Abstract
itrification is regarded as a widely used technology for cryopreservation of oocytes and embryos. The
objectives were to determine effective cryoprotective agents (CPAs), concentration of CPAs and steps
for vitrification of immature oocytes, in vitro matured oocytes and in vivo produced embryos of cows.
To determine an effective CPA, comparison was made among ethylene glycol (EG), di-methyl sulfoxide
(DMSO) and EG+DMSO for vitrification of oocytes and embryos. To determine an effective
concentration, comparison was made among 7.5% supplementation in vitrification solution (VS) 1 and
15.0% in VS 2, 10.0% and 15.0%, and 10.0% and 20.0% combination of EG+DMSO for vitrification of
oocytes and embryos. To determine an effective step, comparison was made between two [7.5% (VS 1)
and 15% (VS 2), EG+DMSO] and three steps [7.5% (VS 1), 10% (VS 2) and 15% (VS 3), EG+DMSO] for
vitrification of oocytes and embryos. To determine an effective warming step, comparison was made
between two steps [1 M (WS 1) and 0.5 M sucrose (WS 2] and three steps [1 M (WS 1), 0.5 M (W3 2) and
0.25 M sucrose (WS 3)] for warming of vitrified oocytes. The vitrified oocytes and embryos were
warmed in holding medium (HM) supplemented with 1 M sucrose (WS 1) for 1 min followed by
warming in HM supplemented with 0.5 M sucrose (WS 2) for 5 min. Nonvitrified oocytes and embryos
were used as control and the cryotop was used as cryodevice. The efficacy of vitrification of immature
oocytes was determined by evaluation of IVM with respect to extrusion of 1st polar body. The efficacy
of vitrification of matured oocytes was determined by evaluation of cleavage rate of presumptive
zygotes after IVF. The efficacy of vitrification of embryos was determined by evaluation of pregnancy
rates in cows after transfer of embryos. The maturation rate after vitrification of immature oocytes did
not vary among EG (48.5%), DMSO (44.2%) and combination of EG+DMSO (51.3%) although
numerically higher proportion of oocytes matured when EG+DMSO was used. The maturation rate
after vitrification of immature oocytes did not vary among combined use of EG+DMSO (49.2%) at a
concentration of 7.5% (VS 1) and 15% (VS 2), 10% and 15% (42.9%), and 10% and 20% (38.9%) although
higher proportion oocytes matured when EG+DMSO at 7.5% and 15% concentration was used. The
maturation rate did not vary between two steps (49.2%) and three steps (41.1%) of vitrification of
immature oocytes. The maturation rate did not vary between two steps (46.2%) and three steps
warming of vitrified immature oocytes (42.8%). The cleavage rate after vitrification of mature oocytes
did not vary among EG (38.1%), DMSO (33.0%) and combination of EG+DMSO (39.6%) although
numerically higher proportion of zygotes cleaved when EG+DMSO was used. The cleavage rate after
vitrification did not vary among combined use of EG+DMSO (38.7%) at a concentration of 7.5% and
15.0%, 10.0% and 15.0% (35.9%), and, 10.0% and 20.0% (33.0%) although higher proportion of zygotes
cleaved when EG+DMSO at 7.5% and 15.0% concentration was used. The cleavage rate was
significantly (P<0.05) higher when vitrification of mature oocytes was done using two steps (38.7%)
than that of three steps counterpart (28.7%). The cleavage rate did not vary between two steps (39.9%)
and three steps warming of vitrified mature oocytes (36.7%). After vitrification, the maturation rates of
immature oocytes (38.9% to 51.3%) and cleavage rates of mature oocytes (28.7% to 39.6%) were
significantly (P<0.05) lower than that of control (maturation 79.3%; cleavage 60.0%). The pregnancy rate
after transfer of in vivo produced vitrified embryos was significantly (P<0.05) higher when combination
of EG+DMSO was used (15.0%) than that of either single use of EG (0.0%) or DMSO (0.0%). Moreover,
the pregnancy rate did not vary between combined use of EG+DMSO and control embryos (22.2%).
The pregnancy rate of vitrified embryos was significantly (P<0.05) higher (16.7%) when combination of
EG+DMSO at a concentration of 7.5% and 15.0% was used than that of other concentrations (0.0%). The
pregnancy of vitrified embryos was significantly (P<0.05) higher when vitrification of embryos was
done using two steps (16.7%) than that of three steps counterpart (0.0%). In conclusions, EG, DMSO or
combination of EG+DMSO is equally effective for vitrification of bovine oocytes. All tested
concentrations of EG+DMSO are equally effective for vitrification of oocytes. Combined use of
EG+DMSO is better than single use of EG or DMSO, and 7.5% (VS 1) and 15% (VS 2) concentration of
combined EG+DMSO is better than other concentrations for vitrification of embryos. Two steps
vitrification is better than three steps for mature oocytes and embryos. Both steps for warming of
vitrified oocytes are equally effective. Further studies are needed to improve the efficacy of vitrification
of oocytes and embryos as lower efficacy of vitrification was observed than control counterparts.