Establishing gene Amelogenin as sex-specific marker in yak by genomic approach.

Yak, an economically important bovine species considered as lifeline of the Himalaya. Indeed, this gigantic bovine is neglected because of the scientific intervention for its conservation as well as research documentation for a long time. Amelogenin is an essential protein for tooth enamel which eutherian mammals contain two copies in both X and Y chromosome each. In bovine, the deletion of a fragment of the nucleotide sequence in Y chromosome copy of exon 6 made Amelogenin an excellent sex-specific marker. Thus, an attempt was made to use the gene as an advanced molecular marker of sexing of the yak to improve breeding strategies and reproduction. The present study confirmed that the polymerase chain reaction amplification of the Amelogenin gene with a unique primer is useful in sex identification of the yak. The test is further refined with qPCR validation by quantifying the DNA copy number of the Amelogenin gene in male and female. We observed a high level of sequence polymorphisms of AMELX and AMELY in yak considered as novel identification. These tests can be further extended into several other specialized fields including forensics, meat production and processing, and quality control.

Adaptation strategies of yak to seasonally driven environmental temperatures in its natural habitat.

The gradual increase of ambient temperature (TA) at high altitude can cause heat stress as an effect of climate change and may shift the traditional habitat of yak to further higher altitude. Therefore, an attempt has been made in this study to evaluate the thermo-adaptability of yaks to different seasons at high altitude. The adaptive capabilities of yaks were assessed based on different heat tolerance tests in relation to changes in rectal temperature (RT; °F), respiration rate (RR; breaths/min), pulse rate (PR; beats/min), and plasma heat shock protein (HSP) profile. The experiment was conducted in 24 yaks, divided into three groups based on age as calf (n = 8), adult (n = 8), and lactating cow (n = 8). Thermal adaptability was determined by temperature humidity index (THI), dairy search index (DSI), and Benezra's thermal comfort index (BTCI) along with HSP70 profile. The THI was higher (P < 0.01) in summer than winter which increased from lowest (40.87) to highest (61.03) in summer by 20 points, where yaks were under heat load beyond THI 52. The RT (100.09 ± 0.18 °F), RR (21.76 ± 0.18), and PR (59.78 ± 0.32) increased by 23-35%, and this was correlated to the higher values of DSI exceeding 1 in calves (1.35 ± 0.03), lactating cows (1.29 ± 0.04), and adults (1.23 ± 0.32) during summer in comparison to winter (0.98 ± 0.02). The BTCI also showed values greater (P < 0.01) than 2 in calves (3.47 ± 0.27), lactating cows (3.23 ± 0.28), and adults (2.98 ± 0.29) which reflected 49-75% increase in rectal temperature and respiration rate during summer. Further, heat stress was substantiated by threefold higher (P < 0.01) level of plasma HSP70 in calves (189.61 ± 3.90 pg/ml) followed by lactating cows (168.62 ± 3.03 pg/ml) and adults (155.33 ± 2.30 pg/ml) against the winter average of 87.92 ± 3.19 pg/ml. Present results revealed that yaks were experiencing heat stress in summer at an altitude of 3000 m above sea level and calves were more prone to heat stress followed by lactating cows and adults.

Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).

We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P = 0.03; corrected P = 0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO-affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease.

A Non-Reciprocal Autosomal Translocation 64,XX, t(4;10)(q21;p15) in an Arabian Mare with Repeated Early Embryonic Loss.

Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a phenotypically normal 3-year-old Arabian mare that had repeated resorption of conceptuses prior to day 45 of gestation and was diagnosed with REEL. Conventional and molecular cytogenetic analyses revealed that the mare had normal chromosome number 64,XX but carried a non-mosaic and non-reciprocal autosomal translocation t(4;10)(q21;p15). This is a novel translocation described in horses with REEL and the first such report in Arabians. Previous cases of REEL due to autosomal translocations have exclusively involved Thoroughbreds. The findings underscore the importance of routine cytogenetic screening of breeding animals.

Comparative organization and gene expression profiles of the porcine pseudoautosomal region.

The pseudoautosomal region (PAR) has important biological functions in spermatogenesis, male fertility and early development. Even though pig (Sus scrofa, SSC) is an agriculturally and biomedically important species, and its genome is sequenced, current knowledge about the porcine PAR is sparse. Here we defined the PAR in SSCXp/Yp by demarcating the sequence of the pseudoautosomal boundary at X:6,743,567 bp in intron 3-4 of SHROOM2 and showed that SHROOM2 is truncated in SSCY. Cytogenetic mapping of 20 BAC clones containing 15 PAR and X-specific genes revealed that the pig PAR is largely collinear with other mammalian PARs or Xp terminal regions. The results improved the current SSCX sequence assembly and facilitated distinction between the PAR and X-specific genes to study their expression in adult and embryonic tissues. A pilot analysis showed that the PAR genes are expressed at higher levels than X-specific genes during early development, whereas the expression of PAR genes was higher at day 60 compared to day 26, and higher in embryonic tissues compared to placenta. The findings advance the knowledge about the comparative organization of the PAR in mammals and suggest that the region might have important functions in early development in pigs.

Cytogenetic and molecular characterization of Y isochromosome in a 63XO/64Xi(Yq) mosaic karyotype of an intersex horse.

Sex chromosome aberrations commonly lead to abnormal sexual development. Here we cytogenetically and molecularly characterized Y isochromosome in an intersex horse. Blood lymphocyte analysis showed a mosaic karyotype with 96% 63,XO and 4% 64,Xi(Y) cells. Molecular analysis of the isochromosome was carried out by fluorescence in situ hybridization and polymerase chain reaction with male-specific and pseudoautosomal markers from the horse Y chromosome. We found that the isochromosome was monocentric, composed of 2 long arms, carrying 2 sets of genes of the pseudoautosomal region (PAR) and the male-specific region of the Y (MSY), including the SRY - thus being genetically equivalent to Y disomy. Sequence analysis of a 1,955-bp region including the SRY exon, the promoter and the UTRs, revealed no mutations in the aberrant Y. The presence of an intact SRY in a small proportion of cells is the proposed cause for the intersex phenotype. Given that the i(Yq) was present in a mosaic form, both post-zygotic and meiotic mechanisms of its origin were proposed. We speculated that nonmosaic 64,Xi(Yq) karyotypes might be rare or absent because of the likely instability of the i(Yq) during cell division. Genetic and phenotypic implications of Y isochromosome formation in other mammals are discussed in the light of the diversity of Y chromosome organization between species.

The pseudoautosomal region and sex chromosome aneuploidies in domestic species.

The pseudoautosomal region (PAR) is a unique and specialized segment on the mammalian sex chromosomes with known functions in male meiosis and fertility. Detailed molecular studies of the region in human and mouse show dramatic differences between the 2 PARs. Recent mapping efforts in horse, dog/cat, cattle/ruminants, pig and alpaca indicate that the PAR also varies in size and gene content between other species. Given that PAR genes escape X inactivation, these differences might critically affect the genetic consequences, such as embryonic survival and postnatal phenotypes of sex chromosome aneuploidies. The aim of this review is to combine the available information about the organization of the PAR in domestic species with the cytogenetic data on sex chromosome aneuploidies. We show that viable XO individuals are relatively frequently found in species with small PARs, such as horses, humans and mice but are rare or absent in species in which the PAR is substantially larger, like in cattle/ruminants, dogs, pigs, and alpacas. No similar correlation can be detected between the PAR size and the X chromosome trisomy in different species. Recent evidence about the likely involvement of PAR genes in placenta formation, early embryonic development and genomic imprinting are presented.

Adrenal histoplasmosis: An unusual cause of adrenomegaly.

Histoplasmosis is a geographically restricted form of fungal infection. Adrenal involvement is seen in disseminated disease but sometimes it may be the only site of demonstrable disease. Early diagnosis and treatment may save the patient from catastrophic adrenal insufficiency. We present two patients showing bilateral adrenomegaly on ultrasonography and contrast-enhanced CT, and was diagnosed to have histoplasmosis on fine-needle aspiration cytology.

Molecular heterogeneity of XY sex reversal in horses.

Male-to-female 64,XY sex reversal is a frequently reported chromosome abnormality in horses. Despite this, the molecular causes of the condition are as yet poorly understood. This is partially because only limited molecular information is available for the horse Y chromosome (ECAY). Here, we used the recently developed ECAY map and carried out the first comprehensive study of the Y chromosome in XY mares (n=18). The integrity of the ECAY in XY females was studied by FISH and PCR using markers evenly distributed along the euchromatic region. The results showed that the XY sex reversal condition in horses has two molecularly distinct forms: (i) a Y-linked form that is characterized by Y chromosome deletions and (ii) a non-Y-linked form where the Y chromosome of affected females is molecularly the same as in normal males. Further analysis of the Y-linked form (13 cases) showed that the condition is molecularly heterogeneous: the smallest deletions spanned about 21 kb, while the largest involved the entire euchromatic region. Regardless of the size, all deletions included the SRY gene. We show that the deletions were likely caused by inter-chromatid recombination events between repeated sequences in ECAY. Further, we hypothesize that the occurrence of SRY-negative XY females in some species (horse, human) but not in others (pig, dog) is because of differences in the organization of the Y chromosome. Finally, in contrast to the Y-linked SRY-negative form of equine XY sex reversal, the molecular causes of SRY-positive XY mares (5 cases) remain as yet undefined.

Characterization of the bovine pseudoautosomal region and comparison with sheep, goat, and other mammalian pseudoautosomal regions.

The pseudoautosomal region (PAR) is a small region of sequence homology between mammalian X and Y chromosomes and is needed for sex chromosome segregation in male meiosis. The region, though studied as yet in only a few species, shows considerable variation in size and gene content. We have constructed a medium-density gene map for the cattle PAR and the adjacent X-specific region by isolating and mapping 18 BAC clones which contain 20 PAR- and 5 X-specific genes. One BAC clone containing TBL1XY and GPR143 spanned the recently demarcated bovine pseudoautosomal boundary (PAB). Comparing the gene map of cattle PAR with the high-resolution maps of human, horse, and dog PAR allowed to estimate that the size of cattle PAR is approximately 5-9 Mb. BAC end sequence analysis showed that there is a gradient of decreasing GC content from PARter towards the PAB which is consistent with findings in human, mouse, and horse. The 20 PAR- and 5 X-specific cattle genes were mapped also in goat and sheep, showing that PAR in the 3 species is similar in size, gene content, and gene order. For the first time the PAB was determined in goat sex chromosomes. Comparison of cattle, goat, and sheep PAR with homologous regions on human and horse X chromosomes showed a high degree of linkage conservation between all species. However, the most terminal human, horse, and dog PAR gene, PLCXD1, is X-specific in ruminants. Since the human/horse linkage group containing PLCXD1 is of ancestral origin, the location of PLCXD1 can be considered as a de novo event in ruminant sex chromosome evolution. The gene map of the cattle PAR adds to our knowledge about the comparative organization and evolution of the eutherian PAR and aids the sequencing, sequence assembly, and annotation of the terminal region of BTAXq.

Multivitamin supplementation of Wistar rats during pregnancy accelerates the development of obesity in offspring fed an obesogenic diet.

OBJECTIVE: The effect of gestational multivitamin supplementation on the development of obesity in rat offspring fed an obesogenic diet was investigated. DESIGN: Pregnant Wistar rats (n=10 per group) were fed the AIN-93G diet with the recommended vitamin (RV) content or a 10-fold increase (high vitamin, HV). At weaning, 10 males and 10 females, from separate dams, and from each gestational diet group were weaned to the liquid obesogenic diet for 48 weeks post-weaning. MEASUREMENTS: Body weight (BW) was measured weekly, and food intake over 24 h was measured once every 3 weeks for 24 weeks. Every 4 weeks, after an overnight fast, food intake over 1 h was measured 30 min after a gavage of water or glucose. An oral glucose tolerance test (OGTT) was carried out every 3-5 weeks. Post-weaning fasting glucose, insulin, ghrelin, glucagon-like peptide 1 (GLP-1), and systolic blood pressure (SBP) were measured. RESULTS: No difference in BW at birth or litter size was observed. Males and females from HV dams gained 17% (P<0.05) and 37% (P<0.001) more BW at 48 weeks post-weaning, and consumed 18% (P=0.07) and 20% (P<0.05) more food. One-hour food intake after water and glucose preloads was 27% (P<0.01) and 34% (P<0.05) higher in males from HV dams. Fasting ghrelin and GLP-1 were 27 and 32% higher in males from HV dams at weaning (P<0.05). Blood glucose response to the OGTT was greater in both males and females from HV dams at 13 weeks post-weaning (P<0.05), and the insulin resistance index was 76 and 43% higher in females from HV dams at 14 and 28 weeks post-weaning (P<0.05). SBP was 23 and 16% higher at 44 weeks post-weaning in male and females (P<0.01). CONCLUSION: High multivitamin intake during pregnancy increases the phenotypic expression of obesity and components of the metabolic syndrome in both female and male rats fed an obesogenic diet.