The bovine brachyspina syndrome: An attempt to produce affected embryos

1. The bovine brachyspina syndrome: An attempt to produce affected embryos Jørgen S Agerholm1, Mette H Schmidt1, Merete Fredholm2 and Poul Hyttel2 1Department of Large Animal Sciences and 2Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark IntroductionThe brachyspina syndrome (Figure 1) is a congenital lethal syndrome in Holstein cattle originally observed in Denmark (1) with identification of additional cases in Italy (2), the Netherlands (3) and Canada (4). The disorder is morphologically characterized by reduction of the birth weight to around 10 kg despite a gestation period of normal or slightly prolonged length, obvious shortening of the spine, long slender limbs, inferior brachygnathism or micrognathism, renal and gonadal dysplasia and in some case, additional internal malformations (1-4). Reported cases have occurred in a familial pattern consistent with autosomal recessive inheritance. Genomic studies of affected calves performed in a collaboration between researchers at The University of Copenhagen and at the University of Liege, Belgium have identified the genomic region where the defective gene is located and have developed a genetic test based on markers (5). But further development and identification of the causal mutation was hampered by the lack of RNA of affected calves as those found were either stillborn or died shortly after delivery in farms located far away from the universities, thus making isolation of RNA impossible. Analysis of pedigree data of affected breeding lines showed that the number of defective calves delivered at term was significantly lower than expected thus indicating a considerably intrauterine loss (abortion) as known for similar malformations affecting the segmentation of the spine (6).To overcome the problems with lack of RNA of affected calves, a project was established to experimentally produce defective embryos and retrieve embryos before they died.Materials and MethodsTwo non-pregnant cows that had previously given birth to brachyspina affected calves were initially selected for the study. Later a non-pregnant mature heifer was identified based on analyses of an EDTA stabilized blood sample by the recently developed genetic marker test.The animals were housed at the experimental animal facilities at the Faculty of Life Sciences, Taastrup Campus. The animals were superovulated by intramuscular injections of decreasing doses of follicle-stimulating hormone and luteinizing hormone (Pluset®, Caliere, Barcelona, Spain) on days 10, 11and 12 of their oestrous cycle.Oestrous was induced on day 13 with prostaglandin in two animals while one cows developed cystic ovaries and was slaughtered. The remaining two animals were inseminated twice during heat with a 12 h interval with semen from a carrier bull, i.e. 25% of the embryos were expected to be homozygous affected. The ovarian response was evaluated by rectal palpation after one week.The cows were transported to a slaughter house on day 17 after insemination. The pregnant uterus was obtained and flushed. The embryos were isolated from the flushing medium under a stereomicroscope. In one of the cows no embryos were found, while five embryos were isolated from the other uterus. The embryos seemed to be normally developed. Examination of the ovaries showed 2-4 corpora lutea in one animal and 6-8 corpora lutea in the other.DNA and RNA were isolated from the extraembryonic membranes from each of the embryos using standard procedures.ResultsThe embryos were genotyped with the genetic marker test. Three of them were homozygous for the wild type allele and two of them were heterozygous. Bovine brachyspina syndrome in a Danish Holstein calf. The calf is growth retarded with shortening of the spine and prominence of the thoracic spinous processes. Notice inferior brachygnatism and long slender legs. Bar = 15 cm. DiscussionThe experimental production of affected embryos was unsuccessful since none of the embryos were homozygous for the mutation. The number of embryos retrieved from the cows was lower than expected although a cow’s response to hormone treatment is not well predictable. The number of embryos was lower than the number of corpora lutea in the ovaries thus indicating a loss of embryos prior to gestation day 17.Continued research and development within molecular genetics have overcome the obstacles faced in this study regarding the lack of suitable materials for genomic analyses. Consequently we have managed to identify the causal mutation associated with brachyspina syndrome in Holstein cattle and developed a test that is licensed to laboratories worldwide for commercial testing of breeding animals (7).References1. Agerholm JS, McEvoy F, Arnbjerg J. Brachyspina syndrome in a Holstein calf. J Vet Diagn Invest 2006;18:418-422.2. Testoni S, Diana A, Olzi E, Gentile A. Brachyspina syndrome in two Holstein calves. Vet J 2008;177:144-146.3. Agerholm JS, Peperkamp K. Familial occurrence of Dutch and Danish cases of the bovine brachyspina syndrome. BMC Vet Res 2007;3:8.4. Agerholm JS, Delay J, Hicks B, Fredholm M. First confirmed case of the bovine brachyspina syndrome in Canada. Can Vet J 2010, 51, 1349-1350.5. Georges M, Coppieters W, Charlier C, Agerholm JS, Fredholm M. A genetic marker test for brachyspina and fertility in cattle. WO2010/012690 A1.20106. Nielsen US, GP Aamand, Andersen O, Bendixen C, Nielsen VH, Agerholm JS. Effects of complex vertebral malformation on fertility traits in Holstein cattle. Livestock Prod. Sci. 2003, 79, 233-238.7. Charlier C, Agerholm JS, Coppieters W, Karlskov-Mortensen P, de Jong G, Li W, Fasquelle C, Karim L, Cirera S, Cambisano N, Ahariz N, Mullaart E, Georges M, Fredholm M. A deletion in the bovine FANCI gene compromises fertility by causing fetal death and brachyspina. PLoS Genetics, submitted.