Ovarian dynamics in progesterone tablet-induced superovulation in goats assessed by magnetic resonance imaging.

Controlled internal drug-releasing (CIDR) devices are commonly used for superovulation in goats. However, such devices are unavailable in some countries, including Japan. In this technical note, we aimed to explore the efficacy of an alternative superovulation protocol using progesterone tablets in goats. We employed intravaginal progesterone tablets (LUTINAS® Vaginal Tablet 100 mg) following a standard superovulation protocol. Additionally, we assessed the ovarian dynamics using 3T-magnetic resonance imaging (MRI) 1 day preceding the progesterone treatment (Day "-1") and 3 days before the end of treatment (Days 11-13). The ovarian monitoring was successfully performed in the short tau inversion recovery T2-weighted images of MRI, and ovulation was confirmed by the disappearance of follicles on Day 13 post-administration of the tablets. Immediately after ovulation, oviduct flushing yielded a substantial number of oocytes (13.5 ± 1.8 oocytes per animal). These findings provide evidence that the administration of progesterone tablets can serve as a viable alternative for inducing. Additionally, our findings suggest that 3T-MRI is a promising alternative to conventional ultrasonography for monitoring ovarian dynamics following superovulation in experimental goats.

Effects of alpha lipoic acid treatment during in vitro maturation on the development of porcine somatic cell nuclear transfer embryos.

Oxidative stress influences the embryo production efficiency in vitro. We investigated the effects of alpha lipoic acid (ALA) treatment during the in vitro maturation (IVM) period on the porcine somatic cell nuclear transfer (SCNT) embryo production. After IVM, maturation rates of the 12.5- and 25-µM ALA-treated groups were not significantly different from those of the 0-µM ALA-treated group. Compared to those in the 0-µM ALA-treated group, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, in the cytoplasm of matured oocytes in the 12.5-50-µM ALA-treated groups. Apoptosis rate in cumulus cells after IVM was significantly lower in the 12.5-50-µM ALA-treated groups than in the 0-µM ALA-treated group. Blastocyst formation rate was significantly higher in parthenogenetic oocytes treated with 12.5-µM ALA than in the 0-, 25-, and 50-µM ALA-treated groups. Similarly, in SCNT embryos, the 12.5-µM ALA-treated group showed a significantly higher blastocyst formation rate than the 0-µM ALA-treated group. Apoptosis rate in SCNT blastocysts was significantly decreased by 12.5-µM ALA treatment. The results showed that treatment with 12.5-µM ALA during IVM improves porcine SCNT embryo development and partial quality.

Changes in population structure and genetic diversity of Misaki horses between 2015 and 2020.

For the preservation of Misaki horses, changes in the population structure and genetic diversity of the horses for 5 years were analyzed using population and genotype data from 2015-2020. The microsatellite genotyping was performed, and the average number of alleles (Na), expected heterozygosity (He), and observed value (Ho) were calculated. Moreover, the average generation length (GL) was estimated from the population management record. Then, no significant differences in Na, He, and Ho were found between 2015 and 2020, suggesting their genetic diversity had been maintained for 5 years. Moreover, the average GL was estimated as 4.6 years. Compared to other native horses, a short average GL suggesting a rapid generation renewing is a characteristic of the Misaki population.

Genetic characterization of Japanese native horse breeds by genotyping variants that are associated with phenotypic traits.

Concerns have been raised about the loss of genetic diversity in Japanese native horses because of their declining populations. In this study, we investigated the genetic variation of four genes, myostatin (MSTN), ligand-dependent nuclear receptor corepressor like (LCORL), doublesex and mab-3 related transcription factor 3 (DMRT3), and 5-hydroxytryptamine receptor 1A (HTR1A), which are associated with horse phenotypic traits, in six Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, and Yonaguni). MSTN, LCORL, DMRT3, and HTR1A showed polymorphisms in the Kiso; Hokkaido and Noma; Hokkaido; and Kiso, Tokara, and Yonaguni breeds, respectively. The Misaki did not show polymorphisms in any of the genes. This study may serve as a basis for developing future breeding strategies focusing on traits in Japanese native horses.

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Effects of tris (2-carboxyethyl) phosphine hydrochloride treatment on porcine oocyte in vitro maturation and subsequent in vitro fertilized embryo developmental capacity.

Oocyte in vitro maturation (IVM) is a crucial process that determines subsequent in vitro embryo production. The present study investigated the effects of the antioxidant tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCL) on the in vitro maturation of porcine oocytes and in vitro developmental competence of fertilized embryos. Oocytes were matured in IVM medium based on four concentration groups of TCEP-HCL (0, 50, 100, and 200 µM) treatment. 100 µM TCEP-HCL treatment significantly increased the oocyte first polar body extrusion rate, monospermy rate and subsequent in vitro fertilized embryo developmental capacity (cleavage rate, blastocyst formation rate, and blastocyst total cell number) compared to those in the control group. Furthermore, 100 µM TCEP-HCL treatment significantly reduced the levels of reactive oxygen species, significantly increased glutathione levels and mitochondrial content compared to those in the control group. Moreover, 100 µM TCEP-HCL treatment significantly decreased the oocyte apoptosis, blastocyst apoptosis compared to that in the controls. In summary, these results indicate that 100 µM TCEP-HCL treatment improves the quality and developmental capacity of in vitro-fertilized embryos by decreasing oxidative stress in porcine oocytes.