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Short-term storage of in vitro produced bovine embryos
249
Theriogenology
SHORT-TERM
STORAGE OF IN VITRO PRODUCED BOVINE EMBRYOS
M.R. Schneider’~2 , J. Schwartz’, H.-D. Reichenbach’ and J.L. Rodrigues2 ‘BLT, The Bavarian Research Station for Animal Breeding, D-85586 Grub, Germany 2Laboratorio de Embriologia e Biotecnicas de ReproducHo, Faculdade de Veterinaria0JFRGS, 91501-970, Port0 Alegre, RS, Brazil The effect of a short term storage of in vitro produced embryos at room temperature on their developmental capacity has not been extensively studied. The aim of this experiment was to evaluate different media in order to establish ideal conditions for the transportation of in vitro produced (IVP) embryos in plastic straws up to 10 h without influencing their viability. Hatching of blastocysts was assessed after a further period of in vitro culture, as a measure of the developmental capacity of embryos after storage. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM 199 supplemented with 10% estrous cow serum (ECS), FSH (10 &nl) and gentamicin (5 pg/ml) for 24 h at 39 ‘C in 5% COZ, 5% O2 and maximum humidity. Frozen-thawed sperm was prepared by swim-up and the matured oocytes were inseminated with 1~10~ spermatozoa/ml in TALP with heparine (10 &ml). After 20 h of incubation, cumulus cells were removed by vortexing (4 min) and the zygotes transferred to 4-well culture dishes with culture medium (TCM 199 with 10% ECS). Embryos that developed up to the blastocyst stage on Day 7.5 (0 = day of insemination) were aspirated into 0.25 ml plastic straws in one of the following media: PBS+0.4% BSA, bicarbonate-free TCM 199+0.4%BSA and TCM 199+10%ECS (Culture medium). Embryos were stored for 10 h at 20 ‘C in the middle column of the straws. After this period, the blastocysts were washed 3 times and transferred to S-well dishes with culture medium. The hatching rate was assessed after 72 h of in vitro culture. Control blastocysts were allowed to hatch without being removed from their culture dishes. The results are presented in the table below: Table 1. Hatching rates of IVP bovine embryos after storage for 10 h at 20 ‘C Embryos 87
Hatched (%)
82
44 (53.7)aab
Culture medium
84
37 (44.0)b
Control
71
52 (73.2)a
Medium PBS+O,4%BSA Bicarbonate-free
TCMl99+0,4%BSA
54 (62.0)a
a.b Numbers with different superscripts differ (~~0.05). Data were analyzed by chi-square test supplemented with 95% Fleiss C.I. Our results indicate, using in vitro hatching as an endpoint, that IVP bovine embryos can be effectively stored for at least 10 h at 20 ‘C in PBS or bicarbonate-free TCM 199, both supplemented with 0,4%BSA. The storage in culture medium was less sucessml, probably because this medium was unable to maintain pH. This study was supported in part by the Probral Grant Program (CAPESIDAAD)
Theriogenology
SHORT-TERM
STORAGE OF IN VITRO PRODUCED BOVINE EMBRYOS
M.R. Schneider’~2 , J. Schwartz’, H.-D. Reichenbach’ and J.L. Rodrigues2 ‘BLT, The Bavarian Research Station for Animal Breeding, D-85586 Grub, Germany 2Laboratorio de Embriologia e Biotecnicas de ReproducHo, Faculdade de Veterinaria0JFRGS, 91501-970, Port0 Alegre, RS, Brazil The effect of a short term storage of in vitro produced embryos at room temperature on their developmental capacity has not been extensively studied. The aim of this experiment was to evaluate different media in order to establish ideal conditions for the transportation of in vitro produced (IVP) embryos in plastic straws up to 10 h without influencing their viability. Hatching of blastocysts was assessed after a further period of in vitro culture, as a measure of the developmental capacity of embryos after storage. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM 199 supplemented with 10% estrous cow serum (ECS), FSH (10 &nl) and gentamicin (5 pg/ml) for 24 h at 39 ‘C in 5% COZ, 5% O2 and maximum humidity. Frozen-thawed sperm was prepared by swim-up and the matured oocytes were inseminated with 1~10~ spermatozoa/ml in TALP with heparine (10 &ml). After 20 h of incubation, cumulus cells were removed by vortexing (4 min) and the zygotes transferred to 4-well culture dishes with culture medium (TCM 199 with 10% ECS). Embryos that developed up to the blastocyst stage on Day 7.5 (0 = day of insemination) were aspirated into 0.25 ml plastic straws in one of the following media: PBS+0.4% BSA, bicarbonate-free TCM 199+0.4%BSA and TCM 199+10%ECS (Culture medium). Embryos were stored for 10 h at 20 ‘C in the middle column of the straws. After this period, the blastocysts were washed 3 times and transferred to S-well dishes with culture medium. The hatching rate was assessed after 72 h of in vitro culture. Control blastocysts were allowed to hatch without being removed from their culture dishes. The results are presented in the table below: Table 1. Hatching rates of IVP bovine embryos after storage for 10 h at 20 ‘C Embryos 87
Hatched (%)
82
44 (53.7)aab
Culture medium
84
37 (44.0)b
Control
71
52 (73.2)a
Medium PBS+O,4%BSA Bicarbonate-free
TCMl99+0,4%BSA
54 (62.0)a
a.b Numbers with different superscripts differ (~~0.05). Data were analyzed by chi-square test supplemented with 95% Fleiss C.I. Our results indicate, using in vitro hatching as an endpoint, that IVP bovine embryos can be effectively stored for at least 10 h at 20 ‘C in PBS or bicarbonate-free TCM 199, both supplemented with 0,4%BSA. The storage in culture medium was less sucessml, probably because this medium was unable to maintain pH. This study was supported in part by the Probral Grant Program (CAPESIDAAD)