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Effect of clavulanic acid-potentiated amoxycillin on semen quality in dogs
ELSEVIER
EFFECT OF CLAVULANIC ACIDPOTENTIATED AMOXYCLLLIN ON SEMEN QUALITY IN DOGS G.C.W. England Unit of Obstetrics Department of Farm Animal and Equine Medicine and Surgery Royal Veterinaty College University of London Hawkshead Lane, North Mymms Hatfield, Herts, England Received for publication: May 23, 1997 Accepted: August
15,
1997
ABSTRACT Two different dosesof clavulanic acid-potentiatedamoxycillin were evaluated for their effect upon semenquality and libido in dogs. There was no significant difference in either parameterbetween control dogs and dogs treatedwith clavulanic acid-potentiated amoxycillin at either 12.5 mgkg-1 or 25 mgkg-1 orally twice daily for 28 d. Despite the marked effect of certain antibiotic agentsupon spermatogenesis,it appearsthat clavulanic acid-potentiated amoxycillin can be used in dogs at up to twice the therapeutic dosagerecommendedby the manufacturer without a deleterious effect upon semenquality. 0 1999 by Elsevier Science Inc.
Key words; dog, semen,antibiotic, fertility INTRODUCTION A large number of therapeutic agentsare widely used in clinical veterinary practice, and for many of thesethere is little available information concerning their effect upon reproduction. In the male, therapeutic agentsmay disturb semenquality by acting directly upon the seminiferous tubules, or may influence the epididymal phaseof development. While the reproductive steroids are agentswith the greatestpotential for altering spermatozoalproduction, other widely used compounds may have a significant effect upon spermatogenesis. Griseofulvin, for example, has been reported to causeoligospermia in dogs when administer&I in large doses(5). Perhapsmore important, however, are the antibiotic agents which are widely used in clinical practice without regard for any potential effect upon fertility. This is surprising since it has been known for many years that several categoriesof antibiotic such as amphotericin and nitrofurantoin may arrest spermatogenesis(6,11,13). More recently, it has becomeclear that several major classesof antibiotic have significant adverseeffects upon spermatogenesisor spermatozoalfunction (8). Marked effects have been noted following the use of trimethoprim, nitrofurantoins, ofloxacin, doxycycline and minocycline (3,6). Despite these studies there is limited information available concerning the effect of antibiotic agentsupon semenquality of dogs (13), and to date no information appearsto be available concerning the effect on semenquality of clavulanic acid-potentiated amoxycillin, an antibiotic combination that is used in dogs worldwide. The aim of the present study was, therefore, to evaluate the effect of this antibiotic combination upon semenquality in dogs when administeredat the therapeutic dosageand twice the therapeutic dosagerecommendedby the manufacturer. Theriogenology 52:1117-l 122,199s 0 1999 by Elseviir Science Inc.
009~69iX/99&-see front matter PII sO093-691 X(99)00203-4
Theriogenology
1118
MATERIALS AND METHODS Semenwas collected by digital manipulation from 6 healthy previously trained beaglesof unknown fertility in the absenceof a teaserbitch. At the start of the study the dogs were 3 yr old. They were housed in a non-controlled environment with accessto the outdoors. Semenwas collected twice weekly every week, regardlessof the treatment being used or whether the animals were on a treatment/control or rest regimen. The 3 fractions of the ejaculate were collected into 3 separateplastic test tubes via glass funnels. Contamination of the secondfraction with the first fraction did not occur using this method. A drop of the second fraction was placed on a warmed glass slide, and sperm motility of the sample was assessed.The percentagesof spermatozoawithin each of the following categorieswere estimatedto the nearest5%: 0 = immotile spermatozoa;I = poorly motile spermatozoawith no progression; II = motile spermatozoawith sluggish progression; III = motile spermatozoawith fair but slow progression; IV = motile spermatozoawith good progression, and V = rapidly motile spermatozoawith good linear progression (12). The spermatozoalconcentration of the secondfraction was determined using an improved Neubauer haemocytometercounting chamber.The numbers of live and dead spermatozoaand spermatozoalmorphology were examined by counting 100 spermatozoaon a nigrosin/eosin stained smearusing the classification of Christiansen (2). Acrosomal integrity was assessed using the classification of Bwanga (l), who found that nigrosin/eosin staining of acrosomes was more discriminative of acrosomaldamagethan several other methods, although this system was more time consuming. Acrosome status was recorded as either normal or abnormal. Spermatozoawith thickened acrosomes,vesiculated or disintegrating acrosomes,or detached and absentacrosomeswere considered abnormal. Libido was subjectively assessedon a scale of 0 to 4, using the criteria described by Taha (12). On this scale, a score 4 representedrapid erection with thrusting movements, 0 representedno erection, and Grade 1 to 3 values were between theseextremes. The dogs were initially divided randomly into 2 groups of 3 dogs. During each treatment, 3 dogs were treated while 3 others served as the untreated controls. Semenwas collected twice weekly during and between each experimental period. Experiment 1 Clavulanic acid-potentiatedamoxycillin (Synulox PalatableTablets, Pfizer Limited, Sandwich, UK) was administered at a 12.5 mgkg-1 dose orally twice daily for 28 d to 3 dogs (treatmentgroup). Another 3 dogs received no treatment (control group). Semenwas collected and libido scored as described above. Semensampleswere collected and data analyzed for the period of 1 mo prior to treatment, during treatment, and for 2 mo after treatment. Experiment 2 After completion of Experiment 1 (above), semenwas collected twice weekly for 1 further month. The dogs which had been previously treated were then assignedto the control group, while those that had been previously untreated were treated with 25 mgkg-1 clavulanic acid-potentiatedamoxycillin orally twice daily for 28 d. Semenwas collected and libido was scored as described above. The data were analyzed for 1 mo prior to treatment, during treatment,and for 2 mo after treatment.
Theriogenology
1119
Statistical Analysis The libido scoreand semenparameters(percentagecategory V motility (percentage normal motility), percentageof live normal spermatozoaand the total spermatozoaloutput), were assessedusing the analysis of variance, with the results grouped in 2 wk periods. Percentagedata were arcsin-transformed, and comparisons were made within groups by referenceto the pretreatmentperiods and between control and treatment groups using the MannWhitney test. Statistical analysis was performed using Apple Macintosh Software (Multistat 1.Ol, Cambridge, UK). Values were considered to be statistically significant at P&OS RESULTS The dogs remained healthy throughout the period of the study, and no adverse effects were attributed to the antibiotic therapy. Experiment 1 Libido score was assessedas 4 for each dog for the duration of the study period. There was no change in mean semenquality for any parameterin either the control dogs or in dogs receiving clavulanic acid-potentiatedamoxycillin (Figure 1). Dogs in the treatment and control groups did not differ significantly from each other with respectto semenquality and libido score. Experiment 2 There was no change in the libido scorefor any dog during the study. There was also no change in mean semenquality for any parameterfor the control dogs or the dogs receiving the clavulanic acid potentiated amoxycillin (Figure 2). The treatment and control groups were not significantly different from each other with respectto semenquality and libido score. DISCUSSION Over recent years it has becomeclear that significant deleterious alterations in semen quality may be induced by the administration of antibiotic agentsat therapeutic dosages(8). As a result of this, clinicians must be aware of the potential for certain antibiotics to adversely affect fertility in males, and to carefully select appropriate agentsin breeding animals. However, in the dog, there is limited information available about which agentscan be used safely without producing potentially deleterious effects upon fertility. The results of the present study have demonstratedthat the short-term administration of clavulanic acid-potentiated amoxycillin has no significant effect upon semenquality in dogs when administered at up to twice the recommendedtherapeutic dosage.Indeed, throughout the study the semenquality of all dogs remained within limits consistent with fertility in this species (4,7). These data, therefore, provide a useful guide for the clinician considering antibiotic therapy in the breeding dog. Although clavulanic acid-potentiatedamoxycillin can be recommendfor use in stud dogs, consideration should also be given to consequencesof antibiotic administration other than
1120
6 ;a 0
600
“0 I4 &
400
& ‘;d 200
.$
-4
-2
0
2
4
6
8
10
12
Weeks in relation to onset of treatment Figure 1.
Mean (SEM) percentagenormal motility (a), mean percentagenormal spermatozoa(b), and mean total spermatozoaloutput (c) for dogs treated orally twice daily with 12.5 mg/kg clavulanic acid potentiated amoxycillin (-6). and untreated controls (-0-). The bar representsthe period of treatment.
Theriogenology
1121
s 0,1 -4
3
2
-4
-2
-2
0
2
4
0
2
4
6
6
0
6
10
10
12
12
Weeks in relation to onset of treatment Figure 2.
Mean (SEM) percentagenormal motility (a), mean percentagenormal spermatozoa(b), and mean total spermatozoaloutput (c) for dogs treated orally twice daily with 25 mg/kg clavulanic acid potentiated amoxycillin (-•-), and untreated controls t-O-). The bar representsthe period of treatment.
Theriogenology
1122
the potential effect upon semenquality. While the beta-lactamantibiotics (including amoxycillin) are considered to be the safestto use during pregnancy (9), Strom and Linde-Forsberg (10) recently showed that administration of antibiotic to bitches could result in the selection of potential genital tract bacterial pathogens.This effect has not been investigated in the male dog; however, it is clear that the clinician should remain vigilant when considering the use of antibiotic preparations in males that are used for breeding. REFERENCES 1. 2.
Bwanga CO. Cryopreservation of Boar Semen.Studies on Freezing, Packaging and Fertilizing Capacity. PhD Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. 1990. Christiansen IbJ. Reproduction in the Dog and Cat. London: Bailliere Tindall, 1984; 115-123.
3. 4. 5. 6. 7.
Crotty KL, May R, Kulvicki A, Kumar D, Neal DE. The effect of antimicrobial therapy on testicular aspirateflow cytometry. J Urol 1995; 153: 835-838. England GCW, Allen WE. Seminal characteristics and fertility in dogs. Vet Ret 1989; 125: 399. McNall EG. Antibiotics Annual. New York: Antibiotica Inc, 1960; 674-679. Nelson WO, Steinberger F. Effects of nitrofuran compounds on the testis of the rat. Fed Proc 1953; 12: 103 abstr. Gettle EE. Sperm morphology and fertility in the dog. J Reprod Fertil 1993; 47 (Suppl): 257-260.
8. 9.
10. 11. 12. 13.
Schlegel PN, Chang TS, Marshall FE Antibiotics: potential hazardsto male fertility, Fertil Steril 1991: 55: 235-242. Schwarz RH. Considerations of antibiotic therapy during pregnancy. Obstet Gynecol 1981; 58 (Suppl): 95-99. Strom B, Linde-For&erg C. Effects of ampicillin and trimethoprim-sulfamethoxazole on the vaginal bacterial flora of bitches. Am J Vet Res 1993; 54: 891-896. Swierstra EE, Whitefield JW, Foot RH. Action of amphotericin B (Fungizone) on spermatogenesisin the rabbit. J Reprod Fertil 1964; 7: 13 abstr. Taha MB. Studies on SemenComposition and Testicular Function in the Dog, with Particular Reference to the Effect of Season,Age and Exogenous Hormones. PhD dissertation, University of London, 1980. Texter JH, Coffey DS. The effects of amphotericin B on prostatic and testicular function in the dog. Invest Urol 1969; 7: 90-106.
EFFECT OF CLAVULANIC ACIDPOTENTIATED AMOXYCLLLIN ON SEMEN QUALITY IN DOGS G.C.W. England Unit of Obstetrics Department of Farm Animal and Equine Medicine and Surgery Royal Veterinaty College University of London Hawkshead Lane, North Mymms Hatfield, Herts, England Received for publication: May 23, 1997 Accepted: August
15,
1997
ABSTRACT Two different dosesof clavulanic acid-potentiatedamoxycillin were evaluated for their effect upon semenquality and libido in dogs. There was no significant difference in either parameterbetween control dogs and dogs treatedwith clavulanic acid-potentiated amoxycillin at either 12.5 mgkg-1 or 25 mgkg-1 orally twice daily for 28 d. Despite the marked effect of certain antibiotic agentsupon spermatogenesis,it appearsthat clavulanic acid-potentiated amoxycillin can be used in dogs at up to twice the therapeutic dosagerecommendedby the manufacturer without a deleterious effect upon semenquality. 0 1999 by Elsevier Science Inc.
Key words; dog, semen,antibiotic, fertility INTRODUCTION A large number of therapeutic agentsare widely used in clinical veterinary practice, and for many of thesethere is little available information concerning their effect upon reproduction. In the male, therapeutic agentsmay disturb semenquality by acting directly upon the seminiferous tubules, or may influence the epididymal phaseof development. While the reproductive steroids are agentswith the greatestpotential for altering spermatozoalproduction, other widely used compounds may have a significant effect upon spermatogenesis. Griseofulvin, for example, has been reported to causeoligospermia in dogs when administer&I in large doses(5). Perhapsmore important, however, are the antibiotic agents which are widely used in clinical practice without regard for any potential effect upon fertility. This is surprising since it has been known for many years that several categoriesof antibiotic such as amphotericin and nitrofurantoin may arrest spermatogenesis(6,11,13). More recently, it has becomeclear that several major classesof antibiotic have significant adverseeffects upon spermatogenesisor spermatozoalfunction (8). Marked effects have been noted following the use of trimethoprim, nitrofurantoins, ofloxacin, doxycycline and minocycline (3,6). Despite these studies there is limited information available concerning the effect of antibiotic agentsupon semenquality of dogs (13), and to date no information appearsto be available concerning the effect on semenquality of clavulanic acid-potentiated amoxycillin, an antibiotic combination that is used in dogs worldwide. The aim of the present study was, therefore, to evaluate the effect of this antibiotic combination upon semenquality in dogs when administeredat the therapeutic dosageand twice the therapeutic dosagerecommendedby the manufacturer. Theriogenology 52:1117-l 122,199s 0 1999 by Elseviir Science Inc.
009~69iX/99&-see front matter PII sO093-691 X(99)00203-4
Theriogenology
1118
MATERIALS AND METHODS Semenwas collected by digital manipulation from 6 healthy previously trained beaglesof unknown fertility in the absenceof a teaserbitch. At the start of the study the dogs were 3 yr old. They were housed in a non-controlled environment with accessto the outdoors. Semenwas collected twice weekly every week, regardlessof the treatment being used or whether the animals were on a treatment/control or rest regimen. The 3 fractions of the ejaculate were collected into 3 separateplastic test tubes via glass funnels. Contamination of the secondfraction with the first fraction did not occur using this method. A drop of the second fraction was placed on a warmed glass slide, and sperm motility of the sample was assessed.The percentagesof spermatozoawithin each of the following categorieswere estimatedto the nearest5%: 0 = immotile spermatozoa;I = poorly motile spermatozoawith no progression; II = motile spermatozoawith sluggish progression; III = motile spermatozoawith fair but slow progression; IV = motile spermatozoawith good progression, and V = rapidly motile spermatozoawith good linear progression (12). The spermatozoalconcentration of the secondfraction was determined using an improved Neubauer haemocytometercounting chamber.The numbers of live and dead spermatozoaand spermatozoalmorphology were examined by counting 100 spermatozoaon a nigrosin/eosin stained smearusing the classification of Christiansen (2). Acrosomal integrity was assessed using the classification of Bwanga (l), who found that nigrosin/eosin staining of acrosomes was more discriminative of acrosomaldamagethan several other methods, although this system was more time consuming. Acrosome status was recorded as either normal or abnormal. Spermatozoawith thickened acrosomes,vesiculated or disintegrating acrosomes,or detached and absentacrosomeswere considered abnormal. Libido was subjectively assessedon a scale of 0 to 4, using the criteria described by Taha (12). On this scale, a score 4 representedrapid erection with thrusting movements, 0 representedno erection, and Grade 1 to 3 values were between theseextremes. The dogs were initially divided randomly into 2 groups of 3 dogs. During each treatment, 3 dogs were treated while 3 others served as the untreated controls. Semenwas collected twice weekly during and between each experimental period. Experiment 1 Clavulanic acid-potentiatedamoxycillin (Synulox PalatableTablets, Pfizer Limited, Sandwich, UK) was administered at a 12.5 mgkg-1 dose orally twice daily for 28 d to 3 dogs (treatmentgroup). Another 3 dogs received no treatment (control group). Semenwas collected and libido scored as described above. Semensampleswere collected and data analyzed for the period of 1 mo prior to treatment, during treatment, and for 2 mo after treatment. Experiment 2 After completion of Experiment 1 (above), semenwas collected twice weekly for 1 further month. The dogs which had been previously treated were then assignedto the control group, while those that had been previously untreated were treated with 25 mgkg-1 clavulanic acid-potentiatedamoxycillin orally twice daily for 28 d. Semenwas collected and libido was scored as described above. The data were analyzed for 1 mo prior to treatment, during treatment,and for 2 mo after treatment.
Theriogenology
1119
Statistical Analysis The libido scoreand semenparameters(percentagecategory V motility (percentage normal motility), percentageof live normal spermatozoaand the total spermatozoaloutput), were assessedusing the analysis of variance, with the results grouped in 2 wk periods. Percentagedata were arcsin-transformed, and comparisons were made within groups by referenceto the pretreatmentperiods and between control and treatment groups using the MannWhitney test. Statistical analysis was performed using Apple Macintosh Software (Multistat 1.Ol, Cambridge, UK). Values were considered to be statistically significant at P&OS RESULTS The dogs remained healthy throughout the period of the study, and no adverse effects were attributed to the antibiotic therapy. Experiment 1 Libido score was assessedas 4 for each dog for the duration of the study period. There was no change in mean semenquality for any parameterin either the control dogs or in dogs receiving clavulanic acid-potentiatedamoxycillin (Figure 1). Dogs in the treatment and control groups did not differ significantly from each other with respectto semenquality and libido score. Experiment 2 There was no change in the libido scorefor any dog during the study. There was also no change in mean semenquality for any parameterfor the control dogs or the dogs receiving the clavulanic acid potentiated amoxycillin (Figure 2). The treatment and control groups were not significantly different from each other with respectto semenquality and libido score. DISCUSSION Over recent years it has becomeclear that significant deleterious alterations in semen quality may be induced by the administration of antibiotic agentsat therapeutic dosages(8). As a result of this, clinicians must be aware of the potential for certain antibiotics to adversely affect fertility in males, and to carefully select appropriate agentsin breeding animals. However, in the dog, there is limited information available about which agentscan be used safely without producing potentially deleterious effects upon fertility. The results of the present study have demonstratedthat the short-term administration of clavulanic acid-potentiated amoxycillin has no significant effect upon semenquality in dogs when administered at up to twice the recommendedtherapeutic dosage.Indeed, throughout the study the semenquality of all dogs remained within limits consistent with fertility in this species (4,7). These data, therefore, provide a useful guide for the clinician considering antibiotic therapy in the breeding dog. Although clavulanic acid-potentiatedamoxycillin can be recommendfor use in stud dogs, consideration should also be given to consequencesof antibiotic administration other than
1120
6 ;a 0
600
“0 I4 &
400
& ‘;d 200
.$
-4
-2
0
2
4
6
8
10
12
Weeks in relation to onset of treatment Figure 1.
Mean (SEM) percentagenormal motility (a), mean percentagenormal spermatozoa(b), and mean total spermatozoaloutput (c) for dogs treated orally twice daily with 12.5 mg/kg clavulanic acid potentiated amoxycillin (-6). and untreated controls (-0-). The bar representsthe period of treatment.
Theriogenology
1121
s 0,1 -4
3
2
-4
-2
-2
0
2
4
0
2
4
6
6
0
6
10
10
12
12
Weeks in relation to onset of treatment Figure 2.
Mean (SEM) percentagenormal motility (a), mean percentagenormal spermatozoa(b), and mean total spermatozoaloutput (c) for dogs treated orally twice daily with 25 mg/kg clavulanic acid potentiated amoxycillin (-•-), and untreated controls t-O-). The bar representsthe period of treatment.
Theriogenology
1122
the potential effect upon semenquality. While the beta-lactamantibiotics (including amoxycillin) are considered to be the safestto use during pregnancy (9), Strom and Linde-Forsberg (10) recently showed that administration of antibiotic to bitches could result in the selection of potential genital tract bacterial pathogens.This effect has not been investigated in the male dog; however, it is clear that the clinician should remain vigilant when considering the use of antibiotic preparations in males that are used for breeding. REFERENCES 1. 2.
Bwanga CO. Cryopreservation of Boar Semen.Studies on Freezing, Packaging and Fertilizing Capacity. PhD Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden. 1990. Christiansen IbJ. Reproduction in the Dog and Cat. London: Bailliere Tindall, 1984; 115-123.
3. 4. 5. 6. 7.
Crotty KL, May R, Kulvicki A, Kumar D, Neal DE. The effect of antimicrobial therapy on testicular aspirateflow cytometry. J Urol 1995; 153: 835-838. England GCW, Allen WE. Seminal characteristics and fertility in dogs. Vet Ret 1989; 125: 399. McNall EG. Antibiotics Annual. New York: Antibiotica Inc, 1960; 674-679. Nelson WO, Steinberger F. Effects of nitrofuran compounds on the testis of the rat. Fed Proc 1953; 12: 103 abstr. Gettle EE. Sperm morphology and fertility in the dog. J Reprod Fertil 1993; 47 (Suppl): 257-260.
8. 9.
10. 11. 12. 13.
Schlegel PN, Chang TS, Marshall FE Antibiotics: potential hazardsto male fertility, Fertil Steril 1991: 55: 235-242. Schwarz RH. Considerations of antibiotic therapy during pregnancy. Obstet Gynecol 1981; 58 (Suppl): 95-99. Strom B, Linde-For&erg C. Effects of ampicillin and trimethoprim-sulfamethoxazole on the vaginal bacterial flora of bitches. Am J Vet Res 1993; 54: 891-896. Swierstra EE, Whitefield JW, Foot RH. Action of amphotericin B (Fungizone) on spermatogenesisin the rabbit. J Reprod Fertil 1964; 7: 13 abstr. Taha MB. Studies on SemenComposition and Testicular Function in the Dog, with Particular Reference to the Effect of Season,Age and Exogenous Hormones. PhD dissertation, University of London, 1980. Texter JH, Coffey DS. The effects of amphotericin B on prostatic and testicular function in the dog. Invest Urol 1969; 7: 90-106.