Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

Development of a plant conveyance system using an AGV and a self-designed plant-handling device: A case study of DIY plant phenotyping

Plant phenotyping technology has been actively developed in recent years, but the introduction of these technologies into the field of agronomic research has not progressed as expected, in part due to the need for flexibility and low cost. “DIY” (Do It Yourself) methodologies are an efficient way to overcome such obstacles. Devices with modular functionality are critical to DIY experimentation, allowing researchers flexibility of design. In this study, we developed a plant conveyance system using a commercial AGV (Automated Guided Vehicle) as a case study of DIY plant phenotyping. The convey module consists of two devices, a running device and a plant-handling device. The running device was developed based on a commercial AGV Kit. The plant-handling device, plant stands, and pot attachments were originally designed and fabricated by us and our associates. Software was also developed for connecting the devices and operating the system. The run route was set with magnetic tape, which can be easily changed or rerouted. Our plant delivery system was developed with low cost and having high flexibility, as a unit that can contribute to others’ DIY’ plant research efforts as well as our own. It is expected that the developed devices will contribute to diverse phenotype observations of plants in the greenhouse as well as to other important functions in plant breeding and agricultural production.     

All-around 3D plant modeling system using multiple images and its composition

In this study, we developed an all-around 3D plant modeling system that operates using images and is capable of measuring plants non-destructively without any contact. During the fabrication of this device, we selected a method capable of performing 3D model reconstruction from multiple images. We then developed an improved SfM-MVS (Structure from Motion / Multi-View-Stereo) method that enables 3D reconstruction by simply capturing images with a camera. The resulting image-based method offers a high degree of freedom because the hardware and software can comprise commercially available products, and it permits the use of one or more cameras according to the shape and size of the plant. The advantages of the image-based method are that 3D reconstruction can be conducted at any time as long as the images are already taken, and that the desired locations can be observed, measured, and analyzed from 2D images and a 3D point cloud. The device we developed is capable of 3D measurements and modeling of plants from a few millimeters to 2.4 m of height using this method. This article explains this device, the principles of its composition, and the accuracy of the models obtained from it.     

Fruit antioxidant activity, ascorbic acid, total phenol, quercetin, and carotene of Irwin mango fruits stored at low temperature after high electric field pretreatment

Greenhouse-grown tree ripe (TR) and mature green (MG) mangoes (cv. Irwin) were exposed to high electric field treatment before 20 and 30 days of storage at 5 degrees C. MG fruits were allowed to ripen at room temperature after low-temperature storage. Fruit physical quality attributes, ascorbic acid, carotene, quercetin, total phenols, and antioxidant capacity were estimated before and after the storage period. Antioxidant capacity of fruit juice was estimated using the ferric reducing antioxidant power (FRAP) assay. Fruit firmness decreased significantly during storage. Titratable acidity decreased 20 days after storage. Total soluble solids did not change during storage. Antioxidant capacity of fruits remained unchanged up to 20 days of storage period and decreased thereafter. Total phenol and carotenes increased during storage. Antioxidant capacity of fruits was significantly correlated only to ascorbic acids. Peel color and carotenes were higher in TR fruits, whereas titratable acidity and firmness were higher in MG fruits. There was no significant difference in other parameters between the stages of picking. Electric field pretreatment affected the respiration and antioxidant capacity of TR fruits and did not have any significant affect on other parameters. TR mangoes of cv. Irwin are more suitable for low-temperature storage and can be successfully stored for up to 20 days at 5 degrees C without any significant losses in functional properties and quality attributes.     

DNA polymorphism of srRNA gene among Eimeria tenella strains isolated in Japan

DNA polymorphism in twelve starains of Eimeria tenella isolated from various places in Japan was examined using 1.l kb small subunits ribosomal RNA amplified by PCR. Genetic variation was evaluated by random amplification of polymorphic DNA (RAPD) analysis. DNA fingerprint patterns were grouped into two, indicating that at least two DNA polymorphisms exist in Japanese E. tenella strains.     

Effects of body condition score in cows peripartum on the onset of postpartum ovarian cyclicity and conception rates after ovulation synchronization/fixed-time artificial insemination

The aim of this study was to examine whether the nutritional state of cows peripartum was associated with the recovery of ovarian function and conception rates after synchronization of ovulation and fixed-time artificial insemination (OVSYNCH/TAI). The effect of the interval in days from calving to the first ovulation on conception rates after OVSYNCH/TAI was also investigated. Conception rates of cows after OVSYNCH/TAI (n=39) were 43.6%. The conception rates of cows with a body condition score (BCS) of 2.75-3.25 at 30 d postpartum and on the day of OVSYNCH treatment were significantly higher than in cows with a BCS < or =2.5 (P<0.05). The percentage of cows establishing ovarian cyclicity before 55 d postpartum in cows with a BCS of 2.75-3.25 at 30 d postpartum and on the day of OVSYNCH treatment were significantly higher than in cows with a BCS < or =2.5 (P<0.05). The conception rates after OVSYNCH/TAI in cows which recovered ovarian cyclicity within 34 d postpartum were significantly higher than in cows with first ovulation > or =56 d (P<0.05). These results indicated that the nutritional state in cows peripartum influenced the conception rates after OVSYNCH/TAI and the postpartum ovarian cyclicity and also suggested that the conception rates after OVSYNCH/TAI decreased in cows with delayed recovery of ovarian cyclicity.