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Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats
SmallRuminant Research, 7 (1992) 123-133
123
Elsevier Science Publishers B.V., Amsterdam
Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats C.J. Chang~and S.H. Youngb aDepartment of Animal Science, National Chung-Hsing University, Taichung 40223, Taiwan bDepartment of Metabolism, Chungkun Memorial Hospital Linkou, Taiwan (Accepted 23 April 1991 )
ABSTRACT Chang, C.J. and Young, S.H., 1992. Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats. Small Rumin. Res., 7:123-133. Insulin (0.4 IU/kg/d) or saline was injected subcutaneously into six goats at 08:00 for 5 days in a double switch-back design in Expt 1. A Latin square design was used in Expt 2 with four treatments, insulin (0.3 IU/kg/d), glucose (50 g/d in 20% solution), insulin plus glucose, or saline, infused intravenously into eight goats at 2-h intervals from 06:00 to 14:00 for 1 day. Milk yield per day/feed consumed was significantly depressed (P< 0.05) by 0.28 units by insulin in Experiment 1. Average mammary vein glucose concentration was significantly lower (P< 0.05) after the treatment. Goats receiving five treatments insulin single day in Expt 2 had no significantly changed (P> 0.05) milk production and average mammary vein glucose concentration. Infusion of glucose with or without insulin depressed average milk fat content (P< 0.05), while the latter treatment significantlyelevated average mammary glucose concentration (P< 0.05). When the lowest milk production was recorded ( 10:00 to 14:00), the elevations of mammary vein glucose after glucose infusion, and milk fat after insulin were significant (P< 0.05). Fluctuations in precursors for the mammary gland induced by exogenous insulin and glucose can be rapidly accommodated by the organs through a similar mechanism as occurrs during the feeding and fasting cycle.
INTRODUCTION
Blood glucose is the most important precursor for lactose synthesis in the mammary gland. In ruminants, a negligible increase or even decrease in portal blood glucose concentration immediately following feeding has been observed (Chase et al., 1977). Most blood glucose is supplied by gluconeogenesis in the liver, and is mainly regulated through glucagon (Bassett, 1981 ). Ruminal fermentation products, mainly propionate, stimulate insulin secre0921-4488/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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C.J. CHANG AND S.H. YOUNG
tion, therefore permitting simultaneous maximal peripheral anabolism (Bassett, 1978 ). Although, muscle and adipose tissues contribute the largest portion of peripheral metabolism, there was no clear evidence for insulin to divert nutrients away from the mammary gland (Christensen, 1985 ). Veno-arterial concentration variability across the mammary gland is one index of nutrient uptake rate (Baumrucker, 1985 ). Effects of exogenous insulin a n d / o r glucose on lactating ruminants have been expressed in terms of changes in concentrations of jugular vein metabolites (Rook et al., 1965; Call et al., 1972; Cummins and Sartin, 1987; McClary et al., 1988). Since the mammary vein collects blood flowing through the ample capillary bed of the mammary gland, substrate concentrations should provide information on the function of the mammary gland. Little information is found on changes of mammary gland activities in relation to feeding or milking. Two experiments were conducted on lactating Saanen goats on the effects of ( 1 ) five-day insulin administrations, and (2) insulin a n d / o r glucose administrations at 0, 2, 4, 5 and 8 h after the morning milking and feeding on lactation performance and concentrations of mammary vein glucose and free fatty acids (FFA). MATERIALS AND METHODS
In Expt 1, differences in lactation performance due to s.c. injections of insulin on postprandial Saanen goats were determined based on a double switchback design. Insulin (0.4 I U / k g / d , N P H type, Organon, Netherlands) was injected once daily (08:00) for five consecutive days followed by two adjustment days. An equal volume of saline was administrated similarly to control goats. Treatments were switched at the end of the first two seven-day periods so that each group resumed its original treatment for the third and the last period. Six Saanen goats, 1-3 weeks postpartum, each in the first or second lactation and weighing from 45-55 kg, milking 3.5 kg/d, were used. All animals were in good body condition and free from mastitis (judged from a CMT test). They were placed individually in slotted pens at least one month before the experiments at an ambient temperature of 20 _+2 ° C and relative humidity of 75 _+5%. Animals were fed a ration of 30% alfalfa pellet and 70% concentrate (from Yi-Chen Co., Ltd., Taiwan). Composition of ration components are in Table 1. Water and mineral blocks were freely accessible at all times, while pangola hay was provided at about 1 kg/d/pen. Goats were milked twice daily at 06:00 and 18:00, immediately followed by feeding. All animals were trained to eat within one hour when weighbacks were recorded to calculate feed intake. Milk yield and feed intake were determined daily, milk samples were taken during the treatment, and animals were weighed at the end of each period.
CHANGES OF MAMMARYVEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTYACIDS
125
TABLE l Major composition of concentrate and alfalfa pellets and calculated values for the mixed ration ~ Item
Concentrate
Alfalfa pellet
Mixed ration 2
Dry matter Crude protein Crude fat Crude fiber Calcium Phosphorus
89.0 16.4 0.8 10.7 0.80 0.60
89.7 17.8 2.6 21.0 1.30 0.20
89.2 ! 6.8 1.3 13.8 0.95 0.48
~As-fed basis. 2Mixed from 70% concentrate and 30% alfalfa pellet.
To collect blood samples from the mammary vein (subcutaneous abdominal vein), animals were kept standing undisturbed in milking position; animals that were extremely nervous about this procedure were excluded. All experimental goats were accustomed to management and handling prior to the start of experiment. Blood samples were obtained at 06:00, 12:00, and 18:00 at days 1, 3 and 5 of each period. In Expt 2, eight Saanen goats, 12-16 weeks postpartum, were used. Milking, feeding, and management were the same as in Expt 1, except that the average ambient temperature was about 28 _ 2 °C, and higher humidity. Animals were randomly allocated to four groups, insulin, insulin and glucose, glucose, or saline was infused directly into the jugular vein five times per day at 06:00, 08:00, 10:00, 12:00 and 14:00 with evenly divided daily doses of 0.3 IU insulin/kg BW or 50 g glucose (20% in saline, total 250 ml). Treatments changed according to a Latin-square design after each period which consisted of one injection day and two adjustment days. Every injection was adjusted with saline to 50 ml in volume, and took about 5 min to administer. Experimental goats were restrained minimally during this procedure, and only one case of subcutaneous bruise and edema was observed after the experiment. Total milk yield was determined as the sum of five milkings at 08:00, 10:00, 12:00, 14:00 immediately before treatments, and at 17:00. Blood (from mammary vein) and milk samples were withdrawn at similar intervals. Blood samples were kept at 4°C for 4-8 h before serum collection. The following analyses were performed within two months on frozen ( - 30°C) serum samples: concentrations of glucose were determined with the o-aminobiphenyl method (Shibata, 1981 ); serum FFA with chloroform-heptanemethanol and colorized with diphenylcarbazide according to Falholt et al. ( 1973 ). Milk samples were analyzed for lactose with orcinol after deproteinization (Slater, 1957). Milk fat was calculated as creamatocrite multiplied with 0.74 (Fleet and Linzell, 1964). Milk protein was measured by the Lowry
126
C.J. CHANG
AND
S.H. YOUNG
method (Lowry et al., 1951 ) after appropriate dilution to avoid interference from cloudiness. In Expt 1, differences in milk yield, body weight change, feed intake, milk conversion rate (milk yield/feed intake), milk composition, serum glucose and FFA levels between goats receiving insulin and saline were calculated as +_ ( (week 1 + week 3 ) - (2 × week 2 ) )/2. Differences were subjected to analysis of variance with means tested for significance of difference by Duncan's test (SAS, 1985 ). Assuming there was a linear effect of period on lactation (Anderson et al., 1984), the double switch-back design cancelled out period effects. In Expt 2, treatments were regarded as main effects, and hours after morning milking as a continuous variable. All means of measurements were tested for effects of treatments as Expt 1. RESULTS
Differences caused by five-day s.c. injections of insulin on milking performance and concentrations of glucose and FFA in mammary vein are presented in Table 2. While average milk yield was slightly decreased (by 92 g/ d, P < 0.1 ), and feed intake slightly increased (by 78 g/d, P < 0.1 ), milk cont--;g Control
4--+ Insulin
X--X Glucose
/
1O0
._~ .o P
[] --[] I/G
o
C "~
0
O.
-1
-100
__-X.
-200
, 2
, 4
&
' 8
,
,
I
I
I
I
2
4
6
8
t 2
t 4
t 6
t 8
I
I
11
0 v -1
o-,
-2
--I
-3
I
I
I
t
2
4
6
8
i
-3
I
11 Hours
-2
after
morning
J 11
milking&feeding
Fig. 1. Changes of milk yield and milk composition of Saanen goats at different hours after morning milking and feeding with values of 08:00 set as zero. Each point represents mean of 8 Saanen goats. Values with different letters within period differ significantly ( P < 0.05).
C H A N G E S O F M A M M A R Y VEIN C O N C E N T R A T I O N S O F G L U C O S E AND FREE FATTY ACIDS
c"~ ontrol ~*
-I"Insulin 4-
XGlucose X
12 7
I ~ -I/G -I~
15 a 10 "o
o
b
i
-5 0.5
i
i
I
I
i
i
i
i
0.4
0.2
0.0
-0.2
0.4 0,5
t
Hours
after
morning
milking
& feeding
Fig. 2. Changesof mammary vein concentrations of glucose and FFA at different hours after morning milking and feeding with values of 08:00 set as zero. Each point represents mean of 8 Saanen goats. Valueswith different letters within period differ significantly (P< 0.05). version rate was significantly depressed by 0.28 units ( P < 0.05 ) during insulin treatment. Body weight was significantly increased by 1.1 kg/week ( P < 0.05 ) over the same period. The contents of three major milk constituents averaged from six milkings during insulin treatment were not changed. Effects of insulin on mammary vein glucose determined at 2 h prior (06:00), 4 h (12:00) and 10 h (18:00) post insulin injection (08:00) for three days, the results indicate a significant decrease ( P < 0.05 ) of 5 mg/dl, but effects on FFA were insignificant. Results of Expt 2 are in Table 3 and Figs 1 and 2. Overall effects of five injections per day of insulin and/or glucose are in Table 3. Means ( n = 8 ) of total milk yield from five collections and average contents of lactose and milk protein as well as average mammary vein FFA concentrations were not significantly changed by treatments. Infusions of glucose alone and glucose plus insulin significantly lowered average milk fat content by 0.5 and 0.7% ( P < 0 . 0 5 ) , respectively, compared to the control group. Average mammary
128
cJ. CHANG AND S.H. YOUNG
TABLE2 Differences in lactation performance and mammary vein concentrations of glucose and free fatty acids (FFA) between Saanen goats receiving five-day injections of insulin (0.4 I U / k g B W / d ) and saline Item Lactation performance Milk yield ( g / d ) Feed intake ( g / d ) Body weight change (kg/week) Milk yield/Feed intake Milk fat (%) Milk protein (%) Lactose (%) Mammary vein substrates Glucose ( m g / d l ) FFA ( m M )
Difference !
Fisher's p
- 92 + 78 + 1. l* -0.28* 0.04 0.06 -0.07
N.S. N.S. N.S.
- 5* -0.01
N.S.
*P< 0.05 +P<0.10 N.S.: not significant |Difference= (week l +week 3 - 2 × w e e k 2 ) / 2 n=6.
TABLE3 Total milk production, average milk contents and mammary vein concentrations of glucose and free fatty acids (FFA) during the day when insulin (0.3 I U / k g / d ) a n d / o r glucose ( 5 0 g / d ) were administered intravenously Items
Treatment Control
Milk yield (g)~ 1369_+ 473 Milk content ($)2 Fat 4. l _+0.9 a Protein 3.5 _+0.5 Lactose 4.2 _+0.6 Mammary vein substrates Glucose ( m g / d l ) 59 _+5b FFA ( m M ) 1.05_+0.08
Insulin
Glucose
Insulin/glucose
1403_+ 381
1521 _+441
1434_+419
4.0 _+0.6 a 3.6 _+0.7 4.1 _ 0.7
3.6 _+0.6 b 3.3 _+0.6 4.0 _ 0.6
3.4 _ 0.6 b 3.3 _+0.4 4.2 _ 0.4
60 _+6 b 1.13_+0.10
68 _ 5 a 1.08_+0.14
62 _+9 b 1.05_+0.10
W alues represent total yields _+SE of five collections at 08:00, 10:00, 12:00, 14:00, and 17:00 during the injection day, n = 8. 2Values represent means _+SE of contents of milk samples from the above five collections or five blood samples withdrawn at the similar interval. Values followed by different letters within rows differ significantly ( P < 0.05 ).
CHANGES OF MAMMARY VEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTY ACIDS
129
vein glucose increased by 9 mg/dl above the control (P< 0.05) by glucose infusion. In Fig. 1, changes in milk production and contents from those values at 08:00 for each group at various hours post-milking and feeding are shown. Regardless of treatment, experimental goats secreted the lowest amounts of milk between 10:00 and 14:00 and highest amounts at 08:00 and 17:00 among the five milkings measured. Milk production of control goats averaged 309, 228, 214, and 377 g at 08:00, 10:00, 12:00, 14:00 and 17:00, respectively. This downward pattern of milk secretion between two milkings during the day was apparently not related in a linear function, as might be expected, to hours post milking and feeding. Milk fat content largely decreased with time after milking, from 4.8 (08:00) to 3.0% (17:00) for control goats. Meanwhile, lactose and milk protein concentrations were relatively stable, from 4.30 and 3.36 (08:00) to 3.57 and 3.11% (17:00), respectively. There was no significant interaction between treatments and post-milking hours (data not shown), the only difference in parameters testing during the intermilkingperiod was caused by insulin on milk fat content at 10:00 when milk fat ( 5.1% ) was significantly (P<0.05) higher than that of the group receiving insulin plus glucose (3.86%). Fluctuations of levels of mammary vein glucose and FFA from those at 08:00 for goats receiving various infusions in Expt 2 are in Fig. 2. Deviations of mammary vein glucose levels during the intermilking period were not significantly different among the four groups, except at 10:00 and 12:00 in glucose-infused goats. Although equal amounts of exogenous glucose ( l 0 g) were administered at 06:00, 08:00, 10:00, 12:00 and 14:00 in that group, mammary vein glucose level was not significantly elevated at 08:00, 14:00 and 17:00 (P> 0.05 ). While, at 10:00 and 12:00, the increments in mammary vein glucose averaged 12.3 and 8.3 mg/dl above those at 08:00, which were significantly greater than the corresponding values for control goats of 0.8 and 3.6 mg/dl, respectively. The mammary vein glucose level was apparently more responsive to glucose infusion at 10:00 and 12:00 than at 06:00, 08:00 or 14:00. As for FFA levels, the deviation from values at 08:00 within each treatment was smooth along the intermilking hours, and no significant difference in deviations at any hours among groups was observed in this experiment. DISCUSSION
Insulin treatment for five days slightly stimulated feed intake in goats (Table 2), but seemed to dispose larger portions of energy into body store, instead of the mammary gland for milk production, as was indicated by the significantly heavier body weight and slightly lower milk yield induced by insulin (Table 2 ). Therefore, energy partition and production efficiency were affected by insulin unfavorably for milk secretion. Similar observations as in
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C.J. CHANG AND S.H. YOUNG
Experiment 1 have been reported by Kronfeld et al. ( 1963 ), Schmidt ( 1966 ), and Collier et al. (1984). However, direct veno-arterial concentration measurements of major milk precursors across the mammary gland (Christensen, 1985) failed to provide enough evidence that insulin may divert nutrients away from the mammary gland to adipose tissues. Probably, more complicated interactions among organs and other antagonistic hormones working compensatorily were involved. In Experiment 1, mammary vein glucose concentration averaging over the five-day period was significantly depressed by insulin for 5 mg/dl, while no change in FFA concentration was noticable. The degree of hypoglycemia caused by insulin depends on the potency and type of preparation used, the physiological status of animal, and the reading time elapsed from treatment. It is well established that NPH-insulin exerts maximal hypoglycemic effects around 8 h post-injection (Galloway, 1980). The bleeding scheme of Expt 1 covered one sampling 2 h prior to injection and two samplings (4 h and 10 h after injection) almost half-way from this low point, therefore, could better reflect the average level of glucose during the treatment period, rather than the possibly extreme low value found transiently after treatment. For FFA, maximal depression in jugular vein concentration was found earlier and for a shorter duration than glucose (Yang and Baldwin, 1973). Another possible explanation for the characteristically small changes in mammary vein substrates was postulated by Collier et al. (1984) that mammary glands extract substrates at rates proportional to their concentrations in the inlet vessels. The diurnal changes of milk production is seldom discussed in published work. Goats in Expt 2 secreted higher amounts of milk at 08:00 and 17:00 than at 10:00, 12:00, and 14:00. Hours relative to milking seemed to have much greater effects on milk production than feeding or insulin and/or glucose treatments at the specified dosages of this study. It was postulated that the mammary gland could function temporarily as a storage and buffering organ (Zak et al., 1979). Therefore, short-term changes in concentrations of blood-born substrates or hormones, caused by feeding or infusion, would not limit milk synthesis. On the contrary, factors, such as prolactin secretion, are regulated according to long-term changes in environment, therefore, patterns of milking, management scheme, and diurnal cycle may have profound effects on the mammary gland that could superimpose short-term fluctuations in blood precursors. It is known that ruminant gluconeogenesis and lipogenesis rates are maximal at the postprandial stage (Ballard et al., 1969). The release of glucagon, in response to feeding, closely parallels the release of insulin (Brockman, 1978 ), by 4-8 h after feeding and concentrations of insulin and glucagon start to fall. It is likely that during the same period when hepatic gluconeogenesis approaches a lower rate due to lower glucagon level, the mammary gland absorbs less glucose in accordance with decreasing amounts of milk produced.
CHANGES OF MAMMARY VEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTY ACIDS
131
When afternoon milking is approaching, prolactin as well as other possible effectors trigger another cycle of mammary gland activity, hepatic gluconeogenesis is also accelerated to meet the increasing requirements per glucose for lactose synthesis. These actions might explain the almost constant level of mammary vein glucose despite the large variations in milk yields during the day and the various elapsed times post-feeding in control goats of Expt 2. When exogenous glucose is challenged, the insulin:glucagon ratio should be elevated, therefore, hepatic gluconeogenesis is depressed (Prior and Christenson, 1978) in order to sustain the homeostasis of glucose. According to Figs 1 and 2, elevation of mammary vein glucose by glucose infusion was most significant when milk production was the least during the day. It is possible that the already depressed hepatic gluconeogenesis during 4-8 h after feeding had reached such a low rate that a further decrease in response to exogenous glucose infusion became difficult. Since the removal of glucose by the mammary gland for milk production was the same among treatments, the mammary vein glucose level was elevated by glucose infusion at that period. Depression of average milk fat content by glucose infusion (Table 3 ) seemed not to be mediated through the action of insulin since insulin alone elevated milk fat at 10:00 compared to insulin/glucose infusion, although it was not significantly different from controls (Fig. 1 ). Regulation of glucose utilization and synthesis need further investigation. Dosage of insulin, 0.3 I U / k g / d , in Expt 2 was divided into five injections, the resulting effects on concentrations of glucose and FFA in mammary vein and milk yield were too small to be detected. More pronounced influences of insulin or glucose on milk yield or serum substrates were reported by Kronfeld et al. ( 1963 ), Rook et al. ( 1965 ), Schmidt ( 1966 ), Christensen ( 1985 ). Amounts of insulin applied in their research, based on body weight or daily rate, were similar to the present study. It is possible that goats receiving more frequent but smaller doses of insulin or glucose at a time can more rapidly adjust themselves and reverse the changes than goats receiving similar total doses but at higher volume rates and at longer intervals. The significant difference ( P < 0.05 ) in degree of deviation in milk fat content at 10:00 between insulin and insulin/glucose treated goats (Fig. 1 ) implies that insulin is not closely related to glucose regulation as in nonruminants. Fermentation products after feeding, especially propionate, seem to be potent stimulators of insulin secretion (Chase et al., 1977), which can be further confirmed by the postprandial effects of insulin. FFA extracted by the mammary gland are partly incorporated into milk fat and oxidized into energy for maintenance, milk synthesis and secretion (Annison, 1983 ). Concentration of jugular FFA was the lowest 4-6 h after feeding, but increased significantly later and was the highest just before the next feeding (deBoer et al., 1985 ). However, FFA are present at relatively low concentration in blood and arterio-venous differences across the mammary
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gland are small, therefore, it is the volatile fatty acids that are more important than FFA for milk fat synthesis (Dils, 1986). That is probably why concentrations of serum FFA were relatively insensitive to treatments and cycles of mammary gland function compared to glucose.
REFERENCES Anderson, R.R., Salah, M.S., Sheffield, L.G. and McKenzie, W.N. Jr., 1984. Milk production and lactation curve of guinea pigs. J. Dairy Sci., 67: 185-193. Annison, E.F., 1983. Metabolite utilization by the ruminant mammary gland. In: T.B. Mepham (Editor), Biochemistry of Lactation. Elsevier, Amsterdam, pp. 339. Ballard, F.J., Hanson, R.W. and Kronfeld, D.S., 1969. Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed. Proc., 28:218-231. Bassett, J.M., 1978. Endocrine factors in the control of nutrient utilization: Ruminants. Proc. Nutr. Soc., 37: 273-274. Bassett, J.M., 1981. Regulation of insulin and glucagon secretion in ruminants. In: J.M. Forbes (Editor), Hormones and Metabolism in Ruminants. Agric. Res. Counc., London, pp. 65. Baumrucker, C.R., 1985. Amino acid transport systems in bovine mammary tissue. J. Dairy Sci., 68:2436-2451. Brockman, R.P., 1978. Roles of glucagon and insulin in the regulation of metabolism in ruminants-a review. Can. Vet. J., 19: 55-62. Call, J.L., Mitchell, G.E. Jr., Ely, D.G., Little, C.O. and Tucker, R.E., 1972. Amino acids, volatile fatty acids and glucose in plasma of insulin-treated sheep. J. Anim. Sci., 34:767-77 I. Chase, L.E., Wangsness, P.J. and Martin, R.J., 1977. Portal blood insulin and metabolite changes with spontaneous feeding in steers. J. Dairy Sci., 60:410-415. Christensen, D., 1985. Effect of insulin levels on milk fat production tested. Feedstuffs, 124: l 0. Collier, R.J., McNamara, J.P., Wallace, C.R. and Dehoff, M.H., 1984. A review of endocrine regulation of metabolism during lactation. J. Anita. Sci., 59:498-510. Cummins, K.A. and Sartin, J.L., 1987. Response of insulin, glucagon, and growth hormone to intravenous glucose challenge in cows fed high fat diets. J. Dairy Sci., 70: 277-283. deBoer, G., Trenlde, A. and Young, J.W., 1985. Glucagon, insulin, growth hormone, and blood metabolites during energy restriction ketonemia of lactating cows. J. Dairy Sci., 68: 326337. Dils, R.R., 1986. Comparative aspects of milk fat synthesis. J. Dairy Sci., 69: 904-910. Falholt, K., Lund, B. and Filholt, W., 1973. An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chem. Acta, 46:105-110. Fleet, I.R. and Linzell, J.L., 1964. A rapid method of estimating fat in very small quantities of milk. J. Physiol., (London) 175: 12-20. Galloway, J.A., 1980. Insulin treatment for the early 80s: facts and questions about old and new insulins and their usage. Diabetes Care 3:615-622. Kronfeld, D.S., Mayer, G.P., Robertson, J.M. and Raggi, F., 1963. Depression of milk secretion during insulin administration. J. Dairy Sci., 46:559- 563. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem., 193: 265-275. McClary, D.G., Sartin, J.L., Kemppainen, R.J. and Williams, J.C., 1988. Insulin and growth hormone responses to glucose infusion in mature and first-lactation dairy cows. Am. J. Vet. Res., 49: 1702-1704. Prior, R.L. and Christenson, R.K., 1978. Insulin and glucose effects on glucose metabolism in pregnant and nonpregnant ewes. J. Anim. Sci., 46: 201-210.
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Rook, J.A.F., Storry, J.E. and Wheelock, J.V., 1965. Plasma glucose and acetate and milk secretion in the ruminant. J. Dairy Sci., 48: 745-747. SAS, 1985. The GLM Procedure. In: SAS User's Guide: Statistics, SAS Institute Inc. Cary, NC, pp. 470-476. Schmidt, G.H., 1966. Effect of insulin on yield and composition of milk of dairy cows. J. Dairy Sci., 49: 381-385. Shibata, S. 1981. o-Aminobiphenyl-Boric acid method. Glucose. In: Inoue, S. and Matsumoto, I. (Editors), Illustrations of Techniques in Laboratory Animal. Kiyouritsu Publishing Co., Tokyo, pp. 89-90. Slater, T.F., 1957. A rapid method for the determination of small amount of lactose in milk and tissue suspensions of mammary gland. Analyst, 82:818-825. Yang, Y.T. and Baldwin, R.L., 1973. Lipolysis in isolated cow adipose cells. J. Dairy Sci., 56: 366-374. Zak, R., Martin, A.F. and Blaugh, R., 1979. Assessment of protein turnover by use of radioisotope tracers. Physiol. Rev., 59: 407-413.
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Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats C.J. Chang~and S.H. Youngb aDepartment of Animal Science, National Chung-Hsing University, Taichung 40223, Taiwan bDepartment of Metabolism, Chungkun Memorial Hospital Linkou, Taiwan (Accepted 23 April 1991 )
ABSTRACT Chang, C.J. and Young, S.H., 1992. Changes of mammary vein concentrations of glucose and free fatty acids induced by exogenous insulin and glucose, and relation to mammary gland function in Saanen goats. Small Rumin. Res., 7:123-133. Insulin (0.4 IU/kg/d) or saline was injected subcutaneously into six goats at 08:00 for 5 days in a double switch-back design in Expt 1. A Latin square design was used in Expt 2 with four treatments, insulin (0.3 IU/kg/d), glucose (50 g/d in 20% solution), insulin plus glucose, or saline, infused intravenously into eight goats at 2-h intervals from 06:00 to 14:00 for 1 day. Milk yield per day/feed consumed was significantly depressed (P< 0.05) by 0.28 units by insulin in Experiment 1. Average mammary vein glucose concentration was significantly lower (P< 0.05) after the treatment. Goats receiving five treatments insulin single day in Expt 2 had no significantly changed (P> 0.05) milk production and average mammary vein glucose concentration. Infusion of glucose with or without insulin depressed average milk fat content (P< 0.05), while the latter treatment significantlyelevated average mammary glucose concentration (P< 0.05). When the lowest milk production was recorded ( 10:00 to 14:00), the elevations of mammary vein glucose after glucose infusion, and milk fat after insulin were significant (P< 0.05). Fluctuations in precursors for the mammary gland induced by exogenous insulin and glucose can be rapidly accommodated by the organs through a similar mechanism as occurrs during the feeding and fasting cycle.
INTRODUCTION
Blood glucose is the most important precursor for lactose synthesis in the mammary gland. In ruminants, a negligible increase or even decrease in portal blood glucose concentration immediately following feeding has been observed (Chase et al., 1977). Most blood glucose is supplied by gluconeogenesis in the liver, and is mainly regulated through glucagon (Bassett, 1981 ). Ruminal fermentation products, mainly propionate, stimulate insulin secre0921-4488/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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C.J. CHANG AND S.H. YOUNG
tion, therefore permitting simultaneous maximal peripheral anabolism (Bassett, 1978 ). Although, muscle and adipose tissues contribute the largest portion of peripheral metabolism, there was no clear evidence for insulin to divert nutrients away from the mammary gland (Christensen, 1985 ). Veno-arterial concentration variability across the mammary gland is one index of nutrient uptake rate (Baumrucker, 1985 ). Effects of exogenous insulin a n d / o r glucose on lactating ruminants have been expressed in terms of changes in concentrations of jugular vein metabolites (Rook et al., 1965; Call et al., 1972; Cummins and Sartin, 1987; McClary et al., 1988). Since the mammary vein collects blood flowing through the ample capillary bed of the mammary gland, substrate concentrations should provide information on the function of the mammary gland. Little information is found on changes of mammary gland activities in relation to feeding or milking. Two experiments were conducted on lactating Saanen goats on the effects of ( 1 ) five-day insulin administrations, and (2) insulin a n d / o r glucose administrations at 0, 2, 4, 5 and 8 h after the morning milking and feeding on lactation performance and concentrations of mammary vein glucose and free fatty acids (FFA). MATERIALS AND METHODS
In Expt 1, differences in lactation performance due to s.c. injections of insulin on postprandial Saanen goats were determined based on a double switchback design. Insulin (0.4 I U / k g / d , N P H type, Organon, Netherlands) was injected once daily (08:00) for five consecutive days followed by two adjustment days. An equal volume of saline was administrated similarly to control goats. Treatments were switched at the end of the first two seven-day periods so that each group resumed its original treatment for the third and the last period. Six Saanen goats, 1-3 weeks postpartum, each in the first or second lactation and weighing from 45-55 kg, milking 3.5 kg/d, were used. All animals were in good body condition and free from mastitis (judged from a CMT test). They were placed individually in slotted pens at least one month before the experiments at an ambient temperature of 20 _+2 ° C and relative humidity of 75 _+5%. Animals were fed a ration of 30% alfalfa pellet and 70% concentrate (from Yi-Chen Co., Ltd., Taiwan). Composition of ration components are in Table 1. Water and mineral blocks were freely accessible at all times, while pangola hay was provided at about 1 kg/d/pen. Goats were milked twice daily at 06:00 and 18:00, immediately followed by feeding. All animals were trained to eat within one hour when weighbacks were recorded to calculate feed intake. Milk yield and feed intake were determined daily, milk samples were taken during the treatment, and animals were weighed at the end of each period.
CHANGES OF MAMMARYVEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTYACIDS
125
TABLE l Major composition of concentrate and alfalfa pellets and calculated values for the mixed ration ~ Item
Concentrate
Alfalfa pellet
Mixed ration 2
Dry matter Crude protein Crude fat Crude fiber Calcium Phosphorus
89.0 16.4 0.8 10.7 0.80 0.60
89.7 17.8 2.6 21.0 1.30 0.20
89.2 ! 6.8 1.3 13.8 0.95 0.48
~As-fed basis. 2Mixed from 70% concentrate and 30% alfalfa pellet.
To collect blood samples from the mammary vein (subcutaneous abdominal vein), animals were kept standing undisturbed in milking position; animals that were extremely nervous about this procedure were excluded. All experimental goats were accustomed to management and handling prior to the start of experiment. Blood samples were obtained at 06:00, 12:00, and 18:00 at days 1, 3 and 5 of each period. In Expt 2, eight Saanen goats, 12-16 weeks postpartum, were used. Milking, feeding, and management were the same as in Expt 1, except that the average ambient temperature was about 28 _ 2 °C, and higher humidity. Animals were randomly allocated to four groups, insulin, insulin and glucose, glucose, or saline was infused directly into the jugular vein five times per day at 06:00, 08:00, 10:00, 12:00 and 14:00 with evenly divided daily doses of 0.3 IU insulin/kg BW or 50 g glucose (20% in saline, total 250 ml). Treatments changed according to a Latin-square design after each period which consisted of one injection day and two adjustment days. Every injection was adjusted with saline to 50 ml in volume, and took about 5 min to administer. Experimental goats were restrained minimally during this procedure, and only one case of subcutaneous bruise and edema was observed after the experiment. Total milk yield was determined as the sum of five milkings at 08:00, 10:00, 12:00, 14:00 immediately before treatments, and at 17:00. Blood (from mammary vein) and milk samples were withdrawn at similar intervals. Blood samples were kept at 4°C for 4-8 h before serum collection. The following analyses were performed within two months on frozen ( - 30°C) serum samples: concentrations of glucose were determined with the o-aminobiphenyl method (Shibata, 1981 ); serum FFA with chloroform-heptanemethanol and colorized with diphenylcarbazide according to Falholt et al. ( 1973 ). Milk samples were analyzed for lactose with orcinol after deproteinization (Slater, 1957). Milk fat was calculated as creamatocrite multiplied with 0.74 (Fleet and Linzell, 1964). Milk protein was measured by the Lowry
126
C.J. CHANG
AND
S.H. YOUNG
method (Lowry et al., 1951 ) after appropriate dilution to avoid interference from cloudiness. In Expt 1, differences in milk yield, body weight change, feed intake, milk conversion rate (milk yield/feed intake), milk composition, serum glucose and FFA levels between goats receiving insulin and saline were calculated as +_ ( (week 1 + week 3 ) - (2 × week 2 ) )/2. Differences were subjected to analysis of variance with means tested for significance of difference by Duncan's test (SAS, 1985 ). Assuming there was a linear effect of period on lactation (Anderson et al., 1984), the double switch-back design cancelled out period effects. In Expt 2, treatments were regarded as main effects, and hours after morning milking as a continuous variable. All means of measurements were tested for effects of treatments as Expt 1. RESULTS
Differences caused by five-day s.c. injections of insulin on milking performance and concentrations of glucose and FFA in mammary vein are presented in Table 2. While average milk yield was slightly decreased (by 92 g/ d, P < 0.1 ), and feed intake slightly increased (by 78 g/d, P < 0.1 ), milk cont--;g Control
4--+ Insulin
X--X Glucose
/
1O0
._~ .o P
[] --[] I/G
o
C "~
0
O.
-1
-100
__-X.
-200
, 2
, 4
&
' 8
,
,
I
I
I
I
2
4
6
8
t 2
t 4
t 6
t 8
I
I
11
0 v -1
o-,
-2
--I
-3
I
I
I
t
2
4
6
8
i
-3
I
11 Hours
-2
after
morning
J 11
milking&feeding
Fig. 1. Changes of milk yield and milk composition of Saanen goats at different hours after morning milking and feeding with values of 08:00 set as zero. Each point represents mean of 8 Saanen goats. Values with different letters within period differ significantly ( P < 0.05).
C H A N G E S O F M A M M A R Y VEIN C O N C E N T R A T I O N S O F G L U C O S E AND FREE FATTY ACIDS
c"~ ontrol ~*
-I"Insulin 4-
XGlucose X
12 7
I ~ -I/G -I~
15 a 10 "o
o
b
i
-5 0.5
i
i
I
I
i
i
i
i
0.4
0.2
0.0
-0.2
0.4 0,5
t
Hours
after
morning
milking
& feeding
Fig. 2. Changesof mammary vein concentrations of glucose and FFA at different hours after morning milking and feeding with values of 08:00 set as zero. Each point represents mean of 8 Saanen goats. Valueswith different letters within period differ significantly (P< 0.05). version rate was significantly depressed by 0.28 units ( P < 0.05 ) during insulin treatment. Body weight was significantly increased by 1.1 kg/week ( P < 0.05 ) over the same period. The contents of three major milk constituents averaged from six milkings during insulin treatment were not changed. Effects of insulin on mammary vein glucose determined at 2 h prior (06:00), 4 h (12:00) and 10 h (18:00) post insulin injection (08:00) for three days, the results indicate a significant decrease ( P < 0.05 ) of 5 mg/dl, but effects on FFA were insignificant. Results of Expt 2 are in Table 3 and Figs 1 and 2. Overall effects of five injections per day of insulin and/or glucose are in Table 3. Means ( n = 8 ) of total milk yield from five collections and average contents of lactose and milk protein as well as average mammary vein FFA concentrations were not significantly changed by treatments. Infusions of glucose alone and glucose plus insulin significantly lowered average milk fat content by 0.5 and 0.7% ( P < 0 . 0 5 ) , respectively, compared to the control group. Average mammary
128
cJ. CHANG AND S.H. YOUNG
TABLE2 Differences in lactation performance and mammary vein concentrations of glucose and free fatty acids (FFA) between Saanen goats receiving five-day injections of insulin (0.4 I U / k g B W / d ) and saline Item Lactation performance Milk yield ( g / d ) Feed intake ( g / d ) Body weight change (kg/week) Milk yield/Feed intake Milk fat (%) Milk protein (%) Lactose (%) Mammary vein substrates Glucose ( m g / d l ) FFA ( m M )
Difference !
Fisher's p
- 92 + 78 + 1. l* -0.28* 0.04 0.06 -0.07
N.S. N.S. N.S.
- 5* -0.01
N.S.
*P< 0.05 +P<0.10 N.S.: not significant |Difference= (week l +week 3 - 2 × w e e k 2 ) / 2 n=6.
TABLE3 Total milk production, average milk contents and mammary vein concentrations of glucose and free fatty acids (FFA) during the day when insulin (0.3 I U / k g / d ) a n d / o r glucose ( 5 0 g / d ) were administered intravenously Items
Treatment Control
Milk yield (g)~ 1369_+ 473 Milk content ($)2 Fat 4. l _+0.9 a Protein 3.5 _+0.5 Lactose 4.2 _+0.6 Mammary vein substrates Glucose ( m g / d l ) 59 _+5b FFA ( m M ) 1.05_+0.08
Insulin
Glucose
Insulin/glucose
1403_+ 381
1521 _+441
1434_+419
4.0 _+0.6 a 3.6 _+0.7 4.1 _ 0.7
3.6 _+0.6 b 3.3 _+0.6 4.0 _ 0.6
3.4 _ 0.6 b 3.3 _+0.4 4.2 _ 0.4
60 _+6 b 1.13_+0.10
68 _ 5 a 1.08_+0.14
62 _+9 b 1.05_+0.10
W alues represent total yields _+SE of five collections at 08:00, 10:00, 12:00, 14:00, and 17:00 during the injection day, n = 8. 2Values represent means _+SE of contents of milk samples from the above five collections or five blood samples withdrawn at the similar interval. Values followed by different letters within rows differ significantly ( P < 0.05 ).
CHANGES OF MAMMARY VEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTY ACIDS
129
vein glucose increased by 9 mg/dl above the control (P< 0.05) by glucose infusion. In Fig. 1, changes in milk production and contents from those values at 08:00 for each group at various hours post-milking and feeding are shown. Regardless of treatment, experimental goats secreted the lowest amounts of milk between 10:00 and 14:00 and highest amounts at 08:00 and 17:00 among the five milkings measured. Milk production of control goats averaged 309, 228, 214, and 377 g at 08:00, 10:00, 12:00, 14:00 and 17:00, respectively. This downward pattern of milk secretion between two milkings during the day was apparently not related in a linear function, as might be expected, to hours post milking and feeding. Milk fat content largely decreased with time after milking, from 4.8 (08:00) to 3.0% (17:00) for control goats. Meanwhile, lactose and milk protein concentrations were relatively stable, from 4.30 and 3.36 (08:00) to 3.57 and 3.11% (17:00), respectively. There was no significant interaction between treatments and post-milking hours (data not shown), the only difference in parameters testing during the intermilkingperiod was caused by insulin on milk fat content at 10:00 when milk fat ( 5.1% ) was significantly (P<0.05) higher than that of the group receiving insulin plus glucose (3.86%). Fluctuations of levels of mammary vein glucose and FFA from those at 08:00 for goats receiving various infusions in Expt 2 are in Fig. 2. Deviations of mammary vein glucose levels during the intermilking period were not significantly different among the four groups, except at 10:00 and 12:00 in glucose-infused goats. Although equal amounts of exogenous glucose ( l 0 g) were administered at 06:00, 08:00, 10:00, 12:00 and 14:00 in that group, mammary vein glucose level was not significantly elevated at 08:00, 14:00 and 17:00 (P> 0.05 ). While, at 10:00 and 12:00, the increments in mammary vein glucose averaged 12.3 and 8.3 mg/dl above those at 08:00, which were significantly greater than the corresponding values for control goats of 0.8 and 3.6 mg/dl, respectively. The mammary vein glucose level was apparently more responsive to glucose infusion at 10:00 and 12:00 than at 06:00, 08:00 or 14:00. As for FFA levels, the deviation from values at 08:00 within each treatment was smooth along the intermilking hours, and no significant difference in deviations at any hours among groups was observed in this experiment. DISCUSSION
Insulin treatment for five days slightly stimulated feed intake in goats (Table 2), but seemed to dispose larger portions of energy into body store, instead of the mammary gland for milk production, as was indicated by the significantly heavier body weight and slightly lower milk yield induced by insulin (Table 2 ). Therefore, energy partition and production efficiency were affected by insulin unfavorably for milk secretion. Similar observations as in
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C.J. CHANG AND S.H. YOUNG
Experiment 1 have been reported by Kronfeld et al. ( 1963 ), Schmidt ( 1966 ), and Collier et al. (1984). However, direct veno-arterial concentration measurements of major milk precursors across the mammary gland (Christensen, 1985) failed to provide enough evidence that insulin may divert nutrients away from the mammary gland to adipose tissues. Probably, more complicated interactions among organs and other antagonistic hormones working compensatorily were involved. In Experiment 1, mammary vein glucose concentration averaging over the five-day period was significantly depressed by insulin for 5 mg/dl, while no change in FFA concentration was noticable. The degree of hypoglycemia caused by insulin depends on the potency and type of preparation used, the physiological status of animal, and the reading time elapsed from treatment. It is well established that NPH-insulin exerts maximal hypoglycemic effects around 8 h post-injection (Galloway, 1980). The bleeding scheme of Expt 1 covered one sampling 2 h prior to injection and two samplings (4 h and 10 h after injection) almost half-way from this low point, therefore, could better reflect the average level of glucose during the treatment period, rather than the possibly extreme low value found transiently after treatment. For FFA, maximal depression in jugular vein concentration was found earlier and for a shorter duration than glucose (Yang and Baldwin, 1973). Another possible explanation for the characteristically small changes in mammary vein substrates was postulated by Collier et al. (1984) that mammary glands extract substrates at rates proportional to their concentrations in the inlet vessels. The diurnal changes of milk production is seldom discussed in published work. Goats in Expt 2 secreted higher amounts of milk at 08:00 and 17:00 than at 10:00, 12:00, and 14:00. Hours relative to milking seemed to have much greater effects on milk production than feeding or insulin and/or glucose treatments at the specified dosages of this study. It was postulated that the mammary gland could function temporarily as a storage and buffering organ (Zak et al., 1979). Therefore, short-term changes in concentrations of blood-born substrates or hormones, caused by feeding or infusion, would not limit milk synthesis. On the contrary, factors, such as prolactin secretion, are regulated according to long-term changes in environment, therefore, patterns of milking, management scheme, and diurnal cycle may have profound effects on the mammary gland that could superimpose short-term fluctuations in blood precursors. It is known that ruminant gluconeogenesis and lipogenesis rates are maximal at the postprandial stage (Ballard et al., 1969). The release of glucagon, in response to feeding, closely parallels the release of insulin (Brockman, 1978 ), by 4-8 h after feeding and concentrations of insulin and glucagon start to fall. It is likely that during the same period when hepatic gluconeogenesis approaches a lower rate due to lower glucagon level, the mammary gland absorbs less glucose in accordance with decreasing amounts of milk produced.
CHANGES OF MAMMARY VEIN CONCENTRATIONS OF GLUCOSE AND FREE FATTY ACIDS
131
When afternoon milking is approaching, prolactin as well as other possible effectors trigger another cycle of mammary gland activity, hepatic gluconeogenesis is also accelerated to meet the increasing requirements per glucose for lactose synthesis. These actions might explain the almost constant level of mammary vein glucose despite the large variations in milk yields during the day and the various elapsed times post-feeding in control goats of Expt 2. When exogenous glucose is challenged, the insulin:glucagon ratio should be elevated, therefore, hepatic gluconeogenesis is depressed (Prior and Christenson, 1978) in order to sustain the homeostasis of glucose. According to Figs 1 and 2, elevation of mammary vein glucose by glucose infusion was most significant when milk production was the least during the day. It is possible that the already depressed hepatic gluconeogenesis during 4-8 h after feeding had reached such a low rate that a further decrease in response to exogenous glucose infusion became difficult. Since the removal of glucose by the mammary gland for milk production was the same among treatments, the mammary vein glucose level was elevated by glucose infusion at that period. Depression of average milk fat content by glucose infusion (Table 3 ) seemed not to be mediated through the action of insulin since insulin alone elevated milk fat at 10:00 compared to insulin/glucose infusion, although it was not significantly different from controls (Fig. 1 ). Regulation of glucose utilization and synthesis need further investigation. Dosage of insulin, 0.3 I U / k g / d , in Expt 2 was divided into five injections, the resulting effects on concentrations of glucose and FFA in mammary vein and milk yield were too small to be detected. More pronounced influences of insulin or glucose on milk yield or serum substrates were reported by Kronfeld et al. ( 1963 ), Rook et al. ( 1965 ), Schmidt ( 1966 ), Christensen ( 1985 ). Amounts of insulin applied in their research, based on body weight or daily rate, were similar to the present study. It is possible that goats receiving more frequent but smaller doses of insulin or glucose at a time can more rapidly adjust themselves and reverse the changes than goats receiving similar total doses but at higher volume rates and at longer intervals. The significant difference ( P < 0.05 ) in degree of deviation in milk fat content at 10:00 between insulin and insulin/glucose treated goats (Fig. 1 ) implies that insulin is not closely related to glucose regulation as in nonruminants. Fermentation products after feeding, especially propionate, seem to be potent stimulators of insulin secretion (Chase et al., 1977), which can be further confirmed by the postprandial effects of insulin. FFA extracted by the mammary gland are partly incorporated into milk fat and oxidized into energy for maintenance, milk synthesis and secretion (Annison, 1983 ). Concentration of jugular FFA was the lowest 4-6 h after feeding, but increased significantly later and was the highest just before the next feeding (deBoer et al., 1985 ). However, FFA are present at relatively low concentration in blood and arterio-venous differences across the mammary
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gland are small, therefore, it is the volatile fatty acids that are more important than FFA for milk fat synthesis (Dils, 1986). That is probably why concentrations of serum FFA were relatively insensitive to treatments and cycles of mammary gland function compared to glucose.
REFERENCES Anderson, R.R., Salah, M.S., Sheffield, L.G. and McKenzie, W.N. Jr., 1984. Milk production and lactation curve of guinea pigs. J. Dairy Sci., 67: 185-193. Annison, E.F., 1983. Metabolite utilization by the ruminant mammary gland. In: T.B. Mepham (Editor), Biochemistry of Lactation. Elsevier, Amsterdam, pp. 339. Ballard, F.J., Hanson, R.W. and Kronfeld, D.S., 1969. Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed. Proc., 28:218-231. Bassett, J.M., 1978. Endocrine factors in the control of nutrient utilization: Ruminants. Proc. Nutr. Soc., 37: 273-274. Bassett, J.M., 1981. Regulation of insulin and glucagon secretion in ruminants. In: J.M. Forbes (Editor), Hormones and Metabolism in Ruminants. Agric. Res. Counc., London, pp. 65. Baumrucker, C.R., 1985. Amino acid transport systems in bovine mammary tissue. J. Dairy Sci., 68:2436-2451. Brockman, R.P., 1978. Roles of glucagon and insulin in the regulation of metabolism in ruminants-a review. Can. Vet. J., 19: 55-62. Call, J.L., Mitchell, G.E. Jr., Ely, D.G., Little, C.O. and Tucker, R.E., 1972. Amino acids, volatile fatty acids and glucose in plasma of insulin-treated sheep. J. Anim. Sci., 34:767-77 I. Chase, L.E., Wangsness, P.J. and Martin, R.J., 1977. Portal blood insulin and metabolite changes with spontaneous feeding in steers. J. Dairy Sci., 60:410-415. Christensen, D., 1985. Effect of insulin levels on milk fat production tested. Feedstuffs, 124: l 0. Collier, R.J., McNamara, J.P., Wallace, C.R. and Dehoff, M.H., 1984. A review of endocrine regulation of metabolism during lactation. J. Anita. Sci., 59:498-510. Cummins, K.A. and Sartin, J.L., 1987. Response of insulin, glucagon, and growth hormone to intravenous glucose challenge in cows fed high fat diets. J. Dairy Sci., 70: 277-283. deBoer, G., Trenlde, A. and Young, J.W., 1985. Glucagon, insulin, growth hormone, and blood metabolites during energy restriction ketonemia of lactating cows. J. Dairy Sci., 68: 326337. Dils, R.R., 1986. Comparative aspects of milk fat synthesis. J. Dairy Sci., 69: 904-910. Falholt, K., Lund, B. and Filholt, W., 1973. An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chem. Acta, 46:105-110. Fleet, I.R. and Linzell, J.L., 1964. A rapid method of estimating fat in very small quantities of milk. J. Physiol., (London) 175: 12-20. Galloway, J.A., 1980. Insulin treatment for the early 80s: facts and questions about old and new insulins and their usage. Diabetes Care 3:615-622. Kronfeld, D.S., Mayer, G.P., Robertson, J.M. and Raggi, F., 1963. Depression of milk secretion during insulin administration. J. Dairy Sci., 46:559- 563. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem., 193: 265-275. McClary, D.G., Sartin, J.L., Kemppainen, R.J. and Williams, J.C., 1988. Insulin and growth hormone responses to glucose infusion in mature and first-lactation dairy cows. Am. J. Vet. Res., 49: 1702-1704. Prior, R.L. and Christenson, R.K., 1978. Insulin and glucose effects on glucose metabolism in pregnant and nonpregnant ewes. J. Anim. Sci., 46: 201-210.
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Rook, J.A.F., Storry, J.E. and Wheelock, J.V., 1965. Plasma glucose and acetate and milk secretion in the ruminant. J. Dairy Sci., 48: 745-747. SAS, 1985. The GLM Procedure. In: SAS User's Guide: Statistics, SAS Institute Inc. Cary, NC, pp. 470-476. Schmidt, G.H., 1966. Effect of insulin on yield and composition of milk of dairy cows. J. Dairy Sci., 49: 381-385. Shibata, S. 1981. o-Aminobiphenyl-Boric acid method. Glucose. In: Inoue, S. and Matsumoto, I. (Editors), Illustrations of Techniques in Laboratory Animal. Kiyouritsu Publishing Co., Tokyo, pp. 89-90. Slater, T.F., 1957. A rapid method for the determination of small amount of lactose in milk and tissue suspensions of mammary gland. Analyst, 82:818-825. Yang, Y.T. and Baldwin, R.L., 1973. Lipolysis in isolated cow adipose cells. J. Dairy Sci., 56: 366-374. Zak, R., Martin, A.F. and Blaugh, R., 1979. Assessment of protein turnover by use of radioisotope tracers. Physiol. Rev., 59: 407-413.