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.
Effect of glycine on cryopreservation of chicken spermatozoa
Teresa Maria Gliozzi
2007
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
