Semen samples were cryopreserved using the TEST-Yolk Buffer (TYB, Irvine Scientific, Santa Ana, CA, USA). Aliquots of TYB equal to 25% of the sample volume were added to the specimen at room temperature, and mixed gently for 5 min using the Hema-Tek aliquot mixer (Miles Scientific, Elkhart, IN, USA). The procedure was repeated to a final 1:1 (v/v) ratio of the freezing medium to the sample. Further, the samples were dispensed into cryovials (1.5 mL; Corning, Pittsburg, PA, USA) and transferred to the freezer (−20 °C) for 8 min (static cooling), and subsequently to liquid nitrogen vapor (−80 °C) for 2 h (vapor-phase cooling). Finally, the cryovials were stored in liquid nitrogen at −196 °C until use for proteomics analysis.
Test yolk buffer
Manufactured by Irvine Scientific
Sourced in United States
TEST-Yolk Buffer is a sterile, ready-to-use media formulation designed for use in the maintenance of oocytes and embryos during in vitro fertilization (IVF) procedures. The buffer contains a combination of salts, amino acids, and other components to provide a balanced environment for the culture of reproductive cells.
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Lab products found in correlation
5 protocols using test yolk buffer
1
Cryopreservation of Semen Samples
2
Cryopreservation of Normozoospermic Semen
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This is a prospective cohort study. Semen samples were obtained from 184 normozoospermic patients following sperm evaluation performed at FertiClinic, Villa Margherita, from September to December 2014. Patients were randomized in two groups: group A (N = 92 samples) and group B (N = 92 samples). Samples in group A were split into 2 aliquots and cryopreserved adding, respectively, TEST Yolk Buffer (Irvine Scientific, California) and Sperm Freeze (FertiPro, Belgium) after washing and resuspension. On the contrary, group B samples were split into 2 aliquots and cryopreserved adding, respectively, TYB and SF directly to the semen (Figure 1 ). Sperm viability and progressive motility were the outcome measures assessed after thawing by a blinded observer. A single team of biologists coordinated all biological work, ensuring that both freezing protocols and viability and motility assessment were standardized.
3
Cryopreservation and Thawing of Sperm
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Equal amount of freshly prepared sperm suspension from 21 patients in cleavage medium and TEST-yolk buffer (Irvine Scientific, CA, USA) were mixed and dispended into cryotubes. After exposing to nitrogen steam for 5 minutes, cryotubes were stocked in liquid nitrogen [24 (link)]. To thaw frozen sperm solution, cryotubes were warmed at 37°C for 5 min, and then each frozen-thawed sperm suspension was dispensed into 4 vials. To prepare the sperm-wash medium containing H2, a 50, 75 or 100% of cleavage medium saturated with H2 was mixed with the medium equilibrated with 5% CO2. Sperm suspensions were washed for 5 minutes with them, and measured their motility.
4
Semen Cryopreservation and Somatic Cell Purification
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Semen samples were collected by masturbation after 2–5 days of sexual abstinence. Semen analysis was assessed in fresh semen samples according the 2010 World Health Organization’s criteria19 . After the semen analysis, semen samples were frozen following a well-stablished slow freeze protocol20 (link). Briefly, semen samples were mixed in a 1:1 ratio with test yolk buffer (Irvine Scientific, CA, USA) and placed in liquid nitrogen vapors and finally maintained in liquid nitrogen until further analysis.
After thawing the sperm samples a sperm purification protocol that included stringent somatic cell lysis was performed as described previously21 (link). This purification was performed by incubating sperm samples with 0.1% SDS and 0.5% Triton X-100 (in Milli-Q® water), on ice followed by two high volume wash steps and a final optical microscopic examination to verify the somatic cell elimination. All samples were also epigenetically screened (DLK1 locus) to ensure no somatic cell contamination according to a previously published method of our group14 (link).
Total sperm DNA was isolated using a sperm-specific modification to the Qiagen DNeasy (QIAGEN, CA, USA) manufacturer protocol5 (link), and DNA concentration and purity were determined using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA).
After thawing the sperm samples a sperm purification protocol that included stringent somatic cell lysis was performed as described previously21 (link). This purification was performed by incubating sperm samples with 0.1% SDS and 0.5% Triton X-100 (in Milli-Q® water), on ice followed by two high volume wash steps and a final optical microscopic examination to verify the somatic cell elimination. All samples were also epigenetically screened (DLK1 locus) to ensure no somatic cell contamination according to a previously published method of our group14 (link).
Total sperm DNA was isolated using a sperm-specific modification to the Qiagen DNeasy (QIAGEN, CA, USA) manufacturer protocol5 (link), and DNA concentration and purity were determined using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA).
5
Cryopreservation of Human Semen
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Semen samples used for the current study were obtained from the University of Utah tissue bank, after informed consent had been provided according to IRB-approved protocols. Individuals were asked to adhere to general semen collection instructions, which included 2-5 days of abstinence immediately preceding collection. Collected samples were mixed in a 1:1 ratio with Test Yolk Buffer (Irvine Scientific) and stored in liquid nitrogen until they were used in this study.
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