Sperm quality of rats exposed to difenoconazole using classical parameters and surface-enhanced Raman scattering: classification performance by machine learning methods.

Difenoconazole is a fungicide extensively used in agriculture. The aim of this study was to evaluate the effects of difenoconazole fungicide on the sperm quality of rats. Wistar rats were divided into four groups: control and exposed to 5 (D5), 10 (D10), or 50 mg kg bwday (D50) of difenoconazole for 30 days, by gavage. Classical sperm parameters and surface-enhanced Raman scattering (SERS) were performed. Progressive motility, acrosomal integrity, and percentage of morphologically normal spermatozoa were reduced in the D10 and D50 groups in comparison with the control group. Sperm viability was reduced only in the D50 group. Sperm number in the testis and caput/corpus epididymis and daily sperm production were reduced in the three exposed groups. SERS measurements showed changes in the spectra of spermatozoa from D50 group, suggesting DNA damage. In addition, machine learning (ML) methods were used to evaluate the performance of three classification algorithms (artificial neural network-ANN, K-nearest neighbors-K-NN, and support vector machine-SVM) in the identification task of the groups exposed to difenoconazole. The results obtained by ML algorithms were very promising with accuracy ≥ 90% and validated the hypothesis of the exposure to difenoconazole reduces sperm quality. In conclusion, exposure of rats to different doses of the fungicide difenoconazole may impair sperm quality, with a recognizable classification pattern of exposure groups.