Bogado Pascottini, O. | bioRxiv Science

bioRxiv2022

Alternative culture systems for bovine oocyte in vitro maturation: liquid marbles and differentially shaped 96-well plates

In vivo matured oocytes exhibit higher developmental competence than those matured in vitro, but mimicking the in vivo environment by in vitro conditions has been challenging. Till now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems may cause cell flattening and does not allow cumulus expansion in all dimensions, which is less physiological. Therefore, implementing a low-cost and highly effective in vivo-like microenvironment methodology may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and V-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared to 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared to the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption, embryo development, and quality in both systems. None of the surface geometries influenced embryo development. These findings provide important inferences in many aspects of oocyte and embryo development. Further investigation is needed to determine other aspects like toxicity testing and ultrastructural changes in oocytes.

developmental biology↗

bioRxiv2020

Effects of parity, blood progesterone, and non-steroidal anti-inflammatory treatment on the dynamics of the uterine microbiota of healthy postpartum dairy cows

This study evaluated the effects of treatment with meloxicam (a non-steroidal anti-inflammatory drug), parity, and blood progesterone concentration on the dynamics of the uterine microbiome of clinically healthy postpartum dairy cows. Seven primiparous and 9 multiparous postpartum Holstein cows received meloxicam (0.5 mg/kg SC, n = 7 cows) once daily for 4 days (10 to 13 days in milk (DIM)) or were untreated (n = 9 cows). Endometrial cytology samples were collected by cytobrush at 10, 21, and 35 DIM, from which the metagenomic analysis was done using 16S rRNA gene sequence analysis. A radioimmunoassay was used to measure progesterone concentration in blood serum samples at 35 DIM and cows were classified as > 1 ng/mL (n = 10) or [≤] 1 ng/mL (n = 6). Alpha diversity for bacterial genera (Chao1, Shannon-Weiner, and Camargos evenness indices) were not affected by DIM, meloxicam treatment, parity, or progesterone category (P > 0.2). For beta diversity (genera level), principal coordinate analysis (Bray-Curtis) showed differences in microbiome between parity groups (P = 0.01). There was lower overall abundance of Anaerococcus, Bifidobacterium, Corynebacterium, Lactobacillus, Paracoccus, Staphylococcus, and Streptococcus and higher abundance of Bacillus, Fusobacterium, and Novosphingobium in primiparous than multiparous cows (P < 0.05); these patterns were consistent across sampling days. Bray-Curtis dissimilarity did not differ by DIM at sampling, meloxicam treatment, or progesterone category at 35 DIM (P > 0.5). In conclusion, uterine bacterial composition was not different at 10, 21, or 35 DIM, and meloxicam treatment or progesterone category did not affect uterine microbiota in clinically healthy postpartum dairy cows. Primiparous cows presented a different composition of uterine bacteria than multiparous cows. The differences in microbiome associated with parity might be attributable to changes that occur consequent to the first calving, but this hypothesis should be investigated further.

microbiology↗