Reptile Euthanasia — No Easy Solution?

Reptiles are commonly studied in biological research projects, and wildlife researchers may be required to euthanize a reptile if it sustains a severe injury associated with the research. When conducting euthanasia of any animal it is critical to confirm death. Whilst in mammals and birds euthanasia and confirmation of death can easily be accomplished, in reptiles these are not straight forward processes due to reptilian poikilothermic biology and physiology. Many traditional methods of reptile euthanasia are controversial, and recommended methods of acceptable euthanasia vary amongst the different reptilian orders. Physical methods of euthanasia involving hypothermia or decapitation alone are considered inhumane and are not acceptable methods of euthanasia. Injectable pentobarbitone sodium is considered an acceptable method of euthanasia for all reptiles, except large crocodiles and other large reptile species where carcass removal in the wild may be problematic. However, in Australia pentobarbitone sodium is a Scheduled 4 drug with requirements for locked storage, and users other than registered veterinarians must apply for authorization to administer scheduled drugs. Stunning followed by destruction of the brain is considered acceptable with reservations in some small species of snakes and lizards, where these methods can be effectively accomplished. MS222 has been used in several species of reptiles, however further research is required to determine whether this technique is effective and humane across the diverse range of reptilian species. Humane euthanasia in reptiles is not easily achieved and, whilst recognizing limitations in accessing veterinary anaesthetic and euthanasia drugs, it can best be assured by using a two-stage euthanasia process — whereby the reptile is initially anaesthetized, and then euthanized by administration of pentobarbitone sodium or alternatively a combination of decapitation and brain destruction following anaesthesia.

Temporal Expression of SOX9 and Type II Collagen in Spheno-Occipital Synchondrosis of Mice after Mechanical Tension Stimuli

Objective: To associate the expressions of SOX9 and type II collagen during growth in the synchondrosis with and without tensile stress in order to understand the role of these factors in the growth of cartilage in spheno-occipital synchondrosis. Materials and Methods: Sixty 1-day-old male BALB/c mice were randomly divided into experimental and control groups. Each group was subdivided again into five different time points which were 6, 24, 48, 72, and 168 hours. Each subgroup consisted of five mice. Each mouse was sacrificed using an overdose of pentobarbitone sodium. The synchondroses were aseptically removed and incubated in a 24-well plate with or without tensile stress in tissue culture. Tissue sections were stained immunohistochemically to quantitatively analyze the expression of SOX9 and type II collagen. Results: There was a statistically significant increase of 57% (P < .001) in the expression of SOX9 between the experimental and control groups at 24 hours, followed by a significant increase of 44.4% (P < .001) in the expression of type II collagen at 72 hours. Conclusions: SOX9 may play an important role for early differentiation of chondrocytes and increase the expression of type II collagen, a major component of the extracellular matrix, during the growth of cartilage in the spheno-occipital synchondrosis.

Semen collection from mice: electroejaculation

The effects of device type (electrostimulator, function generator or computer-generated waveforms), waveform (square, triangle or sine wave), probe type (ring or strip) and anaesthetic compound (ketamine/xylazine combination or pentobarbitone sodium) were investigated on electroejaculation (EEJ) responses of C57Bl × CBA and C57Bl/6J mice. Ejaculates were analysed for total sperm count and motility variables using computer-assisted sperm analyses. Automated computer-generated waveforms delivered through a sound card were more effective and reproducible compared with waveforms generated by function generator and electrostimulator. Sine waves and triangle waves were found to be more effective in producing ejaculate than square waves. As an anaesthetic, pentobarbitone sodium tended to outperform ketamine/xylazine across waveforms and strains. Strip probes failed to produce any ejaculate regardless of the device or waveform employed. Sperm obtained by EEJ exhibited poor motility and C5Bl/6J mice had lower motility variables than C57Bl × CBA mice.

Sympathoexcitatory influence of a fast conducting raphe-spinal pathway in the rat

Experiments were carried out on 20 pentobarbitone sodium (alpha-chloralose supplemented)-anesthetized, artificially ventilated, and paralyzed rats. The possibility was explored that raphe-spinal neurons with myelinated axons arising in the rostral part of raphe obscurus provide excitatory drive to sympathetic neurons. Electrical stimulation within obscurus was observed to evoke an “early” sympathoexcitatory response compatible with its conduction over such a pathway. The effect of the microinjection of excitatory and inhibitory amino acids [DL-homocysteic acid (DLH) and gamma-aminobutyric acid (GABA), respectively] on the evoked response was studied at the sites of electrical stimulation. The size of the early response was increased by 91.7 +/- 24.4% (n = 7) and depressed by -48 +/- 4.8% (n = 7) by DLH and GABA, respectively. Saline was without effect (-14.5 +/- 12.2%, n = 6). The evoked responses were decreased when blood pressure was raised by administration of phenylephrine (2-6 micrograms/kg iv) and totally suppressed by an increase in blood pressure of 19.3 +/- 4.3 mmHg (baseline 89.1 +/- 2.5 mmHg, n = 7). It is concluded that some cell bodies located in rostral raphe obscurus that project to the spinal cord relay excitatory drive to sympathetic neurons.

Increased plasma glucose levels after Hypnorm® anaesthesia, but not after Pentobarbital® anaesthesia in rats

The effects of the fentanyl fluanisone combination (Hypnorm®) and pentobarbitone sodium (Pentobarbital®) anaesthesia on blood glucose, insulin and glucagon were tested in rats in the fed and fasted state. Blood glucose was measured before and at 10, 20 and 30 min after injection of the anaesthetic agents. At 30 min the rats were sacrificed, and blood was drawn for measurement of glucagon and insulin. Pre-anaesthetic values for insulin and glucagon were established in separate groups of fasted and fed rats. In fasting rats given Hypnorm®, blood glucose and plasma insulin were unchanged while there was a non-significant increase in plasma glucagon. The fasted rats given Pentobarbital® had unchanged blood glucose and plasma insulin and a non-significant depression of glucagon. The fed rats given Hypnorm® had a significant increase in blood glucose at 10 min and nearly a doubling of glucose values at 20 and 30 min ( P<0.001). Glucagon increased far less than in the fasted group, whereas insulin was doubled from preanaesthetic values ( P<0.05). The fed rats given Pentobarbital®, had unchanged blood glucose, a slight nonsignificant depression of glucagon and a significant increase in insulin ( P<0.01). Thus Hypnorm® induced hyperglycaemia in fed but not in fasted rats, probably because more glucose was available in the fed state. Fed animals are a modification of the standard fasted animal model, and may be preferable when exploring hyperglycaemic or other reactions to anaesthetic agents.

Pharyngeal dilation associated with cricothyroid muscle contraction in dogs

The mechanical function of phasic respiratory-related activity of the cricothyroid muscle of the larynx is poorly understood. We studied five adult cross-bred dogs (weight 14–20 kg) deeply anesthetized with pentobarbitone sodium, mechanically ventilated via a tracheostomy, and placed prone with the mouth open. Bilateral cricothyroid muscle contraction was induced by supramaximal electrical stimulation of the external branches of the superior laryngeal nerve. Computerized axial tomography was used to assess effects of cricothyroid muscle contraction. During cricothyroid muscle contraction, oropharyngeal (tip of epiglottis) cross-sectional area increased by 18.0 +/- 3.0% (SE) (P = 0.008), whereas combined left and right piriform recess cross-sectional area increased by 85 +/- 25% (n = 4; P = 0.02) at the midepiglottic level and by 152 +/- 37% (P = 0.01) at the base of the epiglottis. Furthermore, at the base of the epiglottis the maximum horizontal distance between the alae of the thyroid cartilage increased by 21 +/- 8% (P = 0.05). In contrast, lateral glottic diameter decreased by 52 +/- 2% (n = 4; P = 0.01), whereas dorsoventral glottic diameter increased by 18 +/- 5% (n = 4; P less than 0.02). The cricothyroid muscle, therefore, has the capacity to act simultaneously as a pharyngeal dilator and a glottic constrictor and thus may play a role in the control of oropharyngeal as well as laryngeal patency.