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Effects of ketamine or medetomidine administration on quality of electroejaculated sperm and on sperm flow in the domestic cat
Theriogenology 68 (2007) 796–803 www.theriojournal.com
Effects of ketamine or medetomidine administration on quality of electroejaculated sperm and on sperm flow in the domestic cat D. Zambelli *, M. Cunto, F. Prati, B. Merlo Veterinary Clinical Department, Obstetrical and Gynaecological Section, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy Received 22 December 2006; received in revised form 8 June 2007; accepted 8 June 2007
Abstract The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species were evaluated performing three experiments. This is the first study about these topics in the domestic cat. In Experiment 1, ketamine or medetomidine effects on cat sperm quality after collection by electroejaculation (E.E.) have been assessed in nine animals. Results showed that mean sperm concentration was significantly higher ( p < 0.01) after medetomidine than after ketamine administration. In Experiment 2, ketamine or medetomidine effects on sperm flow in 12 electroejaculated cats were studied. Mean sperm concentration and mean total number of spermatozoa resulted significantly higher ( p < 0.01) in medetomidine than in ketamine treated animals. The number of spermatozoa displaced in urethra was significantly higher ( p < 0.01) using medetomidine. No significant differences were observed in percentages of retrograde flow. In Experiment 3, ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection, were evaluated. Data obtained showed a significantly higher ( p < 0.05) number of spermatozoa displaced in urethra after medetomidine than after ketamine injection. In conclusion, E.E. in the cat after medetomidine administration determined a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction and without increasing sperm retrograde flow. # 2007 Elsevier Inc. All rights reserved. Keywords: Ketamine; Medetomidine; Electroejaculation; Sperm; Domestic cat
1. Introduction The domestic cat serves as an important experimental model in the study of endangered wild felids diseases. Assisted reproduction is an essential tool in the safeguard plans of these non-domestic felids that, in captivity, often reproduce poorly and frequently reveal a bad sperm quality [1]. Both semen collection and artificial insemination (AI) in cats have been first
* Corresponding author. Tel.: +39 0512097572; fax: +39 0512097568. E-mail address: [email protected] (D. Zambelli). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.06.008
described more than 30 years ago [2], but these procedures are not a routine in veterinary practice. Sperm collection is essential for AI and IVF [3], besides diagnostic purposes and use in research [4]. Sperm collection in the cat is usually performed by E.E. [5–10] or artificial vagina (A.V.) [11–14], but the first is often considered the method of choice in many clinical and research circumstances [7,15] because it allows sperm collection from every subject that can be safely anesthetised, without previous training. Both E.E. and A.V. permit to obtain whole semen with good quality [5–15]. Also after repeated collections, at intervals of every other day and every 3 days, sperm quality is stable for up to 10 days [16].
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Different anaesthetic protocols have been reported in literature to perform electroejaculation in the cat, but their effects on sperm quality have never been studied. The most commonly used drug resulted to be ketamine HCl, either alone or in association with medetomidine, even if association with xylazine, diazepam, phenotiazine derivatives (e.g. acepromazine maleate) or use of inhalation anaesthetics (e.g. halothane, isoflurane) or propofol have also been reported [1,17–19]. Studies about ketamine and medetomidine pharmacokinetics reported a rapid elimination of these drugs from plasma/serum that occurred with half-lives ranging between 40–60 min [20] and 0.97–1.60 h, respectively [21]. Particularly, it was estimated that ketamine still present in the body, 7 h after administration, is less than 0.1% [20]. a-Adrenergic agents are known to influence erection and ejaculation [22] and ejaculatory reflex is considered to be primarily an a-adrenergically mediated event [23]. It has been reported that xylazine in stallions induces ex-copula ejaculation within few minutes after administration. These ejaculates have a low volume and an extremely high sperm concentration, ideal for freezing [22]. Sperm flow features during ejaculation in cat and dog [14,24] and in other species [25–28] have also been examined. Results obtained indicate that retrograde flow into the urinary bladder is a normal component of the ejaculatory process [25–28] and it is not induced by electrical stimuli during E.E. [14]. In addition, it was reported that anaesthesia of cats with ketamine does not determine spermatozoal displacement into the urinary bladder [14], and, in dogs, administration of xylazine increases retrograde flow [24]. The objective of this study was to determine the influence of ketamine or medetomidine administration on cat sperm quality and their effects on sperm flow with or without E.E. 2. Materials and methods Three experiments were performed to evaluate how ketamine (Ketavet1 100; Farmaceutici Gellini, Latina, Italia) or medetomidine (Domitor1; Pfizer S.r.l., Roma-Latina, Italia) administration affects sperm quality and sperm flow. In all cases, pharmacological restriction and analgesia after drugs administration were assessed on the basis of heart rate, respiration rate, muscle exertion. During sperm collection, if these parameters indicated an inadequate pharmacological restriction or analgesia, cats were excluded from the study.
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A total of 41 European cats were included in the experiments (n = 9 in Experiment 1; n = 12 in Experiment 2; n = 20 in Experiment 3). The toms used were privately owned, 2–7 years old, weighing 3–5 kg, and they were allowed to access outdoors. The study was completed from March to June. The experiments were approved by Ethic-scientific Committee of Alma Mater Studiorum, University of Bologna (Prot.: 7639-X/6-27/ 06/2002). 2.1. Experiment 1 In this experiment ketamine or medetomidine effects on cat sperm quality after collection by E.E. have been assessed. A total of 13 animals were electroejaculated a first time, after ketamine administration, in order to obtain subjects with the same period of abstinence from sexual activity. After a rest period of 4 days, cats were electroejaculated again, following ketamine administration, and 9 of them were included in the experiment, on the basis of sperm mean concentration (>90 106 spermatozoa/mL). The experiment started 4 days after the last sperm collection. Each cat included in the experiment (n = 9), was collected twice, with a rest period of 4 days between the first and the second collection. During the period between sperm collections, the cats were housed in the Obstetrical and Gynaecological Section of Veterinary Clinical Department. The first collection was achieved by E.E. after ketamine administration (20 mg/kg i.m.—K1 group) and, 4 days apart, the second collection was done by E.E. following medetomidine administration (130– 140 mg/kg i.m.—M1 group). E.E. was performed as described by Howard et al. [10]: 3 sets of electrical stimuli (80 in all) with 2–3 min of break between sets. The first set consisted of 10 stimuli at 2 V, 10 at 3 V and 10 at 4 V. The second set consisted of 10 stimuli at 3 V, 10 at 4 V, 10 at 5 V. Finally, the third set consisted of 10 stimuli at 4 V and 10 stimuli at 5 V. The semen ejaculated was collected in a 1.5 mL prewarmed (37 8C) Eppendorf tube and sperm quality was assessed. The following sperm parameters were evaluated: volume (mL), concentration (106 spermatozoa/mL), pH, motility (%), progressive motility (scale of 0–5) [1], viability (%), abnormal spermatozoa (%), spermatozoa with normal acrosomes (%). Sperm volume was determined using a variable volume pipette. Sperm concentration was assessed, after fixation of an aliquot of semen in buffered formole saline, with a phasecontrast microscope at 100 magnification, using a Bu¨rker chamber. Motility was subjectively evaluated as
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percent motility and progressive motility [1] at a phasecontrast microscope (100). Viability was estimated by eosin-nigrosin staining and the numbers of abnormal spermatozoa and of spermatozoa with normal acrosome were established using fast green FCF-rose Bengal staining. Stained smears were observed at an optical microscope counting 200 sperm at 400 (viability) and 1000 magnification (abnormal spermatozoa and spermatozoa with normal acrosome). For each analysis, about 5 mL of semen was used. Only pH was assessed on total sperm volume.
Number of spermatozoa in the urinary bladder after E.E. = post-E.E. urine volume adjusted concentration. Number of spermatozoa displaced in urethra after E.E. = number of spermatozoa in the urinary bladder after E.E. + total number of spermatozoa in the ejaculate. % of retrograde flow = (number of spermatozoa in the urinary bladder after E.E./number of spermatozoa displaced in urethra after E.E.) 100. 2.3. Experiment 3
2.2. Experiment 2 Ketamine or medetomidine effects on sperm flow in electroejaculated cats were studied. Also the animals (n = 12) used in this experiment were electroejaculated a first time, after ketamine administration, in order to equalize the period of abstinence from sexual activity. After a rest period of 4 days, cats were electroejaculated again, following ketamine administration, and, on the basis of sperm quality, animals were equally divided (6 subjects/group) in order to obtain two homogeneous groups. The experiment started 4 days after the last sperm collection. During the period between sperm collections, the cats were housed in the Obstetrical and Gynaecological Section of Veterinary Clinical Department. Cats were treated with ketamine (20 mg/kg i.m.—K2 group, n = 6) or medetomidine (130–140 mg/ kg i.m.—M2 group, n = 6). When the pharmacological effect was obtained and before performing E.E., 0.5– 1.5 mL of urine (pre-E.E. urine) were collected from each cat by cystocentesis with a 2.5 mL syringe connected to a 22 gauge needle. After E.E., the urinary bladder was emptied and the urine (post-E.E. urine) collected by cystocentesis was evaluated for volume. Sperm concentration in pre-E.E. and post-E.E. urine were assessed. Semen collected by E.E. was evaluated as reported in Experiment 1. As previously reported by Dooley et al. [14], the following parameters were then calculated: adjusted spermatozoa concentration (106 per mL), spermatozoa total number in the urinary bladder after E.E. (106), number of spermatozoa displaced in urethra after E.E. (106), percentage of retrograde flow (evaluated as percentage of spermatozoa displaced that was found in the urine after E.E.). The parameters above mentioned were calculated with the following formulas: Adjusted spermatozoa concentration = sperm concentration in post-E.E. urine sperm concentration in pre-E.E. urine.
This experiment was performed in order to evaluate ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection. Twenty tomcats were included. The animals, randomly divided into 3 groups, were treated with ketamine (20 mg/kg i.m.—K3 group, n = 7), medetomidine (130– 140 mg/kg i.m.—M3 group, n = 7) or not treated (C group, n = 6). From each cat (K3, M3 and C groups) 0.5– 1.5 mL of urine were collected by cystocentesis (urine by cystocentesis) with a 2.5 mL syringe connected to a 22 gauge needle. Then, the urinary bladder was emptied with a 4 Fr tomcat urinary catheter and urine volume (urine by catheter) was measured and the following parameters were determined [14]: Sperm concentration in urine by cystocentesis (106 per mL). Sperm concentration in urine by catheter (106 per mL). Adjusted concentration (106 per mL) = sperm concentration in urine by catheter sperm concentration in urine by cystocentesis. Total number of spermatozoa displaced in urethra = volume of urine by catheter adjusted concentration. 2.4. Statistical analyses Statistical analyses were performed using a software package (Statistica for Windows—Stat Soft). pValues < 0.05 were considered significant. Values are expressed as mean S.D. Experiment 1: differences were counted with a paired t-test or a Wilcoxon test, depending on the distribution of the data. Experiment 2: data obtained from preliminary collection, in order to obtain two homogeneous groups, were analysed with a t-test for independent samples after evaluating normal distribution. Data from evaluation of sperm collected by E.E. after ketamine or
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96.56 1.23 96.56 1.50 10.55 5.36 9.77 4.43 93.67 3.80 93.67 4.70 5.00 0.00 5.00 0.00 91.11 4.16 90.00 6.12 198.88 20.70 a 112.66 12.90 b 0.12 0.05 0.11 0.05 M1 (n = 9) K1 (n = 9)
a vs. b, p < 0.01.
Progressive motility Motility (%) pH Concentration (106 per mL)
Sperm parameters evaluated after ketamine administration in order to divide cats into two groups were volume (M2 group: 0.06 0.03 mL; K2 group: 0.05 0.03 mL), concentration (M2 group: 19.67 6.30 106 per mL; K2 group: 19.50 5.29 106 per mL), motility (M2 group: 60.00 6.30%; K2 group: 58.33 8.00%), mean total number of spermatozoa (M2 group: 1.28 0.90 106; K2 group: 1.05 1.07 106). No significant differences ( p > 0.05) were found between groups. Data obtained are reported in Tables 2 and 3. In M2 group, mean sperm concentration (73.16 15.90 106 per mL) and mean total number of spermatozoa (4.51 1.60 106) were significantly higher ( p < 0.01) than in K2 group (18.83 8.63 106 per mL and 1.10 1.07 106, respectively). The number of spermatozoa displaced in urethra in M2 group (20.50 13.30 106) was significantly higher ( p < 0.05) than in K2 group (3.98 2.79 106). No significant differences were found between groups in the percentages of retrograde flow and in other sperm parameters.
Volume (mL)
3.2. Experiment 2
Group
Three cats treated with medetomidine were not electroejaculated because of an inadequate pharmacological restriction or analgesia, evaluated on the basis of heart rate, respiration rate, muscle exertion. Data reported in Table 1 shows that mean sperm concentration and mean total number of spermatozoa were significantly higher ( p < 0.01) in M1 group (198.88 20.70 106 per mL and 25.01 10.40 106, respectively) than in K1 group (112.66 12.90 106 per mL and 13.09 5.73 106, respectively). No other significant differences in semen evaluation were found between groups.
Table 1 Evaluation of sperm collected by electroejaculation after medetomidine (M1) or ketamine (K1) administration in Experiment 1
3.1. Experiment 1
Viability (%)
Abnormalities (%)
3. Results
7.07 0.29 7.01 0.05
Normal acrosomes (%)
Number of sperm (106)
medetomine administration were compared using a ttest for independent samples or a Wald–Wolfowitz runs test, depending on the distribution of the data. Data from evaluation of urine pre- and post-E.E. after ketamine and medetomidine administration were compared using a t-test for independent samples or a Wald–Wolfowitz runs test, depending on the distribution of the data. Experiment 3: differences were counted with ANOVA or Kruskall–Wallis ANOVA, depending on the distribution of the data.
25.01 10.40 a 13.09 5.73 b
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Table 2 Evaluation of sperm collected by electroejaculation after ketamine (K2) or medetomine (M2) administration in Experiment 2 Group
Volume (mL)
Concentration (106 per mL)
pH
Motility (%)
Progressive motility
Viability (%)
Abnormalities (%)
Normal acrosomes (%)
Number of sperm (106)
M2 (n = 6) K2 (n = 6)
0.07 0.02 0.07 0.05
73.16 15.90 a 18.83 8.63 b
7.00 0.36 7.02 0.31
65.00 13.80 61.66 14.00
4.50 0.55 4.50 0.55
83.83 4.12 82.83 4.07
16.83 6.91 18.50 6.32
91.17 4.58 91.83 3.82
4.51 1.60 a 1.10 1.07 b D. Zambelli et al. / Theriogenology 68 (2007) 796–803
a vs. b, p < 0.01.
Table 3 Evaluation of urine pre- and post-electroejaculation (EE) after ketamine (K2) and medetomidine (M2) administration in Experiment 2 Group
M2 (n = 6) K2 (n = 6)
Pre-E.E. urine
Post-E.E. urine
Number of spermatozoa
Volume (mL)
Sperm concentration (106 per mL)
Volume (mL)
Sperm concentration (106 per mL)
Adjusted sperm concentration (106 per mL)
Urinary bladder post-E.E. (106)
Urethra post-E.E. (106)
Retrograde flow (%)
0.86 0.43 0.83 0.39
0.01 0.02 0.00 0.00
35.60 19.46 31.30 17.68
0.79 1.12 0.09 0.10
0.78 1.13 0.09 0.09
15.99 14.79 2.89 3.38
20.50 13.30 a 3.98 2.79 b
58.57 38.15 54.46 45.55
a vs. b, p < 0.05.
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Table 4 Evaluation of urine without electroejaculation after medetomidine (M3) and ketamine (K3) administration in Experiment 3 Group
M3 (n = 6) K3 (n = 6) C (n = 6)
Urine by cystocentesis
Urine by catheter
Number of spermatozoa
Volume (mL)
Sperm concentration (106 per mL)
Volume (mL)
Sperm concentration (106 per mL)
Adjusted sperm concentration (106 per mL)
Urethra (106)
0.84 0.55 0.79 0.38 0.52 0.04
0.18 0.20 0.02 0.02 0.08 0.06
19.50 18.12 20.57 10.55 21.50 5.47
2.18 1.56 a 0.02 0.02 c 0.09 0.07 b
2.007 1.454 a 0.002 0.003 b 0.013 0.015 b
22.12 17.64 a 0.15 0.16 b 0.31 0.36 b
a vs. b vs. c, p < 0.05.
3.3. Experiment 3 Data obtained in this experiment are reported in Table 4. Number of spermatozoa displaced in urethra were significantly higher ( p < 0.05) in M3 group (22.12 17.64 106) than in K3 group (0.15 0.16 106) and in C group (0.31 0.36 106). 4. Discussion The aim of this research was to evaluate the different effects of two commonly used drugs for anaesthesia in the cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species. This is the first study about these topics in the cat. The results obtained in Experiments 1 and 2 showed that medetomidine, used for pharmacological restriction for cat E.E., permitted to obtain a higher mean sperm concentration and a higher mean total number of spermatozoa than in ketamine anaesthetised cats, without increasing the percentage of retrograde flow. The mean sperm concentration was higher in Experiment 1 than in Experiment 2 and it could be explained as a consequence of the animal selection in the first experiment (only cats with >90 106 spermatozoa/mL were used). During sperm collection, pharmacological restriction and analgesia after drugs administration were not assessed on the basis of a specific pain scale, but clinical parameters as heart rate, respiration rate and muscle exertion were used. This permitted to observe clinical modifications referable to inadequate analgesia in three animals treated with medetomidine, that were not included in the study. In the present study a rest period of 4 days between semen collections was chosen, on the basis of the results reported about the effect of ejaculation intervals on semen quality [16], and of ketamine and medetomidine pharmacokinetic (half-lives: 40–60 min [20] and 0.97– 1.60 h, respectively [21]). Since the rest period chosen
in this study was long enough to ensure a lack of drug influence, and repeated semen collections by E.E. in the same cat alternatively using the two drugs have been done (unpublished preliminary data) without noticing semen quality modifications, it was reasonable to assume that the order of anaesthetic administration (ketamine–medetomidine or vice versa) for semen collection does not modify sperm quality in the cat. Thus in Experiment 1, ketamine was used first in all cats. Results obtained in that experiment also confirmed the assumption, being sperm quality similar for collections after medetomidine or ketamine administration. The only difference found in sperm quantity was attributable to the different mechanisms of action of the drugs, and it was the cue for Experiment 2. Effects of a-adrenergic agents on sperm flow in dogs [24] and cats [14] and their use to induce or improve excopula ejaculation in other species [22,29–32] has been previously reported. In aspermic men [33], a-adrenergic agonists administration permitted to achieve anterograde ejaculation and, in dogs, combined use of a-adrenergic agents and b-adrenoreceptor antagonists increased spermatozoal output [14,24]. Other Authors [29–31] reported the possibility to pharmacologically obtain, after a-adrenergic agents administration, ejaculates of low volume and extremely high sperm concentration in domestic stallions or in injured horses that are unable to mount. Findings of these studies and results of our Experiments 1 and 2 induced us to evaluate, in the cat, ketamine and medetomidine effects on urethral sperm flow, without any stimulation for sperm collection (Experiment 3). Adrenergic agents action on a-adrenoreceptors determine the contraction of vas deferens and participate in the contraction of the trigone and sphincter of the urinary bladder during ejaculation [34–37]. This could be an explanation for the higher number of spermatozoa released in the urethra after medetomidine administration than after ketamine administration, in Experiment 3.
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The very high concentration of spermatozoa (22.12 17.64 106) in urethra observed after medetomidine administration in Experiment 3 suggested the possibility to collect spermatozoa in the cat using a new method, successively tested with good results [38]. In the domestic cat, a-adrenergic agents administration only determined the release of spermatozoa in urethra [38], without induction of ejaculation, while it has been reported in the horse [29–31]. No significant differences in the percentage of retrograde flow were observed after medetomidine or ketamine administration. This is not in accordance with results previously obtained in the dog [14], where a2adrenergic agonists have a facilitatory effect on retrograde flow of spermatozoa into the bladder, even if retrograde flow is considered a normal component of the ejaculatory process and its magnitude is not influenced by electrical stimulation during E.E. [14]. In conclusion, sperm collection in the cat by E.E. after medetomidine administration permitted to obtain a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction, an adequate analgesia (in 21 out of 24 cats) and without differences in the percentage of sperm retrograde flow. References [1] Howard JG. Feline semen analysis and artificial insemination. In: Kirk-Bonagura, editor. Kirk’s current veterinary therapy. XI. Small animal practice. Philadelphia: WB Saunders; 1992. p. 929–38. [2] Sojka NJ, Jennings LL, Hamner CE. Artificial insemination in the cat (Felis catus L.). Lab Anim Care 1970;20:198–204. [3] Goodrowe KL, Wall RJ, O’Brien SJ, Schmidt PM, Wildt DE. Developmental competence of domestic cat follicular oocytes after fertilization in vitro. Biol Reprod 1988;39:355–72. [4] Sojka NJ. Management of artificial breeding in cats. In: Morrow DA, editor. Current therapy in theriogenology: diagnosis, treatment and prevention of reproductive diseases in large and small animals. 2nd ed., Philadelphia: WB Saunders; 1986. p. 805–8. [5] Scott P. Cats. In: Hafez ES, editor. Reproduction and breeding techniques for laboratory animals. Philadelphia: Lea and Febiger; 1970. p. 205. [6] Platz CC, Fallis T, Demorest N, Seager SWJ. Semen collection, freezing and insemination in the domestic cat. In: Proceedings of the 8th international congress on animal reproduction artificial insemination, vol IV; 1976. p. 1053–6. [7] Platz CC, Seager SW. Semen collection by electroejaculation in the domestic cat. J Am Vet Med Assoc 1978;173:1353–5. [8] Johnstone IP. Electroejaculation in domestic cat. Aust Vet J 1984;61:155–8. [9] Pineda MH, Dooley MP, Martin PA. Long term study on the effects of electroejaculation o seminal characteristics of the domestic cat. Am J Vet Res 1984;45:1038–40. [10] Howard JG, Brown JL, Bush M, Wildt DE. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary–gonadal
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deferens as revealed by selective agonists and antagonists. Br J Pharm 1980;71:445–58. Borda ES, Peredo H, Gimeno MF. Alpha adrenergic stimulation modified prostaglandins release in vas deferens. Prostaglandins 1983;26:701–10. Trachte GJ. Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens. Prostaglandins 1987;33: 25–35. Barwin BN, McKay D, Jolly EE, et al. Retrograde ejaculation. In: Emperaire JC, Audebert A, Hafez ESE, editors. Homologous artificial insemination (AIH). Boston: Martinus Nijhoff Publishers; 1980. p. 127–37. Zambelli D, Prati F, Merlo B, Cunto M. Collection of semen by urethral catheterization after pharmacologically induced spermatozoa releasing in the domestic cat. In: Proceedings of the 5th Biannual Congress. European Veterinary Society for Small Animal Reproduction (EVSSAR); 2006.p. 300.
Effects of ketamine or medetomidine administration on quality of electroejaculated sperm and on sperm flow in the domestic cat D. Zambelli *, M. Cunto, F. Prati, B. Merlo Veterinary Clinical Department, Obstetrical and Gynaecological Section, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy Received 22 December 2006; received in revised form 8 June 2007; accepted 8 June 2007
Abstract The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species were evaluated performing three experiments. This is the first study about these topics in the domestic cat. In Experiment 1, ketamine or medetomidine effects on cat sperm quality after collection by electroejaculation (E.E.) have been assessed in nine animals. Results showed that mean sperm concentration was significantly higher ( p < 0.01) after medetomidine than after ketamine administration. In Experiment 2, ketamine or medetomidine effects on sperm flow in 12 electroejaculated cats were studied. Mean sperm concentration and mean total number of spermatozoa resulted significantly higher ( p < 0.01) in medetomidine than in ketamine treated animals. The number of spermatozoa displaced in urethra was significantly higher ( p < 0.01) using medetomidine. No significant differences were observed in percentages of retrograde flow. In Experiment 3, ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection, were evaluated. Data obtained showed a significantly higher ( p < 0.05) number of spermatozoa displaced in urethra after medetomidine than after ketamine injection. In conclusion, E.E. in the cat after medetomidine administration determined a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction and without increasing sperm retrograde flow. # 2007 Elsevier Inc. All rights reserved. Keywords: Ketamine; Medetomidine; Electroejaculation; Sperm; Domestic cat
1. Introduction The domestic cat serves as an important experimental model in the study of endangered wild felids diseases. Assisted reproduction is an essential tool in the safeguard plans of these non-domestic felids that, in captivity, often reproduce poorly and frequently reveal a bad sperm quality [1]. Both semen collection and artificial insemination (AI) in cats have been first
* Corresponding author. Tel.: +39 0512097572; fax: +39 0512097568. E-mail address: [email protected] (D. Zambelli). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.06.008
described more than 30 years ago [2], but these procedures are not a routine in veterinary practice. Sperm collection is essential for AI and IVF [3], besides diagnostic purposes and use in research [4]. Sperm collection in the cat is usually performed by E.E. [5–10] or artificial vagina (A.V.) [11–14], but the first is often considered the method of choice in many clinical and research circumstances [7,15] because it allows sperm collection from every subject that can be safely anesthetised, without previous training. Both E.E. and A.V. permit to obtain whole semen with good quality [5–15]. Also after repeated collections, at intervals of every other day and every 3 days, sperm quality is stable for up to 10 days [16].
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Different anaesthetic protocols have been reported in literature to perform electroejaculation in the cat, but their effects on sperm quality have never been studied. The most commonly used drug resulted to be ketamine HCl, either alone or in association with medetomidine, even if association with xylazine, diazepam, phenotiazine derivatives (e.g. acepromazine maleate) or use of inhalation anaesthetics (e.g. halothane, isoflurane) or propofol have also been reported [1,17–19]. Studies about ketamine and medetomidine pharmacokinetics reported a rapid elimination of these drugs from plasma/serum that occurred with half-lives ranging between 40–60 min [20] and 0.97–1.60 h, respectively [21]. Particularly, it was estimated that ketamine still present in the body, 7 h after administration, is less than 0.1% [20]. a-Adrenergic agents are known to influence erection and ejaculation [22] and ejaculatory reflex is considered to be primarily an a-adrenergically mediated event [23]. It has been reported that xylazine in stallions induces ex-copula ejaculation within few minutes after administration. These ejaculates have a low volume and an extremely high sperm concentration, ideal for freezing [22]. Sperm flow features during ejaculation in cat and dog [14,24] and in other species [25–28] have also been examined. Results obtained indicate that retrograde flow into the urinary bladder is a normal component of the ejaculatory process [25–28] and it is not induced by electrical stimuli during E.E. [14]. In addition, it was reported that anaesthesia of cats with ketamine does not determine spermatozoal displacement into the urinary bladder [14], and, in dogs, administration of xylazine increases retrograde flow [24]. The objective of this study was to determine the influence of ketamine or medetomidine administration on cat sperm quality and their effects on sperm flow with or without E.E. 2. Materials and methods Three experiments were performed to evaluate how ketamine (Ketavet1 100; Farmaceutici Gellini, Latina, Italia) or medetomidine (Domitor1; Pfizer S.r.l., Roma-Latina, Italia) administration affects sperm quality and sperm flow. In all cases, pharmacological restriction and analgesia after drugs administration were assessed on the basis of heart rate, respiration rate, muscle exertion. During sperm collection, if these parameters indicated an inadequate pharmacological restriction or analgesia, cats were excluded from the study.
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A total of 41 European cats were included in the experiments (n = 9 in Experiment 1; n = 12 in Experiment 2; n = 20 in Experiment 3). The toms used were privately owned, 2–7 years old, weighing 3–5 kg, and they were allowed to access outdoors. The study was completed from March to June. The experiments were approved by Ethic-scientific Committee of Alma Mater Studiorum, University of Bologna (Prot.: 7639-X/6-27/ 06/2002). 2.1. Experiment 1 In this experiment ketamine or medetomidine effects on cat sperm quality after collection by E.E. have been assessed. A total of 13 animals were electroejaculated a first time, after ketamine administration, in order to obtain subjects with the same period of abstinence from sexual activity. After a rest period of 4 days, cats were electroejaculated again, following ketamine administration, and 9 of them were included in the experiment, on the basis of sperm mean concentration (>90 106 spermatozoa/mL). The experiment started 4 days after the last sperm collection. Each cat included in the experiment (n = 9), was collected twice, with a rest period of 4 days between the first and the second collection. During the period between sperm collections, the cats were housed in the Obstetrical and Gynaecological Section of Veterinary Clinical Department. The first collection was achieved by E.E. after ketamine administration (20 mg/kg i.m.—K1 group) and, 4 days apart, the second collection was done by E.E. following medetomidine administration (130– 140 mg/kg i.m.—M1 group). E.E. was performed as described by Howard et al. [10]: 3 sets of electrical stimuli (80 in all) with 2–3 min of break between sets. The first set consisted of 10 stimuli at 2 V, 10 at 3 V and 10 at 4 V. The second set consisted of 10 stimuli at 3 V, 10 at 4 V, 10 at 5 V. Finally, the third set consisted of 10 stimuli at 4 V and 10 stimuli at 5 V. The semen ejaculated was collected in a 1.5 mL prewarmed (37 8C) Eppendorf tube and sperm quality was assessed. The following sperm parameters were evaluated: volume (mL), concentration (106 spermatozoa/mL), pH, motility (%), progressive motility (scale of 0–5) [1], viability (%), abnormal spermatozoa (%), spermatozoa with normal acrosomes (%). Sperm volume was determined using a variable volume pipette. Sperm concentration was assessed, after fixation of an aliquot of semen in buffered formole saline, with a phasecontrast microscope at 100 magnification, using a Bu¨rker chamber. Motility was subjectively evaluated as
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percent motility and progressive motility [1] at a phasecontrast microscope (100). Viability was estimated by eosin-nigrosin staining and the numbers of abnormal spermatozoa and of spermatozoa with normal acrosome were established using fast green FCF-rose Bengal staining. Stained smears were observed at an optical microscope counting 200 sperm at 400 (viability) and 1000 magnification (abnormal spermatozoa and spermatozoa with normal acrosome). For each analysis, about 5 mL of semen was used. Only pH was assessed on total sperm volume.
Number of spermatozoa in the urinary bladder after E.E. = post-E.E. urine volume adjusted concentration. Number of spermatozoa displaced in urethra after E.E. = number of spermatozoa in the urinary bladder after E.E. + total number of spermatozoa in the ejaculate. % of retrograde flow = (number of spermatozoa in the urinary bladder after E.E./number of spermatozoa displaced in urethra after E.E.) 100. 2.3. Experiment 3
2.2. Experiment 2 Ketamine or medetomidine effects on sperm flow in electroejaculated cats were studied. Also the animals (n = 12) used in this experiment were electroejaculated a first time, after ketamine administration, in order to equalize the period of abstinence from sexual activity. After a rest period of 4 days, cats were electroejaculated again, following ketamine administration, and, on the basis of sperm quality, animals were equally divided (6 subjects/group) in order to obtain two homogeneous groups. The experiment started 4 days after the last sperm collection. During the period between sperm collections, the cats were housed in the Obstetrical and Gynaecological Section of Veterinary Clinical Department. Cats were treated with ketamine (20 mg/kg i.m.—K2 group, n = 6) or medetomidine (130–140 mg/ kg i.m.—M2 group, n = 6). When the pharmacological effect was obtained and before performing E.E., 0.5– 1.5 mL of urine (pre-E.E. urine) were collected from each cat by cystocentesis with a 2.5 mL syringe connected to a 22 gauge needle. After E.E., the urinary bladder was emptied and the urine (post-E.E. urine) collected by cystocentesis was evaluated for volume. Sperm concentration in pre-E.E. and post-E.E. urine were assessed. Semen collected by E.E. was evaluated as reported in Experiment 1. As previously reported by Dooley et al. [14], the following parameters were then calculated: adjusted spermatozoa concentration (106 per mL), spermatozoa total number in the urinary bladder after E.E. (106), number of spermatozoa displaced in urethra after E.E. (106), percentage of retrograde flow (evaluated as percentage of spermatozoa displaced that was found in the urine after E.E.). The parameters above mentioned were calculated with the following formulas: Adjusted spermatozoa concentration = sperm concentration in post-E.E. urine sperm concentration in pre-E.E. urine.
This experiment was performed in order to evaluate ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection. Twenty tomcats were included. The animals, randomly divided into 3 groups, were treated with ketamine (20 mg/kg i.m.—K3 group, n = 7), medetomidine (130– 140 mg/kg i.m.—M3 group, n = 7) or not treated (C group, n = 6). From each cat (K3, M3 and C groups) 0.5– 1.5 mL of urine were collected by cystocentesis (urine by cystocentesis) with a 2.5 mL syringe connected to a 22 gauge needle. Then, the urinary bladder was emptied with a 4 Fr tomcat urinary catheter and urine volume (urine by catheter) was measured and the following parameters were determined [14]: Sperm concentration in urine by cystocentesis (106 per mL). Sperm concentration in urine by catheter (106 per mL). Adjusted concentration (106 per mL) = sperm concentration in urine by catheter sperm concentration in urine by cystocentesis. Total number of spermatozoa displaced in urethra = volume of urine by catheter adjusted concentration. 2.4. Statistical analyses Statistical analyses were performed using a software package (Statistica for Windows—Stat Soft). pValues < 0.05 were considered significant. Values are expressed as mean S.D. Experiment 1: differences were counted with a paired t-test or a Wilcoxon test, depending on the distribution of the data. Experiment 2: data obtained from preliminary collection, in order to obtain two homogeneous groups, were analysed with a t-test for independent samples after evaluating normal distribution. Data from evaluation of sperm collected by E.E. after ketamine or
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96.56 1.23 96.56 1.50 10.55 5.36 9.77 4.43 93.67 3.80 93.67 4.70 5.00 0.00 5.00 0.00 91.11 4.16 90.00 6.12 198.88 20.70 a 112.66 12.90 b 0.12 0.05 0.11 0.05 M1 (n = 9) K1 (n = 9)
a vs. b, p < 0.01.
Progressive motility Motility (%) pH Concentration (106 per mL)
Sperm parameters evaluated after ketamine administration in order to divide cats into two groups were volume (M2 group: 0.06 0.03 mL; K2 group: 0.05 0.03 mL), concentration (M2 group: 19.67 6.30 106 per mL; K2 group: 19.50 5.29 106 per mL), motility (M2 group: 60.00 6.30%; K2 group: 58.33 8.00%), mean total number of spermatozoa (M2 group: 1.28 0.90 106; K2 group: 1.05 1.07 106). No significant differences ( p > 0.05) were found between groups. Data obtained are reported in Tables 2 and 3. In M2 group, mean sperm concentration (73.16 15.90 106 per mL) and mean total number of spermatozoa (4.51 1.60 106) were significantly higher ( p < 0.01) than in K2 group (18.83 8.63 106 per mL and 1.10 1.07 106, respectively). The number of spermatozoa displaced in urethra in M2 group (20.50 13.30 106) was significantly higher ( p < 0.05) than in K2 group (3.98 2.79 106). No significant differences were found between groups in the percentages of retrograde flow and in other sperm parameters.
Volume (mL)
3.2. Experiment 2
Group
Three cats treated with medetomidine were not electroejaculated because of an inadequate pharmacological restriction or analgesia, evaluated on the basis of heart rate, respiration rate, muscle exertion. Data reported in Table 1 shows that mean sperm concentration and mean total number of spermatozoa were significantly higher ( p < 0.01) in M1 group (198.88 20.70 106 per mL and 25.01 10.40 106, respectively) than in K1 group (112.66 12.90 106 per mL and 13.09 5.73 106, respectively). No other significant differences in semen evaluation were found between groups.
Table 1 Evaluation of sperm collected by electroejaculation after medetomidine (M1) or ketamine (K1) administration in Experiment 1
3.1. Experiment 1
Viability (%)
Abnormalities (%)
3. Results
7.07 0.29 7.01 0.05
Normal acrosomes (%)
Number of sperm (106)
medetomine administration were compared using a ttest for independent samples or a Wald–Wolfowitz runs test, depending on the distribution of the data. Data from evaluation of urine pre- and post-E.E. after ketamine and medetomidine administration were compared using a t-test for independent samples or a Wald–Wolfowitz runs test, depending on the distribution of the data. Experiment 3: differences were counted with ANOVA or Kruskall–Wallis ANOVA, depending on the distribution of the data.
25.01 10.40 a 13.09 5.73 b
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Table 2 Evaluation of sperm collected by electroejaculation after ketamine (K2) or medetomine (M2) administration in Experiment 2 Group
Volume (mL)
Concentration (106 per mL)
pH
Motility (%)
Progressive motility
Viability (%)
Abnormalities (%)
Normal acrosomes (%)
Number of sperm (106)
M2 (n = 6) K2 (n = 6)
0.07 0.02 0.07 0.05
73.16 15.90 a 18.83 8.63 b
7.00 0.36 7.02 0.31
65.00 13.80 61.66 14.00
4.50 0.55 4.50 0.55
83.83 4.12 82.83 4.07
16.83 6.91 18.50 6.32
91.17 4.58 91.83 3.82
4.51 1.60 a 1.10 1.07 b D. Zambelli et al. / Theriogenology 68 (2007) 796–803
a vs. b, p < 0.01.
Table 3 Evaluation of urine pre- and post-electroejaculation (EE) after ketamine (K2) and medetomidine (M2) administration in Experiment 2 Group
M2 (n = 6) K2 (n = 6)
Pre-E.E. urine
Post-E.E. urine
Number of spermatozoa
Volume (mL)
Sperm concentration (106 per mL)
Volume (mL)
Sperm concentration (106 per mL)
Adjusted sperm concentration (106 per mL)
Urinary bladder post-E.E. (106)
Urethra post-E.E. (106)
Retrograde flow (%)
0.86 0.43 0.83 0.39
0.01 0.02 0.00 0.00
35.60 19.46 31.30 17.68
0.79 1.12 0.09 0.10
0.78 1.13 0.09 0.09
15.99 14.79 2.89 3.38
20.50 13.30 a 3.98 2.79 b
58.57 38.15 54.46 45.55
a vs. b, p < 0.05.
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Table 4 Evaluation of urine without electroejaculation after medetomidine (M3) and ketamine (K3) administration in Experiment 3 Group
M3 (n = 6) K3 (n = 6) C (n = 6)
Urine by cystocentesis
Urine by catheter
Number of spermatozoa
Volume (mL)
Sperm concentration (106 per mL)
Volume (mL)
Sperm concentration (106 per mL)
Adjusted sperm concentration (106 per mL)
Urethra (106)
0.84 0.55 0.79 0.38 0.52 0.04
0.18 0.20 0.02 0.02 0.08 0.06
19.50 18.12 20.57 10.55 21.50 5.47
2.18 1.56 a 0.02 0.02 c 0.09 0.07 b
2.007 1.454 a 0.002 0.003 b 0.013 0.015 b
22.12 17.64 a 0.15 0.16 b 0.31 0.36 b
a vs. b vs. c, p < 0.05.
3.3. Experiment 3 Data obtained in this experiment are reported in Table 4. Number of spermatozoa displaced in urethra were significantly higher ( p < 0.05) in M3 group (22.12 17.64 106) than in K3 group (0.15 0.16 106) and in C group (0.31 0.36 106). 4. Discussion The aim of this research was to evaluate the different effects of two commonly used drugs for anaesthesia in the cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species. This is the first study about these topics in the cat. The results obtained in Experiments 1 and 2 showed that medetomidine, used for pharmacological restriction for cat E.E., permitted to obtain a higher mean sperm concentration and a higher mean total number of spermatozoa than in ketamine anaesthetised cats, without increasing the percentage of retrograde flow. The mean sperm concentration was higher in Experiment 1 than in Experiment 2 and it could be explained as a consequence of the animal selection in the first experiment (only cats with >90 106 spermatozoa/mL were used). During sperm collection, pharmacological restriction and analgesia after drugs administration were not assessed on the basis of a specific pain scale, but clinical parameters as heart rate, respiration rate and muscle exertion were used. This permitted to observe clinical modifications referable to inadequate analgesia in three animals treated with medetomidine, that were not included in the study. In the present study a rest period of 4 days between semen collections was chosen, on the basis of the results reported about the effect of ejaculation intervals on semen quality [16], and of ketamine and medetomidine pharmacokinetic (half-lives: 40–60 min [20] and 0.97– 1.60 h, respectively [21]). Since the rest period chosen
in this study was long enough to ensure a lack of drug influence, and repeated semen collections by E.E. in the same cat alternatively using the two drugs have been done (unpublished preliminary data) without noticing semen quality modifications, it was reasonable to assume that the order of anaesthetic administration (ketamine–medetomidine or vice versa) for semen collection does not modify sperm quality in the cat. Thus in Experiment 1, ketamine was used first in all cats. Results obtained in that experiment also confirmed the assumption, being sperm quality similar for collections after medetomidine or ketamine administration. The only difference found in sperm quantity was attributable to the different mechanisms of action of the drugs, and it was the cue for Experiment 2. Effects of a-adrenergic agents on sperm flow in dogs [24] and cats [14] and their use to induce or improve excopula ejaculation in other species [22,29–32] has been previously reported. In aspermic men [33], a-adrenergic agonists administration permitted to achieve anterograde ejaculation and, in dogs, combined use of a-adrenergic agents and b-adrenoreceptor antagonists increased spermatozoal output [14,24]. Other Authors [29–31] reported the possibility to pharmacologically obtain, after a-adrenergic agents administration, ejaculates of low volume and extremely high sperm concentration in domestic stallions or in injured horses that are unable to mount. Findings of these studies and results of our Experiments 1 and 2 induced us to evaluate, in the cat, ketamine and medetomidine effects on urethral sperm flow, without any stimulation for sperm collection (Experiment 3). Adrenergic agents action on a-adrenoreceptors determine the contraction of vas deferens and participate in the contraction of the trigone and sphincter of the urinary bladder during ejaculation [34–37]. This could be an explanation for the higher number of spermatozoa released in the urethra after medetomidine administration than after ketamine administration, in Experiment 3.
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The very high concentration of spermatozoa (22.12 17.64 106) in urethra observed after medetomidine administration in Experiment 3 suggested the possibility to collect spermatozoa in the cat using a new method, successively tested with good results [38]. In the domestic cat, a-adrenergic agents administration only determined the release of spermatozoa in urethra [38], without induction of ejaculation, while it has been reported in the horse [29–31]. No significant differences in the percentage of retrograde flow were observed after medetomidine or ketamine administration. This is not in accordance with results previously obtained in the dog [14], where a2adrenergic agonists have a facilitatory effect on retrograde flow of spermatozoa into the bladder, even if retrograde flow is considered a normal component of the ejaculatory process and its magnitude is not influenced by electrical stimulation during E.E. [14]. In conclusion, sperm collection in the cat by E.E. after medetomidine administration permitted to obtain a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction, an adequate analgesia (in 21 out of 24 cats) and without differences in the percentage of sperm retrograde flow. References [1] Howard JG. Feline semen analysis and artificial insemination. In: Kirk-Bonagura, editor. Kirk’s current veterinary therapy. XI. Small animal practice. Philadelphia: WB Saunders; 1992. p. 929–38. [2] Sojka NJ, Jennings LL, Hamner CE. Artificial insemination in the cat (Felis catus L.). Lab Anim Care 1970;20:198–204. [3] Goodrowe KL, Wall RJ, O’Brien SJ, Schmidt PM, Wildt DE. Developmental competence of domestic cat follicular oocytes after fertilization in vitro. Biol Reprod 1988;39:355–72. [4] Sojka NJ. Management of artificial breeding in cats. In: Morrow DA, editor. Current therapy in theriogenology: diagnosis, treatment and prevention of reproductive diseases in large and small animals. 2nd ed., Philadelphia: WB Saunders; 1986. p. 805–8. [5] Scott P. Cats. In: Hafez ES, editor. Reproduction and breeding techniques for laboratory animals. Philadelphia: Lea and Febiger; 1970. p. 205. [6] Platz CC, Fallis T, Demorest N, Seager SWJ. Semen collection, freezing and insemination in the domestic cat. In: Proceedings of the 8th international congress on animal reproduction artificial insemination, vol IV; 1976. p. 1053–6. [7] Platz CC, Seager SW. Semen collection by electroejaculation in the domestic cat. J Am Vet Med Assoc 1978;173:1353–5. [8] Johnstone IP. Electroejaculation in domestic cat. Aust Vet J 1984;61:155–8. [9] Pineda MH, Dooley MP, Martin PA. Long term study on the effects of electroejaculation o seminal characteristics of the domestic cat. Am J Vet Res 1984;45:1038–40. [10] Howard JG, Brown JL, Bush M, Wildt DE. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary–gonadal
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deferens as revealed by selective agonists and antagonists. Br J Pharm 1980;71:445–58. Borda ES, Peredo H, Gimeno MF. Alpha adrenergic stimulation modified prostaglandins release in vas deferens. Prostaglandins 1983;26:701–10. Trachte GJ. Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens. Prostaglandins 1987;33: 25–35. Barwin BN, McKay D, Jolly EE, et al. Retrograde ejaculation. In: Emperaire JC, Audebert A, Hafez ESE, editors. Homologous artificial insemination (AIH). Boston: Martinus Nijhoff Publishers; 1980. p. 127–37. Zambelli D, Prati F, Merlo B, Cunto M. Collection of semen by urethral catheterization after pharmacologically induced spermatozoa releasing in the domestic cat. In: Proceedings of the 5th Biannual Congress. European Veterinary Society for Small Animal Reproduction (EVSSAR); 2006.p. 300.