sheep in order to measure the distribution of blood flow in half the udder. Values

Transcription

1 J. Phyeiol. (1980), 302, pp With 1 text-figure Printed in Great Britain THE DISTRIBUTION OF BLOOD FLOW IN THE UDDER OF THE SHEEP AND CHANGES BROUGHT ABOUT BY COLD EXPOSURE AND LACTATION BY G. E. THOMPSON From the A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT (Received 8 August 19719) SUMMARY 1. Microspheres have been injected into an external iliac artery of conscious female sheep in order to measure the distribution of blood flow in half the udder. Values were obtained from five non-lactating ewes in a neutral environment and in a cold environment. Values were also obtained from five lactating ewes in a neutral environment days after parturition. 2. In non-lactating sheep in a neutral environment, blood flow to half the udder averaged 11X7 ml. min- of which 43 % was distributed to lobulo-alveolar tissue, 10 % to skin covering the udder, 2 % to the teat, 20 % to inguinal adipose tissue and 25 % to lymph tissue. 3. Cold exposure significantly reduced udder skin and teat blood flow, by approximately the same amount as it reduced leg skin blood flow, but significantly increased blood flow to adipose tissue in the udder, so total blood flow to the udder was not affected. 4. In lactating sheep, blood flow to half the udder was significantly higher, averaging 357 ml. min-. Flow to the teat was slightly greater than in non-lactating sheep in a neutral environment, but flow to lobulo-alveolar tissue was much greater and accounted for 98 % of total udder blood flow in lactating sheep. INTRODUCTION The udder contains a variety of tissues and most measurements of total udder blood flow (e.g. Reynolds, Linzell & Rasmussen, 1960) have given values which are the sum or the mean of the individual blood flows to these tissues. In non-lactating animals skin, adipose and lymph tissues make up a larger part of the total weight of the udder and might be expected to receive a significant portion of its total blood flow. The present experiments were undertaken to partition blood flow to the udder of sheep, using radioactively-labelled microspheres. These were injected into the external iliac artery; corrosion casts of this artery have shown that one of its branches, the pudendoepigastric artery, supplies the tissues of the sheep's udder with the possible exception of the extreme caudal region where a branch of the pudendoepigastric, the caudal mammary artery, anastomoses with the perineal artery which is a branch of the internal iliac artery (Tanudimadja, Getty & Ghoshal, 1968). It is known that blood vessels cross the uddder mid line, and when animals are /80/ $ The Physiological Society.

2 380 G. E. THOMPSON prepared for chronic measurements of blood flow through half the gland these vessels are ligated (Linzell, 1960). In the present experiments microspheres injected into one half of the udder were traced in the contralateral side and estimates of blood flow between the two halves were made. Measurements of blood flow through the udder in lactating goats have shown that mild acute cold stress has no effect on total blood flow but moderate cold stress reduces it (Thompson & Thomson, 1977). In the present experiments the effect of an acute cold stress on blood flow to individual udder tissues has been studied in non-lactating sheep. The effect on udder skin blood flow is interesting because udder surface temperature of sheep does not change much with changing environmental temperature and this has led to the suggestion that it is not under vasomotor control (Smith, Heath & Ingram, 1978). The teat was considered apart from udder skin in view of the suggestion that blood flow to the teat may control its local temperature in extremely cold environments (Nisbet, 1956). Measurements of blood flow to tissues in the udders of non-lactating and lactating conscious sheep have also been made in the present experiments. METHODS Animadl8. A total of ten Clun Forest ewes were used. Five of these were non-lactating and cyclic in the breeding season, and five were lactating and anoestrous. Four of the non-lactating animals were parous. One of the lactating animals was suckling twins and the others had single lambs; experiments were carried out between 27 and 36 days after parturition when lactation is likely to be near its peak (Peart, Edwards & Donaldson, 1972). The non-lactating animals were fed 1000 g day-" chaff and 450 g day-' concentrated food. The lactating animals were fed 1000 g day-1 chaff and 1300 g day-1 concentrate. Water was available at all times. All ewes had fleeces clipped to make coat depth approximately 5 mm, and were housed in a temperature of 'C. Experiments were carried out in separate neutral or cold chambers, which the sheep and lambs were accustomed to. The non-lactating ewes were also accustomed to the procedure for measuring their oxygen consumption. General anaesthesia of ewes was induced with pentobarbitone sodium (Sagatal, May and Baker Ltd, Dagenham) and maintained with fluothane (I.C.I., Macclesfield) in a closed circuit. A polyethylene tube (i.d. 0-8 mm, o.d. 1-2 mm, Dural Plastics, Dural, Australia), for injection of microspheres, was inserted in a saphenous artery and its tip advanced to the dorsal aorta. Cold saline injected into this cannula produced a fall in temperature of a thermistor placed in the lumen of the femoral artery of the contralateral leg. The cannula was then gradually withdrawn, until injectate did not flow down the opposite leg, and its position fixed with a suture that tied around the saphenous artery. A similar polyethylene tube, for withdrawal of microspheres and blood, was inserted through a purse-string suture in the wall of the femoral artery and its tip advanced 6 cm before it was fixed to the purse-string suture (Fig. 1). The animals recovered rapidly from this operation and experiments were carried out 1-4 days later. Experimental procedure. Non-lactating ewes were each placed in both neutral ( ) and cold (0± 1) 'C environmental temperatures. A preliminary experiment on one non-lactating ewe, in which rectal, ear-skin and back-skin temperatures and oxygen consumption were measured at a variety of environmental temperatures, indicated that skin vasodilatation was maximal in this neutral temperature, and vasoconstriction and oxygen consumption were stimulated in this cold temperature. The sequence of environmental treatment was allocated randomly, three animals being exposed to the control environment first and two animals to the cold environment first. The second environmental treatment immediately followed the first, the animals remaining in each environment for 2 hr during which oxygen consumption was measured continuously, and at the end of 2 hr microspheres were injected. Three sheep were injected with 85Sr-labelled microspheres in the control environment and 141Ce-labelled micro-

3 UDDER BLOOD FLOW 381 spheres in the cold environment, and the other two sheep vice versa. After the second injection of microspheres the animals were injected with a lethal dose of pentobarbitone sodium (Euthatal, May & Baker Ltd) followed by a saturated solution of potassium chloride. Lactating ewes were placed in a neutral ( C) environmental temperature for about 2 hr, with their lambs free to suckle, before 85Sr-labelled microspheres were injected. The lambs were then removed and the ewes killed as before. Kidney, lung and the various tissues within right and left halves of the udder, were dissected from each carcass. Between 5 and 10 g of lobulo-alveolar tissue in the perineal area was separated from the rest of the lobulo-alveolar tissue. Dissectable adipose tissue was not found under the External iliac Internal iliac Deep femoral Deep circumflex iliac Dorsal aorta XI 4 '!V- Femoral Microspheres * Saphenous to udder 'l ' Cannula for injection of microspheres - Cannula for withdrawal of blood and microspheres Fig. 1. Diagram representing a lateral view of the arterial supply to the hind quarters and udder of one side of the sheep. skin covering the udder, but an inguinal fat pad was found, lying between the abdominal wall and the lobulo-alveolar tissue of the udder. A cylinder of skin was cut from the top of the leg on the side of the animal that was injected with microspheres. All tissues were weighed and minced. The positions of the arterial cannulae were checked during dissection; the tip of the infusion cannula lay in the external iliac artery close to the origin of the deep circumflex iliac artery, and the tip of the withdrawal cannula lay in the femoral artery close to its origin from the external iliac artery. The length of the external iliac artery was measured, and ranged from 8-5 to 10.5 cm. Meagurerments. Oxygen consumption was measured using a head cage attached to an open circuit and a paramagnetic oxygen analyser (Model OA 184, Servomex Ltd, Crowborough). Blood flows from the external iliac artery to the udder tissues and to leg skin were measured using the technique devised by Makowski, Meschia, Droegemueller & Battaglia (1968) that employs a syringe, which withdraws blood and microspheres from the circulation at a known rate, as a 'reference organ'. Between 1 6 x 106 and 2-3 x 106 microspheres (3M Company, St Paul, U.S.A.), designated 15 ± 3 Itm in diameter and contained in ml. saline with a drop of Tween 80, were injected into the external iliac artery. The injection syringe was held against a high-speed vibrator immediately before injection to ensure even suspension of the microspheres. Before the injection began, and continuing for more than 30 s after it had finished, downstream arterial blood and microspheres were withdrawn from the femoral artery using a syringe pump. Samples of blood and tissues were put into vials, filled to a height of 2 cm, and their radioactivity measured with a A-counter (Model 80000, LKB-Wallac, Turku, Finland). Pulse-height discriminators on the counter were set to count the 145 kev photopeak of 141Ce and the 513 kev

4 382 G. E. THOMPSON photopeak of 86Sr. "'1Ce produced negligible radioactivity in the "Sr photopeak, but the channel for counting the 14'Ce photopeak had to be narrowed to reduce counts from 86Sr to about 14 % of the counts at 513 kev. This figure was checked at each counting, and corrected for. Microspheres which passed from the injected half of the udder to the opposite half were counted and blood flow across the mid line was calculated. It was then assumed that there was a similar flow of blood, which did not contain microspheres, from the uninjected to the injected half of the udder. This flow was corrected for weight differences between the two halves and then total flow to the udder tissues was calculated by adding this value for flow from the contralateral external iliac artery to the value for flow from the ipsilateral external iliac artery. Statistic. The conventional paired t test was used to estimate the statistical significance of differences between values obtained from the non-lactating ewes when they were exposed to neutral and cold environments. The unpaired t test was used to compare values from nonlactating ewes and lactating ewes in a neutral environment. Non-lactating and lactating ewes RESULTS in a neutral environment The combined weight of the udder tissues was greater in lactating than in nonlactating animals. This was mostly due to a greater weight of lobulo-alveolar tissue but the weights of lymph tissue, teat and of skin covering the udder were also greater (Table 1). In non-lactating ewes in the neutral environment, blood flow to half the udder averaged 11*7 ml. min-' and 43 % of this flow was to lobulo-alveolar tissue, 10 % to skin covering the udder, 2 % to the teat, 20 % to adipose tissue and 25 % to lymph tissue. The lymph nodes comprised only 1-3 % of the combined weight of the udder tissues (Table 1) and the blood flow per unit weight of lymph tissue was very high, averaging 18-6 ml. 10 g'1 min-. Blood flow per unit weight was not significantly different in leg skin, udder skin and teat. The values for udder tissue and leg skin blood flow are measures of 'capillary' or 'nutritive' flow because appreciable numbers of microspheres must have passed through the tissues supplied by the external iliac artery and were lodged in lung tissue. Negligible radioactivity was found in kidney tissue, suggesting that few microspheres passed through the lung and were recirculated. Blood flow to the udder half was much greater in lactating than non-lactating animals. In lactating animals this averaged 357 ml. min' of which 98 % was to lobulo-alveolar tissue. The increase in lactating animals was due to increased weight of secretary tissue and to a large increase in blood flow per unit weight of secretary tissue. Blood flow to the teat was also significantly greater in lactating than nonlactating sheep. This was mostly due to the increased weight of teat; blood flow per unit weight of teat tissue was slightly, but not significantly, increased. Blood flow per unit weight of lymph tissue was lower in lactating animals, averaging 6-3 ml. 10 gel min', but this is not significantly lower than the value for non-lactating animals. The appearance of microspheres in the half of the udder not injected was sometimes marked but sometimes so small as to be similar to their appearance in kidney tissue. However, the percentage contribution of blood flow across the midline to total blood flow in the udder tissues has been calculated, uncorrected for recirculation of microspheres. In non-lactating ewes the rates ipsilateral: contralateral flow

5 UDDER BLOOD FLOW averaged 94:6 in lobulo-alveolar tissue, 94:6 in adipose tissue, 97:3 in lymph tissue, 86:14 in skin and 94:6 in teat tissue. In lactating ewes blood flow across the midline was less important and the means were 99:1 in lobulo-alveolar tissue, 98:2 in adipose tissue, 99:1 in lymph tissue, 92:8 in skin and 99:1 in teat tissue. TABLE 1. Body weights, tissue weights and blood flows (Q) in half the udder of five non-lactating and five lactating sheep (means ±s.e. of mean) Non-lactating Body weight (kg) 56-1 ± 2-23 Udder half weight (g) ± Secretory tissue weight (g) 96-6 ± Adipose tissue weight (g) Lymph node weight (g) 2-1 ± 0-59 Teat weight (g) 241± 0-18 Skin weight (g) 13-6 ± 1-23 Udder half Q (ml:10 g-' min-') Secretory tissue Q (ml. 10 g-' mimd) 0-58 ± Adipose tissue Q (ml. 10 g-1 min-') 0.55 ± Lymph node Q (ml. 10 g-1 min') 18-65± Teat Q (ml.10 g-' min-') 0-82 ± Skin Q (ml.10 g-' min-) 0-90 ± Udder half Q (ml.min-') 11-68± Secretory tissue Q (ml. min-') 4-88 ± Adipose tissue Q (ml. min') Lymph node Q (ml. min-') Teat Q (ml. min-') Skin 0 (ml min-') * P < 0-05, ** P < 0-01, *** P < Lactating *** *** ±048* 3.8±0-50* 29-1 ± 3.64** 6-69 ± 0.818*** 7-57 ± 0 909*** 0-80 ± *** ± *** * TABLE 2. The effect of cold exposure on total oxygen consumption and blood flow (Q) to half the udder tissues and to leg skin, of five non-lactating sheep Neutral ( ) Cold (0 + 1 OC) Difference environment environment +S.E. Oxygen consumption (ml. min') ± 28** Udder half Q (ml. min-') Secretory tissue Q (ml. min') Adipose tissue Q (ml. min-') * Lymph node Q (ml. min-) Teat Q (ml. min1) * Udder skin Q (ml. min') * t Leg skin Q (ml. 10 g'1.min-') * t values for only four animals. * P < 0-05, ** P < It cannot be satisfactorily decided if the internal iliac artery, via the perineal artery, contributed to udder blood flow in the caudal region. In three non-lactating animals pieces of secretary tissue taken from the region of the anastomosis between the caudal mammary artery and the perineal artery contained fewer microspheres per unit weight than the rest of the secretary tissue, which suggests dilution of microspheres from the external iliac artery with blood from the internal iliac artery, but in the remaining non-lactating and lactating animals there was no obvious difference. 383

6 384 G. E. THOMPSON Non-lactating ewes in neutral and cold environments Cold exposure increased the total oxygen consumption of non-lactating ewes by 45 % of the value obtained in a neutral environment, and reduced leg skin blood flow to 47 % of the value obtained in a neutral environment (Table 2). Cold exposure did not significantly alter total udder blood flow but there was a redistribution of flow within the udder. Udder skin and teat blood flow were reduced by approximately the same amount as was leg skin flow, but adipose tissue blood flow was increased. Blood flow to lobulo-alveolar tissue and lymph tissue was not significantly affected. DISCUSSION Blood flow from the external iliac artery to the tissues of the udder and to leg skin has been measured using 15 #tm diameter 141Ce- and 85Sr-labelled microspheres. The possibility that there may also be some blood supplied to the udder from the internal iliac artery has not been ruled out but this is unlikely to be an important part of the total flow. Microspheres of this diameter are known to pass through arterio-venous anastomoses but not capillaries (Hales, 1974) and so the values obtained are for 'capillary' or 'nutritive' flow. The presence of microspheres in the lung in the present experiments does not demonstrate the existence of arteriovenous anastomoses in the udder because they may have reached the lung via leg skin, which is relatively rich in arterio-venous anastomoses (Molyneaux, 1965). Blood flow to lobulo-alveolar tissue and lymph tissue in the udder did not change during cold exposure but reductions in udder skin and teat blood flow were approximately balanced by increased flow to adipose tissue, and total blood flow to the udder was not affected. These results suggest that vasoconstriction in udder skin is a mechanism for reducing heat loss in much the same way as it does in the skin on many other regions of the body. There was no evidence, at this particular cold temperature, of a local control of capillary blood flow to the teat. The observed increase in blood flow to inguinal adipose tissue agrees with previous observations of an increased flow to perirenal adipose tissue in cold-exposed sheep (Hales, Bennett & Fawcett, 1976). It remains possible that when cold stress causes a decrease in total udder blood flow, as it does in lactating goats when they are cold-exposed sufficient to increase their oxygen consumption by 46 % (Thompson & Thomson, 1977), it may reduce blood flow to lobulo-alveolar tissue, and thus reduce milk synthesis in the lactating animal. Values for total flow to the udder have been previously obtained, from pregnant sheep, using an electromagnetic flowmeter (Burd, Lemons, Makowski, Battaglia & Meschia, 1975) and, from lactating sheep, by applying the Fick principle to measurements of the rate of secretion of the amino acid methionine in milk and differences in concentration of methionine in arterial blood and blood from one of the veins, the superficial epigastric, that drains the udder. The values for total right and left flow that were obtained in these experiments on lactating sheep averaged min- (Davis & Bickerstaffe, 1978) compared to the mean of min' obtained in the present experiments. Large variations in udder size and blood flow, between individual sheep, were encountered in both studies.

7 UDDER BLOOD FLOW 385 Measurements of blood flow to mammary lobulo-alveolar tissue have been compared in anaesthetized virgin and lactating rats, using 3H20 (Chatwin, Linzell & Setchell, 1969) 86RbCl and 25,sm l69yb-labelled microspheres (Hanwell & Linzell, 1973), and also in conscious rats (Hanwell, 1972). The values for blood flow per unit weight of lobulo-alveolar tissue that were obtained from sheep in the present study are lower than those previously reported for rats but the two species show similar changes due to lactation. In anaesthetised virgin or non-lactating parous rats, values of about 1P5 ml. 10 g-1 min- have been obtained (Chatwin et al. 1969; Hanwell & Linzell, 1973; Hanwell, 1972), and in anaesthetized and conscious lactating rats 8-10 ml. 10 g-1 min- (Hanwell, 1972). The mean values for conscious sheep are 0-6 ml. 10 g'l min- when dry and 7-6 ml. 10 g-l min-' at a time that was estimated to be near peak lactation. A greater blood flow per unit weight of body skin has been observed in lactating than in non-lactating anaesthetized rats (Chatwin et al. 1969; Hanwell & Linzell, 1973) but no such difference was apparent in the present study on conscious sheep. The rats may have been in an intermediate state between peripheral vasoconstriction and vasodilatation in which case the lactating animals, because of their greater metabolic heat production, would be most dilated. The present experiments were conducted in an environmental temperature in which the non-lactating, and therefore probably the lactating animals also, were fully vasodilated. The values for blood flow per unit weight of inguinal lymph node tissue are very high and lymph node blood flow accounted for a surprisingly large fraction of total udder blood flow in the non-lactating ewe. In the lactating ewe, but perhaps not the non-lactating ewe, flow of blood between right and left udder halves was so small that, for most experimental purposes, it would not be necessary to ligate the vessels crossing between the two halves. In lactating sheep, 98 % of udder blood flow is through lobulo-alveolar tissue and metabolic studies involving measurements of total udder blood flow and difference, in concentration of substances, such as glucose, between arterial and mammary venous blood (e.g. Davis & Bickerstaffe, 1978) can reasonably be interpreted in terms of uptake by lobulo-alveolar tissue. In non-lactating sheep non-secretory tissues contribute much more to udder blood flow and this will make it difficult to compare mammary metabolism in lactating and non-lactating sheep. I would like to acknowledge, with thanks, the help given by M. Bacon, Jane Goode, N. Howell and D. Neal. REFERENCES BtuRD, L. I., LEMONS, J. A., MAxowsKI, E. L., BATTAGLIA, F. C. & MEsCHIA, G. (1975). Relationship of mammary blood flow to parturition in the ewe. Am. J. Phy8iol. 229, CHATWIw, A. L., LnqzELL, J. L. & SETCHELL, B. P. (1969). Cardiovascular changes during lactation in the rat. J. Indoor. 44, DAvis, S. R. & BIcxmsTusmr, R. (1978). Mammary glucose uptake in the lactating ewe and the use of methionine arterio-venous difference for the calculation of mammary blood flow. AuWt. J. biol. Sci. 31, HAtes, J. R. S. (1974). Radioactive microsphere techniques for studies of the circulation. Clin & exp. Pharmacol. & Phy8iol. suppl. 1, PHY 302

8 386 G. E. THOMPSON HALEs, J. R. S., BENNETT, J. W. & FAWCETT, A. A. (1976). Effects of acute cold exposure on the distribution of cardiac output in the sheep. Pfltiger8 Arch. 366, HANRELL, A. (1972). Cardiovascular aspects of lactation in the rat. Ph.D. Dissertation, University of Cambridge. HANWELL, A. & LiNzELL, J. L. (1973). The time course of cardiovascular changes in lactation in the rat. J. Phy8iol. 233, LwzErL, J. L. (1960). Mammary gland blood flow and oxygen, glucose and volatile fatty acid uptake in the conscious goat. J. Phyeiol. 153, M.owsxi, E. L., MEscHIA, G., DRoEGEMuLLER, W. & BATTAGLIA, F. C. (1968). Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero. Circulation Res. 23, MOLYNEAUX, G. S. (1965). Observations on the structure, distribution and significance of arteriovenous anastomoses in sheep skin. In Biology of the Skin and Hair Growth, ed. LYNE, A. G. & SHORT, B. F., pp Sydney: Angus and Robertson. NISBET, A. M. (1956). Arterio-venous anastomoses in the teat of a cow. Nature, Lond. 178, PEART, J. N., EDWARDS, R. A. & DONALDSON, E. (1972). The yield and composition of the milk of Finnish Landrace x Blackface ewes. I. Ewes and lambs maintained indoors. J. agric. Sci., Camb. 79, REYNOLDS, M., LINzELL, J. L. & RASMUSSEN, F. (1968). Comparison of four methods for measuring mammary blood flow in conscious goats. Am. J. Phy8iol. 214, SMITE, R. E., HEATH, M. E. & INGRAM, D. L. (1978). Role of the udder in heat loss from the sheep. J. thermal Biol. 3, TANUDIMADJA, K., GETTY, R. & GHOSHAI, N. G. (1968). Arterial supply to the reproductive tract of the sheep. Iowa St. J. Sci. 43, THOMPSON, G. E. & TitoMsoN, E. M. (1977). Effect of cold exposure on mammary circulation, oxygen consumption and milk secretion in the goat. J. Phyeiol. 272,