THE EFFECT or PHOTOPERIOD AND THYROID ACTWITY ON GROWTH 0? ma (mar; swam mews SYANELLU§ (RAFINESQUE) Thesis {or the Degree of M. 5. MICHIGAN STATE UNIVERSITY Willard Louis Cross 19 6 2 -_—._ LIBRARY Michigan State University ABSTRACT THE EFFECT OF PHOTOPERIOD AND THYROID ACTIVITY ON GROWTH OF THE GREEN SUNFISH LEPOMIS CYANELLUS (RAFINESQUE) by Willard Louis Gross The effect of photoperiod was evaluated in four independent experiments, three of which were performed in conjunction with the effect of thyroid activity. The fish were held in light-tight aquaria at constant temperatures under four photoperiods ‘(8-hour constant, 16- hour constant, variable increasing photoperiod 8 to 16 hours, and variable decreasing photoperiod 16 to 8 hours). Increment in weight and total length, rate of food consumption, and efficiency of food con- version were determined. Results of the first four experiments demonstrated that photoperiod does have an effect upon growth in both length and weight of the green sunfish. Generally, a greater growth occurred in fish held at longer photoperiods. Significant differences in growth in terms of weight gain occurred in two experiments. Growth in weight was better associated with photoperiod than was total length. Significant correlations between the rate of food consumption and growth at given photope riods indicate that photoperiod mediates its effect through increased appetite of the fish. Food conversion was generally most efficient under an increasing photoperiod and least efficient under a decreasing photoperiod. I Results of the four experiments demonstrated that varying photo- period has a greater effect upon growth than a constant photoperiod. An increasing photoperiod (8 to 16 hours) stimulated [growthabove that Willard Louis Gros s for a constant 16-hour photoperiod and a decreasing photoperiod (16 to 8 hour) depressed growth below that of a constant 8-hour photoperiod. Differences in growth of fish given injections of artificial thyroxine (hyperthyroid), radioiodine (hypothyroid), and saline solution (control) demonstrated an effect of thyroid activity on growth in length and weight. Hyperthyroid fish attained the greatest growth and hypo- thyroid fish the least. The differences between the thyroidal groups were not statistically significant, but consistent results were obtained in the experiments. Thyroid activity had no effect on food consumption, but an increased efficiency of food conversion was generally associated with greater thyroid activity. Using the rate of loss of radioiodine from the head region as an index of thyroid activity, an effect of photoperiod on thyroid activity was demonstrated. Greater thyroid activity was associated with short photoperiods and low thyroid activity with long photoperiods. THE EFFECT OF PHOTOPERIOD AND THYROID ACTIVITY ON GROWTH OF THE GREEN SUNFISH LEPOMIS CYANELLUS (RAFINESQUE) By Willard Louis Gros s A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENC E Department of Fisheries and Wildlife 1962 6 ’ Wig/w. Wt“ ACKNOWLEDGMENTS The writer wishes to extend his sincere appreciation to Dr.- Eugene W. Roelofs and Dr. Paul 0.. Fromm under whose guidance this study was undertaken and without whose assistance and encouragement it would not have been possible. He is also indebted to Dr. Philip J. Clark for his advice on statistical analyses, to Dr. Jack R. Hoffert for his advice and help in many areas, and to his fellow graduate students, particularly Darrell King, for their suggestions and help. The writer also wishes to thank his wife, Lorraine, for her patience and understanding throughout the period. The writer's participation in the study was made possible by a Research As sistantship made available through a- National Science Foundation Grant (G-10757). *>l<********>l<* ii TABLE OF CONTENTS Page INTRODUCTION ........... . ....... . ..... 1 Seasonal Growth Patterns in Fish ......... . . . . 1 Factors Influencing Growth . . ...... . ....... 2 Effect of Temperature on Growth ...... . . ..... 2 Effect of Photoperiod on Growth ...... . . ..... 3 Endogenous CyCles of Growth of Fish ........... 4 Role of the Thyroid Gland and Its Relation to Growth in Fish .................... . . . . 5 Effect of Photoperiod on Thyroid Activity ........ 7 Summary . ................. . ...... 8 METHODOLOGY ......................... 9 Experimental Fish ............ . . . . . . . . 9 Aquaria and Accessories . . . . ............. 10 Control of Photoperiod .................. 10 Control of Temperature .................. ll Thyroidal Conditions .......... . . . . ..... 12 Pre-experimental Acclimation and Conditioning ..... 14 Physical Measurements .......... . ....... 15 Food and Feeding Routine ................. l6 Histology .......................... 18 Mortality and Disease ................... 18 Determination of Thyroid Activity . . . . ........ 19 RESULTS AND DISCUSSION ................... 22 Effect of Photoperiod ................... 22 Results of Increment in Weight ........... 22 Results of Increment in Total Length ........ 27 Results of Growth Expressed as a Percentage . . . 31 Effect of Photoperiod on Growth in. Length and Weight. . . ................. 36 Effect of Varying Photoperiod on Growth. . . . . . 38 ~ Effect of Prior History of Photoperiod on Growth . 40 iii TABLE OF CONTENTS - Continued . Page Effect of Photoperiod on the Seasonal Growth Cycle of Fish ................. 41 Effect of Photoperiod on Food Consumption . . . . 42 Effect of Photoperiod on Efficiency of Food Conversion .............. . . . A . 45 Effect of Thyroid Activity ................. 50 Results of Increment in Weight ........... 50 Results of Increment in Total Length ........ 53 Effect of Thyroid Acticity on Growth in Length and Weight............ .. 53 Effect of Thyroid Activity on Food Consumption. . 61 Effect of Thyroid Activity on Efficiency of Food Conversion ........ . . . . . . . . . 62 Effect of Photoperiod on Thyroid Activity ..... . . . 64 Comparison of Thyroid Activity of the Green Sunfish withOther Fish ........... . . . . . . .. . 74 SUMMARY .‘ ........................... 77 CONCLUSIONS .......................... 77 LITERATURE CITED ...... . ............... 79 APPENDICES .......... . ...... . ........ 84 iv LIST OF TABLES TABLE . The size range and mean total length and weight of the fish at the beginning of each of the experiments ..... . Dates, median temperature and maximum deviation in temperature between aquaria for each of the experi- ments ..................... . . . . . . . Results of a statistical analysis of the differences in the mean increment in weight of a fish over the entire experiment ........ . . . ...... . . . . . . . . Percent gains or losses in length and weight for the second three weeks compared to the first three weeks for all photoperiods of each experiment. . . . . . . . . . Average increment in weight and total length of fish under each photoperiod for the third three-week period of the fourth experiment ................. . The average rate of food consumption (percent body weight) of fish under each photoperiod of each six-week experiment . . . ................ . . . . Rates of food consumption (expressed as percent body weight) for the first and second three-weekperiods of increasing and decreasing photoperiods in each experi- ment . . . . .............. . ........ .. Coefficients of correlation between rate of food con- sumption and average increment in weight and total _ length per tank of fish in each six-week experiment . . Average efficiency of food conversion (percent) for each photoperiod of each experiment over the entire experiment, and the rank of the values in order of de- creasing efficiency for each experiment. ........ Page 10 12 25 39 41 43 44 45 46 LIST OF TABLES - Continued TA BLE 10. 11.. 12. 13. 14.. 15. 16.. 17. Efficiency of food conversion (percent) for the first and second three-week periods of each photoperiod in each experiment ............... . . . Coefficients of correlation between efficiency of food conversion and average increment in weight and total length per tank of fish in each six week experiment The average rate of food consumption (percent body weight) of a tank of fish under each thyroidal condition of each six-week experiment .......... . . Average efficiency of food conversion (percent) for a tank of fish under each thyroidal condition of each six- week experiment .................... Regression coefficient and output halftime of each fish and the mean value for each photoperiod ........ Regression coefficients (2) for each fish of the control and hypothyroid groups under each photoperiod in the second experiment ..... . . . . . . . . . . . . Mean regression coefficients (2) and output halftimes for control fish of the 8-hour and 16-hour photoperiods at the beginning and the end of the second experiment . Average output halftime for control fish under each photoperiod at the end of the second experiment . . . vi Page 47 48 61 63 65 69 73 76 LIST OF FIGURES FIGURE 1. Average increment in weight (grams) of a fish under each photoperiod of the four experiments ........ . Average increment in total length (millimeters) of a fish under each photoperiod of the four experiments . . Growth in weight of a fish expressed as the percent gain of the average initial weight of a fish under each photoperiod ........................ .. Growth in total length of a fish expressed as a percent gain of the average initial length of a fish under each photoperiod ...... . . . . . ..... . .- . . . . . Average increment in weight (grams) of a fish under each thyroidal condition of the three experiments . . Average increment in total length (millimeters) of a fish under each thyroidal condition of the three experi- ments . ....................... . Average. index of thyroid activity of the control and hypothyroid groups under each photoperiod of the second experiment . . . . ......... . . . Semilog plot of the output of radioiodine from the head region of green sunfish maintained under constant 8-hour photoperiod and 16-hour photoperiod for two weeks ........................... . Graph of the average regression coefficient of the con- trol fish and hypothyroid fish under each photoperiod of the second experiment . . . . . . ..... . . . . vii Page 23 28 32 34 51 54 58 66 7O APPENDIX A. LIST OF APPENDIC ES Page Mean measurements and increments in total length and weight of fish in each tank of the first three experiments. Initial total length and weight and increment in total length and weight of each fish of the fourth experiment ................ 85 Rates of food consumption (percent body weight) for each tank of fish for each three-week period and the six-week period of each experiment . . . . 91 Efficiency of food conversion (percent) for each tank of fish for each three-week period and six-week period of each experiment ...... . . . . . . . 96 viii INTRODUCTION The objective of this investigation was to obtain a more precise understanding of the effect of photoperiodicity and thyroid activity on growth of fish. The experimental animal was the green sunfish Lepomis cyanellus Rafine sque . Seasonal Growth Patterns in Fish Growth in length and weight of fish is continuous throughout their life, the rate of growth declining with age. . However, this growth occurs at a seasonal rate on an annual basis in many fish. Seasonal growth patterns have been found in yellow perch (Langford and Martini, 1941), river carpsucker (Bucholz, 1957), warmouth (Larimore, 1957), white- sucker (Spoor, 1938), bluegill (Anderson, 1959; Beckrnan, 1943; Sprugel, 1954), smallmouth bass (Brown, 1960), rock bass (Brown, 1960),. brook trout (Cooper, 1953; McFadden, 1961), and green sunfish (Hubbs and Cooper, 1935). The period of maximum growth varies slightly but, in general, growth is greatest from April to July, declines slowly during August and September, drops rapidly the latter part of September and October, and almost ceases during the period November to March. Swift (1955;1961) found a slightly different annual growth cycle in 3-year- old and yearling brown trout in England. The fish had a maximum growth rate in spring (May to June) and usually a smaller increase in growth rate in autumn (September and October) with a low growth rate during winter and mid- summer. These studies indicate that growth bears a definite relationship to the seasons. Factors Influencing Growth The regular variation in the growth patterns of different species is probably due to rhythmic changes in environmental factors. Several environmental factors listed by Brown (1957) as affecting growth are temperature, light, chemical factors, space factors and availability of food. Chemical and space factors vary throughout the year, but these changes are usually very minor and would not account for the repeated annual growth cycle each year. Availability of food can influence growth if the supply of food becomes critical at times. However, this situation does not affect most fish with great regularity under natural conditions. The two factors which appear primarily responsible are temperature and light (photoperiod) or an interaction of these two factors. Seasonal temperature changes are nearly in phase with changes in photoperiod in temperate regions making it difficult to evaluate these effects individually under natural conditions. Effect of Temperature on Growth Since fish are poikilothermous animals, one would expect that temperature would affect their growth. . Literature referring to the effect of temperature on growth of poikilotherms is reviewed by Pickford and Atz (1957). There appears to be an optimal temperature for growth above or below which there is a decline in growth. An exception is the brown trout for which Brown (1946b) and Swift (1955) found two Optimal temperatures. . From his studies on the effect of temperature on the seasonal growth rate of the bluegill, Anderson (1959) concluded that temperature was the primary factor influencing seasonal growth. 7 Swift (1959; 1961) also concluded that temperature was the primary factor influencing the seasonal growth rate of brown trout. Temperature is believed to affect the growth of fish by influencing food consumption and metabolism, especially respiration and digestion. Temperature may not be the only factor affecting the seasonal growth of fish. If the theory of an optimal temperature were true, one would expect an increased growth rate in fall when the optimal tempera- ture is again reached. This does occur in brown trout (Swift, 1961) but is not generally true for other species. In a. laboratory study under constant temperature and variable photoperiod corresponding to different seasons of the year, Anderson (1959) found a variation in the potential growth of the bluegill which was independent of temperature. The great- est increments in length at each temperature coincided with a photo- period comparable to that for the period of May 30 to June 28 for Michigan. Although he had no direct evidence, Anderson suggested that this difference involved an endocrine mechanism. Effect of Photoperiod on Growth The effect of photoperiod on the growth pattern of fish has not been adequately studied. Much of the difficulty lies in the similarity in variations of temperature and day-length. Most studies have involved only the effect of constant photOperiod on growth. Brown (1946a) found that in brown trout kept at 11. 50 C. , under standard conditions, the average specific growth rates were significantly lower with 12 or 18 hours of "standard light than with 6 hours of light per day. Bjorklund (1958) studied the effect of photoperiod on growth of goldfish. There was no significant difference between the growth of fish held in darkness, constant 10-hour day, and on what he termed a constant 16-hour day which began with a ll 3/4-hour day decreased to a 10-hour day over a period of 11 days and then increased in 15 minute increments over a period of 23 days to a 16—hour day where it remained constant for a period of 38 days. The greatest increments in length and weight occurred in fish held in total darkness. Growth was inversely related to photoperiod which Bjorklund felt was due to differences in the activity of the fish. . Eisler (1957), in studies of the influence of light on early growth of Chinook salmon, found a significantly greater growth in fish reared at different light intensities than those reared in the dark. Anderson (1959) performed an experiment on the effects of photoperiod using two groups of four fish each; the first group was held on a constant 15'- hour photo— period and the other group on a constant 10-hour photoperiod for a period of six weeks. No significant differences in gain of length or weight were found; although, the average values were slightly greater for the short day group. Swift (1955) suggested that the seasonal growth rate might be better correlated with photoperiod than temperature; however, he later found no relationship between photoperiod and the seasonal growth rate of brown trout. In these studies, the effect of a varying photo- period upon growth was not considered. Swift (1961) states that it is important to try to distinguish between the effects of day to day changes in photoperiod length and the effect of the length of the photoperiod when it is constant from day to day. All other studies of photoperiod have involved its effect upon maturation of gonads or temperature resistance in fish. _ Endogenous Cycles of Growth in- Fish One investigator suggests that an endogenous seasonal growth pattern may exist. Brown (1946a) maintained brown trout for more than a year in a constant environment (11. 50 C. , constant 12-hour photo- period) and found a cycle of seasonal growth and maturation of the gonads at the same time of year as under natural conditions. The seasonal growth rate decreased to a minimum in October and November, rose to a maximum in February, fell gradually throughout the summer to August, and then decreased markedly. This pattern is somewhat similar to that found by Swift (1955). Further studies by Swift (1961) have re- vealed a slightly different seasonal pattern. 7 He discusses the work of Brown and concludes that an endogenous cycle does not exist. Role of the Thyroid Gland and Its Relation to Growth of Fish Despite the large amount of research on the thyroid of poikio- therms, the role of the thyroid gland in fish is still not clearly understood (Gorbman, 1959). The literature has been reviewed by Gorbman (1959), Lynn and Wachowski (1951), and more fully by Pickford and Atz (1957). Thyroid activity or thyroid hormone in fish has been associated with sexual development, Iosmoregulation, calcium and phosphorus metabolism, fin regeneration, development of epidermis and subcutaneous tissue, and transformations such as the smolt transformation in salmonids and meta- morphosis in the lamprey. The control of maturation by the thyroid and the synergistic action between thyroid hormone and growth hormone is well-known in mammals. Several investigators have attempted to show a similar role in fish. Much controversy exists in the literature as to whether the thyroid exerts a calorigenic effect. Investigations of thyroidal function in fish, have involved a variety of methods such as injection or immersion in water containing anti-thyroid drugs, injection or feeding of thyroxine or thyroid powders, radiothyroidectomy, or simply a comparison of groups in different environments. It appears that the contradictory results obtained may in part be due to the variation in methods. Recent investigations not reviewed by Pickford and Atz afford evidence of an effect of the thyroid on growth. Barrington, Barron and Piggins (1961) noted an increased growth of rainbow trout given thyroxine and thyroid powder. Three inde- pendent experiments by Bjorklund (1958) gave conflicting results. One experiment involving injections of thiourea and of thyroxine retarded growth in both instances. In two other experiments, transitory increases in length and weight were observed in fish injected with thyroxine and also in fish injected with triiodothyronine. Bjorklund felt that the negative results of his first experiment were the result of use of pharmaceutical doses and concluded that the thyroid had a calorigenic effect. Hoar (1952) suggested that the iodine content of the water may limit growth and repro- duction in populations of fish. He cites, as an example, the alewife, a marine species, which has become landlocked in the Great Lakes. The freshwater fish is only a little more than one-half the size of the marine relative; the thyroid is extremely hyperplastic or occasionally atrophic compared with the thyroid of the marine relative. Seasonal fluctuations in thyroid activity have been studied by several investigators. Seasonal variations have been found in the minnow Phoxinus (Barrington and Matty, 1954; Fortune, 1955), brown trout (Swift 1955; 1959), salmon parr (Hoar, 1939) and the killifish Fundulus (Berg, Gorbman and Kobayashi, 1959). Periods of peak activity seem to vary with species. In the above investigations, the results were correlated with spawning activity. Seasonal fluctuation in thyroid activity has not been extensively studied with respect to growth. Swift (1955) found that the peak activity of the thyroid corresponded to maximum activity of trout. He suggested that the increased locomotor activity of the fish led to an increase in the amount of metabolites needed for mainte- nance which reduced the amount available for growth thereby reducing the growth rate. In the investigations cited above, the peak activity of the thyroid, or one of the peaks where two peaks were observed, occurred in spring. This is the usual period of maturation of gonads in fish which are predominently spring spawners. However, this is also the period of rapid growth for many species. The maximum thyroid activity observed in yearling trout (immature) cannot be accounted for by maturation of the gonads. These results plus those cited previously dealing with the influence of thyroid hormone on growth indicate a possible effect of the thyroid gland on the seasonal growth pattern. . Effect of Photoperiod on Thyroid Activity Another aspect of thyroid physiology involves factors affecting thyroid activity. Leloup and Fontaine (1959) state that among the numerous ecological and ethologic factors able to influence iodine metabolism in lower vertebrates are genital maturity, nutrition, environmental concen- tration of iodine, salinity, season, photoperiod, amphibiosis and desic- cation. These factors have been investigated to varying extents. The effect of photoperiod has not been extensively investigated. Several investigations have demonstrated an effect of light on thyroid activity, although the results at times are contradictory. Buser and Blanc (1949) as quoted by Pickford and Atz (1957) found no effect of continuous illumi- nation on thyroids of Ameiurus nebulosus. Rasquin (1949) found that the thyroid of Astanax mexicanus undergoes hypertrophy when the fish are kept in total darkness. 7 Further studies on this species by Rasquin and Rosenbloom (1954) demonstrated that the hypertrophied thyroids of fish exposed to darkness could be restored to normal by returning the fish to light. More recently, Robertson (1958), as quoted by Hoar (1959), found a slightly greater uptake of radioiodine in goldfish maintained on a short- day basis (8 hours of light) compared with those maintained on a long day (16 hours of light). Baggerman (1959), in studies of the migration of juvenile coho salmon, found that day-length influenced the time of induction of the migration disposition. Fish held under an 8-hour day retained a preference for freshwater which was associated with a low level of thyroid activity since thiourea treated fish responded similarly. Fish held under a 16-hour day exhibited an earlier change in preference for salt water than fish held under an 8-hour photoperiod. This change in preference was associated with a high level of thyroid activity since thyroxine=treated fish responded similarly. In studies of the seasonal production of thyroxine by the thyroid, Berg e_t £11. (1959) suggest photo- period as a possible mechanism of thyroid control. . The pattern of production appeared directly related to photoperiod although this effect was not tested separately. These results lead the investigator to believe that a possible interaction may exist between photoperiod and thyroid activity which has an effect upon growth. . Summary A review of literature indicates that the effects on growth of photoperiod and thyroid activity or their interrelationship are unsettled problems. Few of the past studies involved an extensive investigation of the effect of photoperiod, and no studies involved a measurement of the effect of a varying photoperiod. An analysis of the role of the thyroid gland frequently involved the use of anti-thyroid drugs which are known to have toxic side effects. Some reported effects of the thyroid on growth most probably represent the response to pharmaceutical rather than physiological doses. . In this study, an effort was made to hold constant such variables as temperature, availability of food, and space factors in order to determine more accurately the effect of photoperiod and thyroid activity on growth of fish. . This study represents the results of four separate experiments. Each of the first three experiments involved a combined study of the effect of photoperiod and thyroid activity. The fourth experi- ment involved the use of only "normal" fish to further assess the effect of photoperiod. In conjunction with the second experiment, a study was made of the thyroid activity of the fish using the rate of loss of radio- iodine from the head region as an index of thyroid activity. METHODOLOGY Experimental Fish The experimental animal used throughout the study was the green sunfish. Lepomis (lanellus Raf. This species was selected because it is readily accessible, well adapted to aquarium life, and is believed to have a seasonal growth pattern. A seasonal growth pattern was indicated by the work of Hubbs and Cooper (1935) who showed the presence of a spawning mark on the scales of the green sunfish which was much nearer the following winter annulus than the preceding one. The fish were obtained from two sources. Fish for the first experiment were obtained from Burke Lake in the Rose Lake Experiment Station, Clinton County, Michigan. . Fish for all the other experiments were obtained from the private ponds of Dr. Peter Tack,. Clinton County, Michigan. . Fish ranged from 2 to 4 years in age, as determined by scale reading. Both mature and immature fish were used. The size range varied slightly in each experiment depending upon the size of fish captured prior to the experiment. The size range and the mean total length and weight of the fish in each experiment are given in Table 1. In each of the first three experiments, 120 fish were used. Four photoperiods were included in this study. There were 10 fish in each thyroidal condition (hyperthyroid, hypothyroid, and control) under each of the photoperiods. The fourth experiment involved only 60 fish, 15 fish under each photoperiod (5 in each tank). All fish were distributed on a random basis. 10 Table 1. The size range and mean total length and weight of the fish at the beginning of each of the experiments. Expt. Total Length (mm.) Total Weight (gm.) Range Mean Range Mean 1 68- 110 89.1 5.2-23.4 13.5 2 82-110 96.4 7.6-25.3 16.6 3 81-98 89.1 7.5-16.6 11.6 4 89 - 103 89.8 9.0 - 20.4 12.8 Aquaria and Accessories Four light-tight aquaria of approximately loo-gallon capacity were used in the study. Each aquarium had a separate filtering apparatus using glass wool and activated charcoal and having a filtration rate of 60 gallons per hour. Each aquarium represented one of the four photo- periods used in the study. Each was subdivided, by screen dividers, into three compartments (designated hereafter as tanks) approximately 15 by 21 by 12 inches. The water supply was tap water from the wells of Michigan State University. . Control of Photoperiod The four photoperiods used in this study were assigned to the aquaria on a random basis. The photoperiods given below were used in the first three experiments: (1) a constant 8-hour photoperiod throughout the experiment; (2) a constant 16-hour photoperiod throughout the experi- ment; (3) a variable photoperiod increasing from 8 to 16 hours; and (4) a variable photoperiod decreasing from 16 hours to 8 hours. An experi- ment lasted six weeks. In the aquaria having variable photoperiod, 11 changes in day-length were made at the rate of 1 hour over a 5-day period. This was accomplished by changing the dayalength 15 minutes on the first and second day, making no change the third day, and then changing the day-length 15 minutes again on the fourth and fifth day. The lights were controlled by 24—hour timers. The fourth experiment differed slightly from the first three. A regular six-week experiment was performed using the above photo- periods; at the end of the six weeks, the experiment was continued for another three weeks holding the variable photoperiods constant at the day-length that they finished the first six week period. This was done so that a comparison could be made between the growth of fish held at the two constant photoperiods throughout the experiment and the growth of fish at similar photoperiods but having a prior history of a varying photoperiod. The light source in the first experiment consisted of two 25-watt frosted bulbs placed at each end of the aquarium. This did not provide an even distribution of light to all tanks and also caused a heating problem. Therefore, a three-foot fluorescent fixture, with a 30—watt cool white tube, was installed overhead in the hood covering each aquarium. The fixture was covered with translucent polyethylene. This resulted in a more even distribution of light in all tanks of the aquarium and was used in all other experiments. Control of Temperature The temperature of the aquaria was maintained as nearly con- stant as possible by adjustment with cool or warm water in the daily cleaning routine. The temperatures in the aquaria varied closely with the temperature in the laboratory. As a result, temperatures at which the four experiments were run differed slightly depending upon the season of the year. The median temperature for each experiment and 12 the maximum deviation between aquaria is given in Table 2. Table 2. Dates, median temperature and maximum deviation in temperature between aquaria for each of the experiments. Expt. Dates Median Temp. Maximum Deviation Between Aquaria 1 8/24/60 - 10/4/60 78° :1: 4° F.1 2.0° F. 2 7/13/61 - 8/22/61 770140 F. 1.50 F. 3 9/23/61 .. 11/3/61 77°i3° F. 2.00 F. 4 2/10/62 -. 4/12/62 740120 F. 1.00 F. 1Variation from median during experiment. Heaters were installed in the aquaria for the'third and fourth experiments to maintain the water temperatures above the laboratory temperature and comparable to the previous experiments. Temperature variations between the aquaria were infrequent, small and never directional;one aquarium was never consistently higher than another in any of the experiments. Thyroidal Conditions There were three thyroidal conditions used in the experiment, each of which was assigned to one of the tanks of an aquarium on a random basis. An attempt was made to produce a hypothyroid condition by radio- thyroidectomy of fish with carrier-free radioiodine (1131). . Fish in the first experiment received a single intraperitoneal injection of 100 micro- curies (volume .05 cc.). . Fish in the second experiment also received 100 microcuries given in two injections, of 50 microcuries each, into 13 the peritoneal cavity, one week apart. In the third experiment, the fish were given a single intraperitoneal injection of 200 microcuries. Thyroid follicles were present in the lower jaw of fish of the second experiment. Therefore, the dosage was increased in the third experiment. Thyroid follicles were also found in fish of the third experiment. Using histological criteria, no difference could be demon- strated between the control and thyroidectomized fish in both the second and third experiments. No thyroid follicles were found in fish from the first experiment, but there is some question whether complete thyroidectomy had been obtained in view of the findings of the second and third experiments. An evaluation of the hypothyroid condition was made on the fish at the end of the second experiment using the rate of loss of radioiodine from the head region as an index of thyroid activity. The results of the study demonstrated an inhibition of thyroid activity in the radioiodine injected fish compared with the controls. Statistical analysis showed a significant difference. Therefore, although follicles were present, a "hypothyroid" condition was evident in all experiments. Fish in the first experiment were injected with radioiodine one week prior to use in the experiment. The fish in all other experiments were injected three weeks prior to the start of the experiment to allow for utilization of reserve stores of thyroxine in the tissue. . Simpson, Asling and Evans (1950) found 20 days is required for utilization of reserve stores of thyroxine in the rat. A hyperthyroid condition was produced by injection of Na-L- thyroxine solution. The solution was prepared by dissolving Na—L- thyroxine in sodium hydroxide; it was then neutralized with hydrochloric acid and (diluted- with distilled water to a concentration of 100 micro- grams of Na-L—thyroxine/. 05cc. The dosage given to the fish was 100 micrograms L-thyroxine/fish/week. This dosage is based on a study 14 by Hoffert and Fromm (1959) in which two-year-old rainbow trout held at 130 C. were found to have a thyroxine secretion rate of . 303 micro- grams L-thyroxine/lOOgms./day. It was desired to give the fish as much thyroxine as possible while still maintaining a physiological dose. It was felt that 100 micrograms of L-thyroxine per week would constitute a factor several times the secretion rate and still constitute a physio- logical dose. All injections had a volume of . 05 cc. and were made intraperitoneally. This dosage was used in the three experiments in which the effect of the thyroid was evaluated. In order to overcome bias due to weekly handling of hyperthyroid fish, the control fish and hypothyroid fish received intraperitoneal injections of isotonic saline solution each week at the same time the hyperthyroid fish were injected. The injection was . 05 cc. of 0.6% saline solution. All injections were made with a 0. 25 cc. syringe fitted with a 22 gauge needle. Pre-experimental Acclimation and Conditioning The fish were captured 4 to 8 weeks prior to an experiment and were therefore completely adapted to aquarium life and the feeding of artificial food. All fish were treated for parasites and disease in one or more of the following ways: formalin treatment 1:4000 solution for approxi- mately 45 minutes; saline treatment 3. 0% solution for approximately five minutes; terracycline treatment 0. 02% solution for 24 hours. These treatments were given shortly after the fish were collected. The acclimation of the fish to a given photoperiod and constant temperature conditions, in the experimental aquaria, varied in the dif- ferent experiments. Fish in the first experiment were held at a photo- period of 12 hours of light per day for a period of three weeks prior to the start of the experiment. It was later felt that it would be more 15 advantageous to acclimate the fish to the photoperiod that they would begin an experiment. Therefore, in subsequent experiments fish in the constant 8-hour aquarium and variable increasing photoperiod aquarium were held at a 8-hour day-length; the constant 16-hour aquarium and variable decreasing photoperiod aquarium were held at a 16-hour day-length. Fish were acclimated under these conditions for 3 weeks, 1 week and 4 weeks prior to the second, third, and fourth experiments, respectively. An attempt was also made to acclimate the fish to receiving injections prior to the start of an experiment, except for the first experi- ment. The fish were given their respective injections each week during the three week acclimation period in the second experiment. . In the third experiment, all fish received their respective injection twice before the start of the experiment. This also allowed the establishment of a hyper- thyroid condition before the start of an experiment. Physical Measurements Measurements were made of the total length, standard length, and weight of each fish. Measurements of total length and standard length were made to the nearest millimeter on a standard measuring board. Weight was measured to the nearest 0. 1 gram on a triple-beam balance after the fish had been blotted with absorbant paper to remove excess water. Prior to taking measurements, all fish were anesthetized with tricaine methano sulfonate (M. S. 222). All fish were given a three-minute treatment in 3% saline solution after measurements were taken and the fish revived. . Measurements were made at the start of the experiment, end of the third week, and at the end of the sixth week. In the case of the fourth experiment, measurements were also taken at the end of the ninth week. 16 Growth in this study was considered as any increment (gain), in total length or weight. The fish in the first three experiments were not marked and the data are therefore expressed as the average increment in total length and weight per fish in a tank. In the fourth experiment, the fish were marked so that growth of individual fish could be determined. The percent increase in growth over the initial length and weight was also determined to remove any influence of average size of the fish in a tank. 7 Food and Feeding Routine Two types of food were used in this study, a commercially pre- pared, dry pelleted food and frozen beef liver. 1 Fish in the first experi- ment were fed the pellets. There was a high incidence of disease and a high mortality in the experiment. Dr. Allison (personal communi- cation), fish pathologist for the state of Michigan, felt that part of the problem may have been dietary and recommended the use of beef liver. Frozen beef liver was fed in the second and third experiments. In these two experiments, occasional periods of cessation of growth were observed which could be correlated with periods of clouding of the aquarium water. It was felt that the clouding of the water may have resulted from the liver. The feeding of pellets and liver to fish in two other aquaria in the labora- tory resulted in periodic clouding of the aquarium fed the beef liver. , Under the circumstances, it was decided to feed pellets once more in the fourth experiment. . Fish were fed daily allowing 1/2 hour to 1 hour for feeding. They~ were given as much as they would consume. The amount of excess food was determined, the food and waste removed by siphon, and the tanks filled with fresh water. The method of determining the amount of excess food varied with the two types of food. . In the first experiment, the number of pellets 17 remaining on the bottom of the tank was recorded. The average number of pellets in one gram of food was determined and the weight of the excess food determined by dividing the number of pellets counted as waste by the average number of pellets in a gram of food. . In the fourth experiment, the number and size of the pellets remaining in the tank were noted and an equal number of pellets of nearly equal size were weighed on an electric balance. When the fish were fed beef liver, the excess food was removed, blotted, and weighed to the nearest 0. 1 gram on a triple-beam balance. Food consumption was recorded by providing a jar of food for each tank of fish. The jar of food was weighed at the start of the experi- ment, third week, sixth week, and also the ninth week in the case of the fourth experiment. The difference in the weight of a jar between two periods minus the weight of the excess food represented the amount of food consumed by a tank for that period. The rate of food consumption is expressed as a percentage of the mean total body weight of a tank of fish per unit of time, and is calcu— lated by the formula: Food consumed by a tank of fish (gms.) 0 Ave. total body weight of tank (gms.) x 1 0 R. F. C. = (% body weight) Inasmuch as the food consumed by individual fish was not known, body weight had to be based upon the total body weight of all fish in a tank. The average total weight of the fish in a tank, in a given period, is con-. sidered to be the mean of the total weight at the beginning and end of each period of measurement. - Measurements were made on a 3-week and 6-week basis. . For purposes of evaluation, the entire tank of fish is considered a single "organism. " The value represents the rate of food consumption for a particular tank. 18 The efficiency of food conversion was also determined on a per- tank basis by the following formula: Total increment in weight of tank Eff. of Food Conversion (%) = Focicglnc38n)sumed by tank (gms ) x 100 The value obtained expresses the percent efficiency with which food is utilized for growth by a particular tank. Histology The branchialregions of the lower jaw of radiothyroidectomized fish and control fish were sectioned to determine the degree of thyroid- ectomy. The tissue was fixed in Dietrich's fix for 24 hours, embedded in paraffin, sectioned at 6 to 15 microns, and stained using the standard hematoxin- eo sin technique . Mortality and Dis ea 5 e Mortality occurred in all four experiments. The highest incidence of mortality and disease occurred in the first experiment during which 12 fish died (10% mortality). Mortality in the second experiment was limited to 3 fish (2. 5% mortality). The third experiment had a mortality of 8 fish (6. 6% mortality). In the fourth experiment, only 1 fish died (1 . 6% mortality) in the nine-week period. Mortality in the first and third experiments was due to a type of fin rot which attacked the caudal and pectoral fins. . Death usually ensued after the fin was completely eroded away. Attacks on the weakened fish by other fish at times hastened death. Mortality in the second and fourth experiment could not be explained by disease. Those of the second experiment may have been the result of handling or faulty inject- ing. In the fourth experiment, the mortality appeared due to attacks by 19 other fish. Other minor infections occurred but these were not believed to have caused any mortality. The only treatment applied during an experiment was in the first experiment. The fish were fed pellets containing the antibiotic sulfamera- zine. This did not appear to be effective and was discontinued. All fish which died during the course of an experiment were en- tirely eliminated from the experiment for purposes of analysis of growth. The weight measurements of these fish were used, during the period they survived, for the purpose of determining the rate of food consumption and efficiency of food conversion. Fish observed to be in poor condition, at the time measurements were taken, were eliminated from an experi- ment. Determination of Thyroid Activity The rate of loss of radioiodine from the head region of fish, after injection of a tracer dose of radioiodine, was taken as an index of thyroid activity. The method is similar to that employed by Swift (1955). Measurements of thyroid activity were made before and after the second experiment. Counts were detected with a sodium-iodide crystal scintillation tube (Nuclear Instrument Corp. Model DS-l), and counts were recorded by use of a count rate meter (Nuclear-Chicago Model 1620) and an Esterline Angus Strip-Recorder. The Esterline strip-recorder was not employed for counting at the end of the experiment. All fish were marked with numbered flutter tags so that individual fish could be recognized and individual records maintained. Measurements of radioactivity were made 32 v_i_v_o using a flow type apparatus with the fish held in a glass tube narrowed at one end for positioning of the fish. The fish were oriented upstream with the flow 6f water entering at the narrow end of the tube. The tube was then placed 20 in position next to the scintillation tube which lay horizontally on a table. The region of the fish which was counted comprised the head region from about the eye down and posterior to the edge of the opercle. The scintillation tube had a collimated head with an opening one inch in diameter. The region which was examined therefore varied slightly with the size of the fish. The radioactivity of the fish is expressed in terms of percent injected dose. This is determined by dividing the activity of the fish, in counts per minute (c.p.m. ), by the activity of a standard in c.p.m. after both values have been corrected for background. This procedure cor- rects for isotope decay, as well as standardizes the geometry if the standards are counted in the same position as the sample. The activity of the standard is determined from the average of two standards which are counted several times during the counting period. The effect of the glass tube in which the fish were held is negligible. The radioactivity, in percent injected dose, determined each day was plotted against time (days after injection) on a semilog scale. The regression coefficient (slope of the line), fitted by the method of - least squares, was taken as the index of thyroid activity. A larger negative regression coefficient indicates a more active gland. . This holds true if time is allowed for the loss of extrathyroidal iodine. Prior to the start of the second experiment, a group of four normal fish were acclimated to each of the two initial photoperiods, constant 8-hour and constant l6-hour. The fish were placed under the photoperiod on June 8, 1961, acclimated for 12 days after which they received an intraperitoneal injection of 10 microcuries of radioiodine. . Counting was initiated 8 days after injection and continued for 8 consecutive days. One fish died between the time of injection and when counting was initiated. The two standards for this period were prepared by placing 1/5 of the injected dose in each glass planchet. A casein solution was added to bind 21 the iodine and prevent volatilization. The glass planchets were then dried under a heat lamp. Counts of the two standards were made at the beginning and end of the counting period each day. The thyroid activity was determined for the control and hypothyroid fish, under each of the four photoperiods, at the end of the second experi- ment. This study involved 73 fish; 36 fish were controls and 37 were hypothyroid fish. The fish were given a tracer dose of 10 microcuries of radioiodine, intraperitoneally, on August 23, 1961. Counting was started 10 days after injection and continued each day for 8 days except for the 6th day. One fish of the group died 6 days after the start of counting. The two standards for this period were prepared with the same amount of radioiodine that was injected into the fish. Other than this, the standards were prepared in the same manner as at the start of the experiment. Counts were taken of the standards at the start, middle, and end of each counting period. RESU LTS AND DISCUSSION Effect of Photoperiod The effect of photoperiod on growth in length and weight, food consumption, and efficiency of food conversion was determined. . The data were analyzed from three points of view: 1) differences between the four photoperiods over an entire experiment, 2) comparison of the first and second three-week periods of the two varying photoperiods, 3) a comparison of the two constant photoperiods. In the first three experiments, the value for each photoperiod was determined by com- bining the three thyroidal conditions under each photoperiod. Results of Increment in Weight The average increment in weight per fish under each photoperiod is given in Figure 1. The data for the average increment in weight per fish in each tank for the four experiments is presented in Appendix A. Results of the second and third experiments must be interpreted with caution, as these two experiments experienced periods of inhibited growth which was believed due to the feeding of liver as discussed previously. Those photoperiods particularly affected in the second experiment were the first three week period of the decreasing photoperiod and the second three week period of the 16-hour photoperiod. Those affected in the third experiment were the second three-week period of both the constant photo- periods. Periodic clouding of the water occurred in all aquaria during the third experiment rendering the results somewhat questionable. A comparison of the results of the four photoperiod groups for all experiments shows that the fish in the increasing photOperiod had the 22 23 .9395 ooufi become one pounded: mommofi ueomonmm: Hen— ocu mo 9.on o5 3 mega; one meonm ponoumm .pownom Moo? 63m 93 uo>o uswfioa’ cw deem use «comoumon umfi guano mo mod. 05. um moddmer 9:... .3395 moans. pcooom ofi ”Ems? cm cfimw exp musomonmou pomomocme mad.» 2: new Goflnom .8999 05 mmxoos? ooafi umnfl 65 “ammo? 5 5mm o5. manomeumou pomofioco v.39, o5 new use. Aomm mo :ofluom .833 oFH. .mueoemnomxo ado“ o5. Ho pomnomouoxm nose .8de sea m mo Aegeawv “Ammo? Gm “Cogouocfi owmno>< .H oufimfim 24 wdwmeeuoop pneumeoo mammmonofi “gumcoo mammmouoop «Gmumnoo mammmonofi undamnoo u: wAIA: an A: u: oHAlw a: w .3 mAln: an 3 a: 34$ a: w , . 84 mm; mm; \\\ n’ou . . mw.N . mo.o . H m an m . S. m e wed $6 3.2 1N o grOl H on. N 0H .m n>.m mm.N Ho .N _ . o>.o om m om.o mm.o wo.h uaegfiuomxm fiudoh ueoemuomxmfi THEE mafimmonoop uempmdoo mcwmmouocm aneumcoo mammmonoop aneumeoo mcmmmonocm addumcoo .3 mix: a: A: a: 31¢ a: w HS @4me as A: .5 oHAlw u: w m~.~ mo.m >v.~ mo.~ Ho.H wN.N mN.H mN.H om .o . MN .m S .N ow.o oo.~ N@.m mm .m j on . m am .e om .e unoghemxm pnooom ewe 30820me amhwh 25 greatest growth in weight in all but the first experiment in which those in the 16-hour photoperiod had the greatest growth. A two-way analysis of variance of the effect of photoperiod and thyroid activity on the mean increment in weight was performed on the first three experiments to determine if any of the differences were statistically significant. 1 In the fourth experiment, a one-way analysis of variance was performed using the individual increments of each fish. These results are given in Table 3. A Tukey Multiple Range Test was applied to the first and third Table 3. Results of a statistical analysis of the differences in the mean increment in weight of a fish over the entire experiment. Expt. F ratio df Significance 1 11. 17 (3, 6) 1% 2 1. 84 (3, 6) no significant difference 5. 14 (3,6) 5% 4 0 . 58 (3, 55) no significant difference experiments to determine which photoperiods were significantly different. In the first experiment, the l6-hour photoperiod was significantly dif- ferent from the decreasing photoperiod at the 1% level and significantly different from the 8-hour and increasing photoperiods at the 5% level. In the third experiment, 16-hour, increasing photoperiod and decreasing photoperiod were all significantly different from the 8-hour photoperiod at the 5% level. The effect of photoperiod may also be evaluated by a comparison of the gain in weight between the first and second three week periods, of the varying photoperiods (see Figure 1). Under the increasing 26 photoperiod, there was a greater gain the second three weeks (12 to 16 hours light) than the first three weeks (8 to 12 hours light) in three of the four experiments. This did not occur in the third experiment for reasons stated previously. . Similarly, there was a decrease in the weight gain the second three weeks (12 to 8 hr. light) compared with the first three weeks (16 to 12 hr. light) of the decreasing photoperiod group. These differences are especially significant since in the first experiment there was an increased growth the second three weeks for all photoperiods except the decreasing photoperiod, and in the second and fourth experi- ments there was a decrease in increment the second three weeks in all photoperiods except the increasing photoperiod. 1 The differences in the average increment in weight between the first and second three—week periods of the varying photoperiods, in the four experiments, were tested by the Student's "t" test for matched observations. There was no signifi- cant difference between the two 3—week periods for either the increasing or decreasing photoperiod. A comparison was also made of the average increment in weight of a fish between the two constant photoperiods. Over an entire experi- ment, the gain in weight was greater for fish in the 16-hour than the 8- hour photoperiod in three of the four experiments. This did not occur in the second experiment; however, here growth was greatly inhibited the second three week period of the 16-hour photoperiod whereas the 8-hour photoperiod appeared to be unaffected. . Since all fish of the fourth experiment were marked, it was possible to make a better statistical analysis of the effects of photoperiod. The results of a one-way analysis of variance for the individual incre- ments of the fish under each photoperiod has been presented in Table 3. To determine if there was a significant difference in the growth of fish in different tanks under the same photoperiod, a hierarchical analysis of variance (Simpson, Roe, and Lewontin, 1960) was performed. 27 Equal replications are necessary for the analysis; since one fish was lost in the 8-hour photoperiod, one fish from each tank under each photo- period was randomly omitted. There was no significant difference in increment in weight between the different photoperiods or between the tanks under each photoperiod. Using the individual increments of the marked fish, differences in increment between the two 3-week periods of the varying photoperiods were also analyzed by the "t" test for matched observations. There was no significant difference, at the 5% level, between the two 3-week periods of increasing photoperiod group. Under a decreasing photoperiod, the increment in weight between the two 3-week = 2.977 df =14). periods was significant at the 1% level (t =- 5. 68 > t 995 Results of Increment in Total Length The average increment in total length of the fish under each photo- period is given in Figure 2. The average increment in total length of a fish in each tank is presented in Appendix A. The differences in length increment between photoperiods, over an entire experiment, conform to a degree with differences in weight increment. The greatest gain in length occurred in the same photoperiod as the greatest gain in weight in all but the second experiment in which the 8-hour photoperiod group had the greatest gain in length. The photoperiods have the same order of rank for length and weight in each experiment except for the third and fourth experiments in which there was a slightly greater gain in length by fish under a decreasing photoperiod than under 16-hour photoperiod. However, measurements were made only to the nearest millimeter and the slight differences may be due to experimental error. The inhibition of growth in weight that occurred in the second and third experiments also occurred with respect to length; therefore the results of the second and third experiments are again somewhat questionable. Generally, differences in gain in length of fish between the photoperiods of an 28 63qu Joe? 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A two-way analysis of variance was performed in the first three experiments to determine if differences in increment in length were significantly different. The analysis showed that a significant difference occurred only in the third experiment (5% level). A one-way analysis of variance of the fourth experiment demonstrated no significant difference. A comparison of increment in length between the two 3-week periods of the varying photoperiods demonstrated a greater gain with longer photoperiods although the results were not as consistent as for increase in weight. Under an increasing photoperiod, the average length increment of a fish was greater the second three weeks than the first three weeks in two of the four experiments. There also was a decrease in gain in length the second three weeks compared with the first three weeks for fish under a decreasing photoperiod in three of the four experi- ments. In the second experiment, there was basically no change. The mean increment in total length of a fish under each photoperiod for the four experiments was analyzed by the "t" test for matched observations. There was no significant difference between the two 3-week periods under either of the varying photoperiods. A comparison of the fish under 8-hour photoperiod and the 16-hour photoperiod showed a tendency for greater gains in length under a l6-hour than an 8-hour photoperiod. The second experiment is again the exception. A statistical analysis of increment in total length was also made for the first six weeks of the fourth experiment. A one-way analysis of variance was performed based on increments in length of the individual fish. To determine if differences among the different tanks of fish existed, a hierarchial analysis of variance was also performed. There was no significant difference between photoperiods. The effect of tanks was also negligible. 31 The difference in growth of fish between the two 3-week periods of increasing photoperiod was not significant when analyzed by the ”t" test. However, the difference in growth between the two 3-week periods of the decreasing photoperiod group was significant at the 1% level (t=7.57 >t..995' = 2.977, df = 14). Results of Growth Expressed as a Percentage Since the fish in each experiment were distributed on a random basis, slight differences in the average initial total length and weight of fish occurred in each tank. Anderson (1959) found that there was a tendency for larger fish to have greater growth increments. A comparison of the growth of marked fish of the fourth experiment showed this to be generally true. Size hierarchies existed in almost all tanks in each experiment, and this factor had a profound influence on the growth of individual fish. The growth of fish in length and weight was therefore calculated as a per- cent gain of the mean initial total length or weight for each of the three- week periods and over a six-week period. Increment in weight calculated on this basis is given in Figure 3 and for increment in total length in Figure 4. The results are nearly the same as observed for actual incre- ments in length and weight. The only noticeable differences occurred in the fourth experiment. The percent growth in weight for the 16-hour photoperiod group was very nearly the same as for increasing photoperiod group, and the percent growth of the decreasing photoperiod group is less than it appears to be in terms of actual increment in weight. . Over- all, the growth is so similar, that the effects of the average initial length and weight of the fish in a tank was believed negligible and that the actual increments accurately describe the growth of the fish. 32 .3303 mount. vacuum 05. poppies“ mommofi uaomenaop use. one mo $2 on» o“ mogm> one mmonm posoumm .poflnom Moo? xwm o5 no>o anew “coupon map pcomoumon ado. node mo mop oat Hm wedged, oFH .mxoeB e923 pcooom eat 5mm “coupon 93 manomonmon o3d> pomomoco 05 one :ofinom gonad on» ”weave? eons» “mum mg» Gmmm unmouom o5 mucomonmon odd; pmmofioco 93 new yea ea» mo coflunom .833 9;. .powuemouaoam Aomo Hopes 3mm .m mo uflmmoB H.333 owmnoxfim of mo Gfimw unoonom on“ we pemmonmxo 3mm .m mo ”Emmosp cm #3090 .m madman 33 mcfimmonoop uemumeoo wemmmouofi 33.300 wnwmmonoop 33980 mcfimmmiofi udeumcoo H3 wAla: .3 f .3 film .3 w .3 IA .3 a: .3 £1 .3 w W560.” $0.3m medium mém wméd ante “:63 \W\W\W $8- a - 31m pm. 3 pm .mL «.1 o 2.2 mo.~N mm.m~ . he .vm cm .3 a m e a... ow .Se Ho.om aw.wm uqofimnomxm gasoh «dofimnemxm p.338 mcwmmouoop ucdumcoo memmmouoefi 3.3980 mnwmmonoov padumcoo wdwmmehofi unmumqoo .3 @413 .3 a: .3 QHAIw .23 w .3 win: ..3 a: .3 Si .3 w $0.0 EYE 3m.w mde 332 wodw cud cod .m omimfi no.2 oodfi 3;. 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Nww Sv wan mo .m - 00.0 mm; om.“V 00$ 05d 00.0 mwzv ¢0.m Hm . . 0 . 0H 5 mm .n mm 0 v0.0 X. .o 00.6: unmefinmmxm vacuum «noguomxfi “warm 36 Effect of Photoperiod on Growth in Length and Weig_h_t_ The results of the four experiments certainly suggest that photo- period has an influence on growth in length and weight of green sunfish. Greater growth is associated with longer photoperiods and increasing photoperiods at constant temperature. The results are not statistically significant for all four experiments; however, the consistent results of growth, particularly in weight, under each photoperiod in each of the four experiments and the consistent differences between the two 3-week periods of the varying photoperiods certainly afford strong evidence of an effect of photoperiod. The black of statistical significance is probably due in part to the large variance in the growth of the fish due to the size hierarchies established in the tanks and the use of various sized. fish. . Increment in weight appears to be more closely correlated with photoperiod than increment in total length. Differences in increment in weight between the two 3-week periods of the varying photoperiods were correlated with changes in photoperiod with only one exception. . In-length increment, there were three exceptions to this correlation. . It appears that growth in length is less dependent upon photoperiod than is weight. The findings of this study are in agreement with the results of Eisler (1957) and Tryon (1942) both of which used very small fish in their study. . Eisler (1957) studied the growth of chinook salmon fingerlings in darkness and under various constant light intensities for 12 weeks. There was a significant difference in growth in length of fish reared at different light intensities compared with fish reared in the dark; the fish in the light attained a much greater growth in length. Increase in weight was also greater (57%) for fish reared in light. No significant differences occurred between the different light intensities. Tryon. (1942) compared the growth of cutthroat fingerlings reared in hatchery troughs which were covered or exposed to natural light conditions for-a period of 50 days. 37 A significant increase in growth (1% level) occurred in the fish reared in open troughs. . . Other investigations described in the literature in which older, mature fish were usedhave shown either no relationship or an inverse relationship between growth and photoperiod. The work of Bjorklund (1958) on goldfish and Brown (1946a) on brown trout, which were dis- cussed previously, showed an inverse relationship. Anderson.(l959) in studies of the bluegill found no relationship, although the mean gain in weight and length was slightly greater for the short day period (10-hour light). . Swift (1961) studied the specific growth rate inlength of yearling brown trout under natural conditions in which temperature and photo- , period were recorded at monthly intervals, and also under constant environmental conditions with the same fish being exposed. fora period of four weeks to a 4-, 8-, and 12-hour light period. In the study under natural conditions, Swift (o_p_. git.) felt that a closer relationship existed between the annual seasonal growth cycle of length and the annual temperature cycle than with the cycle of photoperiod. He remarked that under the constant environmental conditions (data not given) specific growth rate in length showed no consistent response to photoperiod. To this investigator's knowledge, the present study is the first in which a positive correlation between growth and photoperiod has been found for older fish (2- to 4- year-old fish). It would be of interest to determine whether there is any variation in the response of different sized fish to photoperiod. The reasons for the dissimilarity in the results reported in this study and those reported in the literature are unknown. . Since each involved different species, one might feel that various species respond differently. This does not appear to be the case when comparing Anderson's (1959) work on the bluegill and the present study of the green sunfish. The bluegill and green sunfish are closely related species attaining 38 approximately the same growth, and occupying the same niche in nature. The conclusions of Anderson ((311. git.) are based on a limited number of fish (3 under each photoperiod) and over a limited period of time (30 days). _ A larger sample of fish held. for a longer period of time may. have yielded different results. There may be a lag period before the effects of photo- period are apparent. This was not particularly true for the present study, but a period of 8. weeks elasped before an effect was apparent in the work of Eisler (1957). . Brown (1946a) held trout for periods of two and five months to obtain significant differences. The study by Bjorklund (1958) may have been influenced by the limited food supply given the fish (approximately 1. 7% of body weight daily). Under the circumstances, differences in activity of the fish would have a much greater influence on their growth rate thanon that of fish given an unlimited food supply. EffeCt of Varying Photoperiod on Growth An attempt was made to determine whether a varying photoperiod has a greater effect upon growth than a constant photoperiod. As pre- viously stated, Swift (1961) felt that a- study was necessary to distinguish between the effect of day to day changes in day-length and the effect of day-length when it is constant from day to day. That a varying photo- period has a greater influence on growth is apparent in this study. . In the second, third and fourth experiments, fish exposed to an increasing photoperiod attained the greatest increment in weight. The influence of varying photoperiod is also apparent in Table 4 which (showsthe percent gains or losses the second three weeks over the first three weeks (wt. gain second 3 weeks wt. gain first 3 weeks greatest the second three weeks for fish under an increasing photoperiod. x 100). The percent gains in weight were In the third experiment in which there was a reduction in growth rate the second three weeks under all photoperiods, the reduction was least for the fish under increasing photoperiod. . For fish held under a 39 Table 4. Percent gains or losses inlength and weight the second three weeks compared to the first three weeks for all photoperiods of each experiment. Expt. 8 hr. 8-16 hr. 16 hr. 16-8 hr. percent percent percent percent Weight 1 ‘ +lOO.8O +151.56 +2.1.57 -58.40 2 -4.34 +112.24 -73.59 -15.04 3 -l40.56 -12.04 -lO3.15 —55.73 4 -16.55 +13.90 -16.62. -85.66 Total Length 1 -7.62 +17.39 +21.58 -36.87 2 -4.35 +ll.70 -76.79 +1.38 , 3 -78.37 -32.45 -77.22 -24.87 4 -40.98 -12.64 -30.00 -7l.l9 decreasing photoperiod, there was a general reduction in weight gain the second three weeks (indicated by a percent loss in Table 4). . In the first and fourth experiments, the reduction in weight (percent loss) was greater under the decreasing photoperiod than any other photoperiod. These differences in the percent gain or loss between the two 3-week periods were not as apparent for total length; however, as mentioned previously, growth in total length is possibly somewhat independent of the effect of photoperiod. Another point suggesting a greater influence of the varying photoperiods is shown in the study of the effect of the prior history of photoperiod on growth which will be discussed in the next section. 40 In general, data from all four experiments suggest that decreas- ing photoperiod has a depressing effect upon growth, but the possibility of a stimulating effect of increasing photoperiod is not well-shown. The first experiment indicates that l6-hour photoperiod acts as the greatest stimulant to growth. The fourth experiment suggests that increasing photoperiod stimulates growth. . Fish in the 16-hour photoperiod generally had the greatest growth the first three weeks (Figures 1 and 2). If growth had occurred during the last half of the second and third experiments at a. rate comparable to that occurring in the first half, the greatest growth may have been attained in this photoperiod. The reliability of the data for the second and third experiments has been discussed previously. Effect of Prior History of Photoperiod on Growth The fourth experiment was extended for three weeks beyond the normal six weeks for an experiment. The photoperiod of each of the two I varying photoperiods was held constant during this period» (increasing photoperiod held at 16-hour day-length and decreasing photoperiod at an 8-hour day-length). This was done to permit a comparison of the growth of fish with a prior history of a varying photoperiod with growth of fish held at a constant photoperiod throughout the experiment. . The mean increments in length and weight for the last three-week period of each photoperiod are given in Table 5. Differences between the two aquaria held at the same constant photoperiod were analyzed by means of the Student's "t" test. The fish having a prior history of an 8-hour photoperiod (Group 8C) had a significantly greater growth in length and weight (t = 3.43> t ..995 ‘ = 2.771 and t = 4.29 > 2.771, df = 27) than fish with a prior history of a decreasing photoperiod (Group 8V). . Fish with a prior history of an increasing photoperiod (Group 16V) had a significantly greater gain in weight (t = 2.75 > t.995 = 2.048, df T-‘- 28) than fish having a prior history of a constant 16-hour photoperiod (Group 16C). 41 Table 5. Average increment in Weight and total length of fish under each photoperiod for the third three-week period of the fourth experiment. Description of Group . Weight in Total Length grams in mm. Group 8C - 8-hour constant during 3. 21** 4.629** entire expt. Group 8V - 8-hour constant previously 0.43 1. 93 decreasing 16-8 hour . Group 16C - 16-hour‘ constant 3r ,, 1 . 93 3. 07 during entire expt. Group 16V - 16-hour constant previously 3. 62* 4. 86 increasing 8-16 hour. a: Denotes significance at the 5% level (see text) >i<* Denotes significance at the 1% level (see text) The difference in gain in length between the 16V and 16C groups was not significant by the normal two-tailed test for significance, however the gain -was significantly greater by a one-tailed test for the group having a prior history of an increasing photoperiod (16V) compared to the l6-hour photoperiod (16C). The results show that the prior history of photo- period does have an effect upon growth, an increasing photoperiod tending to stimulate growth and a decreasing photoperiod tending to suppress growth. These results offer further evidence of an effect of avarying photoperiod on growth as mentioned in the previous section. Effect of Photoperiod on the Seasonal Growth Cycle of Fish Since photoperiod has been shown to have an effect upon growth, it appears that it plays a role in the annual seasonal growth cycle of fish. 42 Temperature is probably primarily responsible for the seasonal cycle with photoperiod playing a less important role. It appears that the extremely rapid rate of growth which occurs in spring (April to June) may well be due to a synergistic action between increasing photoperiod and temperature. The decrease which occurs during the summer (July and August) during which temperatures are still increasing or are at amaxi- mum may be due to the decreasing photoperiod or a combination of decreasing photoperiod and temperatures above the optimal level for growth. . In view of the great regularity with which the seasonal cycle occurs and the variation in optimal temperature ranges of different species of fish, it may be that photoperiod is the dominant factor affecting the cycle. A study by Evans it a}. (1962), on respiration of trout tissue, showed that photoperiod was temperature dependent and its effect evident only at high temperatures. The' rapid decrease in growth rate of fish during autumn is probably due to an interaction of both decreasing photo- period and falling temperatures. In winter, temperature appears to be the predominant factor since substantial growth occurred at low photo- periods and high temperature in the laboratory. The above explanation of the annual seasonal growth cycle in fish is largely conjecture. Further studies are necessary to resolve the interaction of photoperiod and temperature on growth. Effect of Photoperiod on Food Consumption The amount of food consumed by a fish increases with the size of a fish. As a result, food consumption was calculated as a percentage of the body weight by the formula previously given. . The rate of food con- sumption for each experiment is given in Table 6. The rate of food con- sumption as calculated for each tank of fish is given in Appendix B. The results given in Table 6 are not comparable between experiments due 43 to the feeding of different types of food. The second and third experi- ments are the only two in which conditions were similar. Table 6. The average rate of food consumption (percent body weight) of fish under each photoperiod of each six-week experiment. Expt. .8 hr. _ 8-16 hr. 16 hr. 16-8 hr. percent percent percent percent 1__ 38.78 42.64 57.24 44.67 2 83.70 77.06 87.11 67.60 3 76.55 91.70 103.01 79.05 4 44. 35 55.15 58.74 55.74 In all four experiments, the rate of food consumption was great- est in the l6-hour photoperiod. . In three of the four experiments, the rate of food consumption was lowest in the 8-hour photoperiod. These results do not agree with the general growth pattern of the fish over the 6 weeks of each experiment (Table 6 and Figures 1 and 2). It is believed that the variability, may be due to differences in activity of the fish in the various photoperiods. In the 16-hour photoperiod the longer light condition allows greater activity over a 24-hour period. This increase in activity increases the overall energy expenditure thereby increasing 1 the maintenance requirements. Roberts, as reported by Anderson.(1959), found a 30% reduction in respiration of sunfish held. at a. 9—hour photo- period compared to fish held at a 15-hour photoperiod. The rate of food consumptionis directly related to photoperiod. This is apparent from a view of Table 6 as well as from the comparison of the rate of food consumption between the two 3-week periods of vary- ing. photoperiods given in Table 7. In general, low rates of food 44 Table 7. Rates of food consumption (expressed as percent body weight) for the first and second three-week periods of increasing and decreasing photoperiods in each experiment. Expt. 8-16 hr. Photoperiod 16-8 hr. Photoperiod lst 3 Wks. 2nd 3 Wks. lst 3 Wks. 2nd 3 Wks. (8-12'hr.) (12-16 hr.) (16-12 hr.) (12-8 hr.) percent percent percent percent 1 20.79 23.60 24.00 20.13 2 37.64 42.72 40. 35 27.46 3 52.52 39.93 44.87 33.45 4 31.67 24.67 39.58 14.76 consumption are associated with short photoperiods and high food consump- tion with longer photoperiods. . Exceptions to this relationship occur in the'increasing photoperiod of the third and fourth experiments. . Few studies of the effect of photoperiod on growth described in the literature have involved an analysis of food consumption. Anderson (1959) found no significant difference in the food consumption of fish held at a 10-hour or 15-hour photoperiod. Although the rate of food consumption-for the six-week periods did not appear closely correlated with increment in length, and weight, there is a close correlation with increment in weight, This can be seen in a comparison of increment in weight and rate of food consumption between the two 3-week periods of the varying photoperiods (Figure 1 and Table 7). . The results of a correlation analysis of rate of food consumption with gain in weight and with gainin length of each tank of fish in each experiment is given in Table 8. The correlation between food consumption and increment in weight is significantly different from zero in every 45 experiment. Total length was not closely correlated with the rate of food consumption; possibly some other factor may be involved in determining increment in length. Table 8. . Coefficients of correlation between rate of food consumption and average increment in weight and total length per tank of fishin each six-week experiment. Expt. . Correlation Coeff. Correlation Coeff. Ave- Weight Increment Ave. T- Length Increment 1 .787** .. .576 2 .750** .025 3 .766**. .613* 4 . 814**>:< . 205 N = 12 * Denotes significance at the 5% level. >'.<* Denotes significance at the 1% level. *** . _ . Denotes Significance at the 0. 5% level. The results indicate that the effect of photoperiod on growth, particularly increment in weight, is partially mediated through increase in the appetite "of the fish. . This is shown by the direct relationship of food consumption and photoperiod and by the relationship of food con- sumption and increment in weight. . Effect of Photoperiod on Efficiency of Food Conversion The efficiency of food conversion was calculated on a per-tank basis and is expressed as the percentage of food which was utilized by the group of fish for growth. The higher the value the greater is the 46 efficiency of conversion. The average efficiency of food conversion for the three tanks of fish under each photoperiod of each experiment is given in Table 9. The values calculated for each tank of fish are given in Appendix C. The efficiency of food conversion among experiments are not comparable because of the different types of food fed. The efficiency of the first and fourth experiments appear very great because of the feeding of dried commercial pellets. The values in the second and third experiments are lower and more reasonable since the liver fed in these experiments was weighed on a wet weight basis. Table 9. Average efficiency of food conversion (percent) for each photo- period of each experiment over the entire experiment, and the rank of the values in order of decreasing efficiency for each experiment. ; m Expt. 8 hr Rank 8—16 hr. Rank 16 hr. Rank 16-8 hr.. Rank percent percent percent percent 1 57.05 III 59.25 11 68.72 I 41.03 IV 2 26.60 11 28.59 I 23.94 111 17.11 IV 3 7.44 IV 23.48 I 20.97 III 21.63 11 4 70.73 111 82.38 I 74.16 11 62.27 IV In general, it appears that efficiency of food conversion is directly related to photoperiod, fish under longer photoperiods having greater efficiency. This is shown in the greater efficiency of food conversion attained by fish in the 16-hour photoperiod compared with fish in the 8-hour photoperiod in three of the four experiments (Table 9). . Except for the increasing photoperiod of the third experiment and decreasing photoperiod of the second experiment, greater efficiency is attained by fish in the three-week period with the longer photoperiod in both the 47 increasing and decreasing photoperiods (Table 10). Differences in efficiency of food conversion between the two 3-week periods of varying photoperiod were not statistically significant. A two-way analysis of variance, performed in conjunction with the effect of thyroid, revealed that differences in efficiency of food conversion between photoperiods of each of the first three experiments were significantly different only in the third experiment. In this experiment, the significant difference was due to the unexplainable low value obtainedvfor the 8-hour photo- period group. . A one-way analysis of variance of the data of the fourth experiment also revealed no significant difference. Although statistically there are no differences in efficiency of food conversion, the consistant response obtained-in the separate experiments certainly suggest that real differences between photoperiod groups may exist. Table 10. Efficiency of food conversion (percent) for the first and * second three-week periods of each photoperiod. of each experiment Expt. ' 3-‘wk. 8hr". 8-16 .hr. 16 hr. 16-8 7hr. period percent percent percent percent 1 47.07 40.60 74.03 53.47 1 2 68.71 74. 17 65.17 27.71 1 24.13 19.63 30.68 15.95 2 2 29.44 35.95 13.06 13.72 1 19.29 25.60 32.27 32.58 3 2 —-* 21.58 —-* 7.69 l 68.46 76.25 76.54 78.40 4 2 73.65 88.63 71.51 25.49 l, Loss of weight occurred, no calculation of food conversion possible. 48 The data indicate that growth is directly related to efficiency of food conversion. This is shown by a comparison of the average efficiency of food conversion for the six week period (Table 9) with growth in weight for the same period (Figure 1). The same relation- ship is shown in similar comparisons of the two 3-week periods of the varying photoperiods. Comparable results are not as apparent for comparisons with total length. However, a correlation analysis of the efficiency of food conversion with gain in total length shows that a close correlation exists. The results of correlation analyses are given in Table 11. The correlation coefficient was significantly different from zero in all experiments for both length and weight. The close correlation of efficiency of food conversion with total length indicates that growth in total length is primarily influenced by this factor. Table 11. Coefficients of correlation between efficiency of food con- version and average increment in weight and total length per tank of fish in each six-week experiment. m Expt. Correlation Coeff. Ave. . Correlation Coeff. Ave. Weight Increment Total Length Increment 1 - 1.904*** .888*** 3 ~944*** .821*** 3 .860*** ,899*** 4 .674* .668* N = 12 *** . .. Denotes Significance a-t the'O. 55% level. a): Denotes significance at the 5% level. 49 It appears that the effects of photoperiod are mediated through an increase in efficiency of food conversion as well as an increase in the appetite of the fish. This is not supported by statistical evidence, but the relationships between length of photoperiod, increased growth in length and weight and increased efficiency of food conversion certainly suggest that the effect of photoperiod on growth of fish is also initiated through increased efficiency of food conversion. . Evidence that photo- period may influence the respiratory metabolism is given by Evans et a_._l. (1962), in which. studies of oxygen consumption of tissue of rainbow trout showed a 16% higher metabolic rate in fish acclimated to. an 8-hour photoperiod than fish acclimated to a 16-hour photoperiod at 160- C. These differences were not reflected in trout acclimated to the same photoperiods at 8? C. indicating that the effect is temperature dependent. A comparison of the rank of each photoperiod in each experiment for all four experiments suggests that varying photoperiod may have a greater effect on efficiency of food conversion than a constant photoperiod (Table 9). In three of the four experiments, data from the increasing photoperiod groups showed the greatest efficiency of food conversion. . Conversely, the decreasing photoperiod groups had the poorest efficiency. Thissituat'ion is also reflected in a comparison of the efficiency of food conversion for the first and second 3-week periods of each experiment (Table 10). The increasing photoperiod group-had an increased efficiency the second 3-week period compared with the first 3-week period in three of the four experiments. . It also had a greater efficiency of conversion than any other photoperiod the second three-«weeks. Under decreasing photoperiod, there was a general decrease in efficiency of the group the second 3-week period. . In the first and fOurth experiments, which are perhaps most reliable, the efficiency of 9food conversion was poorest during the secondthr‘ee-weeks in the decreasing photoperiod groups. These results are in keeping with the previous suggestion that varying photoperiod may 50 have a greater effect upon growth than a constant photoperiod. . Further studies with better controlled conditions are necessary to resolve this issue. . Effect of Thyroid Activity The effect of thyroid gland upon growth in length and weight, food consumption, and efficiency of food conversion was evaluated in the first three experiments. All values are an average of the respective "thyroid" groups under each of the four photoperiods. The results are therefore based upon a larger sample than the results for photoperiod. Results of Increment in Weight The average increase in weight of a fish under each thyroidal condition is presented in Figure 5. . It is assumed that factors which re- sulted in inhibition of growth (second and third experiments) affected each thyroidal condition equally and did not give rise to differences in growth between groups. The results of the effect of thyroid activity on increment in weight of a fish were consistent throughout the three experiments. The greatest increase in weight occurred in the hyperthyroid group and the smallest in the hypothyroid group; the control group had an inter- mediate growth. Differences in increment in weight of fish in each thyroidal group were tested by a two-way analysis of variance in conjunction with the effect of photoperiod. There was no significant difference in gain in weight between thyroidal groups in any of the three experiments. The relationship of increase in weight with increasing thyroid activity per- sisted in all periods except the second 3-week period of the first experiment. The increase in weight of the hyperthyroid group was 14. 0%, 15. 3%, and 37. 9% greater than that for the controls in each experiment. The incre- ment in weight of the hypothyroid group was 14.0%, 5. 7%, and 5. 4% less than that of the controls. 51 Figure 5. Average increment in weight (grams) of a fish under each thyroidal condition of the three experiments. The lower portion of the bar and the value enclosed represents the gain in weight the first three- week period; the upper portion and the value enclosed represents the gain in weight the second three-week period. Values given at the top of each bar represents the gain in weight over a six-week period. 52 .. First Experiment . 5. l4 ‘ 4. 51 3. 87 2. 61 2. 88 2 . 49 2.53 1.63 1.38 Hyper- HYPO' thyroid Control thyroid Second Experiment 4. 06 3. 52 3. 32 .__fl. 1. 9O 1. 68 l . 56 2. 16 1 . 84 1. 76 Hyper- Hypo- thyroid Control thyroid Third Experiment 2. 80 0. 33 2.03 1.92 0. 32 2 2 . 47 1. 71 1. 65 Hyper- HYPO- thyroid Control thyroid 53 Results of Increment in Total Length The average increment in total length of a fish under each thyroidal condition of the three experiments is given in Figure 6. . In the second and third experiments, increase in length was directly related to thyroid activity; the greatest increase occurring in the hyperthyroid group, least in the hypothyroid group, with the controls intermediate. In the first experiment, the control group attained the greatest incre- ment in length, but the hypothyroid group still showed the smallest growth in terms of body length. A two-way analysis of variance per- formed in conjunction with the effect of photoperiod showed no significant difference in increment in length between the three thyroidal groups in any of the three experiments. The increase in total length of the hyper- thyroid group compared with the control group was 9. 5% less in the first experiment, 4.4% greater in the second experiment and 3. 6% greater in the third experiment. The increase in total length of the hypothyroid group was uniformly less (13. 1%, 12. 1%, and 5. 7% respectively) than that of the controls in the three experiments. The data suggest that growth in terms of increase in body length may be directly related to thyroid activity. However, this relationship is not as evident as for weight. , Effect of Thyroid Activity on Growth in Length and Weight VMuch work has been done on the effect of the thyroid gland on growth and differentiation in fish using different methods and contraditory results have been obtained. A . With the use of antithyroid drugs, Dales and Hoar (1954) retarded the growth of chum salmon fry and Scott (1953) obtained similar results with zebrafish. . Fortune (1955) reared Phoxinus 1aevis from the egg in 0. 5% thiourea and found no effect upon growth. . Effects of antithyroid drugs on physiological processes other than growth are reported by Pickford and Atz (1957). 54 Figure 6. Average increment in total length (millimeters) of a fish under each thyroidal condition of the three experiments. The lower portion of the bar and the enclosed value represents the gain inlength the first three-week period; the upper portion and the enclosed value represents the gain in length the second three-week period. . Values given at the top of the bar represent the gain in length over the six- week period. 55 First Experiment 8. 35 7. 55 7. 25 3 . 44 4. 19 3. 88 4. 11 4. 16 3. 37 Hyper- Hypo- thyroid Control thyroid Second Experiment 8. 03 7. 69 6. 76 3. 43 3 . 59 2. 76 4 . 60 4 . 10 4. 00 Hyper- Hypo- thyroid Control thyroid Third Experiment . l 6- 7 6-48 6.11 2. 15 2. 24 l . 94 4 . 56 4. 24 4.17 'Hyper- Hypo- thyroid Control thyroid 56 Other investigators have studied the effect of the thyroid using artificial thyroxine. Dales and Hoar‘ (1954) found a retardation of growth 'of chum salmon fingerlings given thyroxine. Smith and Everett (1943) found no difference in growth of normal and thyroxine-treated immature guppies (Lebistes). Barrington it al. (1961), obtained an increased growth inlength and weight of yearling rainbow trout immersed in a thyroxine solution. Treatment with mammalian thyroid powder has also produced conflicting results. Grobstein and Bellany (1939) reported aninhibition of growth. . Smith and Everett (1943) found no effect upon growth. . Hooper (1961) found no effect upon growth of immature guppies fromrfeeding mammalian thyroid powder. Hooper (1952) obtainedan increased growth in guppies immersed in water to which thyroid powder had been added. Barrington e1 a_l. (1961), obtained a marked stimulating effect upon growth inlength and weight from feeding of thyroid powder. The results of this study using thyroxine~ are generally contra- dictory tothose found in the literature. This discrepancy may be due to the method employed. . In the studies cited above, using artificial thyroxine, the fish were immersed in a solution of water and thyroxine. . Although Pickford and Atz (1957) state that it makes little difference if thyroid hormone is injected, administered orally or added to aquarium water, such a difference may actually exist. Positive results were obtained in this study by injection. . Bjorklund (1958) also obtained a transitory increase in length and weight of goldfish injected, with‘thyroxine and . triiodothyronine in two independent experiments. ! Only one study in the literature relates to the effect of radio- thyroidectomy on growth of fish. . LaRoche and Leblond (1953) found no 1difference in growth (in weight) of thyroidectomized fish and controls after 10 months. However, an analysis of his data shows that after 5 months, slight differences did exist between the controls and radiothy- roidectomized fish. The mean body Weight for two groups of controls 57 was 40 grams, and the mean body weight for two groups of thyroidectom- ized fish was 36 grams. This difference amounts to a 10% reduction in growth of the radiothyroidectomized fish. At the end of the experiment, one lot of radiothyroidectomized fish still had 10% lesser growth than the controls. There was no difference in the second radiothyroidectomized lot and controls. Although thyroidectomy was persistent to the end of the experiment, the difference in the two lots of radiothyroidectomized fish may be explained by the diet fed the fish. The group of fish which had been radiothyroidectomized and which had attained the same growth as the controls were fed a diet containing iodine; whereas the group of thyroidectomized fish which had a lesser growth than the controls were on an iodine-free diet. Pickford and Atz (1957) state that total removal or destruction of the thyroid gland does not wholly abolish the synthesis of thyroid hormone. It appears that a small amount of thyroxine can be synthesized in the total absence of thyroid tissue, although the site of this extra thyroidal function has not been identified. 7 In view of the diet fed the fish, one might conclude that radiothyroidectomy did retard growth as was found in the present study. Since a histological analysis of the lower jaw of radiothyroidec- tomized fish in the second experiment revealed the presence of thyroid follicles, a measurement of thyroid activity was made of the control and hypothyroid fish in this experiment in conjunction with a study of the effects of photoperiod on thyroid activity. The rate of loss of radioiodine from the head region was used as an index of thyroid activity. The fish were marked so that measurements could be made of each fish. . The average thyroid activity of each group under each photoperiod is pre- sented in Figure 7. Values for the individual fish are given in Table 15. The radiothyroidectomized group had a lower thyroid activity than the control group under each photoperiod. The differences in thyroid activity between the two groups are 33% less in the 8-hour phOtoperiod, . 30% less 58 .mdoum fiomm MOM 00:33.0 mucowoflwooo Gmemoummu mwmum>m 05. ohm pan. Ludo 00 mos. on: as Go>wm mogm> .ucoewuomxo paooom 05. mo 03.39393 some pops: masonm Rosina—09»: paw Honucoo ofi mo >fi>30m @3508. m0 News“ emano>< .5 oudwfim 59 waflmmmuomv . .3 wAlo H 292:: nongm HOSGOU mhof 03.. 35.95300 . HA 3 38?: nomknm HoyucoU , in: ... mwé... Bobs: nomzm 33:00 h a wcflm‘mon 0G“ ..Hs ca: ONH .u mdoficflnoo . A: w 39:23 ...an HonuGoU . 0:... mmH... a5... 60 in the 16-hour photoperiod, 15% less in the increasing photoperiod, and 54% less in the decreasing photoperiod. A two-way analysis of variance performed in conjunction with photoperiod showed a significant difference at the 1% level (F = 11.08 > F.99 (1, 55) = 7.08). Although complete thyroidectomy was not attained in the second and third experiments, it is assumed that a hypothyroid condition did exist in all experiments. . Had the investigator been able to accomplish complete thyroidectomy, it is felt that greater retardation of growth may have been attained. One factor which is unexplained is the fact that the increased dose of 2.00 microcuries failed to have a greater inhibitory effect upon growth than in the first and second experiments when less radioiodine was used. ‘The fish which received injections of thyroxine did not have a "true" hyperthyroid condition. . Sections of the lower jaw of these fish revealed the presence of thyroid follicles. If control of the thyroid occurs through the thyroid- stimulating hormone - thyroicine' (TSH-TH) balance in fish as in mammals, as postulated by Chavin (1956), one would expect the epithelial cells to atrophy or become very squamous, which did not appear to be true in this study. . Nevertheless, a hyperthyroid condition was evident in this study. The dose of exogenous thyroxine was based on the thyroid secretion rate for trout and was corrected for size of the fish and temperature. It may be that a sufficient period of time was not allowed for the degeneration of the thyroidal follicular cells. It is pos- sible that if a hyperthyroid condition had been achieved to a greater degree, even greater growth may have been attained. . The mechanism by which the thyroid may affect growth in fish is unknown. A study by Pickford reported in. Pickford and Atz (1957) points to a synergistic action with growth hormone such as occurs in mammals. She refers to an experiment in which hypophsectomized Fundulus were given injections of hake growth hormone, mammalian TSH, and a combination of the two hormones over a five week period. Growth in length 61 and weight was stimulated by the hake growth hormone but not by TSH. Greater growth was attained by the group given a combination of the two hormones. . The percent increase for growth hormone alone was 21. 5%, for weight and 5. 24% for length compared to 35. 8% for weight and 7. 18% for length in the group receiving a combination of the two hormones. The difference in the percent increase in length between the two groups was statistically significant. A synergistic action between these two hormones may exist, or as Hoar (1957) postulates, thyroxine may stimu- late the release of endogenous growth hormone in some manner. The Effect of Thyroid Activity on Food- Consumption The average rate of food consumption for a tank of fish under each thyroidal condition is given in Table 12. r The results are not com- parable between experiments because of the use of different diets. Table 12. The average rate of food consumption (percent body weight) of a tank of fish under each thyroidal condition of each six- week experiment. Expt. Hype rthyroid Control Hypothyroid percent ‘ percent percent 1 _ 50.92‘ 44.07’ 44.14 2 . 84.79 74.07 75.87 3 97.01 88.94 78.14 p The rate of food consumption was greatest for the hyperthyroid group in all three experiments. 7 However, there was very little difference in the rate of food consumption between the control and hypothyroid condition, except for the third experiment. A two-way analysis of variance 62 (performed for each experiment in conjunction with the effect of photo- period) showed no significant difference between food consumption in the three thyroidal conditions. Several investigations (reviewed by Pickford and Atz, 1957) showed increased locomotor activity after treat- ment with thyroxine. . The increase in the rate of food consumption may , be due to an increase in appetite as a result of increased activity. Thyroid activity peg s_e_does not appear to influence food consumption since decreases in food consumption did not always occur in the hypo- thyroid condition. No data on the effect of thyroid activity on the rate of food con- sumption of fish could be found in the literature. . Most studies have involved the effects of thyroid activity on fat, protein, and carbohydrate metabolism. Hoersch gt a_._l.. (1961), in studies of the thyroid secretion rate in sheep, found no correlation between the thyroid secretion rate and food consumption. The present study indicates that the effect of the thyroid in promoting growth of fish is not mediated through an increase in appetite (food consumption). . Effect of Thyroid ActivitLon Efficiency of Food Conversion The efficiency of food conversion (percent) for a tank of fish under each thyroidal condition is given in Table 13. The results are not comparable between experiments for reasons stated previously. The hyperthyroid group showed the greatest efficiency of food conversion in all three experiments and the hypothyroid the poorest efficiency. A two-way analysis of variance (performed in conjunction with the effects of photoperiod) showed no significant differences between the thyroidal groups in any of the three experiments. Although the differences are not great, it appears that the effect of the thyroid is mediated through increased efficiency in. food conversion. The greater efficiency of the 63 Table 13. Average efficiency of food conversion for a tank of fish under each thyroidal condition of each. six-week experiment. Expt. Hyperthyroid . Control Hypothyroid percent percent percent 1 60.78 60.49 ' 51.31 2 25.12 24.73 23.12 3 21.97 17.83 17.14 hyperthyroid group further substantiates the theory that the increased food consumption of this group is due to the increased activity and that there is no effect of the thyroid on appetite (food consumption) of fish. _ Only one reference was found which related the effect of thyroid activity on efficiency of food conversion. Bjorklund (1958) calculated gain in weight 7 food consumed efficiency of food conversion determined in this study. . Bjorklund (op. c_i_t_.) .a~ coefficient of growth ( x 100) which is the same as the found an increased» coefficient of growth in fish treated with thyroxine and in. fish treated with triiodothyronine (40. 8% and 42. 31%; respectively) compared with controls (31. 7%) over a 20-day period. . Over a 70-day ' period, these differences in the coefficient of growth were not apparent. . In a second experiment, a similar increased efficiency was found for , about 20 days following injection of thyroxine and triiodothyronine (44. 3% and 44.2%,respectively) compared with saline injected controls (30. 2%). r The transitory nature of the efficiency of growth can probably be explained by the feed-back mechanism between thyroxine and thyroid- stimulating-hormone which is believed to control thyroid activity. A positive correlation between efficiency of food conversion and thyroid secretion rate has been found in sheep (Hoersch it a_._1. , 1961). 64 Whether the thyroid exerts a calorigenic effect in poikilotherms is still a matter of conjecture. The results of this study on the effect of thyroid activity on the efficiency of food conversion certainly indicate that thethyroid gland does have some metabolic effect in fish. . How this effect occurs awaits further study on the manner in which thyroxine and its analogs enter the biochemical chain. . Effect of Photoperiod on Thyroid Activity The investigator was interested in determining if therewas an effect of photoperiod on thyroid activity. It is possible that the effect of photoperiod on growth may be mediated through the thyroid gland. . It was not possible to test for an interaction between photoperiod and thyroid activity in this study because the growth of individual fish was not known. Only a mean measurement was obtained for a tank of fish and no estimate of the variance within a group was possible, thus preventing a measure- ment of interaction. . It was therefore decided to estimate the thyroid activity of the fish in the second experiment using radioiodine. . The use of the rate of loss from the head region of fish has been reported) by _ other investigators (Swift, 1955,. 1959; Fromm and Reineke, 1956; Hoffert and Fromm, 1959). Thyroid activity was measured for 7 fish, 4. acclimated to an 8-hour photoperiod and 3 to a l6-hour photoperiod, prior to the start of the second experiment. At the end of the -six-week.experime,nt,.— measure- ments of thyroid activity were made using 73 fish (36 control and 37 hypo- thyroid). Measurements of the radioactivity in the fish were not made until the ninth day after injection of the tracer dose in the group of 7 fish and not until the tenth day after injectionasin the 73—fish group. . This lapse of time between. injection and measurements of activity was provided to 65 allow for the loss of extra-thyroidal iodine taken up by the fish. Data presented by Hoffert and Fromm (1959) indicate that the loss of radio- iodine from the head region of trout shows an initial rapid loss followed by a less rapid loss. The initial rapid loss is believed to involve chiefly extra-thyroidal iodine and the less rapid loss represents a true rate of release of radioiodine from the thyroidal tissue. Fromm and Reineke (1959) suggest that to obtain true iodine output rates, only data collected subsequent to the eighth day after injection should be used. Studies of thyroid activity in fish prior “to the beginning of the second experiment showed no difference between fish acclimated for two weeks to a 8-hour photoperiod and those acclimated to a l6-hour photo-.- period. . A semilog plot of the loss of radioiodine for the two groups is given in Figure 8. . Extrapolation of the output curve to zero time indicates thata slightly greater uptake occurred in the 8-hour photoperiod than in the 16-hour photoperiod. The regression coefficient (index of thyroid activity) and output halftime for each fish and the mean for each group is presented in Table 14. Table 14. Regression coefficient and output halftime of each fish and the mean value for each photoperiod. Fish Regression Coeff. Output Halftime (days) 8-Hour Photoperiod 4832 .1389 4. 99 4833 . 0920 7. 54 4842 .1662 5. 96 4843 . 1064 6. 52 ‘ Mean .1134 6.14 16-Hour Photoperiod 4836 . 0772 8. 98 4838 . 1892 3. 66 4839 . 0711 9. 75 Mean . 1125 6.16 66 Figure 8. . Semilog plot of the output of radioiodine from the head region of green sunfish maintained under constant 8-hour photoperiod (solid line) and constant 16-hour photoperiod (broken line) for two weeks. Counting was started the ninth day after injection. Dotted line represents extrapolation back to zero time. Percentage of Initial Dose 100 90 J 80 70 6O 50‘ 40 30 20‘ O‘QWGO 67 l n A A A A I ¥ I l r r 7 .3 9 10.11.12" Days afte r- Injection 2. U 13 A I 14 15 l6 -l7 «1» 18 68 The output halftime refers to the time required for the removal of onevhalf of the radioiodine (corrected for decay) originally present in the thyroidal area. The results of one fish under the 16-hour photoperiod (4838) appears to be inconsistent with the other two fish. - Except for this fish, it would appear that the thyroid activity is lower for fish maintained on a 16-hour photoperiod. The regression coefficients for the individual fish at the end of the second experiment are given in Table 15. The average regression coefficient of fish under each photoperiod and the two thyroid conditions are given in Figure 9. . The results of the control groups indicate that photoperiod may have an effect upon thyroid activity. . The fish under constant 8-hour photoperiod had the highest average index of thyroid activity and the constant 16-hour photoperiod the lowest. The values for the two varying photoperiod groups were intermediate. The increasing photoperiod group, which had been maintained at a 16-hour day-length since the end of the experiment, had an average index “of thyroid activity lower than that of the decreasing photoperiod group and approached the value obtained for the 16-hour photoperiod. The decreasing photoperiod group, which had been maintained at a 8-hour day-length since the end of the experiment, had an average index of thyroid activity which approached that of the 8-hour photoperiod group. Extrapolation of the output curves back to zero time indicated a greater theoretical uptake in the 8-hour photoperiod group than in the 16-hour photoperiod group (129% and 105%, re spectively) . Interpretation of the data for the hypothyroid group regarding the effect of photoperiod on thyroid activity is questionable because of dif- ferences in the degree of thyroidectomy that may have been attained in the various fish. A . A one-way analysis of variance on the data for the control fish showed there was no significant difference in thyroid activity of the fish under the various photoperiods. 69 Table 15. Regression coefficients (2) for each fish of the control and hypo- thyroid groups under each photoperiod in the second experiment. Continuous Increasing Continuous Decreasing 8 hr. Photo. 8-16 hr. Photo. 16hr. Photo. 16-8 hr. Photo Fish _b Fish l_) Fish 2 Fish 2 Control Fish 4828 -. 204 4914 - 118 4944 .089 4934 -. 169 4829 —. 132 4915 - 146 4945 .152 4935 -. 163 4830 -. 159 4916 - 218 4946 .148 4936 -. 140 4831 -. 141 4917 - 225 4947 . 231 4937 -. 141 4832 -. 202 4918 - 108 4948 .175 4938 - 151 4833 -. 198 4919 - 106 4949 .150 4939 - 116 4834 -. 174. 4920 - 124 4950 .148 4940 -. 131 4838 -.238 4921 -.103 4951 .119 4941 -.254 4922 -. 150 4952 .093 4942 -.243 4923 - 172 4943 -.148 bdean - 1786 »b4ean - 1525 Ldean 1450 bdean - 1656 Hypothyroid Fish 4839 -. 135 4905 - 169 4953 .097 4924 -.056 4840 -.124 4907 + 010 4954 . 075 4925 -. 157 4841 -.098 4908 - 162 4955 .107 4926 -.084 4842 -. 154 4909 - 130 4956 .081 4927 -.039 4901 -.008 4910 -. 192 4957 .183 4928 -.072 4902 -. 144 4911 -.073 4958 .035 4929 -. 113 4903 -.086 4912 -. 166 4959 .085 4930 -. 199 4904 -. 145 4913 -. 150 4960 .091 4931 -.008 4906 -.181 4961 .115 4932 +.051 4962 .138 4933 -.076 Mean - .1195 Mean - .1294 Mean .1009 Mean — . 0753 7O ~Figure 9. 1 Graph of the average regression coefficients of the control fish (solidvline) and hypothyroid fish (broken line) under each photo- period of the second. experiment. Regres sion Coefficient .20.. .184 .16- .14“ .12-1 .10-1 .08« .06" .04-1 .02" 71 I I l l r 8 .‘hr. 8—916 1hr. 16 1hr. 16—98 hr. .Constant Increasing . Constant Decreasing Photoperiod Photoperiod . Photoperiod Photoperiod 72 Results of studies of the effect of light on thyroid activity found. in the literature areiinconsistent although most of them indicate light may _ have an effect. Only one study has been directly concerned with the effect of photoperiod on thyroid activity. . Grant (31: a_._l.. (1961) studied the effect of light on thyroidal uptake of injected radioiodine by newts. Groups of experimental animals were kept in continuous darkness, a 12-hour day, and constant illumination. . Those kept under constant illumination had a very low uptake (0. 8-2. 8%) whereas those kept on a. 17.-hour diurnal condition and in continuous darkness showed initial high uptakes (6-22%). The latter two groups exhibited a leveling trend after a. week. and after four weeks they approximated the levels of animals kept under continuous illumination. Using histological criteria, Rasquin (1949) and Rasquin and Rosenbloom (1954) demonstrated that a hypertrophy of the thyroid occurred in Astanax mexicanus kept in darkness but that normal thyroid follicles were restored by exposure of the fish to light. . Baggerman~(l960), in studies of the migration of four species of Pacific salmon, felt that photoperiod was the external stimulus affecting the thyroid-pituitary system which in turn effected the induction of the migration disposition. . Hoar (1959) stated that both thyroid activity and photoperiod affect temperature resistance in goldfish although it is not known whether there is an interaction between these two factors.. Work of Robertson. (1958) as quoted from Hoar (1959) indicates that there may be an interaction. . She found that fish maintained on a short—day basis (8 hours of light) had a greater uptake of radioiodine than fish maintained on. a long-day basis (16-hours of light). . Since high uptake of radioiodine is associated with greater thyroid activity, her findings are in agreement with those pre- sented here for green sunfish. Berg e}: a_._l. (1959) felt that the seasonal pattern of thyroxinogenesis found in Fundulus may have been influenced by photoperiod although this was not studied separately. . Temperature was not a factor since all fish used were acclimated to and maintained at 73 a constant temperature. . Swift (1955) found that thyroid activity was more closely correlated with photoperiod than temperature although further studies by him led him to draw other conclusions. To the investigator's knowledge, this study is the first in which an attempt has been'made to evaluate the effects of different photoperiods on thyroid activity in fish. Studies have been carried out on the effect of different photoperiods in sheep. Hoersch e1: 11. (1961) found that 4,. 8, and 12 hours of light per day suppressed thyroid activity whereas increased light beyond 12 hours stimulated thyroid function. Their data are contrary to the data presented here for fish although measurements were not made using the same tech- nique or at comparable photoperiods. A comparison of the thyroid activity of the fish held at the two constant photoperiods was made prior to the start and at the end of the second experiment. . The mean regression coefficients and output half- times are given in Table 16. An increase in thyroid activity occurred in Table 16. Mean regression coefficients ()3) and output halftimes (T%-) for control fish of the 8-hour and 16-hour photoperiods at the beginning and the end of the second experiment. w Beginning of End of Experiment Experiment Photoperiod 2 Output T%- 13 Output Ti- 8-hour -. 1125 6.14 days -.1786 3.88 days 16-hour - .1134 6.16 days -.1450 4. 78 days both‘photoperiod groups during the experiment. A greater increase in activity occurred in the 8-hour photoperiod group. A Student's "t" test was performed to determine if the differences in thyroid activity were significant. The increase in thyroid activity in the 8-hour photoperiod 74 group was significant at the 1% level (t = 3.48 > t.9g5‘ = 3.169, df = 10). The difference in thyroid activity in the 16-hour photoperiod group was not significant. . It appears that short photoperiods stimulate thyroid activity and that there is an inverse relationship between photoperiod and thyroid activity. This is contrary to the findings of the effect of photoperiod on growth where greater growth occurs in fish under longer photoperiods. One might infer that the effect of photoperiod on growth is not mediated through the thyroid gland. The conclusions are tentative because the values obtained at the beginning of the experiment are based on a small sample size and a greater size range was involved in the determinations at the end of the experiment. . Further study, involving a larger sample and determinations on individual fish are necessary to resolve this issue. . It would be of interest to determine the thyroid activity of individual fish at different intervals during a varying photoperiod. . Comparison of Thyroid Activity of the Green Sunfish with Other Fish The percent uptake of radioiodine obtained in this study is incon- sistent with those reported in the literature for many fresh water teleosts. Berg it a_.__l. (1959) made a study of I131 uptake by eight fresh water teleosts and obtained values varying from 1.6% for Lepomis gibbosus to as high as 33. 1% for Umbra pygmaeus. . Some of the variation may have been due to seasonal factors. 2 Of particular interest were the results obtained for Lepomis gibbosus (pumpkinseed) which is closely related to the green sunfish. Berg it :11. (pp. c_1t.) found maximum uptakes for this species of 1. 6% and 3. 5% at 12 and 20 hours, respectively, after injection. Because of the low uptake of radioiodine by this species, the investigators felt that it had one of the least efficient thyroids of fresh water fish. 75 Gorbman (1959) inferred from these data that thyroxine was produced very slowly in this species if at all. In the study prior to the beginning of the second experiment, the average percent injected dose remaining in the green sunfish nine days after injection was 31% for the 8- hour photoperiod group and 21% for the l6-hour photoperiod group. . Extrapolating the output curve back to 24 hours and ignoring any extra—thyroidal accumulation of iodine, the percent uptake for the 8-hour group and 16—hour group is 76% and 52% respectively. . The study performed at the end of the second experiment was under almost identical conditions as those of Berg (it a_._l. (1959). They obtained an uptake of l. 6% for July and 3. 5% for August at temperatures of 230 and 240 C. in the pumpkinseed. This green sunfish study was made during early September (Sept. 2-9) at temperatures of 740-780 F. (23. 30-25. 69 C. ). . Extreme variability (4. 6%-47%) occurred in the percent injected dose remaining 10 days after injection; the mean percent injected dose remain- ing was 25%. . Extrapolating the output curve back to 24 hours after injection, the values ranged from 35% to over 100%; the mean percent in- jected dose was 100%. The results of this indicate that the green sunfish has a very efficient thyroid gland. . If one considers the uptake 24 hours after injection, such as when the maximum uptake occurred in the pumpkin- 1131 uptake than any of seed, the green sunfish has a greater efficiency of the fresh water teleosts studied by Berg gt a_l. (13. (2.3. ). Greater values were obtained for hypothyroid fish in this study than those obtained by Berg e_t a_._l. (2p. c_it..) The average content of 1131 for the hypothyroids 10 days after injection was 18% of the injected dose and 33% when extrapolated back to 24 hours after injection. Variations are known to occur between species, but the differences found here between the green sunfish and pumpkinseed which occur together in many waters appears so extreme that some other factor such as experimental methods must be responsible. 76 The biological halftime of radioiodine obtained for the green sun- fish was very short. The average output halftime for the controls under each photoperiod is given in Table 17. . Few studies reported in the Table 17. Average output halftime for control fish under each photoperiod at the end of the second experiment. Continuous Increasing » Continuous Decreasing 8-vhr. 8-16 hr. 16 -hr. 16-8 hr. . Photoperiod . Photoperiod Photoperiod . Photoperiod 3. 88 days 4. 54 days 4. 78 days 4. 18 days literature have involved in yijr—o determinations of thyroid activity in fish. Fromm and Reineke (1959) studied iodine output rates of rainbow trout in conjunction with the effects of thyroidectomy on oxygen consumption and obtained an output halftime of 15. 5 days at approximately 150 C.. Hoffert and Fromm (1959) determined the halftime output rate of rainbow trout and obtained a value of 37 days at approximately 150C. Differences be- tween the results reported in the literature and those reported here for the green sunfish may be due to the habitat preferences of these two species. The green sunfish is a warm-water fish whereas the rainbow trout is typically a cold-water fish. The influence of temperature on the thyroid function of fish is controversial (Leloup and Fontaine, 1960); however it appears reasonable to believe that warm water fish may have a greater thyroid activity than cold water fish.. Further study of .‘this matter is warranted. ’ 77 Summary Three of the four independent experiments performed involved a determination of the effect of photoperiod and thyroid activity on growth in total length and weight, rate of food consumption and efficiency of food conversion. The fourth experiment involved only the effect of photoperiod on the above measurements. There were four photoperiods (constant 8- hour, constant l6-hour, variable increasing 8 to 16 hours and variable decreasing 16 to 8 hours) and three thyroidal conditions (hyperthyroid, hypothyroid and control) used in this study. . Differences between the effects of a varying photoperiod and constant photoperiod were determined. The effect of photoperiod on thyroid activity was determined by measuring the rate of loss of radioiodine from the head region of the fish in the second experiment. . Conclusions Although, for the most part, statistical evaluation of the data obtained in this series of experiments indicated few significant differences, the repetition of trends in the data warrants the following conclusions: 1.. Photoperiod does influence growth of fish held at constant temperature. Growth expressed as gain in body weight or percent gain in weight as well as gain in total lengthvaried directly with the length of the photoperiod. Gain in total length appeared to be less dependent upon photo- period than did Weight gain. 2. The rate of food consumption and efficiency of food conversion were highest in fish held at longer photoperiods suggesting that the effect of photoperiod on growth is mediated through an increase in appetite and efficiency of food conversion. 3. A varying photoperiod had a greater influence upon growth than a constant photoperiod. Growth was enhanced by increasing the photo- period and depressed by a gradual decrease in photOperiod. 78 4. Growth of green sunfish appeared to be related to the avail- ability of thyroxine. Hyperthyroid fish exhibited greater growth and hypothyroid fish less growth than normal controls. Differences in growth measured by gain in total length showed a lesser correlation with the three“ "thyroidal" groups than gain in weight. 5.. No differences in food consumption occurred between the hyper- thyroid, hypothyroid and control groups. 6.. Efficiency of food conversion was-found to be greatest in the hyperthyroid fish and least in the hypothyroid fish, although the differences are small. 7. 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Wachowski. 1951. 1 The thyroid gland and its functions: in cold-blooded vertebrates. Quart- Rev. Biol. , 26: 123-168. McFadden, J. T. 1961. A population study of the brook trout Salvelinus fontinalis. . Wildlife Society Publication, Wildlife Monogratho. 7, 73p. Pickford, G. E., and J. W. Atz. 1957. The physiology of the pituitary gland offishes.‘ New York 2001. Soc. ,. New York. 613 p. Rasquin, P. 1949. The influence of light and darkness on the thyroid and the pituitary activity of the characin Astyanax mexicanus and its cave derivatives. Bull. Amer. Mus- Nat. Hist., 93(7): 501-531. Rasquin, P. , and L.. Rosenbloom. 1954. - Endocrine imbalance and tissue hyperplasia in teleosts maintained in darkness. 1 Bull. Amer. Mus. Nat. Hist., 104(4): 363-425. Roberts, J. Influence of photoperiod on respiration of sunfish. Quoted by R. 0. Anderson, 1959. Robertson, G. B. 1958. . Temperature resistance and thyroid activity in goldfish maintained under controlled photoperiods. University of British Columbia. (unpublished thesis). 9 . Scott, J.. L. 1953. The effects of thiourea treatment upon the thyroid, pituitary and gonads of the zebra fish Brachydanio rerio. Zoologica, N- Y., 38: 53-62. 83 Simpson, G. G., A. Roe, and R.. C. Lewontin. 1960. Quantitative Zoology. Harcourt, Brace and Company, NeW'York, 440p. Simpson, M. E., C. W. Asling, and H- M. Evans. 1950. . Some endocrine influences on skeletal growth and differentiation. . Yale Journ. Biol. Med. 23: 1-27. Smith, D. C. and G.. M. Everett. 1943. The effect of thyroid hormone on growth rate, time of sexual differentiation and oxygen consumption in Lebistes reticulatus. Journ.. Exptl. Zool. 94: 229-240. Spoor, W. A. 1938. Age and growth of the sucker, Catostomus commer- sonnii (Lacepede), in Muskellunge Lake, Vilas County, Wisconsin. Trans. Wisc. Acad. Sci. , Arts and Lett., 31: 457-505. Sprugel, G. , Jr. 1954. Growth of bluegills in a new lake, with particular reference to false annuli. Trans. Amer. Fish.. Soc. , 83: 58-75. Swift, D. R. 1955. Seasonal variations in the growth rate, thyroid gland activity and food reserves of brown trout (Salmo trutta Linn. ). Journ.. Exptl. Biol., 32(4): 751-764. Swift, D. R. 1959. Seasonal variation in the activity of the thyroid gland of yearling brown trout Salmo trutta. Journ. Exptl. Biol. 36(1): 1 20-125 . Swift, D. R. 1961. . The annual growth- rate cycle in brown trout (Salmo trutta Linn.) and its cause. Journ.. Exptl. Biol. , 38(3): 595-604. Tryon, C. A. , Jr. 1943. . The effect of covering hatchery troughs-on the growth of cutthroat trout (Salmo c1arkii.). Trans. Amer. Fish. Soc., 72: 145-149. A PPENDICES 84 APPENDIX A Mean measurements and increments in total length and weight of fish in each tank of the first three experiments. Initial total lengthrand weight and increment in total length. and weight of each fish of the fourth experiment. .85 86 ans—... oazv 3N3» om.N Mg .m on...“ .Em CH3 ow6. omA: owfi. «.mé... 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Hm; om.m 08.: omé. mm.o~ 08.x: wmdfi 33:: wméh 0880 3.3330. mo... omtv vm.o O¢.N $m.O O©.H ®¢.wd 003:”: m©.h.~ ON.®© Hatha Omfib @MOHKAAHHQQKAEH 00Mu0m000£n~ ndomwuofi fim00000fl , 33¢ wwfi nwd oogv 30.: .56 mném mméo: wad: mN.No: 3a.: wmdo 3002309an N80 omd mim om.m omé cod £04m oofig mwdd 23:: 3%.: owKa 3.3000 8 .2. 8.8 8 .m 85 8.: 8.0 8.: 8.3: 3.2 8.2: 3.2 8.8 Emftmfim 0030m000£m 000$ 31w 9:000:05 wad. omd mm; 32m owd ow.N 08;: 08.30 Sim: om.mo mmzz omdm Hofffioam 3.0 mw.o end :.m 0w; N80 08.: mméb: 00.2 NN.NoH 00.: omfio #03000_ 8.0 8.2 8.0 8.... mm .m 8.3 3.3 8.2: 3.2 8.2: 3.2 8.3 203.03%: 0030m000£nm udomuw 0505:3000 . Em . SE . Em . SCH .. Em . 88 . Em . SE . Em . 5E . Em . an: £033.50 0 .03 .4.8 JCS .1H.8 .03 .178 .03 .178 .0? .178 .03 .178 0:003 o H0>O 0:003 m cam 0:00? m um: . M003 50 M00? 0.3m H0055 30:00:03,0002 00080000002 0002 0.00530me 0coo0m 0H3 mo x008 £00m a: Lmfih .0 mo 3mg? 0:0 suwc0d 0008 a: 0:050:05 0:0 200320050002 G002 88 00.3 00.0 00.0 00.3 30.0 30 .0 00.0 00.0 30.0 .80 3;? 00.0 0¢.0 00.0 00.0 00.0 00.0 00.0 00.0 :43 00.0 00.0 00.0 .83 43.0... 33035 0 .350 00.0 00.3 00.0: 03.3 03.0: 00.3 03.0 33.0 00.0 03.0 00.0 00.0 00.0 00.0 00.3 03.0 00.3 33.0 $0.0: 00.0 00.0: 00.3 30.0: 00.0 .cfim . .uflcn .»>P :H.H. 30003 0 0:0 00.0 00.0 00.0 00.0 00.0 00.0 00.3 00.0 00.3 03.0 00.3 00.0 00.0 00.0 00.3 0¢.¢ 30.0 00.0 00.0 00.0 00.0 00.0 00.3 00.0 .80 .59“ 3:5 4.3.8 3303 0 “ma unmgouofi 532 00.v3 00.00 00.03 00.00. ¢¢.¢H 0¢.00 00.03 0v.00 00.0H 00.00 00.03 00.¢0 00.03 00.00 00.03 00.00 00.03 $0.303 00.03 00.¢0 03.03 00.00 30.03 00.00 Jhw .cQQH 45> {H.B . Mom? £00 *0 . 3 H 00 . 00 03050032303 00.: 00.00 HOSGOU mm . 2 3. .00 Boigtoam 03.390095” H.903: 03 353330 0 om 4 H $ .2. 333895 3 .2 2 .3 3:80 E .2 E .3 2832205 003008.003 H503 0:03 mafimMonomQ 30.¢3 00.00 00.03 00.00 00.00 0¢.¢0 00.03 00.¢0 00903 00.00 30.03 00.30 00.0H 00.00 0w.mH o~.wm 00.03 00.00 oH.mH o¢.m0 00.03 00.30 00.03 00.00 .95 .55 55> 40.5 . fie? Em mo .2 om .H m 20.23895 3 .2 ow .3 3:80 mo . 2 mm .00 Bobfifimfim 0032300033 H503 03:0 mmwmmmuocH mm .fi ow .oo Refifiofim $0.: 3.3 33.80 3. . S 3. .mm Boifiumfim 00TH 23000.03 . H903 u 0 m 505.330 0 650 .86 cofiflucoU .03 ...H ...H. 1... 333 unmgmudmmoz Gmoz acmewummxm UHEH 05. mo «Bah. nomm cw swam .m 00 5033 95 5.98.4 3.30.0 cw ”2580.353 can 305035032 532 Iitial Weight and Increment in Weight for Each Fish of the Fourth Experiment ..‘4 0 0 0 0 23 000000 000000 000000 000000 00 000000 000000 000000 000000 .5.) 01.14 0 0 0 0 :3 000000 000000 000000 000000 M0 000000 000000 000000 000000 .0 .0 .0.0 H N M \O 0‘ .0 x x 0 J‘ 0 rhé 0 0 0 '23 $000000 $000000 “3000000 {3000000 00 [4000000. {-4000000 8000000 H000000 Ad 0 0 0 0 ...3 000000 000000 000000 000000 m ...... ...... 0.... 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I wofiuomgogm 9503-0 mdodcflno x3 m x3 o x3 m x3 m 33:: is x3 c x3 m x3 m 32.3 xam x3 o x3 m x3 m 353 mm 3 m am in gm “3 E m $3..” 25 ”E in $20. 95. a: ”28832530 Minnow 23 00 03h 00mm .80 Amumuofififigv £9810 0.30.0. 5 £850.35 05m £09810 030,0. 33¢ APPENDIX B Rates of food consumption (per cent body weight) for each tank of fish for each three week period and the six week period of each experiment. 91 92 Data from First Experiment Food Consumption (Percent Body Weight) for the 6~Week Period Condition Hype rthyroid Control Hypothyroid Weighted Mean Photo. Food Consumption (Percent Body Weight) for the First 3-Week Period Condition Hype rthyroid Control Hypothyroid 8 hr. 45.12 35.99 34.56 38.78 8hr 21.83 17.32 17.67 8-16 hr. 16 hr. 16-8 hr. Weighted Mean Thy. 43.15 62.93 47.60 50.02 43.72 55.76 39.33 44.07 41.06 53.05 47.00 44.14 42.64 57.24 44.67 8-16 hr. 19.54 22.36 20.36 16 hr. 29.40 23.03 23.52 16-8 hr. 26.35 20.97 24.61 Food Consumption (Percent Body Weight) for the Second 3*Week Period Condition Hype rthyroid Control Hypothyroid 8 hr. 25.47 21.39 18.41 8-16 hr. 24.00 25.22 21.71 16 hr. 32.60 33.51 30.09 16-8 hr. 20.14 18.19 22.17 93 Data from Second Experiment Food Consumption (Percent Body Weight) for the 6-WeekPeriod Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Weighted Mean Thy. Hyperthyroid 99. 56 81. 40 99. 94 56.15 84. 79 Control 71.48 76.53 73.80 74.35 74.07 Hypothyroid 78.51 72.95 86.71 72.06 75.87 ' Weighted Mean Photo. 83.70 77.06 87.11 67.60 Food Consumption.(Percent Body Weight) for the First 3-Week Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. . Hyperthyroid 58. 72 42. 74 64. 56 31. 05 Control 39.16 37.75 46.63 46.17 Hypothyroid 43. 36 32.16 53. 38 43. 77 Food Consumption (Percent Body Weight) for the Second 3""Week Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Hyperthyroid 41. 37 40.91 34.07 25.19 Control 33. 14 40. 15 25.69 28.49 Hypothyroid 37.08 48.18 32.18 28. 63 94. /"\ Data from Third Experiment Food Consumption (Percent Body Weight) for the 6-Week Period Condition Hyperthyroid Control Hypothyroid Weighted Mean Photo Food Consumption (Percent Body Weight) for the First 3'Week Period Condition Hype rthyroid Control Hypothyroid 8 hr. 83.78 82.47 63.14 76.55 8 hr. 47.10 42.40 46.98 8-16 hr. 16 hr. 16-8 hr. 117.12 110.17 71.40 84.94 99.92 88.39 72.13 98.82 76.86 91.70 103.01 79.05 8-16 hr. 16 hr. 16-8 hr. 72.06 74.04 35.23 45.39 64.89 52.33 39.67 67.69 46.33 Weighted Mean Thy. 97.01 88.94 78.14 Food Consumption (Percent Body Weight) for the Second 3‘Week Period Condition . Hype rthyr oid Control Hypothyroid 8 hr. 33.77 46.16 18.17 8-16 hr. 16 hr. 16-8 hr. 42.31 . 31.89 34.00 39.77 31.18 34.35 32.72 28.25 31.87 95 Data from Fourth Experiment Food Consumption (Percent Body Weight) for the 6-WeekPeriod T ank 1 2 3 Weighted -Mean 8 hr. 45.60 40.56 46.61 44.35 8-16 hr. 51.63 58.98 54.29 55.15 16 hr. 57.43 60.77 57.79 58.74 16-8 hr. 48.13 54.59 64.04 55.74 Food Consumption (Percent Body Weight for the First 3-Week Period Tank 1 2 3 8 hr. 28.87 23.60 28.10 8-16 hr. 30.38 31.90 32.58 16 hr. 34.86 36.09 37. 76 16-8 hr. 33.56 38.75 44.40 Food Consumption (Percent Body Weight) for the Second 3’Week. Period Tank 1 2 3 8 hr. 19.75 17.40 18.99 8-16 hr. 22.65 28.77 22.22 16 hr. 22.52 25.35 24.93 16-8 hr. 11.31 14.81 18. 38 Food Consumption (Percent BodyWeight) for the Third 3"Week Period Tank 1 2 3 8 hr. 21.13 18.19 16.32 8-16 hr. 24.01 28.98 23.69 16 hr. 13.21 18. 31 18.84 16-8 hr. 9.23 9.36 10.42 APPENDIX C Efficiency of food conversion (percent) for each tank of fish for each three-week period and six-week period of each experiment. 96 97 Data from First Experiment Percent Efficiency of Food Conversion for the 6-Week Period Percent Efficiency of Food Conversion for the First 3—Week Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Hyperthyroid 63.06 51.18 79.49 72.59 Control 39.45 37.97 69.70 50. 33 Hypothyroid 20.48 34.01 71.14 34. 17 Percent Efficiency of Food Conversion for the Second 3*Week Period Hyperthyroid 80.30 61.53 47.90 26.57 Control 61. 69 86.04 75. 31 37.66 Hypothyroid 58. 27 74. 50 73. 11 20. 20 Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Weighted Mean Thy. Hyperthyroid 72. 78 57. 20 61.39 51.30 60.78 ~ Control 51.63 63.96 73.31 44.17 60.49 Hypothyroid 39.94 56.11 72.34 27. 33 51.31 Weighted Mean Photo. 57.05 59.25 68.72 41.03 98 Data from Second Experiment Percent Efficiency of Food Conversion for the 6-Week Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr.- Weighted Mean Thy. Hyperthyroid 30. 74 28.85 23.86 17.61 25.12 Control 28.22 31.47 17.31 21. 21 24.73 Hypothyroid 18.24 25.04 29.62 10.71 23.12 Weighted Mean Photo. 26.60 28.59 23.94 17.11 Percent Efficiency of Food Conversion for the First 3"Week Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Hyperthyroid 30. 91 16.86 29. 08 9.84 Control 23.39 23.35 26.50 19.98 Hypothyroid 13.71 18.87 36. 47 15.84 Percent Efficiency of Food Conversion for the Second 3"Week Period Condition 8 hr. .8-16 hr. 16 hr. 16-8 hr. Hyperthyroid 30. 53 39.95 15.05 11.76 Control 33. 38 38. 22 1.64 23.06 Hypothyroid 23. 32 29. 37 19. 59 20. 16 99 . Data from Third Experiment Percent Efficiency of Food Conversion for the 6'Week Period Percent Efficiency of Food Conversion for the Second 3"Week. Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Hyperthyroid 23.24 32. 37 31.85 48. 52 Control 19.03 22.72 32.59 29.28 Hypothyroid 15.41 16. 38 32.43 25. 17 Percent Efficiency of Food Conversion for the Third 3"Week‘ Period Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. .Hyperthyroid **** 21. 98 **** 10.. 39 Control **** 22.99 **** 9. 23 Hypothyroid **** 19. 27 1. 95 2. 83 >:<>:<>:<* Weight loss occurred; no calculation of food conversion possible. - Condition 8 hr. 8-16 hr. 16 hr. 16-8 hr. Weighted Mean Thy. Hyperthyroid 8.19 28 . 03 20. 42 28. 84 21. 97 Control 6.65 21.93 19.71 20.90 17.83 Hypothyroid 7.47 17.74 22.76 16. 24 17. 14 Weighted Mean Photo. 7.44 23.48 20.97 21.63 100 Data from Fourth Experiment Percent Efficiency of Food. Conversion for the 6-Week Period Tank .8 hr. .8-16 hr. 16 hr. 16-8 hr. 1 61.25 85.02 66.75 47.24 2 73.62 88.56 80.72 64.99 3 76.45 73.40 73.66 70.42 Percent Efficiency of Food Conversion for the First 3'Week Period Tank 8 hr. 8-16 hr. 16 hr. 16-8 hr. 1 60.34 74.33 72.70 63.65 2 70.31 80.88 84.50 84.49 3 74.42 73.44 71.33 84.32 Percent Efficiency of Food Conversion for the Second 3~Week< Period Tank 8 hr. .8-16 hr. 16 hr. 16-8 hr. 1 62.60 _96.44 59. 12 0.93 2 77.49 95.08 76.49 19.87 3 78.96 73.35 76.59 43.24 Percent Efficiency of Food Conversion for the Third 3'Week Period 'Tank 8 hr. 8-16 hr. 16 hr. 16-8 hr. 1 88.02 57.70 43.25 §¥¥** 2 98.21 74.90 64.83 44.99 3 82.07 64.75 64.19 27.75 4444* Weight loss occurred; no calculation of food conversion possible. NIVERSITY LIBRARIES Hill 1 "1111111111111!1|“! l 3 12931306188]