Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources.

BACKGROUND: Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated. RESULTS: Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs. CONCLUSION: In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Exploring genetic diversity and population structure of Punjab goat breeds using Illumina 50 K SNP bead chip.

Pakistan has 35 goat breeds. Moreover, the province of Punjab has highest goat population constituting 37% of country's total population with seven goat breeds including Beetal, Daira Deen Panah, Nachi, Barbari, Teddi, Pahari, and Pothwari. The diversity study of breeds warrants the documentation of breeds particularly using genome wide panel of markers, i.e., SNP chip. The objective of the current study was to fill this gap of information. Therefore, in current study we collected total of 879 unrelated goat blood samples along with data on body weight measurements; genomic DNA was extracted, and genotyping was carried out using 50 K SNP bead chip. Quality control measures were performed in Plink 1.07. Genetic diversity was observed among studied populations using heterozygosity and pairwise FST estimates, principal component analysis, admixture analysis in Plink software with visualization in Clumpak, and constructing phylogenetic tree in Mega 7 software. Moderate to high level of heterozygosity was observed among the studied populations. Coefficient of inbreeding varied from 0.0186 ± 0.0327 in Pahari to 0.183 ± 0.0715 in Barbari. Barbari and Daira Deen Panah had quite higher level of inbreeding coefficient as compared to all other breeds with value of 0.183 ± 0.0715 and 0.1378 ± 0.0741, respectively. PCA identified three steps of subdividing the seven goat breeds at various levels of K. All the seven breeds made independent clusters at various levels of PCA. Admixture analysis revealed the distinctness of Teddi and Barbari breeds. Genetic sub-structuring was observed in the admixture patterns of Beetal breed. Moreover, high level of genetic admixture was observed in Nachi, Pahari, Pothwari, and Daira Deen Panah breeds. Admixture results were further interpreted by calculating pairwise FST values. Our results provided first insights about genetic diversity of Pakistani goat breeds based on genomic data. To conclude, the enriched goat breed diversity in Pakistan could provide valuable genetic reservoir for national breeding schemes.

Genetic Susceptibility to Chronic Liver Disease in Individuals from Pakistan.

Chronic liver disease, with viral or non-viral etiology, is endemic in many countries and is a growing burden in Asia. Among the Asian countries, Pakistan has the highest prevalence of chronic liver disease. Despite this, the genetic susceptibility to chronic liver disease in this country has not been investigated. We performed a comprehensive analysis of the most robustly associated common genetic variants influencing chronic liver disease in a cohort of individuals from Pakistan. A total of 587 subjects with chronic liver disease and 68 healthy control individuals were genotyped for the HSD17B13 rs7261356, MBOAT7 rs641738, GCKR rs1260326, PNPLA3 rs738409, TM6SF2 rs58542926 and PPP1R3B rs4841132 variants. The variants distribution between case and control group and their association with chronic liver disease were tested by chi-square and binary logistic analysis, respectively. We report for the first time that HSD17B13 variant results in a 50% reduced risk for chronic liver disease; while MBOAT7; GCKR and PNPLA3 variants increase this risk by more than 35% in Pakistani individuals. Our genetic analysis extends the protective role of the HSD17B13 variant against chronic liver disease and disease risk conferred by the MBOAT7; GCKR and PNPLA3 variants in the Pakistani population.

Genomic Analysis Suggests KITLG is Responsible for a Roan Pattern in two Pakistani Goat Breeds.

The roan coat color pattern is described as the presence of white hairs intermixed with pigmented hairs. This kind of pigmentation pattern has been observed in many domestic species, including the goat. The molecular mechanisms and inheritance that underlie this pattern are known for some species and the KITLG gene has been shown associated with this phenotype. To date, no research effort has been carried out to find the gene(s) that control(s) roan coat color pattern in goats. In the present study, after genotyping with the GoatSNP50 BeadChip, 35 goats that showed a roan pattern and that belonged to two Pakistan breeds (Group A) were analyzed and then compared to 740 goats of 39 Italian and Pakistan goat breeds that did not have the same coat color pattern (Group B). Runs of homozygosity-based and XP-EHH analyses were used to identify unique genomic regions potentially associated with the roan pattern. A total of 3 regions on chromosomes 5, 6, and 12 were considered unique among the group A versus group B comparisons. The A region > 1.7 Mb on chromosome 5 was the most divergent between the two groups. This region contains six genes, including the KITLG gene. Our findings support the hypothesis that the KITLG gene may be associated with the roan phenotype in goats.

Epidemiological survey of bovine gammaherpesvirus 4 (BoHV-4) infection in cattle and buffalo from Pakistan.

This study was conducted to evaluate the prevalence of bovine gammaherpesvirus 4 (BoHV-4) among healthy cattle and buffaloes as well as those associated with different diseases (respiratory tract infection, mastitis and reproductive tract infection) in District Chakwal, Pakistan. Blood, swab and milk samples of cattle and buffaloes were randomly collected from different areas of Chakwal. DNA was isolated from the samples and subjected to nested PCR using thymidine kinase gene primers. Out of 300 samples (200 blood, 50 swab and 50 milk samples) from both species (cattle and buffalo), an overall prevalence of BoHV-4 of 3.33% was obtained. Samples from cattle showed a higher species-specific prevalence (4.16%) than samples from buffalo (2.78%). One sample out of 50 swab samples and 1 out of 50 milk samples were also positive for BoHV-4. DNA sequencing of a positive PCR product from cattle confirmed that the sequence was from the thymidine kinase gene of BoHV-4. Phylogenetic analysis also revealed close similarities with other BOHV-4 thymidine kinase sequences. To detect BoHV-4 antibodies, an indirect ELISA was also performed. Two hundred blood samples were also collected from the same animals in nonanticoagulant-containing tubes for the isolation of serum and were subjected to indirect ELISA. Sixteen samples (8%) were positive for BoHV-4 antibodies. This study will be useful in further diagnoses of BoHV-4 in Pakistan and in devising measures to control the spread of BoHV-4.

Genome wide association study identifies novel candidate genes for growth and body conformation traits in goats.

Pakistan is third largest country in term of goat population with distinct characteristics of breeds and estimated population of 78.2 million. Punjab province has 37% of country's total population with seven important documented goat breeds namely Beetal, Daira Din Pannah, Nachi, Barbari, Teddi, Pahari and Pothwari. There is paucity of literature on GWAS for economically important traits i.e., body weight and morphometric measurements. Therefore, we performed GWAS using 50 K SNP Chip for growth in term of age adjusted body weight and morphometric measurements in order to identify genomic regions influencing these traits among Punjab goat breeds. Blood samples were collected from 879 unrelated animals of seven goat breeds along with data for body weight and morphometric measurements including body length, body height, pubic bone length, heart girth and chest length. Genomic DNA was extracted and genotyped using 50 K SNP bead chip. Association of genotypic data with the phenotypic data was performed using Plink 1.9 software. Linear mixed model was used for the association study. Genes were annotated from Capra hircus genome using assembly ARS1. We have identified a number of highly significant SNPs and respective candidate genes associated with growth and body conformation traits. The functional aspects of these candidate genes suggested their potential role in body growth. Moreover, pleiotropic effects were observed for some SNPs for body weight and conformation traits. The results of current study contributed to a better understanding of genes influencing growth and body conformation traits in goat.

Somatostatin can alter fertility genes expression, oocytes maturation, and embryo development in cattle.

This study was designed to investigate the effect of somatostatin on oocytes maturation and subsequent embryo development in cattle. Bovine granulosa cells separated from oocytes, cultured for 24 h and transfected with pEGFP-N1 vector with mouse SST gene (Experimental) and with out plasmid transfection (Control). RT-PCR and Real-Time PCR were used to estimate the expression of bovine receptors of androgen, estrogen beta, growth hormone, and follicular stimulating hormone. Culture media concentrations of hormones were measured by kits using radioimmunoassay. COCs aspirated from ovaries were co-cultured with granulosa cells layers (transfected or control) at 38.5 degrees C in CO(2) incubator for maturation. We found a significant (2.37X) increase in estrogen receptor beta expression in experimental group. There was a decrease in androgen receptor, growth hormone releasing hormone receptor, and follicular stimulating hormone receptor (P < 0.05). But, 96 h of post transfection, culture media concentration of estradiol-17beta was increased significantly (P < 0.05) and testosterone, growth hormone and follicular stimulating hormone showed opposite trend (P < 0.05) in experimental groups. Co-culture of somatostatin transfected granulosa cells with oocytes, reduced the maturation rate from 70% to 66% but had no effect on subsequent fertilization and embryo development.

First detection and molecular characterization of avian polyomavirus in young parrots in Pakistan.

Avian polyomavirus (APV) infection, also called as budgerigar fledgling disease (BFD) causes various health problems in many psittacine species which may cause untimely death. The aims of this study were to investigate, for the first time, the detection, molecular characterization and phylogenetic analysis of avian polyomavirus (APV) in Pakistani psittacine birds. In an aviary a disease similar to APV was found and 90% of the nestlings died within a few weeks. Seven to ten-day-old parrot nestlings (n = 3) from the aviary were presented with feather abnormalities, plumage defect and were clinically depressed. Birds died at 11th, 14th and 16th day of age. Samples of hearts, livers, spleen, feathers and kidneys were collected from the dead birds. Samples were analyzed for the presence of APV DNA by using PCR. APV VP1 gene was partially sequenced, and phylogenetic analysis was performed. The APV strain was similar to those previously reported in other areas of the world. The results of this investigation indicate presence of a high frequency of APV infections in psittacine birds in Pakistan.

Development of amplified fragment length polymorphism (AFLP) markers for the identification of Cholistani cattle.

The identification issue of livestock can be resolved by using molecular identification tools that are acceptable to preserve and maintain pure breeds worldwide. The application of a molecular identification methodology is more important for developing nations, e.g., Pakistan, where uncontrolled crossbreeding has become a common practice and the import of exotic animals and germplasm is ever increasing. This presents a risk to local breeds as also stated by the FAO. Therefore, the current study was designed to develop standard molecular markers for Cholistani cattle to ascertain their purity for breeding purpose. In this study 50 and 48 unrelated males were sampled for Cholistani and each crossbred cattle, respectively. Candidate molecular markers present in Cholistani but absent in crossbred cattle and vice versa were detected using the amplified fragment length polymorphism (AFLP) method. Eleven markers were developed and were converted to single nucleotide polymorphism (SNP) markers for genotyping. The allele frequencies in both breeds were determined for discrimination ability using polymerase-chain-reaction-restriction-fragment-polymorphism (PCR-AFLP). The probability of identifying the Cholistani breed was 0.905 and the probability of misjudgment was 0.073 using a panel of markers. The identified markers can ascertain the breed purity and are likely to extend the facility for breed purity testing before entering into a genetic improvement program in the country.

Ontogeny of mRNA expression of somatostatin and its receptors in chicken embryos in association with methylation status of their promoters.

The present study was designed to investigate the ontogeny and tissue distribution of somatostatin and its five receptor subtypes (SSTR1-5) mRNA expression in embryonic chicken (Gallus gallus). Brain, gonads (male), intestine, kidney, liver, muscle, stomach and yolk sac membrane (YSM) of chicken embryos on the embryonic (E) ages of 10, 16 and 21days (right before hatch) were investigated. Bisulfite sequencing PCR (BSP) was performed to determine the methylation status of the promoter region of all the six genes in the liver. Somatostatin (SST) was predominately expressed in intestine, brain and gonads (male) with different ontogenic patterns. The highest expression in intestine was detected at E10. There was ontogenic shift from intestine to brain as development progressed. Expression pattern of SSTRs in brain, intestine and kidney was similar to human embryonic expression. In liver, the ontogenic expression pattern of SST and its receptors was associated to methylation status of the respective promoters. Methylation of site Sp1 determines expression level of SST, SSTR1, SSTR2 and SSTR3 while site a is important in governing the expression of SSTR4 and SSTR5. The results show that ontogenic expression profile of chicken SST and SSTRs is time and tissue specific.

Evolutionary history of the somatostatin and somatostatin receptors.

Somatostatin and its receptors have a critical role in mammalian growth through their control pattern of secretion of growth hormone, but the evolutionary history of somatostatin and somatostatin receptors are ill defined. We used comparative whole genome analysis of Danio rerio, Carassius auratus, Xenopus tropicalis, Gallus gallus, Monodelphis domestica, Homo sapiens, Sus scrofa, Bos taurus, Mus musculus, Rattus norvegicus, Canis lupus familiaris, Ovis aries, Equus caballus, Pan troglodytes and Macaca mulatto to identify somatostatin and somatostatin receptors in each species. To date, we have identified a minimum of two genes of somatostatin and five somatostatin receptor genes in mammalian species with variable forms. We established a clear evolutionary history of the somatostatin system and traced the origin of the somatostatin system to 395 million years ago (MYA), identifying critical steps in their evolution.

Development and evaluation of a novel DNA vaccine expressing inhibin alpha (1-32) fragment for improving the fertility in rats and sheep.

Inhibin is an important protein hormone in regulating folliculogenesis. Immunization against inhibin can improve follicle developments. The objective of present study is to investigate inhibin DNA immunization as a potential tool for improving follicle development and litter sizes of female animals. In our study, the inhibin DNA vaccine was constructed with inhibin alpha (1-32) fragment inserted into the C termination of HBsAg-S. Ninety rats and forty sheep were immunized with inhibin DNA vaccine. In rats, immunization against inhibin resulted in increase of positive sera ratio (P<0.01). The treatment was accompanied by a significant increase in the total number of mature follicles of >0.8mm in diameter on the onset of estrous cycle after twice immunization (31.0+/-3.9 in the test groups versus 27.4+/-5 in control groups) and after third immunization (35.2+/-6.7 in the test groups versus 30.3+/-5.2 in control groups). Litter sizes were significantly (P<0.05) bigger in rats treated with inhibin DNA vaccine (12.7+/-4.5 n=6) than in control (9.8+/-4.5). In sheep, twinning rate in test groups (39.2%) was significantly higher than that in control groups (10%) after immunization (P<0.05). These results indicated that inhibin was an important factor in improvement of fertility in rats and sheep, and demonstrated that DNA immunization against inhibin could induce more mature follicles resulting in increased litter sizes. Our results revealed that inhibin DNA vaccine may be an alternative to the use of exogenous gonadotrophins for increasing ovarian follicular development and improving animal fertility.