Investigating the impact of pigmentation variation of breast muscle on growth traits, melanin deposition, and gene expression in Xuefeng black-bone chickens.

The blackness traits, considered an important economic factor in the black-bone chicken industry, still exhibits a common phenomenon of significant difference in blackness of breast muscle. To improve this phenomenon, this study compared growth traits, blackness traits, and transcriptome of breast muscles between the High Blackness Group (H group) and Low Blackness Group (L group) in the Xuefeng black-bone chickens. The results are as follows: 1) There was no significant difference in growth traits between the H group and the L group (P > 0.05). 2) The skin/breast muscle L values in the H group were significantly lower than those in the L group, while the breast muscle melanin content exhibited the opposite trend (P < 0.05). 3) A significant negative correlation was observed between breast muscle melanin content and skin/breast muscle L value (P < 0.05), and skin L value exhibiting a significant positive correlation with breast muscle L value (P < 0.05). 4) The breast muscle transcriptome comparison between the H group and L group revealed 831 and 405 DEGs in female and male chickens, respectively. This included 37 shared DEGs significantly enriched in melanosome, pigment granule, and the melanogenesis pathway. Seven candidate genes (DCT, PMEL, MLANA, TYRP1, OCA2, EDNRB2, and CALML4) may play a crucial role in the melanin production of breast muscle in Xuefeng black-bone chicken. The findings could accelerate the breeding process for achieving desired levels of breast muscle blackness and contribute to the exploration of the mechanisms underlying melanin production in black-bone chickens.

Genes influencing deposition of melanin in breast muscle of the Xuefeng black bone chicken based on bioinformatic analysis.

The Xuefeng black bone chicken (XFBC) represents an important poultry genetic resource. However, the darkness in breast muscle is heterogeneous. The molecular genetic mechanisms underlying melanogenesis of breast muscle in XFBC remains unclear. This study used RNA-seq to compare the difference in transcriptome between hyperpigmentation and hypopigmentation of breast muscle. Six cDNA libraries were constructed for hyperpigmentation and hypopigmentation groups in XFBC. We identified 395 differently expressed genes (DEGs) between hyperpigmentation and hypopigmentation group (P < 0.05, |log2FC|≥1). Gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated several differentially enriched biological functions and pathways involved in melanogenesis of the breast muscle. Gene set enrichment analysis (GSEA) GO analysis identified two significant gene sets, including the pathways of pigment metabolic process and transmembrane receptor protein tyrosine kinase activity. GSEA-KEGG analysis identified the process of tyrosine metabolism and several genes related with melanogenesis in breast muscle of the XFBC. The protein-protein interaction network was constructed and eight genes were clustered in the module. We identified nine hub genes, including TYR, TYRP1, DCT, GPR143, MLANA, SLC24A5, GPNMB, MLPH, and EDNRB2. Taken together, the DEGs and hub genes identified in the study provide a solid basis for the study of the genetic regulatory mechanisms involved the melanogenesis in the breast muscle of the XFBC.

Lung transcriptome analysis for the identification of genes involved in the hypoxic adaptation of plateau pika (Ochotona curzoniae).

The plateau pika, a typical hypoxia-tolerant mammal lives 3000-5000 m above sea level on the Qinghai-Tibet Plateau, has acquired many physiological and morphological characteristics and strategies in its adaptation to sustained, high-altitude hypoxia. Blunted hypoxic pulmonary vasoconstriction is one such strategy, but the genes involved in this strategy have not been elucidated. Here, we investigated the genes involved and their expression profiles in the lung transcriptome of plateau pikas subjected to different hypoxic conditions (using low-pressure oxygen cabins). A slight, right ventricular hypertrophy was observed in pikas of the control group (altitude: 3200 m) vs. those exposed to 5000 m altitude conditions for one week. Our assembly identified 67,774 genes; compared with their expression in the control animals, 866 and 8364 genes were co-upregulated and co-downregulated, respectively, in pikas subjected to 5000 m altitude conditions for 1 and 4 w. We elucidated pathways that were associated with pulmonary vascular arterial pressure, including vascular smooth muscle contraction, HIF-1 signalling, calcium signalling, cGMP-PKG signalling, and PI3K-Akt signalling based on the differentially expressed genes; the top-100 pathway enrichments were found between the control group and the group exposed to 5000 m altitude conditions for 4 w. The mRNA levels of 18 candidate gene showed that more than 83% of genes were expressed and the number of transcriptome The up-regulated genes were EPAS1, Hbα, iNOS, CX40, CD31, PPM1B, HIF-1α, MYLK, Pcdh12, Surfactant protein B, the down-regulated genes were RYR2, vWF, RASA1, CLASRP, HIF-3α. Our transcriptome data are a valuable resource for future genomic studies on plateau pika.

Modulation of intestinal morphology and microbiota by dietary Macleaya cordata extract supplementation in Xuefeng Black-boned Chicken.

Antibiotics are commonly overused to prevent livestock from diseases and to increase production performance. As potential substitutes of antibiotics, plant extracts have attracted the attention of researchers. It was known to all that addition of Macleaya cordata extract (MCE) to the food could advance immunity, intestinal health and animal performance. Thus, it was conducted to investigate the influence of MCE (0, 100, 150 and 200 mg/kg, with six replicate pens/treatment and 24 hens/pen) on intestinal morphology and microbial diversity in different intestinal segments in Xuefeng black-boned chicken in this study. The results showed that MCE supplement (100, 150 and 200 mg/kg) significantly diminished (P < 0.05) the crypt depth of the jejunum as compared to basal diet group. The 100 mg/kg group displayed a marked increase (P < 0.05), compared with 0 and 200 mg/kg group, in ileum microbial diversity as represented by the Shannon's index. In the cecum, treatment of MCE significantly decreased (P < 0.01) the Firmicutes, but Deferribacteres in 200 mg/kg MCE group were significantly raised (P < 0.05). In conclusion, we found that MCE improved intestinal morphology and reduced the crypt depth in jejunum. Together, addition of 200 mg/kg MCE modulated intestinal microbiota, increased beneficial bacteria such as Lactobacillus. Adding 100 mg/kg MCE to diet increased bacterial community diversity and relative abundance in jejunum and ileum, but had no effect on cecum microbial diversity.

Black Soldier Fly (Hermetia illucens) Larvae Meal Modulates Intestinal Morphology and Microbiota in Xuefeng Black-Bone Chickens.

The addition of Hermetia illucens larvae meal (HILM) to the feed could contribute to particular antimicrobial and intestinal health in animal husbandry. This study was conducted to investigate the effects of HILM on intestinal morphology and microbial diversity in different intestinal segments of Xuefeng black-bone chickens. All of 432 birds (45 weeks old) were randomly assigned to four equal groups with six replicates and 18 hens in each replicate: (A) basal diet, (B) basal diet with 1% HILM, (C) basal diet with 3% HILM, and (D) basal diet with 5% HILM. The results showed that, compared with the basal diet group, the HILM supplement significantly increased the abundance-based coverage estimator (ACE) and Chao index in cecum (p < 0.05). Diet with 1% HILM significantly increased the villus height (VH) of the duodenum (p < 0.05) and cecum microbial diversity as represented by the Simpson index (p < 0.05). In particular, 1% HILM displayed a markedly increase in the genus unclassified Bacteroidales (cecum, p < 0.05). A basal diet with 3% HILM markedly increased the beneficial genus Romboutsia (jejunum, p < 0.05). Also, principal component analysis (PCA) cluster analysis showed that 3% of HILM was more individual than other groups (p < 0.05). However, 5% HILM decreased the VH and the ratio of villus height to crypt depth (VH/CD) of the jejunum and increased beneficial bacteria such as Staphylococcus (p < 0.05), which was regarded as pathogenetic genera. In conclusion, we found that HILM improved intestinal morphology and increased microbiological diversity and species abundance. Together, dietary supplementation of 1 or 3% HILM might benefit the intestinal morphology and intestinal microbiota of Xuefeng black-bone chicken. However, the addition of 5% HILM could decrease VH and the ratio of VH/CD of the jejunum and increased pathogenetic genera. HILM was an excellent protein substitute for Xuefeng black-bone chickens, which could meet the nutritional requirements under the condition of less feed. These results provide information for HILM meal as an alternative source of soybean meal in Xuefeng black-bone chickens' feed.

Effects of Macleaya cordata extract on laying performance, egg quality, and serum indices in Xuefeng black-bone chicken.

The abuse of antibiotic growth promoters (AGPs) in feed has led to drug resistance and ecological damage would threaten human health eventually. Natural plants have become a hotspot in the research and application of substituting AGPs because of their advantages of safety, efficiency, and availability. This study was conducted to investigate the effects of Macleaya cordata extract (MCE) in the diet of Xuefeng black-bone chicken on laying performance, egg quality, and serum indices. In this study, 576 birds (47-week-old) were evenly distributed between 4 treatments with 6 replicates of 24 hens each. The control group was fed a basal diet without MCE and the remaining groups received 100, 150, or 200 mg/kg MCE for 84 d. Results revealed that the strength and thickness of the eggshell increased significantly with the dietary addition of MCE (P < 0.05). The serum concentrations of glutathione peroxidase increased in the MCE groups (P < 0.01). Simultaneously, progesterone, follicle stimulating hormone, estradiol as well as serum luteinizing hormone levels also increased with the addition of MCE (P < 0.05). Compared with the control group, supplementation of MCE significantly decreased the tumor necrosis factor-α and interleukin-6 levels (P < 0.01). In summary, it was concluded that diet addition of 200 mg/kg MCE ameliorated egg quality, enhanced anti-oxidation and immune activity, and regulated hormone secretion of Xuefeng black-bone chicken.

The sequence and characterization of mitochondrial of Lepus oiostolus (Lagomorpha: Leporidae).

Lepus oiostolus is widely inhabited in the Qinghai-Tibet Plateau. So far, little mitochondrial genome information of this genus has been described. To grasp a better comprehension on the molecular basis of L. oiostolus, we obtained the complete mitochondrial DNA genome sequences of this species. The mitogenome was 17,320 bp in length, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 noncoding regions. The complete mitochondrial genome of L. oiostolus would be of great utility in the phylogenetic analysis of the Lagomorpha and also provide meritorious insights into the deeper problems of the phylogenic analysis.

Comparison of overfed Xupu and Landes geese in performance, fatty acid composition, enzymes and gene expression related to lipid metabolism.

OBJECTIVE: The aim of this study was to compare overfeeding performance, fatty acid composition, blood chemistry, enzymes and genes expression overfed Xupu and Landes geese. METHODS: Sixty male Xupu geese (80 d) and Landes geese (80 d) were selected. After a period of one-week of pre-overfeeding, Xupu and Landes geese were overfed three meals of 550 and 350 g/d, respectively, of a high-carbohydrate diet in the first week of the overfeeding period. The next week, geese were given four meals of 1,200 and 850 g/d, respectively, over 8 to 14 d. Finally, geese were given five meals of 1,600 and 1,350 g/d, respectively, for the last two weeks. RESULTS: After overfeeding for 28 d: Compared with Landes geese, Xupu geese liver weight and liver-to-body weight ratio decreased (p<0.05), while final weight, slaughter weight, total weight gain, abdominal fat weight, and feed-to-liver weight ratio increased (p<0.05). The levels of elaidic acid (C18:1t9), oleic acid (C18:1n-9), eicosenoic acid, and arachidonic acid in the liver of Xupu geese significantly increased (p<0.05), and the levels of myristic acid and stearic acid significantly decreased (p<0.05), while methyleicosanoate acid significantly increased (p<0.05). Xupu geese had higher plasma concentrations of triglyceride and very low density lipoprotein cholesterol (p<0.05), and decreased activities of alanine aminotransferase, aspartate aminotransferase, and lipase (LPS) (p<0.05). Landes geese had higher LPS activity (p<0.05), but lower cholinesterase activity (p<0.05) when compared with Xupu geese. The mRNA expression levels of fatty acid dehydrogenase (FADS) gene, elongase of longchain fatty acid 1 (ELOVL1) gene, ELOVL5, and acyl-Co A: cholesterol acyltransferase 2 (ACAT2) gene were significantly upregulated (p<0.05) in Landes goose when compared with Xupu geese. CONCLUSION: This study demonstrates that the liver production performance of Landes geese was better than that of Xupu geese to some extent, which may be closely related to LPS activity, as well as the expression of FADS, ELOVL1, ELOVL5, and ACAT2.

A Novel Zebrafish Larvae Hypoxia/Reoxygenation Model for Assessing Myocardial Ischemia/Reperfusion Injury.

Strategies to reduce reperfusion injury after ischemia have been considered in clinical practice, but few interventions have successfully passed the proof-of-concept stage. In this study, we developed a novel zebrafish larvae hypoxia/reoxygenation (H/R) model to simulate myocardial ischemia/reperfusion injury (MIRI), with potential utility as a drug screening tool. After H/R treatment, videos of transgenic [Tg(cmlc:EGFP)] larval zebrafish hearts were captured using a digital high-speed camera, and the heart rate, diastolic area, systolic area, and total fraction of area changed were quantified. The mRNA expression of tnnt2, bnp, and hif1α was quantified, and red blood cells (RBCs) were detected by O-dianisidine staining. We found that a decline in cardiac contractility occurred in zebrafish larvae 48 h after hypoxia treatment. Reoxygenation for 2-5 h after 48 h of hypoxia caused heart dysfunction in zebrafish larvae, and were determined to be the optimum conditions for simulating MIRI similar to mammalian models. Our results indicated that heart dysfunction after reoxygenation in zebrafish larvae was accompanied by an upregulated gene expression of a number of myocardial injury biomarkers and increased numbers of RBCs. In conclusion, the novel larval zebrafish H/R model developed in this study could be used for rapid in vivo screening and efficacy assessment of MIRI therapeutics.

Montmorillonite improved the intestinal mucosal barrier functions of laying hens in late production.

This study was conducted to investigate the effects of dietary supplementation with montmorillonite (MMT) on performance, intestinal endotoxin concentration, gut mucosal oxidation status, intestinal morphology and permeability, and immunological barrier function of laying hens during late production. Four hundred and eighty 75-week-old laying hens (Lohmann Brown) were randomly assigned to five treatments with eight replicates per treatment and 12 hens in each replicate. The hens were fed the basal diet supplemented with 0 (control), 0.3, 0.6, 0.9, or 1.2 g MMT/kg for 70 days. Compared with the control, supplemented with 0.9 g MMT/kg increased egg mass significantly (p < 0.05) during weeks 1-5 of the experiment. Supplemented with 0.6 and 0.9 g MMT/kg also increased the endotoxin concentration in the ileal digesta (p < 0.05), but decreased the MDA concentration in the ileum significantly (p < 0.05). The T-AOC in the jejunum of the group fed 0.3 g MMT/kg was significantly increased (p < 0.05). Compared with the control, the villus height:crypt depth of ileum from the groups fed 0.6, 0.9, and 1.2 g MMT/kg increased significantly (p < 0.05). The sIgA concentration of jejunum in the groups fed 0.6 and 0.9 g MMT/kg was higher (p < 0.05) than the control. The MMT supplementation linearly increased (p < 0.05) the mRNA expression of claudin-1 and claudin-5 in the jejunum. Dietary MMT supplementation down-regulated the mRNA expression of NF-κB P65 and TNF-α in the jejunum in a linear and quadratic manner (p < 0.05). The IL-1ß mRNA expression of jejunum in the group fed 0.6 g MMT/kg was lower (p < 0.05) than the control. In conclusion, dietary supplementation with MMT may improve the gut barrier functions and suggests that 0.9 g/kg of MMT in diets may be the optimal supplemental level for laying hens in late production.

DNA barcodes and their characteristic diagnostic sites analysis of Schizothoracinae fishes in Qinghai province.

The Qinghai-Tibetan Plateau (QTP), the source and upper reaches of many Asian rivers, are crisscrossed by rivers and dotted with lakes. Schizothoracinae fishes, species native to the QTP, are distributed widely through these rivers and lakes. Over the past decades, ecological protection has become increasingly intense. The rapid acquisition of the genetic information and accurate gene sequence database are assumed to play an important role in the conservation of species diversity and biodiversity. In this study, 153 COI sequences (648bp in length) covering 13 species in 8 genera of Schizothoracinae fishes in Qinghai Province were used to determine whether barcode could identify Schizothoracinae species accurately. The average Kimura two parameter (K2P) genetic distances within and among species were 0.35% and 8.83%, respectively. The maximum K2P distance within species was observed in Gymnocypris eckloni (1.36%) while minimum K2P distance among species was observed between Chuanchia labiosa and Schizopygopsis pylzovi (0.23%). Overlaps existed in K2P distance intra- and inter- species based on both the genes. Eleven groups with 9 single-species groups and 2 multi-species groups were identified through Automatic Barcode Gap Discovery System, which were consistent with the overlaps of K2P distance. 96.7% as the accurate ratio for COI barcode was calculated and high solution was observed in the phylogenetic trees based on COI gene and Cyt b gene. Except for the similar results based on two genes above, COI barcode was more economical than Cyt b gene. The SOM model successfully predicted characteristic-diagnostic sites at species level: 36 characteristic-diagnostic sites from eight species, in which 12 from Gmnodiptychus pachycgeilus, 2 from Platypharodon extremus, 7 from Ptychobarbus kaznakovi, 2 from Schizopygopsis anteroventris, 2 from Schizopygopsis malacanthus, 3 from Schizopygopsis malacanthus chengi, 3 from Schizothorax dolichonema and 5 from Schizothorax lantsangensis. Our results show that Schizothoracinae fishes can be identified validly by using COI DNA barcode. Thirty-six characteristic-diagnostic sites were proposed to be applied into works of species identification for the Schizothoracinae fishes in Qinghai Province.

Effects of Bacillus subtilis C-3102 on production, hatching performance, egg quality, serum antioxidant capacity and immune response of laying breeders.

To investigate the supplemental effects of Bacillus subtilis C-3102 on the production, hatching performance, egg quality, serum antioxidant capacity and immune response of laying breeders, a total of 480 Xuefeng black-bone (25-week-old) hens were randomly assigned into four treatment groups: Hens fed the basal diets with 0 (CON), 3.0 × 105 (BS-1), 6.0 × 105  cfu/g (BS-2) and 9.0 × 105 (BS-3) cfu/g of B. subtilis C-3102. As the B. subtilis C-3102 level increased, egg weight (linear, p < 0.01; quadratic, p = 0.003), fertility (linear, p = 0.021; quadratic, p = 0.059), hatchability (linear, p = 0.038; quadratic, p = 0.119) and yolk colour (linear, p = 0.006; quadratic, p = 0.021) increased in a linear or quadratic manner. Yolk index increased quadratically (linear, p = 0.054; quadratic, p = 0.017), and eggshell thickness (linear, p = 0.036; quadratic, p = 0.128), the activity of GSH-Px (linear, p = 0.024; quadratic, p = 0.078), the concentration of IgM (linear, p = 0.016; quadratic, p = 0.056) and the level of AIV-Ab (linear, p = 0.034; quadratic, p = 0.103) in the serum increased linearly as dietary supplementation of B. subtilis C-3102 increased. The results showed that dietary treatments did not affect egg production, feed conversion ratio, egg mass, hatchability of fertile eggs, eggshell-breaking strength, egg-shape index, yolk percentage, Haugh unit, T-SOD, T-AOC, MDA, IgA and IgG concentrations and the level of NDV-Ab in the serum. In conclusion, dietary supplementation of 9.0 × 105  cfu/g B. subtilis C-3102 in laying breeders diets may be a feasible means of effectively increasing egg weight, fertility and hatchability, and improving egg quality such as eggshell thickness, yolk index and yolk colour. Besides, B. subtilis C-3102 can enhance the activity of GSH-Px, the concentration of IgM and the level of AIV-Ab in the serum.

Testosterone-retinoic acid signaling directs spermatogonial differentiation and seasonal spermatogenesis in the Plateau pika (Ochotona curzoniae).

During evolution, animals optimize their reproductive strategies to increase offspring survival. Seasonal breeders reproduce only during certain times of the year. In mammals, reproduction is tightly controlled by hypothalamus-pituitary-gonad axis. Although pathways regulating gametogenesis in non-seasonal model species have been well established, molecular insights into seasonal reproduction are severely limited. Using the Plateau pika (Ochotona curzoniae), a small rodent animal species native to the Qinghai-Tibetan plateau, as a model, here we report that seasonal spermatogenesis is governed at the level of spermatogonial differentiation. In testis of the reproductively dormant animals, undifferentiated spermatogonia failed to differentiate and accumulated in the seminiferous tubules. RNA-seq analyses of the active and dormant testes revealed that genes modulating retinoic acid biogenesis and steriodogenesis were differentially regulated. A single injection of all-trans retinoic acid (ATRA) reinitiated spermatogenesis and inhibition the function of RA-degrading enzyme CYP26B1 for 10 days induced spermatogonial differentiation. Strikingly, testosterone injection reinitiated spermatogenesis in short day adapted animals. Testosterone provides a permissive environment of RA biogenesis and actions in testis, therefore, indirectly controls spermatogonial differentiation. Collectively, these findings provide a key mechanistic insight regarding the molecular regulation of seasonal reproduction in mammals.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep.

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.

Microsatellite Loci Analysis Reveals Post-bottleneck Recovery of Genetic Diversity in the Tibetan Antelope.

The Tibetan antelope (chiru, Pantholops hodgsoni) is one of the most endangered mammals native to the Qinghai-Tibetan Plateau. The population size has rapidly declined over the last century due to illegal hunting and habitat damage. In the past 10 years, the population has reportedly been expanding due to conservation efforts. Several lines of evidence suggest that the Tibetan antelope has undergone a demographic bottleneck. However, the consequences of the bottleneck on genetic diversity and the post-bottleneck genetic recovery remain unknown. In this study, we investigate the genetic variation of 15 microsatellite loci from two Tibetan antelope populations sampled in 2003 (Pop2003) and 2013 (Pop2013). A higher level of genetic diversity (NA, 13.286; He, 0.840; PIC, 0.813; I, 2.114) was detected in Pop2013, compared to Pop2003 (NA, 12.929; He, 0.818; PIC, 0.789; I, 2.033). We observe that despite passing through the bottleneck, the Tibetan antelope retains high levels of genetic diversity. Furthermore, our results show significant or near significant increases in genetic diversity (He, PIC and I) in Pop2013 compared with Pop2003, which suggests that protection efforts did not arrive too late for the Tibetan antelope.

Changes of hemoglobin expression in response to hypoxia in a Tibetan schizothoracine fish, Schizopygopsis pylzovi.

Fishes endemic to the Qinghai-Tibetan Plateau are comparatively well adapted to aquatic environments with low oxygen partial pressures (hypoxia). Here, we cloned the complete cDNA of hemoglobin (Hb) α and ß from the Tibetan schizothoracine fish Schizopygopsis pylzovi, and then investigated changes in Hb mRNA and protein levels in spleen, liver and kidney in response to hypoxia. We applied severe hypoxia (4 h at PO2 = 0.6 kPa) and moderate hypoxia (72 h at PO2 = 6.0 kPa) to adult S. pylzovi. Changes of Hb expression under hypoxia, together with the investigations of spleen somatic index, kidney somatic index and Hb concentration in circulation, suggest that the kidney may not only serve as the erythropoietic organ, but also act as the major blood reservoir in S. pylzovi. From this perspective, the transcriptional activity of Hb in S. pylzovi, as reflected in the kidney, was turned down quickly after the onset of severe hypoxia, while under moderate hypoxia the transcriptional activity of Hb showed upregulation for a short time, but then the transcriptional machinery was turned down slowly on prolonged exposure. Notably, the changes in Hb protein levels in spleen, liver and kidney in response to severe and moderate hypoxia were not in line with the changes in mRNA levels, which are related with the blood reservoir in the kidney. Tibetan schizothoracine fish, at least S. pylzovi, show a particular response of the transcription regulation of Hb to moderate hypoxia, which is different from that of other fish species.

Signatures of functional constraint at Fgfr1a Genes in schizothoracine fishes (Pisces: Cypriniformes): The dermal skeleton variation adapted to high-altitude environments.

Fishes harbor a huge resource of phenotypic diversity and are useful for understanding the genetic basis of morphological variation. However, it is difficult to transfer classical genetic mapping to most non-model species. In this study, we performed a comparative sequence analysis of Fgfr1a to first interpret the evolution of this candidate scale-loss gene in 15 schizothoracine fishes with various scale phenotypes. While considerable amino acid (AA) substitutions were observed, molecular evolution analysis indicates that the overall coding regions were subject to functional constraint. We also identified extra copies of Fgfr1a in 4 scale-loss fishes and detected accelerated evolution in one AA substitution specific to these duplicates. We speculate that Fgfr1a had accumulated mutations in the ancestral lineage of scale-loss schizothoracine fishes before experiencing duplication events, which was further followed by the diversification of species. In silico mutation analysis predicted deleterious effects of the mutations while no disruptive molecular mechanism was detected. Collectively, our results highlight the important role of Fgfr1a gene in the adaptive evolution of schizothoracine fishes during their radiation in the Qinghai-Tibetan Plateau.

The complete mitochondrial genome sequence of Platypharodon extremus (Cypriniformes: Cyprinidae).

Platypharodon extemus is a monotypic species of Schizothoracine fishes and it was listed as Endangered species in the "China Red Data Book (Pisces)", Vulnerable (V) by the National Environmental Protection Agency and Endangered Species Scientific Commission. So far, little mitochondrial genome information of this genus has been described. In this study, we obtained the complete mitochondrial DNA genome sequences of this species. The mitogenome was 16,668 bp in length, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 noncoding regions. The base composition of this mitochondrial genome was 28.6% A, 27.3% T, 18.2% G, 25.9% C, with a high A + T content (55.9%). The complete mitochondrial genome of P. extremus would be of great utility in the phylogenetic analysis of the schizothoracine fishes and also provide meritorious insights into the deeper problems of the phylogenic analysis.

Genetic diversity and population structure of black Dahe pig based on DNA sequences analyses of mitochondrial and nuclear genes.

To investigate the genetic diversity and population structure of black Dahe pigs, we collected 175 samples from 5 local populations and sequenced them using a combination of two selected molecular markers for mitochondrial cytochrome b and Major Histocompatibility Complex (MHC) DRB. Overall, the results of AMOVA and phylogenetic tree and gene flow analyses detected high levels of gene flow among the five populations, particularly individual pigs from Dahe town (Pop1) or Yingshang town (Pop2) to other populations (Pop3, Pop4, and Pop5). The genetic diversity analyses showed that the diversity indices of the five populations did not vary significantly, but they were much lower than those of other Chinese pig species. These results suggest that distinct gene flow, unstable population pattern, and lower genetic diversity have been influenced mainly by human introductions for economic ends. These findings provide genetic information that could be used for the preservation and further genetic improvement of the black Dahe pig, as well as an important reference for the evaluation, conservation, and utilization of the genetic resources of this breed.

Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 µg/mL at 3200 m to 0.44±0.03 µg/mL at 3950 m and 0.24±0.01 µg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment.