6. In this protocol, we explained that embryos should remain
3 min in well 3 of the vitrification dish (VS1). Equine in vivoderived embryos may present a large range in size (200 to
3000+ μm in diameter). Larger embryos may need a longer
incubation time in well 3 (VS1). Our recommendation is that
the embryos should be continuously observed during incubation in VS1 and should be moved to well 4 (VS2) only when
the embryo shows a complete dehydration of the trophectodermal cells. Embryos can remain in VS1 from 3 to 8 min.
7. Using a sterile 21-G needle, make a hole on both walls of the
0.5 mL straw, next to the cotton plug. This opening in the
straw will allow the liquid nitrogen to enter the straw and will
avoid the straw from breaking during warming.
8. During warming it is extremely important to ensure the WS1 is
at 37
C, for embryo thawing/warming at a sufficiently high
rate and avoiding ice formation. Therefore, prepare the warming dish as illustrated in Fig. 2b in advance, and place it on the
thermal plate before needed (10–15 min). The solution temperature should be measured, using a small probe, and if
needed the thermal plate settings should be adjusted to attain
the desired solution temperature.
References
1. Choi YH, Velez IC, Riera FL, Rolda `n JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS,
Hinrichs K (2011) Successful cryopreservation
of expanded equine blastocysts. Theriogenology
76:143–152
2. Bruyas JF, Battut I, Pol JM, Botrel C, Fieni F,
Tainturier D (1995) Quantitative analysis of
morphological modifications of day 6.5 horse
embryos after treatment with four cryoprotectants: differential effects on inner cell mass and
trophoblast cells. Biol Reprod Monogr Ser
1:329–339
3. Legrand E, Bencharif D, Barrier-Battut I,
Delajarraud H, Corniere P, Fie ´ni F,
Ainturier D, Bruyas JF (2002) Comparison of
pregnancy rates for days 7–8 equine embryos
frozen in glycerol with or without previous
enzymatic treatment of their capsule. Theriogenology 58:721–723
4. Diaz F, Bondiolli K, Paccamonti D, Gentry GT
(2016) Cryopreservation of day 8 equine
embryos after blastocyst micromanipulation
and vitrification. Theriogenology 85:894–903
5. Sanchez R, Blanco M, Weiss J, Rosati I,
Herrera C, Bollwein H, Burger D, Sieme H
(2017) Influence of embryonic size and manipulation on pregnancy rates of mares after transfer
of cryopreserved equine embryos. J Equine Vet
Sci 49:54–59
6. Vanderzwalmen P, Bertin G, Debauche C,
Standaert V, Bollen N, van Roosendaal E,
Vandervorst M, Schoysman R, Zech N (2003)
Vitrification of human blastocysts with the
Hemi-Straw carrier: application of assisted
hatching after thawing. Hum Reprod
18:1504–1511
7. Vajta G, Holm P, Kuwayama M, Booth PJ,
Jacobsen H, Greve T, Callesen H (1998) Open
pulled straw (OPS) vitrification: a new way to
reduce cryoinjuries of bovine ova and embryos.
Mol Reprod Dev 51:53–58
522
Carolina Herrera
3 min in well 3 of the vitrification dish (VS1). Equine in vivoderived embryos may present a large range in size (200 to
3000+ μm in diameter). Larger embryos may need a longer
incubation time in well 3 (VS1). Our recommendation is that
the embryos should be continuously observed during incubation in VS1 and should be moved to well 4 (VS2) only when
the embryo shows a complete dehydration of the trophectodermal cells. Embryos can remain in VS1 from 3 to 8 min.
7. Using a sterile 21-G needle, make a hole on both walls of the
0.5 mL straw, next to the cotton plug. This opening in the
straw will allow the liquid nitrogen to enter the straw and will
avoid the straw from breaking during warming.
8. During warming it is extremely important to ensure the WS1 is
at 37
C, for embryo thawing/warming at a sufficiently high
rate and avoiding ice formation. Therefore, prepare the warming dish as illustrated in Fig. 2b in advance, and place it on the
thermal plate before needed (10–15 min). The solution temperature should be measured, using a small probe, and if
needed the thermal plate settings should be adjusted to attain
the desired solution temperature.
References
1. Choi YH, Velez IC, Riera FL, Rolda `n JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS,
Hinrichs K (2011) Successful cryopreservation
of expanded equine blastocysts. Theriogenology
76:143–152
2. Bruyas JF, Battut I, Pol JM, Botrel C, Fieni F,
Tainturier D (1995) Quantitative analysis of
morphological modifications of day 6.5 horse
embryos after treatment with four cryoprotectants: differential effects on inner cell mass and
trophoblast cells. Biol Reprod Monogr Ser
1:329–339
3. Legrand E, Bencharif D, Barrier-Battut I,
Delajarraud H, Corniere P, Fie ´ni F,
Ainturier D, Bruyas JF (2002) Comparison of
pregnancy rates for days 7–8 equine embryos
frozen in glycerol with or without previous
enzymatic treatment of their capsule. Theriogenology 58:721–723
4. Diaz F, Bondiolli K, Paccamonti D, Gentry GT
(2016) Cryopreservation of day 8 equine
embryos after blastocyst micromanipulation
and vitrification. Theriogenology 85:894–903
5. Sanchez R, Blanco M, Weiss J, Rosati I,
Herrera C, Bollwein H, Burger D, Sieme H
(2017) Influence of embryonic size and manipulation on pregnancy rates of mares after transfer
of cryopreserved equine embryos. J Equine Vet
Sci 49:54–59
6. Vanderzwalmen P, Bertin G, Debauche C,
Standaert V, Bollen N, van Roosendaal E,
Vandervorst M, Schoysman R, Zech N (2003)
Vitrification of human blastocysts with the
Hemi-Straw carrier: application of assisted
hatching after thawing. Hum Reprod
18:1504–1511
7. Vajta G, Holm P, Kuwayama M, Booth PJ,
Jacobsen H, Greve T, Callesen H (1998) Open
pulled straw (OPS) vitrification: a new way to
reduce cryoinjuries of bovine ova and embryos.
Mol Reprod Dev 51:53–58
522
Carolina Herrera