# Short-term impacts of embryonic thermal manipulation in mule duck, a kinetic study: new tools for metabolic programming

Charlotte Andrieux, S. Biasutti, Ll. Zwick, M. Marchand, Stéphane Panserat, M. Houssier

► To cite this version:

Charlotte Andrieux, S. Biasutti, Ll. Zwick, M. Marchand, Stéphane Panserat, et al. Short-term impacts of embryonic thermal manipulation in mule duck, a kinetic study: new tools for metabolic programming. BMC Genomics, 2025, 26 (1), pp.1010. 10.1186/s12864-025-12192-7. hal-05355976

HAL Id: hal-05355976

https://hal.science/hal-05355976v1

Submitted on 9 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH

Open Access

<figure><img src="image_2.png" /></figure>

Check for
updates

# Short-term impacts of embryonic thermal manipulation in mule duck, a kinetic study: new tools for metabolic programming

C. Andrieux¹, S. Biasutti², LL. Zwick¹, M. Marchand¹, S. Panserat¹ and M. Houssier¹*

**Abstract**

**Background** Temperature changes during embryogenesis, through a process called embryonic thermal programming, can modify the long-term thermotolerance of broilers or the hepatic metabolism of mule ducks. This study focused on the very short-term impacts on mule duck livers of such a programming which consist of increasing the incubation temperature by +1°C, 16h/24h, from the 13th to 27th day of embryonic development. Using fluidigm technology, we analysed the impact of this temperature change on the relative expression of 81 genes involved in various metabolic pathways closely or remotely related to the fattening of the liver of mule ducks.

**Results** Expression changes were first assessed during temperature increase (from 30 min to 7h, then 3 days, 7 days and 11 days after the start of modification), and at hatching (3 days after the end of temperature modification) in comparison with a group of animals not exposed to this programming.

First, zootechnical measurements confirmed that the moderate discontinuous increase in embryonic temperature led to a drop in internal temperature and hatch weight, but did not reduce hatchability.

Secondly, gene expression in all tested metabolic pathways was affected throughout the study, except for cell proliferation and epigenetic marks, which were only modulated during the thermal stimulus. The most strongly and durably modulated pathways, with significant changes in the expression of several genes at multiple sampling points, were lipid metabolism (*DGAT2*, *CEPT1*, *GPAT1*, *ACOX1*), cellular stress (*HSPA5-HSP70*), and thyroid hormone regulation (*NCOR*). Some genes such as *SCD1* (lipid synthesis), *DIO3* (thyroid hormone regulation), *HSPA2-HSP70*, *HSBP1*, *HSP90AA1* and *IL18* (cellular stress) also showed modulation even after the thermal stimulus ended.

Interestingly, the expression of genes involved in epigenetic and cell proliferation was only slightly affected by the temperature increase, except for *ELP3* (epigenetic marks) which was significantly modulated at two points.

**Conclusion** This study is the first to show the short-term impact of increased egg incubation temperature on gene expression in mule duck liver, from the start of the stimulus to hatching. These results could provide a valuable starting point for understanding the mechanisms of embryonic thermal programming that modulate hepatic metabolism in mule ducks.

**Keywords** Mule duck, Thermal manipulation, Programming, Metabolism

Full list of author information is available at the end of the article

<footnote>*Correspondence:
M. Houssier
marianne.houssier@univ-pau.fr</footnote>
## Introduction

Early environmental stimuli are capable of modifying phenotype, in the short or long term, in animal life [1]. This phenomenon, known as programming [2], has been used in animal production and more specifically in the poultry industry to improve breeding performance [3]. Various stimuli can be applied to program a phenotype such as different colored light during incubation [4–6], variable availability of specific nutrients in the egg [7, 8], or different egg incubation temperature [9, 10]. These early exposures can influence various physiological pathways, such as muscle growth [9–11], global health [12, 13] or the ability to withstand heat stress [14, 15]. In mule duck, it was recently shown that an increase in temperature, from 1 to 1.7 °C during embryogenesis, resulted in higher foie gras production after a 2-week-forced-fed [16, 17].

These phenotypic changes are generally associated with molecular changes such as protein expressions in broiler muscles [18], DNA methylations in broiler brain [19] or transcriptional modulations in chicken heart, brain or spleen [19–21] or in duck liver [16, 22]. The transcriptional effects of embryonic thermal programming are often measured at two stages of the process: firstly, just after the temperature change, and secondly, after the programmed phenotype has been revealed, i.e. often much later in the animal's life. For the first stage, the genes studied are mainly involved in stress perception, belonging in particular to the family of heat shock proteins [23], whereas for the second stage, measurements often focus on the expression of genes involved in revealing the programmed phenotype [17, 22, 24]. These measurements reflect the transcriptional responses at a particular, arbitrarily chosen time point following stimuli.

The objective of this study was to evaluate how embryonic thermal programming affects the expression of genes involved in diverse cellular pathways in the liver of mule ducks, beginning 30 min after the initial thermal stimulus, over several days during the programming phase, and 3 days following its end. To this end, mule duck eggs were subjected to a discontinuous (16h/24h) increase in temperature of 1 °C during the 13th to the 27th embryonic day (E13-E27) in parallel with a control group with no temperature change, before being transferred to the same hatchery. Mortality at different stages, final hatchability, sex ratio, internal temperature, and body weight were measured at hatching (E30). Gene expression was assessed at multiple time points: from 30 min to 7 h after the onset of the temperature increase at E13, then at 3, 7, and 11 days after the start of the temperature increase (E16, E20, and E24), corresponding to the programming period. A final gene expression measurement was taken 3 days after the end of the temperature increase, at hatching (E30).

## Materials and methods

### Animals

The experiment was divided into two individual trials (Fig. 1). For both trials, eggs from the two groups were collected on the same day and stored at room temperature for 3 days before incubation (genotype H85, provided by Grimaud Frères Selection company, Roussay, France). The eggs were randomly divided in two (second trial) or four (first trial) identical incubators for two incubation treatments (SOLOGNE model of LA NATIONALE reconditioned model with a capacity of 2,430 eggs). All incubators were maintained at a constant temperature of 37.6 °C with an average relative humidity of 63.2%, from the embryonic day 0 (E0) to embryonic day 13th (E13) (24h/24). Incubator temperature, relative humidity (RH) and ventilation were automatically regulated. Moreover, the temperature and the RH were confirmed by records every 15 min by two independent sensors (LoRa® SPY U) in each incubator. All eggs in both groups were subjected to automatic 90° rotation every three hours and were manually sprayed for daily cooling from embryonic day 8 until the end of incubation, by opening the incubator doors for a few minutes at 2 pm. This cooling was applied just before the daily temperature increase, with the aim of facilitating the concomitant rise in relative humidity for TM group.

For the first trial (Fig. 1a), at embryonic day 13 (E13), the temperature in two of the four incubators was increased by 1 °C from 8:00 a.m. with an average relative humidity (RH) of 60.9% (Thermal Manipulated or TM group, 250 eggs) while the control incubators were maintained at 37.6 °C and 60.1% RH (control group, 250 eggs). The eggs were then collected from the first two incubators (1 control and 1 TM incubator) at 30 min, 2 h, 4 h, and 7 h after the temperature increase, and from the other two incubators of each group at 1 h, 3 h, and 5 h, in order to avoid significant temperature fluctuations caused by opening the doors during sampling. The embryos were euthanized by decapitation, in accordance with ethical principles [25], to allow for the collection of liver samples. To ensure consistent handling conditions, the 15 eggs from each group were temporarily transferred to a mobile incubator maintained at 37.6 °C, and dissections were performed alternately: one control egg/ one TM egg.

For the second trial (Fig. 1b), from E13 to E27 the temperature in one of the two incubators was increased by 1 °C each day from 4:00 p.m. to 8:00 a.m. with an average RH of 63.6% (TM group, 750 eggs), while the other incubator was maintained at 37.6 °C with 63.2% RH (control group, 750 eggs) throughout the entire incubation period. Liver samples were then taken at E16, E20 and E24 during the programming period at 5 p.m, 1 h after the start of daily temperature rise (when the incubation temperature
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Experimental design. For both trials (a and b), the incubation temperature was maintained at 37.6°C throughout the incubation period for the control group. In the TM (thermal manipulated) group, a temperature increase of 1 °C, 16h/24 was applied from embryonic day 13 to 27 (E13-E27), illustrated by a bold dotted line. In the first trial (a), liver were sampled 30 min, 1 h, 2 h, 3 h, 4 h, 5 h and 7 h after the start of the thermal stimulus. In the second trial (b), liver samples were taken 3 days, 7 days and 11 days after the start of the temperature increase, corresponding to embryonic days 16, 20 and 24 (E16, E20 and E24). A final sample was taken at hatching (E30), 17 days after the start of the stimulus in both groups. At hatching, body weight, hatchability, sex ratio and internal temperature were also recorded</figcaption></figure>

was reached and stable). At day 28 (E28), a candling was
performed to assess mid-embryonic mortality (mortal-
ity between embryonic day 7 to 28), and all viable eggs
were moved into the same hatchery (BRETAGNE model
of LA NATIONALE, reconditioned model with a capac-
ity of 2,520 eggs) maintained at 37.3°C and 80% of RH
for three days. From E28 to E30, newly hatched duck-
lings were recorded once per day. Unhatched ducklings
at E30 were counted to determine late mortality, rate of
fertilized eggs (not shown, (total number of eggs put in
incubation – number of eggs unfertilized identified dur-
ing candling)/total number of eggs put in incubation) and
hatchability (calculated as the ratio of the total number of
hatched ducklings to the number of fertilized eggs set in
the hatcher, multiplied by 100) while final liver samples
were taken from ducklings born at E30 in both groups, 3
days after the end of the programming period.

Sampling and measurements

In the first trial whole livers (8 per condition) were col-
lected 30 min, 1 h, 2 h, 3 h, 4 h, 5 h and 7 h after the first
day of temperature increase (E13). In the second trial,
whole livers (8 per condition) were collected 1 h after the
daily temperature increase (5 p.m.), 3, 7 and 11 days after
the temperature increase (E16, E20 and E24) and three

days after the end of the temperature increase, at hatch-
ing (E30). Similar samples were taken at the same times
for the control group, not exposed to the temperature
increase. The livers were immediately immersed in liquid
nitrogen before being stored at –80 °C.

The sex of all hatched ducklings (433 in the control
group and 475 in the TM group), the body weight of dry
male ducklings (237 in the control group and 255 in the
TM group), and the cloacal temperature of approximately
100 male ducklings in each group were recorded at E30,
by using digital express thermometers (Digital Express
Thermometer from Gilbert).

RNA extraction, reverse transcription, fluidigm and relative
expression measurement

The TRIzol method (Invitrogen/Life technologies) was
used to extract the total RNA from 50 mg of frozen liver
tissue. Total RNA samples were normalized at 100 ng/µL
after concentration analysis by spectrophotometry (opti-
cal density at 260 nm) using a Biotek EPOCH 2 micro-
plate reader (Take 3 Plate). RNA reverse transcription
(RT) was performed with 1µg of RNA mixed with Super
Script (Life Technologies SAS) and RNAseOUT (Life
Technologies SAS). The RT mix was completed by an
exogenous RNA (Luciferase, Promega, 1 pg) to have an
exogenous control. The RT was done in the CFX384 (Bio-Rad, USA) according to the following program: 25°C/5 min, 55°C/60 min, 70°C/15 min.

Gene expression levels were determined by high throughput real-time quantitative PCR from Fluidigm (Gentyane platform, Clermont-Ferrand, France). Two 96 well plates were prepared, one with samples (cDNA) at 5 ng/µL and the second with gene primers at 20 µM, listed in Supplement Table 1. Primer validations were carried out in two steps: their efficiency ranging from 1.85 to 2 was calculated from cascade dilution of a cDNA pool and their specificity was confirmed by amplicon sequencing. The sample amplifications were realized in two steps by using the Fluidigm method. A first pre-amplification with Preamp Master Mix (Fluidigm) was done following program: 95 °C/10 min and 14 cycles of 95 °C/15 s and 60 °C/4 min. Pre-amplified samples were treated by exonuclease (NEB) before a low EDTA buffer (10mM Tris-HCL, 0.1 mM EDTA) dilution and before moving samples to a 96 × 96 chip for Fluidigm Gene Expression Array. The reaction was made using 20 × EvaGreen (Interchim) dye following the program:70 °C/2400 s, 60 °C/30 s, 30 cycles of 96 °C/5 s and 60 °C/20 s. Real time quantitative PCR results were analyzed using the Fluidigm real-time PCR analysis software v.4.1.3.

A initial list of genes of interest was compiled from previous studies conducted on different species concerning metabolic pathways of interest such as stress, inflammation or energy metabolism. 95 genes were selected for Fluidigm analysis and ultimately only those validated for specificity and efficiency were retained for gene expression analysis. Initially, 8 replicates per condition were deposited to calculate a relative expression mean, and only genes for which at least 6 replicates were finally detected were retained for statistical analysis. A total of 81 genes were kept for the hour and the daily kinetics.

The relative expression was normalized with four reference genes (ActB, *Luciferase*, *STAB1* and *USP9X* for the first trial, and *HPRT1*, *Luciferase*, *STAB1* and *UPS9X* for the second trial), selected according to the function GeNorm from the package *CtrlGene* of R (v.3.6.2). Moreover, the relative expression was calculated according to the expression $2^{-\Delta\Delta Ct}$ [26] with $\Delta Ct$ sample = Ct target gene - Ct reference genes (mean of 4 previous genes) and $\Delta\Delta Ct$ = $\Delta Ct$ sample - $\Delta Ct$ reference. The reference that determined the $\Delta Ct$ reference value corresponded to the average of Ct across all groups.

## Statistical analysis

Statistical analyses were carried out using R & Rstudio version 3.6.2. Hatchability, sex ratio and mortalities were compared by using a Chi-squared test. Student (parametric) or Wilcoxon (non parametric) tests were done depending on normality and homoscedasticity. A student

test was applied for body weights at hatch and a Wilcoxon test was done for internal temperatures at hatch. Tests have been adapted to compare the relative expression between groups for the two trials (n = 6 minimum) for each timing (2 ways analysis was excluded due to the flattening of the TM effects).

The data are presented as the average ± standard error of deviation (SD). In every case, differences between the groups were considered statistically significant when *p* < 0.05.

## Results

### Short-term transcriptional impacts of thermal manipulation in the liver

The first trial enabled the analysis of the very short-term transcriptional impact of an increase in incubation temperature, based on an hourly kinetics at E13, with the results presented in Fig. 2. The heat map highlights significant differences in relative gene expression between the two groups at each specific time point, indicated by the appearance of red/brown colored bands, according to the color key. Overall, the analysis reveals a very moderate transcriptional impact of the temperature increase, particularly after 30 min, 2 h, 3 h, and 4 h of exposure, with fewer than 6 genes significantly affected out of the 81 tested. Cellular pathways related to proliferation and epigenetic modifications are among the least affected by the temperature increase, in contrast to those involved in cellular stress, thyroid hormone synthesis, or carbohydrate metabolism, for which at least 50% of the tested genes were significantly modulated. It is worth noting that the expression of many genes is never affected by the temperature increase (light pink/yellow lines, representing 44 out of the 81 tested genes, i.e., 54%). The expression of a few genes (10 out of the 81, 12.3%), such as *TET2* (epigenetics), *ATG18* and *HSP60* (cellular stress), *USP16* and *ALB* (thyroid hormone synthesis), *ACOX1* and *CEPT1* (lipid metabolism), as well as *CREBP2*, *ENO1*, and *PDHA1* (carbohydrate metabolism), was affected at two of the seven time points tested, whereas the remaining altered genes exhibited transient changes at a single time point.

The overall relative expression measured during the second trial, on daily kinetics (E16, E20, E24 and E30) is represented by a second heat map with the same color key in Fig. 3. As shown by the predominance of yellow dots, the expression of a large number of genes is not affected at all by the thermal stimulus, but 37 genes (46.6% of all potential target genes studied) are nevertheless significantly affected at least at one point. Among these target genes, 8 (9.9% of all potential target genes studied) were significantly affected at least at 2 kinetic points, with a maximum of 3 time points.
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Hourly kinetics of hepatic gene expression at E13. The heat map represents an overview of the 81 genes studied, presented by cellular pathway for the 7 sampling points at E13, the first day of temperature increase (30min, 1 h, 2 h, 3 h, 4 h, 5 h and 7 h after the modification). Student or Wilcoxon tests were applied, depending on the conditions to be met, to compare the control and TM groups for the same sampling point. The absence of a significant difference is shown in orange/yellow, while red/brown indicates a significant difference between the two groups according to the color key</figcaption></figure>

For the subsequent analysis, we grouped the 31 genes that were modulated at least once at the time points E13-1h, E16, E20, E24, and E30. To simplify the analysis, only the 1-h time point from the hourly kinetics at E13 was retained, as it is one of the time points with the highest number of temperature-responsive genes, and because the daily kinetics time points were also collected one hour after the onset of the temperature increase.

The results concerning the expression of genes involved in the energy metabolism pathway for each of these time points are shown in Table 1. The expression of 13 genes involved in carbohydrate and lipid metabolisms was significantly affected by the increase in temperature out of a total of 35 genes measured (37%). Most of them were upregulated (except for *GLUT1* at E13, and *CEPT1* at E24), suggesting an overall activation of energy metabolism. The maximum impact is measured 3 days after the

start of the programming period (E16) with the increase of 8 genes, involved in each of the pathways studied. Three days after the end of the thermal stimulation (E30), at hatching, the expression of 3 genes involved in lipid synthesis (*DGAT2*, *SCD1* and *GPAT1*) was still up-regulated in programmed animals (TM) compared to control.

Regarding the expression of genes involved in thyroid hormone synthesis, the results shown in Table 2 highlighted an overall increase, except for *DIO3* at E30, three days after the end of thermal stimulation. E16 is still the stage at which the highest number of genes is affected by the temperature increase, with 3 genes up-regulated out of the 4 identified targets.

Changes in the expression of genes involved in the heat shock response, autophagy and inflammation are presented in Table 3. Among the 10 genes significantly affected at some point, half were already affected within
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Daily kinetics of hepatic gene expression. The heatmap provides an overview of the relative expression of the 81 genes studied, presented by cell pathway for the three sampling points collected during the programming period at 3, 7, and 11 days after the onset of temperature increase (E16, E20, E24), and 3 days after the end of the programming period (E30). Student or Wilcoxon tests were applied, depending on the conditions to be met, to compare the control and TM groups for the same sampling point. The absence of a significant difference is shown in orange/yellow, while red/brown indicates a significant difference between the two groups according to the color key</figcaption></figure>

the first hour of temperature increase, and 4 are still affected at E30, corresponding to the hatching day.

Table 4 summarizes the results concerning the expression of genes involved in proliferation and epigenetic pathways. The 4 genes identified as targets of thermal programming in these two pathways (out of 16 tested) had their expressions modulated seven days after the onset of temperature modification (E20). Only *ELP3*, involved in the epigenetic pathway, is still affected at E24, and none of them are affected at E30 anymore.

## Incubation and hatching data

During the trial 2, no significant difference was observed between the two incubation treatments for embryonic mortalities (mid and late), hatchability or male proportion with average (for both groups combined) of 9.4%, 3.1%, 81.5% and 55.2% respectively (Table 5). However, ducklings subjected to thermal programming presented

significantly lower body weight (48.8 against 50.2 g) and lower internal temperature (39.9 against 40.1 °C) at hatch compared to control group.

## Discussion

This study aimed to investigate the impact of increased incubation temperature in mule duck eggs, to gain a better understanding of short-term transcriptional and physiological changes in order to propose new tools for thermal programming in ducks. To this end, two kinetic analyses were conducted: one on an hourly scale and the other on a daily scale following the temperature increase.

## Consolidation of knowledge on thermal manipulation effects on hatching performance

At the end of the embryonic period, we measured the impact of thermal stimulation at the biological level. The increase of 1 °C between E13 and E27 for 16 h per day did
<table><caption>**Table 1** Relative expression of temperature-modulated genes involved in liver metabolism</caption><thead><tr><th rowspan="2">Pathways</th><th rowspan="2">Gene</th><th rowspan="2">Total number of genes measured</th><th colspan="3">During thermal stimulus</th><th colspan="3">E16 (3 days)</th><th colspan="3">E20 (7 days)</th><th colspan="3">E24 (11 days)</th><th colspan="3">After thermal stimulus E30 (17 days)</th></tr><tr><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th></tr></thead><tbody><tr><td colspan="18"><strong>CARBOHYDRATES</strong></td></tr><tr><td>Transport</td><td>GLUT1</td><td>3</td><td>1.64±0.85</td><td>0.82±0.23 *</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td>ns</td><td></td></tr><tr><td>Oxydation</td><td>CREB2</td><td>7</td><td>ns</td><td></td><td>0.73±0.21</td><td>1.23±0.34 **</td><td></td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ATF4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="18"><strong>LIPIDS</strong></td></tr><tr><td>Synthesis</td><td>DGAT2</td><td>10</td><td></td><td></td><td></td><td>0.45±0.15</td><td>2.23±0.62 **</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td>ns</td><td></td><td>0.83±0.35</td><td>2.00±0.72 **</td><td></td></tr><tr><td></td><td>ACC</td><td></td><td>ns</td><td></td><td>0.62±0.33</td><td>1.54±0.76 *</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr><tr><td></td><td>CEPT1</td><td></td><td>ns</td><td></td><td>0.72±0.35</td><td>1.29±0.44 *</td><td></td><td>ns</td><td>ns</td><td></td><td>1.31±0.35</td><td>0.88±0.33 *</td><td></td><td>ns</td><td></td><td>1.10±0.62</td><td>1.84±0.29 **</td></tr><tr><td></td><td>SCD1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr><tr><td></td><td>ME</td><td></td><td>ns</td><td></td><td>ns</td><td></td><td></td><td>0.9±0.20</td><td>1.27±0.19 ****</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td>0.82±0.25</td><td>1.53±0.67 **</td></tr><tr><td>Oxydation</td><td>GPAT1</td><td></td><td>ns</td><td></td><td>0.61±0.09</td><td>1.37±0.46 *</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr><tr><td></td><td>ACOX1</td><td>9</td><td>ns</td><td></td><td>0.68±0.27</td><td>1.33±0.53 *</td><td></td><td>0.95±0.26</td><td>1.30±0.29 **</td><td></td><td>0.94±0.23</td><td>1.20±0.47 *</td><td></td><td>ns</td><td></td><td></td><td></td></tr><tr><td></td><td>ALD-</td><td></td><td></td><td></td><td>ns</td><td></td><td></td><td>ns</td><td></td><td></td><td>1.22±0.58</td><td>2.15±1.32 *</td><td></td><td>ns</td><td></td><td></td><td></td></tr><tr><td></td><td>H3A2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>ALD-</td><td></td><td>0.86±0.47</td><td>1.40±0.48 *</td><td></td><td>ns</td><td></td><td>ns</td><td></td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr><tr><td>transport</td><td>LDLR</td><td>3</td><td>ns</td><td></td><td>0.72±0.50</td><td>1.70±0.84 *</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr><tr><td colspan="18"><strong>REGULATORS</strong></td></tr><tr><td></td><td>LXRA</td><td>3</td><td>ns</td><td></td><td>0.78±0.14</td><td>1.20±0.40 *</td><td></td><td>ns</td><td>ns</td><td></td><td>ns</td><td></td><td></td><td></td><td>ns</td><td></td><td></td></tr></tbody></table>

The relative expression of genes involved in liver metabolism and significantly affected at least at one point by the temperature change is listed for the 5 sampling points (E13-1h, E16, E20, E24 and E30). The total number of genes measured for each cellular pathway is indicated

Student or Wilcoxon tests were applied, depending on the conditions to be met. Effect: statistical result of the comparison between the control group and the programmed group for the same time point; ns: non significant, *, p < 0.05, **, p < 0.01, ***, p < 0.001. « → not determined (not enough samples to apply tests). n = 6 to 8
<table><caption>**Table 4** Relative expression of temperature-modulated genes involved in proliferation and epigenetic</caption><thead><tr><th rowspan="2">Pathway</th><th rowspan="2">Gene</th><th rowspan="2">Total number of genes measured</th><th colspan="10">During thermal stimulus</th><th colspan="3">After thermal stimulus</th></tr><tr><th colspan="2">E13 (1 h)</th><th colspan="2">E16 (3 days)</th><th colspan="2">E20 (7 days)</th><th colspan="2">E24 (11 days)</th><th colspan="2">E30 (17 days)</th><th colspan="2"></th></tr><tr><th></th><th></th><th></th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th><th>Control</th><th>TM</th><th>Effect</th></tr></thead><tbody><tr><td rowspan="2">Proliferation</td><td>FGFR2</td><td>6</td><td>ns</td><td></td><td>ns</td><td></td><td>0.81±0.47</td><td>1.66±0.63</td><td>**</td><td>ns</td><td></td><td>ns</td><td></td><td></td></tr><tr><td>HGF</td><td></td><td>ns</td><td></td><td>ns</td><td></td><td>0.80±0.36</td><td>1.51±0.53</td><td>**</td><td>ns</td><td></td><td>ns</td><td></td><td></td></tr><tr><td rowspan="2">Epigenetic</td><td>ELP3</td><td>10</td><td>ns</td><td></td><td>ns</td><td></td><td>1.55±0.38</td><td>0.69±0.34</td><td>***</td><td>1.03±0.27</td><td>1.40±0.67</td><td>**</td><td>ns</td><td></td></tr><tr><td>TYMS</td><td></td><td>ns</td><td></td><td>ns</td><td></td><td>0.81±0.33</td><td>1.51±0.65</td><td>*</td><td>ns</td><td></td><td>ns</td><td></td><td></td></tr></tbody></table>

The relative expression of genes involved in proliferation or epigenetic and significantly affected at least at one point by the temperature change is listed for the 5 sampling points (E13-1h, E16, E20, E24 and E30). The total number of genes measured for each cell pathway is indicated.

Student or Wilcoxon tests were applied, depending on the conditions to be met. Effect: statistical result of the comparison between the control group and the programmed group for the same time point; ns: non significant, *<0.05, **<0.01, ***<0.001, n=6 to 8

## Cellular stress pathways are quickly impacted by the temperature increase

The first major group of genes to respond to the increase in temperature, within the first hour, belongs to the cellular stress pathway and primarily involves genes related to the heat stress response such as *HSPA2-HSP70*, *HSP60*, *HSF3* and *HSPA5-HSP70*. These results confirm the rapid sensitivity of this cellular pathway to different types of stress, as already shown in poultry exposed to heat shock [7, 29] or embryonic thermal conditioning [19, 23]. Given that transcriptomic changes in HSP expression can be recovered within 48 h [30, 31], it is interesting to note that the expression of several genes in this group (*HSPA2-HASP70*, *HSP90AA1*, *HSBP1*) was still affected by thermal programming three days after the end of the application of the thermal stimulus. This observation may reveal a different temporal response to this precise type of thermal stimulus in mule ducks, with a longer transcriptomic mark, or it may also highlight other well-described types of hatching-induced stress in birds [32–34]. However, the expression of the three genes involved in the heat shock response pathway, still significantly altered at E30, is decreased compared to the control group. This could mean that thermal programming has already increased the ducklings' ability to cope with hatching stress (particularly in terms of thermal regulation), reducing the need for cellular protection normally provided by HSPs [35, 36]. This could be reflected particularly by the expression of the *HSP90AA1* gene, a major player in the stress response system [37, 38], which is increased at E16 and E20, and decreased at E30.

Moreover, the autophagy pathway, involved in intracellular degradation under normal or stressed conditions [39, 40], also responded quickly after the increase in temperature, with an increase in *WIPI1* expression at E13 but was completely shut down at E30. Only the expression of *IL18*, a pro-inflammatory cytokine involved in the acute stress response [41], was still increased at hatching. It has been previously demonstrated that chickens subjected to embryonic thermal manipulation exhibited an early increase in *IL18* expression in the spleen during a subsequent heat shock compared to control animals [20]. This early response has been interpreted as possibly contributing to improved heat tolerance by increasing the expression of genes involved in the cell repair mechanism, and could similarly in this study, indicate an enhanced stress response during hatching. Nevertheless, the impact of embryonic thermal manipulation on the inflammatory response to hatching stress needs to be further investigated (only the expression of one gene was retained here).
<table><caption>**Table 5** Incubation and hatch measurements</caption><thead><tr><th></th><th>Mid-embryonic mortality (%)</th><th>Late mortality (%)</th><th>Hatchability (%)</th><th>Male proportion (%)</th><th>Weight at hatch (g)</th><th>Internal temperature at hatch (°C)</th></tr></thead><tbody><tr><td>Control</td><td>10.0</td><td>3.2</td><td>78.8</td><td>55.7</td><td>50.2<sup>a</sup>±4.5</td><td>40.1<sup>a</sup>±0.5</td></tr><tr><td>TM</td><td>8.7</td><td>3.0</td><td>84.2</td><td>54.7</td><td>48.8<sup>b</sup>±4.1</td><td>39.9<sup>b</sup>±0.4</td></tr><tr><td>Number of animals in Control and TM group</td><td>501 and 554</td><td>451 and 506</td><td>433 and 475</td><td>433 and 475</td><td>237 and 255</td><td>101 and 98</td></tr></tbody></table>

Mid-embryonic mortality (E28), late mortality (E30), hatchability and male proportion were compared for the two incubation conditions by Chi² tests during the second trial. Body weight was compared by t-test and internal temperature by Wilcoxon test

## Energy metabolism is the most affected by the temperature increase

The second measurement point (E16), three days after the start of the temperature increase, revealed a marked increase in the expression of most genes involved in carbohydrate (CREB2 ATF4) and lipid (DGAT2, ACC, CEPT1, GPAT1, ACOX1) metabolisms or their regulations (LXRA). This overall activation of the energy pathway in the liver was accompanied by increased expression of three genes involved in thyroid hormone pathway (NCOR, JMJD1C and DIO3). NCOR (corepressor) and DIO3 (deiodinase) are known to promote the inactive form of thyroid hormone [42–44] while JMJD1C is a coactivator of the thyroid hormone receptor [45, 46] and is involved in energy metabolism, in particular glycolysis and the oxidative process [47]. The concomitant increase in thyroid pathway activators and inhibitors could highlight a compensatory regulatory mechanism, but more importantly indicates that the thyroid pathway is a key target for embryonic thermal programming in mule duck. In particular, DIO3 has already been identified as a recurrent target of embryonic thermal programming in mule ducks, both at the end of the thermal stimulus or at the end of the overfeeding period [22]. SCD1, CEPT1 and GPAT1 involved in lipid synthesis, or LXRA involved in transcriptional regulation of lipid metabolism [48] have also previously been highlighted as programming targets in the liver of thermally programmed mule ducks [16, 22, 49]. This transcriptional response, induced by the temperature change specifically during the embryonic period corresponding to the peak maturation of liver functions [33], may reflect an adaptation to the metabolic demands observed at this stage and represent an initial mechanism of programming. Besides, although the peak of the transcriptional response to the thermal stimulus is clearly visible three days after the onset of the temperature increase (E16), the expression of several genes is affected throughout embryogenesis and even after hatching (E30). This sustained transcriptional response demonstrates that hepatic metabolism is directly affected by thermal stimulus during embryogenesis, and confirms that metabolic programming is measurable.

## Cell proliferation and epigenetic marks pathways are moderately and transiently activated by thermal manipulation

Finally, the last two groups of genes to be affected by the rise in incubation temperature, predominantly at E20, are those involved in cell proliferation and epigenetics. FGF and HGF are two growth factors that play fundamental role in liver development [50, 51] or liver regeneration [52–55]. Previously, high expression of HGF and FGF receptor 2 (FGFR2) was measured between days 12 and 20 of embryogenesis in mule ducks [33], highlighting their possible activity in the development and growth of liver tissue at this stage. It was suggested that a change in temperature during this period might influence the final number of liver cells in the mature organ, as previously shown in chicken muscle cells [11, 18]. This hypothesis, supported here by a temperature-induced increase in FGFR2 and HGF gene expression, should now be verified by measurements of liver size or cell number. Indeed, although hyperplasia does not appear to contribute to fatty liver enlargement during overfeeding [56], it is conceivable that an increased number of hepatocytes at hatch may promote liver fattening, and then participate to the programming phenotype observed in overfed mule ducks [16, 17].

Then, only 2 out of the 9 tested genes involved in epigenetics mechanisms are affected by the thermal manipulation at E20 and E24. First, *TYMS* is an actor in one-carbon metabolism that provides methyl groups known to be linked to epigenetic mechanisms [57, 58]. The increase in its expression several days after the start of the incubation temperature change could reflect a transient activation of the DNA methylation process that could be involved in the programmed phenotype measured at hatching, but also when ducks are overfed. Secondly, elongator protein 3 (*ELP3*) is a histone acetyltransferase which facilitates transcript elongation by opening access for the RNA polymerase II to numerous genes [59, 60]. In humans, *ELP3* has been shown to regulate *HSP70* gene expression, involved in heat stress response [61], notably through modulation of histone H3 acetylation [62]. Here in duck embryos, we first measured
a decrease in *ELP3* mRNA expression 7 days after the onset of temperature increase, followed by an increase after 11 days, confirming the sensitivity of this histone acetyltransferase to temperature change. However, these changes in *ELP3* gene expression were not accompanied by a similar modulation of *HSP70s* mRNA, suggesting that in ducks, *ELP3* and *HSP70* are not directly linked. To better understand the role of *ELP3* during temperature increases in ducks, it would now be interesting to measure histone acetylation activity under these conditions, as well as later in the animal's life to assess the persistence of these potential epigenetic marks and their actual role in the programmed phenotype observed.

## Conclusion

Although sporadically, all the metabolic pathways suspected of being targets of embryonic thermal programming were affected by the thermal stimulus at different time points during the temperature increase and even up to 3 days afterwards. This study represents only one aspect of the mechanisms involved in metabolic programming induced by an increase in incubation temperature in ducks. Further studies will be required to determine the modulations of protein expression corresponding to the identified mRNAs, enzyme activities, or epigenetic marks. Moreover, the targeted analysis of 'candidate' genes using the Fluidigm technique provides only a partial answer to the question of which metabolic pathways are directly affected by temperature. A more comprehensive approach, such as RNA sequencing, should therefore be considered for future investigations.

However, this study has highlighted for the first time a short-term transcriptional response kinetics of hepatic cells to embryonic thermal stimulus. In particular, it revealed the rapid and sustained induction of heat shock proteins, the activation of a large number of genes involved in metabolism, and the low proportion of genes involved in epigenetic processes affected by temperature. These results could serve as new tools to better understand the mechanisms of embryonic thermal programming.

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12864-025-12192-7.

Supplementary Material 1

Supplementary Material 2

## Acknowledgements

We thank all the technicians of the Experimental Waterfowl Breeding Station (Avipôle, INRAE, Artigüère, France) for their daily care of the animals and for ensuring proper experimental conditions.

## Authors' contributions

**Andrieux C.** contributed to conceptualization, data curation, formal analysis, methodology, validation, visualization, writing—original draft, writing—review & editing. **Biasutti S**. contributed to data curation, formal analysis—original draft **Zwick LL.** contributed to conceptualization, writing—review **Marchand M.** contributed to, data curation, formal analysis—original draft **Panserat S**. contributed to conceptualization, methodology, investigation, validation, visualization, writing—review **Houssier M.** contributed to conceptualization, data curation, formal analysis, methodology, validation, visualization, writing—original draft, writing—review & editing.

## Funding

This work was supported by grants from "Comité interprofessionnel des palmipèdes à Foie Gras (CIFOG- agreement 2020/05) and from "Comité Départemental des Landes" (CD40).

## Data availability

The datasets used and/or analysed during the current study are available in the GEO database under the reference GSE303902 (publicly accessible from August 3, 2025) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303902](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303902).

## Declarations

### Ethics approval and consent to participate

All experimental procedures were in accordance with the French national guidelines for the care of animals for research purposes. The protocols were approved by the committee of the Care and Use of Animals of the Grand Sud-Ouest (n°73) under the file reference APAFIS#25163-2020062311266761 v5.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

### Author details

Received: 19 June 2025 / Accepted: 6 October 2025

Published online: 07 November 2025

## References

1. Lucas A. Programming by early nutrition: an experimental approach. J Nutr. 1998;128(2):4015-4065.

2. Vaiserman A, Koliada A, Lushchak O. Developmental programming of aging trajectory. Ageing Res Rev. 2018;47:105-22.

3. Andrieux C, Petit A, Collin A, Houssier M, Métayer-Coustard S, Panserat S, et al. Early phenotype programming in birds by temperature and nutrition: a mini-review. Front Anim Sci. 2022;2:755842.

4. Halevy O, Piestun Y, Rozenboim I, Yablonka-Reuveni Z. In ovo exposure to monochromatic green light promotes skeletal muscle cell proliferation and affects myofiber growth in posthatch chicks. Am J Physiol-Regul Integr Comp Physiol. 2006;290(4):R1062-70.

5. Riaz MF, Mahmud A, Hussain J, Saima. Impact of dichromatic lighted incubation on hatching result and post-hatch performance of broiler chickens. Trop Anim Health Prod. 2024;56(4):146.

6. Rozenboim I, Biran I, Chaiseha Y, Yahav S, Rosenstrauch A, Sklan D, et al. The effect of a green and blue monochromatic light combination on broiler growth and development. Poult Sci. 2004;83(5):842-5.

7. Han G, Yang H, Bungo T, Ikeda H, Wang Y, Nguyen LTN, et al. In ovo L-leucine administration stimulates lipid metabolisms in heat-exposed male, but not female, chicks to afford thermotolerance. J Therm Biol. 2018;71:74-82.

<footnote n="1">¹Université de Pau et des Pays de L'Adour, INRAE, NUMEA, Mont de Marsan 40000, France</footnote>

<footnote n="2">²Université de Pau et des Pays de L'Adour, IUT Génie Biologique, Mont de Marsan 40000, France</footnote>
8. Kalantar M, Hosseini SM, Hosseini MR, Kalantar MH, Farmanullah, Yang LG. Effects of in ovo injection of coenzyme Q10 on hatchability, subsequent performance, and immunity of broiler chickens. *BioMed Res Int.* 2019;2019:1-8.

9. Piestun Y, Druyan S, Brake J, Yahav S. Thermal manipulations during broiler incubation alter performance of broilers to 70 days of age. *Poult Sci.* 2013;92(5):1155-63.

10. Piestun Y, Halevy O, Shinder D, Ruzal M, Druyan S, Yahav S. Thermal manipulations during broiler embryogenesis improves post-hatch performance under hot conditions. *J Therm Biol.* 2011;36(7):469-74.

11. Halevy O, Krispin A, Leshem Y, McMurtry JP, Yahav S. Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks. *Am J Physiol Regul Integr Comp Physiol.* 2001;281(1):R302-309.

12. Jha R, Singh AK, Yadav S, Berrocoso JFD, Mishra B. Early nutrition program- ming (in ovo and Post-hatch Feeding) as a strategy to modulate gut health of poultry. *Front Vet Sci.* 2019;6:82.

13. Taha-Abdelaziz K, Hodgins DC, Lammers A, Alkie TN, Sharif S. Effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: A review. *Vet Immunol Immuno- pathol.* 2018;201:1-11.

14. Al-Rukibat RK, Al-Zghoul MB, Hananeh WM, Al-Natour MQ, Abu-Basha EA. Thermal manipulation during late embryogenesis: effect on body weight and temperature, thyroid hormones, and differential white blood cell counts in broiler chickens. *Poult Sci.* 2017;96(1):234-40.

15. Al-Zghoul MB, Dalab AES, Yahya IE, Althnaian TA, Al-ramadan SY, Ali AM, et al. Thermal manipulation during broiler chicken embryogenesis: Effect on mRNA expressions of Hsp108, Hsp70, Hsp47 and Hsf-3 during subsequent post-hatch thermal challenge. *Res Vet Sci.* 2015;103:211-7.

16. Andrieux C, Marchand M, Larroquet L, Veron V, Biasutti S, Morisson M, et al. Optimization of embryonic thermal programming confirms increased liver fattening in mule ducks and changes in lipid metabolism. *Front Physiol.* 2023;14:1142398.

17. Massimino W, Davail S, Bernadet MD, Pioche T, Tavernier A, Ricaud K, et al. Positive impact of thermal manipulation during embryogenesis on foie gras production in mule ducks. *Front Physiol.* 2019;10:1495.

18. Piestun Y, Harel M, Barak M, Yahav S, Halevy O. Thermal manipulations in late-term chick embryos have immediate and longer term effects on myoblast proliferation and skeletal muscle hypertrophy. *J Appl Physiol.* 2009;106(1):233-40.

19. Vinoth A, Thirunalasundari T, Shanmugam M, Uthrakumar A, Suji S, Raj- kumar U. Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken. *Cell Stress Chaperones.* 2018;23(2):235-52.

20. Saleh KM, Al-Zghoul MB. Thermal Manipulation during Broiler Chicken Embryogenesis Modulates the Splenic Cytokines' mRNA Expression. *Jordan Journal of Biological Sciences.* 2019;595-601. Volume 12, Number 5, December 2019 ISSN 1995-6673 Pages 595-601

21. Saleh KMM, Tarkhan AH, Al-Zghoul MB. Embryonic Thermal manipulation affects the antioxidant response to post-hatch thermal exposure in broiler chickens. *Animals.* 2020;10(1):126.

22. Massimino W, Andrieux C, Biasutti S, Davail S, Bernadet MD, Pioche T, et al. Impacts of embryonic thermal programming on the expression of genes involved in foie gras production in mule ducks. *Front Physiol.* 2021;12:779689.

23. Al-Zghoul MB, Ismail ZB, Dalab AES, Al-Ramadan A, Althnaian TA, Al-ramadan SY, et al. Hsp90, Hsp60 and HSF-1 genes expression in muscle, heart and brain of thermally manipulated broiler chicken. *Res Vet Sci.* 2015;99:105-11.

24. Loyau T, Métayer-Coustard S, Berri C, Crochet S, Cailleau-Audouin E, Sannier M, et al. Thermal manipulation during embryogenesis has long-term effects on muscle and liver metabolism in fast-growing chickens. *Wu SB*, éditeur. *PLoS ONE.* 2014;9(9):e105339.

25. Ministère de l'agriculture, de l'agroalimentaire et de la forêt. Arrêté du 1er février 2013 fixant les conditions d'agrément, d'aménagement et de fonctionnement des établissements utilisateurs, éleveurs ou fournisseurs d'animaux utilisés à des fins scientifiques et leurs contrôles. *Journal Officiel De La République Française.* 2013. https://www.legifrance.gouv.fr/loda/id/JO RFTEXT000027037983/

26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. *Methods.* 2001;25(4):402-8.

27. Andrieux C, Biasutti S, Barrieu J, Morganx P, Morisson M, Coustham V, et al. Identification of different critical embryonic periods to modify egg incubation temperature in mule ducks. *Animal.* 2022;16(1):100416.

28. Loyau T, Berri C, Bedrani L, Metayer-Coustard S, Praud C, Duclos MJ, et al. Thermal manipulation of the embryo modifies the physiology and body

composition of broiler chickens reared in floor pens without affecting breast meat processing quality. *J Anim Sci.* 2013;91(8):3674-85.

29. Cedraz H, Gromboni JGG, Garcia AAP, Farias Filho RV, Souza TM, Oliveira ER de, et al. Heat stress induces expression of HSP genes in genetically divergent chickens. *Gallyas F*, éditeur. *PLOS ONE.* 2017;12(10):e0186083.

30. Farcy E, Voiseux C, Lebel JM, Fiévet B. Transcriptional expression levels of cell stress marker genes in the Pacific oyster Crassostrea gigas exposed to acute thermal stress. *Cell Stress Chaperones.* 2009;14(4):371-80.

31. Visala Rao D, Boyle GM, Parsons PG, Watson K, Jones GL. Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes. *Mech Ageing Dev.* 2003;124(1):55-69.

32. Christensen VL. Development during the first seven days post-hatching. *Avian Biol Res.* 2009;2(1-2):27-33.

33. Massimino W, Davail S, Secula A, Andrieux C, Bernadet MD, Pioche T, et al. Ontogeny of hepatic metabolism in mule ducks highlights different gene expression profiles between carbohydrate and lipid metabolic pathways. *BMC Genomics.* 2020;21(1). Disponible sur: https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-020-07093-w. Cité 23 Sept 2021.

34. Whittow GC, Tazawa H. The early development of thermoregulation in birds. *Physiol Zool.* 1991;64(6):1371-90.

35. Feder ME, Hofmann GE. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. *Annu Rev Physiol.* 1999;61(1):243-82.

36. Lindquist S. The heat-shock response. *Annu Rev Biochem.* 1986;55(1):1151-91.

37. Mazaira GI, Daneri-Becerra C, Zgajnar NR, Lotufo CM, Galigniana MD. Gene expression regulation by heat-shock proteins: the cardinal roles of HSF1 and Hsp90. *Biochem Soc Trans.* 2018;46(1):51-65.

38. Prodromou C. Mechanisms of Hsp90 regulation. *Biochem J.* 2016;473(16):2439-52.

39. Mizushima N. Autophagy: process and function. *Genes Dev.* 2007;21(22):2861-73.

40. Ravanan P, Srikumar IF, Talwar P. Autophagy: the spotlight for cellular stress responses. *Life Sci.* 2017;188:53-67.

41. Wigley P, Kaiser P. Avian cytokines in health and disease. *Rev Bras Ciênc Avicola.* 2003;5(1):1-14.

42. Astapova I. Role of co-regulators in metabolic and transcriptional actions of thyroid hormone. *J Mol Endocrinol.* 2016;56(3):R73-97.

43. Darras VM, Verhoelst CHJ, Reyns GE, Kühn ER, der Geyten SV. Thyroid hormone deiodination in birds. *Thyroid.* 2006;16(1):25-35.

44. Mendoza A, Hollenberg AN. New insights into thyroid hormone action. *Pharmacol Ther.* 2017;173:135-45.

45. Katoh M, Katoh M. Comparative integromics on JMJD1C gene encoding his- tone demethylase: conserved POU5F1 binding site elucidating mechanism of JMJD1C expression in undifferentiated ES cells and diffuse-type gastric cancer. *Int J Oncol.* 2007;31(1):219-23.

46. Katoh M, Katoh M. Identification and characterization of TRIP8 gene in silico. *Int J Mol Med.* 2003;12(5):817-21.

47. Lynch JR, Salik B, Connerty P, Vick B, Leung H, Pijning A, et al. JMJD1C-medi- ated metabolic dysregulation contributes to HOXA9-dependent leukemo- genesis. *Leukemia.* 2019;33(6):1400-10.

48. Ulven SM, Dalen KT, Gustafsson JÅ, Nebb HI. LXR is crucial in lipid metabo- lism. *Prostaglandins Leukot Essent Fatty Acids.* 2005;73(1):59-63.

49. Andrieux C, Marchand M, Larroquet L, Veron V, Biasutti S, Barrieu J, et al. Fast- ing/refeeding: an experimental model to study the impact of early thermal manipulation on hepatic metabolism in mule ducks. *Am J Physiol-Regul Integr Comp Physiol.* 2023;324(1):R45-57.

50. Shin D, Monga SPS. Cellular and molecular basis of liver development. *In: Prakash YS*, éditeur. *Comprehensive Physiology. 1re* éd. Wiley; 2013. p. 799-815. Disponible sur: https://onlinelibrary.wiley.com/doi/10.1002/cphy.c1 20022. Cité 30 Juill 2025.

51. Zaret KS. Liver specification and early morphogenesis. *Mech Dev.* 2000;92(1):83-8.

52. Böhm F, Köhler UA, Speicher T, Werner S. Regulation of liver regeneration by growth factors and cytokines. *EMBO Mol Med.* 2010;2(8):294-305.

53. Michalopoulos GK. Liver regeneration. *J Cell Physiol.* 2007;213(2):286-300.

54. Padrissa-Altes S, Bachofner M, Bogorad RL, Pohlmeier L, Rossolini T, Böhm F, et al. Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice. *Gut.* 2015;64(9):1444-53.

55. Speicher T, Siegenthaler B, Bogorad RL, Ruppert R, Petzold T, Padrissa-Altes S, et al. Knockdown and knockout of β1-integrin in hepatocytes impairs liver
regeneration through inhibition of growth factor signalling. Nat Commun. 2014;5(1):3862.

56. Benard G, Bengone T, Prehn D, Durand S, Labie C, Benard P. Physiology of ducks during force-feeding: study of hepatic steatosis. Bull Académie Vét Fr. 2006. Tome 159 - N°1 www.academie-veterinaire-france.fr https://agris.fao.org/search/en(providers/122415/records/647367a853aa8c89630cdc82

57. Clare CE, Brassington AH, Kwong WY, Sinclair KD. One-carbon metabolism: linking nutritional biochemistry to epigenetic programming of long-term development. Annu Rev Anim Biosci. 2019;7(1):263-87.

58. Coppèdè F, Stoccoro A, Tannorella P, Migliore L. Plasma homocysteine and polymorphisms of genes involved in folate metabolism correlate with DNMT1 gene methylation levels. Metabolites. 2019;9(12):298.

59. Abo-Ismail MK, Vander Voort G, Squires JJ, Swanson KC, Mandell IB, Liao X, et al. Single nucleotide polymorphisms for feed efficiency and performance in crossbred beef cattle. BMC Genet. 2014;15(1):14.

60. Wittschieben BØ, Otero G, De Bizemont T, Fellows J, Erd judgment-Bromage H, Ohba R, et al. A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol Cell. 1999;4(1):123-8.

61. Flanagan SW, Ryan AJ, Gisolfi CV, Moseley PL. Tissue-specific HSP70 response in animals undergoing heat stress. Am J Physiol-Regul Integr Comp Physiol. 1995;268(1):R28-32.

62. Han Q, Hou X, Su D, Pan L, Duan J, Cui L, et al. hELP3 subunit of the elongator complex regulates the transcription of HSP70 gene in human cells. Acta Biochim Biophys Sin. 2007;39(6):453-61.

63. Piestun Y., Shinder D., Ruzal M., Halevy O., Brake J. and Yahav S. Thermal Manipulations During Broiler Embryogenesis: Effect on the Acquisition of Thermotolerance. 2008 Poultry Science 87:1516-1525 https://doi.org/10.3382/ps.2008-00030

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.