Development of IVF Porcine Embryos in Microwell Culture System

Animals · 2025-08-28

We investigated the development of IVF porcine embryos incubated in microwells of Well-of-the-Well (WOW) culture dishes. In Experiment 1, 500 embryos were cultured under five different culture conditions: 10 individual embryos in microwells covered by 50 μL PZM-3 medium (50 μL WOW); 10 embryos together in a well of 50 μL PZM-3 (50 μL Well); 10 embryos in microwells covered by 20 μL PZM-3 (20 μL WOW); 10 embryos in a well of 20 μL PZM-3 (20 μL Well); and 10 embryos in a 20 μL drop of PZM-3 in traditional dishes (control). In Experiment 2, the best-performing treatment group from Experiment 1 (50 μL WOW) was further compared to control using a total of 350 embryos. There was no difference in the percentage of cleaved embryos among the groups in both experiments. In Experiment 1, the percentage of blastocysts from 50 μL WOW (42.00 ± 6.29%) was higher (p = 0.001) than in 50 μL Well (29.00 ± 4.58%), 20 μL WOW (13.00 ± 2.13%), 20 μL Well (20.00 ± 2.98%) and control (25.00 ± 5.22%). Similarly, in Experiment 2, blastocyst formation and nuclei number in the blastocysts from 50 μL WOW (37.86 ± 3.95% and 38.97 ± 1.80) were higher (p = 0.040 and p = 0.017) than in the control (28.10 ± 2.64% and 33.21 ± 1.56). The results indicate that individual culture in microwells improves the development of IVF pig embryos.

The signal that stimulates mammalian embryo development

Frontiers in Cell and Developmental Biology · 2024-09-17 · 10 citations

Embryo development is stimulated by calcium (Ca 2+ ) signals that are generated in the egg cytoplasm by the fertilizing sperm. Eggs are formed via oogenesis. They go through a cell division known as meiosis, during which their diploid chromosome number is halved and new genetic combinations are created by crossing over. During formation the eggs also acquire cellular components that are necessary to produce the Ca 2+ signal and also, to support development of the newly formed embryo. Ionized calcium is a universal second messenger used by cells in a plethora of biological processes and the eggs develop a “toolkit”, a set of molecules needed for signaling. Meiosis stops twice and these arrests are controlled by a complex interaction of regulatory proteins. The first meiotic arrest lasts until after puberty, when a luteinizing hormone surge stimulates meiotic resumption. The cell cycle proceeds to stop again in the middle of the second meiotic division, right before ovulation. The union of the female and male gametes takes place in the oviduct. Following gamete fusion, the sperm triggers the release of Ca 2+ from the egg’s intracellular stores which in mammals is followed by repetitive Ca 2+ spikes known as Ca 2+ oscillations in the cytosol that last for several hours. Downstream sensor proteins help decoding the signal and stimulate other molecules whose actions are required for proper development including those that help to prevent the fusion of additional sperm cells to the egg and those that assist in the release from the second meiotic arrest, completion of meiosis and entering the first mitotic cell division. Here I review the major steps of egg formation, discuss the signaling toolkit that is essential to generate the Ca 2+ signal and describe the steps of the signal transduction mechanism that activates the egg’s developmental program and turns it into an embryo.

Reduction of cortical pulling at mitotic entry facilitates aster centration

Journal of Cell Science · 2024-03-12 · 6 citations

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture

Cellular Reprogramming · 2024-01-23 · 4 citations

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

Author Reply to Peer Reviews of Reduction of cortical pulling at mitotic entry facilitates aster centration

2023-12-18

Production of Cloned Pigs by Handmade Cloning

Methods in molecular biology · 2023-01-01 · 4 citations

Reduction of cortical pulling at mitotic entry facilitates aster centration

bioRxiv (Cold Spring Harbor Laboratory) · 2023-03-21

Abstract Although it has been studied for more than a century, the question of how one cell divides into two equal parts is still not fully resolved. Zygotes have provided much of the mechanistic insight into how the mitotic apparatus finds the center of the cell since the centrally-located mitotic apparatus is created from a large sperm aster that forms at the cortex and thus far from the zygote center. Here we show that in ascidians, the sperm aster extends throughout the cytoplasm during interphase yet remains located near the cortex and does not migrate towards the zygote center. It is only at mitotic entry, when the sperm aster has duplicated and the mitotic apparatus is being assembled, that most of the migration and centration occurs. This temporal pattern of centration behavior is mirrored by primate zygotes (including human). The current mechanisms of aster centration include cytoplasmic pulling that scale with microtubule (MT) length, MT pushing against the proximal cortex or MT-based cortical pulling. However, it is not yet known whether and how these 3 mechanisms are coordinated to prevent aster migration during interphase and trigger migration at mitotic entry. By monitoring quantitatively all three mechanisms (cytoplasmic pulling, pushing and cortical pulling) we have discovered that cortical pulling is switched off as the zygote enters mitosis while both cytoplasmic pulling and proximal cortical pushing remain active. Physical simulations could recapitulate both the static and migratory aspects of sperm aster and mitotic apparatus behavior. We therefore surmise that the reduction in cortical pulling at mitotic entry represents a switch that allows proximal cortical pushing forces and cytoplasmic pulling forces to center the nascent mitotic apparatus. Graphical abstract Highlights Sperm aster/mitotic apparatus centration occurs at entry into first mitosis MT-based cortical pulling is active during interphase and switched off at mitotic entry Loss of cortical pulling at mitosis entry facilitates centration of the aster MT-based cytoplasmic pulling is active during both interphase and mitosis Agent-based simulations advocate the need for cytoplasmic pulling, a switch in cortical pulling and a minor role of pushing for aster centration at mitotic entry.

Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa

Theriogenology · 2023-02-02 · 11 citations

Early Pregnancy Markers in the Serum of Ewes Identified via Proteomic and Metabolomic Analyses

International Journal of Molecular Sciences · 2023-09-13 · 5 citations

The diagnosis of ewes’ pregnancy status at an early stage is an efficient way to enhance the reproductive output of sheep and allow producers to optimize production and management. The techniques of proteomics and metabolomics have been widely used to detect regulatory factors in various physiological processes of animals. The aim of this study is to explore the differential metabolites and proteins in the serum of pregnant and non-pregnant ewes by proteomics and metabolomics. The serum of ewes at 21, 28 and 33 days after artificial insemination (AI) were collected. The pregnancy stratus of the ewes was finally determined through ultrasound examination and then the ewes were grouped as Pregnant (n = 21) or N on-pregnant (n = 9). First, the serum samples from pregnant or non-pregnant ewes at 21 days after AI were selected for metabolomic analysis. It was found that the level of nine metabolites were upregulated and 20 metabolites were downregulated in the pregnant animals (p < 0.05). None of these differential metabolomes are suitable as markers of pregnancy due to their small foldchange. Next, the proteomes of serum from pregnant or non-pregnant ewes were evaluated. At 21 days after AI, the presence of 321 proteins were detected, and we found that the level of three proteins were upregulated and 11 proteins were downregulated in the serum of pregnant ewes (p < 0.05). The levels of serum amyloid A (SAA), afamin (AFM), serpin family A member 6 (SERPINA6) and immunoglobulin-like domain-containing protein between pregnant and non-pregnant ewes at 21-, 28- and 33-days post-AI were also analyzed via enzyme-linked immunosorbent assay (ELISA). The levels of SAA and AFM were significantly higher in pregnant ewes than in non-pregnant ewes, and could be used as markers for early pregnancy detection. Overall, our results show that SAA and AFM are potential biomarkers to determine the early pregnancy status of ewes.

Adapting the program redesign model for a student‐centered curricula renewal in animal science

Natural sciences education · 2023-02-14

Abstract Departmental‐level curricular redesign is a vital issue in higher education, especially as departments work to move toward more student‐centered teaching and learning environments. However, there is a lack of guidance on the steps to take or potential solutions to challenges faced by faculty and departments undergoing the curricular redesign process. While there are studies that share frameworks and different processes, there are few approaches that emphasize student‐centered learning, as well as being flexible, theory‐based, and data driven. In this paper, we share an overview of an adapted curricular design framework and the detailed process a Department of Animal Sciences is taking to approach departmental curricular redesign. We highlight items to consider prior to beginning a department level curricular redesign, the challenges, potential solutions, and next steps in the process.