Propofol

All surgeries were performed by the senior author, unblinded to the grouping. Animals were hemicastrated five months (154 days) following treatment with the implant or placebo. Orchiectomy of the right testis was performed under general anesthesia with propofol 2–4 mg/kg (CP-Pharma, Burgdorf, Germany) and ketamine 1 mg/kg (CP-Pharma, Burgdorf, Germany), after premedication with dexmedetomidine 5 µg/kg (CP-Pharma, Burgdorf, Germany) and levomethadone 0.2 mg/kg (MSD, München, Germany) maintained by isoflurane (1%) and following general surgical principles. The testes were immediately processed within the next minutes after surgery. After removal of the respective testis, the paraumbilical area was surgically prepared (scrubbed, disinfected) at the location of the implant. Following preparation, a skin incision was made where the implant could be palpated, and the implant was carefully surgically removed in total. In the case that the implant broke, specific care was taken to ascertain complete removal of all pieces. Afterwards, the previous implantation site was flushed with sterile saline. The incision was closed with a skin suture using Vicryl 3/0 (Ethicon, Norderstedt, Germany). All dogs received 4.0 mg/kg carprofen (Rimadyl^®, Zoetis Deutschland GmbH, Berlin, Germany) for three days.

On D149, the left (second) testis was surgically removed under general anesthesia in all dogs by the senior author. The dogs were premedicated with dexmedetomidine 5 µg/kg (CP-Pharma, Burgdorf, Germany) and levomethadone 0.2 mg/kg (MSD, München, Germany), followed by the administration of propofol 2–4 mg/kg (CP-Pharma, Burgdorf, Germany), and ketamine 1 mg/kg (CP-Pharma, Burgdorf, Germany) maintained by isoflurane (1%). Immediately after surgery (D149), the testes were trimmed in pieces of approximately 1 cm³ and fixed in Bouin’s solution. Following washing with 70% ethanol and embedding in paraffin, the sections were cut, hematoxylin–eosin stained, and mounted with Roti^® Histokitt (Carl Roth, Karlsruhe, Germany). A histological assessment of spermatogenesis was performed at 400× magnification (Zeiss West, Oberkochen, Germany [20 (link)]).