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

BERNAL JUAREZ, B.; Varadi, E.; Drobnyaak, A.; Hogervorst, T.; Liptoi, K.; Voets, I.; de Wit, A.; Santiago-Moreno, J.; Hawken, R.; de Kovel, C.; Hiemstra, S.-J.; Woelders, H.; Vegi, B.

doi:10.1101/2025.07.18.664799

bioRxiv · 10.1101/2025.07.18.664799

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

BERNAL JUAREZ, B.·Varadi, E.·Drobnyaak, A.·Hogervorst, T.·Liptoi, K.·Voets, I.·de Wit, A.·Santiago-Moreno, J.·Hawken, R.·de Kovel, C.·Hiemstra, S.-J.·Woelders, H.·Vegi, B.

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Abstract

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.

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BERNAL JUAREZ, B., Varadi, E., Drobnyaak, A., Hogervorst, T., Liptoi, K., Voets, I., de Wit, A., Santiago-Moreno, J., Hawken, R., de Kovel, C., Hiemstra, S.-J., Woelders, H., Vegi, B.. 2025-07-19. Tailoring a glycerol-free cryopreservation protocol with anti-freeze (glycol)proteins for commercial and native breeds of chicken. https://doi.org/10.1101/2025.07.18.664799

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