Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick‐Up: the effects of follicle stimulation and in vitro maturation

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick‐Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non‐stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H₂O₂ levels at the metaphase‐II stage and intracellular Ca²⁺ levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re‐distribution in non‐stimulated OPU‐derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non‐stimulated OPU in terms of ATP content, cytoplasmic H₂O₂ levels, base Ca²⁺ levels and the frequencies and amplitudes of Ca²⁺ oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.     

An abnormal birth in bovine suspected of being caused by Peaton virus first occurred in Shikoku region,Japan

Peaton virus (PEAV) is a type of arthropod-borne virus (arbovirus) belonging to the genus Orthobunyavirus, much like Akabane virus and Aino virus. These arboviruses cause stillbirth and congenital malformations of fetuses in ruminants. In Japan, abnormal birth in bovine caused by PEAV were reported in Okinawa, Kyushu, and Chugoku regions, but it has never been reported in Shikoku region. The abnormal birth occurred in 2020 in Ehime Prefecture (Shikoku region) and suspected of being caused by PEAV from results of clinical signs, pathological findings, and virus neutralization test using PEAV. However, PEAV was not detected and isolated. This report describes the case of abnormal birth in bovine suspected of being caused by PEAV first occurred in Shikoku region, Japan.     

Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro

The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.     

Construction of artificial promoters highly responsive to iron deficiency

Iron deficiency-responsive element 1 (IDE1) and IDE2 are cis-acting elements that are responsible for Fe-deficiency-inducible and root-specific expression of the barley (Hordeum vulgare L.) gene IDS2 (Fe-deficiency-specific clone no. 2). Using these cis-acting elements, we aimed to construct super-promoters that would induce prominent gene expression in the roots of Fe-deficient rice plants (Oryza sativa L.). Modules containing IDE1 and IDE2 of the IDS2 promoter were used as repeats or were linked to the Fe-deficiency-responsive promoter of barley IDS3, and were connected to known enhancer-like sequences. Five artificial promoters, as well as the native promoters of barley IDS2 or IDS3, were connected individually upstream of β-glucuronidase (GUS) and were introduced into rice. Transgenic rice plants were grown under control or Fe-deficient conditions, and GUS expression was analyzed. The artificial promoter that contained one module of IDE1 and IDE2 conferred strong Fe-deficiency-inducible GUS expression to the roots of rice plants. Each of the five artificial promoters induced a similar level of GUS expression in Fe-deficient roots, which did not exceed the GUS expression driven by the native IDS2 or IDS3 promoter. Artificial and native promoters induced GUS expression in response to Fe-deficiency in leaves, although the level of expression was lower than that in roots. Histochemical observations revealed that GUS expression driven by artificial and native promoters was spatially similar, and expression was dominant within vascular bundles and root exodermis. These findings suggest that there is coordinated expression of the genes that are involved in Fe-deficiency-induced Fe uptake in rice.     

Delay in Cleavage of Porcine Embryos after Intracytoplasmic Sperm Injection (ICSI) Shows Poorer Embryonic Development

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.     

Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares

Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares.     

Molecular mapping of quantitative trait loci for domestication traits and β-glucan content in a wheat recombinant inbred line population

Genetic maps are useful for analysis of quantitative trait loci (QTLs) and for marker-assisted selection (MAS) in breeding. A simple sequence repeat (SSR) marker linkage map of common wheat was constructed based on recombination inbred lines (RILs) derived from a cross between Chinese Spring and spelt wheat. The map included 264 loci on all wheat chromosomes covering 2,345.2 cM with 962, 794.6, and 588.6 cM for the A, B, and D genomes, respectively. Using the RILs and the map, we detected 42 putative QTLs on 15 chromosomes for ear length, spikelet number, spike compactness, kernel length, kernel width, kernel height and β-glucan content. Each QTL explained 4–45% of the phenotypic variation. Five QTL cluster regions were detected on chromosomes 1A, 5AL, 2B, 2D, and 4D. The first QTLs for β-glucan content in wheat were identified on chromosomes 3A, 1B, 5B, and 6D.