Uptake of glucose and cholesterol by the ovary of sheep and cattle and the influence of arterial LH concentrations

Animal Reproduction Science 64 (2000) 199–209

Uptake of glucose and cholesterol by the ovary of sheep and cattle and the influence of arterial LH concentrations A.R. Rabiee a,∗ , I.J. Lean b a

Department of Veterinary Science, University of Melbourne, Werribee, Vic. 3030, Australia b Bovine Research Australasia, PO Box 660, Camden, NSW 2570, Australia

Received 4 April 2000; received in revised form 5 September 2000; accepted 5 September 2000

Abstract Time series analysis methods were used to evaluate the relationships between the uptake of glucose and cholesterol, and arterial luteinizing hormone (LH) concentrations. Classical arterio-venous difference methods were applied to study ovarian uptake of metabolites. Arterial and venous samples (n = 20) were obtained from six cows and nine sheep every 10 min. There were highly significant positive cross-correlations of 0.5 for cattle and 0.8 for sheep between the uptake of glucose and cholesterol at lag 0. All individual cross-correlations were significant for sheep. Uptake of these metabolites was not significantly associated with arterial LH concentrations in the cows. This study suggests that glucose may promote cholesterol uptake into the ovarian cells or vice versa. This study is the first to identify such a relationship. If these findings are repeated, the possibility exists that control of the oestrous cycle and fertility may be achieved by seeking a common regulator of uptake of these metabolites or by uncoupling the association between glucose and cholesterol. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cholesterol; Glucose; Cattle ovary

1. Introduction Improved knowledge of the metabolic demands of the ovary could lead to the development of new methods of controlling reproduction in animals. There are only a limited number of studies describing ovarian energy metabolism, and these studies were primarily conducted in vitro. The sources for energy used by the ovary are the subject of conflicting reports. It is ∗ Corresponding author. Tel.: +61-397420484; fax: +61-397420400. E-mail address: [email protected] (A.R. Rabiee).

0378-4320/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 0 ) 0 0 2 0 8 - 6

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known that blood cholesterol concentrations and steroid hormones synthesis are positively related to energy intake and health of animals (Velhankar, 1973), and lower cholesterol and glucose concentrations after calving have been associated with an increased number of days from calving to conception (Kappel et al., 1984). The demands of reproductive tissue for energy and cholesterol, and the metabolism of these in reproductive tissues are important aspects of metabolism to understand. It has been suggested that lipids are the main energy source used by the ovary (Flint and Denton, 1970), but more recent (Chase et al., 1992) and some older studies (Gafvels et al., 1987) suggest that glucose is a major energy source for the ovary. It was demonstrated that glucose uptake by the corpus luteum (CL) plays a role in its energy metabolism in vitro in rats (Gafvels et al., 1987). Studies (Chase et al., 1992) of glucose metabolism in the bovine CL in vitro also indicated that glucose was a major energy source used by the ovary, and that physiological state, stage of the oestrous cycle and time after calving significantly alters in vitro rates of uptake and metabolism of glucose by luteal tissue. The presence of glucose has been found to markedly stimulate the incorporation of carbon from acetate into sterol and steroid in ovarian tissue in vitro in the rat (Flint and Denton, 1969). In vitro studies also show a positive relationship between ATP and pregnenolone synthesis in ovarian mitochondria (Uzgiris et al., 1970; Robinson and Stevenson, 1971). We reported (Rabiee et al., 1997) that glucose was the primary source of energy and that free fatty acids were not a significant source of energy for the bovine ovary in situ. There are few reports on the effects of gonadotrophins on aspects of the cellular metabolism other than those involved directly in the steroidogenesis. Armstrong and Greep (1962) reported that administration of luteinizing hormone (LH) increased uptake of glucose and production of lactic acid by the luteinized rat ovaries in vitro. Investigations regarding the site in the metabolic pathway stimulated by LH, and the effect of LH on incorporation of cholesterol into steroids have often yielded varied or conflicting results. LH may influence progesterone synthesis through a pathway that does not involve cholesterol as an intermediate or precursor (Savard et al., 1965). The influence of LH on cholesterol uptake and de novo synthesis of cholesterol in luteal tissue remains controversial. Time series analysis methods allow an evaluation of relationships between the uptake of metabolites by the ovary and the role of LH in influencing the uptake of metabolites. The objective of this study was to examine relationships between the uptake of glucose and cholesterol and arterial concentrations of LH both in sheep and cattle in vivo.

2. Materials and methods Classical arterio-venous difference methods (Niswender et al., 1975; Wise et al., 1982) with modifications were applied to study ovarian uptake of metabolites in ewes and cows (Rabiee et al., 1997a,b). The methods described by Wise et al. (1982) were used to measure ovarian uptake of metabolites in cows (Rabiee et al., 1997b). Absolute values for metabolites and LH concentrations in cows were previously reported (Rabiee et al., 1997b). The experimental methods used in the cows follows.

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2.1. Experimental animals Nine ewes were used in this study. Blood samples in ewes were taken when two ewes were in proestrus, two ewes were in oestrus, four ewes were in dioestrus and one ewe had no obvious luteal or follicular structure in either ovary. The method of vessel cannulation and the significance of the uptake of metabolites have been previously reported in ewes (Rabiee et al. 1997a,b). Six Holstein-Friesian cows, 5–7-year-old, from a group of normally cycling cattle were used in this experiment. All of the cattle were lactating. Live weights were in the range of 450–620 kg. 2.2. Adaptation to stanchion, diet and preoperative care During the 2 weeks before housing, cows were accustomed to halters and handling. After this initial period, the animals were held in stanchions that allowed free movement up and down, but not backward or forward. Cows were milked twice daily at approximately 07.30 h and 16.30 h in their stalls. During this period, cows were monitored for successful adaptation to the environment and food by observing feed intakes and milk production. The cows were fed to appetite three times a day at regular intervals. The ration contained lucerne hay (43%), barley (17%), whole cottonseed (10%), cottonseed meal (10%), biscuit (18%) and meat meal (2%). The ration was formulated to provide: 16% crude protein, 11.2 MJ of metabolic energy/kg (ME) and 17% crude fiber (CF). All cows were fed this diet from 10 days prior to surgery. Before surgery, oestrus was synchronized in cows by insertion of a controlled internal drug release (CIDR) (EAZI-breed CIDR B; Riverina Artificial Breeders, Pty. Ltd. Sydney, NSW, Australia) for 10 days. The cows were injected with Prostianol (2 ml of Prosolvin; Intervet, Boxmeer, Holland) on the day CIDR devices were removed. Surgery on all cows was conducted during the luteal phase (7–8 days after oestrus). 2.3. Surgical procedures One day before surgery, a polyvinyl catheter (3.0 mm o.d., 2.0 mm i.d.; Dural Plastics and Engineering, Sydney, NSW, Australia) was inserted into the jugular vein of each cow under local anaesthesia. Water and feed were restricted for 6 h before surgery. The ovaries were palpated per rectum for determination of the side of the CL, and cows were sedated with 15 mg of 2% xylazine (Rompun; Bayer Australia Limited, Sydney, NSW, Australia). Anaesthesia was induced with guaifenesin (Giafen® ; Parnell Laboratories, Sydney, NSW, Australia) and thiopentone sodium (Pentothal® ; Boehringer-Ingelheim, Sydney, NSW, Australia). Surgical anaesthesia was maintained by a closed system inhalation procedure using 2–4% Halothane (Baxter Health Care, Old Toongabbie, NSW, Australia) and oxygen (4–5 l/min). 2.3.1. Facial artery cannulation The facial artery was utilized for arterial blood sampling due to its superficial location and ease of catheterization. The methods described by Wise et al. (1982) were used to catheterize the facial artery.

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2.3.2. Cannulation of the ovarian vein and placement of blood flow probe The flank area was prepared surgically, and the reproductive tract was exposed via a vertical flank incision just anterior to the paralumbar fossa. To facilitate exteriorization of the reproductive tract, 10 ml of Clenbuterol (Planipart; Boehringer-Ingelheim) was injected intravenously. The tract was exteriorized, and the ovarian branch of utero-ovarian vein was identified. A branch of the ovarian vein near the hilus was dissected from the adventitia, and a small incision was made, allowing a polyvinyl catheter (1.0 mm i.d., 1.5 mm o.d.) to be introduced 4–5 cm into the vein. The cannula was sutured in situ with fine silk. The cannula was exteriorized through the original flank incision and placed in a pouch which was sutured to the side of the animal (Rabiee et al., 1997b). Catheters were flushed daily with sterile heparinized saline (500 IU/ml). Operations were conducted in the luteal phase, and luteal ovaries were cannulated. Blood samples were taken during the luteal phase, and if an ovulatory follicle was observed by ultrasound examination in the cannulated ovary, blood samples were also taken during follicular phase. To measure the ovarian blood flow (OBF), a 2 cm section of the artery just above the uterine anastomosis was dissected free of the peritoneum, and a 4R transit-time (TT) ultrasonic blood flow probe (Transonic System Inc., Ithaca, NY) was placed around the ovarian artery. Antibiotics were administered (Roscocycline-10; A/S Rosco-Denmark, Denmark) at 24 h intervals for 3 days following surgery. 2.4. Blood flow measurements The OBF was measured using an ultrasonic TT flowmeter (Transonic Inc.). A 4R-series probe was used for measurement of blood flow. Data were obtained from the blood flow meter continuously, and were averaged over 5 m intervals by a data logger (Tain Electronics; Melbourne, Vic., Australia). After 2–3 days of surgery, blood samples were taken from the cows in which OBF was monitored continuously. Blood samples were taken during the luteal phase for all cows, and during the follicular phase for two cows that had regressed CL and ovulatory follicles in the cannulated ovaries. Arterial and venous blood samples (n = 20) were taken from each cow every 10 min. Arterio-venous (A −V) differences in concentrations of metabolites and OBF were used to estimate the uptake of cholesterol and glucose. Ovarian uptake of metabolites per minute was calculated using the following formula: Ovarian uptake per minute = (A − V difference) × OBF 2.5. Analyses of blood samples 2.5.1. Metabolites and arterial LH All blood samples were collected in plastic tubes containing heparin, and immediately centrifuged at 1000 × g for 20 min. The plasma samples were stored at −20◦ C. Samples were analyzed for cholesterol, glucose and arterial LH. Plasma glucose was determined with an autoanalyzer using Boehringer Mannheim Gmbh reagent (Boehringer Mannheim Gmbh, Sydney, NSW, Australia) according to the method of Schmidt (1961). Cholesterol was measured according to the procedure described by Allain

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et al. (1974) with an autoanalyzer (Cobas Mira, Roch Diagnostic Systems, Switzerland) using the Roch reagent (Roch Diagnostic systems, NJ, USA). The uptake of cholesterol and glucose in sheep and cows have been reported (Rabiee et al., 1997a,b).The radio immunoassay for LH used in these studies was a double antibody assay based on the method of Scaramuzzi et al. (1970). The antiserum to LH was provided by Prof. D.R. Lindsay and Prof. G.B. Martin, Department of Animal Science and Production, University of Western Australia. The antiserum (UWA 3B) was raised in rabbits, and the cross-reactivity of this antiserum with NIH bLH B5 was 100%. The minimum binding of tracer in the absence of LH was 35%. Nonspecific binding was consistently below 5% of added tracer. Inter-assay and intra-assay coefficients of variation were 11.6 and 6.3%, respectively. 2.6. Statistical analysis Time series methods of analysis (Chatfield, 1989; Shumway, 1988) were used to analyze the relationship between the uptake of glucose and cholesterol by the ovary and arterial LH. These statistical procedures were developed by Prof. Shumway, Dr. Pope and Dr. Weber (personal communication) in association with the authors. The mean, linear or quadratic trends of mean values for metabolites were removed to produce series for each cow that were approximately stationary using the time series analysis procedure of STATGRAPHICS (1989). Cross-covariances of detrended data for each cow were calculated using BMDP (1992), and cross-correlations of each variable at different lags were calculated using cross-covariance and variance at lag 0 series, with the following formula: R12 (m) Cross-correlation = √ R11 (0) × R22 (0) where R12 is the cross-covariance at lag m, and R11 and R22 the variances at lag 0 of the two series. The cross-covariances were calculated from the following function R12 (m, m + k) = Cov(R1m , R2m+k ) where R12 (m, m + k) is the cross-covariance at time (m) and time (m + k) for series R1 and R2 , respectively, for each cow R1m is the residual for series R1 at time (m) after detrending to produce an approximately stationary series, and R2m+k the residual for series R2 at time (m + k) after detrending to produce an approximately stationary series. The cross-correlation function is used to standardize the cross-covariance coefficients, which depend on the units in which the two series are measured (Chatfield, 1989). Each lag represents a difference of 10 min. Mean cross-correlations presented in Tables 1–5 and Figs. 1–2 represent the mean cross-correlations from the six cows and nine ewes for the two variables at each of the lags examined. 3. Results The time series cross-correlation between each two variables are presented in a tabular form to indicate the strength of association between the uptake of metabolites by the ovary and arterial LH concentrations. Cross-correlations between metabolites were also presented graphically to display cross-correlations for metabolites at different lags.

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Table 1 Cross-correlations between the uptake of glucose and cholesterol by the ovary in the luteal phase for six cows Cow

Lag −4

C1 C2 C3 C4 C5 C6 Mean ∗

−3

−2

−1

0

1

2

3

4

0.92∗∗∗

0.02 0.16 −0.06 0.23 0.22 −0.10

−0.15 −0.0004 −0.03 0.02 0.04 −0.06

0.09 0.07 0.21 −0.10 0.02 0.10

0.029 0.19 0.18 −0.13 0.06 0.017

−0.16 0.12 −0.06 −0.14 −0.06 −0.03

−0.01 −0.08 −0.15 0.06 −0.03 −0.2

−0.21 0.05 0.30 0.25 0.21 −0.001

0.24 0.27 0.73∗∗ 0.44∗ 0.37

−0.05 0.06 −0.03 0.11 −0.05 −0.08

0.06

−0.06

−0.07

0.10

0.50∗∗∗

−0.006

P < 0.05;

∗∗

P < 0.01;

∗∗∗

0.08

−0.03

0.07

P < 0.001.

Table 2 Cross-correlations between the uptake of glucose and cholesterol by the ovary in the follicular phase for two cows Cow

Lag −4

−3

−2

−1

0

1

2

3

4

C1 C2

−0.11 −0.18

−0.14 0.19

0.12 0.04

0.08 −0.10

0.81∗∗∗ 0.94∗∗∗

−0.31 −0.10

−0.23 −0.006

−0.19 0.14

−0.02 −0.21

Mean

−0.14

0.03

0.08

−0.01

0.87∗∗∗

−0.20

−0.12

−0.03

−0.11

∗∗∗

P < 0.001.

Table 3 Cross-correlations between the uptake of glucose and cholesterol uptakes by the ovary at different stages of the estrus cycle for nine ewesa Sheep

Lag −4

S1 S2 S3 S4 S5 S6 S7 S8 S9 Mean a

−3

−2

−0.0008 −0.04 0.0 −0.07 −0.12 0.07 0.02 −0.11 0.03

0.11 −0.07 −0.04 0.18 −0.07 −0.07 −0.06 −0.16 −0.02

0.009 0.40 0.02 0.01 −0.10 −0.21 0.28 0.21 −0.09

−0.03

−0.02

0.06

−1

0

0.11 −0.15 −0.34 0.10 0.06 −0.23 −0.60∗∗ −0.28 0.26

0.60∗∗

0.80∗∗∗ 0.95∗∗∗ 0.52∗ 0.96∗∗∗ 0.81∗∗∗ 0.96∗∗∗ 0.95∗∗∗ 0.70∗∗

−0.03 −0.24 −0.16 0.15 −0.12 −0.46∗ −0.47∗ 0.29

−0.12

0.81∗∗∗

−0.07

1 0.45∗

2

3

4

0.26 0.16 −0.05 −0.07 0.05 −0.21 0.17 0.17 −0.07

−0.002 0.03 −0.01 0.17 −0.04 −0.007 −0.20 −0.17 0.11

0.09 −0.01 −0.004 −0.15 −0.08 0.10 0.02 −0.08 0.10

0.05

0.0001

−0.002

S1 and S2 were in proestrus, S3 and S4 in oestrus, S5–S8 in diestrus, and S9 had no luteal or follicular structure. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

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Table 4 Cross-correlations between transovarian uptake of glucose and arterial LH in the luteal phase for six cows Cow

Lag −4

C1 C2 C3 C4 C5 C6 Mean ∗

−3

−2

−1

0.13 0.05 −0.04 0.10 −0.04 −0.02

0.03 0.02 −0.09 0.05 −0.004 −0.09

−0.28 −0.19 −0.09 0.06 −0.23 0.18

−0.20 −0.20 0.17 0.20 0.14 0.37

0.03

−0.008

−0.09

0.08

0 0.46∗ −0.39 0.26 0.22 0.50∗ −0.23 0.14

1

2

3

4

0.10 0.13 −0.13 0.01 0.03 0.21

−0.09 0.007 0.09 −0.15 0.21 0.21

−0.08 −0.25 0.26 0.15 0.10 0.16

0.004 0.14 −0.17 −0.18 0.04 0.12

0.06

−0.007

0.06

0.04

P < 0.05.

Table 5 Cross-correlations between transovarian uptake of cholesterol and arterial LH in the luteal phase for six cows Cow

C1 C2 C3 C4 C5 C6 Mean ∗∗

Lag −4

−3

−2

−1

0

1

2

3

4

0.09 0.001 0.02 0.0 0.02 −0.11

−0.009 0.05 −0.19 0.22 0.03 −0.17

−0.20 −0.21 0.15 0.21 −0.29 −0.09

0.02 −0.02 0.19 0.36 −0.10 0.02

0.38 −0.33 0.02 −0.12 0.58∗∗ 0.04

0.007 −0.21 −0.07 −0.17 0.29 0.03

−0.04 −0.14 0.12 0.09 0.10 0.14

−0.09 −0.07 0.0 −0.34 0.03 0.11

−0.05 −0.04 0.02 0.10 −0.01 0.16

−0.01

−0.07

0.08

0.09

−0.02

0.04

−0.06

0.03

0.005 P < 0.01.

Fig. 1. Mean cross-correlation between the uptake of glucose and cholesterol in the luteal phase for six cows.

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Fig. 2. Mean cross-correlation between the uptake of glucose and cholesterol at different stages of the estrus cycle for all ewes.

Correlations between the uptake of metabolites were examined for individual cows and sheep. Cross-correlations between glucose and cholesterol uptakes by the ovary and arterial LH in individual ewes and cows at different stages of the oestrous cycle are given in Tables 1–3. Mean cross-correlations (MCC) between cholesterol and glucose uptakes by the ovary were significant at lag 0 in cattle in the luteal and follicular phases (MCC = 0.5 and 0.87, P < 0.01 and 0.001, respectively; Fig. 1), and in sheep (MCC = 0.81, P < 0.001; Fig. 2). Although, three cows did not have significant cross-correlations between the uptake glucose and cholesterol, these correlations were consistently significant for all sheep. There was some evidence of negative feedback in sheep, as significant negative cross-correlations between glucose and cholesterol at lags −1 and +1 were observed. Table 4 shows cross-correlations between glucose and arterial LH for all cows. There was no significant difference between mean cross-correlations for glucose uptake and arterial LH at lag 0 (MCC = 0.14, P = 0.13), but cross-correlations were significant for two cows at lag 0. Cross-correlations between cholesterol uptake and arterial LH are presented in Table 5. Mean cross-correlations between cholesterol uptake and arterial LH were not significant (MCC = 0.096, P > 0.2), but were significant in one cow only at lag 0. There was no similar data available for the sheep to assess these relationships.

4. Discussion Time series analytical methods are useful for examining relationships among variables when repeated measurements have been made on the same individuals. Time series crosscorrelation analysis methods allowed the examination of relationships between ovarian uptake of glucose and cholesterol and arterial LH regardless of the level of the uptake of metabolites. Absolute values for uptake of metabolites, effect of the stage of oestrus cycle on ovarian uptake of metabolites and significance of values for individual cows were

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reported earlier (Rabiee et al., 1997a,b). In brief, we found that there was significant uptake of glucose and significant output of lactate by the ovary. There was no significant uptake of free fatty acids. In the current study, we report correlations between the uptake of glucose and cholesterol during the sampling period. Significant cross-correlations between glucose and cholesterol in cattle and sheep at lag 0 show that the uptake of cholesterol and glucose are strongly linked (Figs. 1 and 2). There was remarkable consistency in the results in all sheep, and good consistency in the results from cattle. Other in vitro studies suggest that glucose uptake may be a necessary condition for cholesterol uptake by the ovary. Glucose uptake by the rat CL plays an important role in ovarian energy metabolism (Gafvels et al., 1987). Studies of glucose metabolism in the bovine CL indicated that the uptake and rate of metabolism of glucose were higher in tissue obtained from growing CLs (Chase et al., 1992). This suggests that the requirements for steroid hormone precursors depend on the simultaneous availability of glucose. Glucose inclusion also stimulated, markedly, the incorporation of carbon from acetate into sterol and steroids in the rat ovarian tissue in vitro (Flint and Denton, 1969). It could be postulated that the simultaneous uptake of cholesterol and glucose might have resulted from a gonadotrophic hormonal action. There are conflicting in vitro data evaluating this proposition. A stimulatory effect of LH treatment on cholesterol uptake was observed only in the presence of glucose (Flint and Denton, 1960), but ATP directly stimulated side-chain cleavage activity in the mitochondria from superovulated rat ovary. The effect of ATP is apparently similar to the stimulatory effect of gonadotrophin in mitochondrial cholesterol utilization, although the enhancement of the rate of steroidogenesis by ATP and gonadotrophin was not additive (Stevenson et al., 1985). It appears that ATP promotes both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for cholesterol utilization from endogenous substrate (Stevenson et al., 1985). The lack, however, of significant correlations between arterial LH and the uptake of cholesterol or glucose by the ovary for the pooled cow data does not support this postulated mode of action. In vitro addition of LH does not augment the transformation of labeled cholesterol to progesterone to the same extent as NADP and glucose-6-P (Savard et al., 1965). Both NADP+ and glucose-6-P increase the transformation of labeled cholesterol to progesterone by 3–30-fold. Similarly, addition of gonadotrophins to incubating human and calf ovarian slices produced no stimulation of glucose oxidation (Field et al., 1960). We found no significant relationship between arterial concentrations of LH and the uptake of cholesterol and glucose. This supports our previous finding (Rabiee et al., 1997a,b) that exogenous GnRH had no significant effect on the uptake of glucose and cholesterol by the ovary. Further, the addition of LH to incubated bovine ovarian tissue caused a highly significant increase in the synthesis of progesterone without affecting the rate of glucose uptake (Armstrong and Greep, 1962). Other investigators (Field et al., 1960) similarly found that addition of gonadotrophins in vitro to incubating human and calf ovarian slices produced no stimulation of glucose oxidation. Savard et al. (1965) proposed that LH may influence progesterone synthesis through a pathway that does not involve cholesterol as an intermediate or precursor. It can be concluded that LH did not stimulate progesterone synthesis in this study, or significantly increase glucose or cholesterol uptake. It remains to be determined whether naturally occurring LH pulses increase glucose and cholesterol uptake.

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The uptake of cholesterol and glucose may be influenced by trophic hormones, including the peptide hormones such as IGF-I, insulin or other growth factors. The plasma concentrations of IGF-I and insulin are low during periods of negative energy balance after calving (Gluckman et al., 1987; Lucy et al., 1992). Because these growth factors are critical for the development of ovarian structures and functions (Adashi et al., 1985; Hammond et al., 1988), these may affect transovarian uptake of glucose and cholesterol, and steroidogenesis. Insulin addition, however, did not increase glucose uptake by the rat lutein tissue in vitro (Armstrong and Greep, 1962). Further studies are required to examine the effect of these peptide hormones on ovarian metabolism, and to examine the effect of 2-deoxy-d-glucose, a metabolic inhibitor of glucose on cholesterol uptake in the ovary in vitro. Our findings showing strong correlations between glucose and cholesterol uptake by the ovary, if repeated, suggest that methods to control or modify ovarian function could be developed through manipulating the uptake of cholesterol or glucose. The possibility that uptake of metabolites by other organs may be similarly linked should be investigated.

Acknowledgements The authors are grateful to USDA Animal Hormone Program, USA (National Hormone and Pituitary Program), for providing the bLH used in these experiments. The authors appreciate the helpful assistance given by A. Pope, N. Weber, W.W. Thatcher and R Shumway for the statistical analysis. These studies were undertaken at the University of Sydney.

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