SPERMATOZOA FROM THE ISOLATED CAUDA

EPIDIDYMIDIS OF RABBITS AND SOME

EFFECTS OF ARTIFICIAL CRYPTORCHIDISM*
T. D. GLOVER
Department of Veterinary Anatomy, University of Liverpool
{Received gtk July 1959)
Summary, ( ) Fourteen adult male rabbits have been subjected to
unilateral isolation of the tail of the epididymis in the abdomen, with
subsequent retention of the organ in an abdominal position. Isolation
of the contralateral cauda epididy midis in each rabbit was effected in the
scrotum as a control measure. (2) Estimates of the proportion of dead and
decapitated spermatozoa as well as the incidence of spermatozoa with
coiled tails, at different levels of the tubule distal to the point of isolation,
have demonstrated a delayed response of so-called mature spermatozoa
to the conditions of experimental cryptorchidism. (3) Quantitative
assessments of these criteria have suggested that spermatozoa in the
body of the epididymis are more susceptible to degeneration than
those in the tail of the epididymis, but it is pointed out that more
evidence is yet required on this matter. (4) The overall results are dis¬
cussed in relation to previous work concerning the origin of similar
sperm abnormalities.

INTRODUCTION
In an earlier paper (Glover, 1958), it was shown that an increased proportion
of dead and decapitated spermatozoa were to be found in the ejaculated semen
of rabbits after 9 to 11 days of artificial cryptorchidism. The origin of these
degenerate spermatozoa was at that time in doubt, but more recent experi¬
ments involving ligated epididymides (Glover, 1959) have shown that many
spermatozoa in the cauda epididymidis appear to die and become decapitated
as a result of the conditions imposed by experimental cryptorchidism. A cumula¬
tive effect of increased temperature upon spermatozoa in this region has thus
been postulated, but closer examination of the cauda epididymidis in rabbits
discloses variation in the diameter of its tubule which detracts from it being
regarded as a simple anatomical entity, despite the demonstration of a con¬
stancy in histological structure (Nicander, 1957). Furthermore, no clear evidence
has hitherto been available to indicate whether spermatozoa throughout the
cauda epididymidis react in the same way during artificial cryptorchidism.
* Read at the Annual
Conference, 1959, of the Society for the Study of Fertility.
121
122 T. D. Glover
The present work reports on the characteristics of spermatozoa taken from
different levels of the distal regions of the epididymis and from the vas deferens
after their isolation from the more proximal parts of the tubule. By means of
this more detailed scrutiny, an attempt has been made to discover the extent

Text-fig. ia. A diagram of the testis and epididymis of the rabbit to illustrate the anatomical
arrangement of the distal regions of the tubule. Observe the spiral arrangement of the
lower part of the corpus epididymidis around a fibrous cord (see text). =
testis.
= head of epididymis. E =
body of epididymis. D distal part of corpus
=

epididymidis (isolated in this experiment together with the cauda epididymidis

and vas deferens). C =
cauda epididymidis. V vas deferens.
=

Text-fig. i b. A diagram to illustrate the method of isolation of the tail of the epididymis
and levels of the tubule from which spermatozoa were withdrawn. I vas deferens. 2
= =

widest part of cauda epididymidis. 3 proximal region of cauda epididymidis.

=

4 =
distal part of corpus epididymidis.

to which morphological characteristics of spermatozoa taken from any one

level of the distal tubule are representative of spermatozoa in the cauda
epididymidis as a whole.
ANATOMICAL CONSIDERATIONS

The epididymis of the rabbit is typical of that of many other mammals in

that it appears to have a head, a body and a tail. The narrow tubule of the
 PLATE

Fig. i. Radiograph of the testis and epididymis of a

rabbit after injection of the arteries with Micropaque.
C.C. capital arteries. T. testicular artery. E.E.
= = =

descending epididymal arteries. V. vasal artery.

=

(Facing p. 123)
 Spermatozoa in Artificial Cryptorchidism 123
corpus epididymidis lies in close proximity to the testis throughout most of its
length, but in this species it ultimately continues posteriorly free from the
testis. In this part, the convolutions of the tubule are intimately bound in spiral
fashion by a fairly strong fibrous cord which extends from the inferior pole of
the testis. The tubule next widens to become what is apparently the first part of
the tail of epididymis, and a further widening gives rise to a large convoluted mass
which turns forward and is attached to the more proximal convolutions by
strong fascia. Finally the tubule continues as an independent ductus deferens
(Text-fig. 1 a). In the present experiment, interruption of the epididymis was
effected opposite the inferior pole of the testis so that a short length of the
body of the epididymis was isolated together with the tail of the epididymis
and the vas deferens (see Text-fig. ib). Therefore spermatozoa withdrawn from
Point 4 in the diagram must be regarded as having been taken from the lower
portion of the corpus epididymidis.
The blood supply to the upper parts of the epididymis in rabbits stems from
the main testicular artery (see PI. 1, Fig. 1). Several capital arteries pass to the
head of the epididymis, whereas the body of the tubule is supplied with blood
via separate descending vessels. A relatively discrete artery runs roughly parallel
to the antero-lateral surface of the body of the epididymis, and a smaller vessel
courses down the ventro-medial border. These two arteries join the much larger
vasal or deferential artery on the surface of the cauda epididymidis. Despite
interruption of the descending epididymal arteries, therefore, the distal part
of the body of the epididymis and the tail should receive an adequate blood
supply (see also PL 1, Fig. 1).
The course of the testicular artery in the rabbit has previously been des¬
cribed in detail (Harrison, 1948) and its superficial position on the surface of
the testis is worthy of note in carrying out ligations of the epididymis, because
the vessel runs close to the epididymis on the dorso-lateral surface of the testis.
Caution was therefore to be observed in order to avoid damage to this artery
when attempting a low ligation of the epididymis.

METHODS
Fourteen rabbits were used in the experiment; they were of mixed strain, varied
from 2.0 kg to 3.2 kg in weight, and were housed in wire mesh cages at 68° F.
Constant access to drinking water and food (Diet 18, as supplied by Oxo Ltd)
was provided. Rabbits were anaesthetized by intravenous injection of sodium

pentobarbitone (Veterinary Nembutal solution, Abbott Laboratories) by the

method described by Cross (1955)·
To isolate the distal part of the epididymis, two ligatures were applied around
the body of the epididymis immediately above the tail, and the intervening
segment removed. This was carried out unilaterally in the abdomen of each
rabbit and the testis and epididymis secured in an abdominal position as
previously described (Glover, 1958). The contralateral epididymis was ligated
in the scrotum, so that the structures remained in situ. By this means each
animal acted as its own control. Ten of the rabbits were sacrificed at 2-day
intervals between 3 and 21 days after operation. As a check on the response, the
A*
124 3n D. Glover
remaining four rabbits were killed at random intervals between these days,
namely on the 4th, 8th, 10th and 14th days after operation. Spermatozoa were
withdrawn from four different levels of the tubule in each rabbit as illustrated
in Text-fig. ib.
For examination of the spermatozoa, the tubule was carefully cut at the
required level and the incised portion lightly touched with a microscope slide.
Aqueous nigrosin-eosin solution (eosin Y [G. T. Gurr] .67 %, nigrosin [G. T.
Gurr] . %, dissolved in distilled water, 30 ml being made up fresh as required)
was added to the spermatozoa on the slide before a smear was made. When
the tail of the epididymis was incised, its contents flowed out freely and six
drops of stain were added. But spermatozoa did not emerge so profusely from
the vas deferens or more proximally from the narrow part of the tubule close
to the ligature; for these samples, therefore, one or two drops of stain were
adequate for obtaining a satisfactory distribution of spermatozoa on the slide
for counting. On each slide, the percentage of dead spermatozoa was estimated
together with the incidence of decapitated spermatozoa and the proportion
of spermatozoa with coiled tails. Two separate counts of 100 spermatozoa were
made on each slide wherever possible.

RESULTS
In order to assess the overall response of spermatozoa in the cauda epididy¬
midis to artificial cryptorchidism, estimated mean values for the incidence of
dead and decapitated spermatozoa taken from two levels within the region
(Levels 2 and 3) were used. Estimates of the percentage of spermatozoa with
coiled tails were treated similarly. For comparing the response at four different
levels of the tubule (see Text-fig. ia), mean values in groups of animals were
taken, as illustrated in Tables 1 and 2.

THE EFFECT OF ARTIFICIAL CRYPTORCHIDISM ON SPERMATOZOA IN THE ISOLATED

CAUDA EPIDIDYMIDIS

Mean values for characteristics of spermatozoa from two levels of the tail of
the epididymis showed that an increased proportion of dead or eosinophilic
spermatozoa occurred in the region after about 8 or 9 days of experimental
cryptorchidism. Moreover, the percentage of dead cells increased progressively
with the time of retention in the abdomen, though quantitative variation of
response in individuals was considerable. Similar results were obtained from
estimates of the incidence of decapitated spermatozoa in the cauda epididymidis.
Quantitatively, the effect of decapitation was not as conspicuous as that of
eosinophilia, but despite this, the trend of a progressive increase in the occur¬
rence of decapitated forms was undoubtedly a real one.
Mean values for spermatozoa with coiled tails indicated that the incidence of
this abnormality in the cauda epididymidis was not significantly influenced by
the conditions of artificial cryptorchidism when the tubule was ligated.
In samples taken from control organs up to 21 days after operation, no change
 PLATE 2

Fig. 2

Fig. 3
Spermatozoa taken from the cauda epididymidis. Left: control side. Right: cryptorchid side. Cells
stained with nigrosin-eosin. X 990.
Fig. 2. Seven days after isolation.
Fig. 3. Fourteen days after isolation.

(Facing p. 125)
 Spermatozoa in Artificial Cryptorchidism !25
in the morphology of spermatozoa or in the incidence of potentially active
cells could be demonstrated. Hence, in animals destroyed up to about 7 days
after operation, no very clear difference was evident between spermatozoa
from abdominally retained epididymides and those from the control sides.
After this time, however, the effects of abdominal retention became increasingly
obvious (PI. 2, Figs. 2 and 3). These results are illustrated graphically in Text-
figs. 2a and 2b.
OPERATION

7 ß 9 10 11 a 14 IS 7 ß 9 IO II 13 14 IS
DAYS DAYS

Text-figs. 2A and 2b. Characteristics of spermatozoa from the tail of the epididymis. Mean values have been
used of estimates from Levels 2 and 3 (see Text-fig. 1). Cryptorchids =-. Controls =-.

If it assumed that in spite of ligation of the epididymis, migration and

were
evanescence of spermatozoa in the distal part of the tubule continued, it might
be argued that degenerate spermatozoa originated at a point proximal to the
cauda epididymidis (viz. at Level 4) and were transported to lower levels.
For this reason, quantitative estimates were made of the characteristics of
spermatozoa from four different levels of the tubule at various stages after
operation. These results are shown in Tables 1 and 2.
THE RESPONSE OF SPERMATOZOA AT DIFFERENT LEVELS OF THE TUBULE DISTAL
TO THE AREA OF LIGATION

Mean percentages of dead and decapitated spermatozoa shown in Tables 1 and

2 indicate a rather higher degree of sperm degeneration at Level 3 (proximal
part of the cauda epididymidis) than at Level 2 (distal region of the cauda
epididymidis) in animals killed up to 8 days after their operation. The differences
in estimates are, however, not statistically significant at the 5 % level using the
test described by Moore (1957). On the same basis, the proportion of decapi¬
tated spermatozoa recovered from vasa deferentia did not exceed that in samples
withdrawn from epididymides, except during the period of 9 to 14 days after
ligation in control organs. Nevertheless, more dead spermatozoa were evident
in the vas deferens of controls than in the cauda epididymidis between 9 and
21 days after ligation.
 T. D. Glover

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 Spermatozoa in Artificial Cryptorchidism 127
In the cryptorchid condition, the percentage of dead and decapitated
spermatozoa in samples taken from Level 4 (distal part of the corpus epididy¬
midis) was generally greater than in those withdrawn from Level 3 up to a
period of 14 days. This was not observed in animals killed between 14 and 21
days. In control organs, however, an excess of degenerating spermatozoa in
the body of the epididymis just distal to the ligatures was characteristic and the
differences in estimates at Level 4 and Level 3 were thus clearly significant
throughout.
Quantitative estimates of the characteristics of spermatozoa above the
ligatures are as yet incomplete, but it may be stated here that considerable
death and decapitation was noted in most abdominally retained epididymides.

DISCUSSION
Confirmatory evidence has been produced in this experiment that degenerate
spermatozoa are to be found in the isolated cauda epididymidis of rabbits as a
result of its retention in the abdomen; it is also clear that the onset of degenera¬
tion of the spermatozoa is delayed. That many spermatozoa were obtained
from different levels of the isolated area of the tubule suggests that adverse
effects of artificial cryptorchidism act upon spermatozoa throughout the
entire length of the distal part of the tubule, and that the appearance of
degeneration is not wholly due to the interchange of spermatozoa from one
level to another.
The index primarily used for the response of spermatozoa was an assessed
proportion of dead and decapitated spermatozoa; admittedly, metabolic
changes which might not be recognized on this basis could precede the demon¬
strated response and thereby influence fertility at an earlier stage. Nevertheless,
more actively motile spermatozoa could be recovered from these lower
regions
of the epididymis during the first week of retention in the abdomen than sub¬
sequently, and it would thus seem that spermatozoa in the cauda epididymidis
possess a certain resistance to the effects of artificial cryptorchidism. Even when
spermatozoa began to succumb to the adverse conditions, degeneration
appeared to be a gradual and progressive process.
Estimates of the characteristics of spermatozoa taken from two levels of the
cauda epididymidis indicated that the response to cryptorchidism was similar
throughout the tail of the epididymis. It would seem, therefore, that in experi¬
mental cryptorchidism the characteristics of spermatozoa taken from any one
point in the tail of the epididymis might be fairly representative of those
throughout the whole area. At the same time, it is to be remembered that in
intact animals some interchange of spermatozoa from the body of the epididymis
might occur which could effect quantitative estimates in samples withdrawn
from the cauda epididymidis.
A higher incidence of dead spermatozoa in the vas deferens than in the
cauda epididymidis of controls after 9 days of ligation is to some extent commen¬
surate with the suggestion of Young (1931) that spermatozoa in the vas deferens
are the most aged. But conclusions drawn from the present experiment need
to be tempered by consideration of the possible influence of ligation of the tubule.
128 T. D. Glover
A distinct preponderance of both dead and decapitated spermatozoa was
apparent in most samples taken from the lower part of the body of the epididy¬
mis up to 14 days of experimental cryptorchidism. That this was not evident
after longer periods of abdominal retention was probably due to an overall
increase in dead and decapitated spermatozoa throughout the tubule which
could overshadow any original excess in this area. At first glance, the result
might seem to indicate a greater susceptibility of more immature spermatozoa
to the effects of cryptorchidism, particularly when it is recalled that sperms in
the body of the epididymis proximal to the ligatures showed a high degree of
degeneration after retention in the abdomen. Yet while this may be so, the
imputation is confused by considerable disintegration occurring in the corpus
epididymidis of controls just distal to the ligatures.
This finding lends itself to a number of interpretations. It is tempting to
dismiss disintegration near the ligatures as being purely due to trauma, but
more interesting possibilities cannot on the present evidence be altogether
excluded.
Obstruction of the normal flow of spermatozoa by the method adopted in
this experiment might in itself cause spermatozoa immediately below the liga¬
tures to degenerate. Though perhaps a little imaginative, the suggestion might
be made that by virtue of an interrupted sperm stream a normal process of
wastage of spermatozoa in the lower part of the corpus epididymidis could be
revealed, which under normal circumstances might be hidden from view by
an overwhelming number of normal spermatozoa perpetually descending the
ductus epididymis. These possibilities are now being further explored.
The present work does not indicate whether the degeneration of spermatozoa
was directly due to an increased environmental temperature, but this could be
tested by using an alternative method of raising the temperature of the epididy¬
mis. Whatever the cause, however, the observed degeneration of spermatozoa
in the tail of the epididymis is not in accordance with a number of previous
claims. Phillips & McKenzie (1934) believed that, in the guinea-pig, artificial
cryptorchidism exerted no effect upon the morphology of spermatozoa in the
epididymis ; this would suggest that morphological abnormalities ofspermatozoa
resulting from experimental cryptorchidism originate entirely in the testes.
Gunn (1936) found that, in rams, decapitated spermatozoa resulting from
insulation of the scrotum appeared in ejaculates at a time which corresponded
to the passage of indian ink through the epididymis. He therefore concluded
that the delay in appearance of these abnormalities represented the time of
migration of spermatozoa through the epididymis. Walton & Uruski (1946)
reached similar conclusions after subjecting rabbits to low atmospheric pres¬
sures. The present results do not substantiate such a claim being made as far
as experimental cryptorchidism in rabbits is concerned. As has been previously
suggested (Glover, 1959), some of the first decapitated spermatozoa found in
the ejaculates of cryptorchid rabbits are likely to originate in the tail of the
epididymis. To this it may now be added that the vas deferens provides another
place of origin of disintegrating spermatozoa. It is therefore suggested that
whilst the delay in appearance of degenerating spermatozoa in ejaculates of
rabbits after artificial induction of cryptorchidism might prove to coincide with
 Spermatozoa in Artificial Cryptorchidism 129
the time of migration of spermatozoa through the tubules of the epididymis,
it is rather an indication of the resistance of so-called mature spermatozoa to
the effects of retention in the abdomen.

ACKNOWLEDGMENTS
The work was aided by a grant from the Agricultural Research Council. My
thanks are due to Mr W. J. McCabe for technical assistance and his care of
the experimental animals, and to Mr W. Lee for the photography. I also wish
to thank Dr E. W. Macmillan for his preparation of the specimen shown in
PI. 1, Fig. 1.

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