Sperm cryopreservation by the pellet method in chickens, turkeys and pheasants: a comparative study

The feasibility of avian sperm cryopreservation by the pellet method was studied in chicken, turkey and pheasant semen. The cryopreservation pellet procedure used for chicken and turkey semen was previously reported by Tselutin et al. in 1999 and 1995 respectively. The procedure used for chicken semen was also applied to pheasant semen. Semen samples were collected by dorso-abdominal massage from Mericanel della Brianza (local Italian breed) chicken breeders, Hybrid Large White turkey breeders and Ring-necked pheasant breeders. In brief, ejaculates were pooled and diluted in prefreezing diluent, equilibrated at 5 °C, added with dimethylacetamide (DMA) and further equilibrated for few minutes at 5 °C and finally directly dropped into a liquid nitrogen bath to form frozen pellets. Thawing was performed at high temperature for few seconds in water bath for chicken and turkey semen, and on aluminum plate for pheasant semen. Details of the cryopreservation procedure used according to the species are summarized in table 1. Sperm quality was assessed in fresh semen soon after collection (time 0) and in frozen/thawed semen pellets (time FT). According to the lab equipments, sperm motility was measured by the Accudenz procedure in turkey and pheasant semen, and by CASA system in chicken semen; sperm viability was measured by the eosin-nigrosin staining in chicken and pheasant semen, and by SYBR14/PI dual staining in turkey semen. The proportional recovery of viable and motile spermatozoa after freezing/thawing was calculated. Table 1. Cryopreservation procedure in chicken, turkey and pheasant semen. Cryopreservation procedure Chicken Turkey Pheasant Diluent Lake’s (1981) Tselutin’s (1995) Lake’s (1968) Dilution rate 1:2 1:4 1:3 Equilibration (min) at 5 °C 20 60 10 DMA (%) 6 8 6 Equilibration (min) with DMA at 5 °C 1 5 5 Drop volume (µl) 100 80 80 Thawing temperature (°C) 60 75 50 In all species, both viability and motility of spermatozoa were greatly reduced after cryopreservation. The proportion of total viable spermatozoa was 83, 77 e 84 % on time 0 and 32, 32 and 16 % on time FT in chicken, turkey and pheasant semen respectively. The proportion of viable spermatozoa recovered after freezing/thawing was similar in chicken (39%) and turkey (41%) semen, and much lower in pheasant semen (20%). In the chicken, the proportion of motile spermatozoa decreased from 52 % on time 0 to 19 % on time FT; therefore, 43 % of motile spermatozoa was recovered after cryopreservation. Optical density measured in the sperm motility Accudenz procedure was 0.496 and 0.270 in turkey and pheasant semen respectively on time 0, and decreased to 0.294 and 0.045 respectively on time FT; therefore, the recovery rate of the sperm mobility was 59 % in the turkey and 17 % in the pheasant. Sperm sensitivity to cryopreservation was different among species. Turkey spermatozoa showed the best recovery rates for both viability and motility; chicken spermatozoa showed a higher sensitivity compared to turkey spermatozoa causing greater loss in sperm motility; pheasant spermatozoa were the most sensitive gametes to cryopreservation and showed the highest decreases in both viability and motility. Large variability was measured in sperm quality parameters of frozen/thawed semen pellets and further studies are suggested to standardize the cryopreservation pellet procedure in order to improve the recovery of viable and motile sperm after storage.