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Gonadal and Extragonadal Sperm Reserves after Unilateral Vasoligation in Rabbits
Gonadal and Extragonadal Sperm Reserves after Unilateral Vasoligation in Rabbits K. L. MACMILLAN, PH.D.,* C. DESJARDINS, PH.D.,t K. T. KIRTON, PH.D.,:!: and H. D. HAFS, PH.D.
after vasoligation has been a subject of considerable controversy. Bouin and Ancel pointed out the disparity of results of others with different species and observed that vasoligation resulted in degeneration of guinea-pig spermiogenic tissue. Steinach reported that vasectomy in rabbits induced an initial decline in spermatogenesis, but that partial recovery occurred 3 weeks later. Moore and Quick considered that their results in rabbits did not support either of these conclusions: they suggested that vasectomy reduced spermatogenesis only if the surgery altered the normal scrotal position of the testis. All the conclusions in these early reports were based on testicular histologic evidence. Amann estimated daily sperm production in vasectomized bulls and concluded that unilateral vasectomy did not alter sperm production in the testes on either side. His data led to the suggestion that the main site of bull sperm resorption was the cauda epididymidis. This is in agreement with Albert's proposal that resorption in fluid and sperm from the epididymis would prevent obstructive necrosis of the testis after vasoligation. The present experiment was designed to determine the effects of unilateral vasoligation on the gonadal and extragonadal sperm reserves of rabbits, and to determine whether the epididymis of this species is capable of resorbing sperm.
THE FATE OF SPERMATOGENESIS
MATERIAL AND METHODS
Under Surital anesthesia, a scrotal incision was made and the left deferent duct was ligated, excluding blood vessels, within 3 mm. of the From the Animal Reproduction Laboratory, Dl'partml'nt of nairy, Michigan State Uniwrsity, East Lansing, Mich. Approved by the Director of the Michigan Agricultural Experiml'nt Station, as Paper 4138. *Present address: New Zealand Dairy Board, Awahuri, Palmerston North, New Zealand. tPresent address: The Jackson Laboratory, Bar Harbor, Me . .;:NIH Predoctoral Fellow. Present address: The Upjohn Co., Kalamazoo, Mich.
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epididymis in each of 25 sexually mature rabbits. The rabbits were regularly examined throughout the experimental period to be certain that the testis remained in the scrotum without adhesions. At 1, 2, 3, 4, and 5 weeks after vasoligation, 5 rabbits were castrated. The testicular parenchymal tissue was weighed and homogenized in 50 mi. of 0.85% saline for 2 min. The caput-corpus and cauda epididymides were weighed and homogenized in 25 mi. and 50 mi. saline, respectively. Sperm numbers were determined hemacytometrically by previously reported procedures.;; Five rabbits were castrated as 0-day controls at the outset of the experiment and 10 rabbits were subjected to the entire surgical procedure except for the actual ligation. Five of the latter 10 rabbits were castrated 1 week and 5 were castrated 2 weeks later as sham-operated controls. The total number of sperm per testicular or epididymidal sample was calculated from the average of 4 hemacytometric counts, 2 made independently by each of 2 people. Analyses of variance compared differences between data obtained on the ligated left side and the unligated right side in the 25 rabbits which had been subjected to unilateral ligation. Orthogonal polynomial analyses were used to determine the nature of response trends with interval of vasoligation. Data from the control rabbits were analyzed to determine whether differences existed between left and right sides of the reproductive tract. Similar analyses of the data from the 1-week and 2-week sham-operated rabbits tested whether the surgery, apart from ligation, affected gonadal and extragonadal sperm reserves. RESULTS
Average weights for the right (control) and left (ligated) sides for testes and caput-corpus and cauda epididymides in each group of rabbits are presented in Table 1. Although ligation of the left deferent duct caused a small decline in average weight of left testes ( p < 0.05), the weight trends with duration of ligation within right or left sides were not significant. Neither did ligation affect significantly the average weight of the caput-corpus epididymides, but the weekly weight averages for both the ligated and normal sides showed a linear increase with duration of vasoligation ( p < 0.05). The most dramatic effect of ligation was on cauda epididymides, which were significantly heavier on the ligated side ( p < 0.01) where average weight increased linearly with duration of vasoligation ( p < 0.001 ) . Averages for total and concentration of testicular sperm (Table 2) re.
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TABLE 1. Weights of Testes, Caput-Corpus Epididymides, and Cauda Epididymides after Unilateral Vasoligation ( gm.) Epididymis Duration of ligation (wk.)
'Pestis
Cauda
Caput-corpus
Normal
Ligated
Normal
Ligated
Normal
Ligated
1.00 0.97 1.39 1.53 1.34 1.20
1.02 0.88 0.88 1.32 1.38 1.07
0.15 0.15 0.19 0.22 0.21 0.20
0.15 0.15 0.22 0.22 0.23 0.22
0.25 0.27 0.32 0.34 0.32 0.32
0.25 0.32 0.43 0.56 0.61 0.53
0 1 2 3 4 5
TABLE 2. Concentration of Testicular Sperm and Total Testicular Sperm after Unilateral Vasoligation (Mean ± S.E. ) Duration of ligation (wk.)
0 1 2 3
4 5
'l'otal (X 101 /testis)
Concentration (X 101 /gm.) Normal
Ligated
8.6 ± 0.8 8.4 ± 0.9 9.4 ± 0.8 7.9±0.5 9.2 ± 0.6 8.6 ± 0.4
8.5 ± 0.4 5.8 ± 1.4 3.3 ± 1.8 6.0 ± 1.5 7.2±0.9 6.5 ± 1.7
Normal
8.5 8.4 13.4 12.3 12.4 10.1
±0.9 ± 2.0 ± 2.8 ± 2.1 ± 1.0 ±0.7
Ligated
8.7 ± 1.1 5.9 ± 1.8 4.5 ± 2.7 9.5 ± 2.9 9.9 ± 1.3 7.4±2.0
vealed that vasoligation resulted in reduced sperm numbers on the ligated side (p < 0.001) during the first 2 weeks of ligation, but there appeared to be some recovery from the second to the fifth weeks as demonstrated by a significant quadratic response with duration of ligation (p """' 0.06). In contrast, average sperm concentration on. the right side showed no significant change with time. Concentration of testicular sperm was much less variable than total testicular sperm within any group of 5 rabbits, presumably because the total sperm criterion was affected by differences in the size of the rabbits and their testes. Therefore, concentration of testicular sperm is a more sensitive criterion for measurement of treatment differences. Vasoligation reduced the number of sperm in the caput-corpus epididymides (p < 0.05) after 2 weeks (Table 3). But, like testicular sperm, the caput-corpus epididymidal sperm recovered to a normal number after the second week (p < 0.01). The weekly averages on the unligated side did not change significantly with time. In contrast to the caput-corpus epi-
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TABLE 3. Sperm Numbers in Caput-Corpus and Cauda Epididymides after Unilateral Vasoligation ( X 107 ) Duration of ligation (wk.)
0 1 2 3 4 5 SE*
Caput-oorpus
Cauda
Total
Normal
Ligated
Normal
Ligated
Normal
Ligated
3.8 2.9 5.9 5.5 3.7 4.3 1.1
4.2 1.2 2.8 5.7 5.1 3.5 1.0
25.0 24.1 19.5 17.8 18.ti 15.8 4.1
23.5 28.4 43.5 67.7 83.2 63.8 11.4
28.8 27.0 25.5 23.3 22.3 20.1 4.7
27.6 29.6 46.3 73.2 88.2 67.3 11.6
• Standard errors for N = 5.
didymides, vasoligation produced a fourfold increase in numbers of sperm in cauda epididymides (p < 0.001), while cauda epididymides on the unligated side exhibited a linear decline in sperm numbers with time (p = 0.07). When the data in Table 3 were combined, for total epididymal sperm, unilateral ligation was associated with a significant increase in epididymal sperm numbers ( p < 0.001), while the normal side exhibited an insignificant reduction. Total epididymal sperm on the ligated side did not change appreciably during the first week of ligation, but tripled from the first to the fourth weeks and then declined to the fifth week, exhibiting a significant cubic response (p ~ 0.05). Data from rabbits after sham ligation of deferent ducts revealed no dramatic effect of this surgery. Of the seven criteria listed (Table 4), only the number of sperm in cauda epididymides 2 weeks later was significantly affected by the sham surgery ( p < 0.05), and the dimension of this response was much smaller than the changes exhibited in the vasoligated bucks (Table 3). DISCUSSION
Data from sham-operated bucks demonstrated that responses observed following vasoligation in the other bucks were not merely due to trauma of surgery, and consequently it was considered unnecessary to extend the sham-operational period beyond 2 weeks. Data from the sham-operated bucks and from the 0-week control group supported the conclusion that left and right halves of male rabbits' reproductive tracts do not differ appreciably.
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TABLE 4. Weights and Spenn Contents of Testis and Caput-Corpus and Cauda Epididymides after Sham Ligation of Left Deferent Duct Weeks of ligation 2
1
rriterion
Normal
Sham
Normal
Sham
Testis weight (gm.) Sperm/gm. testis (X 107) Testicular sperm (X 107 ) Caput-corpus weight (gm.) Caput-corpus sperm ( X107 ) Cauda weight (gm.) Cauda sperm ( X 107 )
1.8 12.4 21.6 0.2 4.6 0.4 18.9
1.8 10.4 18.6 0.3 4.9 0.4 20.2
1.4 12.8 18.4 0.2 8.8 0.4 30.3
1.5 12.2 18.0 0.~
9.4 0.4 34.8
Vasoligation caused a reduction in average testis weight within the first 2 weeks and cauda epididymides on the ligated side were visibly enlarged. This enlargement was linear with time of ligation, at least through 4 weeks, indicating that testicular contributions and epididymal secretions accumulated in this organ for at least 4 weeks after ligation. Moore and Quick used histologic observations to evaluate the effects of vasectomy on spermatogenesis and concluded that vasectomy did not arrest spermatogenesis. We have also observed mitotic divisions and spermatozoa in the lumina of seminiferous tubules at all periods after vasoligation. However, these histologic observations are difficult to quantify and do not preclude the possibility that the level of spermatogenesis may have been affected. Numbers of testicular sperm are more easily quantified and provide a good estimate of level of spermatogenesis. Either total testicular sperm or sperm per gram of testicular parenchymal tissue may be used as a response criterion, but results of this experiment reveal that the latter is preferable. It is less variable, apparently, because it is not influenced by size of testis. Similarly, Almquist and Amann reported that the concentration of testicular sperm was less variable than the total in bulls. Unilateral ligation of the deferent duct reduced testicular sperm concentration only on the ligated side. However, these changes in sperm concentration followed a quadratic response with duration of ligation, suggesting that, as with testis weight, the initial decline was followed by partial recovery. Longer intervals of ligation could have resulted in more complete recovery, because Moore and Quick reported that spermatogenesis was not disrupted even 6 months after va!!«;lCtomy. These workers were critical of the results of Steinach, who suggested that vasectomy resulted in degeneration of the testis followed by a regeneration 2 weeks later. Moore and
VoL. 19,No.6, 1968
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Quick inferred that this decline in spermatogenesis and the subsequent regeneration observed by Steinach was due to temporary retention of the testis in the peritoneal cavity. In the present experiment, the rabbits were regularly examined to ascertain that the testes remained in the scrotum. Thus, our results concur with those of Steinach. However, it should be noted that the degree of response to ligation varied considerably among rabbits. Whereas the epididymides on the vasoligated side contained increased sperm numbers, sperm numbers in the contralateral epididymides declined. On the ligated side, sperm numbers in the caput-corpus epididymides initially declined during the first week, increased from the first to the third week, and then declined again from the third to the fifth week. The initial decline was probably due to the reduction in testicular sperm at that time. However, as large numbers of sperm accumulated in the cauda epididymides, more were apparently retained in the caput-corpus region at the third week. The reduction in sperm numbers in the caput-corpus epididymidis during the final week was associated with a similar decline in the cauda epididymidis. The greatest accumulation of sperm in the cauda epididymidis occurred 4 weeks after ligation, when sperm numbers in this organ amounted to nearly 400% of those of the controls at 0 weeks. However, in terms of sperm per gram of caudal tissue, there was only a 50% increase, suggesting that the cauda epididymidis may accommodate the increased sperm numbers by growth as well as stretching. Superficially, the decline i:q,total epididymidal sperm from the fourth to fifth weeks of ligation seemed contradictory because it occurred at the same time that testicular sperm numbers (i.e., spermatogenesis) were returning toward normal levels. Since spermatogenesis was not arrested, the decline in total epididymidal and particularly in caudal sperm numbers was probably due to resorption of sperm from the cauda epididymides, as suggested by Orgebin-Crist. Almquist and Amann suggest that the cauda epididymidis is also the major site of sperm resorption in the bull. The unoperated right side of the reproductive tract could not be used as a control for the ligated left side because right-side epididymal sperm numbers were not maintained at constant levels. Average sperm content of the right cauda epididymides and sperm per gram of right caudal tissue declined 37% and 57%, respectively. These declines were not associated with any significant changes in the average weight of the cauda epididymides. Since ligation of the contralateral deferent duct failed to affect testicular sperm numbers (i.e., spermatogenesis) significantly on the unoperated side, sperm must have entered the unligated epididymis at a
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relatively constant rate-a conclusion which is supported by data on sperm content of caput-corpus epididymides on the unligated side. Therefore, the decline in total sperm content and sperm concentration in the cauda epididymides on the unligated side was due either to increased sperm resorption at that location or to increased loss of sperm via the deferent duct. The ratio between total epididymal sperm and total testicular sperm in sexually rested animals should reflect the sperm storage role of the epididymis relative to the supply of sperm from the testis. Changes in this ratio should reflect changes in losses of epididymal sperm, either due to resorption or via the deferent duct. The average ratios of cauda epididymidal sperm to testicular sperm on the unoperated right side (Fig. 1) declined significantly ( p < 0.005) with increasing duration of ligation on the left side. A similar curve for total ratios of epididymidal to testicular sperm possessed shape (slope) nearly identical to that in Fig. 1, indicating that the decline in the ratio involving total epididymal sperm was primarily due to changes in the sperm content of the cauda epididymides. The dramatic changes in epididymidal sperm numbers caused by ligation of the contralateral deferent duct reveal that unilateral ligation increases the loss of epididymidal sperm on the unligated side. We believe that this is the first demonstration of such an interaction between the right and left epididymides. Since sperm content of the deferent duct on the unligated side of the tract was not considered in this study, the decline in the ratios (Fig. 1) may have been due either to increased resorption of spe• from the cauda or to increased loss via the deferent duct. Immunologic assay of blood serums from similar animals for sperm-specific antibodies may help to resolve this question. ()
i:::
~ ;IE
~
3
Fig. 1. Ratios of caudal epididymidal to testicular sperm on unoperated side after unilateral ligation of deferent duct. Vertical bars represent standard errors.
Q; ~
5 ~
2
~ ...,'
~
!
I
Q
a ffi ~
§
0
o
I
2
3
4
5
~ WEEKS OF UNILATERAL LIGATION
VoL. 19, No.~6, 1968
SPERM RESERVES
989
Although the curve in Fig. 1 is statistically linear ( p < 0.05), the greatest change occurred between the first and second week after ligation. Thereafter, the declines were slight. Since there was little change in these ra.tios during the first week after ligation, the decreases apparent in Fig. 1 were probably not associated with surgical trauma. Rather, the large decrease in ratios of epididymidal to testicular sperm during the second week was associated with a large increase in caudal and total epididymidal sperm numbers on the ligated side. Subsequent changes in these averages on the ligated side had relatively little effect on epididymidal sperm loss on the normal side. If the ratios of epididymidal to testicular sperm had been influenced by the rate of spermatogenesis in the testis on the ligated side, the trend toward recovery of sperm numbers in this testis during the last 3 weeks should have been associated with concomitant recovery of the ratios in Fig. 1. The data support the conclusion that when sperm content of a cauda epididymidis is increased by about 50% by unilateral vasoligation, sperm losses from the cauda epididymidis on the unligated side are dramatically increased. Further increases in sperm numbers in the epididymidis on the ligated side have little effect on sperm losses from the contralateral side. SUMMARY
Testicular and epididymidal sperm numbers were determined in groups of 5 rabbits 1, 2, 3, 4, and 5 weeks after unilateral vasoligation. Epididymidal sperm numbers increased on the ligated side for the first 4 weeks and then declined during the fifth week. Testicular sperm numbers on the ligated side were significantly reduced although partial recovery occurred by the fifth week. Unilateral vasoligation did not affect testicular sperm numbers on the unoperated side. Sperm numbers in the cauda epididymidis on the ligated side increased fourfold during the 4 weeks after vasoligation. However, unilateral vasoligation caused a significant loss of sperm from the epididymidis on the unoperated side, particularly from the cauda epididymidis. Animal Reproduction Laboratory Department of Dairy Michigan State University East Lansing, Mich. 48823
REFERENCES I. ALBERT, A. "The Mammalian Testis." In Sex and Internal Secretions (ed. 3). Young, W. C., Ed. Williams & Wilkins, Baltimore, 1961, p. 323. 2. ALMQUIST, J. 0., and AMANN, R. P. Reproductive capacity of dairy bulls. II. Go-
990
3. 4.
5. 6. 7. 8.
MACMILLAN ET AL.
FERTILITY & STERILITY
nadal and extra gonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. ] Dai1·y Sci 44:1668, 1961. AMANN, H. P. Reproductive capacity of dairy bulls. III. The effect of ejaculation frequency, unilateral vasectomy, and age on spennatogenesis. Amer ] Anat 110: 49, 1962. BouiN, P., and ANCEL, P. Recherches sur les cellules interstitielles du testicule chez les mammiferes. Arch Zool Exper Gen 1:431, 1903. KIRTON, K. T., DESJARDINS, C., and HAFS, H. D. Distribution of sperm in male rabbits after various ejaculation frequencies. Anat Rec 158:281, 1967. MooRE, C. R., and QUicK, W. J. Properties of the gonads as controllers of somatic an!! psychical characteristics. VII. Vasectomy in the rabbit. Amer ] Anat 34:311, 1924. 0RGEBIN-CRIST, M. C. Delayed incorporation of thymidine 3 H in epithelial cells of the ductus epididymidis of the rabbit. ] Reprod Fertil 8:259, 1964. STEINACH, E. Verjungung durch experimentelle Neubelebung der altenden Pubertat sdriise. Wilhelm Roux'Arch Entwicklungsmech Organ 46:557, 1920.
after vasoligation has been a subject of considerable controversy. Bouin and Ancel pointed out the disparity of results of others with different species and observed that vasoligation resulted in degeneration of guinea-pig spermiogenic tissue. Steinach reported that vasectomy in rabbits induced an initial decline in spermatogenesis, but that partial recovery occurred 3 weeks later. Moore and Quick considered that their results in rabbits did not support either of these conclusions: they suggested that vasectomy reduced spermatogenesis only if the surgery altered the normal scrotal position of the testis. All the conclusions in these early reports were based on testicular histologic evidence. Amann estimated daily sperm production in vasectomized bulls and concluded that unilateral vasectomy did not alter sperm production in the testes on either side. His data led to the suggestion that the main site of bull sperm resorption was the cauda epididymidis. This is in agreement with Albert's proposal that resorption in fluid and sperm from the epididymis would prevent obstructive necrosis of the testis after vasoligation. The present experiment was designed to determine the effects of unilateral vasoligation on the gonadal and extragonadal sperm reserves of rabbits, and to determine whether the epididymis of this species is capable of resorbing sperm.
THE FATE OF SPERMATOGENESIS
MATERIAL AND METHODS
Under Surital anesthesia, a scrotal incision was made and the left deferent duct was ligated, excluding blood vessels, within 3 mm. of the From the Animal Reproduction Laboratory, Dl'partml'nt of nairy, Michigan State Uniwrsity, East Lansing, Mich. Approved by the Director of the Michigan Agricultural Experiml'nt Station, as Paper 4138. *Present address: New Zealand Dairy Board, Awahuri, Palmerston North, New Zealand. tPresent address: The Jackson Laboratory, Bar Harbor, Me . .;:NIH Predoctoral Fellow. Present address: The Upjohn Co., Kalamazoo, Mich.
982
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SPERM RESERVES
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epididymis in each of 25 sexually mature rabbits. The rabbits were regularly examined throughout the experimental period to be certain that the testis remained in the scrotum without adhesions. At 1, 2, 3, 4, and 5 weeks after vasoligation, 5 rabbits were castrated. The testicular parenchymal tissue was weighed and homogenized in 50 mi. of 0.85% saline for 2 min. The caput-corpus and cauda epididymides were weighed and homogenized in 25 mi. and 50 mi. saline, respectively. Sperm numbers were determined hemacytometrically by previously reported procedures.;; Five rabbits were castrated as 0-day controls at the outset of the experiment and 10 rabbits were subjected to the entire surgical procedure except for the actual ligation. Five of the latter 10 rabbits were castrated 1 week and 5 were castrated 2 weeks later as sham-operated controls. The total number of sperm per testicular or epididymidal sample was calculated from the average of 4 hemacytometric counts, 2 made independently by each of 2 people. Analyses of variance compared differences between data obtained on the ligated left side and the unligated right side in the 25 rabbits which had been subjected to unilateral ligation. Orthogonal polynomial analyses were used to determine the nature of response trends with interval of vasoligation. Data from the control rabbits were analyzed to determine whether differences existed between left and right sides of the reproductive tract. Similar analyses of the data from the 1-week and 2-week sham-operated rabbits tested whether the surgery, apart from ligation, affected gonadal and extragonadal sperm reserves. RESULTS
Average weights for the right (control) and left (ligated) sides for testes and caput-corpus and cauda epididymides in each group of rabbits are presented in Table 1. Although ligation of the left deferent duct caused a small decline in average weight of left testes ( p < 0.05), the weight trends with duration of ligation within right or left sides were not significant. Neither did ligation affect significantly the average weight of the caput-corpus epididymides, but the weekly weight averages for both the ligated and normal sides showed a linear increase with duration of vasoligation ( p < 0.05). The most dramatic effect of ligation was on cauda epididymides, which were significantly heavier on the ligated side ( p < 0.01) where average weight increased linearly with duration of vasoligation ( p < 0.001 ) . Averages for total and concentration of testicular sperm (Table 2) re.
984
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TABLE 1. Weights of Testes, Caput-Corpus Epididymides, and Cauda Epididymides after Unilateral Vasoligation ( gm.) Epididymis Duration of ligation (wk.)
'Pestis
Cauda
Caput-corpus
Normal
Ligated
Normal
Ligated
Normal
Ligated
1.00 0.97 1.39 1.53 1.34 1.20
1.02 0.88 0.88 1.32 1.38 1.07
0.15 0.15 0.19 0.22 0.21 0.20
0.15 0.15 0.22 0.22 0.23 0.22
0.25 0.27 0.32 0.34 0.32 0.32
0.25 0.32 0.43 0.56 0.61 0.53
0 1 2 3 4 5
TABLE 2. Concentration of Testicular Sperm and Total Testicular Sperm after Unilateral Vasoligation (Mean ± S.E. ) Duration of ligation (wk.)
0 1 2 3
4 5
'l'otal (X 101 /testis)
Concentration (X 101 /gm.) Normal
Ligated
8.6 ± 0.8 8.4 ± 0.9 9.4 ± 0.8 7.9±0.5 9.2 ± 0.6 8.6 ± 0.4
8.5 ± 0.4 5.8 ± 1.4 3.3 ± 1.8 6.0 ± 1.5 7.2±0.9 6.5 ± 1.7
Normal
8.5 8.4 13.4 12.3 12.4 10.1
±0.9 ± 2.0 ± 2.8 ± 2.1 ± 1.0 ±0.7
Ligated
8.7 ± 1.1 5.9 ± 1.8 4.5 ± 2.7 9.5 ± 2.9 9.9 ± 1.3 7.4±2.0
vealed that vasoligation resulted in reduced sperm numbers on the ligated side (p < 0.001) during the first 2 weeks of ligation, but there appeared to be some recovery from the second to the fifth weeks as demonstrated by a significant quadratic response with duration of ligation (p """' 0.06). In contrast, average sperm concentration on. the right side showed no significant change with time. Concentration of testicular sperm was much less variable than total testicular sperm within any group of 5 rabbits, presumably because the total sperm criterion was affected by differences in the size of the rabbits and their testes. Therefore, concentration of testicular sperm is a more sensitive criterion for measurement of treatment differences. Vasoligation reduced the number of sperm in the caput-corpus epididymides (p < 0.05) after 2 weeks (Table 3). But, like testicular sperm, the caput-corpus epididymidal sperm recovered to a normal number after the second week (p < 0.01). The weekly averages on the unligated side did not change significantly with time. In contrast to the caput-corpus epi-
VoL. 19, No. 6, 1968
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TABLE 3. Sperm Numbers in Caput-Corpus and Cauda Epididymides after Unilateral Vasoligation ( X 107 ) Duration of ligation (wk.)
0 1 2 3 4 5 SE*
Caput-oorpus
Cauda
Total
Normal
Ligated
Normal
Ligated
Normal
Ligated
3.8 2.9 5.9 5.5 3.7 4.3 1.1
4.2 1.2 2.8 5.7 5.1 3.5 1.0
25.0 24.1 19.5 17.8 18.ti 15.8 4.1
23.5 28.4 43.5 67.7 83.2 63.8 11.4
28.8 27.0 25.5 23.3 22.3 20.1 4.7
27.6 29.6 46.3 73.2 88.2 67.3 11.6
• Standard errors for N = 5.
didymides, vasoligation produced a fourfold increase in numbers of sperm in cauda epididymides (p < 0.001), while cauda epididymides on the unligated side exhibited a linear decline in sperm numbers with time (p = 0.07). When the data in Table 3 were combined, for total epididymal sperm, unilateral ligation was associated with a significant increase in epididymal sperm numbers ( p < 0.001), while the normal side exhibited an insignificant reduction. Total epididymal sperm on the ligated side did not change appreciably during the first week of ligation, but tripled from the first to the fourth weeks and then declined to the fifth week, exhibiting a significant cubic response (p ~ 0.05). Data from rabbits after sham ligation of deferent ducts revealed no dramatic effect of this surgery. Of the seven criteria listed (Table 4), only the number of sperm in cauda epididymides 2 weeks later was significantly affected by the sham surgery ( p < 0.05), and the dimension of this response was much smaller than the changes exhibited in the vasoligated bucks (Table 3). DISCUSSION
Data from sham-operated bucks demonstrated that responses observed following vasoligation in the other bucks were not merely due to trauma of surgery, and consequently it was considered unnecessary to extend the sham-operational period beyond 2 weeks. Data from the sham-operated bucks and from the 0-week control group supported the conclusion that left and right halves of male rabbits' reproductive tracts do not differ appreciably.
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TABLE 4. Weights and Spenn Contents of Testis and Caput-Corpus and Cauda Epididymides after Sham Ligation of Left Deferent Duct Weeks of ligation 2
1
rriterion
Normal
Sham
Normal
Sham
Testis weight (gm.) Sperm/gm. testis (X 107) Testicular sperm (X 107 ) Caput-corpus weight (gm.) Caput-corpus sperm ( X107 ) Cauda weight (gm.) Cauda sperm ( X 107 )
1.8 12.4 21.6 0.2 4.6 0.4 18.9
1.8 10.4 18.6 0.3 4.9 0.4 20.2
1.4 12.8 18.4 0.2 8.8 0.4 30.3
1.5 12.2 18.0 0.~
9.4 0.4 34.8
Vasoligation caused a reduction in average testis weight within the first 2 weeks and cauda epididymides on the ligated side were visibly enlarged. This enlargement was linear with time of ligation, at least through 4 weeks, indicating that testicular contributions and epididymal secretions accumulated in this organ for at least 4 weeks after ligation. Moore and Quick used histologic observations to evaluate the effects of vasectomy on spermatogenesis and concluded that vasectomy did not arrest spermatogenesis. We have also observed mitotic divisions and spermatozoa in the lumina of seminiferous tubules at all periods after vasoligation. However, these histologic observations are difficult to quantify and do not preclude the possibility that the level of spermatogenesis may have been affected. Numbers of testicular sperm are more easily quantified and provide a good estimate of level of spermatogenesis. Either total testicular sperm or sperm per gram of testicular parenchymal tissue may be used as a response criterion, but results of this experiment reveal that the latter is preferable. It is less variable, apparently, because it is not influenced by size of testis. Similarly, Almquist and Amann reported that the concentration of testicular sperm was less variable than the total in bulls. Unilateral ligation of the deferent duct reduced testicular sperm concentration only on the ligated side. However, these changes in sperm concentration followed a quadratic response with duration of ligation, suggesting that, as with testis weight, the initial decline was followed by partial recovery. Longer intervals of ligation could have resulted in more complete recovery, because Moore and Quick reported that spermatogenesis was not disrupted even 6 months after va!!«;lCtomy. These workers were critical of the results of Steinach, who suggested that vasectomy resulted in degeneration of the testis followed by a regeneration 2 weeks later. Moore and
VoL. 19,No.6, 1968
SPERM RESERVES
987
Quick inferred that this decline in spermatogenesis and the subsequent regeneration observed by Steinach was due to temporary retention of the testis in the peritoneal cavity. In the present experiment, the rabbits were regularly examined to ascertain that the testes remained in the scrotum. Thus, our results concur with those of Steinach. However, it should be noted that the degree of response to ligation varied considerably among rabbits. Whereas the epididymides on the vasoligated side contained increased sperm numbers, sperm numbers in the contralateral epididymides declined. On the ligated side, sperm numbers in the caput-corpus epididymides initially declined during the first week, increased from the first to the third week, and then declined again from the third to the fifth week. The initial decline was probably due to the reduction in testicular sperm at that time. However, as large numbers of sperm accumulated in the cauda epididymides, more were apparently retained in the caput-corpus region at the third week. The reduction in sperm numbers in the caput-corpus epididymidis during the final week was associated with a similar decline in the cauda epididymidis. The greatest accumulation of sperm in the cauda epididymidis occurred 4 weeks after ligation, when sperm numbers in this organ amounted to nearly 400% of those of the controls at 0 weeks. However, in terms of sperm per gram of caudal tissue, there was only a 50% increase, suggesting that the cauda epididymidis may accommodate the increased sperm numbers by growth as well as stretching. Superficially, the decline i:q,total epididymidal sperm from the fourth to fifth weeks of ligation seemed contradictory because it occurred at the same time that testicular sperm numbers (i.e., spermatogenesis) were returning toward normal levels. Since spermatogenesis was not arrested, the decline in total epididymidal and particularly in caudal sperm numbers was probably due to resorption of sperm from the cauda epididymides, as suggested by Orgebin-Crist. Almquist and Amann suggest that the cauda epididymidis is also the major site of sperm resorption in the bull. The unoperated right side of the reproductive tract could not be used as a control for the ligated left side because right-side epididymal sperm numbers were not maintained at constant levels. Average sperm content of the right cauda epididymides and sperm per gram of right caudal tissue declined 37% and 57%, respectively. These declines were not associated with any significant changes in the average weight of the cauda epididymides. Since ligation of the contralateral deferent duct failed to affect testicular sperm numbers (i.e., spermatogenesis) significantly on the unoperated side, sperm must have entered the unligated epididymis at a
988
MACMILLAN ET AL.
FERTILITY
& STERILITY
relatively constant rate-a conclusion which is supported by data on sperm content of caput-corpus epididymides on the unligated side. Therefore, the decline in total sperm content and sperm concentration in the cauda epididymides on the unligated side was due either to increased sperm resorption at that location or to increased loss of sperm via the deferent duct. The ratio between total epididymal sperm and total testicular sperm in sexually rested animals should reflect the sperm storage role of the epididymis relative to the supply of sperm from the testis. Changes in this ratio should reflect changes in losses of epididymal sperm, either due to resorption or via the deferent duct. The average ratios of cauda epididymidal sperm to testicular sperm on the unoperated right side (Fig. 1) declined significantly ( p < 0.005) with increasing duration of ligation on the left side. A similar curve for total ratios of epididymidal to testicular sperm possessed shape (slope) nearly identical to that in Fig. 1, indicating that the decline in the ratio involving total epididymal sperm was primarily due to changes in the sperm content of the cauda epididymides. The dramatic changes in epididymidal sperm numbers caused by ligation of the contralateral deferent duct reveal that unilateral ligation increases the loss of epididymidal sperm on the unligated side. We believe that this is the first demonstration of such an interaction between the right and left epididymides. Since sperm content of the deferent duct on the unligated side of the tract was not considered in this study, the decline in the ratios (Fig. 1) may have been due either to increased resorption of spe• from the cauda or to increased loss via the deferent duct. Immunologic assay of blood serums from similar animals for sperm-specific antibodies may help to resolve this question. ()
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Fig. 1. Ratios of caudal epididymidal to testicular sperm on unoperated side after unilateral ligation of deferent duct. Vertical bars represent standard errors.
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VoL. 19, No.~6, 1968
SPERM RESERVES
989
Although the curve in Fig. 1 is statistically linear ( p < 0.05), the greatest change occurred between the first and second week after ligation. Thereafter, the declines were slight. Since there was little change in these ra.tios during the first week after ligation, the decreases apparent in Fig. 1 were probably not associated with surgical trauma. Rather, the large decrease in ratios of epididymidal to testicular sperm during the second week was associated with a large increase in caudal and total epididymidal sperm numbers on the ligated side. Subsequent changes in these averages on the ligated side had relatively little effect on epididymidal sperm loss on the normal side. If the ratios of epididymidal to testicular sperm had been influenced by the rate of spermatogenesis in the testis on the ligated side, the trend toward recovery of sperm numbers in this testis during the last 3 weeks should have been associated with concomitant recovery of the ratios in Fig. 1. The data support the conclusion that when sperm content of a cauda epididymidis is increased by about 50% by unilateral vasoligation, sperm losses from the cauda epididymidis on the unligated side are dramatically increased. Further increases in sperm numbers in the epididymidis on the ligated side have little effect on sperm losses from the contralateral side. SUMMARY
Testicular and epididymidal sperm numbers were determined in groups of 5 rabbits 1, 2, 3, 4, and 5 weeks after unilateral vasoligation. Epididymidal sperm numbers increased on the ligated side for the first 4 weeks and then declined during the fifth week. Testicular sperm numbers on the ligated side were significantly reduced although partial recovery occurred by the fifth week. Unilateral vasoligation did not affect testicular sperm numbers on the unoperated side. Sperm numbers in the cauda epididymidis on the ligated side increased fourfold during the 4 weeks after vasoligation. However, unilateral vasoligation caused a significant loss of sperm from the epididymidis on the unoperated side, particularly from the cauda epididymidis. Animal Reproduction Laboratory Department of Dairy Michigan State University East Lansing, Mich. 48823
REFERENCES I. ALBERT, A. "The Mammalian Testis." In Sex and Internal Secretions (ed. 3). Young, W. C., Ed. Williams & Wilkins, Baltimore, 1961, p. 323. 2. ALMQUIST, J. 0., and AMANN, R. P. Reproductive capacity of dairy bulls. II. Go-
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3. 4.
5. 6. 7. 8.
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FERTILITY & STERILITY
nadal and extra gonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. ] Dai1·y Sci 44:1668, 1961. AMANN, H. P. Reproductive capacity of dairy bulls. III. The effect of ejaculation frequency, unilateral vasectomy, and age on spennatogenesis. Amer ] Anat 110: 49, 1962. BouiN, P., and ANCEL, P. Recherches sur les cellules interstitielles du testicule chez les mammiferes. Arch Zool Exper Gen 1:431, 1903. KIRTON, K. T., DESJARDINS, C., and HAFS, H. D. Distribution of sperm in male rabbits after various ejaculation frequencies. Anat Rec 158:281, 1967. MooRE, C. R., and QUicK, W. J. Properties of the gonads as controllers of somatic an!! psychical characteristics. VII. Vasectomy in the rabbit. Amer ] Anat 34:311, 1924. 0RGEBIN-CRIST, M. C. Delayed incorporation of thymidine 3 H in epithelial cells of the ductus epididymidis of the rabbit. ] Reprod Fertil 8:259, 1964. STEINACH, E. Verjungung durch experimentelle Neubelebung der altenden Pubertat sdriise. Wilhelm Roux'Arch Entwicklungsmech Organ 46:557, 1920.