Pelage measurement

2 2.3.1. Primary coat length measurement:

Primary (Summer and/or Winter) fibre length was measured every 2 weeks from a sample of at least 10 hairs in situ. A wooden rule was used to measure the hairs to an accuracy of ± 0.1cm. Measurements were taken adjacent to a shaved lOOcm^ lower- flank site positioned behind the front leg of each animal (Fig. 2.1). Evidence of any new coat growth between the bases of the established coat hairs was recorded and samples of fully grown winter and summer coat hairs were taken on 26 May and 18 September 1993.

2.2.3.2. Skin biopsying:

Skin biopsies were removed at weekly intervals from within the lOOcm^ lower-flank site (Fig. 2.1). Similar areas on both flanks were used so the sampled area was alternated with a contralateral site. This protocol, with a two-week interval between repeat sampling of the same site, allowed healing of the previous biopsy scar. Before sampling, the lOOcm^ area was shaved with electrical clippers and the skin sterilised using pevidine surgical scrub (BK Vetinary Products Ltd, Bury St. Edmunds, U.K.). 2ml of lignocaine hypochloride (Lignavet: C-Vet Ltd., Bury St. Edmunds, Suffolk, U.K.), local anaesthetic, was injected subcutaneously into the centre of the sterilised patch. This was left 5 min to take effect, before a biopsy was taken adjacent to the site of injection using a 6 mm biopsy punch (Stiefel laboratories (U.K.) Ltd., Woobum

Green, Buckinghamshire, U.K.). The biopsy was lifted clear of the skin using blunt forceps and scissors were used to trim off trailing strands of dermis. The biopsy was placed into a universal tube containing neutral buffered formalin (BDH Laboratory Supplies Ltd., Poole, Dorset, U.K.) and the biopsy wound was sprayed with aerosol antibiotic (Alamycin: Norbrook laboratories (G.B.) Ltd. London, U.K.). The animal received a final intramuscular dose of long acting antibiotic (Duphapen LA: Solvay Duphar Veterinary Ltd., Hedge End, Southampton, U.K., or Terramycin LA: Pfizer Ltd., Sandwich, Kent, U.K.). The dose administered was calculated from live weight

FIGURE 2.1: A mature red deer hind showing the position of the site used for coat measurement.

(Duphapen LA: lml/20kg : Terramycin LA: 1ml/10kg). The formalin-fixed biopsies were stored at room temperature before analysis.

2.2.3.3. Analysis of biopsies:

Skin biopsies were analysed under low power (10 X) magnification using a dissection microscope and described according to the following criteria:

i). Number of primary coat hairs per biopsy. ii). Type of hair (Summer or Winter coat).

iii). Phase of the hair growth cycle (Anagen or Telogen). iv). Presence or absence of underwool.

Primary coat densities, represented in this study as hairs/cm^, were calculated from the skin biopsies that were 6 nun in diameter and therefore contained an area of skin

0.28cm^. To validate this method as an accurate estimate of total coat density, a fixed area (4cm^) of summer coat was clipped from each animal on 28 April 1993. The density of coat (hairs/cm^) within this larger site was compared to the estimate of coat density made from 6 mm skin biopsies taken 6 April 1993 from sites next to the clipped

patches. The average density (± SEM) of summer coat hairs within the clipped patches was 33.9 ± 3.0 hairs/cm^ (n=8 ) and was not significantly different (P > 0.05) to the

estimate of coat density of 32.8 ± 2.7 hairs/cm^ (n=8) calculated from biopsies.

Therefore, the density of hair within the 0.28cm^ area of skin removed by biopsy was assumed to be representative of the larger area of coat. Photographs were taken of biopsies selected at intervals in the annual coat cycle.

2.2.3.4. Histology:

After examination, selected formalin-fixed biopsies were sent away to The Royal Free Hospital, Hampstead, London. The samples were processed to paraffin wax in a VIP tissue processor (Bayer Diagnostics, Tissue-Tek / VIP 2000). Due to the fibrous nature of red deer skin an extended 28h program was used for the processing procedure.

The tissues were dehydrated using an ascending series of alcohols, cleared in xylene and incorporated with paraffin wax.

Biopsies were removed from the processor and wax embedded and orientated for both transverse and longitudinal sectioning. Serial 7pm sections were cut on a rotary microtome and mounted on poly-L-lysine (Sigma Chemical Co. Ltd. Poole, Dorset, U.K.) coated slides. The sections were stained using either Weigert’s iron haematoxylin and eosin. Van Geison’s trichrome or the Sacpic method (Auber, 1952; Drury and Wallington, 1967; Nixon, 1993), to visualise aspects of hair follicle structure. The colour differences observed in a selection of tissues using the three different staining methods are shown in Table 2.1.

Table 2.1: A comparison of the three different stains used to visualise tissue components in the annual coat growth cycle in red deer. (After Auber, 1952; Drury and Wallington, 1967; Nixon, 1993).

Tissue Type

Method of staining Haematoxylin

and Eosin

Sacpic Van Geisen’s Trichrome

Club Hair - Yellow Yellow

Club Hair Border Pale Pink Orange Dark Yellow

Collagen Pale Pink Blue Deep Red

Inner Root Sheath Purple Red Purple

Keratin Orange-Red Yellow Yellow

Nucleus Blue-Black Blue-Black Brown-Black

Outer Root Sheath Purple Pale Green Purple

Smooth Muscle Deep Pink Green Yellow

Primary hair follicles from summer and winter coats were described according to phase of the hair cycle. A nine point scale constructed by Nixon et a l (1993) based on the description and nomenclature of Chase et a l (1951) and Chase (1965) was used for this purpose.

2.2.4. Endocrine Techniques:

2.2.4.1. Blood sampling:

Blood samples were collected by jugular venepuncture using the method of restraint described previously (see Section 2.2.2). The samples were collected at weekly intervals between lOOOh and 1200h using a 19 gauge needle with a 10ml syringe, and immediately heparinised in screw topped 1 0ml tubes containing lithium heparin coated

plastic beads (LH/10ml-15IU Heparin: Sarstedt, Numbrecht, Germany). Blood was centrifuged at 2,500rpm for 5 min and the plasma separated and stored at -20°C until assayed.

2.2.4.2. Prolactin iodination:

Ovine PRL (oPRL: NIADDK oPRL-I-2, NIDDK, HHPP, University of Maryland School of Medicine, Rockville, MD, USA) was iodinated by the Chloramine-T method (Greenwood et a l, 1963). This method was developed to allow rapid preparation of labelled peptide hormones of high specific activity (200-300pCi/pg).

Before the start of the main procedure lOOpg of oPRL was weighed out and diluted in 500pl of 0.1 M sodium bicarbonate (pH 9). 25pl aliquots of the solution (containing 5pg oPRL) were removed to vials, snap frozen and stored at -40°C.

A vial containing a 25|nl aliquot of oPRL solution was thawed and 0.25mCi (Amersham International pic.. Little Chalfont, Buckinghamshire, U.K.), was added. Immediately, 4mg of Chloramine-T (Sigma Chemical Co. Ltd., Poole, Dorset, U.K.) was dissolved in 10ml of protein-free assay buffer (0.05M sodium dihydrogen orthophosphate pH 7.4 containing 0.15M sodium chloride). The solution was then diluted 1:10 in 0.5M phosphate buffer (0.5M sodium dihydrogen orthophosphate pH 7.2 containing 0.15M sodium chloride) before 20pl was removed to the reaction vial. The vial was vortex mixed and left for 5 min to react. The reaction was stopped by adding 200pl of a 3% solution of bovine serum albumin (BSA: fraction V powder, 96-99% albumin. Sigma Chemical Co. Ltd., Poole, U.K.) in 0.5M phosphate buffer. The contents of the vial were removed to a Sephadex G disposable column (PD-10:

Pharmacia Ltd., Uppsala, Sweden), and a further 10ml of 3% BSA/0.5M phosphate buffer was added to the column in 1ml aliquots. Each aliquot or fraction was collected into a separate polystyrene LP4 tube (Luckham Ltd., Burgess Hill, Sussex, U.K.), positioned beneath the column. The radioactivity of lOpl samples of each fraction was recorded over 2 min using a Cobra Auto-Gamma counter (Model# 5005: Packard Ltd., Canberra, Australia). Fractions 3 and/or 4 contained the oPRL-I^^^ complex and were retained and stored at 4°C.

2.2.4.3. Prolactin radioimmunoassay:

Prolactin concentrations in cervine plasma samples were measured by a double antibody radioimmunoassay. This involved a competitive interaction between the hormone in a plasma sample and a known amount of radioactively (F^^) labelled hormone, for specific antibody binding sites. Since this is a competitive reaction, the number of counts of labelled hormone bound to the antibody is inversely proportional to the concentration of unlabelled hormone in the plasma sample. The actual concentrations are interpolated from these counts and a standard curve is produced by assaying samples containing known concentrations of unlabelled hormone. The assay used in this work was a modification of an assay developed to measure PRL in ovine plasma and the original validation and protocol is described in McNeilly and Andrews (1974).

(a) Materials and reagents:

Unless otherwise stated, all reagents were obtained from B.D.H. Chemicals Ltd., Poole, Dorset, U.K.

(!) Assay buffer (1% BSA/PBS).

The buffer used for all dilutions in the assay, was a 1% solution of BSA in 0.05M sodium dihydrogen orthophosphate (pH 7.4) containing 0.15M sodium chloride and 0.1% sodium azide (used as a preservative). The buffer was made up in deionised-distilled water and stored at 4°C.

(îi) Prolactin standard.

Ovine PRL (NIH-oPRL-S13) was initially dissolved in NIH buffer (0.03M sodium bicarbonate, 0.15M sodium chloride, 1% BSA. pH 10.8), diluted to a concentration of 200pg/l and stored in 150|il aliquots at -20°C. Aliquots were removed and serially diluted in assay buffer to give 10 dilutions from 200|ig/l to 0.39pg/l.

(iii) Quality control plasma samples.

Pooled red deer plasma with low (c. 0.9pg/l), medium (c. 6 pg/l) and high (c. 40pg/l)

levels of PRL were stored in 250|il aliquots at -20°C until required.

(iv) Plasma samples.

These were stored at -20°C until assayed.

(v) First antibody.

Rabbit anti-ovine PRL (R51) (McN 2532 - bleed no.51: M.R.C. Unit of reproductive biology, Edinburgh, U.K.) was stored at 1:100 dilution in lOOpl aliquots at -20°C. The aliquots were thawed and diluted in assay buffer to provide a working concentration of

1:1 00 , 0 0 0 in the assay.

The dilution of antibody used in the assay was determined by measuring the maximum binding ((Bq- NSB)/T) over a range of antiserum dilutions (Table 2.2).

Table 2.2: Summary of a maximum binding study to determine the optimum dilution of first antibody (R51) in the PRL assay.

First antibody dilution % (B,-NSB)/T 1:25,000 52.9% 1:50,000 46.6% 1:100,000 34.9% 1:200,000 22.9% 1:400,000 14.0% 1:800,000 8.1%

dilution of antiserum together with a set a total counts (T). Highest binding was observed at 1:25,000 dilution (52.9%), although the dilution chosen for use in the assay was 1:100,000. This dilution gave sufficiently high binding of 34.9%, while remaining economical with antiserum stocks.

(vi) Tracer.

f^^-oPRL was diluted with assay buffer to give a solution of 10,000 to 15,000 counts per min (cpm) per lOOpl. Stock aliquots of tracer were stored at 4°C.

(vii) Second antibody.

Separation of bound and free tracer was achieved using a donkey anti-rabbit serum bound to iron oxide particles (SAPIJ). The solid phase antibody particles bound to the first antibody/tracer complex and settled to the bottom of the assay tube under centrifugation of 2,400rpm at 4°C for 5 min. The antibody was diluted 1:40 in WHO buffer (0.08M di-sodium hydrogen phosphate, 0.02M sodium dihydrogen orthophosphate, 0.15M sodium chloride pH 7.2, with 0.1% gelatin and 0.01% sodium azide), supplemented with 0.3% (w/v) hydroxypropylmethyl cellulose (HPMC: Sigma Chemical Co., Poole, Dorset, U.K.). The HPMC increased viscosity in the buffer, maintaining the heavy solid phase antibody particles in suspension to maximise binding.

0 .5 ml of antibody was added to each tube (except total counts) and incubated at room

temperature for 45 min.

Initial assay trials using first antibody R51, with second antibody (SAPU), were run over 2 and 3 days. Binding was good when run as a two-day assay, but better displacement of standard and greater assay sensitivity was achieved by running the assay over three days (Fig. 2.2).

(b) Assay protocol:

Each assay consisted of up to 114 plasma samples, with 3 Qc’s and one standard curve (a maximum of 288 tubes).

On the first day, 50pl of sample, Qc, standard or buffer (maximum binding tubes only) was pipetted into polystyrene LP4 tubes and 200pl of first antibody (R51) was

FIGURE 2.2: Specimen standard curves for the prolactin assay using first antibody R51 and second antibody SAPU in a two-day assay (O) and three-day assay (□). Concentration of standard is expressed on a logarithmic scale and plotted against %

binding (B/B^ x 100). % B / B r OO w 100-1 80- 60- 40 - 2 0- 0 - J 0.1 1 10 100 1000 Prolactin (ng/tube)

added. NSB tubes received 250|j1 buffer only. All tubes were then incubated overnight

at 4°C. After 24 h, lOOpl of tracer was added to all tubes before incubation continued at 4°C. After a further 24 h, 500pl of second antibody (SAPU) was added to all tubes (except total counts), and incubated at room temperature for 45 min. 500|il of saline was then added before centrifugation at 2400rpm for 5 min. The supernatants in each tube were aspirated off, leaving a pellet containing the bound fraction. The tubes were then counted for 2 min using a Cobra Auto-Gamma Counter (Model# 5005: Packard Ltd., Canberra, Australia).

Results are expressed as pg/1 PRL in the blood plasma.

(c) Assay range, sensitivity and precision:

The mean (± SEM) estimated concentration of standard at 20% binding was 43.53 (± 2.09) |ig/l; at 50% was 4.06 (± 0.22) pg/l; and at 80% was 1.00 (± 0.05) pg/l (n=7).

Assay sensitivity was defined as 2 standard deviations from the mean of the maximum binding tubes (B J. Mean sensitivity was 95.9% (% B /B J, equivalent to 0.27 (± 0.04) pg/1 PRL in the plasma (n=7). All samples with a percentage binding greater than this, were recorded as the mean limit of sensitivity. Any sample with fewer counts than the highest PRL standard (200pg/l) was double diluted in assay buffer and reassayed.

Inter-assay precision, expressed as the coefficient of variation for repeated determinations of the quality controls was 19.61% at 1.21pg/l (LQc), 15.88% at 2.71pg/l (MQc), and 18.36% at 39.12pg/l (HQc), (n=7). Intra-assay precision, expressed as the coefficient of variation for repeated determinations of PRL in plasma samples within an assay, was 12.55% at l.Olpg/1 (LQc), 8.64% at 2.62pg/l (MQc), and 13.04% at 40.42pg/l (HQc), (n=15).

The mean (± SEM) for non specific binding (NSB/T), was 3.70 (± 0.11)%, (n=7). The mean (± SEM) for maximum binding tubes ((B^ - NSB)/T) was 26.40 (± 0.55)%, (n=7).

2.3. STATISTICAL ANALYSIS:

Student’s t-test was used to compare the mean diameter and depth of primary and secondary follicles within the skin in the anagen and telogen phases of the hair cycle. The diameters of dermal papillae of primary summer and winter coat follicles in anagen and telogen were also compared.

The position of the peak and nadir of the annual cycle in plasma PRL concentration was recorded for each animal as days relative to 1 January. The mean values were then converted to the date with SEM expressed in days. Mean PRL concentrations at the peak and nadir of the cycle were also recorded.

The onset of the spring rise in PRL was defined as the first of 4 consecutive plasma samples with concentrations greater than twice the mean level for each hind between 6

January and 25 February 1993 (inclusive). Linear correlations between the spring rise in PRL and the reactivation of the summer coat follicles within the skin were calculated using Cricket Graph Program (Cricket Software Incorporated, Philadelphia, PA, USA).

2.4. RESULTS: