Determinación del potencial de membrana mitocondrial mediante citometría de flujo durante el proceso de criopreservación de espermatozoides epididimarios de alpacas

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

El objetivo de este estudio fue demostrar, mediante la citometría de flujo, que el porcentaje de espermatozoides de alpaca con alto potencial de membrana mitocondrial (PMM) se reduce significativamente luego del proceso de criopreservación. Se trabajó con 41 testículos de alpacas obtenidos del Camal Municipal de Ninacaca, Pasco, Perú. Se recuperaron los espermatozoides de la cola de los epidídimos con un dilutor en base a leche descremada y se congelaron solo aquellas muestras con mínimos de 30% de motilidad y 50x10(6) espermatozoides/ml. La evaluación del PMM se realizó antes y después del proceso de criopreservación. Cada muestra se incubó durante 10 minutos a 38 °C con MitoTracker Deep Red (100 nM) para determinar el porcentaje de alto PMM, mediante citometría de flujo con imágenes. El efecto de la criopreservación en el porcentaje de espermatozoides con alto PMM fue evaluado mediante la prueba de T-student pareada. Así mismo, se correlacionaron los porcentajes de motilidad y espermatozoides con alto PMM. Se obtuvo un 49.82 ± 12.41 y de 34.97 ± 9.96% de alto PMM en muestras antes y después del proceso de criopreservación (p<0.05). Se encontró una correlación positiva (r = 0.62; p<0.0001) entre motilidad y PMM. Se concluye que el PMM se reduce significativamente luego del proceso de criopreservación, parámetro relacionado a la motilidad espermática.

Translated abstract

The aim of this study was to demonstrate, by flow cytometry, that the percentage of alpaca spermatozoa with high mitochondrial membrane potential (MMP) is significantly reduced after the cryopreservation process. Forty-one alpaca testicles were obtained from a local slaughterhouse in the Ninacaca district, Pasco, Peru. The spermatozoa were recovered from the tail of the epididymis with an extender based on skim milk and only those samples with minimum 30% motilityand 50x10(6) spermatozoa/ml were cryopreserved. The MMP evaluation was carried out before and after the cryopreservation process. Each sample was incubated for 10 minutes at 38 °C with MitoTracker Deep Red (100 nM) to determine the percentage of high MMP by imaging flow cytometry. The effect of cryopreservation on the percentage of spermatozoa with high MMP was evaluated by a paired t-student test and the percentage of motility and sperm with high MMP were correlated. A 49.82 ± 12.41 and 34.97 ± 9.96% of high MMP were obtained in samples before and after the cryopreservation process (p<0.05).Apositive correlationwas found (r = 0.62, p<0.0001) between motility and PMM. It is concluded that MMP is significantly reduced after the cryopreservation process, a parameter related to sperm motility.

Related collections

Most cited references 14

Record: found
Abstract: found
Article: not found

Mitochondrial activity of frozen-thawed spermatozoa assessed by MitoTracker Deep Red 633.

Szabolcs Nagy, Triin Hallap, Ulle Jaakma … (2005)

The present study was conducted to find a more objective method of evaluating sperm quality than the current subjective motility evaluations by testing the applicability of a novel fluorescent probe, Mitotracker Deep Red 633 (M-22426), for measuring the mitochondrial activity of spermatozoa both after freezing/thawing (PT) and after swim-up selection (SU), using flow cytometry (FC). The results from FC were compared to those of conventional microscopic motility evaluations and of computer-assisted sperm analysis (CASA) as well as to the fertility obtained after AI in the field. Semen from six Estonian Holstein bulls, processed when the sires were aged 3, 5, and 7 years, was included in the experiment. Sperm motility (measured either subjectively or by means of CASA) was always significantly (p<0.01-0.001) higher in the spermatozoa recovered by SU, for any of the age groups considered, or even when combining the age groups. Motility (measured subjectively) after SU was significantly (p<0.05) higher when bulls reached 7 years of age, compared to earlier collection ages, but no differences were registered between ages for CASA-assessed motility, either after SU or immediately PT. The numbers of spermatozoa with high red fluorescence also increased after SU: p<0.05 (for semen from bulls aged 3 years), p<0.001 (5 years), p<0.001 (7 years), and p<0.001 when all age groups were combined. The proportion of spermatozoa with high mitochondrial activity as determined by Mitotracker Deep Red 633 correlated with sperm motility (p<0.01) both PT and after SU, but not with the fertility results. In conclusion, MitoTracker Deep Red 633 seems to be a reliable marker for frozen-thawed bovine semen viability both PT and after SU.