Developmental competence of porcine blastocysts produced in vitro

The establishment of in vitro embryo production (IVP) system in pigs enables us to generate viable embryos with a quality equal to that of in vivo derived embryos. This technology contributes not only to developments in reproductive physiology and agriculture but also to biotechnologies for producing cloned or genetically modified pigs. The birth of piglets from in vitro matured and fertilized embryos at the two- to 4-cell stage was first achieved about 10 years ago, but it was only quite recently that piglets were produced after the transfer of IVP blastocysts. This improvement to the blastocyst stage of the in vitro culture system after in vitro maturation and fertilization can be expected to play a part in the development of an advanced IVP system. Here, we discuss the developmental ability of porcine embryos produced by our improved IVP system and the utilization of this technique in the new biotechnology.     

Bovine hepatocyte growth factor and its receptor c-Met: cDNA cloning and expression analysis in the mammary gland

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine that plays a crucial role in the embryonic and postnatal development of various organs including the mammary gland. We cloned bovine HGF and its c-Met receptor cDNAs, and examined their expression during mammary gland development in dairy cows. The 2.5-kbp HGF cDNA clone contained a 2190 bp open reading frame coding a 730 amino acid protein, while the 4.8-kbp c-Met cDNA clone contained a 4152 bp open reading frame coding a 1384 amino acid protein. The bovine HGF and c-Met sequences exhibited more than 87% identity with those of other mammals. RT-PCR analysis revealed ubiquitous expression of both HGF and c-Met mRNAs in various bovine tissues tested. HGF mRNA was detected only in the inactive stage of bovine mammary gland development and not in the developing, lactating, and involuting stages, while c-Met mRNA was detected in the inactive and involuting stages. Immunohistochemical analysis demonstrated that the c-Met protein was found on mammary epithelial cells in the inactive, developing, and involuting stages, and on myoepithelial cells in all stages. These results suggest pivotal roles of HGF and c-Met in the development of bovine mammary gland.     

Magnetic resonance imaging of alveolar echinococcosis experimentally induced in the rat lung

Pulmonary alveolar echinococcosis (AE) caused by the metacestode of Echinococcus multilocularis is a lethal zoonosis and is a lesion secondarily induced by hematogenous dissemination from hepatic AE lesions. In the present study, a hematogenous pulmonary AE model was experimentally induced in rats by the injection of echinococcal larval tissue homogenate to the tail vein, and then the pathological and diagnostic aspects of pulmonary AE were examined by magnetic resonance imaging (MRI). Histological primary, mature and degenerated AE lesions were observed 5, 18 and 50 weeks after injection, respectively. These lesions were discriminated as signal-void, hypointense and hyperintense regions in T1-weighted MRI (T1WI), respectively. The change in signal intensity in T1WI might reflect the content of proteinaceous fluid as a result of AE cyst degeneration. Western blot analysis of sera with antibodies of two epitopes (Em18 and Em16) of E. multilocularis provided evidence for AE infection in the early stage. T1WI in combination with Western blot analysis could possibility become definitive and early signs of hematogenous pulmonary AE infection.     

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

Identification and functional analysis of alleles for productivity in two sets of chromosome segment substitution lines of rice

Using two sets of chromosome segment substitution lines (CSSLs) of crosses between cvs. ‘Koshihikari’ and ‘Kasalath’ (Ko/Ka) and between ‘Koshihikari’ and ‘Nona Bokra’ (Ko/NB), respectively, we have identified alleles for ten traits related to productivity (e.g., harvest index and biomass) in rice (Oryza sativa L.). A total of 43 chromosome regions affecting traits (CRATs) in Ko/Ka CSSLs and 40 in Ko/NB CSSLs were detected. Among them, ten and 18 CRATs in Ko/Ka and Ko/NB CSSLs, respectively, had positive effects. A CRAT for harvest index (HI) with a positive allele from ‘Kasalath’ on chromosome 6 (tentatively named HI6) increased the HI by 25 % relative to ‘Koshihikari’, raising it to the theoretical upper limit in rice (0.6). Functional analysis using CSSLs with HI6 indicated that HI6 reduced the size of the lower parts of the plant, which is not important for production, while maintaining the size of the other organs related to production (e.g., flag leaf and panicle), resulting in improved nitrogen (N) use efficiency. These results suggest an ‘ideal plant type’ with improved N use efficiency that can sustain higher yields. A CRAT for the SPAD (soil plant analysis development) value, which is a chlorophyll meter value commonly used as an indicator of leaf N content and strongly associated with the source ability of a leaf, with a ‘Nona Bokra’ allele on chromosome 4 increased the value by 13 % relative to ‘Koshihikari’ with no loss of leaf area. These CRATs can be used for the improvement of rice productivity.     

Peripartum back fat thickness of multiparous Holstein-Friesian cows with displacement of the abomasum or ketosis

To establish a method to predict postpartum diseases using prepartum back fat thickness (BFT), the peripartum BFTs of 54 healthy multiparous cows before calving, which were diagnosed with postpartum displacement of the abomasum (DA), clinical ketosis or subclinical ketosis were compared with those of healthy cows from 8 weeks before the expected calving date to 8 weeks after calving. The peripartum BFTs of the cows with DA or clinical ketosis were significantly higher than those of healthy cows. The peripartum BFTs of the cows with subclinical ketosis were not significantly higher than those of the healthy cows.     

Mating behavior increases workload of the heart in Thoroughbred stallions

To evaluate the influence of mating behavior on cardiac function, changes in heart rate (HR), electrocardiogram (ECG), hematocrit (Hct) and serum concentration of alpha-atrial natriuretic peptide (alpha-ANP) were evaluated in 10 clinically sound Thoroughbred stallions before and after mating behavior. The stallions were submitted twice to experimental pseudomating in the same month in 2009 and 2010. Measurements and blood samples were collected at a stable before mating (baseline) and at a covering yard before and after mating. ECG was recorded by a Holter-ECG system. Arrhythmias were detected in 5 stallions before or after mating behavior. Minimum HR (HRmin), maximum HR (HRmax) and HR recorded when the stallions entered into yard (HRent) and ejaculated (HRejc) were 34.2 ± 3.7, 168.9 ± 14.2, 141.8 ± 35.3 and 142.6 ± 27.3 beats/min, respectively. Time from entrance into the yard to ejaculation (mating time; MT) ranged from 30 to 2,103 sec and was highly correlated with HRent (r=-0.82) and the time required for attaining HRmax after entrance into the yard (dT HRmax) (r=0.87). Hct and serum alpha-ANP concentration significantly increased after ejaculation (60.0 ± 3.2%, P<0.0001, and 1.54 ± 0.61 ng/ml, P=0.0353) compared with the baselines values (46.9 ± 4.4%, 1.40 ± 0.60 ng/ml). HRent and Hct were significantly higher in the stallions with an MT of less than 5 min (n=5) compared with those (n=5) with an MT of more than 5 min (P=0.0324 and P=0.0082). Mating behavior increases the workload of the heart in Thoroughbred stallions.     

Influence of precipitations and sunshine hours on yield of paddy field grown wheat (Triticum aestivum L.) in Northern Kyushu,Japan

ABSTRACT

Relationships between wheat (Triticum æstivum L.) yield components and growing season weather conditions (e.g. temperature, precipitation, and sunshine duration) were investigated for a rice-wheat rotation paddy system situated in Northern Kyushu, Japan over a 17-year period (2000–2016). A 1.0 mm increase in precipitation from 21 to 32 days after sowing decreased wheat yield by about 27 kg ha^?1 at early seedling stage with one to two leaves (Zadoks (Z) growth stage Z1.1–1.2). Number of tillers and spikes showed significant negative correlations to the amount of precipitation during the period. An hour increase in mean daily sunshine hours from 94 to 111 days after sowing resulted in an 328 kg ha^?1 rise in grain yield at stem elongation stage with the first and second node detection (Z3.1–3.2). The grain number per square meter also showed strong positive correlations to sunshine hours during the same period. However, this positive effect of sunshine was negated when precipitation exceeded about 30 mm during the early seedling stage. Consequently, precipitations at early seedling stage and sunshine hours at stem elongation stage mostly determined wheat yield in Northern Kyushu paddy field. Increasing precipitations during the early seedling stage highlights the pressing need for effective paddy field drainage management from wheat sowing through harvest.

Abbreviations: CP: Chikushi Plains; FARC: Fukuoka prefectural Agriculture and Forestry Research Center; SARC: Saga prefectural Agricultural Research Center; DAS: days after sowing