History and preoperative examinations
A 4-month-old female Simmental calf was presented with polymelia. At first view, the malformation (in the following text termed “accessory limb”) appeared attached to the left upper part of the normal scapula and separated into two rudimental lower limbs distally (Fig. 1). The thoracic part of the spine was scoliotic and lordotic. At presentation the calf was alert and in a good body condition. Except for a slightly increased respiratory frequency (34 breaths per minute) probably due to excitement, the clinical examination revealed an otherwise healthy animal with an adequate state of physiologic development according to its age. Haematology and blood chemistry showed no abnormalities.
As dorso-ventral and latero-lateral radiographs showed significant overlapping of anatomical structures, it was decided to perform computed tomography (CT) to get more detailed information. After premedication with intramuscular (IM) xylazine hydrochloride (0.1 mg/kg; Xylasol 2% Dr. E. Graeub AG, Bern, Switzerland) and intravenous (IV) butorphanol tartrate (0.05 mg/kg; Morphasol 1% Dr. E. Graeub AG, Bern, Switzerland), anaesthesia was induced with IV ketamine hydrochloride (3 mg/kg; Narketan 10% Vetoquinol AG, Bern, Switzerland). and thereafter maintained with isoflurane (Attane™ Isoflurane ad us. vet., Provet AG, Lyssach, Switzerland) delivered in 100% oxygen under pressure-controlled mechanical ventilation.. Monitoring included pulse oximetry, side stream capnography, electrocardiogram (ECG) and oscillometric blood pressure. Computed tomography was performed in sternal recumbency. Anaesthesia was uneventful and recovery was smooth.
Computed tomography findings
Computed tomography images (Fig. 2a, b) revealed a polymelia originating from the area of the thoracic spine with formation of a single rudimentary scapula and humerus and paired rudimentary antebrachia and distal limbs. Multiple malformations of the thoracic spine between the 5th and 8th thoracic vertebrae were present. A spina bifida of T6 to T8 with fusion of the right sided spinous processes, a malformation of the dorsal spinous processes of T5 to T8, and a focal scoliosis and lordosis due to an asymmetric T7 hemivertebra were diagnosed. Due to the limited soft tissue contrast of CT, no statement concerning the status of the neuronal and vascular structures could be made.
Neurologic examination and nociceptive withdrawal reflex testing
A neurologic examination revealed no abnormal findings. As surgical amputation of the accessory limbs was planned, it appeared particularly important to assess whether a noxious stimulus applied to the accessory limb could be perceived and, if this was the case, to describe the nocifensive reaction evoked. Pin prick stimulation as well as pinching did not elicit any withdrawal of the accessory limb, rather stamping of the ipsilateral normal limb. Due to this unexpected reaction it was decided to quantitatively evaluate the nociceptive withdrawal reflex of both the normal and the accessory front limbs to confirm the presence of a common spinal reflex pathway before surgical intervention was started. After shaving, cleaning and degreasing of the skin two self-adhesive stimulation electrodes were placed latero-proximal to the fetlock over the nervus digitalis communis, parallel to the nerve itself, on the normal front extremity and also on the corresponding anatomical location on the accessory limb for sensory nerve stimulation. The anode was placed in the distal position. The paired recording electrodes for surface electromyography were placed over the deltoid muscle of the ipsilateral, normal front limb to record its reflex muscle activity after direct (normal limb) and indirect (accessory limb) electrical stimulation. The ground electrode was placed over the back.
Normal and accessory limbs were tested following the same order, starting with the normal limb. To define the reflex threshold, a standard stimulus consisting of constant current train-of-five 1 mS pulses at 200 Hz was applied starting at the intensity of 2 mA and increasing in steps of 0.5 – 1 mA until a reflex response was elicited. To confirm the threshold, stimulation was repeated three times at the current where the first electromyographic reflex activity had appeared. Afterwards the current was increased in 0.5–1 mA steps to define the stimulus evoking a reflex with the shortest latency. The results for the normal limb showed initial reflex activity at a current of 4 mA, with a latency of 80 mS, a reflex duration of 40 mS and an amplitude of 81 μV. The shortest latency reflex (31 mS) was seen with a stimulation intensity of 11 mA, a duration of 68 mS and an amplitude of 209 μV. The results from the accessory limb showed an initial reflex at 5 mA, with a latency of 112 mS, a duration of 57 mS and an amplitude of 84 μV. The shortest latency reflex (43 mS) was seen with a stimulation intensity of 20 mA, a duration of 61 mS and an amplitude of 248 μV (Fig. 3).
Surgical treatment, anaesthetic and analgesic management
Three days before surgical excision of the accessory limb, a therapy with gabapentin (2.5 mg/kg, twice daily, per os; Neurontin 100 mg, Pfizer, Berlin, Germany) was started and continued until discharge from the hospital (24 days of treatment).
Haematology and blood chemistry values were all within physiologic ranges the day before surgery. Food but not water was withheld the evening before surgery, and during the fasting period the calf was kept under maintenance fluid infusion with Ringer lactate solution (2 mL/kg/hr.; Ringer-Lösung “Bichsel”, Grosse Apotheke Dr. G. Bichsel AG, Interlaken, Switzerland). On the day of surgery benzylpenicillin sodium (30,000 IU/kg IV; Penicillin Natrium ad us. vet., Streuli Pharma AG, Uznach, Switzerland), flunixine meglumine (1.1 mg/kg IV; Finadyne ad us vet., MSD Animal Health GmbH, Luzern, Switzerland) and a tetanus antitoxin (3000 IU SC; Tetanus-Serum Intervet ad us. vet., MSD Animal Health GmbH, Luzern, Switzerland) were admistered 30 min before anaesthetic premedication with xylazine hydrochloride (0.15 mg/kg IM) and methadone (0.1 mg/kg IV; Methadon, Streuli Pharma AG, Uznach, Switzerland). Anaesthesia was induced with IV diazepam (0.05 mg/kg; Valium 5 mg, Roche Pharma AG, Reinach, Schweiz) followed by ketamine hydrochloride (3 mg/kg IV). The induction was smooth and endotracheal intubation was performed using a 14 mm endotracheal tube. The animal was placed into right lateral recumbency and connected to a circle breathing system. Volume-controlled mechanical ventilation was started immediately with an inspiratory volume of 10 mL/kg. Isoflurane in 100% oxygen was administered, combined with systemic constant rate infusions of ketamine hydrochloride (0.6 mg/kg/hr) and lidocaine hydrochloride (3 mg/ kg/hr., after a bolus of 1.5 mg/kg over 5 min; Lidocain 2% Streuli ad us. vet., Streuli Pharma AG, Uznach, Switzerland). Methadone boli (0.05 mg/kg IV) were admistered slowly over 5 min every hour and as one IM injection at the end of the procedure. Ringer lactate solution was administered at a rate of 10 mL/kg/hr. Monitoring included ECG, capnography, non-invasive oscillometric as well as continuous invasive (auricular artery) arterial blood pressure measurements. The arterial blood gas analysis results were always within physiologic ranges.
After surgical preparation, a ring block with 20 mL bupivacaine hydrochloride 0.5% (Carbostesin 0.5%, AstraZeneca GmbH, Wedel, Germany) was performed proximal to the planned incision lineTwenty minutes later, an ellipsoid incision was made at the height of the scapulo-humeral joint of the accessory limb, the joint was dissected and the major part of the limb removed. The distal part of the remaining additional scapula, including the acromion, was cut through using an oscillometric saw. The exposed bone marrow of the scapula was covered with a hemostyptic bone wax. The sharp edges of the bone were abraded, and a continuous suction drain was placed. The fascial layer was closed and a wound catheter, previously flushed with bupivacaine hydrochloride 0.5%, was placed subcutaneously with its tip located at the highest point of the wound. Splash local anaesthesia was performed with 20 mL of bupivacaine hydrochloride 0.5%. The skin was closed and the wound covered with a sterile bandage. The calf was then allowed to wake up. Recovery was fast and uneventful. The animal was calm and showed no signs of pain.
Intravenous maintenance fluid therapy was kept up until the next day, when the calf was adequately drinking and eating again. Systemic analgesic therapy included continuation of gabapentin, flunixine meglumine 1.1 mg/kg IV once per day, and buprenorphine hydrochloride (Bupaq ad us. vet., Streuli Pharma AG, Uznach, Switzerland) 0.01 mg/kg IV or IM every 6 h. with a total of 20 mL bupivacaine hydrochloride 0.5% was administered via the wound catheter every 4–6 h for three days. Pain assessment was regularly performed and included assessment of the calf’s overall general condition, occurrence of behavioural changes (respiratory changes, reluctance to move or restlessness, excitement or depression, looking towards the wound, anorexia), recording of physiologic parameters such as heart rate and respiratory rate, reaction to wound palpation (large area/punctual palpation), and subjective pain evaluation (no pain to non-bearable pain). Based on these observations, the interval between the application of local anaesthesia was adapted and additional buprenorphine hydrochloride (0.01 mg/kg IM) was administered on two occasions (during the first morning and the second night post-surgery). After removal of the wound catheter (3 days after surgery) and fading of the last bupivacaine hydrochloride injection, the calf showed increased reaction to wound palpation especially to punctual palpation over a period of 24 h. Nevertheless, it was otherwise in good condition and showed no additional signs of pain when left undisturbed. Flunixine meglumine and buprenorphine hydrochloride therapy was stopped 4 days postoperatively. Mild reaction to wound palpation was present until discharge from the hospital.
Wound healing was adequate, and the suction drain was removed three days after surgery. Antibiotic therapy (benzylpenicillin sodium, 30,000 IU every 8 h IV) was maintained over 5 days and sutures were removed 10 days postoperatively. The calf was discharged from the hospital 21 days after surgery in good health conditions.
Anatomic dissection
A gross anatomical dissection was performed on the removed accessory limb. The extremity consisted of two left front limbs sharing one common scapula and one common humerus. Further distally, the bones included ossa antebrachii lacking typical characteristics and partially fused ossa carpi. Metacarpal bones and phalanges were typical as well as the metacarpophalangeal and interphalangeal articulations. No discernible muscle tissue could be identified, and tendons were firmly combined with fascia or periost. Only rudimentary tendons of the lateral digital extensor muscle and of the deep digital flexor muscle could be distinguished. On the palmar side, two metacarpal arteries and one single vein were recognizable as well as the equivalents of the common digital dorsal artery, vein and nerve.
Genetic analysis
A genetic analysis was performed using genomic desoxyribunucleic acid (DNA) isolated from a skin biopsy of the accessory limb, hair roots from the body, and EDTA stabilized blood of the affected calf. Genotyping of 12 autosomal microsatellite DNA markers using standard methods [10] revealed, at several loci, the presence of a third allele (Fig. 4). The peak size of this additional marker allele varies between the three DNA sources. The lowest peak size was observed in DNA derived from blood cells, and a much higher signal intensity in DNA originating from the hair roots and skin of the accessory limb. These findings revealed the presence of cells with different genotypes, which indicated a chimeric condition of the affected calf known in bovine freemartin pregnancies. A heterosexual twin pregnancy was excluded because a polimerase chain reaction (PCR) showed absence of the SRY gene in all available DNA samples.
