High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.
We describe a bovine embryo culture system that supports repeatable high development in the presence of serum or BSA as well as under defined conditions in the absence of those components. In the first experiment, embryo development in SOF with amino acids (SOFaa), sodium citrate (SOFaac) and myo-inositol (SOFaaci) and with BSA or polyvinyl alcohol (PVA) was compared with that in a M199 granulosa cell co-culture (M199 co-culture). Subsequently, development and cell numbers of blastocysts cultured under defined conditions in SOFaaci with PVA (SOFaaci-PVA), or under undefined conditions in SOFaaci with 5% cow serum (SOFaaci-CS) or M199 co-culture were compared. The repeatability of culture results in SOFaaci-CS was checked by weekly replicates (n = 30) spread over 11 months. The viability of embryos developed in SOFaaci-PVA was estimated by transfer of morphologically good blastocysts (n = 10) to synchronized recipients. In the second experiment, the effect of omitting CS or BSA from IVM and IVM-IVF on subsequent embryo development in SOFaaci-PVA or in SOFaaci-CS was investigated. Blastocyst development in SOFaa-PVA, SOFaac-PVA, SOFaa-BSA and M199 was 16 +/- 3b, 23 +/- 2ab, 30 +/- 8a and 36 +/- 7a%, respectively (Pab < 0.05). Additional inclusion of myoinositol resulted in 42 +/- 1a% blastocysts in SOFaaci-PVA vs 19 +/- 3b% in SOFaac-PVA, 47 +/- 7a% in SOFaac-BSA, and 36 +/- 7a% in M199 co-culture, respectively (Pab < 0.01). In 30 replicates, the average cleavage and blastocyst rates of oocytes in SOFaaci-CS were 87 +/- 4 and 49 +/- 5%, respectively. Five normal calves were produced after transfer of 10 blastocysts developed in defined culture medium (i.e., SOFaaci-PVA). Defined IVM or IVM-IVF (i.e., in absence of CS and BSA) reduced cleavage rates (83 +/- 3 and 55 +/- 3% vs 90 +/- 1% in presence of CS; P < 0.01). Subsequent embryo development in SOFaaci-CS was not affected in either of these defined conditions. However, cleavage and blastocyst rates under completely defined IVP conditions were 54 +/- 7 and 19 +/- 4%, respectively. It was concluded that under defined culture conditions, addition of citrate and myo-inositol improved blastocyst development to rates comparable to those obtained with serum, BSA or co-culture and that the quality of blastocysts was not affected by the absence of serum or BSA. However, serum was essential during IVM/IVF for normal fertilization and subsequent high blastocyst development.
Holm
P
P
Embryo Technology Center, Danish Institute of Agricultural Sciences, Tjele, Denmark.
Booth
P J
PJ
Schmidt
M H
MH
Greve
T
T
Callesen
H
H
D000596:Amino Acids; D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000494:pharmacology; D002417:Cattle; D002951:Citrates / Q000494:pharmacology; D018920:Coculture Techniques; D015925:Cryopreservation; D003470:Culture Media; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D006107:Granulosa Cells / Q000166:cytology; D007294:Inositol / Q000494:pharmacology; D008297:Male; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D012662:Semen Preservation; D000077559:Sodium Citrate; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D015053:Zygote / Q000166:cytology / Q000502:physiology
D000596:Amino Acids; D001798:Blood Proteins; D002951:Citrates; D003470:Culture Media; D000077559:Sodium Citrate; D007294:Inositol