Ketamine

Drugs and materials used were ketamine (Ceva Animal Health, Australia), xylaxine (Troy Laboratories Pty, Ltd, Australia), heparin (Hospira, Germany), bovine serum albumin (Sigma, USA), and fresh frozen plasma (Australian Red Cross). D. siamensis, P. textilis, B. arietans, B. gabonica and C. vegrandis venoms were obtained from Venom Supplies (Australia). D. russelii venom was a gift from Professor A. Gnanadasan (University of Colombo). E. ocellatus venom was a gift  from the Liverpool School of Tropical Medicine. For procoagulant assays, venom (1 mg/mL) was prepared in 0.5% bovine serum albumin/tris-buffered saline and stored at −20 °C. Dilutions were prepared in 0.5% BSA/TBS immediately before use.
Animal experiments were approved by the Monash University Ethics Committee (MARP/2014/097 and MARP/2017/147). All experiments were performed in accordance with relevant guidelines and regulations.

Immunization by the intramuscular (IM) route with pgDE7h was performed in a total volume of 100 μL per dose, with 50 μL administered in each paw. Mice receiving electroporation were previously anesthetized intraperitoneally with a mixture of 75 mg/kg ketamine (Ceva Santé Animale, Libourne, France) and 10 mg/kg xylazine (Ceva Santé Animale, Libourne, France). For electroporation, the electrode CUY560-5-0.5 was used, consisting of a pair of parallel fixed needles 0.5 mm in diameter with a space of 5 mm between them. The electrode was inserted into the anterior tibial muscle shortly after inoculation of the vaccine. Six electric pulses of 130 V each were applied with a duration of 450 milliseconds. Electrical pulses were delivered using the NEPA21 SuperEletroporator equipment (NepaGene, Ichikawa City, Japan). The therapeutic gDE7-based protein vaccine (PTN) was administered following a regimen of two SC doses, at day 3 and 7 after cisplatin treatment. Each dose contained 30 μg of the gDE7 protein admixed with 50 μg of the poly(I:C) (pIC; InVivoGen, San Diego, USA) adjuvant, diluted in saline solution (total volume of 100 μL), and inoculated in the right rear flank of the mice.

Random assignment was done to determine which testis to remove in the rats undergoing unilateral orchiectomy. The animals were sedated via an intraperitoneal injection (IP; 125μl/100 g bodyweight) with a mixture of 0.33mg/ml medetor (83255102; Virbac, Leuven, Belgium) and 50mg/ml ketamine (6905306; Ceva Santé Animale, Machelen, Belgium). In adult rats, an incision of 1 cm was made in the scrotum, while in prepubertal rats the incision was made in the abdomen and peritoneum. A double ligature of digestible vicrylTM Plus thread (VCP303H; Ethicon, Norderstedt, Germany) was placed around the vas deferens and the blood supply before removing the testicle. After removal of the testis, the scrotum or abdomen and peritoneum were closed using Catgut absorbable surgical sutures (2151512; SMI, St Vith, Belgium). Subcutaneous injections of 2.5% Baytril (0.1ml; 79828047; Bayer, Antwerpen, Belgium) were given to avoid post-operative infections. Busulfan was administered IP in two doses of 10mg/kg bodyweight (154906; ICN Biomedicals inc, Irvine, USA) with a time interval of fourteen days [15 (link)].