acid, most of the fat which is absorbed appears in the lymph draining the forestomachs are small. As the animal grows older, its diet changes and

Transcription

1 THE ABSORPTION OF FAT IN SHEEP AND LAMBS. By T. J. HEATH and BEDE MORRIS. From the Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra. (Received for publication 24th October 1961) The absorption of fat has been studied in adult sheep and in young lambs with chronic lymphatic fistuloe. When maize oil or olive oil was injected into the gut, the rate of lymph flow decreased during the next hour. The flow rate increased subsequently as fat absorption proceeded and two peaks of both flow rate and fat concentration occurred in adult sheep at 3-5 hr. and 5-8 hr. following the injection of the fat. In lambs, the lymph flow increased to a peak between 2 and 3 hr. while the peak concentration of fat in the lymph occurred between 5 and 8 hr. In adult sheep, the increase in the concentration of total esterified fatty acids was accompanied by an increase in the concentration of phospholipids in the lymph. In lambs, the concentration of total esterified fatty acids was significantly correlated with both the concentration of phospholipids and free fatty acids in the lymph during fat absorption. When 14C tripalmitin was injected into the abomasum or duodenum of lambs, most of the absorbed radioactivity was recovered in the intestinal lymph. In adult sheep significantly less of the labelled fat which was absorbed appeared in the lymph. When the fat was given into the rumen, absorption was slow and took place over a period of several days. More than 90 per cent of radioactivity in the lipids of the intestinal lymph was in the form of neutral glycerides. Small amounts of activity appeared in the lymph phospholipids (0-5 9 per cent) and in the lymph non-esterified fatty acids ( per cent). The significance of the lymphatic system in the transport of absorbed fat in sheep is discussed. IT has been shown in several species of monogastric animals that the lymphatic system plays a major role in the transport of long-chain fatty acids from the gut. When fat labelled with 14C is fed either as triglyceride or as the free acid, most of the fat which is absorbed appears in the lymph draining the intestines [Bergstrom and Borgstrom, 1955]. There is, however, little information concerning the absorption of fat in ruminant animals. The young suckling ruminant ingests relatively large amounts of fat during the first few months of life. In these young animals, the abomasum or glandular stomach is large and well developed whereas the forestomachs are small. As the animal grows older, its diet changes and the forestomachs develop with the increasing intake of cellulose. It is not known if these changes in the anatomy of the gut affect the pattern or pathway of fat absorption. The experiments described in this paper were carried out to assess the significance of the lymphatic system in the transport of absorbed fat in suckling lambs and in adult sheep. Some observations were also made on the lipid content of plasma and the flow and composition of thoracic, intestinal and hepatic duct lymph in sheep while fat absorption was taking place. 157

2 158 Heath and Morris MATERIALS AND METHODS Merino ewes and lambs were used for all the experiments. The ages of the ewes, which varied from two-tooth to full-mouth were estimated to be between 11 and 4 years. Intestinal and hepatic lymph ducts were cannulated in four ewes and thoracic ducts were cannulated in eleven ewes. The surgical techniques used have been described previously [Lascelles and Morris, 1961]. At the same time as the lymph ducts were cannulated, polythene tubes were inserted into the gut at various levels. These were held in place by purse-string sutures and were led out through stab incisions in the abdominal wall. The location of the tubes varied in different animals, being either in the abomasum and duodenum (about 6 in. distal to the pylorus) or in the rumen. A polythene tube, inserted into an external jugular vein in each sheep, enabled frequent blood samples to be taken without disturbing the animal. After the sheep had recovered from the effects of the anaesthetic, they were placed in metabolism cages and were fed on lucerne hay and a mixture of lucerne chaff, oaten chaff and grain oats. Water was given ad libitum throughout the whole of the experimental period. A salt-lick was also provided. All the sheep were standing and eating within 6 hr. of the operation. The experiments were started hr. after the operation, provided clinical signs such as ruminal movements, temperature, heart rate and respiration rate were normal. Four sheep with thoracic duct fistule were rejected because they were not clinically normal, or because the flow of lymph was partly or completely obstructed due to the formation of clots in the cannulke. The lambs used were 2-3 weeks old and were being suckled by their mothers. The intestinal lymph ducts were cannulated, and polythene tubes fixed into either the abomasum or the duodenum. After the operation, the lambs were not restrained in any way, but were kept separate from their mothers and bottle fed with a mixture of 2 parts of whole cow's milk and 1 part of water (fig. 4). Treatments.-Experiments were carried out in adult sheep to measure the changes which occur in the intestinal lymph, hepatic lymph and plasma following the feeding of 25 ml. of olive oil or maize oil. The fat was warmed to 370 C. and was injected into the abomasum through the indwelling polythene tube. Samples of lymph were collected at hourly intervals for the first 8 hr., and at 12, 18 and 24 hr. after giving the fat. The absorption of triglycerides from the gut was studied in adult sheep and lambs. Glyceryl-tri-1-14C palmitate (The Radiochemical Centre, Amersham) was dissolved in olive oil at 370 C. Two ml. of olive oil containing 20 yc. of radioactivity were injected into either the abomasum or the duodenum of eight adult sheep. Samples of lymph were collected at frequent intervals until they contained no radioactivity. In two sheep, 60 ftc. of 14C tripalmitin were dissolved in 4 ml. of olive oil at 37' C. and injected into the rumen. In experiments with lambs, 20 itc. of 14C tripalmitin were dissolved in 1 ml. of olive oil and injected into either the abomasum or duodenum through the indwelling polythene tubes. In one lamb, a stomach tube was passed and the fat injected through this tube. The lambs were given a drink of ml. of diluted cow's milk after the radioactive fat. Collection of Samples.-Blood samples were obtained from the external jugular vein in the sheep and the lambs and the plasma immediately separated from the cells. Lymph samples were collected and stored in the refrigerator. Where necessary the lymph was collected into chilled tubes and frozen immediately. Powdered heparin was used as an anticoagulant. Samples of faeces, which were free from contamination by urine, were also collected.

3 Fat Absorption in Sheep Chemical Analyses.-The following measurements were made on samples of lymph and plasma. 1. Total esterified fatty acids by the method of Stern and Shapiro [1953]. 2. Non-esterified fatty acids by the method of Dole [1956]. 3. Total cholesterol by the method of Rappaport and Eichorn [1960] or the method of Kingsley and Schaffert [1949]. 4. Phospholipids by the method of Zilversmit and Davis [1950]. 5. Protein by the method of Gornall, Bardawill and David [1949]. Measurements of Radioactivity.-The radioactivity in samples of lymph was measured by direct plating. The activity in the lymph lipids was measured after extraction in 20 volumes of 3: 1 ethanol-ether mixture. The fseces which were collected during the experiment were dried and weighed. Samples were finely ground up with a pestle and mortar and the lipids extracted by boiling in 20 volumes of 2: 1 chloroform-methanol mixture for 30 min. The extract was allowed to stand for 1 hr. and portions were plated directly onto planchets. All measurements of radioactivity were made with a thin mica end-window GM counter. Activities were corrected for self-absorption to zero mass. The determinations of radioactivity were considered accurate to ± 5 per cent. The Partition of the 14C Label between the Lipid Constituents of the Lymph. Phospholipids were precipitated from a solution of the lymph lipids in petroleum ether, using acetone and magnesium chloride as described by Borgstrom [1952 a]. In later experiments, silicic acid columns were used to separate the phospholipids from other lymph lipids [Borgstrom, 1952 a]. The chloroform eluates from the silicic acid columns were evaporated to dryness and the lipids dissolved in about 30 ml. of petroleum ether. These solutions of lipids in petroleum ether were added to alkaline ethanol in separating funnels, and the non-esterified fatty acids separated from the neutral glycerides by partitioning between the petroleum ether and alkaline ethanol phases [Borgstr6m, 1952 b]. Any monoglycerides which were present may have contaminated the non-esterified fatty acid fractions [Borgstrom, 1952 b]. 159 RESULTS The Effect of Feeding Fat on the Rate of Flow and the Composition of Intestinal Lymph in Sheep.-The changes in flow rate and fat concentration in the intestinal lymph followed one another closely. When 25 ml. of olive oil was injected into the abomasum of sheep, the lymph flow decreased by an average of 42 per cent during the 1st and 2nd hr. (fig. 1). After the 2nd hr., the flow increased rapidly and reached a peak 3-5 hr. after the fat was injected. The maximum rate of flow was an average of 50 per cent higher than the rate of flow before the fat meal. The flow then decreased but rose again to a second peak after 5-8 hr. During the next 18 hr. the flow gradually returned to the pre-feeding level. Although this response in lymph flow occurred when either maize oil or olive oil was injected into the abomasum, it was best seen when maize oil was given. When a smaller volume (2 ml.) of olive oil was injected into the abomasum or duodenum, the response was essentially the same, but the lymph flow returned to the pre-feeding level within 8 hr. The lymph flow did not change significantly when 4 ml. of olive oil was injected into the rumen. The concentration of fat in the lymph decreased during the first 2 hr. following the injection of the fat. It then increased to a maximum about 1 hr. before the maximum rate of lymph flow occurred. During the next

4 160 Heath and Morris 1-2 hr. it decreased but increased again to a second peak between 5 and 8 hr. The concentration of fat in the lymph had returned to near the pre-feeding level by about 24 hr. The output of fat in the lymph followed the pattern of 120 T. E. F.A. Concentration ~ Lymph flow Hours FIG. 1.-Changes in the flow rate and total esterified fatty-acid concentration of intestinal lymph in an adult sheep after the injection of 25 ml. olive oil into the abomasum. TABLE I. CHANGES IN THE RATE OF FLOW, THE CONCENTRATION AND OUTPUT OF PROTEIN AND TOTAL ESTERIFIED FATTY ACID IN THE INTESTINAL LYMPH OF A SHEEP AFTER 2 ml. OF OLIVE ORL WAS INJECTED INTO THE DUODENUM OR ABOMASUM. Site of injection Time after injection (min.) { Abomasum { Lvmph flow (ml./min.) Protein concentration (g. per cent) Protein Total fat output concentration Total fat (mg./min.) output ( IEq/ml.) (peq/min.) Although the rate of lymph flow varied during the lymph flow very closely. absorption of fat, the concentration of protein remained relatively constant. The output of protein in the lymph thus varied with the flow rate (Table I). During the absorption of fat, the concentration of phospholipids, nonesterified fatty acids and total cholesterol in the intestinal lymph was always

5 Fat Absorption in Sheep 161 higher than the concentration in the hepatic lymph or the plasma (fig. 2). The increase in concentration of esterified fatty acids in the lymph was associated with an increase in the concentration of phospholipids. During the period of maximum fat absorption, the concentration of phospholipids in the intestinal lymph increased by up to 110 per cent. The regression relating phospholipid concentration to total esterified fatty-acid concentration was.6-q -0 -i cl K 0) I LL~ LU i 0.8 E 0.6 u Hours FIG. 2.-The concentration of non-esterified fatty acids and phospholipids in the intestinal lymph (e), hepatic lymph (A) and plasma (A) of a sheep during fat absorption. statistically significant in all experiments (p < 0.01 in experiments with olive oil and p < in experiments with maize oil). The regression of non-esterified fatty-acid concentration on total esterified fatty-acid concentration was significant (p < 0.01) in the experiments with maize oil, but was not significant in experiments with olive oil. The changes in the concentration of cholesterol in the intestinal lymph were significantly correlated with changes in the total esterified fatty-acid concentration in one sheep which received injections of maize oil and olive oil into the abomasum 24 hr. apart. The absorption of fat from the gut had no effect on the rate of flow or composition of liver lymph. In sheep with intestinal or thoracic duct

6 162 Heath and Morris fistulae, the concentration of lipids in the plasma did not change significantly during fat absorption. The Effect of Feeding Milk on the Flow and Composition of Intestinal Lymph in Lambs.-Three lambs with intestinal lymphatic fistule were starved for 8 hr. and then given a drink of ml. of diluted cow's milk which contained 6-10 g. of fat. The lymph flow increased rapidly and doubled during the first 2 hr. after the milk was given. This increase in flow. 10( E 5- Protein output > 2 Protein concentration 30 "I E 20 -* x 0- T. E. F.A. Concentration E 0.1. *... I Hours FIG. 3.-The concentration and output of protein and total esterified fatty acid in the intestinal lymph of a 3-week-old lamb given a drink of 200 ml. Of milk containing 1 ml. of olive oil. was associated with a fall in protein concentration in the lymph but the protein output rose. It was thought likely that the absorption of water directly from the gut into the intestinal lymphatics contributed to the fall in protein concentration in the lymph [Korner, Morris and Courtice, 1954]. The fat concentration in the lymph increased rapidly and reached a maximum several hours after the lymph flow reached its peak. It appeared that fat absorption in lambs continued over a period of at least 10 hr. as the concentration of fat in the lymph had not returned to the pre-feeding level within this time (fig. 3). As with adult sheep, the concentration of phospholipids and non-esterified fatty acids varied with changes in the concentration of total esterified fatty acids in the lymph. The regressions relating phospholipid concentration to total esterified fatty-acid concentration were significant in each experiment (p < ). The regression of total cholesterol

7 IC,. 4. A 3-wX((,1-(ol 1(1hamb N itll (ill Int(stilldll lypilpiati fistula ai)i (1ini)nd-elling tube ill tl( (1ihiomsunSiiiii. The laub bus been giveu (I fat iuieal ani mi ilky 1yImphI is collectinlg illto the( tesst-tulbe. Th1es test-tulbe is carr((ied pl'a cl i conltaine(r filled( tied aro11 (1 the buttoeks is is'l to collct tbe fwees. 't ble Theap110kinl Photoguraph taketn on tie fourth (lidy after o)p)eratioll. 'l'o faec paac 162

8 Fat Absorption in Sheep 163 concentration on total esterified fatty-acid concentration was significant in two out of three experiments. The Absorption of 14C Tripalmitin in Sheep.-When 14C tripalmitin was injected into the abomasum or duodenum of sheep with intestinal lymphatic fistulae, radioactivity appeared in the lymph by the end of the 1st hr. The absorption of fat continued for periods of up to 53 hr. after the 14C tripalmitin CD Hours FIG. 5.-The appearance of radioactivity in the intestinal lymph of a sheep after 14C tripalmitin dissolved in olive oil was injected into the abomasum. The percentage of the total esterified fatty acids in the lymph which was derived from the injected fat is shown, together with the cumulative percentage recovery of labelled fat in the lymph. * =Radioactivity in lymph. Counts/min./ml. A Lymph fat specific activity Injected fat specific activity * =Cumulative percentage recovery of labelled fatty acids. was given. Most of the labelled fat appeared in the lymph in the first 6 hr. (fig. 5). In one sheep, four experiments were carried out in which the labelled fat was given alternately into the abomasum or duodenum over a period of 14 days. The amounts of radioactive fat recovered in the intestinal lymph and from the fasces are shown in Table II. Not more than 90 per cent of the labelled fat which was injected into the abomasum or duodenum was recovered from the intestinal lymph and feeces. It was thought possible that some of the lymph draining from the intestines may have entered the thoracic duct through other lymphatic channels and escaped collection. In an attempt to collect all the lymph draining from

9 164 Heath and Morris the intestines, the thoracic duct was cannulated in five sheep and the absorption of 14C tripalmitin was studied. Between 21 and 44 per cent of the labelled fat which was fed was recovered in the thoracic duct lymph. This was not significantly different from the recovery in the intestinal duct lymph (Table II). A variable percentage of fat appeared in the feeces. Again, not more than 90 per cent of the total radioactivity was recovered in the lymph and feeces. The absorption of radioactive fat injected into the rumen was studied in two sheep with thoracic duct fistule. In these experiments no radioactivity TABLE II.-THE RECOVERY OF 14C TRIPALMITIN IN THE LYMPH AND FAECES OF ADULT SHEEP AND LAMBS. THE RADIOACTIVE FAT WAS INJECTED INTO THE RUMEN, ABOMASUM OR DUODENUM IN DIFFERENT EXPERIMENTS AND THE LYMPH COLLECTED FROM THE INTESTINAL DUCT OR THE THORACIC DUCT. Experiment Sheep 2 Sheep 3 Sheep 4 Sheep 4 Sheep 4 Sheep 4 Sheep 5 Sheep 8 Sheep 9 Sheep 12 Sheep 13 Sheep 14 Sheep 15 Lamb 1 Lamb 3 Lamb 3 Lamb 5 Lamb 5 Lymphatic cannulated Thoracic duct Thoracic duct Thoracic duct Thoracic duct Thoracic duct Thoracic duct Thoracic duct Site of injection Abomasum Abomasum Abomasum Abomasum Abomasum Rumen Rumen Fed by stomach tube Abomasum Abomasum Percentage of injected fat recovered Lymph Faeces * Lymph clotted in the cannula before absorption was complete. appeared in the thoracic duct lymph for the first 3 hr. After this period, the radioactivity increased to a maximum at about 22 hr. (fig. 6). Activity was still present in the thoracic duct lymph of one of these sheep 86 hr. after giving the fat. The lymph clotted in the cannula of the other sheep at the beginning of the 4th day of the experiment. Up to this time, 36 per cent of the radioactive fat had been recovered in the lymph. Only 10 per cent of the radioactive fat was recovered in the feeces during the next 4 days. The Absorption of 14C Tripalmitin in Lambs.-When 14C tripalmitin was injected into the abomasum or duodenum of lambs, radioactivity appeared in the intestinal lymph by the end of the 1st hr., and reached a maximum before the end of 3 hr. (fig. 7). The recoveries of radioactive fat are given in Table II Total recovery in faeces and lymph Percentage absorbed activity recovered in lymph *

10 Fat Absorption in Sheep 165 In one experiment in which a lamb was given 14C tripalmitin through a stomach tube, radioactivity did not appear in the intestinal lymph until 6 hr. after the fat was given. The rate of lymph flow and fat concentration however, increased during the first 3 hr. after the fat was given. The peak of absorption occurred at 17 hr., and radioactivity remained in the lymph for 54 hr. The pattern of fat absorption thus resembled that seen when fat was injected into the rumen in adult sheep (fig. 6) , 12 A 8A 4 A Hours Hours FIG. 6.-The average output of radioactivity per minute in the thoracic duct lymph of an adult sheep and a 3-week-old lamb. The sheep was given 60 MC. Of 14C tripalmitin into the rumen and the lamb 20,ic. by stomach tube. * =Adult sheep. A =Lamb. The Distribution of 14C Tripalmitin Activity between the Lipid Constituents of the Lymph.-When 14C tripalmitin was injected into the abomasum or duodenum of sheep, more than 90 per cent of the activity in the lymph was present as neutral glycerides. An average of 1.2 per cent of the total activity was recovered in the phospholipids and an average of 3.6 per cent in the non-esterified fatty acids. In two experiments, no activity was detected in the lymph phospholipids. In experiments in which 14C tripalmitin was injected into the rumen, the lymph phospholipids contained an average of 5.9 per cent of the total activity and the lymph non-esterified fatty acids contained an average of 4-5 per cent. In experiments with lambs, an average of 0-6 per cent of the total activity was recovered in the lymph

11 166 Heath and Morris phospholipids and 1 M1 per cent in the non-esterified fatty acids. The remainder of the activity was present as neutral glycerides. As none of the sheep or lambs were in a post-absorptive state when the radioactive meal was given, radioactive fat which was absorbed into the lymph was always diluted with exogenous fat. An approximate estimate of the percentage of the total esterified fatty acids in the lymph which was K -~ Hours FIG. 7.-The average output and the cumulative percentage recovery of radioactivity in the intestinal lymph of a 3-week-old lamb after the injection of 20,uc. 14C tripalmitin into the abomasum. * =Output of radioactivity. * =Cumulative percentage of radioactivity recovered. derived from the injected fat was obtained by comparing the specific activity of the fat in the lymph with the fat which was fed. During the period of most rapid absorption, up to 40 per cent of the fat in the intestinal lymph of sheep (fig. 5), and up to 51 per cent of the fat in the intestinal lymph of lambs came from the injected 14C tripalmitin in olive oil. DIscussIoN The Changes in the Flow and Composition of Lymph during Fat Absorption.-When fat is fed to rats, cats and rabbits, the rate of lymph flow from the intestines increases as fat is absorbed into the lymph [Borgstrom and Laurell, 1953; Morris, 1954; Simmonds, 1955]. The concentration of fat in the lymph however, reaches a maximum several hours after the maximum rate of lymph flow. The changes in flow rate and fat concentration in the

12 Fat Absorption in Sheep 167 lymph of lambs were similar to those seen in monogastric animals. The response in adult sheep, however, was different. There was a characteristic bimodal response in both lymph flow and fat concentration when the fat was injected into the abomasum or duodenum. When small amounts of fat were injected into the rumen no regular response in lymph flow was seen. Simmonds [1955 and 1957] investigated the effect of fat absorption on lymph flow in the rat. From results obtained with various pharmacological agents which produced changes in intestinal motility, Simmonds concluded that alterations in intestinal motility had little effect on the changes in lymph flow which were observed during fat absorption. The exact stage of fat absorption at which the increase in lymph flow is produced is not known. It is suggested [Simmonds, 1955] that the changes in lymph flow are due to some humoral effect which influences blood flow and lymph formation in the intestinal mucosa during the early stages of fat absorption. This stimulus apparently occurs at different times in adult sheep and produces two peaks in the rate of lymph flow. Presumably, when fat was given into the rumen it was modified in some way by the ruminal organisms or it was released into the duodenum so slowly that no response in flow rate occurred. It has been shown in several species of animals that the increase in fat concentration which occurs in the lymph during fat absorption is accompanied by an increase in the concentration of phospholipid [Bollman, Flock, Cain and Grindlay, 1950; Borgstrom, 1952 c; Morris, 1954]. It appears that this increase is due to the synthesis of phospholipid in the intestinal mucosa. The phospholipids are associated with the chylomicron fraction of the lymph and probably play an important part in stabilizing the large amounts of triglyceride which appear in the lymph. In sheep as in other animals, only a small proportion of the absorbed fatty acids were carried in the lymph phospholipids. The concentration of cholesterol and phospholipid in the intestinal lymph was always higher than the concentration in the hepatic lymph or in the plasma. In the post-absorptive state, the lipids in lymph are derived from the plasma as constituents of the capillary filtrate [Morris, 1954; Courtice and Morris, 1955]. Under these circumstances, their concentration in the lymph depends on the permeability of the blood capillaries in the area in which the lymph is formed. It appears that in the sheep, considerable amounts of exogenous or newly synthesized phospholipid and cholesterol are present in the intestinal lymph at all times. Fat Absorption in Sheep and Lambs.-The events which follow the ingestion of fat in ruminant animals are not clear. Garton, Hobson and Lough [1958] and Garton, Lough and Vioque [1959] have shown that ruminal organisms can hydrolyze triglycerides both in vivo and in vitro. It is also known that hydrogenation of long-chain unsaturated fatty acids occurs in the rumen [Garton, 1960]. Thus it appears that fat which enters the rumen may undergo extensive modification before it passes into the abomasum and small intestine. When 14C tripalmitin was injected into the abomasum or duodenum, a small percentage of the absorbed fat appeared in the lymph as non-esterified

13 168 Heath and Morris fatty acids. These may have been produced by hydrolysis of the triglycerides in the intestinal mucosa or in the lumen of the gut. If hydrolysis of triglycerides does occur in the intestinal lumen of sheep, this must be followed by incomplete esterification of the absorbed fatty acids within the cells of the intestinal mucosa [Borgstrom and Tryding, 1956]. The role of bile and pancreatic juice in ruminant fat digestion however are not known. It appears that the absorption of fat in sheep and lambs is qualitatively similar to monogastric animals, and the lymphatic system plays an important part in the transport of absorbed fat from the gut. When the rumen is by-passed and fat introduced directly into the abomasum or duodenum, it is absorbed promptly. If the fat is injected into the rumen, absorption occurs slowly over a period of days. The difficulty in deciding the quantitative importance of the lymphatic route in the transport of absorbed fat from the gut lies in collecting all the lymph draining from the intestines. In the experiments in which the thoracic duct was cannulated, the maximum recovery of the absorbed fat was only 87 per cent. In some experiments the recovery was much lower. It is possible that some of the fat may have entered the blood directly through lymphatic-venous anastomoses. Anastomotic channels of this type have been demonstrated in other animals [Silvester, 1912; Job, 1918]. In the adult sheep, the thoracic duct lymph contains large numbers of red cells which may gain access to the lymph within heemolymph nodes [Heath, Lascelles and Morris, 1961]. In lambs, the recoveries of absorbed fat in the lymph were higher than in adult sheep and this may be because lymphatic-venous anastomoses are less common in young animals [Forgeot, 1907]. The possibility that fat may be modified in some way, and enter the blood-stream directly, must also be considered. Most of the sheep and lambs were returned to the paddock after the lymphatic cannulse were blocked up. One lamb remained stunted and failed to grow for some weeks, but the other sheep and lambs recovered and put on weight. It appeared that even in the animals with thoracic duct fistulae, accessory lymph channels had opened up. The lower recoveries of fat in the lymph of some animals which were given second feeds of radioactive fat suggested that these accessory channels begin to develop quite early. ACKNOWLEDGMENTS We should like to thank Mrs. H. Kabau and Miss L. Palmer for their technical assistance.

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