THE MALE RABBIT. II I. DETERMINATION OF DAILY SPERM PRODUCTION BY MEANS OF

TESTICULAR HOMOGENATES 1

R. P. AMANN AND J . T. LAMBIASE, JR. The Pennsylvania State University, University Park 2

T ESTICULAR spermatid reserves have been quantified for bulls, buffalo-bulls, deer, boars, rabbits, guinea pigs, roosters and hu- mans. Generally, a modification of the tech- nique described by Amann and Almquist (1961) has been used. Daily sperm produc- tion can be calculated by dividing the value for testicular spermatid reserves by a time divisor which is the number of days of produc- tion these reserves represent (Amann and Almquist, 1962). Based on knowledge of the duration of one cycle of the seminiferous epi- thelium 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 di- visor of 3.27 days was not valid for other spe- cies or homogenization techniques. When sam- pling 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 produc- tion of rabbits. Estimations of the time divisor as determined by morphologic and autoradio- graphic methods are compared since auto- radiographic studies might be impractical with some animals.

Methods

Experiment 1. Testicular and epididymal homogenates prepared by the usual method (Amann and Almquist, 1961) contain particu- late 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 Re- search 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 assis- tance.

2 Dairy Breeding Research Center, University Park, Penn- sylvania, 16802.

heads during hemacytometric counts. Anti- foaming compounds and anionic detergents were used in preliminary tests aimed at reduc- ing this interference and also foaming. Inclu- sion of Span 80, Dow Antifoam C or Dow An- tifoam FR-10 reduced foaming more than use of Triton X-100, but emulsified fat sometimes obscured the cells. However, with Triton X- 100 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 trans- ferred 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 Merthio- late to retard bacterial proliferation. The saline-Triton-Merthiolate fluid will be re- ferred to as STM. Each epididymis was minced on a watch glass and then homoge- nized 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, imme- diately 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 sim- ilarly. The same three observers counted all homogenates through phase contrast micro- scopes. 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-

369

370 AMANN AND

bers counted, were used in analyses of vari- ance.

Experiment 2. This experiment was designed to determine the number of days' sperm pro- duction represented by the spermatids counted in testicular homogenates. Twenty-two ran- dom-bred, 9-mo.-old, New Zealand White rab- bits were trained to ejaculate into an artificial vagina. During a 7-day pre-experimental pe- riod, 11 males were sexually rested (SR) and 11 were ejaculated at a frequency of one ejac- ulate 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 sex- ually rested.

Rabbits were killed by sodium pentobarbi- tal, 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 sec- ond 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 perpen- dicularly to the long axis into three unequal pieces. The parenchyma of the central one- half 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 homoge- nate and rapidly dried using a hot plate and fan. The smears were fixed in absolute meth- anol 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 Tissue- mat and sectioned at 5~. Sections from both poles were mounted on each of five slides which were deparaffinized preparatory to au- toradiography.

The coded slides with testicular tissue sec- tions 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 de- veloped 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 seminifer- ous epithelium was determined using auto- radiograms of testicular sections. The eight stages of the cycle of the seminiferous epithe-

LAMBIASE JR.

lium have been described by Swierstra and Foote (1963). For six sections per testis, three from each pole, 100 essentially round semini- ferous 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 as- sociated with at least 4 grains. From these data on the relative frequencies of the eight stages of the cycle of the seminiferous epithe- lium and knowledge of the stage containing the most advanced labeled germ cells, the dur- ation of one cycle of the seminiferous epithe- lium was calculated for each testis (Amann et al., 1965).

The time of initial appearance, incidence and degree of spermatid labelling were deter- mined by evaluating five autoradiograms for each testicular homogenate. A total of 1,000 randomly selected spermatid nuclei per ho- mogenate, 200 per slide, were classed as hav- ing 0, 1-2, 3-5, 6-8, 9-11, 12-14, 15-17, 18 20 or ~20 grains. Spermatids associated with 0, 1 or 2 grains subsequently were con- sidered to be unlabeled.

Results Determination o/Sperm Reserves. Data on

testicular spermatid and epididymal sperma- tozoan reserves from Experiment 1 are sum- marized in table 1. No significant difference was found between homogenization times of 1 and 5 rain. or between counts obtained im- mediately and 4 days after homogenization. This was true for both testicular and epididy- real tissue. For epididymal tissue, higher counts (P~.01) were obtained for samples containing Triton X-100. The Triton level x homogenization time interaction (P~,05) for testicular tissue indicated that 5 min. homog- enization reduced sperm counts in saline but not in STM.

Spermatid reserves for individual testes (2.42 gm.) homogenized for 1 min. in STM averaged 388 while those (2.45 gin.) in saline averaged only 361+16x 106 spermatids. Similar values for individual epididymides were 966 and 826-+-88 x 106 sperm. Use of Triton X-100 ap- parently facilitated accurate counting by eli- minating particulate matter. Based on the difference in counts between contralateral tis- sues from individual males, up to 28% of the cells might have been obscured when saline was used for homogenization.

DAILY SPERM PRODUCTION BY THE RABBIT

TABLE 1. INFLUENCE ON CELL COUNT OF TRITON X-100, HOMOGENIZATION TIME AND THE INTERVAL BETWEEN HOMOGENIZATION AND COUNTING"

371

Testis homogenized in Epididymis homogenized in

Count Homo. NaC1 STM b NaC1 STM b interval time (left) (right) Mean (left) (right) Mean

0 day i min. 72 76 74 81 93 87 5 min. 65 77 71 87 104 95 Mean 68 76 72 84 98 91

4 day 1 min. 70 76 73 83 99 91 5 min. 66 77 71 86 107 97 Mean 68 77 72 84 103 94

Mean 1 min. 71 76 74 82 96 89 5 min. 65 77 71 86 106 96 Mean 68 76 72 84 101 92

"Mean number of cells (X10 -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. How- ever, when expressed on the basis of sperma- rids per gram of testis tissue, the values were significantly lower when Triton was omitted. Testes homogenized in STM and saline con- tained 162--+6 and 143 106 spermatids/ gram. Presently, STM is used for preparing all testicular and epididymal homogenates in this laboratory. For rabbits, testes are ho- mogenized only 1 rain. while finely minced portions of the excurrent ducts are homoge- nized 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 subse- quent calculations.

Primary spermatocytes are the most mature germ cells which will incorporate thymidine- 3H into their DNA. Subsequently, the time represented by about three cycles of the semi-

TABLE 2. RELATIVE FREQUENCIES OF THE STAGES OF THE CYCLE

OF THE SEMINIFEROUS EPITHELIUM (%)

Stage Expt. 2 Amann (1968) Mean + S.E.

I 26.9 26.9 26.9-+-0.2 I I 14.0 12.8 13.6-4-0,2

I I I 9.4 10,2 9 .7+0,1 IV 12.9 12,1 12.6__0,2 V 3.7 3.4 3 .6

VI 13.5 15.3 14 .1+0.2 VI I 11.2 10,9 11.1~/-0,1

V I I I 8.4 8.4 8 .4~0.2 No. testes 44 22 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 VII 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-sec- tions 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 epi- thelium 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 seminifer- ous 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 EPITHELIUM ~

Days after thymidine-:~H

injection

Stage of cycle

No. testes I II I I I IV V VI VII VIII

25.5 4 51 66 40 8 7 0 0 0 26.0 6 50 48 53 30 24 4 0 0 26.5 6 59 43 79 32 8 3 0 0 27.0 6 61 56 61 60 35 11 1 0 27.5 6 49 60 48 55 56 24 7 0 28.0 6 46 64 48 44 54 35 8 0 28.5 6 26 60 67 54 68 50 28 2 31.5 4 0 6 22 23 47 40 53 68

a Percentage of tubule cross-sections of a specific stage which contained at least five labeled spermatids of the older generation.

menta l e jaculat ions appeared to have no influ- ence on the durat ion of the cycle. However , for nine of the 20 rabb i ts there was an apparent difference, averaging 0 .37 days, be- tween testes in the durat ion of one cycle. The var iab i l i ty w i th in and among rabb i ts in the percentages of tubule cross-sections conta in ing labeled spermat ids is i l lustrated in table 4. Var iat ion among testes in the progression of labeled spermat ids was greatest for the 26.0- day group. Based on all data, the first labeled spermatozoa should be released f rom the semi- n i ferous tubules 30.0 days after thymid ine-3H in ject ion in the average rabbit . However, as suggested by the data in table 4, the first la- beled sperm might be released as ear ly as 28.5 days or as late as 31.5 days af ter thymid ine- 3H inject ion.

Inspect ion of avai lable data (R. P. Amann, unpublished data; Amann et al., 1965; Orge- b in-Cr ist , 1964, 1965; Swierstra and Foote, 1965) for four capi ta epid idymides removed

30.0 days and eight capi ta removed 31.0 days after thymid ine-~H inject ion, revealed that the first labeled spermatozoa usual ly must en- ter the epid idymis 30.5 days af ter in ject ion. However, the exact t ime of init ia l ent ry is not un i fo rm even wi th in a rabb i t and may occur even later than 32.0 days post in ject ion (R. P. Amann, unpublished data).

Time Divisor Jot Daily Sperm Production. The mean incidence of labeled spermat ids in test icular homogenates for each interval a f ter in ject ion of thymid ine-3H is shown in figure 1. A l though several labeled spermat ids were de- tected in four of the six 2 7.0-day homogenates, not unt i l 27.5 clays after in ject ion did each of the test icular homogenates conta in numerous labeled spermat ids. Analyses of test icular his- tology, summar ized in table 3, ind icated that 27.0 days after in ject ion labeled spermat ids usual ly were just enter ing stage VI of the cycle of the semini ferous epithel ium.

Us ing these data, three est imates were made

TABLE 4. EXAMPLES OF VARIATION IN THE PROGRESSION OF LABELED SPERMATIDS THROUGH THE CYCLE OF THE SEMINIFEROUS EPITHELIUM ~

Days after Stage of cycle Duration thymidine-3H Rabbit of one

injection and side b I II I I I IV V VI VII VIII cycle (days)

26.0

28.5

226 L 65 25 54 67 56 15 0 0 10.3 R 59 23 62 55 56 8 0 0 10.3

212 L 69 62 64 18 29 1 0 0 10.3 R 28 27 50 16 4 0 0 0 10.4

229 L 30 76 46 12 0 0 0 0 11.0 R 48 72 43 13 0 0 0 0 11.0

211L 60 73 51 36 46 35 2 0 10.7 R 41 72 76 45 62 46 12 0 10.7

218 L 1 33 61 42 66 52 24 0 10.7 R 13 52 67 48 84 50 31 0 10.4

207 L 14 51 72 76 70 67 59 12 10.2 R 25 78 74 79 84 50 39 2 10.2

.L Percentage 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 1 X/24 hr.

DAILY SPERM PRODUCTION BY THE RABBIT 373

A

240 g o

,E

E

_J rl ~ I , I ~ I I

25.5 26.5 2Z5 28.5 51.5 Day postinjection

Figure 1. The time of initial appearance of la- beled spermatids in testicular homogenates.

of the time divisor necessary for calculating daily sperm production from testicular ho- mogenates.

(a) Stages VI, VII and VIII represent 33.6% of the duration (10.45 days) of one cycle of the seminiferous epithe- lium. t ime d iv i sorz (0.336) (10.45 ~ 3.5 days

(b) Labeled spermatids first appear in tes- ticular homogenates 27.0 days after in- jection and are released from the germ- inal epithelium at 30.0 days. time divisor ~ 30.0-27.0 ~ 3.0 days

(c) Labeled spermatids first appear in tes- ticular homogenates 27.0 days after in- jection and enter the caput epididy- midis 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 ho- mogenization fluid greatly reduced the amount of particulate matter in the cellular suspen- sions and thus facilitated accurate hemacyto- metric counting. Nevertheless, STM probably should be tested before use with other species. Secondly, the time divisor necessary for con- verting spermatid reserves to daily sperm pro- duction was determined for rabbits.

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 paren- chyma (Lambiase and Amann, 1968). These values are considerably greater than those of 201 x 106/testis and 94 x 106/gin. of testis pa- renchyma reported by Kirton et al. (1967) for 25 rabbits and the maximum value of l l0x 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 ob- tained at this laboratory. Thus, the total tes- ticular 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 ho- mogenization, (c) the extent of destruction of elongated spermatids during homogenization and (d) the variability associated with hema- cytometric counting. When the homogeniza- tion and counting procedures described herein are used, probably only the time divisor ma- terially 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 sperma- rids in paraffin sections was found to be sim- ilar 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 dura- tion 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 injec- tion, that the time divisor was 5.44 days for

374 AMANN AND LAMBIASE JR.

rabbits. This value is drastically different from that of 3.33 days reported herein. Part 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. Orgebin- Crist (1968) concluded that elongated sperm- atids 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 data indi- cated that these stages represent 37.2% of one cycle and Swierstra and Foote (1963) re- ported that stages V to V I I I represent 40.6% of the cycle.

The value used for the duration of one cycle of the seminiferous epithelium is the other major difference between our data and those of Orgebin-Crist (1968). Mean values of 10.45 and 11.2 days were used, respectively. Other values reported are 10.7 days (Amann et al., 1965), 10.9 days (Swierstra and Foote, 1965) and 10.3 days (Orgebin-Crist, 1965). Data presented herein suggest that these mean val- ues may 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 data based on 91 rabbit 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. For 40 testes, revised values ranging from 3.09 to 3.84 days were calculated, using individual testis data for cycle length and the relative duration of the eight stages of the cycle of the seminifer- ous epithelium. We conclude that to convert rabbit testicular spermatid reserves to daily sperm production the time divisor of 3 .43 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 rab- bits injected with thymidine-aH were used to determine the time divisor necessary to calcu- late daily sperm production from testicular

spermatid reserve data as obtained from tes- ticular homogenates. The duration of one cycle of the seminiferous epithelium averaged 10.45 days, but for 9 of 20 rabbits there was an apparent 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 sperm- atozoa per day.

L i te ra ture Ci ted

Amann, R. P. 1968. The male rabbit. IV. Quantita- tive 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. Reproduc- tive capacity of dairy bulls. VIII. Direct and in- direct 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 epi- didymal transit in rabbits injected with thymi- dine-3H. J. Reprod. Fertil. 10:169.

Kirton, K. T., C. Desjardins and H. D. Hafs. 1967. Distribution of sperm in male rabbits after var- ious 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 epi- didymis 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 synthe- sis 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 (rab- bit, guinea-pig, albino rat and fowl). Indian J. Vet. Sci. 36:109.