Evaluation of a Collagenase Generated Osteoarthritis Biomarker in Naturally Occurring Canine Cruciate Disease

Objective— To determine the clinical value of a novel osteoarthritis (OA) biomarker in detecting canine cruciate disease.
Study Design— Cross sectional clinical study.
Animals— Dogs (n=22) with cranial cruciate ligament (CCL) rupture and 12 control dogs.
Methods— Concentrations of collagenase-generated cleavage epitope of type II collagen (Col2-3/4C_{long mono}, or C2C) in serum, urine, and joint fluid were compared between a group of dogs with CCL rupture and a control group. Correlation of C2C concentrations to the clinical stage of stifle OA was also evaluated.
Results— There were no significant differences in C2C concentrations in serum, urine, and joint fluid between groups ( P >.05). Subjective scores of lameness, joint effusion, osteophytosis were significantly more severe in the CCL rupture group compared with the control group ( P <.05). There was no significant correlation of C2C concentrations with clinical stage of stifle OA ( P >.05).
Conclusion— This OA biomarker did not detect pathology associated with CCL rupture. Our results suggest that collagenase-specific degradation of type II collagen in articular cartilage may not be involved in the early stage of naturally occurring canine cruciate disease, and that pathology associated with naturally occurring CCL rupture is different from that of experimental OA model.
Clinical Relevance— C2C is not clinically useful in detecting CCL rupture in dogs.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Physicochemical properties of the thermal gel of water-washed meat in the presence of the more soluble fraction of porcine sarcoplasmic protein

We investigated the physicochemical properties of the thermal gel of water‐washed pork meat (WWM) in the presence of the soluble fraction of porcine sarcoplasmic protein (SP) obtained with ammonium sulfate at 75 percent saturation. Two precipitated fractions of SP were obtained at 0–50 percent and 50–75 percent saturation, named SP‐f1 and SP‐f2, respectively, and the soluble fraction obtained at 75 percent saturation, SP‐f3, was used. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis showed that SP‐f3 contained mainly glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), while SP‐f1 and SP‐f2 had other SPs such as phosphorylase b, enolase, actin and phosphoglycerate mutase. The gel strength of WWM was greater when SP‐f3 rather than one of various animal proteins such as bovine plasma (BP), egg white, or whey protein isolates (WPI), was added and SP‐f3 had a gel‐enhancing effect as good as that of polyphosphate (PP). The gel strength of WWM with added SP‐f3 increased significantly with NaCl at 0.15 mol/L or more, but not in the absence of NaCl (0 mol/L). The effect of SP‐f3 was evident at neutral pH and maximum gel strength was obtained at a pH above 6.0. Differential scanning calorimetric (DSC) analysis showed that an endothermic peak corresponding to myosin heads in WWM shifted to a lower temperature with the addition of SP‐f3, as in the case of PP, though there was no such shift in the presence of other animal proteins (BP, egg white and WPI), suggesting that SP‐f3 increases the gel strength of WWM through the dissociation of actomyosin similar to PP. Scanning electron microscopy (SEM) revealed wall‐like structures among the protein strands in the WWM gel matrix in the presence of SP‐f3. The results of DSC and SEM indicated that the formation of a gel network in meat products is reinforced with GAPDH in SP after the interaction between GAPDH and myofibrillar protein.     

Tooth brushing inhibits oral bacteria in dogs

In this study, scaling, polishing and daily tooth brushing were performed in 20 beagle dogs, and the number of oral bacteria was determined using a bacterial counter. The dogs were randomized into the scaling (S), scaling + polishing (SP), scaling + tooth daily brushing (SB) and scaling + polishing + tooth daily brushing (SPB) groups. Samples were collected from the buccal surface of the maxillary fourth premolars of the dogs immediately after scaling and every week thereafter from weeks 1 to 8. Throughout the study, the number of bacteria was significantly lower in the SB and SPB groups compared with the S group. The findings suggest that daily tooth brushing inhibited oral bacterial growth in the dogs.     

Improvement of the emulsifying properties of porcine sarcoplasmic proteins with a low-molecular weight surfactant by the two-step emulsification method

The emulsifying properties of porcine sarcoplasmic protein (SP) with or without low-molecular weight surfactants were examined. The emulsifying activity (EA) of SP was weaker than that of sodium caseinate (CA), blood plasma (BP) and whey protein isolates at a protein concentration of 0.5–3%. Sodium chloride decreased the EA of SP. The EA of SP was about one quarter that of CA in the presence of 3% sodium chloride. As homogenization time increased, the EAs of SP and BP decreased. The EA of SP was not influenced by sucrose stearate ester (SE) with a hydrophilic-lipophilic balance (HLB) value of 16 (SE-16) but was decreased by those with HLB values of 5 and 1. Low-molecular weight surfactants such as SE-16, Tween 80 and sodium cholate improved the EA of SP with the two-step emulsification method: corn oil was previously emulsified with the surfactant before addition of SP and homogenization. In the case of the addition of SE-16 as primary emulsifier, the EA was about twice as high as that of SP alone. The same effect was shown in the emulsions formed by CA and soy protein isolate. These results indicate that the emulsifying properties of meat protein would be improved by the addition of a low-molecular weight surfactant in a two-step emulsification.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Formation of zinc protoporphyrin IX in Parma-like ham without nitrate or nitrite

Zinc protoporphyrin IX (ZPP) is a characteristic red pigment in meat products that are manufactured without the addition of a curing agent such as nitrate or nitrite. To examine the effects of impurities such as mineral components in sea salt on the formation of ZPP, we manufactured Parmatype dry-cured hams that were salted with refined salt or sea salt and examined the involvement of oxidation-reduction potential (ORP) in the formation of ZPP. The content of ZPP was increased drastically after 40 weeks. Microscopic observation showed strong fluorescence caused by ZPP muscle fiber after 40 weeks. Conversely, heme content varied considerably during processing. ORP increased during processing. However, there was no obvious difference between ham salted with refined salt and that salted with sea salt. Therefore, it was concluded that impurities in sea salt were not involved in the formation of ZPP.     

Estimation of daily urinary potassium excretion using urinary creatinine as an index substance in prepartum dairy cows

In the present study, the daily excretion of potassium (K) in urine (urinary K(UK)) was estimated from a 6 h urine sample using urinary creatinine (UC) as the index substance. All urine was collected from six pregnant Holstein cows at 6 h intervals for 24 h on 3 days of the 4th, 2nd and final week before the expected date of parturition. In total, 72 6 h urine samples were obtained. Daily UC excretion (mg/day per kg bodyweight (BW)) was almost the same for the three sampling days. Daily UC excretion varied among cows from 22.1 to 24.3 mg/day per kg BW with a mean of 22.8 mg/day per kg BW with no significant difference. Thus, daily UC excretion was confirmed to be constant throughout the prepartum period with no differences among individuals. The concentration ratios of K to creatinine ((UK mg/dL)/(UC mg/dL) (UK/UC)) correlated strongly to the hourly K excretions (mg/h per kg BW) (r = 0.952, P < 0.01) in the 6 h urine samples. The differences in the UK/UC ratio between sampling periods were not significant within each cow. Therefore, daily UK excretion (mg/day) can be estimated using the equation: daily UK excretion (mg/day) = daily UC excretion (mg/day per kg BW) × BW (kg) × 6 h urine sample UK/UC, where daily UC excretion can be a given value.