Background/Objectives: Non-surgical neutering strategies have long been pursued for male animals. A previous study from our group showed that magnetic nanoparticle hyperthermia (MNH) applied directly to the testicles is a promising non-surgical sterilization method for male animals, causing testicular atrophy and complete disappearance of seminiferous tubules by the end of a 56 day observation. This long-term study was conducted to verify the long-term efficacy and safety of the method. Methods: Wistar rats treated with testicular MNH received an intratesticular injection of a magnetic fluid composed of manganese-ferrite nanoparticles functionalized with citrate (MnFe₂O₄-citrate) and were subsequently subjected to an alternating magnetic field. Reproductive parameters and animal health were evaluated by blood tests and abdominal ultrasound for 12 months. Results: All MNH-treated animals presented testicular degeneration and atrophy, together with severely reduced or undetectable serum testosterone levels. By the end of the experiment, all but two animals had no identifiable gonads. The only two animals still displaying gonadal-like structures were azoospermic, and histopathology revealed the remaining tissue was non-functional. The procedure was well-tolerated and MNH-treated animals presented no long-term side effects. Hemogram, ALT, AST, urea and creatinine levels were within the normal parameters for Wistar rats over the 12 month period. The liver, spleen, kidneys and lungs had normal structures as revealed by abdominal ultrasound and histopathological exams, with no nanoparticle accumulation in the organs over the long term. Conclusions: In conclusion, testicular MNH caused irreversible infertility in rats in a single application, with no adverse effects on general animal health.

In search of a non-surgical alternative for male animal sterilization, this study investigated the use of gold-coated maghemite nanoparticles (γ-Fe₂O₃@Au) functionalized with citrate to produce testicular photohyperthermia (PHT). Wistar rats received an intratesticular injection of the fluid containing the nanoparticles (150 µL/testicle) followed by testicular irradiation with an LED light (808 nm). Testicular temperature was maintained at ~45 °C for 15 min. The results demonstrated a significant reduction in testicular volume and weight and sperm motility and normal morphology in PHT-treated animals, together with histopathological degeneration of seminiferous tubules. No treatment-related side effects or signs of systemic toxicity were observed. The biodistribution of the gold (Au) and iron (Fe) from the nanoparticles showed that the testes were the primary site of nanoparticle accumulation until day 56 post-treatment with possible renal excretion of Au. These findings support the prospect of testicular PHT mediated by γ-Fe₂O₃@Au nanoparticles as a neutering method for male animals.