PRNP gene polymorphism frequencies for comparing possible vulnerability to BSE in Chinese bovine population.

Among the numerous transmissible spongiform encephalopathies (TSEs), bovine spongiform encephalopathy (BSE) is the most well-known TSEs. It is a potential Creutzfeldt-Jakob (CJD) disease mutation that can be transferred through cattle to humans. In several animals, the prion protein gene (PRNP) is recognized to take active part in TSE vulnerability or tolerance. Previous studies have found indels polymorphism in PRNP gene promoter and intron1 region linked to BSE vulnerability. It's linked with 23 bp indels polymorphism in putative promoter and 12 bp indel in intron 1 of the PRNP gene. The aim of this study was to compare the allele, genotype and haplotype frequencies of PRNP indel polymorphisms in Zhongdian Yak (Bos grunniens) (YK), Zhongdian Yellow cattle (Bos taurus) (YC) and Zhongdian Yakow (Bos primigenius taurus × Bos grunniens) (PK) with worldwide reported healthy or affected BSE cattle, in order to assess their potential resistance to BSE. A comparison of Chinese bovine populations with healthy and BSE-affected German and Swiss cattle from globally was conducted, and result indicating significant difference (p < .001) between healthy and affected cattle. Additionally, as compared to prior studies with Chinese bovine population, the significant results were found. In this study, the allelic frequency D23 finding high deletion in all analyzed Chinese bovine species, and haplotype D12-D23 exhibited a less significant inclination toward susceptibility to BSE.

Association of hormone-sensitive lipase (HSL) gene polymorphisms with the intramuscular fat content in two Chinese beef cattle breeds.

Hormone-sensitive lipase (HSL) was considered as an essential enzyme in glucolipid metabolism. It has been proposed to be a lead candidate gene for genetic markers of lipid deposition in livestock. The aim of this study was to identify sequence variants (SVs) of the bovine HSL gene and evaluate the relations to intramuscular fat in two indigenous Chinese beef cattle breeds. Expression analysis by quantitative real-time polymerase chain reactions (qPCR) indicated that expression levels of bovine HSL gene were highest in the perirenal fat and heart within two different age stage (adult and calf), respectively. Five SVs were identified by direct DNA sequencing, which included four missense mutations (g.16563C>T, g.16734G>A, g.16896A>G, g.17388G>T) in exon 8 and a synonymous mutation (g.17402C>T) in exon 9. Population genetic analysis showed that except for g.16563C>T and g.17402C>T, all the other detected SVs strongly affected the bovine intramuscular fat content (P < 0.01 or P < 0.05). The individuals with Hap5/5 diplotypes (CC-GG-GG-GG-CC) was highly significantly associated with intramuscular fat content than the other diplotypes (P < 0.01). The above results suggested that the HSL gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection in Chinese cattle breeds.

Genetic variants in MYF5 affected growth traits and beef quality traits in Chinese Qinchuan cattle.

Myogenic factor 5 plays actively roles in the regulation of myogenesis. The aims of this study are to identify the evolution information of MYF5 protein among 10 domestic and mammalian animals, to uncover the expression patterns of MYF5 gene in calves and adults of Qinchuan cattle, and to expose the genetic variants of the MYF5 gene and explore its effect on cattle growth traits and beef quality traits in Qinchuan cattle. The bioinformatics results showed that the MYF5 proteins highly conserved in different mammalian or domestic animals apart from chicken. The expression level of MYF5 gene in the heart, muscle, lung, large intestine and liver was greater than that of other tissues. PCR amplicons sequencing identified four novel SNPs at g.5738A>G, g.5785C>T and g.5816A>G in the 3rd exon region and g.6535A>G in the 3' UTR. Genotypic frequencies of g.5785C>T was harshly deviated from the HWE (P < .05). Genetic diversity was low or intermediate for the four SNPs and those SNPs were in the weak linkage disequilibrium. Association analysis results indicated g.5785C>T, g.5816A>G and g.6535A>G significant effect on growth performance and beef quality traits of Qinchuan cattle. H1H3 diplotype had greater body size and better beef quality. All the results implicate that the MYF5 gene might be applied as a promising candidate gene in Qinchuan cattle breeding.

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry.

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.

The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes.

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus-oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.

Influence of multi-enzyme preparation supplemented with sodium butyrate on growth performance blood profiles and economic benefit of growing rabbits.

The present study was carried out to explore the impacts of dietary supplementation of enzyme mixture with sodium butyrate on the growth performance, carcass traits, blood profile and economic benefit in two breeds of weanling rabbits adapted to survive in Egypt (New Zealand White and Rex). One-hundred and twenty weaned male rabbits (New Zealand White and Rex) of 6 weeks of age and 770.5 ± 20 g body weight were allotted randomly into four groups in a factorial arrangement. The obtained results indicated that there were non-significant differences in all growth performance traits, blood profile and economic parameters due to the breed effect. However, there were significant differences in most of carcass traits due to the breed effect except total giblets and New Zealand White breed showed the highest value of these parameters including dressing % (p < .01), forequarter and loin % (p < .001) and hindquarter % (p < .003) compared with Rex breed counterparts. The effect of the treatment and its interaction with the breed significantly (p < .05) improved body weight gain, feed consumption and carcass traits (percentage of dressing, forequarter, hind quarter and lion). However, final body weight and feed conversion ratio were not significantly influenced. Supplementing a diet with treatment significantly decreased blood triglycerides, cholesterol and the ratio between albumin and globulin (A/G ratio), while increased blood total protein and globulin. Although higher feed cost and total costs in treated groups than control ones in each breed, they showed higher total return and net return. Rex non-treated rabbit breed showed the lowest profitability measures compared with other groups. In conclusion, dietary supplementation of multi-enzyme with sodium butyrate is highly recommended in growing rabbits due to their beneficial effects on the growth performance and profitability.

First Report of Single Nucleotide Polymorphisms (SNPs) of the Leporine Shadow of Prion Protein Gene (SPRN) and Absence of Nonsynonymous SNPs in the Open Reading Frame (ORF) in Rabbits.

Prion disorders are fatal infectious diseases that are caused by a buildup of pathogenic prion protein (PrPSc) in susceptible mammals. According to new findings, the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) is associated with prion protein (PrP), promoting the progression of prion diseases. Although genetic polymorphisms in SPRN are associated with susceptibility to several prion diseases, genetic polymorphisms in the rabbit SPRN gene have not been investigated in depth. We discovered two novel single nucleotide polymorphisms (SNPs) in the leporine SPRN gene on chromosome 18 and found strong linkage disequilibrium (LD) between them. Additionally, strong LD was not found between the polymorphisms of PRNP and SPRN genes in rabbits. Furthermore, nonsynonymous SNPs that alter the amino acid sequences within the open reading frame (ORF) of SPRN have been observed in prion disease-susceptible animals, but this is the first report in rabbits. As far as we are aware, this study represents the first examination of the genetic features of the rabbit SPRN gene.

Comparative Analysis of PRNP Gene Indel Polymorphism and Expression among Zhongdian Yellow Cattle, Zhongdian Yak, and Their Hybrids.

Bovine spongiform encephalopathy (BSE) is a fatal disease in cattle caused by misfolded prion proteins and linked to indel polymorphisms in the promoter and intron 1 of the PRNP gene. The aim of this study was to determine the allele, genotype, and haplotype frequencies of PRNP indel polymorphisms and to investigate the effect of PRNP gene expressions of 23 bp and 12 bp indels via polymerase chain reaction (PCR) in Zhongdian Yak (Bos-grunniens) (YK), Zhongdian Yellow cattle (Bos-taurus) (YC), and Zhongdian Yakow (Bos-primigenius taurus × Bos-grunniens) (PK). Resultant high allelic frequencies were found in 23- and 12+, while haplotype frequencies were very low in 23+/12 in YK, YC, and PK. PRNP expression was higher in the +-/-- diplotype of the PK and (mean ± SE) was 3.6578 ± 1.85964. Furthermore, two variable sites were investigated-a 23 bp indel polymorphism holding AP1 binding site and a 12 bp indel polymorphism holding SP1 binding site. Additionally, reporter gene assays revealed a link between two proposed transcription factors and lower expression levels of the +/+ allele compared with the -/- allele. The expression level of PRNP was shown to be dependent on two indel polymorphisms in the bovine PRNP promoter, which includes binding sites for RP58 and SP1 transcription factors. These findings raised the possibility that the PRNP genotype may contribute to the high variation in PRNP expression.

The characteristics of proteome and metabolome associated with contrasting sperm motility in goat seminal plasma.

Sperm motility is an index tightly associated with male fertility. A close relationship between seminal plasma and sperm motility has been confirmed. This study was to assess the protein and metabolite profiles of seminal plasma obtained from adult goats with high or low sperm motility using the proteomic and metabolomic strategies. In total, 2098 proteins were found. 449 differentially abundant proteins (DAPs) were identified, and 175 DAPs were enriched in the high motility group. The obtained DAPs primarily exist in cytoplasma and extra-cellular portion. The Gene Ontology enrichment analysis demonstrated the main functional roles of these DAPs in regulating biological process, metabolic process of organic substances, cellular-metabolic process, primary-metabolic process, metabolic process of nitrogen compounds, etc. Additionally, the Kyoto-Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these DAPs were primarily involved in phosphatidylinositol signaling system, salivary secretion, proteasome, apoptosis, mitophagy-animal, etc. Aided by the parallel reaction monitoring technology, the abundance changing pattern of 19 selected DAPs was consistent with that of the corresponding proteins obtained by TMT. A total of 4603 metabolites were identified in seminal plasma. 1857 differential metabolites were found between the high motility group and the low motility group, and 999 metabolites were up-regulated in the high motility group. The KEGG analysis demonstrated the primary involvement of the differential metabolites in metabolic and synthetic activities. In conclusion, we first established the proteome and metabolome databank of goat seminal plasma, detecting some proteins and metabolites which may affect sperm motility. This study will be valuable for understanding mechanisms leading to poor sperm motility.

Trehalose modifies the protein profile of ram spermatozoa during cryopreservation.

As a magical oligosaccharide, trehalose has been revealed to enhance the post-thaw quality of stock semen. However, information regarding the cryoprotective mechanism of trehalose during cryopreservation has not yet been determined. This study was designed to observe the effects of trehalose on the proteome of ram frozen spermatozoa by applying the isobaric tag for relative and absolute quantification (iTRAQ) strategy combined with parallel reaction monitoring (PRM). A total of 1269 proteins were identified. Among them, there were 21 differentially expressed proteins (DEPs), with 9 up-regulated proteins and 11 down-regulated proteins in spermatozoa frozen with trehalose. These DEPs were primarily located in nucleus, cytoplasm, and extracellular region. The Gene Ontology (GO) enrichment analysis demonstrated the involvement of the DEPs in signal transduction, ion binding, oxidoreductase activity, response to stress, and catabolic processes. Based on the STRING analysis, tight functional correlations were observed between 6-phosphogluconate dehydrogenase, fructose-bisphosphate aldolase A isoform 1, 14-3-3 protein epsilon, tyrosine-protein kinase Fer, and beta-hexosaminidase subunit alpha precursor. Furthermore, 10 DEPs were verified using PRM, confirming the accuracy of the iTRAQ data acquired in this study. In conclusion, trehalose can modify the protein profile of ram spermatozoa during cryopreservation, which may be associated with its cryoprotective effects. Additionally, trehalose may function on frozen spermatozoa through antioxidation, involvement in glycolysis, and increment of spermatozoa tolerance to various stresses.

The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation.

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 µg/mL AFPIII (AFP-1), 10 µg/mL AFPIII (AFP-10), 50 µg/mL AFPIII (AFP-50), and 100 µg/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 µg/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 µg/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 µg/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.

Flavonoid bioactive compounds of hawthorn extract can promote growth, regulate electrocardiogram waves, and improve cardiac parameters of pulmonary hypertensive chickens.

The effect of orally administered hawthorn flavonoid extract (HFE) on growth, electrocardiographic waves, and cardiac parameters of pulmonary hypertensive chickens reared at high altitude (2,100 m above sea level) was examined. A total of 225 one-day-old, mixed broiler chicks (3 treatments with 5 replicates and 15 chicks per each, totally 75 birds/treatment) were assigned to 3 experimental groups: 0, 0.1, and 0.2 ml of HFE per 1 L of drinking water. Birds were administered the drinking water HFE treatments for 42 D. At an age of 28 and 42 D, electrocardiograms were undertaken and cardiac parameters such as the RV:TV, RV:BW, and TV:BW, and indicators of PHS on selected birds were measured. The final BW of chickens receiving the HFE at 0.2 ml/L was greater (2,579 ± 64 g) than that of birds receiving 0.1 ml/L (2,497 ± 62 g) and 0 ml/L (2,323 ± 57 g). Therefore, no supplemented group had a lower final BW than others (P < 0.05). Amplitudes of S and T waves in 0.1- and 0.2-ml/L HFE consumed groups at 28 and 42 D of age decreased compared with that in the control group (P < 0.05). The HFE reduced the heart weight and RV:TV, RV:BW, and TV:BW ratios when supplemented in drinking water at 0.1 and 0.2 mL/L compared with 0 mL/L (P < 0.05). In conclusion, supplementation of HFE in drinking water can reduce the PHS and incidence of cardiac disorders. Owing to the positive effect of HFE on cardiac parameters that mediated through flavonoids bioactive compounds, this product can be used to prevent complications of pulmonary hypertension and disarray of electrocardiographic waves in broiler chickens reared at high altitude.

Isolation and characterization of the major histocompatibility complex DQA1 and DQA2 genes in gayal (Bos frontalis).

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identifiedMHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest thatduplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.

Deletion/insertion polymorphisms of the prion protein gene (PRNP) in gayal (Bos frontalis).

Resistance to fatal disease bovine spongiformencephalopathy (BSE), due to misfolded prion protein in cattle, is associated with a 23-bp indel polymorphism in the putative promoter and a 12-bp indel in intron 1 of the PRNP gene. Gayal (Bos frontalis) is an important semiwild bovid species and of great conservation concern, but till today these indel polymorphisms have not been evaluated in gayals. Therefore, we collected 225 samples of gayals and evaluated the genetic indel polymorphism in the two regions of this PRNP gene. The results revealed high allelic frequencies of insertions at these indel sites: 0.909 and 0.667 for, respectively, the 23 bp and 12 bp indels, both also with significant genotype frequencies (χ2: 9.81; 23 bp and χ2: 43.56; 12 bp). At the same time, the haplotype data showed indel polymorphisms with extremely low deletion (0.01) in both regions of the PRNP gene. We compared these data with those reported for healthy and BSE-affected cattle (Bos taurus) breeds from two European countries, Germany and Switzerland, and significant difference (P <0.001) was observed between BSE-affected as well as the healthy cattle. Further, our data were also extensively compared with previous reports on BSE and highly significant (P<0.001) outcomes were observed. This result suggested negligible genetic susceptibility to BSE in gayals. To the best of our knowledge, this study is the first comprehensive deciphering information about the PRNP indel polymorphisms of 23 bp and 12 bp in gayals, a semiwild species of China.