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The comet assay or single cell gel electrophoresis (SCGE) has become a recognized method for detecting DNA damage in a variety of vertebrate cell types, including spermatozoa. The comet assay is used in several domestic animal species to investigate the potential damage induced by common semen handling practices, such as cryopreservation. The aim of the present study was to study DNA fragmentation in chicken spermatozoa after processing for cryopreservation, in F15 Hubbard strain selected for meat production. Semen collected from 7 males was diluted in modified Lake's pre-freezing extender, frozen in pellets and thawed in water bath at 60°C. Comet assay and viability (SYBR14-PI) were performed on fresh semen soon after collection and dilution, and after thawing. The proportion of spermatozoa with damaged DNA significantly increased from 5.7% (fresh semen) to 28.3% (frozen-thawed semen) during cryopreservation. The proportion of viable spermatozoa significantly decreased from 77.5% to 20.4% in fresh and frozen-thawed semen respectively. Sperm DNA damage was much lower compared to the decrease in viable sperm following cryopreservation, therefore, damages to nuclear DNA are not considered as a major cause of cryoinjury in chicken semen. However, we propose that comet assay is suggested as a useful tool in multiparametric sperm assessment to investigate sperm sensibility to cryopreservation in chicken.

Cryopreservation of chicken semen by the pellet method was studied in two genetic types, a local Italian breed (Mericanel della Brianza, MB) and a strain selected for meat production (Hubbard F15, HF15). The pellet cryopreservation procedure includes different steps of semen processing and different conditions were studied in three consecutive trials to optimize semen processing according to the genetic type. Sperm viability (ethidium bromide fluorescent staining) and motility (subjective method and CASA system) were recorded before and after freezing/thawing. Presence of glycine into the prefreezing Lake's diluent, first equilibration time at 5C, DMA concentration and equilibration time at 5C, and drop volume to form frozen semen pellets significantly affected semen quality after freezing/thawing. The final optimised procedure was similar in both genetic types, exception for DMA concentration. The procedure includes: dilution of semen to 1.5x109/mL in prefreezing Lake's diluent enriched with 50mM glycine; 20 min equilibration time at 5C; DMA addition to 6 and 9% final concentration in MB and HF15 respectively; 1 min equilibration time at 5C; 100 mL drop volume to form frozen semen pellets; thawing in water bath at 60C for 10 s. Semen collected from selected HF15 strain was more sensitive to cryopreservation compared to semen collected from MB local breed. Recovery rates in viability and motility after freezing/thawing were 30 and 20% respectively in HF15 males and 37 and 32% respectively in MB males. The effect of cryopreservation on sperm DNA integrity was studied in both genetic types. DNA integrity was assessed by COMET assay; the proportion of sperm with undamaged DNA and the proportion of DNA in the tail of damaged sperm were calculated. Cryopreservation significantly increased the proportion of sperm with DNA fragmentation; in contrast, the proportion of tail DNA did not change. Undamaged sperm significantly decreased from 94% in fresh semen to 80% in cryopreserved semen in MB local breed, and from 96 to 73% in HF15 strain. The decrease in undamaged DNA sperm was much lower compared to the decrease in viable sperm following cryopreservation; therefore, damages to nuclear DNA are not considered as a major cause of cryoinjury in chicken semen. The effect of cryopreservation on sperm susceptibility to in vitro induced peroxidation (TBARS assay) was studied in semen collected from MB males. MDA production following in vitro induced peroxidation did not significantly increase in spermatozoa after cryopreservation (31 versus 40 mcM/109). Finally, the effect of cryopreservation pellet procedure on semen bacterial contamination was studied in MB males. Total bacteria count and the presence of the following bacteria was determined: Gram-positive and Gram-negative species, Salmonella and Campylobacter. Total bacteria count decreased from 106 in fresh semen to 104 CFU/mL in frozen/thawed semen. Enterococcus (S.) faecali, Staphylococcus spp. and Bacillus spp. were isolated in fresh semen, whereas only Enterococcus (S.) faecali was isolated after cryopreservation. Bacteria isolated in fresh semen were common bacteria from the environment and enteric tract. Cryopreservation decreased contamination, however, Enterococcus faecali was still present.

Aim - The addition of the amino acid glycine into the diluent used for cryopreservation of fish [1] and goat [2] semen was reported to improve sperm motility. In the present study the effect of glycine during freezing/thawing of chicken spermatozoa was studied. Materials and Methods -Semen was routinely collected by abdominal massage twice a week from 18 Mericanel della Brianza male chicken breeders. All ejaculates were pooled and splitted into two samples (L and LGly) diluted 1:2 as follows: L) in Lake's diluent; LGly) in Lake's + 50mM glycine diluent. The following freezing procedure was then used: semen was cooled to 4°C for 20 minutes, added with dimethylacetamide (DMA; 6% final concentration) and equilibrated for 1 minute at 4°C. Then, semen was frozen by direct dropping into a liquid nitrogen bath. Frozen semen pellets were transferred into cryovials, stored into liquid nitrogen tank, and thawed in water bath at 60°C for 10 seconds. Sperm quality was assessed on fresh semen soon after dilution (time 0), after the equilibration time with DMA (time 21) and after thawing (time TH). The following sperm quality parameters were measured: sperm movement parameters (Hobson Sperm Tracker software); damaged spermatozoa (modified ethidium bromide procedure); viable morphologically normal, viable morphologically abnormal, and dead spermatozoa (nigrosin/eosin staining). The experimental protocol was repeated on different days of semen collection to increase the number of replicates within each treatment. The data were analysed by repeated measure analysis of variance using the GLM procedure of SAS. Results - Chicken spermatozoa were strongly damaged during the freezing/thawing procedure, as expected. A significant progressive decrease in the proportion of viable normal spermatozoa and in some movement parameter values (VCL, MAD and ALH) was found from time 0, to time 21 and to time TH. The proportion of damaged, dead and motile spermatozoa did not show a significant change from time 0 to time 21, and a significant negative change occurred after thawing. The addition of the amino acid glycine into Lake's diluent did not significantly affect sperm parameters; however, the proportion of viable normal spermatozoa was higher in LGly compared to L samples (50.9 vs 47.5 %; P = 0.06) . The interaction glycine*time was significant for the linearity (LIN) and straightness (STR) values of sperm movement and different trends of the two parameters were found according to the treatment during cryopreservation procedure. Both LIN and STR values significantly increased from time 0 to time 21 in L samples, and then decreased to the initial values after thawing. In contrast, LIN and STR values did not show significant changes during the cryopreservation procedure in LGly samples. Conclusion - The inclusion of glycine into the diluent prevented significant changes in the quality movement of chicken spermatozoa during freezing/thawing procedure; therefore, the initial values were kept constant until thawing. Similar results were reported in cryopreserved fish spermatozoa and it was suggested that glycine may improve sperm integrity by direct interaction with plasma membrane phospholipids [1]. References - [1] HE S., WOODS L.C. (2003) Effects of glycine and alanine on short-term storage and cryopreservation of striped bass (Morone saxatilis) spermatozoa. Cryobiology, 46: 17-25. [2] KUNDU C.N., DAS K., MAJUMDER G.C. (2001) Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system. Cryobiology, 41: 21-27.

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.

The major polyunsaturate in phospholipids of chicken spermatozoa is docosatetraenoic acid, 22:4n-6, which is positively correlatable with sperm motility and fertility. The potential for dietary manipulation of sperm fatty acid in order to improve male fertility has been extensively studied in the chicken. The effects of diets enriched in n-3 and n-6 long chain polyunsaturates have been investigated in different trials using different oil sources and levels of oil inclusion. The 22:6n-3 and 22:5n-3 content of avian spermatozoa is increased by supplementing the feed with fish oil (rich in 22:6n-3) and linseed oil (rich in 18:3n-3) respectively. The 22:4n-6 content is also increased by supplementing the feed with evening primrose oil (rich in 18:3n-6) in association with high level of vitamin E (200 mg/kg) and with arasco oil (rich in 20:4n-6). The effects of these fatty acid manipulations on sperm quality and /or fertility are reviewed. Both n-3 and n-6 rich diets showed a positive effect on sperm movement during the reproductive period and an age-dependent positive effect on fertility. Reported effects of n-6 rich diets on semen production have been variable with 20:4n-6 rich diet having a positive effect on semen volume and thus on total sperm number and 18:3n-6 rich diets having a negative effect on semen concentration. Spermatozoa enriched in 22:5n-3, or 22:6n3 or 22:4n-6 result in significantly higher fertility values following artificial insemination compared to control spermatozoa; however, such a positive effect is age dependent and observed at 37 to 42 weeks, but not in older birds. The n-6 fatty acid composition of chicken spermatozoa is recognised as a specie-specific characteristic. The fundamental relation between dietary lipid, spermatozoa fatty acid composition and thus sperm quality and fertility can be seen as having a potential commercial application.