Effect of β‐Carotene Supply During Close‐up Dry Period on the Onset of First Postpartum Luteal Activity in Dairy Cows

The aim of this study was to examine the effect of β‐carotene supply during the close‐up dry period on the onset of first postpartum luteal activity in dairy cows. Twelve cows were supplied with 2000 mg of β‐carotene (20 g Rovimix^®β‐Carotene containing 10%β‐carotene; DSM Nutrition Japan K.K., Tokyo, Japan) by oral administration daily from day 21 before expected calving date to parturition. Fourteen cows (control) did not receive β‐carotene supplementation. Blood samples were obtained on days 21, 14 and 7 before expected calving date and on days 1, 7, 14, 21 postpartum. When the plasma progesterone concentration exceeded 1 ng/ml by day 21 postpartum, luteal activity was assumed to have been initiated. The result showed that serum β‐carotene concentrations in the β‐carotene cows were higher than in the control cows during the experimental period (p < 0.01). The number of cows with the onset of luteal activity by day 21 postpartum was 9/12 in the β‐carotene cows and 4/14 in the control cows (p < 0.05). Retinol, certain metabolic parameters and metabolic hormones concentrations did not differ between β‐carotene and control cows. In addition, serum retinol concentration in β‐carotene cows without luteal activity was lower than in β‐carotene cows with luteal activity (p < 0.05), and serum gamma‐glutamyl transpeptidase concentration in β‐carotene cows with luteal activity (p < 0.05) and control cows without luteal activity (p < 0.01) was higher than in control cows with luteal activity. In conclusion, β‐carotene supply during the close‐up dry period may support the onset of luteal activity during early lactation in dairy cows.     

Hemodynamic and Electrocardiographic Effects of Magnesium Sulfate in Healthy Dogs

We investigated the effects of graded dosages of magnesium given i.v. to anesthetized dogs on blood pressure, cardiac output, and electrophysiology. Magnesium was infused at 0.12 mEq/kg/minute until ventricular fibrillation occurred naturally or was provoked by programmed electrical stimulation or until arrest of the sinuatrial node in 8 dogs. Plasma total magnesium concentrations doubled in 1 minute of that infusion rate, and a hemodynamically safe plasma concentration of 12.2 mEq/L was achieved after 16 minutes of infusion. Heart rate, inotropy, lusitropy, and cardiac output increased up to a cumulative infusion dosage of magnesium of 1.0-2.0 mEq/kg, which produced plasma magnesium concentrations of 8.5-12.2 mEq/L (n = 5). Above the cumulative infusion dosage, inotropy decreased and lusitropy increased until death occurred between cumulative infusion dosages of 5.9 mEq/kg and 10.9 mEq/kg. Arterial pressure and vascular resistance decreased, and PQ interval and QRS complex increased, in a dose-dependent fashion. The relationship between ionized and total magnesium was y = 0.624x - 0.542 (r2 = .986), where y is ionized and x is total magnesium in mEq/L in 3 dogs. In conclusion, a cumulative infusion dosage of 0.1-0.2 mEq/kg of magnesium may be given without changing hemodynamic parameters, but with higher cumulative infusion doses heart rate accelerates. Hemodynamic parameters except those related to blood pressure continued to increase to a cumulative infusion dosage of 2.0 mEq/kg. At higher cumulative infusion dosages dogs became hypotensive and the PQ interval was prolonged. However, dangerous arrhythmias were not provoked until a total dosage of 3.9 mEq/kg.     

Quantitative analysis of <Emphasis Type="Italic">Ichthyobodo salmonis</Emphasis> an ectoparasitic flagellate infecting juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> in hatcheries

Ichthyobodosis caused by the ectoparasitic flagellate Ichthyobodo salmonis is a significant cause of mortality in juvenile chum salmon Oncorhynchus keta reared in hatcheries of northern Japan. The present study established a real-time quantitative polymerase chain reaction assay (qPCR) of I. salmonis ribosomal DNA (rDNA) using SYBR Green. This assay allows monitoring of parasite infections for the epidemiological study and control of ichthyobodosis in hatcheries. qPCR showed high reproducibility for measurements between 1.0 and 1.0 × 10⁸ rDNA copy/μl. There was a significant positive correlation between the number of parasites and the amount of I. salmonis rDNA. A survey using the qPCR assay indicated that infection by I. salmonis was present in 23 of 87 hatcheries; parasite loads were estimated to be between 50 and 750 parasites/g juvenile body weight. These results demonstrate that our qPCR assay enables the surveying of juvenile chum salmon reared in hatcheries for infection by I. salmonis.     

Plasma cell-free DNA in canine lymphoma patients as a novel material for genotyping

Canine lymphoma is a disease with high morbidity and poor long-term prognosis, despite a high response rate to chemotherapy. In this study, we focused on liquid biopsy, in which small amounts of substances from body fluids were analysed, to determine whether cell-free DNA (cfDNA) in the plasma can be used as a biomarker for lymphoma in dogs. We found that 23 patients with lymphoma had significantly higher cfDNA concentrations than the 12 healthy dogs (median 2360 ng/mL versus 299 ng/mL, p < .0001). Polymerase chain reaction for antigen receptor rearrangement (PARR) was also employed using cfDNA from the lymphoma group to investigate whether cfDNA could be used for the detection of genetic clonality of lymphomas, as well as the genomic DNA (gDNA) extracted from an original lesion in each case. The correlation of the PARR results between cfDNA and gDNA was observed in 100% of B-cell lymphomas (10/10), 77.8% of T-cell lymphomas (7/9), and 100% of other types of lymphomas (4/4), respectively. These results indicate that plasma cfDNA levels are increasing in canine lymphoma patients, that cfDNA concentration can be a novel diagnostic tool, and that it can be used as a diagnostic tool for PARR.     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

Expression of mRNA for the angiopoietin-tie system in granulosa cells during follicular development in cows

Recent findings indicate that the changing profile of angiopoietins (ANPT) and their receptor Tie2 are closely associated with development and regression of the vascular network in the cyclic ovary. We previously reported that mRNA expression for the ANPT-Tie system in theca interna changes during bovine follicular development and atresia, and both ANPTs affect steroidogenesis in the preovulatory follicle. The aim of this study was to investigate mRNA expression for ANPT1, ANPT-2 and Tie2 in granulosa cells (GC) during follicular development in the cow. Bovine follicles were classified according to the estradiol-17beta (E(2)) concentration in follicular fluid (FF) as follows: (1) E(2)<0.5, (2) 0.5180 ng/ml FF. Semi-quantitative RT-PCR analysis revealed that the expression of ANPT-1 mRNA was not detected in most of the follicle with E(2)<5 ng/ml (diameter of 5-10 mm), but clearly detected in all follicles with E(2)>5 ng/ml (diameter of >10 mm). The mRNA expression for ANPT-2 was drastically decreased in the follicles with E(2)>5 ng/ml. Tie2 mRNA expression remained unchanged at the different stages of follicular development. The present data show that ANPT-1 becomes predominant in the follicle producing high levels of E(2), indicating the possible switch-over from ANPT-2 (antagonist) to ANPT-1 (agonist). Thus, the result suggests that the ANPT-Tie system in bovine GC may stimulate E(2) secretion rather than angiogenesis in the late stages of follicular development.     

Tumour Necrosis factor-α Receptors are Present in the Corpus Luteum Throughout the Oestrous Cycle and During the Early Gestation Period in Pigs

To determine the physiological significance of tumour necrosis factor‐α (TNFα) in the regulation of luteal functions in pig, this study was conducted to identify the presence of functional TNFα receptors in porcine corpora lutea (CL) throughout the oestrous cycle and the early gestation. The CL were isolated from pigs on days 4, 6, 8, 12 or 15 of the oestrous cycle (n=3; day 0 = oestrus) and days 15, 20 or 25 of gestation (n=3; day 0 = mating). A Scatchard analysis revealed the presence of a high‐affinity binding site for TNFα in all samples (dissociation constant; 2.7 ± 0.51 to 5.8 ± 0.50 nM ). The concentration of TNFα receptors was higher on day 15 of the oestrous cycle than on days 4 and 8 of the oestrous cycle (p < 0.05). Furthermore, TNFα receptor concentrations in the CL on days 15, 20 and 25 of gestation were significantly lower than on day 15 of the oestrous cycle (p < 0.05). On day 9 of the oestrous cycle, exposure of cultured luteal cells to 0.06–60 nM TNFα stimulated prostaglandin (PG) F_2α and PGE₂ secretion in a dose‐dependent manner (p < 0.05). These results indicate that functional TNFα receptors are present in the porcine CL throughout the oestrous cycle and early gestation, and suggest that TNFα plays one or more physiological roles in regulating CL function throughout the oestrous cycle and the early gestation period. In addition, TNFα receptor concentration in the CL of the late luteal stage (day 15) of the oestrous cycle was higher than on the respective day in the early pregnant pig, suggesting that TNFα plays a role in accomplishing luteolysis in the porcine CL.     

Correlation of serum concentration of alpha 1-acid glycoprotein with lymphocyte blastogenesis and development of experimentally induced or naturally acquired hepatic abscesses in cattle.

Changes in serum alpha 1-acid glycoprotein (alpha 1AG) concentration in cattle with hepatic abscesses were observed, and function of alpha 1AG was evaluated, particularly its influence on cellular immune response. Test cattle (n = 4) were inoculated with Fusobacterium necrophorum, control cattle (n = 2) were inoculated with inactivated bacteria, and naturally affected cattle (n = 11) were found in a slaughterhouse. Determination of alpha 1AG was made by use of a single radial immunodiffusion method. The action on lymphocyte blastogenesis was determined by [3H]thymidine incorporation. Cultured lymphocytes from healthy cattle were treated with variable concentrations of alpha 1AG purified from serum obtained from cattle with hepatic abscesses and suppression of blastogenesis stimulated by each of 3 mitogens was measured. In cattle with experimentally induced abscesses, serum alpha 1AG concentration increased for 7 to 10 days after F necrophorum inoculation, its change being parallel to that of sialic acid. High concentration of alpha 1AG was found in naturally affected cattle and was highly correlated to sialic acid concentration. Suppression of lymphocyte blastogenesis in cattle with experimentally induced hepatic abscesses was highly correlated to serum alpha 1-AG concentration.