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Volume 19, Issue 1 (January 2017) 19, 107–112; DOI:10.4103/1008-682X.183378

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

Fabrizzio Horta1, Hamida Alzobi2, Sutthipat Jitanantawittaya2, Sally Catt2, Penny Chen2, Mulyoto Pangestu2, Peter Temple-Smith2

1 Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia; Department of Obstetrics and Gynaecology, Unit of Reproductive Medicine, Clínica Las Condes, Lo Fontecilla 441, Las Condes, Santiago, Region Metropolitana, 7550000, Chile,
2 Education Program in Reproduction and Development, Department Obstetrics and Gynaecology, Monash University, Clayton VIC 3168, Australia

Correspondence: F Horta MS., MClinEmb (fabrizzio.horta@gmail.com)

Date of Submission 23-Oct-2015 Date of Decision 09-Feb-2016 Date of Acceptance 25-Apr-2016 Date of Web Publication 15-Jul-2016


This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

Keywords: cryopreservation; epididymal sperm; in vitro fertilization/intracytoplasmic sperm injection; reproductive techniques; sperm DNA damage; vitrification

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