Short- and long-term sex-dependent effects of a
nutritional supplement after hatching on growth,
metabolism and gut microbiota in broiler chickens

Christine Leterrier, Anne Collin, Florent Kempf, Olivier Zemb, Laurent

Cauquil, E. Cailleau-Audouin, Pascal Chartrin, C. Parias, J. Delaveau, C.

Rat, et al.

► To cite this version:

Christine Leterrier, Anne Collin, Florent Kempf, Olivier Zemb, Laurent Cauquil, et al. Short- and long-term sex-dependent effects of a nutritional supplement after hatching on growth, metabolism and gut microbiota in broiler chickens. British Poultry Science, 2025, 66 (5), pp.672-685. 10.1080/00071668.2025.2465350. hal-04998526

HAL Id: hal-04998526

https://hal.inrae.fr/hal-04998526v1

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SHORT TITLE

Growth, Metabolism, and Microbiota Composition after an Early Feed Supply

TITLE

Short- and Long-Term Sex-Dependent Effects of a Nutritional Supplement after Hatching
on Growth, Metabolism and Gut Microbiota in Broiler Chickens

C. Leterrier*<fnref n="1" />, A. Collin#, F. Kempf†, O. Zemb§, L. Cauquil§, E. Cailleau-Audouin#, P. Chartrin#,

C. Parias*, J. Delaveau||, C. Rat||, K. Germain¤, L.A. Guilloteau#

* INRAE, CNRS, Université de Tours, PRC, 37380, Nouzilly, France,

# INRAE, Université de Tours, BOA, 37380 Nouzilly, France,

†INRAE, Université de Tours, ISP, UMR 1282, 37380 Nouzilly, France

§INRAE-INPT-ESUPAT, Université de Toulouse, GenPhySE, 31326 Castanet-Tolosan, France

||INRAE, PEAT, 37380 Nouzilly, France,

¤INRAE, UE1206 Elevage Alternatif et Santé des Monogastriques, Domaine du Magneraud, 17700

Saint-Pierre d'Amilly, France

**Keywords:** growth, stress, physiology, metabolism, gut microbiota, hatching

<footnote n="1">¹Corresponding author: christine.letierrier@inrae.fr</footnote>
ABSTRACT

1. In chicken production, a delay occurs between hatching and the chicks' placement in the rearing building. We analysed the short- and long-lasting effects of this experience on growth, metabolism, and gut microbiota composition and tested whether a nutritional supplement (SUP) could mitigate these effects.

2. Chicks were placed directly in a rearing room (Control) or were exposed to a 24-h period without feed and water before being placed (Delayed). During the 24-h period, half of each group was provided with a SUP.

3. The Delay effect reduced body weight until Day 27 in females (p = 0.017) and Day 34 in males (p = 0.015). On Day 1, the Delay reduced plasma triglyceride (TG) concentration and increased uric acid (UA), total antioxidant status (TAS) and liver thiobarbituric acid reactive species in both sexes (p < 0.050 for each). On Day 34, the delay increased UA concentration and TAS only in females. It increased the α-diversity of caecal microbiota in males (Day 12 p = 0.036; Day 34 p = 0.038) and the β-diversity in females on Day 34 (p = 0.008). Changes in microbiota composition in both males and females were observed until Day 34 at the genus level.

4. The SUP reduced breast percentage in males on Day 34 (p = 0.017). On Day 1, the SUP increased glucose concentration in Delayed and Control males (p = 0.001) and females (p = 0.002). The decrease in TG and increase in UA concentrations and TAS in Delayed chicks were only mitigated by the SUP in females. On Day 34, The SUP reduced the haptoglobin-like activity in Delayed and Control males (p = 0.041) and increased the TG concentration in Control males (p = 0.016). The SUP had few effects on the caecal microbiota.

5. The delayed placement resulted in an immediate chick metabolism adaptation at the expense of growth. It induced long-lasting effects on growth, metabolism, and caecal microbiota composition. Chicks could benefit from a nutritional supplement for short-term metabolism mitigation; however, the effects are variable and sex-dependent.
**Introduction**

Numerous studies have shown that early events can have a lasting impact on the lives of individuals (plants and animals). In animals, stressful events experienced after birth can affect growth and health up to adulthood. Such effects have been demonstrated in mammals, with rearing conditions after birth, maternal care, and many social factors having consequences beyond puberty (Dutcher et al. 2020). In precocial birds, which are the single largest type of birds used for poultry production, it might be thought that these influences were less critical because the young chicks have greater food autonomy since they have vitelline reserves and can look for food on their own. However, their thermoregulation abilities are limited, thus leaving them vulnerable to cold stress when fasted (Davison and Lickiss 1979).

In poultry production, studies have investigated the stress experienced by chicks after hatching due to a delay between hatching and pulling, i.e., opening the incubator (Bergoug et al. 2015; Boyner et al. 2021; Boyner et al. 2023; Willemsen et al. 2010), and another significant delay of up to 72 h between pulling and placing hatchlings in the rearing house (Bergoug et al. 2013; Chen et al. 2023; Hollemans et al. 2018; Ivarsson et al. 2022; Xu et al. 2022). During this time, chicks are manipulated numerous times and are subjected to ambient temperatures that may be suboptimal. Previous experiments have shown that a 48-h delay could induce behavioural changes and have consequences on health, especially on pododermatitis and performances, up to 21 days old or up to slaughter age (Bergoug et al. 2013; Hollemans et al. 2018; Jacobs et al. 2016; Valros et al. 2008). Adverse effects can also be observed with a delay of only 38 h between exiting the incubator and placement in the rearing house (Ivarsson et al. 2022). As little as a 10-h delay can have consequences on performance up to slaughter (Bergoug et al. 2013), while changes in growth and metabolism up to slaughter age have been shown with a 24-h delay (Foury et al. 2020; Guilloteau et al. 2019; Ivarsson et al. 2022).
To avoid these detrimental effects, transport conditions and duration should be optimised; when this is not possible, several strategies can be deployed. Some solutions try to mitigate the effects of this potentially harmful delay by treating after hatching to limit the development of infectious disorders (use of antibiotics or antiseptics in drinking water), and/or by making vitamins and micronutrients available that may strengthen chick immunity, or by using phyto-therapeutic formulations for antiseptic effects. An alternative is to provide chicks with a nutritional supplement (SUP) during this transport period to reduce the duration of fasting and thus help the chicks maintain their metabolic and thermal balance. This strategy has been studied in some experiments (Gonzales et al. 2003; Simon et al. 2014; van de Ven et al. 2011); however, few studies have investigated lasting changes in energy metabolism, antioxidant, and inflammatory statuses. In the present experiment, we tested this option by providing chicks with a complex mixture of micronutrients and a probiotic (Pediococcus acidilactici) during the delay before placing them in the rearing house. We used a 24-h delay before placement in pens with room conditions mimicking transport, and we analysed the immediate and long-lasting effects of providing such a mixture during this 24-h period on growth, metabolism, redox and inflammatory statuses, and caecal microbiota. We tested the hypothesis that this SUP would mitigate the negative consequences of delayed placement.

# Materials and Methods

## Ethics Approval

All procedures used in these experiments were approved by the local ethics committee (Comité d'Ethique en Expérimentation Animale Val de Loire, Tours, France; permission APAFIS#12462015072215422490v2) and carried out in accordance with current European legislation (EU Directive 2010/63/EU).
## Model of a Postnatal Negative Experience in Chicks

The chicks were reared at the Experimental Poultry Facility (PEAT, INRAE, 2018, 37380 Nouzilly, France, doi:10.15454/1.5572326250887292E12). They were allocated to groups according to a 2 × 2 factorial arrangement, resulting in four treatments described below. Six pens of 16 chicks were used for each treatment. The schedule of the experiment is tabled in Supplementary Data 1.

Ross 308 chicks were sex-sorted after opening the incubator, wing-tagged, weighed, and vaccinated against infectious bronchitis (IB) (Nobilis IB 4/91 R, Intervet, Beaucouzé, France) by the conjunctival route. The chicks were then either placed immediately in pens in the rearing facility after their removal from the incubator (Control, 96 males and 96 females, placed in 12 pens of 16 chicks each) or removed and placed in four transportation boxes (48 birds per box) for a period of 24 h before being placed in the rearing facility (Delayed, 96 males and 96 females). The Delayed chicks were subjected to irregular movements and variable room temperatures (Guilloteau et al. 2019): 32°C (30 min), 21°C (90 min), 32°C (30 min), and then 21°C with alternating cycles of box movement (M) and immobility (I) for 24 h. One cycle consisted of 45 min (M), 15 min (I), 30 min (M), and 30 min (I). These conditions were chosen to imitate closely the actual suboptimal conditions experienced by broiler chickens in transport. After the 24 h spent in the transportation boxes, Delayed chicks were placed in 12 pens (16 chicks/pens) in the same house as the controls.

## Provision of a Nutritional Supplement

Half of both Control and Delayed chicks received no SUP during the first 24 h after opening the incubator, while the other half received a single dose of the SUP either in their trough in 6 pens (Control chicks with SUP) or in the transportation boxes (Delayed chicks with SUP). This SUP was prepared with 500 g of pellets (Chick'Boost ND, NewBorn Animal Care, France) mixed with 75 mL of water with a probiotic (*Pediococcus acidilactici*, Lallemand, SAS, France). The pellets consisted of highly digestible protein concentrate (soluble fish protein concentrate, soy
concentrate, corn gluten meal); amino acids; glucose; organic acids (sorbic and citric acids); mannan oligosaccharides; β-glucan; algae extracts; medium-chain fatty acids; minerals; vitamins A, B3, B6, B12, C, D3, E, and K3; pantothenic acid; folic acid; biotin; choline; iron; copper; zinc; manganese; iodine; cobalt; selenium; and water. The analytical composition was as follows: moisture (28% minimum), crude protein (21% minimum), fat (1.7%), crude fibre (2.3%), ash (4.5% to 5%), lysine (11.2 g/kg), and methionine (4.2 g/kg). A single dose of probiotic was provided at the same time as the SUP. The probiotic used was *Pediococcus acidilactici* since it was previously used to supplement chicken feed and resulted in increased weight gain (Jha et al. 2020). The probiotic solution consisted of 400 mg Bactocell® Drink/L (Bactocell® Drink ND, 2.5 10⁹ IU *Pediococcus acidilactici/g*, Lallemand SAS, France); 75 mL of this solution was spread over 500 g of the nutritional supplement, and they were then stirred together. The amount of hydrated pellets provided in the transportation boxes of Delayed chicks with SUP or in the troughs of the Control chicks with SUP was 2.5 g per chick on average (120 g per box and 40 g per trough), i.e., 2.17 g of pellets with 0.33 mL of the probiotic solution. This SUP was spread over the diet in the trough in Control pens with SUP at placement. It was provided in one of the two transport boxes before closing the box. The SUP provided in the boxes was completely eaten at the end of the delay period in Delayed chicks, and that provided in the troughs of Control chicks with SUP was also eaten 24 h after distribution.

The experimental design was thus based on four groups of chicks: Control without SUP, Control with SUP, Delayed without SUP, and Delayed with SUP.

## Rearing Conditions

The chicks were reared in 2 × 3 m² pens under standard temperature and light conditions with *ad libitum* access to water. Wood shavings were used as litter with a wire mesh platform and a perch for environmental enrichment. Chicks had *ad libitum* access to feed without anticoccidial drugs.
They were fed a standard starting diet placed in hopper feeders until Day 12, then a growing diet from Day 12 to Day 20 and a finishing diet until slaughter (Table 1).

## Growth and Health

Body weight (BW) was measured on Days 0, 1, 6, 12, 20, 27, and 34. The weighing days correspond to the end of the delay and to diet changes (Days 12 and 20). Moreover, supplementary weighing days were placed in order to get intervals from 6 to 8 days of life to monitor growth. We checked that the chicks' BW after opening the incubator was not different among the 4 groups or between males and females. One male and one female per pen were slaughtered on Day 12 (48 chicks, 12 per group), and 72 chickens (3 per pen, 18 per group, half males and half females) were slaughtered on Day 34. The yolk sac was weighed on Day 1, and the occurrence of residual yolk was measured on Days 12 and 34. The breast muscle percentage was calculated 1 day after slaughter on Day 34 from the ratio of the pectoralis major muscle weight to slaughter body weight in one male and one female per pen. Infectious diseases were checked for through a clinical examination of individuals during weighing on Days 6, 12, 20, 27, and 34. Gait scores were quantified in 3 chickens by pen on Day 28 using gait scoring adapted from Kestin et al. (1992).

## Feed and Water Intake

Feed intake was measured on Days 12, 20, and 34 in order to calculate the feed conversion ratio. Feed intake was recorded per pen, while BW was recorded individually. The gain-to-feed ratio was calculated based on calculated BW gain and feed intake per pen. Water intake was measured in each pen during the first 2 days after placing chicks: from the placement of control chicks to 24 h and from 24 h to 48 h in Controls, and from 24 h (placement of Delayed chicks) to 48 h and from 48 h to 72 h in Delayed chicks.
Physiological Parameters

Blood samples were collected from 12 chicks per group on Days 1 and 13 (48 chicks; 1 male and 1 female per pen) and 18 chickens per group on Day 34 (72 chickens, half males and half females, 3 chickens per pen). Blood samples were collected at the occipital sinus in sodium heparin tubes for metabolite, redox, and inflammation markers' analyses and dried tubes for creatine kinase (CK) activity and antibody titres' analyses. Commercial kits were used to determine various measures to assess energy metabolism, antioxidant, and inflammatory status. Plasma glucose concentration (mg/L) was determined following the REF 61269/61270 kit's instructions (Thermo Fisher Diagnostics SAS, Asnières sur Seine, France) using the sequence glucose oxidase – peroxidase – chromogen that absorbs strongly at 505 nm (MG981780). Triglyceride concentration (mg/L) was determined in accordance with the REF 61236 kit's instructions (Thermo Fisher Diagnostics SAS) using the sequence lipase – glycerokinase – glycerol-3-phosphate oxidase – peroxidase – chromogen that absorbs strongly at 505 nm (MG981786). Uric acid concentration (mg/L) was determined following the REF 61923 kit instructions (Thermo Fisher Diagnostics SAS) using the sequence uricase – peroxidase – chromogen that absorbs strongly at 520 nm (MG981788). Total plasma antioxidant activity was determined through total antioxidant status (TAS, mmol/L) in line with the manual NX 2332 kit's instructions (NX 2332, Randox Laboratoires, Roissy, France) using a Trolox standard curve and measuring the absorbance of samples at 600 nm. Protocols were used in accordance with supplier instructions and adapted to the automated Thermo Scientific Arena 20XT photometric analyser (Thermo Fisher Scientific, Courtaboeuf, France). Lipid peroxidation was determined using spectrophotometry of thiobarbituric acid reactive substances (TBARS) as previously described for the liver and adapted for plasma (Fourny et al. 2020). TBARS were expressed as nmol of malondialdehyde per mL plasma. Haptoglobin-like activity described as PIT54 in chicken (Wicher and Fries 2006) was evaluated in plasma in accordance with the PHASE3TM Haptoglobin assay cat. No. TP-801 kit's instructions using a colorimetric assay at 600 nm (TP-801, Tridelta Development Limited, Maynooth, Ireland), measuring the inhibition of
haemoglobin peroxidase activity expressed as mg/mL. Plasma creatine kinase (CK) activity (IU/L) was measured at 340 nm by spectrophotometry following the kit's instructions (92207 CKNAC kit BIOLABO, ABLIANCE, Compiègne, France). CK activity was not measured on Day 1 but only on Days 13 and 34.

Immune system activity was assessed by measuring the antibody titres specific to the infectious bronchitis (IB) vaccine that were present in the serum after vaccination. The serum was sampled at hatching (n = 23), on Day 12 (n = 48, 1 female and 1 male/pen), and on Day 34 (n = 72, half females, half males, 3 chickens/pen). Antibody titres were determined by ELISA using the ID Screen® IB Indirect kit and the protocol (IBVS ver 0416) described by the supplier (ID.vet, Grabels, France). Antibody titres were expressed as log₁₀ ( titre) = 1.00 × log₁₀ (S/P) + 3.630 with S/P = (Absorbance<sub>Sample</sub> – Absorbance<sub>Negative Control</sub>) / (Absorbance<sub>Positive Control</sub> – Absorbance<sub>Negative Control</sub>); titre = 10<sup>log₁₀ ( titre)</sup>.

## Caecal Microbiota

Caecal contents were collected after slaughter on Days 12 (n = 48, 12 per group) and 34 (n = 72, 18 per group), half each from females and males. The digesta were collected without scraping the epithelium in order to obtain luminal bacteria. They were stored at -80°C. Microbial DNA extraction was performed using the QIAamp DNA mini-kit (ref #51306, Qiagen Inc., Courtaboeuf, France) as previously described (Mignon-Grasteau et al. 2015). Briefly, 25 mg of thawed digestive content were mixed in 1 mL of lysis buffer and homogenised at maximum speed (frequency 30.sec⁻¹) with 0.4 g of sterile zirconium beads in a tissue lyser mixer Retsch MM400 for 3 min, followed by heating at 70°C for 15 min. After centrifugation (16 000 g, 5 min at 4°C), the supernatant was conserved at ambient temperature, and 300 µL of lysis buffer were added. The homogenisation steps were repeated on the pellet and were followed by a second centrifugation (16 000 g, 5 min at 4°C). The two supernatants were pooled and homogenised for the DNA purification and
filtration steps. Proteinase K and AL buffer were added to the supernatants, and the mixture was heated at 70°C for 10 min to remove proteins. The sample was transferred to a tube containing pure ethanol for purification using a QIAamp column as described by the manufacturer. The sample was then eluted in AE buffer (10 mM Tris-Cl; 0.5 mM EDTA; pH 9.0; Qiagen Inc., Courtaboeuf, France). DNA purity was assessed with a NanoDrop spectrophotometer based on the 260/280 and 260/230 O.D. ratios.

PCR amplification of the bacterial 16S rRNA gene on DNA extracts was carried out using the primers designed to amplify the V4-V5 regions (Forward: CTTTCCCTACACGACGCTCTTCCGATCTGTGYCAGCMGCCGCGGTA; Reverse: GGAGTTCAGACGTGTGCTCTTCCGATCTCCCCGYCAATTCMTTTRAGT). The PCR program used consisted of an initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 60 s, 65°C for 40 s, 72°C for 30 s, and a final extension step of 72°C for 10 min. PCR product size was checked with 2% agarose gel electrophoresis before the sequencing step.

The 16S rRNA gene sequencing used V4-V5 region full-length reads that were obtained using Illumina Miseq 250-bp paired-end reads. The resulting PCR products were purified and loaded onto an Illumina MiSeq cartridge in accordance with the manufacturer's instructions. Using PhiX control following the manufacturer's instructions, the quality of the run was checked internally, and with the help of the previously integrated index, each pair-end sequence was assigned to its sample. Each pair-end sequence was assembled using Flash software (Magoc and Salzberg 2011) with at least a 10-bp overlap between the forward and reverse sequences, which allowed a 10% mismatch (Lluch et al. 2015). The absence of contamination was checked with a negative control during the PCR (water as template). The corresponding sequences were uploaded on NCBI with the access number (PRJNA879333).

The 16S rRNA gene sequences were clustered in operational taxonomic units (OTU) using Swarm (Mahé et al. 2015). Additional filtering steps based on an abundance criterion (i.e., the minimal OTU abundance was $5 \times 10^{-5}$), chimaera detection, and comparison to a PhiX contaminant
database were performed using FROGS (Escudié et al. 2018). Lastly, the OTU taxonomic
assignation was performed using a BLAST comparison with the SILVA 16S 132 database
followed by careful manual curation. The OTUs presenting abundance higher than 0.001 (for a
total of 110 OTUs) were selected. Chao1, Shannon, and inverse Simpson α-diversity indexes were
calculated using the R package vegan v2.6-2. The β-diversity was measured using the Bray–Curtis
index, computed using Phyloseq 1.34.0 (McMurdie and Holmes 2013): the differences were
assessed using Permanova tests computed using the adonis2() function of the R package vegan
v2.6-2 (Oksanen et al. 2022).

## Statistical Analyses

The data were analysed with XLSTAT (2024.2.2) software for Windows. Each pen was considered
as a statistical unit. The normality of residual distribution was checked with a Shapiro–Wilk test,
and log transformation was used to reach data normality if needed. A preliminary 3-way ANOVA
was first carried out using the Delay effect (Control or Delay), the SUP effect (SUP or no SUP),
the Sex effect, the two-by-two interactions, and the 3-way interaction. Since interactions with the
sex effect were significant in many parameters on Days 1 and 34, a 2-way ANOVA was used in
males and females separately at each age to test the influence of the Delay and SUP effects and
the interaction between both effects. This 2-way ANOVA was carried out using the following
statistical model: $Y_{ijk} = \mu + a_i + b_j + ab_{ij} + e_{ij}$, where $Y_{ij}$ is the dependent variable, $\mu$ the overall
mean, $a_i$ the Delay effect (Control or Delay), $b_j$ the SUP effect (SUP or no SUP), $ab_{ij}$ the two-by-
two interaction, and $e_{ij}$ the residual error term. Post-hoc comparisons were then carried out with a
Tukey test for multiple mean comparisons. The chi-square test was used to test the occurrence of
vitellus residual. The results are presented in the text and the tables as means with their standard
deviations. The differences among means were considered statistically significant when *p* was
lower than 0.05.
# Results

## Growth and Health

In the females, body weight (BW) was reduced by the Delay effect from Day 1 to Day 27, but not anymore on Day 34 (Table 2). This Delay effect on BW did not interact with the SUP effect, which was not significant whatever the age. The breast muscle percentage at slaughter was not affected by any effect (Table 2).

In males (Table 3), BW was reduced by the Delay effect until Day 34. The interaction between the Delay and SUP effects was significant on Days 6 and 12. Indeed, the provision of the SUP reduced the decrease in BW induced by the Delay on Day 6 and reduced BW on Day 12 in Control chicks. It also reduced the *P. major* percentage at slaughter age in both Control and Delayed chickens (Table 3).

The percentage of yolk sac weight related to BW was not influenced by any effect on Day 1 (mean ± sd: 4.5 ± 1.1% in females and 5.6 ± 1.9% in males). On Day 13, yolk residues were present in 12.5% of females and 25% of males. These occurrences were not different between females and males and were not affected by any effect. On Day 34, residual yolk was still present in 37% of the Control chickens, without significant differences between females and males (45% in females and 27% in males), but not in any Delayed birds ($\chi^2 = 11.1, p = 0.001$). The occurrence of this residue was not affected by the SUP effect ($\chi^2 = 0.547, p = 0.456$).

No infectious diseases occurred during this experiment. The mean gait score measured on Day 28 was 2.25 ± 0.48, the mortality rate at slaughter age was 1.4%, and both parameters were not affected by the delay or SUP effects. The titre of antibodies against infectious bronchitis was not affected by any effect (Day 0, titre = 1050 ± 738; Day 12: 1387 ± 551; Day 34: 877 ± 466).
Feed Intake, Feed Conversion Ratio, and Water Intake

The feed intake between Days 0 and 12 was lower in Delayed than in Control chickens (mean ±
sd: 277 ± 14 g vs 308 ± 12 g in Control pens; *p* < 0.001) but was not affected significantly by the
SUP effect or the interaction between the delay and SUP effects. Feed intake during the following
periods and the total intake were not affected by any effect (mean Day 12–20 intake: 548 ± 31 g;
mean Day 20–34 intake: 1500 ± 113 g; mean Day 0–34 intake: 2341 ± 115 g). The feed conversion
ratio was reduced in the Delayed chickens between Days 12 and 20 only, and it was reduced by
the SUP effect between Days 20 and 34 (Table 4). The feed conversion ratio calculated from Day
0 to Day 34 was not influenced by Delay or SUP effects or their interaction.

Water intake related to BW when the chicks were placed was affected by the interaction between
the Delay and SUP effects (Figure 1). On the first day after placement, water intake was higher in
Delayed than Control chicks, and this difference was even greater in Delayed chicks with SUP
than in Delayed chicks without SUP in the transport boxes (Figure 1A). On the second day after
placement, only Delayed chicks with SUP drank more than the others (Figure 1B).

<figure><img src="image_2.png" /><figcaption>Figure 1 – Water intake during the first and second days in place</figcaption></figure>
Physiological Parameters

On Day 1, many physiological parameters were modified by the Delay or SUP effect or their
interaction. In females, plasma glucose concentration was influenced by the interaction between
the two effects with an increase in glycaemia in Delayed chicks with SUP compared to Delayed
chicks without SUP which was superior to Control chicks (Table 5). There was no interaction
between the Delay and SUP effects on the plasma triglyceride concentration, but a decrease was
observed in Delayed compared to Control chicks and in chicks with SUP compared to those
without SUP. On the contrary, plasma uric acid concentration and TAS were affected by the
interaction between Delay and SUP effects: they were higher in Delayed than in Control chicks
and higher in chicks that received SUP than those that did not in the Delayed chicks only. Blood
TBARS were not influenced by any effect, but in the liver, they were increased by the Delay effect,
with greater values for the Delayed than for the Control chicks. Haptoglobin-like activity was not
influenced by any of the studied factors (Table 5). In males, plasma glucose concentration was not
affected by the delay but was increased by the SUP in both Control and Delayed chicks, without
interaction between the two effects, contrary to females (Table 6). The plasma triglyceride
concentration was lower in Delayed than in Control chicks, while uric acid concentration, TAS in
the plasma, and TBARS in the liver were increased by the Delay effect. These parameters were
not affected by the SUP effect. Blood TBARS and haptoglobin-like activity were not influenced
by any effect.

Mid-lasting effects occurring on Day 12 were less numerous than on Day 1. In females, the physiological parameters were no longer affected by the Delay and SUP effects (Supplementary Data 2).

In males, only the uric acid concentration was increased in chickens with SUP compared to those
without (No SUP: 66.8 ± 8.0 mg/L; SUP: 75.6 ± 7.9 mg/L; *p* = 0.014). The other physiological
parameters were not affected by the Delay and SUP effects (Supplementary Data 2).
Long-lasting effects of Delay and SUP were different between females and males on Day 34. In females, plasma uric acid concentration and TAS were higher in Delayed than in Control groups (Table 7). Only plasma CK activity was influenced by the interaction between Delay and SUP effects, but this did not lead to significant *post-hoc* differences between groups.

In males, the triglyceride concentration was influenced by the interaction between Delay and SUP effects: it was increased in Control chickens with SUP but not in Delayed chickens (Table 8). Uric acid concentration and TAS were not increased by the Delay effect as in females, but the haptoglobin-like activity was reduced by the SUP effect in both Control and Delayed chickens ($p = 0.041$; Table 8).

## Caecal Microbiota

On Day 12, the $\alpha$-diversity indexes were not affected by any effect in females, and we did not observe any significant differences for the test performed over the Bray-Curtis $\beta$-diversity index between the Delay and SUP categories (Supplementary Data 3). No difference among the groups was significant at the phylum level (Firmicutes: 96.9 ± 3.5%, Tenericutes: 2.1 ± 3.5%, Bacteroidetes: <0.01%; Supplementary Data 3). At the genus level, only the abundance of the *Eubacterium coprostanoligenes* group was higher in Control (1.80%) compared to Delayed females (0.61%; $p = 0.015$).

On Day 12, in males, the Chao1 index was higher in Delayed compared to Control chicks (Figure 2A) without an effect of SUP or interaction between both effects. The other $\alpha$-diversity indexes were not affected by the Delay effect, the SUP effect or their interaction (Supplementary Data 3). In a similar fashion, no difference was found for the test performed over the $\beta$-diversity indexes between the Delay and SUP categories (Supplementary Data 3). In line with this, no difference among the groups was significant at the phylum level (Firmicutes: 97.6 ± 2.5%, Tenericutes: 1.3
± 2.4%, Bacteroidetes < 0.01%; Supplementary Data 2). At the genus level, the abundance of the Ruminococcaceae UCG-014 was higher in Control (15.02%) compared to Delayed males (8.28%; *p* = 0.050), without any effect of SUP or of the interaction between both effects.

<figure><img src="image_3.png" /><figcaption>Figure 2 – Chao1 indexes of the gut microbiota in males on Days 12 and 34</figcaption></figure>

On Day 34, the Chao 1 index in females was not affected by any effect (Supplementary Data 2).
The test performed over the Bray–Curtis β-diversity index indicated that the Delay categories presented significant differences for the females on Day 34 (*p* = 0.008, Supplementary Data 2).
The relative abundance of the Firmicutes phylum was decreased by the Delay effect, while the abundance of the Bacteroidetes phylum was increased by this effect (Table 9). The Tenericutes phylum was increased by the SUP effect (Table 9).

At the genus level, *Faecalibacterium* and *Ruminococcaceae* UCG-005 were decreased by the Delay effect, while *Alistipes* was increased (Figure 3).
Figure 3 – Relative abundance of the *Faecalibacterium*, *Ruminococcaceae-UCG-005*, and *Alistipes* genera in females on Day 34

<figure><img src="image_4.png" /></figure>

On Day 34, in males, the Chao1 index was increased in Delayed chickens, as it was on Day 12 (Figure 3B), while the Bray–Curtis β-diversity index indicated no significant effect of the Delay effect or SUP provision (Supplementary Data 3). No difference among the groups was significant
at the phylum level (Firmicutes: 88.8 ± 5.4%, Tenericutes: 1.1 ± 0.8%, Bacteroidetes: 9.9 ± 5.6%; Supplementary Data 3). At the genus level, the relative abundance of Ruminococcaceae UCG-014 was decreased by the Delay effect, while Erysipelatoclostrium was increased by this effect (Figure 4). The abundance of the bacterium ic1391 species was enhanced by the Delay (0.40% in Control vs 0.78% in Delayed; *p* = 0.002). The abundance of the Clostridiales bacterium 60-7e species (0.40%) was affected by the interaction between the Delay and SUP effects (*p* = 0.043); however, the *post-hoc* test did not establish significant differences between groups.

<figure><img src="image_5.png" /><figcaption>Figure 4 – Relative abundance of the Ruminococcaceae-UCG-005 and *Erysipelatoclostridium* genera in males on Day 34</figcaption></figure>
Discussion

Much research has shown long-term impacts of delays in placing chicks in rearing rooms on performance, metabolism, and physiology (Beauclercq et al. 2019; Guilloteau et al. 2019; Jacobs et al. 2016; Khosravinia 2016), and this study confirms these results. Moreover, this experiment demonstrates that providing chicks with a nutritional supplement before placement had multiple effects on growth, physiology, and caecal microbiota, immediately after the provision and during later life. However, these effects were variable and were very influenced by the sex of the bird.

Effects of the Delay before the Chicks' Placement

At the end of the delay period, large differences were observed between the Delayed and Control chicks, thus confirming this delay's stressful effect. BW in Delayed chicks was lower than in Control chicks (24.1% in females and 21.3% in males), and this can be explained by the difference in feed intake, but also by greater water loss revealed by the higher water intake after placement in Delayed than in Control chicks. The blood parameters also confirm the stressful effect of delaying placement: a decrease in triglyceride concentration and increases in liver TBARS and plasma uric acid and TAS were present in both sexes. The reduction in triglyceride concentration observed in Delayed chicks probably resulted from the hepatic lipid metabolism and consequently from lipid oxidation, as shown by the increase in TBARS. Differences in liver metabolism were also found in a previous study with a 24-h delay (Foury et al. 2020) and with 72-h post-hatch fasting, which demonstrated the high impact of fasting on the liver (Chen et al. 2023) and on lipid metabolism (Xu et al. 2022). The absence of an external nutritional source for Delayed chicks after hatching also resulted in increased uric acid related to increased protein catabolism to supply the chick with energy, as observed in day-old chicks exposed to severe (10°C) and mild (20°C) cold stress (Davison and Lickiss 1979). Moreover, an increase in antioxidants (uric acid, TAS) was observed, as we reported in a previous experiment (Foury et al. 2020). These two studies were
carried out on two different broiler lines (Hubbard and Ross 308) and showed that the delay
procedure was repeatable and induced immediate metabolic adaptation in chicks. In the present
experiment, as in a previous study with the same delay procedure (Guilloteau et al. 2019), there
was no difference in the weight of yolk sac residues (around 5% of BW) between Delayed and
Control chicks at the end of the delay period, which suggests an equal resorption rate whether feed
was provided or not under our conditions. This is in line with previous data (Vieira and Moran
1999) that showed that the yolk resorption during a 24-h delay period represents only a 2-g
decrease, which is small compared to the 12-g difference in BW between Delayed and Control
chicks. Thus, yolk resorption cannot explain all the metabolite differences on Day 1, despite
apparently increasing plasma electrolytes and contributing to chick adaptation (Khosravinia 2016).
The modifications induced by delayed placement were also accompanied by long-term effects that
had a small impact on the major blood metabolites analysed. The delayed placement had no mid-
lasting effects occurring on Day 12 in both females and males, but the long-lasting effects observed
on Day 34 were different in females than in males. On Day 34, uric acid and TAS were increased
by the delay only in females, the other physiological parameters not being modified. However,
long-term modifications were previously observed in the blood transcriptome, particularly in
males, demonstrating that the impact of delayed placement on genes involved in oxidative stress,
growth, and bone metabolism lasted until slaughter at Day 34 (Foury et al. 2020).

Delayed placement had marked consequences on BW up to Day 27 in females and slaughter age
(Day 34) in males, as previously observed (Guilloteau et al. 2019), but this factor did not affect
muscle yield and health parameters in our experiment. The relative growth gap was approximately
10% compared to the Control group on Day 12, and it can be explained by the lower feed intake
in Delayed birds from hatching to Day 12, but this difference in BW gradually narrowed. This
compensatory growth was also related to a higher feed efficiency revealed by a lower feed
conversion rate between Days 12 and 20 and at slaughter age, the difference between Delayed and
Control chickens still being significant only in males. This difference between the sexes is in
accordance with a recent experiment demonstrating that BW was more impacted by early-life
events in males than in females (Elfwing et al. 2015; Guilloteau et al. 2024).

The post-mortem examination carried out at slaughter age revealed that the yolk sac had been
totally resorbed in all Delayed chickens and only in 63% of Control chickens, whatever the SUP
treatment. Yolk sacs that are not completely resorbed can harbour microorganisms and promote
infectious problems, since the translocation of maternal microbiota from blood to ovarian follicles
might be the source of microbiota in the yolk and, consequently, in chicks (Mandal et al. 2020).

This reduction in the number of chickens with yolk residues in the Delayed group would suggest
that a short delay before placement could reduce the risk of abdominal infection.

Since stress in early life influences gut microbiota composition in birds (Ballou et al. 2016; Kraimi
et al. 2022) the consequence of the delayed placement on caecal microbiota was thus analysed. On
Day 12, only the α-diversity of caecal microbiota was increased by the delayed placement in males,
but not in females. A previous study in which access to feed was delayed for 48 h did not detect
this difference, probably because sex was not taken into account (Proszkowiec-Weglarz et al.
2022). However, a difference in gut microbiota between Delayed and Control females was
observed on Day 12 (decrease in the relative abundance of *Eubacterium coprostanoligenes* group).

This is interesting, since it could explain the differences in the molecules found between faeces
from Control and Delayed chickens using gas-chromatography and liquid-chromatography
coupled with mass spectrometry (Beauclercq et al. 2019). The *Eubacterium coprostanoligenes*
group is known to regulate plasma cholesterol concentration in laying hens (Li et al. 1996), and its
abundance in the gut is modified by probiotics in broilers (Zeng et al. 2021). In males, the effect
of the Delay on microbiota composition on Day 12 was different than in females, with a reduction
in the abundance of the Ruminococcaceae-UCG-014 genus, which is known to be a major butyrate
producer and highly sensitive to reactive oxygen species (Rychlik 2020; Wang et al. 2023). This
could be a consequence of the redox imbalance observed during these 12 days of the postnatal
period in males. Experiments have already shown that chickens' microbiota is influenced by early
stressful events (Ballou et al. 2016; Proszkowiec-Weglarz et al. 2022); however, the evidence of
the important differences in the consequences between females and males needs more
investigation.

The impact of the delayed placement on caecal microbiota composition was still present at slaughter age. In females, this effect was even more pronounced at this age than on Day 12, since on Day 34, the β-diversity was modified, and the abundances of the Firmicutes phylum and the *Faecalibacterium* and Ruminococcaceae-UCG-005 genera were decreased by the Delay effect. *Faecalibacterium* produces short-chain fatty acids that have anti-inflammatory effects during fibre fermentation in the gut (Martín et al. 2023; Rychlik 2020), and this reduction may have negative consequences on gut health, since *Faecalibacterium* has even been suggested as a biomarker of health in humans (Li et al. 2024). On the contrary, the abundance of the Bacteroidetes phylum and the *Alistipes* genus increased, which is in line with changes seen in quail submitted to chronic mild stress (Kraimi et al. 2022) and could suggest that the delay had a long-term effect that could unbalance chickens' gut microbiota.

At slaughter age (Day 34), the Delay effect still increased α-diversity in males, as seen on Day 12, and this effect significantly altered the abundances of the Ruminococcaceae-UCG-005 and *Erysipelatoclostridium*. These results are consistent with previous experiments that demonstrated that the Ruminococcaceae family originated from environmental sources rather than from the hens and would thus be more or less abundant depending on early differences in chick environment (Kubasova et al. 2019). However, we did not find any mention of the influence of early life events on the Erysipelotrichaeae family.

The differences observed between both sexes are in line with experiments demonstrating sex-
related differences in microbiota composition and the way these microbiota are modified (Cui et
al. 2021). The effects of early stress on metabolism and microbiota are known to vary more widely
according to sex (Park et al. 2021; Rincel et al. 2019), and we confirmed this phenomenon in
chickens in the present study and a previous experiment (Foury et al. 2020).
Effects of the Nutritional Supplement

One immediate major effect of the SUP was that Delayed chicks increased their water intake after placement. This intake may have been stimulated by the high dose of electrolytes in the SUP and the increase in plasma glucose concentration in the Delayed male and female chicks. This may be a very positive effect of the SUP, since this water intake limits chick dehydration and stimulates metabolic activity and gut transit. However, our data does not enable us to determine whether thirst was stressful for the chicks or even detrimental.

Providing the SUP did not significantly increase feed intake throughout the rearing period in the Delayed chickens and did not affect BW in Delayed females, but it positively impacted BW on Day 6 in Delayed males. This result in males is consistent with a previous study showing that using aqua agar to replace water for post-hatched chicks during transportation increased BW at 21 days of age (Incharoen et al. 2015). Between Days 20 and 34, the SUP significantly reduced FCR, possibly due to compensatory growth after the reduction in BW on Day 12, but this did not lead to a reduction in FCR over the whole rearing period. However, the mitigation was temporary, and the SUP even had negative effects on BW on Day 12 in Control males and resulted in reduced breast muscle yield in both Control and Delayed males on Day 34. These negative effects of the SUP in males cannot be related to differences in caecal microbiota composition, since the SUP increased the Tenericutes phylum only in females on Day 34, without consequences at the genus level. However, to know whether providing a SUP could be beneficial to some chicks, it could be interesting to check this feed intake individually in each chick and investigate the relationship with further growth and physiological parameters with new monitoring devices (Pampouille et al. 2021).

The SUP resulted in different effects on metabolism according to the chick's sex and age. In females, the SUP effect increased triglyceride concentration in both Control and Delayed female chicks on Day 1, since the supplement probably reduces the use of triglycerides as an energy source thanks to the provision of medium-chain fatty acids. The SUP increased glycaemia in Delayed and
not in Control female chicks, suggesting that, at this stage, the SUP's effect on energy metabolism was very strong; the glucose concentration was indeed higher in Delayed females with SUP than in Control females. This may be linked to the glucose supplementation, but also to gluconeogenesis favoured by protein and amino acid provision in the SUP. The SUP appears then to have a major effect on energy metabolism during the first day of life. Uric acid and TAS were also increased by the SUP in Delayed and not in Control female chicks, suggesting that it amplified their antioxidant status, probably in response to an increase in the need for antioxidants due to exacerbated nutrient oxidation. The SUP had no subsequent effect on blood parameters in females on Days 12 and 34.

In males, the SUP's only effect on Day 1 was an increase in glycaemia in both Control and Delayed chicks, confirming a major effect of the SUP on energy metabolism during the first day of life. An increase in uric acid induced by the SUP was found on Day 12 in males, while it occurred as early as Day 1 in females. It can be pointed out that this increase in uric acid concentration was in line with such an increase induced by probiotic supplementation in chickens (Jansseune et al. 2024).

Contrary to females, the SUP effect was still significant on Day 34 in males, with an increased blood triglyceride concentration in Control but not Delayed chickens and a reduction in the haptoglobin-like-protein activity in both Control and Delayed chickens, suggesting that males were more susceptible to long-term effects of the SUP provided before the placement. Hence, our results point out that nutritional supplementation's effects on chicks are different between the sexes in the short and longer terms.

Under our conditions, providing the SUP including the probiotic *Pediococcus acidilatici* had almost no consequences on the intestinal bacterial composition, since the only change was an increase in the Tenericutes phylum in females on Day 34. Overall, the SUP's effects on the microbiota's composition were weak, possibly because the quantity ingested was low (mean intake was 2.5 g) and was provided only once. When probiotics are provided for weeks, effects on microbiota composition can be seen up to 5 weeks of age (Redweik et al. 2020), and the effects of a single dose of probiotics have been demonstrated when an *in ovo* injection was used or when
chicks were inoculated at 2 h post-hatch, but the dose received was much greater in these studies
than in the present one (Arreguin-Nava et al. 2020; Baldwin et al. 2018).

Even though providing chicks with a nutritional supplement did not mitigate all the parameters affected by the experimental placement conditions, it appeared to provide an immediate real energy source, especially to delayed-placed chicks, and could influence their metabolism and performance differently depending on their early life experiences and sex.

Conclusion

Our results confirm previous studies on the long-lasting effects of a 24-h delay in placing chickens
in poultry houses on their metabolism and performance and highlight changes in oxidative stress
and gut microbiota composition. Our study reveals that i) the chick's metabolism immediately
adapts to delayed placement at the expense of growth; ii) long-lasting benefits could exist, such as
faster resorption of the yolk sac and a higher antioxidant status; and iii) the provision of a
nutritional supplement during the first day of life can mitigate some of the negative effects of
delayed placement in the short term only. However, the effects of the delay and the supplement
are highly sex-dependent and need further investigation that would consider this sex effect. These
results therefore suggest that chicks' hatching and transport conditions should be better controlled
and improved to respect the chicks' needs and avoid potential long-lasting risks of metabolic and
gut microbiota disorders and economic impacts. The sexing of the embryo could help improve
early-life conditions for female and male chicks. Alternatively, developing on-farm hatching
systems should be promoted to alleviate the negative consequences of the chicks' delayed
placement.

Author Contributions
CL and LG designed the study with the help of AC. CL and LG performed the experiments with the technical help of ECA, CP, and AC for tissue sampling and physiological parameter analyses. JD and CR performed the delay procedure. The DNA microbial extraction and PCR steps were carried out by KG and CD. OZ and LC were in charge of 16S rRNA gene sequencing and FK performed the statistical analysis of microbiota data. CL and LG wrote the manuscript. All the authors reviewed and approved the final manuscript.

**Declaration of Interest**

The authors declare that they have no competing interests/conflicts of interest.

**Acknowledgements**

We thank Frédéric Mercerand and Harold Rigoreau, who were in charge of animal rearing, and Nicolas Besne for the diet production (Experimental Poultry Facility, PEAT, INRAE, 2018, 37380 Nouzilly, France, doi:10.15454/1.5572326250887292E12). We are very grateful to Christèle Dupont and Alice Hondelatte for microbiota DNA extraction and to Sabine Crochet, Emmanuelle Schifano, and Paul Constantin for their help in data collection. The English was reviewed by ProofreadingServices.com

**Funding**

This research was supported by a grant from the Integrated Management of Animal Health Metaprogram of INRAE for the GISA-WHELP project (www.gisa.inra.fr/en).

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PROSZKOWIEC-WEGLARZ, M., K.B. MISKA, L.E. ELLESTAD, L.L. SCHREIER, S. KAHL, N. DARWISH, P. CAMPOS, and J. SHAO. 2022. "Delayed Access to Feed Early Post-hatch Affects the Development and Maturation of Gastrointestinal Tract Microbiota in Broiler Chickens." *BMC Microbiology* 22 (1). https://doi.org/10.1186/s12866-022-02619-6.

REDWEIK, G.A.J., K. DANIELS, A.J. SEVERIN, M. LYTE, and M. MELLATA. 2020. "Oral Treatments with Probiotics and Live Salmonella Vaccine Induce Unique Changes in Gut Neurochemicals and Microbiome in Chickens." *Frontiers in Microbiology* 10. https://doi.org/10.3389/fmicb.2019.03064.

RINCEL, M., P. AUBERT, J. CHEVALIER, P.A. GROHARD, L. BASSO, C. MONCHAUX DE OLIVEIRA, J.C. HELBLING, et al. 2019. "Multi-hit Early Life Adversity Affects Gut Microbiota, Brain and Behavior in a Sex-Dependent Manner." *Brain, Behavior, and Immunity* 80: 179-192. https://doi.org/10.1016/j.bbi.2019.03.006.

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Tables

<table><caption>**Table 1.** Diet composition and nutrient level of dietary raw materials</caption><thead><tr><th></th><th>Starting diet<br>Days 0–12</th><th>Growing diet<br>Days 12–20</th><th>Finishing diet<br>Days 20–34</th></tr></thead><tbody><tr><td colspan="4"><strong>Ingredient composition (g/kg, air-dry basis)</strong></td></tr><tr><td>Maize</td><td>24.80</td><td>292.58</td><td>415.00</td></tr><tr><td>Wheat</td><td>500.00</td><td>204.10</td><td>200.20</td></tr><tr><td>Soybean meal</td><td>247.00</td><td>312.20</td><td>273.00</td></tr><tr><td>Soybean oil</td><td>55.90</td><td>50.00</td><td>50.00</td></tr><tr><td>Wheat distillery by-product</td><td>30.00</td><td>—</td><td>—</td></tr><tr><td>Rapeseed meal</td><td>50.00</td><td>100.00</td><td>—</td></tr><tr><td>Extruded soybean</td><td>50.00</td><td>30.00</td><td>20.00</td></tr><tr><td>DL-methionine</td><td>3.25</td><td>1.50</td><td>1.50</td></tr><tr><td>Calcium carbonate</td><td>7.25</td><td>8.90</td><td>7.90</td></tr><tr><td>Dicalcium phosphate</td><td>19.00</td><td>18.50</td><td>16.70</td></tr><tr><td>Vitamin-mineral premix<fnref n="1" /></td><td>5.00</td><td>5.00</td><td>5.00</td></tr><tr><td>Salt</td><td>4.00</td><td>4.00</td><td>4.00</td></tr><tr><td>Lysine HCl</td><td>2.80</td><td>2.20</td><td>1.70</td></tr><tr><td>Threonine</td><td>1.00</td><td>5.00</td><td>5.00</td></tr><tr><td colspan="4"><strong>Calculated nutrient composition</strong></td></tr><tr><td>ME (kcal/kg)</td><td>3060</td><td>3100</td><td>3200</td></tr><tr><td>Crude protein (%)</td><td>22.00</td><td>21.00</td><td>19.00</td></tr><tr><td>Lysine (g/kg)</td><td>13.02</td><td>12.00</td><td>10.70</td></tr><tr><td>Methionine (g/kg)</td><td>6.40</td><td>6.00</td><td>5.80</td></tr><tr><td>Methionine+cystine (g/kg)</td><td>10.18</td><td>8.50</td><td>8.10</td></tr><tr><td>Tryptophan (g/kg)</td><td>2.95</td><td>2.50</td><td>2.00</td></tr><tr><td>Threonine (g/kg)</td><td>8.91</td><td>8.27</td><td>7.90</td></tr><tr><td>Calcium (g/kg)</td><td>10.46</td><td>10.50</td><td>9.50</td></tr><tr><td>Available phosphorus (g/kg)</td><td>4.48</td><td>4.20</td><td>4.00</td></tr></tbody></table>

<footnote n="1">¹Each kg of vitamin-mineral premix contained: 2 mg propyl gallate (E310); 329 mg ethoxyquin (E324); 3 000 000 IU vitamin A; 860 000 IU vitamin D₃; 20 000 IU vitamin E; 1000 mg vitamin K₃; 1000 mg vitamin B₁; 1 600 mg vitamin B₂; 20 000 mg vitamin B₃; 5 000 mg vitamin B₅; 1400 mg vitamin B₆; 5.2 mg vitamin B₁₂; 600 mg folic acid; 60 mg biotin; 110 000 mg choline; trace elements: 4000 mg Cu; 10 000 mg Fe; 18 000 mg Zn; 16 000 mg Mn; 400 mg I; 40.0 mg Se.</footnote>
<caption>**Table 2.** Body weight (BW, g) until Day 34 and breast muscle percentage at Day 34 (%) in females (m ± sd)</caption>

2

<table><thead><tr><th>Delay</th><th>Supply</th><th>BW-Day 1</th><th>BW-Day 6</th><th>BW-Day 12</th><th>BW-Day 20</th><th>BW-Day 27</th><th>BW-Day 34</th><th>Breast %-Day 34</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>54.1 ± 1.4 a</td><td>151.8 ± 7.4 a</td><td>357 ± 10 a</td><td>865 ± 20 a</td><td>1424 ± 35 a</td><td>2162 ± 62</td><td>17.75 ± 0.76</td></tr><tr><td>Supply</td><td>53.6 ± 2.3 a</td><td>150.3 ± 8.0 a</td><td>348 ± 23 a</td><td>851 ± 50 a</td><td>1431 ± 47 a</td><td>2148 ± 51</td><td>17.37 ± 1.17</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>40.6 ± 0.5 b</td><td>129.6 ± 5.3 b</td><td>314 ± 8 b</td><td>814 ± 21 b</td><td>1374 ± 38 b</td><td>2115 ± 59</td><td>16.94 ± 0.73</td></tr><tr><td>Supply</td><td>41.1 ± 0.7 b</td><td>137.2 ± 6.0 b</td><td>322 ± 20 b</td><td>821 ± 40 b</td><td>1384 ± 58 b</td><td>2121 ± 77</td><td>17.84 ± 1.53</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.011</td><td>0.017</td><td>0.161</td><td>0.751</td></tr><tr><td>Supply</td><td>0.926</td><td>0.191</td><td>0.964</td><td>0.820</td><td>0.661</td><td>0.873</td><td>0.630</td></tr><tr><td>Delay * Supply</td><td>0.405</td><td>0.068</td><td>0.218</td><td>0.479</td><td>0.950</td><td>0.711</td><td>0.245</td></tr></tbody></table>

3

4 The means with different letters across lines are significantly different (p < 0.05).
<caption>**Table 3.** Body weight (BW, g) until Day 34 and breast muscle percentage at Day 34 (%) in males (m ± sd)</caption>

6

<table><thead><tr><th>Delay</th><th>Supply</th><th>BW-Day 1</th><th>BW-Day 6</th><th>BW-Day 12</th><th>BW-Day 20</th><th>BW-Day 27</th><th>BW-Day 34</th><th>Breast %-Day 34</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>53.4 ± 1.1 a</td><td>152.5 ± 3.7 a</td><td>370 ± 10 a</td><td>913 ± 30 a</td><td>1533 ± 67 a</td><td>2355 ± 99 a</td><td>16.84 ± 1.17 a</td></tr><tr><td>Supply</td><td>52.1 ± 2.0 a</td><td>147.4 ± 5.7 a</td><td>352 ± 10 b</td><td>893 ± 33 a</td><td>1560 ± 44 a</td><td>2416 ± 85 a</td><td>16.40 ± 0.48 b</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>41.2 ± 0.8 b</td><td>128.2 ± 4.7 c</td><td>324 ± 11 c</td><td>830 ± 22 b</td><td>1454 ± 33 b</td><td>2248 ± 74 b</td><td>17.25 ± 1.02 a</td></tr><tr><td>Supply</td><td>41.8 ± 0.9 b</td><td>138.6 ± 4.2 b</td><td>335 ± 6 c</td><td>861 ± 35 b</td><td>1505 ± 65 b</td><td>2331 ± 92 b</td><td>15.03 ± 1.24 b</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.007</td><td>0.015</td><td>0.343</td></tr><tr><td>Supply</td><td>0.484</td><td>0.340</td><td>0.368</td><td>0.667</td><td>0.095</td><td>0.061</td><td>0.017</td></tr><tr><td>Delay *</td><td>0.086</td><td>0.002</td><td>0.001</td><td>0.055</td><td>0.581</td><td>0.757</td><td>0.090</td></tr></tbody></table>

7

8 The means with different letters across lines are significantly different (p < 0.05).
<table><caption>**Table 4.** Feed conversion ratio (m ± sd)</caption><thead><tr><th rowspan="2">Delay</th><th rowspan="2">Supply</th><th colspan="4">FCR</th></tr><tr><th>Day 0 - 12</th><th>Day 12 - 20</th><th>Day 20 - 34</th><th>Day 0 - 34</th></tr></thead><tbody><tr><td colspan="6"><strong>Control</strong></td></tr><tr><td>No</td><td></td><td>1.131 ± 0.015</td><td>1.262 ± 0.041 a</td><td>1.310 ± 0.043 a</td><td>1.269 ± 0.021</td></tr><tr><td>Supply</td><td></td><td>1.141 ± 0.015</td><td>1.268 ± 0.051 a</td><td>1.279 ± 0.036 b</td><td>1.255 ± 0.032</td></tr><tr><td colspan="6"><strong>Delayed</strong></td></tr><tr><td>No</td><td></td><td>1.131 ± 0.010</td><td>1.233 ± 0.010 b</td><td>1.337 ± 0.073 a</td><td>1.282 ± 0.047</td></tr><tr><td>Supply</td><td></td><td>1.129 ± 0.008</td><td>1.238 ± 0.018 b</td><td>1.273 ± 0.048 b</td><td>1.243 ± 0.033</td></tr><tr><td colspan="6"><strong>P-Value</strong></td></tr><tr><td>Delay</td><td></td><td>0.186</td><td>0.048</td><td>0.618</td><td>0.955</td></tr><tr><td>Supply</td><td></td><td>0.437</td><td>0.712</td><td>0.037</td><td>0.069</td></tr><tr><td>Delay*Supply</td><td></td><td>0.230</td><td>0.997</td><td>0.441</td><td>0.396</td></tr></tbody></table>

The means with different letters across lines are significantly different (p < 0.05).
<table><caption>**Table 5.** Physiological parameters in females on Day 1 (m ± sd)</caption><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/g)</th><th>Haptoglobin***<br>(mg/mL)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>2065 ± 66 b</td><td>1003 ± 384 c</td><td>56.29 ± 16.92 c</td><td>1.00 ± 0.22 c</td><td>1.36 ± 0.15</td><td>0.54 ± 0.08 b</td><td>1.03 ± 0.50</td></tr><tr><td>Supply</td><td>2044 ± 120 b</td><td>1396 ± 392 a</td><td>60.75 ± 14.62 c</td><td>0.99 ± 0.21 c</td><td>1.39 ± 0.11</td><td>0.64 ± 0.13 b</td><td>1.13 ± 0.38</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>1931 ± 117 c</td><td>435 ± 70 d</td><td>81.66 ± 32.89 b</td><td>1.29 ± 0.37 b</td><td>1.48 ± 0.09</td><td>0.87 ± 0.20 a</td><td>1.33 ± 0.34</td></tr><tr><td>Supply</td><td>2292 ± 144 a</td><td>1120 ± 383 b</td><td>134.54 ± 39.79 a</td><td>1.95 ± 0.44 a</td><td>1.39 ± 0.24</td><td>1.05 ± 0.44 a</td><td>1.47 ± 0.37</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.240</td><td>0.005</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.440</td><td>0.002</td><td>0.064</td></tr><tr><td>Supply</td><td>0.002</td><td>0.001</td><td>0.022</td><td>0.024</td><td>0.618</td><td>0.196</td><td>0.455</td></tr><tr><td>Delay * Supply</td><td>0.001</td><td>0.140</td><td>0.048</td><td>0.021</td><td>0.379</td><td>0.679</td><td>0.917</td></tr></tbody></table>

15

* TAS: total antioxidant status; ** TBARS: thiobarbituric acid reactive species; *** Haptoglobin: haptoglobin-like protein.

17 The means with different letters across lines are significantly different (p < 0.05).

18
19

<caption>**Table 6.** Physiological parameters in males on Day 1 (m ± sd)</caption>

20

<table><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/g)</th><th>Haptoglobin***<br>(mg/mL)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>1859 ± 200 b</td><td>881 ± 420 a</td><td>42.83 ± 4.50 b</td><td>0.81 ± 0.19 b</td><td>1.34 ± 0.25</td><td>0.79 ± 0.26 b</td><td>0.91 ± 0.61</td></tr><tr><td>Supply</td><td>2102 ± 145 a</td><td>1287 ± 454 a</td><td>63.20 ± 15.42 b</td><td>1.07 ± 0.14 b</td><td>1.36 ± 0.22</td><td>0.78 ± 0.43 b</td><td>1.02 ± 0.33</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>1876 ± 120 b</td><td>438 ± 69 b</td><td>106.35 ± 44.00 a</td><td>1.77 ± 0.60 a</td><td>1.45 ± 0.17</td><td>1.26 ± 0.61 a</td><td>1.05 ± 0.54</td></tr><tr><td>Supply</td><td>2169 ± 193 a</td><td>658 ± 287 b</td><td>98.79 ± 55.78 a</td><td>1.72 ± 0.60 a</td><td>1.54 ± 0.11</td><td>1.27 ± 0.68 a</td><td>1.30 ± 0.33</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.546</td><td>0.003</td><td>0.004</td><td>0.0002</td><td>0.136</td><td>0.037</td><td>0.311</td></tr><tr><td>Supply</td><td>0.001</td><td>0.059</td><td>0.670</td><td>0.570</td><td>0.604</td><td>0.777</td><td>0.390</td></tr><tr><td>Delay * Supply</td><td>0.715</td><td>0.859</td><td>0.357</td><td>0.412</td><td>0.740</td><td>0.918</td><td>0.740</td></tr></tbody></table>

21

22

23

The means with different letters across lines are significantly different (p < 0.05).

<footnote>* TAS: total antioxidant status; ** TBARS: thiobarbituric acid reactive species; *** Haptoglobin: haptoglobin-like protein.</footnote>
<table><caption>**Table 7.** Physiological parameters in females on Day 34 (m ± sd)</caption><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/mL)</th><th>Haptoglobin***<br>(mg/mL)</th><th>Creatine kinase<br>(IU/L)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>2145 ± 109</td><td>677 ± 193</td><td>50.61 ± 10.80 b</td><td>0.91 ± 0.07 b</td><td>1.51 ± 0.40</td><td>1.45 ± 0.08</td><td>0.97 ± 0.20</td><td>16372 ± 7181 a</td></tr><tr><td>Supply</td><td>2103 ± 82</td><td>718 ± 415</td><td>47.19 ± 6.39 b</td><td>0.88 ± 0.10 b</td><td>1.63 ± 0.43</td><td>1.22 ± 0.26</td><td>0.86 ± 0.20</td><td>15313 ± 6184 a</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>2125 ± 55</td><td>885 ± 311</td><td>55.42 ± 10.99 a</td><td>1.02 ± 0.10 a</td><td>1.57 ± 0.39</td><td>1.38 ± 0.30</td><td>0.94 ± 0.20</td><td>12468 ± 5381 a</td></tr><tr><td>Supply</td><td>2130 ± 99</td><td>512 ± 104</td><td>63.63 ± 18.16 a</td><td>1.03 ± 0.17 a</td><td>1.18 ± 0.61</td><td>1.37 ± 0.33</td><td>0.79 ± 0.39</td><td>17503 ± 9712 a</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.922</td><td>0.990</td><td>0.048</td><td>0.012</td><td>0.127</td><td>0.755</td><td>0.644</td><td>0.976</td></tr><tr><td>Supply</td><td>0.611</td><td>0.165</td><td>0.639</td><td>0.852</td><td>0.498</td><td>0.300</td><td>0.228</td><td>0.991</td></tr><tr><td>Delay*Supply</td><td>0.528</td><td>0.087</td><td>0.262</td><td>0.650</td><td>0.294</td><td>0.320</td><td>0.890</td><td>0.016</td></tr></tbody></table>

26

* TAS: total antioxidant status; ** TBARS: thiobarbituric acid reactive species; *** Haptoglobin: haptoglobin-like protein.

28 The means with different letters across lines are significantly different (p < 0.05).
29

<caption>**Table 8.** Physiological parameters in males on Day 34 (m ± sd)</caption>

30

<table><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/mL)</th><th>Haptoglobin***<br>(mg/mL)</th><th>Creatine kinase<br>(UI/L)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>2078 ± 99</td><td>471 ± 126 b</td><td>48.64 ± 10.26</td><td>0.90 ± 0.11</td><td>1.62 ± 0.52</td><td>1.38 ± 0.35</td><td>0.95 ± 0.30 a</td><td>17870 ± 7274</td></tr><tr><td>Supply</td><td>2188 ± 121</td><td>704 ± 133 a</td><td>49.60 ± 5.89</td><td>0.93 ± 0.05</td><td>1.56 ± 0.34</td><td>1.19 ± 0.22</td><td>0.72 ± 0.18 b</td><td>14660 ± 7686</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>2172 ± 89</td><td>646 ± 83 ab</td><td>51.10 ± 8.90</td><td>0.93 ± 0.09</td><td>1.48 ± 0.51</td><td>1.47 ± 0.47</td><td>0.88 ± 0.32 a</td><td>10734 ± 3455</td></tr><tr><td>Supply</td><td>2195 ± 71</td><td>613 ± 145 ab</td><td>49.73 ± 3.62</td><td>0.89 ± 0.06</td><td>1.74 ± 0.52</td><td>1.42 ± 0.22</td><td>0.81 ± 0.17 b</td><td>19949 ± 8472</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.214</td><td>0.414</td><td>0.682</td><td>0.919</td><td>0.726</td><td>0.235</td><td>0.668</td><td>0.094</td></tr><tr><td>Supply</td><td>0.109</td><td>0.063</td><td>0.948</td><td>0.902</td><td>0.718</td><td>0.387</td><td>0.041</td><td>0.878</td></tr><tr><td>Delay * Supply</td><td>0.287</td><td>0.016</td><td>0.712</td><td>0.337</td><td>0.368</td><td>0.620</td><td>0.534</td><td>0.413</td></tr></tbody></table>

31

32

33

The means with different letters across lines are significantly different (p < 0.05).

<footnote>* TAS: total antioxidant status; ** TBARS: thiobarbituric acid reactive species; *** Haptoglobin: haptoglobin-like protein.</footnote>
<table><caption>**Table 9.** Composition of gut microbiota at the phylum level in females on Day 34</caption><thead><tr><th rowspan="2">Delay</th><th rowspan="2">Supply</th><th colspan="2">Firmicutes</th><th colspan="2">Bacteroidetes</th><th colspan="2">Tenericutes</th></tr><tr><th>(%)</th><th>(%)</th><th>(%)</th><th>(%)</th><th>(%)</th><th>(%)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>89.15</td><td>3.24 a</td><td>9.85</td><td>3.23 b</td><td>0.75</td><td>± 0.16 b</td></tr><tr><td>Supply</td><td>90.42</td><td>± 3.87 a</td><td>8.56</td><td>± 3.99 b</td><td>0.87</td><td>± 0.18 a</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>86.87</td><td>± 4.22 b</td><td>12.25</td><td>± 4.23 a</td><td>0.75</td><td>± 0.33 b</td></tr><tr><td>Supply</td><td>83.55</td><td>± 7.43 b</td><td>15.05</td><td>± 7.20 a</td><td>1.23</td><td>± 0.51 a</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.035</td><td></td><td>0.038</td><td></td><td>0.198</td><td></td></tr><tr><td>Supply</td><td>0.619</td><td></td><td>0.711</td><td></td><td>0.035</td><td></td></tr><tr><td>Delay * Supply</td><td>0.271</td><td></td><td>0.318</td><td></td><td>0.199</td><td></td></tr></tbody></table>

36

37 The means with different letters across lines are significantly different (p < 0.05).
**List of Figure Captions**

**Figure 1.** Water intake during the first (A) and second (B) days in place related to body weight when the chicks were placed (mL/g). Mean ± sd. The bars with different letters are significantly different (*p* < 0.05).

**Figure 2.** Chao1 indexes of the gut microbiota in males on Day 12 (A) and Day 34 (B). Mean ± sd. The bars with different letters are significantly different (*p* < 0.05).

**Figure 3.** Relative abundance of the *Faecalibacterium*, *Ruminococcaceae-UCG-005*, and *Alistipes* genera in females on Day 34. Mean ± sd. The bars with different letters are significantly different (*p* < 0.05).

**Figure 4.** Relative abundance of the *Ruminococcaceae-UCG-005* and *Erysipelatoclostridium* genera in males on Day 34. Mean ± sd. The bars with different letters are significantly different (*p* < 0.05).
Supplementary Data 1

Schedule of the Experiment

<table><thead><tr><th rowspan="2">Age</th><th rowspan="2">Actions</th><th colspan="2">Control</th><th colspan="2">Delay</th></tr><tr><th>No supply</th><th>Supply</th><th>No supply</th><th>Supply</th></tr></thead><tbody><tr><td>D0 (Time 0)</td><td>Opening of the incubator</td><td colspan="4">384 chicks</td></tr><tr><td>D0 (Time 0 to 2H)</td><td>Chick identification, sexing, weighing<br>Vaccination against Infectious Bronchitis<br>Blood sampling for infectious bronchitis titers</td><td colspan="4">23 chicks</td></tr><tr><td>D0 (Time 6H)</td><td>Distribution of 192 chicks into 12 pens of 16 chicks per pen<br>Weighing of feed and water in the pens<br>Distribution in 4 transport boxes with 48 chicks per box</td><td>6 pens<br>96 chicks</td><td>6 pens<br>96 chicks</td><td>2 boxes<br>96 chicks</td><td>2 boxes<br>96 chicks</td></tr><tr><td>D0 to D1<br>(Time 6H to 30H)</td><td>Box movement and changes in temperature<br>i.e early negative experience<br>Provision of a supply in the feeder in each pen<br>Provision of a supply in half chicks</td><td colspan="4">All boxes<br>192 chicks</td></tr><tr><td>D1 - Time 30H</td><td>Weighing of all chicks<br>Blood sampling and sacrifice<br>Blood sampling and sacrifice</td><td colspan="4">6 pens<br>1 box</td></tr><tr><td colspan="2"></td><td colspan="4">All chicks (n = 384)</td></tr><tr><td colspan="2"></td><td>1 female and 1 male per pen<br>12 chicks</td><td>12 chicks</td><td colspan="2">3 females and 3 males per box<br>12 chicks</td></tr><tr><td>D2</td><td>Weighing of feed and water in the pens</td><td colspan="4"></td></tr><tr><td>D3</td><td>Weighing of feed and water in the pens</td><td colspan="4"></td></tr><tr><td>D6</td><td>Weighing of all chicks</td><td colspan="4"></td></tr><tr><td>D12</td><td>Weighing of all chicks and feed<br>Feed change<br>Blood and organ sampling</td><td colspan="4">1 female and 1 male per pen<br>12 chicks</td></tr><tr><td>D20</td><td>Weighing of all chicks and feed<br>Feed change</td><td colspan="4">3 chicks per pen<br>18 chicks</td></tr><tr><td>D34</td><td>Weighing of all chicks and feed<br>Blood and organ sampling<br>Half males and half females</td><td colspan="4">18 chicks</td></tr></tbody></table>
Supplementary Data 1

Schedule of the Experiment

<table><thead><tr><th rowspan="2">Age</th><th rowspan="2">Actions</th><th colspan="2">Control</th><th colspan="2">Delay</th></tr><tr><th>No supply</th><th>Supply</th><th>No supply</th><th>Supply</th></tr></thead><tbody><tr><td>D0 (Time 0)</td><td>Opening of the incubator</td><td colspan="4">384 chicks</td></tr><tr><td>D0 (Time 0 to 2H)</td><td>Chick identification, sexing, weighing<br>Vaccination against Infectious Bronchitis<br>Blood sampling for infectious bronchitis titers</td><td colspan="4">23 chicks</td></tr><tr><td>D0 (Time 6H)</td><td>Distribution of 192 chicks into 12 pens of 16 chicks per pen<br>Weighing of feed and water in the pens<br>Distribution in 4 transport boxes with 48 chicks per box</td><td>6 pens<br>96 chicks</td><td>6 pens<br>96 chicks</td><td colspan="2">2 boxes<br>96 chicks</td></tr><tr><td>D0 to D1<br>(Time 6H to 30H)</td><td>Box movement and changes in temperature<br>i.e early negative experience<br>Provision of a supply in the feeder in each pen<br>Provision of a supply in half chicks</td><td colspan="2">6 pens</td><td colspan="2">All boxes<br>192 chicks</td></tr><tr><td>D1 - Time 30H</td><td>Weighing of all chicks<br>Blood sampling and sacrifice</td><td colspan="4">All chicks (n = 384)</td></tr><tr><td>D2</td><td>Weighing of feed and water in the pens</td><td colspan="2">1 female and 1 male per pen</td><td colspan="2">3 females and 3 males per box</td></tr><tr><td>D3</td><td>Weighing of feed and water in the pens</td><td colspan="2">12 chicks</td><td colspan="2">12 chicks</td></tr><tr><td>D6</td><td>Weighing of all chicks</td><td colspan="2">12 chicks</td><td colspan="2">12 chicks</td></tr><tr><td>D12</td><td>Weighing of all chicks and feed<br>Feed change<br>Blood and organ sampling</td><td colspan="2">12 chicks</td><td colspan="2">12 chicks</td></tr><tr><td>D20</td><td>Weighing of all chicks and feed<br>Feed change</td><td colspan="2">12 chicks</td><td colspan="2">12 chicks</td></tr><tr><td>D34</td><td>Weighing of all chicks and feed<br>Blood and organ sampling<br>Half males and half females</td><td colspan="2">18 chicks</td><td colspan="2">3 chicks per pen</td></tr></tbody></table>
## Supplementary Data 2

### Supplementary Data 2A on physiological parameters in females on Day 12 (m ± sd)

<table><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/mL)</th><th>Haptoglobin***<br>(mg/mL)</th><th>Creatine kinase<br>(IU/L)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>2222 ± 127</td><td>403 ± 48</td><td>59.38 ± 8.50</td><td>1.03 ± 0.09</td><td>1.51 ± 0.40</td><td>0.74 ± 0.11</td><td>0.55 ± 0.24</td><td>763 ± 471</td></tr><tr><td>Supply</td><td>2221 ± 107</td><td>464 ± 86</td><td>64.69 ± 14.48</td><td>1.00 ± 0.16</td><td>1.62 ± 0.43</td><td>0.61 ± 0.07</td><td>0.59 ± 0.18</td><td>1626 ± 1181</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>2219 ± 42</td><td>505 ± 73</td><td>63.05 ± 5.92</td><td>1.01 ± 0.05</td><td>1.57 ± 0.39</td><td>0.84 ± 0.17</td><td>0.53 ± 0.25</td><td>618 ± 258</td></tr><tr><td>Supply</td><td>2189 ± 132</td><td>423 ± 159</td><td>70.08 ± 15.41</td><td>1.07 ± 0.23</td><td>1.18 ± 0.61</td><td>0.76 ± 0.18</td><td>0.51 ± 0.30</td><td>968 ± 513</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.695</td><td>0.471</td><td>0.357</td><td>0.693</td><td>0.335</td><td>0.097</td><td>0.662</td><td>0.270</td></tr><tr><td>Supply</td><td>0.724</td><td>0.806</td><td>0.214</td><td>0.917</td><td>0.478</td><td>0.104</td><td>0.934</td><td>0.056</td></tr><tr><td>Delay * Supply</td><td>0.749</td><td>0.095</td><td>0.159</td><td>0.499</td><td>0.207</td><td>0.454</td><td>0.761</td><td>0.927</td></tr></tbody></table>

* TAS: Total antioxidant status; ** TBARS: Thiobarbituric acid reactive species; *** Haptoglobin: Haptoglobin-like protein.

### Supplementary Data 2B on physiological parameters in males on Day 12 (m ± sd)

<table><thead><tr><th>Delay</th><th>Supply</th><th>Glucose<br>(mg/L)</th><th>Triglycerides<br>(mg/L)</th><th>Uric acid<br>(mg/L)</th><th>TAS*<br>(mmol/L)</th><th>TBARS**<br>(nm MDA/mL)</th><th>TBARS in liver<br>(nm MDA/mL)</th><th>Haptoglobin***<br>(mg/mL)</th><th>Creatine kinase<br>(IU/L)</th></tr></thead><tbody><tr><td rowspan="2">Control</td><td>No</td><td>2150 ± 97</td><td>511 ± 187</td><td>64.01 ± 8.90 b</td><td>0.99 ± 0.10</td><td>1.52 ± 0.42</td><td>0.64 ± 0.13</td><td>0.48 ± 0.25</td><td>690 ± 290</td></tr><tr><td>Supply</td><td>2308 ± 167</td><td>463 ± 64</td><td>74.74 ± 7.09 a</td><td>1.09 ± 0.09</td><td>1.25 ± 0.49</td><td>0.77 ± 0.21</td><td>0.38 ± 0.19</td><td>730 ± 277</td></tr><tr><td rowspan="2">Delayed</td><td>No</td><td>2259 ± 56</td><td>473 ± 39</td><td>69.62 ± 6.61 b</td><td>1.06 ± 0.13</td><td>1.53 ± 0.37</td><td>0.66 ± 0.08</td><td>0.50 ± 0.32</td><td>885 ± 654</td></tr><tr><td>Supply</td><td>2243 ± 163</td><td>436 ± 102</td><td>76.52 ± 9.28 a</td><td>1.14 ± 0.12</td><td>1.53 ± 0.41</td><td>0.88 ± 0.33</td><td>0.38 ± 0.28</td><td>1032 ± 403</td></tr><tr><td rowspan="3">P-Value</td><td>Delay</td><td>0.677</td><td>0.497</td><td>0.274</td><td>0.196</td><td>0.412</td><td>0.591</td><td>0.967</td><td>0.230</td></tr><tr><td>Supply</td><td>0.191</td><td>0.365</td><td>0.014</td><td>0.064</td><td>0.464</td><td>0.125</td><td>0.324</td><td>0.462</td></tr><tr><td>Delay * Supply</td><td>0.118</td><td>0.911</td><td>0.567</td><td>0.759</td><td>0.442</td><td>0.881</td><td>0.899</td><td>0.652</td></tr></tbody></table>

* TAS: Total antioxidant status; ** TBARS: Thiobarbituric acid reactive species; *** Haptoglobin: Haptoglobin-like protein. The means with different letters are significantly different (P < 0.05).
## Supplementary Data 3

### Supplementary Data 3A on α-diversity of the caecal microbiota in females on Day 12 and Day 34

<table><thead><tr><th rowspan="2"></th><th colspan="4">ANOVA</th></tr><tr><th>mean ± sd</th><th>Delay</th><th>SUP</th><th>Interaction</th></tr></thead><tbody><tr><td colspan="5"><strong>Day 12</strong></td></tr><tr><td>Chao1 index</td><td>265 ± 54</td><td>0.513</td><td>0.790</td><td>0.063</td></tr><tr><td>Shannon index</td><td>3.99 ± 0.26</td><td>0.590</td><td>0.457</td><td>0.472</td></tr><tr><td>InvSimpson index</td><td>26.76 ± 10.33</td><td>0.740</td><td>0.492</td><td>0.995</td></tr><tr><td colspan="5"><strong>Day 34</strong></td></tr><tr><td>Chao1 index</td><td>398 ± 32</td><td>0.170</td><td>0.581</td><td>0.756</td></tr><tr><td>Shannon index</td><td>4.20 ± 0.20</td><td>0.156</td><td>0.389</td><td>0.398</td></tr><tr><td>InvSimpson index</td><td>22.01 ± 6.81</td><td>0.072</td><td>0.532</td><td>0.642</td></tr></tbody></table>

### Supplementary Data 3B on α-diversity of the caecal microbiota in males on Day 12 and Day 34

The Chao1 indexes in males are provided in the results paragraph.

<table><thead><tr><th rowspan="2"></th><th colspan="2">Control</th><th colspan="2">Delay</th><th colspan="3">ANOVA</th></tr><tr><th>No SUP</th><th>SUP</th><th>No SUP</th><th>SUP</th><th>Delay</th><th>SUP</th><th>Interaction</th></tr></thead><tbody><tr><td colspan="8"><strong>Day 12</strong></td></tr><tr><td>Shannon index</td><td>3.8 ± 0.3</td><td>3.9 ± 0.4</td><td>4.2 ± 0.2</td><td>4.0 ± 0.4</td><td>0.108</td><td>0.795</td><td>0.340</td></tr><tr><td>InvSimpson index</td><td>21.0 ± 12.4</td><td>23.5 ± 11.0</td><td>27.7 ± 11.6</td><td>24.6 ± 11.4</td><td>0.443</td><td>0.919</td><td>0.542</td></tr><tr><td colspan="8"><strong>Day 34</strong></td></tr><tr><td>Shannon index</td><td>40.7 ± 0.2 a</td><td>4.2 ± 0.1 a</td><td>4.3 ± 0.1 a</td><td>4.2 ± 0.2 a</td><td>0.175</td><td>0.920</td><td>0.039</td></tr><tr><td>InvSimpson index</td><td>17.1 ± 6.6</td><td>21.9 ± 4.2</td><td>18.9 ± 3.1</td><td>18.5 ± 5.6</td><td>0.712</td><td>0.308</td><td>0.219</td></tr></tbody></table>

The means with different letters are significantly different (P < 0.05).
<figure><img src="image_6.png" /><figcaption>Supplementary Data 3C on β-diversity of the caecal microbiota in females and males on Day 12 and Day 34</figcaption></figure>
Supplementary Data 3D on the effects of the delay and of the SUP on the composition of the caecal microbiota in females and males on Day 12 and Day 34

Composition of caecal microbiota in females on Day 12

<table><thead><tr><th rowspan="2">Females on Day 12</th><th>Control without SUP</th><th>Control with SUP</th><th>Delayed without SUP</th><th>Delayed with SUP</th><th colspan="3">ANOVA</th></tr><tr><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>Delay</th><th>Supply</th><th>Interaction</th></tr></thead><tbody><tr><td><strong>Phylum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Firmicutes</td><td>97.426 ± 1.093</td><td>96.404 ± 3.999</td><td>98.572 ± 1.308</td><td>95.246 ± 5.743</td><td>0.997</td><td>0.155</td><td>0.443</td></tr><tr><td>Bacteroidetes</td><td>0.001 ± 0.001</td><td>0.025 ± 0.045</td><td>0.005 ± 0.010</td><td>0.006 ± 0.007</td><td>0.435</td><td>0.181</td><td>0.232</td></tr><tr><td>Tenericutes</td><td>1.112 ± 1.092</td><td>2.890 ± 4.049</td><td>0.516 ± 0.977</td><td>3.954 ± 5.449</td><td>0.871</td><td>0.081</td><td>0.565</td></tr><tr><td><strong>Class</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacilli</td><td>0.022 ± 0.022</td><td>0.019 ± 0.024</td><td>0.021 ± 0.007</td><td>0.024 ± 0.023</td><td>0.831</td><td>0.997</td><td>0.754</td></tr><tr><td>Bacteroidia</td><td>0.001 ± 0.001</td><td>0.025 ± 0.045</td><td>0.005 ± 0.010</td><td>0.006 ± 0.007</td><td>0.435</td><td>0.181</td><td>0.232</td></tr><tr><td>Clostridia</td><td>97.426 ± 1.093</td><td>96.404 ± 3.999</td><td>98.572 ± 1.308</td><td>95.246 ± 5.743</td><td>0.997</td><td>0.155</td><td>0.443</td></tr><tr><td>Erysipelotrichia</td><td>1.439 ± 1.243</td><td>0.661 ± 0.372</td><td>0.886 ± 1.191</td><td>0.770 ± 0.590</td><td>0.565</td><td>0.252</td><td>0.393</td></tr><tr><td>Gammaproteobacteria</td><td>1.385 ± 1.241</td><td>0.408 ± 0.195</td><td>0.846 ± 1.181</td><td>0.717 ± 0.593</td><td>0.760</td><td>0.153</td><td>0.268</td></tr><tr><td>Mollicutes</td><td>1.112 ± 1.092</td><td>2.890 ± 4.049</td><td>0.516 ± 0.977</td><td>3.954 ± 5.449</td><td>0.871</td><td>0.081</td><td>0.565</td></tr><tr><td><strong>Order</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillales</td><td>0.500 ± 0.567</td><td>1.563 ± 1.953</td><td>0.433 ± 0.570</td><td>0.941 ± 1.593</td><td>0.530</td><td>0.161</td><td>0.613</td></tr><tr><td>Bacteroidales</td><td>0.001 ± 0.001</td><td>0.025 ± 0.045</td><td>0.005 ± 0.010</td><td>0.006 ± 0.007</td><td>0.435</td><td>0.181</td><td>0.232</td></tr><tr><td>Clostridiales</td><td>76.981 ± 5.316</td><td>77.275 ± 8.078</td><td>80.012 ± 5.050</td><td>75.126 ± 3.885</td><td>0.854</td><td>0.343</td><td>0.286</td></tr><tr><td>Enterobacteriales</td><td>1.385 ± 1.241</td><td>0.408 ± 0.195</td><td>0.846 ± 1.181</td><td>0.717 ± 0.593</td><td>0.760</td><td>0.153</td><td>0.268</td></tr><tr><td>Erysipelotrichales</td><td>6.203 ± 4.177</td><td>3.439 ± 0.911</td><td>4.959 ± 3.246</td><td>5.430 ± 1.967</td><td>0.752</td><td>0.338</td><td>0.181</td></tr><tr><td>Lactobacillales</td><td>13.742 ± 4.936</td><td>14.127 ± 9.478</td><td>13.168 ± 3.021</td><td>13.750 ± 3.687</td><td>0.844</td><td>0.842</td><td>0.967</td></tr><tr><td>Mollicutes RF39</td><td>1.112 ± 1.092</td><td>2.890 ± 4.049</td><td>0.516 ± 0.977</td><td>3.954 ± 5.449</td><td>0.871</td><td>0.081</td><td>0.565</td></tr><tr><td>Sphingomonadales</td><td>0.054 ± 0.019</td><td>0.252 ± 0.464</td><td>0.040 ± 0.023</td><td>0.053 ± 0.024</td><td>0.276</td><td>0.280</td><td>0.339</td></tr><tr><td><strong>Family</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillaceae</td><td>0.500 ± 0.567</td><td>1.563 ± 1.953</td><td>0.433 ± 0.570</td><td>0.941 ± 1.593</td><td>0.530</td><td>0.161</td><td>0.613</td></tr><tr><td>Christensellaceae</td><td>0.037 ± 0.086</td><td>0.061 ± 0.065</td><td>0.098 ± 0.141</td><td>0.089 ± 0.154</td><td>0.358</td><td>0.882</td><td>0.733</td></tr><tr><td>Clostridiaceae 1</td><td>0.089 ± 0.076</td><td>0.154 ± 0.120</td><td>0.196 ± 0.084</td><td>0.096 ± 0.126</td><td>0.571</td><td>0.677</td><td>0.065</td></tr><tr><td>Clostridiales vadinBB60 group</td><td>2.325 ± 3.504</td><td>1.880 ± 1.280</td><td>2.997 ± 1.711</td><td>1.461 ± 1.076</td><td>0.885</td><td>0.266</td><td>0.536</td></tr><tr><td>Defluvitaleaceae</td><td>0.061 ± 0.060 a</td><td>0.182 ± 0.096 a</td><td>0.179 ± 0.113 a</td><td>0.116 ± 0.054 a</td><td>0.459</td><td>0.410</td><td>0.015</td></tr><tr><td>Eggerthellaceae</td><td>0.022 ± 0.022</td><td>0.019 ± 0.024</td><td>0.021 ± 0.007</td><td>0.024 ± 0.023</td><td>0.831</td><td>0.997</td><td>0.754</td></tr><tr><td>Enterobacteriaceae</td><td>1.385 ± 1.241</td><td>0.408 ± 0.195</td><td>0.846 ± 1.181</td><td>0.717 ± 0.593</td><td>0.760</td><td>0.153</td><td>0.268</td></tr><tr><td>Enterococcaceae</td><td>0.023 ± 0.032</td><td>0.005 ± 0.011</td><td>0.007 ± 0.010</td><td>0.014 ± 0.015</td><td>0.645</td><td>0.490</td><td>0.125</td></tr><tr><td>Erysipelotrichaceae</td><td>6.203 ± 4.177</td><td>3.439 ± 0.911</td><td>4.959 ± 3.246</td><td>5.430 ± 1.967</td><td>0.752</td><td>0.338</td><td>0.181</td></tr><tr><td>Family XIII</td><td>0.011 ± 0.020</td><td>0.029 ± 0.035</td><td>0.044 ± 0.056</td><td>0.017 ± 0.030</td><td>0.515</td><td>0.778</td><td>0.158</td></tr><tr><td>Lachnospiraceae</td><td>33.595 ± 7.022</td><td>31.683 ± 6.594</td><td>30.405 ± 11.937</td><td>30.107 ± 5.039</td><td>0.478</td><td>0.741</td><td>0.809</td></tr><tr><td>Lactobacillaceae</td><td>13.719 ± 4.923</td><td>14.121 ± 9.487</td><td>13.161 ± 3.017</td><td>13.735 ± 3.685</td><td>0.845</td><td>0.840</td><td>0.972</td></tr><tr><td>Multi-affiliation</td><td>0.006 ± 0.007</td><td>0.003 ± 0.006</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.051</td><td>0.446</td><td>0.446</td></tr><tr><td>Peptstreptococcaceae</td><td>0.060 ± 0.124</td><td>0.088 ± 0.116</td><td>0.033 ± 0.036</td><td>0.011 ± 0.013</td><td>0.160</td><td>0.924</td><td>0.488</td></tr><tr><td>Rikenellaceae</td><td>0.001 ± 0.001</td><td>0.025 ± 0.045</td><td>0.005 ± 0.010</td><td>0.006 ± 0.007</td><td>0.435</td><td>0.181</td><td>0.232</td></tr><tr><td>Ruminococcaceae</td><td>40.799 ± 11.742</td><td>43.192 ± 4.708</td><td>46.033 ± 12.001</td><td>43.230 ± 3.680</td><td>0.477</td><td>0.956</td><td>0.483</td></tr><tr><td>Sphingomonadaceae</td><td>0.054 ± 0.019</td><td>0.252 ± 0.464</td><td>0.040 ± 0.023</td><td>0.053 ± 0.024</td><td>0.276</td><td>0.280</td><td>0.339</td></tr><tr><td>Unknown family</td><td>1.112 ± 1.092</td><td>2.890 ± 4.049</td><td>0.516 ± 0.977</td><td>3.954 ± 5.449</td><td>0.871</td><td>0.081</td><td>0.565</td></tr><tr><td><strong>Genus</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Unknown genus</td><td>18.268 ± 3.223</td><td>17.600 ± 5.849</td><td>17.853 ± 3.896</td><td>16.290 ± 3.681</td><td>0.627</td><td>0.531</td><td>0.801</td></tr><tr><td>G-Faecalibacterium</td><td>5.916 ± 9.354</td><td>9.797 ± 7.162</td><td>13.856 ± 8.888</td><td>8.992 ± 6.910</td><td>0.296</td><td>0.884</td><td>0.204</td></tr><tr><td>Ruminococcaceae UCG-014</td><td>9.059 ± 7.460</td><td>7.512 ± 5.612</td><td>7.310 ± 7.134</td><td>10.822 ± 7.923</td><td>0.790</td><td>0.738</td><td>0.392</td></tr><tr><td>Lactobacillus</td><td>13.719 ± 4.923</td><td>14.121 ± 9.487</td><td>13.161 ± 3.017</td><td>13.735 ± 3.685</td><td>0.845</td><td>0.840</td><td>0.972</td></tr><tr><td>Multi-affiliation</td><td>7.956 ± 9.392</td><td>6.147 ± 3.994</td><td>6.237 ± 4.772</td><td>7.578 ± 5.217</td><td>0.955</td><td>0.927</td><td>0.541</td></tr><tr><td>Alistipes</td><td>0.001 ± 0.001</td><td>0.025 ± 0.045</td><td>0.005 ± 0.010</td><td>0.006 ± 0.007</td><td>0.435</td><td>0.181</td><td>0.232</td></tr><tr><td>[Ruminococcus] torques group</td><td>4.874 ± 2.577</td><td>4.723 ± 4.172</td><td>4.057 ± 2.236</td><td>4.173 ± 1.737</td><td>0.561</td><td>0.988</td><td>0.909</td></tr><tr><td>Erysipelatodiscidium</td><td>4.557 ± 3.792</td><td>2.500 ± 1.397</td><td>3.859 ± 3.641</td><td>3.504 ± 1.458</td><td>0.895</td><td>0.307</td><td>0.468</td></tr><tr><td>Ruminococcaceae UCG-005</td><td>0.006 ± 0.007</td><td>0.084 ± 0.173</td><td>0.152 ± 0.265</td><td>0.041 ± 0.064</td><td>0.443</td><td>0.808</td><td>0.168</td></tr><tr><td>[Eubacterium] coprostanoligenes group</td><td>1.451 ± 1.451 a</td><td>2.139 ± 1.471 a</td><td>0.589 ± 0.461 b</td><td>0.630 ± 0.535 b</td><td>0.015</td><td>0.422</td><td>0.477</td></tr><tr><td>Butyricicoccus</td><td>3.424 ± 1.559</td><td>2.864 ± 1.789</td><td>3.659 ± 2.354</td><td>2.458 ± 1.999</td><td>0.915</td><td>0.281</td><td>0.691</td></tr><tr><td>Ruminiclostridium 5</td><td>2.681 ± 2.130</td><td>2.183 ± 1.169</td><td>2.203 ± 1.016</td><td>2.980 ± 2.010</td><td>0.816</td><td>0.838</td><td>0.357</td></tr><tr><td><strong>Species</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alistipes sp.</td><td>0.001 ± 0.001</td><td>0.017 ± 0.034</td><td>0.000 ± 0.001</td><td>0.004 ± 0.005</td><td>0.360</td><td>0.174</td><td>0.363</td></tr><tr><td>bacterium ic1391</td><td>0.635 ± 0.744</td><td>0.426 ± 0.333</td><td>0.573 ± 0.072</td><td>0.190 ± 0.133</td><td>0.391</td><td>0.096</td><td>0.613</td></tr><tr><td>Clostridiales bacterium 60-7e</td><td>1.169 ± 1.822</td><td>0.494 ± 0.626</td><td>0.251 ± 0.242</td><td>1.156 ± 0.875</td><td>0.771</td><td>0.793</td><td>0.084</td></tr></tbody></table>

The means with different letters across lines are significantly different (P < 0.05).
Composition of caecal microbiota in males on Day 12

<table><thead><tr><th rowspan="2">Males on Day 12</th><th colspan="3">Control without SUP</th><th colspan="3">Delayed without SUP</th><th colspan="3">ANOVA</th></tr><tr><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>Delay</th><th>Supply</th><th>Interaction</th></tr></thead><tbody><tr><td><strong>Phylum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Firmicutes</td><td>97.678 ± 2.488</td><td>96.857 ± 3.921</td><td>98.766 ± 0.698</td><td>97.170 ± 2.095</td><td>0.512</td><td>0.263</td><td>0.716</td><td></td><td></td></tr><tr><td>Bacteroidetes</td><td>0.000 ± 0.001</td><td>0.003 ± 0.004</td><td>0.001 ± 0.002</td><td>0.005 ± 0.008</td><td>0.493</td><td>0.085</td><td>0.642</td><td></td><td></td></tr><tr><td>Tenericutes</td><td>1.666 ± 2.651</td><td>2.445 ± 4.028</td><td>0.788 ± 0.595</td><td>0.484 ± 0.287</td><td>0.169</td><td>0.813</td><td>0.592</td><td></td><td></td></tr><tr><td><strong>Class</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacilli</td><td>6.874 ± 16.776</td><td>0.029 ± 0.025</td><td>0.027 ± 0.016</td><td>0.013 ± 0.011</td><td>0.328</td><td>0.329</td><td>0.330</td><td></td><td></td></tr><tr><td>Bacteroidia</td><td>0.000 ± 0.001</td><td>0.003 ± 0.004</td><td>0.001 ± 0.002</td><td>0.005 ± 0.008</td><td>0.493</td><td>0.085</td><td>0.642</td><td></td><td></td></tr><tr><td>Clostridia</td><td>90.562 ± 19.820</td><td>96.857 ± 3.921</td><td>98.766 ± 0.698</td><td>97.170 ± 2.095</td><td>0.317</td><td>0.577</td><td>0.353</td><td></td><td></td></tr><tr><td>Erysipelotrichia</td><td>0.865 ± 0.535</td><td>0.666 ± 0.974</td><td>0.418 ± 0.298</td><td>2.327 ± 2.023</td><td>0.216</td><td>0.087</td><td>0.038</td><td></td><td></td></tr><tr><td>Gammaproteobacteria</td><td>0.566 ± 0.444</td><td>0.609 ± 0.988</td><td>0.369 ± 0.306</td><td>2.223 ± 1.872</td><td>0.128</td><td>0.046</td><td>0.056</td><td></td><td></td></tr><tr><td>Mollicutes</td><td>1.666 ± 2.651</td><td>2.445 ± 4.028</td><td>0.788 ± 0.595</td><td>0.484 ± 0.287</td><td>0.169</td><td>0.813</td><td>0.592</td><td></td><td></td></tr><tr><td><strong>Order</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillales</td><td>1.824 ± 2.869</td><td>0.661 ± 0.767</td><td>0.363 ± 0.645</td><td>0.125 ± 0.227</td><td>0.124</td><td>0.273</td><td>0.466</td><td></td><td></td></tr><tr><td>Bacteroidales</td><td>0.000 ± 0.001</td><td>0.003 ± 0.004</td><td>0.001 ± 0.002</td><td>0.005 ± 0.008</td><td>0.493</td><td>0.085</td><td>0.642</td><td></td><td></td></tr><tr><td>Clostridiales</td><td>74.807 ± 13.133</td><td>76.992 ± 14.033</td><td>78.171 ± 3.888</td><td>71.582 ± 11.885</td><td>0.829</td><td>0.643</td><td>0.360</td><td></td><td></td></tr><tr><td>Enterobacteriales</td><td>0.566 ± 0.444 b</td><td>0.609 ± 0.988 a</td><td>0.369 ± 0.306 b</td><td>2.223 ± 1.872 a</td><td>0.128</td><td>0.046</td><td>0.056</td><td></td><td></td></tr><tr><td>Erysipelotrichales</td><td>4.556 ± 3.772</td><td>5.893 ± 4.804</td><td>5.942 ± 1.605</td><td>6.230 ± 4.182</td><td>0.583</td><td>0.605</td><td>0.738</td><td></td><td></td></tr><tr><td>Lactobacillales</td><td>16.492 ± 12.258</td><td>13.312 ± 6.694</td><td>14.289 ± 2.609</td><td>19.234 ± 12.016</td><td>0.630</td><td>0.819</td><td>0.298</td><td></td><td></td></tr><tr><td>Mollicutes RF39</td><td>1.666 ± 2.651</td><td>2.445 ± 4.028</td><td>0.788 ± 0.595</td><td>0.484 ± 0.287</td><td>0.169</td><td>0.813</td><td>0.592</td><td></td><td></td></tr><tr><td>Sphingomonadales</td><td>0.066 ± 0.032</td><td>0.057 ± 0.042</td><td>0.049 ± 0.025</td><td>0.104 ± 0.168</td><td>0.681</td><td>0.534</td><td>0.386</td><td></td><td></td></tr><tr><td><strong>Family</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillaceae</td><td>1.824 ± 2.869</td><td>0.661 ± 0.767</td><td>0.363 ± 0.645</td><td>0.125 ± 0.227</td><td>0.124</td><td>0.273</td><td>0.466</td><td></td><td></td></tr><tr><td>Christensenellaceae</td><td>0.028 ± 0.042</td><td>0.012 ± 0.020</td><td>0.192 ± 0.219</td><td>0.021 ± 0.032</td><td>0.077</td><td>0.057</td><td>0.110</td><td></td><td></td></tr><tr><td>Clostridiaceae 1</td><td>0.120 ± 0.146</td><td>0.093 ± 0.029</td><td>0.170 ± 0.168</td><td>0.151 ± 0.184</td><td>0.374</td><td>0.700</td><td>0.940</td><td></td><td></td></tr><tr><td>Clostridiales vadinBB60 group</td><td>1.565 ± 1.184</td><td>2.535 ± 1.436</td><td>1.374 ± 1.446</td><td>0.694 ± 0.623</td><td>0.055</td><td>0.774</td><td>0.113</td><td></td><td></td></tr><tr><td>Defluvitaleaceae</td><td>0.108 ± 0.114</td><td>0.240 ± 0.388</td><td>0.204 ± 0.129</td><td>0.105 ± 0.083</td><td>0.828</td><td>0.853</td><td>0.205</td><td></td><td></td></tr><tr><td>Eggerthellaceae</td><td>0.015 ± 0.009</td><td>0.029 ± 0.025</td><td>0.027 ± 0.016</td><td>0.013 ± 0.011</td><td>0.759</td><td>0.992</td><td>0.057</td><td></td><td></td></tr><tr><td>Enterobacteriaceae</td><td>0.566 ± 0.444 b</td><td>0.609 ± 0.988 a</td><td>0.369 ± 0.306 b</td><td>2.223 ± 1.872 a</td><td>0.128</td><td>0.046</td><td>0.056</td><td></td><td></td></tr><tr><td>Enterococcaceae</td><td>0.032 ± 0.033</td><td>0.006 ± 0.004</td><td>0.005 ± 0.006</td><td>0.005 ± 0.005</td><td>0.056</td><td>0.076</td><td>0.064</td><td></td><td></td></tr><tr><td>Erysipelotrichaceae</td><td>4.556 ± 3.772</td><td>5.893 ± 4.804</td><td>5.942 ± 1.605</td><td>6.230 ± 4.182</td><td>0.583</td><td>0.605</td><td>0.738</td><td></td><td></td></tr><tr><td>Family XIII</td><td>0.015 ± 0.022</td><td>0.037 ± 0.072</td><td>0.062 ± 0.061</td><td>0.015 ± 0.014</td><td>0.530</td><td>0.537</td><td>0.097</td><td></td><td></td></tr><tr><td>Lachnospiraceae</td><td>27.182 ± 7.350</td><td>30.920 ± 14.009</td><td>31.072 ± 6.216</td><td>32.924 ± 7.854</td><td>0.450</td><td>0.473</td><td>0.808</td><td></td><td></td></tr><tr><td>Lactobacillaceae</td><td>16.459 ± 12.269</td><td>13.306 ± 6.693</td><td>14.283 ± 2.607</td><td>19.226 ± 12.015</td><td>0.628</td><td>0.816</td><td>0.299</td><td></td><td></td></tr><tr><td>Peptococcaceae</td><td>0.002 ± 0.005</td><td>0.000 ± 0.000</td><td>0.002 ± 0.003</td><td>0.018 ± 0.045</td><td>0.335</td><td>0.456</td><td>0.333</td><td></td><td></td></tr><tr><td>Peptstreptococcaceae</td><td>0.050 ± 0.087</td><td>0.030 ± 0.051</td><td>0.035 ± 0.065</td><td>0.125 ± 0.244</td><td>0.479</td><td>0.530</td><td>0.336</td><td></td><td></td></tr><tr><td>Rikenellaceae</td><td>0.000 ± 0.001</td><td>0.003 ± 0.004</td><td>0.001 ± 0.002</td><td>0.005 ± 0.008</td><td>0.493</td><td>0.085</td><td>0.642</td><td></td><td></td></tr><tr><td>Ruminococcaceae</td><td>45.736 ± 7.631</td><td>43.125 ± 18.693</td><td>45.059 ± 6.212</td><td>37.528 ± 15.056</td><td>0.560</td><td>0.350</td><td>0.647</td><td></td><td></td></tr><tr><td>Sphingomonadaceae</td><td>0.066 ± 0.032</td><td>0.057 ± 0.042</td><td>0.049 ± 0.025</td><td>0.104 ± 0.168</td><td>0.681</td><td>0.534</td><td>0.386</td><td></td><td></td></tr><tr><td>Streptococcaceae</td><td>0.001 ± 0.001</td><td>0.000 ± 0.000</td><td>0.001 ± 0.002</td><td>0.003 ± 0.007</td><td>0.362</td><td>0.792</td><td>0.374</td><td></td><td></td></tr><tr><td>unknown family</td><td>1.666 ± 2.651</td><td>2.445 ± 4.028</td><td>0.788 ± 0.595</td><td>0.484 ± 0.287</td><td>0.169</td><td>0.813</td><td>0.592</td><td></td><td></td></tr><tr><td><strong>Genus</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Unknown genus</td><td>15.274 ± 4.480</td><td>19.854 ± 5.524</td><td>13.298 ± 2.881</td><td>14.962 ± 3.393</td><td>0.059</td><td>0.083</td><td>0.405</td><td></td><td></td></tr><tr><td>Faecalibacterium</td><td>8.527 ± 9.859</td><td>4.652 ± 5.037</td><td>14.579 ± 7.536</td><td>6.461 ± 9.365</td><td>0.253</td><td>0.087</td><td>0.532</td><td></td><td></td></tr><tr><td>Ruminococcaceae UCG-014</td><td>14.151 ± 9.028 a</td><td>15.886 ± 9.612 a</td><td>9.073 ± 6.180 b</td><td>7.487 ± 6.125 b</td><td>0.050</td><td>0.982</td><td>0.612</td><td></td><td></td></tr><tr><td>Lactobacillus</td><td>16.459 ± 12.269</td><td>13.306 ± 6.693</td><td>14.283 ± 2.607</td><td>19.226 ± 12.015</td><td>0.628</td><td>0.816</td><td>0.299</td><td></td><td></td></tr><tr><td>Multi-affiliation</td><td>3.387 ± 1.358</td><td>10.016 ± 15.282</td><td>6.668 ± 4.017</td><td>3.739 ± 1.294</td><td>0.650</td><td>0.575</td><td>0.157</td><td></td><td></td></tr><tr><td>Alistipes</td><td>0.000 ± 0.001</td><td>0.003 ± 0.004</td><td>0.001 ± 0.002</td><td>0.005 ± 0.008</td><td>0.493</td><td>0.085</td><td>0.642</td><td></td><td></td></tr><tr><td>[Ruminococcus] torques group</td><td>6.703 ± 3.417</td><td>6.080 ± 2.420</td><td>6.427 ± 3.186</td><td>6.489 ± 2.506</td><td>0.956</td><td>0.816</td><td>0.776</td><td></td><td></td></tr><tr><td>Erysipelatoclostridium</td><td>3.787 ± 3.429</td><td>4.351 ± 4.899</td><td>5.024 ± 1.696</td><td>4.919 ± 3.667</td><td>0.547</td><td>0.878</td><td>0.823</td><td></td><td></td></tr><tr><td>Ruminococcaceae UCG-005</td><td>0.004 ± 0.004</td><td>0.189 ± 0.460</td><td>0.238 ± 0.291</td><td>0.006 ± 0.007</td><td>0.820</td><td>0.832</td><td>0.075</td><td></td><td></td></tr><tr><td>[Eubacterium] coprostanoligenes group</td><td>1.170 ± 1.013</td><td>1.675 ± 1.257</td><td>1.027 ± 0.513</td><td>0.749 ± 0.751</td><td>0.173</td><td>0.767</td><td>0.313</td><td></td><td></td></tr><tr><td>Butyricicoccus</td><td>2.717 ± 1.645</td><td>3.313 ± 2.840</td><td>1.931 ± 1.091</td><td>2.877 ± 0.924</td><td>0.413</td><td>0.304</td><td>0.814</td><td></td><td></td></tr><tr><td>Ruminiclostridium 5</td><td>2.322 ± 1.769</td><td>1.515 ± 0.364</td><td>1.956 ± 0.809</td><td>2.923 ± 1.443</td><td>0.310</td><td>0.875</td><td>0.091</td><td></td><td></td></tr><tr><td><strong>Species</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alistipes sp.</td><td>0.000 ± 0.000</td><td>0.002 ± 0.004</td><td>0.001 ± 0.002</td><td>0.003 ± 0.007</td><td>0.588</td><td>0.299</td><td>0.987</td><td></td><td></td></tr><tr><td>bacteriumic 1391</td><td>0.357 ± 0.427</td><td>0.583 ± 0.688</td><td>0.990 ± 0.591</td><td>0.898 ± 0.807</td><td>0.086</td><td>0.801</td><td>0.551</td><td></td><td></td></tr><tr><td>Clostridiales bacterium 60-7e</td><td>0.181 ± 0.152</td><td>0.396 ± 0.594</td><td>0.357 ± 0.379</td><td>0.470 ± 0.427</td><td>0.474</td><td>0.350</td><td>0.770</td><td></td><td></td></tr></tbody></table>

The means with different letters across lines are significantly different (P < 0.05).
Composition of caecal microbiota in females on Day 34

<table><thead><tr><th rowspan="2">Females on Day 34</th><th colspan="3">Control without SUP</th><th colspan="3">Delayed without SUP</th><th colspan="3">ANOVA</th></tr><tr><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>Delay</th><th>Supply</th><th>Interaction</th></tr></thead><tbody><tr><td><strong>Phylum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Firmicutes</td><td>89.147 ± 3.239 a</td><td>90.417 ± 3.869 a</td><td>86.870 ± 4.216 b</td><td>83.554 ± 7.432 b</td><td></td><td></td><td>0.035</td><td>0.619</td><td>0.271</td></tr><tr><td>Bacteroidetes</td><td>9.854 ± 3.229 b</td><td>8.559 ± 3.938 b</td><td>12.254 ± 4.227 a</td><td>15.049 ± 7.204 a</td><td></td><td></td><td>0.038</td><td>0.711</td><td>0.318</td></tr><tr><td>Tenericutes</td><td>0.747 ± 0.161 b</td><td>0.871 ± 0.180 a</td><td>0.747 ± 0.326 b</td><td>1.225 ± 0.510 a</td><td></td><td></td><td>0.198</td><td>0.035</td><td>0.199</td></tr><tr><td><strong>Class</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacilli</td><td>0.026 ± 0.021</td><td>0.022 ± 0.010</td><td>0.016 ± 0.026</td><td>0.023 ± 0.031</td><td></td><td></td><td>0.655</td><td>0.913</td><td>0.548</td></tr><tr><td>Bacteroidia</td><td>9.854 ± 3.229 b</td><td>8.559 ± 3.938 b</td><td>12.254 ± 4.227 a</td><td>15.049 ± 7.204 a</td><td></td><td></td><td>0.038</td><td>0.711</td><td>0.318</td></tr><tr><td>Clostridia</td><td>89.147 ± 3.239 a</td><td>90.417 ± 3.869 a</td><td>86.870 ± 4.216 b</td><td>83.554 ± 7.432 b</td><td></td><td></td><td>0.035</td><td>0.619</td><td>0.271</td></tr><tr><td>Erysipelotrichia</td><td>0.226 ± 0.154</td><td>0.131 ± 0.077</td><td>0.112 ± 0.086</td><td>0.149 ± 0.169</td><td></td><td></td><td>0.368</td><td>0.582</td><td>0.223</td></tr><tr><td>Gammaproteobacteria</td><td>0.198 ± 0.154</td><td>0.081 ± 0.067</td><td>0.080 ± 0.094</td><td>0.137 ± 0.168</td><td></td><td></td><td>0.560</td><td>0.572</td><td>0.112</td></tr><tr><td>Mollicutes</td><td>0.747 ± 0.161 b</td><td>0.871 ± 0.180 a</td><td>0.747 ± 0.326 b</td><td>1.225 ± 0.510 a</td><td></td><td></td><td>0.198</td><td>0.035</td><td>0.199</td></tr><tr><td><strong>Order</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillales</td><td>0.486 ± 0.305 b</td><td>0.387 ± 0.330 b</td><td>1.612 ± 1.451 a</td><td>1.162 ± 0.826 a</td><td></td><td></td><td>0.014</td><td>0.445</td><td>0.625</td></tr><tr><td>Bacteroidales</td><td>9.854 ± 3.229 b</td><td>8.559 ± 3.938 b</td><td>12.254 ± 4.227 a</td><td>15.049 ± 7.204 a</td><td></td><td></td><td>0.038</td><td>0.711</td><td>0.318</td></tr><tr><td>Clostridiales</td><td>82.100 ± 3.811 a</td><td>82.787 ± 5.115 a</td><td>77.617 ± 5.974 b</td><td>76.325 ± 7.397 b</td><td></td><td></td><td>0.030</td><td>0.898</td><td>0.677</td></tr><tr><td>Enterobacteriales</td><td>0.198 ± 0.154</td><td>0.081 ± 0.067</td><td>0.080 ± 0.094</td><td>0.137 ± 0.168</td><td></td><td></td><td>0.560</td><td>0.572</td><td>0.112</td></tr><tr><td>Erysipelotrichales</td><td>4.367 ± 1.261</td><td>4.366 ± 1.269</td><td>4.149 ± 1.687</td><td>3.860 ± 1.417</td><td></td><td></td><td>0.539</td><td>0.805</td><td>0.807</td></tr><tr><td>Lactobacillales</td><td>2.194 ± 0.480</td><td>2.878 ± 2.397</td><td>3.492 ± 1.753</td><td>2.207 ± 1.858</td><td></td><td></td><td>0.668</td><td>0.682</td><td>0.188</td></tr><tr><td>Mollicutes RF39</td><td>0.747 ± 0.161 b</td><td>0.871 ± 0.180 a</td><td>0.747 ± 0.326 b</td><td>1.225 ± 0.510 a</td><td></td><td></td><td>0.198</td><td>0.035</td><td>0.199</td></tr><tr><td>Sphingomonadales</td><td>0.028 ± 0.021</td><td>0.050 ± 0.040</td><td>0.032 ± 0.030</td><td>0.012 ± 0.005</td><td></td><td></td><td>0.141</td><td>0.942</td><td>0.072</td></tr><tr><td><strong>Family</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillaceae</td><td>0.486 ± 0.305 b</td><td>0.387 ± 0.330 b</td><td>1.612 ± 1.451 a</td><td>1.162 ± 0.826 a</td><td></td><td></td><td>0.014</td><td>0.445</td><td>0.625</td></tr><tr><td>Bifidobacteriaceae</td><td>0.011 ± 0.018</td><td>0.008 ± 0.009</td><td>0.004 ± 0.010</td><td>0.015 ± 0.034</td><td></td><td></td><td>0.988</td><td>0.650</td><td>0.451</td></tr><tr><td>Christensenellaceae</td><td>0.375 ± 0.109</td><td>0.348 ± 0.130</td><td>0.278 ± 0.136</td><td>0.380 ± 0.114</td><td></td><td></td><td>0.518</td><td>0.468</td><td>0.213</td></tr><tr><td>Clostridiaceae 1</td><td>0.004 ± 0.007</td><td>0.022 ± 0.055</td><td>0.013 ± 0.025</td><td>0.003 ± 0.005</td><td></td><td></td><td>0.683</td><td>0.756</td><td>0.267</td></tr><tr><td>Clostridiales vadimBB60 group</td><td>1.232 ± 0.648</td><td>1.466 ± 1.025</td><td>0.894 ± 0.258</td><td>2.285 ± 2.158</td><td></td><td></td><td>0.641</td><td>0.125</td><td>0.268</td></tr><tr><td>Defluvitaleaceae</td><td>0.163 ± 0.079</td><td>0.166 ± 0.055</td><td>0.136 ± 0.055</td><td>0.110 ± 0.062</td><td></td><td></td><td>0.131</td><td>0.659</td><td>0.586</td></tr><tr><td>Eggerthellaceae</td><td>0.016 ± 0.006</td><td>0.013 ± 0.007</td><td>0.012 ± 0.017</td><td>0.008 ± 0.009</td><td></td><td></td><td>0.313</td><td>0.530</td><td>0.903</td></tr><tr><td>Enterobacteriaceae</td><td>0.198 ± 0.154</td><td>0.081 ± 0.067</td><td>0.080 ± 0.094</td><td>0.137 ± 0.168</td><td></td><td></td><td>0.560</td><td>0.572</td><td>0.112</td></tr><tr><td>Enterococcaceae</td><td>0.056 ± 0.096</td><td>0.034 ± 0.032</td><td>0.072 ± 0.167</td><td>0.018 ± 0.012</td><td></td><td></td><td>0.995</td><td>0.357</td><td>0.699</td></tr><tr><td>Erysipelotrichaceae</td><td>4.367 ± 1.261</td><td>4.366 ± 1.269</td><td>4.149 ± 1.687</td><td>3.860 ± 1.417</td><td></td><td></td><td>0.539</td><td>0.805</td><td>0.807</td></tr><tr><td>Family XIII</td><td>0.247 ± 0.081</td><td>0.266 ± 0.064</td><td>0.208 ± 0.059</td><td>0.274 ± 0.110</td><td></td><td></td><td>0.648</td><td>0.212</td><td>0.491</td></tr><tr><td>Lachnospiraceae</td><td>16.964 ± 2.131 b</td><td>17.597 ± 3.538 b</td><td>22.219 ± 4.116 a</td><td>20.484 ± 3.467 a</td><td></td><td></td><td>0.008</td><td>0.695</td><td>0.403</td></tr><tr><td>Lactobacillaceae</td><td>2.113 ± 0.472</td><td>2.699 ± 2.531</td><td>3.418 ± 1.680</td><td>2.189 ± 1.865</td><td></td><td></td><td>0.594</td><td>0.666</td><td>0.231</td></tr><tr><td>Multi-affiliation</td><td>0.293 ± 0.264</td><td>0.961 ± 1.028</td><td>0.610 ± 0.699</td><td>0.494 ± 0.862</td><td></td><td></td><td>0.813</td><td>0.390</td><td>0.226</td></tr><tr><td>Peptococcaceae</td><td>0.039 ± 0.029</td><td>0.042 ± 0.020</td><td>0.021 ± 0.021</td><td>0.059 ± 0.059</td><td></td><td></td><td>0.986</td><td>0.181</td><td>0.242</td></tr><tr><td>Peptostreptococcaceae</td><td>0.063 ± 0.056</td><td>0.090 ± 0.110</td><td>0.053 ± 0.091</td><td>0.061 ± 0.065</td><td></td><td></td><td>0.585</td><td>0.617</td><td>0.781</td></tr><tr><td>Rikenellaceae</td><td>9.854 ± 3.229 b</td><td>8.559 ± 3.938 b</td><td>12.254 ± 4.227 a</td><td>15.049 ± 7.204 a</td><td></td><td></td><td>0.038</td><td>0.711</td><td>0.318</td></tr><tr><td>Ruminococcaceae</td><td>62.719 ± 3.091 a</td><td>61.828 ± 5.323 a</td><td>53.183 ± 5.692 b</td><td>52.174 ± 8.490 b</td><td></td><td></td><td>0.001</td><td>0.701</td><td>0.981</td></tr><tr><td>Sphingomonadaceae</td><td>0.028 ± 0.021</td><td>0.050 ± 0.040</td><td>0.032 ± 0.030</td><td>0.012 ± 0.005</td><td></td><td></td><td>0.141</td><td>0.942</td><td>0.072</td></tr><tr><td>Streptococcaceae</td><td>0.024 ± 0.051</td><td>0.145 ± 0.289</td><td>0.002 ± 0.005</td><td>0.000 ± 0.000</td><td></td><td></td><td>0.178</td><td>0.335</td><td>0.319</td></tr><tr><td>unknown family</td><td>0.747 ± 0.161 b</td><td>0.871 ± 0.180 a</td><td>0.747 ± 0.326 b</td><td>1.225 ± 0.510 a</td><td></td><td></td><td>0.198</td><td>0.035</td><td>0.199</td></tr><tr><td><strong>Genus</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Unknown genus</td><td>15.767 ± 2.142 b</td><td>14.926 ± 2.438 b</td><td>19.240 ± 4.030 a</td><td>19.423 ± 5.233 a</td><td></td><td></td><td>0.015</td><td>0.829</td><td>0.737</td></tr><tr><td>Faecalibacterium</td><td>23.155 ± 7.698 a</td><td>17.428 ± 7.662 a</td><td>12.591 ± 5.167 b</td><td>11.943 ± 2.367 b</td><td></td><td></td><td>0.004</td><td>0.217</td><td>0.322</td></tr><tr><td>Ruminococcaceae UCG-014</td><td>10.176 ± 2.796</td><td>13.653 ± 3.596</td><td>13.260 ± 4.115</td><td>13.153 ± 4.665</td><td></td><td></td><td>0.421</td><td>0.297</td><td>0.268</td></tr><tr><td>Lactobacillus</td><td>2.113 ± 0.472</td><td>2.699 ± 2.531</td><td>3.418 ± 1.680</td><td>2.189 ± 1.865</td><td></td><td></td><td>0.594</td><td>0.666</td><td>0.231</td></tr><tr><td>Multi-affiliation</td><td>3.552 ± 0.870</td><td>3.466 ± 1.119</td><td>3.770 ± 1.157</td><td>3.027 ± 0.747</td><td></td><td></td><td>0.786</td><td>0.316</td><td>0.425</td></tr><tr><td>Alistipes</td><td>9.854 ± 3.229 b</td><td>8.559 ± 3.938 b</td><td>12.254 ± 4.227 a</td><td>15.049 ± 7.204 a</td><td></td><td></td><td>0.038</td><td>0.711</td><td>0.318</td></tr><tr><td>[Ruminococcus] torques group</td><td>1.611 ± 0.784</td><td>2.904 ± 1.893</td><td>2.476 ± 1.256</td><td>1.928 ± 1.018</td><td></td><td></td><td>0.918</td><td>0.493</td><td>0.099</td></tr><tr><td>Erysipelatodostridium</td><td>1.829 ± 0.747</td><td>1.816 ± 0.634</td><td>2.283 ± 0.921</td><td>2.102 ± 1.498</td><td></td><td></td><td>0.379</td><td>0.816</td><td>0.840</td></tr><tr><td>Ruminococcaceae UCG-005</td><td>7.043 ± 7.763 a</td><td>8.872 ± 4.602 a</td><td>2.762 ± 2.779 b</td><td>4.265 ± 3.111 b</td><td></td><td></td><td>0.041</td><td>0.421</td><td>0.937</td></tr><tr><td>[Eubacterium] coprostanoligenes group</td><td>4.252 ± 1.828</td><td>4.287 ± 1.325</td><td>4.171 ± 0.952</td><td>3.599 ± 2.093</td><td></td><td></td><td>0.565</td><td>0.688</td><td>0.650</td></tr><tr><td>Butyricicoccus</td><td>1.290 ± 0.511</td><td>1.130 ± 0.533</td><td>2.546 ± 1.726</td><td>1.263 ± 0.602</td><td></td><td></td><td>0.100</td><td>0.088</td><td>0.178</td></tr><tr><td>Ruminiclostridium 5</td><td>1.153 ± 0.478</td><td>1.847 ± 1.086</td><td>1.186 ± 0.448</td><td>1.504 ± 1.056</td><td></td><td></td><td>0.650</td><td>0.149</td><td>0.584</td></tr><tr><td><strong>Species</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alistipes sp.</td><td>4.332 ± 3.094</td><td>3.711 ± 1.590</td><td>2.984 ± 0.837</td><td>5.894 ± 2.506</td><td></td><td></td><td>0.644</td><td>0.214</td><td>0.062</td></tr><tr><td>bacterium ic1391</td><td>0.489 ± 0.485</td><td>0.322 ± 0.213</td><td>0.259 ± 0.164</td><td>0.305 ± 0.234</td><td></td><td></td><td>0.327</td><td>0.629</td><td>0.398</td></tr><tr><td>Clostridiales bacterium 60-7e</td><td>0.206 ± 0.127</td><td>0.388 ± 0.190</td><td>0.431 ± 0.525</td><td>0.182 ± 0.111</td><td></td><td></td><td>0.935</td><td>0.782</td><td>0.085</td></tr></tbody></table>

The means with different letters across lines are significantly different (P < 0.05).
Composition of caecal microbiota in males on Day 34

<table><thead><tr><th rowspan="2">Males on Day 34</th><th colspan="2">Control without SUP</th><th colspan="2">Delayed without SUP</th><th colspan="3">ANOVA</th></tr><tr><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>mean ± sd</th><th>Delay</th><th>Supply</th><th>Interaction</th></tr></thead><tbody><tr><td><strong>Phylum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Firmicutes</td><td>88.088 ± 4.892</td><td>85.835 ± 6.332</td><td>90.096 ± 4.106</td><td>91.179 ± 5.457</td><td>0.102</td><td>0.788</td><td>0.446</td></tr><tr><td>Bacteroidetes</td><td>10.349 ± 5.141</td><td>12.894 ± 6.904</td><td>8.801 ± 4.011</td><td>7.367 ± 5.505</td><td>0.130</td><td>0.807</td><td>0.385</td></tr><tr><td>Tenericutes</td><td>1.196 ± 1.088</td><td>1.148 ± 1.181</td><td>0.902 ± 0.506</td><td>1.182 ± 0.387</td><td>0.716</td><td>0.746</td><td>0.647</td></tr><tr><td><strong>Class</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacilli</td><td>0.028 ± 0.012</td><td>0.029 ± 0.017</td><td>0.043 ± 0.022</td><td>0.031 ± 0.023</td><td>0.313</td><td>0.462</td><td>0.404</td></tr><tr><td>Bacteroidia</td><td>10.349 ± 5.141</td><td>12.894 ± 6.904</td><td>8.801 ± 4.011</td><td>7.367 ± 5.505</td><td>0.130</td><td>0.807</td><td>0.385</td></tr><tr><td>Clostridia</td><td>88.088 ± 4.892</td><td>85.835 ± 6.332</td><td>90.096 ± 4.106</td><td>91.179 ± 5.457</td><td>0.102</td><td>0.788</td><td>0.446</td></tr><tr><td>Erysipelotrichia</td><td>0.338 ± 0.318</td><td>0.094 ± 0.032</td><td>0.158 ± 0.127</td><td>0.242 ± 0.204</td><td>0.845</td><td>0.337</td><td>0.059</td></tr><tr><td>Gammaproteobacteria</td><td>0.306 ± 0.313</td><td>0.063 ± 0.017</td><td>0.138 ± 0.122</td><td>0.215 ± 0.207</td><td>0.920</td><td>0.319</td><td>0.061</td></tr><tr><td>Mollicutes</td><td>1.196 ± 1.088</td><td>1.148 ± 1.181</td><td>0.902 ± 0.506</td><td>1.182 ± 0.387</td><td>0.716</td><td>0.746</td><td>0.647</td></tr><tr><td><strong>Order</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillales</td><td>1.656 ± 1.409 a</td><td>0.329 ± 0.287 a</td><td>0.489 ± 0.431 a</td><td>0.695 ± 0.588 a</td><td>0.238</td><td>0.104</td><td>0.030</td></tr><tr><td>Bacteroidales</td><td>10.349 ± 5.141</td><td>12.894 ± 6.904</td><td>8.801 ± 4.011</td><td>7.367 ± 5.505</td><td>0.130</td><td>0.807</td><td>0.385</td></tr><tr><td>Clostridiales</td><td>79.508 ± 5.904</td><td>78.092 ± 7.828</td><td>81.108 ± 4.459</td><td>80.614 ± 5.184</td><td>0.408</td><td>0.699</td><td>0.852</td></tr><tr><td>Enterobacteriales</td><td>0.306 ± 0.313</td><td>0.063 ± 0.017</td><td>0.138 ± 0.122</td><td>0.215 ± 0.207</td><td>0.920</td><td>0.319</td><td>0.061</td></tr><tr><td>Erysipelotrichales</td><td>3.953 ± 1.031</td><td>3.911 ± 1.702</td><td>4.448 ± 0.992</td><td>3.810 ± 0.837</td><td>0.689</td><td>0.491</td><td>0.546</td></tr><tr><td>Lactobacillales</td><td>2.971 ± 2.073</td><td>3.503 ± 2.741</td><td>4.052 ± 5.080</td><td>6.060 ± 3.454</td><td>0.220</td><td>0.387</td><td>0.613</td></tr><tr><td>Mollicutes RF39</td><td>1.196 ± 1.088</td><td>1.148 ± 1.181</td><td>0.902 ± 0.506</td><td>1.182 ± 0.387</td><td>0.716</td><td>0.746</td><td>0.647</td></tr><tr><td>Sphingomonadales</td><td>0.032 ± 0.025</td><td>0.031 ± 0.019</td><td>0.021 ± 0.010</td><td>0.026 ± 0.021</td><td>0.329</td><td>0.794</td><td>0.661</td></tr><tr><td><strong>Family</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacillaceae</td><td>1.656 ± 1.409 a</td><td>0.329 ± 0.287 a</td><td>0.489 ± 0.431 a</td><td>0.695 ± 0.588 a</td><td>0.238</td><td>0.104</td><td>0.030</td></tr><tr><td>Bifidobacteriaceae</td><td>0.010 ± 0.007</td><td>0.011 ± 0.010</td><td>0.028 ± 0.019</td><td>0.013 ± 0.013</td><td>0.071</td><td>0.216</td><td>0.145</td></tr><tr><td>Christensenellaceae</td><td>0.259 ± 0.171</td><td>0.241 ± 0.085</td><td>0.298 ± 0.043</td><td>0.251 ± 0.041</td><td>0.560</td><td>0.428</td><td>0.727</td></tr><tr><td>Clostridiaceae 1</td><td>0.002 ± 0.003 b</td><td>0.006 ± 0.011 b</td><td>0.052 ± 0.061 a</td><td>0.031 ± 0.056 a</td><td>0.039</td><td>0.627</td><td>0.466</td></tr><tr><td>Clostridiales vadinBB60 group</td><td>1.479 ± 0.728</td><td>1.672 ± 0.624</td><td>1.721 ± 1.065</td><td>1.109 ± 0.551</td><td>0.614</td><td>0.511</td><td>0.213</td></tr><tr><td>Defluvitaleaceae</td><td>0.136 ± 0.084</td><td>0.157 ± 0.046</td><td>0.195 ± 0.047</td><td>0.164 ± 0.039</td><td>0.169</td><td>0.816</td><td>0.278</td></tr><tr><td>Eggerthellaceae</td><td>0.019 ± 0.012</td><td>0.018 ± 0.012</td><td>0.015 ± 0.007</td><td>0.018 ± 0.015</td><td>0.667</td><td>0.840</td><td>0.767</td></tr><tr><td>Enterobacteriaceae</td><td>0.306 ± 0.313</td><td>0.063 ± 0.017</td><td>0.138 ± 0.122</td><td>0.215 ± 0.207</td><td>0.920</td><td>0.319</td><td>0.061</td></tr><tr><td>Enterococcaceae</td><td>0.093 ± 0.169</td><td>0.031 ± 0.053</td><td>0.018 ± 0.017</td><td>0.098 ± 0.118</td><td>0.930</td><td>0.849</td><td>0.118</td></tr><tr><td>Erysipelotrichaceae</td><td>3.953 ± 1.031</td><td>3.911 ± 1.702</td><td>4.448 ± 0.992</td><td>3.810 ± 0.837</td><td>0.689</td><td>0.491</td><td>0.546</td></tr><tr><td>Family XIII</td><td>0.214 ± 0.120</td><td>0.297 ± 0.105</td><td>0.187 ± 0.039</td><td>0.195 ± 0.075</td><td>0.096</td><td>0.232</td><td>0.326</td></tr><tr><td>Lachnospiraceae</td><td>18.230 ± 4.994</td><td>17.028 ± 3.664</td><td>19.115 ± 2.062</td><td>18.528 ± 1.611</td><td>0.395</td><td>0.522</td><td>0.825</td></tr><tr><td>Lactobacillaceae</td><td>2.846 ± 2.015</td><td>3.440 ± 2.689</td><td>4.033 ± 5.085</td><td>5.959 ± 3.501</td><td>0.211</td><td>0.390</td><td>0.648</td></tr><tr><td>Multi-affiliation</td><td>0.263 ± 0.386</td><td>0.374 ± 0.354</td><td>0.423 ± 0.133</td><td>0.133 ± 0.138</td><td>0.725</td><td>0.444</td><td>0.094</td></tr><tr><td>Peptococcaceae</td><td>0.037 ± 0.041</td><td>0.040 ± 0.025</td><td>0.039 ± 0.022</td><td>0.046 ± 0.025</td><td>0.737</td><td>0.676</td><td>0.880</td></tr><tr><td>Peptostreptococcaceae</td><td>0.058 ± 0.066</td><td>0.140 ± 0.143</td><td>0.074 ± 0.045</td><td>0.099 ± 0.101</td><td>0.757</td><td>0.188</td><td>0.477</td></tr><tr><td>Rikenellaceae</td><td>10.349 ± 5.141</td><td>12.894 ± 6.904</td><td>8.801 ± 4.011</td><td>7.367 ± 5.505</td><td>0.130</td><td>0.807</td><td>0.385</td></tr><tr><td>Ruminococcaceae</td><td>58.831 ± 9.520</td><td>58.138 ± 8.286</td><td>59.005 ± 3.626</td><td>60.058 ± 3.927</td><td>0.712</td><td>0.949</td><td>0.758</td></tr><tr><td>Sphingomonadaceae</td><td>0.032 ± 0.025</td><td>0.031 ± 0.019</td><td>0.021 ± 0.010</td><td>0.026 ± 0.021</td><td>0.329</td><td>0.794</td><td>0.661</td></tr><tr><td>Streptococcaceae</td><td>0.032 ± 0.072</td><td>0.032 ± 0.058</td><td>0.000 ± 0.000</td><td>0.003 ± 0.002</td><td>0.125</td><td>0.916</td><td>0.949</td></tr><tr><td>unknown family</td><td>1.196 ± 1.088</td><td>1.148 ± 1.181</td><td>0.902 ± 0.506</td><td>1.182 ± 0.387</td><td>0.716</td><td>0.746</td><td>0.647</td></tr><tr><td><strong>Genus</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Unknown genus</td><td>16.453 ± 4.674</td><td>13.947 ± 2.795</td><td>15.312 ± 2.083</td><td>14.656 ± 2.851</td><td>0.872</td><td>0.247</td><td>0.493</td></tr><tr><td>Faecalibacterium</td><td>19.877 ± 9.932</td><td>14.347 ± 4.916</td><td>21.549 ± 3.712</td><td>23.067 ± 6.871</td><td>0.075</td><td>0.477</td><td>0.217</td></tr><tr><td>Ruminococcaceae UCG-014</td><td>12.943 ± 5.137</td><td>11.851 ± 2.226</td><td>11.187 ± 1.896</td><td>12.382 ± 3.195</td><td>0.660</td><td>0.970</td><td>0.415</td></tr><tr><td>Lactobacillus</td><td>2.846 ± 2.015</td><td>3.440 ± 2.689</td><td>4.033 ± 5.085</td><td>5.959 ± 3.501</td><td>0.211</td><td>0.390</td><td>0.648</td></tr><tr><td>Multi-affiliation</td><td>3.382 ± 1.784</td><td>4.025 ± 1.577</td><td>3.726 ± 1.057</td><td>3.134 ± 0.824</td><td>0.629</td><td>0.963</td><td>0.281</td></tr><tr><td>Alistipes</td><td>10.349 ± 5.141</td><td>12.894 ± 6.904</td><td>8.801 ± 4.011</td><td>7.367 ± 5.505</td><td>0.130</td><td>0.807</td><td>0.385</td></tr><tr><td>[Ruminococcus] torques group</td><td>2.114 ± 0.696</td><td>2.134 ± 0.452</td><td>2.299 ± 0.663</td><td>2.458 ± 0.961</td><td>0.393</td><td>0.763</td><td>0.814</td></tr><tr><td>Erysipelatodocstridium</td><td>2.293 ± 1.093 b</td><td>1.835 ± 0.941 b</td><td>2.848 ± 0.563 a</td><td>2.663 ± 0.339 a</td><td>0.045</td><td>0.333</td><td>0.676</td></tr><tr><td>Ruminococcaceae UCG-005</td><td>4.558 ± 3.973 a</td><td>9.798 ± 7.966 a</td><td>3.388 ± 1.675 b</td><td>2.480 ± 1.990 b</td><td>0.036</td><td>0.266</td><td>0.120</td></tr><tr><td>[Eubacterium] coprostanoligenes group</td><td>2.239 ± 1.200</td><td>3.817 ± 1.556</td><td>3.692 ± 0.920</td><td>3.724 ± 0.757</td><td>0.163</td><td>0.102</td><td>0.115</td></tr><tr><td>Butyricicoccus</td><td>2.229 ± 0.610</td><td>1.680 ± 1.337</td><td>2.095 ± 0.961</td><td>1.841 ± 0.514</td><td>0.972</td><td>0.295</td><td>0.696</td></tr><tr><td>Ruminiclostridium 5</td><td>1.387 ± 0.474</td><td>1.317 ± 0.406</td><td>1.125 ± 0.165</td><td>1.268 ± 0.499</td><td>0.360</td><td>0.829</td><td>0.529</td></tr><tr><td><strong>Species</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Alistipes sp.</td><td>3.501 ± 1.578</td><td>4.012 ± 1.118</td><td>4.410 ± 1.125</td><td>3.068 ± 1.458</td><td>0.975</td><td>0.454</td><td>0.105</td></tr><tr><td>bacterium ic1391</td><td>0.435 ± 0.219 b</td><td>0.360 ± 0.300 b</td><td>0.864 ± 0.335 a</td><td>0.701 ± 0.214 a</td><td>0.002</td><td>0.296</td><td>0.695</td></tr><tr><td>Clostridiales bacterium 60-7e</td><td>0.237 ± 0.145</td><td>0.593 ± 0.649</td><td>0.487 ± 0.301</td><td>0.188 ± 0.147</td><td>0.617</td><td>0.851</td><td>0.043</td></tr></tbody></table>

The means with different letters across lines are significantly different (P < 0.05).