y,-globulin which is present in high concentration in colostrum. Subsequently, flow

Q. Jl exp. Physiol. (1968) 53, 415-421 LYMPH FLOW AND PROTEIN COMPOSITION OF THORACIC DUCT LYMPH IN THE NEWBORN CALF. By A. D. SHANNON and A. K. LASCELLES. From Dairy Research Foundation, University of Sydney, University Farms, Camden, N.S.W. 257. (Received for publication 1st May 1968) The changes in the flow and protein composition of thoracic duct lymph have been studied in unanesthetied calves during the first 6 days after birth. Thoracic duct-venous shunts were established in four calves (average body weight 35-2 kg.) within 3 hr. of birth. The calves recovered quickly from the operation and were able to suck strongly within 1-2 hr. of its completion. Lymph flow during anmesthesia varied between 9-15 ml./hr. and by 1-2 hr. after the operation, immediately before first feeding, flow had risen to values of 3-45 ml./hr. There was a marked increase in flow and globulin concentration during the 8-hr. period after the first feeding, peak values observed being 85 ml./hr. and 2-72 g. per cent, respectively. Fractionation of colostral whey and lymph on G-2 and DEAE Sephadex columns indicated that the increase in globulin in the lymph was due to the absorption of y,-globulin which is present in high concentration in colostrum. Subsequently, flow and protein content decreased quite rapidly and by 24 hr. after first feeding the values were relatively constant. The results suggested that the absorption of y-globulin had virtually ceased by 24 hr. There was a slower decrease in lymph flow and globulin content over the subsequent 4-6 days. IN earlier studies the high flow and protein output in thoracic duct lymph of milk-fed calves (7-14 days of age) compared with adults was noted [Shannon and Lascelles, 1967], and in subsequent studies it was shown that most of the fluid and protein in thoracic duct lymph was derived from the intestinal lymphatic duct [Shannon and Lascelles, 1968]. It was thought that the high flow of lymph from the thoracic duct was associated with the absorption of large quantities of fat. This was confirmed by the finding that the flow and protein output of lymph of the thoracic and intestinal ducts was substantially reduced when calves were fed skim milk instead of whole milk [Shannon and Lascelles, 1969]. During the first 12-24 hr. of life the calf is subject to changes which would be expected to have marked effects on lymph formation. Thus, soon after birth the calf ingests colostrum which is rich in fat and immunoglobulin, both of which would be absorbed by way of the lymphatics. Moreover, the plasma protein content will increase as a result of the absorption of dietary immunoglobulin and over the first few days there may also be a change in blood volume. In the present study, an attempt has been made to determine the significance of these early events on lymph formation in the major capillary beds by following the changes in flow and protein composition of lymph from the thoracic duct of calves from birth to 6 days of age. 415

416 Shannon and Lascelles MATERIALS AND METHODS Animals. - Thoracic duct-venous shunts were established in four cross-bred Friesian calves (one female and three males). The average weight of the calves at birth was 35-2 kg. (range 33-6 to 36-3 kg.). The calves were taken from their dams within 5 min. of birth and kept indoors. Blood volume determinations were carried out on another three cross-bred Friesian calves (one female and two males). Ancesthesia. - The calves were anaesthetied as described by Shannon and Lascelles [1967]. Briefly, anaesthesia was induced with a small dose of pentothal sodium (Abbot) and maintained with cyclopropane and oxygen in closed circuit. Induction of anaesthesia in calf 1 was carried out within 15 min. of birth and on intubation large amounts of frothy mucinous fluid emerged from the endotracheal tube, resulting in a serious impedence to the passage of air. This frothy fluid had to be removed by suction before anaesthesia could be continued in a satisfactory manner. In view of this experience, anaesthesia was induced in the remaining three calves 45-6 min. after birth when it was predicted that the reabsorption of fluid from the lungs would be complete. Anaesthesia in these three calves was satisfactory. Recovery from the anaesthetic was rapid with the animals standing steadily within 6-12 hr. of the operation. Surgical Technique. - Cannulation of the thoracic duct and recirculation of lymph into the jugular vein were carried out as previously described by Shannon and Lascelles [1967]. Post-operative Care. - On completion of the operation the calves were placed on straw bedding in a 6 ft. x 6 ft. indoor pen. A recovery period of 1-2 hr. was allowed after the operation before the calves were fed. The approximate volume of lymph taken at each collection was replaced in calf 1 with sterile -9 per cent saline. This calf died suddenly 18 hr. after the operation. An acute mineral imbalance was suspected and in the remaining calves a sterile, balanced electrolyte solution [McSherry and Grinyer, 1954] was used to replace the fluid loss incurred by the collection of lymph. Each calf was given 5 mg. chloromycetin intramuscularly each day for 3 days after the operation. Management and Feeding. - The calves were not fed after the operation until a strong suckling reflex had returned. Each calf was fed 1-141. (-25 gal.) of colostrum at the first feeding and a similar amount of colostrum at 5, 14 and 2 hr. after the first feeding. The calves were fed utiliing a calf nipple and the calves' natural suckling reflex until they were strong enough to stand and drink from a bucket. Subsequently, the calves were fed 2-27 1. (-5 gal.) of whole milk at 12 hr. intervals beginning 26-3 hr. after the first feeding. The colostrum and milk fed to each calf in the first 48 hr. of life were obtained from the calf's own dam. Collection of Samples. - The collection and subsequent treatment of samples were similar to that described by Shannon and Lascelles [1967]. After disconnecting the shunt lymph was collected into a measuring cylinder for a period of 2-3 min. Analytical Determination. - Total protein and albumin were estimated in the lymph samples by the procedure described by Gornall, Bardawill and David [1949], as modified by Shannon and Lascelles [1968]. Fractionation of colostral whey, serum and lymph samples on G-2 and DEAE Sephadex (Pharmacia, Uppsala) columns was carried out as described by Mackenie, Outteridge and Lascelles [1966] and Mackenie [1968]. RESULTS The calves rapidly recovered from the operation and were conscious and able to suck within 1-2 hr. of its completion. During the operation lymph flow varied between 9-15 ml./hr. but rapidly increased to values of 3-45

Lymph Flow in Newborn Calves 417 ml./hr. by 1 hr. after the operation. The lymph before feeding was judged to be free of chylomicrons by its transparency after centrifugation and by the absence of visible particles on dark ground microscopic examination. L._ -J y E llj I- C) C-) L- cn) 9 8 7 6 5 4 1 3 F 2 3 2 5 2 1 5 1. 5 _ I I I I I I I Ia Ia I I I I a I I I a. I I I. I I I A R 2 4 6 8 1 12 14 t TIME (HR) t 16 18 2 t 22 24 FIG. 1. Showing the change in lymph flow and concentration of globulin and albumin in lymph immediately before and during the -24 hr. period after first feeding. The points plotted are means± standard errors for the results of three calves. Arrows indicate the times at which the calves were fed colostrum. A A Flow (ml./hr.) O O Globulin concentration (g. per cent) * * Albumin concentration (g. per cent) A =During ansesthesia R =During the recovery period The change in the flow, albumin and globulin concentration of lymph for three calves from the time of cannulation of the thoracic duct, towards the end of the operation, until 24 hr. after first feeding is illustrated in fig. 1. There was a marked increase in flow and globulin concentration of lymph

418 Shannon and Lascelles during the first 8 hr. after the calves were first fed. Albumin concentration tended to decrease during this period. This was followed by a decrease in both flow and globulin concentration and by 24 hr. after first feeding both had fallen to levels which, although higher than the original levels, were now relatively constant. Similar trends were also evident in another calf up until the time of its sudden death 18 hr. after the first feeding. Over the next 4 days there was a gradual decrease in flow and globulin concentration, and an increase in albumin concentration (Table I). There was a vast increase in the output of globulin in lymph from 2-5 g./hr. immediately before first feeding to 23-1 g./hr. 8 hr. later. The greatest output of globulin occurred within the first 12 hr. after the calves were first fed even though colostrum was also fed to the calves at 14 and 2 hr. An analysis of variance and covariance was carried out on the results for flow and globulin concentration of lymph collected at 4-hr. intervals from -24 hr. after first feeding. The analysis of variance indicated that there were significant changes in lymph flow and globulin concentration with time after feeding. These changes were described by a curve with only one significant inflection in the case of lymph flow and by curves with either one or two significant inflections in the case of globulin concentration (cf. fig. 1). The correlation between flow and globulin concentration as they varied with time after feeding was also significant (R + *82, P <-1). There were no other significant correlations. Fractionation of Lymph Samples. - Lymph samples collected before feeding, and again 8 hr. after first feeding when the output of globulin in lymph was at a peak, were fractionated on Sephadex G-2 and DEAE Sephadex columns. The optical density profiles for the fractionation on G-2 are shown in fig. 2. Particularly noticeable was the absence of the 7S peak in the fractionation on -2 Sephadex of lymph collected before feeding (fig. 2a). This contrasts with the dominance of this peak in the lymph collected 8 hr. after feeding (fig. 2b). The results of fractionation of lymph and colostral whey on DEAE Sephadex indicated that the change in profile seen in fig. 2b was consistent with the absorption of yl-globulin, which is the major immunoglobulin of bovine colostrum [Murphy et al., 1964; Pierce and Feinstein, 1965]. Samples of lymph collected at 24 hr. after first feeding when the globulin output in lymph had decreased to a reasonably stable level (cf. fig. 1) were also fractionated on G-2 (fig. 2c). Although the presence of chylomicrons in the lymph at this time resulted in a tremendous exclusion peak, the 7S peak was now greatly reduced in sie and was in fact smaller than the albumin peak. DisCUSSION A most striking finding in the present study was the high flow and protein output of thoracic duct lymph of the calves immediately before first feeding. Indeed the flow was only slightly less than that observed in the same calves at 6-7 days of age when they were being fed whole milk twice daily. It is

Lymph Flow in Newborn Calves 419 TABLE I. FLOW AND ALBUMIN AND GLOBULIN CONCENTRATION IN THORACIC DUCT LYMPH OF THE CALVES AT VARIOUS PERIODS AFTER FIRST FEEDING. The valume presented are means ± 8tandard errors derived from the result8 of three calve8. The number of observation8 at each time interval i8 8hown in parenthei8. Hours after first feeding (6) 1-12 (27) 13-24 (18) 145-156 (36) Lymph flow (ml./hr.) 38-8 ±24-693-1 35-6 618-8±38-42-5 ±2- Albumin Concn. (g. per cent) 1-61 -5 1-44±-2 1-53 ±-3 1-84 ±-3 Globulin ConCn. (g. per cent) -77 +-6 2-12 -16 2-2 -17 1-8+-9-6 -5 4 3 2 1 I- -J 1 4 1 2 1. 8 6-4 2 7-5 3-1 8 6 4 2 5 1 15 EFFLUENT VOLUME (ml.) FIG. 2. The optical density profiles for the fractionation, on G-2 Sephadex, of lymph collected from one of the calves (a) immediately before, (b) 8 hr. after and (c) 24 hr. after first feeding. 4 ml. of lymph were fractionated in each case.

42 Shannon and Lascelles therefore of interest to consider the physiological status of new-born calves in relation to lymph formation. Protein output in lymph is a function of capillary permeability, filtration area and effective pressure [Yoffey and Courtice, 1956]. There was no suggestion that the capillary permeability changed significantly with age, because the lymph to plasma ratios for albumin in the calves at birth were similar to those in the same calves when 6-1 days old. On the other hand it was found that the plasma volume (determined on three calves using Evans blue-t. Gurr) at birth was 6-27 per cent higher than in the same calves at 4 days of age, suggesting that filtration area at birth is higher than in calves at an older age. Moreover, it would be expected that the very low plasma protein concentration at birth [Shannon and Lascelles, 1966] would result in a higher effective filtration pressure. This situation changed dramatically some hours after first feeding when a substantial increase occurred in the concentration of total protein in the blood plasma compartment as a result of absorption of colostral immunoglobulin. It was clear from the results of the fractionation of lymph and colostral whey on G-2 and DEAE Sephadex that the increase in output and concentration of globulin in lymph after first feeding was associated with the absorption of colostral immunoglobulin which is predominantly yl-globulin. The decrease in the globulin concentration in lymph from its peak level, 8-12 hr. after first feeding, to relatively constant levels at 24 hr., even though colostrum was fed at 14 and 2 hr., suggests that y-globulin absorption had virtually ceased within 24 hr. of first feeding. The high correlation between flow and globulin concentration of the lymph during the -24 hr. interval after first feeding suggests that the absorption of y-globulin had a profound effect on lymph formation in the intestines. An increase in flow and total protein content of thoracic duct lymph after the feeding of colostral whey to anesthetied new-born calves has also been reported by Comline, Roberts and Titchen [1951]. The flow of lymph reported by these workers was very much less than that observed in the present experiments, which probably reflects the effect of anaesthesia on lymph formation. We suggest that the increase in flow of lymph after the feeding of colostral whey is due to the bulk transfer of water resulting from the passage of immunoglobulin from the gut lumen into the interstitial fluid. The fall in albumin concentration during the first 12 hr. after the initial feeding is in line with the dilution of albumin, derived mainly from the capillary filtrate, with water associated with the absorbed globulin. It has been known for some time that fat absorption promotes lymph formation in monogastric animals [Simmonds, 1955] and recent work at this laboratory has confirmed that this is also true for calves [Shannon and Lascelles, 1969]. Since significant amounts of fat did not appear in the lymph until 18-24 hr. after first feeding, it is suggested that the stimulating effects of fat absorption on lymph flow would only have become important after this time. Notwithstanding, lymph flow slowly decreased from 18-24 hr. after first feeding until the calves were 5 days old. It is therefore

Lymph Flow in Newborn Calves 421 further suggested that the cessation of globulin absorption in particular and also the fall in blood volume would have led to a decrease in lymph formation, while the onset of fat absorption would have increased lymph formation. It is apparent that the first two factors collectively overrode the effects of the latter. ACKNOWLEDGMENTS The authors wish to thank Miss C. Salt and other members of the Dairy Research Foundation staff for able technical assistance. REFERENCES CoMBiNE, R. S., ROBERTS, H. E. and TITCHEN, D. A. (1951). Nature 167, 561. GORNALL, A. G., BARDAWILL, C. J. and DAVID, M. M. (1949). J. biol. CJhem. 177, 751. MACKENZIE, D. D. S. (1968). Aumt. J. exp. Biol. med. Sci. Auot. J. exp. Biol. med. Sci. 46, 273. MACKENZIE, D. D. S., OUTTERIDGE, P. M. and LASCELLES, A. K. (1966). Aust. J. exp. Biol. med. Sci. 44, 181. MCSHERRY, B. J. and GRINYER, I. (1954). Amer. J. vet. Res. 15, 535. MURPHY, F. A., AALUND, O., OSEBOLD, J. W. and CARROLL, E. J. (1964). Arch. Biochem. Biophy8. 18, 23. MURPHY, F. A., OSEBOLD, J. W. and AALUND,. (1965). Arch. Biochem. Biophy8. 112, 126. PIERCE, A. E. and FEINSTEIN, A. (1965). Immunology 8, 16. SHANNON, A. D. and LASCELLES, A. K. (1966). Aust. J. biol. Sci. 19, 831. SHANNON, A. D. and LASCELLES, A. K. (1967). Aust. J. biol. Sci. 2, 669. SHANNON, A. D. and LASCELLES, A. K. (1968). Q. Jl. exp. Physiol. 53, 194. SHANNON, A. D. and LASCELLES, A. K. (1969). Aust. J. biol. Sci. 22. [To be published.] SIMMONDS, W. J. (1955). Aust. J. exp. Biol. med. Sci. 33, 35. YoFFEY, J. M. and COURTICE, F. C. (1956). Lymphatics. Lymph and Lymphoid Tissue. London: Arnold.