Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0–2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.     

Abnormal functions of Leydig cells in crossbred cattle–yak showing infertility

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle–yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle–yak spermatogenesis. Testicular tissues from yaks (1–3 years old) and hybrids (F1–F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle–yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle–yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.     

Two species of seabirds foraged in contrasting marine habitats across the cold-water belt along the coast of northern Hokkaido in the southwestern Okhotsk Sea

To understand the environmental factors affecting the density of foraging seabirds across the cold-water belt in the southwestern Okhotsk Sea, we conducted a 1-day (180-km transect length) shipboard seabird survey off the northeastern coast of Hokkaido during summer in 2019, along with acoustic observations of potential prey (zooplankton and fish) biomass, thermosalinograph measurements, and CTD observations. Planktivorous short-tailed shearwaters Ardenna tenuirostris (66% of total seabirds) and piscivorous rhinoceros auklets Cerorhinca monocerata (28%) were predominant, but foraged in contrasting habitats. A large foraging flock of shearwaters was observed in the cold-water belt zone, including its front with coastal Soya Warm Current Water and the offshore Fresh Surface Okhotsk Sea Water, where surface chlorophyll a concentrations were the highest but not related to their prey (zooplankton) biomass at any spatial scale between 4.6 and 9.2 km. In contrast, the density of auklets was high in the coastal Soya Warm Current Water, where the acoustically determined fish biomass was large, and showed a positive relationship with the fish biomass especially in the lower layer (29–104 m depth) at any spatial scale. This species-specific difference in response to prey biomass might be related to prey-searching behaviors; i.e., rhinoceros auklets search prey underwater visually, but short-tailed shearwater can use both visual and olfactory cues to locate zooplankton patches from the air.

     

Efficiency of the indirect selection and the evaluation of the genotype by environment interaction using Pilodyn for the genetic improvement of wood density in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

The efficiency of the indirect selection for wood density using the Pilodyn in Cryptomeria japonica was studied by comparing the Pilodyn penetration (PP) depth and the direct measurement of wood density in three test sites. The influence of the genotype by environment (G × E) interaction of wood density was estimated using the Pilodyn with common 12 C. japonica clones in 10 test sites in Kanto breeding region in Japan. The PP depth was highly correlated with wood density, and the genetic correlation between them was −0.88. The indirect selection using the Pilodyn realized 87% of the genetic gain obtained by the direct selection of wood density. The G × E interaction in PP depth was small. The ratio of the variance component of the G × E interaction to that of genotype was only 0.096 in the PP depth, whereas it was 0.700 in tree height and 1.410 in diameter at breast height. These findings indicate that the Pilodyn is useful for the genetic improvement of wood density in Cryptomeria japonica. The small G × E interaction in wood density estimated using the Pilodyn indicates that the relative clonal performance in wood density is stable among diverse environments in Kanto breeding region in Japan.     

A black soybean seed coat extract prevents obesity and glucose intolerance by up-regulating uncoupling proteins and down-regulating inflammatory cytokines in high-fat diet-fed mice

Black soybean seed coat extract (BE) is a polyphenol-rich food material consisting of 9.2% cyanidin 3-glucoside, 6.2% catechins, 39.8% procyanidins, and others. This study demonstrated that BE ameliorated obesity and glucose intolerance by up-regulating uncoupling proteins (UCPs) and down-regulating inflammatory cytokines in C57BL/6 mice fed a control or high-fat diet containing BE for 14 weeks. BE suppressed fat accumulation in mesenteric adipose tissue, reduced the plasma glucose level, and enhanced insulin sensitivity in the high-fat diet-fed mice. The gene and protein expression levels of UCP-1 in brown adipose tissue and UCP-2 in white adipose tissue were up-regulated by BE. Moreover, the gene expression levels of major inflammatory cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1 were remarkably decreased by BE in white adipose tissue. BE is a beneficial food material for the prevention of obesity and diabetes by enhancing energy expenditure and suppressing inflammation.     

Fatal aortic endocarditis associated with community-acquired Serratia marcescens infection in a dog

A 12 yr old Dalmatian was referred for evaluation of acute lethargy, fever, neurologic signs, and a recently ausculted heart murmur. Echocardiography in combination with blood cultures resulted in a diagnosis of nonhospital-acquired Serratia marcescens bacteremia and aortic valve endocarditis. Despite early diagnosis and aggressive therapy, the dog failed to respond to antimicrobials and died within 6 hr after admission. Necropsy findings included aortic valve endocarditis, septicemia, and diffuse thromboembolic disease. There was no history of pre-existing underlying disease or immunosuppressive therapy, and the dog had not been hospitalized before referral.     

Quantitative trait analysis of transplanting time and other root-growth-related traits in tomato

Transplanting time is determined by factors such as the field conditions, the age of the seedlings, and the size of the container, but little information is available on the effect of the genetic background on transplanting time. Here, we examined root dry weight (RDW), shoot dry weight, root/shoot ratio (RSR), time at which 50% of the germinated seedlings have expanded cotyledons (CE50), root ball formation (RBW), the length of time from the cotyledon expansion from the first to the last germinated seedling (ES) and transplanting time (TRD) in a BC₁F₆ population derived from Solanum lycopersicum and Solanum pimpinellifolium. All traits exhibited significant correlation with each other, except for RSR, which was only significantly correlated to RDW. A total of eight additive quantitative trait loci (QTLs) were detected for the five traits, i.e., RSR, RBW, CE50, ES, and TRD. One epistatic (QTL × QTL) interaction each was identified for RSR and TRD. QTLs for RSR, RBW, CE50, ES and TRD clustered near marker LEOH37 on chromosome 4. Clustering of ce50-4, rsr4, rbw4, and es4 with trd4 reflected the dependence of transplanting time on root ball formation, growth uniformity and early seedling growth, especially root growth. In addition, several QTLs in this study were mapped to regions where QTLs for days to flowering or number of leaves before the first inflorescence had been identified previously. This suggests that the roots may exert some influence on the flowering time in tomato.     

Detachable Coils for Occlusion of Patent Ductus Arteriosus in 2 Dogs

OBJECTIVE: To describe the use of a detachable coil for transcatheter closure (TCC) of patent ductus arteriosus (PDA) in 2 dogs. STUDY DESIGN: Clinical study. ANIMALS: Two female Pembroke Welsh Corgi dogs with PDA. METHODS: Using fluoroscopic guidance, an 8-mm-diameter coil stent with 5 loops (detachable coils for PDA closure) was inserted via catheterization of the femoral artery. The catheter was passed through the PDA into the pulmonary artery. The coil was withdrawn so that 1.5 loops remained on the pulmonary side of the orifice of the ductus. The rest of the loops were pushed out from the catheter into the ductus. After confirming the correct placement of the coil and the effectiveness of the occlusion, the delivery wire was detached from the coil. RESULTS: Insertion of the coil was easily performed, even without previous experience. Immediate and marked decrease of the cardiac murmur was auscultated. Only slight residual flow was detected by angiography conducted 3 months' postoperatively. The dogs experienced quick and uneventful recovery after coil placement and required minimal postoperative care. Follow-up evaluation of the dogs showed no functional clinical signs of PDA, and no cardiac abnormalities were detected on electrocardiographic, phonocardiographic, and echocardiographic examination. In dog 1, the residual flow had disappeared on the color-flow Doppler echocardiographic examination at 18 months' postoperatively. CONCLUSION: TCC using a detachable coil was easy, safe, and effective in 2 dogs with PDA. The minimal residual shunting observed only by echocardiography seemed hemodynamically insignificant. CLINICAL RELEVANCE: This method can be used as an alternative to traditional surgical methods.