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I‘ in .- '?"“.@3 .'.-we .33....«1r3m’l '3‘1561?‘ l ‘II‘I' hi ‘ n..— I. LIBRARY Michigan State University a".-. r This is to certify that the thesis entitled IMPORTANCE OF COLOSTRUM TO THE BIOLOGICAL ANTIOXIDANT STATUS OF THE NEONATAL PIG AND HIS COMPETENCE TO MAINTAIN HOMEOSTATIS FOLLOWING PARENTERAL IRON ADMINISTRATION presented by MARI LYN JEAN LOUDENSLAGER has been accepted towards fulfillment of the requirements for M.S. degree in Animal] SCIENCE (”WWMW Major professor Dr. ‘E."R. Miller Date June 15, 1984 0-7639 MSUis an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: Place in book drop to LlBRARlES remove this checkout from ”- your record. FINES will be charged if book is returned after the date stamped below. Afr/(recon; " ii i‘. A L’!‘ 1 '— MAGIC 2 l MAI 5,2119? IMPORTANCE OF COLOSTRUM TO THE BIOLOGICAL ANTIOXIDANT STATUS OF THE NEONATAL PIG AND HIS COMPETENCE TO MAINTAIN HOMEOSTASIS FOLLOWING PARENTERAL IRON ADMINISTRATION BY Marilyn Jean Loudenslager A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1984 ABSTRACT IMPORTANCE OF COLOSTRUM TO THE BIOLOGICAL ANTIOXIDANT STATUS OF THE NEONATAL PIG AND HIS COMPETENCE TO MAINTAIN HOMEOSTASIS FOLLOWING PARENTERAL IRON ADMINISTRATION BY Marilyn Jean Loudenslager Fifteen second parity sows were used to determine the effects of vitamin E and selenium supplementation of the sow's diet and to examine the importance of this diet and/or colostrum consumption for the neonatal pig's tolerance to parenteral iron. Supplementation of the sow's diet increased her plasma vitamin E and selenium, but did not increase plasma glutathione peroxidaSe activity. Colostrum had greater concentrations of vitamin E (primarily G-toc0pherol) and selenium than mature milk. Pig's pre-colostral biological antioxidant status was very low, but by two days of age had increased, especially in vitamin E. Pigs performance was not affected by the pre-colostral iron injection, however, their plasma vitamin E was lower and their plasma selenium and glutathione peroxidase activity tended to be higher at two days than that of pigs not receiving iron. Supplementation of the dam's diet maintained a higher vitamin E and selenium level in colostrum and milk and a higher biological antioxidant status in pigs throughout lactation. ACKNOWLEDGEMENTS Upon completion of my Masters of Science Degree I wish to extend sincere thanks to Dr. E.R. Miller. His knowledge, patience and subtle motivational support have made the completion of this degree possible. I am also grateful to my committee members, Dr. J.L. Gill, Dr. M.G. Hogberg, Dr. H.D. Stowe and Dr. D.E. Ullrey. The willingness of the committee to give freely of their time, resources and professional expertise were deeply appreciated. I wish to thank Dr. W.T. Magee and Dr. C.K. Whitehair for their input into the finalization of this project. I am indebted to Dr. P.K. Ku and Phyllis Whetter for the professional assistance and advice given in the laboratory. The diligent typing and exceptional cooperation given by Judy Witwer in the preparation of this manuscript was sincerely appreciated. I also wish to thank my fellow graduate students who assisted in the activities involved in this project. I am especially grateful to Patty Dickerson and Kris Johnson for giving so much of their time, knowledge and understanding to this project. The final thank you is extended with a great deal of love and respect to my family. The understanding and appreciation of the livestock industry brought with me from home have played a important role in my education, but of even greater value has been the love and continuous support I received from my family making the completion of this degree a reality. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . REVIEW OF LITERATURE Colostrum . . . . . . . . . . . . . Importance for immunocompetance newborn. . . . . . . . . . . . for the Composition and compositional changes of colostrum. . . . . . . . . . . Protein . . . . . . . . . . Fat . . . . . . . . . . . . Vitamins. . . . . . . . . . Vitamin A. . . . . . . . Vitamin D. . . . . . . . Vitamin E. . . . . . . Water soluble vitamins. . Minerals. . . . . . . . . . Selenium . . . . . . . . Other minerals . . . . . Others. . . . . . . . . . . Lactose. . . . . . . Carnitine. . . . . . Biological Antioxidants . . . . . . Functions . . . . . . . . . . . Production of free radicals Naturally occuring . . . Iron induced . . . . . . Defense mechanisms . . . Lipid peroxidation. . . . . Initiation . . . . . . . Secondary initiation . Defense mechanism. . . Cyclic propigation . Defense mechanisms . . Vitamin E . . . . . Other antioxidants. Importance of vitamin E and swine. . . . . . . . . . . Reproduction . . . . . . Baby pig survival. . . . Source. . . . . . . . . . . Vitamin E. . . . . . . . Selenium . . . . . . . Requirement . . . . . . . . MATERIALS AND METHODS . . . . . . . SW8. 0 O I O O O O O O O O O O Pigs. O O O O O O 0 O 0 O O O O «>@CDO\anUh>¢>b Laboratory analysis . . Hematology. . . . . Sample storage. . . Plasma analysis . Sow colostrum and milk analysis Statistical analysis. RESULTS AND DISCUSSION. . . Sows . . . . . . . . . Hematology. . . . . Plasma selenium . . Plasma vitamin E. . Plasma glutathione peroxidase sow Mi 1k 0 O O O O O 0 O Selenium. . . . . . Tocopherols in milk and colostrum . Colostrum and milk lipids Tocopherols expressed per gram of lipid Toc0pherol composition in colostrum and milk . . .. . . . . Pigs. . . . . . . . . . Hematology. . . . . Biological antioxidants Plasma iron . . . . weight. 0 O O O 0 0 CONCLUSIONS . . . . LIST OF REFERENCES. . . ii Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table 10. ll. 12. l3. 14. 15. LIST OF TABLES Composition of Diets. . . . . . . . . . . Effects of Dietary E & Se Supplementation on 2nd Parity Sow's Hematology. . . . . . Effects of Dietary E & Se Supplementation on 3rd Parity Sow's Hematology. . . . . . Effects of Dietary E & Se Supplementation on 2nd Parity Sow's Plasma. . . . . . Effects of Dietary E & Se Supplementation on 3rd Parity Sow's Plasma. . . . . . . . Effects of Dietary E & Se Supplementation of Sows on Milk Selenium. . . . . . . . . Effects of Dietary E & Se Supplementation of 2nd Parity Sows on Milk Toc0pherol . . Effects of Dietary E~& Se Supplementation of 3rd Parity Sows on Milk Tocopherol . . Effect of Dietary E & Se Supplementation of Sows on Percent Milk Fat . . . . . . Effects of Dietary E & Se Supplementation of 2nd Parity Sows on Milk Fat Tocopherol Effects of Dietary E & Se Supplementation of 3rd Parity Sows on Milk Fat Tocopherol Effect of Dietary E & Se Supplementation on the Composition of Milk Tocopherol . . Effects of E & Se Supplementation of the Sow's Diet and Time of Iron Injection on Hematology of Offspring . . . . . . . . . Effects of E & Se Supplementation of the Sow's Diet and Time of Iron Injection on Plasma Antioxidant Levels . . . . . . . . Effects of E & Se Supplementation of the Sow's Diet and Time of Iron Injection on Iron Level and Weight of the Offspring. . Effect of Dietary E & Se Supplementation on 2nd Parity Sow's Performance . . . . . Effect of Dietary E & Se Supplementation on 3rd Parity Sow's Performance . . . . iii 50 51 52 58 64 65 69 71 72 76 80 82 9O 92 93 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. 11. LIST OF FIGURES Reaction Involved in Measuring GSH-Px ACtiVitYo O O O O O O O O O O I O O O O O Sow Plasma (2nd Parity) . . . . . . . . . Milk Selenium . . . . . . . . . . . . . . HPLC Trace for Tocopherols in Colostrum . HPLC Trace for Tocopherols in Milk. . Milk Tocopherols Per Gram of Milk (2nd Parity Sows). . . . . . . . . . . . . . . Total Milk Lipids (2nd Parity). . . . . . Milk Tocopherols Per Gram of Fat (2nd Parity SOWS) C O O O O O O O O O O O 0 Change in Milk Tocopherol Composition . . Changes in Sow's Milk and Sow's and Pig's Plasma Selenium Levels (2nd Parity) . . . Changes in Plasma GSH-Px Activity in Pigs (2nd ParitY). O O O O O O O O O O O O O 0 Changes in Sow's Milk and Sow's and Pig's Plasma Tocopherol (2nd Parity). . . . . iv 38 S6 60 62 63 66 7O 73 77 84 85 87 Introduction Parenteral or oral administration of iron to young, nursing pigs is a common management practice in the swine industry. However Scandanavian researchers have reported toxic effects, including waxy muscle degeneration and transudations into the pericardium and thoracic cavities following iron administration to nursing pigs. Death losses, often involving the majority of the litter, occurred 8-12 hours after the administration of a commonly accepted form and level of iron (Lannek et al., 1962: Tollerz. 1973). The toxic effects of iron are thought to be a result of concomitant vitamin E and selenium deficiency in the sow's diet (Tollerz and Lannek, I964). Tollerz (1973) reported that adequate vitamin E and selenium levels and low quantities of polyunsaturated fatty acids in sow's diet prevented iron toxicosis in the nursing pigs. Iron toxicosis has been difficult to produce in the United States when levels of vitamin E and selenium are low in the sow's diet unless massive doses of iron are given to the pigs (Cook et al., 1981). However, there has been a concern that pre-colostral pigs may be especially sensitive to parenteral iron administration. This study was designed to determine the nepnatal pigs' ability to deal with a parenteral iron injection and the role of colostrum in the biological antioxidant status of the newborn pig. Literature Review I. Colostrum A. Importance for immunocompetence for the newborn. The neonatal pig is devoid of immunoglobulins and antibodies against specific pathogens because the epitheliochorial placentation of the fetal pig does not permit their transfer from the dam (Miller, 1982). Furthermore, pigs are not capable of producing their own antibodies until they are two (Wilson, 1974) to three weeks of age (Brown et a1., 1961, Miller et a1., 1962). Therefore, pigs are dependent upon a supplemental source of immunoglobulins, provided to them through colostrum, to protect them from disease during the first weeks of life. Colostrum is known to have a high immunoglobulin content (Jensen and Pederson, 1979). The immunoglobulins and antibodies present in colostrum are produced by the sow's immune system. Therefore, factors affecting the sows immunity in the weeks prior to parturition will determine the antibody content of colostrum (Wilson, 1974). These factors may be exposure of the sow to viruses or vaccination, either of which will cause the sow's immune system to produce antibodies against specific pathogens. The nutritional regimen of the sow may also affect her ability to produce antibodies. A recent review by Nockels (1980) suggests that vitamins A and E play a role in 3 increasing the synthesis of antibodies in animals. Colostral antibodies have an enteric effect (Wilson, 1974), but are also known to be passed into the portal system unchanged (Miller et a1., 1962; Jensen and Pederson, 1979; Brown et a1., 1961) thereby increasing the serum antibody titer of the pig. For the pig to obtain maximum immunological protection, it must consume colostrum within the first 21-36 hours after birth (Miller et a1., 1962; Asplund et a1., 1962; Lecce, 1966; and Wilson, 1974). After this time the composition of the colostrum and the physiology of the gut have changed and limit absorption of intact immunoglobulins (Jensen and Pederson, 1979). A The importance of the consumption of sufficient amounts of colostrum by the neonate within the first hours of life has been demonstrated by Perry and Watson (1967) who reported increased growth rates and lower mortality in 12- hour old pigs with higher serum antibody levels. Blecha and Kelly (1981) supported these data in a study which found that pigs which died prior to 21 days of age had lower serum Y-globulin concentration in their first day of life than those which lived. Colostrum as a source of immunoglobulins to the neonate is very important for both growth and survival. However, it is important to be aware of the other components of colostrum, and to investigate the role they play in 4 enhancing the survival of the neonatal pig. B. Composition and compositional changes of colostrum. The composition of colostrum and milk is highly variable. This variability can be attributed to genetics, stage of lactation, nutrient levels in gestation and lactation diets as well as environmental factors (Pond and Houpt, 1978). 1. Proteins As one would expect in regard to the earlier discussion of colostral immunoglobulins, the protein content of colostrum is much higher than that of mature milk. Perrin (1955) reported 19% protein in sow colostrum as compared to- 6% found in milk by the second week of lactation. 2. Fat The fat content of colostrum and milk are quite variable (2.7-7.7% in colostrum and 3.5-10.5% in milk) as reported by Bowland (1966). Although the total fat content of colostrum is lower than the percent fat in milk, there are several long chain fatty acids that are at a higher level in sow's colostrum. Oleic and linoleic acid are typically high in sow's colostrum then decline as lactation proceeds. Supplementing the sow's diet with corn oil will increase the proportion of linoleic acid in colostrum. Continuing corn oil supplementation throughout lactation will prevent the level of linoleic acid from dropping as 5 colostrum turns to milk (Miller et a1., 1971). This is in agreement with the recent finding of increased levels of linoleic acid in human milk which has been attributed to increased consumption of margarines by the mothers (Jansson et a1., 1981). The increased level of polyunsaturated fatty acids in colostrum and milk may increase the neonate's need for vitamin E. Hasson et a1. (1966) reported a syndrome in premature infants associated with low plasma vitamin E values and high dietary polyunsaturated fatty acids, possibly indicating a need for supplementary vitamin E, whether through dietary sources or injection. This is further emphasized by the recommendation of the Committee on Nutrition, Academy of Pediatrics (1976) that 0.7 IU of vitamin E be provided for every gram of linoleic acid in the infant formula. 2. Vitamins Vitamin A Only small quantities of vitamin A are transferred from the dam to the fetus (Hjarde et a1., 1961). Therefore, the newborn is dependent upon the vitamin A content of colostrum and milk for its needs. Colostrum is an important source of vitamin A for the newborn pig as it has been reported to contain 4 to 7 times as much of the vitamin as mature milk (Braude et a1., 1947; Evans, 1959; Hjarde et a1., 1961 and Nielsen et a1., 1965). The wide variation may be attributed to both the source and quantity of vitamin A in the sow's diet. Nielsen et al. (1965) reported more efficient 6 transfer of the vitamin from the sow's diet to colostrum and milk if it was supplemented as the synthetic form rather than as cod liver oil. Vitamin D Cholecalciferol (Vitamin D3) is not transferred to the pigs via the placenta, but does increase in the plasma of the pigs upon consumption of the dam's milk. The amount of cholecalciferol in milk varies with the amount of supplementation of the dam (Goff et a1., 1984). Vitamin E The extent to which vitamin E is placentally transfered from the sow to the pigs is unclear. Malm et a1. (1976) reported pre-suckled pigs had several fold higher serum tocopherol, (3.6-6.2 pg/ml), levels than their dam, suggesting efficient transfer of the vitamin across the placental membrane. Loosli (1949) also indicated that placental transfer of vitamin E occurred in swine, but the values reported were much lower than those reported by Malm et a1. (1976). Young et a1. (1977) suggested that there was a relatively low rate of transfer accross the placenta, as he found 0.75 ug/ml and 1.16 ug/ml in the serum of unsuckled newborn pigs who were from vitamin E-deficient or supplemented sows, respectively. Placental transfer of vitamin E in humans and rats is low, but recent studies indicate that some transfer does occur. Pazak's (1983) work with rats indicates placental 7 transfer of vitamin E, but it is preferentially incorporated into heart and lung tissue, resulting in very low circulating levels. Martinez et a1. (1981) found that human infants have one-half the concentration of plasma tocopherol as either placental or maternal plasma. They also indicated that plasma total lipids in the infants were very low. From this evidence they suggested that placental transfer of vitamin E is inefficient and the low tocopherol level may be a consequence of low plasma total lipids. Vitamin E deficiency in the newborn and especially in the premature infant can be a very serious problem (Bell et a1., 1979; Harris et a1., 1952; Jansson et a1., 1978; Jansson et a1., 1981) possibly leading to hemolytic anemia (Oski and Barness, 1967). Jansson et a1. (1978) showed that dietary supplementation of low birth weight (<200 g) or pre-term ( 35 wks, gestation) infants with 16.5 mg tocopheryl acetate/day resulted in higher hemoglobin and lower reticulocyte counts at 8-10 weeks of age than infants supplemented with only 1.5 mg tocopheryl acetate/day, indicating that adequate vitamin E in the diet is important to the neonate. Colostrum is a concentrated source of many essential nutrients to help the newborn through the first few days of life. High concentrations of vitamin E in colostrum have been reported in many species, with a dramatic decline as milk is secreted (Jagadeesan and Prema, 1980: Jannson et 8 al., 1981; Malm et al., 1976; Nielsen et al., 1973). The development of high prasure liquid chromatography (HPLC) analytical techniques has allowed separation of alpha- and, beta/gamma-toc0pherols in colostrum and milk. Jansson et al. (1981) found high levels of alpha- and total-toc0pherol in human colostrum and reported a decline in these levels as the composition changed to milk. Total toc0pherol levels have been reported to follow the same pattern in human (Jagadeesan and Prema, 1980) and sow colostrum and milk (Loosli, 1949; Malm et al., 1976; Neilsen et al., 1973). There is a great deal of variation in the toc0pherol concentrations of sow's milk. This variation can be attributed to the composition of the sow's diet. Increasing the vitamin E in the diet of the sow (Loosli, 1949: Malm et al., 1976; Neilsen et al., 1973) or cow (Dunkley et al., 1967) has resulted in an increase of tocopherols in colostrum and milk. Supplementing selenium in the ewe's diet has also been shown to increase the toc0pherol level in milk (Gardner and Hogue, 1967). Polyunsaturated fatty acid supplementation of the dam's diet has a negative effect on tocopherol levels in colostrum (Malm et al., 1976; Neilsen et a1., 1973). Water soluble vitamins Vitamin C and thiamin have been found in higher concentration in colostrum (Braude et al., 1947; Evans, 1959; Pond and Houpt, 1978) than in milk while pantothenic 9 acid and niacin are found in higher levels in milk than in colostrum (Evans, 1959: Pond and Houpt, 1978). Riboflavin is maintained at a constant high level in both colostrum and milk (Braude et al., 1974: Evans, 1959). 4. Minerals Selenium Placental transfer of selenium has been reported in dogs (McConnell and Roth, 1964) when a subcutaneous injection of selenium was given to the dam, and in rats (Pazak, 1983) through dietary selenium supplementation of the dam. Selenium is also made available to the offspring through colostrum and milk. Mahan et a1. (1975) reported colostrum selenium values ranging from 0.043 ppm to 0.106 ppm and milk selenium values of 0.013 to 0.029 ppm. These values represent a four fold greater concentration of colostral selenium than the selenium concentration of milk. The wide variation in both colostrum and milk selenium values can be attributed to a lack of selenium supplementation in gestation and lactation diets, for the low values, and 0.1 ppm dietary selenium supplementation over the same period for the high values. The source of supplemental selenium in the dam's diet affects how efficiently the selenium is incorporated into milk. In ewes, plant forms of selenium were found to be 10 absorbed and incorporated into milk more efficiently than inorganic selenium (Gardner and Hogue, 1967). Jones and Godwin (1963) showed that radioactive selenium which had been incorporated into plants was absorbed by the dam, incorporated into her milk and could be detected in the pup's stomach just four hours after the plant was fed to the dam. They suggested that selenium complexes in plants are readily metabolized by the dam and are normal constituents of milk. The radioactive selenium was found primarily in the protein fraction of the milk. McConnell and Roth (1964) found that a subcutaneous injection of an inorganic selenium was converted to an organoselenium prior to being incorporated, primarily, into the protein fraction of the milk. Several studies have indicated that the dam will maintain nutrient levels in her colostrum and milk at the expense of her own body stores (Gardner and Hogue, 1967; Lane et al., 1984; and Mahan et a1., 1976). Mahan et al. (1976) reported that sows had decreased serum selenium values at the end of lactation when no supplemental selenium was given. Gardner and Hogue (1967) observed a decrease in milk selenium levels three to six weeks into lactation of unsupplemented sheep, suggesting that they had depleted their body stores. They also reported a decrease in milk production at this time. Lane et a1. (1984), who analyzed the mammary gland of rats on high (1.5 ppm) or low (.03 ppm) selenium, found that the selenium content of lactating ll mammary glands in rats on low selenium diets was high, but glutathione peroxidase activity was low. They suggested that the selenium was being partitioned into the milk compartment of the mammary gland, thus making it unavailable as a prosthetic group for glutathione peroxidase. Other minerals Calcium and phosphorus are found in higher concentrations in milk then in colostrum (Braude et al., 1947; Perrin, 1955). In fact, Braude et al. reported an increase in calcium, phosphorus and total ash as lactation progressed. Copper and iron levels are considered deficient in milk and are not responsive to supplementation of these elements in the sow's diet (Pond and Houpt, 1978). 5. Others Lactose is very low in colostrum (Braude et a1., 1974; Perrin, 1955), but increases as mature milk is secreted. The lactose content tends to decline as lactation proceeds (Braude et al., 1947). Carnitine. Pigs are born with subOptimal levels of carnitine as indicated by the blood and liver of the newborn. Colostrum has a high level of carnitine. In fact,. the sow concentrates carnitine in colostrum as indicated by the six to seven fold greater concentration of carnitine in colostrum in relation to the sow's serum level (Kerner et a1., 1983). 12 II. Biological Antioxidants Vitamin E and selenium, as an integral part of glutathione peroxidase, serve as biological antioxidants in animals. An understanding of the cellular and subcellular role of these two nutrients is needed to understand the lesions seen with vitamin E and selenium deficiency in pigs. The understanding of the function of vitamin E and selenium should also explain why various dietary and managerial practices may have a detrimental effect on pigs fed diets with low levels of these nutrients. A. Functions It is well known that oxygen is essential for aerobic organisms to live, but certain forms of this element are lethal to anaerobic organisms (McCord et a1., 1971). Witting (1980), suggests that this difference in tolerance to oxygen between the two types of organisms can be attributed to a defense mechanism in the aerobic organism which allows it to control the level of oxygen breakdown products. This defense mechanism has both enzymatic and non-enzymatic components and can be classified as a biological antioxidant system. A series of reactions, occuring naturally in animal cells, will produce superoxide (0;), hydrogen peroxide peroxide (H202), singlet oxygen ('02) and hydroxyl radicals (HO°), (Diplock, 1981; Ullrey. 1981). The role these oxygen breakdown products play in the initiation of lipid peroxidation, which may lead to tissue damage, has 13 been reviewed by several researchers (Schwarz, 1976; Witting, 1980; Diplock, 1981: Ullrey, 1981). With the production of potentially damaging free radicals in the animal cell, the importance of a properly functioning defense system is clear. To understand the role of biological antioxidants in maintaining this defense system, it is important to understand the proposed mode of action at the cellular level. 1. Production of Free Radicals Naturally occurring. Various enzymatic reactions in the normal metabolic cycle of the cell will produce superoxide and hydrogen peroxide (Fridovich, 1978; Witting, 1980). However, Schwarz (1976) stated that these two breakdown products are not destructive to tissue components or cellular membranes. Hydrogen peroxide and superoxide are thought to react through a Fenton-type reaction catayzed by chelated iron to form the extremely reactive hydroxyl radical (Halliwell, 1978; Thomas et al., 1978; Witting, 1980). Witting (1980) also suggested that superoxide and the hydroxyl radical may react to form singlet oxygen. Hydroxyl radicals and singlet oxygen are thought to initiate lipid peroxidation (Kellog and Fredovich, 1975- Witting, 1980; Diplock, 1981) and I therefore are destructive to tissue (Schwarz, 1976). l4 Iron-induced free radicals Iron has been reported by many researchers to cause vitamin E deficiency-like lesions often resulting in death in young pigs (Tollerz and Gunnar, 1973; Cook et al., 1981; Lannek et al., 1962). The in vivo mechanism of iron's involvement in vitamin E deficiency-like lesions and death is unclear. However Patterson et a1. (1967) suggested that the initial effect of iron on muscle tissue was to potentiate lipid peroxidation. Galberg et a1. (1960) and Dillard's et a1. (1983) work supports this by showing an increase in lipid peroxidation in iron loaded rats. Cook et a1. (1981) found on microscopic examination of muscle tissue extensive degeneration. Histochemically, the degenerated lesions involved mainly the aerobic red muscle fibers. The degeneration of red muscles has previously been described as vitamin E-selenium myopathy in weanling pigs (Ruth and VanVleet, 1974). There are several proposed mechanisms linking iron to lipid peroxidation and cell wall destruction. In a review by Diplock (1981), Cohen (1977) suggested that iron was a catalyst in a series of oxygen related reactions where hydroxy radicals (OH‘) are produced. He also suggested that Fe2+ +02'+ LFeOE]+ may occur where the product of this reaction is known to be a powerful oxidant. Ascorbic acid and Fe+2 were shown to be powerful initiators of lipid peroxidation in vitro (Fukuzawa et a1., 15 1981) but there is controversy whether these reactions actually occur in vivo. Defense Mechanisms To prevent tissue damage it is important to keep the concentrations of hydroxyl radical and singlet oxygen low. This is accomplished by enzymatic biological antioxidants. Superoxide dismutase decreases the level of superoxides by this reaction; 20; + 2H+ 33D H202 + 02, therefore rendering 02 unavailable for conversion to more destructive forms (Witting, 1980). Glutathione peroxidase and catalase are responsible forthe removal of hydrogen peroxide from the reaction pool, so it will not be converted to the hydroxyl radical. Glutathione peroxidase is a selenium dependent enzyme which was shown in 1973 by Rotruck et al. to catalyze, ZGSH+H202 + GSSG + 2H20. This reaction serves an essential biochemical role in an organism, as well as providing a means of keeping the level of H202 low. Dietary imbalances have been shown to affect the activity of superoxide dismutase and glutathione peroxidase. Dietary levels of copper and iron affect superoxide dismutase activity (Williams et al., 1975; Witting, 1980) whereas deficient levels of selenium (Rotruck et a1., 1973; Chow and Tappel, 1974; Witting, 1980; Burk, 1983) and riboflavin (Brady et a1., 1979) will decrease glutathione l6 peroxidase activity. Sklan et a1. (1981) found that chicks fed diets deficient in vitamin E had an increase in both superoxide dismutase and glutathione peroxidase. It is important to note that any factor, dietary or otherwise, which decreases the activity of these enzymes may result in an increased level of hydroxyl radicals and singlet oxygen and therefore promote tissue damage. 2. Lipid Peroxidation Lipid peroxidation involves a cyclic reaction: RH 1'1 R- R; + 02 + R02° R02° + RH + ROOH + R' (Witting, 1980) where RH is a fatty acid in a membrane structure. Initiation The rate of the production.and removal of the non-lipid free radical will determine the rate (r1) of the first step. The variation rate constant is low and less likely to initiate production of organic free radicals when the level of hydroxyl radicals and singlet oxygen is low. Secondary Initiation As the cyclic peroxidation reaction proceeds there will also be a steady increase in organic peroxides (ROOH). A chain branching reaction, 2RO0H + R02' + R0° + H20. 17 will produce organic free radicals which will result in secondary initiation (Rui, 1961; Witting, 1980). Defense Mechanism To minimize the extent of secondary initiations the organic peroxides can be removed from the system by the following reaction 2ROOH + 2GSH + 2ROH + GSSG + H20 (Lawrence and Burk, 1976; Witting, 1980). This reaction is very similar to the one described earlier for the removal of hydrogen peroxides, and as one might expect, it is catalyzed by the same enzyme, glutathione peroxidase. Therefore, the reaction may be affected by the same dietary changes as were described earlier. Burk (1983) stated that glutathione-S- transferase is also capable of catalyzing this reaction. This enzyme does not have selenium as an active site and is therefore not restricted in selenium deficiency. Glutathione-S-transferase activity has in fact been shown to increase in selenium deficiency, possibly in an attempt to compensate for the decrease in glutathione peroxidase activity (Lee et a1., 1981; Buck, 1983). Cyclic Propagation The initiation of lipid peroxidation may not be dependent upon the structure of the fatty acids making up the membrane. However, once peroxidation has begun, polyunsaturated fatty acids (PUFA) tend to oxidize at a 18 faster rate as compared to saturated fatty acids (Uri, 1961; Witting, 1980). Tinberg and Barber (1970) showed that peroxidation in microsomal membranes leads to a decrease in oleic (18:1) and linoleic acids (18:2) and a total disappearence of arachidonic acid (20:4), suggesting a greater peroxidation of fatty acids with a greater degree of unsaturation. Defense Mechanisms Vitamin E Several roles have been suggested for vitamin E in the protection of cellular membranes against peroxidative damage. Diplock (1973) suggested that.a-tocopherol functions as a membrane bound redox substance. Uri (1961) and Witting (1980) stated that.u-tocopherol is an antioxidant and functions by competing with the lipids for a reaction with the lipid peroxy free radicals (RO°), therefore removing the free radicals from the peroxidation reaction. The protective relationship vitamin E has with PUFA is thought to be a consequence of specific complexes between vitamin E and especially arachadonic, but also linolenic and linoleic acids. The complex stabilizes the membranes and prevents degradation of the PUFA (Diplock and Lucy, 1973). Support for this theory can be found in work by Vos et a1. (1973) which showed reduced levels of linoleic and arachadonic acids in the mitochondrial membranes of vitamin E-deficient chicks. 19 Dillard et a1. (1983) used iron to induce lipid peroxidation in rats and found that dietary supplementation of 40 mg/kg diet a-tocopherol or 50 mg/kg diet y-tocopherol would decrease peroxidation. They also found y-tocopherol was only 31% as effective in preventing lipid peroxidation as a-tocopherol. Other membrane components may affect the activity of vitamin E. Fukuzawa et a1. (1981) found that when cholesterol was incorporated into the membrane in large amounts it increased the antioxidant efficiency of a-tocopherol. Other antioxidants Other antioxidants have been shown to be effective in vivo. Follerz (1973) found that ethoxyquin, diphenyl-B-phenylenediamine and methylene blue are effective antagonists against iron induced peroxidation. B-carotene (Ullrey, 1981), BHT, and nitroxide radicals (Hicks and Gebicke, 1981) have also been suggested as antioxidants capable of helping membranes withstand oxidative stress. However Tollerz (1973) found BHT not to be as effective as ethoxyquin, diphenyl-B-phenylenediamine and methylene blue. B. Importance of vitamin E and selenium for swine Vitamin E and selenium play their roles as biological antioxidants at the cellular and subcellular levels. So how does this phenomenon relate back to the pig? What are the 20 typical signs of a failure of the biological antioxidant system? Several articles have discussed the lesions associated with vitamin E and selenium deficiencies in swine (Ullrey et a1., 1970; Ullrey, 1973; Trapp et al., 1970; Young et a1., 1980). The signs which may be observed in pigs indicating a possible E-selenium deficiency are; sudden death, hepatosis dietetica, edema, nutritional muscular dystrophy, possible decrease in reproductive efficiency and increased incidence of mastitis, metritis and agalactia (MMA). Sudden death in weaned pigs, commonly weighing 20-40 kg, may be induced by environmental stress, fighting, or rapid growth. Many times gross lesions are not seen but upon microscopic examination E-selenium deficiency lesions may be seen in the tissue (Ullrey et a1., 1970; Ullrey, 1970; Trapp et a1., 1970). This is supported by Adkins and Evans in Goihl (1984), who found that pigs died within the first two weeks after weaning when vitamin E was adequate but the selenium level in the diet was only 0.025 ppm. Sudden death may also be seen in newborn pigs and finishing pigs if they are severely stressed (Ullrey, 1973). Sudden death following iron administration, for the prevention of anemia, to suckling pigs has also been studied (Tollerz, 1973). The economic implications of E-selenium deficiency were pointed out by Ullrey (1973). He indicated that growing pigs fed a corn-soy diet unsupplemented with vitamin E and 21 selenium had a 15-20% mortality and a 25% morbidity rate. Nutritional muscular dystrophy (NMD), resulting in stiff-gated, sore-muscled pigs, also may occur in E-selenium deficiency. Though NMD may occur in pigs of all ages it is most prominent in fast growing pigs which were 3-5 months old (Young et a1., 1980). Most investigators found E-selenium supplementation does not improve growth rate and feed efficiency (Young et a1., 1980). This was supported by recent Iowa State work which found no significant effect on average daily gain, feed intake or feed efficiency when selenium was supplemented (Goihl, 1984). The clinical signs of E-selenium deficiency in living pigs are often difficult to detect. When the pig is necropsied typical E-selenium deficiency lesions can be seen. Any animal may have one, or a combination of the lesions described. Liver necrosis is signified by a pale, swollen appearance, with focal lesions that give the surface a rough texture. Microscopic examination of the liver usually reveals both normal and degenerative regions. The damaged area of the liver exhibits cell lysis and dilatation of sinusoids with blood giving the appearance of massive intralobular hemorrhage (Ullrey et a1., 1970; Ullrey, 1973). Other lesions typical of E-selenium deficiency are mulberry heart disease and NMD, both resulting from atrophy 22 of the muscle fiber as well as generalized edema. More extensive edema is found in the lungs, mucosa of the spiral colon, subcutaneous tissue and submucosa of the stomach (Trapp et a1., 1970; Ullrey et a1., 1970; Ullrey, 1973). Trapp et a1. (1970) and Ullrey et a1. (1970) also reported some incidence of anemic, jaundiced pigs or pigs with esophagogastric ulcers associated with E-selenium deficiency. Reproduction Vitamin E and selenium supplementation of the sow has been stated in several reviews to decrease the incidence of mastitis-metritis-agalactia (MMA), spraddle-legged newborn pigs and impaired fertility (Ullrey, 1981, Lannek, 1973). Ullrey (1971) in a two year study found that supplementing the sow's diet with vitamin E, and in some cases selenium, significantly decreased the incidence of MMA. This is in agreement with Vale (1983) who saw signs of MMA in five of ten sows fed a vitamin E and selenium unsupplemented diet and no signs of MMA in the sows receiving the supplemented ration. Nielsen et a1. (1973) found that when oxidized herring oil was added to a diet, which was not supplemented with vitamin E, a very pronounced reduction in milk yield occurred, however mastitis and metritis symptoms were not seen. 23 There is a great deal of disagreement in the literature concerning the effect of vitamin E and selenium on litter size at birth and weaning. Vale (1983) found that sows which were supplemented with vitamin E and selenium throughout gestation and lactation had larger litters, weaned heavier pigs and tend to have increased livability of the pigs. In a two year study conducted by Ullrey et a1. (1971) sows were fed diets supplemented with vitamin E, vitamin E + selenium, or vitamin E, selenium and choline. Supplementing vitamin E tended to increase the number of pigs surviving at three weeks and to reduce the incidence of MMA. Mahan et a1. (1975) fed sows one of two diets for two successive parities. One of these diets was deficient in selenium, the other was supplemented with 0.1 ppm selenium. By the second parity the sows on the deficient diet had significantly smaller litters. Malm et a1. (1976) and Young et a1. (1977) found no effect of vitamin E and selenium supplementation on the sow's reproductive performance. In cattle a prepartum selenium injection was effective in reducing the incidence of metritis and cystic ovaries during the early postpartum period (Harrison et al., 1984). Work of this type is limited for swine, however, Young et a1. (1977) reported that dietary levels of vitamin E and selenium did not effect rebreeding of sows. 24 Although the exact effect of low vitamin E and selenium on reproductive performance is not clear, it is certain that vitamin E and selenium are required in the sow's diet. Young et a1. (1977) using a basal diet which consisted of high moisture corn and Malm et a1. (1974) feeding a semi-purified diet, (both diets containing very low levels of natural vitamin E and selenium with no supplementation of these nutrients), reported heavy death losses. Necropsies of the animals showed various vitamin E and selenium deficiency lesions. Supplementation of either vitamin E (60 IU/kg) or selenium (0.6 ppm) to the high moisture corn diet prevented the death losses and the lesions (Young et a1., 1977). Baby pig survival The survival rate of pigs between birth and weaning need to be improved upon. Although there are many factors which contribute to death losses among pigs during these first few weeks of life, there is reason to believe that adequate vitamin E and selenium may enhance the pig's livability. Young et a1. (1977) found that supplementation of the sow's diet with E-selenium in gestation and lactation increased the E-selenium levels of colostrum and milk. Through placental transfer and consumption of colostrum the serum levels of E-selenium were in turn increased in the pigs. If E-selenium were not supplemented in the sow's diet the levels remained very low in the pig. Environmental 25 stress, to which newborn pigs may be exposed, has been suggested to induce E-selenium deficiency lesions in low to borderline pigs (Ullrey et a1., 1970). An Iowa State study supports the livability theory in their finding of a 20% increase in livability from birth to weaning when pigs from sows on selenium-deficient diets were given 100 IU dl-a-tocopherol, It, at birth (Goihl, 1984). In Great Britain and Scandanavian countries E-selenium deficiency has been attributed to the increased sensitivity of pigs to recommended doses of oral or parenteral iron (Lannek et a1., 1962; Patterson et a1., 1967; Tollerz, 1973). The iron toxicity occured when pigs from sows fed diets high in PUFA and low in vitamin E, were given oral [iron (ferrous sulfate or ferrous fumarate) or parenteral iron (iron dextran) in recommended dosages. Affected pigs have clinical signs 8-12 hours after the iron was given. Death follows a few hours later. Not all pigs showing signs of iron toxicosis died (Tollerz, 1973). Postmortem histological examination of the pigs showed waxy muscle ldegeneration and occasionally pericardium and thoracic edema (Arpi & Tollerz, 1965; Lannek et a1., 1962). Tollerz (1973) suggested that iron-induced muscle damage allowed the contents of the cells to enter the blood.» This is in agreement with Patterson et a1. (1967) who suggested that the initial effect of iron was to potentiate lipid peroxidation. Above a limiting amount of muscle 26 peroxides (possibly indicating cell membrane destruction) there was a rapid release of muscle potassium. Death resulted from cardiac arrest and hypercalcemia. Tollerz (1973) found that a-tocopherol and sodium selenite were only effective in preventing iron toxicosis if they were given 24 hours (vitamin E) or several days (selenium) prior to the iron treatment. Synthetic antioxidants, ethoxyquin, diphenyl-B-phenylenediamine and methylene blue were all effective in preventing iron toxicosis when given along with the iron. Iron toxicosis in nursing pigs is much more difficult to produce in the United States.' Cook et a1. (1981) was able to produce some muscle lesions in pigs from sows on an E-selenium inadequate diet, supplemented with 5% cod liver oil when the pigs were given 750 mg of iron from iron-dextran IM. However, it should be noted that no deaths occurred, muscle lesions were seen in only 2 of the 8 pigs treated and the iron injection given was much larger than the recommended dose. Given a recommended dosage of iron as either iron dextran (IM) or ferrous sulfate (oral) no iron toxicosis was seen in piglets born to sows on a low E-selenium diet (Miller et a1., 1973). 27 C. Source 1. Vitamin E Toc0pherols are not synthesized by mammals and therefore become part of mammalian tissue primarily through ingestion of natural occurring tocopherols (found mostly in plants), or from synthetic forms of tocopherol (Machlin,. 1980). There are eight naturally occurring vitamin E compounds. Of these eight compounds dra—t0c0pherol is biologically the most active. The biopotency of the remaining compounds decrease dramatically with day-tocopherol 40%, d-B-tocopherol 10% and dam-tocotrienol 25% as active as d-a-tocopherol (Bieri and McKenna, 1981). Forages are a good source of vitamin E. In fact, before many producers began raising pigs in total confinement, the NRC (1968) stated: "It is unlikely that practical swine diets would be deficient in vitamin E unless the diet contained excessive amounts of highly unsaturated or oxidized fats." Ullrey (1981) attributes two factors to the increased incidence of E-selenium deficiencies in swine. These are (1) the increased practice of having the entire life-cycle of swine in confinement and (2) the low level of E-Se in corn-soy diets in the Midwestern United States. Tocopherol values for corn vary. Bauernfeind and Cort (1974) cited by Ullrey, (1981) reported a-tocopherol at 6 mg/kg and y-tocopherol at 38 mg/kg. Pond et a1. (1971) published average values of 1.5 mg/kg and 20.6 mg/kg for a- and Y-tocopherol, respectively, and Young et a1. (1975) 28 published a value for corn of 9.3 mg/kg of a-toc0pherol. A great deal of variation seen in corn tocopherol values may be due to the post-harvest handling of the corn. Drying corn at either high or low temperatures did not seem to affect the actual tocopherol content of the corn. However, the high temperature did decrease the biopotency of the tocopherol when selenium levels in the corn were low (Pond et a1., 1971). This is supported by Young et a1. (1975) as well as Chow and Draper (1969) who reported little to no destruction of tocopherol in corn with artificial drying. However Chow and Draper (1969) found that both unsaturated fatty acids and vitamin E can be destroyed in corn if it is over-dried, particularly by over-drying at high temperatures. High moisture corn treated with propionic acid or acetic-propionic acid and stored for 230 days showed a dramatic decrease in artocopherol, from 9.3 mg/kg to 1.2 mg/kg (Young et a1., 1975). Chow and Draper (1969) suggested and Young et a1. (1973) agreed that the high moisture content may be the main contributing factor to the decrease in a-tocopherol. Soybean meal is relatively low in a-tocopherol. Both 44 and 48% soybean meals have been reported to have 3 mg/kg (Brunnel et a1., 1968: Ullrey, 1981). Commercial sources of vitamin E are either the acetate or the hydrogen succintate esters of d-a-tocopherol or the acetate ester of dl-a-tocopherol (Ames, 1979). Young et a1. (1975) checked the stability of a-tocopherol acetate in 29 feeds held under various storage conditions and found that the a-tocopherol potency was retained for at least 70 days in artificially dried corn and corn treated with acetic-propionic acid. The potency of a-tocopherol was retained for at least seven days in high moisture ensiled corn. 2. Selenium Selenium can be incorporated into swine diets in a number of different forms ranging from the inorganic sodium selenite (Naz SeO3) and sodium selenate (Naz 3e04) (NRC, 1979), to the organic seleniferous compounds found in plants; Selenium in grain is primarily associated with the protein (Selenium and Nutrition, NRC, 1983). Scott (1973) found that the bioavailability of selenium from naturally occurring feeds was only 30 to 80% that of the Se in sodium selenite for the prevention of exudative diathesis in chickens. The bioavailability of selenium in corn and soybean meal was reported as 83 and 64%, respectively. Groce (1973) found lower serum blood levels of selenium when pigs were fed seleniferous corn as compared to those in pigs fed the same level of Se as sodium selenite. This may be explained by the greater retention of selenium in the tissues when supplied by seleniferous corn (Ku et al., 1972; Groce et a1., 1971, 1973). The low level of selenium in grain is a problem in many areas. The areas of the United States which are typically 30 low in selenium are the Northwest, Northeast, Southeast and regions surrounding the Great Lakes, which includes most of Michigan, Ohio, Indiana, Illinois, and Wisconsin. (Selenium in Nutrition, 1983). The concentration of selenium in grain is dependent upon the type of soil. Sandy loam soil with a low pH will normally produce grain low in selenium. Variety of grain produced may also be a factor in the Se concentration. Across the United States there is a great deal of variation in selenium content of grains. The ranges reported in Selenium in Nutrition (NRC, 1983) for ingredients typically used in swine diets were: soybean meal, .06-1.00 ppm, whole soybeans 0.07-0.90 ppm, corn .01-1.00 ppm and dicalcium phosphate .15-1.00 ppm. Selenium content of Michigan corn normally fall at the lower end of the range with a mean value of .033 ppm and a range of .013-.089 ppm (Groce, 1972). Groce et a1. (1973) found that selenium in a stored selenite-glucose premix was not retained as effectively as that in a fresh premix. They suggested that the selenite was changed to elemental selenium and was not absorbed as efficiently. D. Requirement The NRC (1979) recommended .15 mg selenium and 10-15 IU vitamin E per kilogram of diet for bred gilts and sows and lactating sows. However, it should be recognized that the 31 levels of vitamin E and selenium required are dependent upon a number of dietary factors as well as environmental factors. The common function of vitamin E and selenium as biological antioxidants explains the relationship between the required levels of the two nutrients. A sparing effect was demonstrated by Groce et a1. (1973) who found increased amounts of selenium excreted in the urine when vitamin E was added to the diet. This suggests that selenium is retained to meet the requirement and the excess is excreted in the urine. Feeding diets high in PUFAs will increase the requirement for vitamin E. It has been suggested that the lability of cell membranes varies with the PUFA content of the constituent phospholipids, and that this composition is sensitive to the dietary level of PUFAs. As more PUFAs are incorporated into the membrane, the requirement for membrane-bound tocopherol is increased and therefore the dietary requirement is increased (NRC, 1979). Dam (1962), on the otherhand, suggested that dietary PUFAs accelerate the depletion of tocopherols in feeds and therefore increase the requirement. The presence of kidney beans or peas in the diet inhibits absorption of vitamin E and thereby increases the dietary requirement (Desai, 1966; Huntz and Hogue, 1964; Machlin, 1980), while the addition of synthetic biological antioxidants to the diet will decrease the requirement (NRC, 1979). Excessive heat, cold or other stresses may also 32 increase the dietary requirements for these nutrients (NRC, 33 Materials and Methods Nineteen Yorkshire X Landrace gilts were balanced by litter, placed on two dietary treatments and bred as they came into heat. The diets were formulated from dried high moisture corn. The analysis of the basal diet found 0.78 ug/g DM vitamin E (E) and 0.04 ppm selenium (Se), while the vitamin E and selenium supplemented diet had analyzed values of 56.1 pg E/g DM and 0.12 ppm Se respectively. The gilts were housed in total confinement during gestation and lactation. Five of the sows from the supplemented group and ten of the sows from the basal group remained in the second parity gestation period. These animals were kept on their respective dietary treatments but were switched to a diet formulated with dried shelled corn (Table 1). By analyses the basal and supplemented gestation diets contained 7.1 and 44.6 ug E/g DM and 0.06 and 0.18 ppm Se, respectively whereas the basal and supplemented lactation diets contained 4.7 and 14.9 ug E/g DM and 0.05 and 0.26 ppm Se, respectively. The sows were on the shelled corn diet from their second parity gestation through their third parity lactation. There was three sows on each dietary treatment in the third parity. This thesis reports only second and third parity results. 34 .u:~ .66.90uouoaoa ad‘osooc .ua.&sou i:.o—eu .o:..o>oouuo.uo.:o ucu 95¢: stony a 6. cm .osoouougoo .66 26a ..6 cco ax\m c.26u.> :— m.s. op.>oh; cu ue.c 6.306 9:6 :. caucoeo.oa:u an) ace «on m :.£oad> 6:6 x.§oue co 2:.co.om no. .cuco c::065 >.oc.u —n.aa no auo.a:ouo .uo.c no mx\:. om ocu>36c ca touzoso.:o:u no) can a\:u can co..om:mu .uo.c 6.306 0:» c. on so; ..a oc.>06z ca peacoSoacezn an) ace :x\om a! ~.oo~ coc_o.:oon .35 m.o .o:.co. 1:6 as o. .uocaoo .55 ha .oaozoacoe .35 co .coh. .6: mm .oc.u .7: a.a— .~.= c.8su.> .7! ca. .oc.~o:o .55 N.n~ .c.oo o‘cofia04cooic .0! 0.5— .cdoo.c .3: n.n .:.>a..oa.. .66 mpsa.u> .6. 66.6 .< c.5~..> .ws ~.~ .o..u.:6.n £3.60. oco.ooco§ .a— com .no.c no ax nod mouse..0u osu a:.>.cd:mn It--- 666.66. 666.66. .ooos 66.6 66.6 666 .ozoauo.:o m.c c.o en‘soum a cusoua> .Ea.co.om 6.6 6.6 a...eo.6. :s> 6.6 6.6 ..o6 ... ... ~66:~6-6 666.661.; 6.. 6.. 666-.6-6 o.ozauo:6 26.0.oo.6uocox 6.6. 6.n~ “66:46-6 .ve. .66: coon>om mh.~k 66.~k .mmnwouv 6.66 6o..o:6 662666 on a a c.§eud>+ .666: .0: .ud: coco—floumcu co.ucuoc4 doco.ascuouc~ 66.66. 6.66. .c.os .6.6 6.6 o..66. 6 :.enu.> mc.o c.o 2.x.eouo. ca B:.co.om 6.6 6.6 a.u.eoua. 2p> 6.6 6.6 ..a6 «.. ~.. ~66-~6-6 o:o..u¢.. 6.. 6.. 666-.6-6 causagosa s:.o.ou.6no:oz 6... 6... .666-¢6-6 .vv. .oo: cannaom v~.66 6.66 .66-~6-c :uoo 6...oz- 6:6666 em a m cutouu>+ .mewm .cz .uoz oucoqeoumcn couaouawm. .oco.uccuouc— o.o.6 .6 :6...ooaeoo .. c.6ae 35 Blood samples were taken from sows in late gestation, immediately post-farrowing and at twenty-one days of lactation. Ten milliliters of blood were drawn from the anterior vena cava with a sterilized 3.5 inch, lS-gauge needle and 20 m1 syringe. The blood was transferred to 10 m1 heparinized centrifuge tubes and covered until transported to the lab for further processing. Weights were taken on the sows when they were moved into the farrowing house, after parturition and at 21 days post-farrowing. At parturition the sows were carefully monitored. If farrowing intervals were greater than 20 minutes, 60 U.S.P. units of oxytocin were given IM. If the oxytocin had no effect, the sow was assisted in delivering the pigs. The numbers of mummies, stillbirths and live pigs were recorded at the end of farrowing. Colostrum samples were collected on all sows while farrowing.‘ Milk samples were taken at 2 and 21 days post-farrowing by administering 60 U.S.P. of oxytocin IM, washing the udder with warm water and drying with paper toweling, to stimulate milk ejection. Uniform amounts of milk were stripped from all functional nipples on one side of the sow's udder. Enough milk was obtained at each sampling to fill a one ounce, Lermer poly-opal plastic ointment jar. The full containers were capped and frozen at -20C until analyzed. Jansson et a1. (1981) reported no 36 significant effect of freezing (-20C) on human milk tocopherol levels. B.’ Pigs The pigs were separated from the sows immediately after birth, to prevent suckling. At the end of the farrowing process the pigs were weighed, a 10 ml blood sample was taken and one half of the litter was given 200 mg parenteral iron, in the form of gleptoferron 1M1. Blood samples were taken by laying the pigs on their backs in a V-shaped holder and as restraint was provided by one person, the second person drew the blood from the anterior vena cava using a 1 inch, 20-gauge needle and a 10 ml glass syringe. Blood was put in 10 m1 heparinized centrifuge tubes and covered until they could be taken to the laboratory for further processing. At 2 days of age the pigs were weighed, a 10 ml blood sample was taken in the same manner as described earlier, and the pigs not receiving iron previously were given 200 mg iron in the form of gleptoferron IM to prevent anemia. The pigs were again weighed and bled at 21 days of age. The procedure for bleeding was similar to that described for the younger pigs, but in this case an 18-gauge, 1 1/2 inch needle was used. 1A polysacharide complex of beta-ferric oxyhydroxide and dextran glucoheptonic acid, Burns-Biotec Laboratories, Inc. 37 C. Laboratory Analyses Hematology Packed cell volume (PCV) or hematocrit was determined on heparinized blood samples using the method described by McGovern et a1. (1955). Each blood sample was drawn into a microhematocrit tube, the tube was sealed at one and using a flame, then it was centrifuged in an International Model MB microhematocrit centrifuge at 13,000 X g for 5 minutes. Spun samples were immediately read on an International microhematocrit reader. Hemoglobin (Hgb) concentration was determined by putting 0.02 ml of whole blood into 5 m1 of Drabkins solution. The blood cells lyse, and ferricyanide converts the iron of hemoglobin from the ferrous to the ferric state to form methemoglobin. Methemoglobin reacts with potassium cyanide to form cyanmethemoglobin, a stable pigment with an absorbance which can be measured by a spectrophotometer at a wavelength of 540 nm (Wintrobe, 1980). The absorbance value is then multiplied by a conversion factor, characteristic of the specific Drabkins solution, to obtain hemoglobin concentration in grams per deciliter (g/dl). Mean corpuscular hemoglobin concentration (MCHC) may be calculated using the Hgb and PCV values for each blood sample as shown: MCHC% = (Hgb(g/d1)/PCV%) X 100. The percentage MCHC represents the concentration of hemoglobin in the red blood cells. 38 Sample storage Heparinized blood was centrifuged for 15 minutes at 3000 rpm. The plasma was harvested into 5 m1 falcon tubes, the air was removed from the tubes with a gentle stream of nitrogen and caps were snapped onto the tubes immediately. The tubes were stored in the freezer (-20C) until further analyses could be run. Plasma Analyses Plasma glutathione peroxidase (GSH-Px) activity was determined by the coupled assay of Paglia and Valentine (1967) as revised by Lawrence et a1. (1974). In this assay known amounts of NADPH and glutathione reductase were placed in a 1 m1 cuvette in combination with a potassium phosphate buffer solution. Reduced glutathione (GSH) solution (0.05 ml) was added to the cuvette along with 0.04 ml of plasma which contains an unknown amount of GSH-Px. Finally hydrogen peroxide (H202) was added to the cuvette to initiate the reaction (Figure 1). The decrease in activity Figure 1. Reaction Involved in Measuring GSH-Px Activity H202 GSH NADP GSH-Px Glutathione Reductase H20 + 02 GSSH NADPH 39 Of NADPH (A340) is measured at a wavelength of 340 nm on the Varian 634 spectrophotometer and recorded on the Varian model 9176 chart recorder for five minutes. Since there were constant amounts of all the reaction components except GSH-Px any change in NADPH activity was reflective of the amount of GSH-Px in the plasma. Glutathione peroxidase activity was reported in enzyme units (EU) which are moles of GSH oxidized per minute. The EU for a plasma sample were calculated by: (the change in A340 of plasma/min. - the change in A340 of blank/min.) X 8.0386. The factor 8.0386 is determined by sample size, the molar extinction coefficient of 6.22 x103 for NADPH and the stoichiometry for the reaction, 2 moles GSH per mole of NADPH oxidized. Alpha-tocopherol was determined in plasma samples by a fluorometric procedure developed by Whetter and Ku (1982) from a tissue a-tocopherol procedure (Taylor, 1976). The a—toc0pherol in the sample can be dramatically reduced by oxidation. Therefore, careful precautions were taken to minimize the oxidation process. This was accomplished by extracting the samples in acid washed glassware, keeping the samples on ice throughout the extraction process and displacing the air in the test tubes with nitrogen (N2) prior to vortexing. Duplicate standards were prepared from a stock standard 40 solution1 in absolute ethanol (AR grade) to obtain standards of 0, l, 2, and 4 a—tocopherol/ml. Two millilters of absolute ethanol were added to the test tubes prepared for the plasma samples followed by 1 ml of plasma. One milliliter of deionized distilled water (DDHZO) was added to each of the standards to bring them to equal volume with the sample and all tubes were vortexed for 5 seconds to precipitate the protein. Cyclohexane (2 m1, Eastman Kodak, AR grade) was added to each preparation followed by 20 seconds of vortexing to extract the a-tocopherol from the sample. Centrifugation of the samples in a Damon/IEC Model PR-6000 refrigerated centrifuge at 2070 X g for 15 min. provided complete sedimentation of the sample debris from the cyclohexane X a—tocopherol layer. The a-tocopherol in the cyclohexane will fluoresce at excitation 296 mu and emission 330 mu. This layer was carefully transferred to 2 dram vials, and read in the Aminco-Bowman spectrophotofluorometer set at the appropriate wavelengths. The reading from the spectrophotoflourometer was percent transmission (%T). To calculate the concentration of ortoc0pherol in the plasma, the known concentration of a-tocopherol in the standards and the %T for the standards were used to formulate a curvilinear regression line. The %T for the samples were lStock standard was 2 mg artocopherol/ml in hexane which was stored in the freezer (~20C). 41 then read off of this line to determine plasma o—tocopherol in ug/ml. Plasma selenium (Se) concentration were determined by a spectrofluorometric procedure (Whetter and Ullrey, 1978). Duplicate plasma samples (1 ml) and duplicate standards of 0, 0.05, 0.1, 0.2 ug of Se/ml were digested in 3 m1 of nitric acid (HNO3) and 2 ml of perchloric acid (HC104, and the HHO3 was driven off. Nine milliliters of ethylene diamino tetraacetic acid (EDTA) was used to wash down the sides of the digestion flask and prevent contamination of the sample with other metals. Approximately 1 ml of concentrated ammonium hydroxide (NH4OH) was added to each sample to neutralize the remaining HC104- Cresol red was added to indicate the prOper acid:base balance of the sample. Any adjustment in pH was made by adding drops of NH4OH or HCl (1:9). Five milliters of 2,3,Diaminonaphthalene (DAN) added to each sample complexed with the Se to form diazoselenol, a light sensitive complex. This complex was then extracted into cyclohexane (5 ml), and transferred to a test tube to allow its %T to be read in the Aminco-Bowman spectrophotoflourometer (excitation 376 mu and emission 510 mp). Plasma Se concentration (pg/ml) were read off a curvilinear regression line calculated from similarly processed standards. 42 Plasma iron concentrations were determined by an atomic absorption spectrophotometric procedure (Olson and Hamlin, 1969). Plasma (1 ml) and 20% trichloroacetic acid (TCA) (2 ml) were combined in plastic centrifuge tubes incubated at 90C for 15 min. in a waterbath to precipitate the plasma proteins. The cooled samples were centrifuged at 2070 x g for 15 min. and the clear supernatant was transferred to 5 m1 falcon tubes and read on the IL951 atomic absorption emission spectrophotometer at a wavelength of 248.3 nm. Iron concentration was expressed in ug/ml supernatant. A simple formula was used to correct this volume for volume as well as absorption due to the TCA. Formula: (sample ug/ml - standard ug/ml) X 300 = plasma iron in ug/dl. Sow Colostrum and Milk Analysis Alpha- and beta/gamma- tocopherols were determined in sow's colostrum and milk by reverse phase HPLC (Whetter and Loudenslager, 1984). Ascorbic acid (0.5 g) was weighed into acid washed test tubes to prevent oxidation of the tocopherols. Duplicate standards of 1, 2, 4 and 8 ug of y-and u-tocopherol/ml were prepared from the stock standardsl, methanol and DDHZO, 1The stock standard solutions were 2 mg Y-tocopherol in methanol and 2 mg a-tocopherol/ml in methanol. Both standards were prepared from Eastman products prior to any analysis and stored in a freezer (-20C) for use in all milk-tocopherol analytical procedures. 43 at the beginning of an analytical run and were processed as the samples. Samples were weighed into test tubes, and two milliliters of methanol (HPLC grade) (2.5 for colostrum) - were added to each sample. The air in the test tube was displaced with nitrogen (N2) and the samples were vortexed (60 sec). Deionized distilled water (1.5 ml) was added to each sample, air was displaced from the sample and the sample was vortexed (10 sec) and allowed to set for 5 min. This procedure precipitated the protein in the milk and allowed the fat soluble tocopherols to be extracted more uniformily. Potassium hydroxide (1 ml for milk, 1.5 ml for colostrum) was added to each sample, and the samples were incubated for 15 min. in a 60C waterbath to saponify the fat. The samples were cooled in ice water for 3 min., 3 m1 of HPLC grade n-hexane was added and the samples vortexed (60 sec.) to extract the tocopherols and stop the saponification reaction. Care was taken in the saponification step to assure equal time of exposure of each sample to the KOH. Prolonged exposure to KOH could be very l damaging to the tocopherols and may cause undue variation between samples if unequal exposure times existed. The samples were centrifuged for 15 min. at 2070 X g to remove debris from the hexane layer. The hexane X tocopherol layer was quantitatively transferred from the sample to a 25 m1 Erlenmeyer flask. The hexane was evaporated off in an 44 evaporation oven leaving only the tocopherols in the flask. The toc0pherols were picked up with 1 m1 of methanol and filtered through Millex-HV filter units (0.45 um) into 2 dram, screw capped vials. The samples at this point could be stored under N2 in the freezer for up to two days prior to reading them on the HPLC. The samples were read on HPLC instrumentation from Waters and Associates, Inc. (Milford, Mass). The system consisted of a model 45-M solvent delivery system, a model U6K universal liquid chromatograph injector, and a model 440 absorbance detector. The detector was connected to a Servogor 120 recorder set at 0.25 cm/min. A RCSS Guard-Pak (C18) used as a pre-column, and a Bondapak €18 reverse phase, 3.9 mm X 15 cm column were used on the system. The solvent mobile phase was 95% methanol: water, and was pumped through the system at 1.5 ml/min. The samples were read off of a curvilinear standard regression line as described for plasma tocopherol then corrected for weight differences and reported in ug/g of milk. Milk Se values were determined by the procedure described earlier for plasma Se determination (Whetter and Ullrey, 1978), with a few minor revisions to compensate for the high fat content and viscous consistency of sows milk. Approximately 1 m1 of milk was weighed into the digestion flask and the weight (g) recorded. The final concentrations were then recorded as pg of Se/g of sample to correct for 45 inconsistencies in allotting the thick samples. To eliminate problems associated with the high fat content the samples were allowed to digest slowly, overnight, in the acids (3 m1, HNO3 and 2 m1, HClO4, 70%) before heat was applied to complete the digestion process. The rest of the analysis was carried out as described for the plasma Se analysis. Total fat was determined on duplicate milk and colostrum samples by a procedure modified by Loudenslager and Whetter (1984) from the Roese-Gottlieb method (1980). Approximately one gram of milk (colostrum) was weighed into a test tube and the sample weight recorded. To allow for a more complete fat extraction the fat micelles were broken down by adding 0.2 m1 concentrated ammonium hydroxide (NH40H) and vortexing (10 sec), and the protein was precipitated as a fine white powder by adding 3 ml absolute ethanol and vortexing (l min). The fat was extracted with diethyl ether (3 m1), vortexing (1 min) and petroleum ether (3 ml) and vortexing (1 min) followed by 15 min. of centrifugation at 1440 x g. The ether layer was decanted into a pre-weighed 25 m1 Erlenmeyer flask, using the indentation on the test tube to hold back debris. The substances remaining in the test tube were re-extracted as described above, and the second ether layer was added to the flask with the first. The ether was allowed to evaporate under the hood overnight. The flasks containing the fat 46 were dried in an evaporating oven, removed and weighed. Percent fat was calculated as follows: Sample flask wt. - initial flask wt. X 100 = % fat milk sample wt. Statistical Analyses The sow data were analyzed in a split-plot design, testing the effects of diet and time on each variable measured. In the event of a significant (P<.05) interaction Scheffe's test was used to determine the significance of the diet effect at each time and the time differences for each diet. The pig data for birth and twenty-one days of age were analyzed using a nested, one-way analysis of variance design. In this design pigs were nested within litters, to eliminate a bias associated with non-random allotment of pigs to treatment groups. The measures analyzed for two-day old pigs required a split-plot design to test for diet and iron treatment effects. Once again, a nested analysis was used. When a significant diet X iron interaction occurred Scheffe's test was used to test the differences between means. In this study a difference was considered highly significant if P<.01: significant if P<.05 and nearly significant if P<.10. Probability estimates of .25 were 47 reported to indicate trends, as this study is preliminary and a relatively small number of sows were used. Homogeneous variance is one of the main assumptions underlying statistical analyses. In the analysis for this study homogeneity was tested by Bartlett's and Cochran's tests. If the probability values for the tests were less than 0.05 heterogeneous variance was considered to be present. All statistical analyses were made by using the multiple analysis of variance program which is part of the SPSS1 program package. lSPSS-Statistical Programs for the Social Sciences, Update 7-9. 48 Results and Discussion 1. Sows One of the sows on the basal diet went down during the second parity gestation. She delivered her pigs but she had to be sacrificed after her second week of lactation. A necropsy of the sow found E + Se-deficiency lessions in her muscle, however it was a fractured vertebra that crippled her. This sow and her pigs were not included in the analyses for this study. There was no significant effect of dietary treatment on the hematology of the second and third parity sows (Tables 2 and 3). There was a tendency for second parity sow's hemoglobin and hematocrit to drop at farrowing and remain at a depressed level throughout lactation. This trend however was not seen in the third parity sows. The mean corpuscular hemoglobin concentration decreased significantly in the second parity sows from farrowing to the end of the lactation period. Again, this change was not seen in the third parity sows. The inconsistencies of the hematology findings between parities may be due to the small number of sows represented in the third parity, rendering small changes in blood values undetectable, or the heterogeneous variance seen in the second parity blood values may have created false differences. .Doouuoocfl on hos ucmowmacmwm ozu mnemouonu .pmumfixo oocmwum> moomcomOHouomo .uowo 630m mo newuoucoanmmom mm .m CH moo oocouommwo come mo oucmowmwcmwmn .oeflu uo>o oocmuomwwo cows «0 cocooamwcmfimm 49 o.m @.~ o.~ mm: mz m2 mz noo~m>lm N.~ o.¢m mo.v m.mm mz h.¢m Hmmmm ~.~ ~.mm mo.v ¢.mm mz m.¢m ooucoaoammom ofimv cOMHouucoocou canonOEm: uoaoomsmuou coo: m.wa m.ma m.ma mm: 62 oz 62 nos.m>um h.m m.vm mz m.mm Oa.v H.5m Hmmmm h.m m.¢m m2 v.mm 0A.v m.wm poucoeoammom o.wv u.uooumEo: ¢.N .VoN ¢oN mm: m2 m2 mz nosam>lm H.. H.NH m2 m... mm.v m.ma Hommm H.H m.HA mz m.aa mm.v b.ma poucoanmmom o..6\mo 6.20.6656: mm: mama am we mca3ouumm mm ceaumuuow mama ammo H! we.e >mo~ouoEom n.3om huwuom cam co coHumucoanmmom mm a m humuoaa m0 nuoowwm .m «and? SO .umwo 630m mo c0wumucoanmmom mm .m on mop mocmumwmao come no mocmowmwcmwmn .mE«u uo>o oocoquMMp come mo oocmoflwficmwmh m.o m.o m.o mm: o..v m2 m2 nooam>lm H.m m.mm m2 m.¢m mz m.¢m Hmmmm H.N ¢.mm mz m.vm m2 «.mm ooucmanmmom Amv coHumuucoocoo cwDonOEom Hoaoomomuou coo: o.m~ o.ma o.ma mm: m2 mz m2 Dooam>lm o.m o.mm mz o.mm m2 o.mm Hmmmm o.m m.am m2 m.0m m2 h.mm ooumoEonmom va ufluoouoEom Mofl moN MoN mm: mz m2 m2 nooao>lm m.o N.HH m2 m.HH m2 m.- Hmmmm m.o N.HH m2 m.oH mz m.HH poacosmammom ..o\m~ 6.20.6056: mm: whoa AN llhm wcfi3ouumm Wm dewumumow mama Homo mafia xmoHoumEom n.30m huwumm cum :0 nodumucoanmmom mm a m humumwa mo muoommm .m canoe 51 .umwo 630m mo coflumucoamammom mm .m CH mop monoummwfio cmoE mo mocoOAMMcmamQ .oEau uo>o oocopmwmwo cmoE mo mocmoammcmmmm ¢.¢mma ¢.¢moa ¢.¢mma mm: mz m2 m2 nooam>lm m.oam m.m¢H Ho.v o.moa Oa.v h.o~a Hommm m.oam o.h¢a Ho.v m.mma Oa.v «.maa poucosoammom Aao\m1v couH mEmoam o¢H.o o¢H.o ova.o mm: 0H.v oa.v oa.v nooam>lm mmo.o no.0 o..v m>.o Ho.v hm.o Homom mac.o co.a Oa.v om.o do.v mH.H ooucoEonmom AHE\:MV mmmpflxonom oceanuouoaw mEmmHm oma.o mma.o om~.o mm: Hoo.v Hoo.v Hoo.v nooam>lm mHH.o vo.a wz mm.o Hoo.v Hm.a Hommm maa.o ~¢.H m2 mm.a Hoo.v mm.~ poucmanmmom AHE\mzv m cfiEmuw> mammam Hoo.o Hoo.o Hoo.o mm: Hoo.v Hoo.v Hoo.v nooam>im moo.o mo.o Oa.v ma.o m2 ma.o Hommm moo.o o~.o mz mm.o mz NN.C poucoEonmom AHE\mnv Eowcoaom mEmmHm mm: mama am We mcw30uumm .mm newumumoo mama Home oEfiB manned 6.30m xuwumm com :0 ceaumucoEonmom mm a m huouowa no muoowwm .v wand? .uowo 6306 m0 cofluoucoEoHQmom mm .m on moo oocouommao cows «0 oocmoawficmamn .0569 uo>o mocouomwwo come «0 mocmowmwcmwmm 52 ..66. ..66. ..66. 662 62 62 62 nos.6>u6 6..66 6.66. 62 6.6.. 62 6.6.. .6666 6..66 ..66. 62 ..66. 62 6.66. 6666656.6666 A.o\m1v couH mammam 6..6 6..6 6..6 662 62 62 62 666.6>:6 66.6 66.6 62 66.6 66.v 66.6 .6666 66.6 66.6 62 66.6 66.v 66.. 66.6656.6666 AHE\DMV mumpflxouom ocoflzumuoao mEmo.m . 66.6 . 66.6 66.6 662 .6.v .6.v .6.v 666.6>u6 66.6 66.6 62 .6.6 66.v 66.6 .6666 66.6 66.6 62 66.6 66.v 66.. 6666656.6666 A.E\m:v m c6§6u6> mEmMHE 6666.6 6666.6 6666.6 662 .oo.v mz .o.v noo.6>lm .66.6 66.6 6..v . 6..6 66.v 66.6 .6666 .66.6 66.6 .6.v 6..6 62 6..6 66.6626.6666 AH£\m:v Eowcoaom mannam 662 6666 .6 Alma 66.306666 66 60.666666 666. 66.6 66.6 mEmm.m 6.30m >uaumm cum :0 cofiuoucosoammom om 6 m xumuowa mo muowwwm .m 6.969 53 Plasma values for second and third parity sows are presented in tables 4 and 5. Plasma Se analyses on samples from second parity sows showed a highly significant (P<.001) response to dietary supplementation of vitamin E and Se. The supplemented sows had a much higher level of plasma Se during gestation and maintained this level throughout lactation, whereas the sows on the basal diet showed a highly significant decrease in plasma selenium by the end of the lactation period. The sows who remained on the basal diet into their third parity gestation did not appear to recover their initial plasma selenium level. A nearly significant (P<.10) decrease in plasma selenium occurred during the third parity lactation. Mahan et al. (1975) reported that sows on a Se-supplemented diet maintained their serum selenium value while sows on a non-supplemented diet containing 0.03 and 0.05 ppm natural selenium during gestation and lactation showed a decrease in serum selenium by the end of lactation and did not return to their initial serum selenium value during the subsequent gestation. The trend was similar to that seen in the sows on this study. Mahan et a1. (1975) suggested that this trend may indicate an increased requirement for selenium during lactation, which can be effectively met by supplementing the sow's diet with 0.1 ppm selenium. 54 Sows fed the basal diet in both parities had significantly (P<.001, P<.01) lower plasma vitamin E levels than the supplemented sows. There was a drop in plasma vitamin E levels (P<.001) in sows on both dietary treatments at the second parity farrowing. This same trend was seen in the third parity sows, however, due to the low number of sows represented in this parity the confidence level was only 94%. The rapid decline in plasma vitamin E at parturition may be a result of the very high level of vitamin E secreted into colostrum (Tables 7 and 8). There was no significant change in the sow's plasma vitamin E level throughout lactation. This may also be indicative of the levels of total-toc0pherols the sow passes into the milk. After the initial secretive burst of colostral vitamin E resulting in a depletion of the sow's plasma vitamin E, the milk tocopherol level immediately decreases. The level of tocopherol in milk continues to decline at a much slower rate from the second day post-farrowing to the end of the lactation period, thereby allowing the sow to maintain her plasma vitamin E level. Malm et a1. (1976) reported serum vitamin E values for sows on a vitamin E and lard supplemented diet similar to those reported for the sow in this study. However, the sows on the basal and lard diet had much lower serum selenium values than were indicated here. The drop in tocopherol at parturition and the relatively constant circulating levels 55 of serum tocopherol throughout lactation appeared to be consistent with findings in this study. There was a nearly significant (P<.10) diet effect on plasma glutathione peroxidase (GSH-Px) activity in second parity sows, with the higher levels associated with sows on supplemented diets. The same diet to GSH-Px relationships appeared to exist in the third parity sows, however, the differences were non-significant. Since GSH-Px is a selenium dependent enzyme, it is appropriate to look at the relationship between selenium and GSH-Px over time. In Figure 2 it is clear that plasma selenium levels remain consistently high throughout the reproductive cycle in the supplemented sows. It is also apparent that GSH-Px activity is not highly correlated with the plasma selenium levels in these sows. Plasma GSH-Px decreased significantly (P<.01, P<.02) in both parities at parturition and remained low throughout lactation. Lane et a1. (1984) found that in mice under certain stressful conditions, such as maintaining a pregnancy while in the growth stage and during lactation, that basal levels of dietary selenium (0.03 ppm) were not sufficient to maintain an effective level of GSH-Px activity in the mammary gland. Plasma GSH-Px values were not measured. They also suggested that the decline in GSH-Px activity during lactation was due to a partitioning of selenium into the milk. The graph in Figure 2 indicates a highly Selenium (119/ ml) 56 '28 thuro 2. $06 Plasma 2.3 (2nd Parity) .26 2.5 .24 2.4 '27- .: 53 2.2 .20 - — _____ 2.0 ““4 .13 1 .s .16 ' 1 .s "‘ ~ 50 1.4 .12 n. w 1.2 \ESH. . ‘1 .10 \\‘ ’f/apa—v—‘o‘ 1 0 GSH-Px /”’ - 4 .08 0.3 .4 .06 o 3 '04 lull Supplemented 0.4 Dist .02 H 32:31 Dist 0.2 Late P‘BIflIflflOfl 21 out. Days tut-Ina) xd - use 57 significant (P<.OOl) correlation (r=.68) between the plasma selenium level and plasma GSH-Px activity in the sows on the unsupplemented diet.‘ A significant correlation did not exist between selenium levels and GSH—Px activity in the plasma of the supplemented sows. This was in agreement with work cited in Selenium in Nutrition (NRC, 1983), which states that in humans a strong correlation between blood Se and GSH-Px activity has been observed in individuals consuming low levels of Se (Thompson et al., 1977; McKenzie et al., 1978). However, others have reported a lack of correlation between blood GSH-Px activity and blood selenium level when adequate or high Se levels are consumed (Schmidt and Heller, 1976; Schrauzer and White: 1978). This difference is thought to be due to the non-specific incorporation of selenomethionine into blood proteins (NRC, 1983). A significant correlation did not exist between plasma Se level and GSH-Px activities in the third parity sows on either treatment. The small sample size (3 sows/treatment) may have resulted in inconsistencies in the values reported for the third parity sows. Plasma iron concentration in the sows were analyzed. The results indicate no significant change in plasma iron level in either parity due to the dietary treatment. Plasma iron significantly (P<.Ol) increased from parturition to weaning in the second parity sows, however, these differences were not detected in third parity sows. .umao m.30m mo scaumucmEmammsm mm .m Op 030 mucoumwmwo some «0 mocmoquficmwmn .mefiu um>o oocmummwfio come no wocmowmwcmwmm 58 hooo.o hooo.o >ooo.o mm: mz m2 mo.v nmsam>ua mooo.o mo.o m~.v mo.o oH.v mo.o Hmmmm mooo.o mo.o oH.v no.0 Hoo.v 5H.o omucmEmHaasm Axans m\m1v ssmcmamm xunuma nudge mooo.o mooo.o moco.o mm: mo.v Hoo.v Hoo.v nmsam>um ~ooo.o ~o.o mo.v eo.o Hoo.v oH.o gamma «coo.o mo.o mo.v mo.o Hoo.v mH.o omucmsmaaasm bwawe m\ma~ Eowcmamm >ufiu0m ocoomm mm: ammo Hm .ma .Nwa m .ma assumoaoo some made Eswcoamm waz :0 030m «0 :0wumuc050ammsm mm a m annumfia mo muomwmm .o manna 59 II. Sow Milk Sow‘s milk was analyzed for selenium concentration at three stages: colostrum, two days and 21 days post-partum (Table 6). The second parity sows had significantly higher (P<.001) milk selenium concentration as a result of dietary supplementation with vitamin E and selenium, in colostrum and two day milk. Towards the end of lactation milk selenium levels became more nearly similar, however, supplemented sow's milk was still higher (P<.05) than that of the sows on the basal diet. The third parity sows showed a similar difference but this difference was not statistically significant. Mahan et al. (1975) found a significantly greater dietary effect on milk selenium levels throughout lactation when sows were fed a diet supplemented with 0.1 ppm selenium. This is in agreement with the findings of this study. There was a highly significant decrease in the milk selenium level from colostrum to the two day milk sample in second parity sows regardless of dietary treatment. A less dramatic (P<.05) decline was detected from two to twenty-one days post-farrowing (Figure 3). The third parity sows on the supplemented diet showed a similar pattern of selenium depletion in the milk through lactation. This is in agreement with studies which found colostrum levels of selenium significantly higher than later milk (Mahan et al., 60 Figurea. Milk Selenium- 0.16 '- 0.14 ' --. E 8: Se Supplemented Dlet‘ —- Basal Diet 0.12 0.10 - 0.08 Selenium (pg/g) 0.06 0.04 0.02 Days of Lactation 61 1975). This rapid decline may be a consequence of the declining milk protein level as colostrum changes to milk, as several workers have found that the selenium present in milk is primarily associated with the protein fraction, (Jones and Godwin, 1963; Jones and Godwin, 1969; McConnell and Roth, 1964). Alpha- and 8+y-tocopherols were measured in the colostrum and milk of the sows. The sketches shown in figures 4 and 5 represent typical tracings from the recorder of the HPLC for colostrum and later milk, respectively. The first peak is the 8+Y-tOCOpher01 peak. Separation of these two tOCOpherols was not possible. The second peak represents the a—tocopherol present in the colostrum. In colostrum the B+Y-tocopherol concentration was very low. This was typical of all colostrum samples. However, by two days the 8+Y-tocopherol peak was much larger (Figure 5) and the artocopherol peak, which was very large in colostrum, was much smaller. The values for the as and 8+Y-tOCOpherol peaks were corrected to ug of tocopherol per gram of milk and summed to obtain the value for total tocopherol. All of these values are reported in tables 7 and 8 and are graphically presented in figure 6. There was a highly significant dietary effect reflected in higher levels of total and a-tocopherol and lower concentrations of 3+y-tocopherol in the colostrum and 2 day 62 HPLC Trace For Tocopherols in Colostrum 63 Flgure 5. HPLC Traces For Tocopherols. In Milk 64 .uofio n.30m mo newumucmamammom mm .m ou moo oocmuommwo come no oocoowwwcmwmn .maau um>o mocmquMHo some «0 mocooflmacmfimw ¢.o ¢.o ¢.o mm: Ho.v Ho.v Ho.v nonam>lm m.o m.N mz h.~ Hoo.v m.a Hmmmm m.o m.a m2 .m.a Hoo.v m.H monomeoammow Axaws mo m\mnv aouonmoooalmeamo + comm m.o m.o m.o mm: Oa.v Hoo.v aoo.v nooam>1m m.o m.o mo.v m.a Hoo.v w.¢ Homom m.o ~.H mo.v m.~ Hoo.v h.m nonsmEmHmmom AxHHE mo m\mnv Houmnmoooelmnmad moo m.O Goo "mm—z oa.v Ho.v Hoo.v nooam>lm m.o m.m oa.v m.¢ Hoo.v H.o Homam m.o o.m mo.v 5.0 Hoo.v 0.0a omucmEmammom AXHHE mo m\mnv Houonmoooe H0909. 1 mm: axon Hm mm woo N Wm EsuumoHoo yuan mafia .Houosmoooe sun: :0 msom saunas cam mo coHumucmemaamsm 0m a m sumumua «0 muomumm .5 manna 65 .umfiv n.30m mo coflumucoamammom mm .m Cu 050 mucoumMMflU come no mocmowmwsmfimn .msflu uo>o mocmuommwc come no oucmoamwcmwmm H.O H.O Hoo mm: H0.v H0.v H0.v nmsam>lm m.0 H.m m2 N.m H00.v 0.0 Hmmmm m.0 0.H mz 0.H a00.v ¢.0 coucmanmmsm AxAfiE mo m\mav HoumsmoooelmEEoo + mumm H.O H.O Hoo mm: mz m0.v H00.v nmoam>1m 0.0 0.0 H00.v h.~ H00.v H.m ammmm o.o m.a Hoo.v m.e Hoo.v m.m emuamEmmdmsm Axafle mo m\mav Houmnmoooelmzmam «.0 0.0 «.0 mm: 02 mz H00.v nmsao>lm 0.0 0.m m0.v m.m m2 h.m Homom 0.0 m.~ H0.v 0.0 H0.v N.0H pmusmamammom AxawE mo m\m:v Houocmoooe Houoe mm: mxwo Hm m0 >00 m .Mm Esuumano Home wage Houmsmoooe xHHz co m3om >uwuom 0pm 00 coflumucoemammom mm a m >uouofi0 mo muoommm .m manna Total Tocopherol (pg/9) Tocopheml (#91 9) {3+8 10 o, 02~ 66 Figure 6. Milk Tocopherols Per Gram of Milk (2nd Parity Sows) L I 21 Days of Lactation - [3 + 5- Tocopherol Days of Lactation Total Tocopherol 10 9 a - TOCOpherol (pg/g) a - Tocopherol Days of Lactation Vlt. E and Se Supplemented Diet Basal Dlet 67 milk of E-Se supplemented sows. However, the toc0pherol level of sows on both dietary treatments drop by the second day of lactation. The differences due to dietary treatment diminished by the end of the lactation period for total and aptocopherol but theE3+Y-tocopherol continued to be significantly lower in the supplemented sows throughout lactation. Literature was not found pertaining to the effects of dietary supplementation of E and Se on the specific tocopherols present in milk, however, Malm et al. (1976) showed an increase in colostral and three week milk total tocopherol levels due to supplementation of vitamin E in the sow's diet. Total tocopherol was high in colostrum and dropped significantly (P<.OOl) by the second day of lactation in both second parity, dietary groups. The variation in total tocopherol content of milk was similar to the findings reported for human milk (Jagadeesan and Prema, 1980; Jansson et a1., 1981), bovine milk (Herting and Drury, 1969), and in swine milk (Malm et al., 1976: Nielsen et al., 1973). After the initial drop in tocopherol there was a lesser decline in tocopherol content over the remainder of the lactation period and it appeared that the total milk tocopherol in the supplemented sows may decline at a faster rate than the total tOCOpherol of the sows on the basal diet (figure 6). The 8+y-tQCOpherol was very low in colostrum and shows a significant (P<.001) increase by the second day of lactation 68 and then was maintained at a constant concentration throughout lactation. The third parity sows showed a similar pattern in total and B+Y-tocopherol changes over the lactation period, however the a-tocopherol content of milk declined significantly (P<.001) throughout lactation. The weather was very hot during the time the third parity sows were in the farrowing house resulting in a stressful environment and possibly exerting an additional drain on their circulating a—tocopherol. Ullrey et al. (1970) suggested stress may induce vitamin E and selenium deficiency signs. It has been recommended by Bieri and Everts (1975) that plasma a—tocopherol levels be expressed in relation to plasma lipid levels. Since sow's milk has a relatively high fat content it is also important to express the colostrum and milk tocopherol on a total lipid basis. The percentages of total lipids at the three stages of lactation are reported in table 9 and the second parity results are graphically presented in figure 7. No dietary effect was seen for percent total lipids in second parity sows, however, third parity sows on the basal diet exhibited a significantly (P<.05) greater concentration of milk lipids than sows on the supplemented diet. Total milk lipids were low in colostrum, increased (P<.001) by two days, and remained constant throughout lactation. This pattern is similar to the pattern seen 69 .uwwo m.3om mo :oHumucmfioaonm mm .m o» 050 mocmuwwwwn some 00 mocmoawwcmfimn .mewu uo>o mocmummmac some 00 mocoofimwsmwmw @oN $.N moN mm: 02 mo.v mz omsam>lm 0.H m.HH H00.v 5.5H #00.v 0.0 ammmm 0.H m.HH wz h.ma H00.v 0.0 OmusmEmaszm Amv one xauz assume cadre b.m h.m h.m mm: mz mz mz nosam>lm m.m N.HH m2 m.aa H00.v m.0 Hmnmm m.~ 0.0H mz H.HH H00.v m.0 voucmEQHmmom mm0 uma xamz xuuuma ecoomm mm: uNma am 00 >00 N on EouumoHOU wean made uom xawz unmoumm so m3om mo cofiumucoEmHmmsm mm a m >umuow0 mo muomumu .m manna % Lipids nuammslooioo .4 70 Figure 7. Total Milk Lipids (2nd Parity) -- E 8: Se Supplemented Diet -- Basal Diet 21 Days of Lactation .uofic m.30m mo cofiuoucoEoHQmom mm .m on 000 mucoummmwo 0005 Mo mocmofimwcmwmn .mEHu um>o mocmuomwwc some 00 mocmommwcmwmw 71 m.e~ m.e~ m.¢~ mm: Ho.v Ho.v Ho.v nmsum>1m 0.mm a.- mz eumm mz ~.e~ Hommm 0.am m.0u mz o.>~ mz 0.o~ empamsmaddsm Anna 00 m\mnv HouoanUOEImEEMO + muom n.5ma m.amH n.5mu mm: Ho.v Ho.v Hoo.v nmsam>1a o.mma m.0 mz H.ma Hoo.v m.es Amman o.mma 0.HH m~.v H.0m Hoo.v m.mea cmucmsmaoosm Anna 00 m\m:v Houo£QOOOEIana< e.oau e.osa e.o>a mm: ou.v Ho.v doo.v nasam>im m.m0m m.m~ mz m.pm Hoo.v 5.0m Hmmmm m.m0~ m.m~ mz H.me Hoo.v m.m0u emuamsmamosm Aumm mo m\mzv Houmnmoooe amuoe mm: mung mm We won m we ssuumoaoo ammo meB HOH0SQOUOB pom xawz :0 @300 >uwuom 00m 00 codumucmaoammsm mm a m wumumwo .0H mans? 72 .0000 0.300 00 nodumuc0s0ammsu 00 .0 00 0:0 0000000000 0005 00 00cmowwwcmfimn .0500 u0>0 0oc0u00000 c005 mo 0ocmoflwflcmflmw 0.0 0.0 0.0 00: 00.v 00.v 00.v n0s~0>10 0.00 0.0M 02 0.5a Oa.v 0.00 00000 0.00 m.¢H 02 .v.m~ 00.v 0.5 000008000050 A000 00 0\0:. HOM0SQOUOBIMEEMO + 0000 0.000 m.00m 0.000 mm: 02 m0.v ~00.v n0oam>lm 0.000 m.5 mz v.ma 00.v 0.00 00000 0.H0v 0.00 02 , 5.mm 000.v 5.000 000:0E0Hmmsm A000 00 0\0:v Hou0nmoooeim£mad 0.00m 0.00m 0.00m 00: 02 02 mo.v n0oam>im m.mam 0.00 02 0.00 mo.v 5.05 00000 m.mam 0.00 02 H.Hm 000.v 0.0H0 000005000030 Aumm mo 0\0:v Hou0£moooe #0009 mm: axon am an and a an ssuumoHoo 0000 0509 0000:00009 0mm am“: suflumm 00m «0 cemumucmEmammsm mm a a 00 muomwmm .HH manna Total Tocopherol 019/9) [3 + 5’ - Tocopherol (us/9) 110 100 90 80 70 60 50 4o 30 20 10 73 (2nd Parity Sows) Total Tocopherol P ‘5 \ D! 3 '6 b 0 8 Q 0 0 O l- C: ‘ J 21 Days of Lactation .. 6 + 6’ - Tocopherol l 02 21 Days of Lactation 160 150 140 130 120 110 100 90 Figure 8. Milk Tocopherols Per Gram of Fat (22 - Tocopherol Days of Lactation E 81 Se Supplemented Diet Basal Diet 74 previously for 8+y-tocopherol changes in milk over the lactation period (Figure 6). This may indicate that the B+y~tocopherols vary with the amount of fat in the milk. Total, 0- and 8+Y—tocopherol values are reported as micrograms of tocopherol per gram of lipid in Tables 10 and 11 and are graphically presented in Figure 8. From these figures it is clear that the B+Y-tocopherol concentration in the milk lipid does not change significantly over the lactation period. A nearly significant (P<.lO) increase was detected in the third parity sows. A correlation analysis over the entire lactation period demonstrated that B+Y-tocopherol in milk was highly correlated with the percent total lipid in milk in second (r=.6505,.P<.OOl) and third (r = .9061, P<.00l) parity sows, further proof that 8+Y-tocopherols vary with percent lipids. Total and a—tocopherol were also correlated with total lipids. This calculation indicated that total and a—tocopherol were negatively correlated with percent lipid in both second (r=-.64, P<.001) parity sows. The graphic presentation in Figure 6 illustrate the relationship between milk and tocopherol. Milk total and a-tocopherols decline as colostrum changes to milk while over the same time frame total lipids are increasing. In human milk no correlation was seen between total lipids and total tocopherol (Jansson et al., 1981). Referring to Tables 10 and 11 it is clear that a—tocopherol per gram of lipid is very high in colostrum, 75 but by the second day of lactation this level has significantly dropped (P<.OOl) and remains constant per gram of fat throughout lactation. This is in accordance with Herting and Drury's (1969) findings in bovine milk and the findings in human's milk by Jansson et al. (1981) which suggest that this increased level of a-toc0pherol in colostrum may be due to an increased transport capacity for vitamin B. As would be expected, similar dietary effects are present when the tocopherols are exPressed per gram of lipid as when reported earlier as tOCOpherols per gram of milk. The colostrum and milk values for total tOCOpherol per gram of fat for the E-Se supplemented sows were in agreement with values reported by Malm et a1. (1976) for vitamin E supplemented sows. The values which Malm et al. (1976) reported for non-supplemented sows were much lower than those reported in this study. This difference was most likely due to the semi-purified diets supplemented with either lard or corn oil which were fed to their sows. As total tocopherol changes in concentration from colostrum to milk, so also does the composition of this tocopherol. Table 12 lists the levels of a- and 5+y-toCOpherol as a percentage of total-tocopherol. A graphic presentation of these value are shown in Figure 9. As colostrum changed to milk there was a great increase in the percentage of 8+Y-tocopherol as a percentage of total-tocopherol, and this percentage continued to rise 76 .0000500000 00000 00 0000000000 0 00 0000000x0 000005000090 0.05 0.00 0.00 0.00 0.00 0.00 00000 0.00 0.00 5.00 0.05 0.0 0.00 000:0E000050 x00: 300 000000 00009 0.05 0.00 0.00 0.00 0.00 0.05 00000 o.oe 0.00 0.00 0.00 o.m0 0.00 000:02000000 1>+m is i>+0 10 1»+0 15 000000500009 MM0000£00009 000000£00009 0000 00 0N00 0 530000000 0000 x00: 300 000000 000000 0000:0000? x00: 00 00000000500 0:0 co :00000c0s000020 00+0 0000000 00 000000 .00 00000 77 00.0. .0000 II «05 0023502330 0...... .0 m I l 900 0.0.0:..000... 0...! 3:00.. 9...... Op ON on 00 on, Co Oh .00 60 GOP .0350023003. h+ Q I .00o.\.0.o..0002-0 { 0.000 00 a w 0 0 0 \\ ‘1 I! II In... ‘all. ‘1. ll. L I‘ull‘tol I‘ III ‘I .‘l .1. . II I I I I I 1 I I I! I L 0 0 0 ’ 0.2.9.0000... 0...: 3:00. 0:300 0.03.02.00.00 .obozaooo... 0.....2 :. 00:00.0 .0 050.0. av 00 . 00 2. 00 00 GOP loquoool 10:01, ,0 zuaomd 78 throughout lactation. It was clear that B+y~toc0pherol made up the majority (55-70%) of the toc0pherol found in the milk of sows on the basal diet. With no supplemental vitamin E the vitamin E compounds that were present in the ration came from the corn and soybean meal, which are both higher in Y-tocopherol than in a-toc0pherol (Bauernfeind and Cort, 1974), possibly explaining the higher level of 8+y-tocopherol found in colostrum and milk of the sows on basal diets. The practical importance of this finding is obvious. The vitamin E biopotencies of B— and Y—tocopherol in relation to a—tocopherol are only 40% and 10% respectively (Bieri and McKenna,.l981). Therefore, not only was the pig nursing the sow on the basal diet receiving significantly less total-toc0pherol in the milk, but the majority of the tocopherol was in a less potent form. Ullrey (1981) stated that absorption of Y-tocopherol was about as efficient as a-tocopherol, but the turnover rate of the gamma form was much faster. Pigs continually nursing may be able to maintain a relatively high level of total tocoPherol in their plasma. However, when they are weaned this level may drop more rapidly if the pigs are nursing a sow on the basal diet rather than the supplemented diet. This may explain why Ullrey et al. (1970) reported that frequently one of the first signs of vitamin E and selenium deficiency in swine is the occurrence of sudden death in weaned pigs. 79 III. Pigs Heterogeneous variance was detected for many of the variables measured on the pigs throughout the nursing period. The areas where heterogeneous variance occurred are marked in the tables. Probability values of .01 or .001 can still be considered significant when heterogeneous variance is present, however values greater than 0.01 may not be reliable. The analyses of hematological measures of the pigs at zero, two and twenty-one days are reported in Table 13. There were no significant differences due to the dietary or the iron treatment in the second parity pigs at any stage throughout the nursing period. - Hematocrit values were not significantly different due to diet over the nursing period with the exception of the second parity pigs. Pigs which nursed supplemented sows and were given an iron injection prior to colostrum consumption had a higher (P<.lO) hematocrit value at 2 days of age than pigs which did not receive iron and pigs which received iron but which were nursing sows on the basal diet. Third parity pigs follow the same trend, however significance levels were low. It would be interesting to know if the dam's diet effect was real, and if supplementation of vitamin E and selenium of the sow's diet might result in neonatal, pre-colostral pigs with cells that are more resistant to oxidative stress. 80 .oocodua> n:00:390uou0:c .ucnaueouu :oud ca 9:? .m~.vm. fiduccouuucada usuuflc.uo.soau01:o oscu sad) 3:93—00 cdsud3 aceatb .ucosucouu coho Cu one .o-.vm. >~uceouudcadu houu‘c unannouozsu can» :u¢3 cisuoo cased) accotz .:0uu .uOuso voua379 coats .amN.nav UCUU—uucaau uoz H «26 .uodo ca 9:: succuouudc no oocao¢u4caqmo .uoao .uOuuo eouosra acnozn .=oquasoacou flouuuo—oo ea hound scuuououoc_a mo anew 058 c« :o»« no as com eo>uooou sods) mac noueoueca +b m~.v o.n a..n n.~n an m~.v n.~ o.nn c.~n a m m~.v ov.n h.nn q.an + a m: o.o. m.—n o.dn o nu— mz o.q ~.~n ‘.~n . um m2 s.o— _.nn m.~n I a mn.v ov.v q.na v._n + an m: ~.a ~..n o.—n 0 ~— .a. :C—uouucvocou ciao—3030: uc~soazduou coo: aw.» n.ov n.on o.~n .a m: n.~o_ n._~ o.¢~ u a m2 om.nd m..~ o.n~ + ~ m2 o.o.— ~.en o.nn a mu. m: o.nn o.nn c.0n uu m2 «.am o.nm zv.n~ n N m~.v an.a s.n« =~.m~ + w m: «.o- v.mn a.mn Q ~— Aav aduUOugo: m~.v m.n o..d 5.0— gm . mz h.o_ b—.~ so.o I a m: oo._ 9n.s 95.n . + w m: ¢.o o.o— s.o_ o gnu m: o.« a.~— n.__ .. aw m2 o.m c.h n.~ z a a: oo.~ a.h ~.n + ~ cm: n.m— —.‘. v._~ o pa :6}... sang-.620: 02:2. ._ um: .35. $5.5. 1523?: as it... can. mo o>oa rcaucauuo no >50—3umao: co couuUOacn coh— uo oe‘b ecc so“: n.3ca ozu no coduoucoeo.2c:m cm a a no ouoouum .n— cane? 81 The decline in both hemoglobin and hematocrit from birth to two days can be explained by the 10 ml blood sample taken from each pig at birth and the extensive protein influx into the blood of the pig as a result of colostrum consumption, thereby causing the vascular system to take up water. The effect of supplementing the sow's diet with vitamin E and Se on the biological antixoidant status of the neonatal and the nursing pig is reported in Table 14. Plasma Se in the neonate of sows fed the vitamin E-Se supplemented diet is approximately 0.015 ug/ml greater than for pigs born to sows on the basal diet. This is in agreement with finding of Young et al. (l976) for pigs and Pazak (1983) for rats. Therefore, it appears that placental transfer of vitamin E occurs and this transfer may be increased by supplementing Se in the dam's diet (Pazak, 1983). However, the neonate's plasma selenium levels are relatively low in relation to the plasma level in the dam (~25% supplemented and ~30%, basal). Hyroner-Dabek et al. (1984) monitored the plasma Se levels of women in early and late gestation and at parturition they obtained a sample of umbilical cord blood. Plasma from umbilical cord blood contained approximately 57% less Se than that of plasma from maternal blood samples. They suggested that the decline in maternal plasma Se was due to placental transfer of Se to the young with greater incorporation of the Se into tissue. 82 .amo.vm. xuaccouuuca.o Aucovn‘v XdU—ofluodUd-ufi.—Q .oocaquo> azoccoaOHsuo:. sound: uo‘uvnuomzu 93cm cu.) mess—co casuda acaoZm .ucoeueoua cos“ cu cap sounds uduuoauozza 93cm ca.) a:§:.oo :.:u.3 «cast: .:0u« .uOuuo eouaccu coats ..n~..m. accosuacad- no: a mac .u9.6 .uOuuo concave acaozo .coai osu uo coduad>oo auaeccumn .co‘udsaocoo Esuuaodoo o» Hodge scuuououo9_a no show are cu can. no as coN mo>~soou sous: mwc nounmucchlmb 'I'... -.| 5 mo.v No.5 NN.o ev.o ~N m2 oo.c ag.o 9N.o I N m: oNooo.o a—.o cN.o + N m: voo.c N_.o N..o o uun o_.v oo.c cv.c ow.o .dN mo.v No.o mN~.o moN.o I cN mo.v oocc.o no..o mNN.o + aN o~.v No.c n~.o o~.o co . ~— . A~E\:m~ ouceuxOHOm arc—zuou:_o m2 Nv.m vu.N cN.~ ~N mo.v oo.v 6N.~ Nn.n I N mo.v 0N_.c en.~ co.n + N mz Nc.. oo.o «e.o o .u. mN.v om.m cm.~ o_.N _N «cc.v Na.h :mv.N :NN.m I «N goo.» oNa.o :vN.N :on.v + «N mN.v o_.o «4.: NN.o co ~— ;;.~§\m . u cases.) o—.v do.o Nmo.o oN~.o cufl m2 sooo.o coo.o nmo.o I N am: o~ooo.o Noo.o amo.c + N mo.v Nooo.o voo.o oco.o .. 6 —~— do.v ~oo.o vmo.o o~o.o c-N ‘00.. vooc.o mcvc.o mmoo.o I «N doo.v omoooo.o mee=.o m—No.o + .N mN.v Noc.o anc.: nmo.o o nu ..t\m . sauce—om cos—o: m nan: .cmc: Aom+3u+ oco.uoonc~ 0:4 Nausea scum no u>oc a_s>o; acccuuo‘ucc canada :0 couaooncu co»— uo 92.9 ace no“: n.33m ozu no co.nau:93w_dcsm am a a no succuum .v. 0.309 83 A similar decline in maternal plasma Se was discussed earlier for the sows on this study which were on the basal diet. In this case, it was suggested that the drop in plasma Se was a result of a partitioning of the nutrient to the milk, however, it is conceivable that the decline could be a result of both placental and mammary transfer. In reference to Figure 10 it appears that plasma selenium values for the pigs nursing supplemented sows greatly increased by the second day of life, reflecting the high selenium content of the sow's colostrum. The pigs nursing sows on the basal diet do not have this distinct. increase in plasma selenium. By the second day of life pigs nursing supplemented sows had significantly higher plasma Se levels than pigs nursing sows on the basal diet. The significantly higher plasma Se level is maintained in the supplemented pig's plasma throughout the nursing period. Glutathione peroxidase activity in the neonate's plasma was very low in relationship to the levels found in the plasma of their dams. However, even at very low levels there was a trend toward greater activity in the plasma of pigs from vitamin E and Se supplemented sows. Pazak (1983) found similar differences in pre—colostral rats for blood GSH-Px due to supplementation of the dam's diet with vitamin E and selenium. Plasma GSH-Px activity in the pigs tended to reflect dietary E & Se supplementation of the sows diet throughout the nursing period. The plasma GSH-Px activity Milk Selenium (,ug/g) mod cod cod mod 36 u to c To a no one end «no end .05 Emma € 5.5 III . a... I..- 35 32.05235 8 a m I 3cm ll mama 3 c. a 5.52.8 23 IrirrlITrrllIIIrTTFTIII 3.2.... Ba. , . 22.6.. 53:20...“ 3555 «hi 3cm {sow ten :52 925m 5 «macs-.0 .op 9.32.... ILLILLIJILIILlLilllLLilLl N66 vcd 06.6 mad Opd N v.6 v pd 0 .6 o pd Dad and Vud (Ins/671) mnguegas ewseld Plasma GSH - Px (Ell/ml) '8. .35 .30 .25 .20 .15 .10 .05. 85 Figure 1 1 . Changes in Plasma GSH - Px Activity in Pigs (2nd Parity) -- Supplemented -- Basal Diet O 2 21 Days of Age. 86 change over the nursing period showed a trend similar to that seen for plasma selenium (Figure 11). This may indicate that as selenium becomes available to the pig, through colostrum and milk consumption, glutathione peroxidase activity increases. Vitamin E levels in plasma were very low at birth and in many samples were non-detectable, possibly indicating inefficient placental transfer. Malm et a1. (1976) reported that sows have efficient placental transfer of vitamin E resulting in 2.2 to 19.3 fold higher serum vitamin E levels in neonatal pigs than that of their dam. This report is inconsistent with findings in pigs (Young et al., 1977), rats (Pazak, 1983) and human (Martinez et al.,-l98l) where circulating levels of vitamin E in neonates are low. The conclusions on the efficiency of placental transfer of vitamin E vary. Pazak (1983) found low circulating levels of serum vitamin E in the pre-colostral rat pup, but indicated a preferential incorporation of the vitamin in lung and liver tissue. Therefore, efficient placental transfer was suggested. Martinez et a1. (1981) on the other hand suggested that there was a barrier present between placental and fetal blood making the transfer of vitamin E inefficient. Plasma vitamin E increased about 18 fold in both dietary groups of pigs by 2 days of age, with a greater (P<.001) concentration found in the pigs nursing 87 Milk Total Tocopherol (pg/g) @Q-NOIOVC'ON P O P .20 3an 4. . i=2 IIIIIII .06 32582.35 5 a m I son «25 —N 3 N u q q . w. ................ .I I I ll lllll .I II! o, I I I i I ‘.II .'d’lnhul.’ I I I I '- o L..." i... all}, I I I. [Ida-”I’ll r 3.3.. 95 3331003. miner. obi can {sow new .52. {sow E «09330 .«p 052". P 03' Q‘ he 0' ID V (0 N O P (Inn/611) ImeqdoooL ewsegd 88 supplemented sows. The increase in plasma toc0pherol at 2 days of age was most likely due to the very high level of tocopherol in colostrum. Figure 12 graphically depicts the relationship between sow and pig plasma and milk toc0pherol levels through the end of the nursing period. The decline in colostrum total toc0pherol was nearly the same as the increases in plasma vitamin E level in the pig by 2 days. The decrease in colostrum toc0pherol from parturition to the second day of lactation equaled 5.3 ug/g of milk for sows supplemented with vitamin E and 1.6 ug/g of milk for sows on the basal diet and the increase in plasma vitamin E concentration between parturition and the second day of lactation were 4.5 ug/ml and 2.2 ug/ml for pigs nursing dams fed supplemented and basal diets, respectively. The plasma vitamin E level of the pig declined from 2 to 21 days of age, however, there still tended to be differences present at weaning due to the sow's diet. his is not consistent with the results of Young et al. (1977) who reported increases in plasma tocopherol of pigs throughout the nursing period for sows on diets containing 40.6 and 56.1 ugE/g 0M of and no increase at all in pigs nursing sows on a diet containing 0.5 ug/g DM of vitamin E. The lactation diets fed in this study contained calculated values of 4.7 and 14.9 ugE/g BM, in the basal and supplemented diets, respectively. The levels which Young et al. (1977) reported for the three-week milk from the 89 supplemented sows appear to be lower than the three—week values found in this study, however, from the results of pig plasma in their study it would appear that higher vitamin E supplementation of the sow's diet during lactation may prevent a decline in plasma tocopherol levels by weaning. Second parity pigs which received 200 mg of iron in the form of gleptoferron, IM, prior to colostrum consumption tended to have higher plasma selenium (P<.05), higher plasma GSH—Px activity (P<.05), and significantly lower (P<.01) plasma vitamin E levels at two days of age than pigs that did not receive iron. The decrease in plasma vitamin E may be a result of its interaction with peroxy radicals generated in the process of lipid peroxidation whidh is catalyzed by the iron. This series of reactions has been shown to occur in vitro (Fukuzawa et al., 1980). The increase in plasma GSH-Px activity may be a result of the higher plasma selenium level present in the iron treated pigs, or it may be a response to an increased requirement of the pig for biological antioxidants. Sklan et al. (1981) reported that chicks on vitamin E-deficient diets had higher' GSH-Px activity. Plasma iron was measured in pigs through the nursing period (Table 15). There was a great deal of variation in plasma levels at two days after the iron injection. This variation can be explained by inconsistent time intervals between the time of the iron injection and the two-day blood 90 .mUCMAum> msomcomoumummv .ucmEumwuu couA 0» map Amo.vmv >AucmoAMAcmAm umMMAb umAuomummsm mEmm :qu mCEDAoo cAnuA3 mcmmzm .ucmEumouu couA 0» 0:6 aAoo.vmV >AucmoAMAcmAm HoMMAb umAuomuomsm oEmn SuAB CEDAOU sAnuA3 menus: .couA .uouuo bmumsvn cmmzm .AmN.Amv HomoAMAcmAm #02 fl mzAV .qup 0» map mucoHMMMAO mo oocmoAMAcmAwo .uon .uOuum pmumswm cmmzn .coAumEoncoo Esuunvoo ou quum couummoummAm mo EuOm 0:0 cw cOuA m0 m5 OON cm>Aooou £0A£3 mAm mmumoAbcA +m mN.v m.mA m.¢ m.o «AN m2 A.A o.A A.N I N mz w~.o h.s o.~ + m mz m.o m.A h.A o AAA m2 N.AA m.m >.m AN mz m.o mN.A m>.A I N m2 0A.o mm.A mo.A + N mz m.o m.A m.A 0 AA Amxv urmsz mN.v moA x m ©.mmA m.moA AN mz 00A x m mm.®a mh.mm l «N mN.v mooA x N mm.mAON mA.mNA + «N mz NOA x o m.mm c.Nm 0 AAA mz moA x g h.mNA m.@NA AN mz 00A x @ :m.mm :¢.mm I «N mN.v mOA x m mm.mmAN Em.mmm + «N bmz moA x m m.¢m m.OAA 0 AA AAb\m v couA MSmmAm omsAm> m 9mm: Ammmm Amm+mv+ mcoAuochA om< NwAumm couH mo m>ma cOHA mo oEAe chummmmO 020 Mo unmAmZ pcm Am>mq GOHA co coAuUmncA new ammo n.3om an» no :OnumucmsmAmmsm mm a m «0 muomumm .ms manna 9l sampling. By the end of the nursing period there were no differences in plasma iron levels, between the sow's dietary treatment. Analyses of individual pig weights are reported in Table 15, there appears to be no difference in weight due to the sow's dietary treatment throughout the nursing period. This is in agreement with Malm et al. (1976) who found no difference in birth or weaning weights of pigs from sows fed two levels of vitamin E (0 or 100 lU/kg). Pre-colostral iron injection did not appear to affect the pigs performance as pigs from sows on both dietary treatments had significantly (P<.05) higher two day weights than pigs which did not receive iron. 2 The parameters for reproductive performance are reported in Tables 16 and 17. There was no dietary effect on average litter size at birth, two or twenty-one days of age. Average pig and sow weights were not affected by the sow's dietary treatment. Conclusions Supplementation of the gestation (vitamin E 50 IU/kg, selenium, 0.1 ppm) and lactation (vitamin E 17.5 IU/kg, selenium 0.1 ppm) diets of sows increased the plasma vitamin E and selenium level of the sows, but did not increase the sow's glutathione peroxidase activity. The sows fed diets supplemented with vitamin E and selenium had higher levels .umAp n30m mo coAumucmEmAmmsm mm .m on map mocmuomwAp same no oocmoAMAcmAmn .mEAu um>o mocmumMMAp cmoE mo 00:00AMAcmAmW 92 «.mmp m.~mn m.~m> mm: mz m2 mz nmsAm>Im m.pmA A.mmA m~.v e.omA oA.v h.¢o~ Amman m.hmA m.oo~ m~.v ~.¢mA oH.v m.oA~ woucosmnnmsm Amxv unmsoz 30m nxmo AN chzouumm coAumunoo mung Am.o Am.o Am.o mm: m2 m2 mz nmsAm>Im A¢.o o.o Aoo.v m.A m2 m.A Amman A¢.o o.o Aoo.v m.A m2 m.A coucmsmAnmsm Amxv pzmAmz mAm mmmum>¢ o.o~ o.m~ o.o~ mm: mz . mz mz nmsAa>Im om.o 6.5 oA.v m.m oA.v o.m Amman om.o 6.5 oA.v m.m on.v ~.m omucmsmAnnsm mNAm umuuAA mmmuo>¢ mm: «was Am IME ammo m .IME chaounmm qun mEAB mocmEuOMHGm n.3om auAumm ch co coAumusoEmAmmsm mm a m >umu0Aa no uommmm .OA mAnme 93 .AMAU n3on mo coAumuco80Ammsm mm .m ca 030 mocmumuuAG come no mozmoAMAcmAmn .oEAu uo>o oocmuomqu some «0 mocmoAMAcmAmm A.@Nm A.@Nm A.mNm mm: mN.v m2 m2 nosAm>Im m.mm m.mmA Ao.v o.oNN mz o.mNN Ammmm m.mm h.mNN m2 .o.NmN m2 m.NMN bowcoEmAmmsw .mx. unmnmz 30m n>ma AN msA3ouumm coAumummw mum: m.A mnA m.A mm: oA.v mz mz nmsAm>Im v.0 m.¢ Aoo.v m.A m2 o.A Annmm ¢.o m.~ Aoo.v m.A mz m.A cmucmsmAmmsm Amxv unonz mAm mmmuw>< m.bm m.hm m.hm mm: mz m2 m2 nmsAm>Im m.A o.m m2 m.oA m2 o.AA Amnmm m.A m.m m2 5.0 . mz h.w UmucoEmAmmsm oNAm umuuAd omnum>< mm: mxan Hm .Iwm axon m as chzouumm uuAa oEAE 00cm500mumm n.3om auAumm bum co coAumvcwEmAmmzm mm a m humuon mo uoomum .hA mADme 94 of selenium, total and artocopherol in their colostrum and lower levels of 8+Y-tocopherol throughout lactation than sows fed the basal diet. The biological antioxidants were concentrated in the colostrum of all sows and decreased as colostrum changed to milk. Total and a-tocopherols do not vary with the level of fat in colostrum. However, 8+Y-tocopherol concentration is highly correlated with milk fat concentration throughout lactation. Pigs are born with relatively low circulating levels of biological antioxidants. The level of antioxidants increases by two days of age, primarily due to colostrum consumption. This increase is not as great if pigs are nursing sows whose diets are not supplemented with vitamin E and selenium and their biological antioxidant status remains lower throughout the nursing period than pigs nursing sows whose diet is supplemented with vitamin E and selenium. Iron injection (200 mg of Fe from gleptoferron) given prior to colostrum consumption did not affect the livability or performance of the pigs from either dam dietary group. 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