The CASA analysis.

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.</div

DNA fragmentation index was determined using AOT.

Intact DNA is shown as green cells, and damaged DNA is observed as yellow and red cells by fluorescent microscopy with 100× magnification (A). The DFI was significantly different in all of the groups (B). The assigned letters a, b, c, and d indicate significant differences (P < 0.05). Values are expressed as mean ± SD, Tukey test; n = 25.</p

S6 File -

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.</div

The viability of sperm.

The viable and dead cells are observed in panel A. The Eosin-Nigrosin staining was used, where colorless sperm and complete/partial stained sperm show live and dead sperm, respectively (A). The percentage of viable cells differed in all experimental groups (B). The assigned letters a, b, c, and d indicate significant differences (P < 0.05). Values are expressed as mean ± SD, Tukey test; n = 25.</p

Sperm morphology.

Normal and abnormal morphology of sperm as indicated by Papanicolaou staining; the intact cells and defects of the tail, head, and midpiece in sperm are observed (A), and the percentage of abnormal morphology differs in all of the groups (B). The assigned letters a, b and c indicate significant differences (P < 0.05). Values are expressed as mean ± SD, Tukey test; n = 25.</p

Comparison of MDA, ROS, TAC and mitochondrial membrane potential among the experimental groups.

Comparison of MDA, ROS, TAC and mitochondrial membrane potential among the experimental groups.</p

Membrane integrity of sperm was estimated using the HOS test.

The membrane intact and damaged are observed (A), and the percentage of membrane-intact differs among the experimental groups (B). The assigned letters a, b, c, and d indicate significant differences (P < 0.05). Values are expressed as mean ± SD, Tukey test; n = 25.</p

S3 File -

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.</div

The acrosome integrity was assessed using the fluorescein conjugated lectin Pisum sativum agglutinin (FITC-PSA) staining.

Sperm with intact and reacted acrosome was observed (A), and the percentage of intact acrosome significantly differs in all groups (B). The assigned letters a, b and c indicate significant differences (P < 0.05). Values are expressed as mean ± SD, Tukey test; n = 25.</p

Evaluation of mitochondrial membrane potential.

The panel of flow cytometry was stained with JC-1 for fresh control (A), frozen control (B), frozen trehalose (C), and frozen gentiobiose (D). The top right quadrant represents the percentage of values showing the proportion of sperm with high mitochondrial membrane potential. The bottom right quadrant represents the percentage of values that display the proportion of sperm with low mitochondrial membrane potential.</p