RESUMEN
Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris-based extender supplemented with 100 µm quercetin to a concentration of 15, 30 or 60 × 10(6) spermatozoa/ml, packaged into plastic tubes or 0.5-ml straws and stored at 15°C. Sperm quality was assessed by computer-assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2 O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10(6) spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD-DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10(6) spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10(6) spermatozoa/ml may provide a suitable balance between motility and H2 O2 production to best maintain overall sperm function and should be evaluated in a large-scale AI trial.
Asunto(s)
Quercetina/administración & dosificación , Conejos , Preservación de Semen/veterinaria , Semen/citología , Recuento de Espermatozoides/veterinaria , Acrosoma/ultraestructura , Animales , Apoptosis , Supervivencia Celular , ADN , Peróxido de Hidrógeno/metabolismo , Inseminación Artificial/veterinaria , Masculino , Semen/fisiología , Preservación de Semen/instrumentación , Motilidad Espermática , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Factores de TiempoRESUMEN
Reactive oxygen species, such as hydrogen peroxide, H2O2, can reduce sperm quality during storage. This study evaluated the effect of methionine and quercetin on rabbit sperm quality during liquid storage over 96h. Semen was collected from adult bucks (n=4) and pooled following evaluation. In Experiment 1, pooled ejaculates were diluted with a Tris extender supplemented with methionine (1, 6 or 12mM), quercetin (50 or 200µM) or no antioxidant (control) and then subdivided for storage at 5°C or 15°C. Sperm quality was assessed by CASA (total motility [TM]) and flow cytometry (viability, acrosome integrity and H2O2 production) at 0, 48, 72 and 96h. Experiments were replicated three times. Motility was significantly higher in control samples and lowest following dilution with 200µM quercetin, irrespective of storage temperature. Storage at 15°C improved viability and acrosome integrity compared with 5°C, but produced significantly more H2O2 at 72 and 96h in sperm diluted with methionine or no antioxidant. Quercetin-supplemented spermatozoa exhibited lower levels of H2O2 at both storage temperatures for all incubation times (P<0.05). In Experiment 2, the concentration of quercetin (0, 25, 50, 100 and 200µM) was investigated with additional quality parameters; lipid peroxidation and DNA integrity. All concentrations of quercetin reduced H202 and lipid peroxidation during storage at 15°C, but were not beneficial for TM, viability, acrosome or DNA integrity. Only supplementation with 100 and 200µM quercetin resulted in similar H202 levels at 5°C and 15°C (P>0.05). Overall, quercetin-supplementation to sperm medium provided protection against oxidative stress in 15°C-stored rabbit spermatozoa over 96h.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Quercetina/farmacología , Refrigeración , Preservación de Semen , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Animales , Antioxidantes/farmacología , Células Cultivadas , Masculino , Conejos , Especies Reactivas de Oxígeno/metabolismo , Refrigeración/veterinaria , Análisis de Semen/veterinaria , Preservación de Semen/efectos adversos , Preservación de Semen/veterinariaRESUMEN
Artificial insemination (AI) programmes in the rabbit meat industry require improved longevity of spermatozoa stored in vitro. Two studies evaluated the effects of storage temperature and extender on in vitro quality and fertility of rabbit spermatozoa over 96h of chilled storage. In Experiment 1, three ejaculates were collected from each of five bucks and diluted 1:10 in either Extender A or B, and then divided further for storage at 5°C or 15°C. Sperm motility (MOT) was assessed by CASA at 0, 24, 48, 72 and 96h of storage. Viability, acrosome integrity, mitochondrial membrane potential (MMP), oxidative stress and DNA integrity of the two best extenders were assessed by flow cytometry. Extender B at 15°C gave significantly higher values of MOT and MMP from 24 and 72h, respectively. At 96h, viability, acrosome and DNA integrity were best maintained at 15°C (P<0.05). In contrast, storage at 5°C resulted in lower oxidative stress from 72h. In Experiment 2, a pilot study examined fertility rates of does inseminated with spermatozoa diluted in Extender B and stored at 5°C or 15°C. Sixty seven multiparous does were inseminated with spermatozoa stored for 0h (n=12; control), 48h (n=26) or 72h (n=29). Kindling rates and litter sizes for does inseminated with semen stored for 48h at 5°C or 15°C and 72h at 5°C were similar (P>0.05) to those of the controls; kindling rate dropped following insemination with spermatozoa held at 15°C for 72h, though litter size did not.