'Wv'QAW-fl'c‘. u-Q-uvro-m— 0 O -0- 0. ”-.-.- 09’.g --oo --.; . ”—O‘WW-“- 0.00.0...” —_‘ --- ‘ o. ...”...Oo— .-. Q... -. ———w-.o-——s--oc—--« -— SERUM THY‘ROXINE RESPONSE TO THYROPROTEIN ADMINISTRATION IN. THE DAIRY COW ... . Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY GARY H. SHAW 1972 I ...... ../.:H.J....... .... . u.... . ...‘o....<.w.\Q.Qa t0\.... .. .. c. you II . O,. . _ . o v - .. . .. a . ....... . . ... u I . . .. .. . .. . ... . .o . . - o o... .. _ . . . . .. . .o - . .. . . . .. _ 0-. . . c. .. ._ . . . . a n . . .. . .. u - . . . . . . . ... .-1 0v... .../an.rfI. .. ... ..olnr .OkJ ‘~l«0 — . . . . _ . u . . . . . . . . OJ.- . fi . . c o . . v. . ... I. or. . . . .. ¢ 2 .1 . , . .. u... v . . . o . Jr! on. I a: v. . ....o. v. . .. I v! . . D I 0-. O . .l ..r. . . .. u . v . I. _ l to I ll. . . a .I u o p u . r . O I I . o . . r cl. IIJ . n. r . . . . .— . . . .. . ..r p . I, . a o . a . . . . .I o . I . on .... . . : ... I 7;... . ' . . _. ....»x ....v.... . ...... ..... c........w..w.. ~.A....u$’..~’..(.1&~ "a o. ... LUBRARY 23$". IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII : 3 1293 01070 8208 I ABSTRACT SERUM THYROXINE RESPONSE TO THYROPROTEIN ADMINISTRATION IN THE DAIRY COW BY Gary H. Shaw Twelve non-lactating dairy cows were fed 10 g thyroprotein daily for seven consecutive days. Blood serum samples were taken from six cows during the period of thyro- protein administration. The remaining six cows were sampled the week of withdrawal of therprotein. Serum was analyzed for protein-bound iodine and serum thyroxine. It was found that both protein-bound iodine and serum thyroxine increased with therprotein administration and decreased after with- drawal. Protein-bound iodine overestimated serum thyroxine in thyroprotein fed cows. Of the twenty-one lactating diary cows used in longer term experiments, seven served as controls, seven received 15 g thyroprotein daily for 5 weeks, seven received 15 g thyroprotein daily for 13 weeks. Serum taken before, during and after treatment was analyzed for thyroxine levels. Thyroprotein feeding caused the serum thyroxine level to increase from a control value of 6 ug/lOO ml to a maximum value of over 13 ug/lOO ml. Long term (13 weeks) and Gary H. Shaw short term (5 weeks) therprotein treatment maintained the thyroxine level above controls. Withdrawal of thyroprotein caused a precipitate decline in serum thyroxine levels. The rate of decline, minimum level and subsequent return toward normal was not greatly affected by length of treatment. Milk yields of thyroprotein-fed cattle were in- creased 13% over controls for six weeks, then declined to control values or below. SERUM THYROXINE RESPONSE TO THYROPROTEIN ADMINISTRATION IN THE DAIRY COW BY Gary H. Shaw A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1972 DEDICATION To the. memony 06 my umy dea/L Mike/L and to the gneaxut 06 Mom ii ACKNOWLEDGMENTS The author wishes to sincerely thank Professor E. P. Reineke for his kind guidance and assistance through- out all phases of this study. Without his confidence and encouragement this study would not have been possible. Special thanks are due to Dr. E. M. Convey and Dr. H. A. Tucker for providing the blood serum samples and for helpful counseling, and to Dr. J. W. Thomas for the information on milk yields and for directing the manage- ment of the experimental cattle. Appreciation is also due Dr. J. R. Hoffert for serving on my committee, critically reviewing and improving the manuscript. Thanks are also due to Miss Marilyn Stoltzfus for assisting in the thyroxine analyses and to Miss Connie Worden for the typing of the rough drafts of this thesis. Due to the academic and social interactions with the "guys upstairs" the time spent on this study was an enriching and pleasurable experience for which I am truly appreciative. I am also indebted to the Michigan Agricultural Experiment Station for support on a research assistantship. iii LIST OF LIST OF INTRODUC REVIEW OF LITERATURE MATERIALS AND METHODS Trea RESULTS Preliminary Study Fina DISCUSSI Preliminary Study Fina SUMMARY TABLES . . FIGURES . TION . . . tments . . Preliminary Study Final Study Blood Serum TABLE Analytical Methods 1 Study . Controls . OF CONTENTS Short Term Thyr0prot in Feeding Long Term Thyr0protein Feeding . Milk Production ON 0 O O 0 Milk Production 1 Study . Control . Treated . LITERATURE CITED . APPENDIX A. B. C. REPRODUCTION HISTORY FINAL STUDY 0 O O O O 0 OF THE COWS SERUM THYROXINE LEVELS . . . . . MILK PRODUCTION iv Page vi koxooooooo oo ll l4 14 15 18 21 26 26 27 28 28 29 36 37 45 46 51 LIST OF TABLES Table Page I. Preliminary Thyroprotein Study: Initiation of Thyroprotein . . . . . . . . . . . . . . . 12 II. Preliminary Thyroprotein Study: Withdrawal of Thyroprotein . . . . . . . . . . . . . . . 13 III. Milk as Percent of Pretreatment Level . . . . 22 Figure 1. LIST OF FIGURES Page Serum thyroxine levels in control and short term experimental cows . . . . . . . . . . . . 15 Serum thyroxine levels in control and long term eXperimental cows . . . . . . . . . . . . 20 Milk and percent fat response to therprOtein o o o o o o o o o o o o o o o o o 24 vi INTRODUCTION Thyroid hormones are required for normal milk production in lactating mammals (Kon and Cowie, 1961). Graham (1934a) first reported that secretions of the thyroid gland profoundly influenced lactational performance of dairy cows. Because of excessive costs and limited availability of thyroxine, utilization of this compound to enhance milk and milk fat production was not economically feasible. These restrictions were overcome when Reineke and Turner (1942) reported an i2_yi3£g method for production of thyro- protein which was an economic, orally active source of thyroid hormone. For almost 30 years synthetic thyroprotein has been fed to dairy cattle, yet there have been no direct determi- nations of changes in serum thyroxine resulting from its feeding. This is due largely to the unavailability until recently, of a simple, accurate procedure for serum thyrox- ine determination. The study to be reported was designed to determine changes in the serum thyroxine concentration of lactating dairy cattle before, during, and after inclusion of thyro- protein in the diet. REV IEW OF L ITERATURE Altered thyroid states affecting milk production in dairy cows was first brought to attention by the work of Graham in 19340 He showed that feeding desiccated thyroid gland to thyroidectomized or normal cows could cause a rise in milk and milk fat production (Graham, 1934a). Graham also showed that injections of thyroxine caused increased milk and milk fat production (Graham, 1934b). It was not until an inexpensive source of thyro- active substance was made available that widespread studies could be done. The develOpment of iodinated casein (thyro- protein) (Reineke and Turner, 1942; Reineke 23 $1., 1943) made available an inexpensive source of thyroactive sub- stance. It was shown that thyroprotein contains thyroxine (Reineke and Turner, 1943a) specifically L-thyroxine (Reineke and Turner, 1943b) and many other iodine compounds (Mischler and Reineke, 1970). Many studies were initiated concerning the effects of thyroxine and thyroprotein on dairy cattle,with special emphasis on milk production (Blater 25 31., 1949; Blaxter, 1952; Thomas, 1953; Moore, 1958). The responses in cattle to therprotein are essen- tially the same as after the administration of thyroxine. Oral administration of DL-thyroxine to dairy cows required approximately 16 times the amount given parentally to achieve the same lactational response (Bailey 33 31., 1949). Judging from its effect in causing weight losses in sheep, oral administration was about 12% as effective as parental administration of thyroxine (Turner and Reineke, 1946). From data obtained in sheep by the thyroxine substitution method (Wagner, 1959) it can be calculated that about 11% of the thyroxine in therprotein is absorbed when orally administered. The increased amount of thyroprotein neces- sary for oral administration is apparently not due to inactivation in the rumen (Thurner and Reineke, 1946) or to losses in the feces (Monroe and Turner, 1949). The lactational response to thyroprotein varies greatly due to a number of factors. In the early studies differences in the potency and tests for potency of iodinated protein preparations could account for much of the variance (Reineke, 1946a). Even with standardized preparations there are many factors known to contribute to variations in milk productions. Age (Booth EE.2£°' 1947), breed (Blaxter, 1946), stage of lactation (Herman 25 a1., 1938; Ralston gt_gl., 1940), previous milk yields (Thomas 23 31., 1957), and nutrition (Thomas 33 31., 1949), affect the degree of response to thyroprotein. It has been shown that thyroxine or thyroprotein increase the milk fat content (Graham, 1934; Reece, 1944), decrease the ascorbic acid (Chanda and Owen, 1952; Blaxter, 1952), and cause other changes in milk composition (Reece, 1944, 1950; Sen et_§1,, 1954). Thyr0protein also causes a marked increase in the iodine content of milk (Bartlett gt. 31., 1949). However, no significant amount of thyroxine has been found in milk (Reineke and Turner, 1944; Bruger and Selberbush, 1946). As to be expected of a compound containing thyroxine, thyroprotein will cause a number of metabolic changes, including increased heart rate (Booth and Elvehjem, 1947; Swanson, 1951), decreased body weight (Swanson, 1951, 1954; Moore, 1958), and may induce heat stress (Moore, 1958). A decrease in general health may also be observed (Leech and Bailey, 1953). There are several methods available for measuring thyroid gland activity and thyroid hormone levels. In the substitution method, "thyroid secretion rate is determined as the minimum amount of thyroxine adminis- tered daily that will completely block the release of 131 taken up by the thyroid gland" previously injected I (Post and Mixner, 1961a, p. 2265). This method, based on observations of Wolff (1951) and Perry (1951), first worked out for sheep (Henneman 33 31., 1952) was applied to dairy calves (Lewis et al., 1955; and Hendrich and Turner, 1964). In mature cattle it yields values ranging from 0.15-0.22 mg L-T4/45.4 kg (Anderson, 1971). Another method for measuring thyroid secretion rate (TSR) is the thyroxine pool turnover method. It was intro- duced by Ingbar and Freinkel (1955), applied to sheep (Freinkel and Lewis, 1957), and to cattle by Post and Mixner (1961a). In this method a small amount of radio- active thyroxine (L-T4Il3l) is injected, serum is then sampled at intervals and measurements are made of protein- bound iodine specific activity. In a corollary to this method (Mixner and Lennon, 1958) measurements of the decline in protein-bound iodine (PBI) are made following an initial injection of unlabeled thyroxine. From the decline in PBI or specific activity the thyroxine degradation rate can be calculated. In this method it is assumed that the degrada- tion rate equals the secretion rate (Ingbar and Freinkel, 1955). Values of 1.42-1.53 mg T4/day/cow (Anderson, 1971) for normal lactating dairy cows and 1.59-1.73 mg T4/day/cow in mature nonlactating dairy cows (Post and Mixner, 1961a) were reported. The substitution method and the pool turnover methods have been compared (Post and Mixner, 1961a; Bauman g£_§1,, 1969; Anderson, 1971). Anderson found that the substitution method overestimates the TSR by 12 to 22%. Aside from measuring thyroid secretion rate, there are methods available to estimate serum thyroxine levels. One of the most commonly used is based upon the fact that most of the circulating thyroxine is bound to plasma protein (Blincoe and Weeth, 1967). In this method (Barker and Humphrey, 1950), protein-bound iodine (PBI) is precipitated from serum and the iodine content of the precipitate is determined colorimetrically. This method has been applied to lactating dairy cattle to yield values of: 7.03 ug T4/100 m1 (Lewis and Ralston, 1953); 5.00 ug T4/100 ml (Mixner £2 31., 1962); 5.66 ug T4/100 ml (Bauman EE.§£°' 1969); and 7.47 ug T4/100 ml (Anderson, 1971). The most direct method of measuring serum thyroxine levels (used for the majority of the work in this study) uses the principle of competitive protein binding (Ekins, 1960). The method was altered by the use of a gel- filtration column (Murphy and Pattee, 1964) which was later replaced by an anion exchange resin (Murphy and Jachan, 1965) and finally modified by the use of a resin impreg- nated sponge (Nakajema £5 31., 1966; Kennedy and Abelson, 1967). The principle of this method is given under the section entitled materials and methods. There are a number of other methods available to measure the level of thyroid function. A partial list would include: radioiodine uptake (Greer, 1951), [which has questionable value (Lodge 33 31., 1958)], thyroxine dis- tribution volume and biological half—life (Bauman gt El! 1969), and butanol-extractable iodine (Benotti and Benotti, 1963). Some of the methods mentioned above have been applied to cattle receiving exogenous thyroxine. In two dairy bull calves, injections of 2.5 and 5.0 mg L—T4 caused PBI levels to increase from 3.7 ug/lOO ml to approximately 12 and 21 ug/lOO ml respectively. Using the turnover method PBI was estimated to decline at a rate of 28% and 81%/hr in the same animals (Mixner and Lennon, 1958a). Plasma PBI levels were observed to increase to 10-14 mg/100 ml after the addition of L-thyroxine to the feed of 10 lactating cows. Removal of L-thyroxine from the feed caused PBI levels to drOp below normal for a short period of time (Swanson and McFee, 1959). Injections of thyroxine 50 and 100% above winter secretion rates caused the thyroxine half-life to decline from 2.32 days to 1.05 and 0.88 days respectively (Prema- ' Chandra and Turner, 1961). Similarly injections of thy- roxine 125 and 150% above normal thyroid secretion rates in dairy cattle caused the FBI levels to increase from 3.7 ug/lOO ml to 7.1 and 9.1 ug/lOO ml with a concurrent decrease in the half-life of thyroxine (Bauman 3E Si! 1969). Previous to the work to be presented herein, there is only one report of serum thyroxine levels after thyro- protein administration. Six cows fed 10 g therprotein for 6 days were found to have a mean serum thyroxine level of 9.66 ug/lOO ml as measured by the competitive binding method (Etta and Reineke, 1971). MATERIALS AND METHODS Treatments Preliminary Study Twelve pregnant nonlactating Holstein cows and heifers were assigned to treatment groups. Six cows were fed 10 g thyroprotein1 daily, beginning on day l and con- tinued to receive thyroprotein through day 7. Thyroprotein was withdrawn on day 8. The other six cows were fed thyro- protein daily from day 8 through day 14. Daily blood samples were taken from all 12 cows and assayed for serum thyroxine (T4) and protein-bound iodine (PBI) on days 8 through 14. Final Study Twenty-one lactating Holstein cows (see Appendix A) were assigned to one of three groups of seven cows each (A, B and C). Group A was fed 15 g therprotein (TP) on day 6 through day 97. Group B was fed 15 g thyroprotein on day 6 through 41. The controls (Group C) received no thyroprotein. All three groups were bled on day 0, day 3, and on subsequent dates as recorded in Appendix B. 1Protamone supplied by Agri. Tech. Inc., Kansas City, Mo. All cows were housed in stanchion barns of the Michigan State University Dairy Department. They were fed 10 lbs alfalfa haylage daily, corn silage to appetite (35- 100 lbs/day/cow) and 1 lb grain per 2.2 lbs milk. The grain ration was adjusted every two weeks. The cows were milked twice daily. Milk weights were obtained for 5 days a week. Thyr0protein was fed in dehydrated alfalfa carrier for the first 4 days, but because of incomplete consumption the method was changed such that 15 g of TP was mixed with 1 1b of the usual grain concentrate and fed in the morning, previous to any other feed. This resulted in complete TP consumption. Blood Serum Coccygeal vein blood was maintained at room temper- ature for 2-3 hrs then at 4°C for 9-10 hrs to allow clot formation. Thereafter, it was centrifuged at 2500 X g at 4°C for 10 min. in a Sorvall-RC3 centrifuge. The serum was aspirated and stored at -25°C until assayed. Analytical Methods Protein-bound iodine: Protein—bound iodine (PBI) was quantitated using the method of Barker and Humphrey (1950). Briefly, serum proteins are precipitated with zinc sulfate and NaOH, washed, dried, ashed, and the ash dis- solved in an aqueous mixture of HCl and H2804. Iodide concentration of the resulting solution was estimated 10 colorimetrically by its ability to catalyze the reaction of Ceric Ammonium Sulfate (yellow solution) + Arsenious acid to cerous sulfate (colorless solution). Serum Thyroxine: Serum thyroxine (T4) was measured by the Tetrasorb-125 method.2 Briefly, serum proteins were precipitated with ethanol with the concurrent release of T4 into the supernatant. A measured amount of the supernatant was then dried in polyproplene tubes. Thyroxine-binding 125 globulin (TBG) labeled with I-T4 was added, and allowed to equilibrate at room temperature for 10 min. An initial radioactivity count was then made. Endogenous T4 displaces 125 some of the I-labeled T from the TBG. A resin- 4 impregnated sponge was then added to bind the labeled and unlabeled free T4. The sponge was washed to remove the TBG-bound T4. The ratio of the radioactivity counts in the washed sponge to the initial radioactivity count is prOportionate to the endogenous T4. 2Abbott Radio—Pharmaceuticals, North Chicago, Ill. RESULTS Preliminary Study The preliminary study was initiated to determine to what degree and at what rate thyroprotein (TP) feeding would effect serum thyroxine levels. It was also used to test whether or not protein-bound iodine (PBI) determination could be used as an accurate measure of serum thyroxine levels in thyroprotein-fed cattle. Blood samples from twelve nonlactating dairy cows were taken on days 8 and 14 and subsequently analyzed for serum thyroxine using the PBI method and the competitive binding method. Data for the six cows fed 10 g of therprotein on days 8 through 14 inclusive are shown in Table I. Maximum individual T4 values were obtained on days 9 or 10 (maximum mean value on day 10). The mean serum thyroxine levels then declined, but to a level that was still above the control level of day 8. Six cows were fed 10 g of therprotein for seven days. Their T4 values for the seven days following thyro- protein withdrawal are presented in Table II. The mean serum thyroxine levels declined from day 9, reaching a minimum on day 13, six days after withdrawal of thyro- protein. 11 12 TABLE I Preliminary Thyr0protein Study: Initiation of Thyroproteina Day 8 9 10 11 12 13 14 Meanb PBI T4 Ug/IOO ml 7.25 15.12 10.61 10.88 10.53 10.59 11.31 S.E. 0.55 1.62 0.64 1.32 0.75 0.67 0.51 MeanC T4 ug/lOO ml 7.25 11.33 11.55 8.67 8.85 7.93 9.66 S.E. 0.66 1.01 0.88 0.81 0.53 0.43 0.38 10 g therprotein/cow/day. b . . . . Mean T4 equivalent of the FBI for SIX cows run in duplicate. CMean T4 for six cows determined by the Tetrasorb-125 method. 13 TABLE I I Preliminary Thyroprotein Study: Withdrawal of Thyroprotein Day 8 9 10 11 12 13 14 Meana PBI T4 ug/lOO ml 17.90 14.79 9.50 7.47 7.29b 6.41 6.34 S.E. 0.77 1.09 0.53 0.32 0.52 0.26 0.28 MeanC T4 09/100 m1 10.28 12.74 9.28 6.36 6.00 3.94 5.20 S.E. 0.83 1.09 0.86 0.37 0.43 0.44 0.43 a . . . . Mean T4 equivalent of the PET for SIX cows run in duplicate. Mean of five cows. cMean T4 for six cows determined by the Tetrasorb-125 method. 14 A comparison of the serum thyroxine levels (Tables I and II) determined by the two different methods1 shows that the protein-bound iodine method consistently overestimates the serum thyroxine level as compared with estimates derived from the competitive binding method. Thyroprotein contains about 7% total iodine, including iodide and a series of organic iodine compounds that are closely related to thy- roxine (Mischler and Reineke, 1971). The protein-bound iodine method is considerably less specific (measuring all iodine compounds that will be precipitated with zinc hydroxide) than the competitive binding method (measuring exclusively thyroxine). Thus the protein-bound iodine method would be expected to yield higher values in therprotein-fed cows. Final Study Controls Seven lactating dairy cattle receiving no thyro- protein were assigned to the control group. Blood samples were taken on the same days as the long and short term groups (days listed in Appendix B). Mean serum thyroxine levels are presented in Figures 1 and 2. Individual values are given under the apprOpriate heading in Appendix B. lSince thyroxine is 65.4% iodine by weight, PBI % 0.654 = PBI thyroxine equivalent. 15 The mean serum thyroxine values can be fitted to a linear equation of the form y = a + bx, where y is the mean serum thyroxine value (in pg T4/100 ml) for a particular day, b is the slope, x is the number of days, and a is the y- intercept in pg T4/100 ml. The calculated y-intercept (a) was found to equal 5.43 pg thyroxine/100 ml, and the lepe (b) was found to be 0.0108 pg thyroxine/100 ml/day (P‘<0.01). Short Term Thyroprotein Feeding Seven different lactating dairy cattle were assigned to the short term feeding group (Group B). This group received 15 g thyroprotein on days 6 through 41 inclusive. They did not receive any thyroprotein on day 42 or on any subsequent day. Serum thyroxine (T4) levels were determined on samples taken starting day 0 and on subsequent days as listed for this group in Appendix B. Mean serum thyroxine levels are presented in Figure 1. Individual values are given under the appropriate heading in Appendix B. Pretreatment values determined for days 0 and 3, averaged 5.68 pg T4/100 ml. On the morning of day 6 thyroprotein was fed; by sampling time that afternoon (approximately 12 hours later) the mean serum T4 level had risen to 7.83 pg/lOO ml.’ The serum T4 level continued to rise, reaching a maximum of 13.13 pg/lOO ml on day 12. On day 14 the concentration was down to about 11 pg T4/100 ml; the T4 level continued to decline,reaching a plateau of ‘ l-_'. IND-“RV- " ’ Figure l. 16 Serum thyroxine levels in control and short term experimental cows. Mean serum thyroxine concentration of control and short term thyroprotein (TP) fed lactating dairy cattle. Thyroprotein (15 g/day) fed days 6 through 41, inclusive. Each point represents mean of six animals. l7 l4 0 CONTROL A T.P. SHORT TERM I0 .1! E — O .9 B 6 . 1 l 0 . ' .3 2... cl I L1 l |_LJ 3O 60 90 TI ME , DAYS Figure l 18 approximately 8.3 pg T4/100 ml. Thyroprotein was not fed on day 42; however, the serum T level dropped only 0.32 4 pg/lOO ml from the previous day. On day 43 the T level was 4 down to that of the controls; it continued to decline, going below controls to reach a minimum of 2.33 pg T4/lOO ml on day 47. The level then increased toward the control level, and by the last sampling day (day 52) had reached 4.93 pg T4/lOO ml. Long Term Thyroprotein Feeding Another seven lactating dairy cattle were assigned to the long term feeding group. This group received 15 g thyroprotein on days 6 through 97. They did not receive any thyroprotein on day 98 or on any subsequent day. Mean serum thyroxine levels are presented in Figure 2 and individual values are given in Appendix B. Pretreatment values determined for days 0 and 3, averaged 5.58 pg T4/100 ml. On the morning of day 6 thyro- protein was fed. By sampling time that afternoon (approx- imately 12 hours later) the serum T4 level had risen to 7.28 pg/lOO ml. The serum T4 level continued to rise, reaching a maximum of 13.98 on day 12. On day 14 the level was down to 12.49 pg T4/100 ml; it continued to decline reaching a local minimum of 7.76 on day 29. During the remaining time,while thyroprotein feeding was continued, the cows' mean serum T4 level fluctuated markedly, but it Figure 2. 19 Serum thyroxine levels in control and long term experimental cows. Mean serum thyroxine concentration of control and long term thyroprotein (TP) fed lactating dairy cattle. Thyroprotein (15 g/day) fed days 6 through 97, inclusive. Each point represents mean of six animals. 20 l4"i - T IOL ' I ... . I ' ' E " o _ ' ' 9 - .. .Ei"5 :15.1 :1. a a. F - r? . ' o CONTROL '; 2 a T.P. LONG TERM SO 60 90 TIME, DAYS Figure 2 21 never dropped to control level nor did it ever reach the height of the first maximum level. On day 98 no thyroprotein was fed; the mean serum level was 10.09 pg T4/100 ml. The level had dropped to 7.65 pg/100 ml on day 99 and continued to decrease, going below controls to reach a minimum of 2.51 on day 104. The level then increased to a value not significantly different from the control level of day 112. Milk Production Most studies of thyroprotein administration have been concerned with its effects on milk production. For this reason milk yields from the cows in groups A, B, and C have been included in Appendix C. Changes in milk production with time and thyro- protein administration for groups A, B, and C are presented in Table 3 and Figure 3. The values show the percent milk yield for a given time period as compared with a control period (9/20/71 to 10/3/71). For the first two weeks of treatment, group A responded to thyroprotein feeding by increasing their milk production to 102.2% of their control level. They main- tained a higher percent milk yield (as compared with Group C) until the two week period beginning 12/7/71. From 12/7/71 until 1/3/72, although continuing to receive thyroprotein, the percent milk yield then dropped below that of the 22 TABLE III Milk as Percent of Pretreatment Level Group Date A B c (%) (%) (%) 9/20/71-10/3/71 100.0 100.0 100.0 10/4/71 102.2a 104.7a 93.6 10/18/71 95.5a 100.9a 84.2 11/1/71 97.0a 99.8a 89.3 11/9/71 90.1a 63.6 82.9 11/23/71 75.3a 76.9 72.6 12/7/71 70.1a 76.3 76.5 12/21/71 63.2a 68.7 69.5 1/4/72 48.6 65.0 66.9 a15 g thyroprotein fed during this time period. Figure 3. 23 Milk and percent fat response to thyroprotein. Responses of milk production and fat content to administration of therprotein (TP) in lactating dairy cattle. Thyr0protein dose: 15 g/day. Each point represents mean response of six animals. control ---O---O--- long term (Group A) TP fed days 6-97 —--X---X--- short term (Group B) TP fed days 6-41 25 control group. For the two week period beginning on 1/4/72, during which thyroprotein had been withdrawn, the percent milk yield was well below that of the control group. During the first two weeks of treatment group B responded to thyroprotein feeding by increasing their milk production to 105% of their control level. They maintained their milk production at approximately 100% of their control levels for the next three weeks of therprotein feeding. For the first two—week period that group B did not receive therprotein, their milk production fell well below that of the control group. After this initial decline, their milk production increased so as to parallel that of the control group for the remainder of the study. The milk production of the control group decreased with time, as is to be expected of cattle in the declining stage of lactation. There was a sustained elevation in milk fat (see Figure 3). DISCUSSION Milk Production During the two week period preceding thyroprotein feeding, milk production for groups A and B was quite close (102% and 99%) to that of group C (see Appendix C). The milk production during the first two weeks of treatment, for groups A and B were 111% of group C. This compares favorably with the 109.9% of control reported for cows receiving therprotein at peak lactation (Tucker and Reece, 1961). Premachandra and Turner reported that the maximum response occurred in 3-5 days (1959). In a similar study they also reported a 27% increase (1962a); however, in these studies thyroxine was injected at 50% above the normal thyroid secretion rate. During the second two week period of treatment the milk yields of groups A and B were 116% and 118% of group C, but by the third two week period their milk production was down to 111% of group C. Although group A continued to receive thyroprotein for the remainder of the 13 weeks, its milk production dr0pped toward, then below that of the control group. This decline toward control has been reported to occur 16-18 26 weeks after initiation of thyroprotein feeding (Schmidt 32 al., 1971). Group B received no thyroprotin after November 9, 1971. During the first two weeks after thyroprotein with- drawal their milk production dropped to 76% of the controls. This was a 37% decrease compared to the previous two week period. In a similar experiment Reece (1947) reported a 34% decrease after two weeks of withdrawal. This decline in milk production was reversed during the next sampling period. This rebound effect has been reported to occur after 10-12 days (Premachandra and Turner, 1962b). When thyroprotein was withdrawn from group A, their milk pro- duction declined in a manner similar to group B. However, they were dried off before any rebound effect could be noted. The conclusion is that therprotein administration is only good for short term stimulation of milk production, as suggested by Thomas EE.E£° (1954). Percent milk fat was shown to increase in groups A and B during thyroprotein feeding. The elevation was main- tained in group B even after withdrawal of thyroprotein. Preliminary Study The preliminary study showed that administration of therprotein to cattle would cause marked elevation in serum thyroxine levels. It also demonstrated that protein-bound 28 iodine determination could not be used as an accurate measure of serum thyroxine levels. The competitive binding method was used exclusively in the final study,for this reason. The inability of the protein-bound iodine method to give reliable T4 values is due probably to the many iodinated compounds present in therprotein (Mischler and Reineke, 1970). The inaccuracy is not due to large iodine losses occurring during the chemical processing of the serum (Post and Mixner, 1961b) nor does prolonged serum storage time contribute much variance (Lennon and Mixner, 1957). Final Study Control The mean serum thyroxine levels for the control cows increased with time. This was found to be linear and may be expressed by the equation: mean serum T4, pg/100 ml==(0.0108 pg T4/100 ml/day) (number of days) + 5.43 pg T4/100 ml 7Using this equation the calculated mean serum thyroxine levels on day 100 would be equal to: (0.0108 pg T4/100 ml/day) (100 days) + 5.43 pg T4/100 ml 1.08 pg T4/100 m1 + 5.43 pg T4/100 ml 6.51 pg T4/100 ml. 29 This compares well with the experimental value found for day 100; 6.41 pg/100 ml. Lorscheider (1970) showed that in the rat increased lactation was accompanied by a decrease in serum thyroxine levels. He also showed that cattle at peak lactation had thyroxine levels 59% of dry cattle but by the end of lacta- tion the serum thyroxine had risen to that of the nonlac- tating cattle. The range of mean serum thyroxine values for the control group was 4.68 to 7.10 pg/lOO ml. This is in agreement with values in the literature. Bauman and co- workers (1969), sampling six cows, reported a mean protein- bound iodine value of 3.7 pg/lOO ml, which is equivalent1 to a serum thyroxine level of 5.66 pg/lOO ml. Other values reported are (after conversion to serum thyroxine): 5.00 pg/100 ml (Mixner et al., 1962) and 7.03 pg/lOO ml (Lewis and Ralston, 1953). Treated The long term thyroprotein-fed cows (group A) and the short term therprotein-fed cows (group B) received the same treatment initially. As expected the two groups ini- tially responded in the same manner. Groups A and B reached their maximum levels of 13.98 pg/lOO ml and 13.13 pg/100 ml Since thyroxine is 65.4% iodine by weight PBI pg/lOO m1 % 0.654 = thyroxine equivalent pg/100 ml. 30 on day 12. The maximum levels for groups A and B were not significantly different from each other but were highly significantly greater than control levels (P <0.01). Groups A and B declined from their maximum to reach local minimums of 7.76 and 7.92 pg T4/100 m1 on day 29. This decline from maximum could be due to the saturation of the thyroxine-binding globulin, increased activity of metabolizing enzymes, and d3 nova synthesis of additional metabolizing enzymes. Another factor to be considered here is that the unusually high levels of T4 in the serum would inhibit the release of thyroid stimulating hormone from the pituitary, which in turn would cause inhibition of release of T4 from the thyroid. There is much evidence for this mechanism. In rats administration of thyroprotein was shown to cause suppression of the thyroid as measured by radio- iodine output, radioiodine uptake, radioiodine biological half-time, thyroid weight, and thyroid histology (Stewart, 1966). In cattle the suppression of the thyroid via administration of T4 is the basis of the substitution method described in the literature review (see Premachandra and Turner, 1961). The rates of increase in serum thyroxine from day 6 to day 12 for groups A and B were 1.12 and 0.88 pg/lOO ml/ day. Since at this point both groups had received the same treatment, their rates of increase may be combined yielding an average increase of 1.00 pg T4/100 ml/day° Thyroprotein 31 contains approximately 1% thyroxine (Mischler and Reineke, 1971) of which approximately 10% is claimed to be absorbed when orally administered (Mixner and Lennon, 1958b), there- fore feeding 15 g thyroprotein should be equal to parenteral administration of 15 mg T4. If the volume of distribution for T4 is estimated to be 60 liters (Mixner EE.El" 1962), 15 g therprotein/day should cause an increase in serum T4 of 25 pg/lOO ml/day. This is considerably different from the 1 pg/lOO ml/day found experimentally. There are two factors that could be responsible for this gross difference. First, in the study that determined the oral absorption value of 10%, serum T4 levels were estimated using the protein-bound iodine method. Earlier in this paper it was shown that protein-bound iodine deter- minations are not an accurate measure of serum thyroxine levels. This error is compounded by the fact that in the study under question (Mixner and Lennon, 1958a) large amounts (25 g) of thyroprotein were fed. Secondly, thyroxine, at levels above normal, tends to be cleared faster than at low or nomal levels. Evidence for this can be seen in that there was a progressive decrease in the rate of increase of serum thyroxine levels as they approached the maximum. This may be due to an increase in the activity of the metabolizing enzymes. The increased clearance at high levels would be enhanced by the fact that the thyroxine-binding globulin is soon saturated 32 (Etta and Reineke, 1971) and cannot bind the additional T4 present. During the time from day 30 until thyroprotein was withdrawn groups A and B maintained a relatively constant serum thyroxine level. Since they were absorbing approx- imately 15 mg T4/day,in order to maintain a plateau,they must have been clearing 15 mg T4/day. This reaffirms the statement that at high levels serum thyroxine is cleared faster than at normal levels. A decrease in the half-life (Tl/2) of thyroxine should result from an increase in the clearance rate. Premachandra and Turner (1961) showed that in normal cattle the Tl/2 of thyroxine was 2.32 days. How- ever, if thyroxine was injected at 150% of the normal thyroxine secretion rate the Tl/2 decreased to 1.05 days; at 200% of thyroxine secretion rate the Tl/Z was reduced further, to 0.88 days. Similar results have been reported by Bauman 23.3l3 (1969). From day 30 until day 97 group A received a constant amount of therprotein daily. Instead of showing a level plateau their serum thyroxine levels fluctuated markedly. An inspection of the individual records shows that the fluctuations were asynchronous. An autocorrelation test was performed on the plateau region to test for cyclicity. The only significant results found were that the serum thyroxine levels on a given day were significantly corre- lated to the levels of the previous day. 33 The wide fluctuation observed might be due to variations in the length of time required for the thyroxine to reach absorptive surfaces in the gastrointestinal tract. Thyroprotein has been shown to have an effect on the gastro- intestinal tract of the cow, causing a decrease in the gastrointestinal fill (Swanson, 1954). When thyroprotein was withdrawn from groups A and B, the serum thyroxine level declined in similar manners. ‘Both groups showed little, if any, decline in thyroxine on the day of withdrawal. However, by the second full day off therprotein the level had dropped approximately 2.5 pg/ 100 ml. Both groups then declined rapidly, reaching minimum levels below control of 2.51 and 2.35. Group B reached its minimum 6 days after the last thyroprotein feeding. Group A required 7 days to reach its minimum, but group A was at a higher serum thyroxine level when thyroprotein was withdrawn. The rates of decline for groups A and B from the first day off thyroprotein to the minimum of 1.08 pg T4/100 ml/day and 1.00 pg T4/100 ml/day, were not significantly different from each other. After reaching the minimum level both groups increased their serum thyroxine levels toward normal. This showed that even after prolonged suppression of the thyroid by exogenous thyroxine the thyroid-pituitary system was still able to resume function, and did so in not longer than 2 weeks. Bauman EE.E£° (1965) showed that the thyroid 34 of cattle previously suppressed by thyroxine injection would resume the uptake of labeled iodine when the thyroxine injections were discontinued. One interesting theoretical discussion of effects of therprotein administration on the serum thyroxine levels in cattle has been given by Pipes EE.El° (1959). In this work it is claimed that in a 1000 lb cow secreting 2 mg T4/day the addition of 15.0 g thyroprotein/day would cause drastic changes in the serum thyroxine levels. Specifically, they claimed that under these conditions the total pool of thyroxine would increase from 5.1 mg to 38.6 mg. If it is assumed that the volume of distribution remains constant, then the concentration of thyroxine in the serum would increase approximately sevenfold. Thus, if the original serum thyroxine level was 6.0 pg/100 ml the level after equilibration would be 45.4 pg/lOO m1. In this study 15 g therprotein was shown to cause the mean serum thyroxine levels to increase to approximately 14 pg/100 ml. Thus the theoretical estimation was almost 3 times the actual level achieved. The theoretical model required 23 days to reach equilibration or 13 days to reach 99% of the equilibration value. They also stated that the equilibration level would be maintained at the original peak level. In this study the level at which the plateau was maintained was reached in two days. However, it was shown that the level continued to rise to a maximum, then declined 35 to the plateau. Thus equilibration was not obtained until approximately 3 weeks after the initiation of thyroprotein feeding, even though the level was reached in two days. The theoretical model did predict correctly the rapid decline in thyroxine, and the drop below control levels which was to be expected considering the well known negative feedback mechanism. It appears then, that a theoretical model may be of some use when there is incomplete data, but that it may also be very misleading. SUMMARY . It was found that protein-bound iodine determinations are not good estimates of serum thyroxine levels in thyroprotein-fed cattle. Lactating dairy cattle fed 15 g thyroprotein showed a transient increase in milk production, followed by a decrease after withdrawal. Thyr0protein feeding caused sustained elevated serum thyroxine levels in dairy cattle. The decline in serum thyroxine levels after withdrawal was found to be essen- tially the same in cattle fed thyroprotein for 5 weeks or 13 weeks. Resumption of normal thyroid function after up to 13 weeks of thyroprotein feeding occurred within approx- imately two weeks. Normal post-peak lactating dairy cattle had increas- ing serum thyroxine levels as lactation progressed. 36 LITERATURE C ITED LITERATURE C ITED Anderson, R. R. Secretion rates of thyroxine and triiodothyronine in dairy cattle. J. Dairy Sci. 54:1195-1199, 1971. Bailey, G. L., S. Bartlett and S. J. Folly. Use of L- thyroxine by mouth for stimulating milk secretion in lactating cows. Nature. 163:800-804, 1949. Barker, S. B., and M. J. Humphrey. Clinical determination of protein-bound iodine in plasma. J, Clin. Endocrinology, 10:1136-1141, 1950. Bartlett, 8., S. Rowland and S. Y. Thompson. Proc. 12th Int'l. Dairy Conf. 1:102-109, 1949. Bartlett, 8., A. W. A. Burt, S. J. Folly and S. J. Rowland. Relative galactopoietic effects of 3:5:3 triiodo-L— thyronine and L-thyroxine in lactating cows. 1. Endocrinology, 10:193, 1954. Bauman, T. R., R. R. Anderson and C. W. Turner. Effects of induced mild hyperthyroidism on serum protein-bound iodine, thyroxine distribution volume, and biological half-life of thyroxine-II” in dairy cattle. g. Dairy Sci. 52:245—249, 1969. Bauman, T. R., G. A. Hindery and C. W. Turner. Resumption of thyroid-stimulating hormone secretion as shown by I”1 uptake in cattle after gradual withdrawal of thyroxine. 1. Dairy S21. 48:484-489, 1965. Benotti, J., and N. Benotti. Protein-bound iodine, total iodine, and butanol extractable iodine by partial automation. Clin. Chem. 9:408, 1963. Blaxter, K. L. Experiments with iodinated casein on farms in England and Wales. J, Agric. Sci. 36:117-150, 1946. Blaxter, K. L. Some effects of thyroxine and iodinated casein on dairy cows and their practical significance. Vitamins and Hormones, 10:217-250, 1952. 37 38 Blaxter, K. L., E. P. Reineke, E. W. Crampton and W. E. Petersen. The role of thyroidal materials and of synthetic gastrogens in animal production and an appraisal of their practical use. J. Anim. Sci. 8:307-352, 1949. ' """ ‘— Blincoe, C., and H. J. Weeth. Serum binding of radio- thyroxine in sheep and cattle. J, Anim. Sci. 26(2):372-373, 1967. Booth, A. N., C. A. Elvehjem and E. B. Hart. Some effects of feeding iodinated casein to dairy cows. J, Dairy Sci. 30:443-455, 1947. Bruger, M., and F. F. Silberbush. Metabolic studies in children fed milk from cows receiving synthetic thyroprotein. ‘J. Clin. Endocrinology, 6:565-569, 1946. Chanda, R., and E. C. Owen. The effect of thyroxine and thiouracil on the composition of milk. II. The carotene and vitamin A content. Biochem. J, 51:404-417, 1952. Ekins, R. P. The estimation of thyroxine in human plasma by an electrophoretic technique. Clin. Chim. Acta, 5:453, 1960. Etta, K. M., and E. P. Reineke. Serum thyroxine (T ) and L-thyroxine binding globulin (TBG) of cattl in several physiological states. J. 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A determination of thyroid secretion rate in indi- vidual intact sheep. J. Anim. Sci. 11:794, 1952. Herman, H. A., W. R. Graham, Jr., and C. W. Turner. The effect of thyroid and thyroxine on milk secretion in dairy cattle. Missouri Agric. Exp. Sta. Res., 1938. Hindery, G. A., and C. W. Turner. Effect of administration of L-thyroxine 25 and 50% above secretion rate on lactating cows. J. Dairy Sci. 48:596-601, 1965. Ingbar, S. H., and N. Freinkel. Simultaneous estimates of rates of thyroxine degradation and thyroid hormone synthesis. J. Clin. Invest. 34:808, 1955. Kennedy, J. A., and D. M. Abelson. Determination of serum thyroxine using a resin sponge technique. J. Clin. Path. 20:89, 1967. Kon, S. K., and A. T. Cowie, eds. 1961. Milk: the mammary gland and its secretion. Academic Press, New York, N.Y. 515 pp. Leech, F. B. The galactopoietic effect of iodinated casein: dose response relationships during prolonged treat- ment. J. Endocrinology, 7:42-53, 1950. Leech, F. B., and G. L. Bailey. The effect on the health of lactating cows of treatment with galactopoietic doses of thyroxine or iodinated casein. J. Agric. Sgi. 43:236, 1953. Lennon, H. P., Jr., and J. P. Mixner. Some factors affecting the determination of plasma protein-bound iodine, using the alkaline fusion-ceric sulfate method. J. Dairy Sci. 40:351-355, 1957. Lewis, R. C., and N. P. Ralston. Protein-bound iodine levels in dairy cattle plasma. J. Dairy Sci. 36:33, 1953. 40 Lewis, R. C., E. P. Reineke and J. R. Lodge. A technique for estimating the thyroid secretion rate of dairy cattle. J. Anim. Sci. 14:1250, 1955. Lodge, J. R., R. C. Lewis and E. P. Reineke. Estimating the thyroid activity of dairy heifers. J. Dairy Sci. 40:209-215, 1957. Lodge, J. R., R. C. Lewis, E. P. Reineke and F. D. McGilliard. Thyroidal uptake of Ila by dairy calves. 2. Dairy Sci. 41:641-646, 1958. Lorscheider, F. L. Thyroid function in the lactating rat, cow, and ewe. Ph.D. thesis, Dept. of Physiol., Michigan State University, East Lansing, Mich., 1970. McQuillan, M. T. The prolonged administration of thyroxine to cows with particular reference to the effects on thyroid function and on pituitary thyroprotein hormone. Brit. J. Exper. Path. 29:93-106, 1948. Mischler, T. W., and E. P. Reineke. Thyroid hormone analysis of thyroactive iodinated casein. J. Dairy Sci. 53:233-238, 1970. Mixner, J. P., D. H. Kramer and K. T. Szabo; Effects of breed, stage of lactation and season of year on thyroid secretion rate of dairy cows as determined by the chemical thyroxine turnover method. J. Dairy Jgi. 45:999-1002, 1962. Mixner, J. P., and H. D. Lennon, Jr. Effects of thyro- trophin, L-thyroxine, and L-triiodo-thyroxine on levels of plasma protein-bound iodine in dairy bull calves. J. Dairy Sci. 41:840-848, 1958a. Mixner, J. P., and H. D. Lennon, Jr. Efficiency of absorption of thyroxine in thyroprotein from the gastrointestinal tract of lactating dairy cattle. J. Dairy Jgi. 41:728, 1958b. Moore, L. A. Thyroprotein for dairy cattle. J. Dairy Sci. 41:452-455, 1958. Monroe, R. A., and C. W. Turner. Univ. Missouri Agr. Expt. Sta. Research Bull. 446, 1949. Murphy, B. E. P., and C. J. Pattee. Determination of thyroxine utilizing the property of protein-binding. J. Clin. Endocr. Metab. 24:187, 1964. 41 Murphy, B. E. P., and C. Jachan. The determination of thyroxine by competitive protein-binding analysis employing an anion-exchange resin and radiothyroxine. J. Lab. Clin. M99. 66:161, 1965. Nakajema, H., M. Kuramochi, T. Horiguchi and S. Kubo. A new and simple method for the determination of thyroxine in serum. J. Clin. Endocr. Metab. 26:99, 1966. Perry, W. F. A method for measuring thyroid hormone secretion in the rat with its application to the broassay of thyroid extracts. Endocrinology, 48: 643-650, 1951. Pipes, G. W., and H. Ruppert. Estimation of the L-thyroxine secretion rate of dairy animals. J. Dairy Sci. 38:610, 1955. Pipes, G. W., B. N. Premachandra and C. W. Turner. The biological half-life of L-thyroxine and L- triiodothyronine in the blood of the dairy cow. J. Dairy J31. 42:1606-1614, 1959. Post, T. B., and J. P. Mixner. Iodine losses in the alkaline ashing method for determination of plasma protein-bound iodine. J. Dairy Sci. 44:2203, 1961b. Post, T. B., and J. P. Mixner. Thyroxine turnover method for determining thyroid secretion rates in dairy cattle. J. Dairy Sci. 44:2265-2277, 1961a. Premachandra, B. N., G. W. Pipes and C. W. Turner. Variation in the thyroxine secretion rate of cattle. J. Dairy Sci. 41:1609-1615, 1958. Premachandra, B. N., G. W. Pipes and C. W. Turner. A study of thyroxine secretion in cattle. J..Anim. Sci. 16:1063, 1957. Premachandra, B. N., and C. W. Turner. Elevated thyroxine pool and lactation. J. Anim. Sci. 18:1564, 1959. Premachandra, B. N., and C. W. Turner. Metabolism of thyroxine in hypothyroidism in cattle. Proc. Soc. Exper. Biol. Med. 106:818-819, 1961. Premachandra, B. N., and C. W. Turner. Increases lactation response in cattle with thyroxine 50% above thyroxine secretion rate. J. Dairy Sci. 45:1098-1101, 1962a. 42 Premachandra, B. N., and C. W. Turner. Thyrotropic hormone secretion in cattle after prolonged thyroxine therapy and after complete withdrawal. J. Dairy ESE: 45:1102-1105, 1962b. Ralston, N. P., N. C. Cowsut, A. C. Ragsdale, H. A. Herman and C. W. Turner. The yield and composition of the milk of dairy cows and goats and influenced by thyroxine. Univ. of Mississippi Agric. Exper. Sta., 1940. Reece, R. P. The influence of a synthetic therprotein when fed to dairy cows over a three-week period. J. Dairy Sci. 27:545-550, 1944. Reece, R. P. The influence of a synthetic thyroprotein when fed to dairy cows over an extended period. J. Dairy Sci. 30:313-324, 1947. Reece, R. P. Thyroprotein in the ration of dairy cattle. I. Its influence on milk production, fat test, heart rate and body weight. J. Dairy Sci. 33:126-133, 1950. Reineke, E. P. Thyroactive iodinated proteins. Vitamins and Hormones. 4:207-253, 1946a. Reineke, E. P. Thyroprotein in livestock investigations. Michigan State College Veterinarian, 7(1), 1946b. Reineke, E. P., and C. W. Turner. Formation in vitro of highly active thyroproteins, their biologica assay and practical use. Missouri Agric. Exper. Sta. Bull. 355, 1942. Reineke, E. P., and C. W. Turner. The recovery of crystal- line thyroxine from iodinated casein. J. Biol. Chem. 149:555-561, 1943a. Reineke, E. P., and C. W. Turner. The recovery of L- thyroxine from iodinated casein by direct hydrolysis with acid. J. Biol. Chem. 149:563-570, 1943b. Reineke, E. P., and C. W. Turner. Non-permeability of the mammary gland to thyroid hormone. J. Dairy Sci. 27:739-805, 1944. Reineke, E. P., and C. W. Turner. The quantitative deter- mination of thyroxine in iodinated casein having ‘ thyroidal activity. J. Biol. Chem. 161:599-611, 1945. 43 Reineke, E. P., M. B. Williamson, and C. W. Turner. The effect of progressive iodination followed by incu- bation at high temperature on the thyroidal activity of iodinated proteins. J. Biol. Chem. 147(1):115- 119, 1943. Schmidt, G. H., R. G. Warner, H. F. Tyrrell and W. Hansel. Effect of thyroprotein feeding on dairy cows. J. Dairy Sci. 54:481-492, 1971. Sen, K. C., B. N. Premachandra, G. K. Marthy, N. N. Bastur and D. Narayan. The effect of feeding iodinated casein on yield and composition of milk. Indian J. Dairy Sci. 7:49, 1954. Stewart, M. E. Thyroid recovery after cessation of treat- ment with thyroidal substances. Masters thesis, Dept. of Physiol., Michigan State University, East Lansing, Mich., 1966. Stockl, W., and W. Jo'Chle. Corticosteroid induced changes in plasma amino acids and thyroid activity in dairy cows treated early or late during lactation. J. Dairy Sgi. 54:271-281, 1971. Swanson, E. W. The effect of feeding thyroprotein to dairy cows during the decline of lactation in successive lactations. J. Dairy Sci. 34:1014-1025, 1951. Swanson, E. W. The effect upon milk production and body weight of varying withdrawal periods after thyro- active supplement feeding. J. Dairy §gi. 37: 1212-1219, 1954. Swanson, E. W., and A. F. McFee. Effect of feeding L- thyroxine upon plasma protein—bound iodine in lactating cows. J. Dairy Sci. 42:928, 1959. Thomas, J. W. The use of thyroprotein for milk production. National Research Council Pub. 266:47, 1953. Thomas, J. W., D. V. KOpland, E. A. Keyes and L. A. Moore. A study of the short-term use of iodinated casein for milk production. J. Dairy Sci. 40:128-141, 1957. Thomas, J. W., D. V. Copland, E. A. Keyes, A. G. VanHorn and L. A. Moore. Effects on economy and efficiency of milk production when thyroprotein is fed for a short period of time to milking cows. J. Dairy S21. 37: 877-888, 1954. Thomas, Thomas, Tucker, Turner, 44 J. W., L. A. Moore and J. F. Sykes. Some effects of feeding therprotein to dairy cows during their first lactation. J. Dairy Sci. 32:278-291, 1949. J. W., and L. A. Moore. Thyroprotein feeding to dairy cows during successive lactations. J. Dairy Sci. 36:657-672, 1953. H. A., and R. P. Reece. Thyroid supplementation at peak of lactation. J. Dairy Sci. 44:1751-1753, 1961. C. W., and E. P. Reineke. The relation of the route of administration of thyroxine, thyroprotein, and intermediate products upon their utilization by ruminants. Missouri Agric. Exper. Sta. Res. Bull. 297, 1946. Van Landingham, A. H., H. 0. Henderson and C. E. Weakley, Jr. Wagner, The effect of ioduated casein (protamone) on milk butterfat production and on the ascorbic acid content of the milk. J. Dairy Sci. 27:385-396, 1944. J. F. The hormonal control of reproductive activity in the cycling and anestrous ewe. Ph.D. thesis, Dept. of Physiol. and Pharmac., Michigan State University, East Lansing, Mich., 1959. Wblff, J. Some factors that influence the release of iodine Yousef, from the thyroid gland. Endocrinology, 48:284, 1951. M. K., and H. D. Johnson. Does a diurnal rhythm of thyroid activity exist in dairy cattle? J. Dairy Sci. 49:209-211, 1969. APPENDIX A REPRODUCTION HISTORY OF THE COWS IN THE FINAL STUDY Cow Number 832 833 873 906 935 939 958 1008 1010 1034 1037 1061 1072 1073 1074 1075 1078 1082 1087 1091 1093 REPRODUCTION HISTORY OF THE COWS Group 33 VOwOOOUJOwwwWWWwS’II’wOO APPENDIX A IN THE FINAL STUDY Date Fresh 4/27/71 4/30/71 4/30/71 3/17/71 5/5/71 6/16/71 6/24/71 4/18/71 4/16/71 7/5/71 6/15/71 4/30/71 5/15/71 3/19/71 3/12/71 6/15/71 3/25/71 7/6/71 6/27/71 6/8/71 6/4/71 aDetermined on 9/14/71. Milka per lb. 41.5 63.5 43.5 44.0 64.0 55.5 43.0 46.0 42.5 50.5 60.5 49.0 42.0 42.0 38.5 50.5 39.0 48.0 41.5 49.0 42.0 45 Lactation Number H 64 H r4 H F‘ H r4 H +4 w r4 N 63 p.10 O’cu m 4> a Pre nanc State Due Bred Bred Bred Open Bred Bred Open Bred Open Bred Bred Due Due Due Bred Due Open Bred Bred Bred 4/24/72 8/27/71 9/13/71 9/20/71 9/9/71 9/26/71 9/11/71 9/4/71 8/30/71 4/14/72 4/28/72 3/4/72 9/26/71 3/27/72 8/31/71 9/10/71 9/15/71 APPENDIX B SERUM THYROXINE LEVELS 46 FT .|HIIV 666.6 56.6 66 66.66 66.6 65.66 66.6 65.6 66.6 65.5 65\66\66 666.6 66.6 66 66.66 66.6 66.66 66.6 66.6 56.6 65.5 65\66\66 666.6 66.6 66 66.6 66.66 66.6 66.5 66.6 66.6 66.66 65\66\66 656.6 66.66 66 66.66 66.66 66.66 65.6 56.5 66.6 66.66 65\6\66 666.6 65.6 66 65.66 66.66 65.6 66.6 66.6 66.6 66.66 65\6\66 666.6 66.6 66 55.66 65.6 65.6 65.66 66.6 66.66 66.66 65\6\66 566.6 65.5 66 66.66 66.6 65.66 66.6 66.6 66.6 66.5 65\56\66 666.6 55.66 66 65.66 66.6 55.66 66.6 66.5 66.66 66.66 65\66\66 566.6 66.66 66 66.66 56.66 65.66 66.6 66.6 66.66 66.66 65\66\66 665.6 66.66 66 66.66 66.66 66.66 66.66 66.6 66.66 66.66 65\66\66 666.6 66.66 66 65.66 56.66 66.66 66.66 66.66 66.66 66.66 65\6\66 666.6 66.66 66 66.66 65.66 65.66 66.66 66.66 66.66 65.66 65\6\66 666.6 65.66 6 55.66 66.6 66.66 66.66 66.66 66.66 66.66 65\5\66 666.6 66.6 6 56.6 66.66 66.66 66.5 66.6 65.5 66.6 65\6\66 666.6 66.6 5 66.6 66.66 66.6 65.6 66.6 56.6 66.5 65\6\66 666.6 66.5 6 66.6 66.6 66.6 65.5 56.6 66.6 66.5 65\6\66 666.6 66.6 6 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\6\66 666.6 56.6 6 56.6 66.6 66.6 66.6 66.6 66.6 66.6 65x66\6 .m.6 666: 666 6666 6666 6666 6666 666 666 666 6666 6 300 AHE 00H\mzv mqm>mq HZonmNmB SDmmm Am muonwv mswpmmm Emma mcoq m xHazmmmd 47 666.6 56.6 666 66.6 56.6 66.5 66.6 66.6 66.6 66.6 65\66\6 666.6 66.6 566 66.6 66.6 66.6 66.6 66.5 66.6 66.6 65\66\6 665.6 66.6 666 56.6 66.6 66.6 66.6 65.6 65.6 66.6 65\66\6 666.6 66.6 666 66.6 66.6 66.6 66.6 56.6 66.6 66.6 65\6\6 565.6 66.6 666 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\6\6 665.6 66.6 666 65.6 66.6 56.6 66.6 66.6 66.6 66.6 65\5\6 666.6 66.6 666 66.6 56.5 66.6 66.6 66.6 66.6 66.66 65\6\6 655.6 66.5 66 66.5 66.6 66.6 66.6 56.6 66.6 66.66 65\6\6 666.6 66.66 66 56.66 66.66 66.6 66.6 66.6 66.66 66.66 65\6\6 566.6 66.66 66 66.66 66.66 66.5 66.6 66.66 66.6 66.66 65\66\66 666.6 66.66 66 66.66 56.66 66.66 65.66 66.6 56.66 66.66 65\66\66 565.6 66.6 55 66.66 66.66 55.6 66.6 66.6 66.6 56.6 65x66\66 665.6 66.6 65 56.66 66.6 66.66 66.6 66.5 66.6 65.66 65\5\66 666.6 66.6 66 66.66 66.66 66.66 66.6 66.6 66.6 66.6 65\66\66 666.6 66.6 66 66.6 66.66 66.6 66.6 66.6 66.6 66.6 65\66\66 666.6 56.6 66 66.6 65.6 56.66 56.6 56.5 66.6 66.6 65\66\66 656.6 66.66 66 66.66 66.66 66.6 66.66 65.6 65.6 66.6 65\66\66 566.6 66.66 66 66.66 66.66 66.66 66.66 66.5 66.66 56.66 65\66\66 666.6 66.6 56 66.66 66.66 66.6 66.66 66.5 66.6 66.6 65\66\66 666.6 66.6 66 56.66 66.66 56.6 66.66 66.6 66.6 66.6 65\66\66 .m.m 6662 666 6666 6666 6 3ww66 6666 666 666 666 6666 UmzcflucOUIlAd muonwv mcfiwmmm Hume 0:06 8 4 i 666.6 66.6 66 66.6 66.6 66.6 56.6 66.6 66.6 66.6 65x66\66 666.6 66.6 66 66.6 66.6 56.6 66.6 66.5 66.6 66.6 65\66\66 665.6 66.6 66 65.6 66.6 66.6 66.6 66.6 65.6 65\66\66 566.6 66.6 56 66.6 66.6 66.6 56.6 66.6 66.6 66.6 65\66\66 656.6 66.6 66 66.6 66.6 66.6 66.6 56.6 65.6 56.6 65\66\66 666.6 56.6 66 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\66\66 666.6 65.6 66 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\66\66 665.6 66.6 66 66.6 66.6 66.5 65.6 66.6 66.6 66.5 65\66\66 666.6 66.6 66 66.6 66.66 66.66 56.5 66.6 65.6 66.6 65\6\66 666.6 66.6 66 66.6 66.66 66.66 66.6 66.6 66.5 56.6 65\6\66 665.6 66.6 66 66.66 65.6 66.6 56.6 66.6 66.5 66.6 65\6\66 656.6 66.5 66 66.5 56.6 66.66 66.6 66.6 65.6 66.6 65\56\66 1666.66 666.66 6566 300 666656066 666.6 66.6 66 66.66 66.66 66.6 66.66 65.6 66.6 66.66 65\66\66 666.6 66.66 66 56.66 66.66 56.6 66.66 66.66 66.5 66.66 65\66\66 666.6 66.66 66 55.66 66.66 66.66 66.66 66.66 66.66 66.66 65\66\66 666.6 66.66 66 56.66 66.66 66.66 66.66 66.66 66.6 66.66 65\6\66 656.6 65.6 66 66.6 66.6 66.66 66.66 66.66 66.6 66.5 65\6\66 666.6 66.66 6 66.66 66.6 56.66 66.66 56.66 66.6 66.5 65\5\66 666.6 65.6 6 66.6 56.6 66.6 66.66 66.66 66.66 66.6 65\6\66 666.6 66.6 5 66.6 66.6 66.6 66.6 66.66 66.6 66.6 65\6\66 666.6 66.5 6 66.6 56.5 66.6 66.6 65.6 56.6 65\6\66 666.6 66.6 6 65.6 66.6 66.6 65.6 66.6 56.6 66.6 65\6\66. 666.6 66.6 6 66.6 66.6 66.6 .66.5 66.6 65.6 66.6 65x66\6 .m.6 6662 66a 5666 6566 6566 6666 5666 666 666 666a 6 300 Am QSOHUV mcflcmmm Emma “605m 668 666\666 66m>mq 626xomwma 2:666 49 1' 666.6 65.6 56 66.6 66m6 66.5 66.6 56.6 66.6 66.6 65\66\66 666.6 66.6 66 66.6. 66.6 66.6 66.6 66.6 66.6 66.6 65x66\66 656.6 66.6 66 56.6 66.6 66.5 66.6 66.5 66.6 66.6 65\66\66 666.6 66.6 66 56.6 65.6 66.5 66.6 66.6 66.6 56.6 65\66\66 666.6 66.6 66 66.6 56.6 56.6 66.6 65.6 66.6 66.6 65\66\66 666.6 66.6 66 65.6 66.6 66.5 66.6 66.6 66.6 65.6 65\6\66 556.6 66.6 66 66.6 66.6 66.6 66.6 65.6 66.6 66.6 65\6\66 666.6 66.6 66 66.6 66.6 66.5 66.6 66.6 55.6 66.6 65\6\66 666.6 66.6 66 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\56\66 666.6 66.6 66 66.6 66.6 66.6 66.6 66.6 66.6 56.6 65\66\66 666.6 65.6 66 66.6 66.6 56.6 66.6 66.6 56.6 65\66\66 666.6 56.6 66 66.6 66.6 66.6 66.6 66.6 66.6 66.6 65\66\66 666.6 56.6 66 56.6 66.6 66.5 66.6 66.6 66.6 66.6 65\6\66 666.6 66.6 66 66.6 66.6 66.5 66.6 65.6 66.6 66.6 65\6\66 656.6 66.6 6 66.6 66.6 66.6 66.6 66.5 66.6 66.6 65\5\66 666.6 65.6 6 66.6 66.6 66.5 66.6 66.6 65.6 66.6 65\6\66 666.6 66.6 5 66.5 66.6 66.5 66.6 66.6 66.6 66.6 65\6\66 566.6 66.6 6 66.6 56.6 66.5 66.6 66.6 56.6 66.6 65\6\66 666.6 66.6 6 65.6 66.6 66.5 66.6 66.6 65.6 66.6 65\6\66 666.6 66.6 6 65.6 66.6 66.6 66.6 66.6 65.6 66.6 65\66\6 .m.m :66: 66a 6666 6666 6 3wm66 6566 6566 656 666 0666 AU msouwv maonpsou 66s 666\6:6 mqm>m6 626606666 sommm 50 666.6 66.6 666 66.5 66.6 66.5 66.5 66.6 66.6 66.6 65\66\6 666.6 65.6 566 66.6 66.6 66.5 66.6 66.6 66.6 66.6 65\66\6 666.6 66.6 666 66.5 66.6 66.5 66.6 66.6 66.5 66.6 65\66\6 666.6 66.6 666 66.6 66.6 66.5 66.6 66.6 65.6 66.6 65\6\6 666.6 66.5 666 66.6 66.6 66.6 66.6 56.6 66.6 65.6 65\6\6 666.6 66.6 666 65.6 66.6 66.6 66.6 66.6 66.5 66.6 65\»\6 666.6 66.6 666 66.6 66.6 66.6 66.6 66.6 56.6 66.6 65\6\6 666.6 56.6 66 66.6 66.6 66.6 66.6 66.6 66.5 66.6 65\6\6 666.6 66.6 66 66.6 65.6 56.6 56.6 65.6 66.6 66.6 65\6\6 566.6 66.6 66 66.6 66.6 66.5 66.6 66.6 66.5 66.6 65\66\66 666.6 66.6 66 ,66.5 66.6 66.5 56.6 66.6 66.6 66.6 65x66\66 556.6 66.6 55 66.5 56.6 66.6 66.6 66.6 55.6 66.6 65\66\66 655.6 66.6 65 66.6 66.6 66.5 66.6 66.6 66.5 66.6 65\5\66 566.6 66.6 66 66.6 66.6 66.6 66.6 66.5 66.6 66.6 65\66\66 666.6 66.6 66 65.6 66.6 66.5 66.6 66.6 66.5 66.6 65\66\66 666.6 55.6 66 66.6 65.6 66.6 66.6 66.6 66.6 66.6 65\66\66 666.6 66.6 66 66.6 66.6 66.5 66.6 65.6 66.6 66.6 65\66\66 666.6 56.6 66 66.6 66.6 66.5 66.6 66.5 66.6 66.6 65\66\66 .6.6 6662 666 6666 6666 6 3ww66 6566 6566 656 666 6666 UmDGHHGOOIIAU muonwv macaw HOHDGOU APPENDIX C MILK PRODUCTION 51 6.66 6.66 6.66 5.66 6.6 6.66 6.66 6.66 65\56\6u65\6\6 6.66 6.66 6.66 6.66 6.66 6.66 6.56 6.66 65\6\6u65\66\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.56 65\66\66u65\5\66 6.66 6.66 6.66 6.66 5.66 6.66 6.66 6.56 65\6\66u65\66\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 65x66\66-65\6\66 6.66 6.66 6.56 5.66 6.66 6.66 6.66 6.66 65\6\66-65\6\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.56 65\66\66-65\66\66 6.56 6.66 6.56 6.56 6.66 6.66 6.66 6.56 65\56\66u65\6\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 65\6\66-65\66\6 6.56 6.66 6.66 6.66 6.66 6.66 5.66 6.66 65\66\6u65\6\6 5.66 6.66 6.66 6.66 5.56 6.56 6.66 5.66 65\6\6-65\66\6 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 65\66\6u65\6\6 1666\66 66626 6662 6666 6666 6666 6666 666 666 666 666666 6666 6 260 U NHQZMHH4 A¢ msouwv msflommm cflmuoumoumce 8669 6:06 ZOHBUDaomm MAHZ 52 6.66 6.66 6.66 6.66 6.56 6.56 5.66 .. 65\56\6u65\6\6 6.66 6.66 6.66 6.66 6.56 6.66 6.66 .: 65\6\6u65\66\66 6.66 6.66 6.66 6.66 6.66 5.66 6.66 6606 65\66\66u65\5\66 5.66 6.66 6.66 5.66 6.66 6.66 6.66 6.66 65\6\66u65\66\66 5.66 6.66 6.66 6.56 6.66 6.66 6.66 6.66 65\66\66u65\6\66 6.66 6.66 6.56 6.66 6.66 6.66 6.66 6.56 65\6\66u65\6\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 65\66\66u65\66\66 6.56 6.66 6.66 6.66 6.66 6.66 6.56 6.66 65\56\66:65\6\66 6.66 6.66 5.56 6.66 6.56 6.66 6.66 6.66 65\6\66u65\66\6 6.66 6.66 6.56 6.66 6.56 6.66 6.66 6.66 65\66\6-65\6\6 6.66 6.66 6.66 6.56 6.66 6.66 6.66 6.66 65\6\6u65\66\6 6.66 6.66 6.66 6.66 6.66 6.65 6.66 6.66 65\66\6-65\6\6 2666\66 26626 6662 5666 6566 65666 36666666 5666 666 666 666666 6666 Am moouwv msflpmmm cflmuoumoumne Emma uuosm UODGHHGOUIIZOHfivbnomm MAHZ 53 6.66 6.66 6.66 6.66 6.66 6.66 6.5 5.66 65\56\6u65\6\6 5.66 5.66 6.66 6.66 6.56 6.66 6.6 6.66 65\6\6u65\66\66 6.66 6.66 6.66 6.56 5.66 6.66 6.66 6.66 65\66\66u65\5\66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.56 65\6\66u65\66\66 6.56 6.66 6.66 6.66 6.66 6.56 6.66 6.66 65\66\66u65\6\66 5.66 6.66 5.56 6.66 6.66 6.66 6.66 6.66 65\6\66u65\6\66 6.66 6.66 6.66 6.56 6.66 6.56 6.66 6.66 65\66\66s65\66\66 5.66 6.66 6.66 6.66 5.66 6.66 6.66 6.66 65\56\66-65\6\66 6.66 6.56 I- 6.66 6.66 6.66 6.66 6.66 65\6\66u65\66\6 6.66 6.66 n. 6.56 6.66 6.66 6.56 6.66 65x66\6u65\6\6 6.66 6.66 u- 6.66 6.66 6.66 6.66 6.66 65\6\6-65\66\6 6.66 6.66 5.66 5.66 6.56 5.66 5.66 6.66 65\66\6-65\6\6 6666\66 66626 6662 6666 6666 65666 30W566 6566 656 666 666666 6666 no msouwv HOHDGOO UODQHHGOUIIZOHfiobaomm MAHZ "661 16111“