Short communication: Microbiological quality of raw cow milk and its association with herd management practices in Northern China.

Contamination of raw milk with bacterial pathogens is potentially hazardous to human health. The aim of this study was to evaluate the total bacteria count (TBC) and presence of pathogens in raw milk in Northern China along with the associated herd management practices. A total of 160 raw milk samples were collected from 80 dairy herds in Northern China. All raw milk samples were analyzed for TBC and pathogens by culturing. The results showed that the number of raw milk samples with TBC <2 × 106 cfu/mL and <1 × 105 cfu/mL was 146 (91.25%) and 70 (43.75%), respectively. A total of 84 (52.50%) raw milk samples were Staphylococcus aureus positive, 72 (45.00%) were Escherichia coli positive, 2 (1.25%) were Salmonella positive, 2 (1.25%) were Listeria monocytogenes positive, and 3 (1.88%) were Campylobacter positive. The prevalence of S. aureus was influenced by season, herd size, milking frequency, disinfection frequency, and use of a Dairy Herd Improvement program. The TBC was influenced by season and milk frequency. The correlation between TBC and prevalence of S. aureus or E. coli is significant. The effect size statistical analysis showed that season and herd (but not Dairy Herd Improvement, herd size, milking frequency, disinfection frequency, and area) were the most important factors affecting TBC in raw milk. In conclusion, the presence of bacteria in raw milk was associated with season and herd management practices, and further comprehensive study will be powerful for effectively characterizing various factors affecting milk microbial quality in bulk tanks in China.

Analysis of POU1F1 gene DdeI polymorphism in Chinese goats.

As a member of the POU-domain family, the POU1F1 is a positive regulator for growth hormone, prolactin and thyroid-stimulating hormone b, by binding to target DNA promoters as a dimer in mammals. This study described the polymorphisms at the goat POU1F1-DdeI locus and analyzed the distribution of alleles in 15 indigenous Chinese goat breeds. The PCR-RFLP analysis showed a predominance of the D1D1 genotype and the D1 allele, with average frequencies of 0.550 and 0.790, respectively, irrespective of goat utility type. The D1D2 genotype was the second most frequent, with a mean frequency of 0.371. The distributions of genotypic and allelic frequencies at this locus were found to be significantly different among populations based on a Chi square test (P < 0.001), suggesting that the breed factor significantly affected the molecular genetic character of the POU1F1 gene. The genetic diversity analysis revealed that Chinese indigenous populations had a wide spectrum of genetic diversity at the goat POU1F1-DdeI locus. However, an ANOVA analysis revealed no significant differences in gene homozygosity, gene heterozygosity, effective allele numbers, or polymorphism information content among meat, dairy, and cashmere utility types (P > 0.05). This suggests that the goat utility types had no significant effect on the spectrum of genetic diversity.

Global transcriptional profiling of longissimus thoracis muscle tissue in fetal and juvenile domestic goat using RNA sequencing.

Domestic goats are important meat production animals; however, data from transcriptional profiling of skeletal muscle tissue in goat have thus far been scarce. We used comparative transcriptional profiling based on RNA sequencing of longissimus thoracis muscle tissue obtained from fetal goat muscle tissue (27 512 850 clean cDNA reads) and 6-month-old goat muscle tissue (27 582 908 reads) to identify genes that are differentially expressed, novel transcript units and alternative splicing events. Gene annotation revealed that 15 960 and 14 981 genes were expressed in the fetal and juvenile libraries respectively. We detected 6432 differentially expressed genes and, when considering GO terms, found 34, 27 and 55 terms to be significantly enriched in molecular function, cellular component and biological process categories respectively. Pathway analysis revealed that larger numbers of differentially expressed genes were enriched in fetal myogenesis or cell proliferation and differentiation-related pathways (such as Wnt), genes involved in the cell cycle and the Notch signaling pathway, and most of the differentially expressed genes involved in these pathways were downregulated in the juvenile goat library. These genes may be involved in various regulation mechanisms during muscle tissue differentiation between the two development stages examined herein. The identified novel transcript units, including both non-coding and coding RNA, as well as alternative splicing events increase the level of complexity of regulation mechanisms during muscle tissue formation and differentiation. Our study provides a comparative transcriptome analysis on goat muscle tissue, which will provide a valuable genomic resource for future studies investigating the molecular basis of skeletal muscle development.

Frequency of gray coat color in native Chinese horse breeds.

Gray horses are born colored, and they then gradually lose their hair pigmentation. Tremendous progress has been made in identifying the genes responsible for graying with age in horses in recent years. Results show that gray coat color in horses is caused by a 4.6-kb duplication in intron 6 of the syntaxin 17 gene (STX17), which constitutes a cis-acting-regulatory mutation. However, little is known about the gray phenotype in native Chinese horses. This study was conducted to explore the frequency distribution of the gray mutation in native Chinese horse breeds. A total of 489 samples from 14 native Chinese horse breeds were genotyped for the STX17 duplication using a simplified conventional PCR-based method. The results show that the gray mutation was present in 12 native Chinese horse breeds, except the Balikun and Guanzhong breeds. The Chakouyi and Hequ breeds had the highest frequency of the gray mutation (P(G) = 0.367 and P(G) = 0.274, respectively). There was no significant geographical difference in the distribution of gray coat color across native Chinese horse breeds. Our results suggest that gray is a common coat color in Chinese horses.

Novel Y-chromosome polymorphisms in Chinese domestic yak.

Y-chromosome-specific haplotypes (Y-haplotypes) constructed using single nucleotide polymorphisms (Y-SNPs) in the MSY (male-specific region of the Y-chromosome) are valuable in population genetic studies. But sequence variants in the yak MSY region have been poorly characterized so far. In this study, we screened a total of 16 Y-chromosome-specific gene segments from the ZFY, SRY, UTY, USP9Y, AMELY and OFD1Y genes to identify Y-SNPs in domestic yaks. Six novel Y-SNPs distributed in the USP9Y (g.223C>T), UTY19 (g.158A>C and g.169C>T), AMELY2 (g.261C>T), OFD1Y9 (g.165A>G) and SRY4 (g.104G>A) loci, which can define three Y-haplotypes (YH1, YH2 and YH3) in yaks, were discovered. YH1 was the dominant and presumably most ancient haplotype based on the comparison of UTY19 locus with other bovid species. Interestingly, we found informative UTY19 markers (g.158A>C and g.169C>T) that can effectively distinguish the three yak Y-haplotypes. The nucleotide diversity was 1.7 × 10(-4) ± 0.3 × 10(-4) , indicating rich Y-chromosome diversity in yaks. We identified two highly divergent lineages (YH1 and YH2 vs. YH3) that share similar frequencies (YH1 + YH2: 0.82-0.89, YH3: 0.11-0.18) among all three populations. In agreement with previous mtDNA studies, we supported the hypothesis that the two highly divergent lineages (YH1 and YH2 vs. YH3) derived from a single gene pool, which can be explained by the reunion of at least two paternal populations with the divergent lineages already accumulated before domestication. We estimated a divergence time of 408 110 years between the two divergent lineages, which is consistent with the data from mitochondrial DNA in yaks.

Polymorphism in the 5ꞌ-UTR of the insulin-like growth factor I gene associated with production traits in Chinese cattle.

The insulin-like growth factor I (IGF-1) gene plays important roles in the growth and body composition of animals. Serum IGF1 concentration has been associated with growth traits in many livestock species. We found a polymorphism of cattle IGF1-TasI locus and analyzed the distribution of alleles in three cattle breeds, including Qinchuan, Nanyang, and Chinese Holstein. PCR-RFLP analysis showed that allele A was the dominant allele. The frequencies of allele A varied from 0.84 to 0.97. Distributions of genotypic and allelic frequencies were significantly different among breeds. Polymorphism of the IGF1 gene was significantly affecting hucklebone width at 6 months in the Nanyang breed and protein and fat yield of the third lactation in Chinese Holstein cattle. Individuals with allele C had a significantly higher performance in production traits.

Paternal origins of Chinese cattle.

To determine the genetic diversity and paternal origin of Chinese cattle, 302 males from 16 Chinese native cattle breeds as well as 30 Holstein males and four Burma males as controls were analysed using four Y-SNPs and two Y-STRs. In Chinese bulls, the taurine Y1 and Y2 haplogroups and indicine Y3 haplogroup were detected in seven, 172 and 123 individuals respectively, and these frequencies varied among the Chinese cattle breeds examined. Y2 dominates in northern China (91.4%), and Y3 dominates in southern China (90.8%). Central China is an admixture zone, although Y2 predominates overall (72.0%). The geographical distributions of the Y2 and Y3 haplogroup frequencies revealed a pattern of male indicine introgression from south to north China. The three Y haplogroups were further classified into one Y1 haplotype, five Y2 haplotypes and one Y3 haplotype in Chinese native bulls. Due to the interplay between taurine and indicine types, Chinese cattle represent an extensive reservoir of genetic diversity. The Y haplotype distribution of Chinese cattle exhibited a clear geographical structure, which is consistent with mtDNA, historical and geographical information.

Short communication: A missense mutation in the PROP1 (prophet of Pit 1) gene affects male fertility and milk production traits in the US Holstein population.

In previous studies, we reported significant associations of the POU1F1 pathway genes with reproduction and production traits in several dairy cattle populations. Polymorphisms in genes of this pathway were found to be associated with both female and male fertility traits in dairy cattle. The POU1F1 gene is a direct downstream target for the regulation of the prophet of Pit1 (PROP1) gene, also known as PROP paired-like homeobox 1. Interestingly, the position of PROP1 coincides with a quantitative trait locus affecting ovulation rate in cattle. Therefore, the objective of this study was to investigate whether PROP1 affects fertility and milk production traits in Holstein cattle. Using the DNA pooling sequencing approach, a missense single nucleotide polymorphism that replaces a histidine amino acid with an arginine was detected in exon 3 of PROP1. The arginine allele was found to be associated with a decrease in sire conception rate and an increase in productive life, protein yield, and net merit index in a population of 1,951 Holstein bulls. The transcription factors produced from the histidine and arginine isoforms are known to have different transcription, DNA binding, and regulation activities. As such, we propose that the association of the arginine isoform with decreased bull fertility is likely caused by reduced activity of this allele in male functions. The findings of this study suggest PROP1 polymorphisms as candidates in selection programs for fertility, health, and milk production traits in dairy cattle.

Impacts of single nucleotide polymorphisms and haplotypes in the bovine Dapper1 gene on body weight.

The Dapper1 protein plays important role in multiple developmental processes via negative modulation of the Wnt signaling pathway. We detected variations in Dapper1 in 1185 individuals from 5 Chinese cattle breeds and determined their effects on bovine body weight. Two silent mutations (g.8344C>T and g.8428C>T) in exon 6 along with two substitutions (g.10513A>G and g.10765C>G) in the 3'-untranslated region were detected with DNA pool sequencing and forced polymerase chain reaction-restriction fragment length polymorphism. Haplotype variability and the extent of linkage disequilibrium of the 4 single nucleotide polymorphisms (SNPs) were analyzed, and the results revealed 16 haplotypes and 7 combined haplotypes in the 5 cattle breeds. Statistical analyses indicated that genotypes CC and AA in the g.8344C>T and g.10513A>G loci were associated with heavier body weight at 6 months in the Nanyang cattle population (P < 0.05), and the combined haplotype had consistent significant effects on body weight with a single SNP. Cattle with haplotype combinations H1H5 (CCCTAACC) displayed the heaviest body weight at 6 months compared with that of other haplotypes (P < 0.05). Our results provide evidence that 4 SNPs and haplotypes in Dapper1 may be used for marker-assisted selection in beef cattle breeding programs.

[Developmental expression patterns and association study with growth traits of bovine].

As a member of the basic helix-loop-helix (bHLH) family of transcription factors, Bhlhe40 played an important role in the regulation of adipogenesis, myogenesis and the development of metabolic syndrome. In this study, we investigated the tissue expression patterns and differences of bovine Bhlhe40 gene during three developmental stages. The result showed that Bhlhe40 was ubiquitously expressed in most tissues of cattle. In addition, the expression of bovine Bhlhe40 gene showed a decreasing trend in skeletal muscle and fat during the de- velopmental stages. Then, two novel mutations (EX5 + 114 T>C, L166P and EX5 + 733 A>G, S372S) of bovine Bhlhe40 gene were identified by BamHI and ApaI ACRS PCR-RFLP in 1226 individuals from five indigenous cattle breeds in China. Frequencies of allele C at the BamHI locus varied from 0.210 to 0.340 among breeds, while frequencies of allele G at the ApaI locus varied from 0.030 to 0.114. Association analysis revealed no significant association of ApaI locus with growth traits in Nanyang breed. However, BamHI locus was significantly associated with the average daily lain at 18 months in Nanvans cattle (P < 0.05). This study aimed to lay the groundwork to further study of Bhlhe40 function at myogenesis and adipogenesis in bovine and should significantly contribute to cattle breeding and genetics through MAS program.

Polymorphisms in the Promoter Region of the Chinese Bovine PPARGC1A Gene.

The peroxisome proliferator-activated receptor gamma coactivator-1 alpha protein, encoded by the PPARGC1A gene, plays an important role in energy homeostasis. The genetic variations within the PPARGC1A gene promoter region were scanned in 808 Chinese native bovines belonging to three cattle breeds and yaks. A total of 6 SNPs and one 4 bp insertion variation in the promoter region of the bovine PPARGC1A gene were identified: SNP -259 T>A, -301_-298insCTTT, -915 A>G, -1175 T>G, -1590 C>T, -1665 C>T and -1690 G>A, which are in the binding sites of some important transcription factors: sex-determining region Y (SRY), myeloid-specific zinc finger-1 (MZF-1) and octamer factor 1(Oct-1). It is expected that these polymorphisms may regulate PPARGC1A gene transcription and might have consequences at a regulatory level.

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).

A novel 28-bp insertion-deletion polymorphism within goat PRNP gene and its association with production traits in Chinese native breeds.

In this study, a novel 28-bp insertion-deletion (indel) polymorphism (AJ298878:g.47836-47853insCCTCAGACACTGAGTCTCCCCAACAGCA) was found in goat prion protein (PRNP) gene in 2373 goats from 13 Chinese native breeds. The frequencies of allele "ins" varied from 0.500 to 1.000 in different breeds. The establishment of association of the 28-bp indel polymorphism with production traits was performed in Inner Mongolia white cashmere (IMWC) and Xinong Sannen dairy (XNSN) breeds. Two significant associations between this polymorphism and 1-year-old body mass (P = 0.011) and average body mass (P = 0.024) were observed in IMWC breed, as well as wool thickness of 3-year-olds (P < 0.001). Furthermore, the novel 28-bp indel polymorphism was significantly associated with total solids in the evening (%) (P = 0.009) and milk yield (P = 0.016) in XNSN breed. These findings suggested that the 28-bp indel polymorphism was a potential DNA marker for eliminating or selecting preferred individuals in relation to production traits in goat marker-assisted selection breeding while carrying out preventing scrapie project.

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.

Single nucleotide polymorphisms of the bovine VEGF-B gene and their associations with growth traits in the Nanyang cattle breed.

PCR-SSCP and DNA sequencing methods were applied to reveal single nucleotide polymorphisms (SNPs) in the bovine VEGF-B gene in 675 samples belonging to three native Chinese cattle breeds. We found 3 SNPs and a duplication NC_007330.5: g. [782 A>G p. (Gly112 =) (;) 1000-1001dup CT (;) 1079 C>T (;) 2129 G>A p. (Arg184Gln)]. We also observed a statistically significant association of the polymorphism (1000-1001dup CT) in intron 3 of the VEGF-B gene with the body weight of the Nanyang cattle (p < 0.05). This polymorphisms of VEGF-B gene need to be verified among a larger cattle population before it can be identified as a marker for bovine body weight.

Sequence variants in the bovine PRDM16 gene associated with body weight in Chinese cattle breeds.

As a zinc-finger protein, PR domain containing 16 (PRDM16) controls brown fat determination by stimulating brown fat cell production while suppressing the expression of genes for production of white fat cells; mutations in this domain are associated with myelodysplastic syndrome and leukemogenesis. In our study, polymorphisms in exons 2, 3, 4, 5, 7, 8, and 9 of the PRDM16 gene were detected by PCR-SSCP, DNA sequencing and CRS-PCR-RFLP methods in 1031 cattle of the Chinese breeds: Jiaxian, Nanyang, Qinchuan, and Chinese Holstein. Three mutations (NC_007314.3: g.577 G>T, 614 T>C, 212237 T>C) were detected. Animals with the homozygote genotype had lower body weight and average daily gain than those with the other genotypes. PRDM16 gene-specific SNPs may be useful markers for growth traits for marker-assisted selection programs.

Impact of angiogenic inhibition in the treatment of newly diagnosed and recurrent glioblastoma: a meta-analysis based on randomized controlled trials.

OBJECTIVE: Glioblastoma (GBM) is the most common and aggressive primary malignant tumor of the central nervous system in adults with high recurrence and mortality rates. Although radiotherapy and temozolomide have become the standard therapeutic regimen for GBM as adjuvant chemoradiotherapy after surgical resection, clinical outcomes remain suboptimal. In recent years, targeted antiangiogenic therapy has attracted considerable attention, but its therapeutic efficacy and safety are still controversial. MATERIALS AND METHODS: Randomized controlled trials (RCTs) of chemoradiotherapy with or without bevacizumab for the treatment of glioblastoma were collected by searching on the Pubmed, Embase, Cochrane, Ovid, Scopus, Web of Science, and Google Scholar databases from the date of database establishment to February 2022. Meta-analysis was performed using RevMan 5.3 software after two investigators independently screened the literature, extracted data, and assessed the risk bias of included studies. RESULTS: A total of 7 RCTs were included. The meta-analysis showed that bevacizumab in combination with chemoradiotherapy was superior to chemoradiotherapy alone in terms of progression-free survival (PFS), with a statistically significant difference. Interestingly, bevacizumab in combination with chemoradiotherapy improved PFS more significantly in recurrent glioblastoma than in newly diagnosed glioblastoma. However, for overall survival (OS), the combination of bevacizumab with chemoradiotherapy was similar to chemoradiotherapy alone, which was not significantly different. With regard to safety, the incidence of most adverse events was higher in the combination of bevacizumab and chemoradiotherapy than in chemoradiotherapy alone, especially in terms of hematologic adverse events. CONCLUSIONS: Current evidence suggests that angiogenesis inhibitor-containing chemoradiotherapy regimens are preferentially recommended for patients with recurrent glioblastoma to prolong their progression-free survival, provided that safety is acceptable, but this does not confer a significant benefit on overall patient survival.

Polymorphism of DLK1 and CLPG gene and their association with phenotypic traits in Chinese cattle.

DLK1 and CLPG were located in DLK1-GTL2 imprinted cluster. They all affected muscle growth and meat tenderness. The functional importance of DLK1 and CLPG imply that the variation of the genes could affect the growth traits of animal. PCR-SSCP and sequencing were used to analyze the four loci of DLK1 gene and CLPG gene in 1109 individuals, which belong to eight breeds/species of bovidae, including cattle, buffalo and yak. A synonymous mutation (C451T) was detected in exon 5 of DLK1 in Qinchuan cattle, but didn't change significantly with phenotypic traits. Three genotypes AA, AB and AC of CLPG were identified in Jiaxian cattle. The associations analyst of different genotypes showed that the individuals with genotypes AA and AC had a greater body weight and longer body length than those with genotype AB (P<0.05 and P<0.01, respectively); the AA individuals were different from those AB (P<0.05) in the circumference of cannon bone. No polymorphism was observed in the other populations at other loci. These results were in agreement with the homology analysis: DLK1 and CLPG genes were in a highly conserved.

Molecular characterization and polymorphisms of the caprine Somatostatin (SST) and SST Receptor 1 (SSTR1) genes that are linked with growth traits.

Somatostatin (SST) and its receptors (SSTR1-5) appear to be important in central regulation of many metabolic systems that affect growth, adiposity and nutrient absorption. In this study, we investigated polymorphisms within the caprine SST and SSTR1 genes and determined their relationship with growth traits. As there were no sequence information of the caprine SST and SSTR1 genes, we explored their DNA sequence and genomic organizations. The caprine SST gene is organized in two exons and is transcribed into an mRNA containing 351 bp of sequence coding for a protein of 116 amino acids. Its protein sequences showed substantial similarity (97-99%) to its respective orthologs from cattle, human and mouse. We also cloned and sequenced a 1.2 kb DNA fragment which contained the major part of the coding region and 3' UTR of the caprine SSTR1 gene. We then detected the polymorphisms in these determined sequences by PCR-SSCP and DNA sequencing methods in 459 goats from four breeds. Four SNPs (GU014693:g.647T>C, GU014693:g.844A>C, GU014693:g.970T>C, GU014693:g.1039T>A), segregating as two haplotypes (T-A-T-T and C-C-C-A), were identified in intron 1 of the caprine SST gene and showed the associations to body length and body height (P < 0.05). Two SNPs (GU014695:g.801 C>T, GU014695:g.948 C>T) were identified in the caprine SSTR1 gene. Significant associations between the three genotypes of GU014695:801 C>T and body length, body height, and chest circumference was observed (P < 0.05). These results suggest that the caprine SST and SSTR1 genes are strong candidate genes that influence growth traits in goat.

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.