Endogenous Tissue Inhibitor of Metalloproteinase-2 Levels Are Associated With High-Quality Neat Semen but Unrelated to Sperm Cryoresistance in Bulls

Abstract

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) is part of the tissue inhibitors of the metalloproteinases (TIMPs) family. Its primary function is to regulate the activity of matrix metalloproteinases (MMPs) across various tissues, including those of the reproductive system. This study aimed to quantify the natural levels of TIMP-2 in seminal plasma (SP) and sperm membrane (SM) of bulls, explore potential associations between TIMP-2 levels and semen quality parameters, and examine the relationship between TIMP-2 levels and sperm cryoresistance in bulls. Thirty semen samples from Frieswal breeding bulls were categorized into two groups based on their initial progressive motility (IPM): Good (IPM ≥ 70%; n = 21) and Poor (IPM ≤ 40%; n = 9). The samples were evaluated for their quality parameters at the fresh stage, and TIMP-2 levels were measured in SP and SM using a bovine-specific ELISA kit. Following cryopreservation of Good samples (n = 21), post-thaw motility (PTM) was used to further classify samples into Freezeable (PTM ≥ 50%; n = 14) and Non-Freezable (PTM < 50%; n = 7) groups. In frozen-thawed samples, sperm attributes, kinetics, and functional parameters were assessed, and the results were correlated with retrospective TIMP-2 levels of SP/SM. Our study revealed that the quantified levels of TIMP-2 ranged from 100.27 to 535.95 ng/L in SP and from 0 to 115.78 ng/10 million spermatozoa in SM. TIMP-2 levels in both SP and SM were significantly higher in Good ejaculates compared to Poor ejaculates (p < 0.01). Furthermore, total TIMP-2 levels in the SP/SM of semen samples from bulls showed a positive correlation with fresh semen attributes. However, SP/SM TIMP-2 levels in the Freezeable group did not show any significant differences compared to the Non-Freezable group in post-thaw semen quality attributes, kinetic parameters, and functional tests, except for a significant positive correlation (r = 0.530, p < 0.05) between sperm DNA integrity and SP-TIMP-2 levels. In conclusion, the findings suggested that TIMP-2 can be a positive regulator of semen quality at the neat stage. However, when it comes to the resilience of sperm to cryopreservation, the levels of TIMP-2 do not seem to exert any significant influence in breeding bulls.

Keywords: MMPs; TIMP‐2; bull; cryopreservation; freezability; semen.