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Semen quality evaluation and determination of its fertilizing ability are essential aspects in the practice of assisted reproduction in domestic species. Fertilization is a multifunctional process, thus several tests related to sperm functional competence and multiparametric data analysis are required for a proper evaluation. Despite many studies are available in the literature and much progress has been made, at present a set of tests suitable for the prediction of fertilizing ability is still not available. Flow cytometry (FCM), allowing the analysis of different parameters on several sperm cells in short time, can provide a fair assessment of semen potential fertility. In this study different quality parameters of bull frozen-thawed semen were evaluated by FCM and by computer assisted sperm analysis (CASA). Frozen semen from 18 Holstein-Friesian bulls, 10 batches each, was assessed. The following parameters were tested by FCM: sperm viability (SYBR-14/propidium iodide (PI) staining), acrosomal status (FITC-PNA/PI staining), membrane lipid disorder (Merocyanine 540 hydrophobic dye), lipid peroxidation (BODIPY (581/591) C11 lipophilic probe), mitochondrial membrane potential (JC-1 potentiometric probe). Sperm kinetic was assessed by CASA. The mean values of measured parameters, standard deviations and minimum and maximum values were: proportion of viable sperm: 41.73±10.57% (16.26 to 66.44); live acrosomeintact sperm: 34.34±10.52% (8.52 to 56.55); live low lipid disorder sperm: 22.54±7.62% (6.97 to 49.84); live peroxidized sperm: 0.55±0.92% (0.00 to 8.49); live peroxidized sperm after promoter (FeSO4) treatment: 4.28±2.93% (0.76 to 16.41); high mitopotential sperm: 43.89±10.74% (9.78 to 67.09); motile sperm: 63.43±14.32% (14.50 to 88.50). Values were in general fairly consistent with levels found in the literature. Sperm viability was significantly correlated (P

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.