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This study aimed to investigate the serum metabolomic profile of obese and lean cats as well as obese cats before and after energy restriction for weight loss. Thirty cats, 16 obese (body condition score 8 to 9/9) and 14 lean (body condition score 4 to 5/9), were fed a veterinary weight loss food during a 4-week period of weight maintenance (L-MAINT and O-MAINT). The 16 obese cats were then energy restricted by a 60% energy intake reduction with the same food for a 10-week period (O-RESTRICT). Fasted serum metabolites were measured using nuclear magnetic resonance and direct infusion mass spectrometry after the maintenance period for L-MAINT and O-MAINT cats and after the energy restriction period for O-RESTRICT and compared between groups using a two-sided t-test. Obese cats lost 672 g ± 303 g over the 10-week restriction period, representing a weight loss rate of 0.94 ± 0.28% per week. Glycine, l-alanine, l-histidine, l-glutamine, 2-hydroxybutyrate, isobutryric acid, citric acid, creatine, and methanol were greater in O-RESTRICT compared to O-MAINT. There was a greater concentration of long-chain acylcarnitines in O-RESTRICT compared to both O-MAINT and L-MAINT, and greater total amino acids compared to O-MAINT. Glycerol and 3-hydroxybutyric acid were greater in O-MAINT compared to L-MAINT, as were several lysophosphatidylcholines. Thus, energy restriction resulted in increased dispensable amino acids in feline serum which could indicate alterations in amino acid partitioning. An increase in lipolysis was not evident, though greater circulating acylcarnitines were observed, suggesting that fatty acid oxidation rates may have been greater under calorie restriction. More research is needed to elucidate energy metabolism and substrate utilization, specifically fatty acid oxidation and methyl status, during energy restriction in strict carnivorous cats to optimize weight loss.

IntroductionThe fecal metabolome provides insight into overall gastrointestinal and microbial health. Methods for fecal sample storage in metabolomics research vary, however, making comparisons within current literature difficult. This study investigated the effect of ambient temperature exposure on microbial-derived metabolites of feline fecal samples.MethodsFecal samples were collected from 11 healthy cats from a local boarding facility. Samples were manually homogenized and aliquoted. The first aliquot was frozen at -80°C within 1 hour of defecation, and remaining samples were exposed to ambient temperature for 2, 4, 6, 8, 12, and 24 h prior to freezing at -80°C. Fecal metabolites were quantified using 1H NMR spectroscopy. Fifty metabolites were grouped into six categories (27 amino acids, 8 fatty acids, 5 sugars, 3 alcohols, 2 nitrogenous bases, 5 miscellaneous).ResultsConcentrations of 20 out of 50 metabolites significantly differed due to ambient temperature exposure (7 amino acids, 6 fatty acids, 2 alcohols, 1 nitrogenous base, 4 miscellaneous). The earliest detected changes occurred 6 h post-defecation for cadaverine and fumaric acid.DiscussionThis study shows ambient temperature exposure alters the composition of the feline fecal metabolome, but short-term (up to 4 h) exposure prior to storage in the freezer seems to be acceptable.

Abstract Background Research in humans and mice suggests that obesity influences the abundance and diversity of gastrointestinal (GI) microbiota, and that an “obese microbiome” influences energy metabolism and fat storage in the host. Microbiota membership and composition have been previously assessed in healthy cats. However, research investigating the effects of obesity and weight loss on the cat’s fecal microbiota is limited. Therefore, this study’s objective was to evaluate differences in fecal microbial abundance and biodiversity, as well as serum cobalamin and folate concentrations in obese cats, before and after weight loss, and compare to lean cats. Fourteen lean and 17 obese healthy client-owned cats were fed a veterinary therapeutic weight loss food at maintenance energy requirement for 4 weeks. At the end of week 4, lean cats finished the study, whereas obese cats continued with a 10-week weight loss period on the same food, fed at individually-tailored weight loss energy requirements. Body weight and body condition score were recorded every 2 weeks throughout the study. At the end of each period, a fecal sample and food-consumption records were obtained from the owners, and serum cobalamin and folate concentrations were analysed. DNA was extracted from fecal samples, polymerase chain reaction (PCR) was performed, and products were sequenced using next-generation sequencing (Illumina MiSeq). Results No significant differences in the relative abundance of taxa and in biodiversity indices were observed between cats in either group (P > 0.05 for all tests). Nevertheless, some significantly enriched taxa, mainly belonging to Firmicutes, were noted in linear discriminant analysis effect size test in obese cats before weight loss compared to lean cats. Serum cobalamin concentrations were significantly higher in lean compared to obese cats both before and after weight loss. Serum folate concentrations were higher in obese cats before weight loss compared to after. Conclusions The association between feline obesity and the fecal bacterial microbiota was demonstrated in enriched taxa in obese cats compared to lean cats, which may be related to enhanced efficiency of energy-harvesting. However, in obese cats, the fecal microbial abundance and biodiversity were only minimally affected during the early phase of a standardized weight loss plan.

Abstract Background Feline fecal microbiota analyses can potentially be impacted by a variety of factors such as sample preparation, sequencing method and bioinformatics analyses. Another potential influence is changes in the microbiota from storage of samples prior to processing. This study examined the effect of ambient temperature exposure on the feline fecal microbiota composition. Fecal samples were collected from 12 healthy cats, within 15 min after defecation. Samples were aliquoted and the first aliquot was frozen at −80 °C within 1 hour of defecation. Remaining aliquots were maintained at ambient temperature (20 to 23 °C) and frozen at −80 °C at 6, 12, 24, 36, 48, 72 and 96 h after collection. DNA was extracted from all aliquots, and polymerase chain reaction (PCR). The PCR products were sequenced with next-generation sequencing (Illumina MiSeq). Results No significant differences were observed in alpha and beta biodiversity indexes, as well as relative abundance of different taxa over time (P > 0.05 for all tests between time points). Principal coordinate analyses demonstrated that samples cluster mainly by cat, with no significant differences between time points (AMOVA, P > 0.05; HOMOVA, P > 0.05). Linear discriminant analysis effect size method was performed and failed to detect any enriched taxa, between time points. Random forest algorithm analysis indicated homogeneity across time points. Conclusions Although existing evidence from human fecal storage studies is contradictory, a recent study in companion animals agreed with the current study, demonstrating that maintenance of feline fecal samples at ambient temperature for up to 4 days has no effect on the bacterial membership and structure.

Three-dimensional (3-D) genome organization in the nuclear space affects various genomic functions. Circular chromosome conformation capture (4C-seq) is a powerful technique that allows researchers to measure long-range chromosomal interactions with a locus of interest across the entire genome. This method relies on enzymatic cleavage of cross-linked chromatin and consecutive ligation to create ligation junctions between physically adjacent loci, followed by PCR amplification of locus-specific associating loci. The enzymes used must meet 4C standards because variations in their efficiency and performance may affect the quality of the obtained data. Here we systematically compare the efficiency and reliability of different T4 DNA ligases and PCR DNA polymerases, assessing the most critical and technically challenging steps in 4C. The results of this analysis enable the use of cost-effective enzymes with superior specificity and efficiency for 4C and save time in screening for appropriate primers. This information provides users with flexibility in their experimental design and guidelines for adapting and testing any enzyme of choice for obtaining standardized results.