Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation

Abstract

BackgroundIn order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry.ResultsThere was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters.ConclusionBased on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.

Markers for Sperm Freezability and Relevance of Transcriptome Studies in Semen Cryopreservation: A Review

“…In recent years, several luorescent probes have shown that the cryopreservation process compromises the sperm plasma membrane integrity (PMI), resulting in reduced fertilizing capacity of postthaw semen [3][4][5][23][24][25][26][27][28][29]. Post-thaw sperm PMI has been assessed with diferent luorescent membrane probes, such as the dual SYBR-14 and propidium iodide (PI) assay [25,28] or membrane-permeable substrate carboxyluorescein diacetate (CFDA), a nonspeciic esterase substrate [24].…”

“…The chlortetracycline (CTC) luorescence assay has been used to detect capacitation-like changes in frozen-thawed spermatozoa, which may compromise their fertilizing ability [6,10,[22][23][24]29]. Cryo-induced changes in the acrosome membrane integrity (AMI) have been monitored by speciic Giemsa-staining technique [25] or with luorescent dyes, such as luorescein isothiocyanate (FITC)-conjugated PNA (peanut agglutinin) or conjugated PSA (Pisum sativum agglutinin), known as plant lectins, which bind to glycoproteins in the outer acrosomal membrane [4,5,26,27]. Studies have shown that the utilization of a triple staining-SYBR-14, phycoerythrin-conjugated PNA (PE-PNA), and PI (SYBR-14/PE-PNA/ PI)-to simultaneously evaluate the PMI and AMI of frozen-thawed bull spermatozoa can be used efectively to assess post-thaw semen viability [26][27][28].…”

“…Studies have shown that the utilization of a triple staining-SYBR-14, phycoerythrin-conjugated PNA (PE-PNA), and PI (SYBR-14/PE-PNA/ PI)-to simultaneously evaluate the PMI and AMI of frozen-thawed bull spermatozoa can be used efectively to assess post-thaw semen viability [26][27][28]. In a recent study, it has been demonstrated that FITC-PSA/PI-staining protocol can detect marked deterioration in both the PMI and AMI in frozen-thawed bull spermatozoa [4]. Earlier changes in the membrane permeability of frozen-thawed spermatozoa have been monitored with the calcium-dependent binding of Annexin-V/FITC/PI [21,24,26] or YO-PRO-1 assay [23,25,28], which is an impermeable membrane probe.…”

“…Cryopreservation of semen allows the preservation of good genetic resources and the protection of endangered species [1][2][3][4][5]. While a great deal of eforts has been done over the last several years to improve the semen cryopreservation technology, efective cryosurvival of spermatozoa from various animal species, including the boar and stallion, still remains elusive and a cryobiological enigma [1].…”

“…While a great deal of eforts has been done over the last several years to improve the semen cryopreservation technology, efective cryosurvival of spermatozoa from various animal species, including the boar and stallion, still remains elusive and a cryobiological enigma [1]. Cryo-induced oxidative stress is associated with excess production of reactive oxygen species (ROS), resulting in biochemical and physical damage to the sperm membrane structures and subsequently leading to reduced fertilizing ability of spermatozoa [4][5][6]. In the artiicial insemination (AI) industry, there is a need to optimize the selection strategy for individuals with good freezability, so as to incorporate this information in the breeding program to improve the fertility of post-thaw semen [2,3,7].…”