Camp. Biochem.

Ph_vsiol.

Vol. 102A.

No. 4, pp. 665-667,

1992

0300-9629/92

$5.00 + 0.00

0 1992Pergamon Press Ltd

Printed in Great Britain

PROLACTIN EXTRACTION DURING

AND THE ONSET OF MAMMARY OF PLASMA TRIACYLGLYCEROLS LACTOGENESIS IN THE GOAT

GORDON E. THOMPSON Hannah Research Institute, Ayr KA6 SHL, U.K. Tel.: 0292-76013; Fax: 0292-671052 (Received

21 Junuury 1992)

Abstract-l.

In untreated goats, the onset of mammary extraction of circulating triacylglycerols (TG) at parturition occurred shortly after the peak of prolactin concentration in circulating plasma. Suppressing this peak of systemic prolactin, by acutely treating with bromocriptine, delayed the onset of mammary extraction of TG and secretion of long-chain TG fatty acids. 2. Regular unilateral removal of secretion pre-partum, which brought about an early local onset of TG extraction and secretion of long-chain TG fatty acids in this treated half of the udder, was not assaciated with an increased local concentration of prolactin in secretion in this gland.

MATERIALS

INTRODUCTION

AND METHODS

Animals

The

lactating mammary gland extracts triacylglycerols (TG) from the circulating blood plasma to support secretion of long-chain TG fatty acids in milk (Annison et al., 1967). The onset of mammary extraction of plasma TG naturally occurs at parturition in the goat, when plasma progesterone

levels are low. However, regular manual removal of as much secretion as possible from the gland before parturition can make the onset of TG extraction occur several days before parturition, when circulating progesterone concentrations are moderately high (Thompson, 1991). Pre-partum removal of secretion from the mammary gland, for some unknown reason, produces high concentrations of progesterone within the secretion (Thompson, 1990) providing further evidence against the theory that the onset of mammary extraction of plasma TG during lactogenesis is triggered by release from an inhibiting effect of progesterone. The experiments reported here were undertaken to examine the possibility that the onset of mammal extraction of plasma TG, and secretion of longchain TG fatty acids in milk, is brought about by the hormone prolactin. This possibility has been examined in three experiments. First, measuring mammary extraction of TG and circulating prolactin concentrations before and after parturition in untreated goats. Second, making the same measurements in goats treated with the drug bromocriptine to prevent the normal pre-partum peak of plasma prolactin. Third, regularly removing secretion from one mammary’ gland pre-partum, which brings about an early onset of TG extraction in this “experimental” gland but not in the contralateral untreated “control” gland in the same animal (Thompson, 1990), and measuring the “local” concentrations of prolactin in secretion obtained from the two glands.

A total of eight British-Saanen goats were chronically prepared with a carotid artery and one or both subcutaneous abdominal veins exteriorized in skin-covered loops for collection of arterial and mammary venous blood samples (Linzell, 1960) by needle puncture. Experiments were conducted over three seasons. Goats were penned individually, with water and hay available continuously, and rations of concentrated food given at approx. 08.00a.m. and 16.00 p.m. daily. After parturition, kids sucked their dams for approx. 24 hr then they were separated and the nanny machine-milked at feeding times. Unilateral pre-partum removal of secretion began on day 143 of gestation; as much fluid as possible was taken manually from one mammary gland, and small (approx. 2 ml) samples taken from the contralateral “control” gland, daily at approx. 10.00 a.m. Blood samples were taken from the veins draining right and left glands, and from the carotid artery. After parturition, kids sucked both halves then both halves were machine-milked. Bromocriptine mesylate (Sigma Chemical Co., Poole, Dorset) was prepared daily by dissolving in ethanol: 5 mmol tartaric acid (1: 1, v/v) and 5 mg~goat injected intramu~ularly (Forsyth et al., 1985) at 11.00a.m. each day beginning at times ranging from 6 to 2 days before parturition and ending on day 2 post-partum. Blood samples were withdrawn into aprotinin (Sigma Chemical Co.), mixed with heparin crystals, then centrifuged at 4°C to obtain plasma. Plasma and milk samples were stored at - 18°C until analysed. Anaiyses The TG content of plasma was estimated by measuring the glycerol content, using a fluo~meter, before and after enzymic hydrolysis (Gleeson and Maughan, 1986). The TG fatty acid content of mammary secretion was estimated by thin-layer and gas-liquid chromatography as described previously (Brownhill et al., 1985). Plasma prolactin concentration was estimated by double-antibody radioimmunoassay (Flint et al., 1979) using ovine prolactin antiserum and standards (NIADDK-oPRL-19 donated by NIADDK, Bethesda, MD, U.S.A.). The sensitivity of the assay was 0.1 ng/ml and the intra- and inter-assay coefficients of

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GORDON

E.

variation were 5 and 1I%, respectively. Milk prolactin concentration was estimated by double-antibody radioimmunoassay using suitable dilutions (Malven et at., 1987) of the same reagents. The sensitivity of the assay was 0.1 ng/ml and the intra- and inter-assay coefficients of variation 8 and 17%. Catcularions Extraction of TG from circulating plasma by the mammary gland was calculated from: extraction (%) = (arterial-venous concentration)/(arterial concentration x 0.01). The significance of change in a series of measurements

made over a period of time within a group of animals was estimated using analysis of variance. The significance of differences between pairs of measurements within animals was assessed using the paired f-test; differences between different animals, and the same animals studied in different seasons, were assessed using the unpaired t-test (Snedecor and Cochran, 1956). RESULTS

Untreated goats

Prolactin concentration in arterial plasma of five goats (Fig. 1) changed significantly during the period from 5 days before, to 5 days after parturition (P < 0.05) with peak values occurring in the 2 days before parturition. Mammary extraction of plasma TG did not significantly change between day 5 and

THOMFWN

day 2 pre-partum (P > 0.05), then increased from a mean of 3% on day 2 pre-partum to a mean of 43% on day 1 post-partum (standard error of the difference (SED) = 6.5%; N = 5; P -=zO.OOl).This increase was first apparent in samples taken before parturition in four of the five goats studied. Between day 2 and day 5 post-partum, milk secretion rate increased from 5 1 to 60 g/hr gland (SED = 3.9 g/hr gland; P < 0.05), and secretion of C,, TG fatty acids in milk increased from 4.1 to 5.0 mmolihr gland (SED = 0.28 mmol/hr gland; P < 0.05) in the same interval. gromocript~ne treatment

Prolactin concentration in arterial plasma of five goats injected with bromocriptine (Fig. 1) changed significantly during the period from 5 days before to 5days after parturition (P < 0.01). The mean was slightly decreased on day 2 pre-partum, then values dropped significantly below control values on day 1 pre-partum (P < 0.001) and remained below control vafues until day 3 post-partum. Mammary extraction of plasma TG in bromocriptine-treated goats did not increase at parturition, and was significantly below the control value every day from parturition (P < 0.01) until day 3 post-partum. Milk secretion by bromocriptine-treated goats averaged 22 + 5.2 (standard error of the mean, SEM) g/hr gland (N = 5) on day 2 post-partum, which was significantly below values for untreated goats (51 k 3.8 (SEM) g/hr gland; N = 5; P < O.Ol), and remained below control values until day 5 post-partum. Secretion of Cl8 TG fatty acids in milk of bromocriptine-treated goats on day 2 post-partum, which averaged 1.2 _t 0.59 (SEM) mmol/hr gland, was also below values for untreated goats (4.1 k 0.68 (SEM) mmol/hr gland; P < 0.05) and remained below control values until day 5 post-partum (Fig. 1). The proportion of short-chain (C& to long-chain (C,,) fatty acids in milk TG was not discernibly altered by bromocriptine treatment.

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1

I

P Time (deys)

6

Fig. 1. The concentration of proiactin in arterial plasma, extraction of circulating plasma triacylglycerols (TTG)by the mammary gland and secretion of Cu TG fatty acids in milk relative to the time of parturition (P), in five untreated goats (solid lines) and five goats treated with bromocriptine (broken lines), Points represent means and vertical bars represent 1 SEM.

Pre-partum emptying

Regular removal of as much secretion as possible from one mammary gland before parturition in five goats brought about an extraction of circulating TG by this gland which averaged 18% between day 5 and day 2 pre-partum when secretion of C,, TG fatty acids in milk averaged 0.90 mmol/hr gland. The contralateral control gland in the same animals during the same time interval extracted an average of 5% of the plasma TG (SED= 3.5%; N = 5; P < 0.05). The concentration of prolactin in secretion from the gland that had secretion removed, averaged 252 ng/ml over this period; small samples of secretion taken from the control gland contained higher prola&n concentrations every day in three of the five animals but consistently lower ~on~ntrations in one animal and the mean, 428 ng/ml, was not significantly different (SED = 114 ng/ml; N = 5; P > 0.05).

DISCUSSION

In untreated goats, a peak of prolactin concentration in blood preceded the onset of mammary

Prolactin and extraction of TG from the circulation, which occurred close to the time of parturition. Injecting goats with bromocriptine over a period, to prevent this peak of systemic prolactin, also prevented the onset of TG extraction at parturition and reduced secretion of C,* TG fatty acids in milk below normal. After bromocriptine treatment ceased, and secretion was removed from the gland by machine-milking, its extraction of plasma TG and secretion of C,, TG fatty acids increased to approach normal. Bromocriptine treatment also temporarily milk volume after parturition, as depressed previously decribed (Davis et al., 1983): secretion of C 6 14 TG fatty acids in milk was similarly reduced, in agreement with observations made by Skarda et al. (1982) on the stimulation by prolactin of de nouo fatty acid synthesis in goat mammary explants in vitro. Concentrations of prolactin in secretion from the mammary gland exceeded those in plasma in the late-pregnant goat, as has been previously observed in the late-pregnant cow (Malven er al., 1987). Injecting exogenous prolactin into the lumen of a mammary gland locally stimulates lactogenesis in the rabbit (Fiddler et al., 1971). However, no evidence for a local lactogenic role of prolactin has been found in the present experiments on goats; the early onset of TG extraction brought about by unilateral regular removal of secretion before parturition did not appear to be associated with a local increase of concentration of endogenous prolactin in the fluid in this gland. The experimental results reported here support the view that the onset of extraction of plasma TG by the mammary gland before parturition and suckling, is a consequence of the pre-partum peak of circulating prolactin. However, removal of secretion from the mammary gland can also stimulate the onset of TG extraction, evidently without mediation through the hormone prolactin. Acknowledgemenrs--I thank Margaret McClelland and Jim McCann for their assistance. This research was funded by the Scottish Office Agriculture and Fisheries Department.

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in goats REFERENCES

Annison E. F., Linzell J. L., Fazakerley S. and Nichols B. W. (1967) The oxidation and utilisation of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism and the role of plasma phospholipids and neutral lipids in milk fat synthesis. Biochem. J. 102, 637-647. Brownhill J., Stewart H. J. and Thompson G. E. (1985) Pre-partum milking and the onset of secretion of milk fat in the goat. J. Physiol. 366, 291-298. Davis A. J., Maule-Walker F. M. and Saunders J. C. (1983) The role of prolactin in the control of the onset of copious milk secretion in the goat. J. Physiol. 341, 83P. Fiddler T. J., Birkinshaw M. and Falconer I. R. (1971) Effects of intraductal prolactin on some aspects of the ultrastructure and biochemistry of mammary tissue in the pseudopregnant rabbit. J. Endocr. 49, 459-469. Flint D. J., Sinnett-Smith P. A., Clegg R. A. and Vernon R. G. (1979) Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat. Biochem. J. 182, 421-427. Forsyth I. A., Byatt J. C. and Iley S. (1985) Hormone concentrations, mammary development and milk yield in goats given long-term bromocriptine treatment in pregnancy. J. Endocr. 104, 17-85. Gleeson M. and Maughan R. J. (1986) A simple enzymatic fluorimetric method for the determination of triglycerides in 10 ~1 of serum. Clin. Chim. Acta 156, 97-104. Linzell J. L. (1960) Mammary-gland blood flow and oxygen, glucose and volatile fatty acid uptake in the conscious goat. J. Physiol. 153, 492-509. Malven P. V., Head H. H. and Collier R. J. (1987) Secretion and mammary gland uptake of prolactin in dairy cows during lactogenesis. J. hairy Sci: 70, 2241-2253. Skarda J.. Urbanova E.. Houdabine L-M.. Delouis C. and Bilek J. (1982) Effects of insulin, cortisol and prolactin on lipid, protein and casein synthesis in goat mammary tissue in organ culture. Reprod. Nufr. Develop. 22, 379-386. Snedecor G. W. and Cochran W. G. (1956) Statistical Mefhods, 5th Edn. Iowa State University Press, Ames, IA. Thompson G. E. (1990) Local control of the onset of mammary extraction of plasma triglycerides during lactogenesis in the goat. J. Dairy Res. 57, 489-493. Thompson G. E. (1991) The onset of mammary extraction of plasma triglycerides and circulating levels of progesterone during lactogenesis in the cow and goat. Hormone Metab. Res. 23, 101-103.

Prolactin and the onset of mammary extraction of plasma triacylglycerols during lactogenesis in the goat.

1. In untreated goats, the onset of mammary extraction of circulating triacylglycerols (TG) at parturition occurred shortly after the peak of prolacti...
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