PERIPHERAL BLOOD ANALYSIS AS AN INDEX TO REPRODUCTIVE CONDITION IN THE CANADA. GOOSE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY WILBUR C. JOHNSON 1967 0' . '- 5’. .' ' ' ' “ - ‘2" I...‘ I" " 3333“.“ ' ‘u‘ ULIEILfiékh" . . “' 'A " -' nu 0 III III III III II III III III IIIII IIII III III III 3 1293 10128 674 ABSTRACT PERIPHERAL BLOOD ANALYSIS AS AN INDEX TO REPRODUCTIVE CONDITION IN THE CANADA GOOSE by Wilbur C. Johnson Two exogenous estrogen experiments were conducted, December 1966 and January 1967, using different daily dosage levels of l7-"B"-estradiol. ~Blood serum samples were taken from the brachial vein at three day intervals from each treatment group prior to, during, and after the treatment period. At dosage levels between 125.ug and 1 mg, mean treat- ment values for serum calcium (ng), measured fluorometri- cally, total protein (g1), albumin (gZ), globulin (g1), determined by electrophoretic separation, and the albumin/ globulin ratio (A/G) showed significant (P(.OS) changes from pre-treatment and post-treatment means. There appeared to be a correlation between increasing dosage levels and the rela- tive changes in serum calcium, total protein and the A/G ratio. As a result of estrogen treatment, an average increase in serum calcium to 16.0 mg% or above, an average increase in serum protein to above 5.6 g% and a decrease in the A/G ratio to 0.8 or less were noted. Blood samples were ob- Wilbur C. Johnson tained from adult, two-year, and one-year old geese at weekly intervals during pre-season (unstimulated), pre-egg laying (stimulated) and egg laying (oviposition) time periods. Pre-season values for serum calcium, total protein, albumin, and A/G ratio were obtained from samples taken from December 31, 1966 to February 17, 1967. Pre-egg laying values for the same parameters were obtained from February 24, to March 17, 1967, and egg laying values were obtained during the period of March 24, to April 7, 1967. The adult geese were divided into normal, suppressed - egg laying and suppressed — non-egg laying after observations on behavior and oviposition were recorded. During the pre-season time period there was no significant difference (P>.05) between the five groups. The three adult groups, one year and two year old female geese could not be distinguished on the basis of peripheral blood constituents. During the pre-egg laying period serum calcium was the only parameter to increase significantly over pre- season values. This increase occurred only in the adult groups and there appeared to be a difference among mean cal- cium levels in normal adults and those adults that were con- sidered suppressed. During the egg laying period the normal Wilbur C. Johnson adults appeared to have a higher mean serum calcium level than the suppressed adults. Significant changes in total pro- tein, globulin and the A/G ratio occurred during the egg lay- ing period in adult geese. One and two-year old geese did not show significant changes in any of the parameters measured. The A/G ratio in those adult females that successfully nest- ed did not decrease below 1.00 until approximately one week prior to the oviposition of the first egg. Suppressed non- laying adult females exhibited an increase in serum calcium but the A/G ratio did not decrease below egg laying values obtained from the normal adults. The time, order and mag- nitude of changes in peripheral blood constituents are of prime importance in their use as an index to reproductive condition. Calcium was the first constituent to demonstrate 'decernable changes. Increases in total protein followed and just prior to oviposition the A/G ratio decreased to below 1.00. PERIPHERAL BLOOD ANALYSIS AS AN INDEX TO REPRODUCTIVE CONDITION IN THE CANADA GOOSE By Wilbur C. Johnson A THESIS Submitted to Muchigan State University in partial fulfillment of the requirements for the degree of MASTER.OF SCIENCE Department of Fisheries and Wildlife 1967 ACKNOWLEDGMENTS The author wishes to express appreciation to the following: Dr. Jack S. Wood, Western Michigan University, for his supervision, encouragement, and guidance throughout the study. Dr. L. W. Gysel for his advice and direction during the study. Dr. Peter I. Tack, Dr. George Lauff, and Dr. Robert Ringer for their time and patience in reviewing the manu- script. Special thanks to Dr. Robert G. Wetzel for his editorial comments. Mr. R. D. VanDeusen, Kellogg Bird Sanctuary, for the access to the Kellogg flock and use of the Sanctuary's facilities and equipment. Mr. Mbrton Rickless and Mr. Nick Smyrnios for their help with the analysis of the serum samples. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . . . . . v LIST OF ILLUSTRATIONS . . . . . . . . . . . . . . . vi LIST OF APPENDICES . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 METHODS AND MATERIALS . . . . . . . . . . . . . . . 6 Blood Sampling Technique . . . . . . . . . . . . 6 Serum Analysis . . . . . . . . . . . . . . . . . 7 Calcium analysis . . . . . . . . . . . . . . . 9 Protein analysis . . . . . . . . . . . . . . . 10 Endogenous Estrogen Stimulation EXperiment . . . 10 First Exogenous Estrogen Experiment . . . . . . . 16 Second Exogenous Estrogen Experiment . . . . . . 17 Statistical Analysis . . . . . . . . . . . . . . 18 RESULTS AND DISCUSSION . . . . . . . . . . . . . . 20 Exogenous Estrogen Experiments . . . . . . . . . 20 First exogenous estrogen eXperiment . . . . . . 20 Second exogenous estrogen experiment . . . . . 24 Endogenous Experiment . . . . . . . . . . . . . . 29 Pre-season period . . . . . . . . . . . . . . 30 Pre-egg laying period . . . . . . . . . . . . .' 33 iii Egg laying period . SUMMARY . LITERATURE CITED APPENDIX A APPENDIX B APPENDIX C iv Page 40 54 59 63 68 73 Table LIST OF TABLES Mean pre-treatment (Pt), treatment CTN, and post-treatment (Po) values for calcium (ng), total protein (gZ), albumin (g1), globulin (g%), and the A/G ratio from the first exo- genous estrogen experiment. December 2, 1966 to December 23, 1966 . . . . . . . . . . . . MEan pre-treatment (Pt), treatment CT), and post-treatment (Po) values for calcium (ng), total protein (g1), albumin (g1), globulin (g1), and the A/G ratio from the second exo- genous estrogen experiment. January 14, 1967 to February 4, 1967 . . . . . . . . Mean pre-season (Ps), pre-egg laying (Pe), 'egg laying (E) values for calcium (ng), total protein (g%), albumin (gZ), globulin (g1), and the A/G ratio in adult, one-year and two-year old female Canada geese . Page 21 25 31 Figure 1. LIST OF ILLUSTRATIONS Blood sample being taken from the brachial vein, which has been enlarged by application of pressure above the sample point . . . . . . Water area maintained by mute swans and part of goose flock used in endogenous experiment. February 11, 1967 . . . . . . . . . . . . . . Experimental breeding pen III, approximately 200 ft. long and 150 ft. wide, with one earthen nesting island in the south end . . . Non-experimental pair of geese defending the vicinity of their previous year's nest, MarCh 9, 1967 O O O O O O O O O O 0 O 0 0 Some of the experimental pairs walking the east border fence of wintering pen on erh 9’ 1967 0 O O O O O O O O O O O O 0 Female in foreground with enlarged abdominal region that occurs a short time prior to egg laying . . . . . . . . . . . . . . . . . . . . Pair 7 at nest site at the base of willow tree, Salix _p. (pen 11), May 14, 1967 . . . . An example of aggressive behavior exhibited by the gander of Pair 13 (pen III), March 17, 1967 . . . . . . . . . . . . . . . . . . . . . vi Page 12 13 35 37 42 44 47 LIST OF APPENDICES APPENDIX Page A Tables of raw data and statistical values from First Exogenous Estrogen Experiment . . 63 B Tables of raw data and statistical values from Second Exogenous Estrogen Experiment . 68 C Tables of raw data and statistical values from the Endogenous Estrogen Experiment . . 73 vii INTRODUCTION Wild populations of the Canada goose, Branta canadensis, in the Mississippi flyway normally leave wintering grounds in mid-March, and reach nesting areas by the third week in April. Within two weeks after arrival, breeding pairs have established territories, selected a nesting site, constructed a nest, and produced a complete clutch of eggs (Hanson and Smith, 1950). At a time prior to and during the northward Spring migration of wild pOpulations, potential breeding female geese of the Kellogg flock progress from a state of sexual rest (non-breeding condition) to one of sexual activity (breeding condition). A number of research projects on do- mestic fowl and more recently the mallard duck, Agag platyrhynchos, indicate that there are distinct changes in the levels of certain blood chemical constituents in birds associated with the transition from a non-breeding to breed- ing condition. Smoes (1966), Wood and Hofman (1967) suggested that changes in peripheral blood constituents are related to the reproductive state in the mallard and that analysis of these changes may yield an index to reproductive condition. This study, conducted from December 2, 1966, to April, 1967, was designed to provide data on the normal quantitative changes of selected blood chemical constituents (serum cal- cium, total protein, albumin, globulin and the albumin/ globulin ratio) in the Canada goose and to measure the effects of estrogenic hormones on these constituents. If changes in the selected peripheral blood chemical constitu- ents are decernable, then it may be possible to develope an index to reproductive state that can be used to determine reproductive potential in wild populations. Since the Canada goose is migratory and its geographic range is in temperate North America, the gonadal and be- havioral changes associated with reproduction are probably controlled and regulated by endogenous hormones which are triggered in reSponse to external stimuli (Witschi, 1959). Much evidence now indicates that temperature and photoperiod may act to synchronize an inherent rhythm of activity of the pituitary (Bissonette, 1937; Burger, 1949; Benoit, 1938; Wolfson, 1952, 1959; Welty, 1962). According to Marshall (1955) and others, the anterior pituitary, controlled by the hypothalamus, is the site of gonadotropin secretion that affects ovarian growth and egg laying. The gonadotropins in fowl are follicle stimulating hormone (FSH) and luteinizing hormone (LH). Follicle stimulating hormone stimulates ovarian folli- cular growth which results in increased amounts of gonadal hormones, particularly estrogen, which has been demonstrated to have a pronounced effect on avian metabolism (Nalbandov, 1953; Lorenz, 1954; and Sturkie, 1965). Estrogen treatment in birds has been shown to cause enlargement of oviduct and pronounced changes in blood composition. A recent study on the mallard duck demonstrated both morphological and blood chemical changes induced by estrogen (Hofman, 1966). A number of other researchers have demonstrated similar estro- genic effects, but only those pertinent to this study will be discussed. A marked increase in serum calcium has been noted in the mallard duck when daily doses of 17-"B"-estradiol were administered (Hofman, 1966; Rickless, 1967; Smyrnios, 1967). A number of other investigators have observed similar in- creases in serum calcium in domeStic fowl of both sexes when estrogen treatments were administered (Landauer, 35 al., 1941; Fleischman and Fried, 1945; Common, 25 a1., 1948; Polin and Sturkie, 1958; and Urist, st 21., 1960). The incre- ases were believed to be caused by the estrogen in the presence 4 of parathormone. Benoit, £5 31. (1941) reported that para- thyroidectomized ducks did not demonstrate an elevation in calcium in response to estrogen treatment. Polin and Sturkie (1958) and Urist, gt a1. (1960) reported similar results in the chicken. Estrogen has also been reported to have a profound effect on serum protein. Hofman (1966), Rickless (1967) and Smyrnios (1967) demonstrated a marked increase in total serum protein following estrogen treatment in the mallard duck. Urist (1959) has reported a similar increase in chickens. The increase was related to the globulin fraction; the albumin fraction re- mained stable or slightly lower than pre-treatment values (Hofman, 1966). Hofman (1966) also demonstrated a significant reduction in the albumin/globulin ratio (A¢G) with daily exo- genous estrogen treatment. Smoes (1967) has reported a similar reduction during normal endogenous estrogen stimula- tion. According to Rickless (1967), exogenous and endogenous stimulation increases in globulin can be attributed to the beta fraction; however, under stress conditions, the alpha and gamma fractions increase. .An increase in gamma globulin occurs just prior to egg laying (Rickless, 1967). Because increase in total protein and decrease in A/G ratio occur both under estrogenic stimulation and stress conditions, the two parameters alone can not be used to indicate reproductive state. However, an examination of the subdivisions of the globulin fraction of the serum protein can be used to deter- mine whether the total protein and A/G ratio are reSponding to "stressors" or estrogen (Rickless, 1967). Therefore a qualitative analysis of the globulin frac- tion along with quantitative changes in serum calcium, total protein, albumin, globulin, and the A/G ratio were used in this study to determine the reproductive state and probabil- ity of egg laying in selected female Canada geese. This project was supported by the W. K. Kellogg Biological Station and a grant from the U. S. Fish and Wildlife Service, Contract Number 14-16-0008-773, to Western 'Michigan University. METHODS AND MATERIALS Semi-wild female Canada geese were used in this study. The individuals were selected accopding to age, (one-year, two-year and three-years or older,) from the research flock at the W. K. Kellogg Bird Sanctuary, Hickory Corners, Michigan. One-year female geese are immature, two-year geese are on the verge of sexual maturity, and three-year geese are normally sexually mature (Wood, 1964). Black plastic and aluminum leg bands with routed numerals were used as code numbers for individuals during the blood sampling periods. Primary feathers on one wing were clipped to prevent escape from experimental pens. -§lggd sampling technique To prevent the blood samples from freezing during the first portion of the study, December 31, 1966 to March 10, 1967, all samples were taken in a laboratory. The geese were tranSported by truck from the experimental pens to the lab- oratory in a 3 ft. x 3 ft. x 5 ft. wire holding crate. From March 17, 1967 to April 28, 1967, blood samples were taken in the field. Because of the size of the geese two men were required to obtain the samples. After the bird was removed from the holding crate, its head was placed under its wing to aid in quieting. It was then turned ventral side up and the outside wing extended exposing the underside of the wing. By lifting the axillary feathers and removing the down from the humeral area, the brachial vein was exposed (Fig. 1). Pressure was applied to the brachial vein causing it to enlarge. The sam- pling area was then cleaned with 70% ethanol and a 2 ml blood sample obtained. ‘ External and internal hemorrhaging were minimized by applying pressure with a dry cotton swab directly on the sam- pling point for several seconds. The blood sample was then placed in a labelled 5cc plastic vial and allowed to stand for about 20 minutes. Samples that did not readily clot were cooled in a water bath at IOCCffioraan additional 5-7 minutes. Clotted samples were-centrifuged at 3000 rpm for 10 minutes. The supernatant serum was removed with clean dis- posable pipettes and transferred to labelled plastic vials. All serum samples were stored frozen at -13 C until time of ' analysis. Serum analysis Blood serum samples were not held for analysis more than six weeks. Decker (1966) demonstrated that no material change Figure 1. Blood sample being taken from the brachial vein, which has been enlarged by application of pressure above the sample point. 9 occurred in frozen samples up to that period. The serum was analyzed for calcium, total protein, albumin, and globulin in the following manner: Calcium analysis Calcium levels expressed in mg per 100 m1 (ng) were obtained by the use of a Turner Fluorometer with a primary filter 110-816(2A) and a secondary filter 110-818(2A-12). The calcein reagent was prepared by removing a 7 ml portion from 1000 ml 0.8N KOH, then replacing the 7 ml with calcein, yielding a 1 mg/ml concentration. (Calcein, 3,6-dihydroxy-2, 4-Bis [N,N-carboxy-methyl-aminomethyI] fluran, is a product of the Fisher Scientific Company.) Three mixtures of known calcium concentration (9, l6, and 21 ng) were used to establish standard curves. The fluorometer was adjusted to zero with the 9 mg% mixture and checked against the curve for the remaining two mixtures. A commercial serum (Versatol, 10.7 mg% calcium) was used as a control during the analysis. Five m1 of the calcein reagent was placed in a plastic vial along with a 26 lambda serum sample, shaken ten times and placed in 27 C water bath, oscillated for ten minutes and the fluorescence recorded. 10 Protein analysis Quantitative values for total protein were obtained with the use of an American Optical T.S. Meter. Serum protein fractions, albumin and globulin were separated using cell- ulose acetate strips in a Celmen cell (1.5 ma per strip for 40 min.) with a barbitone barbitol buffer solution (pH 8.6). The strips were then placed in Ponceau S stain for 6 minutes, cleared with 12% acetic acid in 95% ethanol and placed on glass to dry. A Densicord densitometer and integrator were used in the albumin and globulin determinations [grams per 100 ml (g%)] . Endogenous Estrogen Stimulation Experiments This experiment consisted of ten pairs having previous breeding records, and two 18-24 month old females and two 8-12 month old females that were sampled at weekly intervals to obtain data pertaining to normal fluctuations of peri- pheral blood constituents during the progression from non- breeding to breeding conditions. The geese were fed a daily diet of whole corn, wheat, oats, buckwheat and alfalfa pellets. Several feeding loca- tions within the pen were utilized to prevent intraSpecific competition for food. 'Ample grazing areas were available, except during periods of snow cover. Lettuce trimmings were 11 also fed during the winter months. Feeding was postponed on sample dates until after samples were taken. Blood samples were obtained between 11:00 and 12:00 a.m. on Fridays, from December 31, 1966 to April 28, 1967. During the non-breeding season from December 25, 1966 to March 10, 1967, the entire flock was maintained in a 300 ft. x 150 ft. pen in close proximity to the laboratory. Approximately one-half of the pen consisted of water aver- aging in depth from two to three feet. During periods of severe cold, a water area about ten feet in diameter was kept open by a yearling pair of mute swans (Cygnus 2135). An ice saw was used to enlarge the water area once a week (Fig. 2). Ten adult pairs were transferred to breeding pens when they were believed to be progressing toward a reproductively stimulated condition. The experimental breeding pens were approximately 200 ft. long and 150 ft. wide. About one- fourth of each pen consisted of a shallow narrow pond with one small earthen nesting island in the south end (Fig. 3). In Spite of the fact that the ground was still snow-covered and the ice-filled ponds did not thaw until Mhrch 18, 1967, nine of the breeding pairs were placed in their respective breeding pens on March 10, 1967. 12 Figure 2. Water area maintained by mute swans and part of goose flock used in Endogenous estrogen Experi- ment. February 11, 1967. 13 Figure 3. Experimental breeding pen III, approximately 200 ft. long and 150 ft. wide, with one earthen nesting island in the south end. 14 In order to obtain data on suppressed individuals the listed densities and non-random combinations of pairs were used because of facts known from past reproductive seasons and behavior exhibited by the pairs during confinement in the wintering pen. Three pairs were placed in each of the three breeding pens in the following non-random predeter- mined order: .Pairs 5, 12 and 67 were placed in pen I; Pairs 7, 10, 62 were placed in pen II; Pairs 4, 11 and 13 in pen III and Pair 3 were left in the winter pen which was their resident territory the previous year. By using these com- binations one or more of the individual pairs in each pen were expected to be suppressed due to overcrowding and lack of more than one suitable nest site. In breeding pen 1, Pair 12 were considered the dom- inant because of aggressive behavior1 exhibited during the wintering period and the territory size occupied the previous nesting season. Pairs 5 and 67 were believed to be subor- dinate because of lack of aggressive behavior during the wintering period. 1 The degree of aggressiveness of each pair was based primar- ily on the behavior of the gander. 15 In pen 11, Pair 10 were the resident birds (its nesting territory in 1966) and exhibited moderate aggressiveness dur- ing the wintering period. Pair 7 appeared to be one of the most aggressive during the winter period and past nesting records indicated that the gander was probably a dominant bird in the Kellogg flock. Pair 62 appeared to be sub- missive during the wintering period. Pair 13 in pen III appeared to be one of the most dom- inant and aggressive in the experimental flock. They were also the resident nesting pair in pen III in 1966. Pairs 4 and 11 appeared to be subordinate during theiWintering period and neither pair were considered dominant in the ex- perimental flock. On April 12, 1967, those pairs that had not established a territory, nesting site and begun their egg laying cycle were considered reproductively suppressed and were released as pairs into suitable nesting areas where they were no long- er "overcrowded". Pairs 11 and 4 were released on their res- ident territories from 1966. Pairs 62 and 67 were placed in breeding pens where no other pairs of geese were nesting. Blood sampling was continued until egg laying occurred or until the likelihood of egg laying had passed. The experiment was designed so that fluctuations in 16 peripheral blood chemicals could be obtained for one-year, two-year, adult and suppressed adult female geese as they progressed from non-breeding to breeding conditions. The two-year old (18-24 month) and one-year old (8-12 month) female geese-were retained in the wintering pen for the duration of the experiment. Four earthen nesting islands were present in the wintering pen in event that the two-year geese progressed to a reproductive condition. Two full winged ganders paired off with the two-year old females. First exogenous estrogen experiment On November 22, and 25, 1966, ten two-year or older female geese were live trapped and separated into five groups of two each and placed in pens. The five pens were 40 ft. long and 8 ft. wide; water varying in depth from 6 in. to 2 in. covered approximately three-quarters of the area. An 8 ft. x 8 ft. covered shelter was located on the south end of each pen. The experimental birds were fed the same diet as those in endogenous estrogen studies. Feeding was postponed until after blood samples were obtained on sample dates. The experimental geese were maintained at normal temperatures and photoperiod for that time of year. 17 The experiment was conducted during December 2 - 23, 1966. On December 2, two days prior to the first hormone treatment, pre-treatment samples were obtained from all ten geese. Beginning on December 5, additional pre-treatment samples were taken just prior to the administration of 17-"B"-estradiol in peanut oil. Injections were made Sub- cutaneously in the axillary Space. This treatment was con- tinued for 14 consecutive days with the following dosage levels for each of the five groups. Band Experimental numbers group Dosage/day 21, 22 1 1cc peanut oil 8, l8 2 250 mg estradiol 4, 77 3 1.0 mg " 79, 95 4 2.5 mg " 29, 94 5 5.0 mg " Additional blood samples were taken on days 4, 7, 10, and 13 of the treatment period and on day 2 and 4 after the treatment period. All samples were taken between 11:00 and 11:30 a.m., and estrogen injections were made immediately following sampling. Second exogenous estrogen experiment Using the methods described above, ten adult female geese were divided into five groups of two each and admin- 18 istered a daily dose of 17-"B"-estradiol according to the following schedule: Band Experimental numbers group Dosage/day 08, 49 l 0.5cc peanut oil 2, 47 2 125 Mg estradiol 5, 9 3 250133 " 8, 7 4 500 A18 " 4, 23 5 1 mg " The experiment began on January 14, 1967, and concluded on February 4, 1967. Blood samples obtained during the two exogenous exper- iments were analyzed by the same method as those described in the endogenous estrogen studies. Statistical analysis A two-way analysis of variance using f ratios was em- ployed to Show significant differences at the .05 level between the age groups in relation to time periods (pre- season, pre-egg laying, egg laying, suppressed, and post- season means) in the endogenous estrogen studies. A similar analysis was used for values obtained in both of the exo- genous estrogen experiments. The comparison was made be- tween pre-treatment, treatment and post-treatment means for 19 each dosage level. A model 3600 Control Data computer at the Computer center, Michigan State University, was used to obtain the analysis. RESULTS AND DISCUSSION Exogenous Estrogen Experiments Two exogenous estrogen experiments were conducted using 17-"B"-estradiol in a peanut oil carrier. The first exper- iment was conducted December 2 - 23, 1966 and the second, January 14 - February 4, 1967. The two experiments were conducted in the same manner, differing only in the time of year and dosage levels administered (Tables 1 and 2). Pre- treatment samples were taken three days prior to and on the first day of estrogen treatment just prior to its adminis- tration. Treatment samples consisted of four samples taken on days 4, 7, 10 and 13 during estrogen administration. Post-treatment samples were taken 2 and 4 days after cessa- tion of treatment. .Eirsg Exogenous Estrogen Experiment Mean pre-treatment, treatment and~post-treatment values for serum calcium, total protein, albumin, globulin and the A/G ratio are given in Table l. The mean treatment values for serum calcium showed a Significant difference (P<.05) from mean pre-treatment and post-treatment levels in all but the 2507ug group. Mean pre-treatment values for all groups combined was 10.9 mg%; treatment values were 30.6 mg% and thepost-treatment level was 18.8 mg% (Table 2, Appendix A). 20 A! 40 AJG December 2, 1966 A Alb A Glb )6 TP Ca 11.6 0.53 5.0 0.32 2.9 samples Pt T Po 4 8 4 Mean pre-treatment (Pt), treatment (T), and post-treatment (Po) values for nut oil calcium (mg%), total protein (g%), albumin (g%), globulin (g%), and the A/G ratio from the first exogenous estrogen experiment. to December 23, 1966. Bird Estradiol Number of Group number dosage 20 & 21 1 ml pea- 1 Table 1. 21 MQ'O NM“) 000 \Od'x‘l' 1-lv-Iv-I (“Nd- mx‘l'ln COO mom FINN ROM .4an COO ONu—I GONG") Nx'l'w an-m COO OChN Ind'ln NNO thy—IO OI—IH «moo HNN I-It-Ir-I Pt “T Po 8 a 18 250mg 2 9.9 0.94 4.4 0.41 2.4 0.54 1.8 0.25 1.4 0.38 30.9 14.44 6.2 1.06 2.8 0.38 3.3 1.07 0.8 0.20 17.1 5.64 5.6 0.56 3.3 0.61 2.7 0.76 1.3 0.13 4 & 77 1.0 mg 3 QM I—lr-l 000 £000 HOP-I v-INC’) u-Ic-Iln Ou-IO Omu-I H000 MON HWr-I COO 0000 Nc>c3 ~¢~¢<3 «avid» OMO In\OI\ c>F+c> "3".“ tn<3~o H mun O‘x-r ONO H NdI-I I-IwO‘ v—Id'N Pt T Po 4 8 4 29 a. 94 5.0 mg 5 0.129 0.009 0.143 0.219 0.551 0.245 0.083 0.665 0.064 0.035 (Groups) Probability of E Statistic (Time) Significance level 22 Thus a significant increase occurred during estradiol treat- ment and a decrease after treatment was apparent. There was not a statistical difference among groups on the various dosage levels. However, there was an apparent correlation between increased estradiol dosages and calcium elevation. As dosage levels increased from 2501ug to 5 mg, an increase in mean calcium from 12.2 to 48.4 mg% occurred (Table 1). It appears that 2.5 mg of estradiol may have been the maximum effective dosage for the geese, as the 5 mg treatment group did not demonstrate any apparent increases over those given 2.5 mg (Table 1). Mean treatment levels of total protein as compared to pre-treatment and post-treatment levels approached statis- tical Significance at the 0.05 level (Table 1, 0.08). Even though they were not statistically different, there was an increase that appeared to be correlated withhthe dosage level. The total protein levels attained by the two groups receiv- ing 2.5 mg and 5 mg doses did not appear to be different (Table 1, group 4 and 5). The increases in total protein can be primarily attributed to a marked increase in the globulin fraction. While the globulin increase due to treatment was not Statistically Significant at the 0.05 level, it approach- ed that level (Table 1, 0.06). The albumin fraction neither 23 increased nor decreased markedly from pre-treatment values. The relatively stable albumin level and the increase in the globulin fraction resulted in a significant (§<;05) de- crease in the A/G ratio when mean treatment values were com- paredwith pre-treatment and post-treatment values (Table 1). An analysis for differences among dosage levels again was not significant, but mean values for the A/G ratio given in Table 1 show an apparent decrease from control and pre- treatment means at all dosage levels except 2501ug. The lack of statistical differences among the groups on various do- sage levels, even though pre-treatment values were equable (homogeneous), can probably be attributed to the wide range of dosages (2507ug to 5 mg) resulting in a great variability in group reSponse to these levels of estradiol. The Small number of degrees of freedom resulting from the use of mean values rather than individual sample values reduced statis- tical sensitivity. Even without statistical Significance, it was apparent that increases in serum calcium and total protein occurred in female Canada geese at increased eStra- diol levels. The increases in total protein resulted from an increase in the globulin fraction which in turn resulted in the noted decrease in the AIS ratio. 24 With the acknowledged results and problems encountered in the first exogenous estrogen experiment, a second exo- genous estrogen experiment was conducted. Second Exogenous Estrogen Experiment -The range of dosage levels of estradiol was reduced to 125.ug - 1 mg in an attempt to reduce reSponse level and the variability of reSponse among groups. Lower reSponses increase the accuracy of protein analysis; severe lipemia occurs with high doses. Mean treatment period values for all five parameters were Significantly different (P(.05) from means of pre- treatment and post-treatment periods (Table 2, time). The combined means for pre-treatment calcium levels was 11.4 mg%; treatment level, 15.1 mg%; and post-treatment, 14.2 mg%. As in the first experiment, there was a statistically Significant difference in serum calcium during estradiol treatment and an apparent decrease after treatment (Appendix B, Table 2). A significant difference (P<.05) among groups for mean serum calcium was also obtained in this experiment. .Mean treatment values for each of the five groups were 11.6, 12,5, 13.6, 16.4 and 21.4 mg% reSpectively (Table 2). There ap- peared to be a correlation between increased estradiol and January 14, 1967 Mean pre—treatment (Pt), treatment (T), and post-treatment (Po) values for calcium (mg%), total protein (g%), albumin (g%), globulin (g%), and the A/G ratio from the second endogenous estrogen experiment. to February 4, 1967. Table 2. Estradiol Number of Bird Group number .4 Glb A A/G Alb .4 TP Ca samples dosage 11.0 0.15 4.2 0.05 2.9 0.18 1.4 0.18 2.0 0.48 -Pt 08 & 49 % ml pea- 4 1 .4 0.44 2.6 0.30 1.8 0.35 1.5 0.36 0 4.5 0.30 2.7 0.22 1.8 0.33 1.6 0.32 9 4 6 1. 1 8 0. 6 ll 11 T ~Po 8 nut oil 25 InFlN MMN COO «(no HHF“ COMO MNN COO Or-ld' NNN c~q4\ cw~¢cn COO (Dwd' NNN one qmm 000 000000 c§c> Intncfi FINN FIFIF| Pt T Po 2 a. 47 125 41g 2 ®O\\+ \TNM COO O‘Oq' FIFIF'I mum Ndfi .OOO ooxo .Amv wcwzma wwm pom .Ammv wcammH wwmumwa .Ammv commmmumum emu: .m oHan 32 444.4 444.4 -444.4 444.4 444.4 444444 444444444 4‘44 44444444444 444.4 444.4 444.4. 444.4 444.4 44444444 44>44 444444444444 44.4 4.4 44.4 4. 4 44. 4 4 4 44 4 4.4 44.4 4 44 4 4 444 44.4 4.4 44.4 4. 4 44. 4 4 4 44 4 4.4 44.4 4 44 44 4 444444 44-4 44 44.4 4.4 44.4 4. 4 44 4 4 4 44 4 4.4 44.4 4 44 44 44 444 4444-444 4 44 44.4 4.4 44.4 4 4 44 4 4 4 44 4 4.4 44.4 4 44 4 4 444 44.4 4.4 44.4 4 4 44 4 4 4 44 4 4.4 44.4 4 44 44 4 444444 44-44 44 44.4 4.4 44.4 4 4 44 4 4 4 44 4 4.4 44.4 4 44 44 44 444 4444-434 4 44 4. 444 V 444 4- 44.4 V 4.4. 4. 4o 444444 4444.444 4.44 444444 .444 mafia mo .02 News anouw than 444444444 .4 44444 33 mean values are as follows for the normal three-year or older geese: 4.6 3% total protein, composed of 2.9 3% albumin, 1.6 3% globulin and an A/G ratio of 1.9. Wood and Hofman (1967) have established similar values for the non-laying female mallard: 4.8 3% total protein, consisting of 3.2 3% albumin, 1.6 3% globulin and an A/G ratio of 2.0. The values appear quite similar between the five groups in this experiment during the pre-season period. However, subsequent differences during the following two time periods can probably be related to the level and time of stimulation attained by each of the groups. Endogenous Estrogen Experiment: 3224255 laying period As mentioned in the experimental methods for the endo- genous estrogen experiments, it was necessary to transfer the ten three-year or older pairs to breeding pens at a time when they were believed to be progressing from an unstimulated to a reproductively stimulated condition. This transfer was made on March 10, 1967, after the following observations were made indicating that the pairs were entering a reproductively stimulated condition. 1. The return of established non-experimental pairs to the vicinity of their previous years nesting territory occurred on March 9, 34 1967, when 12 pairs were observed defending their last year's territory2 (Fig. 4). The temperature was above 12 C and sunny. 2. Pair isolation of the flock in general was also used as an indirect method of deter- mining the onset of stimulation. Pair isolation was first noticed on February 22, and became more obvious on March 2, 3, and 9. 3. Agonistic behavior on the part of the dom- inant pairs in the experimental flock be- came more frequent during the first week in March, even to the point where threats and fighting occurred when the geese were 'being driven into the holding pen for sampling on March 10. 4. Fence walking by the experimental pairs in the wintering pens occurred on March 4 and 9, indicating that the pairs pro- bably would have returned to their previous 2Past records at Kellogg on nesting site selection by Canada goose pairs indicated that if their nesting attempts were successful they returned to the same territory and often the same nest site year after year. 35 x‘ . 36““: , Figure 4. Non-experimental pair of geese defending the vicinity of their previous year's nest, March 9, 1967. 36 year's nesting territory had they not been retained by the fence (Fig. 5). The use of behavioral characteristics as indices to reproductive stimulation has been questioned. Wood (1964) has suggested that the presence of behavioral patterns re- lated to reproductive stimulation do not necessarily mean that Canada geese will successfully reproduce; conversely, the absence of these patterns does not mean that they are not in a stimulated reproductive condition. .Behavioral motor patterns that occur and are often associated with re- Yank"; I. productively stimulated adult geese are also performed by known immature geese (Wood, 1964; Sherwood, 1966). Thus, the use of behavioral characteristics as an index to repro- ductive stimulation is probably not suitable. In this study the occurrence of behavioral attitudes in the mature pairs occurred from one to three weeks prior to any decernable peripheral blood reSponse. Had the blood samples been analyzed on the day of release in an attempt to determine the precise time for release from the wintering pen, it would have been noted that March l7,was the first sample date on which all of the experimental mature female geese demonstrated an increase in serum calcium over previous samples, this was the first indication of estrogen stimula- 37 -'~0"‘ 49 Figure 5. Some of the experimental pairs walking on the east border fence of wintering pen on March 9, 1967. 38 tion (Tables 1-3, Appendix C). Individual females, 7 and 13, demonstrated marked increases in serum calcium much earlier (March 3). The first individual to demonstrate a decernable change in serum calcium was number 10 on February 24, (Table 1, Appendix C). Thus, subsequent analysis of the data collected substantiates the selection of March 10, as a re- lease date coinciding with the transition from an unstimu- lated to a reproductively stimulated condition. The variability observed in the start of decernable changes in serum calcium for individual females can probably be attributed to individual variation in reSponse to photo- period. However, there was some indication that a relation- ship may exist between social status and the time and the degree of stimulation at the beginning of the reproductive season. On March 10, those females in group one (dominant pairs) had a mean calcium level of 16.6 mg% while those females in group two and three (subordinate pairs) had a mean calcium level of 13.2 mg%. The small number of samples make the interpretation of the data difficult. Mean pre-egg laying serum calcium values for one and two-year old geese were 11.4 and 11.7 mg%, reSpectively (Table 3). These values do not appear to differ from pre- season values, indicating the birds were not reproductively “Tm” 39 stimulated. An analysis of variance for pre-egg laying means of each parameter for all groups (Table 3, 1-5) did not show any significant differences among the groups. The variability about the means during this period, particularly group one, probably accounts for the lack of significance (Table 3). Lack of statistical significance can also be attributed to the heterogeneity of the groups (one-year, two-year and three- year olds or older geese) that are not comparable physiolog- ically during this period. Another important factor in the significance of an F statistic is the degrees of freedom. These were small because an analysis of variance requires independence among the samples. Continuous sampling of a single group results in dependency. This was corrected by using means which are independent. Even though the mean values were not statistically different, there were noticeable differences in the mean ser- um calcium between the adult geese (groups 1-3) and the one and two-year old geese (groups 4 and 5) (Table 5, Appendix C). Mean serum calcium values for the mature geese were 20.5 mg%, 17.0 mg% and 17.4 mg% in groups 1, 2, and 3, reSpectively. The mean values for one-year old and two-year old geese were 11.6 mg% and 11.7 mg%, and are not different from the pre- 49 season means established for these two age groups (Table 5, Appendix C). It is apparent that the older geese were in a stimulated condition while the one-year and two-year females were not. There were no obvious differences between the other parameters measured during this period. Endogenous Estrogen Experiment: Egg laying period Observations were made on 23 non-experimental nesting 7' pairs during the nesting season in 1967. Of the 23 nests : started by these pairs, 95% laid the first egg between. ' March 29, and April 10. Thus, this time period was designa- g ted as the peak egg laying period. Whether or not the ex- perimental pairs began their egg laying cycle between these dates determined whether or not they were considered repro- ductively suppressed during the egg laying period. At this pits in the experiment the adult female geese were divided into three groups: normal (unsuppressed), suppressed (not laying during peak "egg laying" period), and suppressed (non-egg laying after release). Pair behavior, consisting of overt fighting, head shak- ing, threats, and calling were used as a direct method of determining the social status of the pairs in each pen. According to the investigator's observations, dominant pairs of geese exhibit, for the lack of a better term, affirmation 41 bouts which-consist of synchronous gabbling, head shaking and bowing, and threat postures directed toward all other geese. Subordinate pairs were not observed behaving in this manner under the crowded conditions. Prior to laying there was a decernable enlargement of the abdominal region in the female goose (Fig. 6). Daily observations on this condition as well as the results of T "daily egg hunts" were recorded. The non-random predetermined combinations of pairs that I were unequal in social status as described in the methods were used to affect suppression of one or more pairs due to overcrowding and lack of more than one suitable nesting site. In pen I, where Pairs 5, 12 and 67 were placed, only Pair 12 established a nest, produced a clutch of eggs and successfully hatched them (first egg laid on April 5). The nesting territory established by the male of Pair 12 inclu- ded the entire water area and the nesting island. The mean serum calcium, total protein and A/G ratio for goose number 12 during the egg laying period was 31.4 mg%, 5.7 3% and 0.85, reSpectively (Table 1, Appendix C). Since Pairs 5 and 67 did not begin egg laying during the establish- ed egg laying period they were considered suppressed. For comparison, the mean calcium, total protein and A/G ratio 42 Female in foreground with enlarged abdominal Figure 6. region that occurs a short time prior to egg laying. 43 for these two females during this period were 26.4 mg%, 5.5 g% and 1.34 (Table 3, Appendix C). The two suppressed fe- males exhibited calcium and total protein values slightly lower than those for the egg laying female; these values indicate that they were probably reproductively stimula- ted. The increase in total protein was primarily attribu- ted to beta globulin, indicating that the geese were under estrogenic stimulation, however, the maintenance of a high { albumin fraction resulted in an A/G ratio above that of the egg laying females. Some minor fluctuations in gamma glob- 2K ulin were noted, but were not consistent. Pair 67 were released from pen I, but did not successfully nest after release. The combination of pairs in pen II resulted in the establishment of two nesting territories by Pairs 7 and 10. The single nesting island was occupied by Pair 10 and the base of a willow tree (ggli§‘_p.) was the nest site of Pair 7 (Fig. 7). It appeared that these two pairs were equal in social status and the lack of dominance resulted in the division of the available nesting habitat into two equal portions. Both females began their egg sequence on April 7. The remaining pair (62) did not establish a besting territory during the egg laying period and were considered suppressed. 44 Figure 7. Pair 7 at nest site at the base of willow tree, Salix s2. (pen 11), May 14, 1967. 45 However, on April 3, an egg was found next to the feeding trough used by this pair.(three separate feeding troughs were placed in each breeding pen because the aggressive behavior and defended nesting territories of the pairs excluded the subordinate pairs from large portions of the pens). Thus, female 62 was possibly in an egg laying condition at this time. No other "dropped" eggs were found and Pair 62 was P released from the breeding pen on April 12. The mean serum calcium, total protein and A/G ratio for females 7 and 10 during the normal egg laying period were 32.2 mg%, 6.8 g% and 0.86, reSpectively. Means for female of Pair 62 for the same period were 29.0 mg% calcium, 6.6 3% total protein and 0.77 for the A/G ratio. On March 31, four days prior to the date when the egg was found by the feed trough, the individual sample for number 62 indicated that serum calcium was 36.0 mg%, total protein 7.4 g% and the A/G ratio was 0.85 (Table 2, Appendix C). An analysis of the globulin distribution demonstrated that on March 31, there were elevations in gamma, beta, and alpha globulins. The albumin fraction was reduced. These changes correspond with the noted elevation in total protein and reduced A/G ratio. These observations combined with the high calcium level indicate a general egg laying condition. Female 62 46 laid the firstegg in a latent successful nesting attempt on April 18, seven days after release. The enlarged abdominal region of this female also indicated that she was in an egg laying state. However, the other suppressed females that did not lay also exhibited this condition, leading to the conclusion that it is not necessarily indicative of egg laying. Of the three pairs in pen III, only Pair l3 establish- ed a territory and began egg laying during the egg laying period (first egg oviposited on March 31). The territory defended by the gander of Pair 13 included the nesting island and most of the water area. Pairs 08 and 11 appeared to be suppressed during the egg laying period (Fig. 8). The mean serum calcium, total protein and A/G ratio for the goose of Pair 13 during the egg laying period were equal to the values listed for the other three egg laying females in pens I and II (Table 1, Appendix C, egg laying values). Neither of the females of Pairs 08 or 11 were known to lay eggs during the egg laying period and were considered supp- ressed. Both pairs were released from pen III on April 12. Upon examination of the values obtained on April 7 and 12, for goose 11, it was apparent that she was stimulated and within the range of values of the normal egg laying females 47 Figure 8. An example of aggressive behavior exhibited by the gander of Pair 13 (pen III), March 17, 1967. 48 (Table 2, Appendix C). Prior to release, increases in total protein were primarily a result of increases in alpha glo- bulins. Beta globulin increased just prior to, and following release and appeared to be greater than either the alpha or gamma fraction. Female of Pair ll began a normal egg laying cycle three days after release. Similarly, goose 08, on April 7 and 12, had serum calcium and total protein values that were within the range of the normal egg laying female. ” The A/G ratio was not as low and continued to increase in subsequent samples after release (Table 3, Appendix C). An Q analysis of the globulin distribution indicated that goose 08 was in a stimulated, but non-egg laying condition. Beta globulin increased during the egg laying period, however, alpha globulin appeared to exceed beta and gamma globulins during this period. The maintenance of high alpha globulins is probably indicative of prolonged "stress". After release the protein fractions returned to a non-reproductive dondition. Calcium and total protein values decreased after release. These two observations seem to indicate that female 08 was in a non-egg laying condition accompanied by prolonged "stress". She apparently regressed to a non-reproductive condition after release, as indicated by the return of total protein and A/G ratio to pre-season values on April 28. 49 (Appendix C, Table 3). Adult Pair 3 was not transferred to the breeding pens on March 10. By retaining this pair in a situation where there was no competition for a nesting territory, it was thought that they would serve as a "control" for normal egg laying values in the event that none of the other nine adult pairs began their egg laying cycle within the pre- F viously defined peak egg laying period. Four of the nine pairs under "crowded" conditions did begin to lay within the peak egg laying period, as did Pair 3. And comparing 1 the egg laying values obtained from Pair 3 with those of the other four females, it seems apparent that there was no difference between the five females (Table 1, Appendix C). 0n the basis of the similarity in sample values and the time at the onset of the egg laying period, all five geese and their sample values were considered representative of the flock. A comparison of mean "egg laying" values for one and two-year old geese did not show any marked changes from pre- season values (Table 3). The one-year old females were con- sidered immature and no changes were expected. However, a number of investigators have reported that in the populations they studied a percentage of the two-year female geese are 50 mature and successfully nest (Wood, 1964 - less than 5%; Balham, 1950 - less than 7%; Sherwood, 1966 - 20%; Hanson, 1952 - 80%). Only two two-year females were used in this study and neither of them produced any eggs. There was an apparentllatent stimulation in the two-year old geese as serum calcium levels increased on April 21 and 28 (Table 4, Appendix C). Even though there were increases in serum calcium, values for total protein and the A/G ratio did not change from previous samples, indicating that the geese were not in an egg laying condition. No samples were taken after April 28, to determine if the latent stimulation continued. Mean values for serum calcium showed no significant differences (P>&05) among any of the five groups. The mean egg laying values for serum calcium were 33.7, 26.0, 28.3, 12.1 and 12.3 mg% for groups l-5, reSpectively (Table 3). Even though they were not statistically different, there were obvious differences between the normal adult and the one— and two-year old females, and mean values for suppressed adult females were lower than the means of the egg laying a adult. Mean serum calcium values did show a significant dif- ference (P<.05) between pre-season, pre-egg laying and egg laying time periods indicating that endogenous estrogen 51 stimulation at the onset of the reproductive season resulted in a change in the peripheral calcium levels. This signif- icance should not be interpreted as implying that all of the groups are different (Table 5, Appendix C). It is quite apparent upon examination of the means for the one-and two- year olds that there is little difference in mean values among time periods; therefore, significant differences can be attributed only to the adults. Mean total protein values among groups were not signif- icant (P>.05); however, they were significantly different (P<.05) among the time periods. As with calcium, this dif- ference is applicable only to the adult geese. The three adult groups 1, 2 and 3 had mean total protein values of 6.6, 6.1 and 5.8 g%, reSpectively, whereas the one-and two- year old female mean values were 4.7 and 4.4, reSpectively (Table 3). Means for albumin, globulin and the A/G ratio were not significantly different (P>.05) among groups or time periods, but again there were apparent differences in the globulin fraction and the A/G ratio among adults when compared to one- and two-year olds and pre-season values (Table 3). Mean egg laying values for the mallard duck were re- ported as 20.2 mg% calcium, 5.6 g% total protein consisting . ‘14" . l." —.-.R 52 of 2.1 3% albumin and 3.5 3% globulin, with an A/G ratio of 0.60 (Smoes, 1966). In this study the mean egg laying values for the five normal adult female geese were 33.7 mg% calcium, 6.6 3% total protein consisting of 2.8 3% albumin and 3.8 3% globulin and an A/G ratio of 0.88 (Table 3). It appears that the goose has slightly higher calcium and total protein levels during the egg laying period than other Species. -Win3et and Smith (1958) determined that the serum calcium level of the laying Leghorn chicken was 21.5 to 28.1 mg%. Sendroy 25 a1. (1961) found that the laying pigeon exhibited a serum calcium level of 22.6 mg%. Baldini and Zarrow (1952) using 13 female bobwhite quail determined that the mean egg laying value for serum calcium was 29.3 mg% with a range of 23 to 40.2 mg%. Sturkie (1965) states that the total protein level for the laying mallard is 3.5 3%. This value is somewhat lower than the 5.6 mg% reported by Smoes (1966) and is consider- ably lower than the 6.6 mg% established for egg laying Canada geese in this study. Sturkie and Newman (1951) reported a total protein range of 4.6 to 4.9 3%, albumin range of 2.2 to 2.4 3%, globulin range of 2.5 to 2.7 3%, and a range for the A/G ratio of 0.84 to 0.93 for egg laying values in 18- month old White Leghorn chickens. In their study it was A7. A'J'XIV‘LWUKI!’ .I’" 11:" 53 stated that they used the biuret method for total protein and albumin analysis. This method reportedly produces more variable results than electrophoretic separation (Wood and Hofman, 1967). Brandt g; 31. (1951) found that the total protein level for the laying chicken was 5.4 3 0.71 3%. The discrepancies between reported total protein levels for egg laying periods are possibly explainable on differences {1 between Species and methods of analysis. SUMMARY Two exogenous estrogen experiments were conducted using different daily dosage levels of estradiol. Serum samples were collected from each treatment group prior to, during, and after the treatment period. In the first exogenous estrogen experiment, signifi- cant changes (P<.05) attributable to estrogen treatment were found for serum calcium and the A/G ratio. An analysis for differences among treatment groups on increasing dosage j levels was not significant (19.05). The lack of statistical ‘7 differences among the groups was attributed to the wide range of dosages (ZBBJug to 5 mg) that resulted in a great variability in group reSponse; further, the small number of degrees of freedom resulting from the use of mean values rather than individual sample values reducing statistical sensitivity. In the second exogenous estrogen experiment the range of dosage levels were reduced to 1253u8 to 1 mg. Mean treatment values for all five parameters measured were sig- nificantly different (P<.05) from-mean pre-treatment and post-treatment means. Significant differences (P<.05) among treatment groups for serum calcium, total protein, and glo- bulin were also obtained in this experiment. The increases 54 55 in total protein were attributed to an increased globulin fraction. This relationship resulted in a reduction in the A/G ratio. - In both exogenous estrogen experiments there appeared to be a correlation between increasing dosage levels and change in serum calcium, total protein and the A/G ratio. An average increase of serum calcium to 16.0 mg% or above, an average increase in serum protein to above 5.6 3%, and “in! JIY‘I!)AZn a general decrease in the A/G ratio to 0.8 or less due to the increased globulin and reduced albumin were attributed to estradiol stimulation. 3’ It also appears that there were differences in sensi- tivity to exogenous estrogen among the parameters measured. Calcium seemed to reSpond first, followed by increases in serum protein and a decrease in the A/G ratio. The rela- tionship between the sequence and degrees of change in the selected parameters resulting from estrogen stimulation can be seen more clearly in the endogenous estrogen experiment. Weekly blood samples were taken from ten adult, two two-year old and two one-year old female Canada geese from December 31, 1966 to April 28, 1967. Pre-season values for serum calcium, total protein, albumin, globulin and the A/G ratio were obtained from samples taken from December 31, 56 1966 to February 17, 1967. Pre-egg laying values for the same parameters were obtained from February 24, to March 17, and egg laying values were obtained during the period of March 24 - April 7. During the pre-egg laying and egg laying periods the adult geese were divided into normal, suppressed - egg lay- ing and suppressed - non-egg laying groups after observations - “91:13:” ,. on behavior and egg laying were obtained. During the pre—season period there were no significant differences (P>u05) among the five groups and values for all parameters measured were similar. The adult, one-year and two-year old geese could not be distinguished from one another on the basis of the peripheral blood constituents measured. Pre-egg laying values for serum calcium were different among the adult and one-and two-year old geese. The one- and two-year old geese did not demonstrate any signs of reproductive stimulation whereas the normal adult geese demonstrated a mean serum calcium level of 20.5 mg% - almost a th-fold increase over pre-season values. There were no obvious differences among the other four parameters measured during this period, indicating that serum calcium was the first parameter that showed a decernable change over pre- see th¢ ti! duc di: ad1 laj no: we: 01' C3 Si; ra de. th. th bl an ma Wa th 57 season (unstimulated) values. There was some indication that there may be a relationship between social status and the time and degree of stimulation at the beginning of the repro- ductive season. This statement is based on the apparent difference in mean serum calcium values between the dominant adults and the subordinate adult females during the pre-egg laying period . r The mean egg laying value for serum calcium in the normal adult female goose was 33.7 mg%. Suppressed adults were somewhat lower with 28.0 mg% and the one-and two-year old females did not show a significant increase in serum * calcium over pre-season values. During the egg laying period significant changes in total protein, globulin and the-A/G ratio occurred. The A/G ratio in the adult females did not decrease below 1.00 until approximately one week prior to the laying of the first egg. As in the exogenous estrogen experiments, it was apparent that the time, order and magnitude of changes in peripheral blood constituents are of prime importance in their use as an index to reproductive condition. The normal adult fe- males first demonstrated an increase in serum calcium. This was followed by an increase in total protein resulting from the increased globulin fraction and reduced albumin. This 58 in turn resulted in a reduction of the~A/G ratio. Suppressed non-laying adult females demonstrated some reproductive stim- ulation when calcium values increased, but the lack of in- creased total protein and the maintenance of an A/G ratio above egg laying levels indicated that they were not in an egg laying condition. In contrast to the increased beta globulin and reduced albumin of estrogen stimulation, the F? non-laying females tended to demonstrate increases in alpha globulin. Some increases in gamma globulin also occurred but they were not consistent. Immature geese did not demon- [a strate changes in the measured peripheral blood constituents. Cc De LITERATURE CITED Baldini, J. T. and M. X. Zarrow. 1952. Estrogen and serum calcium levels in the bobwhite quail. Poult. Sci. _3._];: 890-8% 0 Balham, R" W. 1950. The behavior of the Canada goose Branta candensis in Manitoba. Ph.D thesis, Univ. of Missouri, Columbia. 229 pp. Benoit, J. 1938. Role des yeux et de la voie nerveuse oculo-hypophysaire dans la gonadostimulation par la 3 lumiere artificelle chez le canard domestique. 1? Comp. Rend. Soc. Biol. ,igg:231-234. ‘ , G. Fabiani, R. Grangaud and U. Clavert. 1941. ‘i Suppression par la parathyroidectomie de 1'action hypercalcemiate du dipropionate d'oestradiol chez le canard domestique. Comp. Rend. Soc. Biol. 135:1606. ‘m: w. .- Bissonette, T. H. 1937. Photoperiodicity in birds. Wilson Bull. .42:24l-270. Brandt, L. W., R. E. Clegg and A. C. Andrews. 1951. The effect of age and degree of maturity on the serum protein of the chicken. J. Biol. Chem. 191:105-111. Burger, J. W. 1949. A review of experimental investiga- tions on seasonal reproduction in birds. Wilson Bull. Common, R. H., W. Bolton and W. A. Rutledge. 1948. The influence of gonadal hormones on the composition of the blood and liver of the domestic fowl. J. Endo- crinol. ‘§:262-273. Decker, E. H. 1965. A study of the fluctuation of some blood chemical constituents in relation to the repro- ductive cycle of the mallard duck. Unpublished master's thesis, Western Michigan University, Kalamazoo, Mich. V'+ 69 pp. 59 60 Fleischman, W. and I. A. Fried. 1945. Studies on mechanism of the hypercholesterolemia and hypercalcemia induced by estrogen in immature chicks. Endocrinol. .3§:406-415. Hanson, H. C. and R. H. Smith. 1950. Canada geese of the Mississippi flyway, with Special reference to an Illinois flock. Ill. Nat. Hist. Surv. Bull. ‘2§(3): 67-210. Hofman, W. F. 1966. The effects of estrogenic and gonado- trophic hormones on normal levels of Some blood chemical constituents in the non breeding female mallard duck. Unpublished master's thesis, Western {3 Michigan University, Kalamazoo, Much. vii + 88 pp. ' Landauer, W., C. A. Pfeiffer, W. U. Gardner and J. C. Shaw. 1941. Blood serum and Skeletal changes in two breeds of ducks receiving estrogens. Endocrinol. 'g§:458. Lorenz, F. W. 1954. Effects of estrogens on domestic fowl and applications in the poultry industry. Vitamins and Hormones. [12:235-275. Marshall, A. J. 1955. Hypothalamic control of pituitary in birds. Endocrinol. .4:75-93. Nalbandov, A. V. 1953. Endocrine control of physiological function. Poultry Sci. ‘3gz88-103. Polin, D. and P. D. Sturkie. 1958. The influence of the parathyroids on blood calcium levels and Shell de- position in laying hens. Endocrinol. 169:778-784. Rickless, M. S. 1967. Influence of some Stressors on peripheral blood reSponse to estrogen Stimulation in the female mallard duck. Unpublished master's thesis, Western Michigan University, Kalamazoo, Mich. vi + 84 PP- Sendroy, J., M. MacKenzie and H. A. Collison. 1961. Serum protein and calcium of pigeons during the reproductive cycle. Proc. of the Soc. for exp. Biol. and Med. 108:641-645. 61 Sherwood, G. A. 1966. Canada geese of the Seney National Wildlife Refuge. U.S. Dept. of Interior. Fish & Wildl. Serv., Mgt. Studies 1 & 2, Seney Natl. Wildl. Raf. Smoes, R. L. 1966. Blood constituent levels associated with transition into reproduction and during egg for- mation in the female mallard duck. Unpublished master's thesis, Western Michigan University, Kalamazoo, Mich. v + 72 pp. Smyrnios, N. G. 1967. A study of dietary affects on peripheral blood constituents of the female mallard PL under estrogen Stimulation. Unpublished master's thesis, Western Michigan University, Kalamazoo, MiCh. vi + 78 pp. Sturkie, P. D. and H. J. Newman. 1951. Plasma proteins of chickens as influenced by time of laying, ovulation, number of blood samples taken and plasma volume. in Poult. Sci. l39(2):240-248. ” . 1965. Avian physiology, 2nd ed. Cornell Univ. Press, New York. xxx + 766 pp. Urist, M., R. Schjieke and J. MCLean. 1960. The partition and binding of calcium in the serum of the laying hen and the estrogenized rooster. Endocrinol. [65:570. Welty, J. C. 1962. The life of birds. W. B. Saunders & Co. Philadelphia, Pa. Winget, C. M. and A. H. Smith. 1958. Changes in plasma calcium concentration during egg formation. Poult. Sci. .31:509. WitSchi, E. 1959. Endocrine basis of reproductive adapta- tions in birds. chap. 31, Comparative Endocrinology, ed. A. Gorbman. John Wiley and Son Inc., New York. Wolfson, A. 1952. Day length, migration and breeding cycles in birds. Sci. Month. [24:191-200. 62 . 1959. Photoperiodism and bird physiology, chap 31, Comparative Endocrinology, ed. A. Gorbman. John Wiley and Sons, New York. Wood, J. S. 1964. Normal development and causes of re- productive failure in Canada geese. J. Wildl. Mgt. _2§_(2) :197-208 . . and W. F. Hofman. 1967. Peripheral blood reSponse to reproductive stimulation in mallards. J. Wildl. Mgt. _3__1__(3) :546-554. APPENDIX A Tables of raw data and statistical values from First Exogenous Estrogen Experiment. 63 10 13 +2 +4 Sample Period (DaYS*) 64 globulin ratio, in the first exogenous estrogen experiment beginning on December 2, 1966 and (3%), albumin (3%), globulin (g%), and albumin/ terminating on December 23, 1966. Changes in serum calcium (mg%), total protein Dosage Bird Number Table l. 21 J. . .r .. PE.” 20 65 Continued Table 1. Sample Period (DayS*) Bird Number +4 13 +2 10 -2 Dosage on) 0000 Aren’t-u 29 ..... 5 mg 94 -2, 1 Pre-treatment values, 4-13 Treatment values, +2, +4 Post-treatment values * 66 0H.o w wN.H nouns .Emm o.m m H.m. N mq.o No.0 N qH.o oBHH QNN H N.m m m.m e w.N m o.m N Hw.o MH.o e um.o guano w.~ H casan< o¢.N w oh.mH nouns .emm w.m m e.N N ¢¢.m w¢.m N mm.oH oEHH m.¢ H ¢.N m N.N e ¢.m m N.m N :Hmuond Hw.H Ne.q e mm.NH macaw w.e H Hmuoa mm.mm w NN.w¢N nouns .SQm m.wH m 0.0m N «N.m ew.aw¢ N mo.mnm «SHE m.0H H o.mN m o.om d m.oH m N.NH N em.~ em.mH~ e NH.er muons m.HH H aSHUHmo SHDmHumum onmnmm Bouooum monSUm mocmHum> com: moHHmm naouw oHanHm> m new: mo mo mo maHH unmaumoua unopcmdoo moouwoa 85m moHumm , .ucmEHHodxm sowouumm maocowoxo umHHm can you mocmHum> mo mHm%Hmcm now open HmoHumHumum .mcmma UOHHSQ mum anouu .N mHan 67 .wuHhmH wwm u m mwdHhmH wwmuona u N ”acmmumnmum u H " oEHH .Hmohnmco u n ”amoku03u u d mwcHhmH wwmuco: vommmumdnm u m mwcHhmH wwm ummmoummnm u N ”pommouddfimcn u H “anouu No.0 w em.o nouns .aum N.H m o.H N Nw.w 00.0 N HN.H mBHH o.H H H.H m o.H e H.H m m.H N ee.~ HH.o e He.o muons H.H H o\< mw.Hn w Ho.mH Hound .Emm w.N m N.¢ N do.m mm.m N wn.¢H mEHH w.H H N.e m m.m q o.N m H.N N He.H eH.m e eeHHH asouo m.H H :HHsnoHu oHumHumum mumnmm Eopmmum mmnmswm oocmHHm> cmmz ©0Humm muouw, mHanum> m new: no mo mo maHH unoaummue ucmpcodmn mmmummn 85m mouuom eoseHucoo .N mHemH [Ft’ILK'L-I2t". :. I l APPENDIX B Tables of raw data and statistical values from Second Exogenous Estrogen Experiment. 68 69 Changes in serum calcium (mg%), total protein (3%), albumin (3%), globulin (3%), and albumin/globulin ratio, in the second exogenous estrogen experiment beginning on January 14, 1967 and terminating on February 4, 1967. Table 1. Sample Period (DaYS*) Bird +4 13 +2 10 -2 Dosage Number 04211.. .I. b apwlm nuTTA.U.A 1.. o r t n o no 8 no 49 1 apwmm numiAAnu.A g m 5 2 .I. 2 14312 1|. apwwm numiAnnb.A g m 5 2 l 7 I... 04312 .1 apwwm CTAGA co m 0 5 9. a, 70 Continued Table 1. Sample Period (DaYS*) Bird Number +4 13 +2 10 -2 Dosage .250 mg Ca 1 F27. ..... 14221 TP 5 81 ..... ..... 4 -2, 1 Pre-treatment values, 4-13 Treatment values, +2, +4 Post-treatment values *1: 71 No.0 w 0N.o Hound .emm e.N m o.N N mm.n «N.o N Ne.o oBHH o.m H m.N m c.N q N.N m o.N N Nm.o no.9 q NH.o macaw N.N H SHBDHH¢ No.0 w wH.o Houum .aum o.m m H.m N 0N.n NH.o N #N.o oaHH w.¢ H q.m m m.m q o.n m m.¢ N :Hououm ee.o~ He.o e mm.H muons e.e H Hence om.m w o¢.oN Hound .Emm N.¢H m H.mH N m¢.m om.mH N Hm.om maHH «.HH H N.NH m m.dH q N.NH m H.NH NN em.e mm.eH e mm.me ssouo H.HH H ssHono oHumHumum mumnmm aopmmnm mommamm mo:MHHm> com: poHHmm asouw mHanHm> .h emu: mo mo mo mBHH unmaummue ucovcmdoa mmmhwmn Bum mousom .ucmBHuodxm cowouumo msoaowoxm pcoomm on» How mocMHHm> mo mHmmHmcm How mump HmoHumHumum .mcwma poHHom new macho .N mHan 72 mwcHhmH wwmucoc hay . .c p .Ehn. F‘. L.!' .onmmH wwm a m mwchmH wwmuond n N ”commomuond u H u oBHH .nmmmumco n m unmohuosu u q pmmmmnmmam u m mwcHhmH wwm Ummmonmdnm n N mummmmndnnmcs u H ”anonw mo.o w N¢.o nonnm .amm N.H m H.H N mc.mH wo.o N om.H mBHH w.H H ¢.H m N.H e ¢.H m N.H N ~e.~ mH.o e om.o muons H.H H o\< No.0 w mm.o nonnm .aum ¢.N m m.N N Hm.oH om.o N oo.H mane w.H H m.N m o.N e N.N m N.N N wH.m mm.o e um.H ssono H.H H eHHsnoHo SHDmHumum onmsmm Eovomnm mmnmSUm moannm> cmmz vOHnmm muonw mHQMHnm> m one: «0 mo mo maHH ucoaumonH ucmucmdmn moonwon anm monnom emsenusoo .N mHnmn APPENDIX C Tables of raw data and statistical values from the Endogenous Estrogen Experiment. 73 C) 4/7 b) 3/3 3/10 3/17 3/24 3/31 Sample Dates 2/9 2/17 2/24 11.0 11.8 11.9 11.8 11.3 10.6 11.4 13.3 14.1 17.7 31.6 globulin (3%), and albumin/globulin ratio, in unsuppressed adult female 12/30 1/6 1/13 Changes in blood serum calcium (mg%), total protein (3%), albumin (3%), Canada geese. Ca Bird Table 1. Number [\q-m mmq 1.3 1.5 1.7 1.2 3 3 0.7 3.27 1.87 2.11 1.91 1.34 2.62 2.81 2.31 1.56 1.72 0.80 0.87 1.7 1.3 1:1 1.6 1.6 Alb Glb A/G [\m 06! 23.8 25.6 33.6 32.6 (“QB O O O HMN u—le whecu 0H0 \‘I'N 11. [\N \‘I'N 11.1 11.1 4.7 3.4 8 4 11.6 4. 3. Ca TP Alb d1 N'rln 54» 0 0 O C>dwN .H 005$ 2 5 .2 2 Ca TP Alb Glb A/G 10 1.9 2.2 1.5 2.2 2.1 2.3 1.5 2.8 2.7 1.32 1.52 1.51 2.20 1.23 1.10 1.10 2.21 1.09 1.31 0.73 .2 4/7 b) 3/3 3/10 3/17 3/24 3/31 a) 2/9 2/17 2124 12/30 1/6 1/13 ~Continued Bird Table 1. Number 75 N common wNO‘MO 0&0ch \DNNd’O m In 0‘ N OOO‘q-lln \OMNu—Im wet-HMO NCON\OO ('0 0') <1 In \OHOHQ’ dNONO‘ O‘LHMNu—I N©MMO H \1' 0‘ O 'U dMNOO OInMNl-n O o o o o o o o o o o H cn~¢ch4H4 o~quaolHl n .m« H N q; . 0.. H w NowNM 50‘an DD 0 o o o o o o o o o a Nd‘Nu—IN wd'Mq—lu—l q-l .—I H >s CU H 00 1-1 OO‘OMu—l Hooowo OD MMNv—IN d'x'fMNr-d b0 F' M 0‘ O A Gone-4N Md'u—lmm O OMNu—QN Mer-JN "U '4 o 0H \‘I' 00 H auwl\.41n r~c>n4oxuw g1 @MNHN HWMHI—I H 00 I: N O «4 \‘I'ONNM \DanO‘N a" OMNe—lq—l v—ld't'fiv—Iv-I H H H DD u—I H 00 [\BNWN olenN Q) o o o o o o o o o o | Od’Mr-lN Hx'l'Mu—IN Q) H H H 9.. w \D \TNInNd' oomoomoo 3 Od'Nu—lw—l O‘ql-Np-IH 'U 0 N 'l" Gamma Mr-lv-lfiw H o o o o o o o o o o o r-lde-‘N :IDMI-lv—l D: C. 43.00 .030 8 COD-oHv—l\ (GnuHr-I\ cu (JE4< oxc>ono erathH ox ¢>¢H3r\ unawohH .4 ¢>rnncn l\~¢Cfir4 77 0‘ Nomi-1H O‘MNu—IN O‘ wNI-HNN Nd’mI-JN 1—1 N OOQQQ u—Id'Nu-h—l N O NN\O\OO Ohmmv-I O Ommq-o QOMMu—l N O ooocoOH ¢mNmFl N H \OHHOv-l 003;“)?! N m ®l\.OO\¢—l DAMN; N NO‘OO‘O \Od’Mv-h—I H WONNO «neamew .4 <1- «Mncqaun Obscwpwu H awrnname HutchHra .H O wr-lfid'o OdNr-IN H N r-ld'I-nO‘m u-ld'Nu-lr-l H 0') coma-Ho u—ld‘NNN v-l iodc) Suppressedperiod d) After release ing per re-season period b) Pre-egg lay ost-season P 3— P Aw “UN \ <7 AA H NO‘ N 59“” 1; «6 1: \1' with H 800 \ 5v q- .0 H'U AN (Gr-1 0\ 0 <1 AA: 83+J .4 00!: m E m 1: '“SE 8 23 0.) 4.41m V oooqa m M Q) 9.1360 Ah H ,DH H (U \ (U “'0 «1 U0“) (0 0H1: 0 O “‘88 8 .1 “H D W A Q) as fled o co OOH.“ H \ BHE CL M v.60 E .094 Cu 50 U) \1 HA N “FIDO” \ 0\oo N HG (fin-let} AN 05 (OH H \1 5.000 N Hv 1‘0 0‘ 0 “U \ CD'UH N GO '0‘!) O .C.‘ m o «4.) H HA1: \ .DNO H 608 HV \0 0 d’ \ 'HGN H .H. CDHO m DCV C> 5.0 fl) HO'U V CUHC‘. N >bocc H ‘11.3 11.9 Table 4. Bird Number 35 Ca 5 3 9 4. 1.25 1.70 1.27 1.61 2.21 2.10 2.21 2.11 1.82 2.10 2.11 - 78 I-ll\ [\N \Od’ asp: .scn NO‘ \‘fN l‘x‘l’ .acn 2 9 2.1 1.3 1.3 1.3 1.1 2.0 1.4 .3 12.1 12.1 11.2 15.8 12.9 12.1 1.41 1.62 1.69 1.19 1.21 2.14 2.61 2.32 2.81 2.52 1.91 2.50 - 0 4 2 7 6 5 0 4 2 11.9 11.3 12.2 12.2 1 89 Ca 1.3 TGICmLCCC .fi QHDQH Continued Table 4. Sample Dates d) b) 12/30 1L6 1/13 2/9 2/17 2/24 3/3 3/10 3/17 3/24 3/31 4/7 4/14 4/21 4/28- Bird Number 4 2 5 5 3 6 8 4 2 6 0 4 3 9 3 .5 12.2 11.8 13.8 16.2 32. 4 3 O‘MN OMN u—l NMl-n r-ld'N q—l d'x'TI-i r-i\'l'M u—i 0mm H [\H HN \O wln I-IH H mm v—IH O \OO HN N mm HN O \OO Hui-I m O‘H . . HI—l H 0‘” OH H [\N HH N \Od’ HH M 0000 C . r-II-I H mm . . HN \O NH . O NH m l-no . C I—‘N HH NH .09 H\ U< 11.6 11.0 10.9 11.1 11.0 10.5 16.2 11.9 10.1 9.8 13.6 14.5 13.1 88 Ca rxtn ~$c6 Nl\ emu: voHnom 050nm oHHmHnm> m - can: mo mo mo mBHH unmaummne ucmvcmdmn moonwmm aDm monnom .ucmEHnmdxm cowonumo msocmwovcm osu now mocmnnm> mo mHahHmcm now mumv HmoHumHumum .mcmma moHnmd 0cm mnono .m mHan 81 .wcHhmH wwm u m mwcHhmH wwoumnm n N mcommwmuonn u H “ oEHH .nmmhumao a n mnmmmuoBu u q mwcH%mH wmmucoc mmmmonQQSm um MwSHmmH wwm 00mmmn005m IN ”commandmamc: u H "030nm HN.0 m 00.H nonno .Emm m.H m 0.N N 0¢.H Nm.0 N No.0 oBHH N.H H N.H m 0.N e N.H m 0.H N H4.o mo.o e oe.o asono m.H H e\< 0m.0 w 0H.m nonnm .amm m.N m ¢.H N 0m.m N¢.H N «0.N mBHH 0.H H 0.H m 0.H e 0.H m m.N N No.H oe.o e He.H muons H.N H cHHsnoHo SHumHumum oanUm Eommonm mmnmmmm mocmHnm> any: moHnom muonw mHannm> m can: mo mo mo mEHH ucoaummnH ucmmcmmmn moonwon 83m monaom poDcHucoo .m mHHMH "I7'11111111111111ITS