Evaluation of sperm
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
An intact plasma membrane is an integral component of, and possibly a biologic/diagnostic indicator for, sperm viability. The underlying principle is that viable sperm contain intact plasma membranes that prevent the passage of certain stains, whereas nonviable sperm have defects within their membranes that allow for staining of the sperm. Several so-called “vital stains” have been employed for this purpose. They include eosin Y, trypan blue, and/ or nigrosin (27). When viewed with either bright field or phase contrast microscopy, these stains allow for the differentiation of viable, non-motile sperm from dead sperm. This procedure may, therefore, play a significant role in determining the percentage of immotile sperm that are viable and available for ICSI. Unfortunately, dyes such as eosin Y are specific DNA probes that may have toxic effects if they enter a viable sperm or oocyte, which precludes the use of these sperm that have been exposed to the dyes for ICSI or insemination. Flow cytometry has also been utilized for the determination of sperm viability. Like vital staining, flow cytometry is based on the principle that an intact plasma membrane will prevent the passage of nucleic acid-specific stains. Some techniques, such as the one described by Noiles et al., employ dual staining, which can differentiate between an intact membrane and a damaged membrane (28). There are no studies that prospectively evaluate sperm viability staining as a predictor of ART outcome.
Semen Analysis
Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh in Male Infertility in Reproductive Medicine, 2019
The staining solution consists of Eosin–Y (1%) and Nigrosin (10%). The slides are labeled and one drop of liquefied semen sample is placed into a Boerner slide well. Two drops of Eosin are added and mixed well followed by two drops of Nigrosin. After mixing well, two smears are made and air dried and a coverslip is placed and sealed with a cytoseal mounting media. The slide is observed under 100× objective. Eosin stains only the dead sperm, turning them a dark pink, whereas live sperm appear white or faint pink (Figure 3.2). The normal range is 58% according to WHO guidelines [3]. For specimens with motility <25%, viability should be ≥25%.
Semen Analysis and Sperm Washing Techniques
Claude Gagnon in Controls of Sperm Motility, 2020
If a sample shows positive in the screening assay, then a complete spermotoxicity titration assay is run. In this situation, the serial dilutions are prepared in the V-well trays, an equal volume of the sperm preparation added (i.e., 25 μl with 50 μl of the 1/4 dilution being discarded first) and incubated for 60 min at 37°C. After this time, 25 to 50 μl of eosin-nigrosin stain (Table 2) is added, mixed, and air-dried smears prepared. A sample dilution is considered positive if the vitality is less than half that seen in the negative control series of wells. Again, a control for direct spermotoxicity of the complement must be included.
Cryoprotective effect of sericin supplementation in freezing and thawing media on the outcome of cryopreservation in human sperm
Published in The Aging Male, 2020
Faranak Aghaz, Mozafar Khazaei, Asad Vaisi-Raygani, Mitra Bakhtiyari
As shown within the Table 3, the significant differences were peered in motility, viability and DNA fragmentation index (DFI) between the groups. Our results indicate that, total motility (A + B) was considerably higher in treated group with 5% sericin (61.66 ± 12.79) than the control group (46.71 ± 15.63) (P < 0.05). No significant difference was seen between the various sericin groups (0.5, 1.5, and 2.5%) and the control group (0% sericin) when morphology was compared. From the results of Eosin-nigrosin staining obtained, viability was significantly higher in treated group with 5% sericin (67.93 ± 10.15) than the control group (53.13 ± 11.1) (P < 0.05). DFI was significantly lower in the treated groups with 5% sericin (15.73 ± 3.69) than the control (26.13 ± 4.50). However, the differences between 0.5, 1, and 2.5% serein groups weren’t significant. Similar to the results observed in the motility analysis, no significant difference was seen in the percentage of viability and DFI when the 0.5, 1, and 2.5% groups were compared with the 0% group.
Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm
Published in The Aging Male, 2020
Maíra Morales Brito, Daniel de Souza Ramos Angrimani, Bruno Rogério Rui, Giulia Kiyomi Vechiato Kawai, João Diego Agostini Losano, Camila Infantosi Vannucchi
To evaluate the integrity of the sperm plasma membrane, eosin/nigrosin staining was performed, allowing differentiation of cells with changes in plasma membrane permeability [20]. In brief, 5 μL of semen and 5 μL of the previously prepared eosin–nigrosin stain were placed in a prewarmed glass slide. The sperm smear was evaluated under light microscopy by counting 200 cells at 1000× magnification. The Fast-Green/Rose-Bengal staining was used to evaluate the acrosomal membrane integrity of spermatozoa [21]. Briefly, 5 μL of semen was mixed with 5 μL of fast green/rose bengal stain in a warmed glass slide. Smears were evaluated under light microscopy at 1000× magnification. For the analysis of sperm DNA integrity, the technique of toluidine blue staining was used [22]. Smears were evaluated under light microscopy at 1000× magnification. Damaged sperm (DNA fragmentation) were stained blue, while intact sperm (DNA integrity) were stainless.
Evaluating the therapeutic effect and toxicity of theophylline in infertile men with asthenoteratozoospermia: a double-blind, randomized clinical trial study
Published in Drug and Chemical Toxicology, 2022
Atena Sadat Azimi, Malek Soleimani Mehranjani, Seyed Mohammad Ali Shariatzadeh, Alireza Noshad Kamran, Ali Asghar Ghafarizadeh
Eosin–nigrosin staining was used to evaluate sperm viability in order to diagnose live sperms from the dead ones. First, 20 µL of semen samples and 40 µL of the eosin solution (Sigma-Aldrich, St. Louis, MO) (0.5%) were mixed thoroughly for 30 s. Then, 60 μL of the nigrosin solution (Sigma-Aldrich, St. Louis, MO) (0.1%) was added and mixed again for another 30 s. Ten microliters of the final mixture was smeared on the slide and after 60 seconds at least 200 cells were calculated by an optical microscope (Olympus CX41, Tokyo, Japan) with a ×1000 magnification. Dead sperms were stained pink by eosin, whereas the membrane integrity of live sperms prevented eosin from entering the cell and therefore remained unstained. Nigrosin served as the background stain (Figure 1).