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Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00743-0
10.1016/j.psj.2024.104164
104164
GENETICS AND MOLECULAR BIOLOGY
Research note: effects of hyperpigmentation of the visceral peritoneum on body weight and selection method in Chinese yellow-feathered broilers
Wang Yan wynew2004@163.com
*†1
Liu Tianfei †
Liu Sijia *
Luo Wei †
Tang Lin *
Li Yin †
He Yanhua †
Shu Dingming †
Qu Hao †
Luo Chenglong chenglongluo1981@163.com
†2
⁎ Guangdong Polytechnic of Science and Trade, Guangzhou 510430, China
† State Key Laboratory of Swine and Poultry Breeding Industry, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2 Corresponding author: chenglongluo1981@163.com
1 Co-corresponding author:

08 8 2024
11 2024
08 8 2024
103 11 10416428 4 2024
30 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
We previously identified a dark blue appearance through the skin of abdomen, especially the colored chicken breeds, called hyperpigmentation of the visceral peritoneum (HVP) which characterized by intense pigmentation of connective tissue in the visceral peritoneum. The HVP has recently garnered increasing attention due to its negative impact on carcass appearance, and been an important concern in the poultry industry, especially for the Chinese yellow-feathered broilers. In this study, we measured the in vivo HVP at different time points, and analyzed the correlation between the HVP in vivo and postmortem. Then, established an accurate and reliable HVP phenotypic measuring method in vivo for early selection in chickens and analyzed the association of phenotypic variations with the in vivo HVP traits with growth traits. The results showed that the in vivo HVP at 21 d of age in chickens have a high heritability (h2 = 0.452) through estimating genetic parameters, and in vivo HVP levels at 21 and 42 d were both significantly associated with those postmortem in chickens, suggesting that directional selection on reducing HVP can be implemented as early as at 21 d in the breeding and production of chickens. Although, we found HVP had no effect on the body weight at 1 d, it could significantly reduce the body weight at 21, 42, 70 d and 91 d in chickens. This suggests HVP not only has a negative effect on carcass traits, but also significantly reduces the production in the poultry industry.

Key words

growth performance
melanin
relevance analysis
chicken
==== Body
pmcINTRODUCTION

Melanin is a macromolecule mainly synthesized by melanocytes, which originate from the neural crest. Additionally, Schwann cell precursors linked to developing nerves are another cellular source of melanocytes in mouse and chicken (Vandamme and Berx, 2019). Among numerous Chinese indigenous chicken breeds, the Silkie as the noteworthy, is characterized by snow-white silky feathers and possess black skin, meat and bones. A defining trait of the Silkie chicken is Fibromelanosis (FM), which leads to extensive pigmentation of the dermal layer of the skin and its connective tissues (Dorshorst et al., 2011). The FM in Silky fowl is associated with increased expression of endothelin 3 (EDN3), genetically caused by an inverted duplication of 2 large genomic regions (>100 kb for each), that are located on Gallus gallus Autosome 20 (Dorshorst et al., 2011).

We previously observed a distinct dark blue appearance through the skin of abdomen and a black connective tissue layer when the skin is removed, particularly in colored chicken breeds. This phenomenon, termed hyperpigmentation of the visceral peritoneum (HVP), is characterized by the intense pigmentation of the connective tissue within the visceral peritoneum (Luo et al., 2013). Despite this observation, the molecular basis of HVP remain elusive. It is noteworthy that the pigmentation in HVP is confined to the chicken peritoneum, which is distinct from that in FM. Notably, the structural variant associated with the FM in Silky chicken were not detected in HVP-affected chickens (data not presented), suggesting that the genetic mechanism underlying HVP and FM are distinct.

The phenomenon of HVP has garnered an increasing attention in the poultry industry recently, owing to its potential risks to the well-being of affected birds and harm to the aesthetic appeal of commercial chicken carcasses. Beyond the aesthetic impact, it remains a lack of clarity regarding whether HVP affects other economically crucial traits like the growth. Addressing this issue has thus emerged as a pivotal challenge within the poultry industry. Broiler producers are particularly eager to explore methods that enable early identification and selection, thereby minimizing the proportion of chickens affected by HVP. In this study, we measured the in vivo HVP at different time points, and analyzed the correlation between the HVP in vivo and the HVP postmortem. Furthermore, we established an accurate and reliable in vivo phenotypic measuring method for HVP, aiming to facilitate early selection in chickens. To evaluate the potential impact of HVP on poultry production, we also analyzed the phenotypic association between the HVP traits in vivo and the growth traits.

MATERIALS AND METHODS

Ethics Statement

All experimental animal procedures were approved by the Animal Care Committee of Institute of Animal Science, Guangdong Academy of Agricultural Sciences (Guangzhou, People's Republic of China, approval number GAAS-IAS-2009-73) before the start of the experiment. The animals involved in the study were treated humanely.

Animals and Sample Collection

All birds in this study were from the Institute of Animal Science, Guangdong Academy of Agricultural Sciences (Guangzhou, China), and belong to the Chinese yellow-feathered broilers. A method of determining of the HVP in chickens was established and performed on 180 birds of the Chinese in digenous chicken breed Huiyang Bearded (HB) chicken. In detail, the HVP was separately measured in vivo at 21 and 42 d, and then HVP was separately evaluated postmortem at 21, 42 and 112 d, with 60 birds for each. In the association analysis, 2 distinct populations were utilized. The first one was HB chicken population, which was half-sibling and encompassed 446 individuals from 2 hatches. The second population was the “HB × HQLA” F15 resource population, consisting of 1651 individuals. Which were obtained through 13 generations of group self-breeding starting from the F2 generation. Of course, the “HB × HQLA” F14-F16 generations were derived from 12 to 14 generations of self-breeding. This “HB × HQLA” F2 population is a 3-generation intercross resulting from reciprocal crossing between 2 distinct founder lines: the HQLA and HB breeds. The “HB × HQLA” F2 individuals were generated as previously described (Sheng et al., 2013). The HQLA chicken breed, specifically the Fast-growing Lingnanhuang Line A, is highly tailored to Chinese palates, having undergone rigorous selection for fast growth and superior meat quality over a span of more than 10 generations. All birds were raised in cages, provided with unrestricted access to food and water, and underwent the same vaccination and immunization program.

The body weights of 446 HB birds were each measured at 1, 21, 42, 70, and 91 d respectively, and sacrificed at 112 d to evaluate the HVP level for each. Experimental chickens were slaughtered utilizing a wet electric stunning technique, subsequently complemented by a neck-cutting procedure to effectively drain the blood. Each of the 1651 “HB × HQLA” F15 individuals was weighed at 1, 21, 42, and 70 d, respectively. The HVP level of each bird was measured in vivo at 21 d, then were batch-sacrificed and HVP-evaluated at 21 d (200 birds), 42 d (200 birds), 70 d (400 birds), 91 d (400 birds) and 119 d (400 birds), respectively. The “HB × HQLA” F14-F16 resource populations separately had 1293, 1574 and 1233 birds, for which the body weights of each bird at 1 and 21 d, and the in vivo HVP level at 21 d, were recorded and evaluated to estimate genetic parameters, including heritability and genetic correlation.

Evaluation of the Chicken HVP in Vivo and Postmortem

To accurately assess the in vivo HVP level in chickens, we identified a standard abdominal region extending from the bird's sternum terminus to the anus, and encompassing the final ribs on both sides up to the pubic margin (designated as the HVP observed area). This area boasts a thin skin layer devoid of significant muscle occlusion, thereby directly observing the HVP level after feather removal. The HVP phenotype exhibits a high variability in chickens according to the color of pigmentation and its concerning area, and thus can be supposed to be a quantitative trait (Luo et al., 2013). Here, we roughly classified the HVP trait into 4 distinct subphenotypes: Phenotype_0 (P_0, Figures 1A and 1B), Phenotype_1 (P_1, Figures 1C and 1D), Phenotype_2 (P_2, Figures 1E and 1F), and Phenotype_3 (P_3, Figures 1G and 1H). These phenotypes represent the absent, the mild, the severe and the highly severe hyperpigmentation, respectively (Figure 1).Figure 1 Classification of the hyperpigmentation of visceral peritoneum in vivo. A, C, E and G represent absent, mild, severe and very severe hyperpigmentation in vivo at 21 d of age in chickens, represented by 0, 1, 2, and 3, respectively; B, D, F and H represent absent, mild, severe and very severe hyperpigmentation postmortem at 21 d of age in chickens, represented by 0, 1, 2 and 3, respectively.

Figure 1

In detail, P_0 indicates the absence of melanin in the visceral peritoneum; P_1 indicates that the melanin-pigmented area exhibiting cyan or light black does not exceed 25% of the observed HVP area, and/or the area exhibiting black or dark black pigmentation accounting for less than 10% of the observed HVP area; P_2 indicates a melanin-pigmented area in cyan or light black hues accounts for 25% to 50% of the total observed HVP area, and/or an area between 10% and 50% displaying black or dark black pigmentation; Lastly, P_3 indicates that the melanin-pigmented area, encompassing cyan, light black, black, or dark black hues, comprises more than 50% of the observed HVP area. In Postmortem HVP level evaluation, the abdominal skin encompassing above HVP observed area was firstly excised to expose the visceral peritoneum, and then were classified into 4 subphenotypes following the same rules implemented in vivo.

Statistical Analysis

In this study, the AI-REML method (Jensen et al., 1996) used for the variance component estimation is based on the univariate mixed linear, and the genetic correlations estimated by the multivariate mixed linear, both implemented by the DMU package (Madsen et al., 2010).

The variance component estimated model was:y=Xβ+Zα+e where y was the vector of observations of the HVP level, β was the vector of fixed effects (sex, 2 levels); α was the vector of animal additive genetic effects; e was the vector of random residuals; and X and Z were corresponding incidence matrices. It was assumed that α∼N (0, Aσ2α), e∼N (0, Iσ2e), where A is the pedigree-based genetic relationship matrix, I is the identity matrix, σ2α is the additive genetic variance, σ2e is the residual variance.

The HB population harboring 446 birds, and the “HB × HQLA” F15 resource population harboring 1,651 birds, were used for the association analysis, respectively. The GLM program from JMP software (SAS Institute Inc., Cary, NC USA) was employed to evaluate the associations between phenotypes of growth traits and the measured in vivo HPV values at 21 d. The model used to analyze the data was assumed to be:Yijklm=μ+Si+Hj+Sk+Dkl+Fm+eijklm

where Yijklm was the phenotypic value of the trait, μ was the overall mean, Si was the effect of the ith sex, Hj was the effect of the jth hatch, Sk was the effect of the kth sire, Dkl was the effect of the lth dam within the kth sire, Fm was the effect of the mth phenotype of hyperpigmentation of the peritoneum in vivo at 21 or 42 d (4 subphenotypes: P_0, P_1, P_2 and P_3) and eijklm was the residual effect. The adjusted phenotypic value was calculated as μ + eijklm.

RESULTS AND DISCUSSION

HVP Evaluation in Vivo

Utilizing the method outlined in the materials and methods section, we evaluated the in vivo HVP level in the HB birds and the “HB × HQLA” F15 resource population. The results showed that, among the 446 HB chickens tested at 21 d, there were 49 birds with P_0 subtype, 229 birds with P_1, 115 birds with P_2, and 49 birds with P_3. When the same cohort was evaluated at 42 d, the distribution shifted to 182 birds with P_0, 145 birds with P_1, 67 birds with P_2, and 52 birds with P_3. Among the “HB × HQLA” F15 individuals, the in vivo HVP level at 21 d revealed 555 birds with P_0, 834 birds with P_1, 179 birds with P_2, and 83 birds with P_3.

HVP trait seems unique to the yellow-feathered broilers compared with the white-feathered broilers that we ever slaughtered. Drawing parallels from the occurrence, migration, and pathway of melanin in eukaryotes, we preliminarily propose that the molecular genetic basis underlying the dominant white feather trait may serve to suppress the production and migration of melanin (Kerje et al., 2004). Accurately assessing the in vivo HVP after 42 d of age in chickens poses significant challenges. As the birds are being mature, their feathers undergo growth and development, resulting in enlarged pores. Forcibly plucking feathers at this stage can lead to bleeding and significant harm to the chicken. Additionally, observing the color of the visceral peritoneum through the skin becomes challenging due to the thickening of abdominal skin and increased subcutaneous fat in chicken. As a result, the in vivo HVP was only tested at 21 and 42 d, and no further measurements were conducted after 42 d.

Genetic Parameters

Based on “HB × HQLA” resource population, we identified the in vivo HVP at 21 d harbored a high heritability (h2 = 0.452, determined through the AI-REML method), indicating a marked influence of genetic factors on HVP at 21d. Notably, the in vivo HVP level at 21 d showed significantly (P < 0.01) positive genetic correlations (rg) with both postmortem and in vivo HVP levels determined at different times points. For instance, it had a strong correlation with the in vivo HVP level at 42 d (rg = 0.595) and the postmortem HVP level at 112 d in the HB breed (rg = 0.534). Additionally, the postmortem HVP level in “HB × HQLA” F15 resource population displayed significant (P < 0.01) positive genetic correlations with those at 42 (rg = 0.603), 70 (rg = 0.648), 91 (rg = 0.556), and 119 (rg = 0.597) d of age. Furthermore, the in vivo HVP at 42 d in HB chickens displayed a strong positive genetic correlation with that of the postmortem at 112 d (rg = 0.620) .

Early selection in poultry breeding practice is most favored, as this can reduce cost and speed up the process of breeding. The findings presented above suggest that in chicken breeding practice, we can prioritize selecting in vivo HVP level at 21 and 42 d to effectively minimize the proportion of chickens exhibiting suboptimal HVP at market-ready ages. The results revealed that among 446 Huiyang bearded chickens, the in vivo HVP testing at 21 d yielded a P_0 proportion of 10.99% and a P_1 proportion of 51.35%. Conversely, at 42 d, the proportion were 40.81% for P_0 and 32.51% for P_1. respectively. It is noteworthy that the timing of HVP selection can be tailored based on the selection pressure within the population. In large chicken populations, conducting HVP testing and selected at 21 d is advisable as the growing feathers are easily removable, and there is minimal abdominal fat deposition, facilitating observation and minimizing harm to the chickens. Additionally, producers favor earlier selection to curtail costs and expedite the breeding process. In smaller chicken populations, the HVP can be assessed and selected at 42 d, effectively contributing to the goal of minimizing the proportion of chickens exhibiting HVP traits at market time. Once the first-generation selection is completed, initiating the 21 d selection process for the second generation can further diminish the percentage of chickens possessing undesirable HVP traits.

Association of Phenotypic Variations With the HVP Level in Vivo With Growth Traits

As we are concerned, results have shown the in vivo HVP at 21 d was significantly associated with certain growth traits in the HB breed as well as the “HB × HQLA” F15 resource population. Additionally, the live body weight at 21, 42, 70, and 91 d for the birds with the P_1 of HVP level were significantly higher than those for the HB breed birds with the P_3 of HVP level (P < 0.05; Table 1). In the “HB × HQLA” F15 resource population, similar significant trends were observed, wherein birds with the P_0 of HVP level consistently exhibited the highest live body weight at 21, 42, and 70 d, and also possessed the greatest shank circumference at 70 d among all the tested birds.Table 1 Association analyses in the Huiyang Bearded and “HB × HQLA” F15 resource population.

Table 1Traits	Phenotypic variations with the in vivo HVP level at 21 d (μ ± S.E.) of the Huiyang bearded	
P-value	0 (n = 182)	1 (n = 145)	2 (n = 67)	3 (n = 52)	
BW21	0.012	159.943 ± 2.340ab	162.253 ± 1.182a	158.637 ± 1.505b	153.811 ± 2.336b	
BW42	0.033	407.488 ± 7.268ab	410.875 ± 3.703a	399.841 ± 4.683b	387.493 ± 7.345b	
BW70	0.022	876.178 ± 11.550ab	885.283 ± 5.859a	877.035 ± 7.487a	844.356 ± 11.534b	
BW91	0.045	1122.442 ± 14.898a	1117.811 ± 7.526a	1102.879 ± 9.579ab	1071.022 ± 14.871b	
DW112	0.318	1094.575 ± 18.473	1112.887 ± 9.514	1097.809 ± 12.060	1076.280 ± 18.959	
AFW	0.357	21.647 ± 1.722	20.044 ± 0.872	19.498 ± 1.119	17.127 ± 1.772	
	
Traits	Phenotypic variations with the in vivo HVP level at 21 d (μ ± S.E.) of the “HB × HQLA” F15 resource population	
P-value	0 (n = 555)	1 (n = 834)	2 (n = 179)	3 (n = 83)	
BW21	1.65E−7	237.858 ± 1.8388a	230.767 ± 1.577b	221.088 ± 2.900c	220.175 ± 4.065c	
BW42	0.001	644.219 ± 5.306a	629.684 ± 4.495b	608.339 ± 8.668c	614.629 ± 14.371abc	
BW70	0.001	1167.401 ± 12.026a	1135.286 ± 10.210b	1094.917 ± 20.607bc	1053.724 ± 35.641c	
BW91	0.282	1391.224 ± 21.395	1361.475 ± 17.254	1309.755 ± 38.955	1309.126 ± 72.933	
SC21	0.487	2.567 ± 0.320	2.860 ± 0.274	2.204 ± 0.505	2.163 ± 0.707	
SC42	0.088	3.296 ± 0.012	3.277 ± 0.010	3.243 ± 0.020	3.247 ± 0.033	
SC70	0.002	3.790 ± 0.014a	3.753 ± 0.012b	3.738 ± 0.023ab	3.635 ± 0.041c	
n: the number of birds studied.

BW21, 42, 70, 91: live body weight at 21, 42, 70 and 91 d of age, g.

SC21, 42, 70: shank circumference at 21, 42 and 70 d of age, cm.

DW: dressed weight at 112 d of age, g.

AFW: abdominal fat weight at 112 d of age, g.

a,b,c Means within a row lacking a common superscript differ (P < 0.05); in the order from a to c, the average values of traits are represented from largest to smallest, with “a” indicating the largest mean value and “c” indicating the smallest mean value.

μ Means the average values of trait measurements.

S.E. means standard error of the mean.

In broiler production, we observed that the proportion of the HVP tends to rise under suboptimal chicken-rearing environments. However, the underlying mechanism behind how the HVP leads to body weight loss in chickens remains elusive. Notably, studies have revealed that melanin in ubiquitous in various organs and tissues, including the peritoneum, across vertebrates, amphibians, reptiles and fish, primarily serving as a protective barrier against the harmful effects of UV light (Dubey and Roulin, 2014). In Cobb broilers, the presence of melanin in the shanks manifest positive correlation to internal pigmentation, especially for the abdominal fat (Crespo and Pizarr, 2006). Although presence of melanin in the skin and connective tissue poses no harm to humans, the traits that include the HVP are considered unappealing to consumer. Notably, the HVP affects live body weight, resulting in poor uniformity among chickens, which may inflict significant economic losses on producers. This underscores the negative impact of HVP not only on carcass traits but also on the overall production in the poultry industry.

DISCLOSURES

We are submitting a research article entitled “Research note: effects of hyperpigmentation of the visceral peritoneum on body weight and selection method in Chinese yellow-feathered broilers.” All authors have read and approved this manuscript for Poultry science. This is an original article and has not been simultaneously presented to any other periodical, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright holder.

ACKNOWLEDGMENTS

This study was supported by the “Youth Top-notch Talent on Science & Technology” of Guangdong Province Special Support Program (2016TQ03N898 ), the Program for Doctoral Scientific Research Start-up Funds of Guangdong Polytechnic of Science and Trade (GDKM2022-94 ), the key research platforms and projects for ordinary universities in Guangdong Province in 2023 (2023ZDZX4098 ), the Horizontal Project of Guangdong Polytechnic of Science and Trade (GDKM-2022-A-49 ).
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