T H E M A L E R A B B I T . III.

D E T E R M I N A T I O N OF D A I L Y
S P E R M P R O D U C T I O N BY M E A N S OF
TESTICULAR HOMOGENATES 1
R. P. AMANN AND J . T. L A M B I A S E , JR.
The Pennsylvania State University, University Park 2
S T I C U L A R spermatid reserves have been
T Equantified
for bulls, buffalo-bulls, deer,
boars, rabbits, guinea pigs, roosters and humans. Generally, a modification of the technique described by Amann and Almquist
(1961) has been used. Daily sperm production can be calculated by dividing the value
for testicular spermatid reserves by a time
divisor which is the number of days of production these reserves represent (Amann and
Almquist, 1962). Based on knowledge of the
duration of one cycle of the seminiferous epithelium and morphology of the spermatids,
Amann and Almquist (1962) concluded that
for dairy bulls testicular spermatid reserves
represented 3.27 days' sperm production.
They cautioned, however, that this time divisor of 3.27 days was not valid for other species or homogenization techniques. When sampling errors associated with hemacytometry
are minimized, the time divisor is the major
potential source of error in determining daily
sperm production from testicular spermatid
reserves (Amann and Almquist, 1961, 1962).
This report describes an improved method
for determining spermatid reserves useful for
most species and establishes the time divisor
necessary for calculating daily sperm production of rabbits. Estimations of the time divisor
as determined by morphologic and autoradiographic methods are compared since autoradiographic studies might be impractical with
some animals.
Methods
Experiment 1. Testicular and epididymal
homogenates prepared by the usual method
(Amann and Almquist, 1961) contain particulate matter which could obscure spermatid
x Authorized for publication on April 16, 1968 as Paper No.
3398 in the Journal Series of the Pennsylvania Agricultural
Experiment Station. This investigation was supported by Research Grant HD-01356-03 from the National Institute of
Child Health and Human Development. Triton X-100 was
kindly provided by Rhom and Haas, Philadelphia. Mrs. N. G.
Borger and Mrs. B. R. Billeb provided skilled technical assistance.
2 Dairy Breeding Research Center, University Park, Pennsylvania, 16802.

369

heads during hemacytometric counts. Antifoaming compounds and anionic detergents

were used in preliminary tests aimed at reducing this interference and also foaming. Inclusion of Span 80, Dow Antifoam C or Dow Antifoam FR-10 reduced foaming more than use
of Triton X-100, but emulsified fat sometimes
obscured the cells. However, with Triton X100 emulsification of fat was not a problem
and interference of particulate matter was
drastically reduced. Little residue remained in
the blender when the homogenate was transferred to a flask. Therefore, the influence of
Triton X-100 on sperm counts was evaluated
in two 2 "~ experiments.
The left testis from each of seven mature
rabbits was freed of its tunica albuginea, cut
into small pieces and homogenized in 100 ml.
of 0.9% NaC1 solution. The right testis was
prepared similarly except that 0.9% NaC1
containing 0.05% ( v / v ) Triton X-100 was
used. Both fluids contained 100 ppm Merthiolate to retard bacterial proliferation. The
saline-Triton-Merthiolate fluid will be referred to as STM. Each epididymis was
minced on a watch glass and then homogenized in 200 ml. of fluid. Homogenization
times of 1 and 5 min. in a semimicro Waring
Blendor were compared for both tissues. After
1 min. of homogenization a 10-ml. sample was
removed and placed in a screw-cap tube. The
remaining tissue was homogenized for 2 min.,
cooled in ice water for 2 min. and homogenized
for a final 2 min.; a second 10-ml. sample
then was removed.
Spermatids or sperm present in homogenates
were counted without further dilution, immediately and after 4 days of storage at 5 ~ C.
Each observer mixed the homogenate and used
a Pasteur pipette to fill one hemacytometer
chamber. The second chamber was filled similarly. The same three observers counted all
homogenates through phase contrast microscopes. If the values for all six chambers were
in poor agreement (range more than 10-12%
of the mean), at least one observer recounted
the sample. Sample means, based on all cham-

370

AMANN AND LAMBIASE JR.

bers counted, were used in analyses of variance.

Experiment 2. This experiment was designed
to determine the number of days' sperm production represented by the spermatids counted
in testicular homogenates. Twenty-two random-bred, 9-mo.-old, New Zealand White rabbits were trained to ejaculate into an artificial
vagina. During a 7-day pre-experimental period, 11 males were sexually rested (SR) and
11 were ejaculated at a frequency of one ejaculate every 24 hr. (1X/24 hr.). At about 9:00
a.m. on day 0 of the experimental period,
shortly after ejaculation if appropriate, all
rabbits were lightly anesthetized with sodium
thiamylal (Surital) and injected I.V. with
0.25 mCi./kg, of thymidine-methyl-3H (6.0
Ci./mM.). Thereafter, all 22 bucks were sexually rested.
Rabbits were killed by sodium pentobarbital, within 5 min. of the specified time, 25.5,
26.0, 26.5, 27.0, 27.5, 28.0, 28.5 and 31.5 days
after injection. One rabbit from the SR and
one from the 1X/24 hr. group was killed after
each interval. The remaining SR rabbits were
killed at 26.0, 27.0 and 28.0 days while a second 1X/24 hr. rabbit was included in the 26.5,
27.5 and 28.5 day groups. Each testis was
weighed (2.78
gin.) and sliced perpendicularly to the long axis into three unequal
pieces. The parenchyma of the central onehalf of the testis was freed from its tunica
albuginea and homogenized in 45 ml. of STM
for 1 min. using a semimicro Waring Blendor.
Five smears were prepared from each homogenate and rapidly dried using a hot plate and
fan. The smears were fixed in absolute methanol for 5 min., washed in running tap water
for 20 min. and air dried. The quarters from
the upper and lower poles of the testis were
fixed in Bouin-Hollande, embedded in Tissuemat and sectioned at 5~. Sections from both
poles were mounted on each of five slides
which were deparaffinized preparatory to autoradiography.
The coded slides with testicular tissue sections or homogenate smears were dipped into
Kodak NTB-3 liquid emulsion. Slides were
sealed in boxes with drying agent and exposed
for 6 wk. at 5 ~ C. Autoradiograms were developed for 2 rain. at 15 ~ C. with Dektol
diluted 1:1 with distilled water. All slides
were stained with Harris hematoxylin.
The duration of one cycle of the seminiferous epithelium was determined using autoradiograms of testicular sections. The eight
stages of the cycle of the seminiferous epithe-

lium have been described by Swierstra and

Foote (1963). For six sections per testis, three
from each pole, 100 essentially round seminiferous tubule cross-sections were scored. The
stage of the cycle comprising the greater part
of the seminiferous tubule cross-section and
the presence or absence of labeled spermatids
were recorded. A tubule was classed as labeled
if five or more spermatid nuclei were each associated with at least 4 grains. From these
data on the relative frequencies of the eight
stages of the cycle of the seminiferous epithelium and knowledge of the stage containing
the most advanced labeled germ cells, the duration of one cycle of the seminiferous epithelium was calculated for each testis (Amann
et al., 1965).
The time of initial appearance, incidence
and degree of spermatid labelling were determined by evaluating five autoradiograms for
each testicular homogenate. A total of 1,000
randomly selected spermatid nuclei per homogenate, 200 per slide, were classed as having 0, 1-2, 3-5, 6-8, 9-11, 12-14, 15-17,
18 20 or ~ 2 0 grains. Spermatids associated
with 0, 1 or 2 grains subsequently were considered to be unlabeled.
Results
Determination o/Sperm Reserves. Data on
testicular spermatid and epididymal spermatozoan reserves from Experiment 1 are summarized in table 1. No significant difference
was found between homogenization times of
1 and 5 rain. or between counts obtained immediately and 4 days after homogenization.
This was true for both testicular and epididyreal tissue. For epididymal tissue, higher
counts ( P ~ . 0 1 ) were obtained for samples
containing Triton X-100. The Triton level x
homogenization time interaction ( P ~ , 0 5 ) for
testicular tissue indicated that 5 min. homogenization reduced sperm counts in saline but
not in STM.
Spermatid reserves for individual testes
(2.42
gm.) homogenized for 1 min. in
STM averaged 3 8 8
while those
(2.45
gin.) in saline averaged only
3 6 1 + 1 6 x 106 spermatids. Similar values for
individual epididymides were 966
and
826-+-88 x 106 sperm. Use of Triton X-100 apparently facilitated accurate counting by eliminating particulate matter. Based on the
difference in counts between contralateral tissues from individual males, up to 28% of the
cells might have been obscured when saline
was used for homogenization.

371

DAILY SPERM P R O D U C T I O N BY T H E R A B B I T
TABLE 1. INFLUENCE ON CELL COUNT OF TRITON X-100, HOMOGENIZATION TIME
AND THE INTERVAL BETWEEN HOMOGENIZATION AND COUNTING"

Epididymis homogenizedin

Testis homogenizedin
Count
interval

Homo.
time

NaC1
(left)

STM b
(right)

Mean

NaC1
(left)

STM b
(right)

Mean

0 day

i min.
5 min.
Mean

72
65
68

76
77
76

74
71
72

81
87
84

93
104
98

87
95
91

4 day

1 min.
5 min.
Mean

70
66
68

76
77
77

73
71
72

83
86
84

99
107
103

91
97
94

Mean

1 min.
5 min.
Mean

71
65
68

76
77
76

74
71
72

82
86
84

96
106
101

89
96
92

" M e a n number of cells ( X 1 0 -6) in 0,02 rnm 3.

b STM is 0 . 9 % NaC1 containing 0.05% Triton X-100 and 100 ppm Merthiolate.

Possibly, differences or lack of differences

associated with Triton level really represented
variation between sides within rabbits. However, when expressed on the basis of spermarids per gram of testis tissue, the values were
significantly lower when Triton was omitted.
Testes homogenized in STM and saline contained 162--+6 and 1 4 3
106 spermatids/
gram. Presently, STM is used for preparing
all testicular and epididymal homogenates in
this laboratory. For rabbits, testes are homogenized only 1 rain. while finely minced
portions of the excurrent ducts are homogenized for a total of 3 min.
Duration of Spermatogenesis. The relative
frequencies of the eight stages of the cycle of
the seminiferous epithelium for the 44 testes
used in Experiment 2 and for 22 other testes
(Amann, 1968) are summarized in table 2.
The mean values, which represent the relative
durations of the stages, were used in subsequent calculations.
Primary spermatocytes are the most mature
germ cells which will incorporate thymidine3H into their DNA. Subsequently, the time
represented by about three cycles of the semiTABLE 2. RELATIVE FREQUENCIES OF
THE STAGES OF THE CYCLE
OF THE SEMINIFEROUS
EPITHELIUM (%)
Stage
I
II
III
IV
V
VI
VII
VIII
No. testes

Expt. 2

Amann (1968)

Mean + S.E.

26.9
14.0
9.4
12.9
3.7
13.5
11.2
8.4
44

26.9
12.8
10,2
12,1
3.4
15.3
10,9
8.4
22

26.9-+-0.2
13.6-4-0,2
9.7+0,1
12.6__0,2
3.6
14.1+0.2
11.1~/-0,1
8.4~0.2
66

niferous epithelium usually is required for

completion of spermatogenesis (Swierstra and
Foote, 1965; Amann et al., 1965). In rabbits,
primary spermatocytes finish DNA synthesis
in mid-stage I (Swierstra and Foote, 1965)
and stage I represents 26.9% of one cycle
(table 2). Therefore, the last thymidine 3H
is incorporated by primary spermatocytes 2.8 7
cycles of the seminiferous epithelium before
the resulting spermatozoa are released from
the seminiferous tubules. Using this value and
the relative duration of the eight stages of the
cycle, the time of initial appearance of labeled,
elongated spermatids in each specific stage
was calculated. For example, stages VII and
V I I I total 0.20 cycle. Thus, 2.87-0.20 or 2.67
cycles after thymidine-3H injection the first
labeled spermatids should appear in stage VII.
The data for the stage actually containing
the most mature labeled spermatids at various
times after injection are summarized in tables
3 and 4. Although stage VI tubule cross-sections occasionally contained a few labeled
spermatids in three of six testes for day 26.0,
on day 26.5 postinjection 1.2 to 5.4% of the
stage VI tubules in all six testes contained
labeled spermatids. A marked incidence of
labeled stage VI spermatids was not apparent
for all testes until day 27.0. For each of 40
testes (those for day 31.5 were excluded), the
duration of one cycle of the seminiferous epithelium was calculated. For a testis removed
27.5 days after injection in which the most
mature labeled spermatids were in stage VII,
the duration of one cycle would be 27.5/
2.67----10.30 days.
The duration of one cycle of the seminiferous epithelium averaged 10.45_+0.04 days and
ranged from 9.92 to 10.98 days. Pre-experi-

372

AMANN

AND

LAMBIASE

JR.

TABLE 3. PROGRESSION OF LABELED SPERMATIDS THROUGH THE CYCLE OF THE

SEMINIFEROUS E P I T H E L I U M ~
Days after
thymidine-:~H
injection

No. testes

II

III

IV

VI

VII

VIII

25.5
26.0
26.5
27.0
27.5
28.0
28.5
31.5

4
6
6
6
6
6
6
4

51
50
59
61
49
46
26
0

66
48
43
56
60
64
60
6

40
53
79
61
48
48
67
22

8
30
32
60
55
44
54
23

7
24
8
35
56
54
68
47

0
4
3
11
24
35
50
40

0
0
0
1
7
8
28
53

0
0
0
0
0
0
2
68

Stage of cycle

a Percentage of tubule cross-sections of a specific stage which contained at least five labeled spermatids of the older
generation.
m e n t a l e j a c u l a t i o n s a p p e a r e d to h a v e n o influence o n t h e d u r a t i o n of t h e cycle. H o w e v e r , for
n i n e of t h e 20 r a b b i t s t h e r e w a s a n a p p a r e n t
difference, a v e r a g i n g 0 . 3 7
days, bet w e e n t e s t e s i n t h e d u r a t i o n of one cycle. T h e
v a r i a b i l i t y w i t h i n a n d a m o n g r a b b i t s in t h e
p e r c e n t a g e s of t u b u l e c r o s s - s e c t i o n s c o n t a i n i n g
l a b e l e d s p e r m a t i d s is i l l u s t r a t e d in t a b l e 4.
V a r i a t i o n a m o n g t e s t e s in t h e p r o g r e s s i o n of
l a b e l e d s p e r m a t i d s w a s g r e a t e s t for t h e 26.0d a y g r o u p . B a s e d o n all d a t a , t h e first l a b e l e d
s p e r m a t o z o a s h o u l d b e r e l e a s e d f r o m t h e semin i f e r o u s t u b u l e s 30.0 d a y s a f t e r t h y m i d i n e - 3 H
i n j e c t i o n in t h e a v e r a g e r a b b i t . H o w e v e r , as
s u g g e s t e d b y t h e d a t a in t a b l e 4, t h e first lab e l e d s p e r m m i g h t b e r e l e a s e d as e a r l y as 28.5
d a y s or as l a t e as 31.5 d a y s a f t e r t h y m i d i n e 3H i n j e c t i o n .
I n s p e c t i o n of a v a i l a b l e d a t a ( R . P. A m a n n ,
unpublished data; A m a n n et al., 1965; O r g e b i n - C r i s t , 1964, 1965; S w i e r s t r a a n d F o o t e ,
1965) for four c a p i t a e p i d i d y m i d e s r e m o v e d

30.0 d a y s a n d e i g h t c a p i t a r e m o v e d 31.0 d a y s
after thymidine-~H injection, revealed that
t h e first l a b e l e d s p e r m a t o z o a u s u a l l y m u s t enter t h e e p i d i d y m i s 30.5 d a y s a f t e r i n j e c t i o n .
H o w e v e r , t h e e x a c t t i m e of i n i t i a l e n t r y is n o t
uniform even within a rabbit and may occur
e v e n l a t e r t h a n 32.0 d a y s p o s t i n j e c t i o n ( R . P.
A m a n n , unpublished data).

Time Divisor Jot Daily Sperm Production.

T h e m e a n i n c i d e n c e of l a b e l e d s p e r m a t i d s in
t e s t i c u l a r h o m o g e n a t e s for e a c h i n t e r v a l a f t e r
i n j e c t i o n of t h y m i d i n e - 3 H is s h o w n in figure 1.
A l t h o u g h s e v e r a l l a b e l e d s p e r m a t i d s were det e c t e d in four of t h e six 2 7 . 0 - d a y h o m o g e n a t e s ,
n o t u n t i l 27.5 clays a f t e r i n j e c t i o n d i d e a c h of
the testicular homogenates contain numerous
l a b e l e d s p e r m a t i d s . A n a l y s e s of t e s t i c u l a r histology, s u m m a r i z e d in t a b l e 3, i n d i c a t e d t h a t
27.0 d a y s a f t e r i n j e c t i o n l a b e l e d s p e r m a t i d s
u s u a l l y were j u s t e n t e r i n g s t a g e V I of t h e
cycle of t h e s e m i n i f e r o u s e p i t h e l i u m .
Using these data, three estimates were made

TABLE 4. EXAMPLES OF VARIATION IN T H E PROGRESSION OF LABELED

THROUGH THE CYCLE OF THE SEMINIFEROUS E P I T H E L I U M ~
Days after
thymidine-3H
injection
26.0

28.5

Stage of cycle
Rabbit
and side b
226 L
R
212 L
R
229 L
R
211L
R
218 L
R
207 L
R

SPERMATIDS

II

III

IV

VI

VII

VIII

Duration
of one
cycle (days)

65
59
69
28
30
48
60
41
1
13
14
25

25
23
62
27
76
72
73
72
33
52
51
78

54
62
64
50
46
43
51
76
61
67
72
74

67
55
18
16
12
13
36
45
42
48
76
79

56
56
29
4
0
0
46
62
66
84
70
84

15
8
1
0
0
0
35
46
52
50
67
50

0
0
0
0
0
0
2
12
24
31
59
39

0
0
0
0
0
0
0
0
0
0
12
2

10.3
10.3
10.3
10.4
11.0
11.0
10.7
10.7
10.7
10.4
10.2
10.2

.LPercentage of tubule cross-sectious of a specific stage which contained at least five labeled spermatids of the older
generation.
bDuring the pre-experimental period, rabbits 207, 212 and 229 had been SR while rabbits 211. 218 and 226 had been
ejaculated 1X/24 hr.

DAILY SPERM PRODUCTION BY T H E RABBIT

A

240

g
o

,E

_J

rl

25.5

26.5
2Z5
28.5
Day postinjection

51.5

Figure 1. The time of initial appearance of labeled spermatids in testicular homogenates.

of the time divisor necessary for calculating

daily sperm production from testicular homogenates.
(a) Stages VI, VII and VIII represent
33.6% of the duration (10.45 days) of
one cycle of the seminiferous epithelium.
t i m e d i v i s o r z (0.336) (10.45 ~ 3.5
days
(b) Labeled spermatids first appear in testicular homogenates 27.0 days after injection and are released from the germinal epithelium at 30.0 days.
time divisor ~ 30.0-27.0 ~ 3.0 days
(c) Labeled spermatids first appear in testicular homogenates 27.0 days after injection and enter the caput epididymidis at 30.5 days.
time divisor ~ 30.5-27.0 ~ 3.5 days
Since there was no reason to exclude any of
the estimates, all three were averaged giving a
mean value of 3.33 days.
Discussion

These studies make two contributions to

techniques for studying testicular physiology.
First, inclusion of Triton X-100 in the homogenization fluid greatly reduced the amount
of particulate matter in the cellular suspensions and thus facilitated accurate hemacytometric counting. Nevertheless, STM probably
should be tested before use with other species.
Secondly, the time divisor necessary for converting spermatid reserves to daily sperm production was determined for rabbits.

373

In this study, spermatid reserves of testes

homogenized in STM were greater than those
for the contralateral testes homogenized in
saline. For 140 rabbit testes homogenized in
STM, the spermatid reserves averaged 368 x
106/testis or 128x 106/gin. of testis parenchyma (Lambiase and Amann, 1968). These
values are considerably greater than those of
201 x 106/testis and 94 x 106/gin. of testis parenchyma reported by Kirton et al. (1967)
for 25 rabbits and the maximum value of
l l 0 x 106 spermatids/gm, of testis reported
by Verma et al. (1966). However, based on
data for 33 adult rabbits, Orgebin-Crist
(1968) found that each testis contained 367 x
106 spermatids or 133 x 106/gm. These latter
values are virtually identical with those obtained at this laboratory. Thus, the total testicular reserves of a sexually mature, New
Zealand White rabbit are about 735x 106
spermatids.
The accuracy of daily sperm production
values calculated from testicular spermatid
reserves is dependent upon: (a) the accuracy
of the time divisor, (b) the efficiency of homogenization, (c) the extent of destruction of
elongated spermatids during homogenization
and (d) the variability associated with hemacytometric counting. When the homogenization and counting procedures described herein
are used, probably only the time divisor materially influences accuracy of the calculated
daily sperm production values.
The morphology and image density of
spermatid nuclei in cryostat tissue sections
examined by phase contrast microscopy and
the apparent Feulgen stainability of spermarids in paraffin sections was found to be similar for spermatids in stages IV to VIII (R. P.
Amann, unpublished data). Thus, Kirton et
al. (1967) concluded that the cells counted in
testicular homogenates represented spermatids
in the last half of stage IV and in stages V
through VIII. The present autoradiographic
data and those of Orgebin-Crist (1968) clearly
show that, for rabbit testes, the elongated
spermatids present in stage IV do not survive
the rigors of homogenization. Until evidence
to the contrary is available, the absolute duration of stages VI, VII and VIII also might be
used as the time divisor for other species.
However, this value must be determined for
each species.
Orgebin-Crist (1968) concluded, based on
autoradiographic data for five testes removed
24.62 to 30.60 days after thymidine-~H injection, that the time divisor was 5.44 days for

374

A M A N N A N D L A M B I A S E JR.

rabbits. This value is drastically different from

that of 3.33 days reported herein. P a r t of this
discrepancy results from the values used for
the relative duration of the eight stages of the
cycle of the seminiferous epithelium. Possibly
deliniation of the stages differed. OrgebinCrist (1968) concluded t h a t elongated spermatids present in stages V to V I I I were counted
and that these stages represent 48.6% of one
cycle. However, our more extensive d a t a indicated that these stages represent 37.2% of
one cycle and Swierstra and Foote (1963) reported that stages V to V I I I represent 40.6%
of the cycle.
T h e value used for the duration of one cycle
of the seminiferous epithelium is the other
major difference between our d a t a and those
of Orgebin-Crist (1968). M e a n values of 10.45
and 11.2 days were used, respectively. Other
values reported are 10.7 days ( A m a n n et al.,
1965), 10.9 days (Swierstra and Foote, 1965)
and 10.3 days (Orgebin-Crist, 1965). D a t a
presented herein suggest that these mean values m a y reflect variation among rabbits. A
pooled, weighted mean value of 10.67 days for
the duration of one cycle of the seminiferous
epithelium was calculated from the available
d a t a based on 91 r a b b i t testes. Use of this
mean value should give the best estimate for
the time divisor; a value slightly longer than
the 3.33 days calculated above. F o r 40 testes,
revised values ranging from 3.09 to 3.84 days
were calculated, using individual testis d a t a
for cycle length and the relative duration of
the eight stages of the cycle of the seminiferous epithelium. W e conclude t h a t to convert
r a b b i t testicular spermatid reserves to daily
sperm production the time divisor of 3 . 4 3
0.03 days should be used.
Summary
Larger sperm reserve values were obtained
when the saline used for homogenizing testes
and epididymides included 0.05% Triton X 100. The amount of particulate matter which
might obscure cells during hemacytometric
counting was reduced. Twenty-two other rabbits injected with thymidine-aH were used to
determine the time divisor necessary to calculate daily sperm production from testicular

spermatid reserve d a t a as obtained from testicular homogenates. The duration of one

cycle of the seminiferous epithelium averaged
10.45
days, but for 9 of 20 rabbits
there was an a p p a r e n t difference, averaging
0.37___0.06 days, between testes. For rabbits,
testicular spermatid reserves were concluded
to represent 3.43___0.03 days production of
spermatozoa. Sexually mature, New Zealand
White rabbits produce about 210 x 106 spermatozoa per day.
Literature

Cited

Amann, R. P. 1968. The male rabbit. IV. Quantitative testicular histology and comparisons between
daily sperm production as determined histologically
and daily sperm output. In manuscript.
Amann, R. P. and J. O. Almquist. 1961. Reproductive
capacity of dairy bulls. I. Technique for direct
measurement of gonadal and extragonadal sperm
reserves. J. Dairy Sci. 44:1537.
Amann, R. P. and J. O. Almquist. 1962. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production.
J. Dairy Sci. 45:774.
Amann, R. P., H. It. Koefoed-Johnson and H. Levi.
1965. Excretion pattern of labeled spermatozoa
and the timing of spermatozoa formation and epididymal transit in rabbits injected with thymidine-3H. J. Reprod. Fertil. 10:169.
Kirton, K. T., C. Desjardins and H. D. Hafs. 1967.
Distribution of sperm in male rabbits after various ejaculation frequencies. Anat. Rec. 158:287.
Lambiase, J. T., Jr. and R. P. Amann. 1968. The
male rabbit. V. Changes in sperm reserves and
resorption rate induced by ejaculation and sexual
rest. J. Animal Sci. In press.
Orgebin-Crist, M. C. 1964. Delayed incorporation of
thymidine-3H in epithelial cells of the ductus epididymis of the rabbit. J. Reprod. Fertil. 8:259.
Orgebin-Crist, M. C. 1965. Passage of spermatozoa
labeled with thymidine-SH through the ductus
epididymis of the rabbit. J. Reprod. Fertil. 10:241.
Or~ebin-Crist, M. C. 1968. Gonadal and epididymal
sperm reserves in the rabbit: estimation of the
daily sperm production. J. Reprod. Ferfil. 15:15.
Swierstra, E. E. and R. H. Foote. 1963. Cytology
and kinetics of spermatogenesis in the rabbit. J.
Reprod. Fertil. 5:309.
Swierstra, E. E. and R. H. Foote. 1965. Duration of
spermatogenesis and spermatozoan transport in the
rabbit based on cytological changes, DNA synthesis and labeling with tritiated thymidine. Am. J.
Anat. 116:401.
Verma, M. C., U. D. Sharma, and G. Singh. 1966.
Studies on sperm production. III. Testicular and
epididymal sperm reserve in small animals (rabbit, guinea-pig, albino rat and fowl). Indian J. Vet.
Sci. 36:109.