Semen cryopreservation is a strategic tool to secure genetic diversity. Research efforts have focused on developing freezing protocols to improve the cryopreservation of rabbit semen by the reduction of sperm cryoinjuries. Since a lack of knowledge about the use of basic extenders, we tested the effect of a commercial extender (CortalapVR ) compared to Tris-citrate-glucose (TCG) on the in vitro post-thaw quality of rabbit semen. Six pools of semen (4 ejaculates/pool) were collected from 40 adult rabbit bucks of Bianca Italiana breed from the Italian Rabbit Breeders Association (ANCI-AIA, Volturara Appula (FG), Italy), an aliquot from each pool was used for the analysis of fresh semen, the remaining part of pooled semen was cooled at 5 C for 90 minutes. Each pool was split into two equal aliquots, and each of them were diluted to a ratio 1:1 (v:v) with a freezing extender composed of TCG or CortalapVR both containing 16% of dimethylsulfoxide and 0.1 M sucrose. The diluted semen was packaged into 0.25 mL plastic straws, equilibrated at 5 C for 45 min. Semen was frozen at heights of 5 cm above liquid nitrogen for 10 min, then straws were transferred into liquid nitrogen for storage at -196 C. Sperm samples were thawed at 50 C for 10 seconds. Sperm motility (phase contrast microscopy), viability (SyBr-PI), acrosome integrity (FITC-PSA) and DNA intactness (Acridine orange) were examined on fresh and post-thawed sperm. Sperm variables among the fresh and cryopreserved semen were compared by ANOVA, followed by Duncan’s comparison test, the level of significance was set at p .05. Results showed that the cryopreservation process impaired the post-thaw quality of rabbit semen compared to fresh semen (p < .05). However, the quality of frozen-thawed semen was affected by the freezing extender. In fact, the post-thaw semen quality was significantly improved in semen samples diluted in the CortalapVR extender compared with TCG for total and progressive motility (43.4 ± 1.4 vs 36.8 ± 1.5 and 36.5 ± 1.1 vs 30.2 ± 1.9), sperm viability (52.5 ± 1.8 vs 44.6 ± 2.1) and acrosome integrity (37.5 ± 0.6 vs 30.9 ± 1.2). The present results show that the CortalapVR extender provided better in vitro condition to preserve sperm integrity (membrane and acrosome) and function (motility) during the cryopreservation process. However, further studies are needed to confirm these results in vivo.
Comparison of two basic extenders on the in vitro post-thaw quality of rabbit semen
IAFFALDANO N.;DI IORIO M.;MANCHISI A.;
2013-01-01
Abstract
Semen cryopreservation is a strategic tool to secure genetic diversity. Research efforts have focused on developing freezing protocols to improve the cryopreservation of rabbit semen by the reduction of sperm cryoinjuries. Since a lack of knowledge about the use of basic extenders, we tested the effect of a commercial extender (CortalapVR ) compared to Tris-citrate-glucose (TCG) on the in vitro post-thaw quality of rabbit semen. Six pools of semen (4 ejaculates/pool) were collected from 40 adult rabbit bucks of Bianca Italiana breed from the Italian Rabbit Breeders Association (ANCI-AIA, Volturara Appula (FG), Italy), an aliquot from each pool was used for the analysis of fresh semen, the remaining part of pooled semen was cooled at 5 C for 90 minutes. Each pool was split into two equal aliquots, and each of them were diluted to a ratio 1:1 (v:v) with a freezing extender composed of TCG or CortalapVR both containing 16% of dimethylsulfoxide and 0.1 M sucrose. The diluted semen was packaged into 0.25 mL plastic straws, equilibrated at 5 C for 45 min. Semen was frozen at heights of 5 cm above liquid nitrogen for 10 min, then straws were transferred into liquid nitrogen for storage at -196 C. Sperm samples were thawed at 50 C for 10 seconds. Sperm motility (phase contrast microscopy), viability (SyBr-PI), acrosome integrity (FITC-PSA) and DNA intactness (Acridine orange) were examined on fresh and post-thawed sperm. Sperm variables among the fresh and cryopreserved semen were compared by ANOVA, followed by Duncan’s comparison test, the level of significance was set at p .05. Results showed that the cryopreservation process impaired the post-thaw quality of rabbit semen compared to fresh semen (p < .05). However, the quality of frozen-thawed semen was affected by the freezing extender. In fact, the post-thaw semen quality was significantly improved in semen samples diluted in the CortalapVR extender compared with TCG for total and progressive motility (43.4 ± 1.4 vs 36.8 ± 1.5 and 36.5 ± 1.1 vs 30.2 ± 1.9), sperm viability (52.5 ± 1.8 vs 44.6 ± 2.1) and acrosome integrity (37.5 ± 0.6 vs 30.9 ± 1.2). The present results show that the CortalapVR extender provided better in vitro condition to preserve sperm integrity (membrane and acrosome) and function (motility) during the cryopreservation process. However, further studies are needed to confirm these results in vivo.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
