Rahmat Rahmat 1, Andi Ni’mahtul Churriyah 1, Dian Tria Fatmila 2, Muhammad Rizal 1, Nursyam Andi Syarifuddin 1, Athhar Manabi Diansyah 3, Muhammad Yusuf 3, Muhammad Fajar Amrullah 4,5, Andi Muhammad Alfian 3 and Miladiarsi Miladiarsi 6
1Department of Animal Science, Faculty of Agriculture, Lambung Mangkurat University, Jl. Jenderal Ahmad Yani, Banjarbaru, South Kalimantan, 70714, Indonesia; 2Animal Science Study Program, Faculty of Agriculture, Universitas Sumatera Utara, Medan 20155, Indonesia; 3Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Indonesia. Jl. Perintis Kemerdekaan 10 Tamalanrea Makassar, South Sulawesi, 90245, Indonesia; 4Doctoral Student of Animal Biomedical Science, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, 16680, West Java, Indonesia; 5Research Center for Applied Zoology, National Research and Innovation Agency, Cibinong Science Center, Jalan Raya Jakarta-Bogor, 16680, West Java, Indonesia; 6Plant Protection Study Program, Faculty of Agriculture, Lambung Mangkurat University, Jl. Jenderal Ahmad Yani, Banjarbaru, South Kalimantan, 70714, Indonesia
*Corresponding author: rahmatr@ulm.ac.id
Breeding soundness examination (BSE) is widely used to evaluate male reproductive suitability, but its relationship with resilience to sperm cryopreservation and sperm molecular characteristics remains insufficiently understood in Peranakan Etawah (PE) bucks. This study compared post-thaw sperm quality and sperm proteomic profiles between PE bucks classified as satisfactory (SAT; n=10) and unsatisfactory (UNS; n=10) using a modified BSE framework. BSE classification was based on body condition score, locomotion score, scrotal circumference, progressive motility, and sperm morphology. Frozen–thawed semen was evaluated for motility recovery and morphology preservation, while sperm proteomic profiles were analyzed using LC–MS/MS followed by differential abundance, multivariate, functional enrichment, and correlation analyses. SAT bucks showed higher total BSE score, locomotion score, progressive motility, motility recovery, and morphology preservation than UNS bucks. Proteomic analysis identified distinct sperm protein signatures between groups, with SAT-associated proteins primarily involved in flagellar organization, axonemal structure, membrane regulation, energy metabolism, and oxidative stress response, including CFAP43, AKAP4, AQP3, ALDOC, CS, FLOT1, SPAG6, DNAI1, and GSTM3. In contrast, UNS-associated proteins, including MSN, ALB, and MMP9, were linked to altered membrane remodeling, the extracellular protein environment, and reduced sperm functional or structural resilience. Correlation analysis showed that selected SAT-associated proteins were positively associated with progressive motility and morphology preservation, whereas UNS-associated proteins were negatively associated with these outcomes. These findings indicate that integrating BSE classification, post-thaw semen assessment, and sperm proteomic profiling provides a more comprehensive framework for evaluating reproductive soundness in PE bucks. The identified sperm protein signatures represent exploratory candidates for future validation in fertility and cryopreservation resilience studies.