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.
Cryopreservation of chicken semen by the pellet method. Study on semen processing, sperm quality, DNA integrity, and bacterial contamination.
Gliozzi T;
2009
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
