I: 1" .VM.‘J¢!.”J . In ENFLUENCE 0F PREGNANCY AND DRY PERIOD “ON {ACTATION IN THE RAT ’ Thesis for the Degree of Ph. D; aNIICHiGA§ STATE ,UNiv-Easm ' '.: i1AX .FAMES PAAPE 1937' v rm This is to certify that the thesis entitled INFLUENCE OF PREGNANCY AND DRY PERIOD ON LACTATION IN THE RAT presented by MAX JAMES PAA PE has been accepted towards fulfillment of the requirements for 7 t / 7.) degree in (2/. ,’ fl, / x // /:)/»Z:,’ / / /2 I. T [I v" £4; 7’" f dill/Lg'dl 15/ Major professor /7 .v/, f! A '4 :5 , Date fig” g» 1‘ Z” :1 [ff/7? // 4/ I. 0469 ABSTRACT INFLUENCE OF PREGNANCY AND DRY PERIOD ON LACTATION IN THE RAT by Max J. Paape The influence of pregnancy on various biochemical parameters of the secretory and connective tissue com- ponents of lactating and post-lactational involuting mammary glands was studied. In addition, the influence of 0-, A-, 8-, 12-, and l6-day dry periods on the carry over of secretory and connective tissue into a second lactation was also studied. The parameters measured were DNA (cell numbers), RNA and RNA/DNA (protein synthetic activity), hydroxyproline (collagen), hexosamine (ground substance) and crude lipid. Concurrent pregnancy from the 8th to the 2Ath day of lactation reduced (P < 0.01) daily litter weight gain,. DNA, RNA, and RNA/DNA ratio of the mammary gland, relative to lactating, non-pregnant (LNP) controls by 32.1, 7.7, 20.5, and 15.0 per cent respectively. Following weaning on the 8, 12, 16, or 20th day‘ of lactation, pregnancy retarded DNA declines after the 12th day of gestation. 0n the 8th day of the dry period, following weaning on the 8th or 12th day of lactation, DNA, RNA, and RNA/DNA ratios of pregnant animals gradually increased until parturition. Max J. Paape Hydroxyproline of the mammary gland for lactating, pregnant (LP) animals was constant from the 8th to the 20th day of lactation, but then decreased 32.8 per cent between the 20th and 2Ath day. On the 24th day, hydroxyproline for LP animals was 32.3 per cent less than corresponding LNP controls. From the 8, 12, 16, and 20th day of lactation to the 4th day after weaning, hydroxyproline content for both pregnant and non-pregnant animals showed changes ranging from a 3.8 per cent increase to a 18.7 per cent decrease. Unlike DNA, whose loss was retarded after the 12th day of pregnancy, hydroxyproline losses were not retarded until the 16th day of pregnancy. Hexosamine of the mammary gland for LP and LNP animals increased 19.7 and 31.0 per cent respectively, from the 8th to the 16th day of lactation but decreased “2.6 and 17.7 per cent, respectively, from the 16th to the 24th day. These quadratic curves (P < 0.01) suggest that net synthesis of hexosamine by the mammary gland occurred at the peak of lactation. Following weaning, pregnancy effectively reduced the loss of hexosamine after the 12th day of gesta- tion, but did not promote net increases until the 20th day of pregnancy. In the second lactation, dry period length did not influence total litter weight gain from the 3rd to the 8th day, but from the 8th to the 16th day those animals given Max J. Paape a A-day dry period produced 22.0 and 9.3 per cent heavier litters than animals given a 0-day or longer (8-, l2-, l6-day) dry periods, respectively. On the first day of the second lactation, animals given an 8— or O-day dry period contained approximately 13 per cent more DNA than rats given l6—, 12-, or 4-day dry periods. 0n the 8th and 16th day of the second lactation mammary glands of animals given 0- or l6-day dry periods contained approximately 1“ per cent less DNA than animals given a A- or 8-day dry period. The various dry periods had no effect on RNA on the first day of the second lactation (P > 0.05). However, on the 8th day, mammary glands of rats given an 8-day dry period contained on the average 15.8 per cent more RNA than glands which received l6- or 0-day dry periods. By the 16th day these l6- and 0-day dry period groups contained 1A.3 and 22.1 per cent less RNA than rats given the 4-day dry period. Length of the dry period had no effect on RNA/DNA ratios. Thus, the dry period effects subsequent lactation by causing loss of cells or preventing mitosis during lactation rather than loss of synthesis per cell. During the second lactation rats which received the 0- or l6-day dry period contained less hydroxyproline, than rats which received the 4-, 8-, or 12-day dry periods. Max J. Paape Length of the dry period did not effect hexosamine on the lst or 8th day of the second lactation (P > 0.05). However, by the 16th day, a linear increase (P < 0.05) with increasing length of the dry period was observed which was related to a linear increase in weight of the adrenal gland. INFLUENCE OF PREGNANCY AND DRY PERIOD ON LACTATION IN THE RAT By Max James Paapeg-~ A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1967 BIOGRAPHICAL SKETCH Max J. Paape was born at Port Chester, New York, on December 26, 1936. He received his elementary education in Our Lady of Mercy Parochial School, and was graduated from Port Chester Senior High School in June, 1954. In September, 195A, he enrolled at The Long Island Agricultural and Technical Institute, Farmingdale, New York, majoring in Animal Husbandry. He was graduated in June, 1956, with the degree of Applied Arts and Science. From September, 1956 to June 1959, he completed his undergraduate requirements leading to a Bachelor of Science in Agriculture Extension at Michigan State University. In January, 1960, he enrolled in the graduate school of Michigan State University. He received the Master of Science degree in March, 1963 and Doctor of Philosophy degree in September 1967, in the Dairy Department with a major in Mammary Gland Physiology. 11 ACKNOWLEDGMENTS The author wishes to express his gratitude and thanks to his major professor, Dr. H. Allen Tucker, for his guidance and help throughout the Ph. D. program. The assistance of the author's past and present colleagues, Drs. C. Desjardins, Y. N. Sinha, K. T. Kirton, K. L. MacMillan and w. w. Thatcher are gratefully acknow- ledged. Special thanks go to Mrs. Helga Hulkonen and Miss Susan Jeffries for their assistance with the tedious chemical assays. Sincere appreciation is extended to Drs. L. D. McGilliard, P. E. Reineke, A. J. Morris, and J. L. Gill for their help in preparing this manuscript, and to Dr. H. D. Hafs for his assistance and encouragement during the author's graduate study. The author is grateful for the financial support provided by Michigan State University through a Graduate Research Assistantship and Research Instructorship. Gratitude is extended to Dr. C. A. Lassiter, Chairman of the Department of Dairy, for making available the facilities and animals. The sacrifice, patience, and encouragement of the author's wife, Susan, during the course of this study is deeply appreciated. iii TABLE OF CONTENTS Page BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . ii- ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . xi LIST OF APPENDICES . . . . . . . . . . . . . . . . . . xii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 3 Development of the Mammary Gland During Pregnancy and During Lactation . . . . .-. 3 Development of the Lactating Mammary Gland During Concurrent Pregnancy . . . . . . . .'. 5 Status of the Mammary Gland During Post- Lactational Involution . . . . . . . . . . 8 Influence of the Dry Period on Subsequent Mammary Gland Performance . . . . . . .-. . . 11 MATERIALS AND METHODS . . . . . . . . . . . .,. . . . . 1“ Experimental Animals and Design . . . . . . . . 14 Influence of Pregnancy on Mammary Gland Development During Lactation and During Post- Lactational Involution . . . . . . . 1“ Influence of Length of the Dry Period on Subsequent Lactation in the Rat . . . . . 18 Biochemical Parameters Measured in the Mammary Glands and Mammary Fat Pads . . . . . . . . . 20 Statistical Analysis . . . . . . . .-. . . .'. . 29 iv Page RESULTS AND DISCUSSION Influence of Pregnancy During Lactation . . . . . 30 Body Weight . . . . . . . . . . . 30 Weight of the Mammary Gland and Extraparenchymal Fat Pad . . . . . . . . . . 32 Daily Litter Weight Gain . . . .‘. . 35 Nucleic Acid Content of the Mammary Gland and Extraparenchymal Fat Pad . . . . 37 Ethanol, Chloroform: Methanol, Ether (ECME) Extract of the Mammary Gland and Extraparenchymal Fat Pad . . . . . . . AA Hydroxyproline Content of the Mammary Gland and Extraparenchymal Fat Pad . . . . . . A6 Hexosamine Content of the Mammary Gland . . . . 52 Weight of the Adrenal Gland . . . . . -. . 53 Influence of Pregnancy During Various Lengths of the Dry Period . . . . . . . . . .,. . . .' 55 Body Weight . . . . . . . . 55 Weight of the Mammary Gland and Extra- parenchymal Fat Pad . . . . . . . . . 57 DNA Content of the Mammary Gland and. Extraparenchymal Fat Pad . .~ . . 61 RNA and RNA/DNA Ratio of the Mammary Gland . . 70 Ethanol, Chloroform: Methanol, Ether (ECME) Extract of the Mammary Gland and Extraparenchymal Fat Pad . . . . 75 Hydroxyproline Content of the Mammary Gland and Extraparenchymal Fat Pad . . . . . . 79 Hexosamine Content of the Mammary Gland . . . . 87 Weight of the Adrenal Gland . . . . . .'. 89 Influence of Length of the Dry Period on Subsequent Lactation . . . . . . . . . . . . 91 Body Weight . . . . . . . . . . . . 91 Weight of the Mammary Gland and Extraparenchymal Fat Pad . . .4. . . . . . . 93 Total Litter Weight Gain . . . . . 96 Nucleic Acid Content of the Mammary Gland and Extraparenchymal Fat Pad . . . . . . . 98 Ethanol, Chloroform:Methanol, Ether (ECME) Extract of the Mammary Gland and Extraparenchymal Fat Pad . . . . . 106 Hydroxyproline Content of the Mammary Gland and Extraparenchymal Fat Pad. . . . . . . . . 109 Hexosamine Content of the Mammary Gland . . . . 11A Weight of the Adrenal Gland . . . . . . . . . . 116 Page SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . 118 LIST OF REFERENCES . . . . . . . . . . . . . . . . . . 127 APPENDICES . . . . . . . . . . . . . . . . . . . . . . 136 vi LIST OF TABLES Table 1. Influence of pregnancy on body weight of lactating rats . . . . . . . . . . . 2. Influence of pregnancy on weight of the mammary gland of lactating rats 3. Influence of pregnancy on weight of the extraparenchymal fat pad of lactating rats 4. Influence of pregnancy on daily litter weight gain of lactating rats. . . . 5. Influence of pregnancy on nucleic acid content of mammary glands of lactating rats . . . . 6. Influence of pregnancy on DNA content of the extraparenchymal fat pad of lactating rats 7. Influence of pregnancy on ECME extract of the mammary gland of lactating rats . . . . . 8. Influence of pregnancy on ECME extract of the extraparenchymal fat pad of lactating rats 9. Influence of pregnancy on hydroxyproline content of the mammary gland of lactating rats . . . . . . . . . . . . . . 10. Influence of pregnancy on hydroxyprolinezRNA ratio of the mammary gland of lactating rats . . .*. . . 11. Influence of pregnancy on hydroxyproline content of the extraparenchymal fat pad of lactating rats . . . . . . . . . . . 12. Influence of pregnancy on hexosamine content of the mammary gland of lactating rats . . 13. Influence of pregnancy on weight of the adrenal gland of lactating rats 1A. Influence of pregnancy on body weight during dry periods of A, 8, 12 and 16 days vii Page 31' 32 3A 36 38 ”3 AA 45 A6 51 51 52 5A 56 Table 15. l6. 17. 18. 190 20. 21. 22. 23. 2A. 25. 26. Influence of pregnancy mammary gland during A, 8, 12 and 16 days Influence of pregnancy extraparenchymal fat periods of A, Influence of pregnancy mammary gland during A, 8, l2 and 16 days Influence of pregnancy extraparenchymal fat periods of A, Influence of pregnancy mammary gland during A, 8, l2 and 16 days Influence of pregnancy on weight of the dry periods of on weight of the pad during dry 8, l2 and 16 days . . . . . on DNA content of the dry periods of on DNA content of the pad during dry 8, l2 and 16 days . . on RNA content of the dry periods of on RNA/DNA ratio of the mammary gland during dry periods of A, 8, l2 and 16 days Influence of pregnancy mammary gland during A, 8, l2 and 16 days Influence of pregnancy extraparenchymal fat of A, Influence of pregnancy on ECME extract of the dry periods of on ECME extract of the pad during dry periods 8, 12 and 16 days . . . . . . . . on hydroxyproline content of the mammary gland during dry periods of Influence of pregnancy A, 8, 12 and 16 days . . . on hydroxyproline content of the extraparenchymal fat pad during dry periods of A, Influence of pregnancy of the mammary gland of A, Influence of pregnancy adrenal gland during A, 8, 12 and 16 days 8, 12 and 16 days on hexosamine content during dry periods 8, 12 and 16 days . . . . . on weight of the dry periods of viii Page 58 60 66 69 71 73 76 78 80 82 88 90 Table 27. 28. 29. 30. 31. 32. 33. 3A. 35. 36. 37. 38. Page Relation between length of the dry period and body weight during the second lactation . . . . . . . . . . . . . . . . . 92 Relation between length of the dry period and weight of the mammary gland during the following lactation . . . . . . . . 9A Relation between length of the dry period and weight of the extraparenchymal fat pad during the second lactation . . . . . . . . . 95 Total litter weight gain from the 3rd to the 8th and 8th to the 16th day of the second lactation after various length dry periods . 96 Relation between length of the dry period and DNA content of the mammary gland during the second lactation . . . . . . . . . . . . 99 Relation between length of the dry period and DNA content of the extraparenchymal fat pad during the second lactation . . . . . 102 Relation between length of the dry period and RNA content of the mammary gland during the second lactation . . . . . . . . . . . . 10A Relation between length of the dry period and RNA/DNA ratio of the mammary gland during the second lactation . . . . . . . 105 Relation between length of the dry period and ECME extract of the mammary gland during the second lactation . . . . . . . . . 107 Relation between length of the dry period and ECME extract of the extraparenchymal fat pad during the second lactation . . . . . 108 Relation between length of the dry period and hydroxyproline content of the mammary gland during the second lactation . . . . . . 110 Relation between length of the dry period and hydroxyproline/RNA ratio during the second lactation . . . . . . . . . . . . . 111 ix Table Page 39. Relation between length of the dry period and hydroxyproline content of the extraparenchymal fat pad during the second lactation . . . . . . . . . . . . . . 113 A0. Relation between length of the dry period and hexosamine content of the mammary gland during the second lactation . . . . . . 115 Al. Relation between length of the dry period and weight of the adrenal gland during the second lactation . . . . . . . . . . . . . . 117 LIST OF FIGURES Figure Page 1. EXperimental design used to study the influence of pregnancy on mammary growth during lactation and during post- latational involution . . . . . . . . . . . . 15 2. Experimental design used for the control of the experiment shown in Figure 1. . . . . . l6 3. Experimental design used to study the influence of length of the dry period on subsequent lactation . . . . . . . . . . . . 19 A. Relation between DNA, RNA, hydroxyproline of the mammary gland and daily litter weight gain for lactating, non-pregnant animals during latter stages of lactation . . . . . . A8 5. Relation between weight of the extra- parenchymal fat pad and mammary gland of pregnant animals during various stages of lactation and involution . . . . . . . . . . . 62 6. Relation between weight of the extraparenchymal fat pad and mammary gland of non-pregnant animals during various stages of lactation and involution . . . . . . . . . . . . . . . . 6A 7. Relation between DNA and hydroxyproline of the fat pad for pregnant animals during various stages of lactation and involution . . . . . . 83 8. Relation between DNA and hydroxyproline of the fat pad for non-pregnant animals during various stages of lactation and involution . . 85 xi Appendix I. II. III. IV. LIST OF APPENDICES Page Composition of rat feed . . . . . . . . . . . . 137 Solutions used in the analytical procedure for nucleic acid . . . . . . . . . . . . . . 1A0 Solutions used in the analytical procedure for hydroxyproline . . . . . . . . . . . . . 1A2 Solutions used in the analytical procedure for hexosamine . . . . . . . . . . . . . . . 1AA xii INTRODUCTION Several species of mammals may become pregnant during lactation. Although one can imagine that such a condition would present physiological stress on the mammary gland and milk synthesis, little is actually known about the influence of pregnancy on the secretory and connective tissue components of the lactating mammary gland. However, pregnancy in cows reduces milk production, especially as gestation progresses. Such an effect lowers the financial return to the dairyman. A basic knowledge of the biochem- ical changes taking place in the lactating mammary gland, as a result of concurrent pregnancy, may contribute to understanding how pregnancy lowers milk yield. Thus, a primary objective of this study was to determine the influence of pregnancy on various biochemical parameters of the secretory and fat pad stromal components of the mammary gland during lactation. During the past three decades, a large body of evidence has accumulated which suggests that a dry period (a period of nonlactation between lactations) is necessary for maximal milk production in the cow. Absence of a dry period will decrease the milk yields of the subsequent lactation. However, the reasons for the necessity of a dry period still remain obscure. To omit a dry period without causing a deleterious effect on subsequent lactation would probably increase yearly production by at least 15 per cent. The changes taking place within the mammary gland during the dry period and how length of the dry period affects the mammary gland during the subsequent lactation might suggest a method whereby the dry period could be reduced or elimi- nated. Thus, a second objective of this study was to in- vestigate the changes taking place in the secretory and con- nective tissue components of the mammary gland during the dry period, and during the adjacent lactation following different dry period lengths. The biochemical parameters studied in the parenchymal- stromal portion of the mammary gland were deoxyribonucleic acid (DNA), ribonucleicacid (RNA), crude lipid, hydroxy- proline, and hexosamine. The DNA was used to estimate total cell numbers; RNA was used as an indicator of protein synthetic activity; and lipid, hydroxyproline (a measure of collagen content), and hexosamine (a measure of ground sub— stance content) were used as measures of the various compo- nents of connective tissue stroma. In addition, the ex- traparenchymal mammary fat pad, removed from the periphery of the mammary gland, was analyzed for total DNA, lipid, and hydroxyproline to provide a better understanding of the changes in stromal elements in the absence of parenchymal cells during mammary gland growth and involution. The rat was chosen as the experimental animal and may serve as a model for future experiments in cows. REVIEW OF LITERATURE Development of the Mammary Gland During Pregnancy and During Lactation DNA levels of the mammary gland as a measure of total mammary cells, have been extensively studied in the rat (Harkness and Harkness, 1956; Greenbaum and Slater, 1957; Shimizu, 1957; McLean, 1958; Smith and Richterich, 1958; Shimizu and Ugami, 1959; Griffith and Turner, 1959, 1961a; Slater, 1961; Moon, 1962, Tucker and Reece, 1963a, 1963b; Munford, 1963; and Baldwin and Milligan, 1966), and in the mouse (Lewin, 1957; and Brookreson and Turner, 1957). Such studies show that growth of the mammary gland continues throughout pregnancy, reaches a maximum about the 10th day of lactation, remains relatively constant to about the 20th to the 2Ath day and then decreases. Tucker and Reece (1963a, 1963b) reported a 261.1 per cent increase in total DNA from the first to the 20th day of pregnancy and a 2A.8 per cent increase during lactation. Traurig (1967) using tritiated thymidine, reported a wave of mammary epithelial cell proliferation during early lactation, which confirmed the total mammary DNA data. The level of total mammary RNA and the RNA:DNA ratio has been used to indicate the state of metabolic activity of mammary tissue with particular reference to protein synthesis (Kirkham and Turner, 1953; Kuretani, 1957; Shimizu, 1957; Slater, 1961; Tucker and Reece, 1963a; and Baldwin and Milligan, 1966). Total mammary RNA, similar to DNA, increased progressively throughout pregnancy and early lactation, remained constant until the 2Ath day, and then decreased. The RNA:DNA ratios were less than 1 during the greater part of pregnancy, indicating little protein synthesis per cell. Ratios increased steadily during the last few days of pregnancy and during lactation to a level of 2 to 3 between the 10th and 20th day of lactation but then subsequently decreased. There is little information concerning the status of the connective tissue stroma during pregnancy and lactation. It has been suggested that the connective tissue actually determines the form of mammary development. In tissue culture, mammary duct cells grow in irregular sheets in the absence of fibroblasts; but an early organization of the epithelium into alveolar—like structures was discernible if fibroblasts were added to the tissue culture media (Las- farques, 1957). Harkness and Harkness (1956) determined that mammary collagen (hydroxyproline) levels were relatively constant throughout pregnancy and lactation, whereas the lipid content increased slightly in late pregnancy but declined during lactation. On the other hand, Wrenn 32:31. (1965) reported a continuous decrease in lipid content throughout pregnancy and lactation. Development of the Lactating Mammary Gland During Concurrent Pregnancy Rats and mice usually exhibit estrus the night following parturition (Blandau and Soderwall, 19Al). If these animals are mated at this time, blastocyst implant- ation is usually delayed (Lataste, 1891; and Enzmann gt_§l., 1932). The length of this delay is dependent on the number of suckling young (Enzmann gt_gl., 1932; and Mantalenakis and Ketchel, 1966). Four or less suckling young will delay implantation two days, whereas five or more suckling young will delay implantation four or more days(Mantalenakis and Ketchel, 1966). A subcutaneous injection of a 0.3-10ug of estrogen on day 5 of lactation will cause implantation at that time and thus prevent the delay in implantation normally encountered (Yoshinaga and Hosi, 1958). Several studies have been made of mammary development in animals lactating while pregnant. Mammary glands of rats simultaneously lactating and pregnant (LP) do not undergo appreciable involution (Reece, 1958). Reece and Warbritton (1953) observed that mammary mitotic activity for LP animals was greater on the 2lst day than on the 15th day of lac- tation. Furthermore, the mitotic activity was much less for the LP animal than for the nonlactating, pregnant animal. When the authors removed litters on the 17th day of lacta- tion in animals simultaneously pregnant, no increase in mitotic activity could be detected A days later. In the lactating mouse, Mizuno (1960a, 1961) studied involution of mammary glands following unilateral ligation Of the inguinal galactophores with continued suckling of contralateral glands. Mammary involution, on the basis of histology, respiratory quotient (R. Q.), and nucleic acid content was markedly retarded by concurrent pregnancy. In another experiment, Mizuno (1960b) reported no significant differences in mammary oxygen consumption, R. Q., or RNA/DNA ratio between lactating, non-pregnant (LNP) and LP mice on the lAth or 19th day of lactation. However, total DNA and RNA were greater in the LP groups. The author concluded that in the mouse, advancing preg- nancy enhanced lactation, and the conditions for mammary growth may coexist with those for synthesis of milk. Tucker and Reece (196A) studied the influence of concurrent pregnancy on lactation between the 18th and 28th day of lactation without regulating implantation time. Pregnancy caused increases in total mammary DNA and RNA on the 18th day of lactation when compared with LNP controls. 0n the 21, 2A, and 28th day concurrent pregnancy had no effect, significant reduction, and no effect, respectively, on nucleic acid content. Within days 2A and 28, total nucleic acid content declined with advancing stages of concurrent pregnancy. When these authors standardized gestation length, they found that rats with seven or more fetuses contained less mammary DNA and RNA than LNP rats. Low numbers of fetuses (six or less), however, caused enhancement of mammary nucleic acid content. Stanley and Reece (1967) did not observe any sig- nificant difference in litter weight gain for the LP rat as compared with LNP controls. Similarly, no significant differences could be found for total DNA, RNA, or RNA/DNA ratio of the mammary gland. Meites and Turner (19A8) reported that concurrent lactation and pregnancy in the rabbit did not alter pituitary prolactin content. The authors concluded that pregnancy does not interfere with lactation as long as demands for nutrients by both the fetuses and mammary glands are met. Bruce (1961) observed in the rat that estrus recurred every 18 days during prolonged lactation, and when mated on the 75th day of lactation, normal pregnancy ensued. Toward the end of pregnancy, lactation always failed despite con— tinuous suckling. This failure to maintain continuous lactation until parturition is in contrast with the results obtained by Mayer (1956). He was able to maintain lacta- tion by frequent exchanges of litters. There is the possi- bility, as pointed out by Bruce (1961), that the eyes of the older litters of Mayer were open by the time the litters from the post-partum matings were born. Thus, the young were no longer wholly dependent on the mother for food, and whether milk secretion was contributing to litter weight gain just prior to parturition is questionable. There are a few studies showing the effect of preg- nancy on lactation in the bovine. Ragsdale e£_al. (1923), and Gaines (1926) reported that pregnancy did not influence lactation until the 5th month, after which time lactation was depressed. Cows pregnant 8 months produce 20 per cent less milk than non-pregnant cows (Gaines, 1926). The total reduction may amount to ADO-8001b of milk if cows are bred during the early months of lactation (Ragsdale et_al., 1923). Status of the Mammary Gland During Post-Lactational Involution Morphological studies of mammary involution following weaning are well known for the rat (Myers and Myers, 1921; Kuramitsu and Loeb, 1921; and Maeder, 1922); for the mouse (Turner and Gomez, 1933a; Cole, 1933; Fekete, 1938; Hooker and Williams, 19A0; and Williams, 19A2); for the guinea pig (Hasselberg and Loeb, 1937); and for the goat (Turner and Reineke, 1936). According to Turner and Reineke (1936), the mode of regression of mammary glands among species is similar. Upon failure to withdraw the products of secretion, the glands become distended with milk which increases the intramammary pressure. This engorgement persists for several days then gradually recedes. The epithelial cells lining the alveoli collapse and disintegrate, many of them being cast into the lumina, the lobules gradually disappear, and finally the point is reached were only the ducts and gland stroma remain. The primary difference among species during involution is the length of time required for the mammary glands to revert to the virginal state. In the mouse, complete re- gression occurred within 13 days after weaning (Cole, 1933; and Williams, 19A2). In the rat, Maeder (1922) stated that the normal virginal condition was approached by the 9th day; but Myers and Myers (1921) reported that 2 to 3 weeks were necessary. Complete involution of the lobule-alveolar system was noted in the guinea pig after 35 days (Turner and Gomez, 1933b) and in the goat after 75 days (Turner and Reineke, 1936). Generally, the duct system of the involuted mammary gland is more ramified than that of a nulliparous animal (Reece, 1958). Histological studies (Emmel et_a1., 19A6; and Silver, 1956) showed that in the early stages, when the alveoli are grossly distended with milk, the capillaries are collapsed, suggesting a reduced blood flow. Cross and Silver (1956) further suggested that the engorgement inhibited or restricted the transport of precursors and metabolites for milk synthesis. Silver (1956) also found that lactation could be reinitiated in rats weaned early in lactation provided the suckling stimulus was resumed within A to 5 days. After this time irreversible changes in the capillary blood supply to the alveoli prevented the re-establishment of mammary function. 10 Changes in the nucleic acid content of the mammary glands of rats and mice during involution after cessation of suckling have been investigated by several workers (Kirkham and Turner, 1953; Oshima and Goto, 1955; Green- baum and Slater, 1957; Griffith and Turner, 1961b; Mizuno, 1961; Anderson and Turner, 1963; Munford, 1963; Tucker and Reece, l963d; and Ota, l96A). In general. these studies show that both DNA and RNA delcine after the suckling stimulus is removed. However, Slater (1962), reported an increase in rat mammary DNA content during the first A8 hours of involution, which may be partially accounted for by a leucocytic invasion (Okada, 1956). Decreases in DNA then occurred after the A8-hour interval. Williams (19A2), Gibson (1930), Fekete (1938), and Mayer and Klein (1961) reported that post-lactational involution of the mammary gland consisted of atrophy and necrosis of the alveoli and small ducts and their replace— ment by fatty, areolar connective tissue. Harkness and Harkness (1956) reported that non-collagenous protein and DNA decreased, whereas collagen and lipid content of the mammary gland did not change during 21 days of involution. 11 Influence of the Dry Period on Subsequent Mammary Gland Performance The effects<3f1ength of the dry period on the next lactation in cows have been reported by several investi- gators (Sanders, 1928; Dix and Becker, 1936; Dickerson and Chapman, 1939; Klein and Woodward, 19A3; Johansson, 1962; and Ackerman gt_a1., 1967). Such studies indicate that dry periods of less than 6 weeks decreased the milk yield during the next lactation; but dry periods longer than 8 weeks produced only slight increases in subsequent lac— tation yields. More recently, Swanson (1962, 1965) reported that cows milked throughout pregnancy produced 30 per cent less milk in the following lactation than their twins which received dry periods of 2 months. Johansson (1962) showed that cows which, on an average have long dry periods are poor producers. Dickerson and Chapman (1939) compared production records following dry periods of different lengths with those of the first lacta- tion. They found that low-producing cows received greater benefit from a long dry period than high producing cows. These workers also found that short dry periods depressed the milk yield of the next lactation more in herds fed below average than in those fed at above—average levels. Swanson (1965) reported that when adequate nutrition was provided to meet the stress of concurrent lactation and pregnancy, twin cows with no dry periods still produced less than their 12 twins which received 60—day dry periods. The author con- cluded that the role of nutrients during the dry period is probably minor. Smith et_al. (1966) suspended milking in two of four udder quarters in each of two cows 10 weeks before partur- ition. After parturition, when all four quarters were milked, yield of the two quarters given a dry period showed an increase in milk yield over that of the two quarters not given a dry period. These authors also con- cluded that the effect of the dry period on subsequent milk yield cannot be due to nutritional factors but appears to be related to changes which originate within the mammary gland itself and take effect before parturition. In a more extensive sutdy, however, Ackerman gt_al, (1967) suspended milking in two of four udder quarters in each of several cows 10 weeks before parturition. They reported comparable milk yield between quarters given a dry period and those receiving no dry period. Gorman and Swanson (1967) reported that cows which received oxytocin injections during a normal dry period produced less milk than animals which received a dry period. These results suggested that oxytocin may be involved in lactation deficiencies following short dry periods. Altman (19A7) reported that a dry period was necessary to allow rapid regeneration of the secretory epithelium 13 before the next lactation. More recently, however, Pardue and Swanson (1963) showed that on the first day of the second lactation, total DNA content of mammary glands receiving no dry period was equal to the total DNA content of mammary glands receiving a 2-month dry period, suggesting that con- tinuous milking may reduce the function of the mammary gland rather than its structure. MATERIALS AND METHODS Experimental Animals and Design Experimental animals were rats of the Sprague-Dawley strain. They were maintained at 2A: 10C and subjected to illumination between 5 a.m. and 7 p.m. All rats received a specially prepared 21.2 per cent protein diet (Appendix I) and water ad libitum Influence of Pregnancy on Mammary Gland Development During Lacta- tion and During Post-Lacta- tional Involution Primiparous pregnant female rats in advanced gestation were co-habited with male rats. Females were separated from males the day following parturition (second day of lactation). On the third day, teats of the thoracic mammary glands were ligated, litter size adjusted to six pups, and the mothers initial body weight recorded. To control blastocyst implan- tation time, female rats received 0.2 ug estradiol-l7-B (Tucker and Reece, 196A) subcutaneously on the fifth day of lactation. Pregnancy was determined at autopsy. If the animals were pregnant, they were placed into one of the experimental groups shown in Figure 1. If the animals were notpregnant, they served as controls and were placed at . random into one of the control groups shown in Figure 2. 1A .pmaafix mums mo nomads monocoa m Hm>noch wcfipmpomalcoz .co>oEop mnoupfia hams» pan scan: mums pcmcwmsq .wcfipmpoma mo genes: monocoo H Hm>pouCH wcapmpomq coapomncfi eOApawHH coauHASpmmm a Hoavmmumm pace cofipamSpsmm m.awa mma mma r3 HJN 1 mmH NNH MNH Hem mma mmH mma mma Hm: mmH mmH mmH mma mma _ How am om SH NH m m m H mama .COHpsHo>cfi HmCOHpmpomHIpmoo wCHLSU cam coapmuomfl weassp npzoaw mamasms co mocmcwmpo mo mocoSHmcH on» hogan on com: cwflmmp Hmpcmefihooxm .H ohswfim l6 .pmHHHx mama mo nomads mouosoa m .Hm>nmch wcfipmuomanoz .pm>oEoH mumpufia pawn» can moan: mama wcfipmpoma.ucmcmonalsoc mo Hones: monocoma .Hm>nopca meanneomq coauooncfi coapmmfiq Hoacmppmm pace coapfipsppmm m.HmH 1 NH IIIImmH H3N mmH .lllmmH IlllmmH Hmm mmH IllmmH ImmH IIIINNH Hm: mmH IIIINNH IlllmmH IIINNH IIIINNH How am om SH NH m m m H when .H onswam CH czonm pcoefipomxm map mo Hospcoo esp pom pom: cmamop ampsoEHHoQKMII.m omsmflm 17 Any lactating, pregnant rats which did not deliver their second litter by the 2Ath i 1 day after parturition were discarded. Litter weights were recorded on the 8, 12, 16, 20 and 2Ath day of lactation. In order to maintain a strong suckling stimulus, whenever a litter reached 16 days of age, they were replaced with a new 12-day-old foster litter. The foster litter weights were recorded, at the beginning (12th day) and end (16th day) of the A-day suckling period. The routine at the time of killing was as follows: 1. An animal was stunned by a blow to the back of the head and then killed by cervical disloca- tion. The animal was weighed. 2. If the animal was pregnant, the fetuses and uteri were removed and weighed, and the weight sub- tracted from the body weight. Fetus size at autopsy was compared with fetuses of known age taken from rats killed on the 8, l2, l6, and 20th day of normal pregnancy. If the fetuses were not of the expected size, indicating delayed implantation, the animal was discarded. 3. The six abdominal-inguinal mammary glands were removed within 10 minutes after stunning, dissected free of extraparenchymal fat and connective tissue, and weighed. The glands were covered with ice-cold 18 0.25 M sucrose and stored at -20°C for subsequent analysis. The extraparenchymal fat (fat pad), trimmed from the periphery of the mammary glands, was weighed, covered with sucrose and stored at -20°C. A. The adrenal glands were removed within 15 minutes after stunning, dissected free of fat and connec- tive tissue and weighed. Influence of Length of the Dry Period on Subsequent Lactation in the Rat Primiparous pregnant female rats in advanced gestation were co-habited with male rats. Females were removed the day following parturition. 0n the third day, teats of the thoracic mammary glands were ligated, litter size adjusted to six pups, and the mothers initial body weight recorded. On the fifth day after parturition female rats received 0.2 ug estradiol-l7-B. During the first lactation simultaneously lactating, pregnant rats were given dry periods of 0, A, 8, l2 and 16 days (Figure 3). Litter weights were recorded on the 8, 12, 16, 20 and 2Ath day <3f the first lactation. The suckling stimulus was maintained by exchanging all l6-day-old litters for new l2-day-old litters. Rats which did not deliver their second litter by the 2Ath i 1 day after parturition were discarded. 0n the third day of the second lactation, litter size was adjusted to six pups. Litter weights were recorded daily l9 .COHumuomH ocoSom .anuoa mum .:0H»MpomH umpam :oHuoowcH coaumeq COHpfihsppmm Hoaumnpmm pave zoapfithnmm HHHx HHHx HHHH HHHx nu II: new: HHquuuununn III cam: HHHx III new: HHHx III III cam: HHHx III III cam: HHHx III III cam: HHHxflflflflflflflfluuflflflukuH llllll 2mm: ‘ HHHx llllll cam: HHHx |||||| cam: HHHx llllllll cam; HHHx llllllll cam: HWHH llllllll new: SH S :m om SH NH S S m H has coaumuomfi cacomm coHSSHomH HmsHm .GOHpMpomH unendomnsm co map on» no camcofl no ooqodamqfi on» madam 0» com: cwfimmp HmucoEHHonMIl.m ohswfim 20 starting on the third day of the second lactation. For each of the various dry periods, 12 rats each were killed on the 1, 8, and 16th day of the second lactation. At the time of killing, the same autopsy routine was followed as outlined for the previous experiments. Biochemical Parameters Measured in the Mammarnylands and Mammary Fat Pads The biochemical parameters measured in the mammary gland and extraparenchymal fat pad were the concentrations of DNA, hydroxyproline, and ethanol, chloroform-methanol, ether (ECME) extract. The level of RNA and hexosamine were measured in the mammary glands only. The samples, which had been stored in 0.25M sucrose at -20°C, were thawed at A°C. The mammary glands were homogenized for 2 minutes in distilled water at A°C to produce final concentrations of 50 mg of tissue per ml for subsequent analysis. The extraparenchymal fat pads were extracted with 95 per cent alcohol for 2A hours, chloroform-methanol (2:1) for 2A hours, and then ether for 2A hours. After each extraction, the lipid solvent was poured into pre-weighed 50 ml glass beakers, the solvent was evaporated in a fume hood and the beaker with the extracted residue weighed. The defatted pads were dried at 37°C for 12 hours, weighed, and pulverized to a fine powder in a micro Wiley mill (Arthur H. Thomas-00., Philadelphia, Pennsylvania) for subsequent analysis. 21 The analytical procedure for nucleic acids was based on modifications of the Schmidt Thannhauser (19A5) procedure described by Tucker (l96A): l. Mammary gland homogenate, containing 100 mg of tissue, was pipetted into a 16 ml plastic centri- fuge tube. Ten ml of 95 per cent ethyl alcohol was added, the tube capped and shaken at room temperature for 12 hours. The tube was then centrifuged at 17,000 rpm for 15 minutes and the supernatant fluid was poured into a pre-weighed 50 m1 glass beaker. Ten ml of methanolzchloroform (2:1) was added to the sample which was shaken for 2A hours, centri- fuged for 15 minutes at 17,000 rpm, and the super- natant fluid was poured into the same beaker. At this point the nucleic acid procedure was started for the mammary fat pad. Twenty mg of the finely—ground fat pad was placed into a 16 ml plastic centrifuge tube and 10 ml anhydrous ether added to both tubes. Tubes were shaken for 12 hours at room temperature and centrifuged for 15 minutes at 17,000 rpm. The supernatant fluid from the homogenate was again poured into the glass beaker. The supernatant fluid from the fat pad sample was discarded. 22 The glass beaker containing the extracted residue of the mammary gland homogenate (EMCE fraction) was placed in a fume hood, the solvent evaporated, and the beaker with the residue weighed. Five ml of ice-cold 10 per cent tricholoracetic acid (TCA) was added to the lipid-free residues of the mammary gland or fat pad portions, mixed, centrifuged for 15 minutes, and the supernatant fluid discarded. This step was repeated. Five m1 of ice—cold 95 per cent ethanol saturated with sodium acetate was added, mixed, centrifuged at 17,000 rpm for 15 minutes and the supernatant fluid discarded. Two ml of 1N potassium hydroxide (KOH) was pipetted into each sample, the tubes capped and placed in a 37°C oven for 15 hours. The tubes were cooled in ice water, 0.5 m1 of ice- cold 10 per cent perchloric (PCA) added, mixed, centrifuged for 15 minutes at 17,000 rpm and the supernatant fluid (RNA fraction) ofthe mammary homogenate saved. This supernatant fluid from the fat pad sample was discarded. Five m1 of ice-cold 5 per cent PCA was added to both precipitates, mixed, centrifuged at 17,000 rpm for 15 minutes and the supernatant fluid for the mammary gland sample saved. The supernatant fluid for the fat pad sample was again discarded. This step was repeated. 10. ll. 23 The combined supernatant fluids (RNA fraction) of the mammary homogenates were brought up to 20 ml with 5 per cent PCA and mixed. One ml of the RNA fraction was then pipetted into a clean 16 m1 test tube containing 2 ml of 5 per cent PCA and 3 ml of freshly prepared orcinol reagent (Appendix II) was added. The tube was then capped and boiled for 30 minutes. The color development was then read in a Beckman DB spectrophotometer after 15 minutes at 670 mu. The RNA content of the sample was calculated from a standard curve derived from pure yeast RNA (Worthington Bio- chem. Corp.). Five m1 of ice-cold 5 per cent TCA was then added to the precipitates of both mammary gland or fat- pad samples from step 8, mixed, incubated at 70°C for 15 minutes, cooled to 5°C, centrifuged for 15 minutes at 17,000 rpm, and the supernatant fluids (DNA fraction) poured into separate 25 ml graduated test tubes. Five ml of ice-cold 5 per cent PCA was added to the precipitate, mixed, centrifuged at 17,000 rpm for 15 minutes, and the supernatant fluids poured into their respective tubes. This step was repeated. 12. The described follows: 1. 2A The graduated test tube containing the super- natant fluid (DNA) from the mammary gland homo- genate was brought up to 25 ml with 5 per cent PCA. The fat pad sample was brought up to 20 ml with 5 per cent PCA. The Optical densities were read in a Beckman DB spectrOphotometer at 268 mu, and the DNA content of the sample was calculated from a standard curve derived from pure highly polymerized DNA (Worthington Biochemical Corporation). analytical procedure for hydroxyproline analysis, by Prockop and Udenfriend (1960), was modified as Mammary gland homogenate, containing 25 mg of tissue, was pipetted into a 16 ml culture tube and 3.5 ml of HCl:H2O (2.0:1.5) added. For fat pads, 5 mg of ground fat pad was placed into a 16 m1 culture tube and A.0 m1 of HCleZO (1:1) added. The tubes were capped tightly and autoclaved at 15 lb pressure for 15—2A hours. The tubes were cooled and the hydrolyzed contents poured into 25 ml graduated test tubes. The cul- ture tubes were rinsed with distilled water to remove any adhering hydrolysate, and the volume in the graduated test tubes adjusted to 8 ml for the mammary gland sample or to 12 ml for the fat pad sample. 25 One ml of resin-charcoal preparation (Appendix III) was added to the hydrolyzed samples, stirred on a Vortex mixer, poured into 16 m1 plastic centrifuge tubes and centrifuged at 17,000 rpm for 10 minutes. One ml (mammary gland) or 0.5 ml (fat pad) was pipetted into 100 ml culture tubes. One drop of l per cent phenolpthalein in 95 per cent ethyl alcohol was added and the pH adjusted to light pink with 1N KOH and then 0.1N KOH. The tubes were adjusted to approximately 8 ml with distilled water and saturated with an excess of potassium chloride. Readjustments to a light pink were made with 0.1 N KOH if necessary. Two ml of borate buffer and one ml of 10 per cent alanine solution (Appendix III) were added. The reaction mixture was then oxidized with 2.0 ml of a freshly-prepared solution of 0.2 M chloramine T in 2-methoxy-ethanol for 20 minutes. The reaction was stopped by the addition of 6 ml of a 3.6 M solution of sodium thiosulfate in water. The mixture was again saturated if neces- sary with potassium chloride. Ten ml of toluene was added and incorporated into the reaction mixture by stirring on a Vortex mixer. The two phases were allowed to separate 10. 11. The described 1. 26 and the top phase (toluene layer) was removed by suction and discarded. Tubes were capped and placed in a boiling water bath for 30 minutes. After cooling, 10 ml of toluene was added and incorporated into the reaction mixture by use of a Vortex mixer. The two phases were allowed to separate and 5 ml of the toluene phase was pipetted into clean 16 ml glass test tubes. Two ml of sulfuric Ehrlich's reagent (Appendix III) was immediately mixed into the sample. The color reaction was then read in a Beckman DB spectrophotometer after 15 minutes and before 60 minutes at 560 mu. The hydroxyproline content of the sample was calculated from a standard curve, derived from pure hydroxy-L-proline (Calbiochem). The standard curve was obtained by taking different dilutions of standard through the same steps as the unknown samples, starting at step 2. analytical procedure for hexosamine determinations, by Boas (1953), was modified as follows: Mammary gland. homogenate, containing 0.2 gm of tissue, was pipetted into a 10 ml graduated test tube. One ml of 2N HCl was added, the tube was capped with a marble, and hydrolyzed at 100°C for 15 hours. 27 The tube was cooled, made up to 10 ml with distilled water, poured into a 16 m1 plastic centrifuge tube and centrifuged at 17,000 rpm for 10 minutes. Five ml of clear supernatant fluid was added to another 16 ml plastic centrifuge tube con- taining Dowex—50, 250—500 mesh resin (Appendix IV), stirred on a Vortex mixer, centrifuged for 5 minutes at 17,000 rpm, and the supernatant fluid discarded. The Dowex-50 was washed twice with two 5 ml volumes of distilled water, centrifuged for 5 minutes at 17,000 rpm and the supernatant fluids discarded. The hexosamine was eluted with four 2 ml washes of 2N HCl, centrifuged at 17,000 rpm for 5 minutes and the supernatant fluids poured into a 25 m1 graduated test tube. The volume was made up to 10 m1 and then poured into a 16 ml plastic centrifuge tube, and centrifuged at 17,000 rpm for 5 minutes. Three m1 of the clear supernatant fluid was pipetted into a clean 25 m1 graduated test tube. Two drops of 0.5 per cent phenolpthalein in 15 per cent ethyl alcohol was added, and the pH adjusted to a pink color with AN sodium hydroxide 10. 28 (NaOH). 0.5N HCl was added until the indicator color just disappeared. One ml of freshly prepared acetylacetone reagent (2 per cent solution, volume per volume, of acetylacetone in freshly prepared 1N sodium carbonate) was added to the sample. The sample was capped and placed in a water bath at 89° to 92°C for A5 minutes. After cooling, 2.5 ml of absolute ethyl alcohol was added, the sample mixed, and 1 ml of hydro- chloric acid Ehrlich's reagent (Appendix IV) added. The tube was made up to 10 ml with absolute ethyl alcohol and thoroughly mixed. One hour after the addition of the Ehrlich's reagent, the optical density was read at 530 mu in a Beckman DB spectrOphotometer. The hexosamine content of the sample was calculated from a standard curve derived from pure D (+) glucosamine hydrochloride (Matheson, Coleman and Bell). The standard curve was obtained by taking different dilutions of standard through the same steps as the unknown sample, starting at step 3. Statistical Analysis Analysis of variance was used to analyze the influence of pregnancy on mammary gland development during lactation. Within the LP or LNP groups, orthogonal polynomial response curves (Ostle, l96A) were made for the five stages of lactation studied. In some cases, orthogonal contrasts (Ostle, 196A) were performed within LP and LNP animals for the five stages of lactation. Analysis of variance was also used to analyze the influence of pregnancy on involution of the mammary gland during the dry period, and the influence of length of the dry period on subsequent lactation. Orthogonal contrasts (Ostle, 196A) were made between days of lactation, and orthogonal polynomial response curves (Ostle, l96A) were made for dry periods within a stage of lactation. RESULTS AND DISCUSSION Influence of Pregnancy During Lactation Body Weight The influence of concurrent pregnancy and lactation on body weight is listed in Table 1. Analysis of variance showed that body weights of lactating, pregnant (LP) animals (2A6 g) were less (P < 0.08) than lactating, non- pregnant (LNP) controls (25A g). LP animals weighed sig— nificantly less (P < 0.05) than corresponding controls only on the 2Ath day of lactation (235 g versus 256 g, respec- tively). These results on the 2Ath day are not in agreement with those of Tucker and Reece (196A). These authors reported that body weights of LP rats were about the same as LNP controls from the 18th to the 28th day of lactation. A possible explanation for this discrepancy is that pregnancy was delayed in Tucker and Reece's experiment and the rats were not as advanced in gestation as were the rats used in the present study. Another explanation could be that the hooded Norway rats used by Tucker and Reece responded differently than the Sprague-Dawley rats used in this present experiment. 30 31 TABLE l.-—Inf1uence of pregnancy on body weight of lactating rats. Day of lactation Final body weighta (s) Lactating, Lactating, pregnant non-pregnant 8 252 :7 2A8 :5 12 2A5 :8 258 :10 16 2A5 16 256 :8 20 250 15 250 i9 2A 235 15° 256 :8 Overall mean 2A6C 25A aMean and standard error of mean. b Significantly less (P < 0.05) than lactating, non- pregnant controls. cSignificantly less (P < 0.08) than lactating, non- pregnant controls. The depression in body weight that I observed on the 2Ath day could be attributed to water loss associated with parturition and/or to advancing pregnancy. If the latter, then no weight loss could be detected during the early stages of pregnancy because the embryos were small in com- parison to the weight of the mother, and, therefore, the nutrients required were insignificant. After the 20th day, however, the fetuses developed sufficiently to require appreciable amounts of nutrients for growth and maintenance. If the animal was lactating, an additional nutrient 32 Jrequirement would be produced and the animal would probably Lise body reserves in order to meet the requirements of both Ipregnancy and lactation. \Neight of the Mammary Gland and IExtraparenchymal Fat Pad The influence of concurrent lactation and pregnancy <3n weight of the mammary gland is shown in Table 2. Weight of‘the mammary gland of LP animals (9.85 g) was less (P < 0.01) ‘than LNP controls (11.65 g). The interaction between day of lactation and pregnancy state was also significant (P < 0.01). 'TABLE 2.--Inf1uence of pregnancy on weight of the mammary gland of lactating rats. Mammary gland weighta (g) Day of Lactation Lactating, Lactating, b b pregnant non-pregnant 8 9.73:0.65 9.39:0.SA l2 11.6Ai0.59 l2.AA:0.83 l6 11.96t0.81 12.9A:1.0A 20 9.83:0.78 ll.99:0.9A 2A 6.09i0.36 11.50:0.A8 Overall mean 9.85C 11.65 a Mean and standard error of mean. bSignificant quadratic regression (P < 0.01) among days of lactation. CSignificantly less (P < 0.01) than lactating, non— pregnant controls. 33 \deight of the mammary gland for LP and LNP animals increased 218.6 and 27.A per cent, respectively, from the 8th to the 216th day but then decreased A9.0 and 11.0 per cent, respec- ‘tively, from the 16th to the 2Ath day of lactation. These = 0.90) from LNP controls (0.90 g) (Table 3). Weight of the fat pad for either LP or LNP animals did not change (P = 0.75) from the 8th to the 2Ath day of lactation. Thus, weight of the mammary fat pad did not parallel the quadratic regression curves observed for mammary gland weight. Linzell (1959) suggested that the mammary gland grows into the fat pad during pregnancy but regresses out of it during involution. In view of this, it was anticipated that weight of the fat pad would decrease during the increase in mammary gland weight and increase during the decrease in mammary gland weight. Since this was not the case, it is possible that the mammary gland did 3A grow into and regress out of the fat pad, but that the extraparenchymal fat pad showed a compensatory growth and regression, thereby maintaining a constant weight. It is also possible that the mammary gland did not grow into or regress out of the fat pad between the 8th and 2Ath day of lactation, and the change in mammary gland weight during lactation was due to an increase or decrease in mammary gland thickness rather than an increase in length and breadth. TABLE 3.--Inf1uence of pregnancy on weight of the extra- parenchymal fat pad of lactating rats. Fat pad weighta (3) Day of lactation Lactating, Lactating, pregnant non-pregnant 8 0.85:0.11 l.0A:0.25 12 1.02:0.15 0.80:0.1A 16 0.99:0.17 0.83:0.12 20 0.8A:0.09 0.93:0.10 2A 0.86:0.13 0.90:0.10 Overall mean 0.91 0.90 8'Mean and standard error of mean. ‘51 35 Daily Litter Weight Gain Litter weight gains per day, a measure of milk pro- duction, from the 8th to the 12th, 12th to the 16th, 16th to the 20th, and 20th to the 2Ath day of lactation are listed in Table A. Daily gain in litter weight for LP animals (6.93 g) was less (P < 0.01) than LNP controls (10.23 g). The interaction between pregnancy state and day of lacta- tion was also significant (P < 0.01). Daily litter weight gains for LP and LNP animals increased 7.A and 2.7 per cent, respectively, from the 8‘12 to 12—16 day period and decreased 119.5 and 2A.3 per cent, respectively,from the 12—16 to 20-2A day period. These changes were (P < 0.01) quadratic responses in both groups. These results are not in agree- ment with Mizuno (1960b) and Stanley and Reece (1967) who reported that pregnancy did not effect litter weight gain for the first 20 days of lactation. However, the LP mice used by Mizuno were nursing 6 pups per 10 mammary glands and the LP rats used by Stanley and Reece were nursing 8 pups per 12 mammary glands. In the present study I adjusted litter size to 6 pups per 6 mammary glands. The nursing young in Mizuno and Stanley and Reece's experiments had an extra milk supply from the additional mammary glands. Thus, the experimental design used by these experimentors may not have met the requirement set forth by Cowie and Folley (19A7), that the validity of using litter weight gain as a quantitative measure of lactation rests upon there being 36 TABLE A.--Inf1uence of pregnancy on daily litter weight gain of lactating rats. Daily litter weight gaina (g) Day of lactation b b Lactating, Lactating, pregnant non-pregnant 8-12 10.0i0.5 10.8*0.7 12-16 10.810.6 11.110.A 16-20 8.710.6 10.6*1.0 20-2A -l.710.5 8.A*0.7 Overall mean 6.9C 10.2 aMean and standard error of mean. bSignificant quadratic regression (P < 0.01) among days of lactation. CSignificantly less (P < 0.01) than lactating non- pregnant controls. sufficient young to use all of the milk secreted by the mother. Also, the eyes of rat and mice pups open at approximatley 16 days of age, and the pups will start to eat solid food and drink water. Therefore, daily gain in litter weight after 16 days of lactation is not a good measure of milk production. To avoid this problem I switched l6-day old litters for 12-day old litters. Mizuno and Stanley and Reece did not. Their litter gain from the 16th to the 20th day may not represent weight gains attributable solely to milk production. 37 My results in rats agree with results obtained in the cow by Ragsdale g£_al. (192A). These authors showed that pregnancy depressed milk yields after the fifth month of lactation. Nycleic Acid Content of the Mammary Gland and Extraparenchymal FatIPad The influence of concurrent pregnancy and lactation on DNA, RNA and RNA/DNA ratio of the mammary gland, relative to LNP controls is given in Table 5. Analysis of variance showed that total DNA of the mammary gland of LP animals (29.0 mg) was less (P < 0.01) than LNP controls (31.5 mg). The interaction between day of lactation and pregnancy state was also significant (P < 0.01). DNA of the mammary gland for LP and LNP animals increased 7.3 and 7.8 per cent, respectively, from the 8th to the 16th day, decreased 10.0 and 8.7 per cent, respectively, from the 16th to the 20th day and from the 20th to the 2Ath day decreased 23.3 per cent for the LP animals but increased 7.9 per cent for the LNP animals. These changes be- tween the 8th and 2Ath day of lactation represented quadratic (P < 0.01) and cubic (P < 0.05) curves for the LP and LNP animals, respectively. Total RNA of the mammary gland of LP animals (1A8.3 mg) was less (P < 0.01) than LNP controls (186.5 mg) (Table 5). The interaction between day of lactation and state of preg- nancy was also significant (P < 0.01). Total mammary gland RNA for LP and LNP animals increased 37 and A7 per cent, respectively, from the 8th to the 16th day, decreased 25.3 .COSHSHOmH go when wcoem Amo.o v mv scammomwop cacao pQSQSMchHmp .coaumpoma no name mcoem AHo.o v my coammohwop ofiummpmsv unavamficwfimo .maomucoo ucmconQIcoc .wcfiumpoma can» Ado.o v mv mwoa hapCHOSQchHmn .cmme no Homno endgamum one cmozm 38 m.m m.SmH m.Hm no.m hm.w:H no.mm ewes Hampo>o m.oH m.m H m.oe m.oe m.ee m.HH H.S o.mmH m.mm H.m H.HS m.mm :m m.ow m.mau :.HH 3.0“ m.oaw m.HH m.S a.mmH w.mm m.m m.mmH m.mm om m.oe m.mHH H.HH m.ow S.HHH o.HH o.> a.mmm S.mm m.S o.MHm S.mm SH m.ou S.mHH S.HH m.OH m.mH N.HH m.m S.mmH S.mm >.m a.mNH >.om NH m.oe a.mH e.HH m.oe S.HHA A.HH m.: m.mmH 0.0m 3.: m.=ma m.om m Awev Awsv Awev AwEv osm> zo_h40 , ON @— N. . m >¢a m>- %‘ < \ e353. ? l / (I2 lHSISM ova 1w 0 .3 I ’— p 2’ \ v 7% o S‘.‘ j T a: O h {D (5) .LHOIBM ONV'IO wkuvw 2 E 3'. 64 Figure 6.--Re1ation between weight of the extraparen- chymal fat pad and mammary gland of non-pregnant animals during various stages of lactation and involution. 65 (“human aw 1v: omz> 20:43.04... no >40 1mm. 9 N. w . in 92o ‘ w .m w v o w a. o. v o o. «m o e o . 1. d J - J n d u d ! ho- . n . 92 c 8r > 5L3: .e no .. .f .\ I .o W Oat n@ W , > a m E. \ \ .s w 9 u... \ / .m w. . z \ m 3 / a .m M \ _ . a e... ( / \ .29. ‘9‘“. E. m. o o... , .m. a... in. 3. .2 66 TABLE l7.--Influence of pregnancy on DNA content of the mammary gland during dry periods of 4, 8, 12 and 16 days. Total DNA Day of (mg) lactation c when weaned Days dry Pregnant Non-pregnant 8 4 15.4:0.7 12.1:O.8 8 15.6:1.7 9.0:0.8 l2 17.9:0.8 7.8:0.5 16 (parturition) 19.9:0.9 5.6:0.4 l2 4 23.0:1.3 l3.5:0.8 8 18.4:1.3 10.0:0.7 12 (parturition) 19.5:0.7 9.2:0.4 16 4 23.5:1.1 13.9:1.4 8 (parturition) 22.5:1.l 10.4:0.6 20 4 (parturition) 19.7:1.3 l4.2:0.9 Overall mean 19.5b 10.6 aMean and standard error of mean. bSignificantly greater (P<0.01) than non-pregnant controls. cDay 1 of pregnancy = day 1 of lactation. 67 non—pregnant animals on the 4th day of the dry period fol- lowing any of the weaning times. This suggests that the hormones before the 12th day of pregnancy were not being secreted in quantities large enough to retard the fall in DNA. This relatively constant per cent decrease in total mammary gland DNA 4 days after weaning lactating, non- pregnant animals on the 8th, 12th, 16th and 20th day, make it apparent that approximately the same per cent decline in total cells will occur regardless of what stage of lactation the non-pregnant animals are in when weaned. Also, Tucker and Reece (l963d) reported that total DNA decreased 70 per cent 12 days after weaning non-pregnant rats on the 20th day of lactation. We observed that total DNA decreased 74 and 72 per cent 12 days after weaning non-pregnant rats on the 8th and 12th days, respectively, of lactation. Our results on the 4th day after weaning LNP animals on the 20th day of lactation also agree with the results of Griffith and Turner (1961b) and Anderson and Turner (1963). They reported 42.0 and 53.4 per cent decreases, respectively, in total DNA 5 days after weaning on the 20th day of lactation. Total DNA of the mammary gland of pregnant animals increased progressively a total of 21.7 per cent during the 16-day dry period after weaning on day 8 of lactation. How- ever, when weaned on day 12, pregnancy failed to stimulate increases in cell number until between 8 days of involution and the end of the dry period when a 5.4 per cent increase 68 in DNA was observed. Similarly, in the 16th day weaning group a decline of 4.1 per cent in mammary DNA was observed between the 4th and 8th day of the dry period. Total DNA of non-pregnant animals from the 4th day of the dry period to the end of the dry period, decreased 53.4, 31.4 and 25.7 per cent, for the 8-, 12- and l6-day weaning groups, respectively. These results indicate that the hormonal condition after the 12th day of concurrent pregnancy was not only sufficient to retard mammary involution during the dry period but to stimulate net mammary cell mitosis during the 16- and l2-day dry periods. The influence of pregnancy on total DNA of the extra- parenchymal fat pad during various length dry periods is summarized in Table 18. Total DNA of the fat pad of pregnant animals (0.76 mg) was not different (P = 0.75) from non- pregnant controls (0.79 mg). On the 4th day of the dry period, following weaning at the 8th, 12th, 16th and 20th day total DNA of the fat pad of pregnant animals decreased 10.6, increased 10.2, 0.3, and decreased 13.7 per cent, respectively. Total DNA of the fat pad of non-pregnant animals during these same times increased 37.8, 32.0, 25.9 and 21.3 per cent, respectively. In general, the changes in fat pad DNA for pregnant and non-pregnant animals paralleled the changes in fat pad weight (Table 16). This same relationship also existed between mammary weight and DNA. Since cell growth in the mammary glands of pregnant 69 TABLE 18.--Inf1uence of pregnancy on DNA content of the extraparenchymal fat pad during dry periods of 4, 8, 12 and 16 days. Total DNAa Day of (mg) lactation b when weaned Days dry Pregnant Non-pregnant 8 4 8 12 16 (parturition) 12 4 8 12 (parturition) l6 4 8 (parturition) 20 4 (parturition) 0.55:0.04 0.89:0.05 0.81:0.08 0.65:0.07 0.78:0.06 0.63:0.10 0.76:0.06 1.14:0.21 0.71:0.07 0.65:0.07 1.27:0.39 0.82:0.09 0.74:0.08 0.70:0.08 0.59:0.06 0.73:0.09 0.75:0.09 0.91:0.10 0.58:0.06 0.74:0.05 Overall mean 0.76 0.79 aMean and standard error of mean. b Day 1 of pregnancy = day 1 of lactation. 70 animals would be expected to be primarily epithelial cell growth and connective tissue cell growth (fibroblasts) in the fat pad, it appears that the factors regulating growth are the same for both types of cells. RNA and RNA/DNA Ratio of the Mammary Gland Total RNA of the mammary gland of pregnant animals averaged 49.1 mg during the various dry periods, and was greater (P < 0.01) than the 18.7 mg average found in non- pregnant controls (Table 19). The interaction between day of the dry period and pregnancy state was also significant (P < 0.01). On the 4th day of the dry period of rats weaned at the 8th, 12th, 16th and 20th day of lactation, total RNA of pregnant animals decreased 76.5, 60.2, 71.2 and 66.7 per cent, respectively, from the 0-day (Table 5) levels. Total RNA for non-pregnant animals decreased 82.9, 87.1, 87.0 and 85.3 per cent, respectively. This striking decrease in total RNA reflects the decreased protein synthesis associated with involution. Like DNA, the relatively constant per cent decreases in total RNA 4 days after weaning the non-pregnant animals at all stages of lactation suggests that the same per cent loss in total metabolic activity of the mammary gland will occur regardless of when the non-pregnant animals were weaned. Also, Tucker and Reece (l963d) reported that mammary RNA decreased 94.4 per cent 12 days after weaning non—pregnant rats on the 20th day of lactation. We observed 71 TABLE l9.—-Inf1uence of pregnancy on RNA content of the mammary gland during dry periods of 4, 8, 12 and 16 days. Body weights (g) Day of lactation C when weaned Days dry Pregnant Non-pregnant 8 4 31.5:2.4 22.0:2.2 8 28.0:4.8 l3.3:l.2 12 35.5:2.1 10.8:O.6 l6 (parturition) 61.1:4.2 12.2:2.2 12 4 69.1:5.3 24.4:1 4 8 37.7:2.6 15.0:2.2 12 (parturition) 53.0:4.0 15.3:1.4 l6 4 61.4:4.5 29.8:3.4 8 (parturition) 61.0:4.4 16.5:1 l 20 4 (parturition) 53.0:4.0 27.2:2.6 Overall mean 49.1b 18.7 aMean and standard error of mean. bSignificantly greater (P < 0.01) than non-pregnant controls. 0Day 1 of pregnancy = day 1 of lactation. 72 that total RNA decreased 91.6 and 92.0 per cent 12 days after weaning non-pregnant rats on the 8th and 12th days, respectively, of lactation. Total RNA of the mammary gland of pregnant animals weaned on the 8th day of lactation increased 54.1 per cent from the 4th to the 16th day. Those pregnant animals weaned on the 12th day, however, did not reach minimal RNA values until the 8th day of the dry period (37.7 mg) and then increased 29.0 per cent to the 12th day dry. Most of the increases in RNA of those rats weaned at the 8th or 12th day were interpreted as probably being associated with initiation of the second lactation rather than being caused by advancing pregnancy. Rats weaned on the 16th day declined only 0.7 per cent between the 4th and 8th day of the dry period. Total RNA of non-pregnant animals between the 4th and last day of the dry period decreased 44.3, 37.2, and 44.7 per cent, respectively. The influence of pregnancy on RNA/DNA ratios of the mammary gland during various length dry periods is shown in Table 20. The overall RNA/DNA ratio of pregnant animals (2.5) during the dry period was higher (P < 0.01) than non: pregnant controls (1.7). The interaction between day of the dry period and pregnancy state was also significant (P < 0.01). Between the day of weaning on the 8th, 12th, 16th and 20th day of lactation and the 4th day of the dry period, RNA/DNA ratios of pregnant animals decreased 53.4, 47.2, 60.3 and 73 TABLE 20.--Inf1uence of pregnancy on RNA/DNA ratio of the mammary gland during the dry periods of 4, 8, 12 and 16 days. ~0- a 1 Day of RNA/DAA " lactation 0 when weaned Days dry Pregnant Non-pregnant 8 4 2.0:O.1 l.8:0.l 8 1.7:0.1 1.5:0.l l2 2.0:0.1 l.4:0.l 16 (parturition) 3.1:0.2 2.1:0 3 l2 4 3.0:0.2 l.8:0.0 8 2.1:O.1 1.4:0.1 12 (parturition) 2.7:0.l l.6:0.1 16 4 2.6:0.1 2.1:0.2 8 (parturition) 2.7:0.1 1.6:0.l 20 4 (parturition) 2.7:0.1 1.9:0.1 Overall mean 2.5b 1.7 a Mean and standard error of mean. bSignificantly greater (P < 0.01) than non-pregnant controls. 0Day 1 of pregnancy = day 1 of lactation. 74 49.2 per cent, respectively; and 57.8, 68.7, 69.5 and 69.5 per cent, respectively, for non-pregnant animals. Thus, the ratios for the pregnant and non-pregnant animals decreased to about 1/2 and 2/3, respectively, of what they were during lactation (Table 5). RNA/DNA ratios of the mammary gland increased slowly throughout pregnancy, be- coming slightly greater in the later stages of pregnancy. These observations correspond quite well with the histo- logical observations of Hammond (1927), Jeffers (1936) and Speert (1948) that the initiation of milk secretion is a gradual process. Between the 4th and last day of the dry period the ratios increased 31.0, 12.7 and decreased 25.2 per cent for non-pregnant animals weaned on the 8th, 12th, and 16th day, respectively. The increases in the RNA/DNA ratios for the non—pregnant animal were not expected especially in view of the results of Tucker and Reece (1963d). Those authors weaned rats on the 2lst day of lactation and reported that ratios on day 12 and 21 of involution were less than at other stages of involution. However, it has been shown that rats will start their estrous cycles 3 to 5 days after weaning (Toyoda, 1964) and that RNA/DNA of the mammary gland are higher during the estrous and metestrous stages of the cycle (Sinha and Tucker, 1967). It is possible that the increases we observed were due to abnormally large numbers of rats being killed by chance during the estrous and metestrous stages of the cycle. 75 Ethanol, Chloroform:Methanol,,Ether (ECME) Extract of the Mammarngland and Extraparenchymal Fat Pad The data showing the influence of pregnancy on ECME extract of the mammary gland during various length dry periods is in Table 21. Total ECME extract of the mammary gland during the dry period in pregnant animals (4.11 g) was greater (P < 0.01) than in non-pregnant controls (3.23 g). The interaction between day of the dry period and pregnancy state was also significant (P < 0.01). For the first 4 days of the dry period, commencing at the 8th 12th, 16th and 20th day of lactation, total ECME extract of pregnant animals decreased 14.7, 4.6, 2.8, and increased 14.4 per cent, respectively. Total ECME extract for non— pregnant controls during this time decreased 5.0, 27.8, 22.8, and 7.1 per cent, respectively. This loss of lipid 4 days after weaning lactating, non-pregnant animals does not agree with the results of Harkness and Harkness (1956). They found that the amount of lipid present in the mammary gland did not change throughout lactation or 21 days of involution. Whether the loss we observed was due to the loss of intra— or extra-cellular bound lipid (Melcher, 1967) or fat cells per g: is not known. From the 4th to the last day of the dry period, for rats weaned on the 8th, 12th, and 16th day of lactation, total ECME extract of the mammary gland of pregnant animals increased 24.3, 10.7, and 4.6 per cent, respectively; for 76 TABLE 2l.-—Inf1uence of pregnancy on ECME extract of the mammary gland during dry periods of 4, 8, 12 and 16 days. .- Total ECME extracta (a) Day of lactation c when weaned Days dry Pregnant Non-pregnant 8 4 3.48:0.19 3.60:0.18 8 3.93:0.33 3.06:0.20 l2 4.34:0.26 2.99:0.23 l6 (parturition) 4.60:0.26 3.35:0.22 l2 4 4.16:0.25 3.04:0.11 8 4.18:0.32 3.43:0.21 l2 (parturition) 4.66:0.22 3.18:0.24 16 4 3.75:0.23 3.08:0.21 8 (parturition) 3.93:0.31 3.15:0.15 20 4 (parturition) 4.03:0.43 3.38:0.18 Overall mean 4.11b 3.23 aMean and standard error of mean. bSignificanlty greater (P < 0.01) than non—pregnant controls. 0Day 1 of pregnancy = day l of lactation. 77 I'D non-pregnant rats decreased 6.9, increased 4.4, and 2. per cent, respectively. From these results, it may be inferred that during the dry period, a reservoir of mammary gland lipid was being deposited in response to pregnancy which would probably be used as an energy source during the second lactation. Total ECME extract of the extraparenchymal fat pad (Table 22) of pregnant animals (0.94 g) was not different (P 2 0.75) from non-pregnant controls (0.92 g). Four days after weaning on the 8th, 12th, 16th, and 20th day of lac- tation (Table 8) total ECME extract of the fat pad increased 1.9, 15.8, decreased 1.3, and increased 14.4 per cent, respectively. Total ECME extract for non-pregnant animals increased 14.6, 1.7, 19.9 and 35.3 per cent, respectively. From the 4th to the 16th, 4th to the 12th, and 4th to the 8th day of the dry period of rats weaned on the 8th, 12th or 16th day, total ECME extract of the fat pad for pregnant animals increased 35.7, 17.5 and 22.5 per cent, respectively. ECME extract of the fat pad for non-pregnant animals during these same periods increased 39.4, 25.7, and 27.7 per cent, (respectively. These changes in ECME extract 4 days after weaning and during the dry periods follow the changes in fat pad weights during these same periods (Table 16). This suggests that lipid mobilization may be responsible for the weight changes observed in the fat pads during these times. 78 TABLE 22.-~Inf1uence of pregnancy on ECME extract of the extraparenchymal fat pad during dry periods of 4, 8, 12 and 16 days. Total ECME extract3 (a) Pregnantb Non-pregnant Day of lactation when weaned Days dry 8 4 8 12 16 (parturition) l2 4 8 l2 (parturition) 16 4 8 (parturition) 20 4 (parturition) 0.68:0.07 0.70:0.11 0.98:0.12 0.76:0.11 1.04:0.10 0.90:0.16 1.05:0.06 1.16:0.11 0.94:0.11 0.78:0.07 1.25:0.09 0.93:0.12 1.14:0.10 1.05:0.13 0.69:0.08 0.83:0.09 Overall mean 0.94 0.92 —— a Mean and standard error of mean. b Day 1 of pregnancy = day l of lactation. 79 Hydroxyproline Content of the Mammary Gland and Extraparenchymal Fat Pad The effects of pregnancy on total hydroxyproline of the mammary gland during the various dry periods are sum- marized in Table 23. The overall mean total hydroxyproline content of the mammary gland of pregnant animals (19.14 mg) was greater (P < 0.01) than non-pregnant controls (17.11 mg). By the 4th day of the dry period, for rats weaned at 8, 12, 16 and 20 days, total hydroxyproline of pregnant animals decreased 8.0, 14.7, increased 3.8, and decreased 18.4 per cent, respectively, from the 0-day (Table 9) levels. Total hydroxyproline of the mammary gland of non—pregnant animals during this same interval decreased 18.2, 18.7, 0.1 and 1.4 per cent, respectively. Unlike DNA, which showed large 30 and 50 per cent decreases 4 days after weaning lactating, pregnant and lactating, non-pregnant animals, respectively, hydroxyproline showed lower per cent decreases. It is suggested that hydroxyproline within the mammary parenchyma may be more resistant to the process of involution than is cell loss. Total hydroxyproline of the mammary gland of pregnant animals increased 1.7 per cent from the 8th to the 16th day of the 8-day weaned group, 1.9 per cent from the 8th to the 12th day of the 12-day weaned group, and 1.4 per cent from the 4th to the 8th day of the l6-day weaned group. Total hydroxyproline of non-pregnant animals during these same 80 TABLE 23.--lnf1uence of pregnancy on hydroxyproline content of the mammary gland during dry periods of 4, 8, 12, and 16 days. Total hydroxyprolinea Day of (mg) lactation C when weaned Days dry Pregnant Non-pregnant 8 4 20.20:1.15 14.9310 86 8 l7.56:l.23 15.9511.09 l2 17.68:l.23 15 10:1.05 16 (parturition) l7.86:1.43 13,79:1.48 l2 4 19.96:1.66 18.93:1 28 8 l7e79il.46 19.11:l.97 l2 (parturition) 18.1311.01 16.82tl 34 16 4 21.25il.39 l9..7:l.98 8 (parturition) 21.55i1.83 16.80tl.48 20 4 (partur1tion)- 19.44:l.05 l9.89:0.81 Overall mean w.-—‘—w ‘ w— ,— 19.14b 17.11 a . Mean and standard error of mean. b controls. Significantly greater (P < 0.01) than non-pregnant CDay 1 of pregnancy = day l of lactation. 81 periods decreased 13.5, 11.9, and 15.0 per cent, respectively- Unlike total DNA of the mammary gland, whose loss was retarded by the 12th day of pregnancy and then increased after the 12th day of pregnancy, hydroxyproline losses were not effectively retarded until the 16th day of pregnancy. Since estrogen stimulates the fibroblast to synthesize collagen (Asboer Hansen, 1958) it is possible that the increase in connective tissue collagen was caused by estrogen, which is thought to be secreted in greater amounts during late pregnancy. The losses in hydroxyproline from the mammary gland following weaning do not agree with the results of Harkness and Harkness (1956). These authors reported that the hydroxy= proline content of the mammary gland did not change from lactation through 21 days of involution. Total hydroxyproline of the fat pad (Table 24) of pregnant animals (0.83 mg) was not different (P 2 0.60) from non-pregnant controls (0.85 mg). Relative to the day of weaning at the 8th, 12th, 16th and 20th day of lactation (Table 11) on the 4th day of the dry period hydroxyproline of the fat pad of pregnant animals increased 5.6, decreased 9.9, 10.5, and increased 3.5 per cent, respectively. Hydroxyw proline of the fat pad of non-pregnant animals increased 23.0, 18.9, 14.7, and 23.2 per cent, respectively. These changes in hydroxyproline for pregnant and non-pregnant animals parallel the changes in DNA (Figures 7 and 8). How: ever, increases in total hydroxyproline of the fat pad after 82 TABLE 24.--Inf1uence of pregnancy on hydroxyproline content of the extraparenchymal fat pad during dry periods of 4, 8, l2 and 16 days. Total hydroxyprolinea Days of (mg) lactation b when weaned Days dry Pregnant Non-pregnant 8 4 0.71:0.05 0.87:0.10 8 0.72:0.07 0.60:0.09 l2 0.97:0.13 0.81:0.10 l6 (parturition) 0.89:0.09 1.24:0.15 l2 4 0.73:0.12 0.74:0.08 8 0.85:0.09 0.93:0.11 12 (parturition) 1.14:0.12 0.76:0.08 l6 4 0.68:0.07 0.75:0.10 8 (parturition 0.80:0.07 0.88:0.14 20 4 (parturition) 0.85:0.12 0.95:0.09 Overall mean 0.83 0.85 a Mean and standard error of mean. bDay l of pregnancy = day 1 of lactation. 83 Figure 7.--Relation between DNA and hydroxyproline of the fat pad for pregnant animals during various stages of lactation and involution. 84 (W) annouaxouoxu ova no «lo: 3: 3 2| :1 3 3 h 3 ‘\ 8 g \ v 6 >- /‘ ‘ < 35"", N 2 o< .— O \ \ o \ . > 1 / O =/ / 2 / «<\ a \ e \ o l (in l 1.} i 71‘ 1, 1 l0 -: ‘ ' - -' - 9 3 d 3 3 3 (0w) VNO ova LVJ 1vlo.L DAYS DRY l6 :2 DAY OF LACTATION WHEN WEANED 85 Figure 8.--Relation between DNA and hydroxyproline of the fat pad for non-pregnant animals during various stages of lactation and involution. 86 (5,!!!) smooaaxxoaom ova lva mo: ' 8 DAYS DRY - O o s.- e e _. e. 8 3. 5 g r I V T l T I r I ‘ I )- x3 8.. 08 £5 < \\ a \ \\ \ // / \ \ \. x ./’> / \\ .\\ I I I I I I I I I to N -. o a: o b, ca no " “ " "' 0' d 0 d d (W) vuo ova lva 1Vlol 12|604 I204 4 , 16 DAY OF LACTATION WHEN WEANED l2 87 weaning lactating, pregnant animals showed a 4-day lag period in relation to DNA. This suggests that the hormones secreted during late pregnancy may stimulate the fibroblasts to synthesize collagen 4 to 8 days following weaning, whereas the hormones secreted during the estrous cycle of the non-pregnant animals stimulate the fibroblasts to synthesize collagen 4 days after weaning. Thus, it appears that growth of the mammary gland produced by pregnancy during a dry period consists of two stages. First, a pro- liferation of epithelial and connective tissue cells occurs after the 12th day of pregnancy and second, a stimulation of the connective tissue fibroblasts by estrogen to secrete collagen occurs after the 16th day of pregnancy. Since the increases in hydroxyproline of the fat pad for both lac- tating, pregnant and lactating, non-pregnant animals has been attributed to recent synthesis by the fibroblasts, indicates that the regressing mammary gland did not leave extra collagen behind in the fat pad. Hexosamine Content of the Mammary Gland' The hexosamine content of the mammary gland of preg- nant animals totaled an average of 4.47 mg, which was greater (P < 0.01) than the average of 2.71 mg for non- pregnant controls (Table 25). On the 4th day of the dry period, after weaning on the 8th, 12th, 16th, and 20th day of lactation (Table 12), total hexosamine of the mammary 88 TABLE 25.--Inf1uence of pregnancy on hexosamine content of the mammary gland during dry periods of 4, 8, 12 and 16 days. Da of lac ation Total hexosaminea when weaned Days dry Pregnantc Non-pregnant 8 4 4.20:0.58 3.35:0.29 8 3.10:0.66 2.07:0.18 l2 2.88:0.28 1.82:0.18 l6 (parturition) 4.02:0.21 1.74:0.20 l2 4 7.39:0.64, 3.74:0.35 8 3.24:0.23 2.60:0.31_ l2 (parturition) 4.43:0.39 1.88:0.17 l6 4 6.31:0.75 4.43:0.32 8 (parturition) 4.63:0.29 2.13:0.14 20 4 (parturition) 4.54:0.45 3.3310.33 Overall mean 4.47b 2.71 a Mean and standard error of mean. b controls. 0Day 1 of pregnancy = day l of lactation. Significantly greater (P < 0.01) than non-pregnant 89 gland for pregnant animals decreased 24.5, increased 4.7,~ decreased 8.8 and 16.5 per cent, respectively. Total hexosamine for non-pregnant animals decreased 30.5, 39.0, 36.7 and 44.3 per cent, respectively. Total hexosamine during each of the weaning groups for both pregnant and non-pregnant animals, continued to decrease until 4 days before the end of each dry period. At this time, hexo- samine of the mammary gland for the pregnant animals showed a tendency to increase but for the non-pregnant- animals it continued to decline. Pregnancy effectively reduced the loss of hexosamine after the 12th day of preg- nancy but did not promote net increases until the 20th day of pregnancy. Weight of the Adrenal Gland Weight of the adrenal gland, as listed in Table 26, of pregnant animals (55.7 mg) was heavier (P < 0.05) than non-pregnant controls (52.5 mg). Four days after weaning on the 8th, 12th, 16th and 20th day of lactation the. adrenal weight for pregnant animals decreased 10.4, increased 2.0, 5.6 and 2.0 per cent, respectively, and increased 3.4, 1.2, 8.4 and decreased 6.6 per cent, respec- tively, for non-pregnant animals. Pregnancy did not influence weight of the adrenal gland during the first 4 days folloWing weaning. Towards the end of the dry period, however, weights of the adrenal of pregnant animals tended 90 TABLE 26.--Inf1uence of pregnancy on weight of the adrenal gland during dry periods of 4, 8, l2 and 16 days. Adrenal gland weighta Day of (mg) lactation 0 when weaned Days dry Pregnant Non-pregnant 8 4 47.0:1.6 49.4:l.3 8 54.5:2.1 53.1:2.2 l2 57.2:3.l 51.5:1.5 l6 (parturition) 53.6:2.3 51.5:1.5 12 4 53.8:2.3 50.0:2.l 8 55.5:l.7 54.1:2.3 12 (parturition) 58.0:3.l 54.7:l.8 16 4 57.9:2.3 54.6:2.0 8 (parturition) 59.2:2.0 55.1:2.2 20 4 (parturition) 60.7:2.5 51.3:l.9 Overall mean 55.7b 52.5 aMean and standard error of mean. b controls. 0Day 1 of pregnancy = day l of lactation. Significantly greater (P < 0.05) than non-pregnant 91 to increase. This may indicate that the adrenal may be responding to the stress of parturition or to the initiation of milk secretion at the start of the second lactation. Influence of Length of the Dry Period on Subsequent Lactation Body Weight Initial body weights on the 3rd day of the first lactation were not different (P < 0.50) among the various treatment groups. Thus, at the start of this experiment, bias attributable to body weight difference was largely eliminated. Body weights on the first day (254 g) of the second lactation (Table 27) were less (P < 0.01) than body weights on the 8th (265 g) or 16th (277 g) days. Average body weight on the 8th day was also less (P < 0.025) than~ average body weight on the l6th.day. On the lst day of the second lactation, a linear increase (P < 0.05) in body weight with increasing dry period length was observed. On the average, rats which received the.l6-day dry period weighed 11.7 per cent more on the lst day than rats which received no dry period during their first lactation. On the 8th and 16 day of lactation length of the dry period did not influence body weight (P < 0.05). Results suggest that the role of nutrition, as-affected by the dry period may be important on the lst day of the second lactation but that this role of nutrition diminishes as lactation progresses. This is in contrast to results obtained in the 92 cow (Swanson, 1965). He reported that cows milked con- tinuously throughout the first lactation remained 20 to 40 pounds heavier throughout the second lactation than cows which received 60-day dry periods. TABLE 27.--Relation between length of the dry period and body weight during the second lactation. Body weighta Preceding (g) drydgggiod Second lactation lst dayd 8th day 7 16th day 16 266:7 266:6 282 :9 12 257:7 268:8 284 :8 8 263:7 259:7 278:11 4 249:6 272:8 281:10 0 235:5 259:5 261 :7 Overall mean 254‘0 2658‘ 277 a Mean and standard error of mean. b Significantly less (P < 0.01) than 8th and 16th day. CSignificantly less (P < 0.025) than 16th day. d periods. Significant linear increase (P < 0.05) among dry 93 Weight of the Mammary Gland and Extraparenchymal Fat.Pad The relation between length Of the dry period and total weight of the mammary gland during the following lactation is shown in Table 28. Mammary weight on the lst day (6.81 g) was less (P < 0.01) than the weight on the 8th (11.38 g) or 16th (13.43 g) day. Weights of the mammary gland on the 8th and 16th days Of the second lactation were 17.5 and 3.6 per cent heavier, respectively, than weights of lactating, non-pregnant animals at the 8th (9.39 g) and 16th day (12.94 g) of the first lactation, respectively, and 14.5 and 11.1 per cent heavier, than lactating, pregnant animals at the 8th (9.73 g) and 16th day(11.95 g)of the first lactation, respectively. Thus, like the cow (Smith, 1959), the mammary gland of the rat increases in size with recurring lactations. Length of the dry period produced no effects (P > 0.05) in mammary- weights on the lst, 8th, or 16th day of the second lacta- tion. The data suggest that length of the previous dry period plays a minor role in subsequent weight changes Of the mammary gland. The relation between length Of the dry period and total weight of the extraparenchymal fat pad is shown in Table 29. Weight of the extraparenchymal fat pad was heavier (P < 0.05) on the lst day (1.18 g) than on the 8th (1.06 g) or 16th (1.03 g) day of the second lactation. 94 These fat pad weights on the 8th and 16th days of the second lactation were 0.2 and 19.6 per cent heavier, respectively, than fat pad weights from lactating, non-pregnant animals at the 8th (1.04 g) and 16th day (0.83 g) of the lst lactation, respectively, and 19.8 and 3.9 per cent heavier, than lactating, pregnant animals at the 8th (0.85 g) and 16th day (0.83 g) Of the lst lactation, respectively. The interaction between length of the dry period and day of lactation was also significant (P < 0.05). On the lst day TABLE 28.--Relation between length of the dry period and weight of the mammary gland during the following lactation. Mammary gland weighta Preceding (g) dry period (days) Second lactation lst day 8th day 16th day 16 7.21:0.41 11.34:O.47 l3.79:l.33 12 7.21:0.41 ll.35:0.85 l3.89:0.60 8 7.04:0.38 11.37i0.37 l3.64:0.82' 4 6.60iO.48 11.5710.65 13.78i0.79 O 6.01:0.36 11.28i0.63 l2.07i0.63 Overall mean 6.81b 11.380 13.43 aMean and standard error of mean. b Significantly less (P < 0.01) than 8th and 16th day. cSignificantly less (P < 0.01) than 16th day. 95 there was a linear increase (P < 0.01) in fat pad weight with increasing length of the dry period. The signifi- cantly lower weight Of the fat pad on the lst day for the 0- and 4—day dry periods did not inhibit the mammary gland from growing (Table 28) al; the subsequent 8th and 16th day of lactation. NO trends (P > 0.05) in fat pad weight due to dry periods could be detected on the 8th or 16th day. This suggests that the effect of dry period length on weight of the fat pad is lost by the 8th day Of the second lactation. TABLE 29.--Re1ation between length of the dry period and weight of the extraparenchymal fat pad during the second " lactation. Fat pad weighta Preceding (g) dry period (days) Second lactation lst dayc 8th day 16th day 16 1.28:0.10 1.16i0.l3 1.16:0.11 l2 l.59i0.l4 0.97:0.12 1.06:0.15 8 l.25i0.ll 1.01i0.09 0.99:0.10 4 0.92:0.16 l.l2iO.l3 0.97i0.ll 0 0.86:0.13 ~q 1.04:0.11, 0.99:0.09 Overall mean 1.18b 1.06 1.03 8Mean and standard error of mean. bSignificantly greater (P < 0.05) than 8th and 16th day. CSignificant linear increase (P < 0.01) with increasing length Of the dry period. 96 Total Litter Weight Gain The relation between length of the dry period and total litter weight gain (milk production) is shown in Table 30. The total gain of the litter from the 8th to the 16th day of the first lactation for lactating, non- pregnant (81.3 g) and lactating, pregnant (84.6 g) animals was 10.4 and 10.3 per cent less, respectively, than litter weight gain from the 8th to the 16th day of the second lactation. Thus, milk production in the rat TABLE 30.--Tota1 litter weight gain from the 3rd to the. 8th and 8th to the 16th day of the second lactation after various length dry periods. Total litter weight gaina Preceding (g) dry period (days) Second lactation 3rd-8th day 8th-16thdayc l6 46.7:3.8 97.1:4.7 12 49.8:2.9 94.9:3.3 8 52.2:1.9 93.8:4.4 4 44.0:4.2 104.7:6.4 0 43.9:3.2 81.7:5.3 Overall mean 47.3b 94,4 a Mean and standard error of mean. bSignificantly less (P < 0.01) than 8th to 16th day weight. CSignificant cubic regression (P < 0.01) among dry periods. 97 continues to increase like it does for the cow (Lush and Shrode, 1950; and Smith, 1959) during subsequent lactations. It'has also been reported (Wrenn, 32491., 1966) that rats stimulated by two pseudo-pregnancies showed a 15 per cent increase in litter weight during the.subsequent lactation. Length Of the dry period had no effect on litter weight gain between the 3rd and 8th day Of the second lactation (P > 0.05). 'Between the 8th-and 16th day, how- ever, those animals given a 4-day dry period produced approximately 22.0 and 9.3 per cent heavier litters than. animals given a 0-day or longer (8-, 12-, 16-day) dry periods, respectively. These data indicate that a 4-day dry period is Optimum for maximal litter weight gains for the rat, and in general agree with results Obtained in the cow (Johansson, 1962) That is, the lactation yield increased with increasing length.of the dry period up to a certain point, but a further increase in length of the.dry period had no beneficial effect on the yield Of milk in the following lactation. This beneficial effect of a 4-day dry period was not anticipated because dry periods had no effect on Weight of the mammary gland on the lst, 8th, or 16th day of lactation. This could mean that mammary glands from rats which received a 4—day dry period contained more secretory tissue than mammary glands from rats which received the 0-, 8-, 12, or l6-day dry periods. Nucleic Acid Content of the Mammary Gland and Extra- parenchymal Fat Pad The relation between length of the dry period and total DNA of the mammary gland during the following lac- tation is summarized in Table 31. Total DNA on the 1st day (20.8 mg) was less (P < 0.01) than total DNA on the 8th (35.4 mg) and 16th day (37.1 mg). There was no difference (P = 0.80) between the 8th and 16th day. The interaction between dry period and day of lactation was also signif- icant (P < 0.05). DNA Of the mammary gland during the 8th and 16th day Of the second lactation was 15.1 and 12.1 per cent higher, respectively, than DNA of lactating, non- pregnant animals on the 8th (30.0 mg) and 16th day (32.6 mg), respectively; and 14.7 and 12.2 per cent higher, than lactating, pregnant animals on the 8th (30.2 mg) and 16th day (32.6 mg), respectively, Of the first lactation. These results, although not strictly applicable, agree with Observations by Wada and Turner (1959), that recurring pregnancies without nursing periods will increase DNA in the mammary gland. On the lst day of the second lactation, those animals given an 8- or O-day dry period contained approximately 13.3 per cent more DNA (P < 0.05) than rats given l6-, 12-, or 4—day dry periods. Greater levels of DNA were expected for rats receiving no dry period because these rats were continually nursing a litter, and suckling could be 99 maintaining the DNA of the mammary gland (Tucker and Reece, 1963). On the 8th and 16th day of the second lac- tation, significant quadratic regression curves (P < 0.01) in response to the various dry periods were Obtained~ (Table 31). On the 8th day, mammary glands of rats which received 0- or 16-day dry periods contained 14.8 and 13.2 per cent less DNA respectively, than rats which TABLE 31.--Relation between length of the dry period and- DNA content of the mammary gland during the second lactation. TotalDNAa Preceding (mg) dry period (daVS) 1st day0 8th dayd 16th daye 16 19.9:0.9 33.4:l.6 35.5:2.1 l2 19.5:0.7 36.0:1.2 37.7:1.9 8 22.5:1.1 38.5:0.7 38.3:1.6 4 19.7:1.3 36.4:1.6 40.3:2.4 0 22.5:1.2 32.8:l.3 33.5:l.4 Overall mean 20.8b 35.4 37.1 aMean and standard error of mean. bSignificantly less (P < 0.01) than 8th and 16th day. cSignificant cubic regression (P < 0.05) among dry periods. dSignificant quadratic regression (P < 0.01) among dry periods. eSignificant quadratic regression (P < 0.01) among dry periods. 100 received an 8-day dry period. By the 16th day of lac- tation mammary glands Of rats which received 0- or l6-day dryperiods contained 16.8 and 11.8 per cent less DNA, respectively, than rats which received a 4—day dry period. Although length Of the dry period had no effect on weight of the mammary gland within a particular day Of lactation (Table 28), it did have an effect on the number of cells in the gland. This suggests that factors other than cell number are accounting for this equality in weight of the mammary gland. The reason for the low number of mammary cells on the 8th and 16th day of the second lactation for rats which received no dry period could be attributed to the involution of the old cells of the first lactation. These cells were prevented from involuting during the first lactation because of the continuous suckling stimulus, and when these cells were carried into_the second lactation, they may have been lost during the first 8 days and not replaced later in lactation by new cells. This confirms the Observations by Altman (1947) that a dry period was necessary to allow rapid regeneration of the secretory epithelium before the next lactation.. For those rats which received l6-day dry periods (weaned on the 8th day of pregnancy) the Opposite situation existed. Since pregnancy was not effective in retarding cell loss until the 12th day of pregnancy (Table 18), the mammary glands which received the l6-day dry period involuted more than mammary glands 101 which received 4—, 8-, or l2-day dry periods. Therefore, these animals had to grow more cells during the dry period and went into the second lactation with proportionally more new cells than the other dry period groups. It is possible that this additional new cell growth during the l6-day dry period expended some of the energy and co-factors required for normal cell growth that should have occurred during the second lactation. If we consider the 4- to 8-day dry period as optimum for the rat, then our results on the lst day of the second lactation agree with results published for the cow (Pardue and Swanson, 1963). That is, on the.lst day of the second lactation, total DNA of the mammary gland for cows given a 2-month dry period in their first lactation, did not differ from total DNA of cows given very short dry periods. If the cow also behaves like the rat during the latter stages of lactation differences in DNA between the two groups of cows would have been noted at the later stages Of lactation. Thus, the stage of lactation chosen for this type Of com- parison is important and the relation one Observes on the lst day of a subsequent lactation will not necessarily be true later in lactation. The relation between length Of the dry period and total DNA of the extraparenchymal fat pad during the following lactation is shown in Table 32. Total DNA Of the fat-pad on the lst day (0.69 mg) was not different (P = 0.90) 102 from the 8th (0.70 mg) and the 16th day (0.65 mg). Total DNA Of the fat pad during the 8th and 16th day of the second lactation was 21.4 and 16.9 per cent higher, respectively, than fat pad DNA of lactating, non-pregnant animals on the 8th (0.55 mg) and 16th day (0.54 mg), respectively, and 12.9 and 12.3 per cent higher than lactating, pregnant animals on the 8th (0.61 mg) and 16th day (0.57 mg), respectively, of the first lactation. NO response curves (P < 0.05) were Obtained due to length of the dry period on the lst, 8th or 16th day of the second lactation. The data suggest that factors which regulate mammary epithelial cell numbers during a second lactation may not be regulating fat pad connective TABLE 32.--Re1ation between length of the dry period and DNA content of the extraparenchymal fat pad during the secOnd lactation. Fat pad weighta Preceding (3) drgagzriod Second lactation lst day 8th day 16th day 0' 0.6210.04 0.82:0.09 0.6310.03 4 0.59:0.06 0.69:0.05 0.64:0.05 8 0.75*0.07 0.69*0.05 0.64:0.06 l2 0.75:0.08 0.65t0.06 0.67*0.09 l6 0.7610.06 0.67*0.06 0.67:0.06 Overall mean 0.69 0.70 0.65 aMean and standard error of mean. 103 tissue cell numbers. The constant number of cells on the lst day of lactation among the various dry periods, does not follow the linear increase in weight of the fat pad with increasing length of the dry period shown to exist at this time (Table 29). Possibly, during the short dry periods, some of the lipid was mobilized out Of the fat cells located in the fat pad, leaving behind intact fat cells with low amounts Of lipid. The relation between length of the dry period and total RNA of the mammary gland during the second lactation is shown in Table 33. Total RNA on the lst day (58.9 mg) was less (P < 0.01) than RNA on the 8th (172.6 mg) and 16th day (247.0 mg), and RNA on the 8th day was less (P < 0.01) than RNA on the 16th day of lactation. RNA during the 8th and 16th day of the second lactation was 25.4 and 7.0 per cent higher, respectively, than RNA Of lactating, non- pregnant animals on the the 8th (128.8 mg) and 16th day (229.8 mg), Of the first lactation, respectively; and 22.2 and 13.8 per cent higher than lactating, pregnant animals on the 8th (134.3 mg) and 16th day (213.0 mg) of the first lactation, respectively. Thus, total protein synthetic activity Of the mammary gland increased with each recurring lactation. The various dry periods had no affect on total mammary gland RNA during the lst day of the second lactation (P > 0.05). However, quadratic response curves for total RNA 104 were obtained on the 8th (P < 0.05) and 16th day (P < 0.01) of the second lactation among the various dry period groups. On the 8th day, mammary glands of rats given an 8-day dry period contained approximately 16 per cent more RNA than mammary glands which received 16- and 0-day dry periods. On the 16th day, mammary glands of rats given a 4-day dry period contained 14.3 and 22.1 per cent more RNA than TABLE 33.--Relation between length Of the dry period and RNA content of the mammary gland during the second lactation. Total RNAa Preceding (mg) dry period (days) Second lactation lst day 8th dayd 16th daye 16 61.1i4.2 159.2ill.l 231.1i20.l l2 53.0:4.0 177.8i 8.7 262.4:16.0 8 61.0:4.4 188.8: 6.0 260.5:1l.5 4 53.0i4.0 178.1il2.1 270.5i2S.4 O 62.1i6.2 159.1i 9.1 210.7i13.8 Overall mean 58.0b 172.60' 247.0 aMean and standard error of mean. bSignificantly less (P < 0.01) than 8th and 16th day. cSignificantly less (P < 0.01)than 16th day. dSignificant quadratic regression (P < 0.05) among dry periods. eSignificant quadratic regression (P < 0.01) among dry periods. 105 mammary glands which received 16- and 0-day dry periods, respectively. In view Of the DNA results, the trends established within a particular stage of lactation among dry period groups were expected. Since fewer mammary cells were formed in animals which received no dry period or very long dry periods, it is reasonable to assume that mammary RNA would also be reduced in these treatment groups. This was confirmed by the RNA/DNA ratios (Table 34). On the lst, 8th and 16th day of lactation, length Of the dry period TABLE 34.--Re1ation between length of the dry period and RNA/DNA ratio of the mammary gland during the second lactation. .RNA/DNA ratioa Preceding dry period (days) Second lactation 1st day 8th day 16th day 16 3.1:0.2 4.7:o.2 6.5:0.3 l2 2.7:0.l 4.9:0.2 6.9:0.2 8 2.7:0.1 4.9:0.2 6.8:0.2 4 2.7:0.1 4.9:0.2 6.6:0.3 0 2.8:0.2 4.9:0.3 6.3:0.3 b 0 Overall mean 2.8 4.9 6.6 aMean and standard error or mean. bSignificantly less (P < 0.01) than 8th and 16th day. CSignificantly less (P < 0.01) than the 16th day. 106 had no effect (P > 0.05) on the RNA/DNA ratios. Thus the various dry periods are not causing reduced synthesis per cell because cells from mammary glands of rats which received the l6- or O-day dry periods were Just as capable of synthesizing protein as were cells which received the u- or 8-day dry period. These data indicate that the dry period is having its effect on subsequent lactation by causing loss of cells or preventing mitosis during the second lactation rather than loss of synthesis per cell. RNA/DNA ratios of mammary glands during the 8th (4.9) and 16th (6.6) day of the second lactation were 12.0 per cent more and 5.6 per cent less,respectively, than ratios for non-pregnant animals on the 8th (u.3) and 16th day (7.0), respectively; and 10.0 and l.“ per cent more, respectively, than ratios for pregnant animals on the 8th (u.u) and 16th (6.5) day, respectively, of the lst lactation. Thus, recurring lactation did not appre- ciably alter the synthesizing capability of each cell. Ethanol, Chloroform: Methanol, Ether (ECME) Extract of the Mammary Gland and Extraparenchymal Fat Pad The relation between length of the dry period and total ECME extract of-the mammary gland is shown in Table 35. Total ECME extract of the mammary gland on the lst day (4.10 g) was not different (P = 0.50) from the ECME extract on the 8th (“.38 g) or 16th day (“.23 g). Total ECME extract of the mammary gland during the 8th and 16th day of 107 the second lactation was 13.5 and 5.7 per cent higher, respectively, than lactating, non-pregnant animals on the 8th (3.79 g) and 16th day (3.99 g), respectively, of the first lactaiton and 6.8 and 8.7 per cent higher than lac- tating, pregnant animals on the 8th (H.08 g) and 16th day (3.86 g), respectively, of the first lactation. Linear increases in ECME extract of the mammary gland with increasing length of the dry period were TABLE 35.-—Relation between length of the dry period and ECME extract of the mammary gland during the second lactation. Total ECME extracta Preceding (g) dry period (days) Second lactation lst dayb 8th day 16th day° 16 A.60:0.26 4.u0:0.39 n.9310.73 l2 H.66i0.22 u.52:0.u5 u.08¢0.l9 8 3.93:0.31 A.05:0.21 A.63:0.32 A 4.03:0.43 4.u3:0.26 4.11:0.30 O 3.26:0.26 U.N9i0.ul 3.38:0.32 Overall mean 4.10 “.38 4.23 a Mean and standard error of mean. b Significant linear increase (P < 0.01) with increasing length of the dry period. cSignificant linear increase (P < 0.05) with increasing length of the dry period. 108 observed on the lst (P < 0.01) and l6thday (P < 0.05) of the second lactation. Thus, at the beginning and towards the end of the second lactation, the shorter dry periods caused mobilization of lipid out of the mammary gland, probably to be used as energy sources in response to the initiation and maintenance of lactation. The relation between length of the dry period and total ECME extract of the extraparenchymal fat pad is shown in Table 36. Total ECME extract on the lst day (0.90 g) TABLE 36.--Relation between length of the dry period and ECME extract of the extraparenchymal fat pad during the second lactation. Total ECME extracta Preceding (g) dry period (days) Second lactation 1st dayb 8th day 16th day 16 1.05i0.06 0.9710.10 0.83:0.05 12 l.lBiO.lO 0.7310.ll 0.85:0.03 8 0.8910.11 0.8110.08 0.82:0.12 u 0.79t0.11 0.80:0.11 0.83:0.03 0 0.6”10.08 O.93tO.ll 0.6Ui0.05 Overall mean 0.90 0.85 0.79 aMean and standard error of mean. b increasing length of the dry period. Significant linear increase (P < 0.01) with 109 was not different (P = 0.25) from the 8th (0.85 g) and 16th (0.79 g) day. A linear increase (P < 0.01) with increasing length of the dry period was observed on the lst day of the second lactation. This paralleled the linear increase in weight of the fat pad with increasing length of the dry period (Table 29). However, DNA of the fat pad was the same regardless of dry period length (Table 32). This 71 suggests either a mobilization of lipid from the fat cell, 1 leaving behind a fat cell devoid of lipid, or a mobilization of extracellular bound lipid, without altering the fat cell H numbers. Hydroxyproline Content of the Mammary Gland and Extraparenchymal Fat Pad The relation between length of the dry period and total hydroxyproline content of the mammary gland during the following lactation is listed in Table 37. Total hydroxy- proline on the lst day (18.60 mg) was less (P < 0.01) than total hydroxyproline on the 8th (22.37 mg) and 16th day (27.37 mg). Total hydroxyproline on the 8th day was less (P < 0.01) than on the 16th day of the second lactation. During the 8th and 16th day of the second lactation total hydroxyproline was 18.“ and 20.6 per cent higher than lactating, non-pregnant animals on the 8th (18.26 mg) and 16th day (21.7“ mg) of the first lactation, respectively, and 1.8 and 25.3 per cent higher, than lactating, pregnant animals on the 8th (21.96 mg) and 16th day (20.““ mg) of the first lactation, respectively. Since mammary glands also weighed more in the second lactation, it is reasonable to llO expect that these glands should contain more collagen, possibly due to a larger connective tissue framework needed in these larger glands. On the first day of the second lactation a quadratic regression (P < 0.05) among dry periods was obtained. TABLE 37. --Relation between length of the dry period and hydroxyproline content of the mammary gland during the second lactation. Total Hydroxyprolinea Preceding (mg) dry period (days) Second lactation lst dayd 8th daye 16th dayf- 16 17.86:1.“3 23.1“il.3“ 28.56i3.16 l2 18.13il.01 26.12sl.26 28.1112.03 8 21.55il.83 23.63il.35 32.20:2.08 u l9.““il.05 2o.37:0.78 25.1uil.u6 0 l6.00i0.92 18.59i1.35 22.8“il.09 Overall mean 18.60b 22.37° 27.37 a Mean and standard error of mean. b Significantly less (P‘< 0.01) than 8th and 16th day. cSignificantly less (P < 0.01) than 16th day. d dry periods. eSignificant linear increase (P < 0.01) with increasing length of dry period. f dry periods. Significant quadratic regression (P < 0.05) among Significant quadratic regression (P < 0.01) among lll Mammary glands of animals which received the 16- and 0-day dry periods contained 17.1 and 25.8 per cent less hydroxy- proline, respectively, than animals which received the 8-day dry period. Since collagen is necessary for future mammary development (Lasfarques, 1957), these lower amounts of hydroxyproline produced on the lst day by those glands which received the 16- and 0-day dry periods, could be one of the reasons why these same glands contained reduced numbers of mammary cells in later lactation (Table 31). The amount of hydroxyproline synthesized per unit of RNA (Table 38) was TABLE 38.--Relation between length of the dry period and hydroxyproline/RNA ratio of the mammary gland during the second latation. Preceding Hydroxyproline/RNA ratioa dry period (days) Second lactation lst day 8th day 16th day 16 0.31 0.15 0.12 12 1.33 0.15 0.12 8 0.35 0.13 0.12 “ 0.36 8 0.12 0.10 O 0.26 0.12 0.11 Overall mean 0.32 0.13 0.11 aMean. 112 highest on the lst day of the second lactation. Also,on the 1st day (flue ratios were lowest for those animals which received 16- and 0-day dry periods. Thus it appears that the mammary gland was synthesizing more hydroxyproline per unit of RNA on the lst day in those rats given the optimal dry periods, in possible preparation for future-mammary parenchymal cell growth.. The low hydroxyproline/RNA ratio for those animals which received 0-day dry periods, suggests that a greater portion of the RNA was being used to code for milk protein rather than structural protein, since these animals were continually nursing a litter. Total hydroxyproline content of the extraparenchymal fat pad (Table 39) on.the lst day (0.87-mg) of the second lactation was not different (P 2 0.“0) from the 8th (0.89 mg) and 16th day (0.81 mg). The interaction between length of the dry period and day of lactation was signif- icant (P < 0.05). Total hydroxyproline during the 8th and 16th day of the second lactation was 2“.7 and 21.0 per- cent higher than lactating, non-pregnant animals on the 8th (0.67 mg) and 16th day (0.6“ mg), respectively; and 2“.7 and 6.2 per cent higher than lactating, pregnant animals on the 8th (0.67 mg) and 16th day (0.76 mg) of the first lactation, respectively. Possibly, the extra hydroxy- proline may account for the larger fat pads observed during the second lactation. 113 On the lst and 16th day of the second lactation, linear increases (P < 0.01) of hydroxyproline content of the fat pad with increasing length of the dry period were obtained. Since these longer dry periods also resulted in heavier fat pads, collagen together with ECME extract is probably contributing to the heavier fat pad weight. TABLE 39.—-Relation between length of the dry period and hydroxyproline content of the extraparenchymal fat pad during the_second lactation, Total hydroxyprolinea Preceding (mg) dry period (days) Second lactation 1st dayb 8th day 16th dayc' 16 0.89i0.09 0.86:0.11~ 0.97:0.10> 12 1.1“i0.12‘ 0.80:0.09 0.96:0.17 8 0.80:0.07 0.8“:0.06 0.77:0.06v “ 0.85:0.12' 0.89i0.09' 0.7“i0.08 O 0.67:0.09 l.07iO.16 0.60:0.05 Overall mean 0.87 0.89 0.81- 8‘Mean and standard error of mean. b increasing length of the dry period. Significant linear increase (P < 0.01) with cSignificant'linear increase (P < 0.01) with increasing length of the dry period. 11“ Hexosamine Content of the Mammary Gland The relation between length of the dry period and total hexosamine content of the mammary gland during the following lactation is shown in Table uo. Total hexo- samine was less (P < 0.01) on the lst day (“.32 mg) than on the 8th (6.39 mg) and 16th day (7.08 mg). Total hexosamine was significantly less (P < 0.06) on the 8th TABLE “0.--Relation between length of the dry period and hexosamine content of the mammary gland during the second lactation. Total hexosaminea‘ Preceding (mg) dry period (days) Second lactation lst day 8th day 16th dayd l6 “.O2i0.21 6.3210.55 7.7711.00 12 “.“3i0.39 6.19:0.“7 6.98:0.36 8 “.63i0.29 6.92:0.50 7.39:0.“2 “ “.5“i0.“5 6.10:0.37 6.73i0.36 0 “.00t0.50 6.“2i0.“7 6.5“:0.37 Overall mean 14.32b 6.39c 7.08 aMean and standard error of mean. b Significantly less (P < 0.01) than 8th and 16th day. cSignificantly less (P < 0.05) than 16th day. d increasing length of the dry period. Significant linear increase (P < 0.05) with 115 day than on the 16th day of lactation. Total hexosamine content of the mammary gland during the 8th and 16th day of the second lactation was 2“.“ and 1.1 per cent higher, respectively, than lactating, non-pregnant animals on the lst (12.80 mg) and 16th (7.00 mg) day of the first lactation; and 12.8 and 2.2 per cent higher, than lactating, pregnant animals on the 8th (5.57 mg) and 16th (6.92 mg) day of the first lactation, respectively. During the second lactation, total RNA was also higher than the first lactation, notably so on the 8th day of the second lactation. Since the ground substance surrounds each cell and serves as the pathway between the cell and bloodstream, an increase in the metabolic activity of the cell (RNA) could presumably increase the amount of ground substance (hexosamine). On the 16th day of the second lactation there was a‘ linear increase (P < 0.05) in hexosamine with increasing length of the dry period (Table “0). The reason for this» linear increase could be related to the increase in adrenal weight (Table “1) at this time. Both estrogen. and mineralocorticoids which are secreted by the adrenal glands have been shown to increase hexosamine levels of- a tissue (Dorfman and Schiller, 1961). It is possible that these larger adrenals are producing more hormones, thus increasing total mammary gland hexosamine. 116 Weight of the Adrenal Gland The relation between length of the dry period and adrenal gland weight during the following lactation is shown in Table “1. There was no difference (P = 0.20) in adrenal gland weight on the lst, 8th and 16th day of the second lactation. Adrenal glands on the 8th (55.1 mg) and 16th (5“.9 mg) day of the second lactation were 13.“ and 8.9 per cent and “.7 and 0.2 per cent heavier than adrenal glands from lactating, non-pregnant or lactating, pregnant animals on the 8th and 16th day (50.0 mg) of the first lactation, respectively. Quadratic response curves in adrenal gland weight in response to length of the dry period were obtained on the 1st (P < 0.01) and 8th (P < 0.05) day of lactation. This adrenal weight change, in response to length of the dry period parallels the DNA and RNA data reported for this experiment. Thus, it appears that size of the adrenal. glands may be associated with mammary gland growth and synthetic activity. On the 16th day of lactation, an- increase in adrenal weight for those animals which received a l6-day dry period, paralleled the increase in total hexo- samine content of the mammary gland. This suggests that hormones elaborated by the adrenal at this time could be responsible for this increase in hexosamine. 117 TABLE “l.--Re1ation between length of the dry period and weight of the adrenal gland during the second lactation. V— Adrenal gland weighta Preceding (g) dry period (days) Second lactation lst dayb 8th dayb 16th day I. 16 53.6:2.3 5“.1i2.2 57.2il.8 12 58.0t3.1 55.1i1.5. 53.2i3.“ 8 59.2:2.o 57.1:2.o 56.3:2.l i “ 60.7:2.5 58.2:2.2 55.1:2.2 a 0 53.8:2.5 51.0il.5 52.7ila6 Overall mean 57.1 55.1 5“.9 aMean and standard error of mean.. bSignificant quadratic regression (P < 0.05) among dry periods. SUMMARY AND CONCLUSIONS The influence of pregnancy on lactation and mammary gland development was studied during various stages of lactation and also at various stages following weaning. Lactating, pregnant (LP) animals weighed less (P < 0.05) than corresponding non-pregnant controls only on the 2“th day of lactation (2“6 g versus 25“ g). This depression in body weight was attributed to water loss associated with parturition and/or to advancing pregnancy. Following weaning at the 8th, 12th,‘l6th and 20th day of lactation, body weights of pregnant animals tended to increase whereas those of non-pregnant animals decreased. Weight of the mammary gland of LP animals (9.85 g) was less (P < 0.01) than lactating, non-pregnant (LNP) controls (11.65 g). Weights of the mammary gland for LP and LNP animals decreased “9.0 and 11.0 per cent, respectively, from the 16th to the 2“th day of lactation. During the first “-day interval following weaning on the 8th, 12th, 16th and 20th day of lactation, weights of the mammary gland of LP animals were depressed “3.“, 17.2, 3“.7, and 32.8 per cent, respectively. Weights of the gland of LNP animals during these times decreased “6.“- 58.0, 56.3, and 5“.7 per cent, respectively. During 118 119 the dry period, pregnancy did not alter weight of the mammary gland before the 12th day of pregnancy. Weight of the extraparenchymal fat pad for either LP (0.91 g) or LNP (0.90 g) animals was the same and did not change (P = 0.75) between the 8th and 2“th day of lactation. Following weaning, fat pads of both pregnant and non-pregnant animals increased in weight. A plot of the relation between weight of the mammary gland and fat pad during various stages of lactation and involu- tion, revealed that the changes for the fat pad were opposite the changes for the mammary gland. This sug- gests that the mammary gland grew into the fat-pad during pregnancy and regressed out of it during involution. Pregnancy reduced (P < 0.01) daily litter weight gain from the 8th to the 2“th day of lactation (6.9 g versus 10.2 g). By the 2“th day of lactation litters from LP animals were losing 1.7 g‘a day, whereas litters from LNP animals were gaining 8.“ g a day. DNA and RNA of the mammary gland for both LP and LNP animals were the same from the 8th to the 20th day of lactation. However, from the 20th to the 2“th day, DNA for LP animals decreased 23.3 per cent, but increased 7.9 per cent for LNP controls, whereas RNA decreased 61.6 per cent for LP animals and also increased 7.9 per cent for LNP controls. These response curves were quadratic (P < 0.01) and cubic (P < 0.01) for LP and LNP groups, respectively. 120 RNA/DNA ratios were the same for both LP and LNP animals up to the 16th day of lactation, but then decreased 52.5 and 12.“ per cent, respectively, from the 16th to the 2“th day. From these results, it was concluded that pregnancy may be exerting its inhibitory effect on lactation in two ways: (1) between the 20th and 2“th.day of lactation it may be reducing the amount of synthesis per cell, and (2) on the 2“th day it may be causing a further decrease in milk synthesis by reducing the total number of mammary cells present. During the period of involution (dry period) following weaning on the 8th, 12th, 16th and 20th day of lactation, DNA, RNA, and RNA/DNA ratios of the mammary gland of pregnant animals were greater (P < 0.01) than non-pregnant controls. During the dry periods pregnancy retarded the fall in DNA after the 12th day of gestation. On the 8th day of the dry period, following weaning on the 8th and' 12th day of lactation, DNA, RNA and RNA/DNA ratios of pregnant animals gradually increased up to the time of parturition. DNA of the extraparenchymal fat pad of LP animals (0.62 g) was greater (P < 0.05) than LNP (0.5“ g) controls. From the 8th to the 2“th day of lactation, for both LP and LNP animals, a relatively constant percentage of DNA in the fat pad (1.3 to 2.6 per cent) was observed relative to 121 total mammary DNA (mammary gland DNA plus extraparenchymal fat pad DNA). Following weaning, DNA of the fat pad of pregnant animals tended to decrease whereas DNA of the fat pad of non-pregnant animals tended to increase. These changes in DNA of the fat-pad paralleled changes in weight of the fat~pad. Hydroxyproline of the mammary gland for LNP animals increased 21.5 per cent from the 8th to the 12th day,» decreased 13.3 per cent from the 12th to the 20th day, and increased l“.8 per cent from the 20th to the 2“th day of lactation. Hydroxyproline for'LP animals was relatively constant from the 8th to the 20th day of lactation, but decreased 32.8 per cent from the 20th to the 2“th day. On, the 2“th day, hydroxyproline for LP animals was 32.3 per cent less than corresponding LNP controls. Although hydroxyproline, DNA, and RNA of the mammary gland for LNP animals increased 1“.8, 7.9, and 7.8 per cent, respectively, from the 20th to the 2“th day of lactation, daily litter- gain decreased 20.9 per cent. From this, it is possible to speculate that the 7.9 per cent increase in DNA may indicate a proliferation of fibroblasts which could be actively synthesizing collagen, accounting for the increases in RNA and hydroxyproline, but that fewer parenchymal cells were available for milk synthesis, causing the 20.9 per cent decrease in daily litter gain. 122 From the 8th, 12th, 16th, and 20th day of lactation to the “th day after weaning, hydroxyproline of the mammary gland for both pregnant and non-pregnant animals showed changes ranging from a 3.8 per cent increase to an 18.7 per cent decrease. This is in contrast to the 30 to 50 per cent decreases in DNA “ days following weaning for both LP and LNP animals. Thus, hydroxyproline appeared to be more resistant to the process of involution than DNA. Also, unlike DNA whose loss was retarded after the 12th day of pregnancy, hydroxyproline losses were not retarded until the 16th day of pregnancy. This late response of hydroxy- proline was attributed to estrogen which, when secreted in greater amounts during late pregnancy, probably stimulated the fibroblasts to synthesize collagen. Following weaning, hydroxyproline of the extra- parenchymal fat pad for both pregnant and non-pregnant' animals increased. However, the increases in hydroxy- proline after weaning lactating, pregnant animals showed a “-day lag period in relation to the increases in fat pad DNA. Hexosamine of the mammary gland for LP and LNP animals increased 19.7 and 31.0 per cent, respectively, from the 8th to the 16th day and decreased “2.6 and 17.7 per.cent, respectively, from the 16th to the 2“th day. These quadratic curves (P < 0.01) indicate that hexosamine, as a measure of the ground substance, was actively synthesized 123 by the mammary gland at the peak of lactation. Following weaning, hexosamine for both pregnant and non-pregnant animals decreased until “ days before the end of each dry period but net synthesis of hexosamine occurred by the time of parturition. From these results it appears that mammary growth following weaning of LP animals consists of the following steps: (1) a proliferation of epithelial and connective tissue cells starting on the 12th day of pregnancy; (2) a stimulation of the connective tissue cells on the 16th day of pregnancy by estrogen to synthesize collagen (hydroxyproline); (3) an increase in cell protein synthetic activity (RNA) starting on the 20th day of pregnancy, in- preparation for the initiation of lactation on the 2“th day (parturition);(“) an increase in the-ground substance (hexosamine) surrounding each cell in response to the increased passage of products between cell and_blood supply. Weight of the adrenal gland (5“.0 mg) for LP animals was heavier (P < 0.07) than LNP controls (51.“ mg). Linear increases in weight of the adrenal gland were observed from. the 8th to the 2“th day of lactation for both LP (P < 0.07) and LNP (P < 0.01) rats. Four days following weaning, no trends could be established for weight of the adrenal gland for either pregnant or non-pregnant animals. Toward the end of the dry periods however, weight of the adrenal l2“ gland for pregnant animals tended to increase. This may indicate that the adrenal gland is responding to the stress of parturition or to the initiation of milk secretion at- the start of the second lactation. In order to determine the influence of various lengths of a dry period on the carryover of mammary gland secretory and connective tissue components into a second lactation, some LP rats were allowed to deliver their second litter and various biochemical parameters of the mammary gland were measured on the 1st, 8th, and 16th day of the second lactation. From the 3rd to the 8th day of the second lactation, dry period length did not influence-total litter weight gain. Between the 8th and 16th day, however, animals given a “-day dry period produced 22.0 and 9.3 per cent heavier litters than animals given 0-day or longer (8-, 12-, l6-day) dry periods, respectively. On the lst day of the second lactation, animals, .given an 8- or O-day dry period contained approximately 13.3 per cent more DNA than rats given l6-, 12-, or “-day dry periods. On the 8th day of the second lactation, mammary glands of rats which received 0- or l6-day dry periods contained 1“.8 and 13.2 per cent less DNA, respec- tively, than rats which received an 8-day dry period. By the 16th day, 0- or l6-day dry period groups contained 16.8 and 11.8 per cent less DNA, respectively, than rats 125 which received a “—day dry peirod. From these results, it~ is evident that the stage.of lactation chosen to study the influence of length of the dry period on DNA of the mammary gland is important, and the relationship one observes on the lst day of lactation will not necessarily be true later in lactation. The various dry periods had no effect on total mammary gland RNA during the lst day of the second lactation (P > 0.05). However, on the 8th day, mammary glands of rats given an 8-day dry period contained on the average, 15.8 per cent more RNA than glands which received 16- and 0-day dry periods. By the 16th day, 16- and 0-day dry period groups contained l“.3 and 22.1 per cent less RNA than rats given the “—day dry period. Length of the dry period had no effect (P > 0.05) on the RNA/DNA ratios on the 1st, 8th, or 16th day of the second lactation. Thus, the short or long dry periods were not causing reduced synthesis per cell because cells from mammary glands of rats which received the l6- or O-day dry periods were Just as capable of synthesizing-protein as cells which received the “- or 8-day dry period. Linear increases in ECME extract (crude lipid) of the mammary gland with increasing length of the dry period were observed on the lst (P < 0.01) and l6th‘day (P < 0.05) of the second lactation. 126 On the lst, 8th, and 16th day of the second lactation, mammary glands of rats which received the 0- or 16-day dry period contained less hydroxyproline, than rats which received the “—, 8-, or l2-day dry periods. Since collagen is necessary for future mammary development (Lasfarques, 1957), these lower amounts of hydroxyproline produced on the lst day, by those glands which received the 16- and 0-day dry periods, could be one of the reasons why these same glands contained reduced numbers of mammary cells in later lactation. 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Abstr., 25:222. 1961.]— APPENDICES 136 APPENDIX I COMPOSITION OF RAT FEED 137 138 The animal ration was composed of the following Iodized salt ingredients: Gound shelled corn (1/8 inch screen) 607.0 Soybean oil meal, 50% protein 280.0 Alfalfa meal, 17% protein 20.0 Fishmeal, 65% protein 25.0 Dried whey 25.0 Pro—strep 20, 0.5“% penicillin and 2.72% streptomycin “.0 Pro-Gen, 20% arsanilic acid 0.5 Vitamin A, 10,000 units/g 36“.0 Irradiated yeast, 9.000 units/g 38.0 Choline chloride 318.0 D, Ca Pantothenate 2.5 Riboflavin 1.5 Nicotinic acid (Niacin) 15.0 Vitamin B-12 (0.1% mannitol trituration) 3.0 DL alpha tocopherol acetate, 250 IU vitamin E/g 8.8 Menadione (vitamin K) 1.0 DL methionine 227.0 Limestone 16.0 Dicalcium phosphate 17.5 lb 1b lb lb OZ 090909090909 139 Manganous sulphate (MnSou.H20) 32.5% manganous 168.9 g Ferrous sulfate (Fe 804.7H2O) 20.9% iron 215.2 g Calcium carbonate (CaCO3) “0.“% calcium 83.8 g Zinc carbonate, basic (ZnCO3) 56.0% zinc “0.2 g Cupric sulfate (CuSOu.5H20) 25.“5% copper 12.9 g Cobalt chloride (CoCl2.6H20) 2“.77% cobalt “.7 g Potassium iodide (KI) 76.“5% iodine 2.2 g This ration gave the following analysis: protein 21.2 % fat 3.9 % crude fiber 2.5 % productive net energy 902 C/lb APPENDIX II SOLUTIONS USED IN THE ANALYTICAL PROCEDURE FOR NUCLEIC ACID 1“0 l“l Orcinol reagent used in the RNA procedure. 1. A stock solution of-l.6% ferric chloride (FeCl2.6H20) was.prepared by adding 1.6 g of ferric chloride to 1 liter of concentrated hydrochloric acid. 2. A 1% solution of orcinol in 1.6% ferric chloride solution, was prepared 1 hour before use. APPENDIX III SOLUTIONS USED IN THE ANALYTICAL PROCEDURE FOR HYDROXYPROLINE l“2 l“3 Resin-charcoal preparation. 1. Potassium 10 Twenty g of analytical grade cation-exchange resin (Ag 1-X8, 200-“00 mesh, chloride form) was mixed with 10 g Norit A. The mixture was washed several times with 6N HCl in a coarse sintered-glass funnel. Mixture was washed with ethanol and ether and then dried to a fine powder. Borate Buffer. 61.8“g of boric acid and 225.00 g of potassium chloride were added to 800 ml distilled water. the pH was adjusted to 8.7 with 10 N KOH and the final volume adjusted to 1000 m1. Alanine Solution. 1. Ten g DL alpha alanine was added to 90 ml of distilled water. The pH was adjusted with 10 N KOH and the final volume adjusted to 100 m1. Sulfuric acid Ehrlich's Reagent. l. 27.“ m1 of concentrated sulfuric acid was slowly added to 200 ml of absolute ethyl alcohol. 120 g of p-dimethylamino-benzaldehyde was added to 200 m1 of absolute ethyl alcohol in a separate large beaker. The acid alcohol was slowly added with stirring to the p-dimethylamino-benzaldehyde. APPENDIX IV SOLUTIONS USED IN THE ANALYTICAL PROCEDURE FOR HEXOSAMINE 1““ l“5 Dowex-SO, 250-500 mesh, cationic exchange resin. 1. The resin was washed several times on a Bfichner funnel with 2N sodium hydroxide, 2N hydrochloric acid and distilled water in that order, and freed of excess moisture by-suction. 2. A 1:1 (weight per volume) suspension of the resin in water was prepared and 5 ml was pipetted into a 16 m1 plastic centrifuge tube, centri- fuged at 17,000 rpm for 5 minutes, and the water decanted off. Hydrochloric acid Ehrlich's reagent. 1. A 2.67% solution (weight per volume) of p-dimethyl- aminobenzaldehyde, in a 1:1 mixture of absolute ethyl alcohol and concentrated hydrochloric acid was prepared. m TYLB 111111“ 447 MITIWITIWIMIAIIUI WIT!“ 3 12 9 3 0 1 0 3