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Retrograde flow of spermatozoa into the urinary bladder of boars during collection of semen by electroejaculation
Theriogenology
41:869-877,
1994
RETROGRADE FLOW OF SPERMATOZOA INTO THE URINARY BLADDER OF BOARS DURING COLLECTION OF SEMEN BY ELECTROEJACULATION
P. A. Martin,‘,’
M. P. Dooley,*
F. B. Hembrough*
and M.H.
Pineda*
‘Veterinary Medical Research Institute *Department of Veterinary Physiology and Pharmacology Iowa State University Ames, IA 50011 Received
for publication: Accepted:
June
28,
November
1993
19,
1993
ABSTRACT Boars that had a catheter implanted in the urinary bladder (n = 11) were used to determine the magnitude of retrograde flow of spermatozoa into the urinary bladder during electroejaculation. The overall mean (& SD) number of spermatozoa in the electroejaculate of boars was 22 f 20 x lo’, with a mean range for individual boars of The overall mean adjusted total number of spermatozoa in the 3 f 3to48 + 13x IO’. post-electroejaculation urine was 1.038 & 2.656 x 1 Og, and the mean percentage of retrograde flow of spermatozoa into the urinary bladder among boars ranged from 0% to 32.69%, with an overall mean percentage of retrograde flow of 7.51 & 17.82%. These findings indicate that in boars electroejaculation is associated with retrograde flow of spermatozoa into the bladder. Key words:
boar, electroejaculation,
retrograde
flow,
spermatozoa,
urine
INTRODUCTION Significant losses of spermatozoa due to retrograde flow into the urinary bladder occur during electroejaculation in nonanesthetized bulls 17), rams (I 6,18) and men (13), and during electroejaculation in anesthetized cats (4,6,1 I) and long-tailed macaques (20). Retrograde flow of spermatozoa into the urinary bladder also occurs during mating in cats (6). during collection of semen with an artificial vagina in cats (6) and rams (16). and during collection of semen by digital manipulation of the penis in dogs (5,l I). For these species, the retrograde flow of spermatozoa into the bladder during ejaculation or electroejaculation results in antegrade ejaculates that do not contain all of the spermatozoa that are displaced from the epididymides and vasa deferentia during seminal emission (4-7,11-13,16,18).
Acknowledgments The authors thank Ms. K. Adams, Ms. L. Peterson, technical assistance. This research was supported in part by Iowa State Grant No. 47 o-23-90-00-2394.
Copyright
0 1994 Butterworth-Heinemann
and Dr. S. Rodolfo-Sioson University
USDA
Formula
for their Fund
Theriogenology
870
The daily sperm output of boars approaches 100% of the daily sperm production - _. and the magnitude of retrograde fiow of spermatozoa into the biadder and postejaculatory retention of spermatozoa in the urethra were negligible when semen was collected by the gloved-hand method (15). Hence, it became of interest to determine whether retrograde flow of spermatozoa into the urinary bladder of boars occurred when semen was collected by electroejaculation. .--
(22-24),
MATERIALS
AND
METHODS
Eleven mature boars (3 Yorkshire: Boars 1, 3 and 10; 3 Duroc: Boars 2, 7 and 8; and 5 crossbred: Boars 4, 5, 6, 1 1 and 12) that had a catheter implanted surgically into the urinary bladder and had been used in a previous study (15) were sexually rested for 4 wk before the start of the present study. The mean (f SD) age and weight of these boars were 18 -C 2 mo and 198 f 28 kg, respectively. During the period of study, the boars were checked daily, and the catheters, which were filled with 12 ml of a solution of 0.1% were checked weekly for patency. Nitrofurazone,a Semen was collected by electroejaculation from each boar, once a week for 3 wk using an electroejaculator (3) that had an adjustable signal generator to control the voltage, slope, duration of each stimulus, and the interval between stimuli. A custom-made rectal probeb was used to apply electrical stimuli to the boar. Before collection of semen, the boars were placed in a farrowing crate, and 20 ml of fluid were withdrawn from the urinary catheter with a syringe and were discarded to eliminate remnants of the Nitrofurazone solution. With a clean syringe, 20 ml of pre-electroejaculation urine were then collected, and a sample of this urine was placed on a microscope slide and examined immediately at x 400 for the presence of motile spermatozoa. Spermatozoa displaying independent movement, including those with undulatory tail movements, were considered motile. To collect semen, each boar was anesthetized with an intramuscular injection of Telazol’ at a dose of 4 mg/kg body weight and returned to his pen before recumbency. Once the boar became recumbent, the prepuce was washed with tap water, cleaned of smegma, and dried with a paper towel. The tip of the penis was grasped with the aid of an atraumatic forcepsd and exteriorized. To facilitate the collection of semen, the penis was wrapped with surgical gauze within 6 inches from the tip and grasped firmly with one hand. Electroejaculation was induced by giving each boar a set of 5 series of 10 electrical stimuli for a total of 50 stimuli, using a sinusoidal wave form at 30 Hz. The first series of 10 stimuli was given at 2 V, the second series at 4 V, the third series at 6 V, the fourth series at 8 V, and the fifth series at IO V. As previously defined (3), the interval between stimuli was 10 set, the slope of stimulus application was 1.5 set, and the duration of each stimulus from 0 V to the corresponding voltage for the series and back to 0 V was 5 sec. Though not systematically recorded, spot measurements of current for the rectal probe ranged from 80 mA at 2 V to 380 mA at 10 V. The seminal fluid and spermatozoa obtained from a boar during the application of the set of 50 electrical stimuli was considered an electroejaculate. The volume and number of spermatozoa were determined
aTechAmerica Group, Inc., Elwood, KS. bBoar Probe, Catalog No. 25-l; manufactured by Lane Manufacturing, to specifications of Dr. M.P. Dooley. ‘A.H. Robins Company, Richmond, VA. dBozeman Uterine Dressing Forceps, 1 O-in: Arista, New York, NY.
Inc., Denver,
CO
871
Theriogenology
for each electroejaculate. The volume of each electroejaculate was measured at the time of collection with a graduated cylinder. The concentration of spermatozoa per milliliter of electroejaculate was determined with a hemacytometer, using diluted samples of semen. For this purpose, the number of spermatozoa within each central counting area of the 2 chambers of the hemacytometer was averaged (151. The volume of the electroejaculate was then multiplied by the average concentration of spermatozoa per milliliter of semen to determine the total number of spermatozoa in the electroejaculate. Immediately after electroejaculation, the bladder was emptied by withdrawing urine through the catheter while the boar was still recumbent. The post-electroejaculation urine was examined for the presence of motile spermatozoa, as described for the pre-electroejaculation urine. The volume of post-electroejaculation urine was measured with a graduated cylinder. The concentration of spermatozoa in undiluted samples of pre- and post-electroejaculation urine was determined, as described for the electroejaculate. If spermatozoa were not found in the duplicate samples, the urine was considered to be azoospermic (I 5). The adjusted concentration of spermatozoa in the post-electroejaculation urine was obtained by subtracting the spermatozoa1 concentration in the pre-electroejaculation urine from the spermatozoa1 concentration in the post-electroejaculation urine. The adjusted total number of spermatozoa in the post-electroejaculation urine was then determined by multiplying the volume of post-electroejaculation urine by the adjusted spermatozoa1 concentration per milliliter of urine. The total number of spermatozoa displaced from their sites of storage in the epididymides and vasa deferentia during electroejaculation was estimated by adding the adjusted total number of spermatozoa in the post-electroejaculation urine to the total number of spermatozoa in the electroejaculate. The percentage of spermatozoa displaced during electroejaculation that was found in the urine was then calculated for each boar and week and was considered to represent the percentage of retrograde flow of spermatozoa into the urinary bladder. Statistical
Analyses
Factorial analysis of variance (211 was used to determine the effects of boar and week of collection on seminal and urinary end points and on the percentage of retrograde flow of spermatozoa into the urinary bladder during electroejaculation. Factorial analysis of variance was also used to compare the spermatozoa1 concentration in the pre- and postelectroejaculation urine and to determine the interactions boar-by-urine collection and week-by-urine collection. Tukey’s w-procedure (211 was used to test for differences between means of end points for which the analysis of variance indicated a significant (P 5 0.05) F ratio. Chi-square analysis (21) was used to test for differences in the ratios of pre- and post-electroejaculation urine collections that had spermatozoa and for differences in the ratios of pre- and post-electroejaculation urine collections that had motile spermatozoa. RESULTS The concentration of spermatozoa in the pre-electroejaculation urine was affected (P < 0.05; Table 11 by boar but not by week of collection (P > 0.11. The concentration of spermatozoa in the post-electroejaculation urine was not affected (P > 0.1; Table 11 by boar or week of collection. The overall mean (+ SD1 spermatozoa1 concentration in the pre-electroejaculation urine (0.17 * 0.53 x 1 OB/ml; Table 11 was not different (P > 0.051 from the overall mean spermatozoa1 concentration in the post-electroejaculation urine (3.31 f 9.00 x 106/ml). However, there was a trend approaching significance
Theriogenology
872
Table
1.
Spermatozoa1 concentration in the urine of boars before and after electroejaculation, adjusted spermatozoa1 concentration, and volume post-electroejaculation urine
Spermatozoa1
Boar ID
Before electroejaculation
1 2
concentration
After electroejaculation
0= 0.31
f
(1 Oefml) in urine
Adjusted concentration
0
0.47a*b
of
0
Volume of postelectroejaculation urine (ml)
480
f
36ga
0.12
f
0.10
0.04
f
0.07
500
f
33ga
0.66
268
f
274a
3
0a
0.74
f
0.66
0.74
f
4
0a
0.35
f
0.55
0.35
+ 0.55
120
+ 18=
5
Oa
2.32
f
1.68
2.32
+
1.68
203
f
140a
3.92
+ 6.22
3.90
f
6.23
830
f
395a*b
295
f
171a
6
0.02
f
0.03a
7
0.05
& 0.09a
8
0.01
4 0.01a
+- 27.86
625
f
571ajb
10
0.12
f
0.15aJJ
2.82
+: 4.61
2.72
f
4.71
293
f
134a
11
1.36
f
1.30b
2.07
+- 1.28
0.71
f
0.61
1502
f
730b
12
0.01
*
0.01a
5.64
rt 5.31
5.63
f
5.31
263
f
126a
Overall mean * SD
0.17
4 0.53
3.31
f
3.16
f
9.02
489
f
484
Boar
0 18.40
f
0 27.87
9.00
18.40
< 0.05
> 0.1
> 0.1
< 0.005
> 0.1
> 0.1
> 0.1
> 0.1
effect Week effect Values are mean f SD; n = 3 weekly collections for each boar. For the overall means, n = 33 observations. There was a trend (P = 0.057) for an effect of urine collection on the spermatozoa1 concentration in the pre- and the post-electroejaculation urine, but the interactions boar-by-urine collection and week-by-urine collection were not significant (P > 0.1). For spermatozoa1 concentration in pre-ejaculation urine and for volume of postelectroejaculation urine, means in the same column which do not have a common superscript letter are different (P < 0.05).
873
Theriogenology
(P = 0.057; Table 1) for an effect of urine collection (pre- vs post-electroejaculation) on spermatozoa1 concentration in the urine and the interactions boar-by-urine collection and week-by-urine collection were not significant (P > 0.1). Of 33 pre-electroejaculation urine collections, 11 (33%) had spermatozoa (data for each weekly collection not shown in tables) and motile spermatozoa were detected in only 2 urine collections for Boar 1 1, whereas 26 of 33 post-electroejaculation urine collections (79%) had spermatozoa (data for each weekly collection not shown in tables) and motile spermatozoa were detected in 17 of the 26 post-electroejaculation urine collections (65%) that had spermatozoa. The ratios of pre- (11/33) and post-electroejaculation (26/33) urine collections that contained spermatozoa were different (P < 0.0005), and for those urine collections that contained spermatozoa, the ratios of pre- (2/l 1) and post-electroejaculation (17126) urine that contained motile spermatozoa were also different (P < 0.01). The adjusted spermatozoa1 concentration in the post-electroejaculation urine was not affected (P > 0.1; Table I) by boar or week of collection. The overall mean adjusted spermatozoa1 concentration in the urine was 3.16 f 9.02 x 100/ml. The volume of post-electroejaculation urine was affected by boar (P < 0.005; Table 1) but not by week of collection (P > 0.1). The mean volume of urine varied (P < 0.05) among boars and ranged from 120 * 18 ml for Boar 4 to 1,502 f 730 ml for Boar ? 1. The overall mean volume of post-electroejacuiation urine was 489 f. 484 ml. The volume of the electroejaculate was not affected (P > 0.1) by week of collection, but there was a trend approaching significance (P = 0.053; Table 2) for an effect of boar. The overall mean volume of electroejaculate was 114 + 45 ml. The total number of spermatozoa in the electroejaculate was not affected (P > 0.1; Table 2) by week of collection, but there was a trend (P = 0.0631 for an effect of boar. The overall mean total number of spermatozoa in the electroejaculate was 22 + 20 x IO’. The adjusted total number of spermatozoa in urine was not affected (P > 0.1; Table 2) by boar or week of collection. The overall mean adjusted total number of spermatozoa in the post-electroejaculation urine was 1.038 f 2.656 x 10’. The percentage of retrograde flow was not affected (P > 0.1; Table 2) by boar or week of collection, but there was considerable variation among and within boars in the percentage of retrograde flow of spermatozoa into the bladder as attested by the magnitude of the standard deviation, which for most boars exceeded that of the corresponding mean. The mean percentage of retrograde flow ranged from 0% for Boars 1 and 7 to 32.69 + 47.19% for Boar 11. The overall mean percentage of retrograde flow of spermatozoa into the bladder of anesthetized boars during electroejaculation was 7.51 f 17.82%. DISCUSSION Spermatozoa1 concentration in the post-electroejaculation urine was not different from that of the pm-electroejaculation urine. Failure to detect statistical significance is ascribed to the considerable variation among boars in spermatozoa1 concentration in the urine collected before and after electroejaculation, as evidenced by the magnitude of the standard deviations (Table I). An after the fact contrast analysis (19) confirmed the trend approaching significance (P = 0.057) for a higher spermatozoa1 concentration in the postelectroejaculation urine than in the pre-electroejaculation urine. The variation in spermatozoa1 concentration among boars was independent of time and urine collection because the interactions week-by-urine collection and boar-by-urine collection were not
Theriogenology
874
Table
2.
Volume of and total number of spermatozoa in the electroejaculate, adjusted total number of spermatozoa in the post-electroejaculation urine, and percentage of retrograde flow of spermatozoa into the bladder of boars
Electroejaculate
Boar ID
Volume
Total no. of spermatozoa
Adjusted total no. of spermatozoa in the post-electroejaculation urine
(ml)
(lo?
(lOS)
1
196
f
26
28
Z!Z26
2
107
f
50
25
f
3
99
Z!Z32
4
149
+ 49
39
+
0 0.008
0.087
f
0.054
16.19
19
0.044
rt 0.069
0.10
f
f
0.150
1.54
+ 0.47
f
7.704
13.74
103
+ 7
20
f
5
0.323
6
84
f
29
15 *
7
4.641
7
85
ZIZ20
14 *
7
f
28
8
111 98
11
124
12
96
Overall mean -i SD
114
41
* 0.01
f
5
10
0
0.01
0.004
9&S 44
Percentage of retrograde flow
0.16
+ 21.05
0
0
3.878
f
3.211
9.63
f
6.25
+ 68
3+3
0.380
+ 0.659
5.62
f
9.74
f
8&S
0.846
t- 0.826
32.69
t
47.19
49
j, 20
ZIZ26.67
+ 20
48
+
13
1.219
*
1.134
3.07
f
3.57
f
22
*
20
1.038
+ 2.656
7.51
f
17.82
45
Boar effect
= 0.053
= 0.063
> 0.1
> 0.1
Week effect
> 0.1
> 0.1
> 0.1
> 0.1
Values are mean * SD; n = 3 weekly n = 33 observations.
collections
for each boar.
For the overall
means,
Theriogenology
875
significant. Furthermore, there were more (P < 0.0005) post-electroejaculation urine collections (26/33) that had spermatozoa than for the pre-electroejaculation urine collections (1 l/33). In addition, there were more (P C 0.01) post-electroejaculation urine collections (17/26) that had motile spermatozoa than pre-electroejaculation urine collections (2/l 1) that had motile spermatozoa. These results indicate that additional retrograde flow of spermatozoa had indeed occurred after the obtainment of the preelectroejaculation urine and during the application of the electroejaculation regimen. However, there apparently were more boars and urine collections in the present study that had a higher spermatozoa1 concentration in the pre-electroejaculation urine than when semen was collected from the same boars by the gfoved-hand method (I 5). Speculatively, it is possible that the boars used in the present study learned to associate the presence of the operator and the herding of boars into the crate with the stimulation and ejaculation induced by the gloved-hand method and responded to that association with spermatozoa1 displacement to the urethra and bladder before collection of the pre-electroejaculation urine. In a previous study (I 5) on retrograde flow of spermatozoa into the bladder of boars during ejaculation induced by the gloved-hand method, we reported an overall mean percentage of retrograde flow of less than 0.2% and an overall mean percentage of total urinary losses of less than 0.5% of the total number of spermatozoa displaced during the preceding ejaculatory process. In the present study, when semen was collected by electroejaculation after induction of anesthesia from the same boars used in the previous study (151, the overall mean percentage of retrograde flow of spermatozoa into the bladder during electroejaculation was 7.51 + 17.82%, with a range of mean percentages of retrograde flow for individual boars from 0% (Boars 1 and 7) to 33% (Boar 11). Furthermore, for 6 of 11 boars, retrograde flow of spermatozoa into the urinary bladder exceeded 3% of the total number of spermatozoa displaced during the electroejaculatory process. These larger percentages-of retrograde flow for anesthetized boars suggest that collection of semen by electroejaculation is associated with increased urinary spermatozoa1 losses when compared with seminal collection by the gloved-hand method. The increase in the magnitude of retrograde flow of spermatozoa into the bladder may be a response to the electrical stimulation applied, to the anesthetic used, or to a combined response to these factors. The volumes of electroejaculates and numbers of spermatozoa in the electroejaculates obtained in the present study were within the normal ranges reported for boars (14), and compare well with the range of values that were obtained from the same boars during collection of semen by the gloved-hand method in a previous study (I 5). In addition, the number of spermatozoa in the electroejaculates obtained in the present study appears to be comparable to the values reported for the number of spermatozoa in boar semen collected by electroejaculation (1 ,I 0). Unfortunately, only a few studies in boars (1.8) provide some information regarding number or characteristics of the electrical stimulus, while others (2,9,26) provide no information other than to indicate that semen was collected by electroejaculation. Thus, we cannot comparatively evaluate the regimen for electroejaculation used in the present study with those used by others (I ,2,8-l 0,26). Hence, systematic studies to define the characteristics of the electrical stimulus and regimen of electroejaculation are needed, if this approach is to be reliably applied as an alternative to the gloved-hand method to collect semen for breeding soundness examination of untrained or untrainable boars.
Theriogenology
876
Although seminal collection by electroejaculation has been attempted in nonanesthetized boars (2,8), electroejaculation must be performed under general anesthesia because of the stressful response of unanesthetized boars to the electrical stimulation and to reduce the risk of injury to the boar and/or operator. General anesthesia with an intravenous injection of sodium thiamylal produced adequate immobilization of the boar (I ,2). However, thiamylal anesthesia requires intravenous injection which can be difficult to give and stressful to boars. Ketamine, an intramuscularly injectable anesthetic, was used to immobilize boars during electroejaculation (I 0,26), but ketamine-anesthetized boars required barbituric anesthetic supplementation to prevent injuries (26) because boars exhibited hyperexcitation during the induction and recovery periods. We used Telazol anesthesia in the present study because it appeared to be a reliable and safe intramuscular anesthetic for pigs (25). In our hands, a single IM injection of Telazol at the dose given, provided a level of anesthesia adequate to induce recumbency, allowed for retraction of the penis from the preputial sheath, and immobilized the boar for the duration of electroejaculation. Xylazine can be administered to boars after completion of electroejaculation (25) to smooth the recovery from Telazol anesthesia. In the present study, we did not use xylazine because xylazine induces retrograde flow of spermatozoa into the bladder of sexually-rested dogs (5,171 and rams (12). REFERENCES
5.
6.
7. 8. 9. IO. 11.
12.
Basurto-Kuba VM, Evans LE. Comparison of sperm-rich fractions of boar semen collected by electroejaculation and the gloved-hand technique. J Am Vet Med Assoc 1981;178:985-986. Clark TL. Electra-ejaculation in the anesthetized and non-anesthetized boar. In: Proceedings Int Pig Vet Sot Cong 1976;D14. Dooley MP, Murase K, Pineda MH. An electroejaculator for the collection of semen from the domestic cat. Theriogenology 1983;20:297-310. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of semen caused by electroejaculation in the domestic cat. In: Proceedings, 10th Int Cong Anim Reprod Artif lnsem 1984; Vol 3: Brief Comm No 363. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after sedation with xylazine. Am J Vet Res 1990;51:1574-1579. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of cats during electroejaculation, collection of semen with an artificial vagina, and mating. Am J Vet Res 1991;52:687-691. Dooley MP, Pineda MH, Maurer RR, Lunstra DD. Evidence for retrograde flow of spermatozoa into the urinary bladder of bulls during electroejaculation. Theriogenology 1986;26:101-109. Dziuk PJ, Graham EF, Donker JD, Marion GB, Petersen WE. Some observations in collection of semen from bulls, goats, boars and rams by electrical stimulation. Vet Med 1954;49:455-458. Evans LE. Electroeiaculation of the boar. In: Morrow DA fed). Current Theraov in Theriogenology 2. -Philadelphia, W.B. Saunders Company, 1986;1037-1040: ’ Evans LE, Ko JCH. Electroejaculation and artificial insemination in Vietnamese potbellied miniature pigs. J Am Vet Med Assoc 1990;197:1366-1367. Frenette MD, Dooley MP, Pineda MH. Effect of flushing the vasa deferentia at the time of vasectomy on the rate of clearance of spermatozoa from the ejaculates of dogs and cats. Am J Vet Res 1986;47:463-470. Hernandez FI, Dooley MP, Pineda MH. Effect of xylazine on retrograde flow of spermatozoa into the urinary bladder of rams. In: 1992 Beef and Sheep Research Report, Iowa State University, ASL-R938, 1992:160-l 64.
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14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26.
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Hirsh IH, Sedor J, Jeyendran RS, Stass WE. The relative distribution of viable sperm in the antegrade and retrograde portions of ejaculates obtained after electrostimulation. Fertil Steril 1992;57:399-401. Larsson K. Evaluation of boar semen. In: Morrow DA led), Current Therapy in Theriogenology 2. Philadelphia, W.B. Saunders Company, 1986;972-975. Martin PA, Dooley MP, Hembrough FB, Pineda MH. Urinary losses of spermatozoa during ejaculation are negligible in boars. Theriogenology 1993;39:945-964. Pineda MH, Dooley MP. Effect of method of seminal collection on the retrograde flow of spermatozoa into the urinary bladder of rams. Am J Vet Res 1991;52:307-313. Pineda MH, Dooley MP. Yohimbine prevents the retrograde flow of spermatozoa into the urinary bladder of dogs induced by xylazine. J Vet Pharmacol Therap, in orass, February 1994. Pineda MH, Dooley MP, Hembrough FB, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of rams. Am J Vet Res 1987;48:562-568. SAS User’s Guide: Statistics. SAS Institute Inc., Cary, NC, 1982;433-506. Schaffer NE, Cranfield M, Fazleabas AT, Jeyendran RS. Viable spermatozoa in the bladder after electroejaculation of lion-tailed macaques (Macaca silenus). J Reprod Fertil 1989;86:767-770. Steel RGD, Torrie JH. Principles and Procedures of Statistics. New York, McGraw-Hill Book Co, Inc. 1960; 109-l 10, 194-21 1, 370-373. 377-398. Swierstra EE. A comparison of spermatozoa production and spermatozoa output of Yorkshire and Lacombe boars. J Reprod Fertil 1968;17:459-469. Swierstra EE. The effect of low ambient temperatures on sperm production, epididymal sperm reserves, and semen characteristics of boars. Biol Reprod 1970;2:23-28. Swierstra EE. Sperm production of boars as measured from epididymal sperm reserves and quantitative testicular histology. J Reprod Fertil 1971:27:91-99. Thurmon JC, Benson GJ, Tranquilli WJ, Olson WA, Tracy CH. The anesthetic and analgesic effects of Telazol@ and xylazine in pigs: Evaluating clinical trials. Vet Med 1988;83:841-845. Thurmon JC, Nelson DR. Christie GJ. Ketamine anesthesia in swine. J Am Vet Med Assoc 1972:160:1325-1330.
41:869-877,
1994
RETROGRADE FLOW OF SPERMATOZOA INTO THE URINARY BLADDER OF BOARS DURING COLLECTION OF SEMEN BY ELECTROEJACULATION
P. A. Martin,‘,’
M. P. Dooley,*
F. B. Hembrough*
and M.H.
Pineda*
‘Veterinary Medical Research Institute *Department of Veterinary Physiology and Pharmacology Iowa State University Ames, IA 50011 Received
for publication: Accepted:
June
28,
November
1993
19,
1993
ABSTRACT Boars that had a catheter implanted in the urinary bladder (n = 11) were used to determine the magnitude of retrograde flow of spermatozoa into the urinary bladder during electroejaculation. The overall mean (& SD) number of spermatozoa in the electroejaculate of boars was 22 f 20 x lo’, with a mean range for individual boars of The overall mean adjusted total number of spermatozoa in the 3 f 3to48 + 13x IO’. post-electroejaculation urine was 1.038 & 2.656 x 1 Og, and the mean percentage of retrograde flow of spermatozoa into the urinary bladder among boars ranged from 0% to 32.69%, with an overall mean percentage of retrograde flow of 7.51 & 17.82%. These findings indicate that in boars electroejaculation is associated with retrograde flow of spermatozoa into the bladder. Key words:
boar, electroejaculation,
retrograde
flow,
spermatozoa,
urine
INTRODUCTION Significant losses of spermatozoa due to retrograde flow into the urinary bladder occur during electroejaculation in nonanesthetized bulls 17), rams (I 6,18) and men (13), and during electroejaculation in anesthetized cats (4,6,1 I) and long-tailed macaques (20). Retrograde flow of spermatozoa into the urinary bladder also occurs during mating in cats (6). during collection of semen with an artificial vagina in cats (6) and rams (16). and during collection of semen by digital manipulation of the penis in dogs (5,l I). For these species, the retrograde flow of spermatozoa into the bladder during ejaculation or electroejaculation results in antegrade ejaculates that do not contain all of the spermatozoa that are displaced from the epididymides and vasa deferentia during seminal emission (4-7,11-13,16,18).
Acknowledgments The authors thank Ms. K. Adams, Ms. L. Peterson, technical assistance. This research was supported in part by Iowa State Grant No. 47 o-23-90-00-2394.
Copyright
0 1994 Butterworth-Heinemann
and Dr. S. Rodolfo-Sioson University
USDA
Formula
for their Fund
Theriogenology
870
The daily sperm output of boars approaches 100% of the daily sperm production - _. and the magnitude of retrograde fiow of spermatozoa into the biadder and postejaculatory retention of spermatozoa in the urethra were negligible when semen was collected by the gloved-hand method (15). Hence, it became of interest to determine whether retrograde flow of spermatozoa into the urinary bladder of boars occurred when semen was collected by electroejaculation. .--
(22-24),
MATERIALS
AND
METHODS
Eleven mature boars (3 Yorkshire: Boars 1, 3 and 10; 3 Duroc: Boars 2, 7 and 8; and 5 crossbred: Boars 4, 5, 6, 1 1 and 12) that had a catheter implanted surgically into the urinary bladder and had been used in a previous study (15) were sexually rested for 4 wk before the start of the present study. The mean (f SD) age and weight of these boars were 18 -C 2 mo and 198 f 28 kg, respectively. During the period of study, the boars were checked daily, and the catheters, which were filled with 12 ml of a solution of 0.1% were checked weekly for patency. Nitrofurazone,a Semen was collected by electroejaculation from each boar, once a week for 3 wk using an electroejaculator (3) that had an adjustable signal generator to control the voltage, slope, duration of each stimulus, and the interval between stimuli. A custom-made rectal probeb was used to apply electrical stimuli to the boar. Before collection of semen, the boars were placed in a farrowing crate, and 20 ml of fluid were withdrawn from the urinary catheter with a syringe and were discarded to eliminate remnants of the Nitrofurazone solution. With a clean syringe, 20 ml of pre-electroejaculation urine were then collected, and a sample of this urine was placed on a microscope slide and examined immediately at x 400 for the presence of motile spermatozoa. Spermatozoa displaying independent movement, including those with undulatory tail movements, were considered motile. To collect semen, each boar was anesthetized with an intramuscular injection of Telazol’ at a dose of 4 mg/kg body weight and returned to his pen before recumbency. Once the boar became recumbent, the prepuce was washed with tap water, cleaned of smegma, and dried with a paper towel. The tip of the penis was grasped with the aid of an atraumatic forcepsd and exteriorized. To facilitate the collection of semen, the penis was wrapped with surgical gauze within 6 inches from the tip and grasped firmly with one hand. Electroejaculation was induced by giving each boar a set of 5 series of 10 electrical stimuli for a total of 50 stimuli, using a sinusoidal wave form at 30 Hz. The first series of 10 stimuli was given at 2 V, the second series at 4 V, the third series at 6 V, the fourth series at 8 V, and the fifth series at IO V. As previously defined (3), the interval between stimuli was 10 set, the slope of stimulus application was 1.5 set, and the duration of each stimulus from 0 V to the corresponding voltage for the series and back to 0 V was 5 sec. Though not systematically recorded, spot measurements of current for the rectal probe ranged from 80 mA at 2 V to 380 mA at 10 V. The seminal fluid and spermatozoa obtained from a boar during the application of the set of 50 electrical stimuli was considered an electroejaculate. The volume and number of spermatozoa were determined
aTechAmerica Group, Inc., Elwood, KS. bBoar Probe, Catalog No. 25-l; manufactured by Lane Manufacturing, to specifications of Dr. M.P. Dooley. ‘A.H. Robins Company, Richmond, VA. dBozeman Uterine Dressing Forceps, 1 O-in: Arista, New York, NY.
Inc., Denver,
CO
871
Theriogenology
for each electroejaculate. The volume of each electroejaculate was measured at the time of collection with a graduated cylinder. The concentration of spermatozoa per milliliter of electroejaculate was determined with a hemacytometer, using diluted samples of semen. For this purpose, the number of spermatozoa within each central counting area of the 2 chambers of the hemacytometer was averaged (151. The volume of the electroejaculate was then multiplied by the average concentration of spermatozoa per milliliter of semen to determine the total number of spermatozoa in the electroejaculate. Immediately after electroejaculation, the bladder was emptied by withdrawing urine through the catheter while the boar was still recumbent. The post-electroejaculation urine was examined for the presence of motile spermatozoa, as described for the pre-electroejaculation urine. The volume of post-electroejaculation urine was measured with a graduated cylinder. The concentration of spermatozoa in undiluted samples of pre- and post-electroejaculation urine was determined, as described for the electroejaculate. If spermatozoa were not found in the duplicate samples, the urine was considered to be azoospermic (I 5). The adjusted concentration of spermatozoa in the post-electroejaculation urine was obtained by subtracting the spermatozoa1 concentration in the pre-electroejaculation urine from the spermatozoa1 concentration in the post-electroejaculation urine. The adjusted total number of spermatozoa in the post-electroejaculation urine was then determined by multiplying the volume of post-electroejaculation urine by the adjusted spermatozoa1 concentration per milliliter of urine. The total number of spermatozoa displaced from their sites of storage in the epididymides and vasa deferentia during electroejaculation was estimated by adding the adjusted total number of spermatozoa in the post-electroejaculation urine to the total number of spermatozoa in the electroejaculate. The percentage of spermatozoa displaced during electroejaculation that was found in the urine was then calculated for each boar and week and was considered to represent the percentage of retrograde flow of spermatozoa into the urinary bladder. Statistical
Analyses
Factorial analysis of variance (211 was used to determine the effects of boar and week of collection on seminal and urinary end points and on the percentage of retrograde flow of spermatozoa into the urinary bladder during electroejaculation. Factorial analysis of variance was also used to compare the spermatozoa1 concentration in the pre- and postelectroejaculation urine and to determine the interactions boar-by-urine collection and week-by-urine collection. Tukey’s w-procedure (211 was used to test for differences between means of end points for which the analysis of variance indicated a significant (P 5 0.05) F ratio. Chi-square analysis (21) was used to test for differences in the ratios of pre- and post-electroejaculation urine collections that had spermatozoa and for differences in the ratios of pre- and post-electroejaculation urine collections that had motile spermatozoa. RESULTS The concentration of spermatozoa in the pre-electroejaculation urine was affected (P < 0.05; Table 11 by boar but not by week of collection (P > 0.11. The concentration of spermatozoa in the post-electroejaculation urine was not affected (P > 0.1; Table 11 by boar or week of collection. The overall mean (+ SD1 spermatozoa1 concentration in the pre-electroejaculation urine (0.17 * 0.53 x 1 OB/ml; Table 11 was not different (P > 0.051 from the overall mean spermatozoa1 concentration in the post-electroejaculation urine (3.31 f 9.00 x 106/ml). However, there was a trend approaching significance
Theriogenology
872
Table
1.
Spermatozoa1 concentration in the urine of boars before and after electroejaculation, adjusted spermatozoa1 concentration, and volume post-electroejaculation urine
Spermatozoa1
Boar ID
Before electroejaculation
1 2
concentration
After electroejaculation
0= 0.31
f
(1 Oefml) in urine
Adjusted concentration
0
0.47a*b
of
0
Volume of postelectroejaculation urine (ml)
480
f
36ga
0.12
f
0.10
0.04
f
0.07
500
f
33ga
0.66
268
f
274a
3
0a
0.74
f
0.66
0.74
f
4
0a
0.35
f
0.55
0.35
+ 0.55
120
+ 18=
5
Oa
2.32
f
1.68
2.32
+
1.68
203
f
140a
3.92
+ 6.22
3.90
f
6.23
830
f
395a*b
295
f
171a
6
0.02
f
0.03a
7
0.05
& 0.09a
8
0.01
4 0.01a
+- 27.86
625
f
571ajb
10
0.12
f
0.15aJJ
2.82
+: 4.61
2.72
f
4.71
293
f
134a
11
1.36
f
1.30b
2.07
+- 1.28
0.71
f
0.61
1502
f
730b
12
0.01
*
0.01a
5.64
rt 5.31
5.63
f
5.31
263
f
126a
Overall mean * SD
0.17
4 0.53
3.31
f
3.16
f
9.02
489
f
484
Boar
0 18.40
f
0 27.87
9.00
18.40
< 0.05
> 0.1
> 0.1
< 0.005
> 0.1
> 0.1
> 0.1
> 0.1
effect Week effect Values are mean f SD; n = 3 weekly collections for each boar. For the overall means, n = 33 observations. There was a trend (P = 0.057) for an effect of urine collection on the spermatozoa1 concentration in the pre- and the post-electroejaculation urine, but the interactions boar-by-urine collection and week-by-urine collection were not significant (P > 0.1). For spermatozoa1 concentration in pre-ejaculation urine and for volume of postelectroejaculation urine, means in the same column which do not have a common superscript letter are different (P < 0.05).
873
Theriogenology
(P = 0.057; Table 1) for an effect of urine collection (pre- vs post-electroejaculation) on spermatozoa1 concentration in the urine and the interactions boar-by-urine collection and week-by-urine collection were not significant (P > 0.1). Of 33 pre-electroejaculation urine collections, 11 (33%) had spermatozoa (data for each weekly collection not shown in tables) and motile spermatozoa were detected in only 2 urine collections for Boar 1 1, whereas 26 of 33 post-electroejaculation urine collections (79%) had spermatozoa (data for each weekly collection not shown in tables) and motile spermatozoa were detected in 17 of the 26 post-electroejaculation urine collections (65%) that had spermatozoa. The ratios of pre- (11/33) and post-electroejaculation (26/33) urine collections that contained spermatozoa were different (P < 0.0005), and for those urine collections that contained spermatozoa, the ratios of pre- (2/l 1) and post-electroejaculation (17126) urine that contained motile spermatozoa were also different (P < 0.01). The adjusted spermatozoa1 concentration in the post-electroejaculation urine was not affected (P > 0.1; Table I) by boar or week of collection. The overall mean adjusted spermatozoa1 concentration in the urine was 3.16 f 9.02 x 100/ml. The volume of post-electroejaculation urine was affected by boar (P < 0.005; Table 1) but not by week of collection (P > 0.1). The mean volume of urine varied (P < 0.05) among boars and ranged from 120 * 18 ml for Boar 4 to 1,502 f 730 ml for Boar ? 1. The overall mean volume of post-electroejacuiation urine was 489 f. 484 ml. The volume of the electroejaculate was not affected (P > 0.1) by week of collection, but there was a trend approaching significance (P = 0.053; Table 2) for an effect of boar. The overall mean volume of electroejaculate was 114 + 45 ml. The total number of spermatozoa in the electroejaculate was not affected (P > 0.1; Table 2) by week of collection, but there was a trend (P = 0.0631 for an effect of boar. The overall mean total number of spermatozoa in the electroejaculate was 22 + 20 x IO’. The adjusted total number of spermatozoa in urine was not affected (P > 0.1; Table 2) by boar or week of collection. The overall mean adjusted total number of spermatozoa in the post-electroejaculation urine was 1.038 f 2.656 x 10’. The percentage of retrograde flow was not affected (P > 0.1; Table 2) by boar or week of collection, but there was considerable variation among and within boars in the percentage of retrograde flow of spermatozoa into the bladder as attested by the magnitude of the standard deviation, which for most boars exceeded that of the corresponding mean. The mean percentage of retrograde flow ranged from 0% for Boars 1 and 7 to 32.69 + 47.19% for Boar 11. The overall mean percentage of retrograde flow of spermatozoa into the bladder of anesthetized boars during electroejaculation was 7.51 f 17.82%. DISCUSSION Spermatozoa1 concentration in the post-electroejaculation urine was not different from that of the pm-electroejaculation urine. Failure to detect statistical significance is ascribed to the considerable variation among boars in spermatozoa1 concentration in the urine collected before and after electroejaculation, as evidenced by the magnitude of the standard deviations (Table I). An after the fact contrast analysis (19) confirmed the trend approaching significance (P = 0.057) for a higher spermatozoa1 concentration in the postelectroejaculation urine than in the pre-electroejaculation urine. The variation in spermatozoa1 concentration among boars was independent of time and urine collection because the interactions week-by-urine collection and boar-by-urine collection were not
Theriogenology
874
Table
2.
Volume of and total number of spermatozoa in the electroejaculate, adjusted total number of spermatozoa in the post-electroejaculation urine, and percentage of retrograde flow of spermatozoa into the bladder of boars
Electroejaculate
Boar ID
Volume
Total no. of spermatozoa
Adjusted total no. of spermatozoa in the post-electroejaculation urine
(ml)
(lo?
(lOS)
1
196
f
26
28
Z!Z26
2
107
f
50
25
f
3
99
Z!Z32
4
149
+ 49
39
+
0 0.008
0.087
f
0.054
16.19
19
0.044
rt 0.069
0.10
f
f
0.150
1.54
+ 0.47
f
7.704
13.74
103
+ 7
20
f
5
0.323
6
84
f
29
15 *
7
4.641
7
85
ZIZ20
14 *
7
f
28
8
111 98
11
124
12
96
Overall mean -i SD
114
41
* 0.01
f
5
10
0
0.01
0.004
9&S 44
Percentage of retrograde flow
0.16
+ 21.05
0
0
3.878
f
3.211
9.63
f
6.25
+ 68
3+3
0.380
+ 0.659
5.62
f
9.74
f
8&S
0.846
t- 0.826
32.69
t
47.19
49
j, 20
ZIZ26.67
+ 20
48
+
13
1.219
*
1.134
3.07
f
3.57
f
22
*
20
1.038
+ 2.656
7.51
f
17.82
45
Boar effect
= 0.053
= 0.063
> 0.1
> 0.1
Week effect
> 0.1
> 0.1
> 0.1
> 0.1
Values are mean * SD; n = 3 weekly n = 33 observations.
collections
for each boar.
For the overall
means,
Theriogenology
875
significant. Furthermore, there were more (P < 0.0005) post-electroejaculation urine collections (26/33) that had spermatozoa than for the pre-electroejaculation urine collections (1 l/33). In addition, there were more (P C 0.01) post-electroejaculation urine collections (17/26) that had motile spermatozoa than pre-electroejaculation urine collections (2/l 1) that had motile spermatozoa. These results indicate that additional retrograde flow of spermatozoa had indeed occurred after the obtainment of the preelectroejaculation urine and during the application of the electroejaculation regimen. However, there apparently were more boars and urine collections in the present study that had a higher spermatozoa1 concentration in the pre-electroejaculation urine than when semen was collected from the same boars by the gfoved-hand method (I 5). Speculatively, it is possible that the boars used in the present study learned to associate the presence of the operator and the herding of boars into the crate with the stimulation and ejaculation induced by the gloved-hand method and responded to that association with spermatozoa1 displacement to the urethra and bladder before collection of the pre-electroejaculation urine. In a previous study (I 5) on retrograde flow of spermatozoa into the bladder of boars during ejaculation induced by the gloved-hand method, we reported an overall mean percentage of retrograde flow of less than 0.2% and an overall mean percentage of total urinary losses of less than 0.5% of the total number of spermatozoa displaced during the preceding ejaculatory process. In the present study, when semen was collected by electroejaculation after induction of anesthesia from the same boars used in the previous study (151, the overall mean percentage of retrograde flow of spermatozoa into the bladder during electroejaculation was 7.51 + 17.82%, with a range of mean percentages of retrograde flow for individual boars from 0% (Boars 1 and 7) to 33% (Boar 11). Furthermore, for 6 of 11 boars, retrograde flow of spermatozoa into the urinary bladder exceeded 3% of the total number of spermatozoa displaced during the electroejaculatory process. These larger percentages-of retrograde flow for anesthetized boars suggest that collection of semen by electroejaculation is associated with increased urinary spermatozoa1 losses when compared with seminal collection by the gloved-hand method. The increase in the magnitude of retrograde flow of spermatozoa into the bladder may be a response to the electrical stimulation applied, to the anesthetic used, or to a combined response to these factors. The volumes of electroejaculates and numbers of spermatozoa in the electroejaculates obtained in the present study were within the normal ranges reported for boars (14), and compare well with the range of values that were obtained from the same boars during collection of semen by the gloved-hand method in a previous study (I 5). In addition, the number of spermatozoa in the electroejaculates obtained in the present study appears to be comparable to the values reported for the number of spermatozoa in boar semen collected by electroejaculation (1 ,I 0). Unfortunately, only a few studies in boars (1.8) provide some information regarding number or characteristics of the electrical stimulus, while others (2,9,26) provide no information other than to indicate that semen was collected by electroejaculation. Thus, we cannot comparatively evaluate the regimen for electroejaculation used in the present study with those used by others (I ,2,8-l 0,26). Hence, systematic studies to define the characteristics of the electrical stimulus and regimen of electroejaculation are needed, if this approach is to be reliably applied as an alternative to the gloved-hand method to collect semen for breeding soundness examination of untrained or untrainable boars.
Theriogenology
876
Although seminal collection by electroejaculation has been attempted in nonanesthetized boars (2,8), electroejaculation must be performed under general anesthesia because of the stressful response of unanesthetized boars to the electrical stimulation and to reduce the risk of injury to the boar and/or operator. General anesthesia with an intravenous injection of sodium thiamylal produced adequate immobilization of the boar (I ,2). However, thiamylal anesthesia requires intravenous injection which can be difficult to give and stressful to boars. Ketamine, an intramuscularly injectable anesthetic, was used to immobilize boars during electroejaculation (I 0,26), but ketamine-anesthetized boars required barbituric anesthetic supplementation to prevent injuries (26) because boars exhibited hyperexcitation during the induction and recovery periods. We used Telazol anesthesia in the present study because it appeared to be a reliable and safe intramuscular anesthetic for pigs (25). In our hands, a single IM injection of Telazol at the dose given, provided a level of anesthesia adequate to induce recumbency, allowed for retraction of the penis from the preputial sheath, and immobilized the boar for the duration of electroejaculation. Xylazine can be administered to boars after completion of electroejaculation (25) to smooth the recovery from Telazol anesthesia. In the present study, we did not use xylazine because xylazine induces retrograde flow of spermatozoa into the bladder of sexually-rested dogs (5,171 and rams (12). REFERENCES
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6.
7. 8. 9. IO. 11.
12.
Basurto-Kuba VM, Evans LE. Comparison of sperm-rich fractions of boar semen collected by electroejaculation and the gloved-hand technique. J Am Vet Med Assoc 1981;178:985-986. Clark TL. Electra-ejaculation in the anesthetized and non-anesthetized boar. In: Proceedings Int Pig Vet Sot Cong 1976;D14. Dooley MP, Murase K, Pineda MH. An electroejaculator for the collection of semen from the domestic cat. Theriogenology 1983;20:297-310. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of semen caused by electroejaculation in the domestic cat. In: Proceedings, 10th Int Cong Anim Reprod Artif lnsem 1984; Vol 3: Brief Comm No 363. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of dogs during ejaculation or after sedation with xylazine. Am J Vet Res 1990;51:1574-1579. Dooley MP, Pineda MH, Hopper JG, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of cats during electroejaculation, collection of semen with an artificial vagina, and mating. Am J Vet Res 1991;52:687-691. Dooley MP, Pineda MH, Maurer RR, Lunstra DD. Evidence for retrograde flow of spermatozoa into the urinary bladder of bulls during electroejaculation. Theriogenology 1986;26:101-109. Dziuk PJ, Graham EF, Donker JD, Marion GB, Petersen WE. Some observations in collection of semen from bulls, goats, boars and rams by electrical stimulation. Vet Med 1954;49:455-458. Evans LE. Electroeiaculation of the boar. In: Morrow DA fed). Current Theraov in Theriogenology 2. -Philadelphia, W.B. Saunders Company, 1986;1037-1040: ’ Evans LE, Ko JCH. Electroejaculation and artificial insemination in Vietnamese potbellied miniature pigs. J Am Vet Med Assoc 1990;197:1366-1367. Frenette MD, Dooley MP, Pineda MH. Effect of flushing the vasa deferentia at the time of vasectomy on the rate of clearance of spermatozoa from the ejaculates of dogs and cats. Am J Vet Res 1986;47:463-470. Hernandez FI, Dooley MP, Pineda MH. Effect of xylazine on retrograde flow of spermatozoa into the urinary bladder of rams. In: 1992 Beef and Sheep Research Report, Iowa State University, ASL-R938, 1992:160-l 64.
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Hirsh IH, Sedor J, Jeyendran RS, Stass WE. The relative distribution of viable sperm in the antegrade and retrograde portions of ejaculates obtained after electrostimulation. Fertil Steril 1992;57:399-401. Larsson K. Evaluation of boar semen. In: Morrow DA led), Current Therapy in Theriogenology 2. Philadelphia, W.B. Saunders Company, 1986;972-975. Martin PA, Dooley MP, Hembrough FB, Pineda MH. Urinary losses of spermatozoa during ejaculation are negligible in boars. Theriogenology 1993;39:945-964. Pineda MH, Dooley MP. Effect of method of seminal collection on the retrograde flow of spermatozoa into the urinary bladder of rams. Am J Vet Res 1991;52:307-313. Pineda MH, Dooley MP. Yohimbine prevents the retrograde flow of spermatozoa into the urinary bladder of dogs induced by xylazine. J Vet Pharmacol Therap, in orass, February 1994. Pineda MH, Dooley MP, Hembrough FB, Hsu WH. Retrograde flow of spermatozoa into the urinary bladder of rams. Am J Vet Res 1987;48:562-568. SAS User’s Guide: Statistics. SAS Institute Inc., Cary, NC, 1982;433-506. Schaffer NE, Cranfield M, Fazleabas AT, Jeyendran RS. Viable spermatozoa in the bladder after electroejaculation of lion-tailed macaques (Macaca silenus). J Reprod Fertil 1989;86:767-770. Steel RGD, Torrie JH. Principles and Procedures of Statistics. New York, McGraw-Hill Book Co, Inc. 1960; 109-l 10, 194-21 1, 370-373. 377-398. Swierstra EE. A comparison of spermatozoa production and spermatozoa output of Yorkshire and Lacombe boars. J Reprod Fertil 1968;17:459-469. Swierstra EE. The effect of low ambient temperatures on sperm production, epididymal sperm reserves, and semen characteristics of boars. Biol Reprod 1970;2:23-28. Swierstra EE. Sperm production of boars as measured from epididymal sperm reserves and quantitative testicular histology. J Reprod Fertil 1971:27:91-99. Thurmon JC, Benson GJ, Tranquilli WJ, Olson WA, Tracy CH. The anesthetic and analgesic effects of Telazol@ and xylazine in pigs: Evaluating clinical trials. Vet Med 1988;83:841-845. Thurmon JC, Nelson DR. Christie GJ. Ketamine anesthesia in swine. J Am Vet Med Assoc 1972:160:1325-1330.