Tailoring a glycerol-free cryopreservation protocol with anti-freeze (glycol)proteins for commercial and native breeds of chicken

This study evaluated the effects of antifreeze proteins AFPI, AFPIII, and AFGP during fresh processing, and freezing-thawing of chicken semen from a commercial broiler breed (BB) and the local breed Yellow Hungarian (YH), using 0.6 mol/L DMA as cryoprotectant. AFPI had small but significant (p<0.05) positive effects on post-thaw sperm viability and motility in BB. For YH semen, three freezing protocols with different dilution rates (4x, 2.3x, 2.1x) were compared. Protocol 1 (dilution rate 4x) gave the best post-thaw viability (P<0.001) and was used further. Fertility rates (FR) of pre-freezing steps were tested by inseminating WL hens (4 AI/hen in 2 weeks, 100 x 10L sp/dose). FRs for A) raw semen, B) chilled semen, C) B + DMA, D) B + AFPI, and E) B + DMA + AFPI were 48.2, 3.7, 31.4, 21.4, and 16.1%, respectively. B was lower than A (p<0.001), while C-E were higher than B (p<0.005). AFPI during freezing gave no advantage compared with DMA, except for improved post-thaw DNA integrity. Inseminations with frozen-thawed semen (8 AI/hen in 3 weeks, 100 x 10L sp/dose) gave low FR with YH semen, regardless of hen type. FR with YH semen was 0% without AFPI and 1.5% with AFPI. The oviduct embryo mortality (18.0%{+/-}1.4) observed across all the different hen groups suggested insufficient number of spermatozoa inseminated. In conclusion, AFPI improved some post-thaw traits, but fertility outcomes remain inconclusive. Preventing pre-freezing fertility loss and increasing sperm dose concentration are required.