98 Safety and effectiveness of a lyophilised, ready-to-use, bovine in vitro maturation medium

The latest IETS report estimated that 1,156,422 in vitro-produced bovine embryos were generated in 2020.These embryos, either from ovum pickup or abattoir-derived oocytes, are the result of the in vitro maturation (IVM), fertilisation, and embryo culture processes. These processes require research and commercial laboratories to either prepare their own culture media, mainly from powder ingredients or by relying on commercial products, presented in liquid format. Liquid media formulations involve limited lifespan and high transportation costs since the cold chain needs to be maintained from the manufacturing company to the consumer to avoid degradation. An alternative approach, which combines low transportation and storage costs with a long lifespan, are the freeze-dried, ready-to-use, culture media. Freeze-drying or lyophilisation is a technology commonly employed for food, vaccines, and biomaterials preservation. Since basic information on the durability and effectiveness of lyophilised culture media is unknown, we hypothesised that (i) lyophilisation does not jeopardise the subsequent in vitro production (IVP) yield, and (ii) lyophilisation retains media efficacy at least six months after preparation. To test our hypotheses, we acquired two ready-to-use, commercial IVM media, one lyophilised (LYO) (NaturARTs-COW-IVM-LYO, EmbryoCloud) and one in liquid format (LIQ) (NaturARTs-COW-IVM). Commercial media were stored at 4°C and used for six months. In parallel, every day of testing the media, we prepared a fresh IVM medium in our laboratory (F) to compare the IVP yield from slaughterhouse ovaries among the different media (F, LYO, and LIQ groups). The experiment was replicated once a month over the course of a six-month period. Detailed information on the IVP protocol used, embryo fixation, and Hoechst 33342 staining is described in Lopes et al. (2019) (Theriogenology 126, 222–229). Data, expressed as the mean ± standard error, were analysed by one-way ANOVA; when significant, values were compared using the Tukey test. Differences were considered significant when P < 0.05. The experiment was replicated six times with a total oocyte number of 412, 378, and 398 in F, LIQ, and LYO groups, respectively. The results showed similar cleavage rates (73.5 ± 2.2, 74.9 ± 2.2, and 77.9 ± 2.1%, for F, LIQ, and LYO, respectively), blastocyst yield on D7 (20.1 ± 2.0, 22.0 ± 2.1, and 20.9 ± 2.0%, for F, LIQ, and LYO, respectively) and on D8 (22.3 ± 2.1, 25.9 ± 2.3, and 27.4 ± 2.2%, for F, LIQ, and LYO, respectively) among groups. There were also no differences for the mean number of cells per blastocyst (112.3 ± 4.3, 113.0 ± 5.0, and 115.4 ± 4.6, for F, LIQ, and LYO, respectively), or the proportion of D8 blastocyst, expanded blastocyst, and hatched blastocysts. The results confirm that (i) the freeze-dried NaturARTs-COW-IVM-LYO medium does not jeopardise the subsequent IVP yield, (ii) its performance is similar to the liquid medium prepared the day of use, and (iii) a shelf-life of at least six months was demonstrated for both the liquid and the lyophilised formats. This work highlights the promising future of freeze-drying culture media, since it could eliminate batch-to-batch variability, facilitate transportation and storage, and could notably increase the shelf-life of the products.

This research was supported by Fundación Séneca, Región de Murcia, Spain (21651/PDC/21).