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Setting an open-access computer assisted sperm analysis (CASA) may benefit the evaluation of motility in mammalian sperm, especially when economic constraints do not allow the use of a commercial system. There have been successful attempts to develop such a device in Zebra fish sperm and the system has been used in very few studies on mammalian spermatozoa. Against this background, the present study aimed at developing an open-access CASA system for mammalian sperm using the horse as a model and based upon the Image J software previously established for Zebra fish sperm. Along with determining the sperm progressive motility and other kinetic parameters (such as amplitude of lateral head displacement), the "results" window was adjusted to simplify subsequent statistical analyses. The path window was enriched with colored sperm trajectories on the basis of the subpopulation they belong to and a number that allowed the sperm track to be associated to the sperm motility data shown in the "results" window. Data obtained from the novel plugin (named as CASA_bgm) were compared with those of the commercial CASA Hamilton-Thorn IVOS Vers.12, through Bland Altman's plots. While the percentage of total and progressive motile sperm, VCL, VAP, VSL, LIN and STR and ALH were in agreement with those obtained with the commercial system, BCF significantly differed between the two systems probably due to their settings. Interestingly, a positive and significant correlation between the percentages of total motile sperm evaluated through CASA_bgm and those showing high mitochondrial membrane potential evaluated by JC-1 staining was found. In conclusion, CASA_bgm ImageJ plugin could be useful and reliable for stallion sperm motility analysis and it is our aim to apply this system to other mammalian species., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatozoa were more prone to undergo acrosome reaction (P<0.05), at the beginning and after 1.5h and 3h of incubation, and also had higher tyrosine phosphorylation of the tail (P<0.001), only at the beginning of the incubation period. Apparently sex sorted stallion spermatozoa are in a more advanced status of membrane destabilization, which could be associated with capacitation, although similar binding ability to porcine oocytes is maintained., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Single layer centrifugation (SLC) is a useful technique to select porcine spermatozoa for further artificial insemination practices. The aim of this study was to determine possible side-effects related to capacitation due to the process. Semen viability, acrosome integrity and capacitation status were determined through fluorescent probes (SYBR14-PI, FITC-PSA, CTC staining) and Hsp70 immunolocalization and protein tyrosine phosphorylation (by western blotting and immunolocalization) in different groups: control, after SLC with Androcoll (AND), after SLC and washing (AND-Wash) and after SLC, washing and storage for 2h at 17°C with 2.5% of seminal plasma (AND-Wash-SP). Neither viability nor acrosome integrity were impaired by the different treatments; as far as CTC staining, we observed a significant increase (p<0.05) in the capacitation related pattern in AND and AND-Wash, while after exposure for 2h to seminal plasma (AND-Wash-SP group), the increase became less evident; the same trend was observed in Hsp70 immunolocalization for the EL pattern. Neither immunolocalization nor western blotting for tyrosine phosphorylated proteins had an increase in capacitated pattern or in phosphorylation status, except for a 25kDa band that increased in AND and AND-Wash groups and decreased in AND-Wash-SP group. SLC using Androcoll-P induces some capacitation-related changes in boar sperm membrane, as demonstrated by CTC staining and Hsp70 immunolocalization. For protein tyrosine phosphorylation, only a 25kDa protein showed some changes that should be investigated further., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI-) and strongly (VAD++ PI-) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI-, early apoptotic) and YO-PRO-1(YP+ PI-, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI-) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI-) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI-) as compared with the control. Post warming incubation for 2h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P-) oocytes while a decrease of the percentage of VAD+/PI- oocytes and a contemporaneous increase of VAD-/PI- oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI-). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes' cryopreservation protocols., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Sorting procedures submit sperm cells to a set of stressful steps that can trigger an increase of ROS production and consequently reduce sorted semen quality. This study evaluated the effect of supplementation with different antioxidants (EGCG, Na pyruvate+catalase, SOD) on acrosome and plasma membrane integrity, activation of caspases (as assayed by FITC-VAD/PI staining) and immunolocalization of Hsp70 of boar spermatozoa after sperm preparation (Hoechst 33342 staining) and sorting procedure. The effect of antioxidants, with or without seminal plasma, on sorted spermatozoa stored for 24h at 15°C was also evaluated. Antioxidants did not exert any preventive action on sperm modification induced by staining and sorting. After 24h of storage at 15°C, sorted samples supplemented with either EGCG or SOD plus seminal plasma showed a significant (p<0.05) increase of the percentage of viable spermatozoa, while no positive effect on the other tested parameters was observed; EGCG seems to exert an inhibition on caspase activation in that a decrease of the number of dead cells FITC-VAD+/PI+ was recorded. In conclusion, our results indicate that EGCG and SOD in association with seminal plasma are effective in exerting some compensatory protection against the detrimental effects of storage of sorted semen while their action is not evident during steps of the sorting procedure., (Copyright © 2010 Elsevier B.V. All rights reserved.)