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Antifertility effect of busulfan and DL-6-(N-2-pipecolinomethyl)-5-hydroxy-indane maleate (PMHI) in coyotes (Canis Latrans )
THERIOCENOLOGY ANTIFERTILITY EFFECT OF BUSULFAN HYDROXY-INDANE MALEATE (PMHI) J.N.
Ste 1
2Department
AND DL-6-(N-2-PIPECOLINOMETHYl,)-',IN COYOTES (CANIS LATRANS)
lflug', C.W. Leathers2 and J.S. Green3 U.S. Department of Agriculture U.S. Sheep Experiment Station Dubois, 11, 83423 and of Veterinary Microbiology and Pathology Washington State University Pullman. WA 99164
Received
for Publication: Accepted:
February 29, 1984 August 13. 1984
ABSTRACT Antifertility effects of busulfan were evaluated using adult coyotes. In addition, antifertility effects of PMHI were evaluated in adult males. Adult males and females were alloted randomly t
Busulfan,
PMHI,
Coyotes,
Antifertility
Effects
ACKNOWLEDGEMENTS This work was in cooperation with the University of Idaho, Moscow, Idaho. The authors appreciate Hoffman-la-Roche, Nutley, New Jersey for their contribution of PMHI; R. Woodruff and J. Rich for assisting with collection of blood samples and surgeries; V. Gunter for typing the manuscript.
NOVEMBER
1984 VOL. 22 NO. 5
533
THERIOGENOLOGY INTRODUCTION Controlling coyote depredation of livestock is a challenging problem which has been studied in various ways (1). Toxicants were used extensively for coyote control until a limited ban in 1972 (2). Current methods of coyote control include shooting and trapping,but non-lethal methods of control such as reproductive inhibitors are being researched (3-6). Relaxing some of the following listed criteria previously considered essential for a successful reproductive inhibitor (7) may increase the likelihood of finding a suitable compound: (a) The reproductive inhibitor must be effective in a single, oral dose. (b) The margin of safety between sterilizing and lethal dosage levels must be acceptable. (c) The antifertility compound must be relatively stable, inexpensive and effective in minute quantities. (d) It must be odorless and tasteless so as not to cause aversion to treated baits. (e) The antifertility compound must be relatively host specific in order to minimize the effects from ingestion by non-target species. (f) It must be effective for relatively long periods during the reproductive cycle. (g) The practical application of reproductive inhibitors will be dependent upon development of host-specific delivery systems. For instance, a single oral dose may not be required, even though it 4s preferred, due to recent advances in development of attractants (8). Cytostatics used for human cancer chemotherapy have also the effect of reducing reproductive function (9). An advantage of alkylates over the synthetic steroid contraceptives is that a more complete and protracted t rilizing effect can be achieved by a single oral dose (10). Busulfan'aF is cne such cytostatic that has been used for treating reproductive disorders in the bitch (11). The limited breeding season of the coyote of only about 2 months (12) may allow the temporary sterility reported for busulfan to be effective in controlling this population. The objectives of this research were to determine the antifertility effects of busulfan on male and female coyotes and to determine if oral PMHI is effective in arresting spermatogenesis. Previously we reported that a single subcutaneous (SC) dose of 5, 10 or 20 mg PMHI/kg body weight (BW) arrested spermatogenesis 15 and 30 days after administration (6). MATERIALS AND METHODS Forty females, 4 females, 5 apart (Jan
adult coyotes were assigned to three groups: no treatment (7 males); single oral dose of 3 mg busulfan/kg BW (Jan 30: 9 males); and two oral doses of 3 mg busulfan/kg BW nine days 30 and Feb 8: 10 females, 5 males).
(a) Busulfan was purchased from Burroughs Wellcome, Research Triangle Park, NC. Trade names are used in this publication solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee of warranty of the product by the U.S. Department of Agriculture or an endorsement by the Department over other products not mentioned.
534
NOVEMBER
1984VOL.22NO.S
THERIOGENOLOGY
All coyotes were observed for 1 hr after treatment and then intermittently for 24 hr for any adverse effects. Jugular blood samples were taken from females every three to seven days from January 1 to April 30, the period surrounding ovarian activity in female coyotes (12). After centifugation the serum was decanted and stored for three to eight b months at -20 C until analysis for progesterone by radioimmunoassay (RIA, 13). Males were hemicastrated (left testicle) at 30 days posttreatment and the right testicle was removed 30 days later. Testes irom control males were removed at the same time. Both testicles and epididymides from each male were fixed in 109 buffered formalin and prepared for histologic examination. Representative hematoxylin and eosinstained sections were examined without prior knowledge of treatment. The progesterone data for the females showing CL development were analyzed by one way ANOVA (14) for the sum of progesterone produced over all bleeding dates, the maximum progesterone value, and the day of year of maximum value. Regression analysi:.bA: used to determine the starting dates of the progesterone rise in each treatment group. The starting point and maximum point periods were analyzed using repeated measures ANOVA procedures (15). Differences in number of females showing lack of CL development between treatment groups were analyzed with chi-square procedures (16). The untreated control males (n=4) served as controls for the PMHI Ten additional adult male coyotes were assigned to two trial also. groups: single oral dose of 2 mg PMHI/kg BW (Jan 30: n=5); and two oral doses of 1 mg PMHI/kg BW given seven days apart (Jan 30 and Feb 6: n=5). All coyotes were observed for adverse eifects and the testes and epidiymides were removed, fixed, sectioned and examined as in the busulfan study. RESULTS Busulfan
Trial
Serum progesterone of the control female coyotes initially increased in late February and remained elevated until mid to late April (Fig. 1). The progesterone profile for females with CL development given a single oral dose of busulfan (3 mg/kg) was similar to that for the untreated the maximum peak concentration of progesterone controls. However, (X+SE) for females with CL development given two doses of busulfan (2x3 mg/kg) nine days apart was less (PcO.05) than that for the untreated controls and the single-dose females (16.9'2.93, 22.4k4.44 and 26.313.19 One female out of the single-dose treatment group nglml, respectively). and four females from the double-dose treatment group had no signs of CL development as indicated by progesterone concentrations depicted in Figure 1.
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THERIOGENOLOGY
Feb
March
April
Days of the month
Figure 1. Serum progesterone during the breeding period of control coyotes (OF), coyotes which developed CLs after a single dose of busulfan (3 mg/kg on January 30, m-----m) coyotes which developed CLs after two doses of busulfan (2 x 3 mg/kg on January 30 and February 8, o------O), or treated coyotes which did not develop CLs (A------A). For females with CL development, only the maximum peak concentration of progesterone for those given two doses of busulfan (16.9k2.93; XirSE)was statistically different than peak progesterone values for controls (22.4k4.44) and single-dose females (26.3k3.19 &ml).
Control, male coyotes. Histologic findings in control coyotes included a normal series of maturing spermatogonia within seminiferous tubules (Fig. 2) at both observation times. Sertoli cells were prominent, and numerous spermatids and mature spermatozoa were located at the luminal edges. Interstitial cells (Leydig) were ovoid, with a finely granular cytoplasm. Each segment of the epididymis (head, body, and tail) was lined by an intact columnar epithelium. Epididymal lumina were filled with densely packed spermatozoa (Fig. 3). Tunic stroma and blood vessels were considered histologically normal.
536
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Figure 2.
Photomicrograph of the seminiferous tuhules from coyote; x 100.
Figure 3.
Photomicrograph of the epididymis from a control coyote; x 100.
NOVEMBER 1984 VOL. 22 NO.5
537
THERIOGENOLOCY
One dose busulfan, 3 ma/kg. At 30 days post-treatment, the seminiferous tubules and epididymides of all coyotes were similar to that of the controls at both observation times. At 60 days post-treatment, histologic changes were similar for all treated coyotes. At least 50% of the seminiferous tubules were characterized by vacuolar degeneration of spermatogonia (Fig. 4). Often only a single basal layer of spermagonial cells was visible, with smudged eosinophilic strands occupying the rest of the generative layer. Occasional spermatids were seen, but orderly spermatogenesis was rare. Most epididymal lumina were empty or contained only a few spermatozoa (Fig. 5). The epididymal epithelium and Leydig cells were considered histologically normal.
Figure 4. Photomicrograph of the seminiferous tubules from a coyote 60 days after treatment with 3 mg of busulfan/kg BW; x 100.
538
NOVEMBER
1984 VOL. 22 NO. 5
THERIOGENOLOGY
Figure
5. Photomicrograph treatment with
of the epididymis 3 mg of busulfan/kg
from a coyote BW; x 100.
60 days
after
Two doses busulfan, 2 x 3 mg/kg. The testes and epididymides of those coyotes at 30 days post-treatment were histologically similar to those of controls. Some epididymal epithelial cells of one coyote contained prominent,clear cytoplasmic vacuoles which were generally localized at the apex of the cell. Typically only one large vacuole was observed in any particular cell with no inflammatory response accompanying this vacuolation. One coyote had seminiferous tubules (Fig. 6) of approximately normal size, but tubules were not patent. Degenerated, sloughed spermatogonia and Sertoli cell debris plugged the tubules. Many tubules only contained a single basally oriented row of nuclei, and a palely basophilic fibrillar and acellular material filled the lumina. SpermaEpididymal lumina were empty, tids and spermatogonia were not seen. similar to most of those shown in Fig. 5. At 60 days post-treatment, the seminiferous tubules of all coyotes given two doses of busulfan were similar to those described for coyotes at 60 days following a single-dose of busulfan (Fig. 4). Three coyotes had vacuoles in their epididymal epithelium. No adverse effects were observed following oral administration of busulfan to male or female coyotes.
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1984 VOL. 22 NO. 5
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THERIOGENOLOCY
Figure
PMHI
6. Photomicrograph of the seminiferous days after onset of treatment with (2x3 mgikg BW given 9 days apart).
tubules from a coyote 30 a double-dose of busulfan
Trial
One dose PMHL, 2 mg/kg. Histologic findings in all five coyotes 30 days post-treatment were similar to those of controls. At 60 days postactive and orderly spermatogenesis was observed in most treatment, Only occasional seminiferous tubule epithelium tubules from two coyotes. Tissue sections of testes and epididymides of the was vacuolated. remaining three coyotes contained densely-packed spermatozoa. Two doses PMHI, 2 x 1 mg/kg. Thirty day post-treatment sections were similar to those of controls (Fig. 2 and 3).
tissue
active and orderly spermatogenesis was At 60 days post-treatment, Only occasional seminobserved in most tubules from three coyotes. About 50% of epididymal iferous tubule epithelium was vacuolated. lumina were empty, and the remainder were filled with densely-packed spermatozoa. Oral administration of 1 or 2 mg/kg PMHI resulted in acute nausea and vomiting within 18 min after treatment in all coyotes except two One of the latter two vomited 3 hr after which received 2 mg/kg PMHI. treatment and the third coyote vomited between 6 and 15 hr after treatment.
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THERIOCENOLOGY
DISCUSSION All control females showed CL development during the study. Eight out of nine females given a single dose and six out of ten given a double dose of busulfan showed CL development. Only those showing functional CL were used in statistical analyses. The initial dates of significant increases in serum progesterone, estimated by regression procedures were 9, 27 and 23 days from the start of treatments (January 30) for the control, single- and double-dose treatment groups. However, a repeated measures analysis over the dates from 6 to 30 days after start of treatment did not show a significant treatment-by-date interaction due to large animal-by-date variation. The maximum peak concentration of progesterone was reduced significantly (P~0.05) in coyotes treated with two doses of busulfan (16.9 ng/ml) compared to control coyotes (22.4 ng/ml) and coyotes treated with one dose of busulfan (26.3 Repeated measures analyses showed that the maximum production ng/ml). time period was from 43 to 61 days after start of treatments, but neither the treatment nor the treatment-by-time interaction was significant. The time of year of maximum serum concentrations of progesterone for each animal varied by only nine days for control, single-and doubledose females, respectively and these were not different. It appears that busulfan has a detrimental effect on reproduction in the female coyote similar to reports for the bitch (11) in which it affected rapidly proliferating cells. Busulfan appears to have a detrimental effect on the testes of the male coyote. There appeared to be complete suppression of spermatogenesis at 60 days after onset of treatment with busulfan. Few spermatids were found in some tubules. The presence of normal spermatozoa in the epididymal lumina was compatible with fertility even at the time the second testicle was removed. However, busulfan should work at the level of spermatogonia so one would expect 75 to 90 days before all sperm would be eliminated from the epididymis. Vacuolar degeneration of some epididymal epithelium was seen only in busulfan-treated coyotes. Thus, the male coyotes in the single- or double-dose busulfan groups all had similar degenerative changes in the second testis consistent with further progression of damage. Oral administration of busulfan did not cause any adverse reactions oral adminiswithin the first hour after administration. By contrast, tration of PMHI (1 or 2 mg/kg) generally resulted in emesis within 18 This adverse reaction to oral PMHI was similar to the emesis min. Diarrhea enduced when PMHI was administered SC at 5, 10 or 20 mg/kg (6). was also observed after SC, but not after oral administration of PMHI This may be due to loss of the majority of PMHI by emesis when (6). The degree of emesis observed after 1 or 2 mg/kg BW PMHI given orally. two coyotes orally was not expected. In a preliminary tiral, we observed that could tolerate up to 4 mg PMHI/kg BW administered orally, but they were given repetitive doses starting with extremely low doses of PMHI Every 2 to 7 days the dosage was increased, first to 0.2 (0.02 mg/kg). mg/kg then to 0.4, 2, 4 and up to 5 mg/kg before emesis was observed. It is probable that the coyotes developed tolerance to the emetic effect adaptability of this technique to field conditions of PMHI, however, appears limited.
NOVEMBER
1984 VOL. 22 NO. 5
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THERIOGENOLOGY
Histologic findings in testes from coyotes treated orally with 2x 1 mg PMHI/kg BW or 2 mg PMHI/kg BW showed incomplete degeneration of seminiferous tubules even when emesis occurred after an extended period. This is in contrast to the arrest of spermatogenesis reported when 5, 10 or 20 mg PMHI/kg were administered SC (6). Apparently, the oral doses of 1 or 2 mg PMHI/kg BW were expelled from the digestive tract before an amount sufficient to arrest spermatogenesis was absorbed. Arrest of spermatogenesis and sterility was observed in one of the coyotes used in the preliminary trial to determine the emetic threshold of orallyadministered PMHI. Therefore, PMHI is apparently effective when given orally if not expelled from the digestive tract before absorption takes place. In preliminary trials, antiemetics (meclizine, dramamine and prochlorparazine) failed to prevent PMHI-induced emesis. Even though PMHI arrested spermatogenesis in coyotes, oral administration is not practical for field use. Further research with busulfan is warranted since busulfan is orally acceptable and affects reproductive function in both male and female coyotes.
REFERENCES 1.
Wade,
D.A. Impacts, incidence and control of predation livestock in the United States, with particular reference to predation by coyotes. CAST, Spec. Publ. -lO:l-20 (1982).
2.
U.S.
Fish and Wildlife Service. Study of Coyote Management Denver, CO, 1978, 168 pp.
3.
Connolly, G. U.S. Fish and Wildlife Service Coyote Control Research.l_r:Proceedings Fifth Great Plains Wildlife Damage Control Workshop, Lincoln, NE, 1982, pp. 132-149.
4.
Green, J.S. Reducing Coyote Techniques.In:Proceedings Control Workshop, 1982,
5.
Balser, D.S. Antifertility Agents Proceedings Second Vertebrate CA, 1964, pp. 133-137.
6.
Antitesticular Stellflug, J.N., Gates, N.L. and Leathers, C.W. activity of DL-6-(N-2-pipecolinomethyl)-5-hydroxy-indane maleate (PMHI) in coyotes (Canis Latrans) Theriogenology -12:345-354 (1979).
7.
Stellflug, J.N., Gates, N.L. and Sasser, R.G. Reproductive Developments Inhibitors for Coyote Population Control: and Current Status. 1s: Proceedings Eighth Vertebrate Pest Conference, Sacramento, CA, 1978, pp. 185-189.
542
Predator Damage in the West: a Alternatives, U.S. Dept. Interior,
Damage to Sheep with Fifth Great Plains pp. 122-131. in Vertebrate Pest Control
NOVEMBER
Non-lethal Wildlife Damage
Pest Control. In: Conference, Anaheim,
1984 VOL. 22 NO. 5
THERIOGENOLOGY
8.
Teranishi, R., Murphy, E.L., Stern, D.J., Howard, W.E. and Farge, Chemicals Useful as Attractants and Repellents for D.B. Coyotes. 12: Worldwide Furbearer Conference Proceedings, eds. J.A. Chapman and D. Pursley, Volume III, Fosburg, MD, 1981, pp. 1839-1851.
9.
Alkylating agents and human spermatogenesis. Miller, D.J. Amer. Med. Ass. __ 217:1662-1665 (1971).
10.
Temporary sterilization Arbeiter, K., Hager, G. and Kposhitz, M.M. of domestic pigeons that have become feral. Zbl. Vet. Med. A. 22:117-141 (1975). --
11.
Arbeiter, K. diseases
12.
Stellflug, J.N., Muse, P.D., Everson, D-0. and Louis, T.M. Changes in serum progesterone and estrogen of the nonpregnant coyote Proc. Sot. Exptl. Biol. and Med. during the breeding season. 167:220-223 (1981). __
13.
Niswender, G.D. specificity 424 (1973).
14.
Gill,
Designs and Analysis of Experiments in the Animal J.L. Vol. 1, 1st ed. Iowa State University Medical Sciences. Ames, IA, 1978, pp. 135-136.
15.
Gill,
Analysis of repeated J.L. and Hafs, H.D. J. Anim. Sci. -33:331-336 (1971). animals.
16.
Gill,
Designs and Analysis of Experiments in the Animal J.L. Vol. 1, 1st ed. Iowa State University Medical Sciences. Ames, IA, 1978, pp. 69-70.
NOVEMBER
.J.
On a new principle for the treatment of reproductive Prakt. Tierarzt >:295-297 (1976). in the bitch.
Influence of the site of congugation on the of antibodies to progesterone. Steroids -22:413-
1984 VOL. 22 NO. 5
measurements
and Press,
of
and Press,
543
Ste 1
2Department
AND DL-6-(N-2-PIPECOLINOMETHYl,)-',IN COYOTES (CANIS LATRANS)
lflug', C.W. Leathers2 and J.S. Green3 U.S. Department of Agriculture U.S. Sheep Experiment Station Dubois, 11, 83423 and of Veterinary Microbiology and Pathology Washington State University Pullman. WA 99164
Received
for Publication: Accepted:
February 29, 1984 August 13. 1984
ABSTRACT Antifertility effects of busulfan were evaluated using adult coyotes. In addition, antifertility effects of PMHI were evaluated in adult males. Adult males and females were alloted randomly t
Busulfan,
PMHI,
Coyotes,
Antifertility
Effects
ACKNOWLEDGEMENTS This work was in cooperation with the University of Idaho, Moscow, Idaho. The authors appreciate Hoffman-la-Roche, Nutley, New Jersey for their contribution of PMHI; R. Woodruff and J. Rich for assisting with collection of blood samples and surgeries; V. Gunter for typing the manuscript.
NOVEMBER
1984 VOL. 22 NO. 5
533
THERIOGENOLOGY INTRODUCTION Controlling coyote depredation of livestock is a challenging problem which has been studied in various ways (1). Toxicants were used extensively for coyote control until a limited ban in 1972 (2). Current methods of coyote control include shooting and trapping,but non-lethal methods of control such as reproductive inhibitors are being researched (3-6). Relaxing some of the following listed criteria previously considered essential for a successful reproductive inhibitor (7) may increase the likelihood of finding a suitable compound: (a) The reproductive inhibitor must be effective in a single, oral dose. (b) The margin of safety between sterilizing and lethal dosage levels must be acceptable. (c) The antifertility compound must be relatively stable, inexpensive and effective in minute quantities. (d) It must be odorless and tasteless so as not to cause aversion to treated baits. (e) The antifertility compound must be relatively host specific in order to minimize the effects from ingestion by non-target species. (f) It must be effective for relatively long periods during the reproductive cycle. (g) The practical application of reproductive inhibitors will be dependent upon development of host-specific delivery systems. For instance, a single oral dose may not be required, even though it 4s preferred, due to recent advances in development of attractants (8). Cytostatics used for human cancer chemotherapy have also the effect of reducing reproductive function (9). An advantage of alkylates over the synthetic steroid contraceptives is that a more complete and protracted t rilizing effect can be achieved by a single oral dose (10). Busulfan'aF is cne such cytostatic that has been used for treating reproductive disorders in the bitch (11). The limited breeding season of the coyote of only about 2 months (12) may allow the temporary sterility reported for busulfan to be effective in controlling this population. The objectives of this research were to determine the antifertility effects of busulfan on male and female coyotes and to determine if oral PMHI is effective in arresting spermatogenesis. Previously we reported that a single subcutaneous (SC) dose of 5, 10 or 20 mg PMHI/kg body weight (BW) arrested spermatogenesis 15 and 30 days after administration (6). MATERIALS AND METHODS Forty females, 4 females, 5 apart (Jan
adult coyotes were assigned to three groups: no treatment (7 males); single oral dose of 3 mg busulfan/kg BW (Jan 30: 9 males); and two oral doses of 3 mg busulfan/kg BW nine days 30 and Feb 8: 10 females, 5 males).
(a) Busulfan was purchased from Burroughs Wellcome, Research Triangle Park, NC. Trade names are used in this publication solely for the purpose of providing specific information. Mention of a trade name does not constitute a guarantee of warranty of the product by the U.S. Department of Agriculture or an endorsement by the Department over other products not mentioned.
534
NOVEMBER
1984VOL.22NO.S
THERIOGENOLOGY
All coyotes were observed for 1 hr after treatment and then intermittently for 24 hr for any adverse effects. Jugular blood samples were taken from females every three to seven days from January 1 to April 30, the period surrounding ovarian activity in female coyotes (12). After centifugation the serum was decanted and stored for three to eight b months at -20 C until analysis for progesterone by radioimmunoassay (RIA, 13). Males were hemicastrated (left testicle) at 30 days posttreatment and the right testicle was removed 30 days later. Testes irom control males were removed at the same time. Both testicles and epididymides from each male were fixed in 109 buffered formalin and prepared for histologic examination. Representative hematoxylin and eosinstained sections were examined without prior knowledge of treatment. The progesterone data for the females showing CL development were analyzed by one way ANOVA (14) for the sum of progesterone produced over all bleeding dates, the maximum progesterone value, and the day of year of maximum value. Regression analysi:.bA: used to determine the starting dates of the progesterone rise in each treatment group. The starting point and maximum point periods were analyzed using repeated measures ANOVA procedures (15). Differences in number of females showing lack of CL development between treatment groups were analyzed with chi-square procedures (16). The untreated control males (n=4) served as controls for the PMHI Ten additional adult male coyotes were assigned to two trial also. groups: single oral dose of 2 mg PMHI/kg BW (Jan 30: n=5); and two oral doses of 1 mg PMHI/kg BW given seven days apart (Jan 30 and Feb 6: n=5). All coyotes were observed for adverse eifects and the testes and epidiymides were removed, fixed, sectioned and examined as in the busulfan study. RESULTS Busulfan
Trial
Serum progesterone of the control female coyotes initially increased in late February and remained elevated until mid to late April (Fig. 1). The progesterone profile for females with CL development given a single oral dose of busulfan (3 mg/kg) was similar to that for the untreated the maximum peak concentration of progesterone controls. However, (X+SE) for females with CL development given two doses of busulfan (2x3 mg/kg) nine days apart was less (PcO.05) than that for the untreated controls and the single-dose females (16.9'2.93, 22.4k4.44 and 26.313.19 One female out of the single-dose treatment group nglml, respectively). and four females from the double-dose treatment group had no signs of CL development as indicated by progesterone concentrations depicted in Figure 1.
NOVEMBER
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THERIOGENOLOGY
Feb
March
April
Days of the month
Figure 1. Serum progesterone during the breeding period of control coyotes (OF), coyotes which developed CLs after a single dose of busulfan (3 mg/kg on January 30, m-----m) coyotes which developed CLs after two doses of busulfan (2 x 3 mg/kg on January 30 and February 8, o------O), or treated coyotes which did not develop CLs (A------A). For females with CL development, only the maximum peak concentration of progesterone for those given two doses of busulfan (16.9k2.93; XirSE)was statistically different than peak progesterone values for controls (22.4k4.44) and single-dose females (26.3k3.19 &ml).
Control, male coyotes. Histologic findings in control coyotes included a normal series of maturing spermatogonia within seminiferous tubules (Fig. 2) at both observation times. Sertoli cells were prominent, and numerous spermatids and mature spermatozoa were located at the luminal edges. Interstitial cells (Leydig) were ovoid, with a finely granular cytoplasm. Each segment of the epididymis (head, body, and tail) was lined by an intact columnar epithelium. Epididymal lumina were filled with densely packed spermatozoa (Fig. 3). Tunic stroma and blood vessels were considered histologically normal.
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1984 VOL. 22 NO. 5
THERIOGENOLOG Y
Figure 2.
Photomicrograph of the seminiferous tuhules from coyote; x 100.
Figure 3.
Photomicrograph of the epididymis from a control coyote; x 100.
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THERIOGENOLOCY
One dose busulfan, 3 ma/kg. At 30 days post-treatment, the seminiferous tubules and epididymides of all coyotes were similar to that of the controls at both observation times. At 60 days post-treatment, histologic changes were similar for all treated coyotes. At least 50% of the seminiferous tubules were characterized by vacuolar degeneration of spermatogonia (Fig. 4). Often only a single basal layer of spermagonial cells was visible, with smudged eosinophilic strands occupying the rest of the generative layer. Occasional spermatids were seen, but orderly spermatogenesis was rare. Most epididymal lumina were empty or contained only a few spermatozoa (Fig. 5). The epididymal epithelium and Leydig cells were considered histologically normal.
Figure 4. Photomicrograph of the seminiferous tubules from a coyote 60 days after treatment with 3 mg of busulfan/kg BW; x 100.
538
NOVEMBER
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Figure
5. Photomicrograph treatment with
of the epididymis 3 mg of busulfan/kg
from a coyote BW; x 100.
60 days
after
Two doses busulfan, 2 x 3 mg/kg. The testes and epididymides of those coyotes at 30 days post-treatment were histologically similar to those of controls. Some epididymal epithelial cells of one coyote contained prominent,clear cytoplasmic vacuoles which were generally localized at the apex of the cell. Typically only one large vacuole was observed in any particular cell with no inflammatory response accompanying this vacuolation. One coyote had seminiferous tubules (Fig. 6) of approximately normal size, but tubules were not patent. Degenerated, sloughed spermatogonia and Sertoli cell debris plugged the tubules. Many tubules only contained a single basally oriented row of nuclei, and a palely basophilic fibrillar and acellular material filled the lumina. SpermaEpididymal lumina were empty, tids and spermatogonia were not seen. similar to most of those shown in Fig. 5. At 60 days post-treatment, the seminiferous tubules of all coyotes given two doses of busulfan were similar to those described for coyotes at 60 days following a single-dose of busulfan (Fig. 4). Three coyotes had vacuoles in their epididymal epithelium. No adverse effects were observed following oral administration of busulfan to male or female coyotes.
NOVEMBER
1984 VOL. 22 NO. 5
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THERIOGENOLOCY
Figure
PMHI
6. Photomicrograph of the seminiferous days after onset of treatment with (2x3 mgikg BW given 9 days apart).
tubules from a coyote 30 a double-dose of busulfan
Trial
One dose PMHL, 2 mg/kg. Histologic findings in all five coyotes 30 days post-treatment were similar to those of controls. At 60 days postactive and orderly spermatogenesis was observed in most treatment, Only occasional seminiferous tubule epithelium tubules from two coyotes. Tissue sections of testes and epididymides of the was vacuolated. remaining three coyotes contained densely-packed spermatozoa. Two doses PMHI, 2 x 1 mg/kg. Thirty day post-treatment sections were similar to those of controls (Fig. 2 and 3).
tissue
active and orderly spermatogenesis was At 60 days post-treatment, Only occasional seminobserved in most tubules from three coyotes. About 50% of epididymal iferous tubule epithelium was vacuolated. lumina were empty, and the remainder were filled with densely-packed spermatozoa. Oral administration of 1 or 2 mg/kg PMHI resulted in acute nausea and vomiting within 18 min after treatment in all coyotes except two One of the latter two vomited 3 hr after which received 2 mg/kg PMHI. treatment and the third coyote vomited between 6 and 15 hr after treatment.
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DISCUSSION All control females showed CL development during the study. Eight out of nine females given a single dose and six out of ten given a double dose of busulfan showed CL development. Only those showing functional CL were used in statistical analyses. The initial dates of significant increases in serum progesterone, estimated by regression procedures were 9, 27 and 23 days from the start of treatments (January 30) for the control, single- and double-dose treatment groups. However, a repeated measures analysis over the dates from 6 to 30 days after start of treatment did not show a significant treatment-by-date interaction due to large animal-by-date variation. The maximum peak concentration of progesterone was reduced significantly (P~0.05) in coyotes treated with two doses of busulfan (16.9 ng/ml) compared to control coyotes (22.4 ng/ml) and coyotes treated with one dose of busulfan (26.3 Repeated measures analyses showed that the maximum production ng/ml). time period was from 43 to 61 days after start of treatments, but neither the treatment nor the treatment-by-time interaction was significant. The time of year of maximum serum concentrations of progesterone for each animal varied by only nine days for control, single-and doubledose females, respectively and these were not different. It appears that busulfan has a detrimental effect on reproduction in the female coyote similar to reports for the bitch (11) in which it affected rapidly proliferating cells. Busulfan appears to have a detrimental effect on the testes of the male coyote. There appeared to be complete suppression of spermatogenesis at 60 days after onset of treatment with busulfan. Few spermatids were found in some tubules. The presence of normal spermatozoa in the epididymal lumina was compatible with fertility even at the time the second testicle was removed. However, busulfan should work at the level of spermatogonia so one would expect 75 to 90 days before all sperm would be eliminated from the epididymis. Vacuolar degeneration of some epididymal epithelium was seen only in busulfan-treated coyotes. Thus, the male coyotes in the single- or double-dose busulfan groups all had similar degenerative changes in the second testis consistent with further progression of damage. Oral administration of busulfan did not cause any adverse reactions oral adminiswithin the first hour after administration. By contrast, tration of PMHI (1 or 2 mg/kg) generally resulted in emesis within 18 This adverse reaction to oral PMHI was similar to the emesis min. Diarrhea enduced when PMHI was administered SC at 5, 10 or 20 mg/kg (6). was also observed after SC, but not after oral administration of PMHI This may be due to loss of the majority of PMHI by emesis when (6). The degree of emesis observed after 1 or 2 mg/kg BW PMHI given orally. two coyotes orally was not expected. In a preliminary tiral, we observed that could tolerate up to 4 mg PMHI/kg BW administered orally, but they were given repetitive doses starting with extremely low doses of PMHI Every 2 to 7 days the dosage was increased, first to 0.2 (0.02 mg/kg). mg/kg then to 0.4, 2, 4 and up to 5 mg/kg before emesis was observed. It is probable that the coyotes developed tolerance to the emetic effect adaptability of this technique to field conditions of PMHI, however, appears limited.
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THERIOGENOLOGY
Histologic findings in testes from coyotes treated orally with 2x 1 mg PMHI/kg BW or 2 mg PMHI/kg BW showed incomplete degeneration of seminiferous tubules even when emesis occurred after an extended period. This is in contrast to the arrest of spermatogenesis reported when 5, 10 or 20 mg PMHI/kg were administered SC (6). Apparently, the oral doses of 1 or 2 mg PMHI/kg BW were expelled from the digestive tract before an amount sufficient to arrest spermatogenesis was absorbed. Arrest of spermatogenesis and sterility was observed in one of the coyotes used in the preliminary trial to determine the emetic threshold of orallyadministered PMHI. Therefore, PMHI is apparently effective when given orally if not expelled from the digestive tract before absorption takes place. In preliminary trials, antiemetics (meclizine, dramamine and prochlorparazine) failed to prevent PMHI-induced emesis. Even though PMHI arrested spermatogenesis in coyotes, oral administration is not practical for field use. Further research with busulfan is warranted since busulfan is orally acceptable and affects reproductive function in both male and female coyotes.
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