The Testicular Cycle of Blue Grouse (Dendragapus Obscurus
THE TESTICULAR CYCLE OF BLUE GROUSE
(Dendragapus obscurus fuliginosus)
AND ITS RELATION TO
AGE, BREEDING BEHAVIOUR AND MIGRATION
by
BENJAMIN R* SIMARD
Diploma in veterinary medecine Universite de Montreal, P.Q.
A thesis submitted in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
in the Department
of Zoology
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
December, 1964- In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of
British Columbia, I agree that the Library shall make it freely available for reference and study* I further agree that per• mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that.copying or publi• cation of this thesis for financial gain shall not be allowed without my written permission*
Department of Zoology •
The University of British Columbia,
Vancouver 8? Canada
Date December 10th ABSTRACT
The testicular cycle of blue grouse (Dendragapus obscurus
fuliginosus) has been studied from the testes of two hundred and
twenty-four birds collected on their summer range on Vancouver Island over the years 1958 to 1964*
For two age classes, adult and yearling, the characteristics of the following cycles were compared and correlated. The cycle of
increase in size of the testes, the cycle of increase in diameter of the seminiferous tubules, the cycle of the development of the stages of spermatogenesis, the cycles of relative and absolute increase of
the interstitium and the cycles of abundance of interstitial cells.
Samples of sperm from these two age classes were compared. Further• more, these cycles were compared with migration, with the period of egg laying in the female, with behaviour, with age of the bird over
two years of age and with body weight.
In all these tests, the yearlings had a shorter breeding
cycle characterised by a slower recrudescence, a delayed.and shorter
breeding period, a faster and an earlier regression. Although the yearlings had smaller testes and a lesser amount of tubular tissue,
they all seemed to develop all the stages of spermatogenesis and to produce fertile sperm. The smaller amount of intertubular tissue
in yearlings was suspected to be correlated with their secretive
behaviour and the smaller growth of the testes of the yearlings was
attributed to an inherited character rather than to inhibition by
the environments, No difference was found in the testicular cycle iii between hooting and silent males and between replacement or territorial yearlings and silent ones.
It was 'concluded that all male birds present on the breeding range were apparently potential breeders. Smaller interstitium and delayed maturity seemed to be the main factors preventing the young birds from adding to the population of breeders. xii
ACKNOWLEDGMENTS;
I am deeply indebted to members of the Department of Zoology,
at the University of British Columbia, who made this study possible.
Dr. J.F. Bendell, my supervisor, obtained financial support and
provided necessary facilities. I am grateful to him also for his helpful criticism while writing the thesis. Dr. P. Ford guided me
in the histology work and Dr. W.S. Hoar and Dr. I. McT. Cowan provided advice and criticism during the study.
This work was supported by a research grant from the National
Research Council of Canada, by financial help from the British Columbia
Department of Recreation, Fish and Game Branch and by a "Bourse de perfectionnement du Ministere de la Jeunesse de la Province de Quebec".
I am grateful to F.C. Zwickel who helped me in the analysis
of some of the material from the field, permitted me to use some of his data, discussed the problems with me and took upon himself the
tiresome task of correcting the manuscript. Acknowledgments are also due to my colleagues P.W. Elliott, A. Lance and I. Stirling and every other student that worked on this project at one time or^ another and who helped me in many ways.
I finally thank my wife Louise who never ceased to give me all the support and help needed. iv
TABLE OF CONTENTS
ABSTRACT ii
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES
LIST OF PLATES x
ACKNOWLEDGMENTS xii
INTRODUCTION 1
LITERATURE REVIEW 3
LIFE HISTORY OF THE BLUE GROUSE 6
STUDY AREA 11
MATERIAL AND METHODS
Material 12
Methods 13
Histological techniques 16
THE TESTES AND ACCESSORY ORGANS 18
RESULTS
1.. Size of the testes 20
Results 20
Statistical analysis 24
Comparisons between different populations 27
Relation of size of the testes to migration 27
Relation of size of the testes to the period of egg laying 29 Relation of size of the testes to body weight 30 V
Relation of size of the testes to territorial behaviour 30
Relation of size of the testes to age 32
Conclusions 33
2. Size of the seminiferous tubules 34
Results 35
Relation of size of the seminiferous tubules to the period of egg laying 38
Relation of size of the seminiferous tubules to
territorial behaviour 38
Relation of size of the seminiferous tubules to age 39
Relation of size of the seminiferous tubules to volume
of the testes 39
Conclusions 42
3. Spermatogenesis 43
Results 4-9 Analysis of semen 51 Relation of stages of spermatogenesis to the diameter of the seminiferous tubule 53
Relation of stages of spermatogenesis to the size of the testes 54
Spermatogenesis in juvenile males 54
Conclusions 54
4. Size of the interstitium 57
Results 59
Relation of the absolute and relative amount of inter- tubular tissue to behaviour 65
Relation of the absolute and relative amount of inter- tubular tissue to age of bird 66
Conclusions 66 5. Histology of the interstitium
Histology of the interstitium of the blue grouse
Results
Conclusions
DISCUSSION
SUMMARY
LIST OF REFERENCES
APPENDIX I
APPENDIX II Vll
LIST OF FIGURES
Figure
1. Seasonal variation in the average volume of the testes of the adult and yearling 22
2. Linear regressions of three parts of the breeding cycle in adults and yearlings, M.Q.L., 1958-1964- 26
3. Size of the testes in two populations. Bendell (1954-), M.Q.L. 1964- 28
4» Size of the testes in two populations. Standing (i960), M.Q.L. 1964 28
5. Number of males observed / hr. / 2 weeks period at M.Q.L. during the summer I960 and the corresponding volume of the testes in adults 31
6. Monthly average and range of the weight of adults and yearling males, M.Q.L., 1959-1962 31
7. Seasonal variation of the average diameter of the seminiferous tubules in adults and yearlings, M.Q.L., 1958-1964 36
8. Relation of the diameter of the seminiferous tubules to the volume of the testis during recrudescence period in adults and yearlings. M.Q.L., 1958-1964 41
9. Range of stages of spermatogenesis for each week of the summer in adults and yearlings, M.Q.L., 1958-1964 50
10. Relation of volume of the testis to stages of spermato• genesis, M.Q.L., 1958-1964 55
11. Relation of the diameter of the seminiferous tubules with stages of spermatogenesis, M.Q.L., 1958-1964 55
12. Weekly average of the percentage of intertubular tissue in adults and yearlings, M.Q.L., 1958-1964 60
13. Weekly averages of the volume in cc of intertubular tissue in adults and yearlings, M.Q.L., 1958-1964 | 62
14. Relation of percentage intertubular tissue to stages of spermatogenesis in adults and yearlings, M.Q.L., 1958-1964 64 Estimated trend of the cycle of the different types of interstitial cells in yearlings
Estimated trend of the cycle of the different types of interstitial cells in adults, M.Q.L., 1958-1964 ix
LIST. OF TABLES:
Table
I Number of pairs of gonads used in this study and their area of origin 12 II Age class and number of males in each age class collected by 1958 to 1964 U III Number of samples of known age birds for each age group 14
IV Statistical summary of the seasonal growth rate of testes of blue grouse, adults and yearlings, Middle Quinsam Lake, 1958-1964 23
V Equations for the regression of the mean of the testes volumes at weekly intervals, over the period of recrudescence, breeding and regression in adults and yearlings 25
VI Comparison of the size of the testes of known age birds with the average of the adult class 33 VII Number of testes examined, weekly average of the size of the tubules and range for adult and yearling age classes 37
VIII Comparison of the size of the tubules of known age birds with the average of the adult class 39 IX Equations of the linear regressions for the size in microns of the tubules against volume of the testis 40 X Average amount of intertubular tissue in percentage and in absolute amount in adults and yearling males during the spring and summer season 61
XI Comparison of the amount of intertubular tissue of known age birds with the average of the adult class 66 XII Data on testes used in this study (APPENDIX ||) 104 LIST OF PLATES
Plate
1» Stage I of spermatogenesis, X4-00. Adult specimen no. 3, taken March 19, 1964 at Wolf Lake. 98
2. Stage II of spermatogenesis, X400. Adult specimen no. 3, taken March 19, 1964 at Wolf Lake. 98
3* Stage III of spermatogenesis, X400. Adult specimen no. 10, taken March 19, 1964 at Wolf Lake. 98
4. Stage IV of spermatogenesis, X400. Adult specimen no. 19, taken April 9, 1964 at Wolf Lake. 98
5. Stage IV of spermatogenesis, X1000, Adult specimen no. 19, taken April 9, 1964 at Wolf Lake. 99
6. Stage V of spermatogenesis, X400. Adult specimen no. 23, taken April 10, 1964 at Wolf Lake. 99
7. Stage V of spermatogenesis, X1000., Adult specimen no. 30, taken April 12, 1964 at Wolf Lake. 99
8. Stage VI of spermatogenesis, X400. Adult specimen no. 42, taken April 20, I960 at M.Q.L. 99
9. Stage VII of spermatogenesis, X200. Adult specimen no. 78, taken May 6, I960 at M.Q.L. 100
10. Stage VIII A of spermatogenesis, X150. Adult specimen no. 163, taken June 13, 1961 at M.Q.L. 100
11. Stage VIII B of spermatogenesis, X200. Adult specimen no. 214, taken August 31, 1963 at M.Q.L. . 100
12. , Winter stage of spermatogenesis, X1000. Yearling specimen no. 215, taken September 1, 1962 at M.Q.L,. 100
13. Spermatozoa, X1280., Sample from aviary bird, taken June 5, 1964. Stain Eosin nigrosin. 10IE
14. Intertubular tissue in February, X400. Specimen no. 284, died February 19, I960 in the aviary. 1°1
15. Large vesicular type of interstitial cell, X1280. Adult specimen no. 3, taken March 19, 1964 at Wolf Lake. 101 xi
Plate :
16. Exhausted vesicular type of interstitial cell, X1280. Adult specimen no. 3, taken March 19, 1964 at Wolf Lake. 101
17. Young and vesicular types of interstitial cells, X1280. Adult specimen no. 78, taken May 6, I960 at M.Q.L. 102
18. Young type of interstitial cell, X1280. Adult specimen no. 124, taken May 24, 1961 at M.Q.L. 102
19. Juvenile type of interstitial cell, X1280. Adult specimen no. 79, taken May 6, I960 at M.Q.L.. 102
20. Interstitial cells at the beginning of appearance of droplets, X1280. Adult specimen no. 100, taken May 12, 1962 at M.Q.L. 102
21. Juvenile interstitial cell full of droplets, X1280, Adult specimen no. 208, taken August 1, 1962 at M.Q.L., 103
22. Juvenile interstitial cell after disappearance of droplets, X1100. Yearling specimen no. 215, taken September 1, 1962 at M.Q.L. 103 INTRODUCTION
7; This study describes-the testicular cycle in the blue grouse
(Dendragapus obscurus fuliginosus (Ridgway)) on its breeding range. The study of the testis was undertaken to contribute to the understanding of the reproductive biology of grouse. It is of broader importance in the relationship between breeding behaviour and population dynamics.
The number and behaviour of blue grouse on the breeding range vary with time of year and age of bird. A second objective therefore was to find the relationship between gonadal activity, age and behaviour of males on the breeding range. To these ends, anatomy and histology of the testis of wild birds were investigated and correlation will be made with migration, seasonal abundance, age and breeding behaviour.
Benoit (1956) grouped the phenomena of the sexual cycle under two headings; l) "phenomenes physiologiques" and 2) "actes de compor- tement". He agreed with Dunan's views:: "II n'y a aucune difference de nature entre ce qu'on appelle instinct et ce que l'on appelle fonction physiologique, si ce n'est que le premier est observable au dehors, et que la seconde ne l'est pas". The breeding behaviour of a bird1 is the result of two groups of factors! external factors (environment) and internal factors (endocrinology, metabolism etc.). The internal factors will be indirectly investigated by two approaches to the anatomy of the testis: l) the end organ function of the testis
(volume of the whole testis, size of the tubules, stages of the germinative epithelium) and 2) the endocrine function of the testis
(volume and percentage of intertubular tissue and sequence of appearance 2
and disappearance of Leydig cells).
The different races of blue grouse have been the object of several studies related to their ecology, numbers and behaviour (Bendell,
1954; Caswell, 1954; Heebner, 1956j Boag, 1958; Mussehl, 1958; Fowle,
I960; Boag, 1964; Elliott, m.s.; Bendell and Elliott, m.s.). Few authors though (Schottelius, 1951; Standing, I960) have studied the histology of the gonads. The preceding works suggested the following questions: Is the different behaviour of adults and yearlings, as well as between individuals, reflected by similar differences in the testis?
Are the yearlings able to fertilize a female? If so, how does the testicular maturation in the yearlings correlate with that of adult males?
This work is a phase of a broader study on the regulatory factors of a blue grouse population led by Dr. J.F. Bendell. Material for this work was collected by different students on Vancouver Island over the years 1958 to 1961. I participated in the field work during the summer of 1962 and the spring of 1964 in order to collect additional material and round out the collection on hand. 3
LITERATURE REVIEW
The anatomy and histology of the reproductive organs of birds
in general are well known. Many studies have been done on domestic
gallinaceous birds. To name a few that have been most useful: Rowan (1929),
Hogue and Schnetzler (1937) and Miller (1937) on spermatogenesis and the
identification of the different types of cells in the germinative
epithelium; Carson and all (1955), Taneja and Gowe (1961) and McDaniel
and Craig (1962) on the evaluation of fertility; Orban (1929)> Pfeiffer
and Kirschbaum (194-5), Allee (1955), Benoit (1956), Marshall (I960) and
Guhl (1962) on the function of the interstitium; Bissonnette (1937),
Hohn (194-7), Johnston (1956) and Johnson (1961) on the reproductive cycle
and sexual behaviour.
Among the works done on wild gallinaceous birds with an annual
reproductive cycle, most studies have been concerned with the ring-
necked pheasant (Phasianus colchicus). Mc Atee (194-5) recognised seven
phases in the sexual cycle of the pheasant. These phases were erected
mainly on data on the behaviour of the bird throughout its annual cycle.
Hiatt and Fisher (194-7), in their, histological study of the testis of the
ring-necked pheasant, divided the annual cycle into three phases:
breeding, regression and recrudescence. They worked with monthly
averages, and concluded that no difference existed between age classes
(adults and yearlings) in the timing or in the amount of increase in
size of the testis. Kirkpatrick (1944- (a)), working in Indiana, noted
that an increase in testicular weight began on February 16, when males were
271 days of age. Kirkpatrick and Andrew (1944-)* working with pen raised
pheasants, found that some chicks had spermatocytes in their tubules in what can be called the second phase of growth, between 81 and 14-6 days of age..
Mackie and Buechner (1963) did not use microanatomy in their study of the reproductive cycle of the male chuckar (Alectoris graeca).
Their four stages, recrudescence, breeding, regression and quiescence are based on testis size, behaviour, interaction and plumage characteristics
The blue grouse, unlike most galliform species, migrate each year between an upland winter range and a lowland breeding habitat. One must then turn to work done on non-galliform species to find detailed studies on migratory species with an annual breeding cycle. Bullough (1942), in his comparison of two different races of starlings (Sturnus vulgaris)« described in detail the histology of the testis. Bissonnette (1930 (a,b)) and Bissonnette and Ghapnick (1930) described the complete annual cycle of the testis in the European starling. They found no indication of any secretory activity in the Leydig cells at any time. Blanchard (194-1) and Blanchard and Erickson (1949) did a very detailed study of spermato• genesis and the interstitial tissue in the white crowned sparrow
(Zonotrichia leucophrys). This study demonstrated clearly thepre- migratory changes in the testes. They described seven stages of testicular development. Each includes the modifications of the germinative epithelium as well as those of the intertubular cells. Johnston (1956) took a different approach to the same problem. In his study of the
California gull (Larus californicus) he did not include the modifications of the interstitial cells in his determination of seven stages of testicular development. Wright and Wright (1944) compared the rate of
sexual maturation of two age groups (yearlings and adults) of red-winged blackbirds (Agelius phoenicus) and correlated their different rate of 5 maturation with different migration and breeding behaviour.
The literature on the interstitial tissue of migratory species is less abundant. Benoit (1922) showed that the volume of interstitial cells ran parallel with the development of secondary sexual characteristics.
Later (1924.) he presented quantitative evidence to show that the endocrine function of the testis remains unimpaired after the destruction of tubular tissue. At least two more detailed studies of the histology of inter• tubular tissue are available: Blanchard and Erickson (194-9) on the white crowned sparrows already mentioned, and Marshall (194-9) on the fulmar
(Fulmaris glacialis):. This last author recognised the same six different types of interstitial cells as Blanchard and Erickson and he correlated the histology of the interstitium with movement, behaviour and molt of the birds on the breeding ground.
As mentioned previously, few studies are available in the literature on the reproductive cycle of male blue grouse. Most authors
(Bendell, 1955; Standing, I960; Schottelius, 1951; Boag, 1964-) have mentioned the relatively larger size of the testis of adults on the breeding range as compared to yearlings. Schottelius (1951) stated that after July 11 no birds, adult or juvenile, had sperm in their testes. He also noted that the presence of sperm corresponds closely to testis measurement. Standing (i960) described stages of spermatogenesis of adults but he did not have enough yearlings to attempt a comparison between the two age groups. He concluded though that "sub-adult males
(yearlings) appear to undergo some sexual stimulation but not to the same degree as adult males"• 6 LIFE HISTORY OF THE BLUE GROUSE
Since we will correlate the events of spermatogenesis with
numbers of birds and breeding behaviour on the summer range it is worth• while to present a short description of the main events of the life history
of the blue grouse on its breeding range. Only those aspects necessary
for a clear understanding of the testicular cycle will be presented here.
Most of these data are taken from Bendell (1954).
The blue grouse, a member of the family Tetraonidae, is a bird
of the new world. It is an inhabitant of the coniferous forests of western North America. Its distribution follows and includes most of the mountain ranges of the west coast and Rocky Mountains, from Yukon Territory
to south-west California and New Mexico (Aldrich, 1963). The species shows
considerable subspecific variation that coincides with the climax forest.
The subspecies fuliginosus. which is present on Vancouver Island and the
Pacific coastal forests of the continent, is the race with which this
study is concerned.
Blue grouse generally inhabit distinct wintering and breeding
ranges and show a characteristic altitudinal migration (Bent, 1932).
Little is known of the species on its wintering range and migratory movements have been rarely observed. Anthony (1903) and Standing (i960) noted that in the spring males migrated in large flocks, moving down onto breeding range on foot and on the wing, and that the males must be the
first to arrive on the breeding range. On his study area at Lower
Quinsam Lake, on Vancouver Island, Bendell observed that the downward
migration of the males and females must have occurred between March
the 6th and April the 13th. The upward migration of the males, on 7 the same area and for the same year, is said to have started the last week of April or the first week of May and to have been near completion by August. Such a pattern was also confirmed by Boag (1958), Hoffman (1956),
Heebner (1956), Standing (I960) and Elliott (m.s.). Bendell proposed that the number of male birds seen per hour, per week, was a good index of the trend of the male population on the breeding range over the summer and arrived at his conclusions on the basis of this kind of data.
As in all species of Tetraonidae, the two outer primaries of the immature blue grouse are not replaced during the post-juvenile molt. It is not until the bird is well over one year of age that these feathers are replaced (Wright and Hiatt, 1943). Two age classes can therefore be distinguished in the spring on the breeding range: the adults, which are beyond two years of age, and the year old birds, referred to here as yearlings. Yearlings have the two outer primaries more pointed, narrower and more mottled than do adults. The adult bird is also heavier than the yearling (Boag, 1958j Standing, I960; Boag, 1964). Adult and yearling males begin molting their primaries simultaneously around the end of May or the beginning of June, well in advance of the start of the molt of the primaries in the females (Boag, 1964).
The male blue grouse shows strong territorial behaviour. Bendell was the first author to describe the territorial behaviour and the high fidelity of the adult male for his territory in the subspecies fuliginosus.
Some time after its arrival on the breeding range the adult male becomes localized on his territory, an area which he will seldom leave until his departure for the winter range in June or July (Caswell, 1954; Heebner,
1956). Very few cases are known of males shifting the site or modifying the size of their territory from one year to another (Elliott, m.s.). 8
Territorial behaviour consists of! isolation upon a well defined area, defense of this area and vocal display activity. The diurnal activity of the male is characterised by two high peaks of activity at the twilight hours of dawn and dusk (Caswell, 1954)• The male blue grouse is most likely polygynous. It does not show sign of pair bonds, does not take part in nest building, incubation or rearing of the young and territorial males will court all female passing on their territory (Simpson, 1935;
Caswell, 1954; Boag, 1958; Elliott, m.s.). At the same time it has been observed in the aviary that the female remains receptive to the male for a few days. All these observations support the view that promiscuity is the rule in the blue grouse.
Yearling males, unlike adult males, show a different behaviour while on the breeding range. They do not normally show much territorial behaviour. They are usually found silent near or on the territory of an adult male, which seems to attract them by its hooting or displaying,
(Elliott, m.s.). Because yearling males were silent, and were found in very small numbers only, by early students, the conclusion was reached that yearlings did not migrate onto the breeding range except in small numbers, or were pushed onto marginal habitat. These facts accompanied by the smaller size of the testes in yearlings suggested to many authors that yearlings were not sexually mature (Bendell, 1954; Boag, 1958; Standing, I960;
Boag, 1964)• New techniques of field work have shown on the study areas at Middle Quinsam Lake and Comox (Zwickel's field notes and personal com• munications) that the yearling males come to the breeding range in much higher numbers than was once thought. Bendell and Elliott (m.s.) estimate the number of yearling males on the breeding range to equal 40$ of the total 9 population. They estimate 10% of the total territorial males to be yearlings.
Recent work at Middle Quinsam Lake has shown that yearling males will replace adult males if the adults are removed from their territories.
From 1959 to 1962 all males on 100 acres of breeding range at Middle
Quinsam Lake were shot. Shooting proceeded from beginning of arrival to end of replacement. Over most of the study area at Middle Quinsam Lake,
10$ of the male population consisted of yearling hooters. On the 100 acres of the removal experiment, 50% were yearling hooters. Thus, when hooting adults were shot, hooting yearlings took their place and hooted and displayed, this raised the question, were the testes of replacement yearlings different from those of adults?
One other approach to the study of testicular cycle was to look for any consequence of interaction among males to regulate their numbers.
According to Carrick (1963), Wynne-Edwards and Jenkins (i960), non-breeders occur in the population of breeding birds as a symptom or consequence of interaction that regulated numbers. Therefore a constant comparison was made between hooting and silent adult males and between silent yearlings shot randomly on the study area and the replacement yearlings shot on the removal plot.
In order to correlate the main events of the breeding cycle of the male with that of the female, the main events in the life history of the female will be given here. The female appears to move at will over a large but defined area that may overlap with the territories of many males. This home range though does not seem to have any direct correlation with the territory of the male (Elliott, m.s.). The fidelity of the adult female to the same home range seems to be comparable to that of the adult male for his territory (Boag, 1964). The nest is built on the home range, it 10 contains an average of six eggs which take an estimated ten to fifteen days to be laid. In 1962, at Middle Quinsam Lake and Comox on Vancouver
Island (Zwickel's field notes), the laying period started during the second week of May and reached a peak by the end of May (based on data from 23 nests). Incubation is known to last for about 25 to 26 days. The peak of hatch for 1962 was calculated to occur in the last week of June. However, laying and hatching were estimated to be a week late in 1962, in relation to most years. The female with her brood wanders on the breeding range for the rest of the summer and starts her fall migration by August or
September. The sex ratio on the summer range is estimated at 1:1 (Bendell,
1954; Bauer, 1962). 11
STUDY AREA
The specimens used for this study came from three study areas:
Lower Quinsam Lake, Middle Quinsam Lake and Wolf Lake. These areas are situated between Campbell River and Merville, on Vancouver Island. They are part of a discontinuous strip of about twenty miles long, south of the 50° north latitude and east of the 126° longitude. These areas have
in common a past history of climax forest, followed by logging and burning. Each of these areas is presently in a different stage of
recovery: from ten year old burn without replantation to various degrees
of reforestation (replanted 1952 to I960) in Middle Quinsam Lake, and
to a very dense vegetation consisting of a twenty year old replanted
growth of Douglass fir Pseudotsuga menziesii at Lower Quinsam Lake. A more detailed description of these areas, except for Wolf Lake, is given
by Bendell (1954) and Elliott (m.s.).
A few birds from the aviary have been used in this study.
A number of blue grouse are being kept in an aviary on the campus of the
University. These birds came originally from the same areas on Vancouver
Island. They were captured as chicks or were artificially hatched from
eggs collected in the field. These birds were used only in small number
and for limited tests. 12
MATERIAL AND METHODS!
Material,-
The material used in this study consisted mostly of a collection of 220 pairs of testes obtained from the breeding range during the spring and summer seasons of the years 1958 to 1964.. Table I gives the number of birds used in this study and their origin.
Table I. Number of pairs of gonads used in this study and
their area of origin.
Lower Quinsam Lake 27
Middle Quinsam Lake 173
Wolf Lake 20
Aviary 4
Total 224-
The birds were collected for diverse reasons by different students working on various projects (Bendell and Elliott, m.s.; Elliott, m.s.; Zwickel, field notes). The gonads of every bird shot were fixed and preserved in either Bouin's fluid or 10% formaldehyde.
Except for birds collected at hunter game checks (8) every specimen was recorded on a data card and an autopsy form. The data card contained the following information:; the identification of the bird given by hour, day, month and year the bird was collected, bandnumber if any, sex, age, activity and behaviour prior to collection, how the bird was killed, exact location on the study area in relation to territories of other birds, or known land marks, and other pertinent observations. The autopsy form contained: the identification of the bird, the band number 13 if any, location, sex, age, weight of the bird with or without crop, organs preserved if any, liquid used for fixation or storage, the method of storage of the skin and carcass, stage of molt, depth of the bursa of
Fabricius, information relative to disease, and other pertinent data.
Methods.
In this study the general age classification as used by Bendell
(1954-) was followed. The term chick is confined to downy young from hatching to the time when they are covered with juvenile feathers in
July. The term juvenile includes birds in juvenile plumage that are progressively molting to the adult-like post-juvenile plumage, which is normally completed by September. Yearlings are birds which have retained the ninth and tenth primary of the juvenile plumage, but are otherwise similar to adults. An adult is a bird that has completed its first post-nuptial molt.
The age of the specimens was determined in the field on the basis of the characteristics of the primary wing feathers. This criterion has been used successfully by a number of students working with this species (Bendell,
1954; Boag, 1958; Henderson, I960; Bauer, 1962; Boag, 1964). In the lab the same technique was used in coordination with the method based on the rectrices. This method is based on width and length of the outer rectrices and was described and used by Van Rossen (1925), Petrides (1942) and Bendell
(1954). Table II gives the number and age of birds collected for each month and each year and used in this study.
Of the 224 specimens of gonad used in this study, 21 were from banded birds. These birds were banded for other studies prior to the time they were collected. Table III presents the number of specimen in each age group.
Table II. Age class and number of males in each age class collected
by month of 1958 to 1964.
1958 1959 1960 1961 1962 1963 1964 Total
Ad Yr Ad Ir Ad Ir Ad Yr Ad Ir Ad Ir Ad Yr Ad Ir
February 1 1 2
March 1 3 2 1 1 7 12 3
April 18 6 1 1 11 1 30 8
May 6 1 2 15 9 22 3 17 9 62 22
June 12 1 8 10 2 5 1 7 5 42 9
July 1 2 2 1 3 10 16 3
August 1 1 3 1 1 5 2
September 5 3 1 1 5 3
Total 20 2 11 2 48 21 30 7 44 18 2 2 18 1 172 52
Table III. Number of samples of known age birds for
each age group.
learlings 52
Two year olds 7
Three year olds 6
Four year olds 4
Five year olds 4
The testicular anatomy and physiology of adult and yearling blue grouse will be compared in the following aspects: 15
1. Size of the testes
2. Size of the seminiferous tubules
3. Spermatogenesis
4. Size of the interstitium
5. Histology of the interstitium.
This method of investigation has been developed after the models of four similar studies done on other species of birds by Wright and
Wright (1944), Marshall (1949), Blanchard and Erickson (1949) and
Johnston (1956). In these four studies, the authors were concerned with comparison of different rates of testicular maturation in different age classes. Blanchard and Erickson did not find differences in the sexual maturation of year old males, and the rest of the male population.
The three other authors found differences in the rate or degree of tubular maturation between different age classes.
The data from the seven years of collection were pooled into one hypothetical year. Because of a very uneven pattern of collection of specimen between years (Table Ii), it was impossible to treat the seven years individually. On the other hand the comparison of the fragmented curves of each year did not show any significant differences.
It was assumed then that all the seven years were physiologically equivalent and therefore the data were pooled into one hypothetical year.
Because of this organisation of the data, there were generally sufficient specimens for each week to assume that the weekly average and the seasonal curve would be a close approximation of the modification of one individual bird. The unit of time called a week is in reality a 16 fourth of a month. The calendar month was divided into four approximately- equal parts containing 8, 7, 8 and 7 or 8 days, respectively.
Histological techniques.
The greater part of this study will be concerned with the microanatomy or histology of the testes. Of the 224 pairs of testes which were used for measuring the seasonal variations in the testicular hypertrophy,
134 were chosen for histological investigation. The selection of specimens for this section was as follows: the best preserved testes of all yearling
specimens throughout the collection were used, all of the testes of adults up to April and during August and September were used, and up to five
specimens, depending on the amount of well preserved material, were selected for each week in the adult series between May and August. Specimens fixed
in Bouin's fluid were always preferred over material fixed in 10$ formalin.
Both ends of the testes were removed and the middle section retained for histological preparation.
Dehydration of the tissue and embedding were done according to the method described by Davenport (i960). From each embedded middle third of the testis six paraffin sections (8 microns in thickness), from two or three different ribbons, were selected and fixed on a slide with albumen. All sections were stained with Heindenhain1s iron haematoxilin,
counter stained with eosin Y (Davenport) and mounted with Permount.
In order to verify the assumption that a few sections from the middle third were representative of the whole testis, one testis was
completely sectioned from one end to the other. One out of every five
sections was stained and mounted. We were not able to find any measurable 17
differences in the size of the tubules or their distribution throughout the whole testis.
When there was a large difference in size between the right and left testis, both were prepared for histological study. 18
THE TESTES AND ACCESSORY ORGANS
The gonads of the male blue grouse consist, according to the season, of two small black or two large grey testes attached by a short mesorchium to the dorsal wall of the abdominal cavity. They lie at the anterior end of the kidneys, which they cover partially during the breeding season. The testes are also in intimate contact at their anterior end with the adrenal glands.
The tunica albuginea, a thin membrane of connective tissue surrounding the testis, forms no septa as found in mammalian testes. The seminiferous tubules start from a blind end and take a tortuous course dorsally. In the cross-section of a testis, the tubules are cut in all planes and have all kinds of shapes.
The accessory organs are the rete testis and the epididymis, the vasa deferentia, the erectile ejaculatory duct and the copulatory organ.
The rete testis and the epididymis are closely attached along almost the full length of the dorsal surface of each testis. The vasa deferentia, two very small straight tubes in their first third of length, leave the testes at their posterior ends. They lie parallel to the ureters, become larger in their second third and become very convoluted before they enter the cloaca. Each vas deferens terminates in an erectile ejaculatory duct which protrudes into the urodeum of the cloacal chamber. Blue grouse, like other galliforms, do not have intromitent organs. Though the complete
anatomy of the copulatory organ or its mechanism of erection in the blue
grouse was not investigated, what was observed when collecting semen
seemed very similar to what Lorenz described for the domestic cock: 19
a pair of round folds with a small knob terminating the groove between them.
There is no lumen and semen from the ejaculatory ducts flows over the surface of the erectile organs during copulation, thence down the groove between the folds. 20
RESULTS
1. Size of the testes
A common criterion for the estimation of avian reproductive condition has been the degree of testicular hypertrophy. Volume and weight of the testes have been regularly used to measure these variations. Volumetric measurement obtained by liquid displacement has been used here because of the simplicity of this method and the facility with which it can be used in the field. Since all authors agree that there is no histological or physiological difference between the larger and the smaller testis of the same bird, the two were not discriminated between in this study.
After removal of the extraneous tissue from the fixed testes they were blotted on paper and immersed together in a graduated cylinder containing the same liquid that contained them previous to measurement.
The combined volume was recorded. One measurement was sometimes taken in the field and two were taken in the lab; the average of three independent measurements was always calculated and used for each specimen.
Some shrinkage could be detected in the testes fixed in 10% formalin but not in those fixed in Bouin's fluid. When one testis was damaged or missing the volume of the intact one was doubled. This involved some error but since either testis can be the larger or the smaller one, no correction could logicaly be calculated. On the other hand it did not happen more than 10 times out of the 224 samples.
Results.
The relationship between age classes and seasonal variation of 21 the average volume of the testes at weekly intervals and a moving average are illustrated in Figure 1. The weekly averages of the volume of the testes of each of the two main age classes were plotted against time.
The number of observations, range, standard deviation and coefficient of variation for each week are summarised in Table 17.
This study indicated a clear difference in size of the testes between the two age classes during all the summer.
The two classes seemed to start from very similar points in the spring. As the first birds arrived on the breeding range, there was very little testicular increase if we assume that one cubic centimeter is the maximum size of the testes reached during the winter. This was unexpected since many other migratory species show much more enlargement in the size of their testes when they arrive on their breeding range (Wright and
Wright, 1944, Johnston, 1956; Blanchard and Erickson, 1949; Bullough,
1942). This might be explained by the fact that the migration of the blue grouse from its winter range to its breeding range is certainly much shorter. It also suggests that for some birds at least, this migration must be accomplished in a very short period of time.
As soon as recrudescence started, the two age classes rapidly separated. In the adult age class, there was certainly no delay in the process of recrudescence of the size of the testes. In yearlings, however, the recrudescence of the size of the testes seemed to proceed in two steps.
First, a slow process that lasted until the second week of April (week no. 4), and second, a rapid recrudescence quite comparable in rate to the complete recrudescence in adults..
There was no doubt about the larger maximum size of the testes Figure I. Seasonal variation in the average volume of the testes of the adult and yearling 23
Table IV« Statistical summary of the seasonal growth rate of testes of
blue grouse, adults and yearlings,. Middle Quinsam Lake, 1958-1964.
Adiilts. Month Week Number Mean Standard Standard Coeffi- Range of of of testes of the error of devia• .cient volume season examined volume the mean tion of in cc (birds) in cc variat» March 1 4 *2 0,040 0.081 40.50 .1 ~ .3 2 8 ,51 •o.ioi 0,286 56*08 .3 -1.2
April 3 2 «9 0*199 0a282 55.25 ,8 -1.0 4 17 1,21 0,100 0,415 37*73 -.5 -1.8 5 5 1«72 0,242 0,541 31,78 1,0 -2,0 6 5 2.09 0,420 0.939 44.93 . 1,0 -3.6 May •7 25 1,54 0.094 0.472 30,65 »8 —2.6 a 18 1.48 0,098 0*416 28,11 .7 -2.3 7 ': 1*63 0*192 0,508 31.16 loO -2.4 10 ' 12 1*29 ':• 0*177 0*615 47,67 * 85-3.0 June 11 u 1.28 0,099 0,371 28,98 .65—1.8 12 9 1,00 0*115 0.345 34«50 06 —1.8 13 5 ,78 0.037 O/O83 10,64 .7 - .9 U U *5?.'. 0*062 0,233 39,49 *4 —1*2 July "• 15 3 &4° 0,067 0,116 24.16 .4 ** .65 16 2 »30 ••3 - .35 17 A »27 0,051 0,102 37,77 .15- .28
18 6 828 0,041 0,100 28«00 ..1 — .4 August 19 1 fl5 20 2 *15 .12— a18 21 1 *15 22 1 Sept* 25 5 »;ii • • 0.004 0.010 10*00 .08- .12 1 _a ;rH ,
March 1 r-i .15 2 .2 April U 3 *38 0,033 0.057 15.40 .3 5 2 .75 ,7 - .8 6 3 ,83 0.033 0.058 6.98 - »9 May 7 5 ,82 0.017 0,038 4.75 .$7 s-1. 0
8 8 ,61 0.046 0o132 22,00 .4 - .8 9 3 .77 0.066 0.115 34*93 .7 - .9 10 6 .58 0„065 .0.160 27.58 .3 - .7 June 11 4 .57 0.110 0,221 38.77 .3 - .8 12 4 •47 0,065 0,131 27«87. «3 - .7 13 1 .2 July 15 1 .1 16 1 «15 18 1- : -: August 21 .1 1 22 fli4 24 reached by adult birds. The location of this maximum though was difficult to place in the yearling age class, because the difference between the averages of the weeks 6 and 7 was obviously not significant (.83 and .82).
On the other hand, the curve of the moving average seemed to indicate a slightly delayed peak in yearlings.,
Although there was a diminution in the size of the testes immediately after they had reached their maximum size, it did not seem to be the start of regression since the laying period had not yet begun, or was just starting. Then a more or less slanted plateau of the size of the testes seemed to be maintained during breeding period down to the first week of June.
The start of regression of the size of the testes seemed to be the best synchronised point between the two age classes. This first week of June was also the approximate date for the start of the molt of the primaries in the two age classes. The minimum size of the testes, at the end of regression, was reached by the yearling class between 4 to 7 weeks before the same point was reached by the adult class.
Statistical analysis.
The testicular size in the adult age class did not seem as homogenous as in the yearling class. The average of the coefficients of variation of mean testis size in the adult age class was almost twice as large as the same average for the yearling class (33.74 to 19.78). This may be i explained by the fact that the yearling class was composed of only one year old birds while the adult class included birds of all ages beyond two years of age. Hence the size of the testes appeared to vary with age beyond one year of age. 25
A linear relationship between size of the testes and time of the year was assumed. Linear regressions were computed for the common logarithms of the averages of the size of the testes at weekly intervals. The breeding cycle was broken into three parts! recrudescence, breeding and regression, on the basis of what was said earlier and with the help of the coefficient of variation, which seemed to be larger around these points of breakage. The equations and their analysis are given in Table V and the regression lines in
Figure 2,
Table V. Equations for the regression of the mean of the testes volumes
at weekly intervals, over the period of recrudescence, breeding
and regression in adults and yearlings.
Age Period Weeks log Y = a - bT R2%
A Recrudescence 1 to 5 .8164- - .2242 T 94.5 15.81 ^ d u Breeding 5 to 11 .4145 - .0278 T 65,2 1.88
1 XX t Regression 11 to 22 1.1665 - .0991 T 97.6 41.11 __
e Recrudescence 1 to 6 -1.001 _ .1581 T 97.0 32.58 ^ a £ Breeding 6 to 11 .1217 - .0334 T 65.4 1.89
1 Regression 11 to 15 1.9850 - .2002 T 97.9 64.17 n g .
From the regression lines we can see that yearlings have a slightly different cycle from the adults in many aspects: slower recrudes• cence, more levelled breeding plateau, shorter regression and earlier winter stage. This analysis though should be supported by histological analysis, which will be done in further tests. For the moment, these conclusions are speculative. Figure 2. Linear regressions of three parts of the breeding cycle in adults and yearlings, M.Q.L., 1958-1964. 27
Comparisons between different populations.
Bendell (1954) recorded a similar curve of the volume of the testes in the blue grouse population at Lower Quinsam Lake (Figure 3).
The population of that area, in 1954, was estimated to be at a density- three to five times that found in the population of this study.
No difference was found between the present population of Middle
Quinsam Lake and the now vanishing population of Lower Quinsam Lake. Of the sixteen samples (weekly averages) that could be compared for the period 1958 to 1964, seven times the sample from Lower Quinsam Lake was larger, eight times it was the reverse and once the two samples were equal.
No patternseither were found in these different samples.
Standing (i960), working with a population of blue grouse
(Dendragapus obscurus pallidus) from central Washington, used semi• monthly averages of the weight of the testes to trace the curve of seasonal variations (Figure 4). Although his curve seemed quite different from ours at first, closer investigation showed that his curve was merely shifted to the left. The maximum size of the testes seemed to be reached in the middle of April in central Washington but was not reached before the end of April in the population of Middle Quinsam Lake.
These facts supported the view that there was no marked difference in the seasonal variations of the size of the testes of male birds from different populations, at different years, and at different densities at the same latitude.
Relation of size of the testes to migration.
Bendell (1954) noted the striking similarity between the decrease in the number of males on the breeding range and the regression Adults. Bendell (1954).
Figure 3. Size of the testes in two populations. Bendell (1954), M.Q.L. 1964.
Figure 4. Size of the testes in two populations. Standing (I960), M.Q.L. 1964. 29 of the testes over the same period of time. The same relationship existed between the number of males present on the breeding range during the summer I960 (Bendell and Elliott, m.s.) and the regression of the volume of the testes for the summers 1958 to 1964 (Figure 5). These authors also noted an earlier departure of the yearling males from the breeding range.
Changes in the testicular volume of the adults were clearly related to the number of birds on the breeding range. Spring migration occurred just before or at the beginning of recrudescence. The maximum conspicuousness of males was reached just before the maximum size of the testes was attained.
From early May to August the size of the testes decreased as the males progressively moved from the breeding range.
Changes in the size of the testes of yearling males showed a similar general pattern. However, its synchronisation was different. If it is accepted that the earlier departure of the yearlings from the breeding range correlates with the faster regression of the size of the testes in this class, then a similar relationship might be assumed in the spring, and a later migration of the yearlings can be expected in the spring.
The data to support this later spring migration in the yearling male class are ambiguous. In I960, the first adult male collected on the breeding range was shot March 29, and the first yearling, on March 31.
The second yearling though was shot only 10 days later. On the other hand, in the spring of 1964, the collection of males on the area of Wolf Lake (with dogs) produced 19 adults before the first yearling was shot about a month later.
Relation of size of the testes to the period of egg laying.
The maximum size of the testes in adult males was reached just 30 before egg laying started. This period began in early May and lasted approximately until the middle of June, In the analysis of the curves of the average volume of the testes, it was assumed that the breeding condition in males could be expressed by a slanted plateau. If this assumption is right, these plateaus occur in adults as well as in yearlings - however at a much lower level in this last class - and cover the full period of egg laying.
This conclusion is made with reservation until more of the physiology of the testis is investigated.
Relation of size of the testes to body weight.
No relation was found between the size of the testes and the weight of the body of individual birds within the same age class.
Between age classes though, there was a general correlation and the heavier adults had larger testes than the lighter yearlings. Monthly averages and range of the body weight for the two age classes are given in Figure 6.
Further analysis showed that yearlings had smaller testes, even if a correction for their smaller body weight was made. If the average size of the testes, in each age class, for the four periods (recrudes• cence, peak, breeding and regression) is correlated with weight for the same points, the indexes in the yearling age class are always approximately half the same indexes in the adult age class.
Relation of size of the testes to territorial behaviour.
The volumes of testes were examined to find if there was a difference in the size of the testes between birds that were hooting and • 1 • ' 1 111 I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 2021 22 23 24 25 Mar. I Apr. ' May ' June I July ' Aug. ' Sep.
Rgure 5. Number of males observed/hr./2 weeks period at M.Q.L. during the
summer I960 and the corresponding volume of the testes In adults.
Figure 6. Monthly average and range of the weight of adults and
yearling males, M.Q.L., 1959-1962. 32 those that were silent when collected. No constant relationship was found between the size of the testes and the activity of the bird immediately prior to collection. The testes of hooting adults were not different in size from the testes of silent adults, and testes of hooting yearlings or replacement yearlings were not different in size from those of silent yearlings. The average of nine out of twenty samples showed a difference in the size of the testes in favor of silent birds, and eleven showed the reverse. No pattern was discernable either in these differences.
Known banded territory holders were often observed as silent birds.
Furthermore, the territorial activity showed two very distinct peaks at dawn and at dusk. Then, since the birds for this study were collected at any time during the day (Appendix), it was not surprising to find no significant differences in volume of gonads of hooting or silent males.
Relation of size of the testes to age.
There was no doubt that yearlings had a testis cycle completely different in magnitude from the cycle of the adults. Even the range of the weekly samples rarely overlapped. We had a very small sample of known age birds beyond two years of age, however, it is interesting to note the results of the following analysis. If the size of the testes of known age birds is plotted on each side of the average of the adult class, the following percentages are obtained, Table VI. It seems then that the size of the testes continued to increase after a bird had reached the adult age class. 33
Table VI. Comparison of the size of the testes of known age birds
the average of the adult class.
Age of bird in year 1 2 3'' 4 5
Number of birds 52 7 6 4 4
Percentage over average H% 50$ 25$ 50$
Percentage on average U% 50$ 25$
Percentage under average 100$ 72$ 50$ 2% 25$
Conclusions.
Thus far we have examined the volume of the testes of blue grouse on their summer range and related it to location, number, age, behaviour and body weight. The yearlings had a different breeding cycle from the adults; smaller testes, a slower recrudescence, a later peak, a shorter breeding period, a shorter regression and an earlier winter stage. The interpretation of these differences will be investigated in further histological studies.
No differences were found in the seasonal variations of the size of the testes of male birds from different populations at different levels of density but at the same latitude.
No direct relationship was found between size of the testes and the weight of individual birds within the same age class.
No differences in the size of the testes were found to correlate with the behaviour of the bird immediately prior to collection, or with the fact that a yearling was hooting or had replaced a removed adult.
There seemed to be a relationship between age and the size of testes after the bird was more than two years of age. 34
2. Size of the seminiferous tubules
The enlargement of the seminiferous tubules in other species has been found by several authors to be responsible for the main increase in the volume of the testis (Wright and Wright, 1944; BlanShard and
Erickson, 1949; Johnston, 1956). This suggests that changes in size of the tubules may be related to the observed difference in the volume of the testis of adults and yearlings.
To explore this relationship, the diameter of the seminiferous tubules in 124 birds was measured. Each bird was sampled by measuring the diameter of 50 seminiferous tubules in a section through its testis.
Ten round or slightly oval shaped tubules were randomly picked for measurement, in each quadrant and in the center of each cross-section. All measurements were taken in microns with an ocular micrometer calibrated to the microscope which- was used. In the slightly oval cross-sections of tubules, only the shorter diameter was measured because it was assumed that the tubule was cut in a tangential plane. The size of the seminiferous tubule for each specimen was determined by the calculation of the average of these fifty measurements for each individual (Appendix; Table XIl).
According to Wright and Wright (1944) who used this technique, this size of sample is large enough to insure that the calculated mean is within 5$ of the true mean, with a probability of 95$.
All the 134 histologically prepared specimens of testes could not be used for this test. Faulty fixation as well as physical distortion of the tubules rendered some of the material unusable for this particular part of the study. We could note that the testes, in order to be fixed 35 properly for histological study, should have been fixed immediately after the death of the bird or within a period of three to four hours, depending on the weather.
Results.
The weekly averages of the diameters of the seminiferous tubules, for each of the two age classes, were calculated. The number of testes examined, the range and the average diameter of the seminiferous tubules for each week and in each age class are summarized in Table VII. The relationships between the diameter of the seminiferous tubules and season and age are illustrated in Figure 7. The weekly moving average of the diameter of the seminiferous tubules in microns, for each age class, were plotted against time.
The seasonal variation in the size of the seminiferous tubules confirmed many points advanced in the preceding test. Despite the fact that yearlings had a much smaller volume of testes than the adults,
Figure 7 shows that the average diameter of their seminiferous tubules reached a maximum comparable to that of the adult during the breeding period.
On-the whole, the yearlings had a shorter tubular increase cycle than the adults. Although they apparently started recrudescence at the same time as the adults, because of a slower rate of increase, they soon lagged behind by about a week. Regression also seemed to start a week earlier in yearlings. We have estimated it to be similar in the analysis of the volume of the testes. This regression approximately one week earlier in the yearlings was maintained almost throughout the • Weekly average in adults.
O Weekly average in yearlings.
2IO — Moving average in adults.
Moving average in yearlings. § no
£ 170 3
1 'SO w a> !fcc I 130 10 | no E a
? 90 k_ > < 70
• » _l I I I ,1 I I L. I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 B 19 20 21 22 23 24 25 Mar. | Apr. | May | June | July | Aug. | Sep.
Rgure 7. Seasonal variation of the average diameter of the seminiferous tubules in adults and yearlings, M.Q.L., 1958-1964. 37
Table VII, Number of testes examined, weekly average of the size of the
tubules and range for adult and yearling age classes.
Adults, Month Week Number Average Range of of testes in in season examined microns microns (birds) March 1 3 84.5 83.1 - 87,8 2 7 120.7 103.0 - 170.5 April 3 2 156.8 143.1 - 181.5 4 16 168.0 135.2 - 209.0 5 4 192.5 159.5 - 231.0 6 3 185.8 150.0 - 236.0 May 7 8 179.0 151.3 - 203.5 8 5 172.0 149.7 - 196.5 9 3 161.0 154.5 - 168.8 10 4 171.2 155.7 - 198.0 June 11 3 188.8 187.0 - 192.5 12 A 175.1 123.2 - 209.0 13 2 164.0 163.1 - 165.0 A 141.6 104.5 - 181.5 July 15 1 154.0 16 1 115.5 17 2 99.0 88.0 - 110.0 18 3 102.5 82.0 - 121.0 August 19 1 99.0 20 2 74.2 71.5 - 77.0 21 1 82.5 22 1 82.5 September 25 5 69.2 55.0 - 77.0
Yearlings. March 1 1 82.5 2 1 99.0 April 4 3 128.8 112.0 • - 155.6 5 2 180.3 170.5 - 191.3 6 3 155.8 143.0 - 165.0 May 7 177.5 154.0 - 198.0 8 6 176.9 132.0 - 203.5 9 3 190.7 181.5 - 198.0 10 3 161.3 137.5 - 176.0 June 11 165.0 137.5 - 165.0 12 165.6 134.5 - 181,5 13 1 143.0 July 15 1 99.0 16 1 99.0 18 1 88.0 September 23 1 82.0 38 regression period; only towards the end dit this difference increase.
Relation of size of the seminiferous tubules to the period of egg laying.
The maximum development of the seminiferous tubules in yearlings seemed well synchronised with the peak period of egg laying. However, no plateau or maximum was maintained throughout this period as in the adult age class. If we super-impose the plateaus of breeding condition, from the study of volume of the testes, over the curves of the size of the seminiferous tubules, we can see that they seem to fit surprisingly well. From the last week of April to the first week of June, the adults seemed to be in full breeding condition while the yearlings, here again, seemed to be in a similar condition from the first week of May to the first week of June. Only with a study of spermatogenesis can the real differences between these plateaus be appreciated.
Although it has been included in the calculations, the high value given for week No. 5 should probably not be considered as a peak because all indications show that there might be an error in aging, in the technique of measuring, or in the fixation.
Relation of size of the seminiferous tubules to territorial behaviour.
No constant difference existed between the size of the tubules and the activity of the bird immediately prior to the time it was shot.
Out of the twenty weekly averages for which samples can be compared, nine times the tubule diameter of hooting adults or replacement yearlings was larger than the samples of the silent birds, ten times it was the reverse, and once the two samples were equal. Furthermore, no distinguishable patterns 39
could be noted where the differences occurred.
Relation of size of the seminiferous tubules to age.
When the size of the seminiferous tubules of the known age adults was plotted each side of the average of the adult age class (Table
VIIl), no constant relationship with age, as shown for volume, could be found. Except for the fact that yearlings had a shorter cycle, there was no relationship between age of the bird and the size of its seminife• rous tubules.
Table VIII, Comparison of the size of the tubules of known age birds
with the average of the adult class.
Age of bird in year 1 2 3 4 5
Number of birds 39 6 3 2 4
Percentage over average 36$ 66$ 66$ 50$
Percentage under average 64$ 34$ 34$ 100$ 50$
Relation of size of the seminiferous tubules to volume of the testes.
It was found that there was no direct relationship between the
size of the testes and the maximum size of the seminiferous tubules in the
two age classes. But within the same age class, there was a relationship
between these two factors.
To illustrate this point, the size of the tubules up to
approximately the end of recrudescence was plotted against the volume of
the testes (for adults, from week no. 1 to week no. 6; for yearlings,
from week no. 1 to week no. 9). A linear relationship was assumed
between the average size of the tubules for each volume unit and the
volume of the testes. The computed linear regressions are illustrated in Figures 8 A and B. The equations and their characteristics are summarized in Table IX, where I equals size of the tubule, and V equals volume of the testis.
Table IX. Equations of the linear regressions for the size in microns
of the tubules against volume of the testis.
Y = a - bV R2% f
Adult Y = 91.51 - 63.03 V 67.35$ 2.31
Yearling Y = 89.52 - 110.23 V 65.1535 1.10
One would not expect a perfect correlation between the size of the testis and the enlargement of the tubules because there are other variable constituents of the testis that may vary also in amount: inter• tubular tissue, blood vessels, tunica albuginea and elongation of the tubules. However, the linear regressions showed that as the size of the testis increased, the size of the seminiferous tubules increased similarly,
A more detailed analysis of this type was carried out by Blanchard and
Erickson (1949) on the white crowned sparrow. Wright and Wright (1944) and Johnston (1956) also found the same relationship.
In the two age classes a general relationship seemed to exist between the two factors. The divergence of the regression line in the adult age class from the diagonal (which would express a direct and proportional relationship) suggested that the increase in non-tubular tissue or elongation of the tubules had a greater influence on the increase in size of the testis of adults than on the increase in size of the testis of yearling birds. However, we have not been able to demonstrate this point statistically. A) Yearlings (23)
i i i i i i i i i i i i i i i i i i J .2 .3 A .5 .6 .7 .8 .9 I.O I.l 1.2 13 U 1.5 1.6 1.7 1.8
B) Adults (28)
I I I i I. I I I I I I I I .1 .2 .3 .4 .5 .6 .7 .8 .9 I.O I.l 1.2 1.3 1.4 1.5 1.6 1.7 1.8
Volume of the testis in cc.
Figure 8 . Relation of the diameter of the seminiferous tubules to the volume of the testis
during recrudescence period in adults and yearlings. M.Q.L., 1958-1964. A2
Conclusions.
From this study of the cycle of the seminiferous tubules it
is suggested that yearling males have a shorter cycle of increase in size of the seminiferous tubules. This was supported by a slower recrudescence, a shorter breeding stage and an earlier and faster regression in the yearling age class. The tubular cycle seemed well synchronised with the period of egg laying although full certainty on this point will not be reached before spermatogenesis is studied. No clear correlation seemed to exist between the size of the seminiferous tubules and the behaviour of the bird immediately before it was shot, or with age of adults over two years of age. Although it was noted that the increase in the size of the
seminiferous tubules was responsible for the larger amount of the increase
in the size of the testis, in both age groups, it was concluded that the
increase of non-tubular tissue and the elongation of the tubules had a greater effect in adults than in yearlings. 43
3. Spermatogenesis
The objective of this part of the study was to find fixed points of spermatogenesis that could be compared in different age classes and correlated with other characteristics of the testis.
The histological details of spermatogenesis have been worked out for several species of passerine and non-passerine birds. In the broad features of spermatogenesis, all authors are in close agreement, although in the definition and delimitation of stages some differences of opinion have arisen. The males of several of the species studied breed in their first and subsequent-years. In these species, no differences in spermato• genesis between one year old and adult birds could be found. Some non- passerine birds however, do not breed until more than one year of age and Marshall (1949), working with the fulmar (Fulmaris glacialis), suggested that spermatogenesis in one year old birds might differ in some way from that of the adult. On the other hand, Johnston (1956) worked with the California gull, a species that does not generally breed before four years of age. He found that all the birds of the immature classes (from one to three years of age) had a cycle of spermatogenesis, but this cycle differed from the cycle of older birds in two ways.
Either by being an incomplete cycle as in one year old birds or by being slightly out of phase as in the two and three years old birds
(Miller, 1937; Clermont, 1958).
Avian spermatogenesis has been described for several species but for clarity it is thought that a short description of the elements of spermatogenesis should be given here. The course of spermatogenesis may be followed from the periphery of the seminiferous tubule towards the lumen, where the maturing spermatozoa are arranged in bunches attached to a Sertoli cell. It was not possible to observe regular helicoidal waves of maturation in the blue grouse. However, groups of adjacent cells mature synchronously, and several stages of spermiogenesis can be observed within a tubule.
Spermatogonia.
Spermatogonia formed a more or less continuous layer next to the tunica propria. The nucleus, about 4*5 microns in diameter, was recognised by a fine network of) chromatin with two or three local condensations i (Plate 5).
Resting spermatocyte.
The resting spermatocyte was very similar at first glance to spermatogonia. They showed on closer examination, however, a slightly larger nucleus of about 5 microns in diameter (Plate 5). The chromatin network was more difficult to see and the chromatin condensations, more numerous, were almost round. Sometimes, the idiozome could.be seen as a thin cap on one side of the nucleus.
Primary spermatocyte.
As maturation proceded the chromatin condensations disappeared and were replaced by a complex network of chromatin threads that gradually became coarser and drew away from one pole of the nucleus
(Plate 5)» The nucleus, about 7.5 microns by that time, was easily recognizable by a very clear pole and a tangled mass of threads at the 45 other pole. The idiozome in the cytoplasm, very evident then, was also at that pole. The frequency with which this stage of synapse was seen, suggested that it must be a very long stage. The process of unravelling in these threads, so that the chromatin came to occupy the entire nuclear space, also seemed to be a slow process.
Secondary spermatocyte.
These cells were much smaller than the primary spermatocytes
(Plate 5). They had a nucleus of about 4.5 microns in diameter, with one to three large chromatin condensations, and a scattering of smaller granules along the nuclear membrane.
Spermatid..
The nucleus, some 3 to 4 microns in diameter, at first had a very similar appearance to the secondary spermatocytes (Plate 7). But the chromatin condensed rapidly along the nuclear membrane and then in the center of the nucleus, which eventually became a very dark sphere of chromatin of about 1.5 microns in diameter. From that sphere, it was presumed, because of its orientation, that the acrosome first started unrolling. Eventually all the head was liberated and surrounded by a very thin membrane. The spermatozoa, already grouped, then fixed themselves on Sertoli cells for further maturation. The tail formation could not be observed with the stains that were used.
Sertoli cell.
These cells were recognized by a large clear nucleus of about
10 microns in diameter (Plate 5). This nucleus often seemed crushed or 46 squeezed between the seminal cells.
Spermatozoa.
Blue grouse sperm were relatively large. Their overall length, including the tail, exceeded 160 microns. The acrosome was barely distinguishable from the head (Plate 13). Under our microscope it was very difficult to determine the point of junction of the three main parts; head, mid-piece and tail. The head was estimated at about 25 microns in length and the mid-piece at 15 microns.
In his work, Johnston (1956: 155) had felt the need to describe anew the stages of spermatogenesis. Because he found that the interstitial cell cycle was not always "in phase" in a given month for any two age groups, the new stages he described are based on the features of the germinative epithelium only. These stages are roughly comparable to those described by Blanchard (1949).
Johnston's stages have been adopted here without any modification for the following reasons: his stages were more detailed for the recrudes• cence period than for the premigratory period, for which'we had no material. It was also found in our material that the interstitial cell cycle was not always "in phase" in a given month for the two age classes.
Stage I - Inactive condition (Plate l).
In the inactive winter condition the testes are at a minimum size and the tunica albuginea is of maximum thickness. There is a basal row of spermatogonia and Sertoli cells next to the basement membrane. A small lumen is almost always present. A few early primary spermatocytes 47 are also visible close to the lumen. This stage is equivalent to stages
1, 2 and 3 of Blanchard (1949).
Stage II - Primary spermatocyte in synapsis (Plate 2).
There is still basically one row of spermatogonia and Sertoli cells and there may be some increase in the number of these cells. Up to about one half of the primary spermatocytes in a given cross-section of a tubule have their chromosomes in synapsis and frequently the chromosomes lie at one side of the nucleus. As in stage I, these primary spermatocytes form an incomplete row of cells, next to the lumen. The lumen is filled with detritus. Blanchard1s stage 4 is the equivalent (Plate 2).
Stage III - Increase of primary spermatocytes in synapsis (Plate 3).
The tubules start to enlarge and the tunica albuginea becomes thinner. Primary spermatocytes have increased in number to the extent that (l) the majority of them are in synapse and (2) they usually form two or more rows next to the lumen. This is similar to Blanchard's stage 5 except that testes with secondary spermatocytes are not included in this stage.
Stage IV - Secondary spermatocytes (Plates 4 and 5).
Predominance of primary spermatocytes in synapsis, plus a few secondary spermatocytes mark this stage. The appearance of secondary spermatocytes is accompanied by a great increase in the size of the tubules and the appearance of a large empty lumen that is gradually lined by secondary spermatocytes. This stage is included in the second part of
Blanchard's stage 5. Stage V - Spermatids (Plates 6 and 7).
Most of the spermatids border the lumen, but a few which are metamorphosing move away from the lumen towards the Sertoli cells. Only rarely are mature spermatozoa present, and then only in negligible number, though a few might be found in the vasa deferentia.
Stage VI - Spermatozoa in bundles. Breeding condition (Plate 8).
Testes and tubules are at a maximum size. The tunica albuginea is thinnest. Spermatozoa are present in large numbers of bundles which are spaced around the tubule. Much cellular material is present in the lumen, but only occasionally are the spermatozoa seen in the lumen.
Evidently some of them have already passed down and some sperm can be found in the vasa deferentia.
Stage VII - Spermatozoa in tubular lumen (Plate 9).
This stage differs from stage VI because here the majority of the sperm bundles have been shed into the lumen of the tubule. Much detritus is also present in the lumen. This may be the beginning of regression. However, since adult testes reach their maximum size in this stage and no noticeable collapse of the tubules is yet evident,
Johnston regards it as a separate stage from regression (Plate 9).
Stage VIII - Regression (Plates 10, 11 and 12).
After the spermatozoa are shed into the lumen, there is a casting off, in succession, of spermatids, spermatocytes and probably spermatogonia and Sertoli cells. Masses of necrotic cells and cellular debris are found in the collapsing lumen of tubules. Yet at the same 49
time, at least some spermatogenesis is still going on because primary
spermatocytes in synapse are evident. Towards the end of regression
only a few primary spermatocytes may be found in the lumen while a basal
layer of Sertoli cells or spermatogonia remains.
Results.
The range of stages of spermatogenesis in each age group and
for each week of the spring- and summer seasons is illustrated in Figure 9.
One hundred and nineteen slides representing the same number of birds were studied. The stage of spermatogenesis for each bird was recorded
as either early or late in a particular stage.
The development of spermatogenesis during the breeding season
correlated well with the curve of the diameter of the tubule for the
same period. A shorter breeding cycle in the yearling age class was
confirmed again by the slower recrudescence of spermatogenesis, shorter
breeding period (6 weeks in yearlings against 9 weeks in adults) and
faster regression.
Some mature sperm were found in the lumen of the seminiferous
tubule during stage VI though signs of full maturity extended up to the
end of stage VII, when very abundant sperm were released from Sertoli
cells and when the lumen of the tubules became full of sperm. In the
early stage VIII, some normal sperm were produced but they were lost in
cellular detritus. If it is assumed that the period of sperm ripening,
in the vasa deferentia, is the same in both age groups, then some yearlings
were in a breeding status comparable to that of the adult starting about
the second week of May. Therefore, there would appear to be no reason why, 11 1 VTTT 0 011011101 n 1010111 ZELT
o o ! 2: Range in adults. CaQO>. b o 32- Range in yearlings. » a I JL I 23456789 IO 12 13 14 15 16 17 18_1 _ 19 20 21 22 23 24 25_L - 26 March I April I May I June I July I Aug. I Sep. Figure 9 . Range of stages of spermatogenesis for each week of the summer in adults and yearlings, M.Q.L., 1958-1964. o if not impaired by psychologic immaturity or dominance of other adult males, some yearlings could not efficiently copulate with a female. The sperm samples from adults and yearlings in the aviary at the University of British Columbia supported this view. By the middle of May in 1964 it was decided to run a crude physical semen analysis with a few birds from the aviary. During the period of preparation for the collection of sperm, a yearling male, on the 29th of May, copulated successfully with an adult female in the aviary. Subsequently this hen laid thirteen eggs, of which four hatched under artificial incubation. Hence in captivity at least, yearling males were fertile. Analysis of semen. The technique for obtaining semen from fowl originated with Ivanove (1913) who squeezed semen from the vasa deferentia of freshly killed birds. Many other different methods have been described since, but all have been replaced by the method of massaging the side of the abdomen below the pelvic bones and squeezing the semen from the bulbous ducts at the base of the copulatory organs (Burrows and Quinn, 1935- 1937$ Lake, 1957). This method was successfully applied to blue grouse, but not always with equally satisfactory results. On June 5, 1964, semen was collected from eight aviary birds, four adults and four yearlings. A good sample amounted to one drop. Two birds gave samples which were too small to be used in this test (one adult and one yearling). Each sample was collected on a warmed slide held at 30°C. The semen was then diluted with an equal amount of citrate buffered solution, (Sodium citrate Na^C.H-OJ^ELO at 32g/ J o ) / Z litre, plus 10$ citric acid to bring the pH of the buffered solution to approximately 6,8 (Campbell, 1956)). Then approximately a double amount of staining solution, Eosin nigrosin (Blom) was added to the sample. After four minutes a small drop of the semen-buffer-stain mixture was made into three successive smears on slides which were also maintained at 30°C (Coffin, 1957). As a result of this method two tests could be madej a differ• ential count of live (unstained) and dead (stained pink) sperm, and a count of the ratio of abnormal sperm since the dark background permitted identification of these forms. Three slides for each bird were examined for each of the two tests, and for each of the six birds (3 adults, 3 yearlings).. No significant differences were found between the samples from the two age classes. The percentage of dead sperm ranged from 2$ to 8$ in the adult, and from 2% to 6$ in the yearling samples. The percentage of abnormal sperm ranged from 24$ to 28$ in the adult, and from 18$ to 27$ in the yearling samples. The high percentage of abnormal sperm in both age groups can probably be explained by the fact that the samples were collected very late in the breeding cycle. In the wild, both age groups would normally have already started regression by the time that sperm was collected in the aviary. Many of the abnormal forms of sperm showed signs of picnosis. It is interesting to note that no differences were found between the sperm collected from the yearling that copulated success- fully with the adult female and the sperm of the two other yearlings, one of which was kept in a pen together with an adult male which dominated him. This yearling never came to the ground from his perch except to feed. Furthermore no differences were found in stages of spermatogenesis between adult birds that were hooting when they were shot and the silent birds, or between replacement or territorial and silent yearlings, or between older birds of five years of age and the younger adults of two years of age. This suggested that all the males present on the breeding range during the summer were potentially breeding males. Relation of stages of spermatogenesis to the diameter of the seminiferous tubule. It was shown that the size of the seminiferous tubule followed a cycle with different timing in each age class. A similar difference was present in the cycle of spermatogenesis. The question arose then how does the size of the seminiferous tubule correlate with stages of spermatogenesis? The range for the size of the tubule was plotted against the stages of spermatogenesis (Figure ll). Stage VIII was divided into two parts, VIII A and VIII B, because of the long period of time included in that stage and the large variations between its beginning and its end. Although there was a good correlation, similar in both age classes, between the diameter of the tubules and the successive stages of spermatogenesis, there was sufficient overlapping between the stages to make inaccurate the use of diameter of the tubule alone as an indicator of a specific stage of spermatogenesis. Relation of stages of spermatogenesis to the size of the testes. It was shown earlier that the size of the testes in the two age classes followed a different cycle (different in timing and total amount). A similar difference in timing at least existed in the cycle of spermatogenesis. The question raised then wass how is the size of the whole testis correlated with stages of spermatogenesis? To answer this question, the volume of the testis was plotted against the stages of spermatogenesis as in the preceding test (Figure 10). There was a good correlation between volume of testes and the stage of spermatogenesis within the same age class, but obviously not between age classes. Here also, the overlapping between stages was too great to make volume of the testis a reliable indicator of the specific stage of spermatogenesis. Spermatogenesis in juvenile males. The testes of two juvenile males collected in September were examined for any sign of start of spermatogenesis. No signs were found and the epithelium of the tubules in these birds was exclusively composed of spermatogonia. Conclusions. It is likely that every male blue grouse over seven months of age and present on the summer range went through all stages of spermatogenesis. Although the period of sperm production was shorter in the yearling than in the adult age class, the yearling apparently reached full breeding status during the period of egg laying. 55 Range in adults 2.6 I Range in yearlings 2.2 o u ,1-8 £ 1.41- cu E o I.O > D .6 I • • I _ I IT III ~SZL ¥K MA YKB Winter Stages of spermatogenesis Figure IO.Relation of volume of the testis to stages of spermatogenesis, M.Q.L., 1958-1964. £220 o 1 180- E I « 140 • o I k_ co rct iioo I 1 I °k_ 60 co a> E o 5 20|- -I u I I H JE I 21 3ZE "2DEA IZLTB Winter Stages of spermatogenesis Figure il .Relation of the diameter of the seminiferous tubules with stages of spermatogenesis, M.Q.L, 1958-1964. 56 That yearlings may be capable of breeding is relevant to what is observed on the breeding range. All adults and yearlings examined showed the same stages of development of spermatogenesis. However, in the spring in the field, only 10$ of the males on territory were yearling birds. If the possession of a territory is essential to breeding and adults excluded yearlings from territories (Bendell and Elliott, m.s.), then adults prevented potentially breeding males from adding to the population of breeders on the summer range.. Moreover, yearlings will replace adults on territory if these are killed (Bendell and Elliott, m.s.). From the comparison of the development of spermato• genesis no difference existed between these replacement yearlings and those that were silent. Hence yearling males may form a floating reserve of breeders that might replace adult males if they are killed. Although increase in size of the seminiferous tubules and in size of the testes showed a relation with stage of spermatogenesis, neither of these increases or relations could be used within the same age class as indicators of a specific stage of spermatogenesis. No sign of early development of spermatogenesis was found in the juvenile males before they left the summer range for the winter range. i 57 4-. Size of the interstitium The cells of the testis which are commonly thought to produce male hormones are the Leydig cells which are located in the intertubular tissue (Benoit, 1924-). The same author (1923) presented quantitative evidence to show that the volume of interstitial cells runs parallel to the development of the secondary sexual characteristics. There is considerable controversy among different authors about the variation in the relative and absolute amounts of intertubular tissue during the breeding season. Blanchard and Erickson (194-9) reviewed the literature on the point and came to the conclusion that the controversy was rather a question of method. They concluded that the bulk of the evidence seemed to point to an absolute increase in the total inter• tubular tissue up to the breeding period. In their own work on the Gambel sparrow, they arrived at the same result. Theoretically, the growth in total volume of intertubular tissue could be due to three processes: to an increase in size of the blood vessels, to the enlargement of individual intertubular cells, and to an increase in the number of these cells. Blanchard and Erickson (194-9: 275) have found these three processes responsible for the total increase. They found an increase in size of blood vessels especially in the later stages of spermatogenesis, an increase in size of the Leydig cells up to the peak of spermatogenesis, and an increase in number of Leydig cells up to the breeding stage. Therefore, assuming that in blue grouse as well as in Gambel sparrow the two parts of the intertubular tissue (the glandular part and the non-glandular part) show a parallel increase in volume, the measurement of the intertubular tissue alone should give us some indication of the activity of the interstitial gland. The first step was to find the amount of intertubular tissue in relation to the whole testis and then, with this value, to calculate the total amount of this tissue in the whole testis. Parts of one hundred and sixteen cross-sections were projected on sheets of paper with a projection drawing mirror attached to a microscope. Two or three microscopic fields were taken from each cross-section, at the most suitable magnification (X10, X100, X270), in order to sample approximately at least 10% of the complete cross-sections. The percentage of inter• tubular tissue was calculated for each drawing and the average then calculated for each cross-section. Measurement of the percentage of intertubular tissue was taken for eighty-two adults and thirty-four yearlings. The outlines of the tubules were traced with a sharp pencil. The pieces of paper with the areas representing the tubules and the non-tubular tissue were cut apart and the two sets of paper fragments were weighed separately. (The paper used showed a variation in weight of about 2%). The values obtained with this technique were subject to several errors. These included; the small sample, shrinkage and distortion during fixation or in the course of preparation of the slides, inaccuracy in drawing the tubules, and a variation in paper thickness, even if only the best cross-sections were used. Nevertheless, the changes in the intertubular tissue were large enough to make such errors relatively unimportant. Results* The weekly averages of the percentage of intertubular tissue in adult and yearling males from spring to summer are illustrated in Figure 12, The weekly averages of the volume of intertubular tissue in adult and yearling males, for the same period of time, are illustrated in Figure 13, The data from which these figures were drawn, number of observations, averages and range, are summarised in Table X, The percentage of intertubular tissue in the testis was greatest during the non-breeding period and proportionately smaller towards the end of spermatogenesis. However, there was still an increase in the absolute amount of intertubular tissue up to at least the beginning of the breeding period (Figure 13), This figure also shows that the higher percentage of intertubular tissue in yearlings was not the result of a larger absolute amount of intertubular tissue. In fact, as Figure 14 shows, the yearlings had a smaller amount of intertubular tissue during all the period spent on the breeding range and this was true even if the difference in their body weight is taken into consideration. As in the three proceeding tests, we found again the same characteristics in the yearling cycle when related to the'adult cycle; slower recrudescence, shorter breeding period and faster regression. In the study of stages of spermatogenesis, it was found that the two age classes were in comparable breeding stage only on one week, the first week of June. If we report this point on the curves of percentage of intertubular tissue (Figure 12), we note that both adults and yearlings had approximately the same amount of intertubular tissue 40 O Figure 12. Weekly average of the percentage of intertubular tissue in adults and yearlings, M.Q.L., 1958-1964. 61 Table X, Average amount of intertubular tissue in percentage and in absolute amount in adults and yearling males during the spring and summer season. Adults. Month Week Number Relative amount Absolute amount"1"1" Range of of of testes of intertubular of intertubular relative season examined tissue in % tissue in cc amount (birds) March 1 , 3 42.20 .067 34.60 - 51.30 2 7 23.73 .106 14.20 - 30.60 April 3 2 19.85 .160 18.80 - 20.90 4 U 19.62 .207 9.90 - 28.20 5 3 13.77 .217 12.40 - 15.30 6 3 14.39 .355 9.40 - 18.65 May 7 8 12.22 .174 7.30 - 18.15 8 5 9.41 .138 6.75 - 13.80 9 3 7.23 .099 4.85 - 9.95 10 4 9.98 .139 8.90 - 12.40 June 11 3 8.23 .084 5.96 - 9.85 12 4 10.90 .083 6.75 - 15.15 13 2 8.40 .063 8.35 - 8.45 14 4 12.74 .074 5.95 - 15.60 July 15 1 9.85 .039 16 1 12.75 .038 17 2 14.27 .030 14.05 - 14.50 18 3 13.18 .042 12.10 - 15.00 August 19 1 13.40 .020 20 2 22.17 .034 18.45 - 25.90 21 1 29.70 .044 22 1 37.50 .037 September 25 5 34.52 .034 28.20 - 41.20 Yearlings. March 2 1 35.40 .076 April 4 2 31.90 .118 ! 27.40 - 36.40 5 2 21.77 .162 20.15 - 23.40 6 3 20.02 .167 18.85 - 21.95 May 7 4 15.85 .129 12.50 - 20.10 8 6 13.56 .089 8.75 - 20.90 9 3 9.78 .078 7.40 - 10.15 10 3 12.30 .060 IO.45 - 13.90 June 11 2 9.95 .041 8.65 - 11.25 12 4 11.90 .056 10.85 - 12.95 13 1 15.25 .030 July 16 1 24.50 .036 18 1 28.40 .028 September 23 1 39-75 .039 calculated by applying the average percentage of intertubular tissue to the average volume of testes for a particular week. re 13. Weekly averages of the volume in cc of intertubular tissue in adults and yearlings, M.Q.L., 1958-1964. and this situation lasted for about two weeks. The first week of June was also the beginning of simultaneous primary molt in both age classes. Therefore, just before or at the beginning of molt, many processes seemed to become synchronised and showed quite similar values; size of the tubules, stage of spermatogenesis, start of recrudescence, percentage of intertubular tissue in the testis, and most likely fall migration. This does not prove any direct link between these facts, but suggests an hypothesis advanced by Blanchard and Erickson (194-9) that isj since these changes have developed in some degree interdependently with migration, the earliest or synchronised appearance of any part of the pattern is to be taken as the initiation of an adjusted and coherent whole. For further comparison between the stages of spermatogenesis and percentage of intertubular tissue, the relative quantity of inter• tubular tissue of each bird was plotted over its corresponding stage of spermatogenesis (Figure 14-). (Stage VIII was separated in early VIII A and late VIII B, because of its large range). In this figure we can see that, except for stages II and III, for which we had a very small, sample, the relative amount of intertubular tissue was approximately the jsame in each stage of spermatogenesis for the two age classes. This suggests that, with a smaller testis, the yearling had to achieve the same equilibrium between tubular and non-tubular tissue to realize a breeding cycle comparable to that of the adult. Theoretically, the growth in total volume of the testis could be due to any one, or a combination of three processes* (l) an increase in diameter of the seminiferous tubule, (2) an increase in length of the seminiferous tubules and (3) an increase in total amount of intertubular tissue. There was abundant evidence that all three processes were 52r Range in adults 48- Range in yearlings 44- 40- 36- 32 28- .1 24- 12- I U TH W I 3ZL ~SK 3ZDL 3ZE Winter Stages of spermatogenesis Rgure 14. Relation of percentage of intertubular tissue to stages of spermatogenesis in adults and yearlings,M.Q.L., 1958-1964. 65 involved. First, it was shown that the increase in tubule diameter runs approximately parallel with the increase in size of the whole testis. Second, since the maximum size of the seminiferous tubule reached by both age classes was the same, the larger amount of tubular material in the testis of the adult could only be due to longer tubules. Third, the absolute amount of intertubular tissue increased also in a cycle similar to the cycle of the testicular increase in volume. This cycle of the intertubular tissue increase was not in phase with the cycle of the increase in diameter of the seminiferous tubules in the yearling class. This caused the linear regression (of diameter of the seminiferous tubules against increase in size of the testis, Figure 8B) in yearlings to be closer to the diagonal since the influence of the growth in intertubular tissue|was almost completely nullified from one end to the other of the tubular cycle. Relation of the absolute and relative amount of intertubular tissue to behaviour. No constant difference in the percentage or the absolute amount of intertubular tissue was found between the samples of hooting and silent adult males or between replacement and silent yearling males. In seventeen weeks for which we had samples of hooting and silent birds for the two age classes, nine times the samples of hooting birds were larger than the samples of silent birds, seven times it was the reverse and once the two samples were equal. No regular pattern was found in any of these differences. Relation of the absolute and relative amount of intertubular tissue to age of bird. It was found in a similar analysis on the size of the volume of the testes that there was a tendency for older birds to have larger testes. Nothing like this was found in the study of the size of the seminiferous tubules. If the absolute amount of intertubular tissue of every known age bird is plotted with the average of the sample for the corresponding week, one can see again a trend towards a* greater amount of intertubular tissue in older birds (Table Xl), Table XI, Comparison of the amount of intertubular tissue of known age birds with the average of the adult class. Age of bird in year 1 2 3 A 5 Number of birds 3A 6 3 2 A Percentage over average 17$ 33$ 50$ 75$ Percentage on average 17$ 50$ Percentage under average 100$ 66$ 66$ 25$ Conclusions. The study of the relative and absolute amount of intertubular tissue revealed that the yearling males had a shorter cycle than the adult males. The recrudescence was slower and longer, the breeding condition was reached later and lasted for a shorter period of time and the regression was faster and earlier in the yearling than in the adult age class. The absolute amount of intertubular tissue in the yearling class was also smaller than in the adult class even if correction was made for the difference in body weight., 67 It seemed that an approximately equal ratio of intertubular tissue to total size of the testes was reached in the two age classes as the same stage of spermatogenesis was reached. The yearlings had approximately the same percentage of inter• tubular tissue as the adults for a very short period of time (two first weeks of June). This period approximately coincided with a similar situation in stage of spermatogenesis and diameter of the tubules. The beginning of June marked also the start of molt of primary feathers and probably the beginning of fall migration. The cycle of the absolute amount of intertubular tissue in yearlings was not synchronised with the cycle of the increase of diameter in the seminiferous tubules in the yearlings. No relation was found between the amount of intertubular tissue and the behaviour of the bird before it was shot. There was a suggestion that the older birds (4- and 5 years old) had more intertubular tissue than the average of the adult class. 68 5. Histology of the interstitium In the first step of this part of the study, variation in quantity of the intertubular tissue were investigated, assuming, on the basis of what Blanchard and Erickson advanced, that the quantitative variation of the intertubular tissue reflected similar quantitative variation in the interstitial cells. In the second step, the variation in quality of the glandular part of the intertubular tissue was investigated. The identification and physiology of the different types of intertubular cells in the testis are also the subject of controversy among different authors. Benoit (1927, 1929) described two phases in the interstitial, or Leydig cells. He also demonstrated a parallelism between the glandular activity of this tissue and the quantity of male hormones produced. He based his argument on a correlative enlargement of the appendages of the head of the cock. Sluiter and Van Oordt (1947), in their work on the cockerel, did not agree with Benoit. They did not see any reason to call cells which are full of lipoid vacuoles, glandular cells. They suggested that lipids were stored in the large vacuolated cells, and suggested that the glandular role was carried on by two smaller fuschinophil non-lipoidal cells. Marshall (1949), in his study of the fulmar, has recognized the same types of cells as Benoit and Sluiter and Van Oordt, but, working with a wild species in which the sequence of sexual behaviour was known, he disagreed with Sluiter and Van Oordt's interpretation and supported Benoit's views. Marshall's work supported the view that the endocrine function was located in the interstitium 69 and that it was the lipoidal cells, not the so-called "secretory" type of cell, which influenced the behaviour of the bird in its prenuptial sexual activities. He described the cycle of the interstitium and the role of the different types of Leydig cells as follows: " In sexually immature fulmars a small partially lipoidal juvenile cell occurs in the intertubular tissue of the testis. This cell develops into the large and strongly sudanophil mature lipoid Leydig cell not before one year and possibly when the bird is just over two years old. Once sexual maturity is reached, the bulk of the Leydig cells appear to belong to the lipoid type throughout the year except for a brief period which begins about the height of spermatogenesis. From the time the sperms have begun to appear, the Leydig cells are seen to be losing their lipoids until only a faint mottling of sudanophil substances remains in the 'cytoplasm. At this period numbers of non-lipoidal or partially lipoidal and strongly fuchsinophil Leydig cells appear. At the stage when free sperms are visible in the tubules and degenerating epithelial products begin to appear, a Leydig cell regeneration begins at odd spots in the interstitium. At the time of testis collapse and an accompanying fatty metamorphosis of the tubular epithelium, there appears a massive new development of small Leydig cells which may be identical with the juvenile cell in the young bird. These soon become partially lipoidal. They become distributed through the denuded interstitium, gain in size and sudanophil content become organised into groups bounded by fibrous connective tissue, and so the interstitial cycle has begun once more." Microscopic study of cross-sections of the testis of blue grouse revealed that most, if not all, the cells identified in the interstitium of other species (fulmar - Marshall (194-9), white crowned sparrow - Blanchard and Erickson (194-9), California gull - Johnston (1956)) could also be recognised in this species. No specific staining method could be used to positively identify these cells because our material was not fixed properly for such determinations. However, on the basis of the descriptions and measurements of these cells made by these other authors, enough similarities were found to make our identification of the three main types of Leydig cells valid. 70 Histology of the interstitium of the blue grouse. A description of the different types of cells present in the interstitium is given here. Only the details that permitted us to differentiate these different types of cells will be presented. 1. Melanoblasts. These were the pigment cells responsible for the very black appearance of the testis when this organ was at its smallest volume. These cells varied considerably in size, shape, and apparent number at least, both between individuals and during the course of.the summer. They were always found in the intertubular tissue and sometimes in the tunica albuginea. Spread out during the breeding period, they contracted and became more spherical late in the fall. They were always large and considerably obscured the architecture of the intertubular tissue because of their opacity. They were filled with small yellowish brown granules less than 0.5 microns in diameter. 2. Fibroblasts. One of the main cellular constituents of the connective tissue, these cells were present in many structures of the testis such as tunica propria, tunica albuginea and blood vessels. These cells could be recognised by their spindle-shaped nuclei. The size and shape of these cells varied with the stress put on the membrane or the tissue in which they were found. 3. Interstitial cells. Three different types of interstitial cells could be recognised. 71 Except for a short period of time, only two types were present at once in the interstitium. They could be differentiated on the basis of nuclear anatomy. a) The vesicular type of cells. Presumably the homologue of the Leydig cell of Benoit (1929) and of the lipoid Leydig cell of Marshall (194-9). This type of cell could be recognised by its large vesicular nucleus which grew to a maximum of 5 or 6 microns in diameter during the spring recrudescence and contained one, or sometimes two, chromatin condensations (Plate 15). Early in the spring a few large empty vacuoles, which presumably had once contained lipids, could be found in the cytoplasm of these cells. Another very similar type of cell could also be found in the interstitium only during the period of recrudescence. It could be distinguished from the vesicular type of cell only by its puffed out nucleus (Plate 16). Since this type of cell was similar in every other respect to the vesicular type of cell, and since it was never found in very large numbers, it was assumed that the puffed out cells could be an exhausted vesicular cell and it was not separated from the vesicular type of cell itself. The interstitium of two birds which died in the! aviary in February were sectioned to check for the presence of large vacuoles in the vesicular interstitial cells. Indeed, the interstitium of these testes was full of large vacuoles and the nucleus of the vesicular cells seemed to be squeezed between these vacuoles (Plate 14), b) The young cells. This second type of cell, present in the interstitium, was most abundant after the start of spermatogenesis and diminished rapidly to vanish by the end of the summer. These cells could be equated with the "juvenile" cells of Marshall on the basis of their size and time of appearance. These cells, about 8 to 10 microns in diameter, had at times very little cytoplasm. The nucleus was always constantj slightly ovoid and about 3 microns in diameter, and it was always stained more darkly by hematoxilin than any other intertubular cells. The chromatin in the nucleus was scattered in many small granules (Plates 17 and 18). These cells never seemed to vary in size during the period they were present in the testis. c) The juvenile cells. This third type of cell, present in the interstitium, appeared in small numbers shortly after the bird had reached full stage of spermatogenesis. They gradually increased in numbers and in size and were the only present type of interstitial cell with the pigment cells in the fall. These juvenile cells fitted the definition of the "glandular" phase of Benoit (1929), the "secretory cell A" of Sluiter and Van Oordt (1947) and the "fuchsinophil Leydig cell" of Marshall (1949). The identification of this type of cell was sometimes very difficult, since it varied in size throughout its existence in the testis. The best feature by which this type was recognised was its nucleus which varied from 3»5 microns to well over 4 microns in diameter in the fall. The shape of this nucleus was not so round or full as in the vesicular type of cell and the chromatin was much more broken into many granules (Plate 19). At, or after, the appearance of sperm in the lumen of the tubules, the cytoplasm of these cells gradually became filled with non-soluble droplets which were stained by hematoxilin (Plate 20)o These droplets became increasingly abundant and larger as regression proceeded (Plate 2l). They eventually obscured all other structures of the interstitium, then suddenly, before the end of regression, completely 73 disappeared (Plate 22). No explanation could be found for this phenomenon. The origin of each of the Leydig cell types could not be explained, but, since no cell divisions were found in the interstitium, the sequence of appearance of each type of cell supported the hypothesis suggested by Marshall (1949). In this hypothesis, the vesicular type would be the secretory, type of Leydig cell. Our young type would be a transitional stage between the exhausted vesicular cells and the juvenile type. This last type would progressively recuperate and become the large vesicular secretory type of the next breeding season. Because of technical limitations, only the relative abundance and time of appearance and disappearance of each type of Leydig cell could be worked out. Sixty-three slides (thirty-eight from adults and twenty-five from yearlings) were selected for this test. Since cellular details were used to identify the different types of cells, only slides that showed the best fixation and the best staining were used. Never• theless, care was taken to use, as far as possible, a representative sample for the whole summer. The method used by Blanchard and Erickson (1949s 268) and Johnston (1956) was adopted to measure the relative abundance of each type of Leydig cell in our material. The degree of abundance was estimated on an arbitrary scale as follows:: 5) Abundant; filling all the intertubular tissue with very few other cells present. 4) Commons: the predominant intertubular cell type, at least ten cells visible in any field at X450 magnification. 3) Fairly commons: one to three cells visible in any field at X450 magnification, or as often as any one other type. 2) Occasionnal: more than ten cells in a given section, l) Rare: up to ten cells in a given section, 0) Absent, Results, A graphic representation of the cycle of each type of Leydig cell in the interstitium of adult and yearling males over the summer is presented in Figures 15 and 16, These graphs,are subject to several biases and should be considered as estimates rather than an accurate measurement of the relationship existing between the different types of Leydig cells. With these restrictions in mind, it is of interest to note that the cycle of each type of interstitial cell appears to be in agreement with Marshall (194-9). It has already been mentioned that in February, birds from the aviary showed an interstitium composed almost entirely of the vesicular type of cells with very large vacuoles. As the adult birds migrated on the summer range, most, but not all of these large vacuoles had disap• peared and the interstitium was again composed almost exclusively of the large vesicular type of cells. It was difficult to establish whether these cells had started to diminish in number by March, but since few exhausted forms were found, it was suspected that this proeess had started. From the end of March, the vesicular cells diminished rapidly and were rarely found in May. In the testes sampled in the beginning of April the first young Leydig cells were found. Few at first, they rapidly increased and became almost the only type of cell found in the interstitium during May, They then disappeared and became vesicular cells young cells Figure 15. Estimated trend of the cycle of the different types of interstitial cells in yearlings I 2,3 4 5 6,7 8 9 IO, II 12 13 14 , 15 16 17 18, 19 20 21 22,23 24 25 Mar. 1 April 1 May 1 June 1 July 1 August 1 Sept. Figure 16. Estimated trend of the cycle of the different types of interstitial cells in adults, M.Q.L.,1958-1964. rare by July. The juvenile cells started to appear very slowly sometime in April, but it was not until July that they were commonly found. They reached their maximum number in August. In yearlings, the sequence of the appearance and disappearance of the three types of interstitial cells was the same as in adults but the timing was different. The vesicular type of cell showed some vacuoles in March, and it was not until the second half of April that the diminution of this type of cell was noticed. From then on they decreased rapidly in number and probably vanished at the same time as in the adults; by the end of May. Young cells were present in the testis of yearlings in March and reached their maximum number in late April. They seemed to start to disappear almost as soon as they appeared and were rarely found in June. The juvenile type first appeared in May, became fairly common in June and was most abundant in July. In yearlings as well as in adults, as the birds approached sexual maturity, the interstitium consisted almost exclusively of vesicular type of interstitial cells. Many authors have demonstrated a parallelism between the cycle of "lipoid Leydig cells" or our vesicular cells and the heightening of sexuality in wild birds. It was shown that yearlings:;had a smaller interstitium than the adults and it was suggested then that this was one reason for the secretive behaviour of yearlings on the breeding range. Another factor can now be suggested; Benoit (1956) made the point that different patterns in the sexual behaviour of birds were under the control of different quantities of sexual hormones. Then, if it is accepted that the cycle of abundance of the vesicular cells in the interstitium reflects the glandular activity of these cells (Orban, 1929J Pfeiffer and Kirschbaum, 1943), the delay in the cycle of interstitial cells of the yearlings seems to mean that they reached sexual maturity later, and probably too late to stimulate territorial behaviour in early spring. At the height of spermatogenesis and the laying period of the female, the vesicular type of cell had almost completely disappeared and the young interstitial cells were very abundant in the interstitium. As in all the other tests of this study, this period was shorter in the interstitial cycle of the yearling males as compared to the adults. The beginning of increase in number of the juvenile type of cell seemed to be synchronised with the beginning of many other processes: regression, in size of the testis, in the size of the tubules, and in spermatogenesis, molt of the primary feathers and, most likely, beginning of the fall migration, at least in certain segments of the male population. The cycle of the juvenile interstitial cells of the yearling males also correlated with their earlier testicular regression in fall. Conclusions. In this study of the different types of cells of the interstitium, most of the types of cells identified by other authors were recognised. The cycle of abundance of these types of interstitial cells (vesicular, young and juvenile) suggested that yearling males went through the complete cycle as compared with the adults, but had a shorter cycle: late recrudes• cence, short breeding period and early regression. 78 It was suggested that the secretive behaviour of the yearlings on the breeding ground could be due not only to a smaller amount of glandular interstitial tissue, but also to a delay in reaching the hormonal threshold of territoriality, which explains the secretive behaviour of these birds. A synchronisation was found between the appearance of the three types of interstitial cells and the three periods of the cycle of every other component of the testis. The presence of the large vesicular type of interstitial cell characterised the recrudescence period. The young type of interstitial cell and the absence of the vesicular type characterised the breeding period. The regression period was characterised by the increase in size and number of the juvenile type of interstitial cell. 79 DISCUSSION . This study described the testicular cycle of blue grouse. Attempts were made to correlate the main parts of their cycle with age, breeding behaviour, and migration. The whole testicular cycle showed all the characteristics of the cycle of a periodic breeder: a short recrudescence, a short breeding period, a longer regression and a very long quiescent period. Starting with the assumption that territorial behaviour is a sign of sexual maturity and breeding, the testicular cycle of the blue grouse was studied in all its components to find a difference that would correlate or help explain the secretive behaviour of the yearling on the breeding range. When one tries to explain the secretive behaviour of yearling males on the basis of these investigations, the following points are to be recalled. In spite of its behaviour and a very much smaller volume of its testes of the size reached by adults), the yearling male passed through every stage of the testicular cycle of the adult male and produced sperm as good as. adult sperm, as far as,'our limited comparison could show. All the tests made in this study showed a delay of two weeks in the recrudescence of the yearlings and an approximately similar delay in the start of the full breeding condition. These observations lead one to suspect that failure to hold territory and presumably to mate in the yearling males is a behavioural pattern that only endocrinological studies can explain. 80 Hormonal balance as such was not investigated in this study, but comparative measurements of the interstitial gland in the two age classes were made. With the help of other studies on this gland, our results can be interpreted as follows. Since we did not find any difference in the value of the sperm between age classes, the smaller total amount of tubular tissue - except for explaining a lesser production of sperm - cannot explain the behaviour of yearlings. Then the smaller interstitium gland which presumably secretes less hormone and a later spring recrudescence in the yearling age class were likely the reasons for the secretive behaviour of the yearlings. In other words, a smaller and later cycle of secretion of male hormone would result in less and too late aggressiveness, and in less and too late competitive ability in the yearling to gain for himself a territory early in the spring (AI1.ee, Forman and Banks, 1955? Gull, 196l). Several authors have suggested that the degree of aggressiveness in male birds was due to the relative amount of male hormone secreted by their gonads. Collias (1950) showed in the pigeon the relationship between the weight of the testes or ovaries and the sequence of behaviour patterns in the reproductive cycle. In a study of free-living pheasants, Collias and Taber (1951) have shown a sequence of behaviour patterns being related to.the recrudescence of the testes. From a dominance order prior to the onset of the breeding cycle, there was a gradual development of intolerance between males which eventually led to the formation of territories and harems. Working on California valley quail, Genelly (1955) confirmed a similar sequence, with the peak of agonistic behaviour coinciding with full recrudescence of the gonads.. Recent works (Hui Way and Hoar, 1963} Davis, 1963; Vandenberg, 1964) have shown that pituitary hormones had also the effect of increasing the aggressiveness in some vertebrates. It was suggested that at least the first stages of aggressive behaviour (territorial) were under the control of LH. Benoit (1956s 182) correlated variations of spermatogenic activity with variations in the secretion of male hormone: 11 Aux variations de l1 activity spermatogene*tique correspondent des variations de la secretion endocrine de 1'hormone male,' "responsable du developpement des caracteres sexuels et de presque tous,..les actes instinctifs du comportement sexuel." and continueds "Les hormones exercent des effets qui sont dans 1'ensemble proportionnels a leur quantite." Gull (i960) also supported this view of a close relationship between the recrudescence of the gonads and the sequence of certain patterns of behaviour. No difference was found in spermatogenesis or histology of the interstitium between replacement or territorial yearlings, and yearlings found silent usually near an adult and presumably suppressed by him. Moreover, the testes of hooting or replacement yearlings:did not grow in size when the adults were removed from their environment. These observations may be explained partially by the late development of the yearling testes which is an inherited character, and partially by the fact that the presence of adult males did not suppress the growth of the yearling testes but the expression of their maturity. Furthermore, if different behavioural sexual patterns are under the influence of different hormonal thresholds, and if territorial behaviour is one of 82 the highest threshold to reach before a bird becomes territorial (Benoit, 1956), then one would expect that the removal of some environ• mental inhibiting factors will permit a bird to reach a high threshold even with a smaller amount of hormone. The removal experiment conducted on the study area of Middle Quinsam Lake (Elliott, m.s.) in one way verified this. The disappearance of territorial adult males, by removing an inhibiting factor on the yearlings, presumably permitted them to reach the territoriality threshold even with a smaller amount of hormone. Further speculation suggests the following hypothesis: in a newly opened area suitable for breeding for blue grouse, the yearlings would breed commonly at least during the first few years after the opening of the area. The small percentage {10% of the male population) of hooting yearlings found outside the removal plots can probably be explained by the same phenomenon, but would be instigated there by natural fatalities in the population of adult males during migration or in the spring. Bendell (1954-) and Bendell and Elliott (m.s.) could not find any surplus birds in a population of males over two years of age on Vancouver Island. This suggested that any bird over two years old found a territory, no matter what the population density (at least at the density studied). Zwickel (personal communication and field notes), working on a small study area on Vancouver Island, and consequently having more frequent observations of individual male, has brought a different element into this picture. Four yearlings banded in the summer of 1962 were back on the breeding range in 1963 as two year old birds. Only one of them seemed to hold a good territory and was observed many times hooting and displaying. The three others were observed a few times but did not seem to hold definite territories and they apparently left the breeding range early. In 1964, these four birds were back on the breeding range again (now as three years old birds) and all four held good territories Of eight yearlings which were banded as silent birds on the breeding range in 1963 and which returned in 1964, only two held good territorie in 1964. Three others were observed hooting only once, and the other three never showed any sign of territorial behaviour. The population on which Zwickel is presently working is estimated at the same density level as the one from Middle Quinsam Lake, from which Bendell and Elliott's conclusions were derived. Independently of these observations it was found in our material that the size of the testis not only varied between adult and yearling age classes, but also varied between the different age groups within the adult class. Furthermore, this difference in volume correlated with a difference in the amount of intertubular tissue. These data supported the notion that blue grouse continue to grow sexually past one year of age and that in a similar way, territorial behaviour becomes stronger with age. Hooker (1944) worked on the history and function of the interstitial ceil from birth to adulthood in bulls. He showed that even if full spermatogenesis was reached by an eight month old bull, it was not until five to seven years of age that the animal reached its maximum weight of testis and its maximum rate of secretion of male 84 hormone. It is not uncommon to find among vertebrates that the dominant males serve most of the females, Scott (194-2) noted that in sage grouse (Centrocercus urophasianus) dominant males (3% of the male population) were responsible for 83% of the copulations observed. The blue grouse being a territorial bird on its breeding range, no dominant order can be easily observed, but Zwickel's data and our observation of a larger interstitium suggest that older birds might be also more efficient breeders because they spend more time on the breeding range and show a stronger territorial display. Several authors have suggested that many yearling males do not migrate onto the breeding range. Wing (194-7) has made references to the "big males" observed on the winter range1 during the summer and to the absence of females. He suggested that they were mature non- breeding males. Beer (personal communication and field notes to Bendell, 1954) stated that in late June he collected ten males at high altitudes in Washington State. On the basis of their plumage, the soft texture of the neck tissue and the small size of their testes he concluded that they were all immature males hatched the preceeding year. However, he did not reject the idea that they could be migrating birds. With more recent data these observations can be interpreted differently. New techniques have shown that yearlings are present on the breeding range in a much greater number than was once believed and that they start their fall migration earlier than the adults (Bendell and Elliott, m.s.). All the yearlings shot on the breeding range during the summer, without exception, seemed to accomplish the full sexual cycle. No specimens from the wintering range during the summer were available, Johnston's work (1956), with which we have drawn many parallels, has shown that, in all probability, no difference existed in the sexual cycle between birds which migrated and those which did not. Johnston rejected the following view of Behle and Selander (1953 s 24-6): "perhaps the lack of development of the gonads is correlated with poor development of the migratory instinct." Refering to the material presented here, one can see that by the end of June the size of the testes of yearlings has almost regressed'to the average size of the winter stage. One should also remember that the study of a population of blue grouse in central Washington (Standing, I960) gives a phenological table that shows two weeks advance over our population in the same reproductive events. Thus, all facts considered, it may be expected that by the end of June, flocks of yearling birds, and perhaps of two year old birds (that have almost terminated their sexual regression and show little, if any, external signs of breeding maturity) be found on the wintering range. Thus what Beer saw might have been yearlings that had migrated and returned early to the winter range. Several cogent arguments support the assumption of a coherence of patterns between migration and the reproductive cycle. It cannot be denied that migration might be a phenomenon wholly unconnected with changes of the gonads or molt. But since migration is a movement towards a nesting site in the spring, it is not surprising that a parallel exists between this movement and the changes of the gonads. With these facts in 86 mind Blanchard and Erickson proposed the following working hypothesis: "these changes have developed in some degree interdependently and the earliest appearance of any part of the patterns is to be taken as the initiation of an adjusted and coherent whole". The testes of juvenile male blue grouse collected in September, contrarily to what was shown by Kirkpatrick and Andrew (1944) for the pheasant, did not show any sign of spermatogenic activity. It is not likely that recrudescence would proceed during the fall migration either. Then we concluded that juvenile birds arrived on the wintering range for their first winter with impuber testes. Thus, if we agree with Benoit (1956: 176) that the preceding sexual status influences the actual one " A un moment donne du cycle reproducteur, l'organisme de l'oiseau se trouve dans un etat physiologique determine. Cet etat est cree 'motive' par les differents facteurs.externes et internes du moment, mais aussi par les etats physiologiques anterieurs" we can conclude that yearlings were later in their recrudescence because they were younger. 87 SUMMARY 1* The testicular cycle of blue grouse (Dendragapus obscurus fuliginosus) was studied on its summer range on Vancouver Island over the years 1958 to 1964. and was compared with the age, breeding behaviour and migration. 2. Study of the volume of the testes of 224 birds showed that adults had a much larger average maximum size of testes (2.09 cc) than the yearling age class, which had an average maximum size of .83 cc at the beginning of the breeding period. 3. The yearlings had not only a smaller cycle of size of testes, but also a delayed and slower recrudescence, a shorter breeding period, and an earlier and faster regression. 4. The breeding period of the cycle of the volume of the testes was about one week shorter in yearlings than in adults. 5. Study of the size of the seminiferous tubules in 124 birds showed that although yearlings had a different cycle in timing, they reached the same maximum size of the seminiferous tubules as the adults. 6. The yearlings had a later and slower recrudescence, a shorter and delayed breeding period, and an earlier and shorter regression of their seminiferous tubules. 7. The breeding period of the cycle of the seminiferous tubules is smaller in the yearlings and do not reach a plateau as in adults from the middle of April to the middle of June, but they have a single peak of development at the middle of May. 8. Study of the development of spermatogenesis in 119 birds showed that all male birds present on the breeding range pass through 8 stages of spermatogenesis but that they were not synchronised in the development of each stage. 9. The yearlings had a shorter cycle of spermatogenesis; later and slower recrudescence, shorter and delayed breeding period, and earlier and faster regression. 10. The full breeding stages were reached by yearlings two weeks after the adults had attained these stages. This period extended from the first week of April to the first week of June in adults, and from the third week of April to the second week of June in yearlings. 11, A crude test of samples of semen from 6 birds of the aviary showed that adults and yearlings most likely had semen of approximately equal value.. 12. In the aviary a yearling bred with an adult female. This hen subsequently laid 13 eggs, of which four hatched under artificial incubation, 13, Study of the intertubular tissue in 116 birds showed'that in percentage or in absolute amount, the cycle of the intertubular tissue in yearlings was shorter and smaller than in adults* 14, The yearlings developped approximately the same percentage of intertubular tissue as the adults for only a short period of time, from the middle of May to the first week of June, Delayed recrudes• cence and earlier regression were also quite obvious. 15. In the cycle of the amount of intertubular tissue in yearlings the 89 delayed peak was not so obvious, but slower recrudescence and faster regression were again quite noticeable. 16. The yearlings never had more than half the absolute amount of intertubular tissue of the adults. This was also true if a correction was made for the difference in body weight between the two age classes. 17. The study of the interstitial cells in 63 birds showed that both adults and yearlings had 3 types of intertubular cells: vesicular, young, and juvenile types. This study showed also that the sequence of appearance of each type of interstitial cells was again supporting a shorter cycle in yearlings. 18. The presence of the vesicular type of interstitial cell in the interstitium of blue grouse corresponded to the period of heightening sexuality in the male. The young type of interstitial cells which seemed the precursor of the juvenile type started to appear shortly after the bird reached the breeding stages of spermatogenesis. The juvenile type of interstitial cells showed an increase parallel to the rate of regression of the testes in each age class. 19. The two last weeks of May and the first week of June seemed to be the only period when the yearlings were in approximately similar physiol• ogical state to the adults. This period also correlated with the start of fall migration in males and the start of primary molt in both age classes. 20. Although a relation of cause and effect is not sought here, the late recrudescence of the testes of yearlings seemed to correlate with a later migration of the yearling to the breeding range, as noted in the spring 1964. The earlier regression of the testes of the yearlings 90 correlated also with the earlier fall migration of the yearlings towards the wintering range. 21. Although the differences in the testicular cycle of the yearlings and adults were very small when they arrived on the breeding range, it seemed that the yearlings were already behind in their cycle. 22. Equations for the volume of the testes for the three parts of the annual breeding cycle (recrudescence, breeding and regression) were calculated for both age classes. 23. Equations showing the relation of tubule diameter to testis size were calculated. They showed that increase in size of the seminiferous tubules was mainly responsible for the increase in size of the testes, but that the amount of intertubular tissue also had an influence. 24-. There was no marked differences between the size of the testes of two populations of blue grouse at different densities, but at the same latitude. On the other hand, there was a two weeks difference in the phenology and size of the testes between our population and another population of blue grouse studied in central Washington State. 25. No relation could be found between the size of the testes and the body weight except for the general one that yearlings, being lighter than adults, had smaller testes. This relation was true even if correction was made for the smaller body weight of the yearlings. 26. Although there was a good correlation, similar in both age classes, between diameter of the tubules and successive stages of spermato• genesis, there was sufficient overlapping between the stages in each age class to prevent the use of this method as indicator of a specific stage of spermatogenesis. 27. There was a good correlation between the volume of the testes and the stages of spermatogenesis within the same age class, but not between age classes. Overlapping between stages of spermatogenesis was too great to make volume of the testes a reliable indicator of a specific stage of spermatogenesis. 28. There was a good correlation between the percentage of intertubular tissue and the stages of spermatogenesis, between both age classes and it seemed that yearlings, as adults, had to attain a similar relative amount of intertubular tissue before they reached a same stage of spermatogenesis. 29. No relation in any component of the testes examined in this study was found between hooting adults or territorial yearlings and silent adults and presumably suppressed yearlings for the same period. 30. Although there was only 21 adults of known age to verify this, there seemed to be a trend in the adult population towards older birds having larger testes and more intertubular tissue, but no differences were found for the size of the seminiferous tubules. 31. The behaviour of most yearlings on the breeding range can be explained partially by a slower rate of sexual growth and a shorter period of sexual maturity than in adults, and partially by the fact that they are dominated by other males. 92 LIST OF REFERENCES ALDRICH, J.W., 1963. Geographic orientation of american tetraonidae. Jour, of Wildl. Mgt., 27: 529-545. ALLEE, W..C, D. Forman and E.M. Banks, 1955. Effects of androgen on dominance and subordinance in six common breeds of (Gallus gallus). Physiol. Zool., 28: 89-115. 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Spermatozoa concentration in the semen of two breeds of fowl estimated by three different methods. Poul. Sci., 4.0s 608-615. VANDENBERG, J.G., 1964.. The effects of photoperiod on testicular activity and aggressive behaviour of starling. Jour of Exp, Zool., 156: 323-330. VAN ROSSEN, A., 1925, Flight feathers as age indicators in Dendragapus. Ibis, Series 12, Is 417-4-22. WING, L., 194-7. Seasonal movements of the blue grouse. Trans N. Amer. Wildl. Gonf., 12s 504-511. WRIGHT, P.L. and R.W. Hiatt, 1943. Outer primaries as age determiners in gallinaceous birds. Auk, 60: 265-266. WRIGHT, P.L. and M.H. Wright, 1944. The reproductive cycle of the male Red-winged Blackbird (Agelaius phoeniceus). Condor, 46s 46-59. WYNNE-EDWARDS, V.C..., 1962. Animal dispersion in relation to social behaviour. Hafner Publ. Comp., New York. Plate 1. Stage I of spermatogenesis, X400. Adult specimen no. 3, taken March 19, 1964- at Wolf Lake. Plate 3« Stage III of spermatogenesis, X400. Adult Plate 4. Stage IV of spermatogenesis, X400. Adult specimen no. 10, taken March 19, 1964 at Wolf Lake. specimen no. 19, taken April 9, 1964 at Wolf Lake. Plate 5. Stage IV of spermatogenesis, X1000. Adult Plate 6. Stage V of spermatogenesis, X400. Adult specimen no. 19, taken April 9, 1964 at Wolf Lake. specimen no. 23, taken April 10, 1964 at Wolf Lake. Plate 7. Stage V of spermatogenesis, X1000. Adult Plate 8. Stage VI of spermatogenesis, X400. Adult specimen no. 30, taken April 12, 1964 at Wolf Lake* specimen no. 42, taken April 20, I960 at M.Q.L. Plate 9. Stage VII of spermatogenesis, X200. Adult specimen no. 78, taken May 6, I960 at M.Q.L. Plate 11. Stage VIII B of spermatogenesis, X200. Plate 12. Winter stage of spermatogenesis, X1000. Adult specimen no. 214, taken August 31, 1963 at Yearling specimen no. 215, taken September 1, 1962 M.Q.L. at M.Q.L. Plate 13. Spermatozoa, X1280. Sample from aviary- Plate 14. Intertubular tissue in February, X400. bird, taken June 5, 1964. Stain Eosin nigrosin. Specimen no. 284, died February 19, I960 in the aviary. Plate 15. Large vesicular type of interstitial cell, X1280. Adult specimen no. 3, taken March 19, 1964 at Wolf Lake. Plate 17. Young and vesicular types of interstitial cell, X1280. Adult soecimen no. 78, taken May 6, I960 at M.Q.L. Plate 19. Juvenile type of interstitial cell, X1280. Adult specimen no. 79, taken May 6, I960 at M.Q.L. Plate 21. Juvenile interstitial cell full of drop• lets, X1280. Adult specimen no. 208, taken August 1, 1962 at M.Q.L. APPENDIX II TABLE XII- Data on testes used in this study. CQ ca x> •aH CD o (D P -p o -P CO ca •H O Oo p 0 S o 0u) o Xi P o cd -P'M H roH CD rH si H bD u °.s faO O O a, Cd X* 0 •H TJ O H o CD ca P 3 CD U -p cd CD P CD G P CD > *H a> t> CD H CD f-l $ +3 496 M 19/ /62 A S .3 a r 19/1450/64 W S .2 83.1 34.60 c W S .2 87.8 h 2l/H15/64; 41.35 173 (1) 22/ 620/60 M Y S .2 82.5 1273.2 172 22/1620/60 M S .1 82.5 51.3 1118.8 26/1210/64 W s .38 103.0 25.15 28/1145/64 W s .3 108.8 30.6 28/1340/64 w s .4 108.3 26.04 29/1330/64 w s .41 107.4 25.65 174 (2) 29/1630/60 M s .5 126.0 17.55 1340.8 175 29/1655/60 M s .5 1378.0 30/1205/58 L 1.2 170.5 14.20 30/1500/64 W s .4 111.0 26.9 176 31/1350/64 M Y S R .2 99.0 38.40 1107.8 A = Aviary S = Silent x = diseased W - Wolf Lake H = Hooting M = Middle Quinsam Lake Y = Yearling L = Lower Quinsam Lake C = Juvenile R = Removal plots 179 (3) 5/1105/60 M H R 1.0 181.5 20.90 1341.4 180 8/1150/60 M S .8 143.1 18.80 1248.9 A 9/1150/64 W S 1.0 165.0 19.55 P r 9/1420/64 W s .6 168.8 20.65 X 185 1 10/ /60 M Y H? .35 112.0 36.40 1200.0 10/1120/64 W s .7 135.2 26.15 10/1440/64 w s 1.1 10/1620/64 w H 1.3 155,3 17.70 186 11/ /60 M H 1.6 1380.0 11/1245/64 w S 1.6 186.7 18.20 11/1305/64 w S 1.1 149.0 28.20 (4) 11/1455/64 1 H .9 160.1 24,95 188 12/1600/60 M H R 1.5 189.5 1373,5 189 12/1650/60 M S R 1.5 146.5 1295.0 12/1010/64 W S 1.5 152.6 15.30 12/1545/64 W S .5 142.1 24*80 190 13/ 940/60 M H 1.2 154.0 21.35 1444.1 14/1340/64 W s 1.6 205.9 20.45 191 14/1445/60 M H R 1.3 187.0 12.40 1304.0 14/1715/64 ¥ Y S .4 155.6 27.40 192 15/ /60 M H R 1.0 181.5 9.90 1394.0 193 15/1225/60 M H 1.8 209.0 15.05 1295.5 835 15/1335/62 M Y S R .4 110.0 950.0 195 (5) 17/1047/60 M Y S R .7 170.5 23.40 1150.0 836 18/1010/62 M S R 2.0 231.0 12.05 1360.0 196 . 18/1150/60 M I S R .8 191.2 20.15 1154.0 197 20/1135/60 M S R 1.0 181.5 15.30 1260.1 198 (5) 20/1220/60 M Hi R 1.8 209.0 13.90 1206.5 337 21/1022/60 M H 2.4 159.5 1373.9 338 21/1125/60 M S 1.4 1359.1 342 A 25/1500/60 M H 3.6 236.0 9.40 1480.1 P 343 r 25/1630/60 M H R 1.95 171.6 15.11 1155.1 jt 1 26/1900/60 S R 2.0 150.0 1393.6 344 1 M 18.65 345 (6) 26/1940/60 M Y H R .8 143.0 18.85 1189.0 348 27/2025/60 M Y S .8 165.0 19.25 1152.0 350 28/1330/60 L H 1.9 1291.3 351 28/2020/60 L Hi 1.0 1282.2 352 28/2110/60 L Y S .9 159.5 21.95 1193.0 353 2/1035/60 M Y H. R .8 154.0 20.10 1212.0 354 2/1125/60 M Y. H. R .8 170..5 16.95 1226.0 364 2/1425/61 M 2 S 1.55 1296.0 839 M 3/1000/62 M & 1.2 203.5 11.10 1301.0 a 840 3/1030/62 M H 2.4 1235.0 7 546 (7) 3/1035/62 M 2 s; R .8 176.0 15.50 1233.0 841 3/1047/62. M H 1.4 1110.0 108 3/1205/62 M 5+ H R 1..2. 176.0 18.15 1318.0 842 3/1343/62 M H 1.4 1176.0 843 3/1402/62 M S 1.65 1296.0 355 3/1915/60 M 2.2 1394.0 4/ 800/61 M H 1.6 664 4/1430/61 M ft 2.4 1279.0 No 14 5/ /61 M Y .8 620 5/1800/62 M 2 S R 1.2 174.5 9.65 1299.0 363 5/2100/60 M H 1.6 I444.O No 9 5/ /61 M 2.0 1330.0 No 12 5/ /61 M 1.7 No 13 5/ /61 M 1.5 No 15 (7) 5/ /61 M .9 1322.0 No 16 5/ /61 M 1.4 1375.0 No 17 5/ /61 M 1.2 1187.0 No 19 5/ /61 M 1.2 1258.0 386 M 6/1430/61 M 2 S R 1.6 187.0 11.25 1262.0 a I6O 365 y 6/2005/60 M H R 1.0 .4 11.80 1286.0 366 6/2050/6O M H R 1.2 151.3 13.00 1254.0 8/1700/60 L S 2.7 1320.0 844 8/2017/62 M Y H R 1.0 198.0 12.50 1150.0 678 8/2025/62 M 3+ H R 1.7 203.5 7.30 1220.0 845 8/2055/62 M Y H R .7 187.0 13.80 1000.5 9/ 940/62 M Y S .5 132.0 15.15 901.0 9/1247/62 M Y S .8 203.5 11.30 1106.0 606 9/1400/61 M H 1.2 149.7 8.65 1344.0 (8) 9/1515/62 M H 2.0 1213.0 9/1930/60 L H .7 1175.0 9/2025/60 L H 2.3 1350.0 370 9/2100/60 M Y H R .9 143.0 20.90 1190.0 371 9/2115/60 M Y H. R .6 192.5 8.75 1062.0 667 10/1205/61 M 1.5 166.5 6.75 1271.0 10/1930/60 L H 1.7 1400.0 10/1940/60 L H 1.2 1250.0 372 10/2115/60 M Y H. R .5 1234.6 373 10/2130/60 M H. R 1.5 1270.0 374 11/1020/60 M Y H R .7 187.0 9.85 1159.0 M 12/1715/60 L H 1.2 1293.0 a y 12/1930/60 L H 1.3 1160.0 (8) 12/2105/62 M H R 1.3 165.7 8.15 376 12/2130/60 M Y H R .5 1172.0 615 14/1215/61 M H 1.2 1113.0 616 14/1555/61 M S 1.2 1263.0 618 14/1630/61 M H 1.2 1171.0 422 14/1725/62 M 4* H 2.3 1362.0 1 14/2030/59 M H R 1.8 181.5 9.70 1300.0 689 15/1055/62 M 3 + H 1.6 1214.0 32 15/1515/62 M 5* H 1.5 196.5 13.80 1289.0 15/2110/62 M Y H R .4 203.5 15.40 1165.0 16/1345/60 L H 1.2 154.5 6.90 1144.0 16/1550/61 M H 2.2 1291.0 17/1220/60 L H 1.6 159.8 4.85 1208.0 636 (9) 17/2000/61 M H 2.4 1299.0 19/1502/61 M Y H R .9 181.5 7.40 1191.0 681 20/ 905/61 M s, 1.0 1230.0 682 20/1510/61 M Y S R .7 198.0 10.15 1317.0 109 21/1650/62 M S 1.4 168.8 9.95 1330.0 (1) (9) 22/ /58 M H 1.6 22/2035/62 M Y S R .7 192.5 11.80 1148.0 673 24/1620/61 M H 1.0 159.7 8.90 1251.0 672 25/1205/61 L H .9 1215.0 10 25/1505/59 M S R 3-0 198.0 9.25 1375.0 (3) 26/ /58 M S .8 U) 26/ /58 M S 1.6 (2) 26/ /58 M Y s .7 AF 1 M 27/1020/60 M Y s .6 1250.0 a s y 28/ 930/62 M .9 155.7 9.35 1101.0 (10) 28/1015/62 M Y s .3 137.5 12.50 1183.0 AF 3 29/1225/60 M Y s .7 1135.0 AF 2 29/1645/60 M s .9 1280.0 (5) 29/ /58 M H 1.5 (6) 29/ /58 M S 1.6 194 30/2100/61 M 3* H 1.2 1359.0 (7) 30/ /58 M H 1.3 32/1007/62 M S .85 171.5 12.40 1188.0 31/1100/62 M Y s. .5 170.5 13.90 1067.0 31/2150/62 M Y H R .7 176.0 10.45 1116.0 15 J 1/1000/59 M H R 1.8 1396.0 u 403 n 1/1000/61 M 3 S . .8 192.5 5.96 946.0 e 16 (11) 1/1035/59 M Y S R .8 192.5 1250.0 110 106 2/1215/60 M 3* S 1.6 52 2/1610/59 L S 1.8 1317.0 2/1645/60 L Y H .5 165.0 11.25 1192.0 24 2/2145/59 M H 1.8 1286.0 (8) 3/ /58 M H 1.2 AF 5 4/1225/60 M H .95 1300.0 AF 6 (11) 4/1230/60 M Y H .7 165.0 1100.0 AF 7 4/1330/60 M H 1.8 1345.0 AF 9 4/1530/60 M H 1.0 1320.0 27 J 4/2100/59 M H 1.2 187.0 8.90 1320.0 u AF 12 n 5/1930/60 M H 1.2 1378.0 e 6/1435/62 M Y S .3 137.5 8.65 1100.0 120 7/2200/62 M 4 H 1.0 187.0 9.85 1196.0 AF 15 8/1030/60 M H .65 I24O.O 29 8/U00/59 M S 1.2 1230.0 484 10/2020/61 M 3 + H ,8 123.2 10.20 11/1255/62 M Y S .5 176.0 10.85 1093.0 54 11/2225/59 L H .9 1388.0 AF 18 13/ 915/60 M H .9 1375.0 AF 20 (12) 13/1020/60 M H 1.2 1260.0 AF 21 13/1035/60 M H .8 1360.0 704 13/2050/61 M Y H .7 134.5 12.85 989.0 415 13/2200/61 M 2 S 1.0 192.5 6.75 1161.0 13/2210/62 M H .9 176.0 11.50 1263.0 41 14/2205/62 M 5* H 1.8 209.0 15.15 1222.0 14/2210/62 M H ^.6 1202.0 (12) 15/1130/62 M I S .3 181.5 12.95 1162.0 15/2230/62 M Y H R .4 170.5 10.95 1229.0 16/1830/62 M I S .2 143.0 15.25 1129.0 721 17/1300/62 M 2 H .7 165.0 8.35 1265.0 56 (13) 17/2000/59 L S .9 1311.0 AF 22 19/1235/60 M S .8 163.1 8.45 1240.0 658 21/ /61 L H? .8 1337.0 22/1200/62 W S? .7 J 24/2200/62 M H .4 104.5 14.70 1229.0 u s; n 25/ 655/61 L .8 1238.0 e (12) 26/ /58 M H .•4 1162.0 (13) 26/ /58 M H .6 1133.0 (14) 26/ /58 M H .4 1091.0 (15) 28/ /58 M H .5 1133.0 (16) (14) 28/ /58 M H .5 1303.0 (17) 28/ /58 M . H .4 1274.0 (18) 29/ /58 M H .5 148.5 14.70 1331.0 (19) 29/ /58 M H 1.2 181.5 5.95 1246.0 (20) 29/ /58 M H .8 1288.0 (21) 29/ /58 M S .6 132.0 15.60 1303.0 (22) 29/ /58 M H .4 1106.0 122 29/1300/59 M S .8 1435.0 (23) V /58 M HI .4 1331.0 638 (15) 4/1735/62 M 2 S .4 154.0 9.85 1298.0 8/1445/60 L Y H. .1 99.0 1108.0 8/1450/61 L H .65 1231.0 131 10/1030/62 M 4 H .3 115.5 12.75 1270.0 61 (16) 11/ 940/59 L Y H .15 99.0 24.50 1036.0 76 12/2045/61 L 4^ H .3 1171.0 436 13/1630/60 M s a 2.9X J 19/1117/62 M H .28 110.0 14.50 1055.0 u 1 20/1208/62 M S .25 1190.0 (17) 20/2045/62 M H .4 1180.0 23/1210/62 M S .15 88.0 14.05 1145.0 24/1035/62 M H .4 121.0 12.45 1304.0 25/1104/62 M H .25 82.0 15.00 1209.0 26/ /61 M .1 (18) 27/1107/60 L S .3 1330.0 27/1327/62 M H .3 1227.0 63 30/1215/59 L Y S .1 88.0 28.40 1151.0 39 31/2025/62 M 5 + H .33 104.5 12.10 1150.0 (19) 1/ 927/62 M S .15 99.0 13.40 1210.0 113 (20) 9/1218/62 M S .12 77.0 18.45 1118.0 A 10/1008/62 M S .18 71.5 25.90 1216.0 u g u 67 s 19/1400/59 L S .15 82,50 29.70 1127.5 820 (21) 19/1945/61 M Y .1 (22) 31/ /63 M Y .14 31/ /63 M S .1 82.5 37.50 (23) 1/ /62 M Y .1 82.0 39.75 (24) 15/ /62 M C .01 55.0 S 15/ /62 M C .05 49.5 P t #10 e 16/ /62 M H .08 77.0 33.65 m M .1 55.0 36.85 #11 b 16/ /62 S e /62 66.0 41.20 #13 r 16/ M H .1 #14 (25) 16/ /62 M S .12 71.0 32.70 #15 16/ /62 M H .1 77.0 28.20 F e b 18/ /61 A Y .09 55.0 51.80 r u 19/ /60 A Y .09 55.0 57.40 a r