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pyruvate during the first 48 hours of culture on the freezability of IVF bovine blastocysts
344
Theriogenology
EFFECTS OF EPINEPHRINE AND LACTATE/PYRUVATE DURING THE FIRST 48 HOURS OF CULTURE ON THE FREEZABILITY OF IVF BOVINE BLASTOCYSTS E. Gomez and C. Diez Consejeria de Agricultura. CIATA. CENSYRA. Aptdo 155. Somio s/n. Gijon. Asturias. SPAIN Lipid contained in embryonic cells can adversely affect survival of frozen/thawed in vitro produced (IVP) bovine embryos (Leibo et al,Theriogenology 43:265,1995). Moreover, glycogen accumulates in excess in cells from IVP mouse embryos (Ozias and Stem, Biol Reprod, 8:467472,1973) and could retain water and render difficult the dehydration when freezing embryos. Mobilizing these stocks could therefore increase the ability of IVP embryos to be cryopreserved and it is known that epinephrine causes lipid and glycogen breakdown in adult tissues. Degradative products of glycogen and lipids could be used by the embryo depending on the presence of other energy substrates in the culture medium. The effects of epinephrine on the freezability of IVP embryos were evaluated. Cumulusoocyte complexes (COCs) were matured in TCM 199, 10% fetal bovine serum (FBS) and FSHp (20 &ml) for 24 h. Culture conditions were 39” C, 5% CO* in air and high humidity. Matured COCs were incubated together with Percoll-separated sperm in Fert-TALP and 10 ug/ml heparin for 20 h. Zygoies were vortexed for 2 min, allotted to each group and cultured in mSOF, 3g/L BSA and 10% FBS. The effect of 1uM epinephrine during the initial 48 h of culture was determined in a 2 x 2 factorial design in presence (+LP) or absence (-LP) of lactate (3.3 mM) and pyruvate (0.3 mM) in mSOF. From 48 h to Day 7, culture in all groups was performed in mSOF+LP without epinephrine. Day 7 blastocysts were slowly frozen in PBS and 1.5 M ethylene glycol, thawed for 5 set in air and 30 set in water at 30 “C and directly rehydrated in PBS. Thawed blastocysts were cocultured for 48 hours with bovine oviductal cells. Morphological normality at thawing and blastocoelic re-expansion were the criteria for survival. Data were analyzed by ANOVA and REGWF test for means. No interactions between epinephrine and LP were detected. TABLE 1: Effects of Epinephrine and Lactate/pyruvate freezing and thawing of bovine blastocysts in vitro.
on production rates and survival after
2”;$ !
% Blastocysts Day 7 35.6 f 3.3a
FZB 67
(+)
244 4
26.0 + 3.Sb
60
Lactate/pyruvate (-)
366 6
22.8 f 3.2a
Lactate/pyruvate (f)
358 6
31.0 + 2.8b
Group Epinephrine
(-)
Epinephrine
% Survival 24 hours 48 hours 27.2 + 3.0 12.1 * 1.3 25.1 + 3.0
8.1 f 1.1
86
30.0 * 3.2
10.9 * 1.0
111
28.0 f 3.1
15.9 f 1.9
N= number of oocytes. R= replicates. FZB= number of frozen/thawed blastocysts. Levels of significancy: “b (~~0.05). Values are me&SE. Both, presence of epinephrine and absence of LP during the first 48 hours of culture significantly decreased blastocyst rate. Survival rate after thawing was not altered by these treatments.
Theriogenology
EFFECTS OF EPINEPHRINE AND LACTATE/PYRUVATE DURING THE FIRST 48 HOURS OF CULTURE ON THE FREEZABILITY OF IVF BOVINE BLASTOCYSTS E. Gomez and C. Diez Consejeria de Agricultura. CIATA. CENSYRA. Aptdo 155. Somio s/n. Gijon. Asturias. SPAIN Lipid contained in embryonic cells can adversely affect survival of frozen/thawed in vitro produced (IVP) bovine embryos (Leibo et al,Theriogenology 43:265,1995). Moreover, glycogen accumulates in excess in cells from IVP mouse embryos (Ozias and Stem, Biol Reprod, 8:467472,1973) and could retain water and render difficult the dehydration when freezing embryos. Mobilizing these stocks could therefore increase the ability of IVP embryos to be cryopreserved and it is known that epinephrine causes lipid and glycogen breakdown in adult tissues. Degradative products of glycogen and lipids could be used by the embryo depending on the presence of other energy substrates in the culture medium. The effects of epinephrine on the freezability of IVP embryos were evaluated. Cumulusoocyte complexes (COCs) were matured in TCM 199, 10% fetal bovine serum (FBS) and FSHp (20 &ml) for 24 h. Culture conditions were 39” C, 5% CO* in air and high humidity. Matured COCs were incubated together with Percoll-separated sperm in Fert-TALP and 10 ug/ml heparin for 20 h. Zygoies were vortexed for 2 min, allotted to each group and cultured in mSOF, 3g/L BSA and 10% FBS. The effect of 1uM epinephrine during the initial 48 h of culture was determined in a 2 x 2 factorial design in presence (+LP) or absence (-LP) of lactate (3.3 mM) and pyruvate (0.3 mM) in mSOF. From 48 h to Day 7, culture in all groups was performed in mSOF+LP without epinephrine. Day 7 blastocysts were slowly frozen in PBS and 1.5 M ethylene glycol, thawed for 5 set in air and 30 set in water at 30 “C and directly rehydrated in PBS. Thawed blastocysts were cocultured for 48 hours with bovine oviductal cells. Morphological normality at thawing and blastocoelic re-expansion were the criteria for survival. Data were analyzed by ANOVA and REGWF test for means. No interactions between epinephrine and LP were detected. TABLE 1: Effects of Epinephrine and Lactate/pyruvate freezing and thawing of bovine blastocysts in vitro.
on production rates and survival after
2”;$ !
% Blastocysts Day 7 35.6 f 3.3a
FZB 67
(+)
244 4
26.0 + 3.Sb
60
Lactate/pyruvate (-)
366 6
22.8 f 3.2a
Lactate/pyruvate (f)
358 6
31.0 + 2.8b
Group Epinephrine
(-)
Epinephrine
% Survival 24 hours 48 hours 27.2 + 3.0 12.1 * 1.3 25.1 + 3.0
8.1 f 1.1
86
30.0 * 3.2
10.9 * 1.0
111
28.0 f 3.1
15.9 f 1.9
N= number of oocytes. R= replicates. FZB= number of frozen/thawed blastocysts. Levels of significancy: “b (~~0.05). Values are me&SE. Both, presence of epinephrine and absence of LP during the first 48 hours of culture significantly decreased blastocyst rate. Survival rate after thawing was not altered by these treatments.