Ovarian dynamics in progesterone tablet-induced superovulation in goats assessed by magnetic resonance imaging.

Controlled internal drug-releasing (CIDR) devices are commonly used for superovulation in goats. However, such devices are unavailable in some countries, including Japan. In this technical note, we aimed to explore the efficacy of an alternative superovulation protocol using progesterone tablets in goats. We employed intravaginal progesterone tablets (LUTINAS® Vaginal Tablet 100 mg) following a standard superovulation protocol. Additionally, we assessed the ovarian dynamics using 3T-magnetic resonance imaging (MRI) 1 day preceding the progesterone treatment (Day "-1") and 3 days before the end of treatment (Days 11-13). The ovarian monitoring was successfully performed in the short tau inversion recovery T2-weighted images of MRI, and ovulation was confirmed by the disappearance of follicles on Day 13 post-administration of the tablets. Immediately after ovulation, oviduct flushing yielded a substantial number of oocytes (13.5 ± 1.8 oocytes per animal). These findings provide evidence that the administration of progesterone tablets can serve as a viable alternative for inducing. Additionally, our findings suggest that 3T-MRI is a promising alternative to conventional ultrasonography for monitoring ovarian dynamics following superovulation in experimental goats.

Intranodal dynamic contrast-enhanced CT lymphangiography and dynamic contrast-enhanced MR lymphangiography in microminipig.

OBJECTIVES: To evaluate the feasibility and image quality of intranodal dynamic contrast-enhanced CT lymphangiography (DCCTL) and dynamic contrast-enhanced MR lymphangiography (DCMRL) in microminipigs. METHODS: Our institution's committee for animal research and welfare provided approval. Three microminipigs underwent DCCTL and DCMRL after inguinal lymph node injection of 0.1 mL/kg contrast media. Mean CT values on DCCTL and signal intensity (SI) on DCMRL were measured at the venous angle and thoracic duct (TD). The contrast enhancement index (CEI; increase in CT values pre- to post-contrast) and signal intensity ratio (SIR; SI of lymph divided by SI of muscle) were evaluated. The morphologic legibility, visibility, and continuity of lymphatics were qualitatively evaluated using a 4-point scale. Two microminipigs underwent DCCTL and DCMRL after lymphatic disruption and the detectability of lymphatic leakage was evaluated. RESULTS: The CEI peaked at 5-10 min in all microminipigs. The SIR peaked at 2-4 min in two microminipigs and at 4-10 min in one microminipig. The peak CEI and SIR values were 235.6 HU and 4.8 for venous angle, 239.4 HU and 2.1 for upper TD, and 387.3 HU and 2.1 for middle TD. The visibility and continuity of upper-middle TD scores were 4.0 and 3.3-3.7 for DCCTL, and 4.0 and 4.0 for DCMRL. In the injured lymphatic model, both DCCTL and DCMRL demonstrated lymphatic leakage. CONCLUSIONS: DCCTL and DCMRL in a microminipig model enabled excellent visualization of central lymphatic ducts and lymphatic leakage, indicating the research and clinical potential of both modalities. KEY POINTS: • Intranodal dynamic contrast-enhanced computed tomography lymphangiography showed a contrast enhancement peak at 5-10 min in all microminipigs. • Intranodal dynamic contrast-enhanced magnetic resonance lymphangiography showed a contrast enhancement peak at 2-4 min in two microminipigs and at 4-10 min in one microminipig. • Both intranodal dynamic contrast-enhanced computed tomography lymphangiography and dynamic contrast-enhanced magnetic resonance lymphangiography demonstrated the central lymphatic ducts and lymphatic leakage.

Functional Reconstruction of Denervated Muscle by Xenotransplantation of Neural Cells from Porcine to Rat.

Neural cell transplantation targeting peripheral nerves is a potential treatment regime for denervated muscle atrophy. This study aimed to develop a new therapeutic technique for intractable muscle atrophy by the xenotransplantation of neural stem cells derived from pig fetuses into peripheral nerves. In this study, we created a denervation model using neurotomy in nude rats and transplanted pig-fetus-derived neural stem cells into the cut nerve stump. Three months after transplantation, the survival of neural cells, the number and area of regenerated axons, and the degree of functional recovery by electrical stimulation of peripheral nerves were compared among the gestational ages (E 22, E 27, E 45) of the pigs. Transplanted neural cells were engrafted at all ages. Functional recovery by electric stimulation was observed at age E 22 and E 27. This study shows that the xenotransplantation of fetal porcine neural stem cells can restore denervated muscle function. When combined with medical engineering, this technology can help in developing a new therapy for paralysis.

Radiological Arterial Anatomy in Mature Microminipigs as a Pre-clinical Research Model in Interventional Radiology.

PURPOSE: To define the radiological arterial anatomy in mature microminipigs as a pre-clinical research animal model in interventional radiology. MATERIALS AND METHODS: Five female microminipigs (weighing 20.9 ± 2.9 kg) were used in this study. Under general anesthesia, computed tomography (CT) angiography was performed using a 16-slice CT scanner. CT was performed 12 s after initiation of an intravenous injection of 40 ml of nonionic contrast media at 3.0 ml/second using a power injector. The transverse CT angiography images were evaluated using a digital imaging and communication in medicine viewer, and the diameters of the following 41 arteries were measured.: ascending aorta, descending aorta, thoracoabdominal aorta, abdominal aorta, pulmonary artery trunk, both pulmonary, brachiocephalic artery, short common bicarotid, both common carotid artery, subclavian, bronchial, internal mammary, celiac, common hepatic, left lateral hepatic, middle hepatic, left hepatic, gastroduodenal, cranial duodenopancreatic, splenic, left gastric, cranial mesenteric, ileocolic , bilateral colic artery, caudal mesenteric, cranial rectal, renal, both external iliac arteries, internal iliac common trunk, and both internal iliac and femoral arteries. RESULTS: The microminipigs' vascular anatomy was the same as domestic pig anatomy and similar to human anatomy. The diameter of the aorta (ascending to abdominal) was 17.1-7.0 mm, iliac and femoral arteries (internal iliac common trunk to femoral artery): 5.5-3.8 mm, pulmonary arteries: 9.3-14.7 mm, and major first aortic branches (e.g., celiac or brachiocephalic artery): 2.2-9.2 mm. CONCLUSION: This study defined the microminipig arterial anatomy in the trunk.

Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

Spatiotemporal imaging of redox status using in vivo dynamic nuclear polarization magnetic resonance imaging system for early monitoring of response to radiation treatment of tumor.

In general, the effectiveness of radiation treatment is evaluated through the observation of morphological changes with computed tomography (CT) or magnetic resonance imaging (MRI) images after treatment. However, the evaluation of the treatment effects can be very time consuming, and thus can delay the verification of patient cases where treatment has not been fully effective. It is known that the treatment efficacy depends on redox modulation in tumor tissues, which is an indirect effect of oxidizing redox molecules such as hydroxyl radicals and of reactive oxygen species generated by radiation treatment. In vivo dynamic nuclear polarization-MRI (DNP-MRI) using carbamoyl-PROXYL (CmP) as a redox sensitive DNP probe enables the accurate monitoring of the anatomical distribution of free radicals based on interactions of electrons and nuclear spin, known as Overhauser effect. However, spatiotemporal response of the redox status in tumor tissues post-irradiation remains unknown. In this study, we demonstrate the usefulness of spatiotemporal redox status as an early imaging biomarker of tumor response after irradiation using in vivo DNP-MRI. Our results highlight that in vivo DNP-MRI/CmP allowed us to visualize the tumor redox status responses significantly faster and earlier compared to the verification of morphological changes observed with 1.5 T MRI and cancer metabolism (Warburg effect) obtained by hyperpolarized 13C pyruvate MRS. Our findings suggest that the early assessment of redox status alterations with in vivo DNP-MRI/CmP probe may provide very efficient information regarding the effectiveness of the subsequent radiation treatment.

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Dynamic contrast-enhanced computed tomography lymphangiography with intranodal injection of water-soluble iodine contrast media in microminipig: imaging protocol and feasibility.

OBJECTIVES: To evaluate the optimal imaging protocol and the feasibility of intranodal dynamic contrast-enhanced computed tomography lymphangiography (DCCTL) in microminipigs. METHODS: The Committee for Animal Research and Welfare provided university approval. Five female microminipigs underwent DCCTL after inguinal lymph node injection of 0.1 mL/kg of iodine contrast media at a rate of 0.3 mL/min with three different iodine concentrations: group 1, 75 mgI/mL; group 2, 150 mgI/mL; and group 3, 300 mgI/mL. The CT values of the venous angle, thoracic duct (TD), cisterna chyli, iliac lymphatic duct, and iliac lymph node were measured; increases in CT values pre- to post-contrast were assessed as the contrast-enhanced index (CEI). Multi-detector row CT (MDCT) and volume rendering images showing the highest CEI were qualitatively evaluated. RESULTS: The CEI of all lymphatics peaked at 5-10 min. The mean CEI of TD at 10 min of group 2 (193.0 HU) and group 3 (201.5 HU) were significantly higher than that of group 1 (70.7 HU) (p = 0.024). The continuity and overall diagnostic acceptability of all lymphatic system components were better in group 3 (3.6 and 3.0, respectively) than group 1 (2.6 and 1.6) and group 2 (3.0 and 2.6) (p = 0.249 and 0.204). CONCLUSIONS: The optimal imaging protocol for intranodal DCCTL could be dual-phase imaging at 5 and 10 min after the injection of 300 mgI/mL iodinated contrast media. DCCTL provided good images of lymphatics and is potentially feasible in clinical settings. KEY POINTS: • Dynamic contrast-enhanced computed tomography lymphangiography with intranodal injection of water-soluble iodine contrast media showed the highest enhancement of all lymphatics at scan delays of 5 and 10 min. • The optimal iodine concentration for intranodal dynamic contrast-enhanced computed tomography lymphangiography might be 300 mgI/mL. • Intranodal dynamic contrast-enhanced computed tomography lymphangiography provided good images of all the lymphatic system components and is potentially feasible in clinical settings.

Sexual precocity in male microminipigs evaluated immunohistologically using spermatogonial stem cell markers.

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.

Endoscopic Ultrasound-Guided Fine-Needle Injection of Hydrogen Peroxide into the Pancreas: Feasibility and Tolerability Study Using a Survival Porcine Model.

Hydrogen peroxide (H2O2) has been reported to be an effective radiation sensitizer for various cancers. A combination therapy comprising fine-needle injection (FNI) of H2O2 under endoscopic ultrasound (EUS) guidance and chemoradiation might improve treatment outcomes of pancreatic cancer; however, there have been no reports thus far. The aims of this study were to evaluate the feasibility and safety of EUS-FNI of H2O2 into the pancreas using a porcine survival model. EUS-FNI was performed in the pancreas of six pigs, which were randomly divided into three groups based on the solution injected: group 1, 2 mL of sodium hyaluronate (control); group 2, 0.5 mL of H2O2; group 3, 2 mL of H2O2. To evaluate any adverse events, blood tests and computed tomography (CT) were performed before and after FNI, as well as days 3 and 7 subsequently. The pigs were necropsied on day 7. Histologic evaluation was performed according to the criteria for experimental acute pancreatitis. EUS-FNI was successful in all pigs. CT immediately after FNI revealed gas formation in the FNI area in groups 2 and 3. No adverse events were revealed by blood tests and CT. Histologic evaluations revealed pancreatitis scores of 5 and 5 in group 1, 7 and 7 in group 2 and 14 and 15 in group 3. EUS-FNI of H2O2 into the pancreas is feasible; however, it could cause pancreatitis. FNI of H2O2 into only the pancreatic tumor might be ideal in minimizing possible adverse events.

Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection.

OBJECTIVE: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 µg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction. RESULTS: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

Magnetic Resonance Imaging of Ovarian Activity in Microminipigs Showing Normal Estrous Cycles.

We investigated whether magnetic resonance imaging (MRI) might be applicable to evaluation of the ovarian activity of microminipigs. Firstly, using three mature microminipigs, we confirmed ovarian position and morphology by laparotomy or laparoscopy, and then acquired MRI images in various patterns to determine the most suitable condition for the acquisition. Next, using four microminipigs, we performed daily MRI, starting 10 days after ovulation and ending 10 days after the subsequent ovulation, as the starting day of standing estrus was taken as day 0. While the ovarian structure could not be discriminated on T1-weighted imaging, it was possible to confirm the follicles during estrus as hyperintense regions on T2-weighted imaging. With chronological MRI, 3-5 follicles were visible on T2-weighted imaging during the interval from day -2 to day 1, and their size immediately prior to ovulation was 3-5 mm. However, confirmation of the presence of small follicles and the corpus luteum was difficult.

Suppression of influenza virus infection by the orf virus isolated in Taiwan.

Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.

Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody.

CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells with a likely role in cell survival, proliferation and differentiation. In order to study the differentiation activity of porcine CD117 hematopoietic cells in vitro and in vivo we prepared an anti-swine CD117 Mab (2A1) with high specificity for flow-cytometrical analysis. The 2A1 Mab did not recognize mouse or human mast cells suggesting that 2A1 is species-specific. Swine bone marrow (BM) CD117+ cells differentiated in vitro mainly into erythroid and monocyte lineages in the methylcellulose-based colony assay. When the swine BM CD117+ cells were transplanted in vivo into immunodeficient NOG (NOD/SCID/IL-2gc-null) mice, a significant amount of swine CD45+ leukocytes, including CD3 positive T cells, were developed in the mice. These results revealed that the swine BM CD117+ cells possess hematopoietic stem/progenitor activity and when monitored in immunodeficient mice or in vitro they can develop into lymphoid, erythroid, and myeloid cells efficiently with the new monoclonal antibody.

Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.

In the present study, some methodological factors affecting the acrosomal staining of frozen-thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome.

Hyperactivated motility of frozen-thawed spermatozoa from fertile and subfertile Japanese black bulls induced by cyclic adenosine 3',5'-monophosphate analogue, cBiMPS.

This study investigated whether a cyclic adenosine 3',5'-monophosphate (cAMP) analogue, cBiMPS, could induce hyperactivated motility in frozen-thawed Japanese Black bull spermatozoa and compared the ability of spermatozoa to undergo hyperactivation between fertile and subfertile bulls. Frozen-thawed spermatozoa from 3 fertile and 2 subfertile bulls were washed, suspended in BO-Hepes medium and incubated in the presence of 0.1 mM cBiMPS for up to 4 h. At 1-h intervals, the spermatozoa were examined for hyperactivated motility. The proportions of spermatozoa showing a circular swimming pattern with asymmetrical flagellar beating and those showing whiplash beating of flagella to the total number of motile spermatozoa were expressed as C% and W%, respectively. The motile spermatozoa % was barely affected by treatment with cBiMPS or the fertility status of the sperm donor, although it gradually decreased in all sperm samples during the 4-h incubation. In the fertile bulls, the C% was 0% at 0 h of incubation but rapidly increased during the 1-h incubation with cBiMPS. It then decreased slightly towards 4 h concomitantly with a gradual increase in W% towards 4 h. In the subfertile bulls, however, the cBiMPS-induced increase of C% was delayed for 1-3 h, although the incubation time-related changes in mean W% were similar between the fertile and subfertile bulls. In the vehicle controls for cBiMPS, the C% and W% were 0% throughout incubation for all the samples examined. The results suggest that hyperactivation of the flagellum can be induced by the cAMP analogue, cBiMPS, in frozen-thawed Japanese Black bull spermatozoa and that induction of hyperactivation may serve as a useful tool for detection of functional abnormality of spermatozoa from subfertile Japanese Black bulls.

Computer tomography diagnosis of meningoencephalocele in a calf.

A 24-day-old female Holstein calf had a soft, painless fluctuating swelling on the median plane in the frontal region, but did not show any clinical symptoms including neurological signs. Computer tomography (CT) distinctly showed the cyst filled with fluid and part of the encephalon. Hence, this swelling was diagnosed as meningoencephalocele, but not meningocele. The meningoencephalocele was successfully repaired surgically. Meningoencephalocele can thus be easily recognised by CT in a calf.

Japanese black cattle with orotic aciduria detected by gas-chromatography/mass-spectrometry.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

Pericardial mesothelioma in a neonatal calf.

We encountered an extremely rare tumor, a pericardial mesothelioma, in a neonatal calf. The patient calf showed severe abdominal distention, and died immediately after birth. The thoracic cavity was contained a huge heart with a large amount of pericardial fluid. A number of granular and cobblestone-like nodules were dispersed over the epicardium and pericardium. The nodules consisted of papillary proliferations of neoplastic cells, and the neoplasm occasionally showed mesenchymal proliferations. Immunohistochemistry revealed that they had the characteristics of mesothelial cells (cytokeratin-and vimentin-positive), and the neoplasm was diagnosed as mesothelioma.