H. J. Zhang

Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

Effect of monochromatic light stimuli during embryogenesis on muscular growth, chemical composition, and meat quality of breast muscle in male broilers

This study was conducted to evaluate the effect of monochromatic light stimuli during embryogenesis on breast muscle growth, chemical composition, and meat quality of male broilers. Fertile broiler eggs (Arbor Acres; n = 1,320) were preweighed and randomly assigned to 1 of 3 treatment groups in 3 modified incubators: 1) control group (in dark condition), 2) monochromatic green light group (560 nm), and 3) monochromatic blue light group (480 nm). The monochromatic lighting systems sourced from light-emitting diode lamps and were equalized at the intensity of 15 lx at eggshell level. After hatch, 120 male chicks from each group were placed in 6 replicates with 20 birds each. All of the birds were housed under white light (30 lx at bird-head level) with a light schedule of 23L:1D. At 21, 35, and 42 d of age, BW and breast muscle weight in the green light group were significantly increased compared with birds in the blue or dark groups (P < 0.05). The breast muscle weight and breast muscle percentages in birds incubated under green light were significantly elevated by 50.39 g (0.76%) and 54.07 g (1.20%) than those in the dark condition or blue group at 42 d of market age (P < 0.05), respectively. In the green light group, feed intake during 0~42 d was higher than that in the other 2 treatment groups (P < 0.05); feed conversion ratio during 0~35 and 0~42 d were lower than that in the dark condition (P < 0.05). No significant differences in the contents of breast moisture, CP, crude fat, and crude ash among all groups were observed (P > 0.05). Green light stimuli tended to increase cooking loss (P = 0.08) and L* value of 24-h meat color (P = 0.09). These results suggest that green light stimuli during embryogenesis enhanced the posthatch BW of male broilers, increased breast muscle growth, and improved the feed conversion ratio, but it did not cause any noticeable changes in breast chemical composition or overall meat quality characteristics.

The local origin of the Tibetan pig and additional insights into the origin of Asian pigs.

Background The domestic pig currently indigenous to the Tibetan highlands is supposed to have been introduced during a continuous period of colonization by the ancestors of modern Tibetans. However, there is no direct genetic evidence of either the local origin or exotic migration of the Tibetan pig. Methods and Findings We analyzed mtDNA hypervariable segment I (HVI) variation of 218 individuals from seven Tibetan pig populations and 1,737 reported mtDNA sequences from domestic pigs and wild boars across Asia. The Bayesian consensus tree revealed a main haplogroup M and twelve minor haplogroups, which suggested a large number of small scale in situ domestication episodes. In particular, haplogroups D1 and D6 represented two highly divergent lineages in the Tibetan highlands and Island Southeastern Asia, respectively. Network analysis of haplogroup M further revealed one main subhaplogroup M1 and two minor subhaplogroups M2 and M3. Intriguingly, M2 was mainly distributed in Southeastern Asia, suggesting for a local origin. Similar with haplogroup D6, M3 was mainly restricted in Island Southeastern Asia. This pattern suggested that Island Southeastern Asia, but not Southeastern Asia, might be the center of domestication of the so-called Pacific clade (M3 and D6 here) described in previous studies. Diversity gradient analysis of major subhaplogroup M1 suggested three local origins in Southeastern Asia, the middle and downstream regions of the Yangtze River, and the Tibetan highlands, respectively. Conclusions We identified two new origin centers for domestic pigs in the Tibetan highlands and in the Island Southeastern Asian region.

Manganese supplementation enhances the synthesis of glycosaminoglycan in eggshell membrane: A strategy to improve eggshell quality in laying hens

This study investigated the effect of dietary Mn supplementation on eggshell quality, ultrastructure, glycosaminoglycan (GAG), and uronic acid content, and mRNA and protein expression of Galβ1,3-glucuronosyltransferase (GlcAT-I). A total of 216 layers (Hy-Line Grey) at age of 50 wk were divided into 3 groups. In the first 8 wk of the 12-wk feeding trial, all groups were fed a basal diet that met all layer nutrient requirements except for Mn. In the last 4 wk, each group was fed 1 of 3 diets supplemented with Mn levels at 0, 25, or 100 mg Mn/kg. Dietary Mn deficiency did not affect the egg performance of layers. Dietary Mn supplementation significantly improved the breaking strength, thickness, and fracture toughness of eggshells (P < 0.05). In photographs of eggshell ultrastructure, the size of mammillary cones and cracks in the outer surface were decreased by dietary Mn supplementation. The contents of GAG and uronic acids in eggshell membrane were significantly increased by dietary Mn addition (P < 0.05). This result was further confirmed by increased mRNA expression and protein expression of GlcAT-I when Mn was added to the diet. This study suggests that dietary Mn supplementation can improve eggshell quality by enhancing the GAG and uronic acid synthesis in the eggshell glands, which can affect the ultrastructure of eggshells.

Effect of electrical stunning current and frequency on meat quality, plasma parameters, and glycolytic potential in broilers

This study was designed to determine the effect of electrical stunning variables (low currents and high frequencies) on meat quality, glycolytic potential, and blood parameters in broilers. A total of 54 broilers were stunned with 9 electrical stunning methods for 18 s using sinusoidal alternating currents combining 3 current levels (35 V, 47 mA; 50 V, 67 mA; and 65 V, 86 mA) with 3 frequencies (160, 400, and 1,000 Hz). Samples for meat quality were obtained from the pectoralis major (PM) and musculus iliofibularis (MI), and samples for glycogen metabolism were taken from the PM and tibialis anterior muscle at 45 min postmortem. The use of high frequency reduced the shear value in PM (400 and 1,000 Hz vs. 160 Hz; P < 0.01) and cooking loss in MI (1,000 Hz vs. 160 and 400 Hz; P < 0.01). The shear value of PM decreased at high frequency (400 and 1,000 Hz) when current was high (50 V, 67 mA and 65 V, 86 mA; P < 0.01) but increased at high frequency (1,000 Hz) when current was low (35 V, 47 mA). Stunning with 1,000 Hz (vs. 160 Hz) caused low glycogen and glycolytic potential in PM (P < 0.05). Plasma corticosterone decreased (P < 0.05) at high currents (≥50 V, 67 mA) but was not affected by changes in frequency. Electrical current interacted with frequency in plasma glucose, redness 24 h postmortem, shear value (PM), pH 24 h postmortem (MI), and glycolytic potential (tibialis anterior; P < 0.05). This study indicated that high stunning frequencies (400 and 1,000 Hz) may improve meat quality without aggregating stress when the current was not too low (>50 V, 67 mA).

Effects of transport stress on blood metabolism, glycolytic potential, and meat quality in meat-type yellow-feathered chickens

To investigate the effects of transport stress on blood metabolism, glycolytic potential, and meat quality in male Chinese Lingnan meat-type yellow-feathered chickens, three hundred sixty 72-d-old birds were randomly assigned to 1 of 5 treatments: unstressed control, 45-min (short-term) transport with 45-min (short-term) recovery, 45-min transport with 3-h (long-term) recovery, 3-h (long-term) transport with 45-min recovery, and 3-h transport with 3-h recovery. Each treatment consisted of 6 replicates with 12 birds each. All birds (except control group) were transported according to a designed protocol. Long-term transport decreased the plasma glucose level (P < 0.001), glycogen concentration in both breast (P = 0.023) and thigh (P = 0.012) muscles, and affected the size of IIb fibers in the tibialis anterior by decreasing the area (P = 0.031) and increasing the density (P = 0.046), drip loss (P = 0.045), and the a* (redness; P = 0.040) value in breast muscle. Long-term recovery after transport also decreased the plasma glucose level (P = 0.018) and showed a trend toward decreased breast glycogen concentration (P = 0.064). These results suggested that transport did not cause any noticeable changes in overall meat quality characteristics, although breast meat drip loss and meat color were affected. A long-term recovery from a period of feed and water deprivation also caused hypoglycemia and did not improve meat quality in Lingnan meat-type yellow-feathered chickens.

Identification of Genes Related to Growth and Lipid Deposition from Transcriptome Profiles of Pig Muscle Tissue

Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of longissimus dorsi muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that CAV2, MYOZ2, FRZB, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and FASN, SCD, ADORA1, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine longissimus dorsi muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.

Hypoxia adaptation and hemoglobin mutation in Tibetan chick embryo

Tibetan chick lives at high altitudes between 2600 and 4200 m with a high hatchability and low land breeds survive rarely with a hatchability of 3.0% under hypoxia of simulated 4200 m. Under hypoxia of whole 21 d, the hatchability of Tibetan chick and Recessive White Feather broiler differed with a greatest disparity from day 4 to 11 and also significantly in other stages except from day 1 to 3. Hypoxia in each stage did not reduce significantly survival rate of this stage except hatchability. These two results indicated that the hypoxia in the early stage had an adverse effect on the later stage. All exons encoding chick hemoglobins were sequenced to analyze gene polymorphism. The functional mutation Met-32(B13)-Leu, related with hypoxia, was found in αD globin chain and the mutation frequency increased with increased altitude. In addition, under hypoxic conditions, the population with higher mutation frequency had a higher hatchability. The automated homology model building was carried out using crystal structure coordinates of chick HbD. The results indicated that the substitution Met-32(B13)-Leu provides a more hydrophobic environment which leads to higher stability of heme and oxygen affinity of hemoglobin. The occurrence of the mutation Met-32(B13)-Leu is related to the origin of Tibetan chick.

Effects of dietary oxidized oil on laying performance, lipid metabolism, and apolipoprotein gene expression in laying hens.

We studied the effects of dietary oxidized oils on serum lipid metabolic indices, estradiol level, and the gene expression of apolipoprotein B-100 and apolipoprotein VLDL-II in laying hens. Hy-Line Grey hens (280 ± 10 d old; average egg production, 90.0 ± 2.5%) were allotted to 1 of 4 dietary treatments, which were supplemented with 0 (control group), 1% (low oxidized group), 2% (moderately oxidized group), or 4% (highly oxidized group) thermally oxidized soybean oil. Each treatment contained 6 replicates, with 12 birds each. The feeding trial lasted for 30 d. Laying performance data were recorded weekly. Other indices were measured on d 0, 2, 6, 14, and 30 of the feeding trial. Hens in the moderately and highly oxidized groups had significantly lowered feed conversion ratios (P < 0.05). Those in the highly oxidized group also had decreased concentrations of serum very low density lipoprotein cholesterol on d 30 (P < 0.05) compared with the very low density lipoprotein cholesterol of hens in the moderately oxidized group. Hens in the moderately oxidized group had significantly increased expression of apolipoprotein B-100 (P < 0.05) from d 6 to 30. Consequently, hepatic triglyceride increased in this group on d 30 (P < 0.05). Serum triglyceride decreased in the moderately oxidized group on d 30 (P < 0.05), which may have been caused by the activation of peroxisome proliferator-activating receptor α. Serum estradiol levels were not significantly affected by oxidized oils at any time of measurement, but were significantly different between d 0 and 30 within the moderately oxidized group. This fact indicated that the effect of oxidized oils on apolipoprotein B-100 might partially be a cumulative result of the increasing secretion of estradiol. The results suggested that oxidized oil may affect the performance of laying hens through the regulation of apolipoproteins and estradiol.

Genome Resequencing Identifies Unique Adaptations of Tibetan Chickens to Hypoxia and High-Dose Ultraviolet Radiation in High-Altitude Environments

Tibetan chicken, unlike their lowland counterparts, exhibit specific adaptations to high-altitude conditions. The genetic mechanisms of such adaptations in highland chickens were determined by resequencing the genomes of four highland (Tibetan and Lhasa White) and four lowland (White Leghorn, Lindian, and Chahua) chicken populations. Our results showed an evident genetic admixture in Tibetan chickens, suggesting a history of introgression from lowland gene pools. Genes showing positive selection in highland populations were related to cardiovascular and respiratory system development, DNA repair, response to radiation, inflammation, and immune responses, indicating a strong adaptation to oxygen scarcity and high-intensity solar radiation. The distribution of allele frequencies of nonsynonymous single nucleotide polymorphisms between highland and lowland populations was analyzed using chi-square test, which showed that several differentially distributed genes with missense mutations were enriched in several functional categories, especially in blood vessel development and adaptations to hypoxia and intense radiation. RNA sequencing revealed that several differentially expressed genes were enriched in gene ontology terms related to blood vessel and respiratory system development. Several candidate genes involved in the development of cardiorespiratory system (FGFR1, CTGF, ADAM9, JPH2, SATB1, BMP4, LOX, LPR, ANGPTL4, and HYAL1), inflammation and immune responses (AIRE, MYO1F, ZAP70, DDX60, CCL19, CD47, JSC, and FAS), DNA repair, and responses to radiation (VCP, ASH2L, and FANCG) were identified to play key roles in the adaptation to high-altitude conditions. Our data provide new insights into the unique adaptations of highland animals to extreme environments.