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Effects of exercise on mammary metabolism in the lactating ewe
Small Ruminant Research EL-SEVIER
Small Ruminant Research 20 (1996) 205-214
Effects of exercise on mammary metabolism in the lactating ewe G. Animut, K.D. Chandler School of Agriculture,
La Trobe University, Bundoora,
Accepted
15 August
*
Vie. 3083, Australia
1995
Abstract Mammary metabolism in multiparous lactating ewes fed either luceme chaffbarley grain chafflupin grain (L:Lu; 70:30) diets was measured while at rest, during exercise on a treadmill at for 60 min, and during 30 min recovery from exercise. The effects of these treatments on alpha-amino nitrogen (a-amino N), non-esterified fatty acids (NEFA) and acetate were measured. oxygen and metabolites was calculated from mammary blood flow and arteriovenous concentration Mammary blood flow was reduced by 25% during exercise. Arterial concentrations of oxygen,
(L:B; 70~30) or luceme 0.7 m s-’ on a 10” slope plasma glucose, lactate, Net mammary uptake of (A - V ),differences.
glucose, lactate, a-amino N and NEFA increased during exercise, whereas acetate concentration either remained unchanged or declined. Mammary
A -
V differences were significantly higher for oxygen, glucose, lactate and NEFA, and tended to be higher for a-amino N and lower for acetate during exercise. The mammary uptakes of oxygen, glucose, lactate and a-amino N were unaffected by exercise, whereas the uptake of NEFA was significantly increased and that of acetate was significantly reduced. The changes in arterial concentrations and mammary uptakes in response to exercise were not significantly affected by the diet. The responses in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat. Keywords:
Ewe; Exercise; Lactation; Metabolism
1. Introduction The lactating mammary gland is dependent upon substrate concentrations in arterial blood and blood flow to sustain milk synthesis. Net uptake for a substrate can be estimated as the product of the arteriovenous concentration difference (A - V) across the gland and blood flow (Linzell, 1974). Whereas environmental factors such as thermal stress, feed restriction (Lough et al., 19901, starvation (Davis and Collier, 1985) and cold stress (Thompson and
* Corresponding author. Tel.: 61-3-9479-2357, fax: 61-3-9471 0224, e-mail: K.Chandler@?latrobe.edu.au. 00921~4488/96/$15.00
Thomson, 1972) have been reported to affect mammary blood flow, and in turn mammary metabolism, there have been no direct demonstrations of the effects of exercise on mammary metabolism. Despite the limited information available in ruminants on the effects of exercise on mammary gland metabolism, the effect of exercise on lactational performance has been studied in dairy cows (Anderson et al., 1979; Lamb et al., 1979; Lamb et al., 1981; Matthewman et al., 1989; Gustafson et al., 1993; Thomson and Barnes, 1993). In most of these studies it was found that walking extra distances prior to milking affected milk composition and reduced yield.
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0921.4488(95)00795-4
G. Animut, K.D. Chundler/Smull
206
In the present study the effects of moderate exercise on mammary gland metabolism were studied in lactating ewes fed diets based on lucerne chaff supplemented with either barley or lupin grain. Respiratory, blood and cardiovascular responses to exercise and the effects of diet on milk yield and composition were also measured.
2. Materials
and methods
2.1. Animals and diet Nine multiparous crossbred Border Leicester X Merino ewes, initially weighing 50-61 kg and in Week 4 or 5 of lactation, were used. All were housed in individual metabolism cages for at least 2 weeks before the experiment. Four ewes were fed lucerne chaffbarley grain (L:B; 70:30, w:w air dry) and five were fed luceme chaff:lupin grain (L:Lu; 70:30, w:w, air dry). The diets were chosen to meet calculated requirements for metabolizable energy (ME) for maintenance and milk production (Anonymous, 1975). The daily allocation was offered in equal portions at intervals of 2 h with ad libitum access to water and mineralized salt block (Cheetham Salt, Geelong, Australia). Animals were machine-milked twice daily at 08:30 h and 16:30 h following intravenous injection of 1 i.u. oxytocin (Syntocinon; Sandoz, Switzerland). Lactation performance measurements for one ewe in the second group were not included in the data set following the development of a mastitic infection towards the end of the study period. 2.2. Animal preparation All animals were trained to walk on a moving belt treadmill several weeks prior to parturition. At approximately 14 days prior to term, ultrasonic blood flow probes, 4 mm in size (Transonics, Ithaca, NY), were placed around an external pudendal artery (Gorewit et al., 1989) under general anaesthesia induced by thiopentone sodium (Intraval Sodium, Arthur Webster, Australia) and maintained with halothane (Fluothane, ICI, Villawood, Australia). A catheter (polyvinyl chloride, 0.86 mm i.d. X 1.27 mm o.d.; Dural Plastics, Sydney) was inserted into a
Ruminant Research 20 (1996) 205-214
femoral artery via the saphenous artery using local anaesthesia (lignocaine hydrochloride, Troy Laboratories, Australia). The ewes were fasted for 24 h before general anaesthesia and were given 2.5 ml penicillin (Ilium Penstrep; Troy Laboratories, Australia) and 5 ml oxytetracycline hydrochloride (Terramycin; Pfizer, West Ryde, Australia) immediately after surgery and each day for a further 3 days. On the day prior to each experiment, further polyvinyl catheters (1.00 mm i.d. X 2.00 mm o.d.) were introduced into the right ventricle via a jugular vein and (1.00 mm i.d. X 1.50 mm o.d.) into one superficial epigastric or mammary vein, using 12 and 14 gauge needles, respectively (Sureflo Intravenous Catheter, Terumo, USA). All catheters were filled with sterile saline (NaCl, 9 g l- ’ ) containing heparin (500 u. 1-l ) and were flushed daily with minimum volumes of heparinized saline. All procedures with animals had been given prior approval by the Animal Experimentation Ethics Committee of the School of Agriculture, La Trobe University. 2.3. Experimental
protocols
On the day of the experiments, each sheep was placed on a treadmill immediately after milking at 08:30 h with its head in a ventilated hood and was given the residue of its feed allowance for the corresponding period of 2 h. The sheep was then allowed to stand for 30 min before measurements and blood sampling commenced. Immediately following this acclimatization period, at 09:OO h, blood samples were drawn simultaneously from the femoral artery and the mammary vein. This procedure was repeated every 10 min for the 30 min rest period. The ewe was then exercised at 0.7 m s-’ (2.5 km hh ’ ) on a 9” slope for 60 min, which provided a moderate level of exercise. Three series of blood samples were taken at intervals of 20 min during exercise and a further three were collected at intervals of 10 min during the subsequent 30 min recovery period. Blood samples were placed on ice in heparinized, capped syringes for measurements of plasma metabolites, and in anaerobically sealed glass syringes for analysis of blood gases. Measurement of blood gases was undertaken within 3 h of sampling, while blood samples taken for metabolite analyses
G. Animut, K.D. Chandler/Small
were centrifuged and the plasma removed at - 20°C pending assays.
and stored
2.4. Measurements 2.4. I. Blood gases and cardio-respiratory parameters The PO,, PCO, and pH of arterial and mammary venous blood were measured with a pH/blood gas analyzer (Corning, Model 178; Medshield, USA). Haemoglobin (Hb) concentration and oxyhaemoglobin saturation (SO,) were measured with an automatic direct-reading photometer (OSM2, Radiometer A/S, Copenhagen, Denmark) as described by Siggard-Andersen (1977) and calibrated for use with sheep’s blood. Blood oxygen content (ml 1-l) was then calculated as Hb(g l- ’ ) X SO, xl .34. Blood packed-cell volume was measured by the conventional microhaematocrit method. Measurement of total body oxygen consumption (VO,) was achieved with a head cage which was ventilated by a pump and an open-circuit calorimetric apparatus. Cardiac output was then calculated from the Fick equation using measurements of total VO, and arterial-right ventricle blood oxygen content (Chandler, 1983). Heart rates were measured by attaching a Cobe CDX III pressure transducer (Cobe Laboratories, USA) to the end of the arterial catheter and recording pulse frequencies on a chart recorder. Body temperature was recorded using a thermocouple (N-type) inserted 10 cm into a jugular vein. Mammary blood flow was measured using an ultrasonic blood flow probe connected to an ultrasonic blood flow meter (model T201D; Transonic System, Ithaca, NY). A data logger (Model 500; Data Electronics, Australia) was coupled directly to the thermocouple and blood flow meter to provide average values over 5 min for temperature and blood flow, with measurements taken every second. The average blood flow over 5 min measured at the time of blood sampling was used for subsequent calculations of nutrient and oxygen uptakes. 2.4.2. Plasma metabolites Glucose concentrations were measured in untreated plasma samples by the method of Bemt and Lachenicht (1974), adapted for use on an autoanalyzer. Plasma lactate concentration was determined
Ruminant Research 20 (1996) 205-214
207
in deproteinized samples by a semi-automated procedure developed from the method described by Gutman and Wahlefeld (1974). Plasma o-amino N in the protein-free supematants was assayed using the autoanalyzer method of Oddy (1974). Plasma NEFA were extracted into heptane using the reagents described by Dole (1956), and subsequently plasma NEFA concentrations were determined by the procedure of Kelley (1965). Plasma acetate concentrations were determined by a modification of the method previously described by Pethick et al. (1981). After freeze-transfer, the sample was allowed to thaw and then transferred into a small vial and dried on a hotplate under a stream of nitrogen gas. The residue was reconstituted in 0.5 ml 5% (w/v) perchloric acid immediately before injection of 3 pl of the processed sample into a gas chromatograph (Model 8410, Perkin Elmer, UK). The column was packed with chromosorb A WA, mesh lOO/ 120 (Microchem. Association, Vie., Australia). Temperatures of injector, detector and column were 2OO”C, 200°C and 150°C respectively, and a nitrogen carrier gas flow of 14 ml min- ’ was maintained. Net uptakes (U> of blood oxygen and plasma metabolites by the mammary gland were calculated as: U = blood or plasma flow X (A - V). For determination of uptakes of metabolites, blood flow was corrected to plasma flow using measured values of PCV. 2.4.3. Milk yield and milk constituents The milk yield of each ewe was measured daily (sum of 08:30 h and 16:30 h milkings) and averaged over a 3-week period. Milk samples were taken on three consecutive days from pooled twice-daily milkings, and were analyzed using an infrared milk analyzer (Milko-Scan Model 133B; N. Foss, Electric, Denmark) for milk fat, lactose, protein and non-fat solids. 2.5. Statistical
analyses
Values for arterial and arteriovenous concentration differences during the three sampling periods tended not to be significantly different during rest or exercise. Therefore means of the triplicate sets of results were used in all subsequent calculations. The
208
G. Animut, K.D. Chandler/
Smull Ruminant Research 20 (1996) 205-214
Table 1 Milk yield (g day- ’ ) and concentrations of milk constituents (g kg- ‘) for 4 ewes fed Luceme:Barley (LB) and four ewes fed 1uceme:hmins (L:Lu)
Milk yield Milk fat Milk protein Lactose Non-fat solids
L:B
L:Lu
1235*64 52.6 + 2.8 47.lkO.8 53.8f0.9 107.9* 1.0
1361&-102 55.6 5 2.3 50.4+1.1 54.0 * 0.5 111.4* 1.2 * l
Values are means f s.e.m.; * P(O.05.
significance of differences between mean values for the parameters measured were determined by analysis of variance and/or r-tests using the package StateView 512 + (Brain Power, California).
3. Results 3.1. Milk yield and composition Milk yields and concentrations of major milk components for ewes fed the two diets are shown in Table 1. Milk yield was not different (P > 0.05), whereas milk protein and non-fat solids were higher for ewes fed L:Lu than for those fed LIB (P < 0.05). The fat and lactose concentrations were similar for ewes fed both diets.
3.2. Respiratory and cardiovascular responses Differences in respiratory and cardiovascular responses due to diet were not significant, so data for all sheep were pooled to gain single mean values. Exercise caused increases in PCV (P < 0.051, Hba (P < 0.051, pHa (P < 0.051, total VO, (P < O.OS>,cardiac output (P < 0.05), heart rate (P < 0.05) and SaO, (P < 0.051, and decreases in PaCO, (P < 0.05) and PmvCO, (P < 0.05). There was a tendency for PaO, and body temperature to increase (P > 0.05). Most values had returned to resting levels within 30 min of recovery (Table 2). 3.3. Mammary blood flow and oxygen uptake Blood flow to the mammary gland decreased by 25% during exercise (P < 0.05) and returned to preexercise values soon after exercise ceased (Fig. 1). The concentration of oxygen in the arterial blood rose (P < 0.05) during exercise and returned to resting values during recovery. This was accompanied by a higher A - V difference of oxygen across the mammary gland during exercise (P < 0.05) which maintained oxygen uptake (Table 3). 3.4. Plasma metabolites Concentrations of metabolites in the arterial plasma during rest, exercise and recovery for ewes
Table 2 Packed-cell volume (PCV), arterial haemoglobin (Hba), arterial pH (pHa), arterial oxyhaemoglobin saturation (SaO,), arterial partial pressure of oxygen (PaO,), arterial and mammary venous partial pressure of CO, (PaCO,, PmvCO,), total oxygen consumption (total VO,), cardiac output, heart rate and body temperature of lactating ewes during rest, exercise and recovery
PCV (%I Hba (g%) pHa SaO, (o/o) PaO, (mmHg) PaCO, (mmHg) PmvCO, Total VO, (ml min- ‘) Cardiac output (1 min - ‘1 Heart rate (beats min- ’ ) Body temperature (“0
n
Rest
Exercise
Recovery
12 13 13 13 13 13 13 11 11 10 7
23.4 f 0.7 a 7.4 f 0.2 a 7.44*0.01 a 95.6 f 0.4 a 108.7 * 0.9 a 38.9 f 0.5 a 45.0 & 0.6 a 298 i 8 a 8.9 f 0.4 a 86*3= 40.34f0.12 a
28.2 f 0.7 b 8.9 f 0.2 b 7.49 f 0.01 b 96.8 f 0.3 b 109.7 5 0.8 ab 30.2 f 1.0 b 36.1 + 1.2 b 764 + 19 b 14.7i0.6 b 177*8 b 40.75 f 0.18 a
23.8 f 0.8 a 7.4 !c 0.2 a 7.45 f 0.01 = 95.5 f 0.4 a 111.6+ 1.0 b 33.7 * 0.9 c 38.4 & 1.0 b 341 f 24 ’ 10.0 f 0.8 a 104*6’ 40.59+0.18 a
Values are means f s.e.m.; n, number of measurements.
Values for individual
parameters
with different
superscripts
differ: abc P(O.05.
G. Animut, K.D. Chandler/SmuN
Ruminant Research 20 (1996) 205-214
Table 3 Mammary blood flow (ml min-‘), arterial ox ygen concentration oxygen uptake (ml min-‘1 during rest, exercise and recovery
Mammary blood flow Arterial oxygen concentration Mammary oxygen A-V difference Mammary oxygen uptake
(ml I-‘),
mammary
oxygen
Rest
Exercise
Recovery
200 f 12 a 94+3a 36+2a 7.1 + 0.6 a
149f8 b 115*3 b 49*2b 7.4 + 0.6 a
198 f 16 a 95f3a 34fla 6.7 * 0.5 a
Values for individual
3.4.1. Plasma glucose During exercise there were sustained increases in arterial plasma glucose concentrations for ewes fed both L:B and L:Lu (P < 0.05). The concentration of glucose rose by 45% for ewes fed L:B and by 27% for ewes fed L:Lu over pre-exercise levels. These elevated concentrations persisted into the recovery period for both diets (P < O.OS>, so that mean concentrations during this period were different from those observed before exercise. The arterial hyperglycaemia during exercise was accompanied by a higher mammary A - V difference (P < 0.05) for both diets in an inverse proportion to mammary blood flow, which led to maintenance of mammary uptake of glucose. No significant differences were observed for arterial concentrations and mammary
parameters
with different
differ: abc P(O.05.
3.4.2. Plasma lactate Plasma lactate increased in response to exercise in both dietary groups (P < 0.05) and declined during recovery, although it remained higher than the resting values for ewes fed L:B (P < 0.05). Mammary uptake of lactate during exercise was not different from the resting state, although exercise did lead to an increase in the A - V difference (P < 0.05). There were higher arterial concentrations of lactate during rest for ewes fed L:Lu compared with those fed L:B (P < 0.05). Mammary A - V differences and uptakes of lactate were significantly higher for the L:Lu diet in all three stages of the experiment.
N (mg per 100 ml), acetate (mmol 1-l)
Arterial concentration
n
Diet
Rest
Exercise
Recovery
Glucose
7 5 7 5 7 5 4 3 6 5
L:B L:Lu L:B L:Lu L:B L:Lu L:B L:Lu L:B L:Lu
2.55 + 0.05 a 2.67 f 13 a 0.70 f 0.06 a 1.11 f 0.10 a 4.57 * 0.15 a 5.21 fO.ll a 1.16~0.11 a 1.37 + 0.12 a 225 f 12 a 240f15a
3.70 + 0.27 b 3.39 f 0.23 b 1.83 f 0.18 b 2.29 f 0.38 b 5.14+0.17 b 5.60 + 0.15 b 1.18 k 0.08 ab 0.99 f 0.12 b 730 f 58 b 590 f 70 b
3.50 f 0.25 3.33 + 0.24 1.20f0.16’ I .38 f 0.25 4.56 f 0.15 5.05 f 0.13 0.93 * 0.05 1.15 f 0.10 378 f 37 ’ 376 k 32 ’
NEFA
superscripts
A - V differences of plasma glucose between the two diets. However, the uptake of glucose by the mammary gland was consistently higher, and significantly so during rest (P < 0.09, for ewes fed L:Lu than for ewes fed L:B.
Table 4 Arterial plasma concentration of glucose (mmol I- ‘), lactate (mmol l- ‘1, a-amino (pm01 I- ’ ) during rest, exercise and recovery for ewes fed two diets
Acetate
l- ‘) and mammary
(ml
11 13 13 I1
fed the two diets are shown in Table 4, and A - V differences across the mammary gland and mammary uptake of metabolites are given in Table 5.
wNH,-N
difference
n
Values are means f s.e.m.; n, number of measurements.
Lactate
A-V
209
Values are means f s.e.m.; n, number of measurements. Values for individual For comparison of the two diets: * P < 0.05; ns, not significant.
parameters
and NEFA
Effect of diet
b b a a a b ab
Rest
Exercise
Recovery
ns
ns
ns
ns
ns
* *
ns
*
ns
ns
ns
ns
ns
ns
with different
superscripts
differ: abc P < 0.05.
210
G. Animut, K.D. Chandler/Smnll
3.4.3. Plasma a-amino N The increase in arterial a-amino N during exercise was significant (P < 0.05) for both diets and concentrations, and returned to resting values within the 30 min recovery period. Although not significant (P > O.OS>, a higher A - V difference was observed during exercise and this offset the lowered blood flow, thereby maintaining mammary uptake. The arterial concentration of a-amino N was significantly higher for ewes fed L:Lu than for those fed L:B during both rest (P < 0.05) and recovery (P < 0.05). The effects of diet on A - V difference were not significant at all stages, but a higher uptake
220
2
I
200 -
2 5
180 -
G zi
20 (1996) 205-214
(P < 0.05) for ewes fed L:Lu than for those fed L:B was observed during exercise. 3.4.4. Plasma acetate Acetate concentration in the arterial plasma was not affected during exercise in ewes fed L:B but was depressed in ewes fed L:Lu (P < 0.05). The mammary A - V difference of acetate was lowered during exercise for ewes fed both L:B (P > 0.05) and L:Lu (P < 0.05). Coupled with reduced blood flow, this resulted in decreased mammary acetate uptake (P < 0.05) for ewes fed both L:B and L:Lu. 3.4.5. Plasma non-esterified fatty acids The increase in arterial NEFA during exercise was significant (P < 0.05) for ewes fed both diets. Concentrations declined during recovery (P < 0.05) but remained higher than the corresponding resting values (P < 0.05). The increase in arterial concentration was followed by an increased mammary A - V difference (P < 0.05) which brought about a marked increase in mammary uptake of NEFA (P < 0.05) during exercise. No significant dietary effect was observed.
r
‘e ._
Ruminant Resewch
160 -
12oL I
I
I
I
I
4. Discussion
I
6
55 1
I
0
20
40
I
I
60
80
Tome from the start of exercise (min) Fig. I. Mammary blood flow, and plasma concentrations of glucose (mmol l- ’ , 0). lactate (mm01 l_ ’ , n ), a-amino N (mg per 100 ml, 0). and NEFA (mm01 l-‘, 0) during rest, exercise (horizontal line) and recovery. Values are means rir s.e.m.
In the present study there was no significant interaction between exercise and diet. However, differences observed in blood metabolites were accompanied by improvements in milk yield and composition, particularly of milk protein for the ewes fed L:Lu. Similar improvements have been reported previously (Sinclair and Gooden, 1989). It is suggested that these results were due to the higher crude protein content and better digestibility of lupins compared with barley (Sinclair and Gooden, 1989). However, lupins have a lower starch content and more fibre compared with barley (Bartsch and Valentine, 1986). The level of exercise used in the present study (0.7 m s-l up a 9” slope) corresponds to a fast walk for sheep, with animals showing no sign of exhaustion after the exercise period. Nevertheless, this rate of work was sufficient to generate a moderate level of hypocapnea and a subsequent respiratory alkalo-
G. Animut, K.D. Chunder/Smull
Ruminant Research
sis. Consequently body temperature increased slightly during exercise, as opposed to a marked increase reported in non-pregnant (Bell et al., 1983b) and pregnant (Lotgering et al., 1983) sheep. Mammary blood flow decreased by a mean of 25% during the exercise period, with an abrupt decline by some 30% during the first 20 min after which it tended to increase during the latter part of the exercise period (Fig. 1). Values returned to control levels after termination of exercise. The decrease in mammary blood flow during exercise was probably mediated by increased sympatho-adrenomedullary activity. Plasma concentrations of catecholamine have been shown to be elevated in exercising sheep (Palmer et al., 19841, and the contractile response of the mammary vasculature to catecholamines is well known (Linzell, 1974). A signifi-
20 (1996) 205-214
211
cant decrease in PmvCO, and a slight increase in alveolar ventilation (Table 2) may also have contributed to the reduction in mammary blood flow during exercise (Linzell, 1974). Any effect of decreased mammary blood flow on mammary oxygen uptake in this study would have been partly offset by the substantial increase in the arterial haemoglobin concentration, and therefore the oxygen carrying capacity, of the arterial blood. In the present study increases in plasma glucose and lactate occurred during exercise. The hyperglycaemia was presumably due to a higher entry rate of glucose (Judson et al., 1976; Leury et al., 19901, via either gluconeogenesis or glycogenolysis. This could be attributed partly to changes in hormonal status and particularly to elevated glucagon concentrations (Brockman, 1979a; Brockman, 1979b; Bell et al.,
Table 5 A-V differences and mammary uptakes of plasma glucose (mm01 I-’ or pmol min- ‘), lactate (mmol 1-l or kmol mitt- ‘), o-amino N acetate (mmol 1-l or kmol mm ‘) and NEFA (pm01 I-’ or kmol min- ‘) during rest, exercise and ( mg per 100 ml or mg mm’), recovery for ewes fed the two diets n
Glucose Mammary
A-V
Mammary
uptake
Lactate Mammary
A-V
Mammary
uptake
a-NH,-N Mammary
A-V
Mammary
uptake
Acetate Mammary
A-V
Mammary
uptake
NEFA Mammary
A-V
Mammary
uptake
Diet
Exercise
Rest
Effect of diet
Recovery
7 5 7 4
L:B L:Lu L:B L:Lu
0.45 + 0.04 a 0.51 f 0.02 a 62k7’ 821k5~
0.59 f 0.06 b 0.60 * 0.03 b 58+8’ 72+5=
0.40 f 0.03 a 0.47 f 0.02 55f7’ 74+6=
7 5 7 4
L:B L:Lu L:B L:Lu
0.13 + 0.06 a 0.36 + 0.04 a 14*8 ab 63k9a
0.31 f 0.07 b 0.65 f 0.15 b 28&7 b 74+25a
0.07 f 0.04 a 0.22 f 0.06 a 6*8’ 45k 16 a
7 5 7 4
L:B L:Lu L:B L:Lu
0.69 0.79 10.4 12.8
0.79 If: 0.08 = 1.01 f 0.11 b 7.8 + 0.9 ’ 11.5f 1.7 a
0.59 + 0.07 a 0.71 f 0.10 a 8.8 + 1.6 a 8.9* 2.1 a
4 3 4 3
L:B L:Lu L:B L:Lu
0.95 + 0.10 a 1.10*0.11 a 142t23 a 183+25a
0.84 f 0.08 ab 0.80 f 0.10 b 80+ 11 b 94+ 15 b
0.71 f 0.05 b 0.96 5 0.09 ab 91 * 12 b 158 f. 18 a
6 5 6 4
L:B L:Lu L:B L:Lu
79f 10 = 61*8a 11+2 a 9f2a
378 4 33 b 293 f 50 b 37_+6 b 38+7 b
k f f *
0.09 = 0.06 ab 1.8 a 1.5 a
Values are means f s.e.m.; n, number of measurements. Values for individual For comparison of the two diets: * P < 0.05; ns, not significant.
157+20c 156 k 24 ’ 22+5a 26 k 7 ab parameters
Rest
Exercise
Recovery
ns
ns
ns
ns
ns
*
*
*
I
ns
ns
l
IlS
ns.
*
ns
*
ns
ns
ns
ns
IlS
ns
ns
ns
ns
ns
with different
superscripts
1
differ: abc P < 0.05.
212
G. Animut, K.D. Chandler/Small
1983a; Brockman, 1987). Exercise-induced hyperlactacidaemia was presumably a consequence of increased glycogenolysis in exercising skeletal muscle. The magnitude and/or patterns of changes in plasma concentrations of arterial glucose and lactate during exercise and the recovery period in the present study were generally similar to those reported for pregnant (Bell et al., 1983a) and non-pregnant (Brockman, 1979a; Brockman, 1979b) non-lactating sheep during comparable levels of exercise. The elevated concentrations of plasma glucose and lactate measured were probably important in terms of maintenance nutrient supply to the mammary gland. Thus there were both increased arterial concentrations and arteriovenous differences which maintained mammary uptake despite a decrease in mammary blood flow during exercise. The two metabolites were higher during recovery than prior to exercise but A - V differences were similar, which is consistent with results of earlier work reported by Miller et al. (1991), who demonstrated that mammary A - V differences for glucose and lactate were not functions of arterial concentrations. Exercise promotes tissue catabolism in sheep, thereby increasing concentrations of free amino acids in the blood (Brockway and Lobley, 1982). On the other hand, it has been found in humans that individual amino acids are metabolised differently during exercise (Felig and Wahren, 1971). The total arterial o-amino N in the present study significantly increased during exercise, resulting in a higher A - V difference which was able to maintain mammary uptake in spite of exercise. This occurred despite the exercise-induced decline in milk protein concentration measured by others workers (Thomson and Barnes, 1993), which may have been due to increased amino acid oxidation, to depression in protein synthesis as has been observed in the whole animal (Brockway and Lobley, 1982), and/or to changes in circulating amino acid profiles (Felig and Wahren, 197 1) during exercise. Acetate concentrations in arterial plasma decreased significantly during exercise in ewes fed L:Lu but not in those fed L:B. This discrepancy might have been due to the small number of observations that were made. When all the observations were pooled, the acetate concentration before exercise was 1.26 + 0.08 mM as compared with 1.lO t_
Ruminant Research 20 (1996) 205-214
0.07 mM (P > 0.05) during exercise. The latter figure agrees with previous findings in pregnant (Chandler and Bell, 1981) and non-pregnant and nonlactating (Judson et al., 1976) sheep. Pethick (19911, on the contrary, reported an increase in acetate concentration in sheep, which he suggested was due to oxidation of NEFA especially during exercise above the anaerobic threshold. However, the moderate exercise level used in the present study might not have been sufficient to cause this. Most of the acetate in the blood is absorbed from the gut, although there is considerable endogenous production (Pethick et al., 1981). A decline in the splanchnic blood flow during exercise (Bell et al., 1983b; Pethick et al., 1991) might have decreased the acetate concentration in the arterial blood. The lower mammary A - V difference during exercise was presumably due to a lower arterial level of acetate during exercise, as acetate A - V is critically dependent on blood concentration (Miller et al., 1991). The decline in mammary A - V differences and blood flow contributed to the reduced mammary uptake of acetate during exercise. Exercise in sheep prompts a dramatic shift to fat mobilization (Pethick et al., 1987; Pethick, 1991). In the horse, increased lipolysis during exercise was suggested to be due to a decrease in insulin and increases in cortisol and ACTH (Miller-Graber et al., 1991); similar hormonal responses have been observed in exercising sheep (Bell et al., 1983a; Shahneh et al., 1994). In the present study, some 2.5-fold increases in arterial NEFA were evident as compared with the 8- 1.5fold increase reported in non-lactating sheep which were more intensively exercised (Pethick et al., 1987; Pethick, 1991). In the present study there was a higher A - V difference of NEFA across the mammary gland which resulted in substantially higher uptake by the mammary gland during exercise. Not only has an increase in NEFA during exercise been reported, but also a substantial change in the composition of NEFA. For example, Pethick et al. (1987) stated that the ratio of oleic acid to palmitic and stearic acids changed from 0.38 at rest to 0.71 during exercise. It is tempting to suggest that the fat composition of milk might change in exercising animals due to a shift in the acetate and NEFA flux on the one hand and a change in the NEFA composition
G. Animut,
K.D.
Chandler/Smull
on the other. Previous research in dairy cows has revealed that the production of milk fat per se was not severely affected by exercise (Thomson and Barnes, 19931, which might have been due to a substantial uptake of NEFA as opposed to acetate. The use of glucose for lactose synthesis is partially dependent on the availability of acetate (Forsberg et al., 1985; Miller et al., 1991). The lowered uptake of acetate recorded here might have reduced the availability of glucose for lactose synthesis, and presumably milk yield, as has been observed in lactating dairy cows during exercise (Thomson and Barnes, 1993). A rise in body temperature during exercise is another stressor which may have resulted in some metabolites being used for thermoregulation, not only in the whole animal but also in the mammary gland. In conclusion, it has been shown that the magnitudes of changes in substrate concentrations and uptakes by the sheep mammary gland during exercise were not significantly affected by the diets used in the present study. However, exercise was shown to have marked effects on mammary metabolism. The changes in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat. Acknowledgements Financial support for this work was provided by a grant from the Australian International Development Assistance Bureau (AIDAB). G. A. was also supported by a scholarship from AIDAB. We gratefully acknowledge the assistance of Professor G.H. McDowell in the preparation of the manuscript, and Zelko Biki and Simon Westbrook in laboratory analyses. References Anderson, M.J., Lamb, R.C. and Walters, J.L., 1979. Effect of prepattum exercise on feed intake and milk production of multiparous cows. J. Dairy Sci., 62: 1420- 1423. Anonymous, 1975. Energy Allowances and Feeding Systems for Ruminants. Tech. Bull. No. 33, HMSO, London, pp. 44-49. Bartsch, B.D. and Valentine, S.C., 1986. Grain legumes in dairy cow nutrition. Proc. Aust. Sot. Anim. Prod., 16: 32-34.
Ruminant
Research
20 (1996)
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213
Bell, A.W., Bassett, J.M., Chandler, K.D. and Boston, R.C., 1983a. Fetal and maternal endocrine responses to exercise in the pregnant ewe. .I. Dev. Physiol., 5: 129-141. Bell, A.W., Hales, J.R.S., King, R.B. and Fawcett, A.A., 1983b. Influence of heat stress on exercise-induced changes in regional blood flow in sheep. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 55: I91 6- 1923. Bemt, E. and Lachenicht, R., 1974. Determination in blood, serum, or plasma with automatic analysers (GOD-PERID~ method). In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 3. 2nd edn. Academic Press, New York. pp. 1215-1222. Brockman, R.P., 1979a. Effect of somatostatin on plasma glucagon and insulin and glucose turnover in exercising sheep. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 47: 273-278. Brockman, R.P., 1979b. Glucagon responses to exercise in sheep. Aust. J. Biol. Sci., 32: 215-220. Brockman, R.P., 1987. Effect of exercise on net hepatic uptake of lactate, pyruvate, alanine and glycerol in sheep. Can. J. Physiol. Pharmacol., 65: 2065-2070. Brockway, J.M. and Lobley, G.E., 1982. The effect of exercise on amino acid oxidation and protein synthesis in sheep. In: A. Ekem and F. Sundstol (Editors), Proc. 9th Symp. Energy Metabolism of Farm Animals. European Association of Animal Production, Lillehammer, Norway, pp. I24- 127. Chandler, K.D., 1983. Fetal and maternal responses to exercise in the pregnant ewe. M. Agric. Sci. Thesis, La Trobe University, pp. 28-43. Chandler, K.D. and Bell, A.W., 1981. Effects of maternal exercise on fetal and maternal respiration and nutrient metabolism in the pregnant ewe. .I. Dev. Physiol., 3: 161-176. Davis, S.R. and Collier, R.S., 1985. Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci., 68: 1041-1058. Dole, V.P., 1956. A relationship between non-esterified fatty acids in plasma and the metabolism of glucose. J. Clin. Invest., 35: l50- 154. Felig, P. and Wahren, J., 1971. Amino acid metabolism in exercising man. J. Clin. Invest., 50: 2703-2714. Forsberg, N.E., Baldwin, R.L. and Smith, N.E., 1985. Roles of glucose and its interactions with acetate in maintenance and biosynthesis in bovine mammary tissue. J. Dairy Sci., 68: 2544-2549. Gorewit, R.C., Aromando, M.C. and Bristol, D.G., 1989. Measuring bovine mammary blood flow using a transit time ultrasonic flow probe. J. Dairy Sci., 72: 19181928. Gustafson, G.M., Luthman, J. and Burstedt, E., 1993. Effect of daily exercise on performance, feed efficiency and energy balance of tied dairy cows. Acta Agric. Stand., Sect. A, Anim. Sci., 43: 219-227. Gutman, 1. and Wahlefeld, A.W., 1974. L-(+)-Lactate. Determination with lactate dehydrogenase and NAD. In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 3. 2nd edn. Academic Press, New York, pp. l464- 1466. Judson, G.J., Filsell, O.H. and Jarrett, I.G., 1976. Glucose and acetate metabolism in sheep at rest and during exercise. Aust. J. Biol. Sci., 29: 215-222.
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Kelley, F., 1965. Improved method for microtitration of fatty acids. Anal. Chem., 37: 1078-1079. Lamb, R.C., Barker, B.O., Anderson, M.J. and Walters, J.L., 1979. Effect of forced exercise on two-year-old Holstein heifers. J. Dairy Sci., 62: 1791-1797. Lamb, R.C., Anderson, M.J. and Walters, J.L., 1981. Forced walking prepartum for dairy cows of different ages. J. Dairy Sci., 64: 2017-2024. Leury, B.J.. Bird, A.R., Chandler, K.D. and Bell, A.W., 1990. Glucose partitioning in the pregnant ewe: Effects of undemut&ion and exercise. Br. J. Nutr., 64: 449-462. Linzell, J.L., 1974. Mammary blood flow and methods of identifying and measuring precursors of milk. In: B.L. Larsen and V.L Smith (Editors), Lactation, Vol. 1. Academic Press, New York, pp. 143-225. Lotgering, F.K., Gilbert, R.D. and Longo, L.D., 1983. Exercise responses in pregnant sheep: Blood gases, temperatures and fetal cardiovascular system. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 55: 842-850. Lough, D.S., Beede, D.L. and Wilcox, C.J., 1990. Effects of feed intake and thermal stress on mammary blood flow and other physiological measurements in lactating dairy cows. J. Dairy Sci., 73: 325-332. Matthewman, R.W., Merritt, J. and Smith, A.J., 1989. Effect of exercise on lactational performance in cattle. Proc. Nutr. Sot., 48: 92A. Miller, P.S., Reis, B.L., Calve& C.C., DePeters, E.J. and Baldwin, R.L., 1991. Patterns of nutrient uptake by the mammary glands of lactating dairy cows. J. Dairy Sci., 74: 3791-3799. Miller-Graber, P.A., Lawrence, L.M., Foreman, J.H., Bump, K.D., Fisher, M.G. and Kurcz, E.V., 1991. Nutrition and physical activity: Dietary protein level and energy metabolism during treadmill exercise in horses. J. Nutr., 121: 1462-1469. Gddy, V.H., 1974. A semiautomated method for the determination of plasma alpha amino nitrogen. Clin. Chim. Acta, 51: 151156.
Ruminant Research 20 (1996) 205-2/4 Palmer, SM., Oakes, G.K., Champion, J.A., Fisher, D.A. and Hobel, C.K., 1984. Catecholamine physiology in the ovine fetus. III. Maternal and fetal response to acute maternal exercise. Am. J. Obstet. Gynecol., 149: 426-434. Pethick, D.W., 1991. Comparative aspects of fuel supply during exercise. Proc. Nutr. Sot. Aust., 16: 197-204. Pethick, D.W., Lindsay, D.B., Barker, P.J. and Northrop, A.J., 198 I. Acetate supply and utilization by the tissues of sheep in viva. Br. J. Nutr., 46: 97- 110. Pethick, D.W., Harman, N. and Chong, J.K., 1987. Non-esteritied long-chain fatty acid metabolism in fed sheep at rest and during exercise. Aust. J. Biol. Sci., 40: 221-234. Pethick, D.W., Miller, C.B. and Hannan, N.G., 1991. Exercise in Merino sheep-the relationships between work intensity, endurance, anaerobic threshold and glucose metabolism. Aust. J. Agric. Res., 42: 599-620. Shahneh, AZ., Gooden, J.M., Rigby, R.D.G. and Wynn, P.C., 1994. The influence of active immunization of animals against adrenocorticotropin on energy expenditure in the exercising sheep. Proc. Aust. Sot. Anim. Prod., 20: 390. Siggard-Andersen, O., 1977. Experiences with a new direct-reading oxygen saturation photometer using ultrasound for hemolyzing the blood. Stand. J. Clin. Lab. Invest., 37(Suppl. 146); 3-8. Sinclair, S.E. and Gooden, J.M., 1989. Effects of barley and lupin supplementation on milk composition and plasma metabolites in lactating ewes. In: D.J. Farrel (Editor), Recent Advances in Animal Nutrition in Australia. University of New England, Armidale, p. 17A. Thompson, G.E. and Thomson, E.M., 1972. Effect of cold exposure on mammary circulation, oxygen consumption and milk secretion in the goat. J. Physiol., 272: 187-196. Thomson, N.A. and Barnes, M.L., 1993. Effect of distance walked on dairy production and milk quality. Proc. N.Z. Sot. Anim. Prod., 53: 69-72.
Small Ruminant Research 20 (1996) 205-214
Effects of exercise on mammary metabolism in the lactating ewe G. Animut, K.D. Chandler School of Agriculture,
La Trobe University, Bundoora,
Accepted
15 August
*
Vie. 3083, Australia
1995
Abstract Mammary metabolism in multiparous lactating ewes fed either luceme chaffbarley grain chafflupin grain (L:Lu; 70:30) diets was measured while at rest, during exercise on a treadmill at for 60 min, and during 30 min recovery from exercise. The effects of these treatments on alpha-amino nitrogen (a-amino N), non-esterified fatty acids (NEFA) and acetate were measured. oxygen and metabolites was calculated from mammary blood flow and arteriovenous concentration Mammary blood flow was reduced by 25% during exercise. Arterial concentrations of oxygen,
(L:B; 70~30) or luceme 0.7 m s-’ on a 10” slope plasma glucose, lactate, Net mammary uptake of (A - V ),differences.
glucose, lactate, a-amino N and NEFA increased during exercise, whereas acetate concentration either remained unchanged or declined. Mammary
A -
V differences were significantly higher for oxygen, glucose, lactate and NEFA, and tended to be higher for a-amino N and lower for acetate during exercise. The mammary uptakes of oxygen, glucose, lactate and a-amino N were unaffected by exercise, whereas the uptake of NEFA was significantly increased and that of acetate was significantly reduced. The changes in arterial concentrations and mammary uptakes in response to exercise were not significantly affected by the diet. The responses in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat. Keywords:
Ewe; Exercise; Lactation; Metabolism
1. Introduction The lactating mammary gland is dependent upon substrate concentrations in arterial blood and blood flow to sustain milk synthesis. Net uptake for a substrate can be estimated as the product of the arteriovenous concentration difference (A - V) across the gland and blood flow (Linzell, 1974). Whereas environmental factors such as thermal stress, feed restriction (Lough et al., 19901, starvation (Davis and Collier, 1985) and cold stress (Thompson and
* Corresponding author. Tel.: 61-3-9479-2357, fax: 61-3-9471 0224, e-mail: K.Chandler@?latrobe.edu.au. 00921~4488/96/$15.00
Thomson, 1972) have been reported to affect mammary blood flow, and in turn mammary metabolism, there have been no direct demonstrations of the effects of exercise on mammary metabolism. Despite the limited information available in ruminants on the effects of exercise on mammary gland metabolism, the effect of exercise on lactational performance has been studied in dairy cows (Anderson et al., 1979; Lamb et al., 1979; Lamb et al., 1981; Matthewman et al., 1989; Gustafson et al., 1993; Thomson and Barnes, 1993). In most of these studies it was found that walking extra distances prior to milking affected milk composition and reduced yield.
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0921.4488(95)00795-4
G. Animut, K.D. Chundler/Smull
206
In the present study the effects of moderate exercise on mammary gland metabolism were studied in lactating ewes fed diets based on lucerne chaff supplemented with either barley or lupin grain. Respiratory, blood and cardiovascular responses to exercise and the effects of diet on milk yield and composition were also measured.
2. Materials
and methods
2.1. Animals and diet Nine multiparous crossbred Border Leicester X Merino ewes, initially weighing 50-61 kg and in Week 4 or 5 of lactation, were used. All were housed in individual metabolism cages for at least 2 weeks before the experiment. Four ewes were fed lucerne chaffbarley grain (L:B; 70:30, w:w air dry) and five were fed luceme chaff:lupin grain (L:Lu; 70:30, w:w, air dry). The diets were chosen to meet calculated requirements for metabolizable energy (ME) for maintenance and milk production (Anonymous, 1975). The daily allocation was offered in equal portions at intervals of 2 h with ad libitum access to water and mineralized salt block (Cheetham Salt, Geelong, Australia). Animals were machine-milked twice daily at 08:30 h and 16:30 h following intravenous injection of 1 i.u. oxytocin (Syntocinon; Sandoz, Switzerland). Lactation performance measurements for one ewe in the second group were not included in the data set following the development of a mastitic infection towards the end of the study period. 2.2. Animal preparation All animals were trained to walk on a moving belt treadmill several weeks prior to parturition. At approximately 14 days prior to term, ultrasonic blood flow probes, 4 mm in size (Transonics, Ithaca, NY), were placed around an external pudendal artery (Gorewit et al., 1989) under general anaesthesia induced by thiopentone sodium (Intraval Sodium, Arthur Webster, Australia) and maintained with halothane (Fluothane, ICI, Villawood, Australia). A catheter (polyvinyl chloride, 0.86 mm i.d. X 1.27 mm o.d.; Dural Plastics, Sydney) was inserted into a
Ruminant Research 20 (1996) 205-214
femoral artery via the saphenous artery using local anaesthesia (lignocaine hydrochloride, Troy Laboratories, Australia). The ewes were fasted for 24 h before general anaesthesia and were given 2.5 ml penicillin (Ilium Penstrep; Troy Laboratories, Australia) and 5 ml oxytetracycline hydrochloride (Terramycin; Pfizer, West Ryde, Australia) immediately after surgery and each day for a further 3 days. On the day prior to each experiment, further polyvinyl catheters (1.00 mm i.d. X 2.00 mm o.d.) were introduced into the right ventricle via a jugular vein and (1.00 mm i.d. X 1.50 mm o.d.) into one superficial epigastric or mammary vein, using 12 and 14 gauge needles, respectively (Sureflo Intravenous Catheter, Terumo, USA). All catheters were filled with sterile saline (NaCl, 9 g l- ’ ) containing heparin (500 u. 1-l ) and were flushed daily with minimum volumes of heparinized saline. All procedures with animals had been given prior approval by the Animal Experimentation Ethics Committee of the School of Agriculture, La Trobe University. 2.3. Experimental
protocols
On the day of the experiments, each sheep was placed on a treadmill immediately after milking at 08:30 h with its head in a ventilated hood and was given the residue of its feed allowance for the corresponding period of 2 h. The sheep was then allowed to stand for 30 min before measurements and blood sampling commenced. Immediately following this acclimatization period, at 09:OO h, blood samples were drawn simultaneously from the femoral artery and the mammary vein. This procedure was repeated every 10 min for the 30 min rest period. The ewe was then exercised at 0.7 m s-’ (2.5 km hh ’ ) on a 9” slope for 60 min, which provided a moderate level of exercise. Three series of blood samples were taken at intervals of 20 min during exercise and a further three were collected at intervals of 10 min during the subsequent 30 min recovery period. Blood samples were placed on ice in heparinized, capped syringes for measurements of plasma metabolites, and in anaerobically sealed glass syringes for analysis of blood gases. Measurement of blood gases was undertaken within 3 h of sampling, while blood samples taken for metabolite analyses
G. Animut, K.D. Chandler/Small
were centrifuged and the plasma removed at - 20°C pending assays.
and stored
2.4. Measurements 2.4. I. Blood gases and cardio-respiratory parameters The PO,, PCO, and pH of arterial and mammary venous blood were measured with a pH/blood gas analyzer (Corning, Model 178; Medshield, USA). Haemoglobin (Hb) concentration and oxyhaemoglobin saturation (SO,) were measured with an automatic direct-reading photometer (OSM2, Radiometer A/S, Copenhagen, Denmark) as described by Siggard-Andersen (1977) and calibrated for use with sheep’s blood. Blood oxygen content (ml 1-l) was then calculated as Hb(g l- ’ ) X SO, xl .34. Blood packed-cell volume was measured by the conventional microhaematocrit method. Measurement of total body oxygen consumption (VO,) was achieved with a head cage which was ventilated by a pump and an open-circuit calorimetric apparatus. Cardiac output was then calculated from the Fick equation using measurements of total VO, and arterial-right ventricle blood oxygen content (Chandler, 1983). Heart rates were measured by attaching a Cobe CDX III pressure transducer (Cobe Laboratories, USA) to the end of the arterial catheter and recording pulse frequencies on a chart recorder. Body temperature was recorded using a thermocouple (N-type) inserted 10 cm into a jugular vein. Mammary blood flow was measured using an ultrasonic blood flow probe connected to an ultrasonic blood flow meter (model T201D; Transonic System, Ithaca, NY). A data logger (Model 500; Data Electronics, Australia) was coupled directly to the thermocouple and blood flow meter to provide average values over 5 min for temperature and blood flow, with measurements taken every second. The average blood flow over 5 min measured at the time of blood sampling was used for subsequent calculations of nutrient and oxygen uptakes. 2.4.2. Plasma metabolites Glucose concentrations were measured in untreated plasma samples by the method of Bemt and Lachenicht (1974), adapted for use on an autoanalyzer. Plasma lactate concentration was determined
Ruminant Research 20 (1996) 205-214
207
in deproteinized samples by a semi-automated procedure developed from the method described by Gutman and Wahlefeld (1974). Plasma o-amino N in the protein-free supematants was assayed using the autoanalyzer method of Oddy (1974). Plasma NEFA were extracted into heptane using the reagents described by Dole (1956), and subsequently plasma NEFA concentrations were determined by the procedure of Kelley (1965). Plasma acetate concentrations were determined by a modification of the method previously described by Pethick et al. (1981). After freeze-transfer, the sample was allowed to thaw and then transferred into a small vial and dried on a hotplate under a stream of nitrogen gas. The residue was reconstituted in 0.5 ml 5% (w/v) perchloric acid immediately before injection of 3 pl of the processed sample into a gas chromatograph (Model 8410, Perkin Elmer, UK). The column was packed with chromosorb A WA, mesh lOO/ 120 (Microchem. Association, Vie., Australia). Temperatures of injector, detector and column were 2OO”C, 200°C and 150°C respectively, and a nitrogen carrier gas flow of 14 ml min- ’ was maintained. Net uptakes (U> of blood oxygen and plasma metabolites by the mammary gland were calculated as: U = blood or plasma flow X (A - V). For determination of uptakes of metabolites, blood flow was corrected to plasma flow using measured values of PCV. 2.4.3. Milk yield and milk constituents The milk yield of each ewe was measured daily (sum of 08:30 h and 16:30 h milkings) and averaged over a 3-week period. Milk samples were taken on three consecutive days from pooled twice-daily milkings, and were analyzed using an infrared milk analyzer (Milko-Scan Model 133B; N. Foss, Electric, Denmark) for milk fat, lactose, protein and non-fat solids. 2.5. Statistical
analyses
Values for arterial and arteriovenous concentration differences during the three sampling periods tended not to be significantly different during rest or exercise. Therefore means of the triplicate sets of results were used in all subsequent calculations. The
208
G. Animut, K.D. Chandler/
Smull Ruminant Research 20 (1996) 205-214
Table 1 Milk yield (g day- ’ ) and concentrations of milk constituents (g kg- ‘) for 4 ewes fed Luceme:Barley (LB) and four ewes fed 1uceme:hmins (L:Lu)
Milk yield Milk fat Milk protein Lactose Non-fat solids
L:B
L:Lu
1235*64 52.6 + 2.8 47.lkO.8 53.8f0.9 107.9* 1.0
1361&-102 55.6 5 2.3 50.4+1.1 54.0 * 0.5 111.4* 1.2 * l
Values are means f s.e.m.; * P(O.05.
significance of differences between mean values for the parameters measured were determined by analysis of variance and/or r-tests using the package StateView 512 + (Brain Power, California).
3. Results 3.1. Milk yield and composition Milk yields and concentrations of major milk components for ewes fed the two diets are shown in Table 1. Milk yield was not different (P > 0.05), whereas milk protein and non-fat solids were higher for ewes fed L:Lu than for those fed LIB (P < 0.05). The fat and lactose concentrations were similar for ewes fed both diets.
3.2. Respiratory and cardiovascular responses Differences in respiratory and cardiovascular responses due to diet were not significant, so data for all sheep were pooled to gain single mean values. Exercise caused increases in PCV (P < 0.051, Hba (P < 0.051, pHa (P < 0.051, total VO, (P < O.OS>,cardiac output (P < 0.05), heart rate (P < 0.05) and SaO, (P < 0.051, and decreases in PaCO, (P < 0.05) and PmvCO, (P < 0.05). There was a tendency for PaO, and body temperature to increase (P > 0.05). Most values had returned to resting levels within 30 min of recovery (Table 2). 3.3. Mammary blood flow and oxygen uptake Blood flow to the mammary gland decreased by 25% during exercise (P < 0.05) and returned to preexercise values soon after exercise ceased (Fig. 1). The concentration of oxygen in the arterial blood rose (P < 0.05) during exercise and returned to resting values during recovery. This was accompanied by a higher A - V difference of oxygen across the mammary gland during exercise (P < 0.05) which maintained oxygen uptake (Table 3). 3.4. Plasma metabolites Concentrations of metabolites in the arterial plasma during rest, exercise and recovery for ewes
Table 2 Packed-cell volume (PCV), arterial haemoglobin (Hba), arterial pH (pHa), arterial oxyhaemoglobin saturation (SaO,), arterial partial pressure of oxygen (PaO,), arterial and mammary venous partial pressure of CO, (PaCO,, PmvCO,), total oxygen consumption (total VO,), cardiac output, heart rate and body temperature of lactating ewes during rest, exercise and recovery
PCV (%I Hba (g%) pHa SaO, (o/o) PaO, (mmHg) PaCO, (mmHg) PmvCO, Total VO, (ml min- ‘) Cardiac output (1 min - ‘1 Heart rate (beats min- ’ ) Body temperature (“0
n
Rest
Exercise
Recovery
12 13 13 13 13 13 13 11 11 10 7
23.4 f 0.7 a 7.4 f 0.2 a 7.44*0.01 a 95.6 f 0.4 a 108.7 * 0.9 a 38.9 f 0.5 a 45.0 & 0.6 a 298 i 8 a 8.9 f 0.4 a 86*3= 40.34f0.12 a
28.2 f 0.7 b 8.9 f 0.2 b 7.49 f 0.01 b 96.8 f 0.3 b 109.7 5 0.8 ab 30.2 f 1.0 b 36.1 + 1.2 b 764 + 19 b 14.7i0.6 b 177*8 b 40.75 f 0.18 a
23.8 f 0.8 a 7.4 !c 0.2 a 7.45 f 0.01 = 95.5 f 0.4 a 111.6+ 1.0 b 33.7 * 0.9 c 38.4 & 1.0 b 341 f 24 ’ 10.0 f 0.8 a 104*6’ 40.59+0.18 a
Values are means f s.e.m.; n, number of measurements.
Values for individual
parameters
with different
superscripts
differ: abc P(O.05.
G. Animut, K.D. Chandler/SmuN
Ruminant Research 20 (1996) 205-214
Table 3 Mammary blood flow (ml min-‘), arterial ox ygen concentration oxygen uptake (ml min-‘1 during rest, exercise and recovery
Mammary blood flow Arterial oxygen concentration Mammary oxygen A-V difference Mammary oxygen uptake
(ml I-‘),
mammary
oxygen
Rest
Exercise
Recovery
200 f 12 a 94+3a 36+2a 7.1 + 0.6 a
149f8 b 115*3 b 49*2b 7.4 + 0.6 a
198 f 16 a 95f3a 34fla 6.7 * 0.5 a
Values for individual
3.4.1. Plasma glucose During exercise there were sustained increases in arterial plasma glucose concentrations for ewes fed both L:B and L:Lu (P < 0.05). The concentration of glucose rose by 45% for ewes fed L:B and by 27% for ewes fed L:Lu over pre-exercise levels. These elevated concentrations persisted into the recovery period for both diets (P < O.OS>, so that mean concentrations during this period were different from those observed before exercise. The arterial hyperglycaemia during exercise was accompanied by a higher mammary A - V difference (P < 0.05) for both diets in an inverse proportion to mammary blood flow, which led to maintenance of mammary uptake of glucose. No significant differences were observed for arterial concentrations and mammary
parameters
with different
differ: abc P(O.05.
3.4.2. Plasma lactate Plasma lactate increased in response to exercise in both dietary groups (P < 0.05) and declined during recovery, although it remained higher than the resting values for ewes fed L:B (P < 0.05). Mammary uptake of lactate during exercise was not different from the resting state, although exercise did lead to an increase in the A - V difference (P < 0.05). There were higher arterial concentrations of lactate during rest for ewes fed L:Lu compared with those fed L:B (P < 0.05). Mammary A - V differences and uptakes of lactate were significantly higher for the L:Lu diet in all three stages of the experiment.
N (mg per 100 ml), acetate (mmol 1-l)
Arterial concentration
n
Diet
Rest
Exercise
Recovery
Glucose
7 5 7 5 7 5 4 3 6 5
L:B L:Lu L:B L:Lu L:B L:Lu L:B L:Lu L:B L:Lu
2.55 + 0.05 a 2.67 f 13 a 0.70 f 0.06 a 1.11 f 0.10 a 4.57 * 0.15 a 5.21 fO.ll a 1.16~0.11 a 1.37 + 0.12 a 225 f 12 a 240f15a
3.70 + 0.27 b 3.39 f 0.23 b 1.83 f 0.18 b 2.29 f 0.38 b 5.14+0.17 b 5.60 + 0.15 b 1.18 k 0.08 ab 0.99 f 0.12 b 730 f 58 b 590 f 70 b
3.50 f 0.25 3.33 + 0.24 1.20f0.16’ I .38 f 0.25 4.56 f 0.15 5.05 f 0.13 0.93 * 0.05 1.15 f 0.10 378 f 37 ’ 376 k 32 ’
NEFA
superscripts
A - V differences of plasma glucose between the two diets. However, the uptake of glucose by the mammary gland was consistently higher, and significantly so during rest (P < 0.09, for ewes fed L:Lu than for ewes fed L:B.
Table 4 Arterial plasma concentration of glucose (mmol I- ‘), lactate (mmol l- ‘1, a-amino (pm01 I- ’ ) during rest, exercise and recovery for ewes fed two diets
Acetate
l- ‘) and mammary
(ml
11 13 13 I1
fed the two diets are shown in Table 4, and A - V differences across the mammary gland and mammary uptake of metabolites are given in Table 5.
wNH,-N
difference
n
Values are means f s.e.m.; n, number of measurements.
Lactate
A-V
209
Values are means f s.e.m.; n, number of measurements. Values for individual For comparison of the two diets: * P < 0.05; ns, not significant.
parameters
and NEFA
Effect of diet
b b a a a b ab
Rest
Exercise
Recovery
ns
ns
ns
ns
ns
* *
ns
*
ns
ns
ns
ns
ns
ns
with different
superscripts
differ: abc P < 0.05.
210
G. Animut, K.D. Chandler/Smnll
3.4.3. Plasma a-amino N The increase in arterial a-amino N during exercise was significant (P < 0.05) for both diets and concentrations, and returned to resting values within the 30 min recovery period. Although not significant (P > O.OS>, a higher A - V difference was observed during exercise and this offset the lowered blood flow, thereby maintaining mammary uptake. The arterial concentration of a-amino N was significantly higher for ewes fed L:Lu than for those fed L:B during both rest (P < 0.05) and recovery (P < 0.05). The effects of diet on A - V difference were not significant at all stages, but a higher uptake
220
2
I
200 -
2 5
180 -
G zi
20 (1996) 205-214
(P < 0.05) for ewes fed L:Lu than for those fed L:B was observed during exercise. 3.4.4. Plasma acetate Acetate concentration in the arterial plasma was not affected during exercise in ewes fed L:B but was depressed in ewes fed L:Lu (P < 0.05). The mammary A - V difference of acetate was lowered during exercise for ewes fed both L:B (P > 0.05) and L:Lu (P < 0.05). Coupled with reduced blood flow, this resulted in decreased mammary acetate uptake (P < 0.05) for ewes fed both L:B and L:Lu. 3.4.5. Plasma non-esterified fatty acids The increase in arterial NEFA during exercise was significant (P < 0.05) for ewes fed both diets. Concentrations declined during recovery (P < 0.05) but remained higher than the corresponding resting values (P < 0.05). The increase in arterial concentration was followed by an increased mammary A - V difference (P < 0.05) which brought about a marked increase in mammary uptake of NEFA (P < 0.05) during exercise. No significant dietary effect was observed.
r
‘e ._
Ruminant Resewch
160 -
12oL I
I
I
I
I
4. Discussion
I
6
55 1
I
0
20
40
I
I
60
80
Tome from the start of exercise (min) Fig. I. Mammary blood flow, and plasma concentrations of glucose (mmol l- ’ , 0). lactate (mm01 l_ ’ , n ), a-amino N (mg per 100 ml, 0). and NEFA (mm01 l-‘, 0) during rest, exercise (horizontal line) and recovery. Values are means rir s.e.m.
In the present study there was no significant interaction between exercise and diet. However, differences observed in blood metabolites were accompanied by improvements in milk yield and composition, particularly of milk protein for the ewes fed L:Lu. Similar improvements have been reported previously (Sinclair and Gooden, 1989). It is suggested that these results were due to the higher crude protein content and better digestibility of lupins compared with barley (Sinclair and Gooden, 1989). However, lupins have a lower starch content and more fibre compared with barley (Bartsch and Valentine, 1986). The level of exercise used in the present study (0.7 m s-l up a 9” slope) corresponds to a fast walk for sheep, with animals showing no sign of exhaustion after the exercise period. Nevertheless, this rate of work was sufficient to generate a moderate level of hypocapnea and a subsequent respiratory alkalo-
G. Animut, K.D. Chunder/Smull
Ruminant Research
sis. Consequently body temperature increased slightly during exercise, as opposed to a marked increase reported in non-pregnant (Bell et al., 1983b) and pregnant (Lotgering et al., 1983) sheep. Mammary blood flow decreased by a mean of 25% during the exercise period, with an abrupt decline by some 30% during the first 20 min after which it tended to increase during the latter part of the exercise period (Fig. 1). Values returned to control levels after termination of exercise. The decrease in mammary blood flow during exercise was probably mediated by increased sympatho-adrenomedullary activity. Plasma concentrations of catecholamine have been shown to be elevated in exercising sheep (Palmer et al., 19841, and the contractile response of the mammary vasculature to catecholamines is well known (Linzell, 1974). A signifi-
20 (1996) 205-214
211
cant decrease in PmvCO, and a slight increase in alveolar ventilation (Table 2) may also have contributed to the reduction in mammary blood flow during exercise (Linzell, 1974). Any effect of decreased mammary blood flow on mammary oxygen uptake in this study would have been partly offset by the substantial increase in the arterial haemoglobin concentration, and therefore the oxygen carrying capacity, of the arterial blood. In the present study increases in plasma glucose and lactate occurred during exercise. The hyperglycaemia was presumably due to a higher entry rate of glucose (Judson et al., 1976; Leury et al., 19901, via either gluconeogenesis or glycogenolysis. This could be attributed partly to changes in hormonal status and particularly to elevated glucagon concentrations (Brockman, 1979a; Brockman, 1979b; Bell et al.,
Table 5 A-V differences and mammary uptakes of plasma glucose (mm01 I-’ or pmol min- ‘), lactate (mmol 1-l or kmol mitt- ‘), o-amino N acetate (mmol 1-l or kmol mm ‘) and NEFA (pm01 I-’ or kmol min- ‘) during rest, exercise and ( mg per 100 ml or mg mm’), recovery for ewes fed the two diets n
Glucose Mammary
A-V
Mammary
uptake
Lactate Mammary
A-V
Mammary
uptake
a-NH,-N Mammary
A-V
Mammary
uptake
Acetate Mammary
A-V
Mammary
uptake
NEFA Mammary
A-V
Mammary
uptake
Diet
Exercise
Rest
Effect of diet
Recovery
7 5 7 4
L:B L:Lu L:B L:Lu
0.45 + 0.04 a 0.51 f 0.02 a 62k7’ 821k5~
0.59 f 0.06 b 0.60 * 0.03 b 58+8’ 72+5=
0.40 f 0.03 a 0.47 f 0.02 55f7’ 74+6=
7 5 7 4
L:B L:Lu L:B L:Lu
0.13 + 0.06 a 0.36 + 0.04 a 14*8 ab 63k9a
0.31 f 0.07 b 0.65 f 0.15 b 28&7 b 74+25a
0.07 f 0.04 a 0.22 f 0.06 a 6*8’ 45k 16 a
7 5 7 4
L:B L:Lu L:B L:Lu
0.69 0.79 10.4 12.8
0.79 If: 0.08 = 1.01 f 0.11 b 7.8 + 0.9 ’ 11.5f 1.7 a
0.59 + 0.07 a 0.71 f 0.10 a 8.8 + 1.6 a 8.9* 2.1 a
4 3 4 3
L:B L:Lu L:B L:Lu
0.95 + 0.10 a 1.10*0.11 a 142t23 a 183+25a
0.84 f 0.08 ab 0.80 f 0.10 b 80+ 11 b 94+ 15 b
0.71 f 0.05 b 0.96 5 0.09 ab 91 * 12 b 158 f. 18 a
6 5 6 4
L:B L:Lu L:B L:Lu
79f 10 = 61*8a 11+2 a 9f2a
378 4 33 b 293 f 50 b 37_+6 b 38+7 b
k f f *
0.09 = 0.06 ab 1.8 a 1.5 a
Values are means f s.e.m.; n, number of measurements. Values for individual For comparison of the two diets: * P < 0.05; ns, not significant.
157+20c 156 k 24 ’ 22+5a 26 k 7 ab parameters
Rest
Exercise
Recovery
ns
ns
ns
ns
ns
*
*
*
I
ns
ns
l
IlS
ns.
*
ns
*
ns
ns
ns
ns
IlS
ns
ns
ns
ns
ns
with different
superscripts
1
differ: abc P < 0.05.
212
G. Animut, K.D. Chandler/Small
1983a; Brockman, 1987). Exercise-induced hyperlactacidaemia was presumably a consequence of increased glycogenolysis in exercising skeletal muscle. The magnitude and/or patterns of changes in plasma concentrations of arterial glucose and lactate during exercise and the recovery period in the present study were generally similar to those reported for pregnant (Bell et al., 1983a) and non-pregnant (Brockman, 1979a; Brockman, 1979b) non-lactating sheep during comparable levels of exercise. The elevated concentrations of plasma glucose and lactate measured were probably important in terms of maintenance nutrient supply to the mammary gland. Thus there were both increased arterial concentrations and arteriovenous differences which maintained mammary uptake despite a decrease in mammary blood flow during exercise. The two metabolites were higher during recovery than prior to exercise but A - V differences were similar, which is consistent with results of earlier work reported by Miller et al. (1991), who demonstrated that mammary A - V differences for glucose and lactate were not functions of arterial concentrations. Exercise promotes tissue catabolism in sheep, thereby increasing concentrations of free amino acids in the blood (Brockway and Lobley, 1982). On the other hand, it has been found in humans that individual amino acids are metabolised differently during exercise (Felig and Wahren, 1971). The total arterial o-amino N in the present study significantly increased during exercise, resulting in a higher A - V difference which was able to maintain mammary uptake in spite of exercise. This occurred despite the exercise-induced decline in milk protein concentration measured by others workers (Thomson and Barnes, 1993), which may have been due to increased amino acid oxidation, to depression in protein synthesis as has been observed in the whole animal (Brockway and Lobley, 1982), and/or to changes in circulating amino acid profiles (Felig and Wahren, 197 1) during exercise. Acetate concentrations in arterial plasma decreased significantly during exercise in ewes fed L:Lu but not in those fed L:B. This discrepancy might have been due to the small number of observations that were made. When all the observations were pooled, the acetate concentration before exercise was 1.26 + 0.08 mM as compared with 1.lO t_
Ruminant Research 20 (1996) 205-214
0.07 mM (P > 0.05) during exercise. The latter figure agrees with previous findings in pregnant (Chandler and Bell, 1981) and non-pregnant and nonlactating (Judson et al., 1976) sheep. Pethick (19911, on the contrary, reported an increase in acetate concentration in sheep, which he suggested was due to oxidation of NEFA especially during exercise above the anaerobic threshold. However, the moderate exercise level used in the present study might not have been sufficient to cause this. Most of the acetate in the blood is absorbed from the gut, although there is considerable endogenous production (Pethick et al., 1981). A decline in the splanchnic blood flow during exercise (Bell et al., 1983b; Pethick et al., 1991) might have decreased the acetate concentration in the arterial blood. The lower mammary A - V difference during exercise was presumably due to a lower arterial level of acetate during exercise, as acetate A - V is critically dependent on blood concentration (Miller et al., 1991). The decline in mammary A - V differences and blood flow contributed to the reduced mammary uptake of acetate during exercise. Exercise in sheep prompts a dramatic shift to fat mobilization (Pethick et al., 1987; Pethick, 1991). In the horse, increased lipolysis during exercise was suggested to be due to a decrease in insulin and increases in cortisol and ACTH (Miller-Graber et al., 1991); similar hormonal responses have been observed in exercising sheep (Bell et al., 1983a; Shahneh et al., 1994). In the present study, some 2.5-fold increases in arterial NEFA were evident as compared with the 8- 1.5fold increase reported in non-lactating sheep which were more intensively exercised (Pethick et al., 1987; Pethick, 1991). In the present study there was a higher A - V difference of NEFA across the mammary gland which resulted in substantially higher uptake by the mammary gland during exercise. Not only has an increase in NEFA during exercise been reported, but also a substantial change in the composition of NEFA. For example, Pethick et al. (1987) stated that the ratio of oleic acid to palmitic and stearic acids changed from 0.38 at rest to 0.71 during exercise. It is tempting to suggest that the fat composition of milk might change in exercising animals due to a shift in the acetate and NEFA flux on the one hand and a change in the NEFA composition
G. Animut,
K.D.
Chandler/Smull
on the other. Previous research in dairy cows has revealed that the production of milk fat per se was not severely affected by exercise (Thomson and Barnes, 19931, which might have been due to a substantial uptake of NEFA as opposed to acetate. The use of glucose for lactose synthesis is partially dependent on the availability of acetate (Forsberg et al., 1985; Miller et al., 1991). The lowered uptake of acetate recorded here might have reduced the availability of glucose for lactose synthesis, and presumably milk yield, as has been observed in lactating dairy cows during exercise (Thomson and Barnes, 1993). A rise in body temperature during exercise is another stressor which may have resulted in some metabolites being used for thermoregulation, not only in the whole animal but also in the mammary gland. In conclusion, it has been shown that the magnitudes of changes in substrate concentrations and uptakes by the sheep mammary gland during exercise were not significantly affected by the diets used in the present study. However, exercise was shown to have marked effects on mammary metabolism. The changes in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat. Acknowledgements Financial support for this work was provided by a grant from the Australian International Development Assistance Bureau (AIDAB). G. A. was also supported by a scholarship from AIDAB. We gratefully acknowledge the assistance of Professor G.H. McDowell in the preparation of the manuscript, and Zelko Biki and Simon Westbrook in laboratory analyses. References Anderson, M.J., Lamb, R.C. and Walters, J.L., 1979. Effect of prepattum exercise on feed intake and milk production of multiparous cows. J. Dairy Sci., 62: 1420- 1423. Anonymous, 1975. Energy Allowances and Feeding Systems for Ruminants. Tech. Bull. No. 33, HMSO, London, pp. 44-49. Bartsch, B.D. and Valentine, S.C., 1986. Grain legumes in dairy cow nutrition. Proc. Aust. Sot. Anim. Prod., 16: 32-34.
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Research
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Bell, A.W., Bassett, J.M., Chandler, K.D. and Boston, R.C., 1983a. Fetal and maternal endocrine responses to exercise in the pregnant ewe. .I. Dev. Physiol., 5: 129-141. Bell, A.W., Hales, J.R.S., King, R.B. and Fawcett, A.A., 1983b. Influence of heat stress on exercise-induced changes in regional blood flow in sheep. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 55: I91 6- 1923. Bemt, E. and Lachenicht, R., 1974. Determination in blood, serum, or plasma with automatic analysers (GOD-PERID~ method). In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 3. 2nd edn. Academic Press, New York. pp. 1215-1222. Brockman, R.P., 1979a. Effect of somatostatin on plasma glucagon and insulin and glucose turnover in exercising sheep. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 47: 273-278. Brockman, R.P., 1979b. Glucagon responses to exercise in sheep. Aust. J. Biol. Sci., 32: 215-220. Brockman, R.P., 1987. Effect of exercise on net hepatic uptake of lactate, pyruvate, alanine and glycerol in sheep. Can. J. Physiol. Pharmacol., 65: 2065-2070. Brockway, J.M. and Lobley, G.E., 1982. The effect of exercise on amino acid oxidation and protein synthesis in sheep. In: A. Ekem and F. Sundstol (Editors), Proc. 9th Symp. Energy Metabolism of Farm Animals. European Association of Animal Production, Lillehammer, Norway, pp. I24- 127. Chandler, K.D., 1983. Fetal and maternal responses to exercise in the pregnant ewe. M. Agric. Sci. Thesis, La Trobe University, pp. 28-43. Chandler, K.D. and Bell, A.W., 1981. Effects of maternal exercise on fetal and maternal respiration and nutrient metabolism in the pregnant ewe. .I. Dev. Physiol., 3: 161-176. Davis, S.R. and Collier, R.S., 1985. Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci., 68: 1041-1058. Dole, V.P., 1956. A relationship between non-esterified fatty acids in plasma and the metabolism of glucose. J. Clin. Invest., 35: l50- 154. Felig, P. and Wahren, J., 1971. Amino acid metabolism in exercising man. J. Clin. Invest., 50: 2703-2714. Forsberg, N.E., Baldwin, R.L. and Smith, N.E., 1985. Roles of glucose and its interactions with acetate in maintenance and biosynthesis in bovine mammary tissue. J. Dairy Sci., 68: 2544-2549. Gorewit, R.C., Aromando, M.C. and Bristol, D.G., 1989. Measuring bovine mammary blood flow using a transit time ultrasonic flow probe. J. Dairy Sci., 72: 19181928. Gustafson, G.M., Luthman, J. and Burstedt, E., 1993. Effect of daily exercise on performance, feed efficiency and energy balance of tied dairy cows. Acta Agric. Stand., Sect. A, Anim. Sci., 43: 219-227. Gutman, 1. and Wahlefeld, A.W., 1974. L-(+)-Lactate. Determination with lactate dehydrogenase and NAD. In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 3. 2nd edn. Academic Press, New York, pp. l464- 1466. Judson, G.J., Filsell, O.H. and Jarrett, I.G., 1976. Glucose and acetate metabolism in sheep at rest and during exercise. Aust. J. Biol. Sci., 29: 215-222.
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Kelley, F., 1965. Improved method for microtitration of fatty acids. Anal. Chem., 37: 1078-1079. Lamb, R.C., Barker, B.O., Anderson, M.J. and Walters, J.L., 1979. Effect of forced exercise on two-year-old Holstein heifers. J. Dairy Sci., 62: 1791-1797. Lamb, R.C., Anderson, M.J. and Walters, J.L., 1981. Forced walking prepartum for dairy cows of different ages. J. Dairy Sci., 64: 2017-2024. Leury, B.J.. Bird, A.R., Chandler, K.D. and Bell, A.W., 1990. Glucose partitioning in the pregnant ewe: Effects of undemut&ion and exercise. Br. J. Nutr., 64: 449-462. Linzell, J.L., 1974. Mammary blood flow and methods of identifying and measuring precursors of milk. In: B.L. Larsen and V.L Smith (Editors), Lactation, Vol. 1. Academic Press, New York, pp. 143-225. Lotgering, F.K., Gilbert, R.D. and Longo, L.D., 1983. Exercise responses in pregnant sheep: Blood gases, temperatures and fetal cardiovascular system. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 55: 842-850. Lough, D.S., Beede, D.L. and Wilcox, C.J., 1990. Effects of feed intake and thermal stress on mammary blood flow and other physiological measurements in lactating dairy cows. J. Dairy Sci., 73: 325-332. Matthewman, R.W., Merritt, J. and Smith, A.J., 1989. Effect of exercise on lactational performance in cattle. Proc. Nutr. Sot., 48: 92A. Miller, P.S., Reis, B.L., Calve& C.C., DePeters, E.J. and Baldwin, R.L., 1991. Patterns of nutrient uptake by the mammary glands of lactating dairy cows. J. Dairy Sci., 74: 3791-3799. Miller-Graber, P.A., Lawrence, L.M., Foreman, J.H., Bump, K.D., Fisher, M.G. and Kurcz, E.V., 1991. Nutrition and physical activity: Dietary protein level and energy metabolism during treadmill exercise in horses. J. Nutr., 121: 1462-1469. Gddy, V.H., 1974. A semiautomated method for the determination of plasma alpha amino nitrogen. Clin. Chim. Acta, 51: 151156.
Ruminant Research 20 (1996) 205-2/4 Palmer, SM., Oakes, G.K., Champion, J.A., Fisher, D.A. and Hobel, C.K., 1984. Catecholamine physiology in the ovine fetus. III. Maternal and fetal response to acute maternal exercise. Am. J. Obstet. Gynecol., 149: 426-434. Pethick, D.W., 1991. Comparative aspects of fuel supply during exercise. Proc. Nutr. Sot. Aust., 16: 197-204. Pethick, D.W., Lindsay, D.B., Barker, P.J. and Northrop, A.J., 198 I. Acetate supply and utilization by the tissues of sheep in viva. Br. J. Nutr., 46: 97- 110. Pethick, D.W., Harman, N. and Chong, J.K., 1987. Non-esteritied long-chain fatty acid metabolism in fed sheep at rest and during exercise. Aust. J. Biol. Sci., 40: 221-234. Pethick, D.W., Miller, C.B. and Hannan, N.G., 1991. Exercise in Merino sheep-the relationships between work intensity, endurance, anaerobic threshold and glucose metabolism. Aust. J. Agric. Res., 42: 599-620. Shahneh, AZ., Gooden, J.M., Rigby, R.D.G. and Wynn, P.C., 1994. The influence of active immunization of animals against adrenocorticotropin on energy expenditure in the exercising sheep. Proc. Aust. Sot. Anim. Prod., 20: 390. Siggard-Andersen, O., 1977. Experiences with a new direct-reading oxygen saturation photometer using ultrasound for hemolyzing the blood. Stand. J. Clin. Lab. Invest., 37(Suppl. 146); 3-8. Sinclair, S.E. and Gooden, J.M., 1989. Effects of barley and lupin supplementation on milk composition and plasma metabolites in lactating ewes. In: D.J. Farrel (Editor), Recent Advances in Animal Nutrition in Australia. University of New England, Armidale, p. 17A. Thompson, G.E. and Thomson, E.M., 1972. Effect of cold exposure on mammary circulation, oxygen consumption and milk secretion in the goat. J. Physiol., 272: 187-196. Thomson, N.A. and Barnes, M.L., 1993. Effect of distance walked on dairy production and milk quality. Proc. N.Z. Sot. Anim. Prod., 53: 69-72.