Aim - The comet assay (single cell gel electrophoresis, SCGE) is a sensitive method for measuring DNA breaks and is widely used to evaluate DNA damage in mammalian spermatozoa. No papers were found in literature reporting the use of comet assay in poultry sperm, apart the study of Kot?owska et al. [1] on turkey semen. The aim of the present study was to set a comet assay protocol [2] to assess DNA integrity of chicken spermatozoa. Materials and methods - 18 males (Italian local breeders-Mericanel della Brianza) were housed in individually battery cages. Semen samples were collected twice a week by abdominal massage, pooled, diluted 1:2 in Lake's diluent and washed by centrifugation in PBS/BSA. Spermatozoa (about 1*106/ml) were embedded in low-melting point agarose gel solution (LMPA, 0.8% in PBS) on a microscope slide precovered with normal-melting point agarose. After the agarose was allowed to solidify at 4°C, a LMPA third layer was stratified. The slides were placed in lysis buffer (pH 10) with 10mM dithiothreitol and 1% Triton X-100 for 1h at 4°C and were then incubated at 37°C in 5 microg/ml of proteinase K in lysis buffer for 1h. The slides were equilibrated in neutral electrophoresis solution (Tris-acetate-EDTA, TAE, pH 7.3) for 20 min. Then electrophoresis was performed at room temperature, at 10V and 8mA, for 20 min. The slides washed in neutralising buffer (0.4M Tris HCl, pH 7.5) were placed in methanol, air dried and stained with ethidium bromide (20 microg/ml in PBS). Stained sperm samples were examined under an epifluorescence microscope (Leica DMLB) at 400x magnification. Within random fields, 200 sperm from each slide were counted and scored for the presence or absence of comets. Images were captured with a Leica DC 300F camera and analyzed by Comet Assay software (CASP Software). Results and conclusion -This preliminary study allowed to develop a SCGE protocol for chicken spermatozoa. Our procedure produced clear comet pictures, with comet head maintaining the typical shape of the sperm head. Sperm tails were often still visible as previously reported in turkey [1]. The proportion of comets found in fresh semen was 10.14% (visual scoring) and the mean percentage of DNA in the comet tail was 7.05% (CASP analysis). The cells were assigned to 5 categories based on the percentage of tail DNA: category 1 (0-10%) = 79.76%; category 2 (10-25%) = 17.19%; category 3 (25-50%) = 2.22%; category 4 (50-75%) = 0.76% and category 5 (>75%) = 0.07%. Our results reported a quite low baseline DNA damage in fresh semen in agreement with previous studies in different species (human, horse, swine and fish), whereas DNA damage observed in turkey semen [1] was higher, probably due to differences in the comet procedure used. The integrity of sperm DNA is important for the success of natural and assisted fertilization and further studies will be developed to evaluate DNA damage in fresh, refrigerate and cryopreserved avian sperm to investigate its relation with fertility. [1] KOT?OWSKA M., DIETRICH G., WOJTCZAK M., KAROL H. & CIERESZKO A. (2007) Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa. Theriogenology, 67: 276-286. [2] SAKKAS D., MOFFATT O., MANICARDI G.C., MARIETHOZ E., TAROZZI N. & BIZZARRO D. (2002) Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biology of Reproduction 66, 1061-1067.
Sperm DNA integrity in chicken assessed by Comet assay: preliminary results.
Gliozzi Teresa Maria;
2007
Abstract
Aim - The comet assay (single cell gel electrophoresis, SCGE) is a sensitive method for measuring DNA breaks and is widely used to evaluate DNA damage in mammalian spermatozoa. No papers were found in literature reporting the use of comet assay in poultry sperm, apart the study of Kot?owska et al. [1] on turkey semen. The aim of the present study was to set a comet assay protocol [2] to assess DNA integrity of chicken spermatozoa. Materials and methods - 18 males (Italian local breeders-Mericanel della Brianza) were housed in individually battery cages. Semen samples were collected twice a week by abdominal massage, pooled, diluted 1:2 in Lake's diluent and washed by centrifugation in PBS/BSA. Spermatozoa (about 1*106/ml) were embedded in low-melting point agarose gel solution (LMPA, 0.8% in PBS) on a microscope slide precovered with normal-melting point agarose. After the agarose was allowed to solidify at 4°C, a LMPA third layer was stratified. The slides were placed in lysis buffer (pH 10) with 10mM dithiothreitol and 1% Triton X-100 for 1h at 4°C and were then incubated at 37°C in 5 microg/ml of proteinase K in lysis buffer for 1h. The slides were equilibrated in neutral electrophoresis solution (Tris-acetate-EDTA, TAE, pH 7.3) for 20 min. Then electrophoresis was performed at room temperature, at 10V and 8mA, for 20 min. The slides washed in neutralising buffer (0.4M Tris HCl, pH 7.5) were placed in methanol, air dried and stained with ethidium bromide (20 microg/ml in PBS). Stained sperm samples were examined under an epifluorescence microscope (Leica DMLB) at 400x magnification. Within random fields, 200 sperm from each slide were counted and scored for the presence or absence of comets. Images were captured with a Leica DC 300F camera and analyzed by Comet Assay software (CASP Software). Results and conclusion -This preliminary study allowed to develop a SCGE protocol for chicken spermatozoa. Our procedure produced clear comet pictures, with comet head maintaining the typical shape of the sperm head. Sperm tails were often still visible as previously reported in turkey [1]. The proportion of comets found in fresh semen was 10.14% (visual scoring) and the mean percentage of DNA in the comet tail was 7.05% (CASP analysis). The cells were assigned to 5 categories based on the percentage of tail DNA: category 1 (0-10%) = 79.76%; category 2 (10-25%) = 17.19%; category 3 (25-50%) = 2.22%; category 4 (50-75%) = 0.76% and category 5 (>75%) = 0.07%. Our results reported a quite low baseline DNA damage in fresh semen in agreement with previous studies in different species (human, horse, swine and fish), whereas DNA damage observed in turkey semen [1] was higher, probably due to differences in the comet procedure used. The integrity of sperm DNA is important for the success of natural and assisted fertilization and further studies will be developed to evaluate DNA damage in fresh, refrigerate and cryopreserved avian sperm to investigate its relation with fertility. [1] KOT?OWSKA M., DIETRICH G., WOJTCZAK M., KAROL H. & CIERESZKO A. (2007) Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa. Theriogenology, 67: 276-286. [2] SAKKAS D., MOFFATT O., MANICARDI G.C., MARIETHOZ E., TAROZZI N. & BIZZARRO D. (2002) Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biology of Reproduction 66, 1061-1067.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
