# Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick

Elske N. de Haas, Ludovic Calandreau, Elisabeth Baéza, Pascal P. Chartrin, Rupert Palme, Anne-Sophie Darmaillacq, Ludovic Dickel, Sophie Lumineau, Cécilia Houdelier, Isabelle Denis, et al.

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## To cite this version:

Elske N. de Haas, Ludovic Calandreau, Elisabeth Baéza, Pascal P. Chartrin, Rupert Palme, et al. Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick. Developmental Psychobiology, 2017, 999 (3), pp.1-10. 10.1002/dev.21504. hal-01483726

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Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick

<table><tr><td>Journal:</td><td>Developmental Psychobiology</td></tr><tr><td>Manuscript ID</td><td>DEV-16-124.R1</td></tr><tr><td>Wiley - Manuscript type:</td><td>Research Article</td></tr><tr><td>Date Submitted by the Author:</td><td>n/a</td></tr><tr><td>Complete List of Authors:</td><td>de Haas, Elske; INRA-CNRS-Université de Tours François Rabelais, INRA Val de Loire<br>Calandreau, Ludovic; INRA-CNRS-Université de Tours François Rabelais, INRA Val de Loire<br>Baéza, Elisabeth; INRA Centre Val de Loire, Unité de Recherches Avicoles<br>Chartrin, Pascal; INRA Centre Val de Loire, Unité de Recherches Avicoles<br>Palme, Rupert; University of Veterinary Medecine, 1210 Vienna, Unit of Physiology, Pathophysiology, and Experimental Endocrinology, Institute of Medical biochemistry, Departement of Biomedical Sciences<br>Darmaillacq, Anne-Sophie; University of caen Basse-Normandy, Groupe Memoire et plasticité comportementale<br>Dickel, Ludovic; University of caen Basse-Normandy, Groupe Memoire et plasticité comportementale<br>Lumineau, Sophie; University of Rennes 1, UMR CNRS 6552 Ethologie animale et humaine<br>Houdelier, Cecilia; University of rennes, UMR6552; Denis, Isabelle; INRA-U1197, Unité de Neurobiologie de l'Olfaction<br>Arnould, Cécile; INRA-CNRS-Université de Tours François Rabelais, INRA Val de Loire<br>Meurisse, Maryse; INRA-CNRS-Université de Tours François Rabelais, UMR 85 Physiologie de la Reproduction et des Comportements<br>Bertin, Aline; INRA-CNRS-Université de Tours François Rabelais, INRA Val de Loire</td></tr><tr><td>Keywords:</td><td>Gallus gallus domesticus, maternal effects, yolk hormones, poly unsaturated fatty acids, neophobia</td></tr></table>
1 Lipids in maternal diet influence yolk hormone levels and post-hatch neophobia in the domestic chick.

3 Elske N. de Haas¹,²,³,⁴, Ludovic Calandreau¹,²,³,⁴, Elisabeth Baéza⁵, Pascal Chartrin⁵, Rupert

4 Palme⁶, Anne-Sophie Darmaillacq⁷, Ludovic Dickel⁷, Sophie Lumineau⁸, Cécilia Houdelier⁸,

5 Isabelle Denis⁹, Cécile Arnould¹,²,³,⁴, Maryse Meurisse¹,²,³,⁴, Aline Bertin¹,²,³,⁴

6

7 ¹INRA Centre Val de Loire, UMR85 Physiologie de la Reproduction et des Comportements,

8 Laboratoire de Comportement, Neurobiologie et Adaptation, 37380 Nouzilly, France ; ²

9 CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, 37380 Nouzilly,

10 France; ³Université François Rabelais de Tours, 37041, Tours, France; ⁴Institut Français du

11 Cheval et de l'Equitation, 37380, Nouzilly, France; ⁵INRA Centre Val de Loire, Unité de

12 Recherches Avicoles, 37380 Nouzilly, France ; ⁶Unit of Physiology, Pathophysiology, and

13 Experimental Endocrinology, Institute of Medical Biochemistry, Department of Biomedical

14 Sciences, University of Veterinary Medicine, 1210 Vienna, Austria ; ⁷Groupe Mémoire et

15 Plasticité comportementale, EA 4259, Université de Caen Normandie UNICAEN, GMPc

16 EA4259, Caen, France ; ⁸UMR CNRS 6552, Ethos, Université de Rennes1, Rennes, France; ⁹

17 INRA, Unité de Neurobiologie de l'Olfaction (NBO, INRA-U1197), 78352 Jouy-en-Josas,

18 France

19 Correspondence to be sent to: aline.bertin@tours.inra.fr

20 Equipe " Comportement, Neurobiologie et Adaptation"

21 INRA Val de Loire, UMR 85 Physiologie de la Reproduction et des Comportements

22 CNRS-UMR 7247 - Université de Tours - IFCE

23 F-37380 NOUZILLY FRANCE

24

25 Running title: maternal diet engenders maternal effects

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We assessed whether the ratio of dietary n-6/n-3 polyunsaturated fatty acids (PUFA) during egg formation engenders transgenerational maternal effects in domestic chicks. We analysed yolk lipid and hormone concentrations, and HPA-axis activity in hens fed a control diet (high n-6/n-3 ratio) or a diet enriched in n-3 PUFAs (low n-6/n-3 ratio) for six consecutive weeks. Their chicks were tested for neophobia during the first week of life. We found higher corticosterone metabolites in droppings of hens fed the diet enriched in n-3 and significantly higher concentrations of yolk progesterone, androstenedione and estradiol in their eggs compared to controls. Chicks of hens fed the n-3 enriched diet showed a lower body mass at hatch than controls and expressed higher neophobia when exposed to a novel object. These results add support to the hypothesis that the nutritional state of female birds produces variation in yolk hormone levels and engender maternal effects.

**Keywords:** *Gallus gallus domesticus*, maternal effects, yolk hormones, poly unsaturated fatty acids, neophobia
52 Understanding the reasons for phenotypic variation is one of the cornerstones of behavioural biology. Non-genetic maternal effects play a key role in generating phenotypic variation in many taxa (Ledón-Rettig, Richards, & Martin, 2013). In birds, prenatal maternal effects can affect the morphology, behaviour and physiology of offspring (Ruuskanen, 2015).
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56 Such effects are partly mediated via variation in yolk-hormone concentrations of maternal origin according to different environmental conditions (Groothuis, Muller, von Engelhardt, Carere, & Eising, 2005). Yolk androgens (mainly testosterone and its precursor androstenedione) have been widely studied in wild bird populations. Differential allocation of androgens into eggs is presumed to be a way to adapt offspring to their hatching environment (Gil, 2008; Groothuis, et al., 2005). Androgen concentrations can vary substantially within and between egg clutches as a consequence of the maternal environment; factors such as social conditions (density, aggressive interactions, mate quality) (Kingma et al., 2009; Pilz & Smith, 2004; Schwabl, 1997), parasitic infection (Müller, Heylen, Eens, Rivera-Gutierrez, & Groothuis, 2013) and predation risk (Coslovsky, Groothuis, de Vries, & Richner, 2012) can influence hormone production. Because yolk hormone levels show such a strong context-dependency within species (Ruuskanen, 2015), the specific environmental cues triggering maternal effects must be elucidated further.

69 Recently, food resource availability has been identified as an environmental factor engendering differential maternal hormone allocation. In the lesser black-backed gull (*Larus fuscus*), pied flycatcher (*Ficedula hypoleuca*) and black-legged Kittiwake (*Rissa tridactyla*) food supplementation prior to, and during oviposition affected androgen quantity in the yolk (Verboven et al., 2003; Verboven, Monaghan, Nager, & Evans, 2010). While in great tits (*Parus major*), no effect of food supplementation on yolk androgens has been recorded (S. Ruuskanen, Darras, de Vries, Visser, & Groothuis, 2016). Manipulation of food resources also shows to increase the within clutch variation of yolk-hormones in canaries (*Serinus*
77 *canaria*) (Jonas Vergauwen, Goerlich, Groothuis, Eens, & Müller, 2012) and zebra finches

78 (*Taeniopygia guttata*) (Sandell, Adkins-Regan, & Ketterson, 2007). These studies

79 demonstrate that the nutritional state of maternal birds during oviposition contributes to

80 differences in yolk hormone concentrations between or within a clutch. In the reported

81 studies, enrichment of the maternal diet was achieved by providing additional protein and/or

82 lipids from different sources (e.g. seeds, boiled eggs). This procedure leaves open the question

83 on the influence of the nutritional composition of the food provided to the mother. To our

84 knowledge the effect of the quality of maternal diet on egg hormone levels and offspring

85 phenotype hasn't been investigated so far in birds.

This question is of particular importance for factory-farmed birds which are fed an unique diet. The domestic chicken (*Gallus gallus domesticus*) is the most abundant bird species on the earth but, despite this importance, many avenues of research remained to be explored in order to advance our knowledge on maternal effects and behavioral development (Dixon, Sparks, & Rutherford, 2016). Housing conditions (Janczak, Torjesen, & Rettenbacher, 2009), unpredictable access to food (Janczak, Torjesen, Palme, & Bakken, 2007; Nätt et al., 2009), maternal social status (Muller, Eising, Dijkstra, & Groothuis, 2002), early social stress (Goerlich, Nätt, Elfwing, Macdonald, & Jensen, 2012) and thermal environment (Bertin et al., 2013) cause variation in yolk androgens and progesterone levels in domestic hens. This caused modifications in growth, feeding behaviors, and emotional reactivity of their chicks (Goerlich, Nätt, Elfwing, Macdonald, & Jensen, 2012; Nätt et al., 2009; Bertin et al., 2013). In domestic hens, maternal stress during egg formation —mimicked with subcutaneous corticosterone implantation— increased plasma corticosterone levels and decreased the synthesis of reproductive hormones which accumulate in the yolk (Henriksen, Groothuis, & Rettenbacher, 2011). In addition, on-farm longitudinal observations showed that parental stress physiology correlate with offspring's anxiety and expression of damaging
102 behavior (de Haas, Bolhuis, Kemp, Groothuis, & Rodenburg, 2014; Rodenburg & de Haas, 2016).

104 In domestic hens we previously showed that adding fish oil to the maternal diet induced food neophobia in the offspring (Aigueperse, Calandreau, & Bertin, 2013). As fish oil is particularly rich in n-3 poly-unsaturated fatty acids (PUFA), this result suggested that PUFA in the maternal diet may directly affect the development of behaviour in the offspring.
108 The aim of the present study was to test this hypothesis and assess whether the ratio between n-6 and n-3 PUFA in the maternal diet during egg formation could engender maternal effects.
110 N-3 and n-6 PUFA unambiguously influence the development of the central nervous system in both mammalian and avian species (Noble & Cocchi, 1989). But these effects are better-understood for mammals. In mammals, PUFAs and their mediators are known to regulate many processes within the brain, such as neurotransmission, cell survival and neuro-inflammation, and also affect hypothalamic-pituitary-adrenal (HPA) axis responses, thereby possibly playing a role in anxiety-like behavior and cognition (e.g. Bazinet & Layé, 2014; Lafourcade et al., 2011). In the domestic chicken, PUFA ratios in the maternal diet are transferred to the egg yolk (Noble & Cocchi, 1990) and subsequently to the tissues of the developing chick embryo (Maldjian, Farkas, Noble, Cocchi, & Speake, 1995). Numerous studies show that the PUFA profile of the developing chick brain reflects the PUFA composition in the yolk (Cherian & Sim, 1993; Anderson, Connor, Corliss, & Lin, 1989) but the consequences on behavioral development remain to be investigated.

122 We analysed yolk lipid and hormone concentrations, and HPA-axis activity in hens fed a control diet (high n-6/n-3 ratio) or a diet enriched in n-3 PUFAs (low n-6/n-3 ratio).
124 Development and neophobia (i.e. fear of novelty) of the hatchlings were analysed. Neophobia is considered to be a temperament trait which can impair an animal's capacity to adapt to new resources, new habitats and express behavioural innovation (Greenberg & Mettke-hofmann,
127 2001; Webster & Lefebvre, 2001). As a high level of neophobia can reduce fitness (Brown, Ferrari, Elvidge, Ramnarine, & Chivers, 2013), this behavioural trait is a growing topic of interest for behavioural biologists (Camín, Martín-Albarracín, Jefferies, & Marone, 2016; Greggor, Thornton, & Clayton, 2015). The precocial domestic chick is a particularly appropriate model in which to analyse developmental influences on neophobia since chicks start food selection soon after hatching (Bertin et al., 2010). In addition, under commercial conditions the refusal to accept new foods leads to a major reduction in feed intake and, subsequently, in growth and animal welfare (Murphy, 1977).

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## 136 Methods

### 137 *Ethics statement*

138 All birds were maintained at the Experimental Unit PEAT of INRA (Nouzilly, France). The Experimental Unit is registered by the ministry of Agriculture with the license number B-37-175-1 for animal experimentation. All experiments were approved by the Ethic Committee in Animal Experimentation of Val de Loire, CEEA Vdl (reference number 02153.02). The CEEA vdl is registered by the National Committee "Comité National de Réflexion Ethique sur l'Expérimentation Animale" under the number 19. All experiments were performed in accordance with the European Communities Council Directive 2010/63/UE. All animals were sold for rehabilitation at the end of the experiment.

### 146 *Maternal hens and diets*

147 Hens (n=40) and diets were provided by the experimental unit PEAT of INRA (Nouzilly, France). Two groups of 20 one-year old White Leghorn laying hens were matched for body weight, and divided into two dietary treatment groups, a control diet and a diet enriched in n-3 FA. Diets were administered for six consecutive weeks. The control diet contained soya-oil
151 (3%) with a n-6:n-3 ratio of 7.99, a value within the range recommended for commercial laying hens (Van Elswyk, 1997). The n-3 diet contained 3% menhaden fish-oil (Sigma Life Science) having an n-6:n-3 ratio of 0.82. Diets were iso-caloric (2820 kcal ME/kg) and iso-nitrogenous (170 g CP/kg), differing only in the composition of PUFAs (electronic supplementary material, table S1). Hens were housed individually in wire-mesh cages of 100 x 100 x 50 cm (l x w x h), with the possibility of tactile, visual, and vocal contact with neighbouring hens. Cages contained wood shavings and a wooden perch on the floor, a nest enclosed by red curtain flaps, a nipple drinker and a feed trough. Water and food were provided ad libitum. Four hens were excluded from the experiment, three due to a lack of habituation to the experimental diets (food conservatism) and one death unrelated to the diet treatment, resulting in n =18 hens per diet group.

162 *Morpho-physiological measurements of hens*

163 All hens were weighed once per week. 24 h-feed intake per hen was measured once a week by calculating the difference in weight of the feeder before and after 24 h. The number of eggs laid was recorded daily. Laying rate was calculated as a percentage of the number of eggs laid per female per day.

167 In order to evaluate HPA activity in chickens, faecal corticosterone metabolite (FCM) concentrations were measured (Rettenbacher, Mostl, Hackl, Ghareeb, & Palme, 2004). In the sixth week of diet treatment, one fresh faecal dropping per hen was collected from the home cage. Urine was removed from the sample as the concentrations of FCM in the liquid portions of droppings are more likely to represent acute HPA activity caused by handling or disturbing the animals during collection (Rettenbacher, et al., 2004). Each sample was homogenized and stored at -20°C. From each sample an aliquot (0.5 g) was extracted with 60% methanol
174 (Mostl, Spendier, & Kotrschal, 2001) and analysed by a cortisone enzyme immunoassay

175 (EIA) validated for chickens and fully described in (Rettenbacher, et al., 2004).

176 *Analysis of egg quality, yolk hormones and yolk lipids*

177 As the vitellogenesis has an average length of 8 days (Lacassagne, 1960), we collected eggs

178 for hormonal and lipid assays during the fifth and sixth weeks of treatment to ensure hens

179 were fully habituated to the diets, and that differences in egg components were a result of the

180 maternal diet treatments. One egg per female was weighed and stored at -20°C for hormonal

181 assay. Eggshells were separated and dried for 24 h and weighed. Frozen yolk was separated

182 from the albumen and weighed. The weight of albumen was calculated by subtracting the

183 weight of the eggshell and yolk from that of the whole egg. We then determined the ratio of

184 each component relative to the egg mass (yolk mass / egg mass; albumen mass / egg mass;

185 shell mass / egg mass) for each female. A total of 18 eggs were collected per group. The

186 concentrations of immunoreactive progesterone, testosterone, androstenedione and oestrogens

187 were analysed by EIAs. Details of the extraction protocol are found in (Guesdon et al., 2011).

188 For a full description of the assays including specific antibodies, see (Palme, Touma, Arias,

189 Dominchin, & Lepschy, 2013). Intra- and inter-assay coefficients of variation were less than

190 10% and 15%, respectively.

191 In order to measure yolk lipid content, three yolks per female were pooled and homogenized.

192 We then determined the total lipid content of the yolk and the percentages of saturated, mono-

193 unsaturated and poly-unsaturated FAs within the lipid fraction. Yolk lipid analysis was

194 conducted by trans-methylation with gas chromatography (Perkin Elmer Autosystem, St.

195 Quentin en Yvelines, France) following the protocol described in (Chartrin, Berri, Lebihan-

196 Duval, Quentin, & Baéza, 2005).
197 During the fourth and fifth week of treatment, hens were artificially inseminated three times (same mix of sperm for all the hens). Two hundred and thirty nine eggs (mean of 5.6 ± 0.4 eggs per female) produced offspring. Eggs were incubated at 37.8°C with 56% relative humidity for 21 days.

201 *Chicks and housing conditions*

202 Forty-eight chicks from hens fed the n-3 enriched diet (n-3 chicks) and 50 control chicks (C chicks) were used in this experiment. Chicks were identified with a metal leg-ring. Chicks were housed in non-sibling pairs but with birds from the same diet treatment. We chose not to mix treatments within each cage as potential unknown differences in chicks' social or emotional behaviour between treatments may mask prenatal effects. Chicks were housed in plastic cages (50 cm x 40 cm x 30 cm) placed in rows with treatments in alternation within a row (two adjacent cages were from different treatments). All the cages were equally distributed between two identical rooms. Cages had wood shavings on the floor and a wire-mesh lid. Food and water were provided *ad libitum*. Chicks were fed a postnatal starter mash with a PUFA ratio similar to the maternal diet. We used this diet so that the PUFA ratio of the chick's diet would not counteract the effects induced by the maternal diet on offspring's brain PUFA levels. This is especially important since precocial birds adapt their feeding behaviour to their mother's diet choices (Hess, 1964). Moreover, when the chick's brain experiences a deficiency in PUFAs due to restriction of the maternal diet, it is able to compensate for this deficiency by altering lipid metabolism and demonstrating preferential uptake of the PUFA lacking in the diet (Anderson, Van Winkle, & Connor, 1992). As chicks rely on the vitelline sac reserve for 2/3 days after hatching, any phenotypic differences during the first days of life between chicks of both maternal diets could therefore be attributed to maternal diet effects. The n-3 diet administered to experimental chicks had an n-6:n-3 ratio of 1.03, and the control diet had an n-6:n-3 ratio of 8.15. Both diets were iso-caloric (2900 kcal ME/kg) and iso-
222 nitrogenous (207.2 g CP/kg) differing solely in PUFA composition (electronic supplementary material, table S1). To determine the sex we looked for the development of the comb at 23 days of age. The body weight of each chick was measured at hatching and at seven days of age.

226 *Behavioural characterization of chicks*

227 To characterize neophobia, behavioural responses to two novel foods and a novel object were assessed with a protocol previously described by (Bertin et al., 2015). Each test was executed at the same age for all chicks. All tests lasted 180 s.

230 Because chicks become distressed when socially isolated (Hocking, Haldane, Davidson, Sandøe, & Kristensen, 2015), we tested cage mates together (n = 24 pairs of n-3 chicks and n = 25 pairs of C chicks). Testing took place in a separate room within an experimental cage that had the same features as their home cage. Testing commenced 90 min after the feeder was removed from their home cage. Pairs were transported to the test room in a 15 cm × 15 cm × 15 cm plastic container, and were gently deposited in an opaque enclosure (20 cm × 6 cm × 20 cm) within the testing cage, opposite the feeding trough. After 30 s, the enclosure was lifted and removed while chicks remained inside the testing cage. The behaviour of one marked focal chick of each pair was recorded by an unseen observer. Focal chicks were chosen randomly at two days of age and were tagged with a blue-coloured mark on the head. The latency (s) to eat (the moment swallowing was observed) and time spent eating (s) were recorded.

242 (i) *Habituation test/familiarization*: at three days of age, chicks were familiarized with the testing cage and handling procedure. As chicks rely on their vitelline sac reserve the first days of life, this test also measured food motivation. The metallic feeding trough of their home cage was placed in the test cage, filled with 200 g of their usual diet.
246 (ii) *Food neophobia tests*: we used these tests to assess the immediate reaction to novel foods.

247 Chicks were exposed to changes in sensory properties of the food. At four days of age, the

248 first test was done with cracked corn-wheat (cereals contained in familiar pellets). Millet

249 seeds (a novel food) was used at five days of age for the second test.

250 (iii) *Object neophobia test*: chicks were tested at six days of age. The novel object was an

251 unfamiliar feeder containing the familiar diet of the test pair.

252 **Statistical analysis**

253 Data were analyzed with Statview (SAS Institute Inc., Cary, NC). The distribution of

254 variables was tested with a Kolmogorov-Smirnov test. Hen and egg data were normally

255 distributed. Groups were compared using a t-test (FCM, mass of egg components, yolk-

256 hormones, yolk-PUFA concentrations) or repeated measures ANOVA (hen body weight, food

257 intake and laying rate) with the main effect of diet, time, and the interaction between time and

258 diet. The number of chicks hatched (hatching success) and sex ratio between diet groups were

259 compared with a Chi-square test. The weight of chicks at hatching and their growth rate

260 during the first week were analyzed via ANOVA. Offspring behavioural data did not follow a

261 normal distribution even after transformation, so the non-parametric Mann-Whitney *U*-test

262 was used to compare groups. Data are presented as mean ± SEM, with significance accepted

263 at *p* < 0.05.

264

265 **Results**

266 *Dietary effects on laying performance of hens*
267 After five weeks of consuming their respective diets, n-3 enriched hens had significantly higher FCM levels than hens of the C diet (398 ± 57 ng/g vs. 99 ± 19 ng/g, t = 2.98, p = 0.006).

270 No effect of diet or interaction between diet and time were observed for body weight, 24h-food intake, or laying rate (see Table 1). For food intake, a time effect was observed (time: $F_{4,33} = 15.3, p < 0.01$). Hen food intake was lower during the first week compared to the second week for both diets (Table 1).

274 >>> INSERT Table 1 <<<

275 *Dietary effects on egg components, yolk hormones and yolk lipids*

276 The eggs of n-3 hens were significantly lighter than those of C hens (54.9 ± 1.25 g vs. 58.0 ± 0.86 g, t = -2.11, p = 0.04). N-3 eggs had a significantly lower yolk mass/egg mass ratio (0.28 ± 0.04 vs. 0.29 ± 0.06, t = 2.47, p = 0.02), and higher albumin mass/egg mass ratio (0.64 ± 0.05 vs. 0.62 ± 0.04, t = -2.07, p = 0.05) than C eggs. Eggshell mass/egg mass ratio did not differ between diets (0.09 ± 0.02 for both diets, t = -0.41, p = 0.68).

281 Compared to C hens, the egg yolk of n-3 hens contained a significantly higher concentration of immunoreactive progesterone (t = 2.96, p = 0.006), oestrogens (t = 5.97, p < 0.0001) and androstenedione (t = 2.01, p = 0.05) with a trend in the same direction for testosterone (t = 1.68, p = 0.10; Figure 1). If instead of yolk hormone concentrations (ng/g) we analyse hormone contents (ng/yolk), the results remained the same except for androstenedione (n-3 eggs vs. n-6 eggs: progesterone: 17897.2 ± 2241.07 ng vs. 12042.04 ± 1635.8 ng, t = 2.1, p = 0.04; oestrogens: 162.5 ± 4.9 ng vs. 134.05 ± 5.4 ng, t = 3.85, p = 0.0005; androstenedione: 785.6 ± 75.06 ng vs. 641.7 ± 94.16 ng, t = 1.2, p = 0.23; testosterone: 42.7 ± 4.7 ng vs. 30.4 ± 5.2 ng, t = 1.77, p = 0.08).
290 The percentage of total lipid content in the yolk did not differ between diets (34.7 ± 0.6 % vs.

291 33.1 ± 1.0%, t = 1.36, p = 0.18). Within the lipid fraction of the yolk, the percentage of

292 saturated fatty acids did not significantly differ between diets (33.6 ± 0.47% vs. 33.7 ± 0.37

293 %, t = - 0.28, p = 0.78). For n-3 hens, the percentage of mono-unsaturated fatty acids was

294 higher, while the percentage of PUFAs was lower in comparison to eggs from the C hens

295 (mono: 49.5 ± 0.62% vs. 44.0 ± 0.65 %, t = - 6.2, p < 0.001, PUFA: 16.7 ± 0.36 % vs. 22.5 ±

296 0.60 %, t = 8.22, p < 0.001). The yolk of eggs from n-3 hens had significantly higher

297 percentages of n-3 PUFAs with a significantly lower n-6/n-3 ratio, while the yolk of eggs

298 from C hens had significantly higher percentages of n-6 PUFAs and a significantly higher n-

299 6/n-3 ratio (Table 2).

>>> INSERT Figure 1 <<<<

>>> INSERT Table 2 <<<<

302 *Dietary effects on hatching success and weight of chicks*

303 Hatching success did not differ between diets (98 hatched out of 111 fertile eggs for the n-3

304 group vs. 125 out of 138 fertile eggs for the n-6 group; chi²-test, p = 0.67), neither did sex-

305 ratio (25 females and 23 males in the n-3 group and 28 females and 22 males in the C group,

306 chi²-test, p = 0.69). N-3 chicks were lighter at hatching than C chicks (39.4 ± 0.4 g vs. 40.9 ±

307 0.5 g, t = -2.29, p = 0.02) but did not differ significantly in growth rate during the first week

308 of life (8.60 ± 0.16 g/week vs. 9.72 ± 1.86 g/week, F<sub>3,93</sub> = 0.66, p = 0.57).

309 *Dietary effects on neophobic behaviour in the chicks*

310 Latencies to eat and the time spent eating did not differ significantly between n-3 and C

311 chicks in the habituation/familiarization test and both food neophobia tests (Table 3). In the
312 novel object test, n-3 chicks demonstrated a longer latency to eat and spent less time eating

313 from the novel object compared to C chicks (Table 3).

314 >>> INSERT Table 3 <<<

315 Discussion

316 The present experiment was designed to test the hypothesis that PUFA in the maternal diet

317 could engender differential allocation of yolk hormones of maternal origin. We found higher

318 corticosterone metabolites, reflecting heightened HPA-activity, in the faecal droppings of

319 hens fed the diet enriched in n-3 compared to control animals. As expected, yolk n-6:n-3 ratio

320 strongly reflected that of the maternal diet. There were significantly higher concentrations of

321 yolk progesterone, androstenedione and oestrogens, as well as a trend for higher yolk

322 testosterone in eggs of hens fed the n-3 enriched diet compared to controls. Chicks of hens fed

323 the n-3 enriched diet showed a lower body weight at hatching and higher neophobia when

324 exposed to a novel object compared to chicks of hens fed the control diet.

325 After six weeks of consuming a diet enriched in n-3 PUFAs, hens had elevated

326 concentrations of corticosterone metabolites in the faeces and elevated levels of yolk

327 hormones compared to hens fed a control diet. Additionally, hens of the n-3 diet laid lighter

328 eggs, with lower yolk/egg mass ratios than controls. Diets were equal in protein and energy

329 content, and were selected to meet the dietary requirements of laying hens (Van Elswyk,

330 1997). Food intake and body weight followed a similar pattern for n-3 and control hens. Thus,

331 differences between groups cannot be explained by differences in the energy content of the

332 diet or differences in food intake. Rather, they demonstrate an influence of n-6/n3 PUFA ratio

333 in the diet on the metabolism and physiology of hens.
334 These results suggest a potential action for the n-6:n-3 PUFA ratio of diet on the HPA-axis of hens as reported in other species (Fisher et al., 2014; Larrieu et al., 2014; Lafourcade, et al., 2011). First, hens of the n-3 diet had FCM levels more than 3-times greater than controls. FCM levels have been shown to reflect HPA activity (Rettenbacher & Palme, 2009).
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336
337
338 Second, in chickens with elevated corticosterone levels due to a corticosterone implant, egg mass, yolk mass and hatchling's weight reduction have also been recorded (Rie Henriksen, Groothuis, & Rettenbacher, 2011; R. Henriksen, Rettenbacher, & Groothuis, 2011) which is comparable to our chicks from hens treated with n-3. Moreover, in birds, environmental stress can induce HPA axis activation, causing a decrease in egg and offspring weight (Hayward & Wingfield, 2004; R. Henriksen, et al., 2011; Hsu, Dijkstra, Darras, de Vries, & Groothuis, 2016). Third, it is known that maternal environmental stress induces similar increases in hens' yolk hormones as we found in the hens fed the n-3 diet (Bertin, et al., 2013; Guibert et al., 2010). In mammals, the effects of n-3 PUFAs on HPA axis functioning are generally beneficial, but not always. As in mice, a deficiency of n-3 PUFA can alter corticosterone secretion via modifications in glucocorticoid receptors (GRs) of the prefrontal cortex, but not in the HPA-axis (Larrieu, Hilal, De Smedt-Peyrusse, Sans, & Laye, 2016). In the chicken, GRs are also found in the pituitary (Bossis, Nishimura, Muchow, & Porter, 2004) and frontal brain areas (Bordone, Schrott, & Sparber, 1997). Glucocorticoid receptors can be modified by stressful conditions in a variety of avian species (Japanese quail (Zimmer & Spencer, 2014), starlings (Dickens, Romero, Cyr, Dunn, & Meddle, 2009), house sparrow (Lattin & Romero, 2014)) and play a role in the lipid metabolism of the white-crowned sparrow (Landys, Piersma, Ramenofsky, & Wingfield, 2004) (Landys, Ramenofsky, Guglielmo, & Wingfield, 2004). Our results on HPA-functioning are a likely consequence of the surplus of n-3 in the experimental diet. This may have resulted in a lack of arachidonic acid (AA), which is essential for metabolic and hormonal functioning (Huang, Leibovitz, Lee, & Millar, 1990).
359 Based on our results, we hypothesize that the ratio of n-6:n-3 PUFAs in the diet influence HPA activity in birds, but the mechanisms require further investigation. It is important to notice that domestic chickens have been raised for food thousands of years ago and were mainly fed with wheat seeds. As wheat seeds are poor in n-3 and rich in n-6 fatty acids, these animals may have developed specific adaptations and, as a consequence, may cope better with high n-6:n-3 ratios than with low n-6:n-3 ratios in the diet.

365 Egg mass and yolk proportion was reduced in n-3 hens. These results are in accordance with those obtained by administering diets enriched with 2%, 3% or 4% fish oil (Gonzalez-Esquerra & Leeson, 2000; Huang, et al., 1990) in which hens' egg mass was found to decrease linearly with increasing percentages of fish oil (high in n-3). Diets enriched with fish oil can increase albumin production and decrease yolk production compared to control diets (Huang, et al., 1990). The n-3 diet we applied contained little linoleic acid (a molecular precursor essential to the production of n-6 PUFA AA). Moreover, AA production appeared to be strongly inhibited by the dominance of docohexaenoic acid (DHA) in the n-3 diet as demonstrated by the amount measured in the yolk. This could severely hamper AA metabolism, resulting in metabolic changes in the hens receiving the n-3 diet, and thereby altering egg composition (Huang, et al., 1990). PUFAs in the maternal diet can thus have an important effect on the amount and constitution of the resources available for embryo development.

378 Moreover, our data showed that fish oil in the diet led to higher concentrations of yolk progesterone, androstenedione and oestradiol and a trend for higher concentrations of yolk testosterone. These results add support to the hypothesis that the nutritional state of female birds causes variation in yolk hormone levels. However, at the present stage, the interpretation of the mechanisms that mediate variations in yolk hormone levels is bound to be speculative. As mentioned previously, it could be that increased levels of yolk hormones are the result of
384 HPA axis activation in the mother. In accordance with this hypothesis; recent studies showed that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk (Henriksen, Groothuis, & Rettenbacher, 2011). The underlying physiological mechanisms remain poorly understood and are barely addressed in the literature. As argued before, it might be possible that corticosterone levels affect the steroidogenic activity of follicles. Another hypothesis could be related to the direct influence of PUFAs and their oxygenated derivatives on steroid synthesis. Several factors of the steroidogenic machinery are directly regulated by AA and AA-derived eicosanoids (Wathes, Abayasekara, & Aitken, 2007). The n-3/n-6 PUFA ratio in the diet largely determines the balance between the various types of oxygenated metabolites deriving from AA or from EPA and DHA (Yates, Calder, & Rainger, 2014). Therefore, the decreased AA/DHA+EPA ratio and the resulting eicosanoid pattern in n-3 supplemented hens may have directly contributed to the observed differences in yolk hormone levels. Consistently, dietary fat (such as those derived from fish oil) in the maternal diet is known to influence egg weight and plasma oestrogen levels (Whitehead, 1995), a role for PUFAs in the hormonal metabolism of birds seems, therefore, a plausible explanation.

We found that n-3 chicks were more inhibited (longer latency to eat and less time spent eating) than C chicks in the novel object test. This result indicates a higher level of object neophobia in n-3 chicks than C chicks. On the other hand, n-3 and C chicks did not differ in food neophobia test. Since the n-3 maternal diet modified the embryonic environment in several ways (yolk mass, yolk hormones and fatty acids), it is difficult to determine which mechanisms and which hormones affected object neophobia. Little is currently known regarding the developmental influence of yolk progesterone and androgens on neophobia. The influence of progesterone is barely investigated. In Northern bobwhite quail (*Colinus virginianus*), a recent study showed that elevated yolk progesterone levels
409 elevate emotional reactivity in neonates (Herrington, Vallin, & Lickliter, 2015). Interestingly, an artificial increase in yolk hormone levels has been found to alter object neophobia but not food neophobia in domestic chicks (Bertin, et al., 2015), but the direction of effects was not the same as in the present study. Lower object neophobia was found in chicks exposed *in ovo* to increased yolk progesterone, androstenedione and estradiol levels compared to controls. Discrepancies in the direction of yolk hormonal effects are also seen in latencies to approach novel objects depending on species and the injected dose (Vergauwen, Eens, & Muller, 2012). Nevertheless, all of these experiments suggest that variation in yolk hormone levels can alter specific aspects of neophobia.

418 The data concerning the effects of environmental conditions during ontogeny on the development of object or food neophobia are inconclusive. In accordance with our findings, zebra finch chicks (*Taeniopygia guttata*) from eggs with artificially elevated yolk testosterone show no difference in latency to eat novel foods than control animals (Tobler & Sandell, 2007). Similarly, in canaries (*Serinus canaria*), increased levels of yolk testosterone was found to not be the primary source of variation in the expression of food neophobia (Vergauwen, et al., 2012). Our data highlight the importance of studying the quality of the maternal diet in order to understand more fully the ontogeny of emotional and behavioural traits involved in adapting to new resources. This could be of particular importance when food resources become scarcer under the projected future of climate change. In captive bird populations (for production or conservation purposes), no specific recommendation is made regarding PUFA quantity and quality in the diet. This study highlights the importance of carefully considering food quality in captive bird populations as they might engender transgenerational effects on development and behaviour. In our study, chicks were all feeding around two/three days after hatching, therefore we cannot totally exclude the possibility that the behaviour of the chicks was influenced by their respective diet. Two points are of
434 importance when considering this hypothesis. First, even when chicks start feeding, the residual yolk continues to be taken up and thereby provides a source of fatty acids of maternal origin (Romanoff, 1960). Second, in the domestic chicken and king penguin (*Aptenodytes patagonicus*) it was consistently found that the proportion of fatty acids in the brain phospholipids was particularly refractory to the effects of the chicks' diet compared to other tissues (Thil, Speake, & Groscolas, 2003; Anderson, et al., 1992). More specifically, high levels of brain n-3 produced by exposure to a maternal diet rich in fish oil are not reversible on a short time period (Anderson, et al., 1992). Although caution must be exercised, we argue that maternal effects more likely contributed to the differences observed in hatchlings' mass and behaviour than the post-natal diet.

444 The present data show maternal diet composition as an overlooked pathway by which the environment could engender large variation in yolk hormones levels. This may play a role in the development of offspring behaviour and phenotype. In farm birds, as unresolved welfare issues are mediated by unknown cues, this study lends support to perspectives in the field of maternal nutrition. Further investigations to the mechanisms are required but this study suggests common principles regarding the regulative action of n-3 and n-6 PUFA on fear-related behavior across vertebrate taxa. Using precocial birds could yield new insights into the mechanism by which the prenatal resources in PUFA influences behavioral development of individuals in vertebrates

454 Acknowledgements

455 All birds were maintained at the PEAT experimental unit, INRA, Nouzilly, France. We are grateful to all members of the unit, particularly P. Ganier and O. Callut for taking care of the birds. The authors are grateful to Dr. C. O'Brien for English proofreading. This work was
458 supported by the French National Research Agency (PReSTO'Cog ANR-13-BSV7-0002-02)

459 and the University François Rabelais of Tours (Post-doctoral grant).

460

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699

700

701

702

703

704

705
<table><caption>Table 1: Mean (± SE) body mass, food intake and laying rate of adult n-3 hens (N = 18) and control hens (N = 18) during dietary treatment.</caption><thead><tr><th>Parameters</th><th>Hens</th><th>Week 1</th><th>Week 2</th><th>Week 3</th><th>Week 4</th><th>Week 5</th></tr></thead><tbody><tr><td rowspan="2">Body mass (g)</td><td>n-3 enriched</td><td>1958.7 ± 33.9</td><td>1854.5 ± 40.6</td><td>1863.8 ± 37.3</td><td>1835.3 ± 35.3</td><td>1856.6 ± 36.5</td></tr><tr><td>controls</td><td>1966.6 ± 67.2</td><td>1880.9 ± 60.9</td><td>1974.1 ± 62.5</td><td>1865.4 ± 60.8</td><td>1873.5 ± 62.3</td></tr><tr><td rowspan="2">24h feed intake (g)</td><td>n-3 enriched</td><td>154.7 ± 23.4</td><td>144.7 ± 7.4</td><td>173.7 ± 16.7</td><td>194.6 ± 22.8</td><td>224.4 ± 28.1</td></tr><tr><td>controls</td><td>133.7 ± 11.8</td><td>169.6 ± 21.1</td><td>161.8 ± 20.5</td><td>219.0 ± 24.5</td><td>265.4 ± 24.3</td></tr><tr><td rowspan="2">Laying rate (number per day)</td><td>n-3 enriched</td><td>0.78 ± 0.07</td><td>0.89 ± 0.03</td><td>0.91 ± 0.02</td><td>0.78 ± 0.04</td><td>0.69 ± 0.06</td></tr><tr><td>controls</td><td>0.71 ± 0.07</td><td>0.82 ± 0.06</td><td>0.77 ± 0.07</td><td>0.69 ± 0.07</td><td>0.65 ± 0.06</td></tr></tbody></table>
<table><caption>Table 2: Mean (± SE) percentages of n-3 and n-6 poly-unsaturated fatty acids in the yolk of eggs of hens fed a diet enriched in n-3 (N = 18) or a control diet (N = 18).</caption><thead><tr><th rowspan="2">Poly Unsaturated fatty acids</th><th colspan="2">Diets</th></tr><tr><th>n-3 enriched</th><th>Control</th></tr></thead><tbody><tr><td><strong>n-6 fatty acids</strong></td><td></td><td></td></tr><tr><td>Linoleic acid (LA), C18:2 n-6</td><td>13.5 % ± 0.23<sup>a</sup></td><td>18.9 % ± 0.47<sup>b</sup></td></tr><tr><td>Arachidonic acid (AA), C20:4 n-6</td><td>0.64 % ± 0.05<sup>a</sup></td><td>1.81 % ± 0.18<sup>b</sup></td></tr><tr><td>Docosatetraenoic acid (DTA), C22:4 n-6</td><td>0.03 % ± 0.005<sup>a</sup></td><td>0.22 % ± 0.02<sup>b</sup></td></tr><tr><td><strong>n-3 fatty acids</strong></td><td></td><td></td></tr><tr><td>α-linolenic acid (ALA), C18:3 n-3</td><td>0.47 % ± 0.02<sup>a</sup></td><td>0.71 % ± 0.03<sup>b</sup></td></tr><tr><td>Eicosapentaenoic acid (EPA), C20:5 n-3</td><td>0.22 % ± 0.02<sup>a</sup></td><td>0.00 % ± 0.0<sup>b</sup></td></tr><tr><td>Docosapentaenoic acid (DPA), C22:5 n-3</td><td>0.15 % ± 0.01<sup>a</sup></td><td>0.07 % ± 0.02<sup>b</sup></td></tr><tr><td>Docosahexaenoic acid (DHA), C22:6 n-3</td><td>1.72 % ± 0.17<sup>a</sup></td><td>0.74 % ± 0.07<sup>b</sup></td></tr><tr><td>Ratio n-6/n-3</td><td>6.15 % ± 0.53<sup>a</sup></td><td>13.9 % ± 0.42<sup>b</sup></td></tr></tbody></table>

Means between columns with a different superscript (a, b) differ (p < 0.05)

718

719

720

721

722

723

724

725

726

727
Table 3: Mean (± SE) Latency to eat and time spent eating during neophobia tests of n-3 chicks (N = 24 pairs) and control chicks (N = 25 pairs) and Mann-Whitney U-test outcome.

730

731

<table><thead><tr><th>Neophobia Tests</th><th>Parameters Measured</th><th>n-3 chicks</th><th>control chicks</th><th>Z</th><th>p-values</th></tr></thead><tbody><tr><td rowspan="2">Habituation</td><td>latency to eat (s)</td><td>40.5 ± 3.3</td><td>48.3 ± 6.5</td><td>-0.66</td><td>0.51</td></tr><tr><td>time spent eating (s)</td><td>48.9 ± 3.8</td><td>52.1 ± 5.2</td><td>-1.07</td><td>0.28</td></tr><tr><td rowspan="2">Novel food Test 1</td><td>latency to eat (s)</td><td>99.1 ± 13.2</td><td>85.4 ± 10.6</td><td>-0.30</td><td>0.76</td></tr><tr><td>time spent eating (s)</td><td>18.4 ± 3.7</td><td>22.8 ± 3.8</td><td>-0.90</td><td>0.36</td></tr><tr><td rowspan="2">Novel food Test 2</td><td>latency to eat (s)</td><td>84.3 ± 13.9</td><td>107.8 ± 10.2</td><td>-0.96</td><td>0.33</td></tr><tr><td>time spent eating (s)</td><td>32.0 ± 9.1</td><td>24.2 ± 5.9</td><td>-0.15</td><td>0.88</td></tr><tr><td rowspan="2">Novel Object</td><td>latency to eat (s)</td><td>174.1 ± 5.9</td><td>155.2 ± 8.5</td><td>-2.11</td><td>0.03</td></tr><tr><td>time spent eating (s)</td><td>2.1 ± 2.0</td><td>14.11 ± 5.1</td><td>-2.27</td><td>0.02</td></tr></tbody></table>

732

733

734

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736
<figure><img src="image_3.png" /><figcaption>738</figcaption></figure>

<caption>739</caption>

<caption>740</caption>
741 **Figure caption**

742 FIGURE 1: Mean (± SE) yolk progesterone (P4), androstenedione (A4), estrogens (E) and

743 testosterone (T) concentrations (ng/g of yolk) in the eggs from n-3 enriched hens (N = 18) and

744 control hens (N = 18). **p** < 0.01; *p* ≤ 0.05; # 0.1 ≤ *p* ≥ 0.05