Lipid Enriched Reduced Nutrient Culture Medium Improves Bovine Blastocyst Formation

Abstract

The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.

Figure 1

Effects of culture environment on embryo development and blastocyst cell numbers. (A) Embryo development in control medium and reduced nutrient (RN) media supplemented lipid-free and lipid-rich BSA (control, n = 587; RN lipid free, n = 573; RN lipid rich, n = 585, the experiments were replicated seven times). The percentage of cleaved embryos was calculated of the total number of cumulus–complex oocytes matured and fertilized (cleaved/COC); the percentage of blastocysts (blastocyst/cleaved) and of hatching/fully hatched blastocysts (hatching/cleaved) of cleaved embryos were calculated. (B) A representative image of a bovine blastocyst stained by CDX2 (red) and SOX2 (green) to assess blastocyst cell numbers. Scale bar, 20 µm. (C) The number of cells in the trophectoderm (TE) and inner cell mass (ICM), and the total number of cells were determined in bovine blastocysts from control (n = 39), RN lipid-free (n = 6) and RN lipid-rich (n = 20) media. Data are reported as mean ± s.e.m. Different superscripts indicate a significant difference between treatments (P < 0.05).

Figure 2

Effect of culture environment on blastocyst gene expression related to fatty acid metabolism (A), glucose metabolism (B) and embryo quality and redox balance (C). The gene expression value was arbitrarily set to 1 for samples from the control medium. Comparisons were made between samples from the control medium with the ones from RN lipid-free and RN lipid-rich media. The experiments were replicated four times. Data are reported as mean ± s.e.m. Different superscripts indicate a significant difference between treatments (P < 0.05).

Figure 3

Mitochondrial copy number and ATP quantification of individual bovine blastocysts produced in different culture environments. (A) The quantification of relative mtDNA copy number per cell of individual blastocysts were compared between control (n = 19), RN lipid-free (n = 18), and RN lipid-rich media (n = 19). The experiments were replicated three times. (B) ATP quantification of individual bovine blastocysts were compared between control (n = 15), RN lipid-free (n = 15), and RN lipid-rich media (n = 15). Data are shown as pmoL/cell obtained by dividing the ATP content of individual blastocysts by the average total cell number for that treatment. The experiments were replicated three times. Data are reported as mean ± s.e.m. Different superscripts indicate a significant difference between treatments (P < 0.05).

Figure 4

Effect of culture environment on individual blastocyst lipid contents. (A) PCA obtained using lipid content of the blastocysts produced with control (n = 14) and RN lipid-rich media (n = 15). (B and C) A total of 12 out of 40 tested lipids were different between blastocysts from the control and RN lipid-rich media. Compounds were annotated based on spectral matching to in-house, NISTv14, 1 – SToP spectral databases (Broeckling et al. 2016).