# Feed additive carriers affected the physiological effects of a lactobacilli-based pro- and postbiotic in broilers

Samuel C.G. Jansseune, Wouter H Hendriks, Marie-Hélène Pinard-van der Laan, Fany Blanc

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Samuel C.G. Jansseune, Wouter H Hendriks, Marie-Hélène Pinard-van der Laan, Fany Blanc. Feed additive carriers affected the physiological effects of a lactobacilli-based pro- and postbiotic in broilers. Animal Feed Science and Technology, 2025, 329, pp.116485. 10.1016/j.anifeedsci.2025.116485. hal-05325044

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# Feed additive carriers affected the physiological effects of a lactobacilli-based pro- and postbiotic in broilers

Samuel C.G. Jansseune<sup>a,b,c,*</sup>, Wouter H. Hendriks<sup>a</sup>, Marie-Hélène Pinard-van der Laan<sup>b</sup>, Fany Blanc<sup>b</sup>

## ARTICLE INFO

**Keywords:**

Chicken
Growth
Carrier
Additive
Probiotic
Postbiotic

## ABSTRACT

To promote growth performance and health of broiler during production, additives, like pro- and postbiotics, are often used and most of them use carriers. This study aimed to investigate if carriers can modulate the physiological effects of two lactobacilli-based pro- (Pro) and postbiotic (Post) additives, and to gain more insight into their physiological effects in the absence of carriers, in broilers fed a non-starch polysaccharide-rich diet reared under practical housing conditions. A total of 1600, one-day-old male Ross 308 chicks were equally distributed among 5 treatments (control, Pro and Post with and without their respective carriers) with 8 replicate pens each and 40 broilers/pen. Growth performance parameters, footpad lesions, and blood plasma physiological parameters related to nutrition and immunity were determined. On d35, Pro increased body weight (BW) (+2.1 %; p = 0.025), while its carrier negated this effect (-1.9 %; p = 0.047). Post did not affect d35 BW but increased the feed conversion ratio (FCR) by 1.3 % (p = 0.041), while its carrier increased BW (+2.4 %; p = 0.013), and lowered FCR (-1.3 %; p = 0.042). Growth performance parameters showed a beneficial effect of Pro but not Post, while the use of their respective commercial carriers had a growth-depressing and -promoting effect, respectively. Carriers also affected the footpad lesion score, and blood parameters related to nutrition and immunity. These results highlight that additive carriers can modify the effects on broiler growth and metabolism of the pro- and postbiotic additives to which they are added. The occurrence of an additive-carrier interaction indicates that carriers for pro- and postbiotic additives for broilers must be carefully selected.

## 1. Introduction

Poultry production has continuously increased over the past decades and is today the most prevalent meat produced and consumed worldwide by humans (OECD/FAO, 2021). To improve broiler growth performance and health, many types of additives [e.g. organic acids, phytochemicals, phytogenics, enzymes, prebiotics, probiotics, symbiotics, postbiotics] are commonly used in commercial

<footnote><sup>a</sup> Animal Nutrition Group, Department of Animal Sciences, Wageningen University & Research, Wageningen 338 6700 AH, the Netherlands</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, INRAE, AgroParisTech, GABI, Rue de la Manufacture, Jouy-en-Josas 78352, France</footnote>

<footnote><sup>c</sup> Idena, 21 rue du moulin, Sautron 44880, France</footnote>

<footnote>* Corresponding author at: Animal Nutrition Group, Department of Animal Sciences, Wageningen University & Research, Wageningen 338 6700 AH, the Netherlands.</footnote>

<footnote>E-mail addresses: samuel.jansseune@wur.nl (S.C.G. Jansseune), wouter.hendriks@wur.nl (W.H. Hendriks), marie-helene.pinard@inrae.fr (M.-H. Pinard-van der Laan), fany.blanc@inrae.fr (F. Blanc).</footnote>
practice. Most of the feed additives contain a substance capable of accepting and holding the active component without segregation or separation over time. These so-called carriers are used as a diluent to improve mixability, fluidizer, water sequester to reduce water activity or for encapsulation of the active component (Djuragic et al., 2009; Sun et al., 2023). Carriers are often composed of organic (e. g. flours, meals, lactose, maltodextrin) or inorganic (e.g. limestone, di-monocalcium phosphates, clay, salt) components (Djuragic et al., 2009) and have been investigated for their effect on premix mixability in feed (Djuragic et al., 2009; Holt and Aldrich, 2021). Not only does a carrier have to be chemically inert but also not have a negative impact on the known physiological effect(s) of the additive component. Carriers, however, may also interact with the feed additive [e.g. clay minerals can bind bacteria (Fomina and Skorochod, 2020)], can provide nutrients (e.g. flour, clay minerals) or can elicit biological effects [e.g. clay minerals (Nadziakiewicza et al., 2019; Damato et al., 2022)]. Carriers have been shown to influence the release of vitamin E (Lauzon et al., 2008) and oil (Quinger et al., 2024) with subsequent effects on broiler growth. In studies investigating the efficacy of additives in animal feeds, only few report that carriers were used (Lauzon et al., 2008; Moquet et al., 2018; Wu et al., 2019; Jansseune et al., 2024a; b; c). Only two studies were found that reported to have corrected the diets for carriers in term of quantity and composition (Moquet et al., 2018; Wu et al., 2019). The lack of correcting for carriers used makes it impossible to discriminate between the actual effects of the feed additive from that potentially exerted by the carrier.

Among the feed additives commonly studied and used in practice to promote animal performance and health are pro- and post-biotics (Humam et al., 2019; Jha et al., 2020). The latter have been reported to be associated with beneficial outcomes on gut health and immunity (Jha et al., 2020), blood physiological parameters (e.g. related to health and nutrition) (Abd El-Hack et al., 2017; Humam et al., 2019) and bone metabolism (Liu et al., 2021; Jiang et al., 2021) in poultry. We previously reported (Jansseune et al., 2024c) that a lactobacilli-based probiotic (Pro) and its derived postbiotic (Post) with their respective commercial carrier (Pro-C and Post-C, respectively), alleviated the growth depression induced by a non-starch polysaccharide (NSP)-rich challenge diet in broilers reared under practical conditions. Yet, this result could not be reproduced in a follow up experiment using the same housing and a similar NSP-rich challenge diet (Jansseune et al., 2024b). Modification of caecal microbiota composition was observed in a diet-dependent manner, as well as effects on bone health and strength, and blood biological parameters related to nutrition and immunity (Jansseune et al., 2024a; c). The composition of the commercial carriers used for Pro-C and Post-C contained ingredients that are commonly used in animal feed additives (incl. toasted corn and clay), and which could have influenced the effects of Pro and Post.

This study aimed to investigate if the presence of carriers can modulate the physiological effects of two lactobacilli-based pro- and postbiotic additives, and to gain more insight into their physiological effects in the absence of carriers (Pro and Post), in broilers fed an NSP-rich diet reared under practical housing conditions. We investigated effects on the growth performance and physiological parameters related to nutrition and immunity, which are regularly affected by pro- and postbiotics.

## 2. Materials and methods

### 2.1. Ethics statement

The experiment was approved by the French Ministry of education, higher education and research (Ministère de l'éducation nationale, de l'enseignement supérieur et de la recherche) under the protocol No. APAFIS #44135-2023071114126771 v5 and carried out according to French legislation.

### 2.2. Birds housing and management

A large batch of one-day-old male Ross 308 broilers originating from 50-week-old broiler breeders was purchased from a commercial hatchery (Couvoir de Cleden, Cleden Poher, France), with 1600 chicks selected, based on individual weights and distributed across eight replicate pens per treatment with 40 broilers each, so that all pens had a similar average chick body weight (BW) (~44.9 ± 2.42 g) and distribution. A randomised complete block design was used. Pens were 1.90 × 1.25 × 0.8 m (L × W × H) with wood shavings as floor covering. They were located along the wall of air entries on one side of a commercial, 1200 m² Colorado-type building. Water and feed per pen were provided *ad libitum* through 5-6 nipple drinkers and one 40 cm diameter Hung pan feeder (Josse, Montauban de Bretagne, France). The photoperiod was 24 h light until d4 and then 20 h from d5 to 37. Ambient temperature started at 32 °C on d1 and, thereafter, gradually reduced linearly to 23 °C on d22. Birds were inspected daily for lethargy, prostration and lameness, and culled if found to be unhealthy. The total duration of the experiment was 37 days (d1 to 37).

As in normal practice, at day of hatch, broilers were spray vaccinated against infectious bronchitis virus (IBV) (Nobilis BI H120, Nobilis, MSD santé animale, Beaucousé, France) and coccidiosis (Paracox-5, Intervet UK Ltd, United-Kingdom), and were vaccinated through drinking water against infectious bursal disease virus (IBDV) (HIPRAGUMBORO G97, Laboratorios Hipra, Amer, Spain) on d17. At d14, an induced immune reaction using vaccines for Newcastle disease virus (NDV) and IBV (Nobilis Ma5 + CLONE 30, MSD Santé Animale, Beaucouzé, France) booster shot was administered via an individual eye drop.

### 2.3. Experimental treatments

Five treatment groups were used (control (Ctrl), Pro and Post, with or without a carrier, -supplemented groups). The pelleted diet was formulated based on commercial standards for nutrients levels for Ross 308 broilers and provided adequate levels of all nutrients to the birds (Table 1). The diet (Ctrl) was supplemented with a Pro or a Post purchased from STI biotechnologie (Maen Roch, France) with or without a carrier (Table 2). Pro is the biomass resulting from the co-fermentation of a milk-based substrate by a mixture of
Lactobacillus rhamnosus CNCM-I-3698 and L. formosensis CNCM-I-3699, and Post is derived from heat-inactivated (high temperature short time) biomass of Pro. Both Pro and Post biomasses originated from a mixture of four production batches containing 3 × 10⁹ colony-forming units per g of biomass after fermentation and were dried as powder on carriers. In this experiment, two different formulations of carriers were used, either a commercial or experimental formulation (Table 2). The commercial form of Pro (Pro-C) and Post (Post-C) corresponded to the products SORBIFLORE® and METALAC®, respectively, which include their specific commercial carriers (I and II, respectively). Toasted corn flour was used as the experimental carrier for the Pro and Post biomass. Toasted corn flour was also present in the Ctrl and substituted for equal amounts of corn flour due to the addition of the Pro and Post, such that the effect of Pro and Post biomasses can be evaluated. Pro and Post biomasses were included in the diet at 50 and 500 g biomass/t as is, respectively (Table 2), from d1 to 14, and at 40 and 400 g biomass/t as is, respectively, from d15 to 37. The toasted corn flour for the control as well as Pro, Pro-C, Post and Post-C additives were added before pelletization. Pellets were 2.5 × 20, 2.5 × 35 and 3.0 × 50 mm (d × l) for starter, grower and finisher diets, respectively. The temperature of the pellets, as measured using an infra-red thermometer directly after the compactor ring ranged between 79 and 81 °C, 77 and 80 °C, and 82 and 84 °C for starter, grower and finisher diets, respectively (data not shown).

## 2.4. Performance data and sample collection

Individual BW and pen feed intake (FI) were recorded and feed conversion ratio (FCR) calculated on d14, 21, 28 and 35. Dead birds were collected daily and weighed to adjust pen FCR. On d35, footpad lesions (FPL) were scored on both legs in all remaining birds (n = 1469) with grade 0 allocated for no lesions, grade 1 for mild lesions (altered lesion covers ≤ 25 % of the footpad) and grade 2 for

<caption>**Table 1**</caption>

Ingredient and calculated composition including energy content of the challenge diet for broilers.

<table><thead><tr><th>Composition</th><th>Starter<br>(0-14 d)</th><th>Grower<br>(14-28 d)</th><th>Finisher<br>(28-37 d)</th></tr></thead><tbody><tr><td><strong>Ingredient (% as is)</strong></td><td></td><td></td><td></td></tr><tr><td>Corn</td><td>13.982</td><td>12.509</td><td>13.000</td></tr><tr><td>Wheat</td><td>28.000</td><td>30.000</td><td>29.949</td></tr><tr><td>Barley</td><td>10.000</td><td>10.000</td><td>10.000</td></tr><tr><td>Rye</td><td>7.500</td><td>12.500</td><td>18.000</td></tr><tr><td>Soybean meal</td><td>32.000</td><td>26.800</td><td>20.700</td></tr><tr><td>Limestone</td><td>1.640</td><td>1.110</td><td>0.850</td></tr><tr><td>Mono calcium phosphate dihydrate</td><td>1.400</td><td>0.860</td><td>0.800</td></tr><tr><td>Sodium chloride 99 %</td><td>0.280</td><td>0.250</td><td>0.260</td></tr><tr><td>Sodium bicarbonate</td><td>0.100</td><td>0.110</td><td>0.130</td></tr><tr><td>Soy oil</td><td>3.890</td><td>4.650</td><td>4.890</td></tr><tr><td>DL-Methionine 99 %</td><td>0.301</td><td>0.274</td><td>0.231</td></tr><tr><td>L-Lysine HCL 98 %</td><td>0.242</td><td>0.257</td><td>0.329</td></tr><tr><td>L-Threonine 98 %</td><td>0.134</td><td>0.129</td><td>0.163</td></tr><tr><td>L-Valine 96.5 %</td><td>0.031</td><td>0.024</td><td>0.051</td></tr><tr><td>L-Isoleucine 90 %</td><td>-</td><td>0.027</td><td>0.067</td></tr><tr><td>L-Arginine 98 %</td><td>-</td><td>-</td><td>0.080</td></tr><tr><td>Premix<sup>a</sup></td><td>0.500</td><td>0.500</td><td>0.500</td></tr><tr><td><strong>Calculated (% as is)</strong></td><td></td><td></td><td></td></tr><tr><td>Dry matter</td><td>88.8</td><td>88.9</td><td>88.8</td></tr><tr><td>Crude protein</td><td>21.5</td><td>19.6</td><td>17.5</td></tr><tr><td>Crude fat</td><td>5.28</td><td>6.02</td><td>6.28</td></tr><tr><td>Starch</td><td>36.9</td><td>40.0</td><td>43.3</td></tr><tr><td>Ash</td><td>6.61</td><td>5.26</td><td>4.65</td></tr><tr><td>Fibre<sup>b</sup></td><td>13.9</td><td>13.7</td><td>13.3</td></tr><tr><td>Total non-starch polysaccharides<sup>b</sup></td><td>12.4</td><td>12.2</td><td>11.8</td></tr><tr><td>Soluble non-starch polysaccharides<sup>b</sup></td><td>3.05</td><td>2.98</td><td>2.85</td></tr><tr><td>Cellulose</td><td>2.86</td><td>2.76</td><td>2.63</td></tr><tr><td>Dig. Methionine</td><td>0.59</td><td>0.54</td><td>0.47</td></tr><tr><td>Dig. Methionine+cystine</td><td>0.93</td><td>0.86</td><td>0.75</td></tr><tr><td>Dig. Lysine</td><td>1.32</td><td>1.20</td><td>1.10</td></tr><tr><td>Dig. Threonine</td><td>1.32</td><td>1.20</td><td>1.10</td></tr><tr><td>Dig. Valine</td><td>0.90</td><td>0.82</td><td>0.75</td></tr><tr><td>Dig. Arginine</td><td>1.29</td><td>1.14</td><td>1.05</td></tr><tr><td>Calcium</td><td>0.98</td><td>0.67</td><td>0.55</td></tr><tr><td>Available phosphorous</td><td>0.48</td><td>0.35</td><td>0.33</td></tr><tr><td>Chlorine total</td><td>0.27</td><td>0.25</td><td>0.27</td></tr><tr><td>Sodium total</td><td>0.15</td><td>0.14</td><td>0.15</td></tr><tr><td>Apparent metabolizable energy (MJ/kg as is)</td><td>12.13</td><td>12.72</td><td>12.88</td></tr></tbody></table>

<footnote><sup>a</sup> Supplied per kg premix: 2000,000 IU retinyl acetate, 500,000 IU cholecalciferol, 10 g DL-α-tocopherol, 460 mg menadione, 400 mg thiamine, 1500 mg riboflavin, 700 mg pyridoxine-HCL, 4 mg cyanocobalamin, 7 g niacin, 2.4 g D-pantothenic acid, 92 g choline chloride, 200 mg folic acid, 40 mg biotin, 53 g FeSO₄·H₂O, 9.6 g CuSO₄·5 H₂O, 28 g MnO, 33 g ZnSO₄·H₂O, 360 mg KI, 112 mg Na₂SeO₃.</footnote>

<footnote><sup>b</sup> Values were calculated from levels reported by Knudsen (2014) and restricted to cereals and soybean meal.</footnote>
<table><caption>**Table 2**

Components and addition rate of probiotic (Pro) and postbiotic (Post) biomasses.ª</caption><thead><tr><th>Item</th><th>Ctrl</th><th>Pro</th><th>Pro-C</th><th>Post</th><th>Post-C</th></tr></thead><tbody><tr><td>Component (% as-is)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Toasted corn</td><td>100</td><td>98.9</td><td>89.0</td><td>90.0</td><td>-</td></tr><tr><td>Untreated biomass</td><td>-</td><td>1.1</td><td>1.1</td><td>-</td><td>-</td></tr><tr><td>HTST<sup>b</sup> treated biomass</td><td>-</td><td>-</td><td>-</td><td>10.0</td><td>10.0</td></tr><tr><td>Carrier I<sup>c</sup></td><td>-</td><td>-</td><td>9.9</td><td>-</td><td>-</td></tr><tr><td>Carrier II<sup>d</sup></td><td>-</td><td>-</td><td>-</td><td>-</td><td>90.0</td></tr><tr><td>Addition rate (g/t feed as-is)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>d1–14 diet</td><td>4500</td><td>4550</td><td>4550</td><td>5000</td><td>5000</td></tr><tr><td>d15–37 diet</td><td>3600</td><td>3640</td><td>3640</td><td>4000</td><td>4000</td></tr><tr><td>Biomass (g/t feed as-is)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>d1–14 diet</td><td>0</td><td>50</td><td>50</td><td>500</td><td>500</td></tr><tr><td>d15–37 diet</td><td>0</td><td>40</td><td>40</td><td>400</td><td>400</td></tr></tbody></table>

severe lesions (altered lesion covers > 25 % of the footpad).

On d21 and 35, four broilers/pen were randomly selected, and blood collected from the wing vein into EDTA-tubes (BD Vacutainer®, Plymouth, England). Within 1 h after collection, an aliquot of 50 µL of blood was mixed with 10 µL of Transfix® (Cytomark Ltd., Buckingham, England) and kept at room temperature for 1–3 days before flow cytometry analysis. Blood tubes were centrifuged (1800 × g, 5 min, room temperature, 80–2 Electronic Centrifuge, YingTai Medical, Guangdong, China) to recover the plasma before storage at -20 °C.

## 2.5. Plasma biochemistry parameters and coloration evaluation

Blood plasma content of P, uric acid, cholesterol, bile acids and triglyceride were analysed by the phosphomolybate, uricase-peroxidase, cholesterol esterase, 3-α-hydroxysteroid-dehydrogenase and lipase/glycerol-phosphate/peroxidase methods, respectively, with a ProVet analyser (Kitvia, Labarthe-Inard, France) according to the manufacturer's specifications.

As a marker of gut integrity (Celi et al., 2019), carotenoid-based colorations of the bird's blood plasma were assessed. The spectra of plasma coloration were obtained by measuring the optical density (OD) values in the 400–550 nm wavelength range with 10 nm intervals using a TriStar2S plate reader (Berthold Technologies, Wildbad, Germany) on 50 µL of blood plasma samples in 96-well half-area microplates. Peak absorption values for carotenoids in blood are at 450 and 470 nm (Hamzic et al., 2015).

## 2.6. Plasma immune parameter evaluation

Total IgA, IgG, and IgM levels were determined in blood plasma samples by indirect enzyme-linked immunosorbent assays (ELISAs) at d21 and 35 as previously described by Lecoeur et al. (2024) using anti-chicken IgA, IgG or IgM as capture antibodies (Cat no. A30–103A, A30–104A and A30–102A, Bethyl Laboratories, Montgomery, USA) and anti-chicken IgA, IgG or IgM antibodies conjugated to horseradish peroxidase as detection antibodies (Cat no. A30–103P, A30–104P and A30–102P, Bethyl Laboratories).

Blood plasma titres against IBV, IDBV and NDV were assessed at d21 and 35 by specific indirect ELISAs (ID Screen® Infectious Bronchitis Indirect 2.0, ID Screen® IBD Indirect and ID Screen® Newcastle Disease Indirect Conventional Vaccines ELISAs, Innovative diagnostics, Grabels, France). Antibody titres calculation and seropositivity threshold were performed following the manufacturer's instructions. Antibody titres were expressed as the antilog of the log₁₀ titre obtained by calculating the sample to positive ratio (S/P = mean of test sample - mean of negative control / mean positive control - mean of the negative control) and using the equation log₁₀ titre = 0.70 (log₁₀ S/P) + 3.700 for IBV, the equation log₁₀ titre = 0.97 (log₁₀ S/P) + 3.449 for IBDV, and the equation log₁₀ titre = (log₁₀ S/P) + 3.52 for NDV. Samples were considered seropositive when they had a titre above 1625, 875 and 993 for IBV, IBDV, and NDV, respectively.

Blood cell counting and identification were performed by flow cytometry using a method adapted from Seliger et al. (2012) and Bílková et al. (2017). Eight µL of solution containing fixed blood cells were stained in 100 µL buffer containing a combination of antibodies: anti-CD45-SPRD (clone LT40, 1 µg/mL), anti-CD4-PE-Cy7 (clone CT4, 0.05 µg/mL), anti-CD8α-AF700 (clone CT8, 0.1 µg/mL), anti-TCRγδ-AF488 (clone TCR1, 1 µg/mL), anti-Bu1-AF647 (clone AV20, 0.25 µg/mL), and Kul01-PE (0.2 µg/mL) (all from SouthernBiotech, Birmingham, USA), and anti-integrin αV/β3-AF405 (clone 23C6, 1 µg/mL, Santa Cruz Biotechnologies). After a 20 min incubation at 4 °C, 392 µL of BD CellFIX solution (BD Biosciences, Franklin Lakes, USA) were added before analysis on a MACSQuant 16 flow cytometer (Miltenyi, Paris, France) allowing analysis of fluorescence intensities and the absolute count of cells. Data analysis was performed using FlowJo V10 software. All antibodies used were titrated for optimal signal/noise ratios and single-stained samples were used to calculate a fluorescence compensation matrix.

<footnote>ª Pro and Post without and with their respective carrier were provided on top of a control (Ctrl) diet.</footnote>

<footnote>ᵇ High temperature short time.</footnote>

<footnote>ᶜ Contains 653 g/kg as is toasted corn, micronized wheat bran, marine algae meal, corn cob, and 2.2 g/kg as-is calcium propionate (Additive ID no. 1a282).</footnote>

<footnote>ᵈ Contains 458 g/kg as is toasted corn, 262 g/kg as-is sepiolite (Additive ID no. E562), corn cob, and 2.2 g/kg as-is calcium propionate (Additive ID no. 1a282).</footnote>
Erythrocytes were distinguished as CD45- integrin αV/β3- cells, while thrombocytes as CD45low integrin αV/β3 + cells. Leukocytes were the other CD45 + cells. Among them, based on forward side scatter (FSC) and side scatter (SSC), heterophils were characterized as large and mostly granular cells. Monocytes were identified as SSCmed KUL01 + cells, and B lymphocytes as Bu1 + cells. Among B cells, blast cells were identified with a higher FSC and SCC. γδ T cells were distinguished as TCRγδ+ cells and their expression of CD8α was evaluated. Among the remaining T cells (Bu1- TCRγδ- lymphocytes), double negative T cells (CD4- CD8α-), T helper cells (CD4 + CD8α-), cytotoxic T cells (CD4- CD8α+) and double positive T cells (CD4 + CD8α+) were identified. Heterophils/Lymphocytes (H/L) and CD4/CD8α ratios were also calculated.

## 2.7. Statistical analyses

All statistical analyses were performed with R v.4.0.3 (R Core Team, 2023). Probability values <0.05 and 0.05≤ p < 0.10 were considered as significant and showing a trend, respectively. Outliers were identified using the z-score procedure and excluded from the dataset. In all analyses, pens were the experimental units.

Treatment effects on means were analysed per contrast of interest (Pro v.s. Ctrl, Post v.s. Ctrl, Pro-C v.s. Pro and Post-C v.s. Post), using the general linear model procedure and type 2 analysis of variance, while their effects on graded parameters were analysed by ordinal logistic regression with the cumulative linked mixed model function for logistic regression (library lme4 v.1.1.31). Treatment effect on mortality was analysed as bounded counts. For the test on seropositivity rate, a binomial distribution was used in the model. All models included a fixed effect of the treatments and a random block factor. For blood biochemical parameters, BW was included as an additional co-variate. Models were tested for residual normality by Shapiro test and, a log or a boxcox transformation was applied if normality was absent. If residual normality could not be reached with these two data transformation procedures, a rank analysis was performed. Equal variances between treatment groups was confirmed with the Levene test, and in case of variance heterogeneity, a CR2 correction was applied.

# 3. Results

## 3.1. Growth performances and footpad lesions

Mortality, expressed as the number of dead birds per pen, averaged 1.6 ± 0.95 (3.9 %) on d35 and was not different (p > 0.10) between dietary treatments. The growth performance parameters of the broiler chickens were affected by Pro, Post, and their respective commercial carriers (Table 3). Compared to Ctrl broilers, Pro increased broiler BW at all ages, with an increase in daily FI (dFI) and no change in FCR (on d35 +2.1 and 3.5 % for BW and dFI, respectively; p = 0.024 and 0.013, respectively). Carrier I (Pro-C v. s. Pro) had, at all ages, no effect on dFI and FCR, but lowered BW (-1.9 % on d35; p = 0.047). Compared to Ctrl birds, Post increased

<table><caption>**Table 3**
Mean body weight, daily feed intake, feed conversion ratio and footpad lesion score.ª</caption><thead><tr><th>Parameter<sup>b</sup></th><th>Ctrl</th><th>Pro</th><th>Pro-C</th><th>Post</th><th>Post-C</th><th>Pooled SEM</th></tr></thead><tbody><tr><td>Body weight (g)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>d14</td><td>385</td><td>399<sup>***</sup></td><td>386<sup>††</sup></td><td>396<sup>**</sup></td><td>400</td><td>6.27</td></tr><tr><td>d21</td><td>832</td><td>852<sup>*</sup></td><td>824<sup>††</sup></td><td>854<sup>**</sup></td><td>845</td><td>13.2</td></tr><tr><td>d28</td><td>1447</td><td>1472<sup>§</sup></td><td>1432<sup>††</sup></td><td>1458</td><td>1471</td><td>22.4</td></tr><tr><td>d35</td><td>2169</td><td>2214<sup>*</sup></td><td>2173<sup>§</sup></td><td>2168</td><td>2219<sup>†</sup></td><td>32.1</td></tr><tr><td>Daily feed intake (g)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>d1-14</td><td>31.9</td><td>33.3<sup>*</sup></td><td>32.7</td><td>32.9<sup>§</sup></td><td>33.1</td><td>1.12</td></tr><tr><td>d15-21</td><td>86.9</td><td>91.4<sup>**</sup></td><td>90.4</td><td>90.4<sup>*</sup></td><td>89.5</td><td>2.58</td></tr><tr><td>d22-28</td><td>129.3</td><td>132.4<sup>§</sup></td><td>130.1</td><td>130.5</td><td>131.8</td><td>3.88</td></tr><tr><td>d29-35</td><td>192.0</td><td>196.4</td><td>193.9</td><td>191.8</td><td>196.6</td><td>4.89</td></tr><tr><td>d1-35</td><td>95.3</td><td>98.6<sup>*</sup></td><td>97.0</td><td>96.6</td><td>98.0</td><td>2.01</td></tr><tr><td>Feed conversion ratio (g/g)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>d1-14</td><td>1.078</td><td>1.090</td><td>1.102</td><td>1.087</td><td>1.081</td><td>0.013</td></tr><tr><td>d15-21</td><td>1.380</td><td>1.418<sup>§</sup></td><td>1.455<sup>†</sup></td><td>1.398</td><td>1.415</td><td>0.033</td></tr><tr><td>d22-28</td><td>1.479</td><td>1.504</td><td>1.515</td><td>1.516<sup>*</sup></td><td>1.482<sup>†</sup></td><td>0.034</td></tr><tr><td>d29-35</td><td>1.862</td><td>1.854</td><td>1.823</td><td>1.893<sup>*</sup></td><td>1.836</td><td>0.050</td></tr><tr><td>d1-35</td><td>1.512</td><td>1.526</td><td>1.530</td><td>1.532<sup>*</sup></td><td>1.512<sup>†</sup></td><td>0.015</td></tr><tr><td>Footpad lesion score (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0</td><td>47.4</td><td>46.3</td><td>29.1</td><td>40.3</td><td>34.2</td><td>-</td></tr><tr><td>1</td><td>47.7</td><td>43.5</td><td>41.4</td><td>50.2</td><td>45.6</td><td>-</td></tr><tr><td>2</td><td>4.9</td><td>10.2</td><td>29.4</td><td>9.5</td><td>20.2</td><td>-</td></tr><tr><td>mean</td><td>0.575</td><td>0.639</td><td>1.003<sup>††</sup></td><td>0.692<sup>§</sup></td><td>0.860<sup>††</sup></td><td>0.088</td></tr></tbody></table>

<footnote>ª 1 Male Ross 308 broilers were fed a control (Ctrl) diet supplemented with a lactobacilli-based probiotic (Pro) and postbiotic (Post) biomass without and with their commercial carriers (C) from day 1 onward.</footnote>

<footnote>ᵇ Body weight and footpad lesion score values are the mean of individual measures while values for daily feed intake and feed conversion ratio are the mean of 8 pen replicates per treatment.</footnote>

<footnote>§, *, **, *** indicates a difference with Ctrl value at p < 0.10, 0.05, 0.01 and 0.001, respectively.</footnote>

<footnote>†, ††, ††† indicates a difference with corresponding Pro and Post value at p < 0.10, 0.05, 0.01 and 0.001, respectively.</footnote>
broiler BW at d14 and 21 in conjunction with a higher dFI. However, at d28 and 35, BW and dFI were not different from the Ctrl, but FCR increased (+1.7 and 1.3%, respectively; p = 0.044 and 0.036, respectively). Carrier II (Post-C v.s. Post) had no effect on growth performance parameters up to d28, but BW was increased on d35 (+2.4%; p = 0.013) and FCR was lowered (-1.3%; p = 0.042) while dFI was not affected. Overall, Carrier II lowered FCR over the 35-day study. On d35, compared to Ctrl, mean FPL score was not affected by Pro and tended to be increased by Post (+19.0%; p = 0.06). Unexpectedly, both commercial carriers resulted in an increased mean FPL score (Table 3), with a 71.9 and 24.6% rise for Pro and Post, respectively.

## 3.2. Blood biochemical parameters

Broiler blood plasma biochemical parameters showed an effect of Pro, Post and their respective commercial carriers (Table 4). On d21, compared to Ctrl, birds fed Pro had lower plasma bile acids (-22.6%; p = 0.004) and tended to have lower plasma P (-5.2%; p = 0.06) concentrations. In addition, Pro tended to decrease plasma cholesterol concentration on d21 (-7.9%; p = 0.06) and to increase it on d35 (+6.5%; p = 0.07). Other parameters were not affected by Pro. On d21, Carrier I (Pro-C v.s. Pro) decreased plasma triglyceride to cholesterol ratio (-14.0%; p = 0.019) and tended to decrease plasma protein concentration (+7.0%; p = 0.05) and to decrease plasma OD at 450 nm (-11.7%; p = 0.08). On d35, Carrier I increased plasma P (+7.2%; p = 0.017), and cholesterol (+8.6%; p = 0.026) concentrations and tended to increase uric acid (+14.5%; p = 0.07) concentrations. Compared to Ctrl, on d21, Post only decreased plasma bile acids (-21.0%; p = 0.011) and, on d35, only increased plasma P (+9.2%; p = 0.003), uric acid (+14.2%; p = 0.025) and cholesterol (+10.8%; p = 0.004) concentrations. On d21, Carrier II (Post-C v.s. Post) increased plasma bile acids (+22.4%; p = 0.042), decreased plasma triglyceride (-11.7%; p = 0.050) concentration and triglyceride to cholesterol ratio (-13.0%; p = 0.034), and tended to decrease plasma P (-5.2%; p = 0.07) concentration. On d35, Carrier II increased plasma triglycerides (+13.1%; p = 0.048), triglycerides to cholesterol ratio (+15.8%; p = 0.005) and OD at 450 and 470 nm (+12.0 and +12.4%, respectively; p = 0.040 and 0.028, respectively).

## 3.3. Plasma immunoglobulins

On d21, compared to Ctrl, Pro decreased plasma IgA (p = 0.023) and IgG (p = 0.038) but not IgM, while Post decreased plasma IgM (p = 0.015) and tended (p = 0.09) to decrease IgA concentrations but had no effects on IgG concentrations (Fig. 1A). A carrier effect was only observed for Post with an increase of IgM (p = 0.006) associated with the Post-C treatment. Plasma total Ig concentrations that were affected at d21 were all non-significant at d35.

## 3.4. Response to vaccination

Overall, broiler seropositivity to NDV on d21 was 10.1% and reached 57.5% on d35. Broiler response to NDV in terms of mean titre and seropositivity rate was not affected by treatments (Fig. 1B). For IBV, broiler seropositivity was 98 and 99.4% on d21 and 35,

**Table 4**

<table><caption>Mean plasma concentration of nutrition-related biochemical parameters of 21- and 35-day-old male Ross 308 broilers.ª</caption><thead><tr><th>Parameter<sup>b</sup></th><th>Ctrl</th><th>Pro</th><th>Pro-C</th><th>Post</th><th>Post-C</th><th>Pooled SEM</th></tr></thead><tbody><tr><td>d21</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P (mM)</td><td>3.06</td><td>2.90<sup>b</sup></td><td>2.97</td><td>3.09</td><td>2.93<sup>†</sup></td><td>0.15</td></tr><tr><td>Uric acid (µM)</td><td>521</td><td>513</td><td>495</td><td>568</td><td>557</td><td>49</td></tr><tr><td>Bile acids (µM)</td><td>24.8</td><td>19.2<sup>**</sup></td><td>18.4</td><td>19.6<sup>*</sup></td><td>24.0<sup>†</sup></td><td>2.9</td></tr><tr><td>Triglycerides (mM)</td><td>1.82</td><td>1.70</td><td>1.57</td><td>1.96</td><td>1.73<sup>†</sup></td><td>0.19</td></tr><tr><td>Cholesterol (mM)</td><td>4.43</td><td>4.08<sup>b</sup></td><td>4.32</td><td>4.36</td><td>4.33</td><td>0.32</td></tr><tr><td>Triglycerides / cholesterol</td><td>0.42</td><td>0.43</td><td>0.37<sup>†</sup></td><td>0.46</td><td>0.40<sup>†</sup></td><td>0.04</td></tr><tr><td>Protein (mg/mL)</td><td>29.8</td><td>29.8</td><td>31.9<sup>‡</sup></td><td>30.7</td><td>31.9</td><td>2.39</td></tr><tr><td>OD<sup>3</sup> 450 nm</td><td>0.597</td><td>0.600</td><td>0.530<sup>†</sup></td><td>0.614</td><td>0.588</td><td>0.073</td></tr><tr><td>OD 470 nm</td><td>0.547</td><td>0.549</td><td>0.490</td><td>0.565</td><td>0.544</td><td>0.065</td></tr><tr><td>d35</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P (mM)</td><td>2.94</td><td>3.07</td><td>3.29<sup>*</sup></td><td>3.21<sup>**</sup></td><td>3.19</td><td>0.15</td></tr><tr><td>Uric acid (µM)</td><td>324</td><td>318</td><td>364<sup>†</sup></td><td>370<sup>*</sup></td><td>353</td><td>40</td></tr><tr><td>Bile acids (µM)</td><td>24.2</td><td>23.3</td><td>24.7</td><td>24.9</td><td>28.5</td><td>3.7</td></tr><tr><td>Triglycerides (mM)</td><td>1.73</td><td>1.82</td><td>1.96</td><td>1.76</td><td>1.99<sup>†</sup></td><td>0.19</td></tr><tr><td>Cholesterol (mM)</td><td>4.17</td><td>4.44<sup>b</sup></td><td>4.82<sup>†</sup></td><td>4.62<sup>**</sup></td><td>4.46</td><td>0.26</td></tr><tr><td>Triglycerides / cholesterol</td><td>0.41</td><td>0.42</td><td>0.41</td><td>0.38</td><td>0.44<sup>††</sup></td><td>0.04</td></tr><tr><td>Protein (mg/mL)</td><td>34.3</td><td>35.0</td><td>37.1</td><td>36.0</td><td>37.1</td><td>2.12</td></tr><tr><td>OD 450 nm</td><td>0.572</td><td>0.611</td><td>0.595</td><td>0.594</td><td>0.665<sup>†</sup></td><td>0.056</td></tr><tr><td>OD 470 nm</td><td>0.526</td><td>0.568</td><td>0.550</td><td>0.548</td><td>0.616<sup>†</sup></td><td>0.051</td></tr></tbody></table>

<footnote>ª Broilers were fed a control (Ctrl) diet supplemented with a lactobacilli-based probiotic (Pro) and postbiotic (Post) biomass without or with their commercial carriers (C) from day 1 onward.</footnote>

<footnote>ᵇ Values are the mean of 32 animals, excluding outliers (maximum one per treatment).</footnote>

<footnote>ᵇ, *, ** indicates a difference with Ctrl value at p < 0.10, 0.05 and 0.01, respectively.</footnote>

<footnote>ᶜ, ¹, †† indicates a difference with corresponding Pro and Post value at p < 0.10, 0.05 and 0.01, respectively.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Plasma immunoglobulin and antibody titres. Violin plot of (A) total blood plasma immunoglobulin (Ig) A, G and M concentrations, and (B) antibody titres against infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) in 21- and 35-day-old male Ross 308 broilers fed control (Ctrl) diet supplemented with a lactobacilli-based probiotic (Pro) and postbiotic (Post) biomass without or with their commercial carriers (C) from day 1 onward. Dots represent individual broilers with the mean per treatment determined from 32 animals, excluding outliers (For Ig, maximum one per treatment except 2 for Ctrl IgA d21; none for the antibody titres); #, * indicates a difference with Ctrl at p < 0.10 and 0.05, respectively. +, †, ‡ indicates a difference with corresponding Pro and Post at p < 0.10, 0.05 and 0.01, respectively. The horizontal line across treatments within a panel of the antibody titre represents the seropositivity threshold. Blue symbols stand for comparisons of means and red symbols for comparison to the seropositivity level.</figcaption></figure>

respectively. IBV titres were affected by treatments at d21 only with an increase for Carrier I (p = 0.026) and for Post v.s. Ctrl (p = 0.018). All broilers were seronegative to IBDV at d21 and 44.4 % were seropositive at d35. On d35, broiler seropositivity to IBDV was lower for Pro (34.4 %; p = 0.019) and Post (37.5 %; p = 0.040) compared to Ctrl (56.3 %), while Carrier I (Pro-C v.s. Pro) tended (p = 0.09) to increase birds seropositivity with 50.0 %.
## 3.5. Blood cell composition

The effects of the dietary treatments on blood cell populations are presented in Table 5. Treatment effects are mostly restricted to d21, with significant effects of the Pro biomass (Pro v.s. Ctrl) and the Carrier I (Pro-C v.s. Pro) while the Post biomass (Post v.s. Ctrl) showed limited effects and Carrier II (Post-C v.s. Post) had no significant effect. Compared to the Ctrl, chickens receiving Pro had higher levels of circulating leukocytes (p = 0.012), both of granulocytes (p = 0.033) and lymphocytes (p = 0.003) subtypes. Among lymphocytes, T cells were most affected with more γδ T cells (p = 0.037), double positive (CD4 + CD8α+; p = 0.050), cytotoxic (CD4-CD8α+, p = 0.07) and helper (CD4 + CD8α-; p = 0.012) T cells. In addition, some activation markers of B and T cells were also found to be increased as blast B cell proportion among B cells and CD8α expression on γδ T cells (p = 0.09). Also, the CD4/CD8 (p = 0.09) and H/L (p = 0.082) both increased with the Pro additive. Birds fed Carrier I (Pro-C v.s. Pro) showed a lower thrombocyte count (p = 0.038) as well as a trend for less lymphocytes (p = 0.06), blast B cells (p = 0.08), T cells (p = 0.07), and lower γδ T cells, γδ T CD8α+, CD4 + CD8α+ and CD4 + CD8α- T cells (p = 0.019, 0.010, 0.024 and 0.026, respectively). At d35, only double negative (CD4- CD8α-) T cells were found in higher proportions in the presence of Carrier I (p = 0.008). The proportion of the latter mature T cells population relative to the total T cells population was reduced while the proportion of the immature CD4- CD8α- T cells increased.

## 4. Discussion

The use of carriers in feed additives is common practice but their potent interaction with the active component has, to the authors' knowledge, not been reported before. It is often not possible to discriminate between the effect of the purported active compound and

<caption>**Table 5**</caption>

Mean plasma concentration of immune cells (/mL) of 21 and 35-day-old male Ross 308 broilers.ª

<table><thead><tr><th>Parameter<sup>b</sup></th><th>Ctrl</th><th>Pro</th><th>Pro-C</th><th>Post</th><th>Post -C</th><th>Pooled SEM</th></tr></thead><tbody><tr><td>D21</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>832231</td><td>851250</td><td>809121</td><td>797660<sup>**</sup></td><td>845105</td><td>63504</td></tr><tr><td>Erythrocytes</td><td>803530</td><td>821018</td><td>783185</td><td>770448<sup>*</sup></td><td>816515</td><td>61500</td></tr><tr><td>Thrombocytes</td><td>23879</td><td>24098</td><td>20627<sup>†</sup></td><td>21660</td><td>23301</td><td>2420</td></tr><tr><td>Leukocytes</td><td>4822</td><td>6134<sup>*</sup></td><td>5309</td><td>5552<sup>‡</sup></td><td>5289</td><td>1002</td></tr><tr><td>Granulocytes</td><td>908</td><td>1493<sup>*</sup></td><td>1114</td><td>1384<sup>‡</sup></td><td>1335</td><td>451</td></tr><tr><td>Monocytes</td><td>111</td><td>105</td><td>110</td><td>104</td><td>96</td><td>32.1</td></tr><tr><td>Lymphocytes</td><td>3812</td><td>4545<sup>**</sup></td><td>4095<sup>†</sup></td><td>4074</td><td>3866</td><td>610</td></tr><tr><td>B cells</td><td>612</td><td>657</td><td>603</td><td>587</td><td>601</td><td>66.5</td></tr><tr><td>Blast B cells</td><td>115</td><td>147<sup>*</sup></td><td>125<sup>†</sup></td><td>124</td><td>121</td><td>25.5</td></tr><tr><td>T cells</td><td>3197</td><td>3885<sup>**</sup></td><td>3488<sup>†</sup></td><td>3484</td><td>3262</td><td>587</td></tr><tr><td>γδ T cells</td><td>141</td><td>172<sup>*</sup></td><td>137<sup>†</sup></td><td>142</td><td>116</td><td>31.4</td></tr><tr><td>γδ T CD8α<sup>+</sup></td><td>42.8</td><td>50.6<sup>‡</sup></td><td>41.5<sup>†</sup></td><td>40.2</td><td>33.6</td><td>10.06</td></tr><tr><td>CD4<sup>+</sup> CD8α<sup>+</sup></td><td>35.7</td><td>57.0<sup>*</sup></td><td>36.7<sup>†</sup></td><td>40.3</td><td>46.3</td><td>15.49</td></tr><tr><td>CD4<sup>+</sup> CD8α-</td><td>831</td><td>1079<sup>*</sup></td><td>868<sup>†</sup></td><td>849</td><td>778</td><td>186</td></tr><tr><td>CD4- CD8α<sup>+</sup></td><td>445</td><td>530<sup>‡</sup></td><td>453</td><td>430</td><td>409</td><td>80.4</td></tr><tr><td>CD4- CD8α-</td><td>1745</td><td>2047</td><td>1993</td><td>2022</td><td>1913</td><td>415</td></tr><tr><td>CD4/CD8</td><td>1.76</td><td>1.98<sup>‡</sup></td><td>1.76</td><td>1.87</td><td>1.70</td><td>0.235</td></tr><tr><td>H/L<sup>c</sup></td><td>0.24</td><td>0.33<sup>‡</sup></td><td>0.27</td><td>0.34<sup>*</sup></td><td>0.35</td><td>0.064</td></tr><tr><td>D35</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>863203</td><td>830977</td><td>875391</td><td>862734</td><td>868992</td><td>40489</td></tr><tr><td>Erythrocytes</td><td>829437</td><td>799873</td><td>842475<sup>‡</sup></td><td>830068</td><td>835724</td><td>39414</td></tr><tr><td>Thrombocytes</td><td>27249</td><td>24677<sup>*</sup></td><td>26061</td><td>25670</td><td>25900</td><td>2041</td></tr><tr><td>Leukocytes</td><td>6517</td><td>6427</td><td>6855</td><td>6996</td><td>7368</td><td>741</td></tr><tr><td>Granulocytes</td><td>991</td><td>1065</td><td>1226</td><td>956</td><td>1097</td><td>240</td></tr><tr><td>Monocytes</td><td>170</td><td>186</td><td>225</td><td>173</td><td>213</td><td>55.4</td></tr><tr><td>Lymphocytes</td><td>5360</td><td>5178</td><td>5410</td><td>5872</td><td>6066</td><td>600</td></tr><tr><td>B cells</td><td>557</td><td>504</td><td>563</td><td>601</td><td>649</td><td>65.6</td></tr><tr><td>Blast B cells</td><td>196</td><td>193</td><td>198</td><td>219</td><td>248</td><td>45.8</td></tr><tr><td>T cells</td><td>4801</td><td>4671</td><td>4844</td><td>5268</td><td>5413</td><td>560</td></tr><tr><td>γδ T cells</td><td>366</td><td>378</td><td>339</td><td>405</td><td>430</td><td>77.7</td></tr><tr><td>γδ T CD8α<sup>+</sup></td><td>67.6</td><td>59.0</td><td>64.0</td><td>74.5</td><td>73.4</td><td>21.54</td></tr><tr><td>CD4<sup>+</sup> CD8α<sup>+</sup></td><td>64.5</td><td>48.4</td><td>46.5</td><td>77.5</td><td>66.3</td><td>21.65</td></tr><tr><td>CD4<sup>+</sup> CD8α-</td><td>1909</td><td>1886</td><td>1863</td><td>2087</td><td>2205</td><td>268</td></tr><tr><td>CD4- CD8α<sup>+</sup></td><td>990</td><td>865</td><td>874</td><td>1050</td><td>1049</td><td>169</td></tr><tr><td>CD4- CD8α-</td><td>1472</td><td>1494</td><td>1721<sup>††</sup></td><td>1602</td><td>1664<sup>‡</sup></td><td>204</td></tr><tr><td>CD4/CD8</td><td>2.27</td><td>2.50</td><td>2.35</td><td>2.11</td><td>2.18</td><td>0.465</td></tr><tr><td>H/L</td><td>0.18</td><td>0.21</td><td>0.23</td><td>0.16</td><td>0.18</td><td>0.043</td></tr></tbody></table>

<footnote>ª Broilers were fed a control (Ctrl) diet supplemented with a lactobacilli-based probiotic (Pro) and postbiotic (Post) biomass without or with their commercial carriers (C) from day 1 onward.</footnote>

<footnote>ᵇ Values are the mean of 32 animals.</footnote>

<footnote>ᶜ Heterophils to lymphocytes ratio.</footnote>

<footnote>§, *, ** indicates a difference with Ctrl value at p < 0.10, 0.05 and 0.01, respectively.</footnote>

<footnote>†, †† indicates a difference with corresponding Pro and Post value at p < 0.10, 0.05 and 0.01, respectively.</footnote>
carrier, as Ctrl diets are not formulated to compensate for carriers, with the exception of Moquet et al. (2018). Here, we investigated if the presence of two carriers commonly used in practice can impact the physiological effects of a probiotic and its associated postbiotic, and sought to gain more insight into the physiological effects of these pro- and postbiotics in the absence of carriers. We used broilers fed an NSP-rich diet to provide challenging conditions as effect become more apparent compared to optimal conditions (Jansseune et al., 2024c). The presence of the commercial carriers in Pro and Post affected broiler growth, FPL score, and immune parameters, thereby reporting to the authors' knowledge, for the first-time a modulatory effect of a carrier on the efficacy of an additive in broilers.

Of note, the current study involved per treatment 8 pens of 40 birds each for the growth performance parameters and 32 broilers per treatment for the blood measurements. This resulted in a very low probability of obtaining false negative, and increased true positive results compared to regular studies in broilers. With exception of Jansseune et al. (2024a); b) c) and Abd El-Ghany et al. (2022), the studies cited here have a median number of pens and birds per pen of 6 and 13.5, respectively, with the number of birds for blood sampling of 6 per treatment. Another specificity of this study is that it was performed under practical housing conditions in a building housing ~16,000 broilers and a diet formulated according to the French standards for male Ross 308 broilers. As a result, our observations are highly relevant to practice when compared to results obtained under high sanitary status of experimental stations. As we used eight replicate pens per treatment, small differences (1-3 %) in growth performance parameters could be identified as statistically significant, and are important when considering practical aspects of broiler production in light of tight economical margins.

## 4.1. Carriers affected pro- and postbiotic effects

Growth performance is a major proxy to assess beneficial or deleterious effects of a feed additive in production animals. The observed beneficial growth-promoting effect of Carrier II in the present study may be partly due to the presence of sepiolite (1.18 and 1.05 g as is/kg of feed from d1 to 14 and 15-37, respectively). In their meta-analysis, Sholikin et al. (2023) reported a dose-dependent improvement of BW and FCR in broilers supplemented with clay minerals (+4.2 g BW and -0.002 units FCR per g of clay minerals/kg feed at final age), with sepiolite having a positive effect on BW (+3.1 % for the starter phase; no data for the finisher phase). Carrier II could have improved fat digestibility in our study as a higher plasma triglyceride concentration, and OD at 450 and 470 nm at d35 was found. The latter wavelengths correspond to the peak absorption values of carotenoids (Hamzic et al., 2015), which can be used as an indirect marker of fat digestibility (Mignon-Grasteau et al., 2020). The apparent negative effect of the Carrier I on BW but not FCR is surprising. Calcium propionate is commonly used as a preservative agent in food and feed, due to its effectiveness against the growth of fungi and bacteria (Zhang et al., 2020). This may have had deleterious effects on the viability of the bacteria in Pro, negating their beneficial effect where this would not be the case for Post C and Carrier II as no viable bacteria are present in Post C.

Other parameters than those associated with performance were also affected by the carriers, including FPL score as well as blood parameters related to nutrition and immunity. Footpad lesions are necrotic lesions caused by skin inflammation (Tellez et al., 2014). They are promoted by high litter and excreta humidity and a high excretion of undigested protein (Swiatkiewicz et al., 2017). The reasons why broilers receiving the additives with their carriers had increased FPL scores remain to be elucidated. The effects on nutrition-related blood parameters were more present for Carrier II than I, potentially due to the different dosage and composition of the carriers. Concerning immunity-related parameters, at d21, modulation in blood cell composition was observed for Carrier I. The latter lowered the count and concentration of mature T cells while increasing the proportion of immature CD4- CD8α- T cells, suggesting a less mature or less active immune system which may be detrimental considering the low immunity of fast-growing broilers (van der Most et al., 2011). This could have contributed to or result from the lower growth rate of Pro-C fed birds. Effects were also different between ages showing that measurements must be performed at multiple time points to avoid misinterpretation. Whether the observed effects are due to the interaction of Pro and Post with their respective carriers or are due to the carriers themselves was not the aim of the current study and remains to be determined.

The carriers were shown to modulate, in a positive (e.g. higher BW or lower FCR) and negative (e.g. lower BW or FCR) manner, the effects of the pro- and postbiotic additives. Carriers in feed additives are rarely reported in scientific articles. In practice, carriers used in additives are subject to change depending on e.g. costs, changes in legislation, and technological reasons. As the current study shows a carrier impact on the effects of the active component in an additive, proper adjustment for carriers must be ensured and reported and changes in the carrier composition of an additive must be tested for the absence of negative effects.

## 4.2. Pro and Post biomasses effects

Supplementing the diet with the probiotic (Pro) led to an increase in growth up to d35 where this occurred up to d21 for the postbiotic (Post). The postbiotic increased FCR after d22 where there was no effect of the probiotic compared to the control. The observed increase in BW of the probiotic may result from a counteraction of the deleterious effects of CD. We reported previously that a diet formulated closely to the current CD but with less rye had adverse effects on growth in two independent experiments (Jansseune et al., 2024b; c). Due to the presence of rye and barley, the CD is rich in NSP. These polysaccharides and particularly soluble NSP increase digesta water holding capacity and viscosity (Bederska-Lojewska et al., 2017). In the literature, rye and barley have been shown to impair performance, increase digesta viscosity and mean retention time, promoting an environment for growth of harmful bacteria (Lazaro et al., 2003; Mehrabadi and Jamshidi, 2019). Aside from soluble NSP, the insoluble NSP (structural) can act as a physical barrier to digestive enzymes, increasing endogenous losses (Knudsen, 2014; Nguyen et al., 2021). To promote growth, the probiotic (Pro) may have counteracted some of the deleterious effects of CD which was also present in the Ctrl fed birds. The deleterious effect of Post on FCR from d22 onwards may result from an imbalanced amino acid uptake from the feed. Indeed, Post fed broilers had an increased level of blood plasma uric acid, a catabolite of amino acid breakdown, when measured at d35. The reason for
this imbalance only for Pro is difficult to explain as it did not occur for Post-C.

Both Pro and Post lowered blood plasma bile acid concentrations at d21 but not 35. Blood bile acid level is the result from complex regulation mechanisms (Arshad et al., 2021). Bile acids are synthesized in the liver, concentrated in the gallbladder, and expelled in the gut to promote fat digestion. Most of the bile acids are reabsorbed by enterocytes (~95 %), thereby minimalizing their losses in the faeces. Bile acids are also biotransformed into secondary bile acids by gut microbiota (Arshad et al., 2021). Thus, modulation of the gut microbiota, their activity, or both could have mediated Pro and Post effects on plasma bile acids. Effects may have been different at d21 and 35.

In the literature pro- and postbiotics are reported for their effects on immune related parameters (Huang et al., 2004; Karimi Torshizi et al., 2010; Humam et al., 2019; Abd El-Ghany et al., 2022; Yin et al., 2023). Here we observed that on d21, Pro increased circulating leukocytes including granulocytes and T lymphocytes (CD4 + CD8α+, CD4- CD8α+ and CD4 + CD8α- T cells) which may indicate a more matured immunity, with activation marker on gamma delta T cells (CD8α+) and B cells (more blast B cells) (Pieper et al., 2011). This may result or have promoted the growth promoting effect of Pro, despite opposing the lower blood Ig A and IgM level observed on d21. The H/L was higher for Pro and a trend for Post at d21 but not d35. A higher heterophil to lymphocyte ratio due to a reduction of the lymphocyte population is indicative of an increased stress of the animal (Lentfer et al., 2015), but as the lymphocyte population increased in our study, a higher stress is unlikely explaining the higher H/L. Post showed minor effects on immune parameters on d21. However, no consistent effect of Pro and Post were observed on d35. This may result from the proximity of the vaccination, or more likely result from maturation of the immune system between d21 and 35 (Song et al., 2021), offering less opportunity for an effect of the additives. The immune response of the birds to vaccination was extremely low as shown by the low proportion of seropositive birds to IBV and NDV which is in line with our previous experiment performed under similar conditions (Jansseune et al., 2024a). In the current study, the local immunity in the gut was not investigated, whereas effects were reported previously with probiotics (Amerah et al., 2013; Peng et al., 2016; Danladi et al., 2022). Further investigations are required to study the impact of pro- and postbiotics on gut immunity.

## 5. Conclusions

Common additive carriers affected the physiological effects of a Pro and Post additive with effects on growth and metabolism. The occurrence of an additive-carrier interaction reported here indicates that care must be taken when selecting carriers for pro- and postbiotic additives for broilers. Furthermore, the results of the current study support the potential interest of Pro to improve broiler growth under a dietary challenging condition. Pro and Post affected also blood biochemical parameters related to nutrition, while they had minor effects on the recorded immune parameters. Further studies are required to determine whether the observations here are also occurring for other types of carriers and additives.

## Disclosures

Although one of the authors (SCGJ) was a PhD candidate employed by Idena, the authors attest that they were completely free to independently design the study and collect, analyse and interpretate the data as well as write the manuscript.

## CRediT authorship contribution statement

**Samuel C.G. Jansseune:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Wouter H. Hendriks:** Writing – review & editing, Methodology, Conceptualization.
**Pinard-van der Laan Marie-Hélène:** Writing – review & editing, Investigation, Conceptualization.
**Fany Blanc:** Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Conceptualization.

## Funding

This study was funded by Idena (Sautron, France), and the "Association Nationale Recherche Technologie" (ANRT) through the Convention Industrielle de Formation par la RecherchE (CIFRE) grant no. 2021-0384. The funding bodies played no role in the design, analysis and reporting of the study.

## Declaration of Competing Interest

Although one of the authors (SCGJ) was a PhD candidate employed by Idena, the authors attest that they were completely free to independently design the study and collect, analyse and interpretate the data as well as write the manuscript.

## Acknowledgments

The authors gratefully acknowledge G. Lemonnier, J. Lecardonnel, N. Bruneau, J.-L. Coville, A. Assez, C. Denis (all INRAE, Jouy-en-Josas, France), A. Canin, J-M. Watier, (all, Idena, Sautron, France), F. Moutel, J.-P. Pontais, S. Moutel, and D. Cherel (all, STI bio-technologie, Saint Etienne en Coglès, France) for their assistance during sample collection, as well as F. Calenge (INRAE, Jouy-en-
Josas, France) and D. Gourichon (INRAE, Nouzilly, France) for their assistance with the animal ethic application.

## Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

## References

Abd El-Ghany, W.A., Abdel-Latif, M.A., Hosny, F., Alatfeehy, N.M., Noreldin, A.E., Quesnell, R.R., Chapman, R., Sakai, L., Elbestawy, A.R., 2022. Comparative efficacy of postbiotic, probiotic, and antibiotic against necrotic enteritis in broiler chickens. Poult. Sci. 101, 101988.

Abd El-Hack, M.E., Mahgoub, S.A., Alagawany, M., Ashour, E.A., 2017. Improving productive performance and mitigating harmful emissions from laying hen excreta via feeding on graded levels of corn DDGS with or without *Bacillus subtilis* probiotic. J. Anim. Physiol. Anim. Nutr. 101, 904–913.

Amerah, A.M., Quiles, A., Medel, P., Sánchez, J., Lehtinen, M.J., Gracia, M.I., 2013. Effect of pelleting temperature and probiotic supplementation on growth performance and immune function of broilers fed maize/soy-based diets. Anim. Feed Sci. Technol. 180, 55–63.

Arshad, M.A., Faiz-ul-Hassan, S.A. Bhatti, M.S. Rehman, W. Yousaf, G. Younus, O. Sizmaz, and M.Q. Bilal. 2021. Supplementation of bile acids and lipase in broiler diets for better nutrient utilization and performance: Potential effects and future implications - A review. Ann. Anim. Sci. 21:757–787.

Bederska-Lojewska, D., Świątkiewicz, S., Arczewska-Wlosek, A., Schwarz, T., 2017. Rye non-starch polysaccharides: their impact on poultry intestinal physiology, nutrients digestibility and performance indices - a review. Ann. Anim. Sci. 17, 351–369.

Bílková, B., Bainová, Z., Janda, J., Zita, L., Vinkler, M., 2017. Different breeds, different blood: cytometric analysis of whole blood cellular composition in chicken breeds. Vet. Immunol. Immunopathol. 188, 71–77.

Celi, P., Verlhac, V., Pérez Calvo, E., Schmeisser, J., Kluenter, A.-M., 2019. Biomarkers of gastrointestinal functionality in animal nutrition and health. Anim. Feed Sci. Technol. 250, 9–31.

Damato, A., Vianello, F., Novelli, E., Balzan, S., Gianesella, M., Giaretta, E., Gabai, G., 2022. Comprehensive review on the interactions of clay minerals with animal physiology and production. Front. Vet. Sci. 9, 889612.

Danladi, Y., Loh, T.C., Foo, H.L., Akit, H., Md Tamrin, N.A., Azizi, M.Naeem, 2022. Effects of postbiotics and paraprobiotics as replacements for antibiotics on growth performance, carcass characteristics, small intestine histomorphology, immune status and hepatic growth gene expression in broiler chickens. Animals 12, 917.

van der Most, P.J., de Jong, B., Parmentier, H.K., Verhulst, S., 2011. Trade-off between growth and immune function: a meta-analysis of selection experiments. Funct. Ecol. 25, 74–80.

Djuragic, O., Slavica, S., Dusica, I., Kokic, B., 2009. Importance of carrier for feed premixes production. Prog. Theor. Exp. Phys. 13, 324–327.

Fomina, M., Skorochod, I., 2020. Microbial interaction with clay minerals and its environmental and biotechnological implications. Minerals 10, 861.

Hamzic, E., Bed'Hom, B., Juin, H., Hawken, R., Abrahamsen, M.S., Elsen, J.M., Servin, B., Pinard-van der Laan, M.H., Demeure, O., 2015. Large-scale investigation of the parameters in response to *Eimeria maxima* challenge in broilers. J. Anim. Sci. 93, 1830–1840.

Holt, D., Aldrich, C., 2021. Evaluation of mixing efficacy of carriers for supplemental nutrient premixes in animal feed. Kans. Agric. Exp. Stn. Res. Rep. 7, 17.

Huang, M.K., Choi, Y.J., Houde, R., Lee, J.-W., Lee, B., Zhao, X., 2004. Effects of lactobacilli and an acidophilic fungus on the production performance and immune responses in broiler chickens. Poult. Sci. 83, 788–795.

Humam, A.M., Loh, T.C., Foo, H.L., Samsudin, A.A., Mustapha, N.M., Zulkifli, I., Izuddin, W.I., 2019. Effects of feeding different postbiotics produced by *Lactobacillus plantarum* on growth performance, carcass yield, intestinal morphology, gut microbiota composition, immune status, and growth gene expression in broilers under heat stress. Animals 9, 644.

Jansseune, S.C.G., Lammers, A., van Baal, J., Blanc, F., Calenge, F., Pinard-van der Laan, M.-H., Hendriks, W.H., 2024b. Lactobacilli-based pro- and postbiotic efficacy are also influenced by other factors than dietary challenging conditions. J. Anim. Physiol. Anim. Nutr. 109, 203–210.

Jansseune, S.C.G., Blanc, F., Lammers, A., van Baal, J., Bruneau, N., Pinard-van der Laan, M.-H., W. H. Hendriks, M.-H., Calenge, F., 2024a. Microbiota but not immune modulation by a pro- and postbiotic was associated with the diet-additive interaction in broilers. Poult. Sci. 103, 104184.

Jansseune, S.C.G., Lammers, A., van Baal, J., Blanc, F., van der Laan, M.-H.Pinard, Calenge, F., Hendriks, W.H., 2024c. Diet composition influences probiotic and postbiotic effects on broiler growth and physiology. Poult. Sci. 103, 103650.

Jha, R., Das, R., Oak, S., Mishra, P., 2020. Probiotics (direct-fed microbials) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: a systematic review. Animals 10, 1863.

Jiang, S., Yan, F.-F., Hu, J.-Y., Mohammed, A., Cheng, H.-W., 2021. *Bacillus subtilis*-based probiotic improves skeletal health and immunity in broiler chickens exposed to heat stress. Anim. Open Access J. MDPI 11, 1494.

Karimi Torshizi, M.A., Moghaddam, A.R., Rahimi, S., Mojgani, N., 2010. Assessing the effect of administering probiotics in water or as a feed supplement on broiler performance and immune response. Br. Poult. Sci. 51, 178–184.

Knudsen, K.E.B., 2014. Fiber and non-starch polysaccharide content and variation in common crops used in broiler diets. Poult. Sci. 93, 2380–2393.

Lauzon, D.A., Johnston, S.L., Southern, L.L., Xu, Z., 2008. The effect of carrier for vitamin e on liver concentrations of vitamin e and vitamin e excretion in broilers. Poult. Sci. 87, 934–939.

Lazaro, R., Garcia, M., Medel, P., Mateos, G., 2003. Influence of enzymes on performance and digestive parameters of broilers fed rye-based diets. Poult. Sci. 82, 132–140.

Lentfer, T.L., Pendl, H., Gebhardt-Henrich, S.G., Fröhlich, E.K.F., Von Borell, E., 2015. H/L ratio as a measurement of stress in laying hens - methodology and reliability. Br. Poult. Sci. 56, 157–163.

Liu, F., Kong, A., Fu, P., Cao, Q.-Q., Tao, K.-S., Liu, D.-Y., Wang, X.-B., Tong, Z.-X., Rehman, M.U., Huang, S.-C., 2021. *Lactobacillus rhamnosus* JYLR-005 prevents thiram-induced tibial dyschondroplasia by enhancing bone-related growth performance in chickens. Probiotics Antimicrob. Proteins 13, 19–31.

Mehrabadi, M., Jamshidi, R., 2019. Effect of antibiotic, probiotic and prebiotic in diets containing barley on performance, digestibility, intestinal morphology, blood parameters and immunological response in broilers. Iran. J. Appl. Anim. Sci. 9, 497–507.

Mignon-Grasteau, S., Beauciercq, S., Urvoix, S., Le Bihan-Duval, E., 2020. Interest in the serum color as an indirect criterion of selection of digestive efficiency in chickens. Poult. Sci. 99, 702–707.

Moquet, P.C.A., Salami, S.A., Onrust, L., Hendriks, W.H., Kwakkel, R.P., 2018. Butyrate presence in distinct gastrointestinal tract segments modifies differentially digestive processes and amino acid bioavailability in young broiler chickens. Poult. Sci. 97, 167–176.

Nadziakiewicza, M., Kehoe, S., Micek, P., 2019. Physico-chemical properties of clay minerals and their use as a health promoting feed additive. Animals 9, 714.

Nguyen, H.T., Bedford, M.R., Morgan, N.K., 2021. Importance of considering non-starch polysaccharide content of poultry diets. Worlds Poul. Sci. J. 77, 619–637.

OECD/FAO. 2021. OECD-FAO agricultural outlook 2021-2030 (OECD/FAO, Ed.).

Peng, Q., Zeng, X.F., Zhu, J.L., Wang, S., Liu, X.T., Hou, C.L., Thacker, P.A., Qiao, S.Y., 2016. Effects of dietary *Lactobacillus plantarum* B1 on growth performance, intestinal microbiota, and short chain fatty acid profiles in broiler chickens. Poult. Sci. 95, 893–900.

Pieper, J., Methner, U., Berndt, A., 2011. Characterization of avian γδ T-cell subsets after *Salmonella enterica* serovar *Typhimurium* infection of chicks. Infect. Immun. 79, 822–829.

Quinger, F., Kern, J., Bosse, A., Seifert, J., Rodehutsford, M., Siegert, W., 2024. Effects of carriers for oils in compound feeds on growth performance, nutrient digestibility, and gut microbiota in broiler chickens. Poult. Sci. 103, 103803.

R Core Team. 2023. R: A language and environment for statistical computing.

Seliger, C., Schaerer, B., Kohn, M., Pendl, H., Weigend, S., Kaspers, B., Härtle, S., 2012. A rapid high-precision flow cytometry based technique for total White blood cell counting in chickens. Vet. Immunol. Immunopathol. 145, 86–99.
Sholikin, M.M., Sadarman, A.Irawan, Sofyan, A., Jayanegara, A., Rumhayati, B., Hidayat, C., Adli, D.N., Julendra, H., Herdian, H., Manzila, I., Hudaya, M.F., Harahap, M.A., Qomariyah, N., Budiarto, R., Krisnan, R., Asmarasari, S.A., Hayanti, S.Y., Wahyono, T., Priyatno, T.P., Ujilestari, T., Negara, W., Wulandari, W., Nahrowi, N., 2023. A meta-analysis of the effects of clay mineral supplementation on alkaline phosphatase, broiler health, and performance. Poult. Sci. 102, 102456.

Song, B., Tang, D., Yan, S., Fan, H., Li, G., Shahid, M.S., Mahmood, T., Guo, Y., 2021. Effects of age on immune function in broiler chickens. J. Anim. Sci. Biotechnol. 12, 42.

Sun, Q., Yin, S., He, Y., Cao, Y., Jiang, C., 2023. Biomaterials and encapsulation techniques for probiotics: current status and future prospects in biomedical applications. Nanomaterials 13, 2185.

Swiatkiewicz, S., Arczewska-Wlosek, A., Jozefiak, D., 2017. The nutrition of poultry as a factor affecting litter quality and foot pad dermatitis - an updated review. J. Anim. Physiol. Anim. Nutr. 101, 14–20.

Tellez, G., Latorre, J.D., Kuttappan, V.A., Kogut, M.H., Wolfenden, A., Hernandez-Velasco, X., Hargis, B.M., Bottje, W.G., Bielke, L.R., Faulkner, O.B., 2014. Utilization of rye as energy source affects bacterial translocation, intestinal viscosity, microbiota composition, and bone mineralization in broiler chickens. Front. Genet 5, 339.

Wu, Y., Wang, B., Zeng, Z., Liu, R., Tang, L., Gong, L., Li, W., 2019. Effects of probiotics *Lactobacillus plantarum* 16 and *Paenibacillus polymyxa* 10 on intestinal barrier function, antioxidative capacity, apoptosis, immune response, and biochemical parameters in broilers. Poult. Sci. 98, 5028–5039.

Yin, Y., Liao, Y., Li, J., Pei, Z., Wang, L., Shi, Y., Peng, H., Tan, Y., Li, C., Bai, H., Ma, C., Gong, Y., Wei, T., Peng, H., 2023. *Lactobacillus plantarum* GX17 benefits growth performance and improves functions of intestinal barrier/intestinal flora among yellow-feathered broilers. Front. Immunol. 14, 1195382.

Zhang, F., Nan, X., Wang, H., Guo, Y., Xiong, B., 2020. Research on the applications of calcium propionate in dairy cows: a review. Animals 10, 1336.