Prematurational Culture with 3-Isobutyl-1-methylxanthine Synchronizes Meiotic Progression of the Germinal Vesicle Stage and Improves Nuclear Maturation and Embryonic Development in In Vitro-grown Bovine Oocytes

The objective of this study was to clarify the effects of prematurational culture (pre-IVM) supplemented with 3-isobutyl-1-methylxanthine (IBMX) on nuclear and cytoplasmic maturation of in vitro-grown bovine oocytes. In experiment 1, oocytes (95 μm in diameter) derived from early antral follicles (0.5–1 mm in diameter) were cultured for 12 days for in vitro growth (IVG). IVG oocytes with a normal appearance were subjected to examinations of diameter and chromatin structure in the germinal vesicle (GV) before IVM. In addition, percentages of metaphase II (M II) were examined after IVM. Regardless of pre-IVM, the mean diameters of IVG oocytes were about 115 μm. The proportions of GV3 (50.0%) and M II stages (80.1%) of IVG oocytes with pre-IVM were higher than those without pre-IVM (28.0 and 49.4%, respectively). In experiment 2, the fertilizability and developmental competence of IVG oocytes were examined. Regardless of pre-IVM, the normal fertilization rates of IVG oocytes were similar (around 70%) but were lower than that of in vivo-grown oocytes (88.0%). Cleavage and blastocyst rates of IVG oocytes with pre-IVM (63.0 and 26.1%, respectively) were higher than those without pre-IVM (45.8 and 12.7%, respectively). The blastocyst rate based on cleaved IVG oocytes with pre-IVM (41.7%) was similar to that of in vivo-grown oocytes (48.7%), although the cleavage rate of IVG oocytes with pre-IVM was lower than that of in vivo-grown oocytes. In conclusion, pre-IVM with IBMX improved the maturational and developmental competences of IVG oocytes, probably due to promotion of their chromatin transition and synchronization of meiotic progression.     

Effect of postactivation treatment with latrunculin a on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged clawn miniature boar

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.     

A retrospective study of inflammatory colorectal polyps in miniature dachshunds

Medical records of dogs with colorectal polyps were retrospectively reviewed, and clinical presentation of inflammatory colorectal polyps in miniature dachshunds was evaluated. Of 33 dogs found to have colorectal polyps, miniature dachshunds were markedly over-represented with 16 dogs (48%), of which 12 (75%) were found to have inflammatory polyps. Multiple polyps localized between the rectum and the descending colon was the most common finding in miniature dachshunds with inflammatory polyps. Twenty dogs (80%) out of 25 miniature dachshunds with inflammatory colorectal polyps responded to immunosuppressive therapy using prednisolone and cyclosporine. The results of this study indicate that miniature dachshunds are predisposed to develop inflammatory colorectal multiple polyps, for which immunosuppressive therapy may be a treatment option.     

Concentrations and specific antibodies to staphylococcal enterotoxin-C and toxic shock syndrome toxin-1 in bovine mammary gland secretions,and inflammatory response to the intramammary inoculation of these toxins

To investigate the pathological role of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) in bovine mastitis, the production of SEs and TSST-1 was investigated in staphylococci isolated from 120 mammary gland secretions (MGS, 51 from no clinical sign-mammary glands and 69 from staphylococcal mastitic-mammary glands) collected from dairy farms where staphylococcal mastitis frequently occurred in Miyagi and Yamagata prefectures from 1997 to 1998. Concentrations of these toxins and specific antibody titers in each MGS were also measured. Furthermore, SEC and TSST-1 were inoculated into lactating mammary glands and inflammatory responses were analyzed. A high percentage of staphylococci including Staphylococcus aureus and coagulase-negative staphylococci isolated from both no clinical sign- and mastitic-MGS produced both SEC and/or TSST-1. The concentration of SEC increased with the severity of the mastitis, and was significantly higher (P<0.05) in acute mastitic-than in no clinical signs-MGS. Titers of specific antibodies to TSST-1 in MGS were significantly higher (P<0.05) than those to SEC, regardless of whether or not the cows were lactating or mastitic. Specific antibodies purified from MGS neutralized each toxin in vitro. A significant increase (P < 0.05) in somatic cell counts was induced by the intramammary inoculation of SEC but not TSST-1. These findings indicated that SEC rather than TSST-1 plays an important role in the pathology of staphylococcal bovine mastitis. The inflammatory activity of TSST-1 was probably neutralized by specific antibodies in MGS.     

Development of X-ray computed tomography for live standing cattle

An X‐ray computed tomography (CT) system for live standing cattle was developed for studying the meat yield, carcass composition and so on. The gantry contained three X‐ray tubes and detectors that corresponded to each X‐ray tube. The system was able to operate while the animal remained standing. The scan area had a diameter of 900 mm. The Musculus longissimus and Musculus trapezius areas, and the back fat thickness in the CT image were evaluated and compared to the actual cross‐section of the carcass using eight cattle. The differences among the muscles, and the subcutaneous and intermuscular fat were easily recognized. The correlation coefficient between the CT image and the actual carcass photograph of the M. longissimus area and the back fat thickness was high (r = 0.84, r = 0.93, P < 0.01). The present study demonstrated that muscle, fat and bone can be clearly imaged from a live standing animal using X‐ray equipment.     

Physiological Races and Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae Isolated from Crisphead Lettuce in Japan

One hundred and sixteen isolates of Fusarium oxysporum f. sp. lactucae obtained from 85 fields in three crisphead lettuce-producing areas in Nagano Prefecture, Japan were typed for races using differential cultivars Patriot, Banchu Red Fire and Costa Rica No. 4. They were also grouped into vegetative compatibility groups (VCGs) using complementation tests with nitrate non-utilizing (nit) mutants. Two California strains reported as F. oxysporum f. sp. lactucum, a type culture of F. oxysporum f. sp. lactucae, and 28 avirulent isolates of F. oxysporum obtained from crisphead lettuce were included for comparison. Among Nagano isolates, 66 isolates were identified as race 1, and 50 as race 2. Race 1 strains derived from Shiojiri and Komoro cities and race 2 from Kawakami village and Komoro city. All isolates of race 2 were biotin auxotrophs, and the race could be distinguished based on its requirement for biotin on minimal nitrate agar medium (MM). Pathogenic isolates were classified into two VCGs and three heterokaryon self-incompatible isolates. Strong correlations were found between race and VCG. All the race 1 strains were assigned to VCG 1 except self-incompatible isolates, and all the race 2 strains to VCG 2. The 28 avirulent isolates of F. oxysporum were incompatible with VCG 1 and VCG 2. California strains was vegetatively compatible with VCG 1, and they were assigned to race 1. Based on vegetative compatibility, these two races of F. oxysporum f. sp. lactucae may be genetically distinct, and F. oxysporum f. sp. lactucae race 1 is identical to F. oxysporum f. sp. lactucum. Received 7 May 2002/ Accepted in revised form 6 September 2002     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002