Background: Research into energy balance and growth has infrequently considered genetic sex, yet there is sexual dimorphism for growth across the animal kingdom. We test the hypothesis that in the chicken, there is a sex difference in arcuate nucleus neuropeptide gene expression, since previous research indicates hypothalamic AGRP expression is correlated with growth potential and that males grow faster than females. Because growth has been heavily selected in some chicken lines, food restriction is necessary to improve reproductive performance and welfare, but this increases hunger. Dietary dilution has been proposed to ameliorate this undesirable effect. We aimed to distinguish the effects of gut fullness from nutritional feedback on hypothalamic gene expression and its interaction with sex. Methods: Twelve-week-old male and female fast-growing chickens were either released from restriction and fed ad libitum or a restricted diet plus 15% w/w ispaghula husk, a non-nutritive bulking agent, for 2 days. A control group remained on quantitative restriction. Hypothalamic arcuate nucleus neuropeptides were measured using real-time PCR. To confirm observed sex differences, the experiment was repeated using only ad libitum and restricted fed fast-growing chickens and in a genetically distinct breed of ad libitum fed male and female chickens. Linear mixed models (Genstat 18) were used for statistical analysis with transformation where appropriate. Results: There were pronounced sex differences: expression of the orexigenic genes AGRP (P < 0.001) and NPY (P < 0.002) was higher in males of the fast-growing strain. In genetically distinct chickens, males had higher AGRP mRNA (P = 0.002) expression than females, suggesting sex difference was not restricted to a fast-growing strain. AGRP (P < 0.001) expression was significantly decreased in ad libitum fed birds but was high and indistinguishable between birds on a quantitative versus qualitative restricted diet. Inversely, gene expression of the anorectic genes POMC and CART was significantly higher in ad libitum fed birds but no consistent sex differences were observed. Conclusion: Expression of orexigenic peptides in the avian hypothalamus are significantly different between sexes. This could be useful starting point of investigating further if AGRP is an indicator of growth potential. Results also demonstrate that gut fill alone does not reduce orexigenic gene expression. Keywords: Sex, Psyllium, AGRP, POMC, Satiety, Growth, Body weight * Correspondence: email@example.com The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, Edinburgh EH25 9RG, Scotland, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Caughey et al. Biology of Sex Differences (2018) 9:20 Page 2 of 12 Background between layers and broilers . We have previously Sexual dimorphism is all around us; differences in plumage, shown that the expression of AGRP mRNA in the arcuate pelage or ornamentation are observed across the animal nucleus is increased many-fold in broiler breeder chickens kingdom with many species also displaying difference in under feed restriction compared to those fed ad libitum, bodysizeand weight . Most commonly, males are larger whereas the anorectic peptide POMC mRNA was rela- than females, but in some cases, the female is the largest tively unchanged . In a line of chickens segregating at sex with many examples in predatory birds, but also cases the cholecystokinin A receptor (CCKAR) locus, the auto- in mammals such as the blue whale and spotted hyena [2– somal genomic locus with the largest effect on growth and 6]. Research into the role of gene products such as agouti- body weight, hypothalamic AGRP expression was higher related protein (AGRP) and pro-opiomelanocortin (POMC) in the animals carrying the high growth allele which had in the control of energy balance in birds, or in mammals, lower CCKAR mRNA and protein expression . Further- has not paid particular attention to the sex of the animals more, hypothalamic AGRP expression in growing chickens studied . Given the large difference in growth between was shown to be responsive to both short- and long-term malesand femalesofmanyspecies,this issurprising, al- food availability . This indicated that the AGRP neu- though as in our own studies on food intake and metabol- rones have a potentially important role in the control of ism, a focus on one sex is sometimes made for industrial feeding behaviour in birds because the level of AGRP relevance [8–10]. In many galliforms, as in most mammals, mRNA represented not only the immediate satiety state of the male grows larger and faster than the female; indeed, the bird but also how far the bird was from its body the domestic chicken displays one of the clearest sexual di- weight if it had not been food restricted . In other morphisms in body weight, with males around 20% heavier words, hypothalamic AGRP expression in a number of dif- and with a clear difference in growth rate long before sexual ferent situations appears consistent in giving an indication or somatic maturity [11, 12]. This dimorphism for body of growth potential as much as short-term motivation to weight holds true across the spectrum of chicken lines: in eat. Therefore, as our primary aim, we wanted to deter- fast-growing meat-type chickens, in crosses between O- mine if genetic sex would be reflected in differences in Shamo game bird and white leghorns and in egg-laying AGRP expression. strains [11, 13, 14]. Selection for growth appears not to have There is currently a great deal of interest in the control in any way altered the sex difference in body weight, and of food intake in the context of overconsumption and the genetic correlation between male and female sibs of obesity in humans whilst in domesticated animals it is par- meat-type chickens is extremely high, with no evidence that ticularly important for the efficient growth and production the sex difference between them has a heritable component of meat. Chicken meat and eggs provide at least a third of . In other words, the differences in body weight are the world’sanimalprotein . Genetic selection in meat- related entirely to the sex chromosomes inherited (in birds type chickens has led to threefold increases in growth and male ZZ, female ZW), and not to any interaction with the feed efficiency . Capitalising on this genetic potential rest of the genome. From a practical point of view, selecting has come with some adverse consequences. The parents either sex for improved growth rate would be equally of these meat-type chickens, known as broiler breeders, effective. become overweight if allowed to feed ad libitum during The central regulation of energy balance is conserved rearing and to a lesser extent through the reproductive between birds and mammals, with the arcuate nucleus of period, leading to poor welfare, decreased productivity the hypothalamus containing one population of neurons and increased morbidity and mortality of up to 31% [22– producing both AGRP and neuropeptide Y (NPY) with 26]. Food restriction is used successfully in the poultry in- another synthesising α-melanocyte-stimulating hormone dustry to control these issues, with peak restriction (αMSH) and other peptides from the POMC gene, and around 25% of the ad libitum intake at 7–14 weeks of age. co-expressing cocaine- and amphetamine-regulated tran- The birds show high levels of food motivation, with broiler script (CART)mRNA [15–17]. The balance of POMC and breeders willing to experience an aversive stimulus to per- AGRP is critical for controlling food intake; POMC neu- form exploratory and foraging behaviour even when there rons produce αMSH which acts on melanocortin 4 recep- was no food reward . These conflicting welfare issues tors (MC4Rs) to inhibit food intake, and contrastingly, became known as the ‘broiler breeder paradox’ [24, 28, AGRP acts as an antagonist on the same receptors to in- 29]. Understanding how growth is controlled and how dif- crease food intake and energy storage [18, 19]. Intra- ferences in growth are genetically determined are, there- cerebroventricular (ICV) injection of AGRP attenuated fore, of key interest. Furthermore, investigating how the the anorectic effect of αMSH on food intake in both layer activity of anorectic and orexigenic neurons is related to and broiler chicks; however, only in layer chicks did AGRP growth is of potential importance in allowing it to be ma- increase food intake under ad libitum feeding conditions nipulated. One potential solution to the welfare problem suggesting the orexigenic effects of AGRP are different of food-restricted broiler breeders experiencing prolonged Caughey et al. Biology of Sex Differences (2018) 9:20 Page 3 of 12 hunger is to move from quantitative restriction to qualita- assigned an individual cage and one of three treatment tive restriction by using dietary diluents to effectively groups. For each of three replicate batches, six birds (n =2 lengthen the feeding period, change behaviour and appar- per treatment group) were transferred to their allocated in- ently increase satiation whilst still restricting body weight dividual cages in a new room on 4 successive days (different [30–34]. The question remains, however, as to whether room for each day) and allowed to acclimatise for 6 days these approaches increase satiation indicators centrally. A with continued commercial feed restriction. Birds were ei- second objective of this study was therefore to investigate ther then released from restriction and allowed to feed ad whether the short-term inclusion of a fibrous bulking libitum (AL), fed the commercial restricted diet ration plus agent, the arabinoxylan fibre source Psyllium, also known 15% w/w ispaghula husk (IH) or maintained on the com- as ispaghula husk, in the diet alters the gene expression of mercial restricted diet ration (FR). The diets were fed for 2. arcuate nucleus neuropeptides in a release from restriction 5 days, and the birds were then killed with an intravenous model . Psyllium in humans and laboratory rodents injection of sodium pentobarbitol. In each batch, dissec- has been shown to have satiating effects, attributed to the tions (n = 6 per day) were performed when the birds were effects of slowing down the absorption of nutrients by in- on average 11 weeks old over 4 days with equal numbers creasing bulk through its action of absorbing of large from the different treatment groups each day. All dissec- amounts of water. Psyllium is also not digestible or fully tions were performed after 14.00 (7 h after lights on) with fermentable due to its complex polysaccharide structure, one bird from each triplet of treatment being sampled se- but it possibly increases the production of short-chain quentially but randomly to minimise the effect of sample fatty acids in the distal gastrointestinal tract which may time. Basal hypothalamic brain tissue (40–100 mg) was dis- have satiating effects [35–37]. sected as previously described  and snap frozen on dry This study therefore had two main aims: firstly, to test ice before being stored at − 80 °C until processed to extract the hypothesis that there is a sex difference in gene ex- RNA. The group size for each treatment was 24. Sex was pression of neuropeptides controlling food intake in the determined at dissection with broadly equal number of hypothalamic feeding circuitry. Secondly, to test the hy- each sex in each treatment. Ten birds (of 72) were lost to pothesis that qualitative food restriction induces a differ- the study (2 AL, 3 FR and 3 IH) due to early illness. ent pattern of gene expression in the arcuate nucleus compared to quantitative restriction; in other words, to Sex effect repeat: To repeat the experiment on the effect of distinguish the effects of gut fullness from nutritional sex and release from quantitative food restriction on the signal feedback on the expression of central orexigenic expression of anorectic and orexigenic peptide genes in the and anorexigenic signals. These two aims were examined basal hypothalamus together to determine if there was any interaction be- Experiment section “Sex and diet” was repeated using the tween sex and dietary restriction, whether qualitative or same line of chickens with the omission of the IH group quantitative, on hypothalamic gene expression. to ensure the sex difference results observed in section “Sex and diet” were repeatable. Two replicate batches Methods were used with dissections of eight birds (n = 4 per treat- Animal experiments ment group) performed when the birds were on average Sex and diet effect: To test the effect of sex and release 12 weeks old over 2 days in each batch. The group size for from quantitative food restriction to qualitative restriction each treatment was 16, designed with equal numbers of on basal hypothalamic neuropeptide gene expression males and females per treatment based on genetic sexing Un-sexed mixed female and male Ross 308 broiler breeders . One bird was lost to the study (AL female) and one were group housed in three batches (hatches, n =24) from AL bird thought initially to be female was male. hatch until 1 week prior to the experiment. Lighting, nutri- tional composition of the food and dietary restriction from Genetically distinct line: To test the effect of sex on basal day-old to 11 weeks of age was implemented in accordance hypothalamic anorectic and orexigenic gene expression in a with the breeders’ 2016 management manual (http://eu. genetically distinct line of chickens aviagen.com/assets/Tech_Center/Ross_PS//308SF-PS-EU-P Female (n = 15) and male (n = 14) birds from the 20th gen- O-EN-16.pdf) and similar to detailed previously . Our eration of a broiler layer hybrid line  were reared in group experiment was conducted when birds were 12 weeks of housing under 14L:10D lighting and 26 °C temperature (am- age which is within the peak periodof foodrestriction for bient) andallowedtofeedadlibitum on a standardgrower broiler breeders (7–14 weeks ofage), apoint wheregrowth diet until they were humanely killed at 10 weeks of age. is almost at maximum and well before sexual maturity Therewerefivepensof birdusedinthestudywhich was which typically occurs at around 20 weeks onward . confounded with hatch. All birds were heterozygotic for the One week prior to the experiment, birds were weighed, previously described CCKAR locus alleles . This line ranked and randomised according to body weight and then whilst not requiring feed restriction to maintain Caughey et al. Biology of Sex Differences (2018) 9:20 Page 4 of 12 reproductive performance in adulthood does benefit from a experiment section “Sex and diet effect” because some of moderate restriction that increases the production of viable these are partially confounded, sequential tests were ob- eggs. Chickens were culled with an overdose of sodium tained testing these factors in four orders: age before and pentobarbital and basal hypothalamus samples dissected as after treatment and sex, and treatment before and after described for experiment section “Sex and diet effect.” sex. LMMs were fitted to all data and to data omitting outliers (as defined by the linear mixed model residuals) to Ethics statement confirm that results for all data reported here are not just All animal experiments were performed under UK Home attributable to the outliers. Post hoc tests were carried out Office Project Licence 70/7909, and birds were humanely by including contrasts in the fixed effects. P values reported killed as specified in Schedule 1 of the UK Animals (Sci- here are the most conservative when alternative models entific Procedures) Act 1986. were fitted. P values are based on approximate F tests when available but otherwise are based on Wald tests. Genstat RNA extraction and reverse transcription (Genstat, 16th–18th editions, Lawes Agricultural Trust, RNA was extracted from up to 100 mg of tissue with TRI- VSN International Ltd.) was used for all statistical analyses. reagent (Ambion, Life Technologies, UK) and Lysing Matrix D tubes using a FastPrep Instrument FP120 (Thermo Elec- Results tron Corporation, UK) and then purified according to man- Effect of sex and diet on basal hypothalamic ufacturers’ instructions using a Zymo Direct-zol™ RNA neuropeptide gene expression plus physiological mini-prep kit (Cambridge BioSciences, UK). RNA concen- parameters tration was read on a NanoDrop Spectrophotometer ND- Food intake and body and organ weight 1000 (LabTech International, UK). RNA (1 μg) was reverse Restricted birds were fed 46 g/day which equates to ap- transcribed using a high capacity cDNA reverse transcrip- proximately 25% of the food intake of the average ad tion kit (Applied Biosystems; Life Technologies, UK) follow- libitum intake (181 g/day) at 12 weeks of age, as previ- ing the manufacturer’s protocol before being diluted 5.5× ously observed . Birds on the IH diet were fed the and stored at − 20 °C. restricted diet containing 15% w/w ispaghula husk (52.9 g/day in total). Real-time polymerase chain reaction (PCR) assays There was no difference in the food consumed in the Primers and assays were as described previously . days after release from restriction between males and fe- males in the AL group (P = 0.792) in experiment section Statistical analysis “Sex and diet effect”. Similarly, there was no significant All graphs and the table show means ± standard errors of difference in body weight between males and females means (SEMs) on the raw data scale, apart from expres- overall (P = 0.787). In experiment section “Sex effect re- sion measures that were standardised by dividing by the peat” again no differences were observed in the food in- housekeeping gene. For experiment section “Genetically take between males (186.33 ± 7.17 g) and females (188.0 distinct line,” expression measures (log transformed) were ± 8.63 g) in the AL group after release from restriction analysed using an unbalanced ANOVA blocking for pen (P = 0.885) and no significant difference in body weight (identical to cull date) to investigate the effect of sex. Stat- between sexes overall (P = 0.801) nor was there an inter- istical analysis for experiment sections “Sex and diet ef- action with treatment was observed (P = 0.930; AL fe- fect” and Sex effect repeat were performed using linear male 1795.5 ± 87.7 g, AL male 1785.3 ± 38.8 g, FR female mixed models (LMM) fitted to bird and organ weights (all 1515.1 ± 58.9 g, FR male 1495.0 ± 41.0 g). log transformed except pituitary), crop content weight, As expected, average body weight varied with treatment feed intake (AL birds only) and expression measures (log group (P <0.001, F = 127.14) with the AL group (1550. 2,46 transformed). In LMMs, random effects were included for 9 ± 28.1 g) average weight significantly higher (P <0.001) batch (identical to the lab day for expression measures), than that for the FR (1229.0 ± 24.3 g) and IH (1225.9 ± 24. the 12 (4 per batch) different days on which the birds were 5g)groups (F = 191.14, 187.30, respectively). There 1,46 dissected (identical to spatial block), and individual birds was no significant difference in the body weights between (the residual). Body weight at post mortem was also inves- the FR and IH groups (P = 0.940). Average crop content tigated in the model, but this had little or no impact on weight varied with treatment (P <0.001, F =60.31) and 2,46 differences between sexes in gene expression so is not re- was significantly higher in the IH fed group (56.14 ± 6. ported here. Fixed effects were included for bird age (ex- 38 g) compared with the FR group (9.68 ± 2.43 g, P <0. periment section “Sex and diet effect” only, fitted as a 001, F = 48.38) but lower in the IH group than the AL 1,46 four-level factor), diet treatment group (AL, FR, IH (ex- group (83.02 ± 4.59 g, P <0.001, F = 15.79). It was 1,46 perimental section “Sex and diet effect” only)), sex and the noted that whilst the restricted birds ate their ration in interaction between sex and treatment group. For less than an hour, birds fed the restricted diet Caughey et al. Biology of Sex Differences (2018) 9:20 Page 5 of 12 supplemented with IH took the entire daytime period of significantly decreased (P < 0.001) compared with FR birds 14 h to finish the ration. in experiment sections “Sex and diet” (Fig. 1A, B), (AGRP There were highly significant (P < 0.001, F = 87.68 F =70.43, NPY F =50.54) and “Sex effect repeat” 2,46 1,51 1,51 pancreas, 75.64 liver, 47.20 gizzard, F = 47.78 proven- (Fig. 2A, B, AGRP F =27.98, NPY F = 21.67). How- 2,47 1,25 1,25 triculus, F = 8.82 empty gall bladder) differences in ever, in the basal hypothalamus of birds fed the IH diet 2,42 organ weights (Table 1) between treatment groups in expression of AGRP (P < 0.001 vs AL, F =81.69) and 1,51 experiment section “Sex and diet effect” with the AL NPY (P < 0.001 vs AL, F = 81.34) mRNA was high and 1,51 group having, on average, a significantly larger liver (P <0. indistinguishable from that for FR birds (Fig. 1A, B). In ex- 001, F = 80.37, 137.19), gizzard (P <0.001, F =23.88, periment section “Sex and diet effect” an inverse pattern 1,46 1,45 F = 94.39), proventriculus (P <0.001, F =56.78, F was observed for the anorectic genes, POMC and CART, 1,46 1,46 1,47 = 83.34), empty gall bladder (P =0.012, F =6.78, P <0. with significantly higher expression seen in the AL birds 1,52 001, F = 17.19) and pancreas (P <0.001, F = 148.06, compared to the FR (POMC, P <0.001, F =23.27; 1,52 1,45 1,51 F = 110.11) than the FR and IH groups. Interestingly, CART, P <0.001, F = 17.61) and IH (POMC, P =0.002, 1,46 1,51 the FR group had significantly larger gizzards (P <0.001, F =11.09; CART, P = 0.001, F = 11.80) birds (Fig. 1C, 1,51 1,51 F = 21.93) and livers (P =0.009, F = 7.38) than the D) and indistinguishable between IH and FR birds. In ex- 1,46 1,46 IH group. In relation to sex, only the liver was significantly periment section “Sex effect repeat” there was no signifi- larger on average in the females compared to the males (P cant effect of treatment groups on POMC (P =0.216) and =0.015, F = 6.30). There were no statistically significant CART (P =0.625) expression (Fig. 2C, D). 1,51 differences in the weights of other organs between sexes or significant interactions between treatment and sex. Sex differences in basal hypothalamic anorectic and orexigenic Effect of release from restriction to an ad libitum diet or gene expression following release from restriction diet containing a non-nutritious bulking agent on basal A number of pronounced sex differences in gene expres- hypothalamic anorectic and orexigenic gene expression sion in the basal hypothalamus were observed. Across the In experiment section “Sex and diet effect” there was a whole of experiment section “Sex and diet effect” the ex- highly significant (P < 0.001) difference between treatment pression of AGRP (P < 0.001, F =34.99) and NPY (P = 1,51 groups in average expression of AGRP, NPY, POMC and 0.002, F = 11.13) mRNA was significantly higher in 1,51 CART (F = 50.45, 44.88, 12.11, 9.86, respectively) in the males compared with females (Fig. 1A, B). For AGRP and 2,51 basal hypothalamus of birds (Fig. 1). Expression of AGRP NPY mRNA, the sex difference in expression was larger and NPY mRNA in the basal hypothalamus of birds re- within the restricted and IH re-fed groups than the AL leased from restriction and allowed to feed AL was group (Fig. 1A, B) although it should be noted that the Table 1 Whole organ weights (mean ± SEM) for broiler breeders after 2.5 days of a different feeding regime. Average organ weights for the ad libitum (AL; n = 21) fed group compared with the food restricted (FR; n = 20) and the food restricted plus 15% ispaghula husk (IH; n = 21) groups. P values are from LMMs with different labels (a, b, c) indicating differences between means from post hoc tests for the main treatment group effect Caughey et al. Biology of Sex Differences (2018) 9:20 Page 6 of 12 Sex P<0.001 Sex P=0.002 Treatment P<0.001 Treatment P<0.001 A AGRP NPY Sex*Treatment P=0.722 Sex*Treatment P=0.775 0.05 0.035 0.045 0.03 0.04 0.025 0.035 0.03 0.02 0.025 0.015 0.02 0.015 0.01 0.01 0.005 0.005 0 0 fmfmfm fmfmfm AL FR IH AL FR IH Sex P<0.001 Sex P=0.254 POMC C CART Treatment P<0.001 Treatment P<0.001 Sex*Treatment P=0.174 Sex*Treatment P<0.001 0.3 0.03 0.25 0.025 0.2 0.02 0.15 0.015 0.1 0.01 b b b 0.05 0.005 0 0 fmfmfm fmfmfm AL FR IH AL FR IH Fig. 1 Gene expression (mean ± SEM) in the basal hypothalamus of male (m) and female (f) broiler breeders following different diets in experiment section “Sex and diet effect” Orexigenic (AGRP (A)and NPY (B)) and anorectic (POMC (C)and CART (D)) gene expression in birds fed ad libitum (AL; n = 19), feed restricted (FR; n = 20) and birds re-fed with ispaghula husk (IH; n =21). P values are from LMMs with different labels (a, b) indicating statistically significant (P < 0.05) differences between means for the treatment group effect and for the sex by treatment group interaction interaction between sex and treatment was not significant To test the effect of sex in basal hypothalamic anorectic for AGRP (P =0.722) or NPY (P =0.775). and orexigenic gene expression in a genetically distinct For anorectic genes in the basal hypothalamus, the chicken line interaction between sex and treatment was highly sig- In view of the dramatic sex differences observed in the nificant (P < 0.001, F = 8.44) for CART mRNA expres- broiler breeders, expression of arcuate nucleus genes 2,51 sion which was significantly higher in the AL male were compared between males and females of an ad libi- group than in all other treatment by sex groups (P <0. tum fed advanced (20th generation) broiler layer hybrid 001, F = 22.71–32.91; Fig. 1A). There was no statisti- line (AIL). Expression of AGRP mRNA was significantly 1,51 cally significant effect of sex (P = 0.254) or sex by treat- higher in males compared with females (P = 0.002, F 1,23 ment (P = 0.174) on POMC mRNA expression in the = 12.90) but NPY expression did not differ (P = 0.425; basal hypothalamus. Fig. 3). For anorectic peptides, there was no difference in The sex difference in expression of orexigenic genes in the mRNA expression of CART (P =0.200) or POMC the basal hypothalamus was repeatable in experiment (P = 0.351) between sexes. section “Sex effect repeat” (Fig. 2). In this experiment, AGRP (P < 0.001, F = 19.32) and NPY (P = 0.003, F Discussion 1,25 1,25 = 10.71) mRNA expression was significantly higher in The observation of the dramatic differences in arcuate males compared to females (Fig. 2A, B). However, unlike nucleus neuropeptide expression between sexes is novel in experiment section “Sex and diet effect” differences and was initially surprising to us. This has not, as far as were not observed in the basal hypothalamic expression we know, been previously investigated or observed in of anorectic genes between sexes (POMC P = 0.181, birds and little researched even in mammals [41–43]. In CART P = 0.532). There were no significant sex by treat- our experiments, both AGRP and NPY expression were ment interactions observed in AGRP (Fig. 2A, P = 0.188), significantly higher in males than females. The sex dif- NPY (Fig. 2B, P = 0.334), POMC (Fig. 2C, P = 0.559), or ferences in orexigenic peptide expression were repeat- CART (Fig. 2D, P = 0.554) mRNA expression in the re- able and, for AGRP, also observed to be higher in males peat experiment. of a genetically distinct line of chickens. The observation POMC mRNA/ LBR mRNA AGRP mRNA/LBR mRNA NPY mRNA/LBR mRNA CART mRNA/ LBR mRNA Caughey et al. Biology of Sex Differences (2018) 9:20 Page 7 of 12 A AGRP b B NPY 0.6 b 1.2 Sex P<0.001 Sex P=0.003 Treatment P<0.001 Treatment P<0.001 0.5 Sex*Treatment P=0.188 1 Sex*Treatment P=0.334 0.4 0.8 0.3 a 0.6 0.2 0.4 0.1 0.2 0 0 Female Male Female Male Female Male Female Male AL FR AL FR POMC CART CD Sex P=0.532 0.3 0.004 Sex P=0.181 Treatment P=0.625 Treatment P=0.216 Sex*Treatment P=0.554 0.0035 0.25 Sex*Treatment P=0.559 0.003 0.2 0.0025 0.15 0.002 0.0015 0.1 0.001 0.05 0.0005 0 0 Female Male Female Male Female Male Female Male AL FR AL FR Fig. 2 Anorectic and orexigenic gene expression (mean ± SEM) in the basal hypothalamus of male and female broiler breeders following release from commercial restriction in experiment section “Sex effect repeat” Expression of mRNA for AGRP (A), NPY (B), POMC (C) and CART (D) in the ad libitum (AL; n = 16) and commercial food-restricted (FR; n = 16) fed groups. P values are from LMMs with different labels (a, b) indicating statistically significant (P < 0.05) differences between means for the treatment group effect of no difference in NPY gene expression in the genetic- growth between males and females; however, in a subse- ally distinct line of chickens may be explained by the fact quent generation of the genetically distinct line (used in they were fed ad libitum for the whole experiment. Al- experiment section “Genetically distinct line”), we have ternatively, because NPY is relatively ubiquitous in the observed significant differences in the growth and brain and serves other functions in contrast to AGRP, weight between males and females on ad libitum feeding which is confined to one group of neurons, the effect of (see Additional file 1 Figure S1). Further evidence in the sex on the NPY mRNA in the arcuate nucleus neurones literature associating the level of AGRP expression with may have been diluted. growth potential is the observation that in chickens seg- These results support existing evidence that AGRP regating at the CCKAR locus, which is responsible for a expression relates closely to the growth potential of the 19% difference in body weight by 12 weeks of age, the bird, as much as to its nutritional state. Our previous birds homozygous for the high growth CCKAR allele had studies found that AGRP expression levels were signifi- higher AGRP expression than those carrying the low cantly higher in birds released from restriction for growth CCKAR allele . Thus, AGRP mRNA expression 2 weeks compared with birds released for 6 weeks prior was higher in birds that grow larger compared to those to cull, and those having been released longest had with the low growth genotype. greater body mass . Thus, AGRP mRNA expression One of the actions of AGRP is to increase food intake; was highest in birds furthest from their growth potential. however, there was no difference in the present study in the In the present study, AGRP mRNA levels were higher in daily food intake between males and females when released males who are known to grow faster and are larger than from restriction. This may be because of the very short- females and therefore would be furthest from their ideal term period of the release from restriction. Equally, this body weight [11, 12]. Experiment sections “Sex and study was on broiler breeders and it has been previously diet effect” and “Sex repeat effect” were performed with observed that an ICV injection of AGRP did not stimulate birds under restriction so no difference was observed in food intake in broiler chicks under ad libitum feeding AGRP mRNA/LBR mRNA POMC mRNA/ LBR mRNA CART mRNA/ LBR mRNA NPY mRNA/ LBR mRNA Caughey et al. Biology of Sex Differences (2018) 9:20 Page 8 of 12 A B 0.30 0.040 0.035 0.25 0.030 0.20 0.025 0.15 0.020 0.015 a 0.10 0.010 0.05 0.005 0.00 0.000 Female Male Female Male CD 0.018 0.60 0.016 0.50 0.014 0.012 0.40 0.010 0.30 0.008 0.006 0.20 0.004 0.10 0.002 0.000 0.00 Female Male Female Male Fig. 3 Anorectic and orexigenic gene expression (mean ± SEM) in the basal hypothalamus of male (n =14) and female (n = 15) broiler layer hybrids (AIL) on an ad libitum diet. Expression of mRNA for AGRP (A), NPY (B), POMC (C)and CART (D). P values are from unbalanced ANOVA with different labels (a, b) indicating statistically significant (P < 0.05) differences between means for the effect of sex hormone (ACTH) and this is heightened by the presence of conditions whereas it did in layers . The authors in that paper suggested the orexigenic effects of AGRP therefore melanocortin-2 receptor accessory protein 2 (MRAP2) maybedifferent betweenthetwobreeds. This is in whilst AGRP acts as an inverse agonist and antagonist on both MC4Rs and MC3Rs . This along with the co- agreement with the present study, where we did not ob- serve higher food intake in males despite their higher AGRP expression of MC4R, MC3R, AGRP, POMC and MRAP2 expression compared with females when allowed to feed ad mRNAs in the chicken hypothalamus indicates they may be important in the control of energy balance in the libitum. This raises the possibility that the actions of AGRP on food intake (in broilers at least) might be separate to its chicken with similar mechanisms of action as observed in effects on growth potential, thus highlighting a key area of mammals and teleosts . The evolutionary conserved ac- tions of AGRP and the melanocortin system on energy bal- interest and investigation in the interaction of AGRP with the central melanocortin system and its impact upon en- ance across vertebrates leadsustohypothesise thatAGRP ergy balance and growth. Mouse models of obesity already neurons may be an integrative centrefor theexpressionof suggest an involvement of AGRP in growth regulation genetic effects on growth potential. through interaction with the melanocortin system especially We observed that POMC and CART expression were the MC4R. Overexpression of AGRP in the mouse results higher in males compared with females in the AL fed in obesity and targeted inactivation of the MC4R causes groups. There has been some discrepancy in the literature obesity with features similar to the agouti obesity syndrome regarding POMC mRNA expression after re-feeding with [44, 45]. More pronounced effects of the melanocortin sys- examples of both increased expression and no effect [48, tem on growth have been observed in teleost fish, with the 49]. It appears that studies of males did see a difference sexual dimorphic difference in growth in zebrafish reversed between restricted and AL fed groups but studies of fe- by overexpression of the agouti-signalling protein, a mela- males did not, which matches the results observed in this nocortin receptor antagonist . In this species, targeted study [49, 50]. Although it should be noted that these sex prevention of the translation of AGRP mRNA resulted in differences in anorectic neuropeptide expression were not decreased larval growth, an effect mediated by the MC4R observed in our repeat study, the experiment sample size because MC4R knockout teleosts were resistant to the was based on a power analysis for AGRP expression growth-supressing effects of AGRP . Furthermore, changes and the statistical power may not have been suffi- recently, it has been shown chicken MC4Rs are equipo- cient to detect changes in the expression of the POMC tently activated by αMSH and adrenocorticotrophic and CART genes. POMC mRNA/LBR mRNA AGRP mRNA/LBR mRNA CART mRNA/LBR mRNA NPY mRNA/LBR mRNA Caughey et al. Biology of Sex Differences (2018) 9:20 Page 9 of 12 In birds, it has been observed that male and female AGRP in the orexigenic neurons. These results are in line somatic cells in gynandromorphs respond in different with other studies in which it was observed that the level ways to a common steroidal milieu with male and female of crop fill, used as a marker of recent food ingestion, was characteristics developing according to the proportion of not associated with hypothalamic expression of AGRP, the respective male and female cells in a tissue [51–53]. POMC or NPY . Ispaghula husk was added to the diet This is known as ‘cell autonomous sexual identity’ as a non-nutritive bulking agent, and our visual observa- (CASI) which suggests that cells appear to ‘know’ their tion that it appeared to physically fill the crop and gut sex and develop according to the genetic sex. This CASI suggested that it acted in this manner. Its effects are likely theory could be used to explain the sexually dimorphic to be comparable to other diet bulking agents such as oat phenotypes observed in birds, but is still controversial as hulls where improvements in behaviour are observed the evidence from gynandromorphs is mainly correl- whilst still limiting growth rate . The AL group had a ational. Yet, there is support for the CASI theory be- significantly increased body weight compared to the FR cause the transplantation of cells from a host prior to and IH group. This increase is unlikely to be entirely at- gonad differentiation will go onto develop a somatic cell tributable to crop content weight as the IH group also had fate but maintain the differentiated gene expression of significantly higher values, but there was no difference in the sex of the donor tissue . However, there is still a their body weights compared with the FR group. This sug- body of evidence that some of these sex chromosome- gested that the IH diet was nutritionally limited as ex- determined phenotypes are also influenced by gonadal pected. Further support for this is the absence of hormones; one of the clearest examples being the chick- significant differences in expected direction (i.e. increasing en’s comb and wattle [54, 55]. In terms of growth in in size) in the majority of organ weights between the FR chickens, research has only been able to investigate the and IH groups compared to the significantly increased influence of gonadal hormones and observed that block- values in the AL group. For example, after 2.5 days, the ing aromatase with Fadrozole in the embryo, so effect- liver was more than double the weight in the AL group ively sex reversing females so they experience male compared to the FR and IH groups (see Table 1 for other hormone milieu but are genetically female, increased organ weights). These points together prove that the growth by day 42 post hatch and apparently attenuated addition of ispaghula husk provided bulk but little or no the difference between males and females . Also, nutrients; indeed, it was observed the IH group took most body weight gain was inhibited by the application of an- of the daylight period to eat their ration whereas the FR drogens in male, female or castrated chickens . In group food was eaten within the first 10 min, suggesting mice which have been engineered to have male (XY) or the bulking effect caused gut fill and slowed down food in- female (XX) chromosome complements but develop take and passage. This is largely the mechanism put for- within each genotype to have male or female gonads, ward for its benefit in treating gastrointestinal mobility there are examples of genetic effects on body weight- issues in humans [61–63]. related traits. Mice carrying female sex chromosomes, ir- In mammals, the vagal afferents are suggested to relay respective of gonadal type, demonstrated greater food mechanoreceptive stimuli to the nucleus tractus solitarius intake during daylight hours and double the amount of in the hind brain and this signalling can be modified by adipose tissue . Evidence of genes responsible for the the action of classic satiety peptides . These afferents genetic effect on gene expression and potential sex- and the pathways they activate in the brain represent an linked genes responsible for the trans effects on those important target for future studies to understand the con- genes were identified . trol of food intake in birds. The role of mechanoreceptors It is clear that there are many interesting questions in the gut and the afferent pathways to the brain may be surrounding what, sex chromosomes or gonadal hor- more important in birds, but relatively little is known of mones, determines sex differences in the expression of their methods of action in birds at present [65, 66]. From the arcuate nucleus neuropeptide genes and potentially what we have observed in these experiments, IH provided growth and body weight. Further research will be needed bulk and slowed food intake but resulted in no alteration to explore these questions. in hypothalamic gene expression compared with restricted The addition of the bulking agent, ispaghula husk, had birds. It seems likely that the inhibitory effect of gut fill on no significant effect on the expression of the orexigenic food intake is achieved by a mechanism independent of an peptides in the basal hypothalamus compared to normal action on AGRP/NPY and POMC/CART neurons. This feed restriction; both groups had significantly higher may be the case only in broiler breeders; as was discussed AGRP and NPY expression than the AL group released above, injection of AGRP had no effect on food intake but from the restricted diet. This strongly suggests that the did increase it in layer chicks . It may be that in bulking agent supplies no mechanosensory signals from broilers in particular, AGRP neuronal activity may actually the gut to the brain to reduce expression of NPY and relate to the state of energy balance and a bird’s growth Caughey et al. Biology of Sex Differences (2018) 9:20 Page 10 of 12 potential as much as to predicting actual intake of food, Environmental Science and Analytical Services Division of the Scottish Government. particularly when there is no food choice as we have seen in a number of paradigms including the present study . Availability of data and materials The datasets generated and analysed during the current study are available in the University of Edinburgh data share repository https://doi.org/10.7488/ds/2293. Conclusion To conclude, we observed clear sex differences in the ex- Authors’ contributions pression of central food regulatory genes, which exist in SB analysed the data for experiment section “Sex and diet effect” along with the advice on the experimental design. AR provided substantial contribution to genetically distinct lines of chickens and are independent of the design and acquisition of data for experiment section “Genetically distinct feeding status. The higher level of AGRP mRNA in males line". NM provided substantial contribution to the acquisition of data for adds further evidence to the idea that the level of its expres- experiment section “Sex and diet effect". PWW was significantly involved in the design and acquisition of data from all experiments. ICD was a major sion in the chicken hypothalamus is an indicator of a bird’s contributor to the designs of the experiment, data collection, data growth potential. Although research into understanding interpretation and writing the manuscript. TB and RB were considerably how AGRP may determine growth is just at its beginning, involved in the experiment designs, with TB providing input on writing the manuscript. SDC was extensively involved in the experimental design, data the role of AGRP in modulating the central melanocortin collection, analysis and interpretation alongside being a major contributor to system is a good candidate mechanism for determining writing of the manuscript. All authors read and approved the final manuscript. long-term growth as well as short-term food intake. Al- Ethics approval though IH bulked out the diet and spread food intake over All animal experiments were performed under UK Home Office Project Licence a longer period, there were no changes in arcuate nucleus 70/7909, and birds were humanely killed as specified in Schedule 1 of the UK neuropeptide gene expression suggesting that mechanosen- Animals (Scientific Procedures) Act 1986. sory signals do not impact upon the expression of satiety Competing interests signals in the brain. However, the use of bulking agents to The authors declare that they have no competing interests. increase time spent showing feeding behaviour without metabolic satiation but whilst restricting growth seems to Publisher’sNote have welfare advantages from previous studies [30–34]. Springer Nature remains neutral with regard to jurisdictional claims in published The mechanism underlying the apparent welfare improve- maps and institutional affiliations. ment currently remains unknown but may involve neural Author details signals from the gut to the brain via vagal afferents. The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, Edinburgh EH25 9RG, Scotland, UK. 2 3 Bioinformatics and Statistics Scotland, Edinburgh, Scotland, UK. Scotland’s Additional file Rural College, Edinburgh, Scotland, UK. School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, England, UK. Additional file 1: Figure S1. Average weekly body weights for male and female chickens in a genetically distinct line. The average weekly Received: 23 January 2018 Accepted: 7 May 2018 body weights for male (n=57) and female (n=52) from broiler layer hybrid line fed on an ad libitum diet. P values are from a repeated measure ANOVA with different labels (*=p<0.05, **p<0.01, ***p<0.001) References indicating differences between means from post hoc tests. (PDF 361 kb) 1. Berns CM. The evolution of sexual dimorphism: understanding mechanisms of sexual shape differences. In: Moriyama PH, editor. Sexual dimorphism. Rijeka: InTech; 2013. p. 1–16. Abbreviations 2. Amadon D. The significance of sexual differences in size among birds. Proc ACTH: Adrenocorticotrophic hormone; AGRP: Agouti-related peptide; AL: Ad Am Philos Soc. 1959;103:531–6. libitum diet; ANOVA: Analysis of variance; CART: Cocaine- and amphetamine- 3. Ralls K. Mammals in which females are larger than males. 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