed relative to BW (mg/g BW) for statistical analyses. The cage was the experimental unit. Growth performance, BW CV and organ weight data was analyzed in 4 wk intervals: 0 to 4, 5 to 8, 9 to 12, and 13 to 16 woa. Data were tested to check the normality using UNIVARIATE plot normal procedure and subsequently statistical analysis was conducted using PROC GLM procedures of SAS 9.4 with diet as a fixed factor. For LPS responses, the data were analyzed using the Proc GLM procedure of SAS 9.4 with diet, LPS, time, and associated interactions as fixed factors. LSmeans were separated using TUKEY method and linear and quadratic responses to FFF and YB was also evaluated. Significance was declared at P < 0.05 and tendency trends P > 0.05 < 0.10 were discussed.

RESULTS

The analyzed chemical composition of the starter, grower, and developer diets are shown in Tables 1 to 3, respectively. The concentrations of gross energy, crude protein, calcium, and phosphorus were comparable between diets within each phase. The concentration of n-3 FA increased and the ratio of n-6 to n-3 FA reduced with inclusion of FFF in each phase. In the starter diets, the concentration of α-linolenic acid (ALA, % of total fat) was 5.26, 7.33, 10.62, and 14.50 for the control, 1 or 3 or 5% FFF diets respectively (Table 1). Corresponding concentrations in the grower diets were 5.66, 7.88, 11.84, and 15.93 (Table 2) and developer diets were 3.83, 6.71, 11.74, and 16.43 (Table 3), respectively.

Growth Performance and Body Weight Uniformity

Birds fed 0.025% YB were heavier (P = 0.049) than birds fed other diets at 4 woa linked to higher BWG (P = 0.032) between 0 and 4 woa (Table 4). As such YB quadratically (P = 0.003) reduced BW and BWG at 4 woa. At 8 woa, FFF linearly (P = 0.009) and quadratically (P = 0.025), whereas YB tended quadratically (P = 0.067) reduce BW. Linear (P = 0.001) and quadratic (P = 0.003) decrease in BWG was observed in response to FFF between 5 and 8 woa. Whereas YB had quadratic tendency (P = 0.084) to increase BWG between 5 and 8 woa. However, between 13 and 16 woa, BWG of birds fed 1% FFF was higher (P = 0.023) than for birds fed any other diet. Diets had no effects (P > 0.10) on BW at 12 and 16 woa, BWG at 9 to 12 woa and the overall 0 to 16 woa. The BW at 16 woa was 1,172, 1,181, 1,153, 1,169, 1,125, 1,180, and 1,181 g/bird for the control, 1% FFF, 3% FFF, 5% FFF, 0.025% YB, 0.05% YB and 0.1% YB, respectively. There were no (P > 0.10) diet effects on feed intake between 0 and 12 woa or in the overall (0–16 woa). Birds fed 0.025% YB had lower (P = 0.003) feed intake than birds fed other diets during 13 to 16 woa, as result YB increased feed intake quadratically (P = 0.016). Feeding FFF tended to increase (P = 0.079) FCR quadratically in 5 to 8 woa but reduced FCR linearly (P = 0.041) in 9 to 12 woa and quadratically (P ≤ 0.034) in 13 to 16 woa. The YB tended to increase FCR quadratically (P < 0.071) in 5 to 8 woa but exhibited tendency for linear and quadratic (P ≤ 0.078) reduction of FCR in 9 to 12 woa. However, there were no (P > 0.10) diet effects on FCR in the overall, 0 to 16 woa. There were no (P < 0.10) diet effects on BW CV in 0 to 4, 13 to 16 and overall, 0 to 16 woa (Table 4). However, between 5 and 8 woa, FFF increased BW CV quadratically (P = 0.018), whereas YB increased BW CV linearly (P = 0.053) and quadratically (P = 0.047)

Table 4.

Effects of feeding flaxseed (FFF) and yeast bioactives (YB) to Lohmann LSL-Lite pullets from hatch through to 16 wk of age (woa) on growth performance and body weight uniformity.¹

		FFF inclusion, %			YB inclusion, %				P-value
										Response to FFF		Response to YB
Item	Control	1	3	5	0.025	0.05	0.1	SEM	Overall	Linear	Quadratic	Linear	Quadratic
Body weight, g/bird
Day 0	34.8	34.7	34.8	34.7	34.8	34.6	34.8	0.07	0.586	-	-	-	-
4 woa	293b	293b	296b	292b	300a	298b	287c	1.18	0.049	0.916	0.373	0.115	0.003
8 woa	688	676	668	656	670	675	667	3.49	0.247	0.009	0.025C	0.138	0.067
12 woa	975	968	963	970	944	980	968	4.94	0.638	0.723	0.722	0.789	0.457
16 woa	1,172	1,181	1,153	1,169	1,125	1,180	1,181	7.50	0.445	0.720	0.911	0.330	0.246
Body weight gain, g/bird
0–4 woa	258c	258c	261b	257c	265a	264a	252d	1.19	0.032	0.953	0.380	0.144	0.002
5–8 woa	395	380	375	365	378	380	380	2.74	0.076	0.001	0.003C	0.121	0.084
9–12 woa	287	293	292	313	293	298	301	2.98	0.653	0.125	0.614	0.150	0.776
13–16 woa	196c	222a	182d	211b	175d	205bc	204bc	4.25	0.023	0.985	0.052C	0.215	0.488
0–16 woa	1,169	1,135	1,168	1,120	1,129	1,124	1,128	8.60	0.685	0.300	0.823	0.249	0.370
Feed intake, g/bird
0–4 woa	448	463	444	456	465	449	450	4.67	0.648	0.930	0.367	0.484	0.552
5–8 woa	1,279	1,287	1,242	1,245	1,271	1,287	1,247	17.85	0.708	0.241	0.935	0.430	0.386
9–12 woa	1,451	1,432	1,448	1,439	1,404	1,456	1,440	7.91	0.699	0.825	0.753	0.853	0.457
13–16 woa	1,608ab	1,589bc	1,594b	1,624a	1,498d	1,570c	1,565c	9.17	0.003	0.513	0.519	0.618	0.016
0–16 woa	4,766	4,747	4,723	4,746	4,674	4,766	4,738	25.59	0.867	0.886	0.836	0.783	0.569
Feed conversion ratio, g/g
0–4 woa	1.74	1.80	1.70	1.77	1.77	1.70	1.78	0.02	0.594	0.868	0.752	0.232	0.569
5–8 woa	3.24	3.38	3.31	3.42	3.40	3.40	3.28	0.05	0.523	0.181	0.079C	0.768	0.071
9–12 woa	5.07	4.89	4.99	4.70	4.80	4.83	4.79	0.04	0.228	0.041	0.015C	0.069	0.078
13–16 woa	8.63	7.62	8.26	7.88	8.31	7.53	7.79	0.14	0.169	0.248	0.034C	0.021	0.322
0–16 woa	4.08	4.16	4.21	4.25	4.21	4.26	4.20	0.04	0.932	0.251	0.599	0.363	0.311
Body weight uniformity (CV), %
0–4 woa	4.93	4.39	4.90	5.57	4.13	4.14	5.52	0.22	0.498	0.367	0.321	0.552	0.107
5–8 woa	3.76	4.48	3.06	5.37	4.79	4.80	5.49	0.24	0.052	0.208	0.018C	0.053	0.047C
9–12 woa	5.98	5.12	4.50	5.19	4.85	4.97	5.28	0.27	0.859	0.344	0.276	0.534	0.311
13–16 woa	4.24	3.00	3.91	4.95	5.46	5.03	5.71	0.29	0.179	0.344	0.123	0.228	0.711
0–16 woa	4.73	4.81	5.38	4.54	4.60	3.54	4.80	0.16	0.068	0.990	0.228	0.514	0.113

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid; enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

^a–dWithin a row, LSmeans without a common superscript differ P < 0.05.

Lymphoid Organs Weight

Birds fed control diet had heavier thymus (P = 0.001) than birds fed other diets at 4 woa (Table 5). Thymus weight at 4 woa decreased quadratically (P = 0.037) in response to FFF and linearly (P = 0.001) and quadratically (P = 0.027) in response to YB. At 8 woa, FFF linearly (P = 0.012) and quadratically (P = 0.018) decreased in thymus weight but YB had no (P > 0.10) effects on thymus weight. However, there were no (P > 0.10) diet effects on thymus weight at 12 and 16 woa or spleen weight at any time. Feeding FFF linearly (P = 0.017) and quadratically (P = 0.034) reduced bursa weight at 4 woa. However, a tendency for quadratic (P = 0.096) increment in bursa weight was observed in response to FFF at 8 woa. Moreover, FFF quadratically increased bursa weight at 12 (P = 0.009) and at 16 (P = 0.069) woa. Specifically, birds fed 1 or 3% FFF had heavier bursa than control and 5% FFF birds at 12 and 16 woa. Supplemental yeast had no (P > 0.10) effects on bursa weight at any age.

Table 5.

Effects of feeding flaxseed (FFF) and yeast bioactives (YB) to Lohmann LSL-Lite pullets from hatch through to 16 wk of age (woa) on lymphoid organs weight (g/kg BW).¹

		FFF inclusion, %			YB inclusion, %				P-value
										Response to FFF		Response to YB
Item	Control	1	3	5	0.025	0.05	0.1	SEM	Overall	Linear	Quadratic	Linear	Quadratic
Thymus
4 woa	10.58a	8.55b	9.20b	8.94b	6.90c	7.49c	6.91c	0.31	0.001	0.094	0.037C	0.001	0.027Q
8 woa	8.86	7.78	7.51	6.05	8.05	8.04	7.19	0.29	0.261	0.012	0.018C	0.130	0.990
12 woa	5.42	5.39	5.67	5.00	5.62	5.97	5.89	0.14	0.680	0.544	0.369	0.323	0.834
16 woa	3.47	3.21	4.18	3.43	3.50	3.51	3.05	0.12	0.117	0.610	0.123	0.240	0.163
Spleen
4 woa	2.16	2.16	2.13	2.07	2.05	2.11	2.21	0.05	0.988	0.654	0.829	0.731	0.479
8 woa	2.02	2.03	2.03	2.07	2.23	2.23	2.14	0.03	0.395	0.705	0.838	0.335	0.148
12 woa	1.96	2.00	2.16	1.96	2.15	2.21	2.20	0.04	0.449	0.762	0.296	0.138	0.398
16 woa	1.88	1.78	1.73	1.92	2.13	1.90	1.90	0.04	0.085	0.876	0.130	0.670	0.150
Bursa
4 woa	7.65	6.81	6.49	5.96	6.97	6.93	7.52	0.19	0.225	0.017	0.034C	0.846	0.194
8 woa	5.05	4.13	4.99	5.16	4.36	4.66	4.81	0.13	0.211	0.391	0.096Q	0.789	0.206
12 woa	3.92	4.35	4.57	3.79	3.87	4.14	4.09	0.08	0.170	0.877	0.009Q	0.390	0.880
16 woa	2.66	3.10	3.08	2.72	2.91	2.68	2.68	0.09	0.447	0.845	0.069 Q	0.776	0.706

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid; Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

^a-cWithin a row, LSmeans without a common superscript differ P < 0.05.

Response to E. Coli Lipopolysaccharide Challenge

There was no interaction between diet and LPS or diet (P > 0.10) on BW over 168 h post-LPS challenge (Table 6). However, the birds subjected to LPS lost (P = 0.044) more body weight (−12.41 vs. −3.44 g/bird) at 168 h postchallenge compared to nonchallenged birds (Table 6). There were no 3-way interactions between diet, LPS and time (P > 0.10) on cloaca temperature (P > 0.10); however, there were interactions between LPS and time (P < 0.001) and LPS and diet (P = 0.006) (Table 7). The interaction (P < 0.001) between LPS and time on cloaca temperature was such that there were no differences between LPS and saline birds at 3 and 24 h postchallenge (Supplementary Table 1). However, at each time point between 6 and 12 h postchallenge, LPS birds showed higher temperature than saline birds (Supplementary Table 1). The LPS and diet interaction was such that birds fed control, 1, 3% FFF, and 0.10% YB showed higher temperature with LPS challenge relative to saline cohorts and birds fed 5% FFF or 0.025%, 0.05% YB diets. As such in LPS challenged birds, FFF increased cloaca temperature quadratically (P = 0.002) with highest value observed for 3% FFF birds (Supplementary Table 2). Supplemental YB had tendency for quadratic decrease (P ≤ 0.072) in cloaca temperature with birds fed 0.025% YB showing the lowest cloaca temperature (Supplementary Table 2).

Table 6.

Body weight (BW) of 15-wk-old Lohmann LSL-Lite pullets upon challenge with to E. coli lipopolysaccharide (LPS) when fed flaxseed (FFF) and yeast bioactives (YB) from hatch.¹

Item	Prechallenged BW, g	Postchallenge BW, g			Postchallenge BW gain, g
		24 h	72 h	168 h	0–24 h	0–72 h	0–168 h
LPS
-	1,162	1,162	1,159	1,150	−0.49	−1.47	−3.44a
+	1,161	1,160	1,159	1,157	−0.74	−3.91	−12.41b
SEM	5.92	5.92	5.96	6.30	0.806	1.566	3.113
Diet
Control	1,182	1,180	1,175	1,165	−2.38	−7.14	−16.66
1% FFF	1,162	1,161	1,158	1,152	−1.53	−4.58	−10.69
3% FFF	1,153	1,153	1,154	1,155	0.39	1.18	2.75
5% FFF	1,143	1,142	1,141	1,138	−0.74	−2.22	−5.19
0.025% YB	1,152	1,155	1,155	1,147	2.92	2.88	−4.81
0.05% YB	1,171	1,169	1,166	1,158	−1.80	−5.41	−12.63
0.10% YB	1,168	1,167	1,165	1,160	−1.18	−3.54	−8.25
SEM	4.15	4.12	4.13	4.39	1.507	2.929	5.824
P-value
LPS	0.843	0.858	0.933	0.414	0.826	0.275	0.044
Diet	0.201	0.300	0.470	0.729	0.219	0.183	0.331
LPS*Diet	0.887	0.892	0.837	0.489	0.628	0.432	0.119

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid; Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

^a,bWithin a column, LSmeans without a common superscript differ P < 0.05.

Table 7.

Cloaca temperature in 15-wk-old Lohmann LSL-Lite pullets upon challenge with to E. coli lipopolysaccharide (LPS) when fed flaxseed (FFF) and yeast bioactives (YB) from hatch.¹

Item	Temperature, °C
LPS
-	41.93a
+	41.69b
SEM	0.03
Diet Control
	41.85b
1% FFF	41.85b
3% FFF	41.94a
5% FFF	41.75bc
0.025% YB	41.68c
0.05% YB	41.74bc
0.10% YB	41.84b
SEM	0.05
Time, h
3	41.73c
6	41.62d
9	41.85b
12	41.74bc
24	42.13a
SEM	0.04
P-value
LPS	<0.001
Diet	0.0003
Time	<0.001
LPS*Diet	0.006
LPS*Time	<0.001
Diet*Time	0.322
LPS*Diet*Time	0.619

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid; Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

^a-cWithin a column, LSmeans without a common superscript differ P < 0.05.

There were no (P > 0.10) interactions between LPS, diet and time on plasma concentration of total protein, albumin, globulin, glucose, cholesterol, uric acid, bile acid, and carbon dioxide (Table 8). There were (P ≤ 0.014) interactions between LPS and time on the concentration of plasma metabolites. Specifically, concentration of total protein, albumin, glucose, cholesterol, sodium, chloride and the ratio of albumin and globulin was lowest at 6 h postchallenge relative to 12 h postchallenge or in saline birds at any time point (Supplementary Table 3). There was diet effect (P ≤ 0.036) on plasma concentration of cholesterol and bile acid such that, birds fed 0.10% YB showed higher plasma cholesterol than birds fed other diets and control birds exhibited higher plasma bile acid than birds fed FFF and YB. Data for the concentration of plasma enzymes and minerals are shown in Table 9. There were interactions between LPS, diet and time on plasma concentration of creatine kinase (CK; P = 0.030) and trend (P = 0.070) for Cl. The LPS birds fed control showed less plasma CK at 6 and 12 h along with those fed 1% FFF, 0.05% YB at 12 h than birds fed FFF, 0.05 and 0.1% YB or non-LPS birds fed control, 1% FFF and 0.025% YB at 6 h (Supplementary Table 4). There was interaction (P ≤ 0.090) between LPS and time on plasma amylase, Gamma-glutamyl transferase (GGT), Glutamate dehydrogenase (GLDH), Na, and Cl. A diet and time interaction (P = 0.004) was such that control birds had higher plasma P at 12 h postchallenge than birds fed control, FFF and 0.05% YB at 6 h or 0.025% YB at 12 h. Interaction (P ≤ 0.03) between LPS and diet was observed for plasma K and Cl. For K, FFF reduced plasma concentration of K linearly (P < 0.001) and quadratically (P = 0.021) and increased concentration of Cl quadratically (P = 0.016) in non-LPS birds only. Yeast reduced plasma K linearly (P < 0.001) and quadratically (P = 0.001) in non-LPS birds and tended to increase concentration of Cl quadratically (P = 0.071) (Supplementary Table 4). There were no (P > 0.10) 3- or 2-way interactions between LPS, diet and time on plasma aspartate aminotransferase (AST), lactate dehydrogenase (LDH), lipase, and Ca. LPS increased AST (P < 0.001), and reduced lipase (P = 0.0002). Birds fed control tended to have higher plasma AST (P = 0.072) than birds fed 3 or 5% FFF or YB diets (Table 9). Moreover, control and 3% FFF birds had higher plasma lipase (P = 0.007) than birds fed 0.05 and 0.1% YB birds.

Table 8.

Concentration of plasma metabolites in Lohmann LSL-Lite pullets upon challenge with to E. coli lipopolysaccharide when fed flaxseed (FFF) and yeast bioactives (YB) from hatch.¹

Item	Total protein, g/L	Albumin (A), g/L	Globulin (G), g/L	A:G ratio	Glucose, mmol/L	Cholesterol, mmol/L	Uric acid, μmol/L	Bile acid, μmol/L	Carbon dioxide, mmol/L
LPS
-	43.87	17.06a	26.82	0.645a	13.12a	3.21b	202.9b	12.41b	15.21
+	43.86	16.43b	27.41	0.608b	12.40b	3.37a	224.5a	18.39a	14.90
SEM	0.329	0.145	0.300	0.009	0.079	0.031	5.864	0.812	0.161
Diet
Control	43.31	16.52	26.79	0.623	12.80	3.33b	228.6	20.49a	15.23
1% FFF	43.78	16.92	26.86	0.642	12.88	3.23bc	211.3	14.16b	15.10
3% FFF	43.19	16.16	27.03	0.609	12.67	3.10c	215.1	14.03b	14.94
5% FFF	44.09	16.75	27.34	0.619	12.85	3.11c	211.8	15.25b	14.91
0.025% YB	43.68	16.92	26.77	0.639	12.73	3.36b	195.8	14.10b	15.11
0.05% YB	43.99	16.97	26.95	0.642	12.83	3.35b	230.0	14.24b	14.96
0.10% YB	45.03	16.97	28.06	0.611	12.56	3.54a	203.3	15.53b	15.16
SEM	0.615	0.270	0.562	0.017	0.148	0.058	10.970	1.519	0.302
Time, h
6	42.78b	15.90b	26.86	0.600b	12.58b	3.06b	204.3b	14.40	14.48b
12	44.96a	17.59a	27.37	0.653a	12.94a	3.52a	223.1a	16.40	15.63a
SEM	0.329	0.145	0.300	0.009	0.079	0.031	5.864	0.812	0.161
P-values
LPS	0.983	0.002	0.162	0.005	<0.0001	0.0003	0.010	<0.0001	0.167
Diet	0.428	0.266	0.666	0.635	0.727	<0.0001	0.270	0.036	0.985
Time	<0.0001	<0.0001	0.238	<0.0001	0.002	<0.0001	0.024	0.082	<0.0001
LPS*Diet	0.343	0.099	0.496	0.536	0.381	0.165	0.689	0.619	0.866
LPS*Time	<0.0001	<0.0001	0.137	<0.0001	<0.0001	<0.0001	0.014	0.018	<0.0001
Diet*Time	0.616	0.709	0.373	0.323	0.779	0.232	0.920	0.894	0.307
LPS*Diet*Time	0.555	0.589	0.309	0.236	0.739	0.297	0.796	0.836	0.716

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

^a,bWithin a column, LSmeans without a common superscript differ P < 0.05.

Table 9.

Concentration of plasma enzymes and minerals in Lohmann LSL-Lite pullets upon challenge with to E. coli lipopolysaccharide when fed flaxseed (FFF) and yeast bioactives (YB) from hatch.¹

	Enzymes2, U/L							Minerals, mmol/L
	Amylase	AST	CK	GGT	GLDH	LDH	Lipase	Calcium	Phosphorous	Sodium	Potassium	Chloride
LPS
-	374.8b	209.9b	2,790.7	19.36a	3.18b	459.7	9.02a	2.29	1.47	151.2a	5.93a	111.3a
+	445.2a	325.2a	2,706.3	17.67b	5.87a	716.5	6.92b	2.27	1.52	148.5b	5.54b	109.2b
SEM	10.47	15.69	179.39	0.277	0.666	128.29	0.392	0.028	0.020	0.264	0.041	0.199
Diet
Control	404.8	349.9a	1,833.6b	18.67	5.58a	560.5	9.09a	2.29	1.55	149.0	5.95a	109.9
1% FFF	412.7	228.1b	2,868.1a	18.08	7.00a	665.7	8.58ab	2.23	1.51	150.2	6.11a	110.7
3% FFF	452.8	235.2b	2,895.8a	19.16	4.34a	452.8	9.69a	2.21	1.49	149.8	5.72b	110.4
5% FFF	405.1	289.5ab	3,273.5a	19.06	4.72a	1,026.2	7.97abc	2.26	1.46	149.9	5.73b	110.4
0.025% YB	395.8	258.7b	2,748.8a	17.85	2.83b	456.0	7.71abc	2.26	1.45	149.5	5.58bc	109.9
0.05% YB	405.6	249.5b	2,864.8a	17.97	2.21b	455.1	6.56bc	2.33	1.50	151.0	5.58bc	110.8
0.10% YB	393.2	261.6b	2,755.1a	18.81	5.00a	500.7	6.22c	2.38	1.48	149.8	5.50c	109.6
SEM	19.59	29.34	335.60	0.519	1.246	240.0	0.734	0.052	0.037	0.495	0.076	0.372
Time, h
6	424.5a	300.8a	4,346.8a	18.60	4.48	822.8a	6.71b	2.29	1.43b	149.4b	5.79a	110.2
12	395.5b	234.3b	1,150.2b	18.43	4.57	353.4b	9.24a	2.26	1.55a	150.4a	5.69b	110.3
SEM	10.47	15.69	179.39	0.277	0.666	128.29	0.392	0.028	0.020	0.264	0.041	0.199
P-values
LPS	<0.0001	<0.0001	0.740	<0.0001	0.005	0.159	0.0002	0.562	0.084	<0.0001	<0.0001	<0.0001
Diet	0.392	0.072	0.121	0.368	0.130	0.604	0.007	0.225	0.554	0.152	<0.0001	0.207
Time	0.051	0.003	<0.0001	0.663	0.924	0.010	<0.0001	0.418	<0.0001	0.007	0.096	0.657
LPS*Diet	0.703	0.789	0.010	0.867	0.255	0.391	0.176	0.599	0.881	0.181	<0.0001	0.031
LPS*Time	<0.0001	0.543	0.186	0.090	0.058	0.185	0.367	0.791	0.413	<0.0001	0.205	<0.0001
Diet*Time	0.424	0.452	0.238	0.646	0.424	0.537	0.264	0.235	0.004	0.581	0.181	0.481
LPS*Diet*Time	0.648	0.490	0.030	0.877	0.250	0.421	0.691	0.692	0.212	0.160	0.268	0.070

¹Co-extruded full-fat flaxseed and pulse mixture (FFF, 1:1 wt/wt), as a source of a-linolenic acid; Enzymatically bio-processed yeast cell walls from Saccharomyces cerevisiae, as a source of yeast bioactives.

²AST: aspartate aminotransferase; CK: creatine kinase; LDH: lactate dehydrogenase; GGT: gamma-glutamyl transferase; GLDH: glutamate dehydrogenase; LDH: lactate dehydrogenase.

^a,bWithin a column, LSmeans without a common superscript differ P < 0.05.

DISCUSSION

Rearing period is critical for lifetime performance of laying hen as pullet development and laying productivity are highly correlated (Pottgüter, 2016). It is thus important to optimize pullet management and nutrition for target BW and flock uniformity for laying hen performance (Leeson and Summers, 2005; Janczak and Riber, 2015; Jongman, 2021). There is limited research on the role of functional dietary components in pullet growth and development. Some effects of FFF or YB were noted on growth performance in some phases in the present study but no effects in the overall (0–16 woa). We previously reported that feeding 2.3% FFF did not have effects on BW in egg type (Akbari Moghaddam Kakhki et al., 2020) or broiler breeder (Thanabalan and Kiarie, 2021) pullets. Another study observed no effect of feeding 5% to 10% flaxseed on pullet growth (Arshami et al., 2010). In contrast to the present study, feeding 1.25% yeast bioactives to Hy-Line W-36 pullets from placement resulted in heavier birds at 12 woa (Elliott et al., 2020). In broiler chickens, yeast cell wall bioactives have been shown to improve growth performance (El-Manawey et al., 2021; Maina et al., 2022) but no effects in other studies (Kiarie et al., 2022; Sedghi et al., 2022). As such responses of yeast supplements in poultry growth are variable, necessitating further research to elucidate of the mode of action. As a pivotal indicator of flock management, pullet body weight uniformity (BW CV) is of great interest at the point of lay (Hudson et al., 2001; Sujatha et al., 2014). A flock with higher uniformity at point of lay attains peak egg production earlier and produces more and heavier eggs in lifetime and will peak higher than a nonuniform flock due to varying degrees of maturity (Hudson et al., 2001; Leeson and Summers, 2005). While there are little scientific data available, pullet flock with ≤10% BW CV at sexual maturity is considered uniform (Arrazola et al., 2019; Thanabalan and Kiarie 2021). The observed BW CV in the present study was well below 10%. However, birds fed 3% FFF had lower BW CV between 5 and 8 woa and tended to increase with YB. These observations are difficult to explain, however, we previously observed that broiler breeders fed 2.3% FFF from placement had a higher BW CV at 22 woa (Thanabalan and Kiarie, 2021).

The lymphoid organs size is indicative of immunocompetence in an animal (Pope, 1991; Martínez et al., 2021). Studies have demonstrated dietary provision of n-3 FA modulated development of lymphoid organs and lymphocyte proliferation in mammalian and avian species through their role in membranes biogenesis (Fritsche et al., 1991; Yaqoob et al., 1994; Sanderson et al., 1995). In the present study, FFF reduced thymus and bursa weight when birds were younger (<8 woa); however, 1% to 3% FFF tended to improve bursa weight in older birds (>12 woa). Feeding broiler breeder pullets 2.3% FFF had no effects on spleen and bursa weight at 5 and 12 woa (Thanabalan and Kiarie, 2021). Single Comb White Leghorn pullets fed sources of n-3 FA (5% linseed or fish oil) at placement had heavier thymus, bursa, and spleen relative to cohorts fed 5% animal fat at 4 woa (Wang et al., 2000). However, at 8 woa pullets fed animal fat had heavier bursa than those fed n-3 FA (Wang et al., 2000). Based on the present and previous studies, it seems plausible the response of lymphoid organs to n-3 FA was dependent on n-3 FA dose, dietary n-3 FA sources (ALA, EPA, DHA) and bird age. However, investigations on the impact of n-3 FA on the functionality of these organs in terms of proliferation, differentiation, and maturation of adaptive immune cells production warrant further investigations. Yeast cell wall extracts are known to exhibit immunomodulatory properties (Leung et al., 2018,2019; Lu et al., 2019). However, we did not observe effects of YB on lymphoid organs in the present study. Similarly, supplementation of 1.25% yeast fermentation product derived from S. cerevisiae in diets fed to Hy-Line W-36 pullets from hatch to 12 woa had no effects on bursa weight. However, supplementation of yeast cell wall derivatives in the dosages tested in the present study are often reported to enlarge lymphoid organs in broiler chickens (Guo et al., 2003; Zhang et al., 2008; Maina et al., 2022), but in contrast to that results, some of other studies yeast cell wall derivatives did not affect to the size of lymphoid organs (Rathgeber et al., 2008; Kiarie et al., 2022). The differing lymphoid organs responses to yeast bioactives may thus be attributed to differences in bird type and composition of tested yeast products.

Lymphoid organs morphology provides limited information on the impact of dietary intervention on immunocompetence. Utility of immunological and/or oxidative stress models that influences physiological and biochemical processes of animal are accepted approaches for evaluating immunomodulatory attributes of a dietary intervention (Kiarie et al., 2019b). As a component of Gram-negative bacteria cell wall, exogenous administration of LPS elicits pro-inflammatory response (Remick et al., 2000). Poultry are exposed to large amounts of LPS through inhalation of dust and colonization of the air sacs with E. coli emanating from gut-fecal route (De Boever et al., 2009; Linden, 2015). Administration of LPS has been found to negatively affect performance and immunometabolism in poultry (Huff et al., 2008; Yang et al., 2008; Munyaka et al., 2012; Jing et al., 2014; Akbari Moghaddam Kakhki and Kiarie, 2021). In the present study, LPS challenge reduced the BWG, administration of LPS elicits production of pro-inflammatory cytokines that increases acute phase proteins and redirects nutrients such as amino acids and minerals from supporting growth (Huff et al., 2008; Yang et al., 2008; Munyaka et al., 2012; Jing et al., 2014; Akbari Moghaddam Kakhki and Kiarie, 2021). However, there were no dietary treatment effects in the present study. Similarly, feeding 2.3% FFF did not reverse negative effects of LPS on laying hen performance and bone metabolism (Akbari Moghaddam Kakhki and Kiarie, 2021). Whereas supplementation of 4.5% fish oil had no effects on growth performance of broiler chickens challenged with LPS (Yang et al. 2008). Broiler chickens injected with LPS on 4 and 9 of life showed reduced body weight gain relative to nonchallenge group on d 21 irrespective of whether they were fed 0 or 500 mg yeast cell wall per kg of feed (Morales-Lopez and Brufau, 2013). Thus, these studies suggested n-3 FA and yeast bioactives have varied effects on the acute phase depression of performance due to an LPS challenge. It is plausible that the higher demand for nutrients exerted by acute phase response at the expense of growth (Klasing and Korver, 1997) cannot be modified by feed additives.

Febrile response is the hallmark of inflammatory response in LPS challenge model (De Boever et al., 2009). In the present study, we evaluated cloaca temperature as a basis of establishing whether one-dose, one-time LPS challenge was effective. We did not observe 3-way interactions between LPS, time points for cloaca temperature measurements and diet. However, we did observe an interaction between LPS and time on cloaca temperature, in which case challenged birds had elevated temperature relative to saline birds at 6, 9, and 12 h postchallenge. Indicating that LPS elevated core-body temperature and as such effective challenge model. Similarly, a single intravenous injection of Salmonella typhimurium LPS elevated core temperature measured at cloaca by 3 h relative to saline control in broiler chickens (Xie et al., 2000). However, there was no difference between LPS and saline birds on cloacal temperature at 12 and 24 h postinjection. Administration of LPS in broiler chickens elevated cloacal temperature 4 h postchallenge (Ahiwe et al., 2019). Increased plasma concentration of pro-inflammatory cytokines such as interleukin 6 precedes the febrile phase in a LPS challenge model in chickens (De Boever et al., 2009). We hypothesized, feedstuffs with immunomodulatory attributes such as n-3 FA and yeast metabolites might downregulate LPS induced fever. However, interaction between LPS and diet indicated exacerbation of fever linked to the dose of FFF and YB. It is difficult to explain these observations. A previous study suggested that dietary n-3 FA from fish oil initialized reduced host natural resistance to Listeria monocytogenes in mice (Puertollano et al., 2004). Yet another study indicated, feeding fish oil attenuated febrile response induced by injection of murine recombinant interleukin-1 (rIL-1) in guinea pigs linked to increased uptake of n-3 FA in plasma phospholipids (Pomposelli et al., 1989). Studies showed that feeding higher levels of n-3 FA diminished chick lymphocyte proliferation and that linseed oil rich in ALA exhibited differences from fish oil rich in EPA and DHA (Wang et al., 2000). Feeding broiler chickens whole yeast or isolated yeast constituents (mixture of cell walls, purified glucans, and purified mannans) had no effects on cloacal temperature upon challenge with Salmonella typhimurium LPS (Ahiwe et al., 2019). The metabolic response to infections and/or injuries is a cascade of complex physiological changes designed to limit the extent and magnitude of the injury and to promote restoration of homeostasis (Romanovsky et al., 1996). It is possible n-3 FA and yeast metabolites can facilitate quicker and a more efficacious innate immune response in the presence of exogenous LPS antigens, however, more research is required to refine doses and other host factors that may influence outcomes.

Changes in plasma biochemistry in response to LPS challenge are indicative of perturbations of metabolism due to immunological stress. An injection of 8 mg/kg of BW in 32-wk-old hens reduced blood concentration of total proteins, albumin, globulin, and T-cell subsets (CD3+, CD4+, CD8+) but upregulated serum heterophils and expression of genes for cytokines spleen and cecal tonsil tissues compared with the saline-injected hens (Munyaka et al., 2012). However, LPS administered to 13- to 17-day-old broiler chickens had no effects on plasma total protein, globulin, immunoglobulins, cholesterol, and triglyceride (Ahiwe et al., 2019). Plasma glucose transiently reduced and then increased at 12 h post-LPS challenge in the present study. Reduced plasma glucose may be due to LPS inflammation anorexia (Freeman, 1970). Cholesterol level was increased, and it was greater at 12 h than at 6 h post-LPS challenge. These results concurred with previous study in which stressed birds exhibited elevated levels of blood cholesterol (Everds et al., 2013). Independent of LPS, FFF reduced plasma cholesterol in alignment with the fact that n-3 FA are known to reduce plasma cholesterol (Connor et al., 1993). Plasma uric acid is an indicator of oxidative stress and kidney function (Rivetz et al., 1977; Jing et al., 2014; Elazab et al., 2022). Furthermore, elevated plasma bile acids can be linked to liver disease (Bromidge et al., 1985). Overall, these parameters indicated that our LPS model induced hepatic and renal response; however, the diets had no effects.

Plasma enzymes are key biochemical markers of hepatic health and function and their elevation in circulation is interpreted as a sensitive indicator of liver injury (Rivetz et al., 1977; Jing et al., 2014; Cha et al., 2020). The LPS challenge elevated plasma AST and GLDH and decreased GGT and lipase in the present study. These observations are similar to previous studies which demonstrated that changes of AST level are related to damage and morphological alteration of liver in turkey and chicken (Curtis and Butler, 1980; Elazab et al., 2022). Plasma CK exhibited interaction between diet and LPS challenge. The changes in plasma CK activities may be related to muscle damage (Hoffmann and Solter, 2008; Lumeij, 2008). We did not investigate specific parameters to ascertain muscle damage in the present study; however, n-3 FA and yeast bioactives can reduce such damage. Previous studies demonstrated that the elevation of GGT represented inflammation response and was linked to hepatic abnormalities (Chen et al., 2013; Shini et al., 2020). Moreover, the measurement of GGT level is utilized as typical liver dysfunction indicator in livestock, including birds (Center, 2007). The plasma electrolytes reduced in response to the LPS challenge in the present study. Among these, potassium had an interaction effect of diet and LPS challenge. However, it is rather difficult to deduce specific relationship between flaxseed, yeast bioactives and LPS on mineral metabolism.

We investigated the role of n-3 FA and yeast bioactives on the growth, and development of lymphoid organs and immunocompetence in egg-type pullets. The tested feedstuffs had a minimal influence on overall pullet growth trajectory but had notable effects on lymphoid organs weight and parameters evaluated post LPS challenge. Further research is required to refine utility of functional feedstuffs in feeding programs for pullets. Moreover, the goal of future research should determine the potential benefits of immunomodulating feedstuffs in rearing phase on egg production and quality throughout a complete lay cycle.