Molecular basis of fertility variation in cryopreserved bull semen: Functional evaluation and expression analysis of IZUMO1, PLCζ, and CatSper genes

Abstract

Semen cryopreservation serves as a pivotal biotechnological tool implemented in artificial insemination programs to accelerate genetic progress in cattle breeding. However, the freeze-thawing cycle can heavily compromise fertilization efficiency due to sublethal cellular, functional, and molecular injuries. This study aimed to evaluate the impacts of cryopreservation on conventional semen parameters and the relative transcript abundance of fertility-associated target genes (CatSper, IZUMO1, and PLCζ) utilizing straws from high-fertility and low-fertility breeding bulls. A retrospective cohort comprising 92 post-thaw semen samples (50 high-fertility and 42 low-fertility) was investigated. Sperm motility kinetics were quantified via Computer-Assisted Sperm Analysis (CASA), while structural viability, plasma membrane integrity, and normal cytomorphology were determined using Eosin-Nigrosin exclusion, hypoosmotic swelling tests, and Diff-Quik staining, respectively. Quantitative real-time PCR (RT-qPCR) was executed to evaluate relative gene expression normalized against the reference gene GAPDH, based on the comparative 2−ΔΔCt methodology. The spermiogram analysis demonstrated a statistically significant downregulation in directional progressive motility (62.7 % vs. 65.4 %; P=0.011), structural viability (78.5 % vs. 80.2 %; P=0.045), and membrane permselectivity (72.2 % vs. 74.6 %; P=0.045) in the low-fertility group, accompanied by a suppression in normal sperm morphology (73.7 % vs. 78.1 %; P=0.0001). Conversely, overall total sperm motility displayed no significant statistical variance between the cohorts (74.0 % vs. 77.9 %; P=0.063). At the molecular level, all three target transcripts exhibited profound downregulations in the subfertile group: a 2.43-fold decrease for IZUMO1, a 2.78-fold decrease for PLCζ, and a 3.45-fold decrease for CatSper, establishing a distinct hierarchy of transcript instability (P<0.001). The study demonstrates that cryopreservation induces high transcript degradation that directly compromises flagellar hyperactivation, gamete fusion, and post-fertilization oocyte activation pathways. These findings suggest that integrating quantitative gene expression analysis with conventional spermiograms offers an invaluable diagnostic modality to diagnose cryptic male subfertility, optimize semen straw selection, and enhance reproductive efficiency in assisted breeding programs.