Research sheep

2.1.1 South Hampshire and Coopworth sheep

The New Zealand South Hampshire (SH) and Coopworth (CPW) sheep used in studies described in consequent chapters were part of the unique South Hampshire CLN6 variant research flock currently maintained by Prof. David Palmer at Lincoln University, Christchurch, New Zealand. The flock is maintained under standard New Zealand pastoral conditions on a 250 hectare research farm at Lincoln University with management and husbandry performed according to NIH guidelines and the New Zealand Animal Welfare Act (1999).

NCL in South Hampshire sheep was first described by Jolly and West (1976) in two affected rams. An experimental flock was established (Jolly et al., 1980) and maintained by crossing homozygous affected rams with heterozygous carrier ewes resulting in 50% affected and 50% carrier offspring each year. The flock has undergone two outbreeding programs in the past 30 years with the introduction of heterozygous ewes attained from crosses between normal Friesian/ Finn and Coopworth ewes and affected South Hampshire rams to increase fecundity, and improve the health and reproductive performance of the sheep flock. The first outcross occurred early in the establishment of the research flock and included the introduction of Friesian and Finn crossbred ewes, whereas the second outcross occurred in 2001 and included Coopworth crossbred ewes (N. Mitchell, pers. comm.).

The sheep from the New Zealand CLN6 variant research flock will be described in the following as South Hampshire despite these previous outbreeding programs. As no normal SH sheep are born in the research flock, CPW sheep maintained at the research farm at Lincoln University Lincoln University under the same conditions are used as normal controls.

The SH and CPW sheep are characterised as either ‘normal’, ‘carrier’ or ‘affected’ for NCL by using a combination of pedigree information, observation of emerging clinical signs and an indirect DNA test (Tammen et al., 2006). Affected NCL sheep were observed for clinical signs at approximately 10 to fourteen months of age, which is the stage when animals tend to develop blindness and neurological signs (Jolly et al,, 1980). Histopathology analysis of needle brain biopsy samples from lambs at 2 to 3 months of age was used for diagnosis (Dickson et al., 1989) prior to development of the DNA test.

The indirect DNA test used to detect NCL was based on a silent mutation (c.822G>A) identified in exon 7 of the CLN6 gene coding sequence (Tammen et al., 2006) where the ‘A’ allele is closely linked to the disease mutation with LOD score of 13.3 (θ=0.01) (Tammen et al., 2006). The control sheep is considered to be unaffected or normal based on a lack of clinical phenotype and histopathology results, if available (Tammen et al., 2006).

A total of 14 South Hampshire and 3 Coopworth sheep were selected for this study with the DNA samples provided by Prof. David Palmer from Lincoln University, Christchurch, New Zealand.

2.1.2 Merino sheep

Two Merino sheep were selected for this study from the NCL research flock at Camden Campus at the University of Sydney, Australia. These animals were maintained under standard Australian pastoral conditions. The flock was established in 1998, initially using advanced reproductive technologies (Multiple Ovulation and Embryo Transfer - MOET, Artificial Insemination - AI and/or estrus synchronisation (Cook et al., 2002). In more recent years the flock has been maintained largely by using natural mating between carrier animals. Concerns about high levels of inbreeding resulted in outcrossing of carrier rams to unrelated Merino ewes obtained from the University of Sydney farms in the year 2008. Procedures performed on the sheep throughout the research project were approved by the University of Sydney Animal Ethics Committee (AEC)

and are in accordance with NIH guidelines, the NSW Animal Research Act (1985) and the Australia Code of Practice for the Care and Use of Animals for Scientific Purposes 7th Edition (NHMRC 2004).

The Merino sheep are characterised as either ‘normal’, ‘carrier’ or ‘affected’ for NCL within the first two months of birth using a direct DNA test (Tammen et al., 2006). The test was developed based on the finding of the disease causing mutation (c.184C>T) in exon 2 of CLN6, which results in a major amino acid exchange (p.Arg62Cys). The DNA test comprised of blood collection, DNA extraction, PCR amplification, enzyme cleavage of amplicons and agarose gel separation to visualise PCR products. As well as the DNA test, the demeanor and clinical signs of sheep were closely observed throughout their lifetime.

Although research efforts were mainly focused on the SH breed of sheep, the Merino sheep flock was also involved, particularly for use as control animals when comparing sequence and genomic information (Chapter 7). The routine husbandry procedures performed on the Merino sheep, which were assisted by the author, included ear tagging newborns for identification, vaccination, bleeding and tail docking within the first month. Blood for DNA testing was collected into 15 mg ethylenediaminetetraacetic acid (EDTA) treated vacutainer tubes (Becton Dickinson, USA) via jugular venipuncture and samples were kept frozen until processed. Animals were weaned at 2 to 3 months of age and subsequently separated according to sex. At 6 to 8 months of age those lambs that were identified as ‘affected’ based on the direct DNA test were separated from the flock and housed in small groups in outdoor or indoor pens to best allow daily observation for behavioural changes and signs of disease progression.

Animals were euthanised at a range of ages (e.g. 3, 6 or 8 months), chosen to reflect the progress of the disease (Cook et al., 2002) using intravenous injection of an overdose of Lethabarb (Sodium penthobarbitone) (60 mg/kg). During post-mortem, weights of brain, liver and kidney were recorded and tissue samples from various organs were collected, preserved or frozen, and stored for future analysis.