The aim of the present study was to measure changes in DNA integrity of chicken spermatozoa after cryopreservation by the pellet method using the single cell gel electrophoresis assay, commonly known as comet assay. This is a sensitive method which determines DNA fragmentation in individual cells, widely used in mammalian spermatozoa. Semen was collected twice weekly by abdominal massage from 18 Mericanel della Brianza (local Italian breed) male chicken breeders. Semen was frozen/thawed according to the cryopreservation pellet method reported by Tselutin et al. (1999). In brief, pooled semen was diluted 1 : 2 and equilibrated at 4C, added with 6% dimethylacetamide, equilibrated for 1 minute at 4C, and finally dropped into a liquid nitrogen bath to form frozen pellets. Semen pellets were thawed in water bath at 60C. DNA integrity was assessed on fresh semen soon after dilution and after thawing: spermatozoa were embedded in low melting point agarose on a microscope slide and lysed by detergent with high salt medium and dithio-DL-threitol at 4C for 1 h and then incubated at 37C for 1 h with proteinase K (modified from Sakkas et al., 2002). Electrophoresis was performed at pH 7.3 for 20 minutes at 10V and 8 mA. After staining with a DNA-binding dye (ethidium bromide), slides were examined under an epifluorescence microscope (Leica DMLB) at 400x magnification. Within random fields, 200 spermatozoa from each slide were counted and scored for the presence or absence of comets. Images were acquired and then analysed by Comet Assay software (CASP). The proportion of spermatozoa with damaged DNA and the proportion of DNA in the comet tail of damaged spermatozoa were calculated. The experimental protocol was repeated on different days of semen collection to increase the number of replicates. The proportion of total viable spermatozoa was also measured by eosin-nigrosin staining. Sperm DNA damages occurred during cryopreservation of chicken semen and the proportion of spermatozoa with undamaged DNA significantly decreased from 93.9% in fresh semen to 80.2% in frozen/thawed semen pellets (P= 0.0001). The proportion of DNA in the comet tail of damaged spermatozoa was not significantly affected by cryopreservation, even if a limited increase was found between fresh (26.8%) and frozen/thawed (30.9%) spermatozoa. In contrast, the proportion of total viable spermatozoa was greatly impaired by cryopreservation and reduced from 82.7% (fresh semen) to 32.5% in stored semen. The present results suggest a low sensitivity of frozen/thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation. Similar low levels of DNA damage were found in cryopreserved bull and boar spermatozoa, whereas remarkable levels of sperm DNA fragmentation were reported in other animals such as the stallion and the monkey. Species differences in the success of semen cryopreservation are well known, and the widening of DNA damage occurring during freezing/thawing might contribute to understand the underlying phenomena.
Effect of cryopreservation on sperm DNA integrity in chicken semen.
Gliozzi TM;
2008
Abstract
The aim of the present study was to measure changes in DNA integrity of chicken spermatozoa after cryopreservation by the pellet method using the single cell gel electrophoresis assay, commonly known as comet assay. This is a sensitive method which determines DNA fragmentation in individual cells, widely used in mammalian spermatozoa. Semen was collected twice weekly by abdominal massage from 18 Mericanel della Brianza (local Italian breed) male chicken breeders. Semen was frozen/thawed according to the cryopreservation pellet method reported by Tselutin et al. (1999). In brief, pooled semen was diluted 1 : 2 and equilibrated at 4C, added with 6% dimethylacetamide, equilibrated for 1 minute at 4C, and finally dropped into a liquid nitrogen bath to form frozen pellets. Semen pellets were thawed in water bath at 60C. DNA integrity was assessed on fresh semen soon after dilution and after thawing: spermatozoa were embedded in low melting point agarose on a microscope slide and lysed by detergent with high salt medium and dithio-DL-threitol at 4C for 1 h and then incubated at 37C for 1 h with proteinase K (modified from Sakkas et al., 2002). Electrophoresis was performed at pH 7.3 for 20 minutes at 10V and 8 mA. After staining with a DNA-binding dye (ethidium bromide), slides were examined under an epifluorescence microscope (Leica DMLB) at 400x magnification. Within random fields, 200 spermatozoa from each slide were counted and scored for the presence or absence of comets. Images were acquired and then analysed by Comet Assay software (CASP). The proportion of spermatozoa with damaged DNA and the proportion of DNA in the comet tail of damaged spermatozoa were calculated. The experimental protocol was repeated on different days of semen collection to increase the number of replicates. The proportion of total viable spermatozoa was also measured by eosin-nigrosin staining. Sperm DNA damages occurred during cryopreservation of chicken semen and the proportion of spermatozoa with undamaged DNA significantly decreased from 93.9% in fresh semen to 80.2% in frozen/thawed semen pellets (P= 0.0001). The proportion of DNA in the comet tail of damaged spermatozoa was not significantly affected by cryopreservation, even if a limited increase was found between fresh (26.8%) and frozen/thawed (30.9%) spermatozoa. In contrast, the proportion of total viable spermatozoa was greatly impaired by cryopreservation and reduced from 82.7% (fresh semen) to 32.5% in stored semen. The present results suggest a low sensitivity of frozen/thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation. Similar low levels of DNA damage were found in cryopreserved bull and boar spermatozoa, whereas remarkable levels of sperm DNA fragmentation were reported in other animals such as the stallion and the monkey. Species differences in the success of semen cryopreservation are well known, and the widening of DNA damage occurring during freezing/thawing might contribute to understand the underlying phenomena.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
