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Melatonin is known to protect sperm against freezing‐inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA‐82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA‐82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin‐supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.

<p>The present study aimed to evaluate the effect of supplementation of INRA-82 semen extender with different cryoprotectants (dimethyl sulphoxide; DMSO vs. dimethyl formamide; DMF) on the quality of white New Zealand rabbit buck spermatozoa. We also investigated the possible association between the synergistic action of DMSO and DMF and their relation with INRA-82 extender composition. Semen was collected and pooled from 8 adult rabbit bucks. Pooled semen samples were diluted 1:1 with INRA-82 extender supplemented with DMSO 8%, DMF 8% or a combination of DMSO 4% and DMF 4%. The diluted semen samples were cryopreserved in 0.25 plastic straws. After thawing, progressive motility, sperm viability, sperm abnormalities, membrane integrity, acrosome status, viability index and DNA integrity were evaluated. The results showed that dilution of rabbit buck semen in INRA-82 supplemented with DMSO and DMF (4% each) before freezing significantly (<em>P</em>&lt;0.05) improved sperm motility (42.00%), percentage of live spermatozoa (45.30%), proportions of spermatozoa with intact acrosome (59.75%) and percentage of spermatozoa with non-fragmented DNA (86.04%), compared to those diluted in INRA-82 supplemented either with DMSO 8% (+9, +10, +5 and +7 percentage points, respectively) or with DMF 8% alone (+18 +18, +12 and +9 percentage points, respectively). In conclusion, dilution of rabbit buck semen before freezing with INRA-82 extender supplemented with a combination of DMSO 4% and DMF 4% improved quality of frozen-thawed New Zealand White rabbit spermatozoa. Furthermore, our results also suggest that supplementation of INRA-82 with DMSO or with DMF alone at higher concentrations deteriorates the sperm quality.</p>

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