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Objective: This study aims to evaluate the effects of dietary supplement of rubber seed oil on production performance, egg quality, and yolk fatty acid composition in laying hens during a 16-week feeding trial period.Methods: Forty-eight 25-week-old laying hens of Hy-Line Brown were randomly divided into three groups. Each group comprised four replicates and each replicate had four birds. Rubber seed oil was incorporated into a corn-soybean meal basal diet by 3.5% (group I), 4.5% (group II), or 0 (control group) and equivalent nutrition was supplied for the test groups and the control group. The performance related values were determined using standard or well established methods.Results: No significant difference was found in the production performance, the egg quality, the composition of saturated fatty acids, and the content of cholesterol and monounsaturated fatty acids in the yolk within the three groups. Interestingly, both test groups achieved a significantly higher content of linoleic acid, α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid and a significantly lower content of arachidonic acid (p

The yak (Bos grunniens) is a ruminant animal with strong regional adaptability. However, little is known about the adaptation of the rumen microbial community of yaks at different altitudes and the adaptation mechanism of the host and intestinal microorganisms to the habitat. We investigated the adaptability of the rumen microorganisms of yaks at high and low altitudes. We also compared and analyzed the abundance and diversity of core microorganisms and those that varied between different animals. The aim was to compare the rumen bacterial and fungal communities of grazing yak living at two elevations. Bacteroidetes, Firmicutes, Ascomycota, and Chytridiomycota were the dominant bacteria in the plateau and low-altitude regions. Significant differences between the dominant microorganisms in the rumen of yaks were evident in the two regions. The proportion of fiber-degrading bacteria was significantly different between yaks dwelling at high-altitude and low-altitude regions. The abundance of starch-degrading bacteria was not significantly different with altitude. Species clustering similarity analysis showed that the rumen microorganisms in the two areas were obviously isolated and clustered into branches. Functional prediction showed significant differences in rumen microbial methane metabolism, starch and sucrose metabolism, ion-coupled transporter and bacterial secretion system at different altitudes. Overall, the results of this study improved our understanding of the abundance and composition of microorganisms in the rumen of yak at different altitudes.

Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen.

The first animal with black traits to be studied was the Black-boned chicken. In China, we identified a flock of sheep with black traits. In the present study, the complete coding sequences of three genes, PSPH, SNRPA1 and TPM1, of Black-boned sheep (Ovis aries) were amplified using the reverse transcription polymerase chain reaction, according to the conserved sequence information for cattle and other mammals and known highly homologous sheep ESTs. The results showed that the Black-boned sheep PSPH gene encodes a protein of 225 amino acids, which has high homology with the PSPH proteins of seven species: cattle (99%), humans (94%), rat (93%), mouse (93%), African clawed frog (77%), Atlantic salmon (70%) and zebrafish (68%). The Black-boned sheep SNRPA1 gene encodes a protein of 255 amino acids, which has high identity with the SNRPA1 proteins of four species: cattle (99%), humans (99%), mouse (97%) and chicken (91%). The Black-boned sheep TPM1 gene encodes a protein of 284 amino acids, which has ...

The Chinese yakow is the offspring of yak (Bos grunniens) and Yellow cattle (Bos taurus). The melanocortin 1receptor gene (MC1R) plays a crucial role in determining coat colour of mammals. To investigate the relationship of polymorphism of the MC1R with coat colour in the Chinese yakow, the coding sequence (CDS) and the flanking region of MC1R were sequenced from 84 Chinese yakow samples and compared with the sequences of the MC1R from other bovid species. A fragment of 1134 base pair (bp) sequences including the full CDS (954bp) and parts of the 5'- and 3'-untranslated regions (162 and 18bp, respectively) of the Chineseyakow MC1R were obtained. A total of 13 single nucleotide polymorphisms (SNPs) including 4 SNPs (T-129C, A-127C, C-106T, G-1A) in the 5'-untranslated region and 9 SNPs (C201T, T206C, C340A, C375T, T663C, G714C, C870T, G871A and T890C) in the CDS were identified, revealing high genetic variability. Four novel SNPs including T206C, G714C, C870T and T890C, which have not been reported previously in bovid species, were retrieved. Within 9 coding SNPs, C201T, C375T, T663C and C870T were silent mutations, while T206C, C340A, G714C, G871A and T890C were mis-sense mutations, corresponding to amino acid changes p.L69P, p.Q114K, p.K238N, p.A291N and p.I297T, respectively. Amino acid sequences alignment showed a more than 96% similarity with other ruminates. However, three classical bovine MC1R loci the E(D), E(+) and e were not retrieved in the Chinese yakow, indicating other genes or factors could be involved in affecting coat colour in this species.

The gene coding for insulin-like growth factor-binding protein 3 (IGFBP3) is important for regulation of growth, development and metabolism in mammals. The present investigation was conducted to study nucleotide polymorphism of the IGFBP3 in gayal (Bos frontalis) and to compare the variations with those which occur in other ruminants. A fragment of 645 base pairs of the IGFBP3 covering a part of exon 2, the complete intron 2 and exon 3 and a part of intron 3 was amplified, sequenced (n=46) and digested (n=79) with HaeIII restriction enzyme from 125 collected gayal samples. Nine single nucleotide polymorphisms (SNPs) [C14T, A122C, C137T, G144C, C155T, G213A, C279A, G334A and G460A] were identified and located in intron 2, revealing high genetic variability. The alignment of nucleotide sequences was found to be very similar to those for other bovid species. Sequencing and HaeIII digestion showed that frequency of alleles C and A [consisting of fragments of sizes 56, 64, 228, 264, 282, 298 and 497 bp (CC genotype)] was 0.96 and 0.04 for the SNP C279A. Moreover, the genotype frequency of the SNP C279A in gayal was compared with that in other ruminants and it appears that this polymorphism may be associated with low fat content and rapid growth in this rare species.

The dominant rumen bacteria in Gayals, Yaks and Yunnan Yellow Cattle were investigated using PCR-DGGE approach. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis 16S rDNA sequences in DGGE profiles were combined to reveal the dominant bacterial communities and compared the differences between those cattle species. DGGE profiles revealed that Gayals had the most abundant dominant bacteria and the lowest similarity of intraspecies between individuals than other two cattle species. A total of 45 sequences were examined and sequence similarity analysis revealed that Gayals had the most sequences appeared to uncultured bacteria, accounting for 85.0% of the total sequences, Yaks and Yunnan Yellow Cattle had 44.4 and 68.8% uncultured bacterial sequences, respectively. According to phylogenetic analysis, the rumen dominant bacteria of Gayals were mainly phylogenetically placed within phyla firmicutes and bacteroidetes, and the known bacteria were mainly belonged to the genera Lachnospiraceae bacterium, Ruminococcus flavefaciens and Clostridium celerecrescens. Moreover, the dominant bacteria of Yaks were also mainly belonged to phyla firmicutes and bacteroidetes, and the known dominant bacteria were including Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Schwartzia succinivorans and Clostridiales bacterium, most of them are common rumen bacteria. In addition, the dominant bacteria in Yunnan Yellow Cattle were belonged to phyla firmicutes, bacteroidetes and Actinobacteria, and the known dominant bacteria containing Prevotella sp., Staphylococci lentus, Staphylococcus xylosus and Corynebacterium casei. Present study first detected Staphylococcus lentus and Staphylococcus xylosus in the rumen of cattle.

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species. To investigate if the MC1R gene is associated with coat color in water buffalo, the coding region of MC1R gene of 216 buffalo samples was sequenced, which included 49 black river buffalo (Murrah and Nili-Ravi), 136 swamp buffalo (Dehong, Diandongnan, Dechang, Guizhou, and Xilin) with white and gray body, and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo. Among the three variation sites found, SNP684 was synonymous, while SNP310 and SNP384 were nonsynonymous, leading to p.S104G and p.I128M changes, respectively. Only Individuals carrying homozygote E(BR)/E(BR) were black. The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele E(BR) or the allele carrying the amino acid p.104S was important for the full function of MC1R. The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R. Above results indicate that the allele E(BR) or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo.

Complete coding sequences of three Black-boned sheep (Ovis aries) genes Rab2A, Rab3A and Rab7A were amplified using reverse transcription polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or other mammals known to be highly homologous to sheep ESTs. The Black-boned sheep Rab2A gene encodes a protein of 226 amino acids which contains the conserved putative RabL2 domain and is highly homologous to the Rab2A proteins of seven other species—cattle (96%), human (83%), Sumatran orangutan (82%), rat (81%), mouse (80%), African clawed frog (72%) and zebrafish (71%). The Black-boned sheep Rab3A gene encodes a protein of 220 amino acids that contains the conserved putative Rab3 domain and is very similar to the Rab3A proteins of four species—cattle (99%), African clawed frog (99%), Western clawed frog (98%) and zebrafish (95%). And the Black-boned sheep Rab7A gene encodes a protein of 207 amino acids that contains the conserved putative Rab7 domain and has high homology with the Rab7A proteins of six other species—human (99%), dog (99%), Sumatran orangutan (99%), zebrafish (97%), rabbit (97%) and African clawed frog (96%). Analysis of the phylogenetic tree has demonstrated that the Black-boned sheep Rab2A, Rab3A and Rab7A proteins share a common ancestor and the tissue expression analysis has shown that the corresponding genes are expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for a further insight into these three sheep genes.

The complete coding sequences of three of sheep genes SLC25A4, SLC25A5 and SLC25A6 were firstly amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Sheep SLC25A4, SLC25A5 and SLC25A6 genes encode three corresponding proteins of 298 amino acids which contain the identically conserved putative mitochondrial carrier protein domain. Sheep SLC25A4 protein has high homology with the SLC25A4 proteins of six species-cattle (99%), human (95%), rat (95%), mouse (94%), dog (94%) and chicken (89%). Sheep SLC25A5 protein has high identity with the SLC25A5 proteins of five species-cattle (100%), dog (99%), mouse (98%), rat (98%) and human (98%). Sheep SLC25A6 protein also has high homology with the SLC25A6 proteins of four species-cattle (99%), human (97%), pig (97%) and chicken (93%). The phylogenetic tree analysis demonstrated that sheep SLC25A4, SLC25A5 and SLC25A6 proteins share a common ancestor. Moreover, SLC25A4, SLC25A5 and SLC25A6 proteins present stronger interaction each other. The tissue expression analysis indicated that sheep SLC25A4, SLC25A5 and SLC25A6 genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.

Libraries of rumen bacterial 16S rRNA gene sequences of Gayals (Bos frontalis) and Swamp buffaloes (Bubalus bubalis) were cloned and sequenced in the present work to compare the bacterial diversity with the third published library of Holstein cow. Sequence similarity of 97% was used as the definition of operational taxonomic unit (OTU). The majority of the 470 sequences retrieved fell into the phyla of low G + C subdivision (329 sequences) and Cytophaga–Flexibacter–Bacteroides (CFB, 123 sequences) with the percentages of 70 and 26.2, respectively. The remaining clones belonged to the phyla of Proteobacter, high G + C gram positive bacteria (HGCGPB) and Spirochaetes, accounting for 3.8% totally. Only 73 clones (25 OTUs, 15.5%) could be closely related to cultured representatives. However, a larger fraction was related to uncultured representatives. Holstein cow may have more representatives of cultural bacteria and there were more uncultured clones for Gayals. The percentage of cultural representatives was 24, 13.3 and 9.5 for Holstein cow, Swamp buffaloes and Gayals, respectively. Twenty-three OTUs of the 236 ones appeared in more than one library, five of which were cultural. Selenomonas ruminantium, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens were found in two different libraries, while Succiniclasticum ruminis and Pseudobutyrivibrio ruminis were found in all three libraries. Some of the animal-specific bacteria that had not been described previously in the ruminal ecosystem, e.g. Allisonella histaminiformans for Gayals and Staphylococcus sciuri for Swamp buffaloes were also recovered.

The complete coding sequences of two sheep genes--LCK and CDK2--were amplified using the rapid amplification of cDNA ends method based on three sheep EST sequences whose translated amino acids contain the domain PTKc_Lck_BIk and S_TKc domain, respectively. The sequence analyses of these two genes revealed that the sheep LCK gene encodes a protein of 509 amino acids which has high homology with the lymphocyte-specific protein tyrosine kinase (LCK) of eight species: bovine (99%), human (96%), dog (96%), Aotus nancymaae (95%), mouse (94%), rat (91%), horse (91%) and chicken (81%). The sheep CDK2 gene encodes a protein of 298 amino acids which has high homology with the cyclin-dependent kinase 2 (CDK2) of ten species: bovine (100%), goat (100%), rat (99%), mouse (99%), Chinese hamster (99%), dog (98%), golden hamster (98%), human (98%), horse (98%) and rhesus monkey (98%). The tissue transcription profile analyses indicated that that the Black-boned sheep LCK and CDK2 genes are generally but differentially expressed in the detected tissues including in tissues including spleen, muscle, skin, kidney, lung, liver and heart. These data serve as a foundation for further insight into these two genes.

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.

The melanocortin 1 receptor gene (MC1R) plays a crucial role in determining coat colour of mammals. To investigate the relationship of polymorphism of the MC1R with coat colour in gayal, the coding sequence (CDS), and the 5′- and 3′-untranslated regions (UTR) of the MC1R were sequenced from 63 samples from the gayal and compared with the sequences of the MC1R from other ruminant species. A sequence of 1,136 bp including the whole CDS (954 bp) and parts of the 5′- and 3′-UTR (164 and 18 bp, respectively) of the gayal MC1R was obtained. A total of nine single nucleotide polymorphisms (SNPs) including four SNPs (c.−129T>C, c.−127A>C, c.−106C>T, c.−1G>A) in the 5′-UTR and five SNPs (c.201C>T, c.583C>T, c.663T>C, c.871A>G and c.876T>C) in the CDS were detected, revealing high genetic diversity. Three novel coding SNPs including c.201C>T, c.583C>T and c.876T>C, which have not been reported previously in bovid species, were retrieved. Within five coding SNPs, c.201C>T, c.663T>C and c.876T>C were silent mutations, while c.583C>T and c.871A>G were mis-sense mutations, resulting in changes in the amino acids located in the fifth (p.L195F) and seventh (p.T291A) transmembrane regions, respectively. The alignment of amino acid sequences was found to be very similar to those for other bovid species. It was demonstrated, using the functional effect prediction, that the p.T291A amino acid replacement could have an effect on MC1R protein function but not for the p.L195F substitution. Using phylogenetic analyses it was revealed that the gayal has a close genetic relationship with the yak. However, three classical bovine MC1R loci the E D , E + and e were not retrieved in the gayal, indicating other genes or factors could affect coat colour in this species.

The gayal (Bos frontalis) is a rare semi-wild bovid species in which bovine spongiform encephalopathy (BSE) has not been reported. Polymorphisms of the prion protein gene (PRNP) have been correlated significantly with resistance to BSE. In this study, the coding region of PRNP was cloned and characterized in samples from 125 gayal. A total of ten single nucleotide polymorphisms (SNPs), including six silent mutations (C60T, G75A, A108T, G126A, C357T and C678T) and four mis-sense mutations (C8A, G145A, G461A and C756G), corresponding to amino acids T3K, G49S9, N154S and I252M were identified, revealing high genetic diversity. Three novel SNPs including C60T, G145A and C756G, which have not been reported previously in bovid species, were retrieved. There also was one insertion–deletion (187Del24) at the N-terminal octapeptide repeat region. Alignment of nucleotide and amino acid sequences showed a high degree of similarity with other bovid species. Using phylogenetic analyses it was revealed that gayal has a close genetic relationship with Zebu cattle. In short, preliminary information is provided about genotypes of the PRNP in gayal. This could assist with the study of the pathogenesis of transmissible spongiform encephalopathies and cross species transmission as well as a molecular breeding project for gayal in China.

The complete coding sequences of three of Black-boned sheep (Ovis aries) genes Sfxn1, Snai2 and Cno were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) according to the conserved sequence information of the cattle or other mammals and known highly homologous sheep ESTs. Black-boned sheep Sfxn1 gene encodes a protein of 322 amino acids which has high homology with the Sfxn1 proteins of five species--cattle 98%, pig 95%, human 95%, rat 93%, and mouse 93%. Black-boned sheep Snai2 gene encodes a protein of 268 amino acids that has high identity with the Snai2 proteins of six species--cattle 99%, pig 94%, human 93%, dog 93%, rat 91%, and mouse 90%. Black-boned sheep Cno gene encodes a protein of 214 amino acids that has high homology with the Cno proteins of four species--cattle 97%, human 75%, mouse 67%, and rat 65%. The phylogenetic tree analysis demonstrated that Black-boned sheep Sfxn1, Snai2 and Cno proteins have close relationship with cattle Sfxn1, Snai2 and Cno proteins. The tissue expression analysis indicated that Black-boned sheep Sfxn1, Snai2 and Cno genes were expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for further insight into these three sheep genes.

Visible pigmentation phenotypes can be used to explore the regulation of gene expression and the evolution of coat color patterns in animals. Here, we performed whole-genome and RNA sequencing and applied genome-wide association study, comparative population genomics and biological experiments to show that the 2,809-bp-long LINE-1 insertion in the ASIP (agouti signaling protein) gene is the causative mutation for the white coat phenotype in swamp buffalo (Bubalus bubalis). This LINE-1 insertion (3′ truncated and containing only 5′ UTR) functions as a strong proximal promoter that leads to a 10-fold increase in the transcription of ASIP in white buffalo skin. The 165 bp of 5′ UTR transcribed from the LINE-1 is spliced into the first coding exon of ASIP, resulting in a chimeric transcript. The increased expression of ASIP prevents melanocyte maturation, leading to the absence of pigment in white buffalo skin and hairs. Phylogenetic analyses indicate that the white buffalo-specific ASIP allele originated from a recent genetic transposition event in swamp buffalo. Interestingly, as a similar LINE-1 insertion has been identified in the cattle ASIP gene, we discuss the convergent mechanism of coat color evolution in the Bovini tribe.