Cryopreservation of semen from genetic resource chicken lines
The objective of the present study was to establish an effective semen freezing procedure to set up a cryobank for chicken breeds and genetic lines of special interest. The fowls used in the experiments originated from White Leghorn lines maintained as conservation flocks. In this study, different protocols were evaluated in terms of differences in the fertilization success. 1 A) Comparison of slow and fast freezing procedures using a cryoprotectant containing Mix 2 (1 part 6.5% Dimethyl formamide + 2 parts 6.5% Methyl acetamide). The slow freezing procedure in a programmable freezer included two steps from 0 °C to -35 °C with a freezing rate of -3 °C/min, and from -35 °C to -130 °C with a freezing rate of -50 °C/ min. Fast freezing was carried out in a box by exposing the straws to liquid nitrogen vapour at 4 to 4.5 cm above liquid nitrogen surface (-40 °C/min). 1 B) Test of insemination procedures with one or two straws with semen frozen slowly with Mix 2, and 2) comparing three cryoprotectants Mix 1 (1 part 6.5% DMF + 1 part 6.5% MA), Mix 2 and MA only (6.5%). The base extender was the trehalose containing HS1-diluent to Hanzawa et al. (2006). The cryoprotectant-diluted semen was packaged in 0.25 ml plastic straws after dilution to a standardized number of spermatozoa (300 million/straw). All samples were thawed in a water bath at 4 °C. The females were inseminated intravaginal with a dose of 300 million (1 straw) or 600 million sperm (2 straws). The respective results were: the slow freezing procedure was superior to fast freezing and resulted in 49.3% compared to 11.5% fertility. There was no significant fertility difference between inseminations with one (43.6%) and two straws (52.1%). The fertility of both Mix 1 (77.3%) and Mix 2 (81.1%) showed higher fertilization rate than MA only (39.8%). In conclusion, the slow freezing procedure with Mix 2 media is an applicable method for establishing a sperm cryobank in chicken genetic resource populations.