Seminal plasma components from fertile stallions involved in the epididymal sperm freezability

Abstract

Background Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. Objective The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze‐thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two‐dimensional difference gel electrophoresis (2D‐DIGE). Materials and Methods Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D‐DIGE. Results After thawing, the proportions of viable and acrosome‐intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D‐DIGE were different between stallions. Discussion These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity. Conclusion The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine‐rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance.

Recent advances in cryotolerance biomarkers for semen preservation in frozen form–A systematic review

“…This phenomenon extends to sperm rich in polyunsaturated fatty acids (PUFA). However, the exact mechanism inherent to functionality of fatty acid in cryotolerance remains unclear, even though cells with higher levels of PUFA have an enhanced level of membrane flexibility and fluidity that is granted by PUFA with many double bonds [ 21 , 22 ].…”

Nitric oxide-targeted protein phosphorylation during human sperm capacitation

“…The selected protein bands were spliced in approximately 2 × 2 mm parts and prepared for the digestion process as previously reported 86 , 87 . The bands were washed twice with Milli-Q water and then twice with 25 mM ammonium bicarbonate buffer pH 8.5 in 50% v/v acetonitrile during 30 min at 37 °C.…”

“…The separation and analysis of the tryptic digests of the samples were performed as previously described 86 , 87 , with a HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler Module and a High-Speed Binary Pump, connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in the MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev.…”

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability

“…Pivotal studies have reported that lauric, myristic, and oleic acids were two-fold more abundant in spermatozoa collected from stallions considered good freezers [ 187 ]. Furthermore, according to Vieira et al [ 188 ], spermatozoa with large amounts of docosahexaenoic acid (DHA) and stearic acid presented with superior post-thaw motility. However, the specific mechanisms underlying the role of fatty acids in sperm cryotolerance remain by and large unknown, even though cells with a high proportion of PUFAs have a higher degree of membrane fluidity and flexibility, which is provided by an abundance of double bonds present in the fatty acid chains [ 189 ].…”