J. Physiol. (1975), 251, pp. 763-773 With 3 text-figure8 Printed in Great Britain
SECRETORY ACTIVITY OF GOAT MAMMARY GLANDS DURING PREGNANCY AND THE ONSET OF LACTATION
By I. R. FLEET, JANE A. GOODE, MAUREEN H. HAMON, M. S. LAURIE, J. L. LINZELL AND M. PEAKER From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 29 January 1975) SUMMARY
1. The volume of the udder and the composition of the secretion have been followed in five goats through pregnancy to the onset of lactation. 2. During the middle of pregnancy udder volume was minimal and there was little or no fluid in the teats. 3. Two stages of commencing secretary activity (lactogenesis) were defined. In the first, starting up to ten weeks pre-partum, udder volume increased and the fluid in the teats changed from an extracellular-fluid-like to a milk-like composition and acquired a high concentration of immunoglobulins. Four goats accumulated several litres of a pre-colostral fluid with a high [lactose] 6-7 weeks pre-partum. 4. Comparison of the rate of increase in udder volume with previous data for the rate of increase in empty udder volume in pregnant goats showed that the rate of secretion, even in the last few days of pregnancy, was only a few per cent of the rate immediately after parturition. 5. In the last 2-3 days of pregnancy there was a three to elevenfold increase in [citrate] in the secretion; this heralded the onset of copious secretion at about the time of parturition. 6. The changes in mammary gland activities are discussed in relation to changes in plasma hormone concentrations during pregnancy. INTRODUCTION
In spite of the immense effort that has been devoted to the study of milk secretion and mammary development, there is a dearth of information about mammary secretary activity at times other than during lactation. Histological signs of secretary activity can be detected from about halfway through pregnancy (see Cowie & Tindal, 1971) but the amount and
I. R. FLEET AND OTHERS 764 composition of the fluid in the glands is a matter of speculation in most species. Since the aqueous phase forms the bulk of milk we have studied the mechanisms responsible for its composition (Linzell & Peaker, 1971a, b). However, the fluid stored in the glands of unmilked goats during late pregnancy differs from normal milk in that the concentrations of sodium, chloride and immunoglobulins are higher while those of lactose and potassium are lower. Also, in contrast to the situation in lactation, the epithelium is 'leaky' in late pregnancy, so that to some extent lactose and ions can pass by a 'paracellular route' down their concentration gradients between secretion and extracellular fluid (Linzell & Peaker, 1973, 1974). Furthermore, mammary ducts are impermeable to lactose and ions in lactation but are permeable in non-lactating, non-pregnant animals (Linzell & Peaker, 1971c). Before attempting to study the mechanisms underlying these phenomena it is clearly necessary to know the nature and time course of the changes in composition of the secretion and in secretary activity during pregnancy and the onset of lactation in this species. Comparison of the data with those for hormonal changes during pregnancy, obtained by ourselves and other workers, suggests that some of the events can be correlated with changes in plasma hormone concentrations. The rapid rise in citrate secretion at term has been described briefly previously (Peaker & Linzell, 1975). METHODS Animals. Five goats were studied during 1973-1974 (Table 1). Milking was ceased abruptly in November 1973, 8-9 months after the previous parturition. Goat Linda was mated 2 weeks before the cessation of milking and the other four at the first oestrus after milking was stopped. All goats became pregnant and there was a period of mammary quiescence between involution of the glands and their regrowth later in pregnancy. Samples. On the day before cessation of milking the glands were evacuated thoroughly by milking following the i.v. injection of oxytocin (100 m-u.), and the udder volume (i.e. empty udder volume) measured by displacement of water (Linzell, 1966). The animals were then studied at approximately weekly intervals and more frequently near term. The udder volume was measured (i.e. full udder volume) and samples (5-10 ml. if possible) were taken aseptically from each teat; the teat was then sealed again with pliable plastic film (Nobecutane spray, B.D.H.). After parturition, when the young had ingested some colostrum, full and empty udder volumes were measured. Analytical methods. Immediately the sample had been taken into a test tube a portion was drawn anaerobically into a syringe from the bottom of the tube and analysed for pH, PCO2 and total C02; from these values the concentrations of H2CO3 and HCO3 were calculated as described by Linzell & Peaker (1975). The remainder of each sample was analysed for fat (Fleet & Linzell, 1964), immunoglobulins (see Peaker & Linzell, 1974), citrate (White & Davies, 1963), protein
MAMMARY SECRETION DURING PREGNANCY
(Udy, 1956 using Orange G dye), phosphate (Technicon method N-4b for dialysable phosphate) and for lactose, sodium, potassium and chloride (Fleet, Linzell & Peaker, 1972). TABLE 1. Details of the goats studied Milk yield (1./day) _A Number of Age at cesat at previous Gestation Number sation start of drying pregperiod of young of lacGoat Fig. Breed off nancies (days) born milking tation Linda Saanen* 4 152 2 (l) 1-06 61 1 Helga Saanen 24 2 155 2 (d) 1-21 2-46 Zsa Zsat jSaanen 44 3 148 2 (??) 1'83 3-10 24 Saanen Cheryl ) 1 2 (&) 1-30 155 2 Hayleyt Windsor§ 44 3 2 ("Y) 148 102 2-70 * Not pure Saanen ancestry. t One gland autotransplanted to neck. t ? stillborn. § In previous publications goats of this breed have been incorrectly referred to as Welsh; they are in fact descended from animals imported from Kashmir in 1823 and kept in Windsor Great Park from 1828 until 1936 when the herd was transferred to the Zoological Society of London (Whitehead, 1972). __
In this paper we report the changes in mammary secretary activity, from the 'dry period' in mid-pregnancy until after parturition; the changes following cessation of milking and during involution will be reported later. Inspection of the results showed that four phases could be distinguished in the period considered, namely the dry period and the sequential accumulation or secretion of pre-colostrum, colostrum and milk. Since the time course of the changes appeared similar in the four Saanen or Saanen cross goats, the data for these were bulked, and are shown in Fig. 1. The data for the Windsor goat Hayley which showed similar changes but with a different time course are shown separately (Fig. 2). Dry period During the middle of pregnancy, from weeks 4-10 to 12-15, the udder volumes were at their minimum (mean 48 % of the empty udder volume before the cessation of milking). There was very little fluid in the glands and in the Windsor goat (Hayley) and a Saanen (Linda) none at all could be obtained by milking. The fluid from the three Saanen goats resembled extracellular fluid rather than milk in appearance and composition except that the immunoglobulin concentration was increasing during this phase (Table 2).
I. R. FLEET AND OTHERS 100 Udcler volume 806060 (% max.) 40 120 80
[CI] (mm) 70 3 100
[Pi I (rng/100 ml.)
~ 160 [Citrate] 120 8 _ (IiiglO00 ml.) 40
[H C 03(mM) 10 [H2*C03](mM) 1*0
pH [Fat] (g/100
(g/100 ml.) [IgG] (g/100 ml.)
Weeks from parturition
Fig. 1. Changes in mammary volume and the composition of the fluid in the gland from 14 weeks before, until after, parturition in four Saanen goats (mean ± s.E. of three to fifteen determinations; the width of the bars indicates the period over which data were bulked).
Pre-colostrum, colostrum and milk formation After 12-15 weeks of pregnancy (approximately 7-10 weeks pre-partum) the udder volume started to increase and, in all except the Windsor goat (Fig. 2), where fluid could not be obtained until two weeks pre-partum, fluid
MAMMARY SECRETION DURING PREGNANCY
20 Uddler volume (L)
[Na] (mM) 90
'lactose] (mM) [CI](mM) [P (mg/100 ml.)
160 80 0
120 L 80 L 40 0 100 [ 60 20 0
[H2CO3] (m M) 0 pH
Pco,(mmHg) Tot:al [COj (mm)
60 685L 45 *25
[1rat] (g/100 ml.) 20
-in] (g/100 ml.)
[Ig,G] (g/100 ml.)
10 12 4
-14 -10 -6 -2 -12 -8 -4 0 Weeks from parturition
Fig. 2. Changes in mammary volume and the composition of the fluid in the glands of the Windsor goat, Hayley. 0, autotransplanted gland; 0, gland in situ.
could be obtained from the teat. However, in all goats the fluid changed in composition from extracellular-like to milk-like in that there were marked increases in [lactose], [K], [phosphate], and decreases in pH, [Na], [Cl] and [HCO3] (Table 2). The immunoglobulin concentration continued to rise and was high
I. R. FLEET AND OTHERS
TABLE 2. Characteristics of mammary sections in goats
IgG Citrate Lactose K+ Na+ C1HCOpH
Dry period Low Low Low Low High High High High
Pre-colostrum Colostrum High High Low High Rising--> > Rising + Falling > Falling Low Falling Low Falling
Milk Very low High High High Low Low Low Low
near term. As noted previously (Linzell & Peaker, 1974) the immunoglobulin concentration decreased when the goats were first suckled or were milked (i.e. colostrum changed to milk), and the composition of the aqueous phase reached that of milk at about the time of parturition; this latter event can be interpreted as the time at which the 'tight junctions' of the epithelium became truly 'tight' (Linzell & Peaker, 1974).
In all animals there was a dramatic rise in the citrate concentration of 3-11 fold in the period 2-3 days before to 2-3 days after parturition. Thus the characteristics of true colostrum in the goat are the high concentrations of citrate and immunoglobulins (Table 2). DISCUSSION
Onset of secretary activity Before discussing the changes that take place from about the middle of pregnancy until after parturition, it is necessary to consider the terms colostrum and lactogenesis because both have been used variously by different authors. Colostrum is a latin word, which still appears in most European languages and refers specifically to cows and is defined by the Shorter Oxford Dictionary as 'the first milk secreted by a mammal after parturition'. Since we have shown previously (Linzell & Peaker, 1974) and in the present work that the composition of the secretion can change markedly shortly before term it is not strictly correct to refer to the fluid in the mammary gland more than a day or two before parturition as colostrum. Although the time of onset of the pre-colostral secretion varies its composition is characterized by the high concentration of immunoglobulins whereas true colostrum has a high citrate concentration as well. This also applies to the cow but in man, where copious milk secretion does not start until about four days after childbirth, [citrate] is low at birth
769 MAMMARY SECRETION DURING PREGNANCY and rises to a maximum on day 4 (Peaker & Linzell, 1975). Thus citrate is indicative of the onset of milk secretion. The term lactogenesis was first used to describe the action of the lactogenic and other hormones in producing visible or histological signs of milk in mammary tissue. Following the introduction of the term galactopoiesis to mean an increase in milk yield during established lactation, lactogenesis began to be used in a second sense - the onset of copious secretion at about the time of parturition by cells already capable of secretion. However, recently workers studying the effect of hormones on mammary explants from pregnant mice have described the appearance of organelles associated with milk synthesis as lactogenesis, although in vivo these can be detected sometime before the onset of copious secretion. Therefore we propose to call the onset of secretary activity, i.e. the gradual appearance of pre-colostrum in the gland, lactogenesis stage I, and the onset of copious milk secretion at about the time of parturition, lactogenesis stage II (see Barry, 1970) In goats lactogenesis stage I was characterized by an increase in udder volume partly due to the slow accumulation of a fluid with an increasing [lactose]. In rats the appearance of lactose in mammary tissue at about the time of parturition has been used as a criterion of lactogenesis (see Kuhn, 1971). Clearly the appearance of lactose could not be used as a criterion for lactogenesis stage II in the goat which is characterized by a rapid rise in [citrate]. Similarly in cows (Hartmann, 1973) and sheep (Hartmann, Trevethan & Shelton, 1973) [lactose] in the fluid starts to rise 10-30 days pre-partum.
Correlation with hormonal changes It is of obvious interest to compare the time course of the changes in mammary growth, size and function with changes in the concentrations of hormones in plasma during pregnancy; this is done in Fig. 3. The Windsor goat Hayley is not included because the time course of changes was different and because hormonal changes have been studied in Saanens, a dairy breed. In view of the differences between these animals and the Windsor goat - a breed not selected for milk yield - the possibility that selective breeding has altered the relative timing of lactogenesis stage I must be considered. Apart from the changes in composition of the fluid, changes in mammary growth (empty udder volumes of goats of this herd during pregnancy, from Linzell, 1966) and in total udder volume are shown in Fig. 3. From the difference between these two curves and the final absolute values for udder and secretion volumes, the rate of secretion pre-partum was calculated and compared with that measured by milking in the first few
1. R. FLEET AND OTHERS
i blood I ,w ) g. min)l I
0 -20 -1l 6 -12 -8 -4 0 -20 -16 -12 -8 -4 -18 -14 -10 -6 -2 +2 -18 -14 -10 -6 -2 +2 Weeks from parturition Stage II Stage I Stage II Stage I Lactogenesis Lactogenesis
Fig. 3. Comparison of mean time course of udder growth, secretary rate and lactogenesis stages I and II with plasma hormone concentrations in the goat. The empty udder volume is recalculated from the data of Linzell (1966), and the rate of secretion pre-partum from the difference between this curve and the present measurements on unmilked glands together with the amount of secretion in the glands at term. Since the measurements of udder volume in the present studies were influenced by retained secretion from the previous lactation, the curves for empty and total volumes are superimposed at the time of minimum udder volume of the unemptied glands (arrow). Prolactin and placental lactogen data from Buttle et al. (1972), oestrogens (total unconjugated) from Challis & Linzell (1971), progesterone for goats carrying twins from Thorburn & Schneider (1972), cortisol from Paterson & Linzell (1971) and mammary blood flow calculated from the data of Reynolds (1969) and Linzell (1974). The line showing the onset of lactogenesis stage I is based on the increase in [lactose] in the fluid (double arrow).
771 MAMMARY SECRETION DURING PREGNANCY days post-partum. Within about a day the rate of secretion increased by approximately 45-fold confirming that the rate of onset of copious secretion (lactogenesis stage II) is rapid. The shapes of the curves of secretory activity and circulating hormone concentrations plotted against time show similarities which could suggest where causal relationships might be sought. For example, the shape of the oestrogen curve bears a marked resemblance to that of udder growth. The rise in immunoglobulins also appears to be correlated with oestrogens - a finding in agreement with the work of Smith & Ferguson (1970) and Peaker & Linzell (1974) who found that exogenous oestrogens promote the secretion of immunoglobulins in non-lactating, non-pregnant cows and in lactating goats respectively. Circulating prolactin levels are negligible until mid-pregnancy and then start to rise steadily. At this time fluid begins to accumulate in the gland and [lactose] in the fluid increases. From these observations one might infer that, in the presence of progesterone and placental lactogen, mammary growth is largely determined by the level of oestrogens while prolactin secretion appears to correlate with the secretary ability of the cells. The systemic trigger for lactogenesis stage II is still unknown but since oestrogen, progesterone and placental lactogen fall precipitously at term, while prolactin and cortisol rise, these data are consistent with the early view of Meites (1953) that oestrogens and progesterone in the presence of prolactin promote growth but inhibit secretion at a high rate, while prolactin acting alone primarily promotes secretion. With data on the patterns of change in mammary function and of hormone concentrations, it is clear that an experimental rather than a correlative approach to the hormonal factors controlling mammary function can now be embarked upon in this species. However, local factors within the gland may over-ride some hormonal events. Thus if goats are milked unilaterally pre-partum, the yield increases and the composition of the secretion changes to that of normal milk in the milked gland only (Linzell & Peaker, 1974) and so an understanding of these processes is also important in elucidating the control of mammary function in pregnancy and at parturition. We are most grateful to the late G. J. Miller and A. Bucke for assistance with the animals, and to Dr A. T. Cowie for discussions on terminology.
I. R. FLEET AND OTHERS REFERENCES
BARRY, J. M. (1970). Review of Lactogenes"i, ed. REYNOLDS, M. & FOLLEY, S. J. Philadelphia: University of Pennsylvania Press. BUTTLE, H. L., FORSYTH, I. A. & KNAGGS, G. S. (1972). Plasma prolactin measured by radioimmunoassay and bioassay in pregnant and lactating goats and the occurrence of a placental lactogen. J. Endocr. 53, 483-491. CHALLIS, J. R. G. & LINZELL, J. L. (1971). The concentration of total unconjugated oestrogens in the plasma of pregnant goats. J. Reprod. Fert. 26, 401-404. CownE, A. T. & TINDAL, J. S. (1971). The Physiology of Lactation. London: Arnold. FLEET, I. R. & LINZELL, J. L. (1964). A rapid method of estimating fat in very small quantities of milk. J. Physiol. 175, 15-17P. FLEET, I. R., LINZELL, J. L. & PEAKER, M. (1972). The use of an autoanalyzer for the rapid analysis of milk constituents affected by subclinical mastitis. Br. vet. J. 128, 297-300. HARTMANN, P. E. (1973). Changes in the composition and yield of the mammary secretion of cows during the initiation of lactation. J. Endocr. 59, 231-247. HARTMANN, P. E., TREVETHAN, P. & SHELTON, J. N. (1973). Progesterone and oestrogen and the initiation of lactation in ewes. J. Endocr. 59, 249-259. KuHN, N. J. (1971). Control of lactogenesis and lactose biosynthesis. In Lactation, ed. FALCONER, I. R., pp. 161-176. London: Butterworths. LINZELL, J. L. (1966). Measurement of udder growth in live goats as an index of mammary growth and function. J. Dairy Sci. 49, 307-311. LINZELL, J. L. (1974). Mammary blood flow and methods of identifying and measuring precursors of milk. In Lactation, vol. 1, ed. LARSON, B. L. & SMITH, V. R., pp. 143-225. New York: Academic Press. LINZELL, J. L. & PEAKER, M. (1971 a). Mechanism of milk secretion. Physiol. Rev. 51, 564-597. LINZELL, J. L. & PEAKER, M. (1971 b). Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretary mechanism. J. Physiol. 216, 683-700. LINZELL, J. L. & PEAKER, M. (1971c). The permeability of mammary ducts. J. Physiol. 216, 701-716. LINZELL, J. L. & PEAXER, M. (1973). Changes in mammary gland permeability at the onset of lactation in the goat: an effect on tight junctions? J. Physiol. 230, 13-14P. LINZELL, J. L. & PEAKER, M. (1974). Changes in colostrum composition and in the permeability of the mammary epithelium at about the time of parturition in the goat. J. Physiol. 243, 129-151. LINZELL, J. L. & PEAKER, M. (1975). The distribution and movements of carbon dioxide, carbonic acid and bicarbonate between blood and milk in the goat. J. Physiol. 244, 771-782. MEITES, J. (1953). Recent studies on the mechanism controlling the initiation of lactation. Revue can. Biol. 13, 359-370. PATERSON, J. Y. F. & LINZELL, J. L. (1971). The secretion of cortisol and its mammary uptake in the goat. J. Endocr. 50, 493-499. PEAxER, M. & LINZELL, J. L. (1974). The effects of oestrus and exogenous oestrogens on milk secretion in the goat. J. Endocr. 61, 231-240. PEAKER, M. & LINZELL, J. L. (1975). Citrate in milk: a harbinger of lactogenesis. Nature, Lond. 253, 464.
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REYNOLDS, M. (1969). Relationship of mammary circulation and oxygen consumption to lactogenesis. In Lactogenesis, ed. REYNOLDS, M. & FOLLEY, S. J., pp. 145-151. Philadelphia: University of Pennsylvania Press. SMITH, K. L. & FERGUSON, L. C. (1970). Bovine colostrum formation: a possible role of estrogen. Fedn Proc. 29, 1455. THORBURN, G. D. & SCHNEIDER, W. (1972). The progesterone concentration in the plasma of the goat during the oestrous cycle and pregnancy. J. Endocr. 52, 23-26. UDY, 0. C. (1956). A rapid method for estimating total protein in milk. Nature. Lond. 178, 314-315. WHITE, J. C. D. & DAVIES, D. T. (1963). The determination of citric acid in milk and milk sera. J. Dairy Res. 30, 171-189. WHITEHEAD, G. K. (1972). The Wild Goats of Great Britain and Ireland. Newton Abbot: Davis & Charles.