Metabolic and developmental consequences of continuous vs. refreshed culture of bovine embryos in BBH7 media

Wills, Jennifer Rose (2013) Metabolic and developmental consequences of continuous vs. refreshed culture of bovine embryos in BBH7 media. MSc(Res) thesis, University of Nottingham.

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In vitro production of bovine embryos is an effective tool for quickly and efficiently improving herd genetics. Previously, detrimental effects of continuous culture (to day 7) on embryonic development have been reported due to a toxic build-up of ammonia. BBH7 is a serum-free in vitro culture media that has been specifically developed to improve the yield and cryo-tolerance of bovine embryos. Currently, bovine embryos are cultured continuously in BBH7 and it is the aim of this thesis to determine whether this culture system is detrimental to embryo development.

Oocytes were aspirated from follicles (4-9 mm) of abattoir-derived ovaries, matured overnight and fertilised with frozen-thawed semen. Putative zygotes were randomly assigned to two treatments; 1) continuous culture in BBH7 for 144 h, and 2) refreshed media at 48 h intervals over 144 h. Spent media was collected and snap-frozen (-196 °C) at each 48 h interval and final 144 h time points. Cleaved of inseminated (48 h post-fertilisation), blastocysts of cleaved (by 144 h), and embryo grade (IETS) were assessed. Furthermore, ammonia concentrations were determined in spent media by an enzymatic ultraviolet method, and sex ratio was determined by polymerase chain reaction.

Zygote and blastocyst yields did not differ between treatments. There was also no effect of treatment on final stage of embryonic development. Ammonia concentrations 1) increased significantly over time in basal media (P=0.014); 2) increased significantly from 0 h (P=0.005) after each 48 h interval, but not between each interval; and 3) were significantly greater in the ‘no change’ group following 144 h (P=0.048) than in the ‘change’ group. Furthermore, sex ratio did not differ between treatments.

Although there is reported evidence of the detrimental effects of ammonia accumulation on preimplantation embryo development during continuous culture, these data suggest there is currently no benefit in refreshing BBH7 at 48 h intervals. Equally, there was no effect of refreshing BBH7 on sex ratio skew. However, the spontaneous breakdown of amino acids in serum-free media may compromise embryo development by increasing circumjacent ammonia concentrations. Finally, effects on amino acid metabolism await analysis, and post-transfer pregnancy rates await experimentation.

Item Type: Thesis (University of Nottingham only) (MSc(Res))
Supervisors: Sinclair, K.D.
Subjects: S Agriculture > SF Animal culture
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Clinical Sciences
Item ID: 13617
Depositing User: EP, Services
Date Deposited: 20 Jun 2014 10:17
Last Modified: 13 Sep 2016 15:03

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