A Novel Approach to Identifying Physical Markers of Cryo-Damage in Bull Spermatozoa

Abstract

Cryopreservation is an efficient way to store spermatozoa and plays a critical role in the livestock industry as well as in clinical practice. During cryopreservation, cryo-stress causes substantial damage to spermatozoa. In present study, the effects of cryo-stress at various cryopreservation steps, such as dilution / cooling, adding cryoprtectant, and freezing were studied in spermatozoa collected from 9 individual bull testes. The motility (%), motion kinematics, capacitation status, mitochondrial activity, and viability of bovine spermatozoa at each step of the cryopreservation process were assessed using computer-assisted sperm analysis, Hoechst 33258/chlortetracycline fluorescence, rhodamine 123 staining, and hypo-osmotic swelling test, respectively. The results demonstrate that the cryopreservation steps reduced motility (%), rapid speed (%), and mitochondrial activity, whereas medium/slow speed (%), and the acrosome reaction were increased (P < 0.05). Differences (Δ) of the acrosome reaction were higher in dilution/cooling step (P < 0.05), whereas differences (Δ) of motility, rapid speed, and non-progressive motility were higher in cryoprotectant and freezing as compared to dilution/cooling (P < 0.05). On the other hand, differences (Δ) of mitochondrial activity, viability, and progressive motility were higher in freezing step (P < 0.05) while the difference (Δ) of the acrosome reaction was higher in dilution/cooling (P < 0.05). Based on these results, we propose that freezing / thawing steps are the most critical in cryopreservation and may provide a logical ground of understanding on the cryo-damage. Moreover, these sperm parameters might be used as physical markers of sperm cryo-damage.

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].…”

“…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].…”

“…Moreover, there is accumulating evidence indicating that the cryopreservation procedure induces apoptotic-like features in bovine spermatozoa, which appeared as ordered events during the freezing-thawing process, such as reduced mitochondrial membrane potential (MMP), increased caspase activation, and modiications in membrane permeability [6,21,24,27,28,30]. Additionally, post-thaw PMI has been assessed with the hypo-osmotic swelling (HOS) test, which evaluates the functional membrane integrity of the acrosome and tail regions when spermatozoa are exposed to hypoosmotic conditions [22,24,29].…”

“…Several studies showed that cryo-induced damage to the MMI of spermatozoa is one the major causes of their reduced fertilizing capacity [4,5,10,24,25,29,33]. These studies detected a marked deterioration in the sperm mitochondrial function following cryopreservation, as reported by the luorescent staining with rhodamine 123 (R123), the lipophilic cationic compound 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzymidazolyl carbocyanine iodine (JC-1), or with ATP measurements by the bioluminescence assay.…”

“…These studies detected a marked deterioration in the sperm mitochondrial function following cryopreservation, as reported by the luorescent staining with rhodamine 123 (R123), the lipophilic cationic compound 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzymidazolyl carbocyanine iodine (JC-1), or with ATP measurements by the bioluminescence assay. The percentages of frozen-thawed spermatozoa with functional mitochondria, as assessed by either the R123/PI or JC-1/PI assay, are highly correlated to motility [5,29,33]. Besides the R123/PI and JC-1/PI assays, there are a plethora of luorescent dyes that can be used to microscopically or cytometrically assess the sperm mitochondrial membrane function (MMF).…”