Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Use of amnion and placenta in neonatal screening for canine GM1-gangliosidosis and the risk of diagnostic misclassifications

BACKGROUND: A closed breeding colony of Shiba dogs with GM1-gangliosidosis is maintained at Hokkaido University (Sapporo, Japan). Neonatal genotyping is essential to control the breeding colony genetically as an animal model for the human disease. OBJECTIVES: The purpose of the present study was to determine the utility of amnion and placenta in the neonatal screening or diagnosis for canine GM1-gangliosidosis. METHODS: Twenty neonatal Shiba dogs of a pedigree with GM1- gangliosidosis were differentiated into 3 genotypes--normal, heterozygous, and affected dogs--by using a previously reported DNA mutation assay. Acid beta-galactosidase activity was measured in amnion and placenta and compared among the 3 genotypes. RESULTS: The level of beta-galactosidase activity in the amnion of affected dogs was negligible and <2% of the mean activity in normal dogs; there was no significant difference among the 3 genotypes. In placenta, beta-galactosidase activity was significantly different among all the genotypes; however, there was wide overlap in enzyme activity between normal and heterozygous dogs. The level of activity in affected dogs was relatively high and >10% of the mean activity in normal dogs. The DNA mutation assay gave correct information about genotype with genomic DNA extracted from amnion but ambiguous information with DNA from placenta. CONCLUSIONS: Amnion and placenta were not useful as enzyme sources in neonatal screening in canine GM1-gangliosidosis because of the risk of misdiagnosis. DNA from amnion is applicable as a template for genotyping, whereas placenta should not be used because canine placenta contains maternal cells.     

Cleaning procedures and cleanliness assessments of bucket milkers and suckling buckets on Japanese dairy farms

Cleanliness of milking equipment is known to be important for the safety of dairy products and to prevent the spread of diseases among cows. We investigated the cleaning procedures of milking equipment and suckling equipment on Japanese dairy farms, and the cleanliness of bucket milkers, suckling buckets, milk receivers, and bulk tanks, using adenosine triphosphate (ATP) bioluminescence test. Bulk tanks (except one bulk tank) and milk receivers were washed by automated cleaning, but all bucket milkers and suckling buckets were washed by manual cleaning. Detergents were often not used to clean bucket milkers and suckling buckets. The log₁₀ transformed relative luminescence units (LRLU) of equipment washed by manual cleaning was higher than equipment washed by automated cleaning. Clean surfaces (≤2.2 LRLU) were only observed on the bulk tank and the milk receiver. More than 50% of the LRLU of the mouthpiece, the rubber packing of claw, and the nipple of the suckling bucket were determined dirty. These results suggest that the cleanliness of the bucket milkers and the suckling buckets washed by manual cleaning was lower than that of the equipment washed by automated cleaning, and may be due to insufficient cleaning procedures.     

Gas chromatographic resolution of organochlorine insecticides on OV-1/OV-17, OV-210/OV-17, and OV-225/OV-17 mixed phase systems.

The resolution of organochlorine insecticides and their related compounds on OV-1/OV-17, OV-210/OV-17, and OV-225/OV-17 mixed phase column systems was investigated. Four BHC isomers and p,p'-DDT, its isomer, and their metabolites (except p,p'-DDE and o,p'-DDE) were resolved on the OV-210/OV-17 system. Heptachlor and heptachlor epoxide were separated on 5% OV-1/2% OV-17 or OV-225/2% OV-17. Resolution of aldrin, dieldrin, and endrin was obtained on 5% OV-210/2% OV-17, p,p'-DDE and o,p'-DDE could not be separated by any of the systems studied.     

Genetic evaluation of heat tolerance in Holstein cows in Japan

We used test‐day records and daily records from provincial weather stations in Japan to evaluate heat tolerance (HT) in Holstein cows according to a random regression test‐day model. Data were a total of 1,641,952 test‐day records for heritability estimates and 17,245,694 test‐day records for genetic evaluation of HT by using milk yield and somatic cell score (SCS) in Holstein cows that had calved for the first time in 2000 through 2015. Temperature–humidity index (THI) values were estimated by using average daily temperature and average daily relative humidity records from 60 provincial Japanese weather stations. The model contained herd–test‐day, with lactation curves on days in milk within month–age group as a fixed effect. General additive genetic effect and HT of additive genetic effect were included as random effects. The threshold value of THI was set to 60. For milk yield, estimated mean heritabilities were lower during heat stress (THI = 78; 0.20 and 0.28) than when below the heat stress threshold (THI ≤ 60; 0.26 and 0.31). For SCS, heritability estimates (range 0.08–0.10) were similar under all heat stress conditions. Genetic trends of HT indicated that EBVs of HT are changing in an undesirable direction.     

Immunohistochemical observation of canine degenerative myelopathy in two Pembroke Welsh Corgi dogs

Immunohistochemistry was performed to assess whether oxidative stress and/or denatured proteins play roles in the pathogenesis of canine degenerative myelopathy (DM). Two Pembroke Welsh Corgi (PWC) dogs with a homozygous mutation (c.118G>A) in the canine superoxide dismutase 1 (SOD1) gene were examined. The pathological features of the dogs were consistent with those of previous cases of DM in PWC. In the spinal lesions, diffuse SOD1 expression was observed in the neurons while no inclusion-like aggregates had formed, which disagreed with the findings of a previous study. A unique inducible nitric oxide synthase (iNOS) staining pattern in reactive astrocytes and a significant increase in ubiquitin immunoreactivity in the spinal lesions were also observed. These findings indicate the involvement of oxidative stress and the accumulation of ubiquitinated proteins in the pathogenesis of canine DM, whereas the role of SOD1 remains unclear.     

Retrospective surveillance of metabolic parameters affecting reproductive performance of Japanese Black breeding cows

This retrospective study was conducted to confirm the relationship between pre- and postpartum metabolic parameters and postpartum reproductive performance and to clarify seasonal characteristics of the metabolic parameters by using our metabolic profile test (MPT) database of Japanese Black breeding herds. In evaluation 1, MPT databases of blood samples from multiparous cows collected prepartum and postpartum were divided into two groups according to calving interval, and each MPT parameter was compared. In evaluation 2, the same MPT databases used in evaluation 1 were divided into two groups according to the sampling period. Significant differences were found in the prepartal total protein and postpartal γ-glutamyltransferase in evaluation 1. In evaluation 2, significant differences were found in the prepartal and postpartal total protein, albumin/globulin ratio, and glucose. Clear seasonal differences in MPT results emphasized the usefulness of the MPT in breeding cattle herds fed home-pasture roughage and suggest that unsatisfactory reproductive performance during hot periods reflects inadequate nutritional content of the diet and possible reduced feed intake due to heat stress.     

Real-time PCR genotyping assay for canine trapped neutrophil syndrome and high frequency of the mutant allele in Border collies

Trapped neutrophil syndrome is an autosomal recessive inherited neutropenia in Border collies. The causative mutation is a 4 base pair deletion in exon 19 of the canine VPS13B gene. In this study, a real-time PCR assay was developed and a genotyping survey was carried out in Border collies in Japan. The carrier frequency was 11.1%, suggesting that the mutant allele frequency is high enough to warrant measures to control and prevent the disease.     

Nephron segment identification in the normal canine kidney by using lectin histochemistry

Lectin-binding patterns in normal canine kidneys were histochemically investigated using eight lectins. WGA, ConA, and RCA-I showed positive signals in glomerular capillary walls, with signals for RCA-I being detected heterogeneously. In tubular segments, signals for WGA, s-WGA, ConA, and RCA-I were distributed widely from proximal tubules to collecting ducts, whereas those for SBA, PNA, DBA, and UEA-I were localized in thin limbs of the loop of Henle, thick ascending limbs, distal tubules, or collecting ducts. Apart from PNA and UEA-I, lectins showed heterogeneous bindings in collecting ducts with the heterogeneity. UEA-I-positive reactions were restricted to those parts of the distal tubules in close proximity to the glomeruli, and in these parts, signals in the macula densa were markedly stronger than in other regions. Based on the present findings, lectin probes, singly or in combination, could be utilized to identify the affected nephron segment in canine renal pathology.     

A comparative study of chronic kidney disease in dogs and cats: induction of cyclooxygenases

The present study investigated whether renal cyclooxygenase (COX) induction is associated with the severity of chronic kidney disease (CKD) in dogs and cats. The collected kidneys were examined histopathologically and immunohistochemically. The immunoreactivities of COX-1 and COX-2 were evaluated quantitatively, and the correlations to the plasma creatinine concentrations, glomerular size, glomerulosclerosis, interstitial fibrosis, and interstitial cell infiltration were evaluated statistically. Immunoreactivities for COX-1 were heterogeneously observed in the medullary distal tubules and collecting ducts; no correlations with the severity of renal damage were detected. Immunoreactivities for COX-2 were heterogeneously observed in the macula densa (MD) regions. In dogs, the percentage of COX-2-positive MD was significantly correlated with the glomerular size. In cats, glomeruli with COX-2-positive MD had significantly higher sclerosis scores than those with COX-2-negative MD. In conclusion, renal COX-2 is induced in canine and feline CKD, especially in relation to the glomerular changes.