Comparison of sperm quality after double slow freezing and double vitrification of stallion sperm

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Intracytoplasmic sperm injection (ICSI) is a valuable tool to optimize the remaining frozen sperm of subfertile or dead stallions. The possibility of thawing, diluting, and re-freezing, conventionally frozen spermatozoa has been previously studied, but not for vitrified sperm. We compared the effect of thawing/warming and re-freezing/re-vitrifying conventionally frozen/vitrified stallion sperm. For this purpose, 10 ejaculates from 2 stallions were divided into two aliquots and submitted to i)slow freezing (SF), extending sperm at 100 × 10 6 sperm/ml in permeable cryoprotectant medium (Botucrio, Betlabs, Lexington, KY), slow cooling for 2 hours and frozen 4cm over liquid nitrogen (LN 2 ) for 10 minutes; ii) or vitrification (VIT), extending sperm in permeable-cryoprotectant free medium (INRA96 + Trehalose 100mM + BSA 1%), cooled for 1 h and plunged directly into LN 2 . Two SF straws were thawed at 37 °C for 30 s, whereas two VIT straws were warmed at 60 °C for 5s. One straw was used for sperm parameters assessment while the second straw was re-frozen (RE-SF) or re-vitrified (RE-VIT) andthawed for sperm analysis. Sperm were analyzed for total (TMOT) and progressive (PMOT) motility (CASA, SCA, Microptic, Barcelona, Spain) and percentage of viable, non-reacted sperm (VNR, PI/PNA staining using flow cytometry). TMOT (80.6 ± 2.2 % vs. 70.2 ± 2.9% and 79.4 ± 2.5% vs. 70.1 ± 3.0%), PMOT (46.9 ± 6.1% vs. 31.3 ± 3.4% and 47.8 ± 6.4% vs. 38.1 ± 3.9%) and VNR (81.4 ± 2.4% vs. 71.2 ± 3.1% and 79.1 ± 3.1 vs. 72.2 ± 3.9%) showed significantly (p<0.05) lower values after SF vs. RE-SF and VIT vs RE-VIT, respectively. RE-VIT showed significantly (p<0.05) higher PMOT values than RE-SF (38.1 ± 3.9% vs. 31.3 ± 3.4%). In conclusion, both re-freezing and re-vitrification decrease sperm parameters after warming. Interestingly, re-vitrified sperm obtained higher progressive motility values than conventionally re-frozen sperm. Although further studies are needed to evaluate the effect of using vitrified sperm for ICSI, re-vitrification seems an interesting alternative for the optimization of valuable stallion ejaculates.