Date of Award

8-2020

Degree Name

MS in Agriculture - Animal Science

Department/Program

Animal Science

College

College of Agriculture, Food, and Environmental Sciences

Advisor

Fernando Campos-Chillón

Advisor Department

Animal Science

Advisor College

College of Agriculture, Food, and Environmental Sciences

Abstract

Cryopreservation of in vivo derived Jersey bovine embryos have resulted in a 10% lower pregnancy rate compared to other dairy breeds. Poor embryo survival after cryopreservation has been partially attributed to the high lipid content of Jersey embryos. In vitro-produced (IVP) bovine embryos have darker cytoplasm than their in vivo-derived counterparts because of higher lipid accumulation. High lipid accumulation is associated with impaired embryo quality. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. L-carnitine is required for transport of fatty acids from the intermembrane space of the mitochondria into the mitochondrial matrix to support the process of β-oxidation, and enhances ATP production. We hypothesized that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin + L-carnitine would reduce lipid content of IVP embryos and vitrification with embryo collapse would improve the cryosurvival of Jersey IVP embryos. The objectives of this experiment were (1) to analyze lipid content of in vivo and IVP Jersey and Holstein cattle embryos, (2) to evaluate the effect of forskolin + L-carnitine added to IVP culture media, and (3) evaluate Jersey IVP survival rates after three cryopreservation procedures. The factorial experimental design for objectives one and two used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forsk/L-C). In vivo produced embryos (n = 27 blastocysts) were collected from superstimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of 1.5mM L-carnitine during maturation and embryo culture with forskolin (10 µM) added at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data was analyzed by ANOVA, and means were compared using Tukey’s HSD. For the third objective, grade 1 Jersey IVP blastocysts (n=356) were divided into six treatments using a 2x3 factorial design comparing intact (IB) vs collapsed blastocoele (CB) and three cryopreservation methods: slow freezing (SF) vs vitrification using open pulled straws (OPS) or cryotop (CT). Slow freezing embryos were equilibrated in 0.7 M glycerol and 0.1 M galactose in holding media for 10 min, held for 10 min at -6°C, seeded after 5 min, decreased to -32 °C at 0.5 °C /min, held at -32°C for 5 min, and finally plunged into liquid nitrogen. Vitrified embryos were equilibrated in 1.5 M ethylene glycol (EG) for 5 min, exposed to 7 M EG + 0.6 M galactose for 30 s while loaded into OPS or placed onto CT, then immediately plunged into liquid nitrogen. SF embryos were thawed in air for 10 s and placed in a water bath at 37°C for 45 s. Vitrified embryos were warmed directly into holding medium at 37°C supplemented with 1.0 M, 0.5 M and 0.25 M galactose for 3 minutes each. Subsequently, embryos were cultured in BBH7 and re-expansion rates were assessed at 24 and 48h post warming and data was evaluated by GLIMX. For objective 1, Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin + L-carnitine lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. For experiment 2, re-expansion rates were higher for CT, than OPS, and SF (85 vs. 66 vs. 72% ± 0.4, respectively; p<0.05). Main effect means for re-expansion were higher for CB than IB (79 vs 68% ± 0.3; p<0.05). In conclusion, IVP embryos have higher lipid accumulation over Holstein in vivo embryos. Addition of forskolin and L-carnitine to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. The CT method and collapsing the blastocoele prior to cryopreservation resulted in higher blastocyst survival rate. Further studies including transfer of embryos to recipients are necessary to corroborate these results.

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