FEED-BACK INHIBITION OF MILK SECRETION: THE EFFECT OF A FRACTION OF GOAT MILK ON MILK YIELD AND COMPOSITION

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1 Quarterly Journal of Experimental Physiology (1988), 73, Printed in Great Britain FEED-BACK INHIBITION OF MILK SECRETION: THE EFFECT OF A FRACTION OF GOAT MILK ON MILK YIELD AND COMPOSITION COLIN J. WILDE, CAROLINE V. P. ADDEY, MICHAEL J. CASEY*, DAVID R. BLATCHFORD AND MALCOLM PEAKER Hannah Research Institute, Ayr KA6 SHL (MANUSCRIPT RECEIVED 29 SEPTEMBER 1987, ACCEPTED 26 NOVEMBER 1987) SUMMARY A milk fraction containing whey proteins of kda was injected into one mammary gland of lactating goats via the teat canal. This fraction produced a temporary dose-dependent reduction in milk yield in the treated gland; the milk yield of the other gland, which received an equal volume of carrier solution, was not affected. Injection of a second fraction, containing whey proteins of > 30 kda, affected milk secretion only at high doses, and this effect was not wholly specific to the treated gland. The kda fraction and the > 30 kda fraction produced similar transient changes in the concentrations of several ions and lactose in milk of the treated gland, but not in that of the untreated gland. These data indicate that a milk constituent present in the kda whey inhibits milk secretion in a temporary and reversible manner. The results are discussed in relation to regulation of milk secretion through local feedback inhibition. INTRODUCTION The rate of milk secretion by a lactating animal is regulated by the frequency of milk removal, a mechanism which acts to match the supply of milk to the demand by the offspring or the milking regime. This control is achieved not only through the release of galactopoietic hormones during suckling or milking (Cowie, Forsyth & Hart, 1980): studies on lactating goats indicate that the rate of milk secretion is also under autocrine regulation, i.e. locally active mammary factors are also involved. When one gland of the udder is milked more frequently, either hourly or thrice daily instead of twice daily (as in the other gland), the rate of milk secretion increases only in the more frequently milked gland (Blatchford & Peaker, 1982; Henderson, Blatchford & Peaker, 1983; Henderson & Peaker, 1984). This local increase, which cannot be due to systemic factors, is not the result of a decrease in physical distension of the gland (Henderson & Peaker, 1984), and is attributable directly to the more frequent removal of milk constituents. Therefore, it appears that milk contains a locally active chemical inhibitor, which decreases milk secretion by negative feed-back. The presence of milk constituents capable of inhibiting milk synthesis is supported by recent studies on the effect of milk fractions on lactose and casein synthesis by rabbit mammary explants in organ culture (Wilde & Gamble, 1984; Wilde, Calvert, Daly & Peaker, 1987 a). These experiments showed that synthesis of milk constituents was inhibited by a milk fraction containing whey proteins with a molecular mass of between 10 and 30 kda. As the inhibitory effects of this milk protein fraction were established in vitro using * Present address: Department of Veterinary Physiology and Biochemistry, University College, Dublin, Eire.

2 392 C. J. WILDE AND OTHERS explants, which reflect but do not entirely reproduce the performance of the tissue in vivo, it is important to determine whether the effect is also realized in the lactating animal. In this study, the putative inhibitory milk fraction was injected into the cistern of one gland of lactating goats. Effects on milk secretory rate and composition are compared with those in the other gland, which received an equal volume of buffered isosmotic sucrose. The effects were also compared with those obtained after the injection of a milk protein fraction that had no inhibitory effect on milk synthesis in vitro (Wilde et al. 1987a). METHODS Animals British Saanen goats were used between weeks 8 and 26 of lactation. They were milked twice daily at approximately and h; the yield of each gland and the time of milking were recorded. At each milking, half of the concentrate ration of 2-0 kg/d (Goat Mix- 1, Edinburgh School of Agriculture, Edinburgh) was given. Hay was available throughout the day. Milk fractions were prepared from fresh milk collected from a second pool of goats at the morning milking. Milk fractionation Fresh milk was defatted by centrifugation (2500 g, 15 C, 20 min) and filtration of the infranatant through glass wool. Defatted milk was centrifuged at g and 15 C for 2 h, yielding a pellet of casein micelles and a clear supernatant containing whey proteins. The whey protein fraction was subjected to ultrafiltration (nominal molecular mass cut-off 30 kda). The retentate volume was reduced by 95 % and washed with 4 volumes of 10 mm-hepes (ph 617) containing 0 3 M sucrose. The filtrate was equilibrated in 10 mm-hepes (ph 6 7) containing 0 3 M-sucrose by diafiltration with a 10 kda filter, and concentrated to 5 % of the original volume of milk using the same filter. Fractions were sterilized by passage through a 0-22,um filter and doses of units of each fraction were prepared in a final volume of ml of sterile buffered sucrose, one unit being defined as the amount of milk fraction derived from 1-0 ml of fresh milk. Intraductal injections For 2 d before intraductal injection and for up to 7 d after, milk samples were collected from both glands at each milking, and stored in vials containing a few drops of formaldehyde. On the day of intraductal injection, the animal was milked at h using oxytocin (0 1 i.u., I.v.; Sandoz Products Ltd, Feltham) to assist in the removal of alveolar milk. The tip of each teat was disinfected with 1-6% (w/v) chlorhexidine gluconate in 70 % (v/v) ethanol, and the whey protein fraction was then injected through the teat canal into the cistern of one gland using a metal catheter. The other gland received the same volume of 10 mm-hepes ph 6-7 containing 0 3 M-sucrose; both glands were then massaged. The injected material remained in the gland for h before the afternoon milking. Analytical methods Milk was analysed for Na+ and K+ using a Corning 480 digital flame photometer (Ciba Corning Diagnostics Ltd, Halstead, Essex). Measurement of Cl- concentration was performed with a Corning 925 chloride analyser. Lactose concentration was measured by a colorimetric method using o- toluidine (Hyvarinen & Nikkila, 1962). Milk protein concentration was measured by the method of Mabon & Brechany (1982). Milk fat content was measured by the method of Fleet & Linzell (1964). Calculations Unilateral effects on milk secretion after intraductal injection of whey protein fraction (test gland) and isosmotic sucrose (control gland) were detected by calculation of the relative milk yields quotient (RMYQ) as described by Linzell & Peaker (1971): RMYQ = (t2 C1)/(t, C2), where cl = yield of control gland on day 1, t, = yield of test gland on day 1, c2 = yield of control gland on day 2 and t2 = yield of test gland on day 2. RMYQ values of < 1 indicate that the milk yield of the test gland had decreased since the previous

3 FEED-BACK INHIBITION OF MILK SECRETION 393 day relative to the control gland, and therefore that the whey protein fraction had caused a unilateral decrease in secretory rate. RESULTS Milk yield Intraductal injection of the kda fraction of whey proteins into one gland of lactating goats caused a temporary reduction in the rate of milk secretion in that gland. The effect was not apparent at the normal afternoon milking following the injection: during this period the secretory rate in all glands treated with the fraction was % (mean + S.E.M.; twenty-four determinations) of that on the previous day. However, the rate of milk secretion during the next milking interval decreased to % (n = 5) and % (n = 5) of pre-injection values after treatment with doses of 100 and 500 units of the fraction respectively. This effect persisted for at least 24 h, and with higher doses was apparent for up to 48 h, although on the second day secretory rate was reduced significantly (P < 0 05) only with the highest dose of 500 units of the kda fraction. The rate of milk secretion in the other gland of the same animals, which were treated with carrier solution, was not affected. An example of the time course of the effect on milk secretion, obtained with an intermediate dose of 300 units of the kda whey fraction, is shown in Fig. 1. The inhibitory effect of the kda milk fraction was quantified by comparing the hourly rate of milk secretion in the 24 h period between the next two afternoon milkings following injection with that in the same gland before the injection. This showed that doses of units (see Methods) of the kda whey fraction each caused a significant reduction (P < 0 05) in the rate of milk secretion (Table 1), and that inhibition of milk secretion increased with increasing doses of inhibitor fraction. RMYQ values of indicated that at each dose the decrease in secretory rate occurred unilaterally in the gland treated with whey fraction. The second whey fraction, containing proteins of > 30 kda, had no effect on milk secretion rate when tested at doses of units (Fig. 1; Table 1). Injection of higher doses of 400 and 500 units each reduced the secretion rate in the test gland by 14% (P < 0 05), markedly less than the effects obtained with similar doses of the kda fraction (Table 1). RMYQ values of > 0 9 indicated that the effect of high doses of the > 30 kda fraction was not unilateral and may therefore have resulted from a systemic effect. Milk composition Inhibition of milk secretion by the kda fraction of whey proteins was accompanied by transient effects on the composition of milk in the treated gland. Unlike the depression of milk secretion, these effects were observed immediately, i.e. at the milking following intraductal injection. An example of the changes observed is shown in Fig. 2. Concentrations of Na+ and Cl- were increased by up to 300 and 100% respectively, and over the same period K+ and lactose concentrations decreased by up to 50 and 35 % respectively. A small increase of 20% in milk protein concentration was also observed after the injection, but this may be attributed to the exogenous protein introduced into the gland: for example, the increase of 0 g/100 ml in milk protein in glands receiving 200 units (equivalent to 1-4 g) of whey fraction represented an increased output of 1 9 g protein at the afternoon milking. Milk fat concentration showed a transient decrease at the milking immediately following the injection. Injection of carrier solution did not affect the ionic composition of the milk (Fig. 2).

4 394 C. J. WILDE AND OTHERS.- c) S.e.- A 110 r Injection 100 F 90 F B 'lor 100 F 90 F CD 80 p 801-0X u) 1.Y 70F RMYQ RMYQ 101i 0* l Day relative to injection Fig. 1. Changes in the rate of milk secretion by goats injected via the teat canal in one mammary gland with 300 units of whey protein fraction (@) and in the other with isosmotic sucrose carrier solution (O). A, kda whey protein fraction; B, > 30 kda whey protein fraction. Mean hourly rates of secretion during each 24 h period are expressed as a percentage of that in the same gland before injection. Day +1 is the period between successive afternoon milkings following injection. *P < 0 05 with respect to before treatment (paired t test). Insets: relative milk yields quotient (RMYQ) comparing relative changes in the yields of the glands on successive days (see Methods). Values are the mean + S.E.M. for five goats. Table 1. Effect of intraductal mammary injection of whey protein fractions on secretory rate in lactating goats kda whey fraction > 30 kda whey fraction Amount injected/dose Secretion Secretion (units)t rate (%)t RMYQ rate (%) RMYQ (5)* (5) (5)* (5) 0 94± (5)* (4) (4)* (5)* (5)* (5)* Solutions containing whey proteins of kda or > 30 kda were injected via the teat canal into one gland, while the other (control) gland received the same volume of buffered sucrose carrier solution. t One unit is the amount of milk fraction derived from I ml of fresh milk. $ The hourly secretory rate during a 24 h period between successive afternoon milkings following injection is expressed as a percentage of that in the same gland during the day before, excluding milk removed after injection of oxytocin. Values are the mean + S.E.M., with the number of determinations in parentheses. *P < 0 05, with respect to before treatment (paired t test). Relative milk yield quotient, as described by Linzell & Peaker (1971). Values of RMYQ of < I indicate a unilateral decrease in secretion rate of the treated gland relative to the control gland.

5 FEED-BACK INHIBITION OF MILK SECRETION 395 I-1 z L Injection E in-. 80 r ' Alk. ot- --Mw wa--a E o z L E E 0 0 -e CIO og Time (d) Fig. 2. Changes in milk composition of glands injected with 200 units of kda whey protein fraction (0) or isosmotic sucrose carrier solution (Ol). Values are the mean for four goats at each milking; for clarity, bars indicating the S.E.M. are shown only for determinations immediately after intraductal injection.

6 396 C. J. WILDE AND OTHERS Although the milk fraction containing serum proteins of > 30 kda did not affect the rate of milk secretion, it did, however, alter the composition of the milk, in a manner similar to that of the other protein fraction (results not shown). DISCUSSION The temporary reduction in milk secretion observed after intraductal injection of a fraction of goat milk supports other evidence (Henderson & Peaker, 1984) that milk secretion is modulated through chemical inhibition by milk constituents. Dose-dependent inhibition of milk secretion was obtained only with the kda whey protein fraction and not with the fraction containing proteins of > 30 kda. These findings are similar to the effects observed when milk fractions were included with rabbit mammary explants in organ culture: the factor(s) in defatted milk which exerted rapid reversible inhibition on lactose and casein synthesis were localized specifically in the kda fraction of the whey proteins (Wilde et al a). This fraction also inhibited milk accumulation in lactating rabbits after intraductal injection, and again the > 30 kda fraction had no effect (Wilde, Calvert & Peaker, 1987b). The change in milk composition which accompanied inhibition of milk secretion was not a specific action by constituents of the kda fraction, as milk composition was affected in a similar manner by the > 30 kda fraction, in many cases in the absence of any effect on milk yield. This indicates that changes in milk synthesis and in milk composition occurred through discrete mechanisms. The evidence from frequent-milking studies indicates that secretion of milk constituents is co-ordinately controlled by the local feedback mechanism, i.e. milk secretion is increased without affecting milk composition (Henderson et al. 1983; Maltz, Blatchford & Peaker, 1984). The changes in milk composition observed here may instead be due to non-specific effects of the whey protein fractions, perhaps through alteration of tight intercellular junctions in the secretory epithelium, thereby allowing some paracellular movement of lactose and ions (see Peaker, 1984). The dose dependence of the effect exerted by the kda protein fraction on milk secretory rate, and the demonstration that this fraction was active when introduced via the teat canal, together suggest that the degree of feed-back inhibition during normal lactation is determined by the concentration of inhibitor in the alveolar lumen. Evidence from other experiments in lactating goats supports this conclusion. First, when milk stored in the cistern of the gland is milked out, but that in the alveolar lumen is not removed, the stimulatory effect of frequent milking is not realized (Henderson & Peaker, 1986), which indicates that the site of inhibitor action is the secretory alveoli. Second, dilution of alveolar milk with isosmotic sucrose leads to an increase in the rate of milk secretion (Henderson & Peaker, 1986), presumably by reducing the concentration of inhibitor present in the alveolar lumen. Changes in inhibitor concentration during milk accumulation and after milking, and the mechanism(s) by which they are achieved, are currently under investigation. We are grateful to Elaine Lamberton, Marian Kerr and Shirley Philp for excellent technical assistance and to T. Hutchison and Mrs Sandra Paton for care of the animals.

7 FEED-BACK INHIBITION OF MILK SECRETION 397 REFERENCES BLATCHFORD, D. R. & PEAKER, M. (1982). Effect of frequent milking on milk secretion during lactation in the goat: relation to factors which limit the rate of secretion. Quarterly Journal of Experimental Physiology 67, COWIE, A. T., FORSYTH, I. A. & HART, I. C. (1980). Hormonal Control of Lactation. Berlin: Springer- Verlag. FLEET, I. R. & LINZELL, J. L. (1964). A rapid method for estimating fat in very small quantities of milk. Journal of Physiology 175, 15-17P. HENDERSON, A. J., BLATCHFORD, D. R. & PEAKER, M. (1983). The effects of milking thrice instead of twice daily on milk secretion in the goat. Quarterly Journal of Experimental Physiology 68, HENDERSON, A. J. & PEAKER, M. (1984). Feed-back control of milk secretion in the goat by a chemical in milk. Journal of Physiology 351, HENDERSON, A. J. & PEAKER, M. (1986). Effects of removing milk from the mammary ducts and alveoli, or of diluting stored milk, on the rate of milk secretion in the goat. Quarterly Journal of Experimental Physiology 72, HYVARINEN, A. & NIKKILA, E. (1962). Specific determination of blood glucose with o--toluidine. Clinica chimica acta 7, LINZELL, J. L. & PEAKER, M. (1971). The effect of oxytocin and milk removal on milk secretion in the goat. Journal of Physiology 216, MABON, R. M. & BRECHANY, E. Y. (1982). The measurement of protein in fresh and stored goats' milk by a dye-binding technique. Laboratory Practice 31, MALTZ, E., BLATCHFORD, D. R. & PEAKER, M. (1984). Effects of frequent milking on milk secretion and mammary blood flow in the goat. Quarterly Journal of Experimental Physiology 69, PEAKER, M. (1984). Secretion of ions and water. In Biochemistry oflactation, ed. MEPHAM, T. B., pp Amsterdam: Elsevier Science Publishers. PEAKER, M. & BLATCHFORD, D. R. (1987). The distribution of milk in the goat mammary gland and its relation to the rate and control of milk secretion. Quarterly Journal ofexperimental Physiology (in the Press). WILDE, C. J., CALVERT, D. T., DALY, A. & PEAKER, M. (1987 a). The effect of goat milk fractions on synthesis of milk constituents by rabbit mammary explants and on milk yield in vivo: evidence for autocrine control of milk secretion. Biochemical Journal 242, WILDE, C. J., CALVERT, D. T. & PEAKER, M. (1987b). Effect of a fraction of goat milk serum proteins on milk accumulation and enzyme activities in rabbit mammary gland. Biochemical Society Transactions 15, WILDE, C. J. & GAMBLE, J. A. (1984). Inhibition of lactose and casein synthesis in rabbit mammary explants by fractions of goat milk. Biochemical Society Transactions 13,