Culture of sheep zygotes in synthetic oviduct fluid medium with different concentrations of sodium bicarbonate and HEPES

Abstract

An investigation was conducted of the viability of sheep zygotes in vivo subsequent to culture in synthetic oviduct fluid medium (SOFM) in which sodium bicarbonate was partly or wholly replaced with HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid); concentrations of 0, 6.25, 12.5, 18.75 and 25 mM HEPES were studied. The addition of HEPES lowered the pH during the culture period, and was associated with a reduction in the percentage of zygotes that developed to blastocysts (range 60.4 to 85.2%); this reduction was significant (P < 0.05) at concentrations of 18.75 and 25 mM. There was also a significant (P < 0.05) reduction in the percentage of zygotes that commenced hatching (range 47.2 to 74.1%) and a significant (P < 0.05) delay in the time of blastocyst formation (range 4 to 7 d) in medium containing 25 mM HEPES. Viability, as assessed by elongation of the trophoblast after transfer to recipient ewes, was similar for zygotes cultured for 3 d either in medium with 12.5 mM HEPES or in medium without HEPES (90.3 and 93.9%, respectively). Extending the culture period to 5 d was associated with a general decline in viability (P < 0.05), with 67.7% of zygotes cultured with HEPES elongating compared with 45.5% for those cultured without HEPES. This study confirms that sheep zygotes can be successfully cultured in SOFM for up to 3 d without loss of viability. The addition of HEPES provides additional buffering capacity but zygote development may be compromised.