NON-PASSERINE BIRDS BRIEF OBSERVATIONS ON THE SEMEN AND SPERMATOZOA OF CERTAIN PASSERINE
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
BRIEF OBSERVATIONS ON THE SEMEN AND
SPERMATOZOA OF CERTAIN PASSERINE AND
NON-PASSERINE BIRDS
P. N. HUMPHREYS
Department of ^oology, University College, Cardiff
(Received 27th November 1970, accepted 20th October 1971)
Summary. The semen of some passerine and non-passerine birds was
examined. That of passerine birds was thick in consistency and con-
tained relatively few spermatozoa, while that of non-passerine species
was more liquid in nature with many spermatozoa.
The two types of spermatozoa were found to differ considerably in
morphology and activity, the passerine form showing a spiral configura-
tion and a helical forward movement. The differences were also
pronounced at the ultrastructural level.
INTRODUCTION
Although the sperm morphology of birds has received attention from the times
of Ballowitz (1888) and Retzius (1909, 1911), there has been little study of the
differences known to exist between the semen of passerine and non-passerine
species. McFarlane (1963, 1971) has shown that there are important differences
in the morphology of avian spermatozoa and considers that these differences
may be of phylogenetic importance. This was the conclusion of Tuzet (1950)
and also of André (1963) who stated that 'the micro-anatomy of the sperm might
be as instructive to the systematist, and sometimes even more so, than the
micro-anatomy of the whole animal'.
The only bird species whose semen has been extensively studied are the
domestic and game species such as the common fowl, Gallus domesticus, duck,
Anas platyrhynchos, turkey, Meleagris gallopavo, goose, Anser anser and, more
recently, Japanese quail, Coturnix japónica (Lake & Smiles, 1952, 1954; Johnson,
1954; Watanabe, 1957; McCartney & Brown, 1959; Szumowski, 1960;
Cooper, 1963, 1969; McFarquhar & Lake, 1964; Lake, 1966, 1969).
The following account represents a preliminary study of the semen and sperm
morphology of some groups of birds which, hitherto, have been little repre¬
sented in the literature.
MATERIALS AND METHODS
The species of birds involved were the mallard, Anas platyrhynchos, Hawaiian
goose, Branta sandvicensis, house sparrow, Passer domesticus, tree-creeper, Certhia
327
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free access328 P. . Humphreys
familiaris, canary, Serinus canaria, robin, Erithracus rubecula, collared dove,
Streptopelia decaocto, jackdaw, Corvus monedula and lapwing, Vanellus vanellus.
Spermatozoa obtained from recently dead specimens of all these species were
taken from the epididymis and vas deferens and both unstained samples and
those stained with Leishman's stain, Heidenhain's haematoxylin and méthylène
blue were examined with the light microscope.
Tissue samples of mature testes were fixed in Bouin's solution, and embedded
in paraffin wax in the conventional manner. Sections were cut at 7 µ , stained
with Heidenhain's iron haematoxylin and Orange G, and examined under
the bright field of an ordinary microscope.
Semen was obtained from the mallard drake and Hawaiian gander by the
abdominal massage method (Johnson, 1954) and from canaries by gentle
squeezing of the cloacal protuberance (Wolfson, 1960) after removal of the
cloacal guard feathers. Both were examined undiluted or diluted with mamma¬
lian Ringer's solution or de-fatted milk, under bright-field or phase-contrast
illumination. Fresh semen was also stained by the nigrosin/eosin method
(Cooper & Rowell, 1958) and dried smears were stained in the same way as
the epididymal specimens.
Testicular material from the house sparrow and ejaculated semen from
the mallard and canary were prepared for electron microscopy by the following
method: the mallard semen was centrifuged for 2 min at 3800 rev./min
(R.C.F. 2100 g) to give a small pellet to which the other procedures could be
applied.
Material was fixed in 3% glutaraldehyde (T.A.A.B.) for 3 hr at 4°C,
washed overnight in m/10 phosphate buffer, pH 7-4, and post-fixed in Millonig's
buffered osmium tetroxide for 1 hr at the same temperature. It was then
washed and dehydrated through successive concentrations of alcohols and
placed in propylene oxide for 30 min at room temperature, to be transferred
through 50/50 propylene oxide/araldite to fresh araldite, having been con¬
tinuously agitated overnight. Embedding in gelatine capsules and incubation
for 12 hr at 60° C took place the next day.
Sections were cut on an L.K.B. Electrotome 3 to silver-grey colour (400 to
1000 Â), and mounted on carbon-coated copper grids. They were then pre-
stained with saturated uranyl acetate in 50 °/0 ethanol (Gibbons & Grimstone,
1960) or 1 % potassium permanganate (Lawn, 1960) and finally stained with
lead citrate (Reynolds, 1963). Stained sections were examined on an A.E.I.
EM6 electron microscope and photographed.
RESULTS
Semen
The seminal fluids of the mallard drake and Hawaiian gander are viscous
liquids, milky white in the mallard but normally colourless in the Hawaiian
gander. When collected by massage, the semen is ejaculated at the base of the
engorged penis; this organ is normally extruded in the geese but not always in
the drakes. Specimens were collected for examination either by means of a
small glass funnel or directly on to a clean glass slide. The semen of both species
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessSemen of certain passerine and non-passerine birds 329
rapidly dehydrates when exposed to air and, in the case of slides, measures
were taken to prevent this
by protection with a cover-slip. Dilution of mallard
semen with equal parts of mammalian Ringer's solution was effective in pre¬
serving sperm motility for at least 3 hr at room temperature. Staining by the
nigrosin/eosin method was usually carried out in the field at the time of
collection, the stain being added to the semen and not vice versa.
The pH of three specimens of undiluted mallard semen was 7-5; no pH
measurements of Hawaiian goose semen were possible owing to frequent
contamination with urates and faecal matter which would have rendered any
measurements invalid.
Good samples of mallard semen showed lively sperm motility and, in an
undiluted specimen, periodic wave formation occurred, with a tendency to
clustering as dehydration proceeded. The sperm density in specimens from two
different birds was of the order of 3-6 106/mm3. Semen from Hawaiian ganders,
however, showed poor to bad motility with no suggestion of wave formation
and a number of epithelial cells were nearly always present with spermatozoa
aggregated around them. Sperm density was very low, being less than
0·5 106/mm3 in six samples examined. In both species, a number of deformed
but motile spermatozoa were found on every occasion, the heads being bent
backwards ('broken-necked') or else assuming a spherical appearance, having
apparently failed to elongate. The latter defect was found more frequently in
mallard semen.
Nigrosin/eosin staining showed approximately 1 % stained 'dead' sperma¬
tozoa; no differences could be detected between the two species in relation to
density. In two or three instances the presence of spermatozoa in the ejaculate of
Hawaiian ganders was only detected after staining and prolonged examination
of the slide.
The semen of all passerine birds so far examined consists of a small droplet,
thick and glutinous in character, and of a brownish pink colouration. Specimens
collected from three male canaries at weekly or bi-weekly intervals were seen
to take the form of a dense mass of typical passerine spermatozoa, with little
accessory fluid. Little initial motility was detected in undiluted seminal
specimens, but the addition of a drop of de-fatted milk activated the sperma¬
tozoa so that nearly all were showing some movement at the end of 3 min.
It was estimated that 2000 to 3000 spermatozoa were present in each droplet
collected. Nigrosin/eosin staining usually revealed a few pink-stained cells,
but, on one occasion, when no collection had been made in the previous 2
weeks, the semen of one bird showed no motility on diluting with milk and
eosin stained nearly all the spermatozoa, many of which showed abnormalities
and some degree of disintegration.
Spermatozoa
The spermatozoa of the Hawaiian gander and mallard drake had an identical
appearance under the microscope which conformed closely with previous
descriptions of domestic fowl spermatozoa (Grigg, 1952; Lake & Smiles, 1952,
1954; Lake, Smith & Young, 1968). Each spermatozoon was an elongated,
flagellate cell measuring about 100 µ in length, with a nuclear diameter of
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free access330 P. JV. Humphreys
about 0·5 µ . This type of spermatozoon is known as 'sauropsid' from its
resemblance to reptilian spermatozoa. The nucleus could be seen to have a
gentle curvature in one dimension. As well as the nucleus and flagellum, an
apical acrosomal region and a mid-piece could be distinguished. In life, the
cells propelled themselves by vigorously lashing the flagellum ; the effect of the
nuclear curvature was to impart a widely spiral projectory. Cells exposed to
Leishman's stain after fixation stained uniformly blue in the mature state
(late spermatids stained pink or red). Live and dead or disintegrating sperma¬
tozoa appeared to stain differentially as usual in nigrosin/eosin ; in the majority
of spermatozoa, the stain was confined to the nucleus, although some cells
were seen in which only the mid-piece was coloured. As disintegration pro¬
ceeded, the nucleoplasm swelled and stained darker, the acrosome and flagel¬
lum resisting disruption longer than the nucleus. Separation of the flagellum
100 ¿¿m
Text-fig. 1. Diagrammatic representation of (a) canary and (b) mallard spermatozoa.
A =
acrosome; =nucleus; M mid-piece; F flagellum; u.m. undulating
= = =
and mid-piece from the rest of the cell was a fairly common abnormality. Some
of the coiled or shortened flagella observed could have been artefacts caused
by the method of preparation or collection. Petitjean (1969, and personal
communication) considers that the majority of sperm deformities found in
domestic gander semen are caused by the method of obtaining semen by
abdominal massage. Fewer are seen when spermatozoa are collected directly
from the vasa deferentia.
The spermatozoa of the passerine birds differed greatly from those described
above. Those obtained from canaries were approximately 300 µ in length
with a spiral conformation (Text-fig. 1 ). The acrosomal portion was very large
in relation to the nucleus. The mid-piece appeared extremely long, and it was
not possible to distinguish any junction with the flagellum. In addition, there
was an undulating membrane which arose from the apex of the acrosome and
extended for the length of the cell. The spermatozoa differed from those of the
mallard in their reaction to Leishman's stain in that the nucleus stained pink
while the acrosome and flagellum stained blue. The nigrosin/eosin technique
used in a milk-diluted sample of semen appeared to work normally. The un¬
dulating membrane was particularly well defined with this stain. Passerine
spermatozoa disintegrated in a different manner from non-passerine sperma¬
tozoa. The large acrosome appeared to be the area most sensitive to degenerative
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessSemen of certain passerine and non-passerine birds 331
change and might be found unattached, leaving a cup-shaped depression in the
anterior end of the nucleus, the last part of the cell to disintegrate.
There was no lashing of the flagellum in passerine spermatozoa as was found
in non-passerine and mammalian spermatozoa. Instead, the whole cell revolved
around its longitudinal axis, the helical structure causing it to move forward
in the manner of a twist-drill. If unobstructed, the revolutions were fast
enough to blur the cell outline ; if partially obstructed, for instance, by a fat
globule from the milk diluent, the cell revolved more slowly; if forward
movement was prevented, the cell showed oscillation and vibration like that
caused by plucking the string of a musical instrument. Bending of the mid-piece
was never seen when the cell was in motion.
A common feature of canary semen was the presence of bundles of sperma¬
tozoa apparently interlocked along their longitudinal margins. Some of these
bundles showed partial separation of individual spermatozoa at their anterior
or posterior ends; occasionally, spirals of amorphous material were found
lying freely in the semen between bundles or single cells. No motility was
ever observed in any of the cells contained in one of these bundles, which
usually consisted of twelve to twenty spermatozoa. Similar bundles have been
observed in dissections of the vasa deferentia of other passerine species, e.g.
house sparrow, jackdaw, robin. The spermatozoa found in this situation
appeared very similar to those of the canary, though the jackdaw spermatozoa
were much smaller,
being about 100 µ in length, and those of the robin were
very much longer with a fairly small, hooked acrosome and a very prominent
undulating membrane. Numbers of late spermatids were always found in the
vas deferens, indicating that there is a maturation process occurring during
passage from the testes to the seminal glomus.
In the stained testis sections, non-passerine spermatozoa were seen to be
more or less evenly distributed on the circumferential Sertoli cells and no
particular pattern of attachment could be ascertained. Passerine spermatozoa,
on the other hand, were placed in evenly spaced bundles around the circum¬
ference of the tubule. The tubular diameters were proportionately greater,
with fewer tubular sections in each microscopic field.
Examination by the electron microscope illustrated further differences in
the two types of spermatozoa. Those of the mallard (PI. 1, Fig. 2) showed an
acrosomal cap at the anterior end, covering a cup-shaped depression in the
nucleus ; within this depression, but separated from the nucleus by an unstained
area, lay a rod-like perforatorium (PL 1, Fig. 4). The nucleus was seen as a
gently curving elongated structure with an electron-dense granular nucleo-
plasm. A double cell membrane could be detected. The posterior end of the
nucleus was markedly concave. The mid-piece (PI. 1, Fig. 5) showed a typical
double centriolar structure with the proximal centriole placed at right angles
to the distal centriole and showed evidence of nine triplet tubules arranged
around the central lumen. The distal centriole extended posteriorly for approxi¬
mately two-thirds of the length of the mid-piece, and the posterior third showed
the anterior roots of the two axial flagellum fibrils. There was a terminal
annulus to the midpiece which was invested with tightly packed mitochondria
and there was a double membrane adherent to the surface. In cross-section, the
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free access332 P. . Humphreys
mitochondria be overlapping, suggesting evidence of a spiral
were seen to
configuration to the mitochondrial sheath, and a typical '9 + 2' fibrillar
arrangement could be seen in the flagella, with evidence of connections
between the outer pairs of fibrils and the two inner axial fibrils as well as with
each other (PI. 2, Fig. 10). No outer accessory fibres could be detected distal
to the annulus (PI. 2, Fig. 7). In the mature spermatozoon, no microtubuli are
visible (Mclntosh & Porter, 1967).
The passerine spermatozoon, as has been seen, is spiral in shape, so that a
complete longitudinal section is difficult to obtain. However, in material
expressed from the seminal glomus of the canary (PI. 1, Fig. 1), the nucleus
was seen as a lightly staining body with a rather
abrupt connection with the
juxta-nuclear junction. A double cell membrane was present. A granular
structure could be seen at the base of the nucleus, partly surrounding the juxta-
nuclear junction (PI. 1, Fig. 6), which is possibly analogous with the cytoplasmic
remnant of some mammalian species. The long midpiece would appear to be a
continuation of a single centriole and in cross-section was seen to consist of
nine peripheral accessory fibres and a '9 + 2' system of axial fibrils (PI. 2, Fig. 9).
A single spiral mitochondrion wound from the nucleus to the midpiece termina¬
tion along most of the length of the midpiece sheath (PL 2, Fig. 8). This would
apparently constitute the substance of the undulating membrane seen under
the light microscope. The acrosome showed a remarkable extension of material
which forms the spiral border described by Furieri (1963); on cross-section,
the border appeared as a long process arising at one side of the circumference ;
a central vacuole containing amorphous material extended into the process
(PL 1, Fig. 3). No mitochondria were to be seen at this level. Large numbers of
bundles of microtubuli were to be found free in the seminal fluid (PL 2, Fig. 11),
and were frequently aggregated into a spiral formation; these microtubuli
were much in evidence in immature spermatids in sections of material taken
from the higher levels of the genital tract. Nicander (1970) regarded them as
important in achieving the spiral configuration of the cell ; during maturation,
the bundles of tubuli are detached from the cell surface and unwind.
EXPLANATION OF PLATE 1
Fig. 1. Longitudinal section of the nucleus and part of the acrosome of a canary sperma¬
tozoon, a, Acrosome; n, nucleus; jn, juxta-nuclear junction. Potassium permanganate,
30,000.
Fig. 2. Longitudinal section of the nucleus and acrosome of a mallard spermatozoon,
a, Acrosome; n, nucleus. Uranyl acetate, 18,000.
Fig. 3. Cross-section of the acrosome of a canary spermatozoon, m, Double cell membrane;
s, spiral border process. Potassium permanganate, 30,000.
Fig. 4. Longitudinal section of the acrosome of a mallard spermatozoon, a, Apical cap;
p, perforatorium ; m, cell membrane. Uranyl acetate, 30,000.
Fig. 5. Longitudinal section of the mid-piece of a mallard spermatozoon, a.c, anterior
centriole; d.c, distal centriole; a, annulus. Uranyl acetate, 24,000.
Fig. 6. Longitudinal section of the juxta-nuclear junction of a canary spermatozoon,
n, nucleus; jnb, juxta-nuclear body; c, central fibrils, m, mitochondrion. Potassium
permanganate, 30,000.
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessPLATE I
(Facing p. 332)
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessPLATE 2
(Facing p. 333)
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessSemen of certain passerine and non-passerine birds 333
DISCUSSION
Semen
There were significant differences in the semen obtained from mallard
drakes and Hawaiian geese. Mallard semen was comparable, in most respects,
with that of domestic fowls and ducks (Szumowski, 1960) ; Hawaiian goose
semen followed the pattern reported for domestic geese (Szumowski & Theret,
1965) in being markedly inferior in quantity and quality. In the captive
population (derived from a very small wild remnant population from Hawaii)
of Hawaiian geese which was sampled, a problem of infertility was known to
exist (Kear, Roberts & Warner, 1967; Humphreys & Kear, 1968; Humphreys,
1969). Too few cases have been thoroughly investigated for any firm conclusion
to be drawn at this stage, but it is interesting that one bird, in the semen of
which no live spermatozoa were detected during a whole season of investiga¬
tion, was found from stud records never to have produced any live progeny
in two previous breeding seasons, although mated to a different female on each
occasion.
Important differences in individual male fertility have been shown to be
common in the domestic goose (Szumowski & Theret, 1965) and to be related
to the mass of the testis when mature and fully spermatogenic, an inherited
characteristic susceptible to alteration by selective breeding (Khranowski,
1959, in Szumowski & Theret, 1965). It was not possible to distinguish dif¬
ferences in the volume or quality of the semen from individual mallard drakes ;
all samples, however, were greatly superior to any obtained from the Hawaiian
geese. The mallard drakes were obtained from a wild population and released
at the end of the season so that no record of testis weight or volume could be
obtained; information from the autopsy data available (Table 1) indicates
that the testis volume of the mallard increases very much more in the breeding
season (1 :145) than does that of the Hawaiian gander (1 :8), although the
respective volumes of the testes between breeding seasons is similar in the two
species. As far as is known, there have been no reports of poor fertility in the
British wild mallard population; the population indices of this species have shown
an upward trend for some years past (Atkinson-Willes, 1970).
EXPLANATION OF PLATE 2
Fig. 7. Longitudinal section of the flagellum of a mallard spermatozoon. Potassium
permanganate, 26,000.
Fig. 8. Longitudinal section of the flagellum of a canary spermatozoon, m, Mitochond¬
rion. Potassium permanganate, 40,000.
Fig. 9. Cross section of the flagellum of a canary spermatozoon, m, Mitochondrion.
Uranyl acetate, 40,000.
Fig. 10. Cross section of the flagellum of a mallard spermatozoon. Uranyl acetate,
40,000.
Fig. 11. Discarded microtubular bundles in canary semen. Potassium permanganate,
40,000.
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free access334 P. jV. Humphreys
Spermatozoa
The morphology of the spermatozoa in birds also poses some interesting
questions. The non-passerine type with perforatorium, double centriolar mid-
piece, mitochondrial sheath and undulating type of motility bears a general
resemblance to the mammalian spermatozoa which have been studied over
many years (Burgos & Fawcett, 1955; Änberg, 1957; Austin & Bishop, 1958;
Blom & Birch-Anderson, 1960; Saake & Almquist, 1964; Austin, 1968;
Table 1
left testis volumes* of mallard and hawaiian geese; data from autopsy
records at the wildfowl trust, slimbridge
Breeding season samples Out of breeding season samples
Mallard Hawaiian Goose Mallard Hawaiian Goose
(27th March to (7th December to (8th August to (10th June to
28th May) 5th April) 17th February) 2nd November)
21,354 943 165 131
15,714 894 131 18
13,876 434 93 10
10,476 333 57
10,476 308 45
7,920 264 21
1,060 188
131
Mean 11,402 434 85 53
Increase 1: 145 1:8
*
Volume (in mm3) calculated as for an elipsoid from the formula: \n a2b, where a = smallest
diameter/2, and b =
greatest diameter/2.
Fawcett, 1970). Sotelo & Trujillo-Cenoz (1958), investigating the spermatozoa
of a number of animals, used the house sparrow as their representative of the
avian group. They considered that its spermatozoa most resembled the sperma¬
tozoa of the urodele newts in the arrangement of the juxta-nuclear body and
undulating membrane, although some other animal spermatozoa possess these
features. The undulating membrane of urodele spermatozoa, however, is not
really analogous with that of passerine spermatozoa, the latter being a mito¬
chondrial spiral and possessing no evidence of having any potentially contractile
elements. McFarlane (1971) finds there to be an evolutionary progression
towards spiralization in avian spermatozoa; at the present time, it is difficult
to envisage any particular advantage within the genital tract that spiralization
could afford.
The differences in the nature of the nucleoplasm, the rôle of the micro¬
tubuli in spermiogenesis and the relationship of morphology to function
would seem to be worthy of further research.
ACKNOWLEDGMENTS
Much of this work was undertaken while the author was the holder of a
Wellcome Research Training Scholarship.
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessof certain passerine and non-passerine birds
Semen 335
Thanks are due to Dr D. K. Roach, Dr G. V. T. Matthews, Dr T. E.
Thompson, Dr P. E. Lake and Dr Janet Kear for their interest and advice
during various stages of this work. Mr T. E. Davies and Mrs C. Stratford
contributed valuable technical assistance, as did Mr A. Stevenson.
The author also wishes to acknowledge the help and facilities given by the
Curator and staff of the Wildfowl Trust, Slimbridge, and especially to thank
Miss E. Temple Carrington for her assistance with the manuscript.
REFERENCES
Änberg, (1957)
A. The ultrastructure of the human spermatozoa. Acta obstet, gynec. scand. 36, Suppl.
2,1.
André, J. (1963) Some aspects ofspecialisation in sperm. In: The General Physiology of Cell Specialisation.
Eds. D. Mazia and A. Tyler. McGraw-Hill, New York.
Atkinson-Wiixes, G. (1970) National wildfowl counts. In: The New Wildfowler in the 1970s. Eds. N. E.
Sedgewick, P. Whitaker and J. Harrison. Barrie & Jenkins, London.
Austin, C. R. (1968) Ultrastructure offertilization. Holt, New York.
Austin, C. A. & Bishop, M. H. W. (1958) Role of the rodent acrosome and perforatorium in fertiliz¬
ation. Proc. R. Soc. B, 149, 241.
Ballowitz, E. (1888) Untersuchen über die Struktur der Spermatozoën, zugleich ein Beitrag zur
Lehne vom feineren Bau der Controktilen Elemente, Theil 1. Die Spermatozoën der Vögel.
Arch, mikrosk. Anat. EntwMech. 32, 401.
Blom, E. & Birch-Anderson, A. (1960) The ultrastructure of the bull sperm. I. The middle piece.
Nord. VetMed. 12,261.
Burgos, M. H. & Fawcett, D. W. (1955) Studies on the fine structure of the mammalian testis. I.
Differentiation of spermatids in the cat (Felis domesticus). J. biophys. biochem. Cytol. 1, 287.
Cooper, D. M. (1963) Artificial insemination of poultry. Br. vet. J. 119, 194.
Cooper, D. M. (1969) The use of artificial insemination in poultry breeding, the evaluation of semen and semen
dilution and storage. In: Fertility and Hatchability of the Hen's Egg. Eds. T. C. Carter and B. M.
Freeman. Oliver & Boyd, Edinburgh.
Cooper, D. M. & Rowell, J. G. (1958) Relations between fertility, embryonic survival and some
semen characteristics in the chicken. Poult. Sci. 37, 699.
Fawcett, D. W. (1970) A comparative view of sperm ultrastructure. Biol. Reprod. Suppl. 2, 90.
Furieri, P. (1963) La morphologia degli spermi di Fringilla coelebs L. e di Tálentela mauritanica L. studia
al microscopio elettronico. Monitore zool. ital. 70-71.
Gibbons, I. R. & Grimstone, A. V. (1960) On flagellar structure in certain flagellates. J. biophys. biochem.
Cytol. 7, 697.
Grigg, G. W. (1952) The morphology of the fowl sperm. Proc. Soc. Study Fert. 4, 15.
Humphreys, P. N. (1969) Notes on study of semen in Hawaiian Geese. (Unpublished, privately
circulated, available Wildfowl Trust Library.)
Humphreys, P. N. & Kear, J. (1968) A preliminary study of the fertility in the Hawaiian gander
Branta sandvicensis. (Unpublished, privately circulated, available Wildfowl Trust Library.)
Johnson, A. D. (1954) Artificial insemination and the duration of fertility in geese. Poult. Sci. 33, 638.
Kear, J., Roberts, H. S. & Warner, R. (1967) The Hawaiian Goose in captivity. (Unpublished,
privately circulated, available Wildfowl Trust Library.)
Lake, P. E. (1966) Physiology and biochemistry of poultry semen. Advances in Reproductive Physiology,
vol. 1, p. 92. Ed. A. McLaren. Logos Press, London.
Lake, P. E. (1969) Factors affecting fertility. In: Fertility and Hatchability of the Hen's Egg. Eds. T. C.
Carter and B. M. Freeman. Oliver & Boyd, Edinburgh.
Lake, P. E. & Smiles, J. (1952) Some observations on the morphology of fowl spermatozoa. Proc. Soc.
Study Fert. 7, 18.
Lake, P.E. & Smiles, J. (1954) The relationship between morphology and function of fowl spermatozoa.
Proc. 10th Wld's Poult. Congr. Department of Agriculture for Scotland. 79.
Lake, P. E., Smith, W. & Young, D. (1968)
The ultrastructure of the ejaculated fowl spermatozoon.
Q_.Jl exp. Physiol. 53, 356.
Lawn, A. M. (1960) The use of potassium permanganate as an electron dense stain for sections of
tissues embedded in epoxy-resin. J. biophys. biochem. Cytol. 7, 197.
McCartney, M. G. & Brown, K. I. (1959) Spermatozoa concentration in three varieties of turkeys.
Poult. Sci. 38, 390.
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free access336 P. . Humphreys
McFarlane, R. W. (1963) Taxonomic significance of avian sperm, Proc. 13th Int. orn. Congr. 1, 91.
McFarlane, R. W. (1971) The ultrastructure and phylogenetic significance of avian spermatozoa. Abst. Ph.D.
thesis, Univ. Florida.
McFarquhar, A. M. & Lake, P. E. (1964) Artificial insemination in quail and the production of
chicken-quail hybrids. J. Reprod. Fert. 8, 261.
McIntosh, J. R. & Porter, K. R. (1967) Microtubules in the spermatids of the domestic fowl. J1. Cell
Biol. 35, 153.
Nicander, L. (1970) Microtubules in spermatids of mammals and birds. J. Ultrastruct. Res. 18, 230.
Petitjean, M. J. (1969) Estimation du pouvoir fécondant des jars; premières difficultés. Bull. Soc.
scient. Hyg. aliment. 57, 100.
Retzius, G. (1909) Die Spermien der Vogel. Biol. Unters. (New Ser.), 14, 89.
Retzius, G. (1911) Zur Kenntnis der Spermien der Vogel. Biol. Unters. (New Ser.), 16, 89.
Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron
microscopy. J. Cell Biol. 17, 208.
Saake, R. H. & Almquist, J. O. (1964) Ultrastructure of bovine spermatozoa. Am. J. Anat. 115, 143.
Sotelo, J. R. & Trujillo-Cenoz, O. (1958) Kinetic apparatus in sperm cells. Z- Zellforsch, mikrosk.
Anat. 48, 565.
Szumowski, P. (1960) Insémination artificielle chez des palmipèdes. Reel. Méd. vét. Éc. Alfort. 136, 1165.
Szumowski, P. & Theret, M. (1965) Causes possibles de la faible fertilité des oies et des difficultés de
son amélioration. Reel. Méd. vét. Éc. Alfort 141, 583.
Tuzet, O. (1950) Le spermazoïde dans la série animale. Revue suisse Z°°t- 57, 433.
Watanabe, M. (1957) An improved technique of the artificial insemination in ducks. J. Fac. Fish.
Anim. Husb. Hiroshima Univ. 1, 363.
Wolfson, A. (1960) The ejaculate and the nature of coition in some passerine birds. Ibis, 102, 124.
Downloaded from Bioscientifica.com at 05/05/2020 04:34:50AM
via free accessYou can also read
