Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring

Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.     

Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets

Pheophytinase (PPH) activity and gene expression of chlorophyll (Chl)-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets were determined. PPH is involved in the dephytylation of Mg-free Chl a, pheophytin (Phy) a. However, in vitro chlorophyllase (Chlase, EC.3.1.1.14) also uses Phy a as a substrate to produce pheophorbide (Pheide) a by dephytylation. For an accurate determination of PPH activity, the PPH protein fraction was separated from Chlase protein by ammonium sulfate precipitation. The protein precipitated by 45-60% saturated ammonium sulfate included a little bit of Chlase activity and was suitable for PPH determination. PPH activity in broccoli florets treated with a UV-B dose of 19 kJ m⁻² was repressed for the first 2 d of storage at 15 °C, whereas it increased gradually with senescence of control broccoli florets. The expression level of BoCLH1 was reduced in broccoli florets on day 4 of storage, while BoCLH2 and BoCLH3 were up-regulated with UV-B treatment. A high BoPAO expression level was found in senescent broccoli florets, and the up-regulation of this gene was delayed by UV-B treatment. The highest expression level of BoPPH was found in the control, and its expression was clearly repressed by UV-B treatment on day 2 of storage. We suggest that the up-regulation of Chl-degrading enzyme genes could be delayed by UV-B treatment, resulting in the suppression of floret yellowing in stored broccoli.     

Carcinogen-induced Thyroid Proliferative Lesions in Wistar Hannover GALAS Rats with Thyroid Dysplasia

Incidences and morphological features of thyroid proliferative lesions induced by carcinogens in Wistar Hannover GALAS rats (GALAS rats) showing normal growth with or without thyroid dysplasia were examined. All thyroid tissue samples were obtained from our recently conducted study using male GALAS rats treated with 5 carcinogens according to the medium-term multiorgan carcinogenicity bioassay protocol (called DMBDD treatment). In the DMBDD-treated rats, thyroid dysplasia was found in 9 out of 114 rats. Follicular cell adenomas were found in 5 out of 9 rats with thyroid dysplasia and in 7 out of 105 rats without thyroid dysplasia. The incidence of adenoma was significantly increased in rats with thyroid dysplasia (55.6%) compared with that in rats without thyroid dysplasia (6.7%). Adenomas in rats with thyroid dysplasia were observed as single or multiple nodules, well demarcated and composed of variously sized vacuolated cells or unvacuolated cells. These histopathological features and staining profiles of luminal colloid for PAS and thyroglobulin, together with PCNA-positive cells, were fundamentally similar to those of rats without thyroid dysplasia. On the other hand, the luminal colloid in adenomas of rats with thyroid dysplasia had a tendency to be poorly stained for T(4) compared with that of rats without thyroid dysplasia. From these findings, it appears that dysplastic thyroids of rats showing normal growth are more sensitive to carcinogens than normal thyroids. In addition, the morphological features of carcinogen-induced thyroid proliferative lesions in GALAS rats with thyroid dysplasia were fundamentally similar to those of rats without thyroid dysplasia, except for the vacuoles and T(4) staining profile.     

Sesquiterpenes from the aerial part of Chloranthus japonicus and their cytotoxicities

Four new sesquiterpenes, chlorajapolides F–I (1–4), along with nine known terpenoids (5–13) were isolated from the aerial part of Chloranthus japonicus. Their structures were elucidated on the basis of spectroscopic analysis, and a lindenane sesquiterpene, named 9-hydroxy-heterogorgiolide, previously isolated from the C. japonicus, was revised as its 8-epimer (1a). Moreover, methanol extract (ME), EtOAc fraction (EF), water fraction (WF), and all isolates (1a, 1–13) were evaluated for their cytotoxicities using two human cancer cell lines.     

Clearance of Theileria sergenti-infected bovine red blood cells in severe combined immune deficiency mice

Clearance of Theileria sergenti-infected bovine red blood cells (Bo-RBCs) from the blood circulation of severe combined immune deficiency (SCID) mice was studied to help understand the mechanisms of anemia developing in cattle infected with T. sergenti. For the clearance test, Bo-RBC samples having 2%, 58%, and 76% parasitemia and, as a control, parasite-free Bo-RBCs were prepared in the Bo-RBC-SCID mouse model. The T. sergenti-infected Bo-RBCs and the uninfected control Bo-RBCs were separately labeled with two, green and red, fluorescent dyes, mixed together, and injected intravenously into SCID mice. The blood samples collected at various time points were observed under a fluorescent microscope, and the numbers of green and red fluorescing RBCs were counted differentially to determine the clearance rates of T. sergenti-infected and uninfected Bo-RBCs. This test clearly demonstrated that the Bo-RBC samples having higher parasitemias were cleared faster from the blood circulation of SCID mice. The results suggest that the intravascular clearance system in SCID mice may have a mechanism by which T. sergenti-parasitized and non-parasitized Bo-RBCs are recognized and cleared differentially.     

Three-dimensional live imaging of bovine embryos by optical coherence tomography

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.     

Interspecific hybrids betweenAllium fistulosumandAllium schoenoprasumreveal carotene-rich phenotype

Interspecific hybridization is an effective method to generate a new crop that gains available functions in a short time. Interspecific hybrids (2n= 16) betweenAllium fistulosumL. (2n= 16) andAllium schoenoprasumL. (2n= 16) were produced by reciprocal crossings through ovary culture, but the hybrids were much fewer in the combination usingA. schoenoprasumas a seed plant. All the hybrids have eight long chromosomes originated fromA.fistulosumand eight short chromosomes originated fromA. schoenoprasum. In addition, the hybridity was confirmed by randomly amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequence (CAPS) analysis of the rDNA internal transcribed spacer (ITS) region. The interspecific hybrids showed a vigorous growth habit; their foliage was slightly bloomy and deep green. The hybrids did not form bulbs, but rather propagated vegetatively by tillering. Carotene contents of the hybrids and both parents were quantified using high-performance liquid chromatography (HPLC). The contents of all edible parts of the hybrids were approximately seven times higher than those of either parent. These results indicate that the hybrid is a new and carotene-rich vegetable ofAlliumspecies.     

First report of Helleborus net necrosis virus isolated from hellebores with black death syndrome in Japan

Helleborus net necrosis virus (HeNNV) in hellebores (Helleborus spp.) has been detected for the first time in Japan. Infected plants had black streaks on fresh leaves and black spots on sepals, which resembled the symptoms of black death disease. The morphology of the virus particles isolated from infected plants was comparable to that of carlaviruses. RT-PCR analysis showed that the entire HeNNV genomic sequence isolated in Japan shared 99% identity with that of HeNNV previously reported in the United States.     

Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna of Japan.     

Three-layered Feedforward artificial neural network with dropout for short-term prediction of class-differentiated Chl-a based on weekly water-quality observations in a eutrophic agricultural reservoir

To effectively manage a eutrophic reservoir, short-term predictions of algae class-differentiated chlorophyll a (Chl-a) were conducted. The study adopted a three-layered feedforward artificial neural network using discrete water environment datasets collected through weekly observations from May to November over seven years (2012–2018). This network was constructed using supervised learning, and the available datasets of a certain observation day were set as the input variables to determine the total Chl-a, Chlorophyceae Chl-a, and cyanobacteria Chl-a that would exist after one week. From the viewpoint of the simplification of the network’s complexity to suppress overfitting, input variables were carefully selected by identifying the important variables related to the seasonal changes in Chlorophyceae and cyanobacteria and eliminating the duplicated expressions of water-quality parameters. However, network downsizing was found insufficient to suppress overfitting. To improve prediction accuracy, dropout was introduced, which stochastically deactivated some nodes in the input and hidden layers in the learning process. The analysis results showed that sufficient short-term predictions of total Chl-a and Chlorophyceae Chl-a may be achieved. The insufficient prediction accuracy of cyanobacteria Chl-a may be overcome using meteorological data as close as possible to the desired prediction day. Consequently, this model may serve as a useful tool for the management of eutrophic reservoirs because short-term predictions of the dominant phytoplankton can be achieved, and the necessary mitigation measures may be accordingly planned.