In cattle, nearly all heifers born co-twin to a male are freemartins, XX/XY chimeras that exhibit a characteristic masculinized phenotype. However, in sheep, while litters containing males and females are common, freemartins are relatively rare. The primary aim of this study was to determine the frequency and features of XX/XY chimerism in female Rideau Arcott sheep. Also, breeding records were used to investigate the effect of litter size and sex ratios, as well as the genetic basis of the condition. Finally, the migration and transcriptional competence of cells of the opposite sex in the XX/XY female and male chimeras was explored. Genomic DNA (gDNA) from peripheral blood cells of ewes was screened by PCR for the male-specific SRY gene. Of 230 lambs screened, 10 were identified as chimeras. Litter size and sex ratio showed no statistically significant effect on the frequency of chimerism. PCR and FISH analysis confirmed the presence of opposite sex cells in female and male chimeras, and in the case of ewes, their migration to tissues other than blood. Transcriptional activity of SRY and AMH was detected in gonads of ewes, whereas XIST expression was detected in white blood cells of chimeric rams. It was concluded that the frequency of sex chromosome chimerism in Rideau Arcott sheep is estimated at 4.35%, with no significant effect of litter size and sex ratio. Moreover, as it was shown that opposite sex cells can migrate to tissues other than blood and be transcriptionally active in chimeric sheep, we speculate on the role they can play in these animals.

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Our previous studies showed that expression patterns of X-linked genes in cultured cells are different from those of their tissues of origin. This investigation analyses the transcription pattern of the X-linked genes BIRC4, GAB3, MECP2, RPS4X, SLC25A6, and XIST in bovine in vitro matured oocytes and in vitro fertilized embryos, and their in vivo counterparts. In vitro-derived pools of mature oocytes and pre-attachment embryos were obtained by: (a) TCM-199/serum with bovine oviductal epithelial cells as co-culture, and (b) synthetic oviductal fluid/BSA. Pools of in vivo-derived morulae and blastocysts were provided by a commercial embryo transfer operation. Total RNA was extracted for quantification of gene-specific transcript levels using real-time quantitative PCR. Statistical analysis was performed using a mixed model factorial ANOVA with alpha = 0.05. The effect of the in vitro environmental conditions on X-linked gene transcription was most evident during the fourth cell cycle, at the period of activation of the embryonic genome, and seemed to be less pronounced at later developmental stages, with the exception of BIRC4. The levels of X-linked genes transcripts in in vivo-derived embryos were lower relative to their in vitro counterparts for all genes tested. Finally, the pattern of expression of XIST in bovine oocytes and embryos was similar to that reported in humans. These results highlight the possibility that X-linked gene expression analysis is a useful tool to monitor the impact of reproductive biotechnologies on the developmental potential of embryos and aid in their improvement.

Infectious diseases are an important cause of economic loss in the agri-food business. This study investigates indicators of bovine high (HR) and low (LR) immune response and their associated patterns of gene expression. Holstein cows were immunized to induce antibody (AMIR) and cell-mediated (CMIR) immune responses. Based on the results of enzyme-linked immunosorbent assay (ELISA) and delayed-type hypersensitivity (DTH), cows were ranked as HR, LR or average (AR) immune responders. For microarray analysis, phenotypic HR and LR status in both groups was confirmed and total RNA from blood mononuclear cells (BMCs) was obtained. RNA from a pool of AR cows was used as a common reference for hybridization to an in-house cDNAmicroarray. Results of microarray analysis showed transcriptional differences in several immune-related genes between the HR and LR groups. Genes identified as differentially expressed include transcription factors, cytokines, MHC, and TCR-related genes. These results can aid in the establishmentof selection programmes based on broad-based disease resistance, aimed at improving general health in cattle herds.

The sex determination system in mammals creates an imbalance between males and females in the number of X chromosomes. This imbalance is compensated through transcriptional silencing of one of the two X chromosomes in female diploid cells by epigenetic modifications. Although common for mammals, X inactivation shows marked species-specific differences in mechanisms and end results, and provides a unique opportunity to study epigenetic regulation of gene expression. The aim of the present study was to establish the expression pattern of selected X-linked genes in bovine fetal muscle tissue and muscle fibroblast cultures in order to follow possible modifications at the transcriptional level attributable to in vitro culture. We used heterologous cDNA microarray hybridization and quantitative real-time PCR to study the pattern of expression of X-linked genes including SLC25A6, GAB3, MECP2, RPS4X, JARID1C, UBE1, BIRC4 and SLC16A2. Quantitative real-time PCR analysis in fetal bovine muscle showed higher transcript levels in females for all X-linked genes tested with the exception of SLC25A6, with differences being significant for RPS4X, JARID1C and UBE1. The expression in fibroblast cultures derived from the same samples differed, with significantly higher levels for UBE1, GAB3 and BIRC4, while the rest of the panel of X-linked genes remained unchanged. The changed expression pattern in vitro, probably reflecting modifications in the epigenetic mechanisms that regulate transcriptional activity and gene silencing in X inactivation, has important implications for the advancement of new biotechnologies such as somatic cell nuclear transfer and stem cell therapy.