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FAC U LTY O F LIF E SC I EN C ES
UNIVERSITY OF COPENHAGEN
The bovine brachyspina syndrome:
An attempt to produce affected embryos
Jørgen S
1
Agerholm ,
1Department
Mette H
1
Schmidt ,
Merete
2
Fredholm
2Department
of Large Animal Sciences and
Sciences, University of Copenhagen, Denmark
and Poul
2
Hyttel
of Basic Animal and Veterinary Sciences, Faculty of Life
Introduction
The 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 Methods
Two 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.
Results
The 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.
Discussion
The 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).
References
1. 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
A1.2010
6. 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.