.} . . .. . . H o~ - y‘-~V.~"unju:u‘ 1w. H flung," nun.-pum.numu:z,un'waumma'ii' . I ‘3 . Z- . _‘., ‘ HUN“ ., _ 71¢. THE—5‘9 l V . i , , ' ' , , .1, ~'r‘ I .q. . 1. . l ". ' ' ' -- , ' 'v'o ! in .~ . ‘. “J" 2.1;. e U _' 7 . ”1.. 2'. are, x) , "at ' / ‘owvrv-«fir-wwqou—uc—v ~—-. -- .fifl... This is to certify that the thesis entitled MASTITIS METRITIS AGALACTIA IN SWINE: Role of Vitamin E and Selenium presented by Oswaldo E. Vale has been accepted towards fulfillment of the requirements for Master _of_Science_degree in iaLhQLQgL. W Major professor Date May 16, 1983 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES . —:—- your record. FINES W111 be charged if book is returned after the date stamped below. 75- “,i. '1’“ .' b q 2‘” IL final. fi|.|\lul ll RHHVXJ \i- / «2:9 ~93 9.;— Pa. MASTITIS METRITIS AGALACTIA IN SWINE: Role of Vitamin E and Selenium by Oswaldo E. Vale A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1983 ABSTRACT MASTITIS METRITIS AGALACTIA IN SWINE: Role of Vitamin E and Selenium by Oswaldo E. Vale The role of vitamin E and selenium (Se) fed to gilts during gestation and lactation in preventing mastitis- metritis-agalactia (MMA) and death losses in baby pigs was determined in 10 gilts fed a basal diet in comparison to 9 gilts fed the same diet supplemented with vitamin E and Se. The corn used in the basal diet was initially high moisture shelled corn that had been stored anaerobic- ally for several months, then dried artificially and used as dried shelled corn. Gilts fed the basal diet had more clinical signs of MMA, and farrowed and weaned smaller litters in which individual pigs were lighter in weight at birth and at 21 days of age than were baby pigs nursing gilts fed supplemental vitamin E and Se. Plasma vitamin E, Se, and glutathione peroxidase (GSH—Px) values were lower at birth and at 21 days in pigs nursing gilts fed the basal diet. Dedicated with love to my parents, my wife, Maria, and my children, Maria Alejandra and Oswaldo Rafael, who encouraged me to finish this study. ii ACKNOWLEDGEMENTS I am very pleased to acknowledge Dr. C. K. Whitehair, my major professor, for guidance and moral support during this research, and for his advice in the preparation of this thesis. I am also most grateful to Dr. E. R. Miller for his generous cooperation, assistance and counsel. . The cooperation of Dr. P. K. Ku and graduate students during the laboratory work in the Department of Animal Science is.greatly appreciated. My thanks also go to the Government of Venezuela for financial support, and to the faculty and staff of the Department of Pathology at Michi- gan State University for the cooperation and training I have received. Finally, I gratefully acknowledge the patience, under- standing, encouragement, and many sacrifices of my wife, Maria. iii TABLE OF CONTENTS Page INTRODUCTION... ......................................... 1 LITERATURE REVIEW................. .......... ............ 3 History and incidence. ............................. 3 Clinical signs..................................... 5 Microbiology......... ......... ......... ..... ....... 6 Pathology.......................................... 7 Experimental production ......... . ........ . ......... 8 Importance of vitamin E and Se in ‘ swine production and diseases.. ..... . ..... .... 10 Role of vitamin E and Se........................... 11 Iron and vitamin E in swine .................. . ..... 13 Vitamin E and prostaglandin........................ 14 Summary..... ...... ......... ......... .... ..... ...... 16 OBJECTIVES .............................................. 17 MATERIALS AND METHODS... ................... . ............ 18 Experimental diets ................................. 18 Experimental animals... .............. . ............. 18 Analytical procedures.. ..... . ..... ....... ..... ..... 20 Hematology....... ....... ...................... 20 Plasma collection ............................. 21 Vitamin E analyses ............................ 22 Selenium analyses.... ..... ....... ........... .. 22 Iron analyses... ..... ...... ...... . ............ 23 Glutathione peroxidase analyses ............... 23 Microbiology ....................................... 24 Pathology .......................................... 24 Statistical analyses ............................... 25 RESULTS ................................................. 26 Incidence of MMA and clinical obser- vations of gilts and baby pigs ................ 26 Vitamin E, selenium, iron, glutathione peroxidase and hematology in gilts during gestation and lactation ................ 30 iv Page Blood plasma parameters, perfor- mance and hematology of baby pigs at birth and at 21 days Of age. 0 O O O O O C O O O O O O O O O O O O O OOOOOOOOOOOOOOOOOOO 32 Microbiology ....................................... 38 Gross pathology. ......... ...... ......... .. ........ . 38 Histopathologyp.v;..... ............................ 41 DISCUSSION... ........ . ....... . ................. . ........ 48 Gilts performance .................................. 48 Blood analyses of gilts.... ....... . ..... . .......... 49 Progeny performances .................... . .......... 50 Blood analyses of baby pigs..... ................... 51 SUMMARY ........................ ' ......................... 53 REFERENCES .............................................. 54 VITA .................................................... 62 APPENDIX ................................................ 63 Table A-1. Plasma selenium, vitamin E, glutathione peroxidase activity and iron individual values of gilts during gestation and lactation.... ............ 63 Table A-2. Plasma selenium, vitamin E, glutathione peroxidase activity and iron individual values of baby pigs at birth nursing gilts fed the basal diet .................................... 64 Table A-3. Plasma selenium, vitamin E, glutathione peroxidase activity and iron individual values of baby pigs at 21 days of age nursing gilts fed the basal diet ....................... ' ......... 67 Table A-4. Plasma selenium, vitamin E, glutathione peroxidase activity and iron individual values of baby pigs at birth nursing gilts fed the vita- min E and Se supplemented diet ................ 70 Table A-5. Plasma selenium, vitamin E, glutathione peroxidase activity and iron individual values of baby pigs at 21 days of age, nursing gilts fed a vitamin E-Se supplemented diet .............. 73 Page Table A-6. Plasma selenium, vitamin E, glutathione peroxidase activity and iron values of pigs per litter at birth and at 21 days of age....... ..... . ...... 76 Table A-7. Liver selenium individual values from 14 pigs necrOpsied ................ 77 vi Table 10 LIST OF TABLES Composition of gestation-lac- tation diets ............... . ................. Reproductive performance of gilts ............ Clinical observations in gilts and death losses of baby pigs .................... Plasma vitamin E, Selenium, iron, glutathione peroxidase and hematology values in gilts during gestation and lactation..... ............................... Plasma vitamin E, Selenium, iron, and glutathione peroxidase values of pigs at birth and at 21 days of age ............... Plasma vitamin E, Selenium, iron, glutathione peroxidase, hematology values and weight of pigs at birth and at 21 days of age ........................ Incidence of stillborn, malformed pigs and mortality from birth to 21 days of age ............................... Gross lesions and microbiology in tissues and organs of pigs during necropsy ..................................... Liver selenium analyses ...................... Histopathological changes of pigs necropsied .............................. vii Page 19 27 28 31 33 35 37 39 40 42 Figure LIST OF FIGURES Page Gilt with metritis...... ......... . ............ 29 Baby pig with syndactylism. ..... . ............. 29 Serofibrinous pericarditis and epicarditis with bacterial colonies ........... 43 Pulmonary edema and inter- stitial pneumonia......... ..... . .............. 43 Serofibrinous pleuritis with bacterial colonies and inter- lobular edema ...... . ..... .... ................. 44 Centrolobular hemorrhages and vacuolating hepatopathy.... .......... . ........ 46 Hepatic necrosis, pyknosis and karyorrhexis ....... . ......... V ............. 46 Fragmentation, granulation and loss of striations in cardiac fibers ................................ 47 Calcification of cardiac fibers ............... 47 viii INTRODUCTION The mastitis-metritis-agalactia (MMA) complex is a serious and economically important disease in swine through- out the world. Swine producer associations and research groups give a high priority to additional research on MMA.1 The disease occurs at parturition, and it was clinically so named in the USA because of the 3 main clinical signs present,2 and referred to by the acronym MMA. However, in recent years, agalactia has been considered as the pri- mary disturbance which directly or indirectly contributes to death losses in young pigs from birth to weaning. Illinois workers estimated that these losses account for about 75% of the preweaning losses, and amounted to over $150 million annually.3 The MMA complex was especially troublesome to swine producers during the 1960's. In recent years, swine prac- titioners and producers mention that, while MMA is still an importan disease, its incidence seems to have declined.4 This decline may be due in part to the increased use of vitamin E and Se in swine rations. In the early 1970's, it was established that growing pigs required supplemental vitamin E and Se to prevent excessive death losses.5_7 Thus, a carry-over effect of a vitamin E and Se deficiency to amturity may have had an effect on improving repro- duction. Whereas it is well-established that the currently used corn-soybean diet fed to growing pigs requires supple- mental vitamin E and Se,8’9 there is limited information on the requirements of these nutrients during reproduc- tion-lactation. Vitamin E has been known to be essential in reproduction, particularly in laboratory type animals for over 60 years.10 Many factors have been suggested as causes of 2,4,11,12 MMA Infectious agents were initially incriminated because of the clinical signs present and the specific pathogens isolated from natural causes.11"16 However, at- tempts to reproduce MMA by giving these agents to suscep- tible sows were unsuccessful.4'l5’17"19 17,18 Thus, the cause is not clearly established. A fundamental contribu- tion to the understanding of a disease is the experimental reproduction of it as it occurs naturally. This has not been achieved for MMA in sows. I selected research on MMA in swine to obtain training which would have direct application to my teaching and research activities in Venezuela. Only a limited amount of research has been conducted on MMA. The facilities, cooperation and expertise were available at Michigan State Universidty to do research on MMA. LITERATURE REVIEW Raising swine is an intensive type of livestock pro- duction, where profitability depends upon rearing large, healthy litters.20 Newborn pigs are more prone to the haz- ards of diseases than other livestock. Important factors contributing to baby pig losses are nutritional deficiencies and infectious agents, or a combination of both.21’22 Proper nutrition for baby pigs begins with an adequate nutrition for the sow during gestation and lactation. A voluminous amount of literature is available on factors 21’22 This literature causing death losses in baby pigs. review emphasizes an important disease (MMA) occurring in swine at parturition which contributes to significant death losses in young pigs.3 History and Incidence During the late 1960's and early 1970's, American investigators described a prevalent and important disease in swine that occurred at or Shortly after parturition. Prior to this time, isolated individual case reports of parturition disorders in sows were described. The early U.S.A investigators' attention as to treatment and pre- vention of the disease was focused on the most obvious clinical signs of mastitis and metritis, and the agalactia 4,14-16 received less attention. Some controversy has existed on the disease through the years as to its cause and clini- cal signs.18 This may be because of variation in clinical signs as influenced by management practices, nutritional 11'12’18 Details programs, environment, and other factors. of a 10-year investigation on a similar post-parturient problem were published in 1960 by Dr. Ringarp, at the Royal 11 Veterinary College in Stockholm, Sweden. This report was initially overlooked by early American investigators.l3-16 In Sweden, the predominant symptom was also agalactia, "11.12 and the disease was called "agalactia toxemia. Most investigators in the U.S.A. now agree that the MMA complex is primarily a lactation failure.4'17-19 The incidence of agalactia is difficult to determine. Backstrom and Morkoc,4 in a recent review article, stated that in most countries during the 1960's, 7 to 17% of far- rowing sows had the disease. In the U.S.A., MMA was ranked as the number one disease problem in swine production.4 Missouri workers reported an incidence of 13.1% of swine agalactia.23 The Michigan State University swine herd was reported to have an MMA incidence of 21.2% in 1969. This was based on a clinical diagnosis of 1.4% mastitis, 19.3% metritis, and 0.8% agalactia.24 There is a tendency for the incidence of MMA to increase with the number of far- 4,11,25 rowings. The highest incidence occurs during 4’11 While there are some vari- 11 the 3rd and 4th farrowings. ations, the disease occurs at all seasons of the year. Ringarp correlated the highest incidence (88.5%) of "aga— lactia toxemia" with poor feeding programs, but no specific deficiency could be demonstrated. Clinical Signs The clinical signs usually appear in the sow as soon as parturition is completed. It is not well-established as to which of the clinical signs is most important. There is agreement that the sow first appears restless and nervous.4’11'17'19:21,22 Other miscellaneous clinical signs include inappetence, constipation and recumbency. Some sows may lie on their udders, preventing the pigs from nursing, but other sows may attempt to nurse the piglets.18'19 Any of the clinical signs of mastitis and metritis are highly suggestive of the disease. The mastitis (with or without metritis) is clinically characterized by edema, firmness, increased temperature and red or purple skin 4'18’19 The metritis referred to of the mammary glands. is characterized by the presence of a uterine muc0purulent discharge which is thicker than normal, and increased in quantity.4’11'l8’19 The normal uterine discharge in the sow is also a mucopurulent type of exudate, but scanty and of smaller volume.11 A wide range of body temperatures in sows with MMA has been reported, but this seems to be unreliable in diagnosing the disease.11 Clinically, one or all signs may be present, and vari- ll,18,19 This ation of symptoms exists from sow to sow. makes the disease difficult to diagnose. The main aspect of MMA is a lactation failure during the first 24 to 60 hours post-farrowing which is not readily detectable.11’18’19 Most authors emphasize that careful observation of the piglets and sow during nursing is essential to determine if milk is available for the litter in order to avoid losses from starvation.4'18’22 Microbiology The microflora associated with MMA in sows has been well-established by isolating and identifying microorganisms 11,12,13,15,26,27 from naturally occurring cases. The micro- organisms most commonly isolated were enterobacteri- acea.4’ll’14-18 Among the isolates, Escherichia coli (E. ll,13,15,16 coli) and Klebsiella occurred most frequently. Escherichia coli has been consistenly found, but isolation of serotypes indicates that it is not the primary pagthogen.15 As dicussed later, attempts to reproduce MMA using mainly 4,17,18 E. coli have not been successful. Thus, the pathogens isolated might have been secondary to other primary causes. Other miscellaneous microorganisms occurring in iso- lated cases of MMA were proteus and staphylococcus.13'15'16 Texas workers reported Mycoplasma hyogenitalium to be a 27 cause of MMA in sows. Pathology Sows with MMA rarely die, unless a severe toxic septi- 18 Therefore, there are no known reports of cemia occurs. characteristic lesions correlated with death. Some workers reported on the lesions of sows with MMA that were slaughtered.ll'25’28’29 Grossly, mastitic changes and generalized edema of 11:25r29 The mammary glands were the udder were present. inflamed, and small foci of necrosis with serohemorrhagic or purulent exudate were noted. Necropsy of 14 sows from spontaneous cases of agalactia revealed that they were 11 There was subcutaneous in a good state of nutrition. and extensive edema of the mammary glands, and excessive serous fluid mixed with blood and fibrin present in abdom- inal and thoracic cavities. In more severe cases of mastitis, the udder had yellow gelatinous masses in the interstitial and subcutaneous tissue. Those cases with metritis had edematous and hemorrhagic uterine endometrium with variable quantities of muCOpurulent fluid. Hepatosplenomegaly was also present.11 Histologically, there was marked vacuolation of uter- ine endometrium and mammary alveolar epithelium. Necrosis and exfoliatiOn of the lining alveolar epithelium.were also present in the mammary tissue. In other organs and tissues, there was vacuolating hepatopathy and centrolobular non-reactive necrosis of hepatocytes.11 Fatty changes were present in the adrenal glands and the myocardium, and there was a waxy degeneration of skeletal muscle fibers. Similar lesions to the above were present in 1 sow slaughtered 11 from 7 experimentally reproduced cases. Purdue University investigators reported that, in 18 sows with MMA, the lesions were very similar to those as described by Dr. Ringarp.29 Experimental Production Many attempts have been made to reproduce MMA in sows in order to clarify the cause and pathogenesis.11'12’15'17'18 Because of the complexity of the problem and diverse opinons on factors involved, various approaches were made to repro- duce the disease. In the U.S.A,, Midwestern investigators attempted to reproduce MMA or agalactia by using either coliform bacterials"17 isolated from natural cases, or E. coli endo- 17'30’31 Iowa workers reported that intravaginal toxins. infusion of E. coli isolated at necrOpsy from 14 natural cases of MMA produced uterine infection in 13 of 16 sows, but clinical evidence of agalactia, mastitis or profuse vaginal discharge was not observed.15 In later work, Iowa researchers noted that, in the necropsies of 13 agalactic sows, coliforms were the most common bacteria associated 14 with mastitis. Other researchers reported on reproducing MMA in a small number of cases by intranasal and intrave- nous inoculation of cultures of Mygoplasma gyogenitalium.25 The role of E. coli endotoxins on MMA was investigated by Wisconsin workers.30 They injected E. 22;; endotoxin into the mammary gland and produced clinical changes simi- lar to those noted in natural cases of agalactia. Workers at Illinois also noted the E. 99;; endotoxin resulted in marked suppression of prolactin and reduced piglet growth rate.4’18 In contrast to the Illinois reports, Missouri workers infused E. coli endotoxin into the jejunum of 12 postparturient sows, and they exhibited no clinical signs of endotoxin absorption or lactation failure. There was no indication of endotoxemia on gross pathology or light . 32 microsc0py. Ringarp also considered the role of microorganisms in agalactia toxemia.11 He examined 127 uterine tampons and 167 milk samples from agalactic sows. The main bacteria isolated were coliforms and fl—hemolytic streptococci. He concluded that no uniform infection could be demonstrated. Ringarp, because of field observations on the incidence of agalactia previously mentioned, gave more attention to the role of the diet in attempts to reproduce the disease 10 by feeding a poor quality feed. The feed (wheat) that he used had been damaged during harvest, and was unsuitably stored. He purchased this feed from a farm in which all 6 sows developed the disease when they farrowed during a 2-month period. The farm had no previous history of aga— lactia toxemia. Ringarp, using this feed experimentally, reproduced the disease in 7 of 9 sows.11 Importance of Vitamin E and Selenium In Swine Production and Diseases Vitamin E was first recognized in 1922, and chemically 33 This vitamin is known to have an 34-36 identified in 1938. antioxidant effect against lipid peroxides, and for many years a deficiency has been associated with myopathy, reproduction problems, and infertility in livestock and poultry.37 Selenium was demonstrated to be an essential 38 micronutrient in 1957. It functions through GSH—Px at subcellular levels within the mitochondria to protect the 39 Thus, these nutrients cells from peroxidative damage. work together to avoid peroxidation of vital phospholipids in cellular and subcellular membranes to preserve the in- tegrity of tissues.4 The role of vitamin E and Se in swine production and diseases has been recently summarized.41 They appear to 35,36,39 function primarily as in vivo antioxidants, and their requirements in swine are influenced by a number ll of factors.42 Increased resistance of chicks and lambs to E. 991; and chlamydia was reported when vitamin E was supplemented in the diet.43 Supplementation of this vita- min in swine rations was reported to enhance the immune response to E. coli bacteria.44 Ohio investigators reported a reduced pig mortality at weaning and increase in the immune humural response when vitamin E and Se were given to weanling swine.45 Lowered levels of GSH-Px activity and decreased microbicidal ability in circulating leukocytes and pulmonary macrophages wer reported in vitamin E and Se deficient animals.41 In addition, experimental work supports the theory that supplementation of these 2 nutrients increases the resistance of pigs to swine dysentery. In 3 experiments, the incubation period was shorter, and the severity of lesions was more pronounced in deficient pigs than in those 46’47 This research supplemented with vitamin E and Se. suggests that these 2 nutrients play an important role in the mechanisms of defense against disease. The Role of Vitamin E and Selenium Prior to about 1970, vitamin E and Se were assumed to have an unimportant role in practical swine production.48 This was summarized in the 1968 Nutrient Reqpirements of Swine with the statements, "The biochemical functions of 12 selenium have not been clarified," and "It is unlikely that practical swine diets would be deficient in vitamin E unless the diet contained excessive amounts of highly unsaturated fatty acids or oxidized fats; therefore, a supplemental source is not needed." Following the 1969 5’6 and and 1970 reports from Michigan State University Purdue University7 researchers on the importance of these 2 nutrients in preventing death losses in young pigs under practical conditions, the 1973 Nutrient Requirements of §g£g§49 states that "It is suggested that 11 IU of vita— min E be added per kilogram of diet until more Specific information is obtained." In addition to the reports on the importance of vitamin E and Se in growing pigs, Michi- gan State University investigators reported that the sup- plementation of these nutrients in swine herds under field conditions reduced the incidence of MMA in sows.6 Experi- mental evidence was also available which indicated that vitamin E supplemented to sows reduced the incidence of MMA from 50 to 14%, and the addition of vitamin E also increased the survivability of baby pigs to 3 weeks of age.50’SL Many investigators have questioned the importance of MMA during the past decade. Some authors mentioned var- ious factors that have changed in modern swine produc- 11’20’30’42 among them, the use of improperly har- 11,42,52 tion, vested and stored feed. 13 Iron and Vitamin E in Swine Iron (Fe) is a vital element for cellular respiration and oxygen transport in the tissues, and forms part of 53 the respiratory enzymatic systems. Pigs have a basic needs for Fe at birth because of an inadequate dietary 39,54,55,56 source from the sow's milk. Thus, pigs have to be supplemented with Fe in order to avoid depletion of body iron and subsequent anemia.S7 Iron injections have at times produced death losses 58-60 These losses were associated with a 61,62 in baby pigs. vitamin E and Se deficiency in baby pigs. Evidence on the protection of vitamin E and synthetic antioxidants 61’63 The rela- against iron toxicosis in pigs was reported. tionship of a low vitamin E level and high level of poly- unsaturated fatty acids in the diet was observed in baby 62 In addition, pigs which had died after Fe administration. British researchers reported that vitamin E and Se defi- cient pigs had low tolerance to Fe injections.64 Iron was demonstrated to increase the rate of free radical peroxida- tion and to enhance vitamin E requirements.65 In recent years, there has been a tendency to reduce labor costs in swine production by giving a single large injection of Fe rather than repeated smaller dosages to baby pigs. This has accounted from cases of iron toxicosis in vitamin E and Se deficient pigs under practical conditions. 14 Vitamin E and Prostaglandin Vitamin E, in addition to having an antioxidant role 34-36 E2_vivo, has also been reported to be involved in prostaglandin (PG) synthesis.66 These cyclic oxygenated fatty acids (arachidonic acid) are active biological com- pounds which function in the maintenance of cellular homeo- stasis.66 They also exert significant effects in a number of physiological and pathological processes, such as blood flow and pressure, platelet aggregation, immune response, 66-68 and gastrointestinal circulatory disturbances. There- fore, it is not unlikely that PG is also involved in several endocrinologic processes. Many investigators have emphasized 4,11,17,18 that MMA is related to endocrine imbalances and 11’18 resulting in physiological gastrointestinal disorders, and pathological effects including a lactation failure. Recent reports were published on a decreased incidence of MMA in gilts induced to farrow with prostaglandin F20<.4 Reduced peristalsis of the gastrointestinal tract (GIT), coprostasis and constipation leading to endotoxemia have been reported in sows with MMA.1‘1’18 Even though these changes were associated with nutritional disturbances, a role of PG as regulators of gastrointestinal circula- tion and physiology is possible. Furthermore, several in- vestigators reported on the role of PG in blood circula- 67,68 tion in GIT, liver, pancreas and spleen. An increase in substances derived from arachidonic acid was also 15 reported in experimental animals with endotoxic shock.69’70 Thus, the pathOphysiology of MMA appears to involve predis- posing factors (vitamin E deficiency), altered endocrine functions and endotoxemia, with infectious agents playing a secondary or aggravating role.17 Summary The literature is clearly supportive that MMA or aga- lactia toxemia is a widespread, important and complex dis- ease in swine. The cause is not established, although the problem has existed for 20 to 25 years. The dieases is associated with production techniques, especially feeding and processing feeds. Various authors have suggested causa- tive factors based mainly on the history and clinical in- formation. Only limited research has been conducted to reproduce the disease as it occurs naturally. Although infectious agents have not been incriminated as a primary cause, there is evidence that they have a secondary or complicating role. European investigators have incriminated the general role of nutrition, but a specific nutrient' deficiency has not been established. In growing pigs, a specific role for vitamin E and Se has been established, and the lesions of the deficiency are not too dissimilar from the lesions described in MMA. In recent years, there has also been evidence that vitamin E has a role in endo- crinology, especially in the synthesis of prostaglandin 16 that are involved in many physiological and pathological events. Thus, research on the role of these nutrients in MMA is justified. OBJECTIVES The objectives of this research were: 1. To produce experimentally the MMA complex as it occurs naturally in sows. To determine the role of vitamin E and Se in preventing MMA or reducing its incidence. To evaluate the role of these 2 nutrients in improving the livability and health of baby pigs from birth to 3 weeks of age. To determine the susceptibility of vitamin E and Se deficient neonatal pigs to Fe injections. To monitor plasma levels of vitamin E, Se, Fe and GSH-Px to establish minimum levels expected in a dietary deficiency of these nutrients in sows during gestation, parturition and lactation. To ascertain plasma, vitamin E, Se, Fe and GSH-Px status of baby pigs from birth to 21 days. To determine the role of vitamin E and Se in swine as being of practical value in improving swine production, especially during late gesta- tion and early growth. 17 MATERIALS AND METHODS Experimental Diets A basal high moisture shelled corn-soybean meal diet was fed to 2 groups of Yorkshire x Landrace gilts in the fall of 1982. The high moisture corn was anaerobically stored for 6 to 8 months, then dried, ground and mixed with the other ingredients of the diet. This diet was re- ported to enhance the production of a vitamin E and Se 51’71 poultry,72 and MMA in sows.11’50’51 deficiency in pigs, One group of gilts (10) was fed the basal diet, and the other group of gilts (9) was fed the basal diet supple- mented with vitamin E (50 IU/Kg) and Se (0.1 ppm). The composition of diets is given in Table 1. Experimental Animals Nineteen crossbred gilts (Yorkshire x Landrace) were divided into 2 groups and fed the experimental diets. Gilts were bred as they came into heat. After mating, gilts were weighed and bled early in gestation, at parturition and at 21 days of lactation, and blood plasma vitamin E, Se, Fe, GSH-Px, and hematology [Hemoglobin (Hgb), Packed Cell Volume (PCV), Mean Corpuscular Hemoglobin Concentration (MCHC)] values were determined. At parturition, data on 18 l9 TABLE 1. COMPOSITION OF GESTATION-LACTATION DIETS a DIETSe __ INGREDIENTS . MSU BASAL + VITAMIN E-Se DRIED HIGH MOISTURE SHELLED CORN 893 892.32 DEHULLED SOYBEAN MEAL 7o 70 MONO-DICALCIUM PHOSPHATE 15 15 CALCIUM CARBONATE (CaCO3) 12 12 SALT 5 VTMb PREMIX 5 VITAMIN E (PREMIX)° --- .18 SELENIUM 90 (PREMIX)d --- .5 Total 1,000 1,000.00 aINGREDIENTS ARE EXPRESSED IN PARTS PER THOUSAND. bVTM, VITAMIN TRACE MINERALS PREMIX, AS IN BULLETIN 537, SWINE FEEDS AND FEEDING, MICHIGAN STATE UNIVERSITY, EAST LANSING. cVITAMIN E (PREMIX) CONTAINS 275,000 IU/KG (SUPPLEMENTED TO SUPPLY 50 IU/KG DIET). SELENIUM 90 (PREMIX) CONTAINS 200 MG/KG (SUPPLEMENTED TO SUPPLY 0.1 PPM). eON ANALYSIS, MSU BASAL CONTAINED 0.78 ug/g VITAMIN E AND 0.04 ug/g Se. SUPPLEMENTED DIET CONTAINED 56.1 ug/g VITAMIN E AND 0.12 ug/gr Se. d 20 Clinical observations, length of farrowing time and rec- tal temperatures of each gilt were monitored daily. There were 175 live pigs obtained from 19 litters. Each pig was weighed, ear-notched, taildocked, and given 200 mg or 200 mg of Fe* intramuscularly at birth. Pig weights were also recorded at 21 days of age. Litters were weaned at 28 days of age. All the pigs were bled at birth and at 21 days, and hematology, plasma vitamin E, Se, Fe, and GSH-Px values were determined. The 42 pigs that died from birth to 21 days of age were necropsied and tissues were collected for microbiology and pathological evaluation. Records on stillborn, malformed and dead pigs were also maintained. Analytical Procedures Hematology Bleeding Procedures. Baby pigs were bled from the anterior vena cava with a sterilized 1 inch, 18-gauge needle and 15 ml syringe. Blood was collected in 10 ml heparinized test tubes. Gilts were also bled by the same technique, using a sterilized 3.5 inch, 18-gauge needle and 20 ml syringe. *Gleptoferron = a polysacharide complex of beta-ferric oxyhydroxide and dextran glucoheptonic acid, Burns-Biotec Laboratories, Inc. 21 Packed Cell Volume (PCV). Hematocrit values were 73 determined by centrifuging heparinized microhematocrit tubes containing blood samples for 5 minutes at 10,000 rpm in an International model MB microhematocrit centrifuge.73 The percent of packed cells was recorded from an Interna- tional microhematocrit reader. Hemoglobin (Hgb). Hemoglobin values were measured by the cyanmetahemoglobin method using a Drabkin's cyanide reagent to lyse the red blood cells. The absorbance of the solution was measured in a spectrophotometer at 540 nm of wavelength.74 Absorbance values were multiplied by a conversion factor in order to obtain the Hgb concentration values in grams per deciliter (g/dl). Mean Corpuscular Hemoglobin Concentration (MCHC). This blood parameter was calculated by using the following fOrmula: Hgb (g/dl) MCHC (%) = x 100 PCV (%) Plasma Collection. Blood samples were centrifuged at 3,000 rpm for 15 minutes in order to separate plasma from cellular elements. Plasma samples were kept in clean 5 ml tubes, the air was displaced with nitrogen gas, and then frozen for determination of vitamin E, Se, Fe, and GSH-Px values. 22 Plasma Vitamin E, Selenium, Iron and GSH-Px Analyses Vitamin E. Plasma vitamin E values were measured by a sensitive fluorometric method for tissue tocopherol 75’76 The method con- as modified for plasma tocopherol. Sisted of precipitating the protein in 1 ml of plasma with 2 ml of absolute ethanol and displacing the air with nitro- gen gas before shaking the samples for 5 seconds. Afterward, 2 ml of cyclohexane were added, air was again displaced, and tubes were shaken for 20 seconds. All the tubes were maintained on ice and treated in the same manner. Test tubes were centrifuged in a Danon/IEC model PR-6000 refrig- erated centrifuge at 3,000 rpm for 15 minutes and the upper layer was pipetted into vials. Duplicates were run for standards and samples. Transmission of samples was read in an Aminco-Bowman spectrofluorometer at 296 nm excitation and 330 nm emission. Values were regressed against micro- grams of dt-tocopherol using a computerized curvilinear program. Selenium (Se). Selenium values were determined by 77 a routine fluorometric method which consisted of digesting 1 m1 of plasma with 2 ml of nitric acid (HNO3) and 2 m1 of percholoric acid (HClO4, 70%) on hot plates in Erlan- meyer flasks. The samples were neutralized and chelated with 3 ml of Ethylene Diamino Tetraacetic Acid (EDTA) (8 ml), and then complexed with 2,3 Diaminonaphthalene (DAN) (5 ml) to extract the diazoselenol into 5 ml of cyclohexane. 23 Deionized water was used to bring the volume up to the neck of the Erlenmeyer flasks, and the upper layer was repipetted into Clean tubes. Duplicates were run for stand- ards and samples. An Aminco-Bowman spectrOphotofluoro- meter set at 376 nm excitation and 510 nm emission was used to read the transmission (T%) and a curvilinear re- gression was performed to calculate Se values in micro- grams per ml (pg/ml). Iron (Fe). Plasma Fe was determined by atomic absorp- tion spectrophotometry using a model IL 951 Atomic Absorp- tion Emission Spectrophotometer at 248.3 nm wavelength and an acetylene flame. The method consisted of precipita- tion of protein contained in 1 ml of plasma with trichloro- acetic acid (TCA) and incubation at 90°C for 15 minutes. Samples were then centrifuged for 15 minutes, and Fe was 78 Iron values were calculated measured in the supernatant. 'in pg per deciliter (Hg/d1). Glutathione Peroxidase (GSH-Px). Plasma GSH-Px ac- 79,80 tivity was determined by the coupled assay for GSH—Px. The method consisted of mixing all the reagents with samples into 1 ml cuvettes. Hydrogen peroxide (H202) was then added, and the contents mixed. The reaction for the mixture was monitored in a Varian 634 spectOphotometer and recorded in a Varian model 9176 chart recorder for about 5 minutes. Enzyme unites were calculated as micromoles of gluta- thione (GSH) oxidized per minute. Increment of absorbance (aAb) 24 was recorded for blanks (AAbB) and samples (AAbS) by readings (R) at 0 minutes (R1) and at 5 minutes (R2) on the charts, and the difference (R1 - R2) was divided by the time (t). After that, AAbB was subtracted from AAbS and multiplied by a factor (8.0386) to obtain the enzyme units (EU) of GSH-Px per ml of sample. The formulas used are as follows: R - R R - R _ 1 2 _ 1 2 ASAbB - t £>Abs t GSH-Px (EU/ml) = (AAbS - AAbB) x 8. 0386 Microbiology During necropsy of baby pigs, microbiology tissue specimens were saved in sterile containers and sent to the Microbiology Department at Michigan State University Veterinary Clinical Center for bacteriological analyses. Tissues collected were lung, liver and heart. Isolation of enterotoxigenic E. gel; from a pig with diarrhea was attempted. Pathology Necropsy was performed on 42 baby pigs at the Michigan State University Animal Health Diagnostic Laboratory (AHDL). Multiple tissues were collected and fixed in 10% buffered formalin. These tissues were later trimmed, parafin embedded, 25 sectioned and stained with hematoxylin and eosin. The pro- cedures are described in the Manual of Histological Methods of the Armed Forces' Institute of Pathology.81 During nec- ropsy, randomized liver samples were saved in plastic con- tainers and frozen for further Se analyses. The values obtained were related to the dry matter (DM) content of the samples and thus expressed as ug/g of DM1. Statistical Analyses A linear regression (y = a + bx)82 was used to calcu- late individual values for vitamin E and Se at each samp- ling. Individual values of each of the blood parameters (PCV, Hgb, MCHC, vitamin E, Se, Fe, and GSH-Px) were deter- mined for the 2 experimental groups of animals. By using the analysis of variance,82 treatment mean values were compared between groups, and their statistical level of Significance (P) was established. RESULTS The reproduction performance of gilts fed the basal diet compared to gilts fed the same diet supplemented with vitamin E and Se, :is summarized in Table 2. Gilts fed the supplemented diet had larger litters and heavier pigs at birth and at 21 days than gilts fed the basal diet. These parameters were significantly different (P < 0.05). In addition, the supplemented gilts weaned more pigs than unsupplemented gilts. The livability to 21 days of age was greater in pigs nursing the supplemented gilts. There were no differences between the 2 treatment groups in weight changes of gilts from postfarrowing until 21 days of lac- tation. Incidence of MMA and Clinical Observations of Gilts and Baby Pigs The incidence and clinical observations of MMA in gilts and baby pig death losses during lactation are sum- marized in Table 3. Five gilts fed the basal diet had some evidence of MMA. Of 3 gilts with signs of MMA, 2 had mas- titis and 1 had metritis (Figure 1). The mastitis was char- acterized by a warm, firm and reddish udder. Two additional gilts had excessive pig death losses with no signs of 26 27 .Qmmm mmB 20mm QMQQDU mmm3 92¢ mDMBmm OBZH mZOU OB DHAHdm mbomw QMBZHZ ImammDm Omlm ZH mBAHw mmmmfi umDmBmm ZH mBAHO mo .02 fl mmmmmazmmfim ZH mmmmEDZO WUZ¢UHmHZOHm ho 4m>mq u vmn de2 ho momma QfidflZfiBm fl Emma Hm. m.~ .m. H.m .oH. m.oH omsmemm emmHm OB osz¢ oo.H S.m m.mm ~.om w .AmwHq mm. «H. H.m m.¢ ox .mwao Hm ea emonz OHS .o>< mo. v.m H.m¢ v.Hm ox .mwma am as emonz mmequ .o>< Ha. mu. m.m m.» mmequ\m»¢O Hm ea mon m>Hq .o>< mo. No. m.H e.H ox .memHm ea emonz on .o>< No. mm. «.mH ~.HH om .memHm ea emonz mmequ .o>m SA. on. m.cH H.m mmequ\zmom mon m>Hq .o>< «H. mm. m.oH m.m mmequ\zmom mon aceoe .o>¢ mmmaqu m.mu m.mu Mozamo emonz oo.s m.v H.mmH n.mmfi one+ Amman om: mmmemsmmam memHO oneoaoomm mBQHw m0 fiOZflEMOhMNQ.W§HBUDQOMQMfi .N mqmda 28 TABLE 3. CLINICAL OBSERVATIONS IN THE GILTS AND DEATH LOSSES OF BABY PIGS DAM'S DIET PARAMETERS MSU BASAL + VITAMIN E-Se NO. GILTS 10 9 NO. WITH MMA SIGNSa 3 o No. WITH AGALACTIA OR b HYPOGALACTIA 2 0 LIVE PIGS 1 DAY 81 91 21 DAYS 65 79 MORTALITY 16 (19.8%) 12 (13.2%) aTwo (2) GILTS HAD CLINICAL SIGNS OF MASTITIS AND ONE (1) HAD METRITIS. bTHESE GILTS HAD EXCESSIVE PIG DEATH LOSSES. _,'.._.._.'—~~ . ' .. 1 '_ : ' -' ; 3 ”1 w...— Fig 1 - Metritis in a gilt fed the basal diet. Note blood clots in vulva and purulent material on the buttocks and floor. Fig 2 — Pig with syndactylism born to a gilt fed the basal diet. Note the curling of the phalanges. 30 mastitis or metritis. There were also 2 supplemented gilts that did not milk as well as expected. This was indicated by a slower growth rate of the pigs until 21 days. Mortal- ity from birth to 21 days was greater in pigs nursing gilts fed the basal diet. There was no difference in appetite of gilts fed either diet at or following parturition of gilts in both groups fluctuated greatly, and no difference could be ascertained as to the influence of the diet. Some of the gilts fed the basal diet seemed to have more diffi- culties during farrowing than the supplemented gilts. Gen- eral observations on the length of time for farrowing did not appear to be different between the treatment groups. There were no obvious differences in the gilt behavior at parturition between the 2 groups, with the exception of 1 unsupplemented gilt which appeared to be more nervous at parturition, and lost the entire litter within 48 hours. This gilt was given 3 piglets from another sow, and raised them successfully. Vitamin E, Selenium, Iron, Glutathione Peroxidase and Hematology Values in Gilts During Gestation and Lactation The plasma vitamin E and Se values of gilts fed either diet‘ were similar during the early gestation (Table 4). However, they were Significantly different (P < 0.05) by late gestation and after 21 days of lactation. The plasma 31 TABLE 4. PLASMA VITAMIN E, SELENIUM (Se), IRON (Fe), GLUTA- THIONE PEROXIDASE (GSH-PX) AND HEMATOLOGY VALUESa IN GILTS DURING GESTATION AND LACTATION PRODUCTION DIETS b C PERIOD MSU BASAL +VITAMIN E-Se SEM P< EARLY GESTATION (30 DAYS) NO. OF GILTS 5 5 Se. ug/ML 0.15, 0.19 .011 .15 VITAMIN E, uG/ML 2.74 2.14 .035 1.00 LATE GESTATION (100 DAYS) NO. OF GILTS 5 5 Se, uG/ML 0.13 0.17 .005 .001 VITAMIN E,_uG/ML 0.87 1.91 .094 .0003 GSH-Px, EUd/ML 1.21 1.01 .010 1.00 Fe, uG/DL 135.68 133.20 .051 1.00 LACTATION (21 DAYS) NO. OF GILTS 10 9 Se, uG/ML 0.08 0.20 .007 .0001 VITAMIN E, uG/ML 0.76 2.02 .127 .0001 GSH-Px, EUd/ML 0.47 0.95 .057 .0004 Fe, uG/DL 102.80 89.60 4.76 .16 Hgb, G/DL 11.5 10.9 .36 1.00 PVC, % 34.7 32.3 1.05 .25 MCHC, % 33.3 35.2 .53 .07 aVALUES ARE EXPRESSED AS AVERAGES (R). INDIVIDUAL VALUES ARE IN APPENDIX TABLE A-l. SEM = STANDARD ERROR OF MEAN. P< = LEVEL OF SIGNIFICANCE. EU = ENZYME UNITS AS MICROMOLES OF GSH OXIDIZED PER MINUTE. b d 32 GSH-Px activity values were significantly lower (P < 0.05) in gilts fed the basal diet at 21 days of lactation as compared to values of gilts fed the supplemented diet. These GSH-Px values were also lower in gilts fed the basal diet at 21 days of lactation in comparison to values during late gestation. Iron values were similar in gilts fed both diets during late gestation and decreased in both groups by 21 days of lactation. The values stayed higher in gilts fed the basal diet. Hemoglobin and PCV values were similar in both treatment groups at 21 days of lactation. However, the MCHC values were higher (P < 0.07) in gilts fed addi- tional vitamin E and Se. Blood Plasma Parameters, Performance and Hematology of Baby Pigs at Birth and at 21 Dayg of Age Average plasma vitamin E, Se, Fe, and GSH-Px values in baby pigs at birth and at 21 days nursing gilts fed either diet are summarized in Table 5. Baby pigs nursing gilts fed the basal diet had lower plasma Se values at 1 day and at 21 days than pigs nursing gilts fed the sup- plemented diet. These plasma Se values were considered to be lower than the borderline for a Se deficiency which is 0.05 ppm. Plasma GSH-Px activity values were similar in both groups at birth and at 21 days. The GSH-Px and Se mean values were positively correlated. Iron values 33 .mmqm2¢m m0 mmmSDZ .MBDZHS mmm QHNHQHxO mmw m0 mmqozomUHZ m4 mBHZD MEwNZm A v Dmn .mua xHozmmmm 2H.mm< mmmequ szeHz mmoq<> 24m: .ma< .Sua .mua .mua mmqm AEDOH>HO2H .mmmeeHn zmmsemm oz< szeHz mmoamm>¢ ma ommmmmmxm sea mm34<>8 .mm. v.HHH Abs. wH.mh Ammv nv.nma Ace. mm.vm qo\o: .om .mn. mom.o AHm. mmH.o Ame. mam.o Amn. mHH.o qz\nbm .xmummo Amp. SH.~ Adm. Hm.H Ame. om.o Amps GH.H qz\o: .m sz Amhv mho.o .mmv owo.o Ame. mvo.o Aam. mmo.o qz\wn .mm on Ha me am mon mo mmmzaz seeoe m m m oH memequ so .02 memo am as use H 94 mama am PM see H as mmmemz + q Axmlmmwv mm <2m 0.05). The livability of pigs nursing gilts fed the basal diet was lower as compared to pigs nursing gilts fed the supplemented diet. Furthermore, the livability of pigs given 400 mg of Fe was reduced in both groups in comparison to pigs given 200 mg. Blood plasma vitamin E, Se and GSH-Px values of pigs nursing gilts fed either diet in general increased from birth to 21 days of age. The differences were significant (P < 0.05) for pigs nursing gilts fed the supplemented diet. Plasma Fe values of pigs nursing gilts fed the basal diet were significantly higher (P < 0.05) than in pigs nursing gilts fed the supplemented diet at birth and at 21 days. There was no significant interaction between Fe and dietary treatments. The incidence of deaths, stillborn, malformed pigs and mortality rates are summarized in Table 7. Gilts fed either diet had no difference as to the percentage of still- born and mummies. The mortality of pigs at 21 days was higher in gilts fed the basal diet than in gilts fed the supplemented diet. 35 Hm. oo.H Hooo. omo. mm.m HS.~ em. vo.H mwaa Hm oo.H oo.H mooo. who. Hm.H mm.H mH.H mH.H wan H Hz\oa .m szaeH> oo.H oo.H Hooo. Hoo. mno. use. mvo. 680. mass Hm oo.H mm. Hooo. moo. mmH. coo. Hmo. mmo. HMO H Hz\oa .mm v.om v.mm m.oh m.mm muse Hm OP 3 .HGHHHm¢>HH oo.H oo.H mH. HS. «.mm o.mm H.nm m.om mseo Hm AH. oo.H oo.H ow. m.mm m.Hm v.Hm H.om was H w .>om oo.H mm. mo. mH. m.mH o.mH H.HH m.HH mama Hm mm. SH. om. NH. m.m H.0H o.oH m.m sea H Ha\o .nmm oo.H mo. mm. mm. oo.m mm.m m8.v AH.m mmao Hm oo.H oo.H mo. «0. nv.H nv.H 8m.H om.H wan H UM .emonz Hm m8 mm mm mama Hm me me me ow sea H mon so .02 wmxa 6E O nzmm oov com 008 com mmemzamam vac OmozeonHonm OHA\os,48m 2H OHE\oz .mm 2H mmum szaeH> + Hawam om: memHo .moe mo mace Hm Be oza sea H a4 mon mo smonz Oza mmmnq<> wooqoeazmm .meummov ammonommm monmeeSHo 20.3 29: .88 2323mm .m 2333 4:95 .6 mamas 36 .NBDZHZ mam QmNHDHxO 3mm. m0 mmA020m0H2 mm mBHZD MSMsz M am .Oh sz Q m0 ZOHBU¢mMBZH mom QUZ¢UHmHZUHw m0 AN>HA fl OhXQ «mmDA<> ZQmH mom mozmonHZUHm m0 AW>MA u 0m «BWHD.2€Q mom muzmonHZOHm m0 AM>HA n Q G U .me2 NO. momma Qmmazmfim u 2mm a .Huo mmoamm>¢ me ommmmmmxm mam mmoH¢>8 oo.H no. ooooo. o.m o.HHH H.8oH >.ooH ~.o~H msao Hm oo.H oo.H muoo. mm.H ~.oh o.on «.mo o.mo wan H Ho\oa .om oo.H oo.H Hooo. moo. mm. mm. mm. om. mwao Hm oo.H oo.H moooo. woo. oH. mH. HH. HH. woo H Hz\mom .xmnmmo omxo on o exam ooo oom oow oom mmemzomam vao umozaonHonm on\oz .mm 2H OHM\oz .mm 2H «mum szmaH>+ Hanan om: memHo Awwscflpcoov .@ Manda 37 .MOHEEDE paw cuonHHHum mo muHHmuuoz Q macaw smmnzom1cuoo poaaosm ousumwoe zoo: COHHQM N.MH NH 5 Ha m omim cwemua> + m.mH 0H 5 HQ 0H Hemem sz moan mmwssoz mmemHQ a coma new moan muaww huwamuuoz .oz nwumeuuoz coaHOMHmz :uonaawum o>HH .oz mama Hm on nuuHm nuuHm He N04 m0 mfima am 09 mBmHm 20mm wBHfldamOZ QZ< .mUHm szmoquz .zmomAAHBm m0 mozmQHUZH .h mammfi 38 Microbiology The microorganisms isolated from pigs that died are given in Table 8. A heavy growth of E. gel; was isolated in tissues of 4 of 7 pigs that had lesions of polysero- sitis, accumulation of excessive seroganguinous fluid and serofibrinous adhesions in body cavities. One of the 4 pigs also hadgfl -hemolytic streptococci. However, the strain of E. coli isolated in all pigs was not demonstrated to be enteropathogenic. Attempts to isolate enterotoxigenic E. coli from a pig with diarrhea were not successful. Gross Pathology The gross lesions and Se analyses of liver samples of pigs necropsied are summarized in Tables 8 and 9 res- pectively. Twenty-seven of 42 baby pigs that died between birth and 21 days of age were necropsied. Pigs from both treatment groups had enlargement of lymph nodes and yel- lowish coloration ofsubcutaneous tissue. However, a yel- lowish to brownish tinge was more often observed in pigs nursing gilts fed the basal diet. These pigs also had a generalized edema and serofibrinous polyserositis. Accumu- lation of excessive serosanguinous fluid in body cavities and pericardial sac were also commonly seen. These lesions were also present in pigs nursing gilts fed the supplemented diet, but they were less prominent. There was 1 malformed TABLE 8. 39 SUMMARY OF GROSS LESIONS AND MICROBIOLOGY IN TISSUES AND ORGANS AT NECROPSY. THESE PIGS WERE INJECTED INTRAMUSCU- LARLY WITH 200 MG OR 400 MG OF IRON. DIETS NO D P . OF a IGS NO. OF GROSS LESIONS AND NECROPSIED MICROBIOLOGY MSU BASAL + VITAMIN E-Se 23 19 15 12 r- PALENESS AND BROWNISH DISCOLORATION OF CARCASSES. ENLARGEMENT AND DARKNESS OF LYMPH NODES AND SPLEEN. DEHYDRATION AND DIARRHEA (2 CASES). SEROSANGUINOUS FLUID IN THORACIC AND ABDOMINAL CAVITIES (7 CASES). SEROFIBRINOUS PLEURITIS, PNEUMONIA AND PERITONITIS (6 CASES). CARDITIS (7 CASES). E;_COLI WAS ISOLATED FROM 4 OF 4 PIGS. SUPERFICIAL ABSCESSES AND LACERATION OF SKIN (2 CASES). MALFORMED PIGS (SYNDAC'I'YLIA) (1 CASE). THIS PIG HAD LESIONS IN THE LIVER CON- SISTENT WITH HEPATOSIS DIETETICA. YELLOWISH OOLORATION 0F CARCASSES. ENLARGED LXMPH NODES. SEROSANGUINOUS FLUID AND FIBRINOUS EX- UDATE IN THDRAX AND ABDOMEN (2 CASES). URIC ACID CRYSTALS IN KIDNEYS (2 CASES). MALFORMED PIGS (1 CASE). aONLY PIGS THAT DIED PROM BIRTH To 21 DAYS OF AGE. HYDROPERICARDIUM AND SEROFIBRINOUS PERI- 40 ZH mmm mmDA€> quQH>HQZH .hI4 mnmdfi Xanmmmm .mHmmm mmBHmS Hmn 20 mmwmmm>¢ mm nmmmmmmxm mmm ZDHZHAmm m0 mmDA¢>w on . H o «H m H 8 more 5.25». + 8H o H mm B .293 om: 2o o3: mmHBém MESH omHmmomomz 83 name no: ES 86m .02 82 .oz .02 .2909 .mem QMHmmOmumz 20mm mflAmZGm VH m0 mMDfl<> Awmv ZDHZHAmm mm>HA .m mqmms 41 pig (syndactylism) (Figure 2) born to a gilt fed the basal diet. At necropsy, this pig had gross lesions suggestive of hepatosis dietetica. The Se concentration in liver sam- ples analyzed were similar in pigs from both treatment groups (Table 9). Lesions suggesting Fe toxicity were not observed grossly in pigs from either group. Histopathology The gross lesions were confirmed microscopically, and confined to body cavities, serous surfaces, reticulo- endothelial system, heart, and skeletal muscles (Table 10). In body cavities and serous surfaces, a serofibrinous and suppurative generalized polyserositis was present in 7 pigs nursing 4 different gilts fed the basal diet. However, these changes were also seen in 2 pigs nursing gilts fed the supplemental diet. A serofibrinous pericarditis and epicarditis with bacterial colonies (Figure 3) were present in pigs nursing gilts fed the basal diet. Pulmonary edema with interstitial pneumonia (Figure 4) and serofibrinous pleuritis with bacterial colonies were also observed (Figure 5). In pigs of both groups, a heavy iron pigment (hemo— siderin) was observed in the spleen, subcapsular sinuses of lymph nodes and the Kupffer cells of the liver. It was less severe in pigs nursing the supplemental gilts. 42 TABLE 10. SUMMARY OF HISTOPATHOLOGICAL CHANGES FROM PIGS NECROPSIED. NO. OF MICROSCOPIC DIETS PIGS NECROPSIED CHANGES ’ SEROFIBRINOUS AND SUPPURATIVE GENERAL- IZED POLYSEROSITIS. DEGENERATIVE MYOCARDITIS WITH LOSS OF STRIATIONS. PULMONARY EDEMA WITH SEROFIBRINOUS PLEURITIS AND PNE ON . MSU BASAL 15 ‘< UM IA SUBEPICARDIAL HEMORRHAGES WITH EPICAR— DITIS AND PERICARDITIS AND BACTERIAL COLONIES. CENTROLOBULAR HEPATIC NECROSIS AND HEM- MORRHAGES WITH EXTRAMEDULARY ERYTHRO- POIESIS. SEVERE VACUOLATING HEPATOPATHY. HEAVY DEPOSITION OF IRON IN LIVER, SPLEEN AND LYMPH NODES. \— SEROFIBRINOUS POLYSEROSITIS. + VITAMIN 12 VACUOLATING HEPATOPATHY. E-Se FOCAL PYELITIS IN KIDNEYS. MILD DEPOSITION OF IRON IN LYMPH NODES AND SPLEEN. Fig 3 — Serofibrinous pericarditis and epicarditis with bacterial colonies (arrow) in a pig from a gilt fed the basal diet. . . . \ "‘nuf. it" . 33,. . .°°P"='.-'-:.~‘ {R1 'i 1. at 7" Fig 4 - The same pig as in Fig 3. Pulmonary edema (arrow) and interstitial pneumonia (a) with accumulation of inflam- matory cells. 44 . eff-i=9" 35,—} wl'x at». "-7 Fig 5 - A lower magnification of the same lung as Fig 4. Note the serofibrinous pleuritis with bacterial colonies and interlobular edema (arrow). 45 Centrolobular hemorrhagic necrosis along with diffuse vacuolating hepatopathy and extramedullary erythrOpoiesiS were also present in the pig with syndactylia (Figures 6 and 7). Degenerative myocarditis with calcification and gran- ulation of fibers were observed in pigs nursing gilts fed the basal diet (Figures 8 and 9). Loss of striations of skeletal muscle fibers was also present. 46 \Or“ 7;??- " I! II}; . t~# ' {31 he?” . . L)- "‘""x" afl't. ' '3 "r‘ #4.?) Fig 6 - Centrolobular hemorrhages (arrow) and vacuolation of hepatocytes. Liver of pig with syndactylism. Note also the extramedullary erythopoiesis (lower center) .0. ’ g , ' r. 3..- 11-7! '"‘ . {.1 0 a} 9. . 3}“ .“2‘? ‘3‘? 9f; 3 .’ f q. 0:41: . ""5."V.a. '3; 9. .' $3,. ‘ ’ tips, ’4? , 04.5,. ’\ . 3 c 3,, i . i O". \m . ‘4 ,. :i a . ,51'5. ' I ) o __ V ‘ 3’ O ‘ 0 Q ’ I K ‘ - l‘ I. . . . ' ( J o I . . .9 O ... g “‘ ’ . O- O ’. , ‘ r .0. o g b a. Q . . \f.‘ .a ’ .0 ’ s o a .4] . a .18 “a . ‘3’ - L .. ‘ u a .0 . . .660 ‘1'. ‘ ‘ i... do 0‘...“" . /-.‘. ' . Q . a {‘3'0 . g ‘ I... . . xi J 0"” .—-"~. ' ‘ ' a, o r - a . .Q . - a ‘ 0 3‘ : a. . o ,a 0 O ‘ . v. . 2 . _ ‘ § I - . (a. ' 1; .L-,. :- . . ~o".0 . - . 1 ’11.? . 0.. o . .\:J . . 5’ a ’ l5) ;' e . Q .C .. .0 g 0 Q o C” . 30 " 5 3 d . . 9 ‘ c. 7;. f In ' 0 $ ‘ . 3., . - . ' . I. c I 1. -4 a ‘ C .0 .15 I. on " Fig 7 - Section from the same liver as Fig 6 (higher mag- nification). Necrosis of hepatocytes evidenced by pyknosis (arrow) and karyorrhexis of nuclei. (8.) . 47 Fig 8 - Fragmentation and granulation of cardiac fibers. Note loss of striations. 9.33.. HH. 1 13.311.521.11. @111 memwt 1:; e. 1n _. .1 ‘ . s. Fig 9 - Higher magnification of the same heart as in Fig 8. Note calcification of cardiac fibers. DISCUSSION This research confirms evidence that vitamin E and Se have a role in reproduction in swine.50'83 It also pro- vides information that a dietary deficiency of these nutri- ents is related to the MMA problem in swine. Additional evidence was obtained that the quality of feed as reported 11,42 by Ringarp has an important role in increasing the requirements of swine for vitamin E and Se. Gilts' Performance The general reproductive performance of gilts sup- ported previous observations that vitamin E and Se in- creased the number of live pigs born and surviving to 3 50 weeks of age. These nutrients also reduced the incidence of MMA in gilts which confirms previous experiments and 50’51 From the general behavior of gilts in observations. both groups, it is concluded that assessment on whether or not gilts had MMA was difficult. Whereas the clinical manifestations (mastitis and metritis) were not as evident . . . . 11,18,19 as antiCipated to occur according to the literature, agalactia was evidenced in the poor growth rate of baby pigs. It was the single most important factor that reduced 4,18,21 the growth rate and caused death losses. This agrees 48 49 with Ringarp's11 emphasis on the lack of milk production and death losses of young pigs as emphasized by Leman.3 It was of interest that vitamin E and Se supplemented gilts farrowed 2 more and weaned 2.3 more pigs per litter than gilts fed the basal diet. While this was not significant (P = 0.11), it is considered an economidally important factor in the profitability to swine producers.3 Although the number of observations in this research was limited, the data suggest that pigs carried to term are reduced when gilts are fed a diet deficient in vitamin E and Se. This confirms the observations of Ohio researchers.84’85 However, it disagrees with previous observations86 in which no impairment in reproductive performance of sows occurred when a corn-soybean diet was fed without additional inor- ganic Se. Blood Analyses of Gilts Blood parameters obtained from both groups of gilts confirmed that plasma vitamin E and Se values depend dir- ectly upon dietary intake of these nurtients.84 These values decreased in gilts fed either diet during gestation which confirms the previous observations85 that in gravid gilts plasma vitamin E and Se values decline during preg- nancy. Vitamin E, Se and GSH-PX values tended to decrease during lactation in gilts fed the basal diet, while in gilts fed the supplemented diet tended to increase. In general, 50 there was a positive correlation between plasma Se values and GSH-Px activity. This confirms previous observations.87 Plasma Fe values were higher in gilts fed the basal diet during lactation. These gilts had lower MCHC values than gilts fed the supplemented diet. The biological explanation of this observation is not known. However, a defective utilization of Fe and a decreased Fe binding capacity might be involved. Prggennyerformances Excessive losses at parturition and a higher mortal- ity of baby pigs (19.8%) during lactation occurred in gilts fed the basal diet. This supports previous observations that piglets nursing Se deficient sows had a higher mor- tality than piglets nursing Se supplemented sows.84 Al- though baby pigs in both groups had a reduced weight at 21 days of age when higher dosages of Fe were given, tol- erance to the different Fe dosages was observed which con- firms previous work.57 This reduction of pig weight was significantly (P = 0.05) different between pigs in each treatment group. The biological implications of this re- sult are not known. While clinical signs of Fe toxicosis in baby pigs were not observed, a greater incidence of infections was noted in pigs nursing gilts fed the basal diet. This supported the observations that a dietary de- ficiency of vitamin E and Se increases susceptibility to 51 infections or reduces the defense mechanisms.41-47 It was of interest that most of the pigs from which E. coli was isolated were pigs which had been injected with 400 mg of Fe. This confirms earlier observations that Fe injec- tions exacerbate E. coli growth.88’89 Blood Analyses of Baby Pigs Plasma vitamin E and Se values in baby pigs suggest that their plasma antioxidant status depends directly on the dietary status of the dams. This antitoxidant status was not influenced by Fe injections given to pigs which confirms previous observations in rats that oxidative stress associated with elevated dietary Fe intake did not increase these values in blood plasma.90 However, a significant increase of Fe values in plasma of pigs nursing gilts fed the basal diet was observed at 21 days as compared to pigs nursing gilts fed the supplemented diet. Plasma Se and GSH-Px values at birth and at 21 days in pigs from either treatment group confirm that genetic factors control this status within red blood cells and the dependency of baby pigs on Se and GSH-Px status of the dam.91 Of particular interest was that plasma vitamin E values did not change much from birth to 21 days and tended to be higher in pigs nursing gilts fed the supplemented diet. This may be due to the fact that pigs nursing supplemented gilts obtained more vitamin E from colostrum. 52 Borderline values for vitamin E were present in the 'baby pigs nursing gilts fed the basal diet (0.90 ug/ml). The Se borderline values for the gilts were estimated to be about 0.80 ug/ml. Borderline values for Se were con- firmed to be similar to the data previously published on the borderline values for Se in pigs (0.03 - 0.07 ng/ml).72 This research provides preliminary information on borderline vitamin E values expected during a dificiency of this nutrient in swine. It also confirms that death losses in neonatal pigs and preweaning mortality have not been improved lately as indicated in previous reports.21'93 Finally, this research suggests that future investigations should be directed toward a better understanding of non- infectious factors influencing the incidence of MMA in swine. This is in agreement with previous observations on the role of infectious agents in MMA.93 SUMMARY The role of vitamin E and Se fed to gilts during ges- tation and lactation in preventing MMA and death losses in baby pigs was determined in 10 gilts fed a basal diet in comparison to 9 gilts fed the same diet supplemented with vitamin E and Se. The corn used in the basal diet was initially high moisture shelled corn that had been stored anaerobically for several months, then dried arti- ficially and used as dried shelled corn. Gilts fed the basal diet had more clinical signs of MMA, farrowed and weaned smaller litters in which individual pigs were lighter in weight at birth and at 21 days of age than were baby pigs nursing gilts fed supplemental vitamin E and Se. Plasma vitamin E, Se and GSH-PX values were lower at birth and at 21 days in pigs nursing gilts fed the basal diet, and these pigs had a higher incidence of infections. Baby pigs nursing gilts fed the basal diet gained less weight following Fe injections than pigs nursing sup- plemented gilts. There was also a high death loss from birth to 21 days of age in pigs nursing gilts fed the basal diet. Supplementing vitamin E and Se to a corn-soybean basal diet composed of dried high moisture corn reduced the incidence of MMA in gilts, and improved the health and performance of baby pigs from birth to 21 days of age. 53 LI ST OF REFERENCES REFERENCES 1. King NB: Contribution and needs of animal health and disease research. Am J Vet Res 42: 1096-1097, 1981. 2. Martin, CE, Elmore RC: Mammary glands, in Leman AD (ed): Diseases of Swine. Ames, Iowa, Iowa State Univer- sity Press, 1981, pp 155-169. 3. Leman AD, Knudsen C, Rodeffer HE, et al: Repro- ductive performances of swine on 76 Illinois farms. J Am Vet Med Assoc 161: 1248-1250, 1972. 4. Backstrom L, Morkoc A: Postparturient dysgalactia in the sow. 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Marks J: Vitamins in health and disease: A guide to vitamins. Tech Publish Co, Ltd, Cambridge, England, 1975, pp 62-65. 36. de Duve C, Hayaiski O: Toc0pherol, oxygen and biomembranes. Biomedical Press. Elsevier, North Holland, Amsterdam, New York, 1978, pp 23-71. 37. Taylor TG: The importance of vitamins to human health. Int Med Publisher, London, 1978, pp 101-107. 38. Schwarz K, Foltz CM: Selenium as an integral part of Factor 3 against dietary necrotic liver degener- ation. J Am Chem Soc 79: 3293, 1957. 39. Fontenot JP, Coppock CE, Goodrich RD, et a1: Mineral tolerance of domestic animals. Acad of Sci, Wash- ington D. C., 1980, pp 242-252, 392-401. S7 40. Hanahan DJ, De Luca H: The fat soluble vitamins. Handbook of lipid research, Vol 2, Plenum Press, New York and London, 1978, pp 133-197. 41. Van Vleet JF: Current knowledge of Selenium- Vitamin E deficiency in domestic animals. J Am Vet Med Assoc 176: 321-324, 1980. 42. Cunha TJ: Swine feeding and nutrition. Academic Press, New York, 1977, pp 127-130. 43. Nockels CF: Protective efforts of supplemental vitamin E against infection. Fed Proc, 38: 2134-2138, 1979. 44. Ellis RP, Vorhies MW: Effect of supplemental vitamin E on the serologic response of swine to an E. coli bacterin. J Am Vet Med Assoc 168: 231-232, 1976. 45. Peplowski MA, Mahan DC, Murray FA, et al: Effect of dietary and injectable vitamin E and selenium in weanling swine antigenetically challenged with sheep red blood cells. J Anim Sci 51: 344-351, 1981. 46. Teige J Jr, Nordstoga K, Aursjo J: Influence of diet on experimental swine dysentery. 1. Effects of a vitamin E and selenium deficient diet supplemented with 6.8% cod liver oil. Act Vet Scand 18:384-396, 1977. 47. Teige J Jr, Saxegaard F, Froslie A: Influence of diet on experimental swine dysentery. 2. Effects of a vitamin E and selenium deficient diet supplemented with 3% cod liver oil, vitamin E or selenium. Act Vet Scand 19: 133-146, 1978. 48. Cunha TJ, Becker DE, Bowland JP, et al: Nutri- ent Requirements of Swine. Nat Acad Sci, Washington D. C., 1968, pp 12-13. 49. Cunha TJ, Baumgardt BR, Bell JM, et al: Nutrient Requirements of Swine. Nat Res Counc (NRC), WashingtonD. C. , 1973, pp 11-12. 50. Ullrey DE: Vitamin E and MMA. Rep of Swine Res Dept of Anim Sci, Michigan State University, East Lansing, 99: 10-13, 1969. 51. Ullrey DE: Vitamin E (Selenium and Choline) in reproduction and MMA. Rep of Swine Res Dept of Anim Sci, Michigan State University, East Lansing 99: 10-13, 1969. 58 52. Young LG, Lun A, Pos J, et al: Vitamin E stab- ility in corn and mixed feed. J Anim Sci 40: 495-499, 1975. 53. Beck WS: Hematology. The Mitt Press, Cambridge, 1978, PP 165-173. 54. Whitehair CK, Miller ER: Nutritional deficiencies, in Leman AD (ed): Diseases of Swine. Ames, Iowa, Iowa State University Press, 1981, pp 656-670. 55. Miller ER: Baby pig anemia and the need for iron. Cont Anim Health News Conf, 1980. 56. Miller ER, Waxler GL, Ku PK, et al: Iron require- ments of baby pigs reared in germ-free or conventional environments on a condensed milk diet. A Anim Sci 54: 106- 115, 1982. 57. Cook RW: IrOn Tolerance in the Young Pig. M. S. Thesis. Dept of Path, Michigan State University, East Lansing, 1974. 58. Arpi T, Tollerz G: Iron poisoned in piglets. Autopsy findings in experimental and spontaneous cases. Acta Vet Scand 6: 360-373, 1965. 59. Behrens H: Pigs poisoned by iron injection. J Am Vet Med Assoc 132: 169-170, 1958. 60. Blandford TB, Lodge GA: Acute hepatic necrosis following an iron dextran injection. Vet Rec 78: 117, 1966. 61. Lannek N, Lindberg P, Tollerz G: Lowered resis- ance to iron in vitamin E-deficient piglets and mice. Nature 195: 1006-1007, 1962. 62. Henriksson K: Poisoning of piglets during iron therapy. Finsk Veterinartids Krift 68: 293-297, 1962. 63. Tollerz G, Lannek N: Protection against iron toxicity in vitamin E-deificnet piglets and mice by vita- min E and synthetic antioxidants. Nature 201: 846-847, 1964. ‘ 64. Ullrey, DE: Vitamin E for swine. J Anim Sci 53: 1039-1052, 1981. 65. Worthington R, Bonnie S: Contemporary develop- ments in nutrition. The CV Mosby Co., St. Louis, 1981, pp 135-158. S9 66. Gryglewski RJ, Vane JR: The evolution of ideas in prostaglandin research, in Ruzyllo E (ed): Materia Me- dica Polona, The Polish J of Med and Phar 12: 159-168, 1980. 67. Gallavan RH Jr, Jacobson ED: Prostaglandins and the splanchnic circulation. Proc Soc Exp Biol Med 170: 391-397, 1982. 68. Takeuchi K, Svanes K, Critshlow J, et al: Pro- staglandins stimulate and inhibit acid secretion in am- phibian fundic mucosa. Proc Soc Exp Biol Med 170: 398- 404, 1982. 69. Reines HD, Halushka PV, Cook JA, et al: Plasma thromboxane concentrations are raised in patients dying with septic shock. Lancet 2: 174-175, 1982. 70. Lipinski B, Macklin LJ: Enhanced susceptibility to endotoxin-induced intravascular coagulation in vitamin E deficiency. Med Sci 9: 122-123, 1981. 71. Sharp BA, Young LG, van Dreumel AA: Effect of supplemental vitamin E and selenium in high moisture corn diets on the incidence of mulberry heart disease and hepa- tosis dietetica in pigs. Can J Comp Med 36: 393-397, 1972. 72. Moran ET, Carlson HC, Pettit JR: Vitamin E-Sel- enium deficiency in the duck aggravated by the use of high- moisture corn and molding prior to preservation. Avian Dis 18: 536-543, 1974. 73. McGovern JJ, Jones AR, Steinberg AG: The hemato- crit of capillary blood. New Eng J Med 253: 308, 1955. 74. Crosby WH, Munn JI, Furth FW: Standardizing a method for clinical hemoglobinometry. U.S. Armed Forces Med J 5: 693, 1954. 75. Whetter PA, Ku PK: Procedure for plasma/serum aC-tocopherol determinations. Dept of Anim Sci, Michigan State University, East Lansing, 1982. 76. Taylor SL: Sensitive method for tissue toco- pherol analysis. Lip 11: 530-538, 1976. 77. Whetter PA, Ullrey DE: Improved fluorometric method for determiing selenium. J Assoc Off Anal Chem 61: 927-930, 1978. 60 78. Olson AD, Hamlin WB: A new method for serum iron and total iron binding capacity by atomic spectrophoto- metry. Clin Chem 15: 438-444, 1969. 79. Paglia DE, Valentine WN: Studies on the quanti- tative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab and Clin Med 70: 158-169, 1967. 80. Lawrence RA, Sunde RA, Schwartz GL, et a1: Glu- tathione Peroxidase Activity in rat lens and other tissues in relation to dietary selenium intake. Exp Eye Res 18: 563-569, 1974. 81. Luna LL: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill Book Co., New York, 1968. 82. Fredman D, Pisani R, Purves R: Statistics. W. W. Norton and Co., Inc., New York, 1978, pp 146-166. 83. Whitehair CK: Role of vitamin E and selenium in swine diseases. J Am Vet Med Assoc 158: 1853-1854, 1971. 84. Mahan DC, Pehale LH, Clive JH, et al: Efficacy of supplemental selenium in reproductive diets on sow and progeny performance. J Anim Scie 39: 536-543, 1974. 85. Piatowski TL, Mahan DC, Cantor AH, et a1: Sel- enium and vitamin E in semipurified diets for gravid and nongravid gilts. J Anim Sci 48: 1357-1365, 1979. 86. Wilkinson JE, Bell MC, Masincupp FB: Effects of supplemental selenium on swine. I. gestation and lac- tation. J Anim Sci 44: 224, 1977. 87. Sivertsen T, Karlsen JT, Froslie A: The rela- tionship of erythrocyte glutathione peroxidase to blood selenium in swine. Act Vet Scand 18: 494-500, 1977. 88. Klasing KC, Knight CD, Forsyth DM: Effects of iron on the anticoli capacity of sow's milk in vitro and in ligated intestinal segments. J Nutr 110: 1914-1921, 1980. 89. Kadis S, Dressen DW, Shotts EB: Relationship between administration of iron to baby pigs and their sus- ceptibility to enterotoxic colibaciosis. Res Inv Rep. Iowa, 1983, pp 33-34. 90. Lee YH, Layman DK, Bell RD, et al: Response of glutathione peroxidase and catalase to excess dietary iron in rats. J Nutr 111: 2195-2202, 1981. 61 91. J¢rgensen FOgd P, Hyldgaard-Jensen J, et al: Glutathione peroxidase activity in porcine blood. Act Vet Scand 18: 323-334, 1977. 92. Underwood EJ: Trace elements in human and animal nutrition. Academic Press, New York, 1971, pp 40-41. 93. Ross RF: Vaccination for control of mastitis and agalactia of the sow. Res Inv Rep. Iowa 1983, pp 31-32. VITA Oswaldo Emiro Vale Echeto was born in Barquisimeto, Lara, Venezuela, on December 19, 1950. He attended Cristo Rey Primary School and Pedro E. Coll High School, and re- ceived a degree in Veterinary Medicine (DVM) from the Uni- versity of Zulia in 1976. After graduation, he worked as a veterinary officer with the Ministry of Agriculture on "Tuberculosis and Bru- cellosis" in the State of Zulia until 1980. He came to the U.S.A. in 1980, and pursued English courses for a year at the University of Toledo, in Ohio, and then commenced graduate studies at Michigan State University in June 1981. He expects to return to Venezuela in the summer of 1983 with the possibility of a university or government position. His address will be Calle 60 No. 3D-80, Sector Bella Vista, Maracaibo, Estado Zulia, Venezuela; or "C0- linas de los Chaguaramos," Calle Autocine, Quinta, VI-VA-VE No. 02-12, Caracas D. F., Venezuela. 62 APPENDIX TABLE A-l. PLASMA SELENIUM (Se), VITAMIN E, GLUTATHIONE PEROXIDASE (GSH-PX) ACTIVITY AND IRON (Fe) INDIVIDUAL VALUES OF GILTS DURING GESTATION AND LACTATION. MSU BASAL DIETa PRODUCTION GILT Se, VITAMIN E GSH-Px Fe PERIOD NO. ugflml HSAml EU/ml Bg/dl Gestationc 209-2 .139 2.95 (30 Days) 211-4 .144 1.30 103-4 .163 2.48 208-1 .192 2.10 148-1 .099 4.76 Gestationc 209—2 .134 1.15 (100 Days) 211-4 .133 .60 103-4 .126 .77 208-1 .131 .83 148-1 .118 .99 Lactation 209-2 .136 1.10 .423 122.1 (21 Days) 211-4 .093 .97 .659 99.0 103-4 .061 .68 .608 100.8 208-1 .079 .33 .442 65.5 148-1 .076 .35 .486 95.3 206-1 .079 .78 .346 86.7 146-3 .056 .86 .193 102.0 211-2 .123 N.D. .824 90.9 147-5 .056 1.43 .299 137.4 107-1 .055 1.14 .441 128.4 + VITAMIN E-Se DIETb GestationC 208-3 .197 2.04 (30 Days) 209—1 .065 1.43 211-1 '.203 2.05 148-2 .208 2.06 103-1 .133 3.11 Gestationc 208-3 .176 1.53 (100 Days) 209-1 .169 1.61 211-1 .177 2.10 148-2 .186 2.10 103-1 .140 2.21 Lactation 208-3 .218 2.54 .771 89.1 (21 Days) 211-1 .176 2.65 1.093 99.6 148-2 .211 1.00 .973 109.8 146-1 .164 1.73 .778 84.0 214-1 .153 1.76 .611 82.5 206-2 .232 2.93 1.471 58.8 211-3 .196 1.66 .892 71.1 147-2 .203 1.69 .695 100.2 107-3 .224 2.18 1.235 111.0 aDried shelled corn-soybean bDried shelled corn-soybean supplemented with E-Se. c = GSH-Px and Fe values were not determined in either group. N.D. = Non-detectable. 64~ TABLE A-Z. PLASMA.SELENIUM (Se): VITAMIN E, GLUTATHIONE PEROXIDASE (GSH-PX) ACTIVITY AND IRON (Fe) INDIVIDUAL VALUES OF BABY PIGS AT BIRTH NURSING GILTS FED THE BASAL DIET. Se Vitamin E GSH-PX Fe No . of Pig "g/ml “g/ml EU/ml Mg/d1 140-1 .107. .36 .161 63.6 140-2 .034 .53 .198 62.8 140-3 .048 .13 .124 54.0 140-10 .057 .47 .164 53.3 140-11 .079 .21 .138 59.1 140-12 .070 .63 .146 69.8 140-13 .104 .32 .125 50.9 141-1 .042 .30 .080 62.70 141-2 .059 .40 .098 2 141-3 .035 .50 .069 a 141-4 .022 .40 .029 a 141-5 .037 .40 .074 53.10 141-6 .035 .65 .015 58.80 141-7 .034 .76 .113 a 141-8 .016 .58 .072 a 141-10 .030 .30 .095 80.40 141-11 .036 .50 .068 a 141-12 .031 .20 .045 a a - values not expressed: not enough plasma 145-1 145-2 145-3 145-4 145-5 145-6 145-10 145-11 145-12 145-13 145-14 145-15 145-16 .039 .039 .031 .047 .049 .019 .029 .022 .026 .023 .027 .032 .029 1.53 2.49 2.33 1.88 .65 1.48 1.36 1.52 2.20 2.68 1.05 1.65 1.58 .088 .096 .072 .064 .088 .056 .104 .088 .080 .016 .064 .072 .072 a = values not expressed; not enough plasma 65 TABLE A-2. (continued) Se . Vitamin E GSH-Px Fe N0. of Pig “gflml “gflml EU/ml ”gldl 146-1 .028 2.68 .152 a 146-2 .029 1.72 .144 8 146-3 .023 2.99 .144 a 146-4 .024 2.76 .136 a 146-5 .022 3.40' .168 a 146-6 .025 2.36 .192 a 146-10 .032 3.00 .217 70.20 146-11 .038 2.44 .201 a a a values not expressed: not enough plasma 147-1 .023 1.87 .128 68.40 147-2 .021 a .096 a 147-3 .014 .42 .056 59.40 147-10 .033 .39 .104 94.80 147-11 .035 1:76 .104 85.20 a - values not expressed; not enough plasma 148-1 .024 a .072 46.92 148-2 .032 a .112 28.02 148-3 .027 a .128 a 148-10 .017 1.21 .088 a 148—11 .021 1.95 .144 39.42 148-12 .017 .64 .088 34.92 148-13 .018 .59 .080 45.65 148-14 .028 1.03 .072 45.65 148-15 .014 2.65 .064 a 148-16 .026 1.44 .144 a 148-17 .054 1.09 .096 a 148-18 .043 3.12 .088 a a a values note expressed; not enough plasma 149-1 .041 .67 .195 96.60 149-2 .026 .72 .120 88.20 149-3 .019 .45 .188 65.10 149-4 .029 .99 .182 64.50 TABLE A-2. (continued) 66 Se Vitamin E GSH-Px Fe No. of Pig. “g/ml (lg/m1 EU/ml liq/d1 150-1 .029 1.25 .120 78.35 150-2 .035 1.31 .088 92.15 150-3 .027 .10 .120 a 150-4 .027 .63 .064 a 150-5 .034 .33 .096 a 150-6 .033 .70 .112 a 150-10 .029 1.79 .096 a 150—11 .029 .10 .120 68.45 ISO-12 .024 .76 .128 64.25 150-13 .027 1.70 .112 63.35 150—14 .033 2.34 .128 54.85 a a value not expressed: not enough plasma 154-1 .021 .50 .088 74.52 154-2 .032 .52 .104 8 154-3 .038 .52 .104 8 154-4 .044 .48 .168 51.42 154-5 .021 .50 .120 a 154-6 .021 .13 .088 a 154-10 .031 .73 .088 62.22 154—11 .030 .95 .104 a a - value not expressed: not enough plasma 163-10 .036 .91 .133 a 163-11 .026 1.81 .158 a 163-12 .023 1.81 .124 a a a value not expressed; not enough plasma TABLE A-3. PLASMA.SELENIUM (Se), VITAMIN E, GLUTATHIONE PEROXIDASE 67 (GSH-Pat) ACTIVITY AND IRON (Fe) INDIVIDUAL VALUES OF BABY PIGS AT 21 DAYS OF AGE NURSING GILTS FED THE BASAL DIET. Se Vitamin E GSH-PX Fe No. of Pig “9/m1 “9/m1 EU/ml ”/61 140-1 .060 1.00 .410 166.5 140-2a -- -- -- -- 140-3 .056 1.35 .410 79.3 140-10 .063 1.29 .487 224.5 140-11 .046 .74 .344 109.4 140-12 .043 .85 .315 183.3 140-13 .063 .30 .328 143.3 (a) Pig 140-2 died before 21 days of age. 141-1 .043 .56 .275 261.0 141-2 .041 .38 .307 167.6 141-3 .040 .50 .195 79.2 141-4 .039 .41 .228 194.4 141-5 .040 .36 .169 114.4 141-6 .055 .28 .201 170.2 141-7 .050 .38 .317 170.8 141-8 .037 .17 .251 175.2 141-10at -- -- -- -- 141-11 .057 .28 .252 32.7 141-12 .071 .55 .235 90.7 (a) Pig 141-10 died before 21 days of age. 145-1 .038 1.90 .305 151.8 145-2 .039 :73 .297 a 145-3 .043 1.79 .233 145.8 145-4 .036 1.50 .273 83.1 145-10 .037 2.48 .289 140.7 145-11 .043 2.07 .210 179.4 145-12 .044 1.00 .185 a 145-13 .044 .82 .265 117.9 145-14 .041 1.09 .153 184.2 145-16 .041 1.99 .169 a a = value not expressed; not enough plasma 146-1 .060 1.11 .337 147.9 146-2 .061 1.44 .273 174.6 146-3 .058 1.29 .281 152.7 146-4 .060 1.62 .265 153.6 146-5 .056 1.17 .337 127.5 146-6 .045 1.54 .209 111.0 146-10 .046 1.42 .249 115.2 68 TABLE A-3. (continued) Se Vitamin E GSH-Px Fe no. of Pig “gflml ug/ml EU/ml “gldl 147-1 .067 1.27 .426 88.8 147-2 .059 .83 .297 -130.8 147-3 .066 1.09 .362 119.7 147-10 .062 1.03 .321 135.0 147-11 .080 1.64 .378 96.3 148-1 .038 1.40 .313 165.3 148-3 .037 .97 .129 144.9 148-10 .035 1.59 .113 138.0 148-11 .033 1.45 .289 150.6 148-12 .052 1.52 .201 159.0 148-13 .039 1.08 .305 131.7 148-14 .052 1.01 .350 166.2 148-15 .040 1.67 .357 108.6 149-1a -- -- -- -- 149-2a -- -- -- -- 149-3a -- -- -- -- 149-4a -- -- -- -- (a) The entire litter died within 3 days of birth. 150-1 .031 .90 .339 129.6 150-2 .028 1.01 .320 a 150-3 .026 .90 .148 156.3 150-4 .037 .85 .360 a 150-4 .027 .83 .326 8 150-6 .026 .79 .307 a 150—10 .029 .70 .212 a 150-12 .043 .90 .365 242.4 150-13 .031 1.05 .195 a a = value not expressed; not enough plasma 154-1 .053 1.14 .341 98.7 154-2 .030 .70 .211 133.2 154-3 .050 .88 .217 128.1 - 154-4 .036 .93 .424 71.0 154-5 .033 1.10 .158 a 154-10 .039 .88 .347 76.5 154-11 .042 ‘1.34 .503 81.6 a a value not expressed; not enough plasma TABLE A-3. (continued) 69 Se Vitamin E GSH-Px Fe No. of Pig “9/m1 "9/m1 EU/ml "9/d1 163-10 .061 N.D. .595 127.8 163-11 .059 .08 .463 118.2 163-12 .064 .02 .396 232.8 N.D. =- Non-Detectable values 70 TABLE A-4. PLASMA SELENIUM (Se), VITAMIN E, GLUTATHIONB PEROXIDASE (GSH-Rx) ACTIVITY AND IRON (Fe) INDIVIDUAL VALUES OF BABY PIGS AT BIRTH NURSING GILTS FED THE VITAMIN E AND Se SUP- PLEMENTED DIET. Se Vitamin E GSH-PX Fe No. of Pig “9/m1 “9/m1 EU/ml us/dl 138-1 .108 2.64 .270 66.20 138-2 .080 2.54 .132 74.40 138-3 .139 2.87 .158 66.45 138-4 .115 1.12 .170 67.35 138-5 .088 1.61 .105 63.75 138-6 .082 2.39 .146 69.45 138-7 .084 2.58 .280 55.50 138-8 .081 1.51 .193 75.00 138-9 .078 1.92 .080 66.30 138-10 .083 1.14 .127 72.30 138-11 .103 3.81 .114 64.50 138-12 .156 1.02 .145 70.80 142-1 *a 1.42 .164 67.7 142-2 "a 1.00 .161 35.4 142-3 .083 .26 .187 58.3 142-4 'a 1.09 .204 51.7 142-5 .086 .66 .153 50.4 142-6 .076 .88 .195 90.0 142-7 .078 .34 .215 a- 142-10 .063 .75 .204 52.5 142-11 .061 .67 .164 45.2 142-12 .057 .50 .135 a“ a = value not expressed; not enough plasma 143-1 .041 2.07 .098 a" 143-2 .049 1.60 .082 57.65 143-3 .033 1.23 .066 3 143-4 .024 1.66 .076 62.45 143-5 .042 2.27 .076 53.15 143-10 .045 2.74 .151 54.35 143-11 .063 2.64 .109 81.65 143-12 .078 2.40 .116 a— 143-13 .026 2.49 .105 49.25 143-14 .055 3.67 .158 a~ a = value not expressed; not enough plasma 71 TABLE A-4. (continued) Se Vitamin E GSH-PX Fe No. of Pig “glml ug/m1 EU/ml ”/81 144-1 .054 .81 .104 63.00 144-2 .037 2.40 .148 61.80 144-3 .059 2.66 .153 81.00 144-10 .046 .63 .096 95.40 144-11 .050 .99 .120 81.60 144-12 .024 1.36 .128 84.00 155-1 .034 1.20 .152 ~a 155-2 .036 3.84 .136 58.02 155-3 .057 .83 .136 56.82 155-4 .041 .52 .144 52.92 155-5 .045 .27 .120 a 155-10 .035 .59 .096 75.42 155-11 .045 .59 .072 57.12 155-12 .032 1.82 .104 'a 155-13 .039 1.58 .120 76.92 155-14 .035 1.62 .136 117.12 155-15 .064 1.91 .144 ‘g 155-16 .034 1.02 .072 'a a a value not expressed; not enough plasma 159-1 .052 .83 .175 69.30 159—2 .048 .99 .127 74.10 159-3 .043 1.10 .164 78.00 159—4 .057 .94 .159 105.00 159-5 .049 .62 .095 93.00 159-10 .049 .80 .121 105.30 159-11 .042 .72 .072 77.70 159—12 .044 1.46 .146 117.30 159-13 .068 1.49 .183 71.40 159-14 .039 1.92 .162 96.60 160-1 .073 1.14 .187 70.62 160-2 .009 1.00 .180 80.92 160-3 .058 .63 .198 a 160-4 .045 .86 .172 a 160—10 .052 .86 .166 50.22 160-11 .063 .73 .275 65.52 160—12 .012 1.40 .129 61.02 160-13 .107 .93 .122 62.52 160-14 .076 1.13 .166 78.12 160-15 .107 .79 .106 63.72 160-16 .101 1.21 .179 87.12 160-17 .039 .83 .169 46.02 a = value not expressed; not enough plasma 72 TABLE A-4. (continued) Se Vitamin E GSH-Px Fe N0. of Pig “gflml “gflml EUIml “gflml 161-1 .100 4.31 .361 a- 161-2 .074 5.13 .337 a” 161-10 .083 4.29 .313 117.9 161-11 .082 4.23 .225 91.8 161-12 .062 2.91 .281 92.7 161-13 .060 3.35 .241 91.2 161-14 .069 1.35 .209 63.9 a a value not expressed; not enough.p1asma 162-1 162-2 162-3 162-4 162-5 162-6 162-7 162-8 162-9 162-10 162-11 162-12 162-13 .038 .065 .047 .046 .022 .049 .044 .039 .048 .039 .042 .046 .039 uwNNUNNwHI-‘wa NNWNIme-‘WO‘UINO‘D bNNwOO‘D-‘O‘Hmflbm .289 .281 .193 .209 .137 .193 .137 .152 .169 .193 .161 .177 .177 112.5 78.3 79.8 66.3 69.3 90.0 64.2 72.8 70.5 62.7 78.3 94.8 93.9 73 TABLE A-S. PLASMA SELENIUM (Se), VITAMIN E, GLUTATHIONE PEROXIDASE (GSH-Px) ACTIVITY AND IRON (Fe) INDIVIDUAL VALUES OF BABY PIGS AT 21 DAYS OF AGE, NURSING GILTS FED A VITAMIN E-Se SUPPLEMENTED DIET. Se Vitamin E GSH-Px Fe No. of Pig “9/m1 “g/ml EU/ml “9/61 138-1 .083 1.26 .233 75.3 138-2 .088 3.31 .378 a 138-3 .078 2.10 .297 8 138-4 .075 1.86 .233 86.4 138-5 .090 2.41 .297 198.8 138-6 .079 1.41 .370 119.4 138-7 .095 1.92 .257 75.0 138-8 .086 3.23 .289 164.1 138-9 .081 2.96 .233 102.3 138-10 .085 3.46 .321 165.3 138-11 .091 1.88 .426 110.4 a = value not expressed; not enough plasma 142-1 .080 2.08 .442 119.1 142-2 .072 2.31 .402 118.2 142-3 .067 2.44 .265 178.2 142-4 .068 1.55 .337 143.4 142-5 .062 1.56 .396 131.4 142—10 .064 2.03 .265 96.6 142-11 .066 1.21 .570 79.8 142-12 .064 2.31 .361 102.3 143—1 .072 1.33 .570 116.1 143-2 .064 1.99 .321 8 143-3 .062 1.12 .394 74.1 143—4 .077 2.06 .401 149.1- 143-5 .074 2.53 .595 79.2 143-10 .077 2.47 .482 123.9 143-11 .076 1.66 .426 86.1 143-12 .054 2.01 .410 127.2 143-13 .077 2.17 .579 109.1 143-14 .103 2.66 .635 a a = value not expressed; not enough plasma 144-1 .077 1.35 .201 134.1 144—2 .058 1.04 .257 108.0 144-3 .065 1.45 .176 104.6 144-10 .068 .92 .378 50.4 144-11 .072 1.25 .217 102.0 144-12 .070 1.58 .370 129.3 74 TABLE A-S. (continued) Se Vitamin E GSH-Px Fe N0. of Pigs “gflml “glml EU/ml u'g/dl 155-1 .066 2.19 .482 86.4 155-2 .066 2.68 .616 97.8 155-3 .072 2.41 .225 109.2 155-10 .066 1.55 .254 71.4 155-11 .074 2.10 .334 96.0 155-13 .069 2.23 .367 85.2 155-14 .069 1.95 .153 114.0 155-15 .067 1.75 .164 82.5 155-16 .054 1.86 .288 90.6 159—1 .061 2.00 .294 a 159-2 .065 1.99 .392 65.1 159—3 .079 2.12 .314 76.8 159-5 .061 2.50 .241 79.2 159-12 .058 2.10 .289 151.5 159—14 .073 2.05 .201 114.0 a a value not expressed: not enough plasma 160-1 .071 1.87 .489 8 160-2 .076 1.59 .370 a 160-3 .099 2.67 .564 102.6 160-4 .064 1.42 .408 a 160-10 .079 2.43 .471 94.2 160-11 .087 1.72 .754 a 160-12 .073 2.85 .505 183.3 160-13 .072 1.69 .389 a- 160-14 .086 2.35 .564 132.6 160-15 .069 2.08 .523 101.4 160-16 .087 2.03 .566 117.4 160-17 .067 1.47 .481 71.4 a = values not expressed; not enough plasma 161-1 .083 2.33 .367 169.2 161-2 .097 3.59 .392 200.7 161-10 .077 2.60 .373 168.3 161—11 .067 3.71 .429 133.8 161-13 .080 2.31 .270 71.1 75 TABLE A-S. (continued) Se Vitamin E GSH-Px Fe No. of Pig ugflml ”gflml EU/ml “gldl 162-1 .073 1.90 .291 96.6 162-2 .077 2.79 .348 119.1 162-3 .059 1.42 .441 88.2 162-4 .069 2.61 .574 83.4 162-5 .071 3.29 .441 102.0 162-6 .076 2.54 .595 117.6 162-7 .088 2.45 .457 a- 162-8 .078 2.23 .280 79.5 162-9 .076 4.06 .246 94.5 162-11 .085 2.49 .585 77.7 162-12 .069 3.09 .434 a 162-13 .064 2.07 .334 91.2 a = value not expressed; not enough plasma 76 .NBDZHS mmm .mflflmdfi XHszmm4 ZH NM4 mHDA¢> AflDQH>HQZH .mmAQde m0 mmmzbz fl 0 V QQNHDHXO Mmmdfl m0 mflAOZOmUHZ mfl mBHZD MEMsz .mmBBHA mum mflUflMfi>¢ mfl Qmmmmmmxm mmd mmoqfl>d Q 0.00 0000 00.00 000. 000.0 0000 000.0 0000 00.0 0000 00.0 0000 000.0 0000 000.0 0000 000 0.000 000 00.00 000 000.0 000 000.0 .00 00.0 000 00.0 000 000.0 000 000.0 000 000 0.000 000 00.00 0000 000.0 000. 000.0 .000 00.0 0000 00.0 0000 000.0 000. 000.0 0000 000 0.000 00. 00.00 000. 000.0 .00 000.0 0000 00.0 000 00.0 0000 000.0 000 000.0 0000 000 0.00 .00 00.00 000 000.0 .00 000.0 .000 00.0 000 00.0 .000 000.0 000 000.0 0000 000 0.000 00. 00.00 000 000.0 000 000.0 000 00.0 .00 00.0 000 000.0 000 000.0 000 000 0.000 000 00.00 000 000.0 000. 000.0 .000 00.0 000. 00.0 .000 000.0 0000 000.0 0000 000 0.000 00. 00.00 000 000.0 00. 000.0 0000 00.0 000 00.0 0000 000.0 000 000.0 000 000 0.000 0000 00.00 0000 000.0 0000 000.0 0000 00.0 .00. 00.0 0000 000.0 0000 000.0 0000 000 0000 00 000 0 0000 00 000 0 0000 00 000 0 0000 00 000 0 .02 000000 00\0: 00 02\ an 00:000 qzxwa 0 202000> 02\0a 00 0000 00:0 202000> + 0.000 000 -uu .00 000.0 000 000.0 00. 00.0 000 00.0 000 000.0 000 000.0 000 000 00.00 00. 00.00 000 000.0 000 000.0 000 00.0 000 00.0 000 000.0 000 000.0 000 000 0.000 000 00.00 000 000.0 000 000.0 0000 00.0 000 00.0 0000 000.0 000 000.0 0000 000 us 000 00.00 000 nu 000 000.0 000 I: .00 00.0 000 u: .00 000.0 000 000 0.000 .0. 00.00 000 000.0 000 000.0 0000 00.0 000 00.0 .00 000.0 000 000.0 0000 000 0.000 000 00.00 .00 000.0 00. 000.0 000 00.0 000 00.0 000 000.0 00. 000.0 000 000 0.000 000 00.00 000 000.0 .00 000.0 000 00.0 .00 00.0 .00 000.0 000 000.0 000 000 0.000 000 00.00 000 000.0 0000 000.0 .000 00.0 0000 00.0 000. 000.0 0000 000.0 0000 000 0.000 .000 00.00 000 000.0 0000 000.010000 00.0 0000 00.0 0000 000.0 0000 000.0 0000 000 0.000 000 00.00 000 000.0 000 000.0 .00 00.0 000 00.0 000 000.0 000 000.0 000 000 0000 00 000 0 0000 00 000 0 0000 00 000 0 0000 00 000 0 .02 000000 00\0: 00 02\000 001000 0z\0: 0 202000> 02\01 00 0000 00000 002 000 00 0000 00 00 020 00000 00 000000 000 0000 00 000000> 0000 2000 020 000>00O0 00010000 0000000000 02000000000 .0 202000> .0000 20020000 020000 .010 00000 77 TABLE A97. LIVER SELENIUM (Se) INDIVIDUAL VALUES FROM 14 PIGS NECROPSIED Sample Liver Sample Se No. Weight (g) mail (as) ug/g DM BASAL DIET 145-6 5.054 19.22 1.23 146-11 5.167 22.18 1.44 148-17 5.165 18.80 1.08 148-16 5.014 24.42 0.78 149—2 5.514 20.18 1.17 149-3 5.185 20.83 1.25 149—4 5.222 23.17 0.83 150-5 5.127 21.96 0.80 138-2 144-? 155-5 159-10 159-11 159-15 5.090 5.394 5.076 5.176 5.072 5.438 + VITAMIN E-Se DIET 22.01 20.31 23.07 19.76 18.29 19.36 0.94 1.09 1.37 1.45 1.67 1.33 a DM = dry matter basis