Standard saline comprised (in mM): NaCl (145), KCl (5), CaCl₂ (2), MgSO₄ (1), D⁺ glucose (10) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10), pH 7.40 at 37˚C (290 ± 5 mOsmol∙kg⁻¹). To investigate Ca²⁺-free conditions, CaCl₂ was omitted and the Ca²⁺ chelator EGTA (1 mM) added; for Na⁺-free saline, N-methyl-D-glucamine (NMDG⁺) replaced Na⁺. Cells were always prepared in standard saline and only exposed to these Ca²⁺- or Na⁺-free solutions for a few minutes. Stock solutions of digitonin and Fura-2-AM were dissolved in DMSO. Fura-2 was obtained from Calbiochem (Merck, Darmstadt, Germany) and other chemicals such as Dulbecco’s modified Eagle’s medium (DMEM) from Sigma-Aldrich (Poole, UK). Ultrapure (Milli-Q, Merck Millipore, Mass, USA) water was used to dilute salines for a 50% HTS. Cell viability was not affected by any of these manoeuvres and remained at >95% (as assessed by Trypan blue which is excluded from viable cells).

Ethical approval for the research was obtained from the Department of Veterinary Medicine (University of Cambridge). Bovine feet (from animals aged 18 - 36 months) and ovine feet (<12 months) from humanely slaughtered animals were obtained from local, UK abattoirs. All tissue samples were refrigerated (5˚C) or frozen (−20˚C), and processed within 72 h of death.

The lower forelimb (below the carpus) of bovids, equids and ovids were skinned, cleaned and frozen (−20˚C, 48 h) before cutting through the MCP joint and cannon bone, longitudinally with a band saw.

After cleaning and thawing, a digital micrometer was used to measure the thickness of the cartilage overlying the load-bearing area. The cartilage was clearly visible and was measured from the articular surface to the delineation between cartilage and bone. Blinded measurements were taken from central positions at the lateral and medial trochlears, and at the upper most part of the condyles. The effects of freezing and thawing were not determined. Longitudinal slices through the frozen joint and cannon bone also allowed comparison of the internal architecture of the metacarpal bone of all species.

Immediately after collection, the MCP joints of bovids (n = 18) and ovids (n = 5) were skinned, cleaned and stored at 5˚C. The joints were opened aseptically in a flow hood within 72 hours of death. Full depth hyaline cartilage shavings from the MCP joint were taken at ambient O₂ tension and placed in DMEM containing penicillin (100 IU∙ml⁻¹), streptomycin (0.1 μg.ml⁻¹) and fungizone (2.5 μg∙ml⁻¹). They were incubated at 37˚C, 5% CO₂ for 16 - 18 h at 20% O₂ whilst matrix was digested with 0.1% (w/v) collagenase type I (16 h) in cattle and sheep. Isolated chondrocytes were re-suspended in DMEM lacking phenol red (which is known to affect pH measurements and subsequently Ca²⁺ levels) and a cell count performed using a haemocytometer. Cell viability was determined by the Trypan Blue exclusion test, at >95% before and after experimental regimes (within 3 h post isolation).

Intracellular Ca²⁺ levels ([Ca²⁺]_i) from multiple individuals (bovids n = 9 or n = 4, ovids n = 3 or n = 4) were measured using Fura-2 [17] [18] . Chondrocytes were incubated with 5 μM Fura-2-AM for 30 min at room temperature followed by 15 min at 37˚C. The chondrocyte suspension was then centrifuged and washed once, and then resuspended in the appropriate experimental saline at a final dilution of 10⁶ cells∙ml⁻¹. They were transferred to a cuvette in a thermostatically regulated fluorimeter (F-2000 Fluorescence Spectrophotometer, Hitachi) and experiments were completed within 3 h of isolation. Fura-2 was alternately excited at 340 nm and 380 nm, and emission intensity was measured at 510 nm for 300 s. The chondrocytes were subjected to a 50% HTS at 60 s. The 340:380 nm fluorescence ratio (R) was converted to Ca²⁺ concentration (nM) using the calibration equation [25] :

[ Ca 2 + ] i = K d × [ ( R − R min ) / ( R max − R ) ] × ( S f 2 / S b 2 )

The dissociation (K_d) of Fura-2 was taken as 224 mM although this may alter with different intracellular environments. R is the 340:380 nm fluorescence ratio of the indicator at an unknown [Ca²⁺]. R_min is the ratio in the absence of Ca²⁺ and R_max is the maximum fluorescence ratio at saturating [Ca²⁺]. The S_f2/S_b2 is the ratio of the long wavelength (380 nm) in the absence of Ca²⁺ (S_f2) and presence of Ca²⁺ (S_b2). The autofluorescence in the absence of dye at the 340 and 380 nm wavelength was subtracted in order to calculate [Ca²⁺]_i [26] .

Results are presented as the means ± standard error of the mean (SEM) for n individual animals unless otherwise stated, and statistical analysis performed by the GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, California, USA). Gross anatomy: Multiple animal joints were used for all measurements (bovids n = 6, ovids n = 5). Cartilage thickness: The average cartilage thickness overlying the medial and lateral trochlears and condyles from the same animals was calculated, and the standard deviation (SD) and SEM determined. An Independent t-test was employed to ascertain statistical significance (P < 0.05) between cartilage thickness at these points in different species. Ca²⁺_i levels: Each experiment was repeated in triplicate using tissue from individual animals (bovids n = 9, n = 4, n = 4 and ovids n = 3, n = 4 and n = 4 respectively for chondrocytes suspended in normocalcaemic, Ca²⁺-free or Na⁺-free salines). However, chondrocytes in suspension tend to descend to the bottom of eppenddorfs over a period of time, and despite attempts to resuspend them this can cause currents that result in a reduction in the number of chondrocytes placed in the cuvette and subsequently, a decrease in the fluorescence signal emitted. This has been reported previously in similar studies and resulted in the data being pooled for analysis. To calculate Ca²⁺_i levels, numerical analysis of ratiometric measurements and [Ca²⁺]_i was performed. The autofluorescence was measured and deducted from 340 and 380 nm wavelength recordings and controls were taken. The results were compared before and after HTS in each case for chondrocytes suspended in normocalcaemic saline (control group), or Ca²⁺-free and Na⁺-free salines (test groups). The mean, percentage increase, SD and SEM were determined. The Student’s paired t-test was utilised to determine statistical significance (P < 0.05) on changes of [Ca²⁺]_i following HTS within control and test groups of the same species. The Independent t-test was employed to ascertain statistical significance (P < 0.05) between resting Ca²⁺ levels and those following HTS for both control and test groups of the same species (dependant on saline) and different species. For example, the resting [Ca²⁺]_i of chondrocytes suspended in standard saline and the resting [Ca²⁺]_i of chondrocytes suspended in Ca²⁺-free saline of one species, or both. The mean ± SEM was expressed in all histograms and statistical significance denoted with an asterisk (*) or hash sign (#).

The distal metacarpal bones of cattle (18 - 36 months) and sheep were very similar in structure (Figure 1 and Figure 2). The growth plate was evident in the bones of individual bovids (n = 6) and ovids (n = 5) indicative of an immature animal. The thickness of cartilage, measured from the articular surface to the subchondral bone was greater in the load-bearing areas of the proximal MCP joint of cattle compared to sheep. The average cartilage thickness was much thinner covering the condyles of cattle (P < 0.001, Independent t-test) and sheep (P < 0.003, Independent t-test) compared to cartilage at the trochlears. Closer examination showed that the thickness of cartilage appeared to vary across the joint of all bovids and ovids in a similar manner (Figure 3). In cattle, for example, the cartilage of the right MCP joint was thicker (0.65 mm) at the abaxial lateral trochlear (outermost), decreased to a similar thickness at the axial lateral and the axial medial trochlears (~0.5 mm) and increased (0.6 mm) at the abaxial medial trochlears (innermost). Measurement of cartilage thickness at the medial (0.37 mm) and lateral (0.26 mm) condyles showed the opposite pattern.

Chondrocytes from cattle and sheep articular cartilage were isolated overnight by collagenase digestion (0.1% w/v) Following this procedure, the number of chondrocytes from 1 g of shavings obtained was similar in bovine (1 ± 0.2 × 10⁷, n = 18) and ovine (1.2 ± 0.3 × 10⁷, n = 5) tissue. Cell viability, ascertained by the Trypan Blue exclusion test was found to be >95% for both species. There were no noticeable differences in the staining or size of chondrocytes.

The intracellular Ca²⁺ levels of bovine and ovine chondrocytes were followed fluorimetrically before and after HTS over a 300 s time course. This is shown in representative traces in normocalcaemic (2 mM Ca²⁺) saline and also in the absence of Ca²⁺ and Na⁺ (Figure 4(a) and Figure 4(b)). Mean values are presented in normocalcaemic saline, Ca²⁺-free and Na⁺-free salines in Figures 5-7 below. Before HTS, the intracellular Ca²⁺ levels were constant. The mean steady state level of Ca²⁺ in bovine (60 ± 7 nM, n = 9) chondrocytes was lower than in ovine chondrocytes (Figure 5). The [Ca²⁺]_i of ovine chondrocytes was much higher (160 ± 27 nM, n = 3) (P < 0.05). HTS was given after 60 s and there was an immediate rise in Ca²⁺ levels in both species until plateau values were reached. The percentage rise in Ca²⁺ was lower in bovine chondrocytes (45 ± 7%) and ovine chondrocytes (80% ± 20%), with P < 0.001 and P < 0.002 (Student’s paired t-test) respectively, compared to resting steady state values. A similar percentage increase means more Ca²⁺ enters the cytoplasm and, or is released by intracellular stores when the resting intracellular Ca²⁺ level is higher. After this point, a plateau was reached and maintained as Ca²⁺ entry and efflux were balanced. Ca²⁺ levels then began to fall after ~200 s (shown in Figure 4(b) for ovine chondrocytes; but similarly for bovine―data not shown) as Ca²⁺ was removed from chondrocytes and steady state levels were restored. Thus, [Ca²⁺]_i in chondrocytes from both species behaved in a similar way to hypotonicity.

Ca²⁺ influx from the extracellular fluid was investigated in bovine (n = 4) and ovine (n = 4) chondrocytes by suspending them in Ca²⁺-free saline (Ca²⁺ replaced by 1 mM EGTA). The absence of Ca²⁺ caused an immediate reduction in the mean steady state Ca²⁺ levels. For bovine chondrocytes, a significant reduction of 50% was observed (P < 0.001, Independent t-test; Figure 6). In ovine

chondrocytes, a reduction of 30% was observed in response to suspension in Ca²⁺-free saline, although the change was not significant. Notwithstanding the absence of extracellular Ca²⁺, HTS evoked a rise in intracellular Ca²⁺ levels, by approximately60% in both species (P < 0.04, Student’s paired t-test; Figure 6). The absolute magnitudes of the Ca²⁺ responses elicited by HTS in chondrocytes from both species, however, were lower than those in controls in the presence of extracellular Ca²⁺. The percentage rise for bovine chondrocytes was slightly higher than that recorded in the presence of extracellular Ca²⁺, whilst the rise was reduced in ovine chondrocytes.

Experiments were performed to investigate the function and role of extracellular Na⁺ in maintaining [Ca²⁺]_i in bovine and ovine chondrocytes. In bovids and ovids, the suspension of chondrocytes in Na⁺-free saline (Na⁺ replaced by 145 mM NMDG⁺) caused a rise in the steady state resting Ca²⁺ levels in comparison to controls (Figure 7). Ca²⁺ levels in bovine chondrocytes (80 ± 12 nM, n = 4) were lower than in ovine chondrocytes. Ca²⁺ levels in ovine chondrocytes (190 ± 19 nM, n = 4) were much higher (P < 0.007, Independent t-test, compared to controls in standard saline). In response to HTS, Ca²⁺ levels increased to a greater extent than controls suspended in standard saline in the presence of extracellular Na⁺ in both species (all P < 0.03, Student’s paired t-test). Although the percentage rise in Ca²⁺ following HTS was higher in bovine (90% ± 24%) chondrocytes compared to ovine (70% ± 12%) chondrocytes, these differences were not statistically significant.