Detection of Clinically Relevant Pain Relief in Cats with Degenerative Joint Disease Associated Pain

Background

Detection of clinically relevant pain relief in cats with degenerative joint disease (DJD) is complicated by a lack of validated outcome measures and a placebo effect.

Hypothesis/Objectives

To evaluate a novel approach for detection of pain relief in cats with DJD.

Animals

Fifty‐eight client‐owned cats.

Methods

Prospective, double‐masked, placebo‐controlled, stratified, randomized, clinical study. Enrolled cats were 6–21 years of age, with owner‐observed mobility impairment, evidence of pain in at least 2 joints during orthopedic examination, and overlapping radiographic evidence of DJD, and underwent a 2‐week baseline period, 3‐week treatment period with placebo or meloxicam, and 3‐week masked washout period. Outcome measures were evaluated at days 0, 15, 36, and 57.

Results

Both groups significantly improved after the treatment period (day 36) on client‐specific outcome measures (CSOM) and feline musculoskeletal pain index (FMPI) (P < .0001 for both); there was no difference between the groups on CSOM or FMPI score improvement. After the masked washout period, more cats that received meloxicam during the treatment period had a clinically relevant decrease in CSOM score (P = .048) and FMPI score (P = .021) than cats that received placebo.

Conclusions and Clinical Importance

Using both a client‐specific and a general clinical metrology instrument, owners of cats with DJD were able to detect evident recurrence of clinical signs after withdrawal of active medication than after withdrawal of placebo, and that this study design might be a novel and useful way to circumvent the placebo effect and detect the efficacy of pain‐relieving medications.     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

Influence of recombinant duck follistatin protein on embryonic muscle development and gene expressions

Follistatin (FST) acts as a positive regulator of muscle development by inhibiting the activities and expression of myostatin. The recombinant duck FST protein was injected into hatching eggs and was also added to the medium of duck myoblast to study its role on duck embryonic muscle development and gene expressions. Duck embryo weight increased 3.49% (p > 0.05) in FST treatment group as compared with control group, but minor effects were found on leg or breast muscle weights of ducklings at 2 days post‐hatching (p > 0.05). Relative expression of Pax7 was upregulated in both leg and breast muscle tissues (p < 0.05), while MyoD was only upregulated in leg muscle (p < 0.05), and Myf5 was only upregulated in breast muscle (p < 0.05). Relative expression of myostatin was downregulated in both muscle tissues researched (p < 0.05). In vitro studies also showed some maker genes relevant to protein synthesis and degradation, cells’ proliferation and differentiation had significant changes in myoblasts after treated with FST. These results suggested that in ovo feeding of recombinant FST protein to duck hatching eggs had an effect on duck embryo development but have less roles on the duck embryonic muscle development.     

Apoptosis‐Like Events and In Vitro Fertilization Capacity of Sex‐Sorted Bovine Sperm

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.     

Leucine markedly regulates pancreatic exocrine secretion in goats

Four goats (30.1 ± 1.3 kg) with common bile duct re‐entrant catheter and duodenal catheter were used to evaluate the effects of duodenal leucine infusion on pancreatic exocrine secretion and plasma parameters with two 4 × 4 Latin square design experiments. In the long‐term infusion experiment, goats were fed twice daily [700 g/day, dry matter (DM) basis] at 8:00 and 18:00 hours and were duodenally infused with 0, 3, 6, 9 g/day leucine for 14 days. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from d 11 to 14 days to encompass a 24‐h day. In the short‐term experiment, goats were infused leucine for 10 h continuously at the same infusion rate with Experiment 1 after feed deprivation for 24 h repeated every 10 days. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The results showed that the long‐term leucine infusion did not affect pancreatic juice secretion, protein output, trypsin and lipase secretion and plasma insulin concentration, but linearly increased α‐amylase secretion. No changes in pancreatic protein and lipase secretion were observed in the short‐term infusion. Pancreatic juice and α‐amylase secretion responded quadratically, with the greatest values observed in the 3 and 6 g/day leucine respectively. Trypsin secretion linearly decreased, while plasma insulin concentration increased linearly with increased leucine infusion. The results demonstrated that duodenal leucine infusion dose and time dependently regulated pancreatic enzyme secretion not associated with the change in plasma insulin concentration.     

An insight into population structure and gene flow within pure‐bred cats

Investigation of genetic structure on the basis of pedigree information requires indicators adapted to the specific context of the populations studied. On the basis of pedigree‐based estimates of diversity, we analysed genetic diversity, mating practices and gene flow among eight cat populations raised in France, five of them being single breeds and three consisting of breed groups with varieties that may interbreed. When computed on the basis of coancestry rate, effective population sizes ranged from 127 to 1406, while the contribution of founders from other breeds ranged from 0.7 to 16.4%. In the five breeds, F_IS ranged between 0.96 and 1.83%, with this result being related to mating practices such as close inbreeding (on average 5% of individuals being inbred within two generations). Within the three groups of varieties studied, F_IT ranged from 1.59 to 3%, while values were estimated between 0.04 and 0.91%, which was linked to various amounts of gene exchanges between subpopulations at the parental level. The results indicate that cat breeds constitute populations submitted to low selection intensity, contrasting with relatively high individual inbreeding level caused by close inbreeding practices.     

Salix tetradenia Hand.‐Mazz: a new natural plant host of ‘Candidatus phytoplasma’

This article reports Salix tetradenia Hand.‐Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer‐simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI‐B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.     

Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain

Cryopreservation, a technique of high potential for culture collections, might offer a solution for reliable supply of microalgae in aquaculture units. Marine microalgae used in aquaculture were cryopreserved under 4, −20 and −80 °C using common cryoprotectants (methanol, dimethylsulfoxide (DMSO), propylene glycol and polyvinylpyrrolidone (PVP)) with promising results for Chlorella minutissima, Chlorella stigmatophora, Isochrysis galbana and Dunaliella tertiolecta. As cryoprotectants usually are toxic above certain concentrations and exposure time, and assuming that low amounts of cryoprotectants will remain in regenerated cultures, an experimental scheme was employed to explore the lower limits of safety for these algae and their primary consumers in hatchery food chains. Results showed that methanol was well tolerated by C. stigmatophora and D. tertiolecta up to a concentration of 1.6% (v/v) while I. galbana could not survive in culture at any concentration and C. minutissima exhibited some 30% of the control's yield at 0.2%. DMSO was highly tolerated up to 1.0% by all strains with the Chlorella strains surviving well up to 2%. Propylene glycol was not only tolerated up to 8% by Dunaliella but induced mixotrophic growth as well, while for Isochrysis it was lethal at any concentration. Among zooplanktonic consumers, brine shrimp Artemia nauplii could tolerate very high concentrations of the tested cryoprotectants, the rotifer Brachionus plicatilis was found sensitive to low amounts of PVP, while the nauplii of the shrimp Penaeus japonicus and the crab Eriocheir sinensis were in general very sensitive to all cryoprotectants and in several cases to much lower amounts than 1%. However, as long as the residues of cryoprotectants are kept below 1% in the regenerated cultures, there will be no problem with the animal consumers.