Comb size, shape complexity and laterality of laying hens reared in environments varying in resource choice.

The comb is an ornament involved in signalling condition in domestic fowl. We hypothesised that comb size, comb shape complexity (i.e., rugosity, the comb perimeter jaggedness), and comb laterality of laying hens would be influenced by the degree of environmental enrichment experienced during juvenile development in the form of resource choice. We conducted a 2 × 2 factorial crossover experiment with pullets reared in pens containing four perches of equal length and four litter areas of equal size. Pullets were exposed to a single choice vs multiple choices of perch and litter types (i.e., all the same vs all different) during Weeks 1-4 (Period 1) and/or Weeks 5-15 (Period 2) of rearing (n = 4 pens/treatment combination) prior to transfer to standard adult laying pens for Weeks 16-27 (Period 3). In Week 27, combs were photographed, and comb laterality (hanging on left or right side) was noted. Using a custom-made image analysis programme, we captured comb area (mm2), perimeter length (mm), and rugosity ((perimeter length - horizontal length) / horizontal length) from comb photographs of 6-7 randomly selected hens/pen. We predicted that hens reared in the multi-choice environment during Periods 1 and 2 would have larger, more complex, and left-side-biased combs than those in the other treatment groups, reflecting lower allostatic load. The predicted comb side bias was based on a possible bias in head posture/movements associated with greater right eye/ear use and left-brain hemispheric dominance. Contrary to our predictions, we detected an overall right-side bias in comb laterality, and no associations between resource choice treatment in Period 1 or Period 2 and comb area, perimeter length, rugosity, or laterality of the adult hens. Thus, variation in allostatic load resulting from the rearing treatments was insufficient to modify the trajectory of comb morphological development, possibly due to a ceiling effect when comparing environmental treatments on the positive end of the welfare spectrum. We found that left-lopping combs had shorter perimeters than right-lopping combs. However, among hens with left-lopping combs, those with larger combs were heavier and had less feather damage, while among hens with right-lopping combs, those with longer-perimeter combs were heavier and tended to have less comb damage. In conclusion, comb characteristics were related to physical condition at the individual level but did not serve as sensitive integrated indicators of hen welfare in response to basic vs enhanced resource choice during rearing.

Morphological characteristics of native chicken of village Chhajjian, Haripur Pakistan.

The present study was conducted to describe the variations in morphological characteristics of different selected populations of indigenous chickens. Five populations of chickens in different (localities) of Chhajjian, KP, Pakistan, were studied based on qualitative traits recorded for a total of 100 chickens. Each of the study populations contains multiple variants of plumage colors and other physical features. The average flock size was observed to be 38. Predominant plumage color was grayish and other mixtures along with different percentages in different localities. Pea comb was the dominant comb type in all localities. Most of the chickens were yellow skinned. Males in all populations were heavier and taller than the females. This recorded variation in morphological traits will help in the conservation of these chickens.

Genome-wide re-sequencing and transcriptome analysis reveal candidate genes associated with the pendulous comb phenotype in domestic chickens.

To determine the causative variations associated with two chicken comb phenotypes, pendulous comb (PC) or upright comb (UC), two pooled genomic DNA samples from PC and UC chickens were re-sequenced by Next-Generation Sequencer, and genome-wide Single nucleotide polymorphisms (SNPs) were detected. Using three selective sweep approaches, FST , θπ, and Tajima's D, with top 5% window values serving as the threshold, a total of 84 positively selective genes (PSGs) were identified. There were no SNPs in exons of the PSGs with significant differences in allele frequencies between the two comb phenotype groups. Then, 515 differentially expressed genes (DEGs) between the PC and UC were identified by RNA-seq. Three genes including CD36 (CD36 molecule), ADAMTSL3 (ADAMTS-like 3), and AOX1 (aldehyde oxidases 1) are overlapped between PSGs and DEGs. After genotyping seven candidate SNPs in the regulatory regions of the three overlapping genes in 120 chickens from two other breeds, two variants (rs14607046 and rs731818051) in the regulatory regions of AOX1 and ADAMTSL3 were found to have significant differences in allele frequency between the PC and UC, suggesting that the two variants may be causative mutations for PC. Overall, our study shed light on the genetic basis underlying the PC phenotype in chickens.

Phenotypic characteristics of upright and pendulous comb among chicken breeds and association with growth rate and egg production.

Upright and pendulous combs commonly exist in most single-comb chicken breeds. Here, the phenotypic characteristics of upright and pendulous combs in chickens and association with growth rate and egg production were analyzed. Phenotypic frequencies of upright and pendulous comb were investigated in five chicken breeds; the phenotypic frequencies of complete pendulous comb (CPC) and partial pendulous comb (PPC) ranged from 10.1% to 29.0% and 21.8% to 65.3%, respectively. CPC hens produced more eggs than PPC hens (P < 0.05) in Nongda-3, Huainan and Wenchang breeds. In Huainan breed, CPC males were heavier than PPC males at 12 and 16 weeks of age, while CPC females were heavier at 24 weeks of age. PPC and CPC chickens have greater (P < 0.05) comb length, comb height and comb index than upright comb (UC) chickens. There was no significant difference in comb phenotypic frequency distribution between the offspring from UC(♂) × CPC(♀) and CPC(♂) × UC(♀); however, it differed (χ² = 45.12, P < 0.01) between offspring from UC(♂) × UC(♀) and CPC(♂) × CPC(♀). These results suggested that the comb phenotype does not appear to be Z-linked; the effective loci influencing the trait could be estimated in a further study.

The role of pleiotropy and linkage in genes affecting a sexual ornament and bone allocation in the chicken.

Sexual selection and the ornaments that inform such choices have been extensively studied, particularly from a phenotypic perspective. Although more is being revealed about the genetic architecture of sexual ornaments, much still remains to be discovered. The comb of the chicken is one of the most widely recognized sexual ornaments, which has been shown to be correlated with both fecundity and bone allocation. In this study, we use a combination of multiple intercrosses between White Leghorn populations and wild-derived Red Junglefowl to, first, map quantitative trait loci (QTL) for bone allocation and, second, to identify expression QTL that correlate and colocalize with comb mass. These candidate quantitative genes were then assessed for potential pleiotropic effects on bone tissue and fecundity traits. We identify genes that correlate with both relative comb mass and bone traits suggesting a combination of both pleiotropy and linkage mediates gene regulatory variation in these traits.

Is sexual ornamentation an honest signal of male quality in the Chinese grouse (Tetrastes sewerzowi)?

We examined the variation in sexual ornamentation of male Chinese grouse (Tetrastes sewerzowi) in the Gansu Province, China, seeking to identify factors involved in whether ornament size and brightness are honest signals of male quality. Compared to unmated males, mated males had significantly larger and redder combs and, although they did not have significantly larger territories, they defended them more vigorously. Mated males had significantly higher blood carotenoid and testosterone levels, significantly better body condition, and significantly lower parasite loads than unmated males. Our findings are thus consistent with the hypothesis that comb size and color are honest signals of better male quality in the grouse, mediated through lower parasite loads and/or higher testosterone levels.

A sexual ornament in chickens is affected by pleiotropic alleles at HAO1 and BMP2, selected during domestication.

Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations.

Social context decouples the relationship between a sexual ornament and testosterone levels in a male wild bird.

In order to maximise fitness individuals should adjust their level of signalling according to their surrounding social environment. However, field experiments showing such adjustment of current signalling associated to changes in social context are lacking. Here, we manipulated levels of male aggressive- and dominance-related displays in a wild bird in our treated area by increasing testosterone levels using implants in a subset of males. We then compared the expression of sexual signals (i.e. comb size) between non-treated red grouse Lagopus lagopus scoticus males from control and treatment areas. We further explored the potential endocrinological mechanism linking social environment and signal expression by analysing testosterone levels in all males. Our treatment successfully increased overall aggressive- and dominance-related behaviours in the treatment area. Furthermore, testosterone-implanted birds increased their comb size as repeatedly shown in previous studies in male red grouse. Interestingly, untreated males living in the treatment area decreased their comb size, whilst increasing testosterone levels. Since comb size is a signal of dominance, untreated males from the treatment area may have perceived themselves as subordinate individuals and decreased their signalling levels to avoid confrontations with testosterone-treated, dominant individuals. In conclusion, our findings show that social context has the potential to regulate sexual signalling and testosterone levels. Our results highlight the role of social context when exploring the link between testosterone and behaviour, as it may reverse the relationship between both traits. Our results suggest that social context affects signalling and testosterone independently.

The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility.

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.

Effects of caponization and different forms of exogenous androgen implantation on immunity in male chicks.

This study determined the caponization effects on the immune responses in male chicks. Different forms of exogenous androgen implantation on male chick immunity were compared. Healthy, uniform male Single Comb White Leghorn chicks were caponized at 3 wk of age. Birds were housed in individual cages (35 x 30 x 40 cm, length x width x height). Each of 27 sham-operated (sham) and caponized (capon) male chickens were used for trial 1. Trial 2 used 60 capons divided into 4 treatments with implants of either 1 mm i.d. x 3 mm o.d. 58 mg of cholesterol, testosterone (TES), 5alpha-dihydrotestosterone (5alpha-DHT), or 19-nortestosterone (19-NorT). The exogenous androgen was implanted immediately after caponization and resupplied every 4 wk for an entire 13-wk feeding trial. The results from trial 1 showed that the relative bursa weight increased compared with the sham treatment (P < 0.05). The 2 wk post-Newcastle disease virus titer and the delayed-type hypersensitivity (DTH) of 48 h post-phytohemagglutinin phosphate (PHA-P) injection were increased compared with the sham treatment (P < 0.05). In trial 2, implanted 5alpha-DHT and 19-NorT could decrease the relative bursa weight in capons (P < 0.05). The 2 wk post-Newcastle disease virus titer in the 5alpha-DHT group was higher than that in the cholesterol group (P < 0.05). The 19-NorT group had the highest (P < 0.05) PHA-P response. Peripheral blood lymphocyte subset population analysis revealed that the percentage of CD4 T cells in the TES group was lower (P < 0.05) compared with that of the 5alpha-DHT group. Differently, the percentage of CD8 T cells in the TES and 19-NorT groups was higher (P < 0.05) than that in the 5alpha-DHT group. Male chicks that were caponized had increased bursa weight and PHA-P response, whereas different forms of exogenous androgen implantation reverted the phenomena in an order of potency of 5alpha-DHT and 19-NorT > TES, and the PHA-P response was TES > 5alpha-DHT >19-NorT.

Effects of caponization and testosterone implantation on immunity in male chickens.

This study examined the effects of caponization using different doses of testosterone (TES) on sexuality, hematology, and immune responses in male chickens. Healthy male chickens were caponized at 12 wk of age and selected at 16 wk of age for a 10-wk experiment. Fifteen intact male and 15 caponized male chickens were assigned to trial 1. In trial 2, ten sham-operated male chickens (sham) and 40 capons (randomly divided into 4 treatments) were implanted with cholesterol (CHOL, 9.24+/-0.36 mg), low TES (5.88+/-0.23 mg), medium TES (9.81+/-0.17 mg), or high TES (16.7+/-0.24 mg) administered at 16, 20, and 24 wk of age. Results from trial 1 showed caponization decreased the comb length, height and weight, and hematocrit (P<0.05) and increased the hemagglutination inhibition (HI; 1 wk postchallenge) and hemagglutination titer after Newcastle disease virus (NDV) and SRBC injections (P<0.05). In trial 2, the medium TES increased the comb length and height as compared with the CHOL group. Only the high TES increased the comb weight (P<0.05). The HI titer (1 wk postchallenge) in the CHOL group was higher than the sham (P<0.05). The medium TES decreased the HI titer (P<0.05) to the level of the sham (P>0.05). The phytohemagglutinin response was higher in the high TES group 24 h postinjection (P<0.05) and in the medium TES 48 h postinjection (P<0.05) as compared with the CHOL group. High dose TES implantation decreased the white blood cell counts as compared with the CHOL and sham groups (P<0.05). It appears that caponization decreased the blood androgen concentration and enhanced the humoral (anti-NDV and anti-SRBC) immune response. Testosterone implantation up to a threshold concentration could inhibit the humoral (anti-NDV) immune response and increase the cell-mediated (phytohemagglutinin) immune response.

Sperm: seminal fluid interactions and the adjustment of sperm quality in relation to female attractiveness.

An important predictor of male fitness is the fertilizing efficiency of their ejaculates. Ejaculates are costly to produce and males are predicted to devote greater resources to copulations with reproductively superior females. It is well established that males allocate different numbers of sperm to ejaculates. However, less is known about how males adjust their sperm quality, which has important implications for our understanding of fertilization and the evolution of sexual strategies. Here we test in the fowl, Gallus gallus, whether males adjust their sperm velocity by differentially allocating seminal fluid to copulations with attractive and unattractive females. To disentangle the contributions of sperm and seminal fluid to sperm velocity, we separated and remixed sperm and seminal fluid from ejaculates allocated to females of different attractiveness. We show that dominant males increase the velocity of the sperm they invest in more attractive females by allocating larger ejaculates that contain seminal fluid that increases sperm velocity. Furthermore, we find weak evidence that males also allocate sperm with higher velocity, irrespective of seminal fluid, to more attractive females.

Ornamental comb colour predicts T-cell-mediated immunity in male red grouse Lagopus lagopus scoticus.

Sexual ornaments might reliably indicate the ability to cope with parasites and diseases, and a better ability to mount a primary inflammatory response to a novel challenge. Carotenoid-based ornaments are amongst the commonest sexual signals of birds and often influence mate choice. Because carotenoids are immuno-stimulants, signallers may trade-off allocating these to ornamental colouration or using them for immune responses, so carotenoid-based ornaments might be particularly useful as honest indicators of immuno-compentence. Tetraonid birds, such as the red grouse Lagopus lagopus scoticus, exhibit supra-orbital yellow-red combs, a conspicuous ornament which functions in intra- and inter-sexual selection. The colour of combs is due to epidermal pigmentation by carotenoids, while their size is testosterone-dependent. In this study, I investigated whether comb characteristics, and in particular, comb colour, indicated immuno-competence in free-living male red grouse. I assessed T-cell-mediated immunity using a standardised challenge with phytohaemagglutinin. Red grouse combs reflect in the red and in the ultraviolet spectrum of light, which is not visible to humans but that grouse most likely see, so I measured comb colour across the whole bird visible spectrum (300-700 nm) using a reflectance spectrometer. I found that males with bigger and redder combs, but with less ultraviolet reflectance, had greater T-cell-mediated immune response. Comb colour predicted T-cell-mediated immune response better than comb size, indicating that the carotenoid-based colouration of this ornament might reliably signal this aspect of male quality.

Changes in sperm quality and numbers in response to experimental manipulation of male social status and female attractiveness.

In promiscuous species, male reproductive success is determined by the interaction between the ability to access and choose females of the highest reproductive quality and, after copulation, the ability to outcompete the ejaculates of rival males. Disentangling the factors regulating the interplay between traits conferring a reproductive advantage before and after copulation is therefore crucial to understanding how sexual strategies evolve. Here we show in the fowl Gallus gallus, where social status determines copulation success, that dominant males produce more sperm than subordinates but that the quality of dominant males' sperm decreases over successive copulations, whereas that of subordinates remains constant. Experimentally manipulating male social status confirmed that ejaculate quality (the number and quality of sperm produced) was a response to the social environment rather than the result of intrinsic differences between dominant and subordinate males. We further show that dominant males responded to variation in female sexual ornamentation, which signals reproductive quality, by adjusting the number and quality of sperm they transferred, whereas subordinate males did not: they transferred ejaculates of similar quality to females with different ornament sizes. These results indicate that trade-offs between traits influencing reproductive success before and after copulation, combined with variation in social dynamics and female quality, may favor the evolution of phenotypically plastic alternative reproductive strategies.

Potential relationships between physical traits and male broiler breeder fertility.

Genetic selection in primary broiler breeders may modify skeletal structure, possibly impeding semen transfer, and could alter the size and degree of fluctuating asymmetry (FA) of bilateral traits associated with fertility. Hence, we hypothesized specific morphometric traits could predict differential fertility. Sixty primary broiler breeder males from Strains A and B (n = 30/strain) were individually housed with an average of 10 females per male. Male fertility and sperm penetration (SP) through the perivitelline layer were estimated on fresh eggs. At 50 wk, BW, keel length (KL), posterior pelvic width and length (PPW, PPL), dorsal pelvic width and length (DPW, DPL), tarsometatarsal length and width (TL, TW), comb length and width (CL, CW), and wattle length, width, and area (WL, WW, WA) were measured. Results indicated that Strain A had smaller BW, KL, WL, WW, WA, CL, CW, PPL, DPL, and DPW. A higher degree of FA was found in Strain A TL and WL (P < 0.05), yet DPW FA was greater for Strain B (P < 0.001). In addition, DPW FA negatively correlated with Strain B fertility (r = -0.369; P < 0.01); however, other FA measurements did not correlate with estimated fertility or SP. Strain A WL correlated with SP (r = 0.383; P < 0.01) and fertility (r = 0.346; P < 0.01). Results indicate DPW alteration may impact semen transfer upon copulation, as Strain A fertility negatively correlated with DPW (r = -0.298; P < 0.05). This research provides evidence that morphometric traits might be useful to predict fertility in broiler breeders.

Single-step divergent selection for male comb shape in two White Leghorn lines.

1. Divergent selection for comb shape (SH, the way that the cockerel bears the comb) was performed in 2 White Leghorn lines in a study aimed at assessing possibilities for improving SH. 2. Line A, selected for large comb size (CS) at 29 weeks of age, had great SH problems whereas Line H, selected for high hyaluronic acid concentration in the comb (HA), had minor SH problems. Multivariate analyses were used to estimate genetic parameters for SH, CS and HA in lines A and H and in a control line (C). 3. Significant direct selection responses for SH were achieved in both lines. There were significant unfavourable correlated responses for CS in both lines. 4. The correlated responses for HA were not significant and were unfavourable in Line A and favourable in Line H. 5. Heritabilities for SH and CS were high in both lines and relatively low for HA. Most of the genetic correlations were in agreement with the correlated responses obtained.

A tradeoff between immunocompetence and sexual ornamentation in domestic fowl.

Females often select their mates on the basis of the size or intensity of sexual ornaments, and it is thought that such traits are reliable indicators of male quality because the costliness of these traits prevents cheating. The immunocompetence handicap hypothesis is a recently proposed mechanistic explanation of these costs and states that males carry ornaments at the expense of their resistance to disease and parasites. The tradeoff between immunocompetence and sexual ornamentation was hypothesized to arise as a consequence of the dual effect of androgens on ornamentation (+) and immune function (-). To test this hypothesis, we compared comb size between male domestic chickens Gallus domesticus of lines divergently selected for antibody responses to sheep erythrocytes (three lines: selected for low response or high response and a control line). The importance of comb size in inter- and intrasexual selection is well established, and comb size is strongly dependent on testosterone level. Comb size was larger in the males of the low line than in the high line, and comb size of control males was intermediate, indicating a tradeoff between ornamentation and immunocompetence. Testosterone (T) levels varied in a similar fashion (TLow > TControl > THigh), suggesting that this hormone could mediate the tradeoff between ornamentation and immunocompetence. These results support the idea that a tradeoff with immune function may constrain the expression of secondary sexual ornaments.

The effect of pyrazine odor on body weight and the weight of various organs in chicks (Gallus gallus domesticus).

Embryonic and posthatch long-term exposure to the odor of 2-methoxy-3-isobutyl-pyrazine (2M3IP) was examined for its potential physiological consequences as reflected in changes in BW and organ weights in domestic chicks (Gallus gallus domesticus). Experiments were run from Day 1 of incubation to the age of 3 wk with a total of 360 fertile chicken eggs. The experimental design consisted of four treatment groups: PP chicks were exposed to 2M3IP during both incubation and posthatch rearing; PC chicks were exposed to 2M3IP during incubation only; CP chicks were exposed to 2M3IP during rearing period only; CC control chicks were not exposed to 2M3IP. Chicks were weighed immediately after hatch and at 3 wk of age, when they were necropsied. Various organs (thyroid, adrenal, testes, comb, liver, spleen, abdominal fat, and the bursa of Fabricius) were removed and weighed. Body weights of both sexes in the PP group were reduced. This reduction was significant in males relative to both CP and CC groups and in females only relative to the CP group. Effects of 2M3IP exposure on the examined organs were as follows: in males, adrenal gland weight significantly increased in the PP group vs all other groups. No weight differences were found between the other inspected organs among the four treatments. In females, comb weight significantly decreased compared with the rest of the groups when 2M3IP exposure occurred during incubation (PC). Further investigation is needed to study the mechanisms that underlie the differential effects of pyrazine odor on male and female chicks.

Comb size and estrogen levels toward the onset of lay in broiler and layer strain females under ad libitum and restricted feeding.

The time course of comb development and estrogen levels were compared in broiler (BX) and layer (LX) females that consumed feed ad libitum (A) or were subjected to quantitative feed restriction (R). The chicks were reared under short photoperiod [(6 h light (L):18 h dark (D)] until 22 wk of age. At this time, photoperiod was increased in one step to 8L:16 D, and then gradually increased until 14L:10D at 34 wk. There was a significant interaction between genetic type and feeding treatment, such that entry into lay of the LX-R and BX-R females was delayed by 1 and 4 wk, respectively, relative to the LX-A and BX-A birds. Mean comb size of LX-A birds began to increase while still under 6L:18D photoperiod; that of the other treatment groups did not begin to increase until photoperiod was shifted to 8L:16D. Comb size of individual LX-A, LX-R, and BX-A birds began to increase about 8 wk prior to individual onset of lay; that of BX-R birds about 11 wk prior to onset of lay. In all groups, estrogen levels remained low until 3 to 4 wk prior to onset of lay, when they showed a sharp increase. Following onset of lay, estrogen levels of all groups remained high. Critical day length of LX-A birds appears to be lower than that of BX-A birds. In addition, feed restriction per se appears to decrease photoperiod responsiveness of BX birds.