161 EFFECT OF BOVINE PITUITARY EXTRACT ON HATCHING AND POST-HATCHING DEVELOPMENT OF IVP BOVINE EMBRYOS

Abstract

The effect of bovine pituitary extract (BPE; BD Biosciences, Inc., San Jose, CA, USA) on development of in vitro-produced (IVP) bovine blastocysts after Day 7 was studied. IVP embryos were produced from in vitro-matured COCs processed from local slaughterhouse ovaries or obtained from Bomed, Inc., Madison, WI, USA. The first 7 days of embryo culture following in vitro fertilization were in G1/G2 medium in 5%O₂ + 5%CO₂ + 90%N₂. Embryos that had reached the compacted morula or early blastocyst stage on Day 7 (29% of total) were identified and randomly assigned to four media treatments (n = 75/treatment, r = 7). DMEM/199 medium (D2) or G2, each supplemented by the addition of BSA, insulin, transferrin, and selenium (ITS), were used with or without BPE at 30 μg mL⁻¹. Extended embryo culture was continued until Day 11. Further development was assessed by counting hatched blastocysts and measuring the size (cross-sectional index) and the number of cells per blastocyst. Size diameters of blastocysts ranged from 200 μm to 400 μm across treatments. Cell counts were performed by staining the fixed blastocysts with Hoechst 33342 and counting the nuclei of squashed blastocysts with a fluorescent microscope. The percentage of hatched blastocysts was significantly greater (P < 0.05) in G2 with BPE (63.7 ± 6.8%) than in G2 alone (29.6 ± 7.3%). D2 with BPE (59.3 ± 6.8%) was not significantly different from D2 alone (56.8 ± 7.9%). Area index gave similar results; G2 + BPE (267 ± 168) was significantly higher (P < 0.05) than G2 alone (76 ± 28), and D2 + BPE (192 ± 139) and D2 alone (155 ± 106) were not different. Cell number gave similar results (P < 0.05); G2 + BPE (303 ± 164), G2 alone (123 ± 47), D2 + BPE (254 ± 166) and D2 alone (233 ± 120). A second experiment randomly assigned 7 day embryos (33% of total) to extended culture in G2 or D2 medium with or without irradiated STO feeder cells and under either 5% O₂ or 20% O₂ (n = 49/treatment, r = 5). All treatments contained BPE and a layer of agarose. Hatched blastocysts were counted on Day 11 and percentages were not different (P < 0.05) for both media without STO in low O₂ (G2 = 75 ± 12 %; D2 = 67 ± 8%) and in air (G2 = 82 ± 19%; D2 = 83 ± 11%). Hatching was not different (P < 0.05) with STO in both media in air (G2 = 73 ± 10%; D2 = 68 ± 15%), but was significantly reduced (P < 0.05) when STO was combined with low oxygen (G2 = 28 ± 14%; D2 = 4 ± 6%). Further culture to 18 days was completed, but many of the embryos collapsed by Day 12 and thereafter grew as lobed bodies and occasionally attached to each other. By 18 days a few embryos continued uncomplicated growth, however, and were composed of as many as 6900 cells. These results indicate that promotion of in vitro bovine blastocyst development in a minimal medium such as G2 requires BPE while a more complex medium, D2, supports further development without BPE. STO feeder cells in combination with low oxygen culture was inhibitory to blastocyst hatching and survival.