200 A.R. Rabiee, I.J. Lean Animal Reproduction Science 64 2000 199–209
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 demon- strated 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 car- bon 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 preg- nenolone 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 re-
ported 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 incorpo- ration of cholesterol into steroids have often yielded varied or conflicting results. LH
may influence progesterone synthesis through a pathway that does not involve choles- terol as an intermediate or precursor Savard et al., 1965. The influence of LH on choles-
terol 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 metabo- lites. 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 metabo-
lites and LH concentrations in cows were previously reported Rabiee et al., 1997b. The experimental methods used in the cows follows.
A.R. Rabiee, I.J. Lean Animal Reproduction Science 64 2000 199–209 201
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 energykg 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 pal-
pated 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 lmin.
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.
202 A.R. Rabiee, I.J. Lean Animal Reproduction Science 64 2000 199–209
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 IUml. 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 ultra-
sonic blood flow probe Transonic System Inc., Ithaca, NY was placed around the ovarian artery. Antibiotics were administered Roscocycline-10; AS 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
A.R. Rabiee, I.J. Lean Animal Reproduction Science 64 2000 199–209 203
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 im- munoassay 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 an-
alyze 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 calcu-
lated 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:
Cross-correlation = R
12
m √
R
11
0 × R
22
where R
12
is the cross-covariance at lag m, and R
11
and R
22
the variances at lag 0 of the two series. The cross-covariances were calculated from the following function
R
12
m, m + k = CovR
1m
, R
2m+k
where R
12
m, m + k is the cross-covariance at time m and time m + k for series R
1
and R
2
, respectively, for each cow R
1m
is the residual for series R
1
at time m after detrending to produce an approximately stationary series, and R
2m+k
the residual for series R
2
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
