of the rabbit testis were obtained.

Transcription

1 FERTILITY AND STERILITY Copyright 1970 by The Williams & Wilkins Co. Vol. 21, No.9, September 1970 Printed in U.S.A. THE MALE RABBIT. IV. QUANTITATIVE TESTICULAR HISTOLOGY AND COMPARISONS BETWEEN DAILY SPERM PRODUCTION AS DETERMINED HISTOLOGICALLY AND DAILY SPERM OUTPUT* R. P. AMANN, PH.D. Dairy Breeding Research Center, The Pennsylvania State University, University Park, Pennsylvania Knowledge of quantitative testicular histology is essential for precise evaluation of the effects of surgery/ 21 drugs,29 or unknown factors. 24 In addition, quantitative data on testicular histology can be used as the basis for estimates of daily sperm production Daily sperm production (DSP) is the total number of sperm produced per day by the two testes. One method for calculating DSP was proposed by Amann and Almquist3 and requires enumeration of the Stage I spermatids in a known volume of testis. Another histologic method, developed by Kennelly, Foote, and Swierstra/ is based on the relative volume of spermatids within the testis. Both methods require knowledge of the duration of 1 cycle of the seminiferous epithelium. Alternatively, DSP can be estimated by counting spermatids in testicular homogenates or by quantitative recovery of the effluent from a cannula placed in the rete tcstis Daily sperm output (DSO) is the total number of ejaculated spermatozoa collected over a period_ of time expressed on a per day basis If a male is ejaculated at a sufficiently high frequency, DSO may approach DSP. With humans, only daily sperm output has been estimatcdy For humans, the most feasible approach *Authorized for publication on December 19, 1969 as Paper No in the Journal Series of the Pennsylvania Agricultural Experiment Station. This investigation was supported by Research Grant HD from the National Institute of Child Health and Human Development. 662 for determining DSP may be histologic analyses of testicular tissue. This paper reports the first study using both histologic methods of analysis to determine the DSP of one group of animals. The values obtained are compared with maximum DSO in ejaculated semen. Concurrently, comprehensive data on the quantitative histology of the rabbit testis were obtained. MATERIALS AND METHODS Twelve New Zealand White rabbits were ejaculated twice successively every 48 hr. from weeks of age. One false mount preceded each cj aculatc. Procedural details and results were reported by Amann and Lambiase. 4 Complete sperm output data were obtained for only 10 buck' because two frequently refused to c.i aculatc during the last 7 weeks of collection. Earlier data were used for these two rabbits. At 54 weeks of age, each of the 12 rabbits was killed with sodium pentabarbital, immediately after collection of two cj aculates, and its reproductive system was removed. Weights of the testes and epididymides were recorded and the extragonadal sperm reserves were determined." Part of each testis was frozen and sectioned at 20 microns in a cryostat microtome. Sections were picked up on glass slides and immediately a cover glass was carefully mounted using 50% glycerol. The minor diameter of 100 seminiferous tubule cross-sections/testis, 25/tissue section, was measured to the nearest 5 ft using

2 September 1970 THE MALE RABBIT 663 phase contrast microscopy and an ocular micrometer. The major portion of each testis was placed in Bouin-Hollande fixative, embedded in Tissuemat, and sectioned at 7 1'- Four sections, each separated from the others by at least 140 1'- were mounted on each slide and sets of slides were stained with periodic acid-schiff or hematoxylin and eosin. All slides were coded to reduce observer bias. To enable correction for the shrinkage of testicular tissue associated with histologic processing, the minor diameter of 25 seminiferous tubules in each of four periodic acid-schiff-stained sections was measured to the nearest 2 1'- For each testis, the mean value obtained by bright field microscopy after paraffin sectioning was compared with the mean minor seminiferous tubule diameter previously measured in the unfixe d cryostat sections. To determine the volume of that portion of the testis actually involved in sperm production, several measurements were made. Volume of the testis parenchyma was considered to be 3 : (testis weight) - (tunica albuginea weight) +- (density of parenchyma). A value of g./cm 3. was used for this density. The relative volumes of the seminiferous tubules and interstitial tissue were determined by Chalkley's technique. 7 A magnification of X 312 and four indicating pointers were used. The area of the mediastinum was avoided while recording 100 hits for each of four sections per testis. After correction for artifact hits, the mean relative volume of seminiferous tubules for each section and testis was calculated. Duplicate evaluations were made for 10 randomly selected testes. The relative volume of the mediastinum testis was estimated from five other testes fixed in toto and then sliced perpendicularly to the mediastinum into eight pieces of similar thickness. Paraffin sections representing each level were prepared and hematoxylin and eosin stained. Complete cross-sections were projected onto bond paper and the outlines of the testis and mediastinum were traced. After cutting out the tracings for 10 sections/ level, the relative volume of the mediastinum at each of the eight levels was determined by weighing individual tracings. The relative frequencies of the eight stages of the cycle of the seminiferous epithelium28 were determined by scoring (at X 500) 200 randomly selected, essentially round, tubule cross-sections in each of four tissue sections per testis. When a tubule cross-section contained more than one stage, it was recorded as being in the stage comprising the greater proportion of the cross-section. Triplicate determinations were made for eight testes. The relative volume occupied by round spermatid nuclei within the testis was determined by Chalkley's technique.7 For each testis, 500 hits were recorded for each of four tissue sections. The ocular contained five pointers and the total magnification was X Hits were recorded as representing: (1) round nuclei of spermatids in Stage I, (2) round nuclei of spermatids in Stages V through VIII, (3) all other tissue within the seminiferous tubules including the lumen, ( 4) interstitial tissue, and (5) artifact or space. For hits on round spermatid nuclei, the stage of the cycle of the seminiferous epithelium for that portion of tubule was determined. Artifact hits were subtracted from the total of 2000 hits/testis and the relative volume of each element within the testis was calculated. Duplicate determinations were made for 10 testes. Concurrently, for each testis the major and minor diameters of 40 randomly selected, whole, round spermatid nuclei, representing those in Stages V-I, were measured to the nearest 0.6 1'- using an ocular micrometer. The number of round spermatid nuclei

3 664 AMANN Vol. 21 was counted (at X 1250) for 10 randomly selected, essentially round, Stage I seminiferous tubule cross-sections for each of four tissue sections per testis. The major and minor diameters of the same Stage I tubules were measured to the nearest 2.4 p. and the diameters of one whole spermatid nucleus were measured to the nearest 0.8 p.. Thus, 40 Stage I seminiferous tubules/ testis were evaluated. These actual counts were corrected to compensate for the inclusion of partial nuclei using the following formula: 1 for each testis was used to calculate the correction factors. The thickness of each histologic section studied was calculated from 10 measurements using a Leitz double beam interference microscope. 6 Knowledge of the duration of 1 cycle of the seminiferous epithelium is essential for any calculation of daily sperm production. The value for rabbits used throughout this report is days.5 Three methods were used to calculate the DSP of each testis. Knowledge of the volume of the sperm-producing portion of True count [ b l thickness of histologic section = o served count [ (section thickness) + V (average diameter /2) 2 - (average diameter I 4) 2 J For 22 testes the mean diameter of a Stage I spermatid nucleus was found to be 6.03 p.. This value was considered a constant, but the mean histologic section thickness the testis, i.e., corrected testis volume, is essential for each method. This was determined by the formula proposed by Amann and Almquist.3 Corrected testis volume ( testis parenchyma) (correction factor) (correction factor for) weight for mediastinum tissue shrinkage = ~--~~--~~ (density of testis) Using the appropriate quantitative histologic data, the DSP of each testih was calculated using each of the three formulas below. Formula 1 (Amann and AlmquisV) ( corrected testis) (percentage of seminiferous) (corrected number of spermatids per) DSP = volume tubules in the testis Stage I tubule cross-sectio[l 1 (duration of 1 cycle of the) ( area of Stage I ) ( thickness of ) seminiferous epithelium tubule cross-section histologic sections Formula 2 (Swierstra 26 ) DSP = (corrected testis volume) (relative volume of Stage I spermatid nuclei) 2 (duration of 1 cycle of the) (relative duration) (volume of a Stage I) seminiferous epithelium of Stage I spermatid nucleus Formula 3 (Swierstra26) DSPa = (corrected testis volume) (relative volume of all spherical spermatid nuclei in Stages V-I) (duration of 1 cycle of the) (relative duration of) (volume of a spherical) seminiferous epithelium Stages V-I spermatid nucleus

4 September 1970 THE MALE RABBIT 665 The relationships among the DSP values determined by each method and their correlation with DSO were analyzed. TABLE 1. Characteristics of the New Zealand White Rabbits Characteristic Mean± S.E. (N = 11) RESULTS Although 12 rabbits were used in this experiment, the daily sperm output of one rabbit declined drastically from a level of about 160 X 106 to a value of 64 X 10 6 during the 54th week. When killed, the weight of the two testes totaled only 3.66 gm. and the extragonadal reserves (including sperm ejaculated just before killing) totaled only 330 X 106 sperm. Therefore, data for this abnormal rabbit were excluded from this report. The 11 rabbits used are characterized in Table 1. Qualitative Testicular Histology. The weight of the individual testes averaged 3.08 ± 0.10 gm., of which the tunica albuginea represented 0.12 ± 0.01 gm. or 3.8 ± 0.2%. Based on the mean parenchyma weight of 2.96 ± 0.10 gm. and the assumed density of gm./cm 3., the parenchymal volume averaged 2.85 cm 3 This volume represented the mediastinum, seminiferous tubules, and interstitial tissue. Considerable shrinkage of testicular tissue was associated with routine histologic processing. The minor diameter of seminiferous tubule cross-sections measured in fresh cryostat sections averaged 225 ± 2 /h whereas that for tubules measured after paraffin sectioning and PAS staining was only 182 ± 2 p.. For the 22 testes, linear shrinkage associated with histologic processing averaged 19.0 ± 1.0%. This represented a volumetric shrinkage of 46.4 ± 2.2%. Thus, the volume of a segment of seminiferous tubule observed in a paraffin section represented only 42-87% (mean = 54%) of what its volume had been during life. The correlation between seminiferous tubule diameters in freshfrozen and in paraffin sections was 0.62 (p < O.Dl). These data suggest that this Age (weeks) Body weight (kg.) Daily sperm output* (106) Testes weight (gm.) Extragonadal sperm reserves (106) Capita-corpora epididymides Caudae epididymides Ductuli deferentia Ejaculated on day killed Total 54± ± ± ± ± ± 62 59± ± ± 140 * Two ejaculates, each preceded by one false mount, were collected every 48 hr.; mean for weeks of age. parameter must be determined for each testis because the extent of shrinkage is large but quite variable. The thickness of individual histologic sections used for quantitative analyses ranged from 2.59 p p. when measured by interference microscopy. The mean thickness of 3.47 p. was markedly less than expected because the microtome had been set at 7.0 p.. A new microtome subsequently tested produced sections with a thickness closer to the indicated value. Nevertheless, the thickness of histologic sections evaluated should be determined if quantitative applications are planned. The volume of the mediastinum testis was found to represent 0.82, 0.91, 1.16, 1.63, and 1.63% of the parenchymal volume for five testes. The mean value of 1.23% was used in subsequent calculations. Generally, the ventral pole of a rabbit testis is smaller in diameter than the dorsal pole. In the dorsal, central, and ventral thirds of these five testes the mediastinum represented 0.79, 1.29, and 1.63% of the parenchymal volume. The seminiferous tubules were found to occupy 86.8 ± 0.5% of the testis parenchyma excluding the mediastinum. Testis

5 ' 666 AMANN Vol. 21 values ranged from %. An analysis of variance revealed that differences among rabbits for the 22 testes in the relative volume occupied by seminiferous tubules were highly significant (p < 0.01). However, differences between testes within rabbits were not significant. Duplicate evaluations for 10 testes gave a mean of 86.4% seminiferous tubules as compared to 86.7% for the first replicate. Differences between replicates or between replicates within testes were not significant (p > 0.05) although the differences among the pooled testis means were highly significant (p < 0.01). Similar data later were obtained when the relative volume of spermatids was determined. These data, based on 2000 hits/testis at a magnification of X 1250, indicated that seminiferous tubules comprised 83.7 ± 0.4% of the volume of the 22 testes whereas for the earlier data, based on 400 hits at X 312 magnification, the mean was 86.8 ± 0.5%. The mean difference between pairs of values was greater than zero (p < 0.01), but values obtained by the two methods were correlated (r = 0.44, p < 0.05). For determinations of the relative volume of seminiferous tubules a relatively low magnification is considered to give more precise data; values obtained at X 312 were repeatable. From these data, total length of the seminiferous tubules, 225 p. in diameter, in a TABLE 2. Relative Frequencies of the Stages of the Cycle of the Seminiferous Epithelium Stage Mean± S.E. (N = 22) Replicate means (N = 8) First Second Third % % % % I 26.9 ± II 12.8 ± III 10.2 ± IV 12.1 ± v 3.4 ± VI 15.3 ± VII 10.9 ± VIII 8.4 ± typical 3.08-gm. rabbit testis was calculated to be about 62 m. Data on the relative frequencies or durations of the eight stages of the cycle of the seminiferous epithelium are summarized in Table 2. Analyses of variance revealed that for each stage the differences in relative frequency among the 11 rabbits and between testes (left vs. right) were not significant (p > 0.05). The variation between testes within rabbits was significant (p < 0.05) for Stages III and VIII and highly significant (p < 0.01) for Stages II and VI. Significant differences (p < 0.05 or p < 0.01) were found among the 22 testis mean values for each stage except Stages I and IV. The relative frequencies calculated from triplicate evaluations of the same eight testes by one person also are in Table 2. Replicate 1 was a portion of the over-all data. Replicate 2 was made using the same slides after all 22 testes had been evaluated and Replicate 3 was made several months later using a different set of slides. These data were converted to angles and the coefficient of repeatability was calculated; it was 0.82 (p < 0.01). Thus, determinations of the frequency of the stages of the cycle of the seminiferous epithelium are repeatable even though there is some variation among testes. The relative volumes occupied by round spermatid nuclei and other testicular components are summarized in Table 3. The elongated spermatid nuclei present in Stages II-VIII were classed with other seminiferous tubule components. Data for the relative volume of all round spermatid nuclei in Stages V-I were evaluated by an analysis of variance. Variation among the 22 testes was significant (p < 0.05), but the differences between testes within rabbits and among rabbits were not significant. The correlation between the relative volume of all round spermatid nuclei in Stages V-VIII and that in Stage I was Physiologically, it is logical that

6 September 1970 THE MALE RABBIT 667 TABLE 3. Relative Volume of Testicular Components Component Mean± S.E. (N = 22) Replicate means (N = 10) First Second Round spermatid nuclei in Stage I Round spermatid nuclei in Stages V -> I Other seminiferous tubule components Interstitial tissue % 2.31 ± ± ± ± 0.44 % % this correlation should be positive and significant. Therefore, it appears that an insufficient area of the testis was studied or that the number of hits was insufficient to give a valid analysis of the area studied. The data obtained apparently were valid. Values from the duplicate determinations for 10 testes, summarized in Table 3, were converted to angles before calculating the coefficient of repeatability. The value of 0.99 (p < 0.01) indicated that these determinations were repeatable. Thus, in future studies the tissue sections studied should be larger in area or separated by a greater distance. The nuclei of the younger generation of spermatids in Stages V-I were essentially spherical. Major and minor diameters averaged 5.88 ± 0.02 and 5.65 ± 0.02 p.. Thus, the mean diameter of 5.76 p. was used in the formula 1/6 1rd3 to determine nuclear volume. The value of p. 3 subsequently was used in all calculations of DSP. An average of round spermatid nuclei was counted in the Stage I seminiferous tubule cross-sections. However, the raw data from which this mean was calculated were corrected to compensate for the inclusion of nuclear fragments. The corrected number of spermatid nuclei per Stage I tubule cross-section was ± 3.7. Values for the 22 testes ranged from An analysis of variance revealed that differences associated with testis (left vs. right), area within the testis, testes within rabbits, and among the 22 testes were not significant (p > 0.05). However, there were differences (p < 0.01) among the 11 rabbits in the number of spermatid nuclei per Stage I cross-section. The major and minor diameters of these same Stage I seminiferous tubules were 217 ± 10 and 196 ± 2 p.. Previously, the minor seminiferous tubule diameter for these testes was found to be 182 ± 2 p. when randomly selected tubules of all stages were measured for the determination of tissue shrinkage. Differences between the two values for the minor diameter of seminiferous tubules in each testis were highly significant (p < O.Dl) when analyzed by a paired t test, but the values were correlated (r = 0.76, p < O.Dl). This difference in the diameter of the seminiferous tubules could reflect cyclic variation or may have resulted from the errors associated with the measurements. Daily Sperm Production. The DSP values calculated using each of the three formulas are compared with actual DSO and with other characteristics of the rabbits in Table 4. The means of 260, 239, 251 X 10 6 for DSP1, DSP2, and DSP3 were not significantly different. However, the mean DSO of 152 X 106 was highly significantly less (p < 0.01) than each of the three mean DSP values. The DSO represented 60 ± 4% of the DSP1, 65 ± 5% of the DSP2, and 61 ± 4% of the DSP3 values. Thus, about 40% of the sperm presumably produced were not obtained even with collection of two ejaculates every other day. The correlations between DSO and DSP were significant (see Table 5). However, neither DSO nor DSP was correlated with body

7 668 AMANN Vol. 21 TABLE 4. Comparisons of Daily Sperm Production Values Calculated by Different Methods with Daily Sperm Output and Other Characteristics Rabbit Body weight Testes Percentage Estimated DSP (10') Observed parenchyma seminiferous DSO, weight tubules weeks 47-54* DSP1 DSP, DSPs kg. gm. JOB t t Mean± S.E ± ± ± ± ± ± ± 11 * Includes sperm recovered by flushing artificial vagina. t Frequently refused to ejaculate during Weeks 47-54; ':alue represents best estimate of DSO based on data for Weeks TABLE 5. Correlation Coefficients DSP1 DSP, DSPs DSO Based on 11 rabbits Body weight Testes paren- 0.74t t 0.87t chyma weight DSO 0.60* 0.59* 0.75t Based on 22 testes Testes paren- 0.66t 0.44t o.8ot chyma weight DSP, 0.84t 0.69t DSPz 0.48t * Statistical significance, p < t Statistical significance, p < weight. The parenchymal weight of the testes was not significantly correlated to body weight of these mature rabbits (r = 0.41, p > 0.05), but was significantly correlated with DSO, DSP1, and DSP3. The relative volume of seminiferous tubules within the testes, a component used in calculating DSP1, was not significantly correlated with DSO (r = 0.34). The high correlation between the values for DSP 2 and DSP3 apparently resulted from the inclusion of the same values for testicular weight and the volume of Stage I spermatid nuclei in both estimates. As discussed above, there was little relation between the relative volume of round spermatid nuclei in Stage I and in Stages V-VIII. From Formulas 1, 2, and 3 and the correlations summarized in Table 5, it is apparent that weight of the testicular parenchyma was the main parameter underlying the magnitude of DSO and all estimates of DSP. Thus, expression of DSP on the basis of testicular weight should be informative because it eliminates the influence of testicular size. Values calculated by the DSPt, DSPz, and DSP3 methods were 43.7 ± 1.7, 40.4 ± 2.3, and 42.0 ± 1.4 X 10 6 sperm/day /gm. of testis parenchyma. On this basis, DSP3 values were correlated (p < 0.01) with those for DSP, (r = 0.69) and DSP2 (r = 0.63), but DSP1 and DSP2 values were not significantly correlated (r = 0.33) for the 22 testes. DISCUSSION Daily sperm production for these 22 rabbit testes did not differ significantly when calculated by three methods. Thus,

8 September 1970 THE MALE RABBIT 669 any of these methods might be expected to give equivalent results if used with testicular tissue from humans or other mammals. Selection of a method might be influenced by several factors. Determination of DSP3 is considerably easier and the values are at least as precise as those estimated for DSP2. It is not necessary to establish the stage of the cycle of the seminiferous epithelium for each spermatid included when determining the relative volume of all round spermatid nuclei. This saves time and eliminates a subjective decision. Swierstra26 also concluded that calculations using all round spermatids (DSP3) were preferable to those using Stage I spermatids or primary spermatocytes. It is simpler to obtain raw data used for calculating DSP3 than that needed for DSP1. In both methods, the shrinkage of tissue during histologic processing must be determined. Regardless of the method used, this requires an initial measurement on the fresh testicular tissue. As reported herein, knowledge of the actual thickness of the histologic sections eventually analyzed is imperative if accurate DSP1 values are to be calculated. Halh3n10 suggested using a methacrylate duplicate of the tissue section for measurements by interference microscopy. Such duplicates could be used if an interference microscope is not immediately available. Data on the relative frequency of round spermatids for use in DSP3 calculations are easy to obtain and are not influenced by thickness of the histologic section as are the more tedious counts of Stage I spermatids used in calculating DSP1. The duration of 1 cycle of the seminiferous epithelium, which must be known to calculate DSP, is established for humans and some other species. Even when this time factor was not available, these methods for quantifying testicular histology have been useful in studies of seasonal breeding animals17 and hormonal factors influencing spermatogenesis.8 The other values necessary to calculate either DSP1 or DSP3 can be obtained without difficulty. In some cases the amount of testicular tissue available may be a limiting factor in quantitative histologic analyses. The area of one or more tissue sections which must be evaluated to get a representative sample of the testis probably differs among species. For example, the seminiferous tubules in bull testes are highly convoluted and, thus, a small region of a single tubule appears several times in the same section.n This configuration necessitates evaluation of a larger area of bull testis than is true for sections of rat testis. These data suggest that the small volume of tissue available from a testicular biopsy may be insufficient for quantitative analyses. However, several workers have concluded that valid data of the type used to calculate DSP 3 can be obtained from good biopsy specimens. Alternative methods for determining DSP include enumeration of elongated spermatids in testicular homogenates.2 5 Values determined for rabbits by this approach are compared with the DSP values reported herein (Table 6). Similar data are not available for the 11 rabbits discussed in this report. The homogenate DSP values of about 25 X 10 6 sperm/day I gm. of testis reported by one group of workers are considerably lower than values of X 106 sperm/day jgm. similarly calculated by two others These latter values are in remarkable agreement with those determined histologically. Probably, values calculated from testicular homogenates or from quantitative testicular histology both are good estimates of DSP. Based on data for 114 of the animals included in Table 6, the DSP of a sexually mature4 New Zealand White rabbit was calculated to average 218 X 10 6 or 38.7 X 1Q6 sperm/day /gm. of testicular parenchyma. The DSP of rabbits may be influenced by illumination or season.19 The influence of external factors on the DSP

9 670 AMANN Vol. 21 TABLE 6. Comparisons among Reported Values for the Daily Sperm Production of Rabbits Based on testicular homogenates Based on testicular histology: Amann (present data) Lambiase and Orgebin- Characteristic Amann Crist Kirton Macmillan et al.* et a!. DSP, DSP, DSPa Experi- Experi- G~up Group ment 1 ment 2 B Swanson and Hafs Age (mos.) D-14 Unknown Unknown Testes parenchyma weight (gm.) t 5.22t Daily sperm production (106) DSP/gm. testis parenchyma (10') :i No. of rabbita The mean testicular spermatid counts reported were converted to DSP using the time divisor of 3.43 days.' The resulting DSP estimates are considered more accurate than the DSP values actually reported by these authors which, when reported, had been calculated using time base divisors of 4.93 and 5.44 days. t Calculated on the basis of 96.2% of total testicular weight. of other animals, including humans, should be evaluated. Other reports confirm that in rabbits the testes produce more sperm than can be recovered by frequent semen collections using an artificial vagina. About 40-50% of the sperm produced are not obtained. It is still uncertain whether under physiologic conditions these unaccounted for sperm are resorbed in the excurrent duct system/ 4 16 voided with urine 15 or eaten in a manner similar to coprophagy.24 The possibility that rabbit sperm are eliminated at irregular intervals via the urethra may explain the low correlation between DSP and DSO. In this study, DSP1 and DSP3 both were more highly correlated with testicular parenchyma weight than with DSO. Using the mean of the DSP1 and DSP3 values for each rabbit, the correlation coefficients were 0.81 and 0.69, respectively. Unfortunately, paired testicular parenchyma weight accounts for only 66% of the variation in sperm production among rabbits. Thus, even though testicular volume can be measured accurately in living rabbits,22 calculation of DSP by regression equation from testicular volume should be restricted to applications in which large differences are anticipated. Similarly, DSO did not prove to be a precise estimator for DSP of rabbits. Less than 48% of the variation in mean DSP was reflected by the DSO of these rabbits. Thus, with rabbits and boars27 DSO may not provide an accurate estimate of DSP as apparently is true for dairy bulls.3 Direct determinations of DSP for humans have not been reported, but accurate estimates probably could be made using biopsy specimens. SUMMARY This study was designed to determine the daily sperm production (DSP) of rabbits and to evaluate two histologic methods for determining DSP of mammalian testes. Data were obtained for 11 New Zealand White rabbits which had been ejaculated twice every second day from puberty until they were killed at 54 weeks of age. Daily sperm output (DSO), based on ejaculated semen, averaged 152 X 106 The tunica albuginea averaged 3.8% of the testis weight of 3.08 gm. whereas the mediastinum comprised about 1.2% of the testis volume. Quantitative histologic analyses revealed that after embedding in paraffin, the volume of testis tissue was only 42-87% of what it had been before fixation. Seminiferous tubules occupied 87% of the testis parenchyma and had a diameter of about 225 p. in fresh tissue. Determinations of the relative frequencies of the stages of the cycle of the seminiferous epithelium and of the relative volume occupied by

10 September 1970 THE MALE RABBIT 671 Stage I spermatids or by all round spermatids were highly repeatable by the same technician. For a typical rabbit, spherical spermatid nuclei occupied 5.7% and other seminiferous tubule components 78.0% of the testicular parenchyma. Each 3.5 p. cross-section through a Stage I tubule contained about 159 spermatid nuclei. Daily sperm production as calcuiated by each of three formulas was similar and the values averaged 250 X 106 or 42 x 10a sperm/day /gm. of testis parenchyma. Thus, the DSO was only 60% of DSP. Although DSP was significantly correlated with both DSO and testicular parenchyma weight, it was concluded that neither of these characteristics gave an accurate estimate of DSP in individual rabbits. If the thickness of histologic sections to be evaluated is actually measured, DSP can be calculated accurately from enumerations of Stage I spermatids. However, the histologic method for determining DSP based on the relative volume of all round spermatid nuclei probably is better because of its relative simplicity. Acknowledgments. Mrs. N. G. Borger provided skilled technical assistance. REFERENCES 1. AMANN, R. P. Reproductive capacity of dairy bulls. III. The effect of ejaculation frequency, unilateral vasectomy and age on spermatogenesis. Amer J Anat 110:49, AMANN, R. P., AND ALMQUIST, J. 0. 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 ALMQUIST, J. 0. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J Dairy Sci 45:774, AMANN, R. P., AND LAMBIASE, J. T., JR. The male rabbit. I. Changes in semen characteristics and sperm output between puberty and one year of age. J Reprod Fertil 14:329, AMANN, R. P., AND LAMBIASE, J. T., JR. The male rabbit. III. Determination of daily sperm production by means of testicular homagenates. J Anim Sci 28:369, BENEKE, G. "Application of Interference Microscopy to Biological Material." In Introduction to Quantitative Cytochemistry, Wied, G. L., Ed. Acad. Press, New York, 1966, p CHALKLEY, H. W. Method for the quantitative morphologic analysis of tissues. J Nat Cancer Inst 4:47, DESCLIN, J., AND 0RTAVANT, R. Influence des hormones gonadotropes sur la duree des processus spermatogenetiques chez le rat. Ann Biol Anim Biochem Biophys 3:329, FREUND, M. Effect of frequency of emission on semen output and an estimate of daily sperm production in man. J Reprod Fertil 6:269, HALLEN, 0. Quantitative analysis of sectioned biological material. J Histochem Cytochem 10:96, HocHEREAU, M. T. Constance des frequences relatives des stades du cycle de!'epithelium seminifere chez le taureau et chez le rat. Ann Biol Anim Biochem Biophys 3:93, KENNELLY, J. J., AND FooTE, R. H. Sampling boar testes to study spermatogenesis quantitatively and to predict sperm production. J Anim Sci 23:160, KIRTON, K. T., DESJARDINS, c., AND HAFS, H. D. Distribution of sperm in male rabbits after various ejaculation frequencies. Anat Rec 158:287, LAMBIASE, J. T., JR., AND AMANN, R. P. The male rabbit. V. Changes in sperm reserves and resorption rate induced by ejaculation and sexual rest. J Anim Sci 28:542, LINO, B. F., BRADEN, A. W. H., AND TuRN BULL, K. E. Fate of unejaculated spermatozoa. Nature (London) 213:594, MACMILLAN, K. L., DESJARDINS, C., KIRTON, K. T., AND HAFS, H. D. Gonadal and extragonadal sperm reserves after unilateral vasoligation in rabbits. Fertil Steril 19:982, MARTINET, L. Modification de la spermatogenese chez le campagnol des champs (Microtus arvalis) en fonction de la duree quotidienne d'eclarirement. Ann Biol Anim Biochem Biophys 6:301, McFEE, A. F., AND KENNELLY, J. J. Evaluation of a testicular biopsy technique in the rabbit. J Reprod Fertil8:141, RGEBIN-CRIST, M. C. Gonadal and epididymal sperm reserves in the rabbit: estimation of the daily sperm production. J Reprod Fertil15:15, PAUFLER, S. K., AND FooTE, R. H. Semen quality and testicular function in.rabbits following repeated testicular biopsy and unilateral castration. Fertil Steril 20:618, PAUFLER, S. K., AND FoOTE, R. H. Spermato-

11 672 AMANN Vol. 21 genesis m the rabbit following ligation of the epididymis at different levels. Anat Rec 164-: 339, PAUFLER, S. K., VANVLECK, L. D., AND FooTE, R. H. Estimation of testicular size in the live rabbit. Int J Fertil14-:188, RoosEN-RUNGE, E. C. Quantitative investigations on human testicular biopsies. I. Normal testis. Fertil Steril 7:251, STEINBERGER, E., AND TJIOE, D. Y. A method for quantitative analysis of human seminiferous epithelium. Fertil Steril19:960, SwANSON, L. V., AND HAFs, H. D. Testicular and epididymal sperm numbers in unilaterally vasoligated rabbits. Proc Soc Exp Biol M ed 131:763, SwrERSTRA, E. E. 'Structural composition of Shorthorn bull testes and daily sperm production as determined by quantitative testicular histology. Can J Anim Sci 4-6:107, SwrERSTRA, E. E. A comparison of spermatozoa production and spermatozoa output of Yorkshire and Lacombe boars. J Reprod Fertil 17:459, SwiERSTRA, E. E., AND FooTE, R. H. Cytology and kinetics of spermatogenesis in the rabbit. J Reprod Fertil5:309, SwrERSTRA, E. E., WHITEFIELD, J. W., AND FooTE, R. H. Action of amphotericin B (Fungizone) on spermatogenesis in the rabbit. J Reprod Fertil 7:13, VoaLMAYR, J. K., ScoTT, T. W., SETCHELL, B. P., AND WAITES, G. M. H. Metabolism of testicular spermatozoa and characteristics of testicular fluid collected from conscious rams. J Reprod Fertil14-:87, VoaLMAYR, J. K., WAITES, G. M. H., AND 'SETCHELL, B. P. Studies on spermatozoa and fluid collected directly from the testis of the conscious ram. Nature (London) 210:861, WHITE, I. G., VoaLMAYR, J. K., AND LARSEN, L. H. "Studies of Rete Testis Fluid Collected from the Living Bull." In Proceedings Sixth International Congress on Animal Reproduction and A. I., Thibault, C., Ed. Institut National de!a Recherche Agronomique, Paris, 1968, p. 79.