Immune function, egg quality and production responses in layer hens fed two different lignocellulose fibre supplements during the early laying period

Abstract The effects on immune function and egg productivity of 2 commercial lignocellulose supplements with similar insoluble and soluble fibre concentrations and water holding capacities, were determined in Hy-Line Brown hens from 20 weeks-of-age for 8 weeks. Hens were fed 1 of 3 diets (6 pens/diet, 3 hens/pen): Control diet – a commercial layer diet; Diet LC1 - Control diet plus 1.5 g Arbocel RC fine/100 g diet; Diet LC2, Control diet plus 1.5 g OptiCell C5/100 g diet. After 4 weeks, heterophil phagocytosis index and percentage (innate immunity), was greater in LC1 (p < 0.05) than Controls. After 8 weeks, absolute and relative weights (g/100 g BW) of LC1 thymus glands were heavier (p < 0.05) than Control; numbers of intestinal Peyer’s patches in LC1 were greater (p < 0.05) than LC2 and Control; total and relative areas of LC1 Peyer’s patches were higher (p < 0.05) than Control. Yolk immunoglobulin Y concentrations of LC1 were higher but not significantly, than Control and LC2. Between 6 and 8 weeks, egg weights and eggs produced/hen per d of LC1 hens were greater (p < 0.05) than Controls. Feeding LC1, but not LC2, during early lay significantly improved immune function and egg productivity compared to Control. As dietary fibre concentrations of LC1 and LC2 were similar, differences in their relative concentrations of chemical components such as cellulose and lignin or types of polysaccharides, may have contributed to the improvements during early lay. HIGHLIGHTS Lignocellulose supplements (LC) had similar soluble and insoluble fibre contents. Hens fed LC1 had improved immune function and egg production compared to Controls. LC2 caused no significant improvement. As fibre concentrations of LC1 and LC2 were similar, differences in concentrations of chemical components such as cellulose and lignin may have contributed to their different effects.


Introduction
Microbial resistance to antibiotics and its implications for the treatment of human diseases has led to the restriction or banning of in-feed antibiotics for improved production of poultry (Laxminarayan et al. 2015).As a result, different sources of dietary fibre, among other products, are being investigated as alternatives for the promotion of poultry health and production (Guzm� an et al. 2016;Yokhana et al. 2016;Hussein et al. 2017;Sozcu and Ipek 2020).
Although dietary fibre is a complex mix of chemical compounds that have different physiological effects on poultry, its impact as a beneficial supplement is frequently attributed to the relative contents of insoluble, non-fermentable fibre (IF) and soluble, fermentable fibre (SF) (Dhingra et al. 2012).The attributes of IF and SF relate primarily to the degree of solubility and retention time of the fibre in the digestive tract (Bach Knudsen 2014;Choct 2015).The benefits of adding IF to poultry diets have often been shown to be greater on growth, production and health than SF (Mateos et al. 2012;Tejeda and Kim 2021).Insoluble fibre from different sources can, however, have different effects on production or physiological processes.Gonz� alez-Alvarado et al. (2008), for example, observed different effects of two IF sources -oat hulls (OH) or soya hulls (SH) -on the gizzard and proventriculus of broilers.Relative weight (RW) of the gizzard and gut retention time of ingesta was greater with OH than SH: the authors suggest that the higher concentration of lignin in OH contributed to these results.On the other hand, SH caused increased RW of the proventriculus and water holding capacity compared with OH (Gonz� alez- Alvarado et al. 2008).Other examples of different responses to supplementation by different IF sources have been reported on weight gain (Guzm� an et al. 2015), nutrient retention (Jim� enez-Moreno et al. 2019), gut microbiota (Zeitz et al. 2019) and immunity (Sadeghi et al. 2015;Hussein and Frankel 2019).
The 2 lignocellulose fibre (LC) supplements (LC1 and LC2), used in the current experiment have been shown to cause differences in the development and immune function of layer pullets from 4 to 8 weeks ofage and from 10 to 18 weeks-of-age (Hussein et al. 2017).The current experiment was designed to determine whether the LC supplements fed from 2 weeks before the start of lay through to 28 weeks-of-age, could improve the immune functions and rates of egg production and egg quality in hens during early lay.
Published analyses of the 2 LC show that ranges of concentrations for IF and SF (all in g/100g) overlap.Range for IF in LC1 is 69.8 − 94.4, and in LC2 it is 86.7 − 94.2: for SF, the range in LC1 is 0.34 − 1.4 and for LC2, it is 1.1 − 1.3 (see Supplementary Table S1).Ranges for water holding capacities are, 6 -7 g/g supplement for LC1 and 3.79 − 7.3 for LC2 (Table S1).
The hypothesis was that hens fed either of the LC supplements for a relatively short period of time from 2 weeks before the start of lay (20 weeks-of-age) through to 28 weeks-of-age, would have improved immune function, egg quality, and production compared with un-supplemented Controls and that the effects of each supplement would not be different.

Experimental birds, housing and diets
Sixty, 16-week-old vaccinated, Hy-Line Brown pullets, were obtained from a single rearing-shed on a grower pullet farm in Victoria, Australia.Pullets were given a commercial grower pullet diet (Table 1, Pullet Grower, Barastoc, Ridley AgriProducts Pty Ltd, Pakenham, 3810, Australia) until 18 weeks-of-age: thereafter they were given a commercial laying hen diet (Table 1, Golden Yolk, Barastoc).At 20 weeks-of-age, hens were checked for physical abnormalities and signs of ill-health.Fiftyfour healthy hens were selected, weighed, and randomly distributed into 3 treatment groups of 6 pens/ treatment, 3 hens/pen.There was no significant difference in live body weights (BW) of the 3 groups at the beginning of the experiment (Table 2) and mean weights were in the upper range of standard weights for the Hy-Line Brown strain (1630 − 1730 g; Hy-Line Brown International 2014).
Hens were all housed within 1 shed in floor pens, 0.9 m x 1.8 m x 1.8 m (width x length x height) on plastic slatted floors without bedding: nest boxes and perches were provided in each pen.Because of the type of flooring and feeders, accurate feed intake measurements and faecal sample collection were not possible.The ambient temperature was between 20 to 23 � C. Light interval was 10 h/d at 16 weeks-of-age and was increased gradually each week to 15 h/d at 28 weeks-of-age (Hy-Line Brown International 2014).The Control Group (C) was fed only Golden Yolk laying hen diet (Barastoc, Table 1); Group LC1 was fed the Control diet supplemented on top with 1.5 g/100 g diet of a commercial LC, Arbocel RC Fine (JRS Co. Inc., Rosenberg, Germany); Group LC2 was fed the Control diet supplemented on top with 1.5 g/100 g diet of the commercial LC, OptiCell C5 (Agromed Austria GmbH, Kremsm€ unster, Austria).Published analyses of Arbocel RC Fine and OptiCell C5 are in Supplementary Table S1.
The crude fibre (CF) concentrations in the three diets were analysed by FeedTest, Agrifood Technology Pty. Ltd., Australia.Control diet contained 6.4 g CF/ 100 g diet; diet LC1 − 7.31 g CF/100 g and diet LC2 − 7.24 g CF/100 g.
Hens in the three dietary treatments were fed according to the recommendations of Hy-Line Brown management guide (Hy-Line Brown International 2014) and fresh water was always available.
The experimental procedures were approved by the Animal Ethics Committee of La Trobe University under the approval number LTU AEC14-16.

Immune function measurements
Four weeks after the start of the experiment, blood samples were collected from 1 hen/pen, randomly selected, for measurement of heterophil phagocytosis and oxidative burst activities.At week 7, blood samples (1 hen/pen) were collected for measurement of lymphocyte proliferation from hens that had not previously been sampled.At week 8, all hens (3 hens/pen, 18 hens/treatment) were individually weighed and then killed with an intravenous overdose of pentobarbital sodium (Lethabarb, Virbac Animal Health, Milperra, NSW, Australia).Primary (thymus glands and bursa of Fabricius) and secondary lymphoid organs (spleen and caecal tonsils) of all hens were weighed and average weight/pen was the unit for statistical analysis.Samples of the small intestine (jejunum plus ileum) were weighed and stored at −20 � C for later measurements on Peyer's patches (PP).
Methods to determine the innate immune function (phagocytosis of latex beads and mitogen stimulated oxidative burst in isolated heterophils), active immunity by mitogen-stimulated lymphocyte proliferation in isolated cells, measurements for primary and secondary lymphoid tissues and organs, and the number and size of PP have been described in detail in Hussein et al. (2017) and Hussein and Frankel (2019).
The concentration of yolk IgY in 2 eggs/pen collected in week 8, was determined by an ELISA assay (IgY Chicken SimpleStep ELISA kit, Product number: ab189577, abcam, Cambridge, UK).Reagents were prepared and procedures were followed according to the manufacturer's instructions and are briefly described.
Thawed yolk samples (2/pen) were individually mixed and about 1 g (accurate to 0.01 g) of each yolk was weighed into a 15 ml screwcap, conical bottom centrifuge tube (Cellstar,Interpath,Heidelberg West,Vic.,Aust.,3081).Cell extraction buffer supplied in the kit was added at exactly two times the weight of the yolk.Samples were homogenised on ice with a 12 mm Polytron homogeniser probe (Kinematica AG, Switzerland) for 1 min then centrifuged at 16,000 x g for 10 min at 4 � C (ScanSpeed To calculate a mean for each pen, three hens/pen were used for measurements of immune organ weights and 1 hen/pen for PP measurements and jejunal and ileal areas.C Control diet -see Table 1.LC1 -Control diet plus 1.5 g/100 g Arbocel RC fine.LC2 -Control diet plus 1.5 g/100 g OptiCell C5.

Refrigerated
Multi-Purpose Centrifuge, LaboGene ApS, Denmark).The supernatant (egg yolk lysate) was stored undiluted in aliquots of about 50 ll at −20 � C until used in the IgY ELISA assay.
On the day the ELISA assay was carried out all samples and reagents (prepared according to manufacturer's instructions) were kept on ice until being equilibrated to room temperature for use in the assay.Standards of IgY were prepared by serial dilution of the reconstituted IgY chicken lyophilised purified protein (IgY standard protein) to give concentrations ranging from 10.0 to 0.16 ng IgY antibody/mL of the supplied diluent.Egg yolk lysate samples were serially diluted with diluent to a final dilution of 1:6 x 10 6 to give an estimated final concentration within the range 1.25 − 2.0 ng IgY/mL.
Blank controls (diluent), standards, and yolk lysate samples were analysed in duplicate in the SimpleStep Pre-Coated 8-well strips supplied in the kit.After adding the antibody cocktail to each well, they were incubated for 1 h at room temperature on a plate shaker at 400 rpm.Each well was washed three times with the wash buffer, everted and blotted onto paper to remove excess liquid.After adding the indicator solution (3,3 0 ,5,5 0 tetramethylbenzidine) to each well, the wells were incubated for 3 min in the dark with shaking at 400 x g.The stop solution was added quickly, and wells were shaken for 1 min.Then absorbance was measured in a microplate reader (EnSpire, PerkinElmer Co, USA), wavelength 450 nm.

Measurements of egg production and quality
All eggs were collected and individually weighed each day.Numbers of eggs laid/pen per d were recorded and number of eggs produced/hen per d calculated for each pen over four, 2-week intervals (0 -2; 2 -4; 4 -6 and 6 -8 weeks after the start of the experiment).Over the same 4 periods, the mean egg weight/pen (g egg/hen per d) was calculated by dividing the total eggs produced per pen in 2 weeks by ( 14x number of hens/pen).
At 4 and 8 weeks after the start of feeding the experimental diets (24 and 28 weeks-of-age), 3 eggs laid during the past 24 h were randomly selected from each pen for measurements of Haugh units and eggshell thickness at room temperature.On the day of collection, unrefrigerated eggs were separately broken onto a glass surface and Haugh units were measured using an Ames micrometre (accuracy of 0.1 mm, Model S-8400, Ames Co., Waltham, MA, USA): direct reading of Haugh unit values was made after adjusting the micrometre for the egg weight including shell (Bish et al. 1984).Egg-shell thickness was measured using a ball pinpoint calliper with an accuracy of 0.01 mm (Model: 40142244, 30 mm, UK).The average of measurements on 3 eggs/pen were used for statistical analyses.
At 8 weeks, following the measurement of Haugh units, yolks of 2 eggs/pen were separated from the albumen, and weights were determined: the yolks were placed in separate sterile 50 mL polypropylene tubes (Greiner Bio-one) and stored at −20 � C for later determination of IgY concentration (see above).The egg-shells were weighed after being air-dried in the laboratory at room temperature for 24 h (Yalc ¸ın et al. 2012).The weight of albumen of each egg was determined by subtracting the weights of the yolk and egg-shell from the weight of the whole egg.

Statistical analyses
Data was analysed with SPSS 23.0 for Windows.The data from layer hens were tested for homogeneity of variances and normality using Shapiro-Wilk's test (Shapiro and Wilk 1965) before being analysed statistically to check for significant differences among treatments.All variables for which data were normally distributed, were analysed statistically with one-way analysis of variance (ANOVA) and if significant, differences between treatment means were tested with a post hoc significance test, Tukey's comparison test.Statistical significance was taken at a value of p < 0.05.
Absolute weight and RW of the bursa of Fabricius and caecal tonsils and lymphocyte proliferation stimulated with mitogen PMA were not normally distributed and were therefore analysed statistically by the nonparametric Kruskal-Wallis (instead of one-way analysis of variance for normally distributed data): when there was a significant difference among treatments Mann-Whitney U test (Gibbons and Chakraborti 2014) was used to compare the mean of each pair of treatments.Egg production (eggs produced/hen per d) and egg weights (g egg/hen per d) averaged over 2 weeks, were analysed on a pen basis (n ¼ 6 pens).Data comparing egg production and egg weight between the first 2 weeks and the last 2 weeks of the experiment (0 -2 and 6 -8 weeks after the start of the experiment), Haugh units and egg-shell thickness in the different treatments between 4 and 8 weeks, were analysed by repeated measures ANOVA and the treatment means were compared with Bonferroni's multiple comparison test.

Live body weights
Live BW of Group LC1 were about 40 g heavier than the maximum weight standard for hens of 28 weeksof-age (1830 − 1950 g; Hy-Line Brown International, 2014): although Group LC1 hens were heavier than Control and Group LC2 hens, the differences were not significant (Table 2).The absence of a significant effect of LC1 on BW of hens is consistent with those of Yokhana et al. (2016) who found that BW of Hy-Line Brown layer hens was not significantly improved after supplementing diets with the LC1 supplement (at 0.8 g/100 g diet) from 19 to 31 weeks-of-age.However, in growing pullets fed diets supplemented with the same 2 LC supplements, BW of 8-week-old pullets was significantly heavier than Controls, and at 18 weeks-ofage only pullets fed the LC1 were heavier than Controls (Hussein et al. 2017).R€ ohe et al. ( 2019) feeding dual purpose hens with Arbocel R found that BW was lower than controls.Guzm� an et al. ( 2016) found no significant difference in BW between controls and hens given diets with straw as an IF supplement, from 17 to 46 weeks: BW of hens given sugar beet pulp (a SF source) tended to be lower than un-supplemented controls.However, Mohiti-Asli et al. ( 2012) and Moradi et al. (2013) using cellulose as an IF source, found BW of broiler breeder hens was significantly decreased compared to controls.Thus, the effects of fibre on weight gain appear to vary considerably between experiments and it is possible other diet components influence or contribute to the effects of a fibre (R€ ohe and Zentek 2021).

Lymphoid organs and tissues
The weights and RW of the thymus glands (primary lymphoid organs) in Group LC1 hens were greater (p < 0.05) than Control hens (Table 2).Although weights in Group LC2 were heavier than those for Controls, the differences were not significant (Table 2).The weights and RW of the other primary lymphoid organ, the bursa of Fabricius, although slightly heavier in Group LC1 than Control, were not significantly different between Groups.The weights of the secondary lymphoid organs, spleen, and caecal tonsils, were not significantly different between the dietary groups (Table 2).
The effects of the 2 LC on lymphoid organs of laying hens were different from those observed in 8-and 18-week-old pullets prior to lay (Hussein et al. 2017).In 8-week-old pullets fed the same 2 LC supplements for 4 weeks, the weights and RW of the primary lymphoid organs, bursa of Fabricius and thymus glands were significantly greater in both treatment groups than Controls (Hussein et al. 2017).However, in the current experiment, only Group LC1 had heavier thymus glands than Controls.In 18-week-old pullets before the start of lay, those fed LC1 supplement had heavier weights of the bursa but not thymus glands (Hussein et al. 2017).
The spleen in 8-week-old pullets (Hussein et al. 2017) fed the LC1 supplement from 4 weeks-of-age was significantly heavier than in Controls whereas in 18-week-old pullets prior to lay (Hussein et al. 2017), and in the 28-week-old laying hens in the current experiment, there were no differences between treatments.In the caecal tonsils of 18-week-old pullets prior to lay (Hussein et al. 2017), unlike in the current experiment, both supplements had significant effects on weight.The responses to the LC supplements may have been due to the different stages of development (maturation and involution) of the bursa of Fabricius and thymus glands (Aire 1973;Ciriaco et al. 2003;Ol� ah et al. 2014).The results reported here suggest that LC1 supplementation during early lay may contribute to reducing the rate of involution of the thymus gland or stimulate its growth and thus help to improve immune function.
There are not many reports on the effects of dietary fibre supplements on development of lymphoid organs in poultry.However, in broilers, positive effects of olive cake or pulp (Valiente et al. 1995;ELbaz et al. 2020), grape pomace (Hosseini-Vashan et al. 2020) and tomato pomace (Hosseini-Vashan et al. 2016) are reported: although no effects are also reported for olive cake (Al-Harthi 2017) and grape pomace (Ebrahimzadeh et al. 2018).It is important to note that phytogens or bioactive compounds are present in olive cake (Uribe et al. 2015), grape pomace (Yu and Ahmedna 2013), and tomato pomace (Perveen et al. 2015) and may contribute independently to any effects of dietary fibre on lymphoid organs.
The numbers of PP in the small intestine of Group LC1 hens were higher (p < 0.05) compared to LC2 and Control hens (Table 2): no difference was observed between Group LC2 and Control hens.The total and relative areas of small intestinal PP of the Group LC1, but not the LC2 hens, were greater (p < 0.05) than those of the Controls.
In younger pullets fed the LC1 supplement from 4-8 or 10-18 weeks-of-age, number and areas of PP were increased: only in those fed LC2 were areas greater than Controls (Hussein et al. 2017).These results, together with the increased number of PP in LC1 hens in the current study, suggest that the LC1 supplement may enhance gut mucosal immunity in hens.A positive contribution of supplement LC1 on the growth of PP could have been due to various mechanisms of action operating singly or in combination.Fibre-related actions that have been shown to beneficially influence the gut-associated immune system and gut physiology are, mechanical effects of fibre on intestinal walls; microbial production of fermentable substrates such as fructo-oligosaccharides or butyrate; changes in the microflora population with either reductions in pathogens or increases in numbers of beneficial bacteria in the gut; improvement of amino acid availability through improved proteolytic enzyme activity (Cao et al. 2003;Friedman et al. 2003;McReynolds et al. 2009;Yokhana et al. 2016;Farran et al. 2017).

Innate immunity
Heterophil phagocytosis index (PI) and percentage (%P) of Group LC1 hens were higher (p < 0.05) at 4 weeks after the start of the experiment (24 weeks-ofage) than Control hens (Table 3) however, there were no significant differences in oxidative burst (DRFU) activity of heterophils of laying hens fed LC1 and LC2 diets compared to those of Controls (Table 3).The innate immune system is the first line of defence against invasive pathogens and heterophil phagocytosis, as part of the system, is an essential mechanism for destroying pathogens before active immunity operates (Kogut et al. 1998;Chuammitri et al. 2011).
The increase in heterophil phagocytosis index (PI) seen in Group LC1 was also observed in 7.5-and 14week-old birds of the same strain (Hussein et al. 2017).However, oxidative burst activity of heterophils, in contrast to effects on the younger birds, was not improved by either LC1 or LC2 hens.This difference may have been due to the ages of the hens used in the 2 studies and to the influence of changed physiological states such as circulating levels of minerals (Lichtman et al. 1983) and hormones (Leitner et al. 1996), that occur with a change from pre-egg laying to egg laying.

Active immunity
Lymphocyte proliferation after 7 weeks on the experimental diets (27 weeks-of-age) was not significantly different between groups although concanavalin A stimulated proliferation of T lymphocytes, lipopolysaccharide stimulated B lymphocytes and phorbol 12myristate 13-acetate stimulated T and B lymphocytes in the LC1 hens were greater than in LC2 and Control hens (Table 3).
Unlike in the current experiment, Hussein et al. ( 2017) observed a significant increase in mitogenstimulated proliferation of T and B lymphocytes in 17week-old Hy-Line Brown pullets fed for 7 weeks with diets containing either LC1 or LC2 supplements.A possible cause of the different responses could have See Table 2 for information on treatment diets C, LC1 and LC2.One hen/ pen was used for heterophil and lymphocyte measurements and for IgY a mean for each pen was obtained from 2 egg yolks.
a,b Means within the same row with different subscript letters are significantly different (p < 0.05). 1 -Oxidative burst measured 1 h after stimulation with phorbol 12-myristate 13-acetate as a change in relative fluorescent unit (DRFU) in hens fed experimental diets for 4 weeks.2 -Mitogen stimulated lymphocyte proliferation in hens fed experimental diets for 7 weeks.Expressed as a percentage of the increase of mitogen stimulated lymphocytes relative to non-stimulated lymphocytes.Mitogens:-ConA -2.5 lg/mL concanavalin A; LPS -3.125 lg/mL lipopolysaccharide from Escherichia coli; PMA -1.25 lg/mL phorbol 12-myristate 13-acetate.
3 -IgY measured in egg yolks from hens fed diets for 8 weeks.
been changes in circulating oestrogen levels that occur with egg production.Differentiation and proliferation of lymphocytes in the thymus and bursa of Fabricius are adversely affected in the late stages of chick embryogenesis by increased levels of oestrogen (Katayama et al. 2012): oestrogen levels (Lee and Bahr 1994) start to increase from about 6 weeks before egg laying: then levels rapidly decline to about half the peak level at about 2 weeks before egg laying (Senior 1974) and remain relatively constant until at least 200 weeks-of-age (Joyner et al. 1987).The differentiation and proliferation of lymphocytes in the thymus and bursa of Fabricius are adversely affected by increased levels of oestrogen in the late stages of chick embryogenesis (Katayama et al. 2012).However, the effects of oestrogens on the immune system are complex and can be affected by age and stage of development of the immune system (Kondo et al. 2004).Other factors resulting from dietary fibre supplements such as changes in the availability of amino acids (Li et al. 2007) or circulating calcium concentrations could also influence lymphocyte proliferation (Chen and Chen 2004).
Although the concentration of IgY in yolks of eggs from Group LC1 was about 10% greater than the Controls, and the amount in whole yolk about 12% greater (Table 3), differences were not significant.However, Sozcu and Ipek (2020) have shown that circulating concentrations of IgY are increased in laying hens given a LC supplement Lignochar, which contains a high concentration of lignin (92.6 g/100 g).The effects they observed were dependent on dietary concentration: when the diet was supplemented with 0.5 or 1.0 kg Lignochar/ton of feed, serum IgY concentrations were increased compared with un-supplemented controls whereas at the highest rate of supplementation, 2.0 kg/ ton of feed, IgY concentration was not significantly different from controls (Sozcu and Ipek 2020).Lee et al. (2013) who fed a diet containing 5% palm kernel meal (to increase dietary fibre) to 73-week-old, hens for 6 weeks, found no effect on serum IgG [IgY] and a nonsignificant decrease in egg yolk IgG.

Egg production and quality
In all 3 treatment groups, egg production increased (p < 0.05) from 20 to 28 weeks-of-age (Table 4) and is consistent with the standards for the Hy-Line Brown strain (Hy-Line Brown International 2014).During the last 2 weeks of the experiment (6 -8 weeks), eggs produced/hen per d was greater (p < 0.05) in LC1 than Controls (Table 4).At earlier time points, although egg production of Group LC1 was higher than that of the other groups, the differences were not significant.For all treatments, egg production was in the upper range of values for the strain in the first 2 weeks of the experiment, while in the last 2 weeks of the experiment only Group LC1 was within the standard range (Hy-Line Brown International 2014).
The weights of eggs in all treatment groups increased (p < 0.05) with age from 20 to 28 weeks (Table 4) and is consistent with standards for the strain (Hy-Line Brown International 2014).Between 4 -6 and 6 -8 weeks after the start of the experiment, Group LC1 laid heavier eggs (p < 0.05) compared to the Controls.Although the eggs from Group LC2 were lighter than those from LC1 hens the differences were not significant nor were the differences in egg weights between LC2 and Controls (Table 4).While egg weights of the 3 groups in the first 2 weeks of the experiment were below the recommended weights, in the last 2 weeks they were in the upper end of the recommended range for all 3 treatment groups (Hy-Line Brown International 2014).
Yolk quality (Haugh units, Table 4) was in the upper range for the standard for Hy-Line Brown strain (Hy-Line Brown International 2014) in all three groups at both time points (4 and 8 weeks after the start of the experiment).No differences in effects of LC1 or LC2 were seen on Haugh units or on egg-shell thickness compared to those of the Controls (Table 4).
Supporting the results obtained here on improved egg production rate in LC1 hens, are those of Sozcu and Ipek (2020) in 18-to 29-week-old layers fed Lignochar high in lignin, those of Incharoen and Maneechote (2013) in 32-to 44-week-old laying hens fed diets supplemented with rice hulls (an IF source) and R€ ohe et al. ( 2019) who fed hens with the IF, Arbocel R. In broiler breeders fed various IF ingredients, improvements in egg production have also been shown (Zuidhof et al. 1995;Enting et al. 2007;Mohiti-Asli et al. 2012;Moradi et al. 2013).The improvements may have been secondary to beneficial effects of IF on digestibility of protein and starch; retention time of digesta in the upper part of the digestive system; absorption of nutrients; weight and activity of gizzard and health of the digestive tract (Rogel et al. 1987;Jørgensen et al. 1996;Svihus and Hetland 2001;Hetland et al. 2005;Jim� enez-Moreno et al. 2009;Mateos et al. 2012;Yokhana et al. 2016).
In the current experiment the lack of effects of the LC supplements on measures of egg quality, egg-shell thickness, absolute weight of egg contents and Haugh units, is contrary to those of Sozcu andIpek (2020), andR€ ohe et al. (2019) for shell weight.They are in agreement with the results of Mohiti-Asli et al. (2012) who reported that cellulose (an IF) did not improve egg quality.
The original hypothesis when the experiment was designed (as was that of Hussein et al. (2017)), was that any effects of the 2 LC supplements would be a consequence of relative IF and SF contents and effects on solubility.However, it is not inconceivable that other differences in the compositions of the supplements (for example the relative concentrations of cellulose and lignin or types of polysaccharides) could have contributed to the effects observed (Gonz� alez -Alvarado et al. 2008;Choct 2015;R€ ohe and Zentek 2021).
In 8-week-old layer pullets fed supplements containing different ratios of cellulose to lignin (C:L) for 4 weeks, differences in immune function were observed (Hussein and Frankel 2019).Pullets fed a supplement containing a C:L ratio of 3:1 had improved immune function, compared to pullets fed a supplement containing a lower C:L ratio of 2:1 and to those fed a supplement of cellulose without lignin (Hussein and Frankel 2019).In the current experiment the LC1 supplement was calculated (from values in Supplementary Table S1), to contain 52.38 g cellulose and 23.36 g lignin/100 g, giving a higher C:L ratio of 2.24:1 than the ratio of 1.3:1 in LC2 (calculated from average values of 40.3 g cellulose and 30.4 g lignin/ 100 g in Table S1).It is, therefore, possible that the different proportions of cellulose to lignin in LC1 and LC2 supplements, rather than insolubility or solubility of the dietary fibre, could have had a contributing effect on the results described here.

Conclusions
Feeding the LC supplements from only 2 weeks before the start of lay and through to 28 weeks-of-age resulted in beneficial effects on immune function and production measurements.However, responses observed in layer hens to the supplements were different: when compared with Controls, LC1 had more beneficial effects on the immune and production measurements, than LC2.It is not possible from this experiment to identify the active components of the 2 LC supplements.The supplements are derived from different plant substrates and have different production methods: although the IF and SF in the 2 LC have similar ranges of concentrations (see Table S1), there are components of the fibre of the LC (such as cellulose and lignin) that have different relative concentrations and it is possible that they could have contributed to the different responses to dietary supplementation.
If specific components of dietary fibre can have specific effects on immune, physiological and production parameters then it is important that those components be identified and their actions defined so as to enable a more targeted approach to the use of dietary fibre to replace in-feed antibiotics.

Table 1 .
Chemical composition (g/100 g) of commercial diets, 'Pullet grower' and 'Golden yolk', used as the Control and basal experimental diets (manufacturer's information a ).Feed analysis and ingredients were obtained from manufacture but for confidentiality reasons the quantities used were not able to be obtained. b

Table 2 .
Live body weights (BW), weights and relative weights (RW, g/100 g BW) of immune organs, Peyer's patches (PP) measurements and jejunal plus ileal areas of Hy-Line Brown layer strain hens given diets containing different types of lignocellulose fibres for 8 weeks from 20 weeks of age (mean, pooled SEM, n ¼ 6 pens).Means within the same row with different subscript letters are significantly different (p < 0.05).

Table 3 .
Measurements of innate immunity (phagocytosis and oxidative burst of heterophils 1 ) and active immune function (proliferation of lymphocytes 2 and egg yolk IgY 3 ) in Hy-Line Brown layer hens fed diets containing different types of lignocellulose fibre for 8 weeks from 20 weeks-of-age.Heterophil functions measured after being fed diets for 4 weeks, lymphocyte proliferation after 7 weeks and egg yolk IgY after 8 weeks.(mean, pooled SEM, n ¼ 6).

Table 4 .
International 20144)gg quality measurements of Hy-Line Brown layer hens fed diets containing different lignocellulose fibre supplements for eight weeks from 20 weeks-of-age (means, pooled SEM, n ¼ 6 pens).See Table2for information on treatment diets C, LC1 and LC2.a, b Means within the same row with different subscript letters are significantly different (p < 0.05).A, B Means within the same column for a parameter at 24 and 28 weeks-of-age with different uppercase superscripts differ significantly (p < 0.05).RW: relative weight (g/100 g whole egg).cStrainaverageeggproduction rate at 20 -22 weeks-of-age is 0.695 and at 26 -28 weeks, 0.953 g (converted from values in hen-d percentage, Hy-Line BrownInternational (2014)).dStrainaverage egg weight at 20 -22 weeks is 51.88 g and at 26 -28 weeks-of-age it is 59.05 g (Hy-Line BrownInternational 2014).e Haugh units at 24 weeks of age average, 96.0 and at 28 weeks-of-age, 94.2 (Hy-Line Brown International, 2014).f After 4 and 8 weeks being fed experimental diets, the means of 2 eggs/pen were used to measure egg quality and weights of egg components.