.v.'...1‘~:'_'~":gi..;-"4' r .. :1 fiEM?mw or was FETAL Mo nzwaom PEG 3. {bitum- Pmfiafions and Scrum Protein Cmnflufiem Thesis €01! ihe PDQ-gr“ of 9h. D. MICHEGAN ESTATE UNIVERSETY fiaifias Gena Wad‘éifii f96§ vac '.'~r&v8 8 l {”121...‘. This is to certify that the thesis entitled Hematology of the Fetal and Newborn Pig I. Cellular POpulations and Serum.Protein Concentra- tions presented by Dallas Gene Waddill has been accepted towards fulfillment of the requirements for (dame c‘i. M622 Major professor % 8/12/60 Date 0.169 r M L [BR A R Y Michigan State Us; ‘1 -12} 'f 'HEMATOLOGY OF THE FETAL AND NEWBORN PIG I. Cellular'POpulations and Serum Protein Concentrations by Dallas Gene Waddill AN ABSTRACT ‘ I Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR or PHILOSOPHY ‘ Department of Animal Husbandry 1960 Approved: ABSTRACT DALLAS GENE WADDILL One hundred twenty-seven fetuses and 80 newborn pigs from 22 female swine were used in this study. The fetuses were studied at approximately 30, 51, 72 and 93 days post- . conception. The newborn pigs were bled immediately after birth. The following measurements were made on the indi- cated number of pigs: Number Measurement of pigs Red cell counts 196 HemOglobin 193 Hematocrit 192 Reticulocytes 196 lean corpuscular volume 189 Mean corpuscular hemoglobin 189 Mean corpuscular hemoglobin concentration 189 Total leucocyte counts 182 White cell differential (Term pigs) 78 Total serum protein 152 Electrophoretic separation of serum proteins 134 Electrophoretic separation of plasma proteins 145 Red cells increased from a mean of 563,000/mm5 of blood at 52 days of age to a mean of 5.5 million/mm5 at birth. The slowest increase in number was from 72 to 93 days. The period of greatest increase came between 95 days ABSTRACT DALLAS GENE WADDILL Mean corpuscular hemoglobin decreased from 51 micromicro- grams at 51 days to 21 micromicrograms at term. All de- creases were significant with the exception of 95 days to term. Mean corpuscular hemoglobin concentration varied within a narrow range. Total white cell numbers increased throughout gesta- tion in the fetal blood. The count increased from 1,455/mm3 of blood at 51 days to 6,269/mm3 at term. A11 increases were significant except the increase between 72 and 95 days. Differential counts showed a relative lymphOpenia and neutrOphilia in the blood of term pigs. NeutrOphils com- prised about 60 percent of the total leucocytes and lympho- cytes 58 percent. Basophils, monocytes and eosinophils were present in very small numbers. Total serum protein concentration in the fetal blood decreased significantly from 51 to 72 days (P¢:.Ol). The concentration increased significantly from 72 to 95 days and from 95 days to term (P‘:.Ol). The concentration was 2.85 grams percent at 51 days and 2.95 grams percent at birth. Electrophoretic separation of serum protein compo- nents established that at 51 days the protein was largely alpha and beta globulins. The relative percent of alpha ABSTRACT DALLAS GENE WADDILL and term. The increases at all ages studied were signifi- cant (P¢:.Ol). Sex differences were not significant. Hemoglobin concentration increased from 6.45 grams percent at 51 days to 8.68 grams percent at 72 days (P<:.Ol). The level decreased to 8.65 grams percent at 95 days then increased to 11.69 grams percent at birth. The blood of males was frequently higher in hemoglobin than females but the difference was not significant. Crossbred pigs had higher hemoglobin levels than purebred pigs at birth (P<.O5). ‘ ‘ Hematocrit percentage increased throughout gestation. It increased from 20.4 percent at 50 days to 57.8 percent at term. A significant increase occurred between 51 and 72 days and between 95 days and term (P‘:.Ol). Sex dif- ferences were not significant at any age studied. This was true also of the breed differences. Reticulocyte percentage was low at all ages studied. The highest mean value was 1.65 percent at 51 days and the lowest .40 percent at 72 days. Mean corpuscular volume and mean corpuscular hemo- globin decreased throughout gestation in the fetal blood. Mean corpuscular volume decreased from 117 cubic microns at 51 days to 67 cubic microns at term. The decrease be- tween all age increments studied was significant (P .Homuon oponmmona n gm .mfimononmonpooao women n Ml.mmpoummmw mafiaaomfla u 9 Adv .Aammav .He no seem an eoaeoaoo an. pamaomaoo one muonpzm Homebom hp oopnomon mosao> qfladnoamuamjmsam mafiannodwtmuw moosaowH on .pnoonom madam H.o amended on do» ouch afloponm Hmpou Ho mosdob Maw mammam I m .adgom u m m Aoomav noaaaa a .m e.oa r.oe m.ma m.o m.m m . Annmav meaeoeeoz a ”a as an as or r.a m . aamawwtwm, » a mm mm Ma w imam m ._ .- flammav heroes on .9 on on r e e.m a sonar . Hdpmafihom . > .m be no ma 0 n m .oa an ArmmHv aoa>eoam a .m am we . an o t m .oa am a no .a on on m ma : m as 00m = no ”a mm mm 0H 0 u m as omm a am .e we om ma 0 n m as end a he a a o o u . m an cos = no ”a no . mm ca 0 u m as one Armamav oaces no s OH no mm o u m as on Adeflmhnm oaaaoaa ocaaanoaw oaaaooam qaaaooamt>MM as one vacuums. . Av m V v afloponm baseman one we» m&q decode Ho Ho ooaonomom unnamed awopohm mammam no swarm Ho gownsnanpman aspen Hence esnom owd .anonkoq one mono“ no mammam ono.adnom ocean mo mosam> connoqon no hfioaanm .m capes 1? utilized methods involving paper electrOphoresis. Knill g3 g1. (1958) showed this was a more rapid procedure re- quiring a smaller amount of serum and giving statistically valid results. Howe (1921) was one of the early pioneers in the study of animal serum. His study was concerned with the newborn calf and the very rapid change which occurred in serum com- position during or shortly after nursing. This change in- volved an increase in a component which at that time was called euglobulin. Newborn calf blood did not contain any of this component until after nursing and colostrum was ingested. This component is now known as gamma globulin. Jameson g5 gl. (1942) found serum of newborn calves contained no gamma globulin and only small amounts of beta globulin before the ingestion of colostrum. During the nursing period the composition of calf serum changed rapidly. Both gamma and beta globulins increased in con- centration initially and then decreased. The concentration of alpha globulin and albumin decreased initially during the nursing period, followed by an increase of albumin. This work was substantiated again by Hansen and Phillips (1947) when it was reported there was an immediate increase in the blood serum gamma globulins following the ingestion of colostrum during the first 24 hours of life. _ Moore g5 g1. (1945b) reported on the blood plasma pro- tein composition of fetal pigs. From four to six components 18 were observed by these workers. Serum albumin has been shown to separate into two fractions (Hewitt, 1956). Barbariak g; 2;. (1958) reported alpha globulin also could be separated into two fractions. This may account for the six components found by Moore gg g1. (1945b). Rutqvist (1958) reported only three components in the blood serum of pig fetuses. The components were albumin, alpha globulin and beta globulin. No gamma globulin was found in this study. , . Pedersen (1944) isolated a new globulin from the blood of calves under two weeks of age. It was reported that serum from fetal calves and sheep contain a large amount of fetuin which makes up the largest part of the total globulin. Rabbit and human fetal blood serum contain very little fetuin. Barboriak g2 g1. (1958) reported on the electr0phoreth: 1 separation of plasma proteins in fetal sheep and goats. They revealed that the fetal plasma lacks gamma globulin and exhibited an additional peak in the area of alpha1 globulin. Thispeak probably corresponded to fetuin. The quantitative concentration of fetal blood plasma proteins seemed to depend largely on the age of the fetus. In the goat fetus, the percent of total proteins and of plasma albumin increased and fetuin decreased with progressing age. The remaining plasma proteins did not seem to be 19 affected. In fetal sheep the percentages of total plasma proteins, plasma albumin and beta globulins increase; levels of fetuin and alphal globulin decreased with age. The per- centage of alpha2 globulins remained unaffected. Meschia (1955) compared the colloidal osmotic pressure of fetal blood in goats and sheep with that of the maternal blood. The colloidal osmotic pressure in the fetal blood plasma at 64 days of age was ca. 100 mm H20; as age ad- vanced the pressure gradually increased to the value of ca. 250 mm H20 near term. Koenig and H0gness (1946) reported fibrinOgen was con- taminated with various globulin components and a small amount of albumin in adult swine plasma. They compared different buffers and found that the resolution of the beta globulin, fibrinogen and gamma globulin in phosphate buffer was superior to veronal buffer; however, the resolution of the alpha1 globulin and albumin was superior in veronal buffer. It was also noticed that in veronal buffers two alpha globulins and two beta globulins were resolved; how- ever, the second beta globulin migrated with the fibrinogen. In phosphate buffer only one alpha globulin and one beta globulin were resolved with a possibility that alphal globulin migrated with the albumin. Deutsch and Goodloe (1945) reported fibrinogen comprised 15.9 percent of the total plasma protein of pigs. They found the characteristic 20 tall peak of fibrinOgen lay in close proximity to that of beta globulin. In some of the plasma samples studied it was impossible to ascertain the amount of particular glo- bulins present, because they were not sufficiently sepa- ' rated. Differences in their mobility in an electric field were too small for good resolution. This was often true in the case of fibrinOgen, in which evidences of this proteinh presence were sometimes seen in the form of a sharp peak superimposed on a mass of other protein. Stenhagen (1958) reported fibrinogen in human blood plasma was an electro- phoretically well-defined protein migrating more slowly than beta globulin but faster than gamma globulin. Appreciable and characteristic specific differences in electrophoretic patterns have been reported for blood plasma from several species under comparable conditions (Moore, 1945a). The pattern obtained depended considerably on the buffer used and it was established that patterns should not be compared unless the conditions of the experi- ments were similar. Perk and Lobl (1959) found a differ- ence in the serum protein between Holstein-Friesian cattle and native Damascene cattle in Israel. The high milk pro- ducing Holstein-Friesian cattle showed a higher total pro- tein and gamma globulin, but a lower albumin/globulin ratio than the hot and dry climate resistant Damascene cattle. In both breeds, alpha and beta globulin patterns were simi- lar. 21 The albumin fraction of the plasma, being responsible for most of the effective osmotic pressure (West and Todd, 1957), constitutes the factor governing the water-retaining capacity of blood plasma. This led Perk and Lobl (1959) to the conclusion, that the prolonged thirst endurance in the Damascene cow may be partly due to higher blood albumin levels as compared with the Holstein. Liquid taken up by drinking and pasture feeding was probably retained in the blood of the Damascene over a longer period. Cartwright g3 gl. (1948) showed a prolonged dietary restriction of protein results in a marked diminution in both the relative and absolute amount of albumin, while a relative increase occurs in globulin, especially alpha globulin. Many investigators have published studies on protein composition of the serum from human infants during fetal life and following birth. Rappoport g5 g1. (1945) found the blood fibrinogen levels to be constant at all ages, and equal to adult values. Total serum protein values incremxd with increasing maturity. Both the albumin and globulin fractions were involved in the increase, but there was a prOportionately greater increase in the globulin fraction. Throughout all of infancy there was a reduction in certain of the globulin fractions, probably gamma globulin. Longsworth g3 gl. (1945) found both the absolute and the relative concentrations of fetal gamma globulin were 22 higher than the maternal values. Orlandini ggygl. (1952) found the mean gamma globulin level in newborn infants slightly higher than in the mothers but the difference was not significant. Orlandini g3 gi. (1955) reported mean cord blood levels of gamma globulin were significantly higher than the level in the mother's blood. The relative gamma globulin concentration after birth showed a definite decrease in the first month of life. Moore g§,gl. (1949) reported serum albumin and gamma globulin increased rapidly during the development of the human fetus, whereas the alpha. and beta globulins remained at'a low level. The high gamma globulin level in newborn infants decreased markedly during' the first month or two of life, whereas the other serum components increased. The relatively high level of fetal gamma globulin in humans was due to the type of placenta. Osborn g§,gl. (1952) reported the human placenta near term was permeable to many antibodies. Pedersen (1944) reported placental transfer of gamma globulin in rodents and man in which the maternal blood was separated from the fetal blood by only one to three layers of cells. In ruminants and swine there are five and six layers of cells respectively separating the maternal and fetal blood (Arey, 1954). Dancis gglgl. (1955) reported evidence for the produc- tion of gamma globulin but not of albumin by the human 25 placenta. Dancis g3 g1. (1957) reported the liver from human fetuses of 5-4 months of age was already capable of synthesizing plasma proteins, exclusive of gamma globulin. They also concluded that at no stage of pregnancy does the placenta synthesize proteins that are electrOphoretically identifiable as albumin or gamma globulin. It was conclu- ded that under normal circumstances the placenta does not contribute significantly to the plasma proteins of the fetus. MATERIALS AND METHODS One hundred twenty-seven fetuses and 80 newborn pigs from 22 female swine were included in this study. Eight— een, nulliparous, Yorkshire gilts, all of approximately the same age and weight were selected from the college station herd. The other four females were Duroc sows which were being used in a Vitamin A study and which had previously given birth to one litter. These sows were being fed an adequate control ration, and were therefore considered normal. The Yorkshire gilts were all self-fed, in confinement (on concrete), ration 1 shown in Table 4a. The Duroc sows were also fed in confinement but were hand- fed ration 2, Table 4b. The Yorkshire gilts were all bred twice during a single estrus to boars of their own breed. Three of the Duroc sows were bred to Yorkshire boars and the other to a Hampshire boar. After conception had been assured by the absence of further estrus, the pregnant individuals were selected at specific periods during gestation, and brought to the Michigan State Veterinary Hospital where caesarean sections were performed to remove the fetuses. The opera- tions were performed at intervals of approximately 50, 51, 72 and 95 days after breeding. The pigs to be studied at term were farrowed naturally and blood samples taken _ 24 - 25 Table 4a. Ration 1 - self-fed to Yerkshire gilts. -: _- Ingredient Percent Corn 6 . Oats 1 . Alfalfa meal 1 . Soybean meal (44%) 1 I-‘OI-‘OOOWI—‘OOQ O OV‘IONUI‘PVTUIOOCD Meat and bone scrap Limestone , Super trace mineral salt Dicalcium phosphate + zinc Vitamin B-supplementa : lb /ton Vitamin A and D mixb . 1b /ton Bl2 supplementC . 1b /ton (a) 2 gm riboflavin, 4 gm pantothenic acid, 9 gm niacin and 10 gm choline per pound of supplement. (b) 4,450,980 I.U. Vitamin A and 1,264,074 I.U. Vitamin D per pound. (0)9 9mg per pound of supplement. Table 4b. Ration 2 - hand-fed to Duroc sows. 4 _:~ Ingredient Percent Oats 54,4 Wheat 50.0 Soybean meal (44%) 8.0 Meat and bone scrap 4.0 Dried corn distillers solubles 2.6 Trace mineral salt 0.5 .Limestone 0.5 Vitamin B supplementa 2.0 lb /ton B12 supplementb 1.0 lb /ton Vitamin D supplementc 4.5 gm /ton Vitamin A supplement (a) 2 gm riboflavin, 4 gm pantothenic acid, 9 gm niacin and 10 gm choline per pound of supplement. (b 9 mg per pound of supplement. go 142,000 I. U. per gm. d 16 micrograms per kilogram bodyweight daily. 26 immediately after birth and before nursing. The number of litters taken at each fetal age varied slightly (30 days - 5 litters; 51 days - 4 litters; 72 days - 4 litters; 95 days - 7 litters). The term litters included three Yorkshire litters and the four crossbred litters from the» Duroc sows. The gilts were anesthetized.with an intravenous in- jection of Surital sodium (thiamylal sodium)‘ into the marginal auricular vein. Anesthesia was maintained throughout the operation with ether or with slow intra.. venous injections of Surital. . The fetuses were removed from the uterus, their posi- tion in the horn recorded and a hemostat placed on the umbilical cord to prevent loss of blood. The pig was weighed and a blood sample taken as quickly as possible. The method of sampling the blood from the fetuses varied with their age, because it was very difficult to obtain blood from the very young fetuses. The data from the 50-day-01d fetuses were limited because of the ex- tremely small amount of blood present, therefore, it was necessary to obtain blood from these pigs before sepa- rating them from the maternal circulation, by incising the umbilical vessels and removing a sample of blood with a heparinized capillary tube. ‘Parke Davis and Co. - Detroit, Mich. 27 Blood was much easier to obtain from 52-day-old fe- tuses. After weighing, the thoracic cavity was opened, exposing the heart, and blood samples were taken directly with pipettes and capillary tubes. The 72 and 95-day-old fetuses, as well as the term pigs were bled from the anterior vena cava according to the method described by Carle and Dewhirst (1942). The pigs were restrained in a dorsal, recumbent position and the blood taken with a 10 cubic centimeter syringe and a 20 or 22 gauge, one inch needle. Each sample was divided into two aliquots; one por- tion was heparinized and the other was allowed to clot. The unheparinized blood was rimmed to hasten clotting and placed in a warm room at 40° C. for a short time to allow complete clot retraction. The samples were placed in an International centrifuge, Model V, size 2, for 50 minutes at 5,000 rpm and the serum removed. If the serum was to be used within one week, it was stored in a walk-in cooler at 4° C. If the length of storage was to be longer than one week, the samples were stored at minus 20° C. The heparinized sample was used for complete blood counts, hemoglobin and hemocrit determinations, while the serum was used for electrOphoretic and total serum pro- tein studies. 28 Red cell counts For red cell counts the heparinized blood was drawn to the 0.5 mark of a "Zero error Hellige tru count“ pi- pette and diluted to the 101 level with .85% saline. The cells were distributed uniformly through the diluted blood by at least a lO-minute rotation on a Bryan-Gary pipette rotor. The counts were made on a Neubauer chamber accord- ing to the method of Ham (1956). Duplicate counts were made and repeated if they varied more than seven percent of the lower count. White cellgcountg For white cell counts the heparinized blood was drawn to the 0.5 mark of a "Zero error Hellige tru count" pipette and diluted to the 11 mark with three percent acetic acid, producing hemolysis of all the nonnucleated erythrocytes. The cells were distributed through the diluted blood in the same method as the red cells. Two sides of the Neubauer chamber were counted and differences were not acceptable if they exceeded 10 percent of the lower count. Nucleated red cells could not be distin- guished from leucocytes in the counting chamber and were counted as white cells in the total white cell count. Swenson g3 g1. (1958) experienced the same difficulty. To correct the white cell counts for the presence of nucleated 29 red cells, 200 cells in each smear were identified as either leucocytes or nucleated erythrocytes. The number of nucleated red cells per 200 cells counted gave a cor- rection factor. White cell differential Differential counts were performed on each of the term pigs. The smears were made as quickly as possible after obtaining the blood from the pig and were stained with Wright's stain according to the procedure of Wintrobe (1956). Phosphate buffer (pH6.8) containing equal vol- umes of M/15 Na2HP04 and M/l5 KH2PO4 was used. The leuco- cytes were differentiated into lymphocytes, monocytes, eosinOphils, baSOphils, non-segmented neutr0phils and polymorphonuclear neutrophils. Two counts of one hundred cells each were made on blood smears from each pig. The method of counting was that described by Ham (1956). The number of normoblasts observed while counting 100 cells was enumerated and the correction for total white cell counts was made by use of the following formula: Corrected white cell count = Total white 100 cell count x 100 + number normoblasts Hematocrit determinations Capillary tubes 75 mm long and 1.2—1.4 mm in diameter were used in hematocrit determinations. Because of the 50 small amount of blood available in the 52-day-old fetuses, one heparinized tube and one plain capillary tube was filled. The samples were spun in an International "hema- crit" centrifuge for five minutes. The hematocrit was determined with an International micro-capillary reader. The serum and plasma from the capillary tubes were used for total serum protein and serum protein electrOphoresis. Hemoglobigdetermination Hemoglobin was determined by the cyanmethemoglobin method of Crosby g§_gl. (1954). The blood was taken with a Sahli pipette (.02 ml) and transferred into five milli- liters of the diluent solution. The diluent used was Drabkin's (1949) solution which contained the following: Nahco3 1.0 gm KCN 50 mg K5Fe(CN)6 200 mg Diluted to 1 liter with double distilled water. The readings were made immediately on a Bausch and Lomb Spectronic 20. Reticulocyte cggggg The procedure described by Ham (1956) was used for the reticulocyte counts. A drop of brilliant cresyl blue solution was spread smoothly on one end of a slide and allowed to dry. The thin film was polished, face downward on smooth paper. A drop of blood was placed over the 51 brilliant cresyl blue, mixed with the edge of a slide, and a smear made. The smear was counterstained with Wright's stain. The counts were made under an oil immersion lens (100K). A cross line reticule was used which divided the field into four quarters. One thousand cells were counted on each slide again using the method of Ham (1956). mov, MCH, MCHCa (a) The mean corpuscular volume was calculated by the following formula: MCV _ Hematocrit (percent) x 10 ‘ " Red cell count (106/mm5) which expressed the average volume of the individual red cells in cubic microns. (b) Mean corpuscular hemoglobin was calculated by the following formula: .. Hemoglobin Kan/100 ml L1 15; MCH _ Red cell count (lOb/mm5) which expressed the average content of hemoglobin of the individual red cell in micromicrograms. (0) Mean corpuscular hemoglobin concentration was calculated by the following formula: Hemoglobin gm/lOO m1) x 1&1 MCHC = . Hematocrit percent) which expressed the average hemoglobin concentration per 100 milliliters of packed red cells in grams. aFormulas from Ham (1956). 32 Serum and plasma_proteins The protein fractions were separated on a Spinco, Model B, paper electrOphoresis system (Spinco Technical Bulletin 6027A) at room temperature. A constant current of three milliamperes per cell was maintained for 16 hours on Spinco number 500-846 paper strips using veronal buffer of pH 8.6 and an ionic strength of 0.075. This buffer was made up of 2.26 grams of di-ethyl barbituric acid and 15.4 grams of sodium diethyl barbiturate in one liter of dis- tilled water. When the buffer was used more than one time the current was reversed within the two cells. After 16 hours, the strips were dried for 30 minutes in a forced draft oven at 110° C. The temperature and time were kept constant because these factors were found to be very critical by Henry 23 al. (1957). These workers reported that for every degree change in temperature be- tween 100-l20° there was a one percent increase in the albumin/globulin ratio. Spinco number 300 pipettes were used for filling the applicator with serum for application to the strips. Ap- proximately .006 milliliter of serum was applied to each strip. One serum sample and one plasma sample were run from each pig. The plasma sample was used to determine the amount of fibrinogen present and served as a check on separation of the other protein fractions. 35 The serum present in the capillary tube from the 52- day-old fetuses was used for both electrOphoresis and total serum protein. In order to get the serum into the pipettes, the capillary tube was broken just above the level of packed cells after making a mark at this point with a small file. The amount of serum necessary for electrophoresis was removed and the tube resealed with a flame. The staining method used was described in the Spinco Technical Bulletin 6027A. The dye used was brom phenol blue (one gram brom phenol blue in one liter of methanol). The rinse consisted of five percent acetic acid. The strips were then blotted and placed in the oven at 110° C. for 15 minutes. After develOpment of basic color, by use of NH4OH, relative intensities of the separated proteins were determined by scanning the stained strip with the Spinco Model RB Analytrol with number five cam. Total serumproteins Because of the small amount of serum available, the biuret determination of serum protein was unsatisfactory. Measurement of light absorption at 280 my, the approximate position of an absorption maximum for proteins has been used in the quantitative estimation of protein concen- trations. The method used in this study was a simple 34 ultraviolet spectrophotometric method, first described by Waddell (1956), which permitted attainment of greater sen- sitivity, accuracy, and specificity because of the use of shorter wave lengths. This method required only small amounts of serum and has been shown to agree closely with results of other methods. The serum was withdrawn from the capillary tubes with a five lambda pipette and diluted to five milliliters (1:1,000) with 0.9 percent NaCl. The readings were made on a Beckman Model DU ultraviolet SpectrOphotometer. Readings were made at wavelengths of 215 mnand at 225 mg. The absorbance at 225 mJLwas subtracted from that at 215 my, This difference multiplied by 144 gave the protein concentration in the solution expressed in micrograms per milliliter. This value was then converted to grams per- cent. RESULTS AND DISCUSSION I. Cellular components Results of analysis of variance are summarized in Table 5. Analyses were run on the effect of age and sex on blood composition. The crossbred litters were not in- cluded in the graphs but are included separately in the tables. Table 6 is a summary of the cellular studies, showing the number of observations, averages of all values for the various ages and ranges of these values. Red cell counts The red cell counts increased steadily from a mean of 560,000/mm3 of blood at 50 days of age to a mean of approx- imately 5.5 million/mm3 at birth. In Table 6 the normal values for the red corpuscles at various ages are presented. The increase in red cell count can be more easily perceived from Figure 1, which shows the increase to be rapid but steady to 72 days followed by a slower rate of increase to 95 days and then a rapid rise from 95 days to birth. The difference in red cell counts between males and females was not significant although there was a tendency for the males to exhibit slightly higher concentrations at the respective age increments with the exceptions of 72 days where they were equal and 50 days where sex was not determined. - 35 - 56 .anmp use mhmv mm pm mama pnonmmwwo hapqmowmflnmflm ADV .mononmmmfld pamowwfiawfimnon I .m.m.z .Amo.uvmv pathogens sapeeoscaemam . .AHo.uvmv peonmmeae sapnwoamaemsm .. .dcooom can» Hmpmosm czam> pmnwm mmpwowdnfl :1: .pman awn» ampwmnw adam> unooom mmmeHUGH =+= Amv §e+ e§+ on.moz we! we+ oe+ oe+ SHmB .m> m0 .Q.m.z .Q.w.z eel eel .Q.m.z .Q.m.z ee+ mm .m> N5 *+ 99+ tel eel §§+ i§+ §§+ NB .m> Hm 000 000 000 .00 omomcz coo §§+ Hm amp om NmNMMu owd ne+ .Q.m.z .Q.m.z .Q.m.z .n.m.z .Q.m.z .Q.m.z S .m> h page qanoam .o.m.2 .>.D.S Iopmamm Ioamm .o.m.m wmddw> nwadomsmnoo mama nomwnmmaoo mmmoflmhmmwfifi Pfimflomaoo HGHSHHmO HO mflmhflwcw mofimfinwb HO hhmafidm om GHQwB 37 . .pmupmmono u N “onwnmxnow u H nv .qmma on» no msonnm pumoqum mum mmmonpnmnmm aw muonsdz va m.¢¢nn.mm Aou.vn.mm om mm.¢alom.oa A®N.vem.ma om m¢.mlmu.¢ Mam.vwo. 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NH (% m8) utquBomaq JO (gmm/suortttm) 'O’H'H 40 The blood of crossbred pigs at birth was found to con- tain higher red cell concentrations than that of purebred pigs. This difference was significant at the five percent level. Hemoglobig_determinatiqng The concentration of hemoglobin exhibited a rapid, highly significant (P Amazomamnoo uses as momqmno .N madman Amhmdv owd . same om on on 4 4 a 1 . omr V w _ .bu .II .0 umes 11 L om .A mos \/ 0d,. fir! O a om - - em mm .0 ma >02 m” om., mm .4mm OOH! _ omav nmm Mean corpuscular hemoglobin Mean corpuscular hemoglobin decreased rapidly from 51 to 95 days followed by a more steady decline until birth (Figure 2). The decreases between 51 and 72 days and be- tween 72 and 95 days were highly significant (P<:.Ol). The decrease from 95 days to birth was not significant. No significant differences were found between males and females or between breeds. Mean corpuscular hemoglobin concentrgtigg The mean corpuscular hemoglobin concentration varied within a narrow range (Figure 2), from a low of 27 percent at 51 days to a high of 52 percent at birth (Table 6). The differences between 51 and 72 days and between 95 days and term were found to be significant. The changes can be more easily perceived from Figure 2. No significant differences were found between sexes or breeds. Total white cells The number of white cells present increased uniformly from 51 to 72 days followed by a slower rate of increase to 95 days and a very rapid increase from 95 days to term (Figure 5). The number rose from a low of 1,455/mm5 of blood to a high of 6,269/mm5 (Table 7). The increase from 51 to 72 days was significant at the five percent level. The change from 72 to 95 days was .mufia prmm meandd Goapmadmom mphooosma as mmquno .m cadwwm Amhmwv cm¢ Ema om on . om - . . 08. a W l 08. m . ooofi (gum/Spuesnoqq) qunoo Itao aqrqy L 80.x. 45 Table 7. Total leucocyte counts at different ages. t Age (days) No. of animals Sex W.B.C./mm3 51 ' 11 F 1,112(251)a 12 M 1.726(524) 72 18 F 2,204(419) 25 M 2.256(295) 95 26 F 2.785(300) 15 M 2.759(159) Termb 15 F 5.175(520) 18 M 7.182(699) Termc 24 F 5.924(277) 2o . M 4.525(514) (a; Standard error of the mean. (b Purebred Yorkshires. (c) Crossbred pigs. not significant but the increase from 95 days to term was highly significant (Table 5). _ Significant sex differences were found only in the term purebred litters where the white cell concentration of males was significantly higher (P<:.01) than that of fe- males. 46 The purebred pigs had significantly higher total white cell concentration than the crossbred pigs. This difference was significant at the one percent level. Differential count The blood of pigs at birth was found to exhibit a distinct lymphopenia and neutrOphilia. NeutrOphils were by far the most numerous cell seen in the counts, com- prising about 60 percent of the total leucocytes (Table 8). Lymphocytes represented approximately 58 percent and the bas0phils, monocytes and eosinOphils combined repre- sented only two percent. ‘ No statistical differences were found between sex or breed in percentages of lymphocytes and neutrophils. The other white cells were present in such highly variable concentrations the differences were judged nonsignificant. Red blood cell concentration, hemoglobin and the volume of packed red blood cells followed the same pattern of development during fetal growth of the pig. Increase of these blood components was slow during the first third of gestation followed by a more rapid rate of increase during the middle third (58 to 76 days). The most rapid increase occurred during the last third of gestation or more specifically the last three weeks (95 days to term) during which time the number of red blood cells increased #7 .qme map mo Moshe unmcnmpm Adv 00. mo. mo. mm. Amm.mvmm.ae Aeo.mvwm a ma ma. no. om. no.a Ama.mvmw.oo Ama.me.mm m ma commemono mm. mo. OH.H mH.H Amm.mvm©.am Amm.mVum 2 ON ma. mm. mm. mm. Ame.mvmm.mm mfiom.mvmm m em onflnmxnow npnflm edema nonpoez maflnm maflnm mmpho pounce maflnmoHpsz mcpho qufimom Iommm. some: Iwmmlnoz Hmpom toQQShHI massage Aaq HeateeMemaae Hate means New Ho .02 ewe .mmflm anonemw Ho asuwoamn flame mean; .w manna 48 fromapproximately 4,000,000/mm5 of blood to approximately 5,600,000/mm5, an increase of 40 percent. Hemoglobin dur- ing this same period increased from 8.65 grams percent at 95 days to 11.69 grams percent at birth, an increase of 55 percent. The volume of packed red cells increased 20 percent from 51.5 percent at 95 days to 57.8 percent at birth. , Mean corpuscular volume which is a measure of the size of red blood cells decreased rapidly from 51 to 72 days followed by a more steady decline until birth. Mean corpuscular hemoglobin decreased rapidly from 51 to 95‘ days followed by a more steady decline until birth. Mean corpuscular hemoglobin concentration, which is a measure of the percent of each red cell occupied by hemoglobin, varied within a rather narrow range, being slightly higher at birth than at 51 days. Eventhough the size of red blood cells was decreas- ing rapidly, this change did not keep pace with the inp creasing number of red cells. The volume of packed red cells, representing the total mass of red corpuscles (con- trolled by number as well as size), increased quite rapidly. During the first one-half of gestation the placenta is growing faster than the fetus as shown by Warwick (1928) in pigs, Elliott gt gl. (1954) in goats and by 49 Barcroft £§.éi- (1959) in sheep. The growth of the pig fetus (expressed in grams body weight) was slow between 50 and 51 days (Figure #), a little faster between 51 and 72 days followed by a rapid increase in body weight from 72 days to term. Thus it seems that for more than half the gestation period the fetus is gravimetrically the less important intra-uterine structure. Warwick (1928) found the greatest weight increase in pig fetuses occurred in the last 20 days of gestation. Erythrocyte numbers, hematocrit values and hemoglobin concentrations keep pace with the fast developing placenta and slow growing fetus up to 72 days when placental de- velopment is near completion. At this time, fetal growth becomes rapid and the blood components do not keep pace, suggesting the growth of fetal body structures becomes the predominant intra—uterine phenomenon. The proportion of blood present in the fetus in comparison to that in the placenta has been found to increase as the fetus grows older (Barcroft and Kennedy, 1959). Blood can be driven through the placenta at a faster rate as pregnancy proceeds because of higher fetal blood pressure. Subjective ex- aminations suggest that more blood in prOportion to weight was present in the pig fetuses at 72 days than at the 51 day period. The blood is flowing more rapidly through the placenta, therefore, the 72-day—old fetus does not require .mwm paw pnmflos Hmumm amosump maAmQOHpmHmm .3 chewam 50 Amends owe. om ou . on x 0m SHOE d i d A OON 00¢ O O \O ( m3) °4M oom OOH l coma 51 that the red cells and hemoglobin maintain the same con- centration in the blood relative to body weight as do 5l-day-old fetuses which have a much smaller prOportion of blood that is moving more slowly. The rapid increase in red cells, hemoglobin and hema— tocrit during the period of 95 days to term is necessary because during this period the placenta is beginning to degenerate (Warwick, 1928 and Elliott gt al., 1954) and the fetal size is increasing rapidly (Figure 4). As the fetus exceeds more and more the weight of the placenta, the amount of oxygen and other nutrients necessary for life of the fetus and the need for excretion of larger amounts of waste becomes more of a problem. Therefore, during the last stage of gestation (95 days to term) the erythrocyte number and hemoglobin level must increase rapidly in order to counteract the relatively anoxemic conditions of late fetal life. The number of erythroblasts was very high at 50 days, making up the majority of red blood cells present at this age. The number decreased rapidly as the fetus developed. Reticulocytes, on the other hand, were present in small percentages at all ages studied. Some of the earlier work- ers (Wintrobe and Shumacker, 1956 and Jones 25 al., 1956) reported very high percentages of reticulocytes at 52 days of age. The ages of these fetuses were estimated from 52 crown—rump lengths, therefore, the estimated age might vary from the real age by a few days. Also in these studies the percentage of both erythroblasts and reticulocytes were re- ported. The total of these two exceeded 100 percent in some cases. It is apparent the reticulocyte values reported were expressed as a percentage of the non-nucleated erythro- cytes. In the present study the percentages of reticulo- cytes were determined by counting 1,000 red cells includ- ing all stages. Thus the large discrepancy between the data presented here and earlier studies was probably due to error in estimating age or differences in the method of expressing the count. In the present study, only those cells in which the baSOphilic material in the cell was precipitated, appearing as a blue network or reticulum, were counted as reticulocytes. The total white cell count followed the same pattern as the red cells and hemoglobin, increasing gradually to 95 days followed by a very rapid increase from 95 days to term. The 95 day count of 2,768 mm5 increased to 6,269 at term, an increase of 126 percent. This large increase was probably due largely to stress. ‘The fetus had become quite large by this time and the placental membranes had become smaller in comparison, which undoubtedly resulted -in a stress upon the fetus and its hemat0poietic organs, which were quite well develOped by this age. The increase 55 in total white cells appears due largely to an increase in neutrophils whichhave been shown to increase during stress (Palmer, 1917b; Luke, 1955b; Gardiner gt al., 1955 and Winqvist, 1954). Kindred and Corey (1950) reported the same increase in white cells of rats during the last few days of fetal life. The term purebred pigs were farrowed during May, June and July and had significantly higher leucocyte concentration.than the term crossbred pigs which were farrowed in October and November. The differential counts on the newborn pigs showed a definite lymphopenia and neutrophilia. Luke (1955c) re- ported the same pattern after injection of adrenocorti- cotrOphic hormone and adrenal cortical extract in pigs. The high neutrOphil count at birth was perhaps due to stress reacting through the adrenal gland. _ II. Serum and plasma proteins The values together with standard errors of the means for total serum proteins are presented in Table 9. In Table 10 the relative percentages of the electrophoretic- ally separated components of serum and plasma are pre- sented. Total serum protein change with age as well as the change in the separated components can be more easily perceived from Figure 5. Total serum proteins at 51 days were present at the rate of 2.85 gm per 100 ml of serum, composed mainly of {Table 9. Total serum protein Age No. of animals Sex Total serum protein gm/lOO ml 51 days 8 F 2.86(.l4)a 9 M 2.80(.O9) 72 days 19 F 2.19(.O6) 21 M 2.50(.06) 95 days 11 F 2.48(.O6) 9 M 2.57(.05) Termb 14 F 2.95(.lO) 19 M 2.92(.05) Termc 25 F 2.99(.oe) 19 M 2.95(.05) (a; Standard error of mean. b Purebred Yorkshires. (c) Crossbred pigs. alpha and beta globulins with somewhat smaller amounts of the other components. Levels of serum gamma globulin varying between 8 and 11 percent were found in the blood of fetal pigs at all ages studied, as well as in the pigs at birth. No gamma globulin was found in the bloodof fetal pigs by Rutqvist (1958) or by Moore 32 al. (1945b). Serum protein distribution 'Table lOa. albumin -globu1in Distribution of serum roteins (%2 B-globulin ‘r-globulin of Sex animals No. A e (dive) CHE 51 AA 'Md’ FHA VV can NW AA 00 HH VV L\d" O‘v-l 19 2O 72 27 17 95 AA (003 HO NO HN AA O\=1’ 00 UV MO 0 O HO Fir-l l5 19 Term (a) Standard error of mean. 55 Plasma protein distribution Table 10b. DistributiOn of plasmapproteins (%) albumin B-globul in d- -gTobul in ‘f—globulin and fibri- of Sex animals No. Age (days) ogen 5:33 E 22.4 21.5 F105 51 12 12 21 72 12 95 1.23 1.1 E 19.8 20.8 3 15.6(1.2) 2.0 0.9 15.4(O.8) E 18.6 18.4 F M 15 19 25 2O Termb M NN . HH vv d’m C‘O‘ r-O 1 F M Termc Standard error of mean. ire pigs Purebred Yorksh Crossbred pigs. E a b E (c 56 57 ( 1m Cor/m3) UIGQOJd muses Ieaom .mdfloponm adnmm paw mwm amped decapop QHAmQOHumHmm .m onsmfim AmKMUv mmd sums mm on om :. ew;} :1 1 EL .t. {-pix QfiHSDOHWI Y 11 3! like... 3.3.11! Liliflhczrst: X11111}... . L OH. .d 9 . J 1 as aspoamum \‘l/ .. 0mm GHEQH< . «Attriii... it)!!! E!!! - I l I w 0 I. - 0mm. 1. 1,. .. B 1 .L S e _I L 0.5“ m .d J caasnoawttulr w. . l on m. u dampond . Hepe . om 58 IRook gt gl. (1951) and Rutqvist (1958) found no gamma glo- ‘bulin present in the serum of pigs at birth, however, Foster gt gt. (1951) reported a small percentage and NOrdbring and Olsson (1957) obtained an average of 7.9 percent. Miller gt at. (1960) found a similar percentage in newborn pigs. The difference between the present study and other work reported may have been due to differences in methods of separation, protein denaturation at point of application, or to differences in distinguishing between the gamma globulin and slow moving beta globulin. The point of separation was quite arbitrary and required con- siderable personal judgment. As shown in Figure 5, the amount of total serum protein decreased rapidly from 51 to 72 days. This decrease was highly significant (P<:.Ol) and was followed by a highly significant increase to 95 days. The increase from 95 days to term was also highly significant. The significant drOp in serum protein from 51 to 72 days was likely due to the change in the amount of blood in the fetal circulation. At 51 days the fetus contained very little blood when com- pared to the amount present at 72 days of age, therefore, even though the total amount of protein present in the serum may have increased the concentration per 100 ml of blood decreased because of dilution with greater amounts of blood. 59 As shown in Figure 5, during the period from 51 days to 72 days, alpha globulin increased whereas, beta globu- lin decreased quite rapidly. Pierce (1955) reported that fetuin possibly has a substituting function for albumin in newborn animals. If fetuin corresponds to thesz-globulin as suggested by Nordbring and Olsson (1957) and Rook gt gt. (1951), then the alpha globulin during fetal life may re- place albumin as the primary factor maintaining osmotic pressure of the blood. During this period when alpha glo- bulin increased rapidly the blood volume increased rapidly also. Meschia (1955) found that colloidal osmotic pres- sure increased greatly during the middle of the second third of the gestation period. Thus it is reasonable that there is a direct relationship between the increase in blood volume of the swine fetus during the second third of gestation and the colloidal osmotic pressure, resulting from the increasing level of alpha globulin present in the blood during this same period. Albumin and gamma globulin remained quite constant during this period. From 72 to 95 days the fetus made rapid growth (Figure 4), exceeding the weight of the placenta (Warwick, 1928 and Elliott gt gl., 1954). Alpha globulin production was rapid during this period in the fetal pig becoming the dominant serum protein component at 95 days as shown, in Table 10. Total serum protein increased during this 60 period due entirely to the increase in alpha globulin. Albumin, which is produced by the fetal liver, decreased during this period. Total serum protein increased significantly from 95 days to term reaching its highest level of 2.95 grams per 100 m1 at birth. Relative levels of alpha and beta globu- lins decreased during this time whereas the relative amount of albumin increased. This increase in albumin accounts for the slight decrease in alpha globulin percentage. Alpha globulin production did not change much during this time although the relative serum concentration did. Beta globulin production did, however, seem to decrease slightly. The relative amount of gamma globulin present through- out fetal life is either produced by the fetus itself, the fetal placenta, or passes from the maternal circulation into the fetus. The anatomical arrangement of the pla- cental membranes of the pig prevents the transfer of large amounts of gamma globulin. The pig has the epithelio- chorial type placenta in which there are several barriers to the transfer of substances across the placental mem- branes. The human fetus has been found to contain a higher percentage of gamma globulin than the maternal blood (Longsworth gt gt.,1945 and Orlandini gt|gl,,l952) and the placenta has been found to be highly permeable to maternal antibody (Osborn gt gt., 1952) especially in the latter 61 ;part of pregnancy. Humans have the hemo-endothelial pla- centa (Arey, 1954) in which there is little barrier be- tween maternal and fetal bloods. This accounts for the relatively high level of circulating gamma globulin and albumin. Gamma globulin and albumin have been found to be the fastest increasing protein components in the blood of the developing human fetus (Moore gt gt., 1949). Paper electrOphoresis of plasma was found to be com- pletely unsatisfactory as a measure of the percentage of fibrinogen or of the separated protein components present. The differences obtained between serum and plasma can be readily seen by comparing the values in Table 10. The re- sults varied quite widely between serum and plasma and the variation was due largely to the fibrinogen present and perhaps in part to the fact that the small amount of plasma present in the capillary tubes could not be mixed before applying to the strips. The same general pattern can be seen in comparing the plasma and serum patterns. Fibrinogen seemed to increase greatly during the last three weeks of gestation as shown in Table 10. Fibrinogen migrated very slowly and was found in close proximity to the gamma globulin. It was impossible to distinguish be- tween these two protein components. Alpha globulin was not separated into two fractions in this study. Miller gt gt. (1960) separated the alpha 62 .fraction.from pig blood into two distinct fractions. This ;phenomenon had been observed by Nordbring and Olsson (1957) .Rook.gt gt. (1951) referred to the alpha1 fraction as component ”X". Pedersen (1944) found a high relative con- centration of a component in the blood of young calves which.was called fetuin. This component has been found in.1arge percentages in the fetal blood of goats and sheep (Barboriak gt|gt., 1958) with the same electrOphoretic mobility as the alphal globulins. Nordbring and Olsson (1957) and Rook gt gt. (1951) suggested that the alpha1 fraction may correspond to the fetuin component of Pedersen (1944) and Barboriak gt gt. (1958). Any of this compound evident in the present study was included in the alpha globulin fraction. Figure 6 shows the electro- phoretically separated pattern at the various ages stu- died. No significant differences were found in total serum protein between males and females. The albumin was sig- nificantly higher at 51 days and at term in the male pigs than in the female pigs. No significant differences were found between purebred and crossbred pigs in either total serum protein or the separated components. ... J klullj 1.1“. ; .. £111...) 1.. 65 B M 8 d. A 51-day fetus 72-day fetus d~ B 95-day fetus Newborn pig Figure 6. ElectrOphoretic patterns of serum proteins from different age fetuses. Total serum proteins at 51 days constituted a con- centration of 2.85 grams per 100 m1 of serum composed mainly of alpha and beta globulins with somewhat smaller- amounts of the other components, as shown above the rela- tive amount of gamma globulin changed very little. The most pronounced change was thetf-globulin which increased from 55.5 percent at 51 days to 48.5 percent in the new- born pigs. The percent of B-globulin decreased through- out fetal life. The relative amount of albumin decreased slightly during the gestation period. The total serum protein decreased from 51 to 72 days then increased to term at which time the level was 2.95 grams per 100 m1. SUMMARY One hundred twenty-seven fetuses and 80 newborn pigs from 22 female swine were used in this study. The fetuses “were studied at approximately 50, 51, 72 and 95 days post- conception. The newborn pigs were bled immediately after birth. The following measurements were made on the indi- cated number of pigs: ‘ Number Measuremegt ~ of pigs Red cell counts 196 Hemoglobin ‘ 195 Hematocrit 192 Reticulocytes ~ 196 Mean corpuscular volume 189 Mean corpuscular hemoglobin 189 Mean corpuscular hemoglobin concentration 189 Total leucocyte counts 182 White cell differential (Term pigs) 78 Total serum protein 152 Electrophoretic separation of serum proteins 154 Electrophoretic separation of plasma proteins 145 Red cells increased from a mean of 56O,OOO/mm5 of blood at 52 days of age to a mean of 5.5 million/mm5 at birth. The slowest increase in number was from 72 to 95 days. The period of greatest increase came between 95 days _ 64 - 65 and term. The increases at all ages studied were signifi- cant (P<:,Ol). Sex differences were not significant. Hemoglobin concentration increased from 6.45 grams percent at 51 days to 8.68 grams percent at 72 days (P‘(.Ol). The level decreased to 8.65 grams percent at 95 days then increased to 11.69 grams percent at birth. The blood of males was frequently higher in hemoglobin than females but the difference was not significant. Crossbred pigs had higher hemOglobin levels than purebred pigs at birth (P<:.05). ‘ Hematocrit percentage increased throughout gestation. It increased from 20.4 percent at 50 days to 57.8 percent at term. A significant increase occurred between 51 and 72 days and between 95 days and term (P<:.Ol). Sex dif- ferences were not significant at any age studied. This was true also of the breed differences. Reticulocyte percentage was low at all ages studied. The highest mean value was 1.65 percent at 51 days and the lowest .40 percent at 72 days. Mean corpuscular volume and mean corpuscular hemo- globin decreased throughout gestation in the fetal blood. Mean corpuscular volume decreased from 117 cubic microns at 51 days to 67 cubic microns at term. The decrease be- tween all age increments studied was significant (P<:.Ol). 66 Mean corpuscular hemoglobin decreased from 51 micromicro- grams at 51 days to 21 micromicrograms at term. All de- creases were significant with the exception of 95 days to term. Mean corpuscular hemoglobin concentration varied within a narrow range. Total white cell numbers increased throughout gesta- tion in the fetal blood. The count increased from 1,455/mm5 of blood at 51 days to 6,269/mm5 at term. All increases were significant except the increase between 72 and 95 days. Differential counts showed a relative lymphopenia and neutrOphilia in the blood of term pigs. Neutrophils com- prised about 60 percent of the total leucocytes and lympho- cytes 58 percent. Basophils, monocytes and eosinOphils were present in very small numbers. Total serum protein concentration in the fetal blood decreased significantly from 51 to 72 days (P<:.Ol). The concentration increased significantly from 72 to 95 days and from 95 days to term (P<<;01). The concentration was 2.85 grams percent at 51 days and 2.95 grams percent at birth. Electrophoretic separation of serum protein compo- nents established that at 51 days the protein was largely alpha and beta globulins. The relative percent of alpha globulin increased to 95 days when it was approximately 67 50 percent of the total protein. Beta globulin decreased in pr0portion to this increase. Albumin percentage reached a low of 17 percent at 95 days then increased to 21 percent at birth. Gamma globulin varied between 8 and 11 percent during gestation. Fibrinogen increase was greatest during the last three weeks of pregnancy. LITERATURE CITED Arey, L. B. 1954. Developmental anatomy. 6th ed. p. 147. Saunders, Philadelphia and London. - Barboriak, J. J., G. Meschia, D. H. Barron and G. R. Cow- gill. 1958. Blood plasma proteins in fetal goats and sheep. Proc. Soc. Exp. Biol. and Med. 98(5): 655. Barcroft, J. and P. Rothschild. 1952. The volume of blood in the uterus during pregnancy. J. Physiol. 76:447. Barcroft, J. and J. A. Kennedy. 1959. The distribution of blood between the fetus and the placenta in sheep. J. Physiol. 95:175. . Barcroft, J., J. A. Kennedy and M. F. Mason. 1959. The blood volume and kindred prOperties in pregnant sheep. J. Physiol. 95:175. Brinkman, R., A. Wildschut and A. Wittermans. 1954. On the occurrence of two kinds ofthaemoglobin in normal human blood. J. Physiol. 80:577. Brinkman, R. and J. H. P. Jonxis. 1955. The occurence of several kinds of haemoglobin in human blood. J. Physiol. 85:117. Byers, J. H., I. R. Jones and J. R. Haag. 1952. Blood hemoglobin values of dairy cattle. J. Dairy Sci. 55:661. Calhoun, M. L. and E. M. Smith. 1958. Hematology and hema- t0poietic organs; Diseases of swine. Chap. 2. .Iowa State College Press. Carle, B. N. and W. H. Dewhirst, Jr. 1942. A method for bleeding swine. J. Amer. Vet. Med. Assn. 101:495. Cartwright, G. E., E. L. Smith, D. M. Brown and M. M. Wintrobe. 1948. ElectrOphoretic analysis of sera of normal and hypoproteinemic swine. J. Biol. Chem. 176 : 585 0 Cooper, G. R. 1945. ElectrOphoretic analysis of mixtures of protein. J. Biol. Chem. 158:727. - 68 - 69 - Craft, W. A. and L. H. Moe. 1952. Statistical observa- tions involving weight, hemOglobin and the pr0portion of white blood cells in pigs. J. Amer. Vet. Med. Assn. 81:405. Craig, R. A. 1950. Anemia in young pigs. J. Amer. Vet. Med. Assn. 76:558. - Crosby, W. H., J. I. Munn and F. W. Furth. 1954. Stan- dardizing a method for clinical hemoglobinometry. U.S. Armed Forces Med. J. 5:695. Dancis, J., N. Brauerman and B. Katchen. 1955. The syn- thesis of serum proteins by human placenta. Amer. J. Dis. Child. 90:558. . Dancis, J., N. Brauerman and J. Lind. 1957. Serum pro- tein in human embryos. J. Clin. Invest. 56:598. Deutsch, H. F. and M. B. Goodloe. 1945. An electro- phoretic survey of various animal plasmas. J. Biol. Chem. 161:1. Doyle, L.P., F. P. Mathews and R. A. Whiting. 1928. Anemia in young pigs. J. Amer. Vet. Med. Assn. 72:491. . Doyle, L. P. 1952. Anemia in young pigs. J. Amer. Vet. Med. Assn. 80:556. ' . Drabkin, D. 1949. The standardization of hemoglobin measurement. Am. Jour. Med. Sci. 217:710. Draper, H. H. and L. W. McElroy. 1949. A study of nu- tritional anemia in suckling pigs. Sci. Agric. 29:570. ’ Elliott, R. H., F. G. Hall and A. St. Huggett. 1954. The blood volume and oxygen capacity of the fetal blood in the goat. J. Physiol. 82:160. Foster, J. F., R. W. Friedell, D. Catron and M. R. Dieckmann. 1951. ElectrOphoretic studies on swine. III. Composition of baby pig plasma and sow's whey during lactation. Arch. Biochem. Biophys. 51:104. Fraseg, S. C. 1958. A study of the blood of pigs. Vet . 4:5. 7O Gardiner, M. R., W. L. Sippel and W. C. MacCormick. 1955. The blood picture in newborn pigs. Amer. J. Vet. Res. 14:68. . Glynn, J. H. 1948. The story of blood. A. A. Wyn, Inc., ' New York. Greatorex, J. C. 1957a. Observations on the haemotology of calves and various breeds of adult dairy cattle. Brit. Vet. J. 115:469. Greatorex, J. C. 1957b. Observations on the haemotology of calves and various breeds of adult dairy cattle. Brit. Vet. J. 115:29. Ham, T. A. 1956. A syllabus or laboratory examinations in clinical diagnosis. Harvard Press. Hansen, R. G. and P. H. Phillips. 1947. Studies on pro- teins from bovine colostrum. l. ElectrOphoretic studies on the blood serum proteins of colostrum free calves and of calves fed colostrum at various ages. J. Biol. Chem. 171:225. Henry, R. J., O. J. Golub and C. Sobel. 1957. Some of the variables involved in the fractionation of serum proteins by paper electrOphoresis. Clin. Chem. 5:49. Hewitt, L. F. 1956. Separation of serum albumin into two fractions. Biochem. J. 50:22. Holman, H. H. 1955. The blood picture of the cow. Brit. Vet. J. 111:440. Howe, P. E. 1921. An effect on the ingestion of colostrum upon the composition of the blood of new-born calves. J. Biol. Chem. 49:115. Jameson, E., C. Alvarez-Tostado and H. H. Sortor. 1942. Electrophoretic studies on new born calf serum. Proc. Soc. Exp. Biol. Med. 51:165. Jones, J. M., M. E. Shipp and T. A. Gonder, Jr. 1956. Changes occurring in the blood picture during fetal life. Proc. Soc. Exp. Biol. Med. 54:875. 71 Kernkamp, H. C. H. 1952. The blood picture of pigs kept under conditions favorable to the production and to the prevention of so-called "anemia of suckling pigs." Univ. Minn. Agr. Exp. Sta. Tech. Bull. 86. IKindred, J. E. and E. L. Corey. 1950. Studies on the blood of the fetal rat. I. Total counts of the red and white corpuscles. Anat. Rec. 47:215. Knill, L. M., T. R. Podleski and W. A. Childs. 1958. Normal values of swine serum proteins. Proc. Soc. Exp. Biol. Med. 97:224. Koenig, V. L. and K. R. H0gness. 1946. ElectrOphoretic analysis of swine plasma and serum. Arch. Biochem. 9:119. Kohler, H. 1956. Knochenmark und Blutbild des Ferkels. 1.” Das gesunde Ferkel. 2. Dgs Ferkel mit spontaner Anamie. Zentralbl. F. Veterinar. Med. 5:559. Longsworth, L. G., R. M. Curtis and R. H. Pembroke, Jr. 1945. The electrOphoretic analysis of maternal and fetal plasmas and sera. J. Clin. Invest. 24:46. Luke, D. 1955a. The differential count in the normal pig. J. Comp. Pathol. Therap. 65:546. Luke, D. 1955b. The reaction of the white blood cells at parturition in the sow. Brit. Vet. J. 109:241. Luke, D. 19550. The effect of adrenocorticotrOphic hormone and adrenal cortical extract on the differ- ential white blood count in the pig. Brit. Vet. J. 109:454. Manresa, M. and S. C. Orig. 1941. HematolOgical studies on cattle: IV. Variations of hemoglobin and white blood cells in the Philippine native breed of cattle. Phil. Agric. 50:575. Matthews, J. and D. A. Buthala. 1956. Effect of repeated freezing and thawing on the electrOphoretic pattern of swine serum. Amer. J. Vet. Res. 17:485. McCay, C. M. 1951. The hemoglobin and total phosphorus in the blood of cows and bulls. J. Dairy Sci. 4:575. 72 Meschia, G. 1955. Colloidal osmotic pressures of fetal and maternal plasmas of sheep and goats. Amer. J. Physiol. 181:1. Miller, E. R., D. E. Ullrey, I. Ackerman, D. A. Schmidt, J. A. Hoefer and R. W. Luecke. 1960. Swine hemato- logy from birth to maturity. (Unpublished research). Mitchell, H. S. 1952. Factors influencing anemia develop- ment in young rats. J. Biol. Chem. 97:xv-xvi. Moore, D. H. 1945a. Species differences in serum protein patterns. J. Biol. Chem. 161: 21. Moore, D. H., S. C. Shenn and C. 3. Alexander. 1945b. The plasma of develOping chick and pig embryos. Proc. .Soc. Exp. Biol. Med. 58:507. Moore, D. H., R. M. Dupan and C. L. Buxton. 1949. An I electrOphoretic study of maternal, fetal and infant sera. Amer. J. Obstet. Gynecol. 57: 512. Nicholas, J. S. and E. B. Bosworth. 1927. The determina- tion of the amount of hemoglobin present in rat fetuses during development. Amer. J. Physiol. 85:499. Nordbring, F. and B. Olsson. 1957. Electr0phoretic and immunological studies on sera of young pigs. 1. Influence of ingestion of colostrum on protein pat— tern and antibody titre in sera from suckling pigs and the changes throughout lactation. Acta. Soc. Med. Upsalien 62:195. Orlandini, T. 0., A. Sass-Kortsak and J. H. Ebbs. 1952. Serum gamma globulin levels in infancy. Amer. J. Dis. Child. 84: 652. Serum gamma globulin levels in normal infants. Pediatrics 16:575. Osborn, J. J., J. Dancis and B. V. Rosenberg. 1952. Studies of the immunology of the newborn infant. III. Permeability of the placenta to maternal antibody during fetal life. Pediatrics 10:450. Palmer, C. C. 1917a. Morphology of normal pigs blood. J. Agr. Res. 9:151. 75 Palmer, C. C. 1917b. Effects of muscular exercise and heat on the blood and body temperature of normal pigs. J. Agr. Res. 9:167. .PederSen, K. O. 1944. Fetuin, a new globulin isolated from serum. Nature 154:575. Pensinger, R. R., E. F. Reber, E. Ersoy and H. W. Norton. 1959. A pooled human serum as a standard and the effects of freezing and thawing on the electro- phoretic pattern of baby pig serum. Amer. J. Vet. Res. 20:180. Perk, K. and K. Lobl. 1959. vA comparative study on the sera proteins and lipids in two breeds of cattle. Brit. Vet. J. 115:411. Pierce, A. E. 1955. ElectrOphoretic and immunological studies on sera from calves from birth to weaning. J. Hyg. 55: 247. Rappoport, M., M. I. Rubin and D. Chaffee. 1945. Frac- tionation of the serum and plasma proteins by salt precipitation in infants and children. 1. The changes with maturity and age. 2. The changes in glomerulonephritis. 5. The changes in nephrosis. J. Clin. Invest. 22:487. Rook, J. A. F., J. Moustgaard and P. E. Jakobsen. 1951. An electrOphoretic study of the changes in the serum proteins of the pig from birth to maturity. Copen- hagen K. Veterinaer-og Landbo-Hejskole 1951:81. Rusoff, L. L. and P. C. Piercy. 1946. Blood studies of Louisiana dairy cows. II. Calcium, inorganic phosphorus, hemoglobin value, erythrocyte count, leucocyte count and differential leucocyte percent- ages. J. Dairy Sci. 29:851. Rutqvist, L. 1958. ElectrOphoretic patterns of blood serum from pig fetuses and young pigs. Amer. J. Vet. Res. 19:25. Scarborough, R. A. 1951. The blood picture of normal laboratory animals. Yale J. Biol. 5:547. Senftleben, O. 1919. Das Blutbild des gesunden Schweines. Monatsh. F. Prakt. Tierheilk. 50:289. 74 Sinclair, R. D. 1955. The influence of radiant energy on hemat0poietic processes in the pig. Sci. Agric. 15:757. Stenhagen, E. 1958. ElectrOphoresis of human blood plasma. ElectrOphoretic prOperties of fibrinogen. Biochem. J. 52:714. Svensson, H. 1941. Fractionation of serum with ammonium sulfate and water dialysis, studied by electrOphore- sis. J. Biol. Chem. 159:805. Swenson, M. J., D. D. Goetsch and G. K. L. Underbjerg. 1955. The effect of the sow's ration on the hemato- lOgy of the newborn pig. Proc. Amer. Vet. Med. Assn. p. 159. Swenson, M. J., G. K. L. Underbjerg, D. D. Goetsch and C. E. Aubel. 1958. Blood values and growth of newborn pigs following subcutaneous implantation of bacitracin pellets. Amer. J. Vet. Res. 19:554. Venn, J. A. J. 1944. Variations in the leucocyte count of the pig during the first twelve weeks of life. J. Comp. Pathol. Therap. 54:172. Von Deseo, D. 1929. Beitrag zur Kenntnis der fetalen Blutentwicklung beim Rinde. Arch. F. d. ges. Physiol. 221:526. Waddell, W. J. 1956. A simple ultraviolet spectro- photometric method for the determination of protein. J. Lab. Clin. Med. 48:511. Warwick, B. L. 1928. Prenatal growth of swine. J. Morphol. 46:59. West, E. S. and N. R. Todd.. 1957. Textbook of bio- chemistry. 2nd ed. Macmillan, New York. Windle, W. F. 1941. Development of the blood and changes in the blood picture at birth. J. Pediat. 18:558. Winqvist, G. 1954. Morphology of the blood and the hemo- t0poietic organs in cattle under normal and some ex- perimental conditions. Acta Anatomica 22:8upplement 21. ' 75 Wintrobe, M. M. and H. B. Shumacker, Jr. 1955. Compari- son of hemat0poiesis in the fetus and during recovery from pernicious anemia. J. Clin.Invest. 14:857. Wintrobe, M. M. and H. B. Shumacker, Jr. 1956. Erythro- cyte counts, hemoglobin and volume of packed red cells, mean corpuscular volume, diameter and hemoglobin con- tent, and proportion of immature red cells in the blood of fetuses and newborn of the pig, rabbit, rat, cat, dog and man. Amer. J. Anat. 58:515. Wintrobe, M. M. 1956. Clinical Hematology. 4th ed. Lea and Febiger, Philadelphia. Wyman, J., J. A. Rafferty and E. Ingalls. 1944. Solu- bility of adult and fetal carbonylhemoglobin of the cow. J. Biol. Chem. 155:275. Zeidberg, L. D. 1929. A quantitative determination of the changes in hemoglobin concentration, volume of red cells, and baSOphilia in the blood of rabbit fetuses at various stages during the last third of pregnancy. Amer. J. Physiol. 90:172. NEE! M” a" U“! "I TI" mm" 7 2 9 7 4| JIHIHIHIH