This study was conducted to determine the effects of 25-hydroxycholecalciferol ( 25-OH-D 3 ) on performance, egg quality, tibia quality, and serum hormones concentration in laying hens reared under high stocking density. A total of 800 45-week-old Lohmann laying hens were randomly allotted into a 2 × 2 factorial design with 2 levels of dietary 25-OH-D 3 levels (0 and 69 μg/kg) and 2 rates of stocking densities [506 (low density) and 338 (high density) cm 2 /hen]. Laying hens were monitored for 16 wk. High stocking density decreased laying rate, egg weight, and feed intake compared with low stocking density ( P < 0.01) during 1 to 8 wk and 1 to 16 wk. Overall, high stocking density increased eggshell lightness value and decreased shell redness and yellowness value, strength, thickness, and relative weight compared with low stocking density ( P < 0.05). Dietary supplementation with 25-OH-D 3 reduced the value of the eggshell lightness and increased its yellowness and eggshells weight ( P ≤ 0.05). The increase in eggshell thickness was more pronounced when 25-OH-D 3 was supplemented to layers under high stocking density (interaction, P < 0.05). Layers under high stocking density had lower ash content and calcium content in the tibia than layers under low stocking density ( P = 0.04); dietary 25-OH-D 3 increased tibia strength compared with no addition ( P = 0.05). Layers under high stocking density had higher serum concentrations of 25-OH-D 3 , corticosterone ( CORT ), lipopolysaccharide ( LPS ), and osteocalcin ( OC ; P < 0.05), lower content of parathyroid hormone ( PTH ) compared with layers under low stocking density ( P < 0.01). Dietary 25-OH-D 3 increased serum concentration of 25-OH-D 3 , carbonic anhydrase ( CA ), and calcitonin ( CT ) ( P < 0.01) and reduced corticosterone, lipopolysaccharide and osteocalcin concentration ( P ≤ 0.05). The increase effect in PTH was more pronounced when 25-OH-D 3 was supplemented to layers under high stocking density (interaction, P = 0.05). Overall, the results gathered in this study indicate that high stocking density result in reducing production performance, shell color and quality, and tibia health, whereas dietary 25-OH-D 3 was able to maintain tibia health and to mitigate the negative impact of high stocking density on productive performance.

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Peroxisome proliferator activated receptor-alpha (PPARα) and Egl nine homolog 3 (EGLN3) play critical roles in facilitating the adaptation to a hypoxic environment. However, the relationship between EGLN3 and PPARα variants and hypoxic adaptation remains poorly understood in Tibetan chickens. To better understand the effects of genetic variation, we sequenced exons of PPARα and EGLN3 in 138 Lowland chickens (LC) from 7 breeds that were located in Emei, Miyi, Shimian, Wanyuan, Pengxian, and Muchuan in the Sichuan province, and Wenchang in the Hainan province (altitudes for these locations are below 1800 meters). Total 166 Tibetan chickens (TC) from 7 subpopulations that were located in Shigatse, Lhoka, Lhasa, Garze, Aba, Diqing and Yushu in the Tibetan area were also sequenced (altitudes greater than 2700 meters). One single-nucleotide polymorphism (rs316017491, C > T) was identified in EGLN3 and was shared by TC and LC with no significant difference for allele frequencies between them (P > 0.05). Six single-nucleotide polymorphisms (SNP1, A29410G; SNP2, rs13886097; SNP3, T29467C; SNP4, rs735915170; SNP5, rs736599044; and SNP6, rs740077421) including one non-synonymous mutation (SNP2, T > C) were identified in PPARα. This is the first report of SNP1 and SNP3. There was a difference between TC and LC for allele frequencies (P <0.01), except for SNP1, SNP4, and SNP5) The fix index statistic test indicated that there was population differentiation between TC and LC for SNP2, SNP3, and SNP6 in PPARα (P < 0.05). Phylogenetic analysis showed that the genetic distance among chickens, finch and great tit were close for both EGLN3 and PPARα. Bioinformatics analysis of PPARα showed that SNP2 leads to an amino acid substitution of Ile for Met, which results in the protein being more likely to be hydrolyzed. Thus, genetic variation in PPARα may play a role in the ability of TC to adapt to a high altitude environment; however we were unable to identify a relationship between polymorphisms in EGLN3 and environmental adaptability.