The reversal of xylazine hydrochloride by yohimbine and 4-aminopyridine in goats

INTRODUCTION Xylazine (an 2-agonist) is the most widely-used agent for chemical restraint in ruminants, including goats. At a dose of 0.02mg/kg administered intramuscularly, xylazine can produce recumbency for 1–2 hours in ruminants, and some residual anorexia and central nervous system (CNS) depression may manifest for up to 24 hours. An antidote for the reversal of xylazine will allow the shortening of the recovery period of goats sedated with xylazine, and therefore reduce the possibility of tympany, regurgitation, pressure damage to nerves or muscles, or other problems associated with the recumbent sedated ruminant. Moreover, because ruminants, and especially goats require small doses of xylazine, an agent to reverse the effects of xylazine will be useful for treating accidental overdoses of xylazine. Studies have shown that yohimbine and 4-aminopyridine antagonise xylazine in sheep, cattle, dogs, cats and some wild animals such as deer and llama. Both drugs reverse xylazine/ ketamine anaesthesia in goats and horses, xylazine/thiopental anaesthesia in goats and xylazine/pentobarbital anaesthesia in horses. Tolazoline and doxapram are effective in antagonising xylazine in goats. However, there is no information available about yohimbine and 4-aminopyridine as antagonists to xylazine in goats. This experiment was planned to study the effects of 4-aminopyridine, yohimbine and their combination in goats treated with xylazine at 2 and 4 times the routine clinical dose. MATERIALS AND METHODS The study involved 24 small East African goats of both sexes weighing 13–25.5 kg and aged 12–36 months. During the study, the goats were housed in indoor pens and were fed with hay, water and mineral licks (Maclic, Twiga Chemicals, Nairobi) ad libitum. Commercial maize bran and maize germ (Unga Feeds, Nairobi) were supplemented regularly. A preliminary study to determine the doses of 4-aminopyridine and yohimbine to be used in the experiments was performed. This involved administering varying doses of these drugs to groups of 5 non-sedated goats. The dose rates were considered adequate if they produced signs of mild CNS stimulation such as trembling, muscle twitching and vocalisation, without signs of extreme stimulation such as convulsions. The study was carried out in 2 phases. During the 1st phase the goats were randomly assigned to 4 groups of 6 goats each. The goats were injected with xylazine (Chanazine, Chanelle Pharmaceutical) intramuscularly at 0.44mg/kg. At the time of maximum sedation, the goats were injected intravenously with 1m sterile water (controls, Group 1), 0.15% 4-aminopyridine at 0.4mg/kg (Group 2), 0.1% yohimbine at 0.25 mg/kg (Group 3), or a combination of both drugs at the same dose rates (Group 4). During the 2nd phase, the combination of 4-aminopyridine (Kyron Lab.) and yohimbine (Kyron Lab.), which produced the fastest recovery in Phase 1, was used to antagonise intramuscular xylazine at 0.88mg/kg in 6 randomly-selected goats. Control animals were not used because of the risk of xylazine overdose. After an overnight fasting period, the goats were weighed before being taken to the study area and given a brief period to adjust to their new surroundings and also recover from any excitement. The heart rate, respiratory rate and rate of ruminal movements were then determined and recorded. The heart rate and rate of ruminal movements were determined by auscultation for 1 and 2 minutes respectively, and recorded as beats/minute and contractions/2 minutes respectively. The


INTRODUCTION
Xylazine (an "2-agonist) is the most widely-used agent for chemical restraint in ruminants, including goats.At a dose of 0.02mg/kg administered intramuscularly, xylazine can produce recumbency for 1-2 hours in ruminants, and some residual anorexia and central nervous system (CNS) depression may manifest for up to 24 hours 13 .An antidote for the reversal of xylazine will allow the shortening of the recovery period of goats sedated with xylazine, and therefore reduce the possibility of tympany, regurgitation, pressure damage to nerves or muscles, or other problems associated with the recumbent sedated ruminant 24 .Moreover, because ruminants, and especially goats require small doses of xylazine, an agent to reverse the effects of xylazine will be useful for treating accidental overdoses of xylazine.
Studies have shown that yohimbine and 4-aminopyridine antagonise xylazine in sheep 9 , cattle 12 , dogs 8,23 , cats 10 and some wild animals such as deer 20 and llama 21 .Both drugs reverse xylazine/ ketamine anaesthesia in goats 14 and horses 11 , xylazine/thiopental anaesthesia in goats 18 and xylazine/pentobarbital anaesthesia in horses 16 .Tolazoline 26 and doxapram 22 are effective in antagonising xylazine in goats.However, there is no information available about yohimbine and 4-aminopyridine as antagonists to xylazine in goats.This experiment was planned to study the effects of 4-aminopyridine, yohimbine and their combination in goats treated with xylazine at 2 and 4 times the routine clinical dose.

MATERIALS AND METHODS
The study involved 24 small East African goats of both sexes weighing 13-25.5 kg and aged 12-36 months.During the study, the goats were housed in indoor pens and were fed with hay, water and mineral licks (Maclic ® , Twiga Chemicals, Nairobi) ad libitum.Commercial maize bran and maize germ (Unga Feeds, Nairobi) were supplemented regularly.
A preliminary study to determine the doses of 4-aminopyridine and yohimbine to be used in the experiments was performed.This involved administering varying doses of these drugs to groups of 5 non-sedated goats.The dose rates were considered adequate if they produced signs of mild CNS stimulation such as trembling, muscle twitching and vocalisation, without signs of extreme stimulation such as convulsions.
The study was carried out in 2 phases.During the 1st phase the goats were randomly assigned to 4 groups of 6 goats each.The goats were injected with xylazine (Chanazine ® , Chanelle Pharmaceutical) intramuscularly at 0.44mg/kg.At the time of maximum sedation, the goats were injected intravenously with 1m sterile water (controls, Group 1), 0.15% 4-aminopyridine at 0.4mg/kg (Group 2), 0.1% yohimbine at 0.25 mg/kg (Group 3), or a combination of both drugs at the same dose rates (Group 4).During the 2nd phase, the combination of 4-aminopyridine (Kyron Lab.) and yohimbine (Kyron Lab.), which produced the fastest recovery in Phase 1, was used to antagonise intramuscular xylazine at 0.88mg/kg in 6 randomly-selected goats.Control animals were not used because of the risk of xylazine overdose.
After an overnight fasting period, the goats were weighed before being taken to the study area and given a brief period to adjust to their new surroundings and also recover from any excitement.The heart rate, respiratory rate and rate of ruminal movements were then determined and recorded.The heart rate and rate of ruminal movements were determined by auscultation for 1 and 2 minutes respectively, and recorded as beats/minute and contractions/2 minutes respectively.The respiratory rate was determined by observation of thoracic and abdominal movement for 1 minute, and recorded as breaths/minute.The presence of pedal and palpebral reflexes as well as noxious stimulation was also determined.The noxious stimuli consisted of pinching with mosquito forceps at the flanks, horn base, scrotal sacs, ventral abdomen and limb extremities.A positive reaction to the noxious stimuli was taken as the withdrawal of the limb for the extremities and twitching of muscles for the other body regions.The reflexes and the reaction to noxious stimuli were recorded as either present or absent.Thereafter, xylazine was injected.The variables were determined again at the time of maximum sedation, and just before the administration of the reversal agents.Sedation was regarded as maximal when the goats became recumbent and could not be aroused easily.
Subsequently, the heart rate, respiratory rate as well as the rate of ruminal movements were monitored at 5, 10, 15, 20, 30, 40 and 50 minutes after the administration of sterile water or the antagonists.The reappearance of reaction to noxious stimulation as well as the reflexes were also assessed at the same time intervals.The standing time was also recorded.This was taken as the time from the injection of the antagonists until the animal could stand unaided when stimulated by hand-clapping, whistling and patting them.The average of the standing times was defined as the mean standing time (MST).
After the animals stood, they were placed in an observation area with hay, maize bran and water available and observed in order to determine the total recovery time.This was taken as the time from injection of xylazine until the time when overt sedation disappeared and the animal could be considered normal in movement and behaviour, including eating and drinking normally.The average of the total recovery times was defined as the mean total recovery time (MTRT).

Statistical analysis
The data for the heart rate, respiratory rate and rate of ruminal movements were analysed using the repeated measures analysis of variance (ANOVA), and the Neuman-Keul's multiple comparison test was used to test for significance.Data for the standing time and total recovery time were analysed using 1-way ANOVA and the Tukey's Student range test used to test for significance of difference.In all analyses, P < 0.05 was considered significant.SAS statistical software was used for the analysis.

RESULTS
Doses of 0.44 mg/kg and 0.88 mg/kg of xylazine were adequate to produce sustained recumbency and marked sedation.Onset of signs occurred within 10 minutes of administration.

Reversal of xylazine at 0.44 mg/kg
Xylazine produced a significant decrease (P < 0.05) in heart rate in all groups (Table 1).The administration of the antagonists produced a significant increase (P < 0.05) in heart rate compared with the pre-antagonist value of the same group, but the heart rate was unaltered in the control group.Groups 2 and 4 had significantly (P < 0.05) higher rates compared with the control group, but despite the increase in heart rate in Group 3, there was no significant difference (P > 0.05) compared with the control group.
Xylazine also produced a significant decrease (P < 0.05) in respiratory rate in all groups of goats (Table 2).After the administration of the antagonist drugs, the respiratory rate increased significantly (P < 0.05) compared with the pre-antagonist rate of the same group, but the respiratory rate was unaltered in the control group.In comparison with the control group, the antagonists increased the respiratory rate significantly (P < 0.05) at some of the times assessed.There was no significant Means with different superscripts are significantly different (P < 0.05).¶ Significantly different (P < 0.05) from the pre-antagonist heart rate.

Time after administration of antagonists (minutes)
Xylazine abolished the ruminal contractions at the time of maximum sedation, and this effect persisted for variable time periods in the groups (Table 3).The ruminal movements were recorded first in Group 4 and last in Group 3.They were not recorded in the control group for at least 40 minutes.At some of the times assessed, Groups 2, 3 and 4 had significantly higher (P < 0.05) rate of ruminal movements compared with the preantagonist value of the same group and also when compared with the control group.
The antagonists significantly decreased (P < 0.05) the MST, whereas the MTRT was significantly (P < 0.05) decreased in Groups 2 and 4 (Table 4).In Group 3, a slight but not significant reduction in the MTRT was noted.The reduction of both variables was greatest in Group 4, and its values were significantly smaller (P < 0.05) than for Group 3.
At the time of maximum sedation, the reflexes were markedly depressed or absent, and there was loss of reaction to the noxious stimulation at the flanks, horn base and ventral abdomen but not at the limb extremities and scrotal sacs.Intravenous administration of the antagonists, alone and in combination, rapidly reversed the 2 variables.

Reversal of xylazine at 0.88 mg/kg
The combination of yohimbine and 4-aminopyridine, which produced the greatest reduction in MST and MTRT in the 1st phase of the study, was used to antagonise xylazine at 0.88 mg/kg.Xylazine significantly decreased (P < 0.05) the heart rate, respiratory rate and rate of ruminal movements from pre-xylazine values of 64.8 ± 3.8, 17.8 ± 1.2, and 2.8 ± 0.3 to 49.0 ± 1.6, 14.0 ± 1.9 and 0 respectively, at the time of maximum sedation.The antagonist combination reversed the xylazine-induced decrease in the heart rate and rate of ruminal movements, but produced a slight improvement in the respiratory rate.At the time of maximum sedation, there was loss of the noxious stimulation at the flanks, ventral abdomen and around the horns, while the reflexes were absent.Both returned rapidly.The MST and MTRT were 11.6 ± 0.9 and 307.5 ± 2.1 minutes respectively.
Neither relapse to recumbency nor marked sedation were observed in either phase of the study.However, some residual sedation persisted for varying lengths of time.Upon standing, the animals in all groups began to eat hay and maize bran immediately when offered.

DISCUSSION
Xylazine at 0.2 mg/kg is adequate to induce marked sedation in ruminants 13 .Goats are more sensitive to xylazine than sheep and cattle 24 .The doses of xylazine used in this study were 2 and 4 times the recommended dose in clinical procedures.The effects produced by xylazine at these doses were generally as expected, and are well-documented 7,13 .
Blockers of the central " 2-adrenoceptors such as yohimbine, tolazoline, piperoxan, idazoxan and atipamezole have been used to antagonise xylazine in various species 3,4,17 .These antagonists act by occupying and interacting with " 2 -adrenergic receptors, thus denying the "2-agonists access to these receptors.In the process, they enhance the release of norepinephrine and other excitatory neurotransmitters 3,17 .They may also be capable of influencing serotonergic, dopaminergic, cholinergic and (-aminobutyric receptors 7 .Yohimbine is a mixed "-adrenoceptor antagonist, but is more specific for the "2-adrenoceptors 3,7 .
Effects of xylazine are also reversed by the analeptics 4-aminopyridine, doxapram and caffeine, which are physiological antagonists to CNS depressants 3,4,17 .The drug 4-aminopyridine acts by facilitating uptake of neuronal calcium ions and enhancing acetylcholine release.It also produces a selective block of potassium ion channels in excitable membranes 2 .
The results obtained in this investigation showed that the administration of 4-aminopyridine, yohimbine or their combination was appropriate to reverse the xylazine-induced bradycardia, bradypnoea and ruminal atony.Similar results were also obtained in cattle 12 and in sheep 9 .At 0.88 mg/kg of xylazine, however, the combination of 4-aminopyridine and yohimbine produced only a slight improvement in the respiratory rates.
Administration of the antagonists, alone and in combination, also reversed xylazine-induced CNS depression, as evidenced by the rapid return of the reflexes.The 2 selected reflexes are among those used to monitor the level of anaesthesia, and their reappearance is usually interpreted as a sign of acquisition of variable degrees of consciousness 25 .This finding is in agreement with the results of other studies.For instance, in xylazinetreated sheep, yohimbine reduced the time to reappearance of the headdrooping reflex 9  1.0 ± 0.0 b, ¶ 1.0 ± 0.0 b, ¶ 1.2 ± 0.2 b, ¶ 3 1.8 ± 0.2 0* 0 a 0 a 0 a 0.3 ± 0.2 a 0.5 ± 0.2 b, ¶ 1.0 ± 0.0 b, ¶ 1.0 ± 0.0 b, ¶ 4 1.5 ± 0.2 0* 0 a 0.2 ± 0.2 a 0.7 ± 0.2 b, ¶ 0.8 ± 0.2 b, ¶ 1.0 ± 0.0 b, ¶ 1.2 ± 0.2 b, ¶ 1.3 ± 0. Means with different superscripts are significantly different (P < 0.05).¶ Significantly different (P < 0.05) from the pre-antagonist rate of ruminal movements.pyridine and their combination were also reported to cause rapid return of front and hind limb withdrawal reflexes in xylazine-treated cattle 12 .
There are conflicting reports on the analgesic effects of xylazine.Some authors have reported persistence of pain even at the maximum depth of sedation 1,19 .Others have reported adequate analgesia 6,15 .The presence of analgesia that varied in intensity and duration in various body regions has been reported 5,13 .It was minimal in the extremities, at the horn base and the flanks.In the present study, the reaction to noxious stimuli was consistently present at the limb extremities and scrotal sacs even in the deeply-sedated animals.Analgesia in other body regions was rapidly reversed by the intravenous injection of the antagonists, alone and in combination.
It is very important in goats, as in any other ruminants, to recover to a standing position as soon as possible after sedation or anaesthesia 24,25 .The hazards of regurgitation, excessive salivation, aspiration and ruminal tympany are present during the recovery period, while the animal is weak and has not regained appropriate control of reflexes and cannot stand up 24 .The antagonists, alone and in combination, significantly reduced the MST, which expresses the time required to gain full control of motor activities.Similar effects have been observed in sheep 9 , cattle 12 and llamas 21 .The reduction was greatest when yohimbine and 4-aminopyridine were combined, followed by 4-aminopyridine.The animals were stimulated to stand by hand-clapping, whistling and patting them.This was necessary because sedated animals tend to remain recumbent if not stimulated to rise.
The antagonists, alone and in combination, also reduced the MTRT, which is the time from the injection of xylazine until overt sedation disappears.The reduction was greatest with the yohimbine/ 4-aminopyridine combination, followed by 4-aminopyridine.Most authors have, however, reported non-significant reductions of the MTRT in xylazine-treated cattle 12 and dogs 8 , and in xylazine/ ketamine-anaesthetised horses 11 and goats 14 .
Overall, the combination of yohimbine and 4-aminopyridine produced better responses.The results of this study demonstrated the superiority of the combination of yohimbine and 4-aminopyridine over the individual drugs in antagonising xylazine in goats.The individual drugs may be used for the same purpose but the recovery may be prolonged.
2 b, ¶ G = group.BX = baseline rate of ruminal movements.BA = ruminal movements after xylazine treatment.*Significantly different (P < 0.05) compared with the pre-xylazine rate of ruminal movements.a,b

the rate of ruminal movements of goats sedated with intramuscular xylazine at 0.44mg/kg
(mean ± SE).

Table 4 : Effects of various antagonists on the mean standing time (MST) and mean total recovery time (MTRT) in goats sedated with intramuscular xylazine at 0.44mg/kg
(mean ± SE).
*Significantly different (P < 0.05) from the control value.