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Background: REAC technology (acronym for Radio Electric Asymmetric Conveyor) is a technology platform for neuro and bio modulation. It has already proven to optimize the ions fluxes at the molecular level and the molecular mechanisms driving cellular asymmetry and polarization., Methods: This study was designed to verify whether this technology could extend spermatozoa life-span during liquid storage, while preserving their functions, DNA integrity and oxidative status. At 0, 24, 48, and 72 h. of storage at 4 °C, a battery of analyses was performed to assess spermatozoa viability, motility parameters, acrosome status, and DNA integrity during REAC treatment. Spermatozoa oxidative status was assessed by determining lipid peroxidation, the activity of superoxide dismutase (SOD), and the total antioxidant capacity., Results: During liquid storage REAC treated spermatozoa, while not showing an increased viability nor motility compared to untreated ones, had a higher acrosome (p > 0.001) and DNA integrity (p > 0.01). Moreover, the analysis of the oxidative status indicated that the mean activity of the intracellular superoxide dismutase (SOD) was significantly higher in REAC treated spermatozoa compared to untreated controls (p < 0.05), while the intracellular concentration of malondialdehyde (MDA), an end product of lipid peroxidation, at the end of the REAC treatment was higher in untreated controls (p > 0.05). The REAC efficacy on spermatozoa oxidative status was also evidenced by the higher trolox equivalent antioxidant capacity (TEAC) found in both the cellular extract (p < 0.05) and the storage media of REAC treated spermatozoa compared to untreated controls (p < 0.0001)., Conclusion: The present study demonstrated that REAC treatment during liquid storage preserves spermatozoa acrosome membrane and DNA integrity, likely due to the enhancement of sperm antioxidant defenses. These results open new perspective about the extending of spermatozoa functions in vitro and the clinical management of male infertility.

Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%-6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P < 0.0001). No significant differences were observed in intracellular ATP concentrations. Additional molecular features revealed that sperm functionality was affected in EXT EY, as demonstrated by lower DNA and acrosome integrity (P < 0.05), and higher lipid peroxidation compared with spermatozoa cryopreserved in EXT LC (P < 0.0001). Results obtained in the heterologous in vitro fertilization test showed that EXT LC better preserved spermatozoa functionality, as demonstrated by the higher fertilization rates compared with the other media (66.2 ± 4.5% for EXT LC vs. 32.7 ± 4.5%, 38.7 ± 4.5%, 39.6 ± 5.2% for EXT, EXT EY, and commercial extender; P < 0.01). The present study demonstrated that lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin-supplemented extenders (P<0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P<0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose-dependent manner. These results are likely to be mediated by its well-known antioxidant properties, even if a direct action of the indolamine cannot be ruled out., (© 2011 The Authors. Journal of Pineal Research © 2011 John Wiley & Sons A/S.)

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 μm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 μmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December - March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 microl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique.

Simple Summary: The oxidative stress in the cell is the result of an imbalance between oxidants and the antioxidant defense system. In particular, oxidative stress in erythrocytes and spermatozoa of small domestic ruminants can negatively affect their productivity and welfare. This review highlights the assay methods for the quantification of ROS and enzymatic antioxidant activity in these cells, in order to help the researchers to evaluate the intracellular oxidative status and antioxidant defenses. These data can be useful in monitoring the health and reproduction of small domestic ruminants. The present review aims to provide an overview of the assay methods for the quantification of ROS and principal enzymatic antioxidants as biomarkers of oxidative stress in erythrocytes and spermatozoa of small domestic ruminants. A complete literature search was carried out in PubMed, Scopus and the World Wide Web using relevant keywords and focusing on the last five years (2018–2023). Among spectrophotometry, fluorometry and chemiluminescence, the most widely used method for ROS assay is fluorometry, probably because it allows to simultaneously assay several ROS, using different probes, with greater economic advantages. Regarding intracellular antioxidant enzymes, recent literature reports only spectrophotometric methods, many of which use commercial kits. The use of a less sensitive but cheapest method is suitable because both erythrocytes and spermatozoa samples are highly concentrated in domestic ruminant species. All methods considered in this review have been found to be appropriate; in general, the differences are related to their costs and sensitivity. Quantification of ROS and enzymatic antioxidant activity in erythrocytes and spermatozoa may find application in the study of the welfare and health status of small domestic ruminants for monitoring livestock production. [ABSTRACT FROM AUTHOR]