Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.     

Risk factors for atrial fibrillation during racing in slow-finishing horses

OBJECTIVE: To determine prevalence of atrial fibrillation (AF) immediately after racing among racehorses that finished well behind the winners and examine potential risk factors for AF in these horses. DESIGN: Case-control study. ANIMALS: 39,302 racehorses representing 404,090 race starts in races sanctioned by the Japan Racing Association between 1988 and 1997. PROCEDURE: Horses that finished > or = 4 (turf races) or 5 (dirt races) seconds behind the winner or that did not complete the race were examined for AF within 5 minutes after the race. Logistic regression and chi2 analyses were used to determine whether sex, age, race distance, race surface, year, or development of epistaxis was associated with development of AF. RESULTS: Estimated minimum frequency of AF was 0.03% (123 instances of AF following 404,090 race starts), and estimated minimum prevalence of AF among racehorses was 0.29% (115 horses with AF among 39,302 racehorses). Estimated frequency of AF among horses that finished slowly or did not finish was 1.39% (120 instances of AF among 8,639 examinations), and estimated prevalence of AF in horses that finished slowly was 1.23% (92 instances of AF among 7,500 horses) or 1.01% when only the first time a horse finished slowly was considered (76 instances of AF among 7,500 horses). Atrial fibrillation was paroxysmal in most horses. Among horses that finished slowly, 4-year-old and older horses and horses that raced on turf were more likely to develop AF. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the likelihood of AF among racehorses that finish slowly is related to age and racing surface.     

Water Movement Characteristics in a Multi-Soil-Layering System

The Multi-Soil-Layering (MSL) system consists of soil units arranged in a brick-like pattern that are surrounded by layers of zeolite or alternating particles with a homogeneous size that allow a high hydraulic loading rate. Characteristics of the changes in the water movement, hydraulic retention time (HRT) and system weight during the wastewater treatment in the MSL system were investigated using a laboratory-scale MSL system (D10 × W50 × H73 cm). As the loading rate increased from 1,000 to 5,500 L m⁻² d⁻¹, wastewater preferentially flowed into the permeable layers in the MSL, which decreased the contact of the wastewater with the soil mixture layers. HRT was inversely related to the loading rate. HRT decreased from 20 to 1 h, as the loading rate increased from 250 to 6,000 L m⁻² d⁻¹. As an indicator of the system condition, the weight variation of the system was determined during the wastewater treatment. When the weight was stable, input and output of wastewater and decomposition of organic matter appeared to be equilibrated. When the weight increased, the system started to clog. Due to clogging, the efficiency of COD and phosphorus removal decreased, while the efficiency of nitrate removal increased.     

Effects of yield-related QTLs SPIKE and GPS in two indica rice genetic backgrounds

Recent progress in rice genomics has promoted the identification of quantitative trait loci (QTLs) associated with yield and its related traits. SPIKE, a QTL controlling spikelet number per panicle, and GPS, a QTL controlling leaf photosynthesis rate, were identical to NAL1. To assess the effect of SPIKE/GPS on yield potential, we compared DNA sequences of these alleles and conducted yield experiments in the field of Japan using the near-isogenic lines NIL-SPIKE (allele from Daringan in IR 64 genetic background), NIL-GPS (allele from Koshihikari in Takanari genetic background), and a chromosome segment substitution line, SL2115 (allele from Koshihikari in IR 64 genetic background). Despite the two SNPs in the promoter regions between Koshihikari and Daringan, both alleles were effective to increase the number of spikelets per panicle both in Takanari and IR 64 backgrounds. However, the extent of the increase was smaller and unstable in Takanari background than that in IR 64 background. In addition, SPIKE/GPS improved percentage of filled spikelets only in the IR 64 background. These results suggest that the effects of SPIKE/GPS alleles are similar but are affected by the difference of the genetic backgrounds. Because the increasing effect of spikelets number per panicle was canceled by the decrease of the number of panicles, which seems to be affected by environmental factors, none of NIL-SPIKE, SL2115, or NIL-GPS significantly out-yielded their parental cultivars. These results indicate the importance to consider genetic backgrounds and QTL-environment interaction toward the future use of SPIKE/GPS.     

Usage of putative chicken U6 promoters for vector-based RNA interference

Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA.     

Association between race history and risk of superficial digital flexor tendon injury in Thoroughbred racehorses

OBJECTIVE: To determine whether race history, including the number of races and total race distance, was associated with risk of superficial digital flexor tendon (SDFT) injury in Thoroughbred racehorses in Japan. DESIGN: Matched case-control study. ANIMALS: 515 Thoroughbred racehorses (case horses) that sustained an SDFT injury during training or racing in Japan during 2002 and 951 horses (control horses) without SDFT injury that were matched with case horses on the basis of age and month of the latest race. PROCEDURE: Variables related to race history were compared between case and control horses by means of conditional logistic regression. RESULTS: The odds of SDFT injury increased as mean race distance and mean body weight at race time increased. Compared with females that had never competed in steeplechase races, males regardless of steeplechase race history and females that had competed in steeplechase races had higher odds of SDFT injury. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that longer mean distance per race, heavier mean body weight at race time, steeplechase experience, and sex (male) increased the risk of SDFT injury in Thoroughbred racehorses.     

Pulsed-field gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp. enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm

Salmonella enterica serovar subsp. enterica Livingstone and serovar Cerro isolates from a commercial egg-producing farm, which had previously been untypeable by pulsed-field gel electrophoresis (PFGE) because of DNA degradation during the PFGE process, successfully gave banding patterns using electrophoresis buffer supplemented with 50 microM thiourea. By PFGE in the presence of thiourea, DNA degradation-sensitive S. enterica serovar Cerro isolates from the commercial egg-producing farm were found to be genetically unrelated to S. enterica serovar Cerro isolates that gave the patterns in the absence of thiourea. Forty-five of 50 (90%) S. enterica serovar Livingstone isolates from the farm showed arbitrarily designated XbaI-digested patterns X1 and X2 that were distinguished by one-band difference and had an identical BlnI-digested pattern. In one of the two layer houses in the farm, the numbers of isolates having the pattern X2 increased from 57% in 1997 to 89% in 1998, whereas virtually all the isolates obtained from the other house in the same period showed the profile X1. This suggests that strains having the pattern X2 might have an advantage to preferentially colonize in the former house.     

Changes of biomechanical characteristics of the bone in experimental tibial osteotomy model in the dog

Experimental osteotomy model of canine tibia was prepared to investigate the changes in biomechanical characteristics during the healing process. After 16 weeks, although radiographs revealed that fracture healing proceeded, the recovery rates of the mechanical parameters ranged from 12.3 to 47.3%, compared to the intact side. After 32 weeks, those recovered to 68.9-93.2%. These results suggested that the biomechanical characteristics of the healing bone could not be recovered sufficiently even after the passage of the healing period which has been empirically proposed from clinical findings. It was also considered that evaluation of the mechanical parameters using this osteotomy model would be suitable for investigating the effect of osteoinductive growth factors on fracture healing.     

Identification of Equine Subclinical Lameness Induced by Pressure to the Sole of Fore- or Hindlimb

Lameness has been investigated in past studies with kinematic analyses, and the features of lameness have been qualitatively and quantitatively analyzed. The output was, however, limited to visible lameness that was scored by experienced clinicians. Subclinical supporting lameness that was induced by subtle pressure to the sole of limb, but that could not be detected by visual check, has yet to be extensively analyzed. To find specific kinematic parameters that were influenced by such subclinical lameness of the fore- or hindlimb, we produced subclinical, subtle and mild lameness in five sound horses by applying slight pressure to the sole. Trot on a treadmill was recorded by a high-speed camera. Symmetric properties of acceleration of the head and the tuber sacrale were disturbed in mild fore- and hindlimb lameness, respectively. In subclinical forelimb lameness, no obvious changes in the symmetric properties of the head, the withers, or the tuber sacrale were observed; however, the vertical lift-off acceleration of the treated forelimb was decreased. In the case of subclinical hindlimb lameness, the lift-off points of both the treated and sound hindlimb shifted posteriad. These findings may contribute to further investigations of subclinical lameness aiming to establish new procedures for early detection of subclinical musculoskeletal disease, which would enable more timely treatment.     

Simulation-based estimation of BSE infection in Japan

The first case of bovine spongiform encephalopathy (BSE) in Japan was found in September 2001. As a result, national BSE surveillance systems in slaughterhouses and farms were introduced between October 2001 and April 2004. All cattle, with the exception of those under 24 months of age that die at farms, now undergo compulsory testing when they die or are slaughtered. The removal of specified risk material (SRM) from all slaughtered cattle and a ban on the feeding of meat-and-bone meal to all farm animals were implemented in October 2001. However, infected cattle that died or were slaughtered before these measures were put into practice could have been a source of infection to other cattle through the rendering process. The slaughtered cattle could also have been a source of infection to humans via SRM that entered the food chain. The purpose of this study was to estimate the number of BSE-infected cattle that could have been a source of infection to cattle and humans before October 2001. Since all typical cases were dairy cattle, this study focused on the dairy cattle population. We developed a simulation model to obtain the year of death and the final disposition of infected cows born in each year from 1996 to 2001. In this model, the dairy cattle population was divided into birth cohorts, and parameters regarding its population dynamics were assumed to be constant. Using this model, the total number of infected cattle in each birth year was estimated by maximum likelihood estimation using data on the number of detected cases from 2002 to 2006. Finally, the number of infected cattle that died or were slaughtered each year was estimated by Monte-Carlo simulation using the same model with the total number of infected cattle estimated by maximum likelihood estimation. It was estimated that the majority of infected cattle that could have been sources of infection before 2001 were born in 1996. The total number born in 1996 was estimated to be 155 (95% confidence interval: 90-275). Of these 155 cattle, 56 died or were slaughtered before October 2001, after the accumulation of infectious agent in their bodies. Only 5 of these 56 cattle were estimated to have been slaughtered. Therefore, the number of infected cattle that could have served as a source of human infection would appear to have been a very limited subset of the BSE-infected cattle in Japan.