Integrating transcriptome and metabolome to identify key genes regulating important muscular flavour precursors in sheep.

Flavour precursors are the basis of meat flavour, and their metabolism is regulated by a variety of enzymes. Thus, it is of great significance to identify the key genes related to meat flavour precursors. In this study, the difference in flavour precursors and transcriptome between Hu sheep and Dorper with different intramuscular fat (IMF) content were investigated using widely targeted metabolomics and RNA-sequencing technologies. Then, the key genes regulating the metabolism of vital precursors were explored by integrating transcriptome and metabolome. Consequently, 594 metabolites were detected in sheep longissimus dorsi, and 76 differentially abundant metabolites (DAMs) were identified between Hu sheep and Dorper. No DAMs were observed between distinct IMF content groups within each breed. A total of 10 lysophospholipids (LPs), including four lysophospholipid ethanolamines and six lysophospholipid cholines, were identified as the main differential precursors between Hu sheep and Dorper. Furthermore, the weighted gene coexpression network analysis uncovered three differentially coexpression modules that were significantly associated with the content of differential LPs in Dorper. From the three modules, GLB1, PLD3, LPCAT2, DGKE, ACOT7, and CH25H genes were identified as key genes regulating the metabolism of LPs. This work provides an insight into understanding the difference in flavour between different sheep breeds, as well as a basis for further exploring the regulatory mechanism of key genes on LPs.

Effect of feeding linseed diet on testis development, antioxidant capacity, and epididymal cauda sperm concentration in Chinese Hu lamb.

Polyunsaturated fatty acids (PUFAs) are essential for mammalian testis development and sperm function. However, PUFAs that are contained in linseed oil are easily oxidized in the diet and biohydrogenated in the rumen. In this study, we investigated the effect of linseed as a source of PUFAs on the antioxidant capacity and testis development in Hu lamb. Seventy-five 3-month-old lambs were randomly assigned to three groups. Within each treatment group, 25 lambs were allocated to five pens (five lambs per pen). The lambs in the control group were fed a control diet without linseed for 42 days from D22 to D63. Group I (BS28) was fed a control diet from D22 to D35 and 8% linseed diet from D36 to D63. Group II (BS42) was fed an 8% linseed diet for 42 days from D22 to D63. After 63-day feeding trial, all lambs except the heaviest and lightest in each pen were humanely slaughtered and investigated. Results revealed that feeding linseed did not affect the body weight, scrotal circumference, and testis weight, whereas feeding linseed for 42 days increased the epididymis weight (37.85 ± 1.61 g vs. 32.09 ± 1.06 g, P < 0.05) compared with the control group. Feeding lambs with linseed for 42 days also significantly upregulated the expression of antioxidative (glutathione peroxidase 4 and copper-zinc superoxide dismutase), steroidogenesis (3ß-hydroxysteroid dehydrogenase and steroid acute regulatory protein), and PUFA metabolism-related genes (fatty acid desaturase 2 and elongation of very long-chain fatty acid protein 2) and proliferating cell nuclear antigen mRNA (P < 0.05). It also increased the relative expression of mitochondrial DNA (P < 0.05), total antioxidant capacity (0.230 ± 0.019 mmol/mgprot vs. 0.175 ± 0.011 mmol/mgprot, P < 0.05), and superoxide dismutase (1661.467 ± 147.117 U/mgprot vs. 1158.891 ± 98.850 U/mgprot, P < 0.05) in testicular tissue but decreased the cholesterol concentration (0.331 ± 0.073 mmol/mgprot vs. 0.671 ± 0.092 mmol/mgprot, P < 0.05) compared with the control group. Therefore, feeding lambs with linseed for 42 days stimulated seminiferous tubule development and increased the number of Sertoli cells (20.71 ± 0.89 vs. 17.6 ± 0.73, P < 0.05), epididymal cauda lumina diameter (638.26 ± 22.32 µm vs. 444.41 ± 34.80 µm, P < 0.05), and the number of sperm in the epididymal cauda (68.91 ± 7.06 × 108/g vs. 36.61 ± 7.50 × 108/g). All these results suggested that feeding linseed in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and PUFA metabolism-related genes; increased the antioxidant capacity in lamb's testis; and contributed to testis development and spermatogenesis.

Copy number variation of ZNF280AY across 21 cattle breeds and its association with the reproductive traits of Holstein and Simmental bulls.

The mammalian Y chromosome gene families in the ampliconic region are expressed predominantly or exclusively in the testis, and their copy number variations (CNV) are significantly associated with male reproductive traits, suggesting they have important roles in spermatogenesis and testicular development. ZNF280AY (zinc finger protein 280A, Y-linked) is a member of the zinc finger protein family and has been identified as a bovid-specific Y-chromosome gene. The current study applied a reliable quantitative real-time PCR method to estimate the CNV of ZNF280AY in 715 bulls across 21 cattle breeds and to further investigate the association of the CNV of ZNF280AY with bull reproductive traits and ZNF280AY mRNA expression levels in adult testis. The results revealed that the median copy number of ZNF280AY was 47, and the copy number varied from 11 to 154, showing significant CNV between and within the investigated cattle breeds. In addition, all 715 bulls were classified into Y1, Y2, and Y3 lineage groups based on a rapid genotyping method described previously. Pairwise comparisons indicated that bulls belonging to the Y1 lineage had a significantly lower median copy number (40) than bulls belonging to the Y2 (52) and Y3 lineages (57). Association analysis revealed that the CNV of ZNF280AY was correlated negatively with the percentage of normal sperm and sperm concentration in Holstein bulls, whereas no significant correlation was observed with ejaculation volume, total sperm count, sperm motility, postthaw motility (PTM), and scrotal circumference in Holstein and Simmental bulls. Furthermore, no correlation was observed between ZNF280AY copy number and ZNF280AY mRNA expression levels in the testis. The current study suggests that the CNV of the ZNF280AY gene family is associated with male reproductive traits and may serve as a valuable marker for early bull fertility selection in Holstein breeding programs.

[A systematic review of international simulation models on the natural history of breast cancer: current understanding and challenges for Chinese-population-specific model development].

Objective: To systematically review the worldwide simulation model studies on the natural history of breast cancer and to summarize related parameters. Methods: A structured literature search was conducted in PubMed and the Cochrane Library to identify articles during 1980-2015. Articles were screened independently by two researchers. Health states in the natural history and relevant parameters were extracted. Results: A total of 36 studies were included for analysis, within the earliest one was published in 1990. Most studies were from Europe and America countries, and 2 studies from China. Markov model was mostly applied to evaluating breast cancer screening programs (n=32). Reported health status included "healthy" (n=36), ductal carcinoma in situ (DCIS, n=17), invasive breast cancer (IBC, n=36), and death (n=27). There were two definite classifications for IBC, tumor size (n=9) and TNM staging (n=9, 3 studies reported transition rates). The median (range) of annual transition rates from DCIS to stage-Ⅰ IBC, Ⅰ to Ⅱ, Ⅱ to Ⅲ, Ⅲ to Ⅳ were 0.279 (0.259-0.299), 0.150 (0.069-0.430), 0.100 (0.060-0.128) and 0.210 (0.010-0.625), respectively. A total of 15 studies reported the mean duration from predinical to clinical stage for IBC was 1.95-4.70 years, which gradually increased with age, and 7 studies reported that for DCIS. Conclusions: Despite closer attention was paid to breast cancer natural history models, in recent years atypical hyperplasia has been neglected. Data on the mean duration of DCIS requires reasonable conversion. Various classifications for IBC exist whereas transition rates are limited. Current findings would be valuable references but challenging for the Chinese-population specific natural history model, development.

[Natural history of breast cancer: a systematic review of worldwide randomized controlled trials of mammography screening].

Objective: To parameterize the 1-year transition probabilities between different health status of the natural history of breast cancer based on the data of randomized controlled trial of X-ray mammography screening worldwide. Methods: Based on the breast cancer screening randomized controlled trials defined by a mammography screening review from the Cochrane 2013 and the International Agency for Research on Cancer, a systematic review was initiated in PubMed by searching names of the key investigators of the trials, combined with the diseases, screening intervention and outcome indicators. If applicable, all the original cumulative incidence rates were converted into one-year transition rate, using the life-table approach considering time length of follow-up. Results: A total of 23 reports from 9 RCTs were included. The data on transition rate between the healthy status to precancerous lesions was absent. The 1-year transition rate from health to carcinoma in situ was 17.78 to 50.21 per 100 000 persons in the intervention group and 9.16 to 26.84 per 100 000 persons in the control group. Correspondingly, the 1-year transition rate from health to breast cancer (including carcinoma in situ and invasive cancer) were estimated as 143.75 to 316.97 per 100 000 persons in the intervention group, and 141.45 to 288.84 per 100 000 persons in the control group. Furthermore, the transition rate from the healthy status to invasive breast cancer was 159.79 to 264.60 per 100 000 persons in intervention group and 170.12 to 255.33 per 100 000 persons in control group. The transition rate from carcinoma in situ to invasive breast cancer varied among different pathological types. Conclusions: The most common natural history states of reported by the included trials involved the full healthy status, carcinoma in situ and invasive breast cancer. The findings of transition rates between different health statuses will be informative for future model development of natural history studies of breast cancer. Information in relation to breast precancerous lesions still limited and needs to be further addressed.

[Economic burden of esophageal cancer in China from 1996 to 2015: a systematic review].

Objective: To explore existing evidence of economic burden of esophageal cancer in China over the past 20 years. Methods: Based on PubMed, CNKI and Wanfang, literatures published from 1996 to 2015 were retrieved with the key words such as " economic burden" , "cost of illness" and so on. Then the information excerpted from those literatures were analyzed after several exclusionary procedures for non-esophageal cancer related literatures. The information about subjects and data source, methodology, main results were structurally abstracted and then analyzed. Quality assessments were conducted independently by two investigators using an 11-item instrument recommended by the Agency for Healthcare Research and Quality (AHRQ) for cross-sectional studies. All the expenditure data were calculated according to year-specific personal health care consumer price index (CPI) of China, the annual growth rate was calculated according to the average speed of growth. Results: A total of 23 studies (21 individual surveys and 2 population-based surveys) were included in the analysis, in which 12 were published over the past 5 years. Among the 21 individual surveys, 17 were hospital-based and the data were obtained through medical record review, and most of which only considered the direct medical economic burden (including the average overall expenditure per patient, per time and per diem). The median expenditure per patient during 1996-2011 ranged from 7 463 to 37 647 yuan (RMB) and the average growth rate was 7.68%. The median medical expenditure per clinical visit during 1996-2013 ranged from 6 851 to 57 554 yuan (RMB) and the average growth rate was 11.89%. The median medical expenditure per diem during 1996-2010 ranged from 225 to 1 319 yuan (RMB) and the average growth rate was 12.53%. The direct medical expenditure per clinical visit varied greatly with area, which were much higher in Beijing, Shanxi and Hubei. In both individual survey and population-based survey, less data about the direct non-medical expenditure and the influence of indirect expenditure on the economic burden were reported. Conclusion: Less data are available on economic burden of esophageal cancer in China over the past 20 years and the data's comparability are poor, especially in terms of population level or indirect burden. Direct medical expenditure is on the rise, and regional differences.

[Economic burden of cancer in China during 1996-2014: a systematic review].

Objective: To explore the current status of research on economic burden of cancer in China from 1996 to 2014. Methods: The key words including cancer, economic burden, expenditure, cost were used to retrieve the literatures published in CNKI and Wanfang (the two most commonly used databases for literature in Chinese) and PubMed during 1996-2014. A total of 91 studies were included after several exclusionary procedures. Information on subjects and data source, methodology, main results were structurally abstracted. All the expenditure data were discounted to year of 2013 value using China's health care consumer price indices. Results: More than half of the included studies were published over the past 5 years, 32 of the studies were about lung cancer. Among the 83 individual-based surveys, 77 were hospital-based and obtained data via individually medical record abstraction, and most of which only considered the direct medical expenditure. Expenditure per cancer patient and expenditure per diem were the most commonly used outcome indicators. Majority of the findings on expenditure per cancer patient ranged from 10 thousands to 30 thousands Chinese Yuan (CNY), with larger disparity in lung and breast cancer (ranged from 10 thousands to 90 thousands CNY), narrower difference in esophageal and stomach cancer (ranged from 10 thousands to 50 thousands CNY), and most stable trend in cervical cancer (almost all the values less than 20 thousands CNY). Without exception, the expenditures per diem for all the common cancers were increasing over the period from 1996 to 2014 (3-7 fold increase). Only 8 population-level economic burden studies were included and the reported expenditure of cancer at national level ranged from 32.6 billions to 100.7 billions CNY. Conclusions: Evidence on economic burden of cancer in China from 1996 to 2014 are limited and weakly comparable, particularly at a population level, and the reported expenditure per patient may be underestimated.

Copy number variation of PRAMEY across breeds and its association with male fertility in Holstein sires.

Multi-copy gene families are especially prevalent in the male-specific region (MSY) of the mammalian Y chromosome. Copy number variations (CNV) of these Y-linked gene families have been shown to affect human and animal fertility. The PRAMEY (Preferentially expressed antigen in melanoma, Y-linked) gene family is a newly identified, bovid-specific Y-linked gene family, which codes for a cancer/testis antigen that is expressed predominantly in testis and various tumors. The PRAMEY gene family is believed to play an important role in spermatogenesis and male fertility in cattle. The objective of this study was to investigate the CNV of PRAMEY within and across breeds and to determine whether CNV was associated with reproductive traits in Holstein bulls. A quantitative real-time PCR method was applied to measure the copy number of PRAMEY among 460 bulls using a Y-linked single copy gene, DDX3Y (DEAD box polypeptide 3, Y-linked), as a reference. The median copy number of PRAMEY was 13, ranging from 2 to 31. Significant variations in PRAMEY copy number were observed among 15 breeds investigated. Holstein bulls had the lowest median copy number (12), whereas Limousin bulls possessed the highest median copy number (26). Furthermore, bulls in the taurine lineage (13) had a significantly lower median copy number than those bulls in the indicine lineage (20). Association analysis revealed that PRAMEY copy number was correlated negatively with scrotal circumference (SC), relative scrotal circumference (RLSC), percentage of normal sperm (PNS), and nonreturn rate (NRR), but had no significant association with postthaw motility (PTM), incubated motility (IM), percentage of intact acrosome (PIA), sire conception rate (SCR), or relative breeding efficiency (RBE). The data from this study indicate that CNV of the PRAMEY gene family is associated with male reproductive traits and may serve as a valuable marker for sire fertility selection at an early age in cattle.

Effects of CSN1S2 Genotypes on Economic Traits in Chinese Dairy Goats.

The aim of this study was to investigate allele frequencies at the CSN1S2 locus in two Chinese dairy goat breeds and the effects of its variation on dairy goat economic traits. Seven hundred and eight goats from Xinong Saanen (XS, n = 268) and Guanzhong (GZ, N = 440) breeds were selected. The milk samples of 268 XS goats were collected during the middle of lactation, body size parameters (708 goats) and daily milk yield (202 goats) were registered. The RFLP (restriction fragment length polymorphism) and SSCP (single strand conformation polymorphism) were used to detect the polymorphisms in CSN1S2. The Hardy-Weinberg (HW) equilibrium and the associations between body size, milk yield and composition and the genotypes were calculated. The results revealed that only A and F CSN1S2 alleles were found in the two Chinese dairy goat breeds. Allelic frequencies of A and F were 0.795, 0.205 and 0.739, 0.261 in Xinong Saanen and Guanzhong population respectively. Xinong Saanen breed was in Hardy-Weinberg equilibrium, while Guanzhong breed deviated from Hardy-Weinberg equilibrium (p<0.05). The association of polymorphism with economic traits indicated that the goats with FF genotype have higher milk fat and total solid concentration than those with AA and AF genotypes (p<0.05).

Characterization of cytochrome b diversity in Chinese domestic horses.

Previous mitochondrial DNA (mtDNA) D-loop and microsatellite studies have shown that Chinese horses have multiple maternal origins and high genetic diversity. To better characterize maternal genetic origins and diversity of Chinese domestic horses, we conducted a comprehensive analysis of 407 complete 1140 bp sequences of the horse mitochondrially encoded cytochrome b (CYTB) gene, including 323 horses from 13 Chinese indigenous breeds and 84 reference sequences from GenBank. A total of 114 haplotypes were identified, of which 73 appeared among the 13 Chinese horse breeds. The high mitochondrially encoded cytochrome b haplotypic diversity suggests multiple maternal origins in Chinese horses.

AGPAT6 polymorphism and its association with milk traits of dairy goats.

As one of the eight members in the 1-acylglycerol-3-phosphate-O-acyltransferase (AGPATs) family, AGPAT6 is a crucial enzyme for the biosynthesis of glycerolipids and triacylglycerol in eukaryotes, as well as catalyzing the conversion from lysophosphatidic acid to phosphatidic acid. AGPAT6 can be considered as a candidate gene for regulating milk composition. DNA sequencing and PCR-RFLP methods were applied to detect genetic variation in the AGPAT6 gene in 549 Chinese dairy goats. Four polymorphisms (NC_007328.3:g.152G>C, 8124G>A, 9263C>G, 16436G>A) were detected in 5'UTR, intron 2, exon 4, and 3'UTR, respectively. For the KpnΙ locus, the frequencies of the AGPAT6-G allele were 0.955 and 0.936 for SN (Xinong Sannen) and GZ (Guanzhong) dairy goat breeds, respectively. In the PCR-RFLP analysis for KpnΙ, EcoRII, NcoΙ, and BglΙ, the frequencies of the G allele of AGPAT6 were 0.955 and 0.936, 0.694 and 0.819, 0.206 and 0.254, 0.729 and 0.623 for SN and GZ dairy goat breeds, respectively. The 9263C>G mutation revealed a synonymous genetic code of Thr (threonine). Associations between the four mutations and milk traits were analyzed in two dairy goat breeds. At the 9263C>G locus, genotype GG and CG individuals showed significantly better milk performance than genotype CC individuals (P < 0.05). Therefore, the G allele is suggested to be a molecular marker for milk production in dairy goats.

The CSN1S1 N and F alleles identified by PCR-SSCP and their associations with milk yield and composition in Chinese dairy goats.

A method was depicted to identify null allele CSN1S1 N and low allele CSN1S1 F of the CSN1S1 gene of goat using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism). First, primer A was designed to amplify the exon 9 of CSN1S1 gene which produced three genotypes AA, AB, and BB. Among these three genotypes, only AA and AB individuals had a cytosine deletion at exon 9 after DNA sequencing, which cannot be used to identify the N and F alleles. Therefore, primer B was used to amplify intron 14 of CSN1S1 of described AA and AB individuals. Genotypes FF, FN and NN were detected within AA individuals and genotypes FO and NO were detected in the above AB individuals. The frequencies of F and N alleles in 708 samples from Xinong Saanen (XS) and Guanzhong (GZ) dairy goat breeds were 0.1139, 0.0927, and 0.2376, 0.1193, respectively. In 268 XS samples, the individuals with NN genotype contained a significant lower protein content than that of other genotypes (P<0.01). Individuals of FF genotype had significant higher milk yield than that of NO genotype in the first milk lactation of 202 XS individuals (P<0.05). Therefore, the variability at CSN1S1 locus contains enough genetic diversity to be potentially useful in improving the quality and production of milk in Chinese dairy goat breeds.

A novel polymorphism of the lactoferrin gene and its association with milk composition and body traits in dairy goats.

Milk composition and body measurement traits, influenced by genes and environmental factors, play important roles in value assessments of efficiency and productivity in dairy goats. Lactoferrin (LF), involved in the efficient expression of protein in milk, is also an anabolic factor in skeletal tissue and a potent osteoblast survival factor. Therefore, it is an important candidate gene for milk composition and body measurement trait selection in marker-assisted selection. We employed PCR-SSCP and DNA sequencing to screen the genetic variations of the LF gene in 549 Chinese dairy goats. A novel single-nucleotide polymorphism (SNP) (G198A in exon II) of the LF gene was detected. The frequencies of the AA genotype were 0.0285 and 0.0261 in GZ and SN populations, respectively. Both populations were found to have low levels of polymorphism and were in Hardy-Weinberg disequilibrium (P < 0.05). We found significant (P < 0.05) associations of the SNP marker with milk protein and acidity in the total population; animals with the AA genotype had higher mean values for milk protein than those with the GA genotype. Animals with genotype AA had higher mean values for withers height than those with genotype GG (P < 0.05). We concluded that this SNP of the LF gene has potential as a genetic marker for milk composition and body traits in dairy goat breeding.

S-novel SNP of alpha-lactalbumin gene in chinese dairy goats.

The alpha-lactalbumin (alpha-LA) plays a key role in lactose synthesis in mammary glands of domestic animals. Mutations in the alpha-LA gene are associated with the milk traits in dairy cattle. In our study, a novel SNP: NO_X06366:g.875 C > T was detected in 708 dairy goat individuals--268 of the Xinong Saanen breed and 440 of Guanzhong breed, which revealed a synonymous mutation in the exon 1 of alpha-LA gene. The Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and sequencing techniques showed that there were three genotypes: CC, CT and TT. Moreover, the chi2-test showed that the genotype frequencies of the two breeds were in good agreement with the Hardy-Weinberg equilibrium (P > 0.05). The relationship of the polymorphism of dairy goat alpha-LA gene with the milk trait and the body size trait was revealed. Individuals with the CC genotype were significantly smaller at chest circumference than those with CT (P < 0.05) in both breeds. But the milk trait and other body size traits of the two dairy goat breeds had no significant association with genotypes studied.