FACTOE$ WHICH ENFLUENCE GASTEOINTESTENAL ABSORPTION (N THE EMMEDEATE POST-RAYAL PEREOD {Bests {or the Deqruo o'f pk. D. MECHEGAN STATE UNIVERSITY Charles H. Long 1964 ABSTRACT FACTORS WHICH INFLUENCE GASTROINTESTII‘JALR‘: ABSORPTION IN THE IMMEDIATE POST-NATAL PERIOD I. Effect of Feeding Protein and Protein-free Diets on Absorption of Whole Proteins in Colostrum-Deprived Pigs.- II. Effect of Increasing Blood Volume on Whole Protein Absorption. III. Changes in Serum and Milk Phosphatase in Five Litters during the Post-Natal Period. by Charles H. Long Three trials were conducted using colostrum-deprived pigs fed pro- tein and protein-free diets to study their ability to absorb whole proteins ' from the gastrointestinal tract. In all 3 trials the pigs on treatment were caught at parturition and removed to locations previously unin- habited by swine. Pigs left on the sow served as controls. When taken from the sow, the pigs were bled and placed in individual boxes heated to 800 C. Pigs were bottle fed either casein-glucose or glucose diets 5 times a day. The rations contained fat, vitamins and minerals in adequate quantities. At either I or 2 weeks of age, the pigs were fed solutions of labeled protein and were killed. In Trials I and II, FITC-labeled 7-globulin was administered and the animals were killed at 6 hours post feeding. In Trial III, a mixture of 7-globulin and colostral whey was given and the pigs were killed 2h hours later. rSerum samples were taken before and after administration of the tagged protein. Tissues were collected from 6 locations in the intestinal tract for histological examination and alkaline phosphatase assay. Paper electrophoretic separations and total serum protein analyses were per- Charles H. Long formed to follow the changes in the fractional distribution and quantity of protein in the sera. Immuno-electrophoresis was used to detect the absorption of 7-globulin and other proteins. Performance of the pigs in these 3 litters varied greatly between litters. Trial I pigs, with the exception of l pig, maintained or in- creased in weight over their weight at birth. In Trial II, all of the animals weighed less when they were killed than they did when they were born. Casein-fed pigs in Trial III generally maintained their birth weight; but the glucose-fed pigs declined in weight. Spleen weights of both treatment groups were significantly lighter than were the controls. In all 3 trials, colostrum-deprived pigs fed the synthetic diets had lower total serum protein concentrations than did the animals nursing the sow. Glucose-fed pigs had even lower serum protein concentrations than did the casein-fed pigs. Both treatments were significantly lower than the controls. Relative serum protein fractions followed similar patterns, but there was some variation among the 3 litters. In Trial I, both the casein- and glucose-fed pigs showed changes in their serum profile as compared to that at birth. In Trial II, in which only the glucose diet was fed, only slight changes in the serum fractions were detectable when compared to the relative ratios at birth. Similar results were seen in Trial III. Intestinal alkaline phosphatase activity was highest in the jejunum- ileum in all 3 trials. Activity was higher in Trial I than in either Trial II or III among the same treatments. Casein-fed pigs had higher tissue activity than did the glucose—fed pigs. In the pigs killed at 1 week of age, the glucose—fed pigs had higher tissue activity than did the Charles H. Long controls. By the second week the activity in the glucose-fed pigs fell below that of the controls. In Trials II and III, insulin was added to the protein mixture and blood glucose concentration was determined at 3 hour intervals for 6 hours. Blood sugar data indicated that insulin was absorbed since blood levels decreased more in the treatment pigs than it did in the controls. Fluorescence within the epithelial cells was the intended criterion of absorption of the labeled protein in Trials I and II. In Trial I, the tissue from the glucose-fed pigs appeared to possess more fluorescence than did the casein-fed pigs, although the intensity was weak in both cases. Glucose-fed pigs in Trial II appeared to have absorbed more fluorescent-labeled material than did the glucose-fed pigs of Trial I. In Trial III, the validity of this criterion was studied by allowing time for absorption to take place, and following the changes in blood serum. Changes in total serum protein and the relative ratios of the serum fractions 2h hours post feeding in Trial III indicated no detectable change in serum proteins. Immunoelectrophoresis of serum before and after feeding the labeled protein indicated thatcx, and B-globulins and albumin were absorbed, but that y-globulin was not. One litter of 12 pigs was used to study the effect of increasing blood volume on whole protein absorption. Pigs caught at birth were divided into the control and treatment.group which received dextran plasma extender to increase their relative blood volume to that level expected at 2h hours of age. All pigs were returned to the sow and changes in weight, total serum protein concentration, and serum protein fractions were followed. The lack of significant differences in the above- Charles H. Long mentioned criteria indicated that absorption of colostral proteins was not affected by changes in blood volume. Alkaline phosphatase activity in the milk of the dams and alkaline phosphatase activity in the serum of pigs in 5 litters was studied during the‘post-natal period. Data were collected from these 5 litters at birth, I, 2 and 7 days of age. Two litters were also studied at lb and 30 days of age. Individual values within a litter were highly variable, but sow litters had higher serum alkaline phosphatase activity than did gilt litters. Activity, generally, but not always, decreased after birth. Negative values of .53 and .33 were obtained when serum activity was correlated with weight and total serum protein, respectively, in these 5 litters. Colostrum and milk from gilts had higher alkaline phosphatase activity than did the sows, although activity in both decreased as lac- tation progressed. The fall in serum activity of the pigs generally followed the decline in milk phosphatase activity. Sera from pigs of h ages were stored either at 50 C for 18 hours or at -20°C for h weeks to study the effect of storage on enzyme activity. Storage at 5° C significantly increased phosphatase activity, while storage at -200 C generally resulted in a significant decrease in serum activity. FACTORS WHICH INFLUENCE GASTROINTESTINAL ABSORPTION IN THE IMMEDIATE POST-NATAL PERIOD I. Effect of feeding protein and protein-free diets on absorption of whole proteins in colostrum-deprived pigs. II. Effect of increasing blood volume on whole protein absorption. III. Changes in serum and milk phosphatase during the post-natal period. By A ‘l' Charles H. Long A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 196u Charles H. Long candidate for the degree of Doctor of Philosophy DISSERTATION: Factors which Influence Gastrointestinal Absorption in the Immediate Post-Natal Period OUTLINE OF STUDIES: Main Area: Animal Husbandry (Animal Nutrition) Minor Areas: Biochemistry, Physiology BIOGRAPHICAL ITEMS: Born: December 20, 1935. Batesville, Arkansas Undergraduate studies: Arkansas State College, 1958-1960 Graduate studies: Michigan State University, l960-l96h EXPERIENCE: United States Army, l95h-l957 Graduate Assistant, Michigan State University, 1960-196h MEMBER: American Society of Animal Science Delta Tau Alpha ii ACKNOWLEDGEMENTS The author wishes to express his gratitude for financial support provided by the following agencies, without which his ambition would never become a reality: Lambda Chi Alpha Fraternity for the alumni scholarship; Michigan State University and Dr. R. N. Nelson for the graduate research assistantship; and the Rackham Foundation for its support of research projects. To Dr. D. E. Ullrey the author would like to give his appreciation for his wise and patient counseling; his constructive criticism always supported the highest standards of scientific investigation and evalua- tion. His encouragement of weekly scientific discussions will not be forgotten. The author is grateful for the example set by Dr. E. R. Miller, whose competence in research has also provided a constant challenge for achievement. Acknowledgement is made to the other members of the guidance committee, Drs. Dorothy Arata, R. W.-Leucke, and R. K. Ringer, for their guidance and reading of the manuscript. Invaluable assistance in many phases of animal care and analytical procedures was provided by Betty Vincent, Frank Corin, Gordon Grossman, John Schoepke, Frank Walters, Bob Struthers, and Chesley Zutaut. The author is indebted to his colleagues, Charles Derrickson and Ben Brent, for time and help offered on many occasions. The secretarial assistance of Kay Butcher has been most helpful; and the careful preparation and typing of the manuscript by Marie Stehlik is appreciated by the author.. For her faith, loyalty, and confidence, the author wishes to thank his wife, Judith. The long-standing encouragement of the author's parents has been inspirational throughout his course of study. iii TABLE OF CONTENTS I. INTRODUCTION .4. . . . . . . . . . . . . . . . . . . . . . . . II. REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . A. Macromolecule Absorption in the Small Intestine . . . . . Serum Protein after Nursing . . . . . . . . . . . . . Acquistion of Passive Immunity . . . . . . . . . . . . . Colostral Globulin Uptake . . . . . . . . . . . . . . . l3 Absorption of Heterologous Proteins . . . ... . . . . . 1h Absorption of Inert Material ... ... . . . . . . . . . 15 Duration of Absorptive Period . . . . ... . . . . . . 16 Proteinuria Associated with Globulin Absorption . . . l8 . l A h Serum Proteins before First Nursing. . . . . . . . . . . 2 8 CD-«lmm-F'UUIUH B. Histological Evidence of Absorption . . . . ... . . . .7. 19 C. Serum and Tissue.Alkaline Phosphatase during Absorption and Factors which Alter Them . ... . . . . . . . . . . . . 21 1. Enzyme Alterations in Disease . . . . . . . . . . . . . 22 2. Origin of Serum Alkaline Phosphatase . ... . . . . . . 22 3. Age and Breed Differences . . . . . . . . . . . . . . . 25 A. Effect of Diet . . . . . .,. . . . . . . . . . . . . . .25 5. Effect of Hormones . . . . . . . . . . . . . . . . . . .26 6. Intestinal.Activity . . . . . . . ... . . . . . . . . .28 7. Milk Phosphatase . . . . . . ... . . . . . . . . . . . .28 8. Studies in Farm.Animals . . . . . . . . . . . . . . . .30 D. Raising Animals Deprived of Colostrum . . . . . . . . . . .31 III. EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . . . . . .3h A.“Effect of Feeding Protein and Protein-Free Diets on Absorption of Whole Proteins in Colostrum-Deprived Pigs . .34 General . . ... . . . . . ... . . . . . . . . . . . . . 3h . Trial I . . . . . . . ... . . . . . . . . . . . . . ... 38 . Trial II . . . . ... . ... . . . . . . . . ... . . . . 39 Trial III . . . . . . ..... . . . . . .1. . . . . ... . 39 Fluorescent Labelling of Proteins ... . . . . . Production of Rabbit Anti-Porcine Immune Sera . . . . . Al . Biological Determinations .,. . . . . . . . . . . . . . M2 Nam-F‘UOI'DH a. Paper Electrophoresis . . . . . . . . . . . . . . ..h2 b. Immunoelectrophoresis . . . . . . ... . . . . . . . #2 c. Preparation of Tissue for Enzyme Analysis . . . . . #7 iv VI. VII. TABLE OF CONTENTS (Continued) d. Tissue Alkaline Phosphatase . . . . . . . ... . . e. Protein Determination . . . . . . . . . . ... . . f. Glucose . . . . . . . . . . . . . . . . . ... . B. Effect of Increasing Blood Volume on Whole Protein Absorption . . . . . . . . . . . . . . . . . . . . .1. General . . . . ... . . . . . . . . . . . . . . C. Changes in Serum and Milk Phosphatase in Five Litters dm‘ing the POSt-Na'tal PeriOd o .o o o .o o o o o o o o o l I General 0 O O O O O ' O ' O O O O O I O O O I O O O O 2. Effect of Storage on Serum.Alkaline Phosphatase . 3. Serum Alkaline Phosphatase . . ... . . . . . . RESULTS AND DISCUSSION ... . . . . . . . . . . . . . . A. Effect of Feeding Protein and Protein-Free Diets on Absorption of Whole Proteins in Colostrum-Deprived Pigs. l 0 Trial I o o o o o o o o o o o o o o o o o o o o o o o 2 0 Trial II o o o o o o o ' o o o o o o o o o o o o o o o 3 0 Trial III 0 o o o o o o - o - o o o o o o , o o o o o o o o B. Effect of Increasing Blood Volume on Whole Protein Absorption . . . . ... . . ... . . . ... .,. . . . . C. Changes in Serum and Milk Phosphatases in Five Litters durmg the POS't-Na'tal PeriOd o o o o to o o o o .0 'o o o 1. Effect of Storage-on Serum Alkaline Phosphatase . SUMMARY . . . . ... ... . . . ... . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . ... APPmDH O O O I O I O 0 O O O O O O O '0 O O O O O Q C O O Page 1+7 1+8 #9 .19 .19 SO SO 51 51 53 53 -53 73 89 - 91 . 9h ~97 .lOl Table Chm-(TU) N 10 ll l2 13 11+ 15 l6 17 18 LIST OF TABLES Observations on the presence of 7-globulin in the newborn. Composition of solids in purified diets . . . . . Weight data on pigs in Trial I . . . . . . . . Total serum protein in Trial I . . . . . . . . . . . Weight data on pigs in Trial II . . . . . . . ... . Total serum protein in Trial II . . . . . . . Blood glucose levels of pigs given insulin in Trial I Weight data on pigs in Trial III . . . . . . Total serum proteins in pigs on Trial III . . . Blood glucose in pigs in Trial III . . . . . . . . Serum phosphatase in pigs in Trial III at feeding and 2"" hows Post-feeding o o o o o o o o o o o o o o o 0 weight changes in pigs on blood volume trial . . . .. Total serum protein in pigs on blood volume trial . Serum protein and hematocrit of pigs on blood volume Serum phosphatase activity in five litters . . . . . Phosphatase activity in colostrum and milk ... . . . Alkaline phosphatase in udder sections . . . . . . . Effect of storage on alkaline phosphatase in serum . of pigs of four ages . . . . . . . . . . . . ... vi I O 0 trial 87 90 90 91 92 91:. 9h .- 96 Figgge l 10 11 LIST OF FIGURES Agar gel electrophoresis vessel used in preparation of immunoelectrophoresis plates . . . . ... . . .,. . ... . . #5 Diagram of the arrangement of agar, buffer, and coolant in the electrophoresis vessel . . . . . . ... . . . . .. . . .h6 Comparison of the serum protein profiles among the zero hour pig, one week old pigs fed synthetic diets, and the sow-fed controls from Trial I . . . ... . . . ... . ... . . . . . 56 Comparison of serum protein profiles among the zero hour pig, pigs fed synthetic diets for two weeks, and the sow-fed controls from Trial I . . . . . . ..... . .1. . . . . . . 57 Immunoelectrophoretic pattern resulting from the incubation of (l. to r.) serum from a one week old control-pig, one week old casein-fed pig, one week old glucose-fed pig and a zero hour pig from Trial I with rabbit anti-prepartum sow serum ..... ... . ... ... w . . . . v . . . ... . . . 59 Comparison of intestinal alkaline phosphatase activity at six locations in the casein- and glucose-fed pigs and the controls at one week of age in Trial I .7. . ... . ..... . 60 Comparison of intestinal phosphatase activity at Six locations in the casein- and glucose-fed pigs and the controls at two weeks of age in Trial I... . . . ... . . . . . . . 6l Comparison of the serum protein profiles among the zero hour pig, glucose-fed pig one week old, and a glucose-fed pig and sow-fed control two weeks old from Trial II . . . .66 Immunoelectrophoretic pattern resulting from the incubation of zero hour sera from h'pigs used in Trial II with rabbit mti-h‘ months pig sew o o o o o o -o -o ‘0 o o o o o 67 Immunoelectrophoretic pattern resulting from the incubation of (l. to r.) serum from a one week old control, one week old casein-fed pig, one week old glucose-fed pig, and a zero hour pig from Trial II with rabbit anti-h months pig serum . . . . . . . . . . . . . . . . . . . . . . . ... . Comparison of intestinal alkaline phosphatase activity at 6 locations in pigs fed the glucose diet for l and 2 weeks and a 2 week old control . . . . . . . . . . . . . . . . . 71 vii LIST OF FIGURES (Continued) Figgre Page 12 Photomicrograph showing the cross section of a villus from a glucose-fed pig in Trial II 6 hours after feeding FITC-labell8d. pI‘Ote'In o o o o o o o o o o o o o o o o o o 72 13 Photomicrograph showing the uptake of fluorescent material by the columnar cells of the colon and passage into the blood vessels . . . . . . . . . . . . . . . . . . . . . . 72 lh Photomicrograph Showing a longitudinal section of a villus which demonstrates the fluorescence seen in the blood vessels 0 O O O O O O O O ‘0 O O O O I O O O O O O O 0 O O 73 15 Comparison of the serum protein profiles among zero hour pig, 1 week old pigs fed synthetic diets, and a sow-fed contrOl from Trial III I O O O O I O O O O O O O O O I O O 76 16 Comparison of the serum protein profiles among the zero hour 2 week old pigs fed synthetic diets, and a sow-fed control fI'Om Trial III 0 o o o o o o o o o o o o o o o o o -o o o o 77 17 Immunoelectrophoretic pattern resulting from the incubation of serum from (1. to r.) a 1 week old control, 2 casein-fed pigs, 2 glucose-fed pigs, and 2 control pigs in Trial III with rabbit anti-h month pig serum . . . . . .,. . . . T9 18 Immunoelectrophoretic pattern resulting from the incubation of serum from (1. to r.) 2 one week old controls, 2 casein-fed pigs, 2 glucose-fed pigs, and another control in Trial III with rabbit anti-h month pig serum 2h hours after feeding the protein mixture . . .-. . . . . . . . . .80 19 A tracing of the immunoelectrophoretic pattern resulting from the incubation of the electrophoresed colostral whey- -globulin mixture with rabbit anti-h months pig serum and anti-prepartum sow serum . . . . . . . . . . . 81 20 Comparison of intestinal alkaline phosphatase activity at 6 locations in the 1 week old casein- and glucose-fed pigs and the controls in Trial III . . . . . ... . . . . . 83 21 Comparison of intestinal alkaline phosphatase activity at 6 locations in the 2 week old casein- and glucose-fed pigs and the controls in Trial III . . . . . . . . . . . . 8h 22 Serum alkaline phosphatase activity from birth to death in the 15 day old casein-fed, glucose-fed, and control pigs in Trial III . . . . . . . . . . . . . . . . . ... . 86 viii Fi 23 2h 25 26 e LIST OF FIGURES (Continued) Photomicrograph of a kidney from a casein-fed pig in Page Trial III showing a fluorescing glomerulus as a result of ' the FITC in the Plasma-. O O O I O 0 ~ 0 O O O O C 0 O O O O Photomicrograph of a section of thymus showing the 88 fluorescent particles which appeared to be associated with the reticuloendothelial cells . ... . . . . . . . . . . . 88 Comparison of serum total protein, serum alkaline phosphatase activity, and weight change in the pigs from 5 litters during the fiI'St month Of life b o‘ o o o o o o o o o 0 Comparison of alkaline phosphatase activity in pig serum.and milk of the dam during the first 2 weeks postpartum . ix .93 -95 Table 10 ll 12 LIST OF APPENDIX TABLES Page Serum protein fractions in Trials I and II at 6 hours post-feeding the labelled protein. . . . . . . . . . . . . l22 Serum protein fractions in Trial III at feeding and 2% hours post-feeding the labelled protein . . . . . . . . . l22 Intestinal phosphatase in pigs of Trials I, II, and III . . . 123 Tissue dry matter of colostrum-deprived and control pigs in Trials I, II, and III . . . . . . . . . . . ... . . . . 12h Serum alkaline phosphatase of pigs in Trial III before and after feeding the labelled protein . . . . . . . . . . 125 Weight and total serum protein in 5 litters . . . . . . . . . 126 weight of individual pigs in litters 65, 66, and 69 . . . . . 127 Weight of individual pigs in litters 70 and 72 . . . . . . . l28 Serum alkaline phosphatase in individual pigs of . litters 65, 66, and 69 . . . . . . . . . . . . . ... . . . 129 Serum alkaline phosphatase in individual pigs of litters 70 and 72 . . . . . . . . . , . . . . . . . . . . 130 Total serum protein in individual pigs of litters 65, 66’ and 69 . C 0 O O O O O O C C C . ‘ C C . C C C O O O C Q 131 Total serum protein in individual pigs of litters 70 811d 72 O O O O O O I O O I O O O O O O O O O O O O O O 132 I. INTRODUCTION Baby pigs are among those animals which are born almost devoid of 7'-globulin. Acquisition of this serum fraction is of prime importance since much of the passive immunity derived from the dam obtained as colostrum is ingested (Brambell, 1958a; Speer E E1” 1959; Miller 2’2 31., 1962). Of considerable significance is the fact that the animal is able to absorb these large protein molecules, unaltered, across cellular mem- branes. Serological (Haliday, 1956b), histological (Comeline gt al., 1951), electrophoretic (Miller gt al., 1962a), and immunochemical (Lecce gt al., 1962) procedures are a few of the techniques used to establish that this absorption does occur. This "absorptive state" is temporary, and the gut soon becomes closed to proteinaceous macromolecules. The factors which bring about this change in the intestinal epithelium and the consequent "gut closure" provide the subject of this thesis. That gut closure is time dependent has been replaced by recent theories which suggest that closure is effected by the amount of colostrum ingested. It is the opinion of some workers (Payne and Marsh, 1962b) in this area of investigation that protein must contact the gut before closure is accomplished. If this is the case, would colostrumpdeprived pigs, maintained on a non-protein diet, retain their ability to absorb large molecules, i.e., 7-globulin, indefinitely? This question provides the basis for the first objective of this study. One of the problems in pursuing the above objective is the diffi- culty of maintaining colostrum-deprived pigs for extended periods of time. Attempts to formulate diets for colostrum-deprived pigs have been prompted primarily by studies of mineral requirements (Lecce, §t_§l., -1- -2- 1961a) and studies of specific-pathogen-free pigs (Berry et'al., 1962). Mortality in these studies was usually high as a result of diarrhea, which was attributed to microbial contamination or inadequacies in the diet or husbandry. Miller, et 21. (1951+, 1957, 1962b, 1961+a, 196%) have used a synthetic diet patterned after the composition of sow‘s milk with much success in rearing early-weaned pigs. Utilization of the liquid form of this diet to rear pigs taken from the sow at birth provides another objective for this thesis. Fluorescent-labeled protein techniques have been used to study whole protein absorption by Payne and Marsh (1962a). Gut segments from pigs starved 106 hours were used by these workers to study the uptake of fluorescein isothiocyanate-labeled proteins by the epithelial cells. Tissue sections from these animals fed the labeled protein contained large droplets of material which possessed fluorescence. This fluores- cence was attributed to the presence of labeled protein. No attempts were made to study the uptake of this labeled protein when it was ad- ministered orally or to ascertain the absorption of protein by criteria other than the presence of fluorescence in the columnar cells. wellman and Engel (196M) have demonstrated protein absorption at 120 hours of age in pigs which were fed boiled sow's milk. The pigs in which gut closure has been studied have usually been starved. Colostrum—deprived pigs which have received all the required dietary nutrients except protein have never been utilized to determine if the gut remains open on such a dietary regimen. This approach was used to see if intestinal cells not contacted by protein would retain their absorptive ability. Much research has been conducted to determine the time of gut closure; yet little has been done to study the mechanism of this phenome- -3- non. If the volume of colostrum ingested is responsible for changing the absorptive state to the non-absorptive state, it would seem that the amount of colostrum ingested would, in some way, affect the blood. Rameriz gt El' (1963) have reported that'the relative blood volume in the newborn pig changes abruptly with the ingestion of colostrum. At 2h hours, which is about the average time reported for gut closure, the relative blood volume changes from 8.6 to 10 percent. The relationship between this rise in relative blood‘volume and absorption of colostral proteins is another area of investigation included in this dissertation. work with mice and rats has shown a 20-fold increase in duodenal alkaline phosphatase activity which reaches a peak at the same time the animal ceases to absorb whole molecules from the gut (Moog, 1955, 1962). Payne and Marsh (1962b) have reported that the same events occur in the baby pig. They state that a 7-fold increase in intestinal phosphatase activity occurs at the time of gut closure. Serum phosphatase activity, on the other hand, is reported to decrease in the neonatal pig (Young and Underdahl, l9h8). Whether these changes in alkaline phosphatase cause, or are the result of gut closure, has not been determined. The changes in serum and intestinal alkaline phosphatase in colostrum-deprived pigs, and serum phosphatase in pigs nursing the sow provide another area of investigation. II. REVIEW OF LITERATURE A. Macromolecule Absorption in the Small Intestine For a short while after birth, several species have the ability to absorb large, intact, protein molecules from the intestine into the blood stream. Electrophoresis, ultracentrifugation, serology, and other analytical procedures have established that absorption without alteration of these molecules can occur. Molecular weights of these compounds approach 200,000, the average value in the literature for 7-globulin. Compounds designated as macromolecular in this review shall have molecular weights of 6,500 or more. Cessation of absorption, or gut closure, shall be defined as that time after which the animal is unable to absorb macromolecules. 1. Serum Proteins before First Nursing Many studies have been conducted to investigate the serum protein profile. Investigations of the fractions with regard to their precipi- tation and sedimentation properties have been reviewed by Cohn (l9hl, 19h6) and Edsall (19h7). The physiological functions of the serum pro- teins are discussed by Howe (1925) and Kazal (1962). Gutman (19h8) has 'reviewed plasma proteins in disease; and Kekwick (1959) has compared the serum proteins of the fetus and young of several species. Fewer exami- nations of protein fractions of pig-serum have been made (Svensson, l9hl; Cooper, l9h5; Deutsch and Goodloe, l9h5; and Koenig and Hogness l9h6). Koenig gt g1., (19h9) have analyzed sheep sera. Investigations of serum proteins in pigs from birth to maturity have been conducted by Jacobsen and Moustgaard (1950) and Miller 2£.§l' (1961). Fetal serum protein -h- -5- studies in sheep were made by Meschia (1955) in which he found that total proteins tended to increase with progressing gestation. Rutquist (1958) and Waddill 23 E1. (1962) have followed serum protein changes in the fetal pig. Little change occurred in total protein concentration, but Waddill did observe an increase in a-globulin and a decrease in B-globulin before birth. -In 19kb, Pedersen isolated a new globulin from fetal serum, calling it fetuin. Rutqvist (1958) noticed a split in the albumin fraction of fetal and newborn pigs. Barboriak gt g1. (1958a,b) working with fetal goats and sheep observed an additional peak in the a, region. They concluded that this was due to the presence of fetuin. The fetal globulin content declined with age. Fetuin has been isolated and found to be an alglobulin which can account for about h0% of total serum pro- tein in the calf. Pirtle and Deyoe (1963) indicated that two major a-globulins were present in newborn SPF pigs. Several workers have questioned the presence of 7-globulin in the newborn. The following table illustrates the lack of agreement on the question. The more recent papers are just as contradictory as were the early ones. Payne and Marsh in 1962a suggested that the densitometer used to scan conventional bromophenol blue paper electrophoretic strips lacked sensitivity. Although greater sensitivity was produced by using nanother dye and filter arrangement, separation was not greatly improved. Immunoelectrophoresis is probably the most sensitive indicator avail- able for routine clinical use. Sterzl gt El’ (1960) have used this pro- cedure to detect a non-antibody 7-globulin in newborn pigs not receiving colostrum. -Immunochemical, sedimentation, and electrophoretic properties of this non-antibody globulin were studied further by Franek gt 31. -6- (1961). Their data indicate that this globulin is electrophoretically identical with 7-globulin, but immunochemically non-identical. They also differ in molecular weight as evidenced by a significantly lower sedimen- tation coefficient, Lecce gt El. (1962) suggested that they, too, had ob- served the non-antibody 7-globulin in their immunodiffusion separations but their published illustrations failed to support this observation in the immature pig. 2. Serum Proteins after Nursing As early as 1935, Earle observed an increase in serum globulin of pigs receiving colostrum. This observation was also made by Foster gt El. (1951) and Barrick §t_gl. (195M). Widdowson and McCance (1956) stated that a rise in serum protein concentration was part of post-natal develop- ment in the pig. Other workers(Nordbringeufl.Cflsson, 1957; McCance and Widdowson, 19593 Payne, 1959; Vesselinovitch, 1959; Lecce and Matrone, 1960; Lecce gt 31., 1961a,b; Lecce and Morgan, 1962; Lecce and Matrone, 1961; Asplund, 1962; and Payne and Marsh, 1962a,b) have also demonstrated ”0 this change in pig serum after absorbing colostral proteins. The s rum ( of newborn SPF pigs has been studied by Pirtle and Deyoe (1963). Changes in lamb serum proteins after the absorption of colostrum have been reported by Charlwood and Thompson (19h8) and McCarthy and McDougall (1953). Jameson gt g1. (19h2), Deutsch and Smith (195b, 1957), Pierce (1955), Hansen and Phillips (l9u7, l9u9), Bangham.§t.§1. (1958), and Balfour and Comeline (1959), have used electrophoresis to follow the changes in calf sera after the ingestion of colostrum. Smith and Erwin (1959) studied the changes in calf serum proteins when colostrum.was in- troduced directly into the duodenum. Varnell gt El. (1960) reported the changes in glyco- and lipo-proteins in normal and colostrum—deprive calves. -7- .TABLE 1. Observations on the Presence of 7-Globulin in the Newborn WOrkers §g§§| Animal Presence Jameson gt g1. 19h2 Calf - Hansen and Phillips 19h9 Calf + Pierce 1955 Calf + Steck 1963 Calf + ‘ Baglioni and Firoetti 1963 ~ Calf + Polson l9h3 Foal - Barboriak gt 91. 1958b Lamb - Barboriak gt g1. 1958a,b Goat - Jacobsen and Moustgaard 1950 Pig - Rook £3 31. 1951 Pig - Nordbring and Olson 1957 Pig + Rutqvist 1958 Pig '- Vesselinovitch 1959 Pig — 'Lecce and Matrone 1960 Pig - . Sterzl §t_gl. 1960 ' Pig + Varne11.§thgl. 1960 Pig + Miller _e_t_ 31. 1961 (Pig _+ Asplund gt El. 1962 Pig - Lecce gt _a_l_. 1962 Pig - Payne and Marsh 1962a Pig + Waddill 1962 Pig + Pirtle and Deyoe 1963 SPF Pigs + -8- Electrophoretic analysis and isolation of the immune globulins by Smith (l9u6a,b) indicated that lacto-globulin is the predominant immune protein in bovine colostrum. The colostral immune globulins and plasma 7 -globulin weresflunnlto be quantitatively equivalent in producing anaphylaxis in guinea pigs. The immune globulins constitute about 10% of the protein of normal colostral whey. In a later study, Smith (19h8) concluded that colostral globulin did not migrate electrophoretically with serum globulin. However, Johnson and Pierce (1959) using electro- phoresis and ultracentrifugation, showed that the globulins were similar. There is usually an increase in plasma volume associated with the absorption of colostral protein. McCance and Widdowson (1959) reported a 30% increase in plasma volume in the first 2h hours of postnatal life. Hansard §t_§1. (1951) studied blood volume increases during neonatal life. Ramirez gt El. (1963) also found an increase in relative blood volume and in plasma protein which they related to the influx offSand 7- globulins from colostrum. Several workers have used immunoelectrophoresis to study the changes in serum protein of the neonatal animal. Brummerstedthansen (1961) and Brummerstedt-Hansen and Hirschfeld (1961) have studied serum variations with age in the Danish Landrace breed of pigs. Lecce and Morgan (1962) and Lecce gt El. (1962) have used this technique to follow serum protein changes resulting from the intake of synthetic diets by pigs. Baglioni and Fioretti (1963) have likewise used this procedure to study the inn gestion of colostrum by calves. 3. Acquisition of Passive Immunity The rabbit, guinea pig, and the human obtain passive immunity as a result of transfer of antibodies from the mother to the young through -9- the placental circulatory system. In the rat, mouse, dog and cat, some antibody transfer occurs before birth as well as through the ingestion of colostrum. In the ruminant, horse and pig there is thought to be no transfer of antibodies across placental membranes (Payne and Marsh, 1962b). Ratner _e_t_ _a_1_. (1927) suggested that the histological difference in the placentae of the various species explains the difference in permeability. Many factors are concerned with placental transfer and morphology of the placenta is only one (Cohen, 1950). Brambell's (1958b) work would suggest—that the blood level of antibodies at the time of birth is directly correlated with development, persistence, and time of withdrawal of the yolk sac into the unbilical cord. In species that have a relatively high level of blood antibody at birth, the yolk sac is exposed to the uterine lumen during most of gestation. In species that have only a slight antibody level at birth, the yolk sac is withdrawn into the unbilical cord early in gestation. This explanation is in contrast to the common opinion that the number of cell layers of the maternal and fetal placentae determines the degree of transfer of antibodies. The ingestion of colostrum with its rich supply of antibodies is the route by which domestic farm animals receive passive immunity (McGirr, 19117). The importance of antibodies as the primary agents of actively or passively acquired immunity is discussed by McMaster (1961). According to Wilson (1962), Ehrlich in 1892 was the first to demonstrate that passive immunity was derived from nursing the mother. Freund (1927, 1930) and Baumgartner (l93h, 1937) have stated that the young of a species has a limited ability to produce antibodies. Dancis 23.2;- (1957) reported that the human fetal liver was capable of synthesizing -10- all the plasma proteins except 7-globulin. This is contradicted by more recent work of Sterzl gt El. (1960) and Franek.§t_§1. (1961) in which they have isolated a fetal 7-globulin from the pig. It will be noted below that several workers have found that young animals begin to synthesize detectable amounts of 7-globu1in at 3 to h weeks of age. Studies by Orcutt and Howe (1922), Pierce (1955) and Olsson (1959b) have demonstrated that antibodies are associated with the globulins of serum. .The antibody level in serum is reflected to some extent by the amount of 7-globulin present. Sell (l96h) determined the 7-g1dbulin con- tent of germ-free guinea pigs to be only % to 1/6 of that of non-germ-free guinea pigs. A review of the therapeutic uses of 7-globu1in has been made by Gross 23 El- (1959). Antibodies are absorbed via the lymphatic as are other large proteins (May and Whaler, 1958). Nelson (1932) and Hoerlein (1952) stated that pigs receive their passively acquired antibodies in the first 2h to 36 hours of life. Nordbring and Olsson (1958b) used antibodies to paratyphoid agglutinins and found that they were absorbed up to 36 hours postpartum in the pig. Speer EE.El° (1959) used antibodies to Escherichia coli. These were not absorbed by the pig in significant amounts beyond 2% hours of age. Miller 33 El. (1962a) observed a similar absorption time using Salmonella pullorum antibodies. Bruner gt gl.~(19h9) reported no absorption of hemagglutinins after 2 days of age. Antibodies to swine influenza virus ‘were demonstrated in pig serum within 30 minutes after the pigs began to suckle (Young-and Underdahl, l9h9). Wellman and Engel (196h) showed antibody absorption at 120 hours after birth in pigs fed on heated cow's milk. Antibody titers to several antigens decline during the first few -11- weeks of life. Young and Underdahl (1950) noted that antibody titer to influenza virus dropped over a period of h weeks. Wellman and Heuner (1960) suggested that the passive immunity acquired from the sow persisted from a few weeks to several months depending on the level of immunity of the sow. Hoerlein (1957), Olsson (1959a), Brown _e_t 3;. (1961) and Miller 33 El. (1962a) have all reported antibody synthesis in the pig by the third week of age. Using pigs nursing "immune" and "non-immune" sows, Hoerlein (1957) showed that passive transfer of antibodies delayed the pigs in synthesizing their own antibodies. Schoenheimer (19h2) gave a half-life of 1% days for pig 7-globulin but Miller gt 21. (1962a) reported 7.5 days. Smith and Little (1923), Mason pp p_l_. (1930), McDiarmid (19%), Smith and Holm (l9h8) and Kaechenbeeck §t_§1. (1962) have all shown absorption of antibodies to various antigens by the calf. Antibody titer in the blood was maximum at 12 hours of age (Kaechenbeeck gt g1., 1962) and decreased over the next few weeks (Smith and Holm, l9h8). Smith (1930) tried to provide passive immunity in calves with serum rather than with colostrum and concluded that a calf would have to drink 2 to 3 liters of serum to match the antibody content of the colostrum it normally receives. The half-life of bovine 7-globulin was 20 and 21 days according to Smith (19h8) and Dixon gt El. (1952), respectively. Chodnik §t_g1. (1960) have demonstrated antibody absorption in the lamb. The titer which developed in the lamb serum depended on the dam's colostral titer. The rat receives antibodies both directly from the mother (Brambell and Halliday, 1956; Brambell, 1958a and Halliday, 1956a) and from colostrum (Halliday, 1956b and Culbertson, 1939a,b). The young rat can -12- still absorb antibodies from milk or immune serum at 20 days of age (Halliday, 1956a). Halliday determined that by far the greater amount of passive immunity is acquired after birth. The rat is more sensitive to the presence of heterologous sera or antibodies than are other species. Halliday (1955, 1957) and Morris (1957) have shown that serum anti- bodies from several other species are poorly absorbed by the rat, and the absorption of rat antibodies in their presence is also greatly reduced. Hemmings and Morris (1959) observed that homologous antibodies were absorbed faster than heterologous antibodies. Halliday and Kekwick (1961) fractionated antisera and titrated the sera to produce evidence that the selection of antibodies by the intestine is related to their location in the serum protein fractions. Campbell gt _a_]_.. (1957) and Sarwar _e_‘_t_ §_l_. (1958) demonstrated that organisms normally present in the mouth of the newborn calf have the ability to provoke the secretion of specific antibodies by the dam's udder. Porterfield and Petersen (1959) found antibodies in milk within 2 hours after lactating cows were infused with the antigen. Dixon gt El. (1961) have studied serum.7-globulin and albumin conCentrations in blood and udder secretions during pregnancy and lactation. -In all udder secretions there were greater amounts of 7-g16bu1in than any other serum protein. Both heterologous and autologous 7-globulins were transferred across the udder. Histological studies showed that the acinar epithelium transported the serum proteins but little or no 7-globulin was formed in the secretory portion of the udder. Larsen £3 31., (195%) also studying-blood serum proteins in the pregnant cow, observed a 10-25% drop in serum protein concentration during gesta- tion which reached a maximum at parturition. -This drop was due to re- -13- ductions in the 32- and 7-globulins. The total amount of globulins in colostrum accounted for the globulins lost from the cow's blood. In human infants, the 7-globulin is slightly higher in the serum of the new- born-than in the mother (Longsworth gt 91., 19h5). The protective mechanism of colostrum is largely an immunological one according to Briggs gt_§1. (1951). Lecce and Beep (1962) disagree With assigning a single function to colostral derived glObulins. They suggest that these globulins are responsble for: ' 1. Promoting localized inhibition of bacteria in the gut. 2. Inhibiting bacteria that do get absorbed. 3. Influencing rapid maturation of the serum profile. A. Colostral Globulin Uptake The absorption of intact proteins by fetuses or newly-born mammals is of unique significance. This absorption of protein differs quanti- tatively from the insignificant absorption in the adult animal (Hogben, 1960). -It is not known if this absorption is due to transient local effects in the intestinal epithelium, or if it represents a more organized process (Carter gt 21., 1959); ' The calf (Howe, 1921; Smith and Little, 192A; Jameson :33 21-: 191.12; Hansen and Phillips, l9h7; McGirr, 19h7; Smith and Holm, 19h8; Comeline, ‘Einél'2 1951; Deutsch and Smith, l95h; Pierce, 1955; Deutsch and Smith, 1957; Bangham gt 21., 1958; Johnson and Pierce, 1959; and Smith and Erwin, 1959), goat (Earle, 1935; McGirr, 191+7; Barbar-iak _e_t_ _a_1_., 1958a; Barbariak _e_t_ _a_1_., 1958b; and Brambell, 1958a), horse (Polson, 19113; and Brambell, 1958a), sheep (Earle, 1935; Charlwood and Thompson, 191+8; Koenig, 19119; McCarthy and McDougill, 1953; and Lecce and Morgan, 1962) and the pig (Jacobsen and Moustgaard, 1950; Foster gt gl., 1951; Rook -1h- gt gl., 1951; Barrick gt g1., 195A; Widdowson and McCance,-1956; Rutqvist, 1958; Vesselinovitch, 1959; McCance and Widdowson, 1959; Bremmerstedt- Hansen, 1961; Lecce and Morgan, 1961; Lecce and Matrone, 1961; Miller _e_t_ _a_l_., 1961; Miller g _a__1_., 1962a; Asplund 2’3 21., 1962; Lecce PIP. a_1_., 1962; Lecce and Morgan, 1962; and Wellman and Engel, 196h) have all been observed to absorb intact colostral globulins. Absorption of intact proteins occurs to a lesser extent in other species. Reid (1900) and Dent and Schilling (1919) demonstrated the absorption of serum proteins by dogs. Bangham and Terry (1957), Halliday and Kekwick (1957), and Brambell _e_t_ _a:_L_. (1961) have demonstrated protein absorption in the rat. Using cattle serum, Stone 33 21. (1957) have tested absorption in several species. Rabbits, puppies, monkeys, guinea pigs and the rat were all found to absorb bovine serum proteins. 5. Absorption of Heterologous Proteins A Heterologous globulins are similarly absorbed by the intestinal epithelium. Hansen and Phillips (l9h9) Observed the presence of cow and‘pig colostral globulins in the blood of kids following the ingestion of cow and pig colostrum. Balfour and Comeline (1959) fed eggs and Steck (1963) fed horse blood serum to observe the absorption of these proteins into calves blood. Barrick gt 21. (l95h)‘recorded the absorption of globulins from bovine serum in the pig. Lecce gt 21. (1961b) and Leece and Morgan (1961) reported the uptake of globulins from bovine colostrum and egg white proteins. Payne (1959) used pig, sheep, cow, dog and human celostrums and Payne and Marsh (1962a) used cow, sheep and human colostrum to study heterologous absorption in the pig. Brambell gt El. (1958) used human and bovine serum and Brambell gt 21. (1961) used I131- labeled bovine globulins in similar studies in rats. Bovine colostrum -15- was also used by Olsson (l959a,b,c) to study absorption of heterologous proteins in the pig. Intact protein absorption is not limited to colostral proteins. To test this statement, a highly purified, easily quantitated substance was needed which would cause some measurable physiological change. Protamine zinc insulin meets these specifications and has been used. Absorption of this hormone is reflected by its hypoglycemic effect. Danforth and Moore (1959) have used insulin to study the effect of soybean trypsin inhibitor. Asplund g a_1. (1962) used insulin to deter- mine gut closure time in pigs receiving colostrum. Both groups of workers were able to demonstrate the movement of insulin across the gut. 6. Absorption of Inert Material Further evidence of non-specific absorption during this neonatal period is provided by studies on the ingestion of inert material by the columnar cells of the mucosa. Balfour and Comeline (1959) fed calves a solution of .2% dextran. These particles were later observed in the lymph. Clark (1959) using saccharated iron oxide and colloidal gold demonstrated the uptake of these particles by the jejunum and ileum but not the duodenum of the rat. Barrnett (1959) using the electron micro- scope noticed that duodenal cells of the rat had engulfed insoluble dye particles. Another type of particle has been used by Sanders and Ashworth (1961). They fed latex spheres, which were 1000 to 2000.3 in diameter, to mice and demonstrated these particles within vesicles in the jejunal epithelium. Lecce 33 El. (1961b) have used a non—protein blood plasma extender polyvinylpyrrolidone (PVP). This compound had a molecular weight of -16- .h0,000. PVP was selected because of its similarity to proteins with regard to molecular size and affect on plasma osmotic regulation. When administered orally to pigs, the PVP was found in the blood and the lack of specificity of the intestine was again illustrated. 7. Duration of Absorptive Period The length of time after birth that the gut retains-its ability to absorb whole proteins varies with the species; and the findings of certain investigators differ. McCarthy and McDougall (1953) reported that lambs can still absorb proteins at 29 hours but not at R8 hours postpartum. Brambell (1958a) stated that lambs still absorb proteins at 36 hours postpartum, while Lecce and Morgan (1962) failed to demonstrate absorption at 2h to 36 hours after birth. Deutsch and Smith (19511) found no protein absorption in calves at h8 hours and Smith.and Erwin (1959) detected none at ho hours postpartum. -According to Steck (1963) protein absorption in calves is restricted to the first 1 to 1% days of life. The foal will still absorb proteins-at 36 hours after birth ’ (Brambe-ll, 1958a). Brambell (1958a) reported that=the pig still absorbs antibodies at 36 hours postpartum. Nelson (1932); Barrick gt 31. (195h); Hoerlein (1952); Speer _e_t a_1. (1959a); Olsson (1959) and Lecce and Matrone (1960) concluded that no intestinal absorption occurs in the pig after 2h hours postpartum. .Lecce and Morgan (1962) concluded that the gut becomes im- pervious to proteins at 2h to 36 hours after birth while Asplund 33 $1." (1962) decided that closure took place between 21 and 27 hours after birth. According to Miller 23 E1. (1962a) absorption of colostral gldbulins.was still occurring at 2h hours of age following a logarithmic decline in absorption rate from birth. Absorption of protein was ob- . -17 - served 120 hours after birth in pigs fed boiled milk (Steck,-l963). Payne and Marsh (1962a) demonstrated absorption in pigs starved for 106 hours. The lymphatics have been found to be the route of absorption of the ingested particles. Alexander gt 2;; (1936) fed dogs egg whites and traced absorption through the lymph. Comeline gt El‘ (1951) studied the absorption of colostrum and its passage through the thoracic duct. No colostrum was detected in the portal vein. .Absorption by the small intestine and transport by the lymphatics was also reported by Brambell (1958a). The finding of a trypsin inhibitor (Laskowski and Laskowski, 1950) in colostrum led to the hypothesis that the physiological role of the .inhibitor is to protect the antibodies of colostrum from being digested and thus to facilitate their absorption (Laskowski and Laskowski, 1951). The concentration of the enzyme declines to non-detectable levels by the 5th day. The colostral enzyme is similar in activity to the pan- creatic enzyme and is twice as active in inactivating trypsin as is the soybean derived enzyme (Laskowski gt gl,,jl952). Barrick gt_§l. (l95h) fed trypsin inhibitor in a milk- 7=—globulin mixture but were unable to improve absorption of 7-globulin. Laskowski 93 El. (1957) demonstrated that the trypsin inhibitor in swine colostrum was 70 times as active as that of human colostrum. The theory that protein absorption is the result of trypsin inhibition was somewhat substantiated when LaskowSki gt al. (1957) promoted the absorption of insulin in intestinal loops of adult rats by administering antitrypsin. Nordbring and Olsson (1958a) were able to increase absorption of B-and 7-globulins at 2h and 36 hours postpartum in pigs fed bovine trypsin inhibitor. Danforth and -18- Moore (1959) also tried to demonstrate intestinal absorption of insulin in the rat using soybean trypsin inhibitor. Insulin was not absorbed in the presence of soy trypsin inhibitor but was absorbed when diisopropyl- fluorophosphate or idole-3-acetate was given. Oeda and Sakaino (1962) have isolated an anti-rennin factor in swine serum. Several compounds have been injected intramuscularly or intra- peritoneally in an attempt to prolong protein absorption in the calf. Cortisone, ACTH, diethylstilbestrol in oil or water, progesterone in oil or water and diethylstilbestrol with progesterone had no effect on extend- ing the absorptive period (Deutsch and Smith, 1957). Aluminum hydroxide gel and probanthine were also fed to prolong absorption. A test globulin was fed at #0 hours postpartum to the calves but no absorption was observed. Payne (1959) has injected, intraperitoneally, 7-globulin from the colostrum of sheep, cows, dogs and humans and increased blood 7-globulin levels in.the pig. The high blood levels failed to alter the absorptive ability of the intestine. 8. Proteinuria Associated with Globulin Absorption In addition to changes in blood properties, changes in the urine have also been noted while absorption of proteins is at a peak. Howe (1921) and Smith and Little (192A) discovered that newborn calves ex- hibited proteinuria after ingesting large quantities of colostrum. Since the proteinuria disappeared within 2A to #8 hours after birth, it was thought to result from small molecular weight molecules spilling out into the urine. A similar observation was made by McCarthy and McDougall (1953). Proteinuria was also reported by Pierce (1959) in calves fed colostrum, but not in calves fed boiled milk. Termination of -19- intestinal absorption from the gut was found to coincide with falling protein levels in the urine. Electrophoretic analysis showed at least 6 components. Two components had a mobility faster than that of albumin and composed 5A% of the total urine protein. These results suggested that the urinary protein was composed of small molecules which were cleared from the circulation by glomerular filtration. This proteinuria was studied further by Pierce (1960) and Pierce and Johnson (1960) in an attempt to identify the specific proteins occurring in the urine. 'The components of the colostrum appearing in the urine were found to be B-lactogldbulins. B. Histological Evidence of Absorption In 1925 Smith observed that epithelial cells from newborn calves contained many vesicles which he believed to contain protein. The vesciles disappeared about the time~that the cessation of protein absOrption occurs. Comeline §t_al. (1951) also made this observation on calves 3 to 2A hours old. When the sections were stained, the vesicles resembled.stained whey proteins. Some globules were present in the lacteals of the villi. It was concluded that during the first 2h hours of life, colostral globulin is absorbed unchanged. .In 1953, Comeline 23 El. performed similar experiments on newborn pigs and kittens. Again staining procedures indicated the presence of a substance not found in the tissue of older animals. Vesicles in the intestinal epithelium of newborn kids and lambs were also demonstrated by Hill and Hardy (1956). Fluorescent labeling of protein has added evidence to support the theories of cellular involvement in whole protein absorption. Payne and Marsh (l962a) administered fluorescein isothiocyanate-labeled -20- globulins from the colostrum of several species to the pig. When viewed under the fluorescent microscope, globules within the cells exhibited fluorescence. The occurrence of these fluorescent particles within the cells was taken as evidence of absorption’of heterologous globulins. In tissue sections of pigs apparently not absorbing protein, the fluor- ~escence occurred outside the villi. Hill (1956) suggested that the cessation of intestinal absorption of immune proteins from colostrum by the newborn of several species co- incides with the onset of gastric protein'development. A histological study of the gastric glands in the abomasum was made. Parietal cells 'which were very sparse at birth increased rapidly during the first AB hours of life. The pH of the abomasal contents was between 6 and T at birth, but gradually decreased to pH 3 to h by 36 hours. wA comparative study in rats showed that gastric glands were not fully developed until the third postnatal week. This corresponds to the extended period of protein absorption in the rat reported by Clark (1959). The author 'concluded that those species in which antibody transfer occurs 23 33232, secretion of active gastric Juices occurs at or before birth. Those species receiving antibodies from the colostrum have delayed gastric protein digestion by retarded development of the gastric glands or by one of the cell types of the gastric glands. Barrnett (1959) observing the uptake of insoluble dye particles, suggested that morphologically the process was identical to pinocytosis. Clark (1959) had also proposed pinocytosis as the method by which part- eiculate material was ingested in mite. Each particle appeared to be in a membrane-enclosed vacuole in the cytoplasm. .These vacuoleS‘werevpart of a system of interconnecting vacuoles and tubules which were con-t -21- tinuous with the apical cell membrane. Clark (1959) further proposed that adult animals may not have lost the capacity for pinocytosis, but rather have become selective as to‘What substance provokes it. Within 72 hOurs after intramuscular injection of cortisone, the cells resemble those of adult animals and no longer ingest proteins. Latex particles fed newborn rats by Sanders and.Ashworth (1961) were traced through the epithelial cells. The particles were carried through the cytoplasm in intact vesicles, discharged into the interstices of the lamina propia to gain entrance into'the lymphatics. The process resembled very much the present day concept of lipid absorption (Wilson, 1962). Bertalanffy (1957) has calculated that 79% of the small intestine is renewed every day. Using s35-labeled methionine, Leblond gt a1. (1957) showed that cells forming in the crypts of the villi move from the base to the tip. The cells move along the villus and are released or pinched off at the zone of extrusion (Leblond and Stevens, l9h8). -As the cells move to the tip of the villus they change gradually from cuboidal to columnar in shape and the microvilli increase in length. According to Quastler and Sherman (1959) this takes about 1 3/h days. Protein in the proliferating cells is highly stable (Lipkin gt 21., 1961). C. Serum and Tissue Alkaline Phosphatases during Absorption and Factors which Alter Them Kay pointed out in 1930 that the phosphatase content of bone, kidney, liver and blood may change under varying conditions of diet, growth and disease. The phosphatases of serum.and of various tissues were considered identical by Kay (1932) and phosphatase activity in the serum was thought due to leakage of phosphatase from the tissues. These early observations indicated that alkaline phosphatase is a rather -22- non-specific enzyme which is affected by several factors. In order to evaluate the changes which take place in intestinal alkaline phosphatase activity during macromolecule absorption, these factors will be reviewed. 1. Enzyme Alterations in Disease Serum alkaline phosphatase can reflect a pathological state in the case of several bone diseases (Kay, 1930). Snyder and Tweedy (19h2) reported that a magnesium deficiency resulted in decreased serum alkaline phosphatase activity. Miller _e_t al- (1962b, l96ha, 1961+b) have elevated. phOSphatase activity by producing calcium, phosphorous and vitamin D deficiencies in the baby pig. An increase in serum alkaline phosphatase was also observed in vitamin D deficient chickens (Correll and Wise, l938). Madsen gt El. (19h7) reported an increased enzyme level in beef cattle due to a vitamin A deficiency. Zinc deficiency also reduces alkaline phosphatase activity (Hoefer gt a__l_., 1960 and Starcher and Kratzer, .1963). Combs §£.Ein (1955) used alkaline phosphatase to establish the nutritional status of pigs on a phosphorous availability study. Elevated serum phosphatase activity also occurs in jaundice if it is hepatic in nature (Cantrow and Miller, l9h8). Phillips (1932) proposed that plasma phosphatase concentrations could serve as a criterion forthe degree of fluorosis in cattle. Mitchell and Edman (1951) were not in agreement with this proposal. Maplesden gt a1. (1960) proved that in rats and rabbits plasma or several tissue levels of alkaline phosphatase were not suitable criteria for the degree of fluorosis. 2. Origin of Serum Alkaline Phosphatase The clinical significance of using alkaline phosphatase as a diagnostic tool was reviewed by Yaguda (1936). Even though serum -23- alkaline phOSphatase analysis is used as a prognostic procedure, the exact chemical nature of its action has not been fully elucidated. Moog (19H6) described alkaline phosphomonoesterase as a salt-soluble substance which consists of a metallic co-enzyme and a protein-like carrier. Bodansky (l93h) and Schlamowitz (195A) suggested that serum alkaline phosphatase had a diverse origin. Bone, intestine, liver and kidney were all believed contributors of the enzyme. Armstrong and Banting (1935) found that removal of the viscera from animals did not affect the serum phosphatase level and concluded that most of the serum phosphatase came from bone. Gould and Schwachman (19h2) and Schlamowitz and Bodansky (1959) also designated the bone as the source of the serum enzyme. Madsen and Tuba (1952) thought that some of the serum enzyme was osseous in origin but noted that there were 2 phosphatases in the liver. Kosman 23 a1. (19A2) presented evidence that the duodenum and jejunum are principle sources of phosphatases secreted into the intestine. Flock and Bollman (1950) ligated the bile ducts in rats and demonstrated that the increase in serum alkaline phosphatase which accompanies the ingestion of fat could be prevented. From this they postulated that bile in some way causes a release or transport of alkaline phosphatase from the intestinal mucosa. Tuba and Robinson (1953) showed that intestinal alkaline phosphatase consists of an adaptive portion which varies with the dietary state and a non-adaptive portion which remains constant during prolonged fasting. Wilson and Wilcox (1963) used enzyme inhibitors to study inbred lines of chickens which exhibited either high or normal serum alkaline phosphatase activities. Sodium borate was used since it was found that _2h_ this compound would inhibit phosphatases more in the high serum line than in the random-bred controls. This inhibitor was used on tissues from the two lines and intestinal phosphatase activity was depressed significantly more than that in bone, liver, and kidney. From this it was suggested that the increased serum phosphatase of the high line had originated in the intestine. Gutman and Jones (l9h9) reported that liver alkaline phosphatase is relatively insensitive to HCN. The pH optimum for the hydrolysis of glycerolphosphate was determined to be 9.85 for chick bone, kidney and liver phosphatase, but 9.2 for intestinal phosphatase. Human alkaline phosphatase of intestine, but not of liver, bone, lung, bile, kidney and spleen was almost completely inhibited by L-phenylalanine but was not so affected by D-phenylalanine (Fishman at. a_1., 1963). These workers interpreted this as evidence for a difference in the catalytic site of the intestinal enzyme from that of the enzyme from other'sources. Harris _e_t _a_l. (1952) and Harris and Mehl (1955) have attempted to purify intestinal alkaline phosphatase. They obtained a preparation from the microsomes of steer intestinal mucosa. Their results indicated that the enzyme may have been in a complexed form, but the compleX'was disrupted by repeated electrophoretic separation. A 200-fold purifi- cation was achieved, but the yield was small. Boyer (1963) electrophoresed alkaline phosphatase from human organs and observed considerable heterogeneity. Ultracentrifuge sedimentation studies suggested that these heterogenous alkaline phosphatases possess similar molecular weights. Three antigenic classes of the nonspecific phosphatases were distinguished: 1. Liver, bone, spleen and kidney -25- 2. Intestinal 3. Placental Numbers two and three were cross-reactive with one another. 3. Age and Breed Differences As early as 1938, Thannhauser et a1. (1938) recognized that animals from different strains differed in their serum phosphatase levels. Serum with high phosphatase activity had the ability to increase the amount of inorganic phosphorous when mixed with normal serum in equal pro- portions. Gutowska.§t_al. (l9h3) related plasma phosphatase levels to productivity in hens. Kunkel _e_t al- (1953) reported differences in enzyme levels in several breeds of beef cattle. Brahman cattle exhibit- ed alkaline phosphatase levels twice as high as did European cattle. Crosses between these cattle of different origin yielded cows with intermediate enzyme activity. Bodansky (193hb) demonstrated in puppies that serum enzyme levels rise during the first 2h hours of life and then decrease with increasing age. Reid gt 21. (19A8a,b) stated that no relationship existed between age and plasma phosphatase levels in bulls. Plasma enzyme concentration appeared to be dependent upon the frequency of ejaculation and more particularly on the number of spermatozoa yielded. The rations did not influence plasma levels. Russoff et_al. (195A) reported that differences were present between 3 breeds of dairy bulls. Chickens from inbred lines were shown to differ in serum alkaline phosphatase levels (Stutts 23.3l') 1957 and Wilcox, 1963). Combs et a1. (1959) found differences also in breeds of swine. Tuba (1953) developed strains of mice with different levels of serum alkaline phosphatase. A. Effect of Diet -26- Dickie £2.21- (1955) and Ross and Batt (1956) have found intestinal phosphatase to be sensitive to casein in the diet. Fasted rats fed several fatty_acids exhibited increased intestinal alkaline phosphatase levels (DiCkie gt al., 1955). Choline when fed with the fatty acids oblit- erated the effect on the intestinal enzyme. Serum alkaline phosphatase in cows grazing winter wheat was investigated by Crookshank gt 21. (1952). Their data indicated that serum levels were highly variable under these conditions. Triantaphyllopoulos and Tuba (1959) Observed changes in phosphatase activity when rats were fed solutions of amino acids. Duodenal phosphatase activity was also increased by several antibiotics (MOOg, 196A). Rats on low protein diets produced a rather constant liver enzyme level (Benditt et al., l9u9), but intestinal phosphatase was decreased (Lawrie and Yudkin, 19h9). Gould (l9hh) and Tuba and Dickie (l95h) increased serum and plasma phosphatase respectively by prolonged feeding of high fat diets. Such a diet has been shown by Nimni (1957) to also increase the level of the liver enzyme. Stenram.(195h) was unable, histochemically, to demonstrate any differences in intestinal alkaline phosphatase activities of rats fed high carbohydrate, fat or protein diets. Bodansky and Jaffee (1931), Neil and Russell (l9uo), Flock and Bollman (19MB), Miller (1950), Jackson (1952), Tuba and Madsen (1952), and Sukumaran and Bloom (1953) have reported a decrease in serum alkaline phosphatase of fasted rats. Ely and Ross (1951) have observed an increase in liver alkaline phosphatase during-fasting. Dickie gt a1. (1955) found no change in duodenal levels of phosphatase upon fasting. 5. Effect of Hormones Li §t_§l. (l9h6) and Li and Evans (l9u7) reported that hypophy- -27.. sectomy, either of male or female rats, decreased plasma alkaline phos- phatase levels. Administration of growth hormone increased the plasma enzyme level in both normal and hypophysectomized rats. ACTH counter- acted this effect only in hypophysectomized animals (Li _e_t_ a_1., 19W). A large increase in alkaline phosphatase of the rat genital tract was produced by Talmage (l9h9) by injecting estradiol. Owen (l9h8) studying the effect of thyroxin injection on dairy cows observed a greatly decreased milk phosphatase level. Normally, duodenal alkaline phosphatase in the mouse increases to a maximum at 20 days after‘birth (Moog, 196A). A premature increase in the duodenal phosphatase activity was obtained by Moog (1953) by administer- ing cortisone after the animals were 8 days old. Peak intestinal activity was observed at 1h days, instead of 20 days postpartum. ACTH, if in- jected after the 12th day of life, caused a 2-day accelerated increase in tissue enzyme activity. Adrenalectomy inhibited phosphatase accumula- tion only if performed before the adrenal cortices began to stimulate enzyme synthesis in the gut. Mbog gt al. (195%), Moog and Thomas (1955) and Moog (1961b) found that those steroids which caused premature phos- phatase levels also resulted in growth inhibition. -Moog and Richardson (1955) have suggested that the functional differentiation of the small intestine of the chick is controlled by secretions from the embryo's own adrenal glands. Shewell and Long (1956) found rats, mice, and rabbits to be cortisone sensitive while monkeys and guinea pigs were resistant to cortisone effects. One effect in these animals not re- sistant to cortisone, was a decreased 7-globu1in level. Halliday (1958, 1959) reported that along with a rise in intestinal alkaline phosphatase in rats there was a decrease in the absorption of whole proteins in the -28- rat. Moog (1961a) has demonstrated the increase in intestinal phos- phatase histochemically. Hydrocortisone injections caused an increase in the level of intestinal invertase (Doel and Kretchmer, 196A). 6. Intestinal Activity Intestinal development and alteration have been studied mostly in the chick and guinea pig. Moog (1950) observed that alkaline phos- phatase accumulated in the chick duodenum from 9 to 17 days of incubation rising to a peak before hatching. Duodenal phosphatase increased if the chick was deprived of food for A8 to 72 hours. -The enzyme was present at the tips of the villi but never at the crypts (Moog, 1950; Kallman, 1951; Richardson 3’2 21-: 1955) of "the chick, mouse (Moog, 1961a) and guinea pig (Moog and Ortiz, 1960). With phenylphosphate as the substrate, duodenal phosphatase activity was maximum at A9 days in the fetal guinea pig. It was shown that the rise in activity was not parallel when both B-glycerol phosphate and phenylphosphate were the substrates. In the fetal pig, Sprague et_al. (1963) Observed the highest intestinal activity at 93 days in the pig fetus. The highest activity appeared in the cranial and mid jejunum-ileum. These 93 day levels were signifidantly lower than those observed at birth. Moog (1961a) reported that alkaline phos- phatase activity was highest in the duodenum and declined to a much lower level in the jejunum-of the-mature mouse. This duodenal activity was higher when phenylphosphate was the substrate than it was when B-glycerol phosphate was the substrate. Jejunal and ileal phosphatases exhibited greater activity when reacting with B-glycerol phosphate. In the preweaning period, alkaline phosphatase activity was uniform throughout the small intestine. 7. Milk Phosphatase -29- The alkaline phosphatases of cow's milk (Chanda and Owen, 1952 and Haab and Smith, 1956) and of the milk of several other species (Buruiana and Badilita, 1938 and Kannan and Basu, 19A8) have been studied. The investigators agree that milk phosphatase is highest during the colostral period and declines before rising again as lactation progresses. Dempsey §t_al. (19A7) demonstrated that alkaline phosphatase was located in the myo-epithelial cells and capillary endothelium of the mammary glands. The presence of the enzyme at the latter site was greatly increased during lactation. Earlier work by Folley and Kay (1935) indicated that milk phosphatase was identical with the phosphomonoesterase of kidney tissue. Alkaline phosphatase of milk is associated with a lipoprotein complex. About 30 to A0% of the enzyme content is absorbed by fat globules. The nature of the lipoprotein particles was investigated by Morton (195A). Further studies by Morton (1953b, 1955) involved the purification of the enzyme. Electrophoretic analysis of the enzyme indicated that at least 3 proteins were present. Chemical analysis and comparison of milk and intestinal phosphatases showed both enzymes to be colorless, unconjugated proteins. The two enzymes differed in their activity-pH relationships. Maximum activity of the purified enzymes was obtained in the presence of magnesium chloride, but zinc and beryllium salts were inhibitory. The evidence indicated that milk and intestinal phosphatases were distinctly different, but had similar specificities. Zittle at 21. (1950) and Zittle and Brigham (1960) have also compared bovine mucosal phosphatase and milk phosphatase. Based on reactions to inhibitors it was suggested that milk phosphatase was more closely re- lated to bone and kidney phosphatase than it was to intestinal phos- -30- phatase. Milk phosphatase was inhibited more by cations and lysine than was intestinal phosphatase. Milk phosphatase was less active than intestinal phosphatase when o-carboxyphenylphosphate served as the sub- strate. The difference in reactivity was attributed to a difference in the enzyme-substrate dissociation of the lipid-enzyme complex. The Michaelis Constants for the two enzymes were identical when the enzyme complexes were disrupted with n-butanol. 8. Studies in Farm Animals Very few studies have been conducted on the alkaline phosphatase levels in farm animals. Young and Underdahl (19A8) studied serum phosphatase levels in the pig at weekly intervals for 56 days. Serum activity decreased up to 50 days of age with considerable variation between animals. Activity was high in the newborn but fell considerably by the 5th day. Enzyme determinations were made on frozen serum samples. A similar pattern of serum phosphatase activity was observed by Earle (1952) in foals and Kunkel §t_al. (1953) in cattle. In Brahman cattle, Kunkel s32 a_1. (195A) found a highly significant correlation (.85) between alkaline phosphatase levels at the beginning of the trial and sub- sequent gain. No significant correlation was observed in the European breeds. Fletcher et a1. (1956) attempted to correlate alkaline phos- phatase with growth in cattle. The correlations ranged from -.35 to .63. Enzyme analyses were made on frozen serum in part of the trials. Bailey 23 El' (1956) have studied several intestinal enzymes in the young pig but did not include alkaline phosphatase. Serum phosphatase levels at 1 and 7 days were correlated by Combs gt a1. (1959) with weaning weight. No significant correlations were established with regard to growth, but the phosphatase levels were higher in females than in males at 1 day of age. -31- D. Raising Animals Deprived of Colostrum The calf deprived of colostrum lacks something which permits in- testinal bacteria to invade the body and multiply in the various organs (Smith and Little, 1922) . McRoberts and Hogan (19%) stated that mort- ality was high when newborn pigs were removed from the sow at birth; and it was assumed that the failure to receive colostrum was at least partly responsible for the deaths. 'Bauriedel.et.§l. (195A) suggested that pigs deprived of colostrum could be raised if they were removed from their natural environment at or before birth without bacterial contamination. (The need for immediate isolation has also been discussed by Young-and Underdahl (1951, 1953). The intestinal tract of the newborn pig-is relatively devoid of microbial grbwth but within 2A hours, these populations reach their greatest density in terms of viable cells per gm. of contents (Wilbur §_t_ 21,, 1960). Hill _e_t_ 21- (1955) reported a significant decrease in mortality by raising colostrum—deprived pigs in separate pens. The difficulties of raising colostrum-deprived pigs have also been discussed by Weybrew et a1, (19A9) in their review of supple- mented milk diets. Owen £3 21- (1961) and Owen and Bell (196A) have attempted to rear colostrum-deprived pigs in a non-isolated environment. They have fed immune globulins from sera to provide passive immunity. Pigs receiving only albumin exhibited a high mortality rate. MOre frequent feeding resulted in greater daily total intake with a lower incidence of diarrhea in pigs on a modified cow‘s milk diet (Berry 2’2 31., 1962). Bustad g a_1. (19A8), Lecce and Matrone (1960), and Lecce gt al. (1961a) have all Observed that pigs deprived of colostrum developed diarrhea at 3 to A days. Anderson and Hogan (19A7) and Bustad -32- gt 21. (l9A8) noticed the formation of a dark brown exudate around the eyes of colostrum-deprived pigs. Serum profiles of animals which received no colostrum are con- siderably altered in comparison to those which nursed their mother. In colostrum-deprived calves, Hansen and Phillips (19A7) noticed that the serum protein fractions did not approach normal until the animals were 8 weeks old. During this time there was a gradual increase in 7-globulin and a concurrent decrease in.a-globulin. Albumin was notice- ably lower in colostrum-deprived pigs at A weeks of age than in the con- trols (Rutqvist, 1958). The 7-globulins were not significantly different from the sow-fed controls. Lecce and Matrone (1960) and Lecce gt_al. (1961a) observed that pigs receiving no colostrum and fed an "amino acid" milk failed to develop a mature serum protein profile. -At 8 weeks of age, Varnell.g§fl§l. (1960) reported higher glycoprotein concen- vtration in colostrum-deprived calves than in nursing controls. A rise in lipoporteins up to 21 days was seen in both groups though less pron- ounced in the colostrum-deprived calves. Payne and Marsh (1962a) com- pared pigs farrowed normally with pigs taken by hysterectomy and reared in isolation. The greatest differences between the nursing pigs and colostrum-deprived pigs were in the total serum protein concentration, 6 gm.-vs.-2.A gm./lOO m1 and in the amount of 7-globulin, 33 mg per 100 ml vs. 2.8 mg per 100 ml, respectively. Two of the more popular diets for raising colostrum-deprived pigs are boiled cow's milk or whole cow's milk. Johnson and Pierce (l9A9) have shown an increase in the 7-globulin fraction of serum of pigs fed boiled milk diets. Hansen and Phillips (l9A9) likewise reported an in- crease in serum-immune proteins of calves fed whole milk. -33~ Other diets have been used with varying degrees of success in rearing colostrum-free pigs. Young and Underdahl (1951, 1953) have reported success with homogenized cow's milk with its butterfat adjusted to 6.5% by adding cream. .A vitamin-mineralnand antibiotic-fortified diet was adequate in a study conducted by Catron et a1. (1953). Pigs fed a synthetic milk diet used by Bustad sic. a_1. (19A8) failed to survive more than 22 days. This diet included 25.5% each of sucrose and lactose, which may have contributed to the severe diarrhea. Lecce and Matrone (1960) have formulated an "artificial" milk and an "amino acid" milk. The former is a casein-lactose diet while the latter is composed of a casein hydrolyzate-lactose mixture. Berry gt a1. (1962) preferred a modified cow's diet over a casein—lactose diet for rearing SPF and colostrum-deprived pigs. A human infant formula was found inadequate for the colostrum-deprived pig. Aureomycin produced some growth stimulation in pigs on synthetic milk diets (Wahlstrom egg _a_l., 1950). For purposes of studying deficiencies and requirements of the pig, some investigators adopted the practice of permitting the pigs to nurse the sow for a few days and then placing them on a synthetic diet. Johnson.§t El. (l9A8) and Miller at al. (1957) have reported this procedure. Miller gt 21. (1957) stated that pigs weaned at A days of age and placed on synthetic diets grew less rapidly than did pigs left on the sow for up to A weeks. From this age pigs on synthetic diets grew more rapidly and exhibited higher serum 7-globulin levels than did pigs left on the sow. Lecce and Matrone (1961) observed normal maturation of serum protein pro- files in pigs when they were removed from the sow at l and A days and placed on cow's milk. III. EXPERIMENTAL PROCEDURES A. Effect of feeding protein and protein-free diets on absorption of whole proteins in colostrum-deprived pigs. l.vGeneral Until recently, gut closure was thought to be time dependent; but Payne and Marsh (1962) and later Lecce and Morgan (1962) demonstrated that closure was affected by feeding and the amount of colostrum ingest- ed. ‘Most of the workers studying gut closure have used pigs which were completely starved, or they have used gut segments from starved pigs. Pigs starved for 106 hours have been used to demonstrate that the gut remains permeable to proteins (open) if no protein is ingested. -These approaches induce many variables other than the lack of protein such as added stress, dehydration, and altered electrolyte levels. .In view of this fact, these experiments were conducted with the following-objectives: a. Develop a diet that will sustain newborn animals not receiving colostrum for extended periods of time. b. Follow weight and serum protein changes in the colostrum-deprived P18- c. Use FITC-labeled protein to ascertain gut permeability under the above conditions. Three trials utilizing 33 pigs from 3 litters were used in this study. All 3 litters were Yorkshire-Hampshire crosses, Pigs were alloted to the treatments on the basis of weight, with the smaller pigs being left on the sow for controls. No attempt was made to balance the sexes between the treatments. Close observation was maintained over the pregnant sows to insure that pigs would be caught as they were born. As the sow farrowed, the pigs were cleaned with paper towels and placed in a clean cardboard box -3h- -35- that was warmed with a heat lamp. As soon as the last pig was farrowed, the pigs selected for treatment were removed from the swine barn to locations in Anthony Hall never before inhabited by swine. Blood samples were obtained from the anterior vena cava of all the animals, using the technique of Carle and Dewhirst (19A2). Blood serum samples were rimmed in the tube and left at room temperature to permit syneresis. The clot was removed and final separation of serum from the cells was accomplished in an International Centrifuge, size 2, Model V, at 2000 x g for 15 min. ~Serum was transferred to glass vials and used fresh for immediate analyses or frozen for future use. After blood samples were taken, 300,000 units of procaine penicillin G1 were injected intramuscularly. Each pig was fed 1 oz. of warmed synthetic milk and placed in a-2A x 30 x 30 in. cardboard box previously heated to 300 C. Wood shavings-and crushed corncobs were used for bedding. .Diets fed the pigs deprived of colostrum were modifications of the ration reported by Miller £3 31. (195A). Composition of the dietary solids is given in Table 2. One kg. of the solids was mixed with A kg. of water and homogenized to yield a final solids concentration of 20 per cent. Cellulose was added to maintain normal fecal consistency and to promote passage of meconium. Tween 80 was used to maintain suspension of the solids. Five kg. of each diet were prepared at a time and stored in brown bottles at 50 C. The pigs were fed with individual baby bottles every A hours from 8:00.A.M. to 12:00 P.M. .After each feeding the bottles were washed, keeping the nipples with their respective bottles. Twice a day 35 mg. of neomycin sulfate2 were added to the diet in the 1Corn States Laboratories, Inc., Omaha, Nebraska. -Biosol, Upjohn Company, Kalamazoo, Michigan. -36- bottle. If diarrhea was observed, the antibiotic was increased to 70 mg. per feeding and continued until improvement was observed. TABLE 2. Composition of solids in purified diets fed the colostrumr deprived_pigs. Protein, % Glucose,_% Casein 30 -- Cerelose 52 82 Lard 10 10 Mineral mixture 6 6 (x-cellulose 2 2 Vitamin mixture + + Tween 80 + + Mineral Mixture KCl 10.0 KI 0.002 FeSOh.2H20 0.7 CuSOu 0.1 CoC03 0.1 MnSOh.H20 0.1 ZnSOu.H20 O.A MgSOh.7H20 5.5 NaHC03 25.0 CaHPOu 28.5 C8003 12.5 Glucose 17.1 Vitamin mixture Thiamine-mononitrate 3 mg/kg diet Riboflavin 6 Nicotinamide A0 Calcium pantothenate 30 Pyridoxine°hydrochloride 2 pnAminobenzoic acid 13 Ascorbic acid 80 a 4t0c0pheryl acetate 10 Inositol 130 Choline chloride 1300 Pteroylglutamic acid 260 meg/kg diet Biotin 5O Cyanocobalamin 100 2-methyl-l,A-naphthoquinone A0 Vitamin A 10,000 IU/kg diet Vitamin D 1,000 3 -37- Labeled protein was administered via a polyethylene stomach tube attached to a 30 ml. syringe. The animals were fasted until they were killed. Whenever possible, urine samples were obtained from the bladder at death for protein analysis. Animals were killed by injecting 5 m1. of saturated MgSOh into the anterior vena cava. After weighing, a midline incision was made and the viscera were quickly removed. Sections of liver, kidney, spleen and thymus were collected first. After the intestine was separated from the mesentary, samples were taken from the duodenum, cranial, middle and caudal jejunumrileum,and cranial and caudal colon. Samples on which alkaline phosphatase were to be determined were frozen at -720 C in an isopentane- dry ice-alcohol bath (Lillie, 195A), and then placed in a freezer at -200 C. Tissue from corresponding locations was blotted dry, and weighed for dry matter determinations. Drying was accomplished by heating the tissue at 1100 C for 2A hours in a forced draft oven. Tissues from these same locations were taken for histological examination. iEthyl alcohol or FAA fixation was used for enzyme or fluorescent preparations, respectively. FAA consists of 80 per cent ethanol, 15 per cent formalin, and 5 per cent glacial acetic acid. Tissues were left in this solution for 18 but not longer than 2A hours. The enzyme fixation technique was the method of Gomori (1952) modified by Lillie (195A). Beta-glycerol phosphate and naphthol.AS phosphatel were used as substrates for alkaline phosphatase. After fixation in FAA, tissues were placed in 70 per cent ethanol prior to fixation and dehydration in the Autotechnicon. Imbedding was in 1Sigma Chemical Company, St. Louis, Missouri. -38- Paraplastl using plastic molds. Sections were cut at 7p and attached to alcohol-cleaned slides with Haupt's albumin adhesive. One section of each tissue was stained with hematoxylin-eosin and one was cleared of paraffin for observation of fluorescence in UV light. A Leitz Orthulux Fluorescence microscope with a BC 38 heat absorbing filter, BG 12 UV filter, dark field condenser, and a UV absorbing eyepiece filter was used to view the fluorescent slides. 2. Trial I A litter of 8 pigs was used in the first trial to study their performance on the glucose and casein synthetic diets. Two pigs on the sow served as controls. The remaining 6 were divided into two groups and placed on the synthetic diets. At 7 days, a casein- and a glucose-fed pig and a control were given 60 ml. of a solution containing FITC—labeled 7-globulin. uAfter 9 days on the trial, one each of the caseinh and glucose-fed pigs received the FITC-labeled protein and were killed. When they were 2 weeks old, the remaining 3 animals were treated in the ~same manner. This 60 ml. of labeled protein was calculated to contain 3 gm. of a commercial y-globulin.2 Blood samples were taken from all animals before the protein was fed and at death. Serum was analyzed for total protein concentration, and paper electrophoretic separations were performed. Immunoelectrophoresis was also performed. Urine samples 'were tested for the presence of protein by the biuret reaction. .Intest- inal tissue samples were collected and preserved by freezing or fixation for enzyme analysis and histological preparations. Samples of intestinal tissue were also collected for drygmatter-determinations. lAloe Scientific Company, St. Louis 3,.Missouri. ‘gArmour Veterinary Company, Kansas City, Kansas. -39- 3. Trial II Since the number of observations in Trial I was so small, a second trial with a litter of 10 was used to further study pigs fed the glucose diet. Three pigs were left on the sow, but 7 received no colostrum and were placed on the synthetic glucose diet. At 1 week of age, 3 of the pigs on the glucose diet were given A0 m1. of FITC-labeled protein plus A00 units of insulin, orally. Blood samples were taken and protected from clotting with EDTA, and the pigs were killed. Blood glucose was determined at 3 and 6 hours post-administration. The remainder of the glucose-fed pigs were treated-in a like manner when they reached 2 weeks of age. The 3 control pigs were also killed at 2 weeks of age, but were treated somewhat differently. A11 3 were fed FITC-labeled protein and insulin with two of them also receiving a trypsin inhibitor. Soybean trypsin inhibitor was administered to one, and eggawhite trypsin in- hibitor was administered to the other at a rate of A0 mg./kg. body weight. The third pig received no inhibitor. Blood glucose was again observed at 3 and 6 hours post-feeding. .Tissue samples were again taken from the intestine. Other blood analyses were the same as reported in Trial I. A. Trial III. In Trials I and II, ultraviolet-microscopic observations of tissue suggested that fluorescent particles were present; but it could not be ascertained that the fluorescent material was attached to protein molecules. In an attempt to clarify this matter, a third trial was conducted. In this trial, the animals were again reared on the glucose and casein diets with- out nursing the mother. Instead of using 7-globulin alone as the test material, a mixture of 7-globulin and colostral whey was labeled and con- -ho- centrated. A litter of 15 pigs was divided into 3 equal groups and either served as controls or were fed the casein or glucose diets. Two animals from each group were given A0 ml. of the FITC-labeled protein mixture plus A00 units of insulin orally at 1 week. The protein mixture was calculated to provide each pig with 3.5 gm. of 7-globulin. Blood samples were taken at 0, 3, and 6 hours for glucose determination. Serum samples were obtained before administration of the protein and at death. The remaining 9 pigs were killed at 2 weeks of age after the same treatment. The labeled protein fed at 2 weeks of age provided each pig with A.O gm. of 7hglobulin. Determinations of total protein concentrations and immunoelectrophoretic patterns were performed on the sera. Intestinal tissues for enzyme and histological preparations were collected. 5. Fluorescent Labeling of Proteins A mixture of equal volumes of protein solution and 0.05 M sodium! bicarbonate-carbonate buffer of pH 8.5 was placed in a flask with a magnetic stirring bar. Fluorescein isothiocyanate (FITC) was added to this solution at a rate of 50 mg- per gm. of protein. This mixture was stirred over night at 50 C before it was passed through a Sephadex Column to separate the non-conjugated FITS. A Sephadex G25l slurry was poured into a chromatographic column half filled with .02 M phosphate buffer of pH 6.5. The column was developed with this buffer and covered with filter paper to prevent disturbance of the gel surface. Five m1. of the protein solution were placed on the column at a time. In about 30 minutes this sample was separated from the nonbound FITC and had moved through the column. Passage through the column resulted in approximately 59 percent increase in sgmple volume.4LConcentration of the sample to the lPharmacia Fine Chemicals, Inc., New York 17, N. Y. -h1- desired protein concentration was accomplished using an LKB ultrafilter as described by Aronsson (1961). Residual FITC in the column was quickly removed by continued flushing with the phosphate buffer. 6. Production of Rabbit Anti-Porcine Immune Sera To induce the production of anti-swine antibodies in rabbits the potassium-aluminum-sulfate method of Proom (19A3) was used. The inject- able precipitate was made from the following mixture: 25 m1. porcine serum or diluted whey 80 ml. distilled water 90 ml. 10 percent KAl(SOu)2.12H20 The pH was adjusted to 6.5 with 5N NaOH. This mixture was centrifuged and the sediment washed twice with isotonic saline. The sediment was then made up to 100 ml. with saline. Merthiolate or phenol was added to retard bacterial activity. The solution was stored in rubber-stoppered serum.bottles at 50 C- Rabbits were injected with this preparation according to the following schedule: Days Procedure 1 5 ml. intramuscularly in each buttock 1A 5 ml. intramuscularly in each buttock 2A 1 ml. raw serum or diluted whey injected . . intraperitoneally ' 3 3A ' Blood collected by heart puncture This schedule was repeated after a rest period of 2 weeks. The rabbit blood was allowed to stand overnight at 50 C to permit syneresis. The clot was removed mechanically and the serum centrifuged. The serum was divided into 1 m1. portions and placed in small glass vials and frozen (--200 C) until needed. The serum used to prepare the precipitate was a pooled sample of at -h2- least 3 individual animals. Each of the following pig antigens were in- jected into 2 rabbits: serum taken from the pig at 0 and 2A hours, at 5 weeks, and at A months; from the prepartum sow; and colostral whey. Colostral whey was produced from mechanically defatted colostrum by rennin coagulation. One-half of a rennetl tablet (.5 gm.) was crushed and dissolved in each 100 ml. of colostrum. The mixture was allowed to stand at room temperature for 6 to 8 hours as the curd formed. The curd was separated from the whey by centrifugation at 12,800 x g at 50 C. Colostral whey was diluted 50 percent by volume to prevent anaphylaxis in the rabbits. 7. Biological Determinations a.-Paper Electrophoresis S Electrophoretic separation was performed on a Spinco, Model R, paper electrophoresis system at room temperature. A constant current of 2.5 ma. per cell was maintained for 18 hours on Schleicher and Schuell 20A3A paper strips. Veronal buffer (with 0.5 percent tergitol) of pH 8.6 and ionic strength of 0.075 was used. The separated proteins were stained with bromphenol blue dye and scanned with a Spinco Model RB analytrol. All samples were run in duplicate. Eight 1 of serum from newborn or colostrum-deprived pigs and 6 X from controls were used. b.11mmuneolectrophoresis Buffers for the agar and electrode vessels were those reported by Hirschfeld (1960). The agar used was Ionagar No. 22. Buffered agar was prepared by mixing 35 m1. of stock agar buffer, 65 m1. of distilled water and .75 gm. of agar. The solution was heated to boiling on a hot plate or in the autoclave at 1210 C for 5 minutes. If the hot plate was used, 1Salada-Shrriff-Horsey, Inc., Woborn, Massachusetts. Consolidated Laboratories Inc., Chicago Heights, Illinois. -h3- the flask was always covered with a watch glass. After bringing the agar to a boil, it was filtered through several layers of cheese cloth onto the slides. Three sizes of glass slides were used: 1 x 3 in. microscope slides, 2 x 3 in. microscope slides, and 3% x A in. lantern slide cover glasses. All slides were washed in 70 percent alcohol, dried with lint-free tissues, and numbered. Several slides were usually prepared at a time 1 was by using flat, rectangular pans. A layer of 1 percent Agar.Agar poured into the pans which were oriented to a particular space on a lab bench. This agar was permitted to solidify in the lab and was then taken to the cold room (50 C) for hardening. The pan was returned to the same spot on the lab bench and the cleaned slides were placed on the level surface of the agar. Hot .75 percent agar was poured over the glass slides. The volume of agar to use was empirically determined to provide an agar depth on the slides of 1 to 1.5 mm. After hardening, the agar over the slides was divided with a scapel at the slide edges and the slides with their agar covering were removed by lifting them out with a spatula. Proteins to be subjected to immunodiffusion were electrophoresed in a chamber constructed by the author. The vessel and its dimensions are shown in Figure 1. Its design resulted from modifying a similar |i—a unit first described by Weime (1959). Construction was of u in. acrylic plastic except for the bottom which was 1/8 in. thick. The pieces were purchased from a local plastics shop and cemented together with methylene chloride. Platinum.wire was used for electrodes which were held in the ends of the vessel by plastic dowels. 1__ The arrangement of a slide and fluids in the vessel isgpictured in lConsolidated Laboratories Inc., Chicago Heights, Illinois. -hh- Figure 2. The same .75 percent agar used on the slides was also used in the ends of the vessel. The agar blocks were prepared by filling the ends of the vessel, permitting solidification, and then removing enough agar to provide Space for the buffer wells. The agar in the vessel could be used several times until salts began to accumulate. This accumulation was evidenced by a rise in amperage on the power supply. The slides were inverted so that there was an agar to agar contact (Figure 2). The center compartment was filled with petroleum ether (30- 600 B.P.) covering the slide with about 5 in. of ether. The power supply was a Heathkitl model IP-32. Electrophoresis was conducted at 125 volts for 30 minutes for microscope slides and A5 minutes for 3% x A slides. The amperage was controlled by regulating the evaporation of the petroleum ether. This was accomplished by adjusting the door on the hood, in which the electrophoresis was performed, to regulate the draft. Wells in the agar on the slides to contain the serum to be electro- phoresed were made with a cut-off hypodermic needle attached by a hose to a vacuum line. The antibody slot, made after electrophoresis had been accomplished, was cut out with a scalpel, using a plexiglas template. After electrophoresis of the antigen, 50 to 70 k of antibody were placed in the slot. Incubation for 12 to 18 hours in a humid chamber placed in the warm room (370 C) resulted in the formation of precipita- tion bands. The slides were then washed in 2 changes of saline over night followed by 2 changes of deionized water for 6 hours. A wet filter paper was placed over the agar surface of the slides which were then dried in the warm room at 370 C. A_, Ponceau S stain was preferred since the red dye allowedgpictures to J'Heath Company, Benton Harbor, Michigan. -h5_ ~h6- 3:2 \\\ /////a / ///VV//// / // ////// // /H /.//. //.»/W/F// Hommob mamohoamofirooam map S @8308 one £8829 98mm mo psoaomrmhho 93 mo amemHQ o \ loo-u- ciao... . . m EUHrm //////// / ./ xravW/a/ // bane \ . 0‘ C . wfi 3:: w: 3...: are . . . on 35o .. .5223.— ‘ o I‘ ...-o- va.” 2:28 33V... 3:93. 32° dam: 2323.353: ..3 -b _h7_ be made in an enlarger. Fixation for 15 minutes in.a 2 percent acetic acid bath preceded the staining. The slides were then stained 30 minutes in .2 percent Ponceau S in 3 percent trichloroacetic acid. Excess stain was removed by clearing for about 15 minutes in a 5 percent acetic acid solution. Time necessary for clearing was determined by the difficulty in removing the excess stain. Photographs were made by placing the stained slides on the film plate of an Elwood enlarger and exposing on Kodabromide h-A paper for 10 seconds at fll. Developing was in Dektol as recommended for this grade paper. c. Preparation of Tissue for Enzyme.Analysis Approximately 1 gm. of thawed wet tissue was weighed on a plasticized weighing paper. Surgical scissors were used to slice the tissue into small pieces. The sample was transferred to a 50 ml. polyethylene centrifuge tube. Twenty ml. of cold deionized water was used to wash the weighing paper free of any residual sample. A Iourdes Volu-mixer fitted with a centrifuge tube adaptor and cutting blade running at approximately 10,000 R.P.M. was used to homogenize the tissue. The resulting slurry of tissue was then centrifuged in a Lourdes Betafuge at 12,800 x g at 50 C for 10 minutes. The supernatant was removed and ice cold deionized water was added to dilute the original sample 100:1. Enzyme activity was determined immediately on duplicate samples of the diluted supernatant. d. Tissue Alkaline Phosphatase Alkaline phosphatase was assayed by the clinical procedure commonly psed.l The procedure was as follows: 01"Sigma 10h Phosphatase Substrate", Sigma Chemical Company, St. Louis, Missouri. Sigma Techn. Bull. 10h:1963. _h8_ 1. One ml. of alkaline buffered substrate (equal proportions of 10.5 pH, 0.1 M glycine buffer and P-nitrophenyl phosphate substratel) was pipetted into a test tube. Due to the number of samples being assayed for-this enzyme, 50 of these tubes were prepared at a time and were frozen at -200 C. When determinations were to be made, tubes were re- moved from the freezer and placed in the 370 C water bath for 5 minutes. 2. Using a lab timer and noting the exact time, 0.1 ml. of the enzyme containing supernatant was pipetted into duplicate reaction tubes. Tissue homogenates were diluted 100:1 to allow the reaction to procede for 30 minutes, and to yield a color which could be read on the spectrophotometer. A blank using the same reagents and 0.1 m1. of water was included in each group of determinations. 3. After 30 minutes, 10 ml. of-0.02 N NaOH were added to each tube to stop the reaction. The samples were mixed on a Vortex mixer. A. Optical density was read on a Beckman DU Spectrophotometer at A10 mp zeroed with a reference blank. 5. Color due to p-nitrophenol was removed from the tubes by adding 2 drops of concentrated HCl. Optical density was again recorded at A10 mm and subtracted from the original reading. This corrected for any inter- ference by tissue particles present in the sample. e- Protein Determinations 'Protein by the biuret procedure was determined on urine samples by the method of Gornwall.gpflal. (19A9). One-half m1. of sample was mixed with 9.5 m1. of saline. Two m1. of this mixture were mixed with A ml. of biuret reagent. The Bausch and Lomb Spectronic 20 was set at zero optical density;with a saline blank. After 30 minutes, the samples were l"Sigma 10A Phosphatase Substrate", Sigma Chemical Company,-St. Louis, Missouri. Sigma Techn. Bull. 10A:l963. _h9_ read and optical density compared with a standard curve prepared from bovine albumin. All samples were assayed in duplicate. Total serum protein was determined by the method of Waddell (1956). Five A of serum were diluted to 5 m1. (1:1000) with 0.85 percent saline. A reading at wavelengths of 215 and 225 mp was made on a Beckman Model DU Spectophotometer. The absorbance at 225 mu was subtracted from that at 215 m u. -The difference when multiplied by 1A.A gave the protein concentration in serum in gm. per 100 m1. f. Glucose The Nelson-Somogyi microtechnique was used for blood glucose (American Association Clinical Chemists, 1953). A Ba(OH)2 supernatant of whole blood was prepared by mixing 0.1 ml. of whole blood with 0.95 ml. of 0.3 N Ba(OH)2 and .95 ml. of 5 percent ZnSOu. The mixture was centrifuged for 10 min. at 2,000 x g. One ml. of alkaline copper reagent and 0.5 m1. of the supernatant were placed in a Folin sugar tube and mixed. A marble was placed on top of the tube and it was autoclaved for 5 minutes at 1210 C. After cooling for l min. in water at room temperature, 1 m1. of arsenomolybdate reagent was added, mixed and the solution was diluted to the 10 ml. mark with distilled water. Optical density was measured at 5A0 mp.using a Bausch and Lomb Spectronic 20. .Concentration of sugar in the unknowns was calculated from glucose standards carried out simultaneously. All determinations were made in duplicate and within 1 hour after the blood samples were taken. B. Effect of increasing blood volume on whole protein absorption 1. General It is the general opinion of the workers who have studied gut closure that protein must contact the gut before closure is accomplished. -Several -50- workers have reported an increase in blood volume in the pig which occurs at about the same time as closure takes place. To study the relationship between closure and the increase in blood volume, 1 litter of 12 Hampshire pigs were caught as they were born. They were randomly divided into 2 groups, 1 of which served as controls. Ramirez 2; a1. (1963) reported an increase in relative blood volume from 8.6 percent to 10 percent in the first 12 hours of life. On this basis, the treatment pigs were weighed and their blood volume increased to 10 percent of their body weight by intraveneous injection of a dextran plasma extender, after which all pigs were returned to the sow. Blood samples were taken at birth, 10, and 2A hours, 3 and 5 days and 3 and 5 weeks of age. .Sera from these samples were analyzed for total serum protein concentration and serum protein distribution by paper electrophoresis. Immunoelectro- phoresis was also used to compare the serum fractions. C. Changes in serum and milk phosphatase in 5 litters during the post-natal period. ‘1. General Intestinal phosphatase in the mouse and rat has been reported to undergo an increase after birth with the maximum activity coinciding with the cessation of absorption of whole proteins from the gut (Moog, 1955). A similar but less pronounced change is reported to occur in the pig at the time of gut closure (Payne and Marsh, 1962b). Serum phosphatase activity has been reported to decrease after birth in several species. ~This study has been conducted to investigate the alkaline phosphatase activity of pig serum and milk of the dam during the first 2 weeks after parturition. Litters from 2 gilts and 5 sows were included in this investigation. The pigs were caught as they were farrowed, weighed, and a blood sample -51- taken before they were returned to the mother. Before the pigs nursed, a sample of colostrum was obtained. Other samples were obtained at 1, 2 and 7 days in all the litters. Two litters were also sampled at 2 and A weeks. The pigs were weighed at every sampling period and milk was collected from the mother; total protein concentration and alkaline phosphatase activity were determined on the serum. Before the milk samples were analyzed for alkaline phosphatase, the fat was removed mechanically after spinning at 12,800 x g in a Lourdes Betafuge at 50 C for 10 minutes. Correlations between serum phosphatase activity, weight and total serum protein were calculated on the mean values of all the litters. .Correlations within each litter were determined between weight and phosphatase activity. 2. Effect of storage on serum alkaline phosphatase Stability of serum.alkaline phosphatase from pigs of A ages was investigated. Samples of 0, 1, 7, and 1A day old pig sera were divided into glass vials all but one of which were frozen at ~20O C. The one remaining vial was placed in the coldroom at 50 C over night for 18 hours. Phosphatase was determined on the sample after storage in the coldroom over night and on the frozen.samp1es at weekly intervals. Serum.activity of the stored samples was compared with that of the fresh serum. 3. Serum Alkaline Phosphatase a. Serum phosphatase activity was determined by the method of Bessey _e_t_ ag. (19A6) using 0.1 ml. of serum. Equal parts of alkaline buffer 1 solution and p-nitrgphenylphosphate were placed in test tubes and kept lSigma Chemical Company, St. Louis, Missouri Sigma 10A Phosphatase Substrate, Sigma Techn. Bull. 10A:l963. -52- frozen at _200 C until needed. Incubation was carried out in a waterbath at 37° c. An equal amount of water replaced the serum for a blank. The reaction was stopped with .02 N NaOH after which the color was read at A10 m u on a Beckman DU Spectrophotometer. After the samples were read the first time, 2 drops of concentrated HCl were added to each tube to dissolve the color. The samples were read and the resulting optical density was subtracted from the first reading. A standard curve was prepared using p-nitrophenol according to the procedure given in Sigma Bulletin 10A. IV. RESULTS AND DISCUSSION A. Effect of Feeding Protein and Protein-Free Diets on Absorption of Whole Proteins in Colostrum-deprived Pigs. As indicated in the EXPERIMENTAL PROCEDURE section, pigs from 3 litters were fed synthetic diets. Very little difficulty was encounter- ed in management of the pigs during the 2 week period. There was some variation between the 3 litters used in these trials and these variations will be mentioned below in the individual trials along with the data obtained. 1. Trial I Data on the 8 pigs from the litter used in Trial I are shown in Table 3. Only 1 of the pigs in Trial I lost weight when the weights at death were compared with birth weights. The treatment pigs which gained weight did not gain as much as the controls on the sow, either at l or 2-weeks of age. Spleen weights were compared because there appeared to . be a significant difference between treatments when the pigs were killed. Both treatments had smaller absolute and relative spleen weights than did the controls. Since there was only 1 animal per treatment, the differences could not be analyzed statistically. Total serum protein data in Table A indicate that the casein-fed pigs had higher serum protein levels than did the glucose-fed pigs. Neither approached the concentration observed in the control animals. -53- -Sh- .spwap so pswaos op o>aeoaow names: soon as amassed mm. om.oa m.:oa + Poem :H oawa Honpcoo 2. ma 0H. mm.m m.om + :mmm :H omma omoosao m NH Ha. mm.m m.Hm + Hmom 4H Coma cwomwo .2 0H Ha. sm.a s.ma + mmwa m omma choosao a Ha Ha. mm.a m.m + mowa m ozwa swomoo m m mm. Om.: in coma N In Hospcoo z :a mo. om.a m.H u wwwa N OHNH omoodao 2 MH ea. cm.m a.» + mews s oema choose a m wzwfiok A.swv mpnwflok .N.swv Amhwcv A.swv pcmavmohe xmm .oz wwm soon soon spoon spoon mo m mm pnwfimb ca pm pm pnwflob .93 comamm nomamm ownwno m pnwwos ow< HdeHnH .H seawawma amen so open amwam: .m mamas -55.. TABLE A. Total serum protein in Trial I. (gmp/lOO ml.) Age (days) Glucose Casein Control 7 2.31 3.66 5.20 9 2.13 3.31 - -- 1A 2 . 02 3 . 27 6.09 Serum.protein fractions of the pigs in Trial I, established by paper electrophoresis, are listed in Appendix Table l and are illustrated graphically in Figures 3 and A. Pigs fed the synthetic diets for one week had serum profiles which generally resembled the profile at birth. There was some indication that the relative proportion of serum fractions had changed in both the casein- and glucose-fed pigs. Increases were -observed in the percent of albumin and B-globulins with the changes being most obvious in the casein-fed pigs. Treatment animals fed for 9 days also exhibited an increase in the albumin fraction. Pigs fed the two synthetic diets for 1A days showed greater dif- ferences between treatments than did the pigs fed for only a week. Glucose-fed pigs, except for some increase in albumin, had a profile very similar to the newborn pig. Pigs fed casein had synthesized some albumin and.a-gldbulin. These animals had a higher proportion of.3-globulin than did the control group. rThe proportion of albumin in the casein- fed pigs approached that of the control. The data in Appendix Table I indicate that some‘y-globulin was present at birth and at other ages in these colostrum-deprived pigs. This is probably an artifact resulting from the trailing of faster moving fractions, particularly albumin. Immunoelectrophoresis, a far more ac- curate analytical tool, showed only a trace of ‘7-globulin present in TIHH.I CONTROL r «c A ,0 mm L 1mm “' A cwcos: n _J l. ‘4 FIGURE 3. Comparison of the serum protein profiles among the zero hour pig, one week old pigs fed synthetic diets, and the sow-fed controls from Trial 1. -56- A CONTROL 7 R d mm [A] I A 4 4 L GLUCOSE M p M "Ill 1 2 WEEKS FIGURE A. Comparison of serum protein profiles among the zero hour pig, pigs fed synthetic diets for two weeks, and the sow-fed controls from Trial I. -57- -58- some of these animals. A comparison of the immunoelectrophoresis pattern of the pigs in Trial I is shown in Figure 5. One week and zero hour sera were incubated with rabbit anti- prepartum.sow serum. The casein-fed pig had more precipitation bands than did the glucose-fed pig, but both exhibited increases in the intensity of the bands from that seen in the zero hour pig. Gamma globulin was present only in the control pig. However, a very weak precipitation band was present in the 7-region on slides of some week old pigs. Similar results were seen also in pigs killed after 2 weeks on the synthetic diets. Alkaline phosphatase activity was determined at 6 locations in the intestinal tract. Activity expressed as mM. per gm. of dry matter per 30 minutes is recorded in Appendix Table 3. Intestinal dry matter concentrations used in these calculations may be found in Appendix Table A. The highest enzyme activity was present in the cranial and mid-jejunum-ileum. Duodenal levels were lower than the levels in the colon. These changes in activity in the pigs fed for l and 2 weeks on the synthetic deits are illustrated in Figures 6 and 7. In the 1 week old pigs, little difference existed between the enzyme activity in gut sections from the casein- or glucose-fed pigs. Both treatments were higher in activity in the jejunum-ileum than were the controls. In the 2 week old pigs, all treatments exhibited lower enzyme activity than 'was present in the 1 week old animals. The glucose-fed pigs were some- what lower in intestinal activity in the upper tract than either the casein-fed pigs or the control pigs. In this litter of pigs it appeared that there was no difference .Essom Bow fisaammoamufipeo weapon ape: H HmHsB 80am wag ado: oaoN m sew wag oomuomoooam oao moo: moo «mag mowlnflomoo 6H0 amok oco “magnaospeoo oHo moo: moo m Sosa Edema A.n op .HV mo cospmpdosfl esp Sosa weflpHSmoa choppmm oflposonaoapomaoocsseH .m mmDUHm . . .. 10: mun/Cm D. M./30 Min . ”A. "g o ‘ :S as 3' a ‘- au§ Sui ‘ E a: . =, 3 ‘ .55." a ! a : I - ’ 5 ' \ 2 4 "Mon," 5! .‘ "Hog" " .‘fi’ ' .’ ,' O FIGURE 7. TISSUE PHOSPHATASE TRIAL l 2 WEEKS Comparison of intestinal phosphatase activity at six locations in the casein- and glucose-fed pigs and the controls at two weeks of age in Trial I. -61- TISSUE PILOSPIIITISE TIIIL l 1 WEEK FIGURE 6. b- a \‘\ s‘ s, ‘ E -I S -: .2.- '~. \ Ire,” ~. ‘ : \ 6 's. '3 ! 2 I?! ‘ . §l ‘3 u\ [:1 ‘3‘. ‘ s! V: \ I a 3': 5 'i ‘ é”// ‘-=. \. 9! ‘.= ' Q . \‘z “/.r--' ii {WNW .‘fir§b T f‘”’ 'T" V V g E- .2- a- o U a .q 0‘ Comparison of intestinal alkaline phosphatase activity at six locations in the casein— and glucose-fed pigs and the controls at one week of age in Trial I. _60- ..n- -62- between the two synthetic diets as to their effect on intestinal phos- phatase activity up to 1 week of age. At 2 weeks of age, however, the glucose diet differed greatly from the casein diet with respect to its effect on intestinal phosphatase. Casein, itself, has been shown by Dickie _e_p _a_l. (1955) and Ross and Batt (1956) to increase intestinal phosphatase. In pigs 2 weeks old the glucose diet may alter this enzyme activity because of the absence of any protein. In this respect, the animals approach a fasting condition which also has been demonstrated to affect intestinal enzyme activity. Lawrie and Yudkin (1919) observed decreased intestinal phosphatase activity on low protein diets. Dickie pp a_l. (1955) found no change in duodenal levels of phosphatase upon fasting. Data obtained in this trial indicate that intestinal phosphatase activity was decreased as the result of feeding the glucose diet for 2 weeks. When intestinal sections from the pigs in Trial I were viewed under the fluorescence microscope, only a weak, very dispersed, fluore- scence was observed. This would be expected to some extent, since the A0 ml. of protein solution fed the animals was distributed throughout the digestive tract. Large, fluorescent particles such as those described by Payne and Marsh (1962a) were not seen. The glucose-fed pigs showed some small particulate fluorescence but the casein-fed pigs did not. A very weak fluorescence was usually present in the control group, also. If the presence of fluorescent particles is used as the criterion for protein absorption, then it appeared that the glucose-fed pigs were able to absorb more of the protein than were the casein-fed pigs. Since all the pigs in Trial I were killed 6 hours after feeding, there was no increase in total serum protein to indicate that absorption -63- had occurred. Urine from all the animals fed the synthetic diets and from the controls fluoresced brightly when placed under a UV light. When the sera from these animals were placed under a UV light, they, too, fluoresced. Biuret determinations were made on all the urine samples to see if the fluorescence in the urine was associated with protein. None of the pigs in this trial had protein in the urine. This indicated that some deconjugation had occurred ig_vixg since the labeled protein solutions were passed through SEPHADEX columns to remove the unbound FITC. Payne and Marsh (1962a) state that free FITC is not absorbed by gut segments, but gave no proof for this contention. These observations would suggest that, if the FITC-protein conjugate is absorbed intact, the mere presence of fluorescence in tissue or body fluids is not 93.22232 evidence of the presence of the original conjugate but simply indicates the location of FITC. 2. Trial II Since it appeared in Trial I that the glucose-fed pigs absorbed more labeled protein, Trial II was conducted in which only the glucose diet was fed. All the pigs fed this diet lost weight. The weights of the animals at birth and at the end of the trial are recorded in Table 5. There was a considerable difference between the litters used in Trial I and Trial II, and this was reflected somewhat by their difference in performance. Relative spleen weights of pigs at two weeks of age fed the synethetic diets were significantly less (P <.05) than those of the controls. Performance was not as good in this trial and some scouring was observed in this litter on the third day of age. This observation has .Amogv mv own mama so mwsm oomnomooSHw soap sowaos_hspnoosMHnwsm moso> Hospeoo owmao>¢x .npasn pm pawsoz op o>spmsos pzwsoz moon as owesnoo *ms. om.m e.msm + omes ms . omss soapcoo 2 mm *mm. mo.s m.oss + mmsm ms sews sospcoo 2 so .m *ms. me.s o.sos + mmmm ms ssw soapcoo 2 mm ,o 1 _ so. om. m.sm . ooms ms omos omooaso 2 sm os. em.s o.o . ooms ms owes moccaso a as os. mm.s m.o . msss ms ooms onoooso a ms ss. ss.s o.ms . ooms ms oems ouooaso a as ss. mm.s m.ms - soss m osms omoosso a mm ms. ms.s o.ss - zoo w ooos omoosso a om ms. ma.s s.s - moms m. osms onoooso 2 ms Pawsos N.ewv magmas: A.swv Awhoov A.awv psospmosa xom .oz wsm soon soon spoon spoon season so a no passes as so so snsosss .93 coosmm seesaw ownmno * pnwfloz ow< oHH HdHH-H- CH mwflnw CO Gunman PQMHM»; ow Egg-H- -65- been made also by Bustad gt El. (l9A8), Lecce and Matrone (1960) and ' Lecce _ep _a_l. (1961a). Total serum protein values are given in Table 6. Total serum protein concentration in the glucose-fed pigs was significantly lower than that of the controls at 2 weeks of age. Total serum protein de- clined between 1 and 2 weeks, but the difference was not significant. TABLE 6. Total Serum Protein in Trial II. (gm./lOO ml.) Age Glucose Control 1 week 2.37 : .OA -- 2 weeks 2.30 i .11 5.90 i .27 Pigs fed the glucose diet in Trial II did not produce a serum protein profile comparable to the pigs in Trial I. Data presented in.Appendix Table l and illustrated in Figure 8 suggest that little change in the relative proportion of protein fractions had occurred from birth to one week of age. There was a small increase in Grand B—globulins and a decrease in albumin. At 2 weeks of age, control pigs had a significantly (P <.01) higher percentage of albumin and a significantly (P (.01) lower percentage of Okglobulin. The 7-globulin figures do not closely agree with immunoelectrophoresis patterns, probably for the same reasons as given in the discussion of Trial I. 'Immunoelectrophoretic patterns of the sera from A pigs in this litter are presented in Figure 9. The antiserum used was rabbit anti-A months pig serum. No 7-globulin was present in these sera. Differences between the glucose-fed and the control pigs are seen in Figure 10. The serum pattern in the pigs on the.synthetic diets resembled the pattern at birth; but an increase in the number and intensity of the I TRIAL 2 COKTIOL q . L r 2 WEE KS CLUCOSE Age-—-—"I’fi‘“/’::\~—’//<<\s-.___ GLUCOSE d I I? A. 1 WEEK M o noun FIGURE 8. Comparison of the serum protein profiles among the zero hour pig, glucose-fed pig one week old, and a glucose-fed pig and sow-fed control two weeks old from Trial II. -66_ .Edsom mam mgpooa suspem psppos muss HH smssH dons mmsm 4 Sosa meow ado: osmN mo COHPonoCs ozp Eosm mcspsdmos dsospom osposo: ospomsoo. .m meUHh .Es-aom msm mas-Laos sighs #3,an nah: HH smflHE scam mung ado: osmn am one .39 ohm-smoos-Hm 30 some» moo «was oomussomoo 30 some» ono aHosp-poo 30 Mom: 020 o sosm season As op .sv wo cospopsnoss one. Seam waspssmos 580.me osposogmospoosooss-sefi .os mmwam J -69- bands had occurred. Control pigs' serum differed from the treatment pigs' in the greater quantity of albumin and the presence of 7-globulin. Similar observations were made with respect to the pigs at two weeks of age. In Trial II, another method was used to study gut permeability in addition to the feeding of the fluorescent-labeled protein. Along with the protein solution, AOO units of insulin were fed and blood glucose concentration was determined at zero, 3 and 6 hours post-administration. Blood glucose levels are presented in Table 7. A depression was seen in both 1 and 2 week old pigs receiving insulin when they were compared with animals not receiving insulin. The gradual decrease in blood sugar seen in pigs not getting insulin was probably due to the fasting conditions of the animals. Due to the small number of animals, the differences were not significant. TABLE 7. Blood glucose levels of pigp given insulin in Trial II. Time after insulin ingestionpphours o 3 6 Effect of insulin ingestion, 1 wk. pigs Insulin Control (I7* 685** 77 66 Insulin treated (2) 8A 55 7A Effect of insulin ingestion, 2 wk. pigs Insulin Control (1) 63 5A A7 Insulin treated (3) 55 A6 A5 Effect of trypsin inhibitors on insulin absorppion Soybean trypsin inhibitor (1) 136 115 115 Egg white trypsin inhibitor (1) 125 87 61 Control (1) 125 81 (died) *Number in parentheses indicate number of pigs. **Blood glucose expressed as mg./lOO ml. -70- One theory for the inability of the older animal to absorb whole proteins is that an anti-trypsin is present in colostrum which declines in activity as lactation progresses (Laskowski and Laskowski; 1951). The three control pigs in Trial II were used to study the effect of two kinds of trypsin inhibitor on the absorption of insulin by pigs that had nursed the sow for two weeks. One pig was given soybean trypsin in- hibitor and another received egg white trypsin inhibitor at the rate of A0 mg. per kg. bodyweight. The third pig served as a control. All 3 pigs received AOO units of insulin. Blood glucose data are presented in Table 7. These data would suggest that the egg white trypsin inhibitor may enhance the absorption of insulin, but since a similar effect was seen in the control pig, the effect was most likely from bleeding. Alkaline phosphatase acitivity in 6 locations in the intestine are recorded in Appendix Table 3. Differences among these individual locations are illustrated in Figure ll. Somewhat less enzyme activity is seen in the pigs in Trial II than was seen in Trial I, in both the treatment and control pigs. Animals in both trials had the highest activity in the middle jejunum-ileum. The difference in enzyme activity between these two trials might be attributed to litter difference. The difference in performance of the 2 litters cn the synthetic diet should also be remembered. Somewhat more fluorescence was present in the tissue sections of the pigs in Trial II than was present in the glucose-fed pigs of Trial I. Fluorescent material was present in the epithelial cells throughout the tract. Even cells in the colon appeared to be taking up fluorescent material. Visually, it could not be determined if this material was the FITC-labeled protein or free—FITC. (Figures 12 and 13). Serum fluores- 10‘ mm/Cm n. M./30 Min 9 . TISSUE 8 ‘ PHOSPHATASE ,. mm 2 6 . s 1 4 . 3 . {N ‘E,; f V; ‘\\\ 4 .5 ’ .\ 2 *‘ ’. oat" ‘ ".- ‘6‘ ’¢‘- \ fo’o fipmaoa pswsos.2o09 as owsosoo -75- .. mw.ss .. u- es owes soascoo 2. o: ms. ws.s now + mosm es osss soapeoo 2 mm ms. sm.m sos + wmom es . owm sonscoo a wm so. so.s ws . ssss as owss onoosso 2 mm so. ow. w . smms ss oess onoosso 2. am so. omw. m - wsss ss omms onooaso 2 mm wo. ms.s o . muss es owns ssonmo a om so. so. ms .+ mmms es owss csonoo .2 wm wo. oso.s ms + msms es ooms csomoo a so ms. mm.m ow + msom s omss soapsoo 2 sm ms. mm.m oos + swsm s owos soapsoo .2 mm so. aw. w . omms s oems omooaso .2 mm so. mom. 4 + msms s omms omoooso 2 sm mo. oo.s ms . mmss s omms csommo .2 mm wo. nws. ss . moms s owms esonno a wm pnwsos. .N.swv mpnwsos A.awv dmhmov Naswv Psossmoaa 2mm .0: wsm soon panama soon as assoc so seams senses mo R on coosmm owsosu R sawsoz so om<. HoHpHsH .93 noosmm .HHH Hosea as mwsm so wsoo pawsmz .m mnm¢a , mu 3 A 1 It COKTIOL . CL ‘A M 1 WEEK CASEIK ._.‘t==”£:-—”=:II—~"'—!=--‘::EE::§-_____. GLUCOSE ‘ % N o noun FIGURE 15. Comparison of the serum protein profiles among zero hour pig, 1 week old pigs fed synthetic diets, and a sow-fed control from Trial III. -76- mu 3 A CONTROL 2 WEEKS M o noun FIGURE 16. Comparison of the serum protein profiles among the zero hour 2 week old pigs fed synthetic diets, and a sow-fed control from Trial III. -77- pv N. J. . -78- patterns seen in Figures 17 and 18. These pictures illustrate the appear- ance of the immunoelectrophoretic pattern before and after the feeding of the labeled protein mixture to a 1 week old pig. Albumin,a and B-globulins were the primary proteins present in the treatment animals before feeding (Figure 16). Gamma globulin was present as well in the control pig. After feeding, still only albumin, aand B-globulins were present in the treatment animals; but there were more and heavier bands present. The increase in albumin was particularly noticeable. There was no indication that 7-globulin was absorbed by these pigs. Some fetal '7-globulin was present. This has also been reported by Lecce and Mbrgan (1962). The immunoelectrophoretic pattern of the protein mixture in- cubated with anti A-months pig serum and anti colostral whey is seen in Figure 19. The mixture was quite high in 7r-globulin concentration but also had somecxand.B-globulins and albumin. TABLE 9. Total serum protein in pigs on Trial III. (gmp/lOO ml.). Age Treatment No. One weeka No. Two weeksET F ~ =F+2A F F+2A Casein 2 3.76:.26**bc 3.92:.20 3 3.911.189:c A.o6:.23 Glucose 2 2.08:.1A** 2.00+.2l 3 2.11:.23** 2.16:.25 Control 2 6.01:.1A -- 6.A6_+:.2l 3 5.80_+_.A8 6.25:..A7 3Feeding and 2A7hours post-feeding. can : standard error. cSignificantly higher total protein than glucose pigs (P <;Ol). *Significantly lower than control at same age (P <.05). **Significantly lower than control at same age (P <.Ol). Insulin was also added to the protein mixture fed the pigs in Trial III. Blood sugar changes were followed and these data are given in Table 10. It appeared that the glucose level was reduced more in the treatment animals than in the controls even though all the changes were significant with respect to the zero hour levels. The failure of the sugar .sdsom wsg £9205 2|H9cw 9sppms 29s: HHH HmssB 2s musm Hos9doo m one ammsm wowuowoodsm m ammsg wowlcsomwo m «Hos9coo oHo Moos H w A.s 09 .Hv Boss Essom so oos9wpdocs o29 Boss mss9H5mms oso99mm os9ososmos9ooaooc5ssH .sH mmbon ‘. ,u.?. .. .oH59xss 2269029 o29 mesooow spasm mason :m Edsom wsm 29202 :ns9no 9sppms 2993 HHH Hosae cs sos9eoo sog9oem one smwsm wowuomoodsm m amwsm oosuesmmmo m smsos9noo oso 2mm: ono m A.H o9 .sv 202m gosom so 2029mndoes m29 Eons mes9s2mos nso99mm os9osonmos9omsoondssH .ws mmwam .\ Y—CLOOULIN and WNET MIXTURE anti-4 anti: fig sm- _ . + / j I O \ _- uti - :0]!!th J!9L_ _ __w ._ FIGURE 19. A tracing of the immunoelectrophoretic pattern resulting from the incubation of the electrophoresed colostral whey—y-globulin mixture with rabbit anti-A months pig serum and anti-prepartum sow serum. -81- _82- level in the glucose-fed pigs to rise again after the 3 hour sample was probably due to the fasted state of the animals and the lack of appreci- able glycogen reserves. TABLE 10. Blood glucose in pigs in Trial III. (mg.[100 ml.). Age Treatment No. Time (hours) (wks.) O 3 6 l Casein 2 93+ 78 15+8** 56+6* Glucose 2 72: T 25EA** 18£A** Control 2 1AA: A . 13010:“:b 99:1** 2 Casein 3 75: 6 29:6“ 3o:5** Glucose 3 T2: 8 2T:l** .21:3** Control 3 135110 9A:6* 87:5** _EMean : standard error. One value, one sample was lost at 3 hours. Significantly lower than zero hour (P <.05). **Significantly lower than zero hour (P <.01). Intestinal alkaline phosphatase activity was determined at 6 loca- tions in the pigs on this trial. These data are reported in Appendix Table 3 and are graphically presented in Figures 20 and 21. Tissue activity in both casein- and glucose-fed pigs was lower in the 1 week old pigs than in the 2 week old pigs. This was just the opposite of the trends observed in Trial I. One week old controls had higher activity than the casein-fed pigs and approached the activity of glucose-fed pigs in the middle jejunumrileum. rThese—controls appeared to have similar activity in the middle and caudal jejunum-ileum. One week old casein-fed pigs had the highest activity at the caudal jejunum-ileum. -Data on the enzyme activity from the intestines of the 2 week old pigs resembled that observed in Trial I. The mouse was reported by Moog (1961) to have the highest activity in the duodenum with decreasing levels in the jejunum. Pigs and mice appear to differ in this respect. Tissue alkaline phosphatase was studied histologically in an 10 l .../c- o. also as. PNOS'NNTNSE TRINL 3 5 . 1 WEEK . 3T . f '3 '6 c 3 g.- .2.- 3_ 3° :2 g 0‘ .“ o“ 03 '38 FIGURE 20. Comparison of intestinal alkaline phosphatase activity at 6 locations in the 1 week old casein— and glucose-fed pigs and the controls in Trial III. _83_ 10* 1 J {mm/Cm D.M./30 min. TISSUE PHOSPHATASE TRIAL 3 2 WEEKS l 1“ e," 1,, 1”, 'l = c: 1: == 1: :3 «3.. :5... =7.. :§.£E =IJE FIGURE 21. Comparison of intestinal alkaline phosphatase activity at 6 locations in the 2 week old casein- and glucose-fed pigs and the controls in Trial III. —8A— -85- attempt to observe changes in enzyme activity. The Lillie modification of the Gomori method which used B-glycerol phosphate as the substrate was preferred to that of the naphtholsAS phosphate procedure reported by Burstone (1958a,b). Neither of the procedures were quantitative enough to suggest differences between the treatments. In all cases the enzyme was located in the brush border of the columnar cells at the tips of the villi. Moog (1950) and Moog and Ortiz (1960) reported this same location for the enzyme in the intestine of the guinea pig and chick. Formalin fixation, which was used in preliminary trials, resulted in a loss of most of the enzyme activity. These results are consistent with those reported by Seligman 23 El. (1951). Serum alkaline phosphatase was determined on this litter at birth, A days and at the l and 2 week treatment periods. These data are pre- sented in Appendix Table 5 and are illustrated in Figure 22. Serum phosphatase was also determined 2A hours after the protein mixture was fed. Differences in serum phosphatase activity were present at A days between the various treatments. In the animals killed at 1 week, both the casein- and glucose-fed pigs had significantly (P<:.Ol) lower serum phosphatase activity than did the controls (Table 11). The same was true for the 2 week old animals. A decrease in activity was seen in all the treatments between the time of feeding and 2A hours later. This is not the result of further fasting since the animals were fed during this period. Some of the effect may be attributed to the bleeding, itself. Tissue sections from all the pigs in Trial III were viewed under a fluorescent microscope for the presence of fluorescent material in the epithelial cells of the villi. As 2A hours had lapsed since the labeled material was fed, no fluorescence was observed in these cells. However, m— z -86- .HHH HwfiaE as mwsm Hos9coo out «oomuomoodaw aoomuesomwo oso 2wo ms o29 n9 29ooo 09 29ssp Boss 299>s9oo omm9osmm02m ocsswxsw Edema a»: uncucluo: :3“ . c b VT—Vjfi 'fiTTVva if V v i ‘7 ~— 3 3 3 an {-2}.- .mm mmDUHm -87- TABLE 11. Serum phosphatase in pigs on Trial III at feeding and 2A hours post-feeding. (gMQ/m1.[pr.)p_ Age One week No. Two weeks Treatment No. F F+2AI_ F F+2A_' Casein 2 11:10.7(31 A.6:o.5 3 2.5:O.A 3.1:O.6 Glucose 2 T.A:O.6 3.9:O.l 3 2.7:O.6 2.A:O.6 Control 2 36.A:1.8** 31.2:o.6**3 18.611. 3** 18.A:3.6** _aMean : standard error. **Significantly greater than either treatment at this time (Pi<.01). the serum fluoresced brightly, as did the urine from these animals when placed under a UV light. The blood vessels in the villi were very distinguishable. Fluorescence which was detectable in the urine was also detectable in sections of the kidney. A glomerulus from the kidney of l of the casein-fed pigs is shown in Figure 23. Blood vessels around the proximal convoluted tubules also contained fluorescent plasma. As in the previous trials, no protein was detected in the urine. Some of the fluorescent material was taken up by the reticuloendo- thelial cells. These cells were observed in some intestinal sections and in the thymus (Figure 2A). Aside from the fluorescence in the plasma, another location of high intensity was the connective tissue. The serosa around the intestine fluoresced brightly. Mancini.§pflgl. (1961) also made this observation when they injected rhodamine-labeled proteins intraveneously into rats. These workers presumed that the injected protein retained its label and that it was stored in the connective tissue. They stated that the strong bonding between the label and the protein is too stable to be disrupted as the complex passes through membranes of cells. They further state that rhodamine never binds to serum.proteins EEHXIK9° In view of the fact that no 7'- globulin could be detected in these animals 2A hours -88- Figure 23. Photomicrograph of a kidney from a casein—fed pig in Trial III, showing a fluorescing glomerulus as a result of the FITC in the plasma. Figure 2A. Photomicrograph of thymus showing the fluorescent particles which appeared to be associated with the reticuloendothelial cells. fut -V..: yvpao Mob ‘. o ‘ v,p.. p VC‘A‘ “ro- ’ urn-J ‘ up”. ”A. a -89- after feeding and that the fluorescence in the urine was not associated with protein, the FITC-protein complex was disrupted somewhere in the organism and the free dye transported across the cellular membranes. In summary, animals deprived of colostrum.were maintained for as long as two weeks on synthetic diets containing a single protein, or no protein. Using the presence of fluorescenceirlthe cell as the criterion for protein absorption, glucose-fed pigs appeared to absorb more protein than the casein-fed pigs. However, the results presented would indicate that in animals reared under these conditions, the presence of fluores- cence in the intestinal cells may not be a valid criterion for the presence of protein. There appeared to be a deconjugation of the dye-protein complex ip'xizg. The results of Trial III suggested that if ‘7-globulin reaches the intestinal epithelium, it is not absorbed. Other serum fractions did appear to be absorbed. B. Effect of Increasing Blood Volume on Whole Protein Absorption. Weight data for the first 3 days of life on the Hampshire litter used in the blood volume trial are presented in Table 12. Mean weights of the 6 pigs in the 2 groups were significantly different only at 72 hours. This lower weight in the controls at 72 hours is probably the result of the large variation in this group rather than any effect from the treatment. Total serum protein levels for 5 weeks are presented in Table 13. Treatment pigs were significantly higher in total serum protein con- centration than the controls at 5 days of age. No explanation can be given for this difference at 5 days. Hematocrit was decreased in the treatment animals only at the 10 and 2A hour sampling periods. Serum protein fractions were also studied in this litter and the data are r!“ L.— -‘ A: TABLE 12. Weight changes in pigs on blood volume trial (gm.) Hoursl_post-treatment Pig Treatment Pig Control no. 0 2A 72 no. 0 2A 72 2 1180 1320 1680 l 13A0 1A80 1880 A 1380 1500 1660 3 1280 1380 1680 6 1080 1280 1680 5 12A0 1A20 1680 8 1A70 1620 1900 7 1270 1320 1060 10 1A00 1580 1800 9 12A0 1A00 1600 12 1A20 l6AO 1960 1 ‘1370 1560 2020 Mean 1321 1A90 1767** 1290 1A26** l653**a S.E. 61.3, 63.A 4_58.6 19.5 3A.2 13A.A aSignificantly different from 72 hour treatment value (P <;O5). **Significantly different from birth value (P <101). TABLE 13. Total serum protein in_pigs on blood volume trial. (gm./lOO'ml.) Age 0 Hr. 10 Hr. 2A Hr. 72 Hr. _5 Days 3 Wks. 5 Wks. Control 2.61.1a 5.0:.3 5.5:.6 5.A:.5 5.11.2 5.2:.1 5.0:.1 Treatment 2.8:.1 A.9:.A 5.A:.2 5.7:.2 5.8:.l** 5.3:.3 5.r:.l aMean :_standard error. **Significant1y greater than control (P <.01). presented in Table 1A. These values represent a mean of 2 samples. Little difference existed between the treatment and control group at any time from birth through 5 weeks of age. The lack of difference between the pigs in which blood volume was increased and the controls in weight, total protein and serum protein fractions indicates that absorption of colostral proteins was not affect- ed. The increase in blood volume reported by Rameriz gp.gl. (1963) apparently was not related to gut closure. VS -91- TABLE 1A. Serum protein fractions and hematocrit of pigs on blood volume trial. Hct ‘ Serum protein fractions, % Age Treatment % Albumin Globulins ix 8 _gy 0 Hour Control A2.9 30911.5a AA.7:1.5 16.6:0.7 6.9:0.7 Dextran A2.9 30.8:1.6 A5.7:1.3 17.110.0 6.3:0.7 10 Hours Control 31.6 l2.3:0.7 ll-3il-2 29.5:l.7 A6.8:2.3 Dextran 26.2b l3.9:0.8 l2.1:l.9 28.8:2.3 A5. _l.8 2A Hours Control 29.8 13510.8 10.6123 26.5:2.6 A9.2:A.0 Dextran 22.8b 1A.3:l.0 10.3_2 3 26.6:2.3 A7.7:2.0 3 Days Control 23.9 2A.0:1.A 1A.9:0.7 26.A:1 2 33.8:2.0 Dextran 23.5 22.5:1.9 lA.8:l.O 2A.5:1 6 38.2:2.A 5 Days Control 21.5 32~5:l-l 17.110.6 25.5:0.A 23.7:2.A Dextran 22.7 32.8:l.6 15.7:0.8 26.2_0.73* 25.2:l.9 3 weeks Control -- 65.5:1.3 10.2:C.5 16.7:0.A 7.6:1.0 Dextran -- 63.3:1.l 11.0:0.A 17.2:O.6 8.A:0.A 5 Weeks Control -— 6A.3:1.A 1A.0:1.1 15.1:O.6 6.6:l.0 Dextran -— 6A.6:0.8 12.1:0.5 15.6:0.A 7.A:0.A aMean : standard error. bSignificantly less than the control (P <.05). cSignificantly greater than the control (P <.01). C. Changes in serum and milk phosphatase in five litters during the post-natal period. Mean litter values of serum alkaline phosphatase for these 5 litters are recorded in Table 15. Individual values were highly variable both within a litter and between litters. Sow litters had higher serum alkaline phosphatase activity at birth than did gilt litters. Activity did not always decline after birth as suggested by Young and Underdahl (l9A8). These workers based their statement on the difference in birth and 5 day samples. If a seven-fold increase in intestinal phosphatase activity occurs as is reported by Payne and Marsh (1962b), it is not very well reflected in serum phosphatase activity. -92- TABLE_;5. Serum phosphatase activity in_5 litters. (uMp/ml./hr.). Litter Agg_(dgys) no. No. 0 l 2 7 1A 30 65 8 Sow 61:9.Aa 72:13.A 51:6.6 23:1.A** 19:0.6** 7:0.7** 66 10 Sow 7918.3 A6:2.5** 37:2.8**22:1.9** 17:1.8** 7:0.2** 69 5 Gilt 19:1.2 30:3.7** 2A:l.3* 2112.5 —- _- 70 8 Gilt 1A:2.A A3:3.6** 22:1.7* 27:5.7** -- -- 72 10 Sow A1:3.2 A0:A.6 70:8.3**12:0.7** -- _- aMean :_standard error. *Significantly different from 0 hour (P <.05). **Significantly different from 0 hour (P <.01). A diagram of changes in serum alkaline phosphatase, weight, and total serum protein is presented in Figure 25. A significant negative correlation of -.53 existed in these 5 litters between weight and serum alkaline phosphatase activity. Total serum protein concentration was also negatively correlated with serum alkaline phosphatase activity (-.33). Weight and total protein data on these 5 litters are recorded in.Appendix Table 6. Colostral and milk changes in alkaline phosphatase activity with stage of lactation are indicated in Table 16. These data represent values obtained from fat-free samples. The activity of colostrum was somewhat higher in gilts than in sows. This difference was not significant due to the small number of observations. The decline after parturition was consistent with the findings of studies involving other Species (Buruiana and Badilita, 1938 and Kannan and Basu, 19A8). Actually, the true enzyme activity in colostrum of milk was some 30 to A0 percent higher than these data indicate in view of the studies reported by Morton (1953a), in which he found about 30 to A0 percent of the enzyme -93- .ooss so spoon sagas one messes naoeess m sons nwso one as mwcmflo PSWHO? dcw 2%9H>Hpod mmmpwflflmOQQ mQHHwMHm Efihmm «QHm9OHQ HwPOP.ESH®m %O QOmHHmQEOO .mm HmDUHh a»: own.-- -.--Immrlr ~ ~ ., , _ .. o V N Ar —........... W \ N Jl1oo’ O ‘ A a“ V 2 ntm \ t \ . cm V e s at. c \ We... ~s o . s .9. v.. 0833Iff. ‘ ' "'W W ------W . --- -"" 90:89.90:‘03....3338331, 2.. e - .- .2 E ...: . a is}... fi 0 3...: .I .2 lo -9h- activity to be associated with the milk fat. TABLE 16. Phogphatase activity in colostrum and milk. (pMZml./3O minutes). Day of lactation No. 0 1 2 7 1A 3 SowS 12.2 7.6 7.6 5.1 2.6 2 Gilts 3A.2 12.2 lA.2 9.A -_ Individual values for alkaline phosphatase activity in the udder sections of 3 animals were also studied. Samples were taken at 0, 2A, and A0 hours; and the activity in the individual sections was grouped in- to the right and left sides. Data obtained in this study are recorded in Table 17. A significant difference between the 2 sides appeared only at 2A hours. This difference was probably the result of unequal amounts of colostrum having been removed from the sections. TABLE 17. Alkaline ppogphatase activity in udder sections. (pM/ml./3O min.). Sample Sections time Right Left Both hours Side side Sides 0 Sow 17:1.1 18:1.A 17:0.8 2A Sow 912.0 6:1.0** 7:1.1 A0 Gilt 910.3 913.5 9:0.8 **Significantly different from opposite side (P<<.Ol) ean : standard error. Changes in pig serum and milk alkaline phosphatase are plotted in Figure 26. 1. Effect of storage on serum alkaline phosphatase Alkaline phosphatase activity was observed to increase in all samples stored at 50 C for 18 hours. In the case of the 2A hour pig serum, a 70 percent increase occurred. Data in Table 18 also indicate .Ed9awm9mom m2003 m 9mssm C29 mesado sow on9 mo mafia out Boson wsm 29 299>s9om omw9ogmmosm oesaoxsm so cOmssmeoo a»: . . - a .ll/ .‘fimhfillllnunlllll.Inlllllnulllll v ..— . .ymwowanm/ . .2 III II. 1 .2” I I II II . cc I “22:32..— “23:2 .--:I--:......:.L— 3. 53:..- .wm mmbUHh -96- that storage at -200 C affected serum activity. Zero hour pig serum activity was significantly different from fresh serum activity only after l week of storage at this temperature. The activities of the other sera were consistently altered with regard to the activity of fresh serum.as the result of storage. Kunkel.e§.al. (1953) in studies with cows, reported serum phosphatase to be stable while frozen for lh days. Wilcox (1963) stated that the enzyme in chicken serum was stable for h weeks. Enzyme determinations reported by Young and Underdahl (l9h8) and Combs §§_al. (1959) were made on frozen samples. TABLE 18. Effect of storage on alkaline phosphatase in serum of pigs of four ages. (uM/mlL/hr.). Stored Serum Fresh 18 hrs. Frozen at -200 C, weeks sample sample <@ Q? C l 2 3 L 0 Hour 28550.7a 1+03~_o.6** 35:0.9** 30:0.2 29:1.5 2911.6 2h Hours 51:1.5 87:1.3** 1+9:1.h 3h:o.9** hh:0.5** 38:3.2** lWeek 2510.9 29:0.1** 20:0.5H 18:0.1** 17:1.9H 15:1.6** aMean: standard error. **Significantly different from fresh sample (P< .01). Serum levels of alkaline phosphatase in the 5 litters studied were highly variable but did decrease during the first 2 weeks of life. Pig serum activity tended to decrease somewhat like the activity in the dam's milk. Enzyme activity was highest in colostrum and decreased as lacta- tion progressed. Serum alkaline phosphatase activity of young pigs seemed to be affected by storage. V. SUMMARY Three trials were conducted using colostrum-deprived pigs fed pro— tein and protein-free diets to study their ability to absorb whole proteins from the gastrointestinal tract. In all 3 trials the pigs on treatment were caught at parturition and removed to locations previously uninhabited by swine. Pigs left on the sow served as controls. When taken from the sow, the pigs were bled and placed in individual boxes heated to 800 C. Pigs were bottle fed either casein-glucose or glucose diets 5 times a day. The rations contained fat, vitamins and minerals in adequate quantities. At either 1 or 2 weeks of age, the pigs were fed solutions of labeled protein and were killed. In Trials I and II, FITC-labelled 7-globulin was administered and the animals were killed at 6 hours post feeding. -In Trial III, a mixture of 7-globulin and colostral whey was given and the pigs were killed 2h hours later. .Serum samples were taken before and after administration of the tagged protein. Tissues were collected from 6 locations in the intestinal tract for histological examination and alkaline phosphatase assay. Paper electrophoretic separations and total serum protein analyses were performed to follow the changes in the fractional dis- tribution and quantity of protein in the sera. Immunoelectrophoresis was used to detect the absorption of 7-globulin and other proteins. Performance of the pigs in these 3 litters varied greatly between litters. Trial I pigs, with the exception of l pig, maintained or in- creased in weight over their weight at birth. In Trial II, all of the animals weighed less when they were killed than they did when they were born. Casein-fed pigs in Trial III generally maintained their birth -97- -98- weight; but the glucose-fed pigs declined in weight. Spleen weights of both treatment groups were significantly lighter than were the controls. In all 3 trials, colostrum-deprived_pigs fed the synthetic diets had lower total serum protein concentrations than did the animals nursing the sow. Glucose-fed pigs had even lower serum protein concentrations than did the casein-fed pigs. Both treatments were significantly lower than the controls. Relative serum protein fractions followed similar patterns, but there was some variation among the 3 litters. In Trial I, both the casein- and glucose-fed pigs showed changes in their serum profile as compared to that at birth. In Trial II, in which only the glucose diet was fed, only slight changes in the serum fractions were detectable when compared to the relative ratios at birth. Similar results were seen in Trial III. Intestinal alkaline phosphatase activity was highest in the jejunumr ileum.in all 3 trials. Activity was higher in Trial I than in either Trial II or III among the same treatments. Casein-fed pigs had higher tissue activity than did the glucose-fed pigs. In the pigs killed at 1 week of age, the glucose-fed pigs had higher tissue activity than did the controls. By the second week the activity in the glucose-fed pigs fell below that of the controls. In Trials II and III, insulin was added to the protein mixture and blood glucose concentration was determined at 3 hour intervals for 6 hours. Blood sugar data indicated that insulin was absorbed since blood levels decreased more in the treatment pigs than it did in the controls. Fluorescence within the epithelial cells was the intended criterion of absorption of the labelled protein in Trials I and II. In Trial I, -99.. the tissue from the glucose-fed pigs appeared to possess more fluorescence than did the casein-fed pigs, although the intensity was weak in both cases. Glucose-fed pigs in Trial II appeared to have absorbed more fluorescent- labelled material than did the glucose-fed pigs of Trial I. In Trial III, the validity of this criterion was studied by allowing time for absorption to take place, and following the changes in blood serum. Changes in total serum protein and the relative ratios of the serum fractions 2h hours post feeding in Trial III indicated no detectable change in serum proteins. Immunoelectrophoresis of serum before and after feeding the labelled protein indicated that a, and B-globulins and al- bumin were absorbed, but that 7-globulin was not. One litter of 12 pigs was used to study the effect of increasing blood volume on whole protein absorption. Pigs caught at birth were divided into the control and treatment group which received dextran plasma extender to increase their relative blood volume to that level expected at 2h hours of age. All pigs were returned to the sow and changes in weight, total serum protein concentration, and serum protein fractions were followed. The lack of significant differences in the above- mentioned criteria indicated that absorption of colostral proteins was not affected by changes in blood volume. Alkaline phosphatase activity in the milk of the dams and alkaline phosphatase activity in the serum of pigs in 5 litters was studied during the post-natal period. Data were collected from these 5 litters at birth, 1, 2 and 7 days of age. vao litters were also studied at 1h and 30 days of age. Individual values within a litter were highly variable, but sow litters had higher serum alkaline phosphatase activity than did gilt litters. Activity, generally, but not always, decreased -lOO- after birth. Negative r values of .53 and .33 were obtained when serum activity was correlated with weight and total serum protein,.respectively, in these 5 litters. Colostrum and milk from gilts had higher alkaline phosphatase activity than did that from the sows, although activity in both decreased as lactation progressed. The fall in serum activity of the pigs generally followed the decline in milk phosphatase activity. Sera from pigs of A ages were stored either at 5° C for 18 hours or at -200 C for h weeks to study the effect of storage on enzyme activity. Storage at 50 C significantly increased phosphatase activity, while storage at -200 C generally resulted in a significant decrease in serum activity. VI. LITERATURE CITED Alexander, H. L., K. Shirley and D. Allen. 1936. 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Percent Age Globulins (days) Treatment No. Albumin a 45 ,1 Trial I O -- 8 ' 34.3 44.3 16.7 4.2 7 Casein 1 36.6 36.6 24.7 2.0 Glucose 1 36.8 41.1 20.0 2.1 Control 1 35.2 14.2 26.5 24.1 9 Casein 1 49.7 27.3 21.0 1 9:: Glucose 1 38.9 36.3 19.8 4.9 14 Casein 1 50.0 30.2 17.0 2.8 Glucose 1 44.5 32.5 21.1 1.9 Control 1 56.0 13.0 18.7 12.4 Trial II 0 -- 10 33.9: 40.5 18.9 6.6 7 Glucose 3 27.6 45.7 20.2 6.5 14 Glucose 4 30.8 38.3 24. 6.5 Control 43, g55.8 .13,5 25.0 8:5 ~ APPENDIX TABLE 2. -Serum proteins of pigs in Trial III at feeding and 24 hours post—feeding the labelled protein. Percent Age . Globulins (days) Treatment No. Sample .Albumin a S 7 7 Casein 2 F - 43.2 34.2 - 18.0 4.4 F + 24 45.1 33.2 17.8 3.9 Glucose 2 F 20.1 47.4 23.4 8.9 F + 24 37.7 20.8 30.2 10.8 Control 2 F 31.8 16.5 29.5 22.2 F + 24 32.7 16.6 33.2 17.4 14 Casein 3 F 46.5 27.7 19.6 5.9 F + 24 48.7 24.2 19.3 . 5.4 Glucose, 3 F 28.4 40.8 21.6 10.7 F + 24 29.3 41.8 20.4 8.3 Control 3 F 44.2 18.1 27.1 10.7 F + 24 44.0 19.4 28.6 7.9 .sflopoam ooHHoemH one mo mnHeommupmog mason asomuapcozep .chposm eoHHopmH one mo meHeoomnpwom mason xflmo whHo.u.mv Hoppcoo aosm peonmmmHo szsonMHsmHm** .Amo.uva Hospcoo Eosm psoHCMMHo ercsOHMchHm* -123- sw. mm.H sm.m Hm.m as. ms.a H Homecoo me. om.H m>.m :m.m **Hm.m HN.H m omoous em. oo.s om.e am.m *som.m om.H m assume as mm.H Hm. mm.m mm.m mw.H sm.H m Hoapeoo mm. mm. oe.H NH.m mm.H :H.H m omoosHo *oo. *mm.H ma.m *ao.H ma.H mo.a m cecnmo s QHHH HeHsB NH. om. em.H e:.m mm. we. m Hospeoo :H me. as. mm.m mm.m mm.H oo.H a onocsao aH mm. we. mm.m oo.m He. we. m omoodHU e eHH Hmflaa MH. mm. mm. :H.: mm. mH.H H Hoapsou mo. mm. we. mm.H :p. mm. H omoodHo mm. mm. :m.m mw.s I: 0H.m H cHomwo 2H mo. mm. He.m mH.w NN.H we. H omoodHU mm.H NO.H mm.m mo.w mm.H ao.m H eHomwo m mH. wH. m:.: oo.m mm.m om.H H Hospcoo mm. Hm. om.m so.OH mm.m ms.H H omoosHo mo.H Hm. Mm.m Sm.m mo.m Hm.H H chmoo a mH HmHaB COHoo SOHoo Hh Hh Hh onm .oz psospmoha Amhoow Undo coho Undo we: mean mw< soapocm HecapncesH ndncas om\.z.o .amxst .HHH can .HH .H manage so nmaawee cnmensmnosm ancaencecH .m mamas mezmmma -12h- APPENDIX TABLE 4. Tissue dry matter of colostrumrdeprived and control 4_pigs in Trials 1, II, and III.‘ Percent Pig Uppera Lower ___> no. tract tract 8 16.9 16.5 9 16.4 16.5 10 16.1 16.5 11 22.9 13.3 12 14.3 13.8 13 15.1 18.0 14 15.7 16.0 15 16.9 ‘ 19.4 16 17.8 16.7 17 16.0 16.7 18 18.1 16.0 19 19.0 14.5 20 16.6 17.1 21 19.6 19.0 22 18.3 17.8 23 17.8 19.2 24 17.0 19.0 25 18.3 19.4 26 18.0 16.6 27 14.2 18.3 28 13.4 18.0 29 16.9 15.9 30 19.7 .13-3 31 16.5 15.4 32 11.4 12.8 33 14.8 14.8 34 16.3 16.1 25 14.0 12.5 36 17.6 16.4 37 17.3 16.1 38 14.7 12.8 39 14.7 16.0 40 15.8 16.4 aUpper tract is that part of the intestine cranial to the cecum. -125- APPENDIX TABLE 5. Serum alkaline phosphatase of pigs in Trial III before and after feeding the labelled protein. (uM./ml./hr.) A‘- Age Pig One weeka__ Two weeks no. Treatment 0 Hr. 4 Days F F+24 F F+24 26 Casein 20.0 9.3 6.4 5.1 29 Casein 25.4 10.5 7.8 4.2 31 Glucose 20.2 8.7 8.0 3.9 32 Glucose 15.0 10.5 6.8 3.9 36 Control 21.0 30.0 38.2 30.6 37 Control 14.2 29.1 34.6 31.8 27 Casein 20.6 13.2 3.2 4.2 28 Casein 12.8 15.5 2.4 3.0 30 Casein 25.4 15.3 2.0 2.2 33 Glucose 23.2 12.0 3.6 3.2 34 Glucose 25.4 5.1 3.0 2.8 35 Glucose 36.0 7.8 1.6 1.2 38 Control 17.2 25.8 19.8 12.2 39 Control 20.4 21.0 20.0 18.0 40 Control 15.4. 23.7 16.0 24.8 aF = Time of feeding protein mixture. F+24 - 24 hours post-feeding of protein mixture. -126- .Hosso osmosspm H.cmmzs --. .. wo.mwm.e so.mws.m mo.mwa.s ma.m:a.m oa me u- .. cH.Hso.m mm.Hmo.m mmmmmm.m mo.HHs.m m os In- In- am.Hmm.s mmnwmm.: Hm.Hsm.: :H.Hms.m m mm 8.4.5... causal. 8.4.8... means... 8. Has... Swarm ca 8 so.+mm.a mo.+mm.s ma.+ma.m om.+os.m sm.+mm.o nmo.+ms.m w mm A.Hs OOH\.emv CHmpoam afisom Hmpoa . -- -- sawmdm 8 was. samba. same... on s. -- -- mm Hmmmm cm Homes mamasma mmmwame m os I.-- In- saanmmmH moammmma mmwmoaa Hanseaa m do moanmomOH mmmnosme smHHsHmm moausoom om+moma nmm+oame 0H mm msm+aomme mem+ooma mmm+omom sma+soma w me name name..- om sH cu. m H o .oz scenes when .mhmppHH wicH :HoHOHm,ESHmm H6PO¢ 6cm inflmz .m mflmafi NHszmm¢ -127- .APPENDIX TABLE 7. weight of individual pigs in litters 65, 66, 69 (gm.). Pig Days no. ,0 A 1 2 7 14 30 Litter 65 1 -- -- 2020 3440 5240 12939 2 -- -- 1580 3200 5020 12258 3 -- -- 71800 3260 5300 12712 4 -- -- 1880 3380 5380 13620 5 -- -- 1880 2980 4520 11350 6 -- -- 1680 3700 5800 12939 7 -- --- i1620 2600 4340 11350 8 -- -- 880 1680 3600 10442 Mean -- --— .1667 3030 4900 12201 Litter 66 4 1500 1780 2120 3200 5320 11577 5 1560 1860 2320 3300 5460 12939 6 880 1010 1400 2160 4060 9307 7 1480 1760 2320 3420 5200 10669 8 1400 1580 2040 3060 5380 11804 9 1080 1300 1640 2460 4420 10215 10 1260 1500 1840 2620 4700 10669 11 1460 1800 2280 3300 5500 12712 12 1280 1840 2300 3360 5920 9761 Mean 1340 1603 2004 2914 4970 10805 Litter 69 1 1480 1440 1540 2020 -- -- 2 1600 1560 1640 2100 -- -- 3 1520 1720 1880 1780 -- -- 4 1180 1180 1260 1640 -- -- 5 1440 1440 1440 2300 -- -- Mean 1444 1468 1552 1968 -- -- ~228- APPENDIX TABLE 8. weight of individual pigs in litters 70 and 72 (gm.). Pig Dgys no. ,0 1 2 7 14 30 “Litter 70 1 1260 1420 1460 2880 -- .-- 2 1480 1680 1820 3200 -- _-_ 3 1280 1540 1760 3400 -- -- 4 1420 1640 1820 3280 _- -_ 5 1440 1680 '1980 3640 -- f__ .6 1240 1400 1640 2900 .-- _- 7 1400 1580 1740 3440 -- -- 8 1220 1400 1620 3120 -- .-- Mean 1342 1542 1730 3232 -- -- .Litter 72 1 1500 1640 1900 . 3320 -- -- 2 1480 1580 1840 2880 -- --- 3 1240 1340 '1560 2920 -- -- 4 1700 1800 2080 3540 -- -- 5 .1720 1840 2280 3840 -- .-- ‘6 1320 1420 1640 '3060 -_ -_ 7 1200 1300 1140 2000 -- .-- 8 1480 1600 1900 2980 -- -- 9 1500 '1620 1840 3140 -- -_ .10 1620 1720 1960 ,3500 »-- .. Mean 1476 1586 1814 3118 -- .-- -129- APPENDIX TABLE 9. Serum alkaline phosphatase in individual pigs of littersgéS, 66, and 69.g£pM/ml.[hr.). Pig Daxs no. 0 1 2 7 14 30 Litter 65 1 34.8 50.4 32.4 16.8 18.2 6.4 2 100.2 111.0 73.2 24.9 19.4 _- 3 98.2 49.2 49.8 26.1 22.0 9.0 4 50.1 42.0 35.4 18.0 19.4 5.2 5 36.0 76.8 48.6 26.1 18.0 9.0 6 75.3 45.0 41.4 22.8 17.8 6.4 7 54.6 54.6 46.2 26.7 20.4 7.8 8 41.1 145. 18.5 18.9 -— 6.8 Mean 61.3 71.8 51.6 22.5 19.3 7.2 Litter 66 4 72.0 47.6 39.0 24.6 20.4 6.8 5 66.0 42.6 27.0 12.6 16.0 5.4 6 51.0 40.2 36.0 24.6 11.0 6.8 7 49.2 36.6 32.4 15.0 8.0 7.0 8 103.2 52.2 42.6 18.6 9.4 6.2 9 107.4 64.2 58.2 28.2 16.0 7.0 10 63.0 41.4 39.0 25.5 19.4 6.2 11 57.6 42.0 27.0 15.0 22.8 8.2 12 120. 48.6 39.0 22.5 21.8 6.0 13 101.4 40.2 34.2 29.1 24.2 7.0 Mean 79.1 45.6 37.4 21.6 16.9 6.7 Litter 69 1 21.0 32.4 27.6 21.0 -— -- 2 15.6 24.0 22.0 21.0 -- -- 3 20.8 43.8 21.4 12.6 -- -- 4 16.2 24. 25.4 21.6 -- -- 5 20.4 24.0 21.0 28.5 -- -- Mean 18.8 29.8 23.5 20.9 -- -- APPENDIX TABLE 10. -130... Serum alkaline phosphatase in individual litters 70 and 72. figM/ml./hr:), pigs of Pig Days, 1 no. 0 1 2 7 14 30 Litter TO 1 15.4 30.0 20.4 42.4 —- __ 2 14.0 40.2 18.6 46.0 -- -- 3 13.0 42.6 24.0 47.8 _- -- 4 9.0 49.2 20.4 25.0 -- -_ 5 12.2 40.2 21.6 11.2 -- __ 6 30.0 63.6 33.6 13.4 -- __ 7 7.4 33.6 18.6 13.4 -- -- 8 12.2 45.0 22.2 13.4 -- -- Mean 14.2 43.0 22.4 26.6 -- -_ Litter 72 1 43.2 61.2 102.6 12.3 -- __ 2 46.2 60.6 109.2 10.8 -- -- 3 34.5 31.2 84.0 15.3 -- -- 4 31.8 30.4 63.6 10.2 -- -_ 5 27.0 38.4 96.6 11.1 -- -- 6 36.3 29.4 48.0 15.6 -- -- 7 57.6 39.6 55.2 12.6 -- __ 8 31.5 25.2 38.4 8.1 _- -- 9 51.9 57.6 52.2 11.4 —- -— 10 49.2 24.6 54.6 12.3 -- __ Mean 40.9 39.8 70.4 11.9 -- -- -l3l- APPENDIX TABLE ll. Total serum protein in individual pigs of litters 65L_66. and 69. (gmillOO m1.) Pig Days no. 0 l 2 7 14 30 Litter 65 1 2.71 6.50 5.77 5.29 4.98 5.00 2 2.71 6.44 5.79 4.97 4.90 -— 3 2.82 5.74 5.38 5.12 4.58 4.62 4 2.64 6.46 5.92 5.57 4.72 4.85 5 2.97 7.29 6.32 5.49 4.68 4.99 6 2.22 6.24 6.01 5.14 5.10 5.10 7 2.86 7.21 5.93 5.47 4.81 4.76 8 2.84 4.92 4.48 4.53 -- 5.16 Mean 2.72 6.35 5.70 5.19 4.82 4.92 'Litter 66 4 2.42 4.90 4.87 4.63 5.13 4.67 5 2.45 4.99 4.40 5.00 5.28 5.01 6 1.98 4.82 4.56 4.79 4.86 4.78 7 2.59 4.63 4.30 4.86 5.61 4.67 8 2.47 4.58 4.71 4.38 4.56 4.74 9 2.20 4.66 4.79 5.17 5.05 4.80 10 2.37 4.84 4.90 4.74 4.68 4.78 11 2.30 4.64 4.80 4.64 4.66 4.57 12 2.60 5.02 4.64 4.77 4.81 4.93 13 2.47 5.56 5.20 5.24 4.92 5.11 Mean 2.38 4.86 4.72 4.82 4.96 4.81 Litter 69 1 2.58 4.79 4.07 4.07 -- --- 2 3.16 5.27 4.36 3.88 -- -- 3 2.91 3.95 5.02 5.17 -- _- 4 2.35 3.54 4.58 4.16 -- -- 5 2.90 4.16 3.71 3.96 -- -— Mean 2.78 4.34 4.35 4.23 -- -- -l32- Total serum protein in individual pigs of litters J0 and 72. @./100 m1.). .APPENDIX TABLE 12. Daxs 30 ll; Pig no. 53811073 90931182 h5h555i45 87:21 091 Q/QJ11QJ 9/7711 2.hl 5.59 5108 5.07 Mean 2 Litter 7 6 156148517. 1 06527330 uhhhhhhhhu 37 751141489 6h. 97.01675 0000000000 l231456780/m 2.hh h.ll 3.77 h.32 Mean