Summary The effect of copper supplementation of pasture fed mares and foals on the copper status of the foals, in terms of plasma, soft tissue and bone copper concentrations and caeruloplasmin activity, was investigated. Twenty‐one Thoroughbred foals from either control mares (n = 9), or copper‐supplemented mares (n = 12) were divided randomly into control (pasture only, n = 10) or supplemented (pasture and oral copper sulphate, n = 11) groups. The pasture diet was grazed by all animals, and contained 4.4–8.6 mg Cu/kg dry matter (DM). The copper supplement for the mares contained copper sulphate equivalent to 0.5 mg Cu/kg liveweight (LW)/day. This daily dose was converted to allow administration as a thrice weekly dose (i.e. multiplied by 7/3) which was given for 13–25 weeks prior to foaling. The supplemented foals, also dosed 3 times a week, received 0.2 mg Cu/kg LW/day at age 21 days, which was increased to 0.5 mg Cu/kg LW/day at 49 days and was continued at this level until euthanasia at 150 days. Foal plasma copper concentration and caeruloplasmin activity increased from birth to 21 days post partum and then plateaued at a concentration similar to the mare, but the rise in these indices was not affected by copper supplementation of the mare or foal. Copper supplementation of the foal increased foal liver copper concentration at 150 days (P<0.03). Copper intake of diets containing approximately 8–28 mg Cu/kg DM is well reflected by liver copper concentration, but is poorly reflected by bone, other soft tissue copper concentrations and circulating copper status indices.

The identification of cell proliferation sites in the wool follicle bulbs of the skin of New Zealand Romney sheep was investigated with two immunocytochemical techniques. These methods were based on the in vivo labelling of DNA synthesising follicle matrix cells with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and a surgical preparation of the skin on the lateral abdominal flank of the sheep. Using a monoclonal antibody to BrdU, an indirect immunoenzyme method and a biotin-streptavidin method were compared for specificity and sensitivity in detecting replicating bulb matrix cells which had incorporated infused BrdU during the S-phase of the cell cycle. The immunocytochemical results for both methods showed a black-brown staining reaction of cell nuclei entering mitosis. The biotin-streptavidin method proved to be more highly specific and sensitive than the immunoenzyme technique. The immunocytochemical detection of cell cycle S-phase is highly suited for studying cell proliferation sites and cytokinetics in wool follicle bulbs and in other mitotically active tissues. Immunocytochemical detection of mitotically active cells has the advantages of high specificity, cost efficiency and rapidity and may be an alternative to methods employing metaphase arresting agents like colchicine or autoradiography.

Effects of selection for high fleeceweight in the New Zealand Romney sheep were investigated in relation to the morphology of individual follicles and changes in the germinative cell population of the follicle bulb. Two-year-old Romney rams, 10 from each of two selection lines (Massey University fleeceweight-selected (FWT) and control (CLT) flock), were run together on pasture for a period from June to early December. At three times during this observation period (June, August and November) skin samples were taken from their midside flanks after local injection of bromodeoxyuridine (BrdU), to assess proliferation of bulb cells and several dimensional measurements of the follicle bulb and dermal papilla. FWT sheep had larger follicle dimensions than CLT sheep during winter and summer, with a greater number of proliferating bulb cells. Both flocks showed a seasonal change in follicle size, with a decline during winter, but the size of the dermal papilla was less affected than the germinative tissue area. Measurements of proliferation density (number of proliferating cells per area/volume of bulb tissue) suggest that changes in proliferation density do not contribute to flock differences in fleece production. However, during summer, FWT showed a 40% advantage over CLT sheep in hourly cell production based on data from three dimensional follicle bulb extrapolation. The different genotypes showed variations in width, as well as area of cortex and inner root sheath (IRS), measured across the top of the dermal papilla. The expression of these differences was further enhanced through seasonal influences, suggesting that there is an interaction between genetic/flock influences and seasonal influences on cell distribution to cortex and inner root sheath.

After establishing an immunocytochemical technique for the identification of BrdU labeled S-phase cells in the follicle bulb of wool fibres, the usefulness of a facilitated intracutaneous injection method for BrdU was investigated. This method enables easy and quick administration of the labelling agent and thus permits in vivo examination of cellular processes in the wool follicle bulb under field conditions. Because of very low dose rates, the local i.c. injection decreases the risk of toxic side effects of BrdU in the skin and more generally in the whole body, that accompany intravenous infusion. The accumulation of BrdU labelled cells over time in the follicle bulb was measured with immunocytochemical detection techniques. Intracutaneously injected BrdU was incorporated immediately into nuclear DNA of proliferating cells, and the labelling effect was confined to an area within 20 mm from the injection site. This allows sequential injections to be made on the same midside area of the sheep without interference of adjacent BrdU pools. Labelled S-phase nuclei accumulated in a linear fashion from 5 min to 24 h. Intradermally administered BrdU has a slow tissue clearance rate, with levels up to 10% of the initial peak still present after 24 h. The BrdU labelling effect showed detectable immunoreactivity at total injection loads as low as 5 8g. The free pool clearance rate in association with strong immunoreactivity suggests that a single intradermal injection can be regarded as comparable to a continuous labelling technique, if cell cycle parameters and growth fractions are to be determined. The method of administering BrdU intradermally opens a new perspective for cell population studies of wool follicles. It allows serial as well as concurrent estimates of cell activity to be made in the same animal, while eliminating difficulties and potentially toxic side effects associated with whole body infusion techniques.

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