Dynamics of a fish-killing dinoflagellate Karenia mikimotoi red-tide captured by composite data sources.

Bloom dynamics of K. mikimotoi during summer 2015 in the Yatsushiro Sea, Japan, which caused fish mortality was studied using field survey data and satellite data. The bloom initially formed in the western area, subsequently appeared in the southern area, and finally expanded to the central area. The field-survey detected the horizontal displacement of the bloom which was also assessed by satellite data. Acoustic backscattering intensity of the current meter captured the modulation of the diurnal vertical migration of K. mikimotoi. After the modulation, K. mikimotoi distributed at a shallower depth in the nighttime than the period prior to the modulation. Factors affecting the modulation are suggested to be the continuous low nutrient conditions. Synchronization between the shallowed distribution during the nighttime and the wind driven surface northeastward current enabled a sudden horizontal transport toward the central area. Satellite and acoustic backscattering data are beneficial subsidiary tools for detecting blooms.

Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN.

Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.

Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm.

It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV-C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.

Efficacy of Abdominal Ultrasonography for Differentiation of Gastrointestinal Diseases in Calves.

This study investigated the clinical efficacy of abdominal ultrasonography for abomasal dilation in three calves, intestinal volvulus in five calves, intussusception in one calf, and internal hernia in one calf. In the abdominal ultrasonograms of the abomasal dilation cases, this disease was commonly characterized by severely extended lumens, including heterogeneously hyperechoic ingesta without intraluminal accumulations of gas. In the animals with intestinal volvulus and intussusception, a to-and-fro flow was observed to be a common ultrasonographic characteristic that led to suspicion of an intestinal obstruction. The use of abdominal ultrasonography for five cases with intestinal volvulus gave no reason to suspect this disease, despite its efficacy in one case, based on an acutely angled narrowing. Although three of five animals with intestinal volvulus had intestinal ruptures, no ultrasonographic evidence could be obtained. When abdominal ultrasonography was used for one case with intussusception, this pathological condition could be strongly suspected, as a "target" sign was observed. This finding supported surgical intervention for this case, followed by treatment with manual reduction, resulting in a favorable outcome. In terms of the differential and definitive diagnosis for various intestinal diseases, abdominal ultrasonography may be poor at providing indicative evidence, but very helpful for confirming intestinal obstruction.

Relationship between GnRH-induced LH increase profiles in the serum and vaginal mucus of Japanese Black beef cows.

This study aimed to investigate the relationship between increases in the luteinizing hormone (LH) profiles in the serum and vaginal mucus of cows induced by gonadotropin-releasing hormone (GnRH). Samples for LH determination were collected from Japanese Black beef cows during estrus, which was induced with a controlled internal progesterone-releasing device and the administration of cloprostenol immediately before GnRH administration and every 30 min from the start of GnRH administration until 6.5 h. The peak serum LH concentration was clearly identified at 2.5 h post-GnRH administration, with serum concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak vaginal mucus LH concentration was identified 4.5 h after GnRH administration. These results indicate that the LH secretion peak in vaginal mucus appeared about 2 h after peak LH secretion in the serum.

Comparative analysis of bilirubin glucuronidation activity in canine and human primary hepatocytes using a 3D culture system.

Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24 h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs.

Gene editing in porcine embryos using a combination of electroporation and transfection methods.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.

Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes.

BACKGROUND: Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated. METHODS: The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1. RESULTS: The blastocyst formation rate of ZP-free zygotes after electroporation was significantly lower (p < 0.05) than that of ZP-intact zygotes. The mutation rate in blastocysts from ZP-weakened zygotes was similar to that in ZP-intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups. CONCLUSIONS: Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.

Ophthalmic findings in a septic calf with the concurrent exhibition of meningitis and endophthalmitis.

The aim of this study was to evaluate the impacts of ophthalmic findings obtained from both macroscopic examination and ocular ultrasonography when diagnosing bovine endophthalmitis. A newborn crossbreed (Japanese black and Holstein breeds) calf was suspected of visual impairment and central nervous system (CNS) symptoms, such as decreased activity and weak drinking performance. This calf was found to display macroscopic signs, such as clouded lens, convergent strabismus, and horizontal nystagmus, in both eyes. On ocular ultrasonography of both eyes, a V-shaped, thickened, hyperechoic structure was present in the anechoic vitreous humors, indicating retinal detachment. The animal died 4 days after the examination. Sepsis was evident in this case, as Escherichia coli was isolated from multiple organs. The autopsy and histological examination revealed meningitis, encephalitis, and secondary hydrocephalus in the CNS, and endophthalmitis and retinal detachment in both eyes. In this case, the ophthalmic findings did not provide definitive evidence for a diagnosis of endophthalmitis. However, this study indicated that retinal detachment might be an ultrasonographic finding that is suggestive of bovine endophthalmitis.

Vaginal stimulation enhances ovulation of queen ovaries treated using a combination of eCG and hCG.

Follicular changes throughout the oestrous phase have been poorly documented in queens because of the location and the small size of ovaries. We investigated follicular development in queens treated with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and evaluated the effects of vaginal stimulation by a tomcat on ovulation induction. A hormonal treatment was administered using a simple crossover design. Four queens were administered 150 IU of eCG (day 1) and 250 IU of hCG on day 5 and 6. Half of the queens were mated with a vasectomised tomcat for 3 days after hCG injection. Ultrasound imaging of the ovaries clamped at a subcutaneous site was performed once a day from day 1 to 7, and on day 13, and the serum concentrations of oestradiol and progesterone were examined on day 1, 5, 7 and 13. The mean number of follicles gradually increased with the eCG treatment and decreased after hCG injection. The ovulation rate of follicles was significantly higher in the vaginal stimulation group (70.0%) than in the control group (42.6%). During the hormonal treatments, the serum concentration of oestradiol and progesterone did not differ between the two groups. Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that eCG and hCG treatment promoted the follicular growth and corpus luteum formation, respectively. The combination of hCG injection with vaginal stimulation by a vasectomised tomcat enhanced the ovulation rate of follicles.

Interannual variations in abundance and distribution of Chattonella cysts, and the relationship to population dynamics of vegetative cells in the Yatsushiro Sea, Japan.

The fish-killing raphidophytes Chattonella spp. have a resting cyst stage. To investigate the abundance and distribution of Chattonella cysts and determine their relationship to the population dynamics of vegetative cells, we conducted field observations from 2002 to 2017 in the Yatsushiro Sea, a semi-enclosed embayment in Japan, and analyzed the data including environmental conditions. Analysis of sediment sampled in the spring (mid-April to early June), shows that cysts are relatively abundant in the northern to middle area, where initial vegetative cells and large blooms are frequently detected. The maximum density of cysts was 616 cysts cm-3 in the northern area in 2016. Mean cyst abundance in the spring varied interannually, ranging from 5 to 138 cysts cm-3. A significant positive correlation between mean cyst abundance in the spring and maximum density of vegetative cells the preceding summer was seen, but no significant correlation was observed the following summer. The first detected date of vegetative cells (FDD) each year, which is likely related to cyst abundance and environmental conditions influencing cyst germination and/or growth characteristics of vegetative cells, also varied interannually from mid-April to early June. Regression analyses showed that FDD tended to be early when cyst abundance and bottom-water temperature were high. However, no significant correlation was observed between mean cyst abundance and bloom timing (the period from FDD to the occurrence date of the bloom), and bloom duration the following summer, as was the maximum density of vegetative cells. Instead, the timing and duration of blooms were correlated significantly with meteorological factors (e.g., solar radiation) for a month after FDD. The results suggest that cyst abundance reflecting the bloom magnitude of the preceding summer contributes to the timing of the appearance of vegetative cells in the year, but that bloom occurrence is likely to be controlled by the growth dynamics of vegetative cells through environmental conditions rather than by cyst abundance. The three distinct peaks in Chattonella cysts and vegetative cells from 2002 to 2017 correspond to the timings just after the El Niño. Large-scale atmospheric variability and its global teleconnection are possibly linked to long-term population dynamics of Chattonella in the area through local meteorological conditions and their life cycle.