Plasma protein increase as a chronological aging factor in healthy toy poodles

Plasma protein increase as a chronological aging factor in healthy toy poodles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plasma protein increase as a chronological aging factor in healthy toy poodles Satoru Ozaki, Yoshiko Honme, Seiichiro Higashi, Kouya Hattori, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5341224/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although extensive research has examined aging markers in larger dog breeds, little is known about small breeds. This study assesses the relevance of aging biomarkers examined in larger breeds and other biological species, focusing on toy poodles (N = 40) as a model of small breeds and retrievers (N = 17) serving as a large-sized reference. Healthy individuals with no significant health declines for up to a year post-data collection were studied for age-related changes in various parameters, excluding disease factors. Our cross-sectional analysis identified significant correlations between age and increases in plasma protein concentration and amylase levels across both breeds, with breed-specific age-related declines in vaccine responses to various viruses observed only in toy poodles. Longitudinal analysis over one year confirmed a significant temporal increase in plasma protein in toy poodles, with a similar, albeit non-significant, trend in retrievers. Unlike in other species, NAD⁺ levels and fecal microbiota showed no age-related changes. Additionally, the previously reported frailty index correlated with age in retrievers but not in toy poodles. Notably, including deceased individuals during the study strengthened correlations. These results suggest plasma protein increase as a chronological aging factor in toy poodles and enhance our understanding of aging in healthy small dog breeds. Plasma protein aging dogs NAD⁺ gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Dogs have been domesticated for over 10,000 years and have long been integral members of human society [ 1 ]. Selective breeding has produced a variety of inbred lineages, resulting in approximately 400 pet dog breeds [ 2 ]. Due to strong artificial selection, domesticated dogs have become one of the most morphologically diverse mammalian species, with significant variation in size among breeds. The average adult dog weight varies more than 30-fold from breed to breed. Based on this size diversity, breeds are classified as large (average adult weight > 25 kg), medium (10–25 kg), or small (< 10 kg) [ 3 ]. Size influences lifespan and aging rates in each breed, with an inverse correlation between size and lifespan [ 4 , 5 ]. Different breeds also exhibit varying rates of age-related diseases [ 6 ]. Canine breed size is positively associated with IGF-1 levels [ 7 ], and slower aging progression in small breeds is attributed to relatively low IGF-1 levels [ 6 , 8 ]. Lowering IGF-1 signaling has been shown to increase lifespan in model organisms [ 9 ]. These findings highlight the need for studies across different breed sizes to better understand aging in pet dogs. Multiple reports exist on factors associated with aging in dogs. It has been reported that DNA methylome analysis can estimate canine chronological age [ 10 , 11 ]. Several studies measuring blood biochemical indices have reported correlations between age and increased levels in total protein, globulin, ALT, albumin, and glucose [ 12 – 15 ]. While these studies have found common relationships between each factor and age, they are limited to large and medium breeds [ 12 , 13 ] or to populations of multiple breeds in which these sizes are predominantly present [ 14 , 15 ]. While the population of small-sized breeds has increased in recent years, it remains largely unclear whether these findings can be adapted to small dogs or if they have different aging profiles. Additionally, many studies are cross-sectional, and there are limited reports analyzing aging-related factors in the same population through longitudinal analysis. It has been reported that in humans, immune function declines with age and vaccine response decrease [ 16 – 19 ]. This decreased reactivity has also been observed for vaccines against COVID-19 across a wide geographic and racial spectrum [ 20 , 21 ], suggesting decreased reactivity to new antigens. Post-vaccination antibody titers have also been reported to decrease in large and medium-sized dogs as they age, but reports on small breeds are limited [ 22 , 23 ]. Frailty is a condition commonly seen in the elderly that increases the risk of falls, disability, hospitalization, and death [ 24 ]. In humans, several indices have been proposed to objectively assess the degree of frailty, and these are used clinically for health management [ 24 – 26 ]. A method for calculating the frailty index has also been proposed for dogs, showing a modest correlation between aging and the index, and a strong association with mortality regardless of age [ 27 ]. However, the number of studies on small dogs is relatively small, and the association is weak [ 27 ]. NAD⁺ (nicotinamide adenine dinucleotide) is a coenzyme involved in energy metabolism, DNA repair, and cell signaling [ 28 – 30 ]. It has been reported that NAD⁺ levels in vivo decrease with aging common in eukaryotes including humans [ 31 – 35 ], rodents [ 35 , 36 ], and nematodes [ 36 ]. In mice, the intake of NAD⁺ precursors increases NAD⁺ levels and improves tissue functions and metabolism [ 36 – 38 ]. However, no reports on the relationship between aging and in vivo NAD⁺ levels in dogs have been published at the time of this manuscript's submission. The composition of the human gut microbiota has been associated with various poor health conditions and diseases such as obesity, diabetes, and inflammatory diseases [ 39 , 40 ]. The human gut microbiome also changes with age, with a decrease in Bifidobacterium and Faecalibacterium and an increase in Enterobacteriaceae reported with aging [ 41 , 42 ]. Additionally, frail humans show a decrease in Faecalibacterium and an increase in Enterobacteriaceae compared to non-frail individuals, exhibiting a trend similar to chronological aging [ 43 , 44 ]. These findings suggest that the human gut microbiota is linked to health and aging. In contrast, studies on the relationship between age and gut microbiota in dogs remain sparse, and consistent trends have not been confirmed [ 45 , 46 ]. Here, we aim to assess changes associated with chronological aging in healthy dogs by measuring multifaceted parameters. Toy poodles were analyzed as representatives of small dogs, and retrievers were analyzed as representatives of large dogs for comparison. We measured blood biochemistry, blood NAD⁺ levels, fecal microbiome, antibody titers as vaccine responses against multiple viruses, echocardiographic measurements, and the frailty index as candidate parameters. All parameters, except for antibody titers, were measured twice with a six-month interval between measurements. We analyzed the relationship between age and various indicators. Indicators with the strongest correlations were subjected to longitudinal analysis over a one-year period to evaluate whether there were consistent changes with age over time. Materials and methods Animals Toy poodles, golden retrievers, and labrador retrievers kept at the same facility in Ibaraki Prefecture, Japan, were recruited for the study with the consent of the owners. We excluded dogs with pre-existing diseases, those diagnosed by a veterinarian with poor health status at the start of the study, and those that died within 24 months from the start due to the possibility of being unhealthy. Animals that became pregnant during the study were excluded upon confirmation of pregnancy. In some analyses, participants were stratified based on their age at the start of the study. Toy poodles under 7 years old and retrievers under 6 years old were classified as "Adult," while participants older than these thresholds were classified as "Senior" [ 23 , 47 ]. The two breeds of retriever were merged in the data analysis. Blood sampling The participants were subjected to fasting from the evening prior to blood collection, with water intake allowed ad libitum. The fasting period was within 12 to 24 hours. Blood samples were collected using a syringe from the cephalic vein. After collection, the blood samples were transferred to silicon-coated blood collection tubes (#365963, Becton, Dickinson and Company, NJ, USA) for biochemical and antibody titer testing, and to evacuated tubes containing sodium fluoride, citric acid, sodium citrate, and EDTA-2Na (VP-FH051K, Terumo, Japan) for NAD + quantification. Plasma biochemical analysis Plasma was obtained by centrifuging the tubes at room temperature at 3000 rpm for 10 minutes, after which the supernatant was collected as plasma. Plasma was kept refrigerated until biochemical analysis and antibody titer measurement. The concentrations of total protein (TP), albumin (ALB), lipase (LIP), calcium (Ca), and total bilirubin (TBIL) were measured by the biuret method, bromocresol green method, DGGR substrate chromogenic method, o-cresolphthalein complexone method, and chemical oxidation method, respectively. Blood urea nitrogen (BUN), creatinine (CRE), blood glucose (GLU), total cholesterol (TC), triglycerides (TG), total bile acids (TBA), and inorganic phosphorus (P) were measured by enzymatic methods. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and amylase (AMY) were measured by the Japanese Society of Clinical Chemistry standardization method. Sodium (Na), potassium (K), and chloride (Cl) were measured by the electrode method. AMY, LIP, ALT, and AST were analyzed using log 10 -transformed values. Quantification of whole blood NAD concentration The processing method for blood samples for NAD + quantification followed the protocol described in a previous report [ 48 ]. After sampling, blood collection tubes were gently inverted several times and 20 µL of collected blood was immediately treated with 980 µL of cold 55% MeOH and transferred to a -80°C freezer for long-term storage. Prior to measurement, stable isotopes of NAD + , nicotinamide adenine dinucleotide-13C5 (Syncom), were added to thawed samples, followed by ultrafiltration with Amicon filters with a molecular weight cutoff of 10 kDa (Millipore). Half of the flow-through was evaporated in a centrifugal evaporator. Samples were dissolved in 1 mL of a 10 mM ammonium acetate solution and further diluted 10-fold for quantification using LC-MS/MS. LC-MS/MS analyses were performed using a Shimadzu HPLC system (Shimadzu, Japan) coupled with a QTRAP4500 mass spectrometer (AB Sciex, MA, USA). Chromatographic separation was achieved on a Hypercarb column (2.1 mm × 100 mm, 3 µm particle size, Thermo Scientific, WI, USA). The column temperature was maintained at 60°C. The mobile phase consisted of 10 mM ammonium acetate with 0.05% (v/v) ammonium hydroxide in water (A) and 0.05% (v/v) ammonium hydroxide in acetonitrile (B), delivered at a flow rate of 0.2 mL/min. The injection volume was 3 µL. The gradient elution started with 5% B for the first 1.8 min, increased to 54% B from 1.8 min to 14 min, then to 90% B from 14.0 min to 14.10 min, held at 90% B until 17.1 min, decreased linearly to 5% B from 17.1 min to 17.2 min, and remained at 5% B until 32.2 min. The MS/MS analysis was performed in positive ESI mode. The detection of NAD + was performed by multiple reaction monitoring. The ions were selected in the first quadrupole (Q1) and collided with nitrogen gas in the second quadrupole (Q2), and the product ions were detected in the third quadrupole (Q3). The m/z value of the precursor ions detected at Q1 was 664.2 (NAD + ) and 669.1 (stable isotope-labeled NAD + ). The collision energy at Q2 was 38 V. The m/z values for the product ions detected at Q3 were both 428.2 (NAD + and stable isotope-labeled NAD + ). Echocardiography Echocardiography was performed using a LOGIQ P6 (GE Healthcare, USA). IVSd (mm), LVIDd (mm), LVIDs (mm), and LVPWd (mm) were measured in M-mode using the right parasternal left ventricular short-axis cross-sectional image in the right lateral recumbent position with hand-holding without instruments. EDV (ml), ESV (ml), EF (%), SV (ml), and FS (%) were calculated from these measurements. Additionally, the E/A ratio was measured using the pulsed Doppler method for inflow blood flow at the mitral valve, using a left parasternal apical quadrant cross-sectional image in the left lateral recumbent position. Each measurement was performed 2–5 times, and the average value was recorded. Abbreviations of each parameter are as follows: IVSd: Interventricular Septal thickness in diastole LVIDd: Left Ventricular Internal Dimension in diastole LVIDs: Left Ventricular Internal Dimension in systole LVPWd: Left Ventricular Posterior Wall thickness in diastole EDV: End-Diastolic Volume ESV: End-Systolic Volume EF: Ejection Fraction SV: Stroke Volume FS: Fractional Shortening E/A ratio: Early diastolic mitral inflow velocity / Atrial systolic mitral inflow velocity ratio Vaccination and measurement of antibody titer At 6 months after the start of the study (6 m), the participants received a vaccination with Vanguard Plus 5CV/L (Zoetis, NJ, USA). This formulation included vaccines for distemper, parvo, and adeno viruses with potencies of over 10 3.5 , 10 6.4 , and 10 5.5 TCID50, respectively. The vaccination history prior to this vaccination was 12 months ago, except for two toy poodles who were excluded from the analysis. Blood samples were collected before vaccination (6 m), 1 month after vaccination (7 m), and 6 months after vaccination (12 m) to measure antibody titers for distemper, parvo, and adeno viruses. Plasma was obtained using the same method described in the biochemical test section. Antibody titers were measured by fluorescence immunoassay. Frailty index The frailty index was evaluated using the method described in a previous report [ 27 ]. Briefly, 33 items related to frailty were assessed by veterinary nurses and a veterinary physician. Some health deficits were assigned a score of 0 (absent) or 1 (present), while other deficits were evaluated using a semi-quantitative scale as absent (score = 0), mild (score = 0.5), or severe (score = 1). The frailty index was calculated by dividing the sum of the scores by the total number of items (33). Fecal sampling and DNA extraction Feces naturally excreted were quickly collected into a feces container (Sarstedt) and frozen until DNA extraction. Whole genomic DNA from feces was extracted according to "protocol Q" from a previous report [ 49 ], with slight modification. Twenty mg of feces was measured into a tube containing 0.3 g of 0.1 mm diameter zirconia beads, and 1 mL of Buffer ASL (QIAGEN, Hilden, Germany) was added. The mixture was vortexed and then held at 95°C for 15 min. After incubation, the sample was shaken 4 times for 1 min at 5 m/s using a FastPrep-24 instrument (MP Biomedicals, USA) and then centrifuged. The supernatant was collected in another tube, and the pellet was resuspended in Buffer ASL. The mixture was incubated, shaken, and centrifuged again, and the supernatant was combined with the previous one. A 1/5 volume of 10 M ammonium acetate solution was added to the supernatant and incubated for 5 min. The mixture was centrifuged, the supernatant was collected, and an equal volume of isopropanol was added. After standing on ice for 30 min, the tube was centrifuged, the supernatant was removed, and the pellet was washed with 70% ethanol. The pellet was dissolved in 200 µL of TE buffer and then treated with RNase and Protease K. Then 200 µL of ethanol was added and vortexed. The solution was placed in a QIAamp spin column (QIAGEN) and centrifuged at 16,000 g for 1 min. The column was flushed with 500 µL of Buffer AW1 (QIAGEN) and then flushed with the same volume of Buffer AW2 (QIAGEN). The column was placed on a fresh tube, and Buffer ATE (QIAGEN) was added. After a 1 min incubation, the column was centrifuged, and the eluent was used as the genomic DNA solution. 16S rRNA gene sequencing library preparation and sequencing The V3-V4 region of the bacterial 16S rRNA gene was amplified using genomic DNA as a template and primers 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’ and 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’ [ 50 ]. Template genomic DNA concentration was measured using Quant-iT™ dsDNA Assay Kits (Thermo Fisher Scientific) and adjusted to 5 ng/µL. 16S Metagenomic Sequencing Library Preparation was performed according to the manufacturer's instructions (Illumina, CA, USA). The DNA library was sequenced using a MiSeq System (Illumina) with a 2 × 300-base-pair protocol. Analysis of 16S rDNA sequencing data Sequencing data were analyzed using Qiime2(version 2020.11) [ 51 ]. To trim the primer region from raw sequences, the Cutadapt plugin in Qiime2 was used [ 52 ]. Sequences without the primer region were processed for quality control, paired-end read joining, chimera filtering, and amplicon sequence variant (ASV) table construction using the DADA2 algorithm [ 53 ]. For each representative sequence of ASV, BLAST [ 54 ] was used to assign the taxonomy based on the SILVA database (version 138) [ 55 ]. After random sampling of 14,000 reads using the feature-table plugin [ 56 ], conversion of compositional data and diversity analysis was performed. Statistical analysis R (ver. 4.3.2) was used for linear mixed model analysis of TP and AMY using the lme4 package. TP and AMY were modeled as a function of the time of measurement, with time included in the model as a fixed effect and individual as a random effect. Permanova analysis with Weighted UniFrac Distance was used to test for significant differences in fecal microbiota β-diversity between groups. GraphPad Prism 8 was used for Mann-Whitney U-test and Spearman's rank correlation analysis. Results Study Subjects Forty toy poodles, eight golden retrievers, and nine labrador retrievers were recruited for this study (Fig. 1 A). Two toy poodles and two retrievers were excluded due to health check results at the start of the study. During the study, six toy poodles and five retrievers were excluded due to death, and four toy poodles and one retriever were excluded due to pregnancy. Twenty-eight toy poodles, four golden retrievers, and five labrador retrievers successfully completed the study. The age of the participants who completed the study ranged from 1 to 14 years at the start. The median ages for toy poodles and retrievers were 6.7 years and 5.3 years, respectively. Based on the threshold, 15 toy poodles and five retrievers were assigned as adults, while 13 toy poodles and four retrievers were assigned as seniors. The average body weight of the completed toy poodles and retrievers at the start were 4.7 and 24.2 kilograms, respectively. Plasma biochemistry tests Blood was collected from each animal at the start of the study (0 m) and six months later (6 m), and 18 plasma biochemical indices were measured (Fig. 1 B). Fifteen of the 18 indices were compared to the reference range [ 57 ], and the mean values of these 15 indices measured in this study were all within the reference range. These results indicate that the blood biochemical indices in the present study population do not deviate significantly from those in other healthy cohorts. Correlation analysis between each indicator and age was performed in both the toy poodle and retriever populations. In the toy poodles, TP and AMY showed a significant (p < 0.05) positive correlation with age at both 0 m and 6 m (Fig. 2 , Fig. S1). TBA and Cl showed significant positive and negative correlations with age at 0 m only, respectively. In retrievers, only Cl showed a significant negative correlation with age at 0 m (Fig. 3 , Fig. S2). TP and AMY, which correlated with age in toy poodles, showed positive correlation coefficients in retrievers, although they were not significant at 0 m and 6 m (Fig. 3 , Fig. S2). These data suggest that blood TP and AMY levels are linked to age, commonly observed regardless of the timing analyzed. Whole blood NAD ⁺ concentration To investigate the relationship between in vivo NAD⁺ concentration and age in dogs, we quantified the concentration of NAD⁺ in whole blood at 0 m and 6 m, and analyzed the correlation with age (Fig. 4 and Fig. S3). The mean concentrations of NAD⁺ in whole blood at 0 m and 6 m were 23.5 and 23.6 µM for toy poodles and 20.6 and 23.3 µM for retrievers, respectively. At 0 m, no significant correlation between age and NAD⁺ was identified in either toy poodles or retrievers (Fig. 4 A, B). There were no significant differences in NAD⁺ concentrations among breeds at 0 m (Fig. 4 C). The same trends were observed at 6 m (Fig. S3). These results indicate that the concentration of NAD⁺ in canine whole blood is not significantly correlated with age and that there are no significant differences among breeds. Frailty index The same frailty index as previously reported [ 27 ] was measured in our cohort and analyzed for correlation with age (Fig. 5 ). We found no significant correlation between frailty index and age at 0 m in toy poodles (p = 0.66, Fig. 5 A), and a positive correlation trend at 6 m (p = 0.0502) (Fig. 5 B). In retrievers, frailty index and age showed a significant positive correlation at both 0 m and 6 m (Fig. 5 C, D). Since frailty index is positively related to mortality [ 27 ], we analyzed the relationship between frailty index and age at 0 m, including individuals excluded due to death, and found that frailty index was positively correlated with age in toy poodles (Fig. S4A). In retrievers, the correlation coefficient with frailty index increased with the addition of deceased individuals (Fig. S4B). These results suggest that frailty index is positively correlated with age in retrievers in the present study population, and that in toy poodles it is weakly related to age in healthy individuals but positively related to mortality. Echocardiography To investigate the relationship between cardiac functions and age, echocardiography was performed at 0 m and 6 m on toy poodles over 8 years old, and correlation analysis between each index and age was conducted (Fig. 6 , S4 ). No significant correlation with age was observed for any of the 10 indices measured. However, EDV showed a trend of positive correlations with age at both 0 m and 6 m, and LVIDd and SV at 6 m only (p < 0.1). These results suggest that cardiac function indices such as EDV may correlate with age in older toy poodles, but the relationship is weak. Vaccine Antibody Titer To examine the relationship between aging and vaccine reactivity, antibody titers were measured before (6 m), one month after (7 m), and six months after (12 m) vaccination. Titers at 6 m reflect the persistence of the vaccine shot from a year ago, titers at 7 m reflect an acute vaccine reaction to a new shot, and titers at 12 m reflect the persistence of that effect. The measurements of the Adult (younger individuals) and Senior (older individuals) groups were compared. In toy poodles, distemper virus vaccine antibody titers were significantly lower in the Senior group at 7 m and tended to be lower at 6 m and 12 m compared to the Adult group (Fig. 7 A). Parvo virus vaccine antibody titers in toy poodles were significantly lower in the Senior group than in the Adult group at all three time points (Fig. 7 B). Adeno virus vaccine antibody titers were lower in Seniors than in Adults at all three time points, although none were significant (Fig. 7 C). In retrievers, in contrast to toy poodles, distemper virus vaccine antibody titers were significantly higher in the Senior group at 6 m, and there were no significant differences between the two groups at 7 m and 12 m (Fig. 7 D). Parvo virus vaccine antibody titers in retrievers were not significantly different at 6 m, 7 m, and 12 m (Fig. 7 E). Adeno virus vaccine antibody titers in retrievers were lower on average in Seniors than in Adults at all three time points and were significant at 7 m (Fig. 7 F). These results suggest that both short-term reactivity and persistence to vaccines decline with age in toy poodles across the three viral vaccines, while no consistent changes were identified in retrievers. Fecal microbiome To evaluate the effects of age on the intestinal microbiota of poodles and retrievers, fecal samples were collected at 0 m and 6 m for microbiota analysis. The predominant bacterial phyla identified were Firmicutes, Bacteroidota, Fusobacteriota, Actinobacteriota, and Proteobacteria (Fig. 8 A). These five taxa have been reported to be predominant in the canine gut microbiota [ 58 , 59 ], suggesting that the individuals in this study have a general canine gut microbiota composition. To examine the effect of age, the β-diversity analysis was performed for the Adult and Senior groups at 0 m and 6 m, and found no significant differences were found between Adult and Senior groups in both breeds at either time point (Fig. 8 B-E). In humans, an age-associated increase in Enterobacteriaceae and a decrease in Bifidobacterium and Faecalibacterium have been reported [ 41 , 42 ]. We compared the occupancy of these taxa in the Adult and Senior groups (Fig. S6). In toy poodles, Enterobacteriaceae was significantly lower in the Senior group than in the Adult group only at 0 m, contrary to the human reports. No significant differences were detected in Bifidobacterium and Faecalibacterium between the groups at 0 m and 6 m. Thus, no significant age-related effects on the fecal microbiome were noted in this cohort. Longitudinal analysis of TP and AMY From these extensive cross-sectional analyses at two time points, TP, AMY, and vaccine antibody titer were found to be significantly correlated with chronological aging in toy poodles. The practicability of antibody titer as an indicator of aging is limited because its value fluctuates depending on the timing of vaccination. Therefore, we examined the validity of TP and AMY as chronological aging factors by following their changes over time. For the population in this study, TP and AMY were also measured at 12 m from the start of the study. TP and AMY values were modeled as a function of measurement time (0, 6, and 12 m), and changes per unit time per month were calculated and tested for significant differences in change over time (Fig. 9 ). The results showed that TP increased significantly over time in toy poodles (Fig. 9 A). In retrievers, TP increased over time, but not significantly (Fig. 9 B). In toy poodles, AMY increased over time, but not significantly (Fig. 9 C). In retrievers, AMY showed little variation over time (Fig. 9 D). These results indicate that in toy poodles, TP significantly increased over the course of a year, in addition to showing a positive correlation with age as observed cross-sectionally at two time points. Discussion The primary objective of this study was to identify factors associated with chronological aging in small-sized dogs. We found that TP and AMY were positively correlated with age, and TP continuously increased over a one-year period in a cohort of healthy toy poodles. Vaccine responses for several viruses declined in the older age groups. The frailty index was positively related to age only when the population included individuals who died during the study. No significant relationship with age was found for whole blood NAD⁺ concentration, echocardiographic indices, or fecal microbiome. Similar trends of an increase in TP and AMY were observed in retrievers. In contrast, vaccine antibody titers showed no consistent changes with age in retrievers. Plasma biochemical indices are among the most frequently investigated in relation to aging in dogs [ 12 – 15 ]. Several indices, including TP, have been positively correlated with age in studies of mixed breeds or single medium or large breeds [ 12 , 14 , 15 ]. However, it is not clear which of these indices are particularly linked to aging. Our findings that TP is consistently correlated with aging in poodles in both cross-sectional and longitudinal analyses indicate that TP may be used as a sensitive aging indicator in small dogs. A similar trend was also observed in retrievers, although the reference population was relatively small. Additional studies are needed to determine whether TP can also be applied to medium and large dogs. Regarding the relationship between the type of protein in the blood of dogs and age, albumin (ALB), the most abundant protein in the blood, typically decreases in late aging or shows no strong correlation with age. In contrast, globulin, the second most abundant protein, generally increases with age [ 13 – 15 ]. In this cohort, we found no significant relationship between age and ALB in toy poodles or retrievers, while globulin was not measured. Chronic kidney disease has been reported to cause hypoalbuminemia in dogs [ 60 ]. It is possible that ALB levels in this study focusing on healthy individuals were not affected because it did not include individuals with hypoalbuminemia. Further studies are needed to determine which protein species account for the age-dependent increase in TP. In the present study, toy poodles showed a decrease in vaccine antibody titers with age, which is consistent with previous reports in other medium and large-sized breeds [ 22 , 23 ]. On the other hand, in retrievers, the vaccination antibody titers of the three viruses responded differently: Adeno virus antibody titers showed an age-related decrease, while Distemper virus and Parvo virus antibody titers showed no consistent age-related relationship. The cause of this is unknown, but it is possible that some individuals were subclinically infected, or vaccine reactivity might be unique to retrievers. Further studies on a larger scale including antibody titer measurements for other vaccine types are needed for a detailed analysis. NAD⁺ levels decrease with age in worms, flies, and mice, and have also been reported to decrease in human tissues such as blood, brain, and skin in data from relatively small populations [ 31 – 33 , 61 ] We were unable to find any prior reports quantifying in vivo NAD⁺ levels in dogs until the time of manuscript preparation. We measured whole blood NAD⁺ levels in the present study and found no significant relationship with age in either of the breeds. The mean NAD⁺ concentration in dogs was 23 µM, which was slightly lower than the human NAD⁺ concentration of 26 µM measured by the same extraction method used in this study [ 48 ]. While these results do not rule out the possibility of a correlation between NAD⁺ levels and aging in other tissues, they do indicate that it is highly unlikely that NAD⁺ levels in whole blood can be used as a sensitive marker of aging in healthy dogs. We evaluated the frailty index and echocardiography as indicators of health-related phenotypes that can be assessed by veterinarians and nurses. Regarding the frailty index, which has been reported to correlate with aging and mortality in a diverse population of large- and medium-sized breeds [ 27 ], no significant relationship with age was found in the toy poodle population we evaluated. However, in an analysis including individuals who died during the study, the frailty index at study entry showed a significant positive correlation with age. In retrievers, the correlation coefficient was also higher in analyses that included deceased individuals. These results suggest that the frailty index might be a stronger predictor of mortality than chronological age. There is little reported common relationship between the fecal microbiome and aging in dogs. In humans, an increase in Enterobacteriaceae and a decrease in Bifidobacterium and Faecalibacterium have been reported with aging [ 41 , 42 ]. However, a comparison of the Adult and Senior groups of poodles in this study found no commonality with these reports. No significant differences in beta diversity were detected between the two age groups. We believe that this group of dogs was characterized by a uniform living environment and a uniform diet, with relatively few confounding factors influencing the gut microbiota. Nevertheless, the lack of a significant correlation suggests that while we do not rule out the possibility that some endemic bacterial species may correlate with aging in certain regions or under certain conditions, it is difficult to use the canine gut microbiota as a sensitive marker of chronological aging. In summary, we found that elevated plasma total protein is a relatively sensitive indicator of chronological aging in healthy small-sized dogs. We also found that NAD⁺ levels and microbiome composition, which have been reported as aging markers in other species, have very low sensitivity as markers of chronological aging, regardless of dog size. Limitations of this study include its single-center design and the limited breeds included. To generalize the aging-related indices to all small dogs, studies should be conducted at multiple institutions and include small dog breeds other than toy poodles. Additionally, the relationship between the aging indices identified in this study and the decline in body function with aging needs to be examined in a longitudinal study over a longer time span. Such further efforts will lead to the development of methods for better understanding and preventing aging in dogs. Declarations Acknowledgements This research was supported by RIKEN Aging Project (to M.Y), Japan Society for the Promotion of Science (JSPS) KAKENHI (20K11553 to T.K.I.), and funding by Meiji Holdings Co., Ltd. Funding This research was supported by RIKEN Aging Project (to M.Y), Japan Society for the Promotion of Science (JSPS) KAKENHI (20K11553 to T.K.I.), and funding by Meiji Holdings Co., Ltd. Data availability 16S rDNA sequencing data is available under accession number DRA019236 (under project number PRJDB18730) from the DDBJ DRA database (https://www.ddbj.nig.ac.jp/dra/index-e.html). Other data that support the findings of this study are available from the corresponding authors, T.K.I and S.O., upon reasonable request. Author contributions T.K.I conceived and S.O. and T.K.I. conceptualized the study. S.O. and T.K.I. designed the experiments and analyzed the data. S.O. developed figures. S.O. performed blood biochemistry and antibody titer analysis. S.O. and S.H. performed blood NAD⁺ measurement. E.M. performed cardio echography. E.M. performed the frailty index measurement. Y.H. performed microbiome sequencing. K.H. performed statistical analysis for microbiome data. M.M. and M.Y. provided intellectual input and essential resources for project completion. S.O. and T.K.I. prepared the manuscript with input from co-authors. Ethics declarations Animal Ethics The protocol was approved by the Laboratory Animal Committee of Meiji Holdings Corporation (Application No. 2020_1000_0034) and was conducted in compliance with experimental facility guidelines. Conflict of interest S.O., Y.H., S.H., K.H., and M.M are employees of Meiji Holdings Co., Ltd. References Bergstrom A et al Origins and genetic legacy of prehistoric dogs. Science, 370, 6516, pp. 557–564, Oct 30 2020, 10.1126/science.aba9572 Parker HG et al Genomic Analyses Reveal the Influence of Geographic Origin, Migration, and Hybridization on Modern Dog Breed Development. 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Supplementaryfigurelegends.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5341224","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374084390,"identity":"5da0ea90-b4d6-4fb9-b4cc-9d9900288695","order_by":0,"name":"Satoru Ozaki","email":"","orcid":"","institution":"Meiji Holdings Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Ozaki","suffix":""},{"id":374084393,"identity":"03bf58aa-fff5-4229-9ac8-f116746080e9","order_by":1,"name":"Yoshiko Honme","email":"","orcid":"","institution":"Meiji Holdings Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yoshiko","middleName":"","lastName":"Honme","suffix":""},{"id":374084394,"identity":"11b5e31b-d07e-4eb2-9cf5-8a9806d22ea0","order_by":2,"name":"Seiichiro Higashi","email":"","orcid":"","institution":"Meiji Holdings Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Seiichiro","middleName":"","lastName":"Higashi","suffix":""},{"id":374084395,"identity":"2ca0fe3a-fd42-478c-aca2-350c92a009a7","order_by":3,"name":"Kouya Hattori","email":"","orcid":"","institution":"Meiji Holdings Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kouya","middleName":"","lastName":"Hattori","suffix":""},{"id":374084396,"identity":"6e2aad6e-843e-4519-9f39-89d040fa63d7","order_by":4,"name":"Masashi Morifuji","email":"","orcid":"","institution":"Meiji Holdings Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Morifuji","suffix":""},{"id":374084399,"identity":"df054082-62ad-491b-b1c9-04428ec37ca6","order_by":5,"name":"Eriko Mizuno","email":"","orcid":"","institution":"TSUKUBA PET Professional School","correspondingAuthor":false,"prefix":"","firstName":"Eriko","middleName":"","lastName":"Mizuno","suffix":""},{"id":374084401,"identity":"11bbaaa8-f0b2-49c2-837d-0c7feea002d0","order_by":6,"name":"Minoru Yoshida","email":"","orcid":"","institution":"RIKEN Center for Sustainable Resource Science","correspondingAuthor":false,"prefix":"","firstName":"Minoru","middleName":"","lastName":"Yoshida","suffix":""},{"id":374084403,"identity":"ae5d3289-219f-4aa0-8745-f7c275a186e6","order_by":7,"name":"Takashi K. 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B, Schedule of the trial.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409111.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/f96003a36da85470ad3f8b99.png"},{"id":68391493,"identity":"857427d0-5289-438e-b82a-d8ee4bed89b5","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72755,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between age and blood biochemical indicators in toy poodles at 0 m.\u003c/p\u003e\n\u003cp\u003eρ represents the Spearman rank correlation coefficient between age and each indicator.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409112.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/a5c1aa1d479f0fd8a4f4585c.png"},{"id":68391495,"identity":"d9a9d46a-9e26-43b9-98bd-4dc200938529","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60106,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between age and blood biochemical indicators in retrievers at 0 m.\u003c/p\u003e\n\u003cp\u003eρ represents the Spearman rank correlation coefficient between age and each indicator.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409113.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/042e429fd7af35ca39cae65d.png"},{"id":68391499,"identity":"86fa0ae3-2266-495e-8db1-b463668254b7","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35326,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between age and whole blood NAD⁺ concentrations at 0 m.\u003c/p\u003e\n\u003cp\u003eA, Correlation between age and NAD⁺ concentration in the whole blood of toy poodles. B, Correlation between age and NAD⁺ concentration in the whole blood of retrievers. ρ represents the Spearman rank correlation coefficient between age and NAD⁺ concentration. C, Whole blood NAD⁺ concentrations in toy poodles and retrievers.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409114.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/921b48aeb482177b4344a7b4.png"},{"id":68391497,"identity":"837f7e79-f1b8-4e03-9024-812d0af2d37f","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36001,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between age and the frailty index.\u003c/p\u003e\n\u003cp\u003eA, Correlation between age and the frailty index in toy poodles at 0 m. B, Correlation between age and frailty index in toy poodles at 6 m. C, Correlation between age and the frailty index in retrievers at 0 m. D, Correlation between age and the frailty index in retrievers at 6 m. ρ represents the Spearman rank correlation coefficient between age and each indicator.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409115.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/89966e4da14fd706705bd16e.png"},{"id":68391805,"identity":"1268b305-bed0-4578-b3eb-1ce0bf00daf7","added_by":"auto","created_at":"2024-11-06 19:40:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53622,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between age and echocardiogram indices in toy poodles at 0 m.\u003c/p\u003e\n\u003cp\u003eρ represents the Spearman rank correlation coefficient between age and each indicator.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409116.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/c4f34c7ef78bb407c10e0287.png"},{"id":68391806,"identity":"340a2c1b-c2e7-40b9-9908-b06a28a4291f","added_by":"auto","created_at":"2024-11-06 19:40:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":60853,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of vaccination on distemper, parvo and adeno virus antibody titers.\u003c/p\u003e\n\u003cp\u003eA, Distemper virus antibody titer of toy poodles. B, Parvo virus antibody titer of toy poodles. C, Adeno virus antibody titer of toy poodles. D, Distemper virus antibody titer of retrievers. E, Parvo virus antibody titer of retrievers. F, Adeno virus antibody titer of retrievers. Y-axis indicates logarithmically transformed antibody titer values. X-axis indicates the number of months since the start of the study. Vaccination was administered immediately after blood collection at 6 months. The Mann-Whitney test was used to test significant differences between the Adult and Senior groups at each time point. **: p \u0026lt; 0.01, *: p \u0026lt; 0.05, #: p \u0026lt; 0.1, ns: p \u0026gt; 0.1.\u003c/p\u003e","description":"","filename":"FiguresOzakietal2409117.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/2708bc9e811bb9834873e31d.png"},{"id":68391501,"identity":"97398b7a-ebd3-4913-b533-7514cc863698","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":159156,"visible":true,"origin":"","legend":"\u003cp\u003eAverage phylum-level composition of bacteria in dog feces.\u003c/p\u003e\n\u003cp\u003eA, Average composition according to breeds at 0 m and 6 m. B, Average composition of bacteria in feces according to age groups of toy poodles at 0 m. C, Average composition of bacteria in feces by age groups of toy poodles at 6 m. D, Average composition of bacteria in feces by age groups of retrievers at 0 m. E, Average composition of bacteria in feces by age groups of retrievers at 6 m. The significance of the difference between the Adult and Senior groups in β diversities was tested using the PERMANOVA test with the Weighted UniFrac Distance. ns: p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/2e3272349bea31858ee93993.png"},{"id":68391502,"identity":"7b2091b9-dcdb-4b24-8acf-0f4bb6141f53","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":49068,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal analysis of blood total protein (TP) and amylase (AMY).\u003c/p\u003e\n\u003cp\u003eA, TP in toy poodles. B, TP in retrievers. C, AMY in toy poodles. D, AMY in retrievers. The values were measured at 0, 6, and 12 m. Dots represent measurements for each individual, and dots connected by lines indicate measurements for the same individual. White bars indicate mean values. Slope and p-value indicate the variability of TP and AMY per month calculated by the mixed model and its significance, respectively.\u003c/p\u003e","description":"","filename":"FiguresOzakietal24091110.png","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/ed228dae1af6fd9d5d6877d1.png"},{"id":80485642,"identity":"ec4fb10c-826e-43ab-ae0c-84c7c8656855","added_by":"auto","created_at":"2025-04-13 15:31:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1300760,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/d6df78f5-9af4-4571-943e-5ba32fedf085.pdf"},{"id":68391498,"identity":"39914066-8718-4d84-ad81-f0413bf668f2","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2365352,"visible":true,"origin":"","legend":"\u003cp\u003eFigures 1-9 and Supplementary Figures 1-7.\u003c/p\u003e","description":"","filename":"FiguresOzakietal240911.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/257168879eef483fdda85419.pdf"},{"id":68391500,"identity":"1082eb74-0b25-411d-8383-7bd9a3eb4f3e","added_by":"auto","created_at":"2024-11-06 19:32:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15149,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5341224/v1/ddfdea6f97b19739358fbc49.docx"}],"financialInterests":"Competing interest reported. S.O., Y.H., S.H., K.H., and M.M are employees of Meiji Holdings Co., Ltd.","formattedTitle":"Plasma protein increase as a chronological aging factor in healthy toy poodles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDogs have been domesticated for over 10,000 years and have long been integral members of human society [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Selective breeding has produced a variety of inbred lineages, resulting in approximately 400 pet dog breeds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to strong artificial selection, domesticated dogs have become one of the most morphologically diverse mammalian species, with significant variation in size among breeds. The average adult dog weight varies more than 30-fold from breed to breed. Based on this size diversity, breeds are classified as large (average adult weight\u0026thinsp;\u0026gt;\u0026thinsp;25 kg), medium (10\u0026ndash;25 kg), or small (\u0026lt;\u0026thinsp;10 kg) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Size influences lifespan and aging rates in each breed, with an inverse correlation between size and lifespan [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Different breeds also exhibit varying rates of age-related diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Canine breed size is positively associated with IGF-1 levels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and slower aging progression in small breeds is attributed to relatively low IGF-1 levels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lowering IGF-1 signaling has been shown to increase lifespan in model organisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings highlight the need for studies across different breed sizes to better understand aging in pet dogs.\u003c/p\u003e \u003cp\u003eMultiple reports exist on factors associated with aging in dogs. It has been reported that DNA methylome analysis can estimate canine chronological age [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several studies measuring blood biochemical indices have reported correlations between age and increased levels in total protein, globulin, ALT, albumin, and glucose [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While these studies have found common relationships between each factor and age, they are limited to large and medium breeds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] or to populations of multiple breeds in which these sizes are predominantly present [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While the population of small-sized breeds has increased in recent years, it remains largely unclear whether these findings can be adapted to small dogs or if they have different aging profiles. Additionally, many studies are cross-sectional, and there are limited reports analyzing aging-related factors in the same population through longitudinal analysis.\u003c/p\u003e \u003cp\u003eIt has been reported that in humans, immune function declines with age and vaccine response decrease [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This decreased reactivity has also been observed for vaccines against COVID-19 across a wide geographic and racial spectrum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], suggesting decreased reactivity to new antigens. Post-vaccination antibody titers have also been reported to decrease in large and medium-sized dogs as they age, but reports on small breeds are limited [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrailty is a condition commonly seen in the elderly that increases the risk of falls, disability, hospitalization, and death [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In humans, several indices have been proposed to objectively assess the degree of frailty, and these are used clinically for health management [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A method for calculating the frailty index has also been proposed for dogs, showing a modest correlation between aging and the index, and a strong association with mortality regardless of age [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the number of studies on small dogs is relatively small, and the association is weak [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNAD⁺ (nicotinamide adenine dinucleotide) is a coenzyme involved in energy metabolism, DNA repair, and cell signaling [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It has been reported that NAD⁺ levels in vivo decrease with aging common in eukaryotes including humans [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], rodents [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and nematodes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In mice, the intake of NAD⁺ precursors increases NAD⁺ levels and improves tissue functions and metabolism [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, no reports on the relationship between aging and in vivo NAD⁺ levels in dogs have been published at the time of this manuscript's submission.\u003c/p\u003e \u003cp\u003eThe composition of the human gut microbiota has been associated with various poor health conditions and diseases such as obesity, diabetes, and inflammatory diseases [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The human gut microbiome also changes with age, with a decrease in \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e and an increase in Enterobacteriaceae reported with aging [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, frail humans show a decrease in \u003cem\u003eFaecalibacterium\u003c/em\u003e and an increase in Enterobacteriaceae compared to non-frail individuals, exhibiting a trend similar to chronological aging [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These findings suggest that the human gut microbiota is linked to health and aging. In contrast, studies on the relationship between age and gut microbiota in dogs remain sparse, and consistent trends have not been confirmed [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we aim to assess changes associated with chronological aging in healthy dogs by measuring multifaceted parameters. Toy poodles were analyzed as representatives of small dogs, and retrievers were analyzed as representatives of large dogs for comparison. We measured blood biochemistry, blood NAD⁺ levels, fecal microbiome, antibody titers as vaccine responses against multiple viruses, echocardiographic measurements, and the frailty index as candidate parameters. All parameters, except for antibody titers, were measured twice with a six-month interval between measurements. We analyzed the relationship between age and various indicators. Indicators with the strongest correlations were subjected to longitudinal analysis over a one-year period to evaluate whether there were consistent changes with age over time.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e Toy poodles, golden retrievers, and labrador retrievers kept at the same facility in Ibaraki Prefecture, Japan, were recruited for the study with the consent of the owners. We excluded dogs with pre-existing diseases, those diagnosed by a veterinarian with poor health status at the start of the study, and those that died within 24 months from the start due to the possibility of being unhealthy. Animals that became pregnant during the study were excluded upon confirmation of pregnancy. In some analyses, participants were stratified based on their age at the start of the study. Toy poodles under 7 years old and retrievers under 6 years old were classified as \"Adult,\" while participants older than these thresholds were classified as \"Senior\" [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The two breeds of retriever were merged in the data analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood sampling\u003c/h3\u003e\n\u003cp\u003eThe participants were subjected to fasting from the evening prior to blood collection, with water intake allowed ad libitum. The fasting period was within 12 to 24 hours. Blood samples were collected using a syringe from the cephalic vein. After collection, the blood samples were transferred to silicon-coated blood collection tubes (#365963, Becton, Dickinson and Company, NJ, USA) for biochemical and antibody titer testing, and to evacuated tubes containing sodium fluoride, citric acid, sodium citrate, and EDTA-2Na (VP-FH051K, Terumo, Japan) for NAD\u003csup\u003e+\u003c/sup\u003e quantification.\u003c/p\u003e\n\u003ch3\u003ePlasma biochemical analysis\u003c/h3\u003e\n\u003cp\u003ePlasma was obtained by centrifuging the tubes at room temperature at 3000 rpm for 10 minutes, after which the supernatant was collected as plasma. Plasma was kept refrigerated until biochemical analysis and antibody titer measurement. The concentrations of total protein (TP), albumin (ALB), lipase (LIP), calcium (Ca), and total bilirubin (TBIL) were measured by the biuret method, bromocresol green method, DGGR substrate chromogenic method, o-cresolphthalein complexone method, and chemical oxidation method, respectively. Blood urea nitrogen (BUN), creatinine (CRE), blood glucose (GLU), total cholesterol (TC), triglycerides (TG), total bile acids (TBA), and inorganic phosphorus (P) were measured by enzymatic methods. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and amylase (AMY) were measured by the Japanese Society of Clinical Chemistry standardization method. Sodium (Na), potassium (K), and chloride (Cl) were measured by the electrode method. AMY, LIP, ALT, and AST were analyzed using log\u003csub\u003e10\u003c/sub\u003e-transformed values.\u003c/p\u003e\n\u003ch3\u003eQuantification of whole blood NAD concentration\u003c/h3\u003e\n\u003cp\u003eThe processing method for blood samples for NAD\u003csup\u003e+\u003c/sup\u003e quantification followed the protocol described in a previous report [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. After sampling, blood collection tubes were gently inverted several times and 20 \u0026micro;L of collected blood was immediately treated with 980 \u0026micro;L of cold 55% MeOH and transferred to a -80\u0026deg;C freezer for long-term storage. Prior to measurement, stable isotopes of NAD\u003csup\u003e+\u003c/sup\u003e, nicotinamide adenine dinucleotide-13C5 (Syncom), were added to thawed samples, followed by ultrafiltration with Amicon filters with a molecular weight cutoff of 10 kDa (Millipore). Half of the flow-through was evaporated in a centrifugal evaporator. Samples were dissolved in 1 mL of a 10 mM ammonium acetate solution and further diluted 10-fold for quantification using LC-MS/MS. LC-MS/MS analyses were performed using a Shimadzu HPLC system (Shimadzu, Japan) coupled with a QTRAP4500 mass spectrometer (AB Sciex, MA, USA). Chromatographic separation was achieved on a Hypercarb column (2.1 mm \u0026times; 100 mm, 3 \u0026micro;m particle size, Thermo Scientific, WI, USA). The column temperature was maintained at 60\u0026deg;C. The mobile phase consisted of 10 mM ammonium acetate with 0.05% (v/v) ammonium hydroxide in water (A) and 0.05% (v/v) ammonium hydroxide in acetonitrile (B), delivered at a flow rate of 0.2 mL/min. The injection volume was 3 \u0026micro;L. The gradient elution started with 5% B for the first 1.8 min, increased to 54% B from 1.8 min to 14 min, then to 90% B from 14.0 min to 14.10 min, held at 90% B until 17.1 min, decreased linearly to 5% B from 17.1 min to 17.2 min, and remained at 5% B until 32.2 min. The MS/MS analysis was performed in positive ESI mode. The detection of NAD\u003csup\u003e+\u003c/sup\u003e was performed by multiple reaction monitoring. The ions were selected in the first quadrupole (Q1) and collided with nitrogen gas in the second quadrupole (Q2), and the product ions were detected in the third quadrupole (Q3). The m/z value of the precursor ions detected at Q1 was 664.2 (NAD\u003csup\u003e+\u003c/sup\u003e) and 669.1 (stable isotope-labeled NAD\u003csup\u003e+\u003c/sup\u003e). The collision energy at Q2 was 38 V. The m/z values for the product ions detected at Q3 were both 428.2 (NAD\u003csup\u003e+\u003c/sup\u003e and stable isotope-labeled NAD\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eEchocardiography was performed using a LOGIQ P6 (GE Healthcare, USA). IVSd (mm), LVIDd (mm), LVIDs (mm), and LVPWd (mm) were measured in M-mode using the right parasternal left ventricular short-axis cross-sectional image in the right lateral recumbent position with hand-holding without instruments. EDV (ml), ESV (ml), EF (%), SV (ml), and FS (%) were calculated from these measurements. Additionally, the E/A ratio was measured using the pulsed Doppler method for inflow blood flow at the mitral valve, using a left parasternal apical quadrant cross-sectional image in the left lateral recumbent position. Each measurement was performed 2\u0026ndash;5 times, and the average value was recorded.\u003c/p\u003e \u003cp\u003eAbbreviations\u003c/p\u003e \u003cp\u003eof each parameter are as follows:\u003c/p\u003e \u003cp\u003eIVSd: Interventricular Septal thickness in diastole\u003c/p\u003e \u003cp\u003eLVIDd: Left Ventricular Internal Dimension in diastole\u003c/p\u003e \u003cp\u003eLVIDs: Left Ventricular Internal Dimension in systole\u003c/p\u003e \u003cp\u003eLVPWd: Left Ventricular Posterior Wall thickness in diastole\u003c/p\u003e \u003cp\u003eEDV: End-Diastolic Volume\u003c/p\u003e \u003cp\u003eESV: End-Systolic Volume\u003c/p\u003e \u003cp\u003eEF: Ejection Fraction\u003c/p\u003e \u003cp\u003eSV: Stroke Volume\u003c/p\u003e \u003cp\u003eFS: Fractional Shortening\u003c/p\u003e \u003cp\u003eE/A ratio: Early diastolic mitral inflow velocity / Atrial systolic mitral inflow velocity ratio\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVaccination and measurement of antibody titer\u003c/h2\u003e \u003cp\u003eAt 6 months after the start of the study (6 m), the participants received a vaccination with Vanguard Plus 5CV/L (Zoetis, NJ, USA). This formulation included vaccines for distemper, parvo, and adeno viruses with potencies of over 10\u003csup\u003e3.5\u003c/sup\u003e, 10\u003csup\u003e6.4\u003c/sup\u003e, and 10\u003csup\u003e5.5\u003c/sup\u003e TCID50, respectively. The vaccination history prior to this vaccination was 12 months ago, except for two toy poodles who were excluded from the analysis. Blood samples were collected before vaccination (6 m), 1 month after vaccination (7 m), and 6 months after vaccination (12 m) to measure antibody titers for distemper, parvo, and adeno viruses. Plasma was obtained using the same method described in the biochemical test section. Antibody titers were measured by fluorescence immunoassay.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFrailty index\u003c/h3\u003e\n\u003cp\u003eThe frailty index was evaluated using the method described in a previous report [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, 33 items related to frailty were assessed by veterinary nurses and a veterinary physician. Some health deficits were assigned a score of 0 (absent) or 1 (present), while other deficits were evaluated using a semi-quantitative scale as absent (score\u0026thinsp;=\u0026thinsp;0), mild (score\u0026thinsp;=\u0026thinsp;0.5), or severe (score\u0026thinsp;=\u0026thinsp;1). The frailty index was calculated by dividing the sum of the scores by the total number of items (33).\u003c/p\u003e\n\u003ch3\u003eFecal sampling and DNA extraction\u003c/h3\u003e\n\u003cp\u003eFeces naturally excreted were quickly collected into a feces container (Sarstedt) and frozen until DNA extraction. Whole genomic DNA from feces was extracted according to \"protocol Q\" from a previous report [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], with slight modification. Twenty mg of feces was measured into a tube containing 0.3 g of 0.1 mm diameter zirconia beads, and 1 mL of Buffer ASL (QIAGEN, Hilden, Germany) was added. The mixture was vortexed and then held at 95\u0026deg;C for 15 min. After incubation, the sample was shaken 4 times for 1 min at 5 m/s using a FastPrep-24 instrument (MP Biomedicals, USA) and then centrifuged. The supernatant was collected in another tube, and the pellet was resuspended in Buffer ASL. The mixture was incubated, shaken, and centrifuged again, and the supernatant was combined with the previous one. A 1/5 volume of 10 M ammonium acetate solution was added to the supernatant and incubated for 5 min. The mixture was centrifuged, the supernatant was collected, and an equal volume of isopropanol was added. After standing on ice for 30 min, the tube was centrifuged, the supernatant was removed, and the pellet was washed with 70% ethanol. The pellet was dissolved in 200 \u0026micro;L of TE buffer and then treated with RNase and Protease K. Then 200 \u0026micro;L of ethanol was added and vortexed. The solution was placed in a QIAamp spin column (QIAGEN) and centrifuged at 16,000 g for 1 min. The column was flushed with 500 \u0026micro;L of Buffer AW1 (QIAGEN) and then flushed with the same volume of Buffer AW2 (QIAGEN). The column was placed on a fresh tube, and Buffer ATE (QIAGEN) was added. After a 1 min incubation, the column was centrifuged, and the eluent was used as the genomic DNA solution.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA gene sequencing library preparation and sequencing\u003c/h2\u003e \u003cp\u003eThe V3-V4 region of the bacterial 16S rRNA gene was amplified using genomic DNA as a template and primers 5\u0026rsquo;-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3\u0026rsquo; and 5\u0026rsquo;-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3\u0026rsquo; [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Template genomic DNA concentration was measured using Quant-iT\u0026trade; dsDNA Assay Kits (Thermo Fisher Scientific) and adjusted to 5 ng/\u0026micro;L. 16S Metagenomic Sequencing Library Preparation was performed according to the manufacturer's instructions (Illumina, CA, USA). The DNA library was sequenced using a MiSeq System (Illumina) with a 2 \u0026times; 300-base-pair protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of 16S rDNA sequencing data\u003c/h2\u003e \u003cp\u003eSequencing data were analyzed using Qiime2(version 2020.11) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. To trim the primer region from raw sequences, the Cutadapt plugin in Qiime2 was used [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Sequences without the primer region were processed for quality control, paired-end read joining, chimera filtering, and amplicon sequence variant (ASV) table construction using the DADA2 algorithm [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. For each representative sequence of ASV, BLAST [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] was used to assign the taxonomy based on the SILVA database (version 138) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. After random sampling of 14,000 reads using the feature-table plugin [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], conversion of compositional data and diversity analysis was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eR (ver. 4.3.2) was used for linear mixed model analysis of TP and AMY using the lme4 package. TP and AMY were modeled as a function of the time of measurement, with time included in the model as a fixed effect and individual as a random effect. Permanova analysis with Weighted UniFrac Distance was used to test for significant differences in fecal microbiota β-diversity between groups. GraphPad Prism 8 was used for Mann-Whitney U-test and Spearman's rank correlation analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStudy Subjects\u003c/h2\u003e \u003cp\u003eForty toy poodles, eight golden retrievers, and nine labrador retrievers were recruited for this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Two toy poodles and two retrievers were excluded due to health check results at the start of the study. During the study, six toy poodles and five retrievers were excluded due to death, and four toy poodles and one retriever were excluded due to pregnancy. Twenty-eight toy poodles, four golden retrievers, and five labrador retrievers successfully completed the study. The age of the participants who completed the study ranged from 1 to 14 years at the start. The median ages for toy poodles and retrievers were 6.7 years and 5.3 years, respectively. Based on the threshold, 15 toy poodles and five retrievers were assigned as adults, while 13 toy poodles and four retrievers were assigned as seniors. The average body weight of the completed toy poodles and retrievers at the start were 4.7 and 24.2 kilograms, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePlasma biochemistry tests\u003c/h2\u003e \u003cp\u003eBlood was collected from each animal at the start of the study (0 m) and six months later (6 m), and 18 plasma biochemical indices were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Fifteen of the 18 indices were compared to the reference range [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and the mean values of these 15 indices measured in this study were all within the reference range. These results indicate that the blood biochemical indices in the present study population do not deviate significantly from those in other healthy cohorts.\u003c/p\u003e \u003cp\u003eCorrelation analysis between each indicator and age was performed in both the toy poodle and retriever populations. In the toy poodles, TP and AMY showed a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) positive correlation with age at both 0 m and 6 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. S1). TBA and Cl showed significant positive and negative correlations with age at 0 m only, respectively. In retrievers, only Cl showed a significant negative correlation with age at 0 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig. S2). TP and AMY, which correlated with age in toy poodles, showed positive correlation coefficients in retrievers, although they were not significant at 0 m and 6 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig. S2). These data suggest that blood TP and AMY levels are linked to age, commonly observed regardless of the timing analyzed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole blood NAD\u003c/b\u003e⁺ \u003cb\u003econcentration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the relationship between in vivo NAD⁺ concentration and age in dogs, we quantified the concentration of NAD⁺ in whole blood at 0 m and 6 m, and analyzed the correlation with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig. S3). The mean concentrations of NAD⁺ in whole blood at 0 m and 6 m were 23.5 and 23.6 \u0026micro;M for toy poodles and 20.6 and 23.3 \u0026micro;M for retrievers, respectively. At 0 m, no significant correlation between age and NAD⁺ was identified in either toy poodles or retrievers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). There were no significant differences in NAD⁺ concentrations among breeds at 0 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The same trends were observed at 6 m (Fig. S3). These results indicate that the concentration of NAD⁺ in canine whole blood is not significantly correlated with age and that there are no significant differences among breeds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFrailty index\u003c/h2\u003e \u003cp\u003eThe same frailty index as previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was measured in our cohort and analyzed for correlation with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We found no significant correlation between frailty index and age at 0 m in toy poodles (p\u0026thinsp;=\u0026thinsp;0.66, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and a positive correlation trend at 6 m (p\u0026thinsp;=\u0026thinsp;0.0502) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In retrievers, frailty index and age showed a significant positive correlation at both 0 m and 6 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Since frailty index is positively related to mortality [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we analyzed the relationship between frailty index and age at 0 m, including individuals excluded due to death, and found that frailty index was positively correlated with age in toy poodles (Fig. S4A). In retrievers, the correlation coefficient with frailty index increased with the addition of deceased individuals (Fig. S4B). These results suggest that frailty index is positively correlated with age in retrievers in the present study population, and that in toy poodles it is weakly related to age in healthy individuals but positively related to mortality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between cardiac functions and age, echocardiography was performed at 0 m and 6 m on toy poodles over 8 years old, and correlation analysis between each index and age was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). No significant correlation with age was observed for any of the 10 indices measured. However, EDV showed a trend of positive correlations with age at both 0 m and 6 m, and LVIDd and SV at 6 m only (p\u0026thinsp;\u0026lt;\u0026thinsp;0.1). These results suggest that cardiac function indices such as EDV may correlate with age in older toy poodles, but the relationship is weak.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eVaccine Antibody Titer\u003c/h2\u003e \u003cp\u003eTo examine the relationship between aging and vaccine reactivity, antibody titers were measured before (6 m), one month after (7 m), and six months after (12 m) vaccination. Titers at 6 m reflect the persistence of the vaccine shot from a year ago, titers at 7 m reflect an acute vaccine reaction to a new shot, and titers at 12 m reflect the persistence of that effect. The measurements of the Adult (younger individuals) and Senior (older individuals) groups were compared. In toy poodles, distemper virus vaccine antibody titers were significantly lower in the Senior group at 7 m and tended to be lower at 6 m and 12 m compared to the Adult group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Parvo virus vaccine antibody titers in toy poodles were significantly lower in the Senior group than in the Adult group at all three time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Adeno virus vaccine antibody titers were lower in Seniors than in Adults at all three time points, although none were significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn retrievers, in contrast to toy poodles, distemper virus vaccine antibody titers were significantly higher in the Senior group at 6 m, and there were no significant differences between the two groups at 7 m and 12 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Parvo virus vaccine antibody titers in retrievers were not significantly different at 6 m, 7 m, and 12 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Adeno virus vaccine antibody titers in retrievers were lower on average in Seniors than in Adults at all three time points and were significant at 7 m (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThese results suggest that both short-term reactivity and persistence to vaccines decline with age in toy poodles across the three viral vaccines, while no consistent changes were identified in retrievers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiome\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of age on the intestinal microbiota of poodles and retrievers, fecal samples were collected at 0 m and 6 m for microbiota analysis. The predominant bacterial phyla identified were Firmicutes, Bacteroidota, Fusobacteriota, Actinobacteriota, and Proteobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). These five taxa have been reported to be predominant in the canine gut microbiota [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], suggesting that the individuals in this study have a general canine gut microbiota composition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the effect of age, the β-diversity analysis was performed for the Adult and Senior groups at 0 m and 6 m, and found no significant differences were found between Adult and Senior groups in both breeds at either time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-E). In humans, an age-associated increase in Enterobacteriaceae and a decrease in \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e have been reported [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We compared the occupancy of these taxa in the Adult and Senior groups (Fig. S6). In toy poodles, Enterobacteriaceae was significantly lower in the Senior group than in the Adult group only at 0 m, contrary to the human reports. No significant differences were detected in \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e between the groups at 0 m and 6 m. Thus, no significant age-related effects on the fecal microbiome were noted in this cohort.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLongitudinal analysis of TP and AMY\u003c/h2\u003e \u003cp\u003eFrom these extensive cross-sectional analyses at two time points, TP, AMY, and vaccine antibody titer were found to be significantly correlated with chronological aging in toy poodles. The practicability of antibody titer as an indicator of aging is limited because its value fluctuates depending on the timing of vaccination. Therefore, we examined the validity of TP and AMY as chronological aging factors by following their changes over time.\u003c/p\u003e \u003cp\u003eFor the population in this study, TP and AMY were also measured at 12 m from the start of the study. TP and AMY values were modeled as a function of measurement time (0, 6, and 12 m), and changes per unit time per month were calculated and tested for significant differences in change over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The results showed that TP increased significantly over time in toy poodles (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). In retrievers, TP increased over time, but not significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). In toy poodles, AMY increased over time, but not significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). In retrievers, AMY showed little variation over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). These results indicate that in toy poodles, TP significantly increased over the course of a year, in addition to showing a positive correlation with age as observed cross-sectionally at two time points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary objective of this study was to identify factors associated with chronological aging in small-sized dogs. We found that TP and AMY were positively correlated with age, and TP continuously increased over a one-year period in a cohort of healthy toy poodles. Vaccine responses for several viruses declined in the older age groups. The frailty index was positively related to age only when the population included individuals who died during the study. No significant relationship with age was found for whole blood NAD⁺ concentration, echocardiographic indices, or fecal microbiome. Similar trends of an increase in TP and AMY were observed in retrievers. In contrast, vaccine antibody titers showed no consistent changes with age in retrievers.\u003c/p\u003e \u003cp\u003ePlasma biochemical indices are among the most frequently investigated in relation to aging in dogs [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several indices, including TP, have been positively correlated with age in studies of mixed breeds or single medium or large breeds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, it is not clear which of these indices are particularly linked to aging. Our findings that TP is consistently correlated with aging in poodles in both cross-sectional and longitudinal analyses indicate that TP may be used as a sensitive aging indicator in small dogs. A similar trend was also observed in retrievers, although the reference population was relatively small. Additional studies are needed to determine whether TP can also be applied to medium and large dogs.\u003c/p\u003e \u003cp\u003eRegarding the relationship between the type of protein in the blood of dogs and age, albumin (ALB), the most abundant protein in the blood, typically decreases in late aging or shows no strong correlation with age. In contrast, globulin, the second most abundant protein, generally increases with age [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this cohort, we found no significant relationship between age and ALB in toy poodles or retrievers, while globulin was not measured. Chronic kidney disease has been reported to cause hypoalbuminemia in dogs [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is possible that ALB levels in this study focusing on healthy individuals were not affected because it did not include individuals with hypoalbuminemia. Further studies are needed to determine which protein species account for the age-dependent increase in TP.\u003c/p\u003e \u003cp\u003eIn the present study, toy poodles showed a decrease in vaccine antibody titers with age, which is consistent with previous reports in other medium and large-sized breeds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. On the other hand, in retrievers, the vaccination antibody titers of the three viruses responded differently: Adeno virus antibody titers showed an age-related decrease, while Distemper virus and Parvo virus antibody titers showed no consistent age-related relationship. The cause of this is unknown, but it is possible that some individuals were subclinically infected, or vaccine reactivity might be unique to retrievers. Further studies on a larger scale including antibody titer measurements for other vaccine types are needed for a detailed analysis.\u003c/p\u003e \u003cp\u003eNAD⁺ levels decrease with age in worms, flies, and mice, and have also been reported to decrease in human tissues such as blood, brain, and skin in data from relatively small populations [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] We were unable to find any prior reports quantifying in vivo NAD⁺ levels in dogs until the time of manuscript preparation. We measured whole blood NAD⁺ levels in the present study and found no significant relationship with age in either of the breeds. The mean NAD⁺ concentration in dogs was 23 \u0026micro;M, which was slightly lower than the human NAD⁺ concentration of 26 \u0026micro;M measured by the same extraction method used in this study [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. While these results do not rule out the possibility of a correlation between NAD⁺ levels and aging in other tissues, they do indicate that it is highly unlikely that NAD⁺ levels in whole blood can be used as a sensitive marker of aging in healthy dogs.\u003c/p\u003e \u003cp\u003eWe evaluated the frailty index and echocardiography as indicators of health-related phenotypes that can be assessed by veterinarians and nurses. Regarding the frailty index, which has been reported to correlate with aging and mortality in a diverse population of large- and medium-sized breeds [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], no significant relationship with age was found in the toy poodle population we evaluated. However, in an analysis including individuals who died during the study, the frailty index at study entry showed a significant positive correlation with age. In retrievers, the correlation coefficient was also higher in analyses that included deceased individuals. These results suggest that the frailty index might be a stronger predictor of mortality than chronological age.\u003c/p\u003e \u003cp\u003eThere is little reported common relationship between the fecal microbiome and aging in dogs. In humans, an increase in Enterobacteriaceae and a decrease in \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e have been reported with aging [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, a comparison of the Adult and Senior groups of poodles in this study found no commonality with these reports. No significant differences in beta diversity were detected between the two age groups. We believe that this group of dogs was characterized by a uniform living environment and a uniform diet, with relatively few confounding factors influencing the gut microbiota. Nevertheless, the lack of a significant correlation suggests that while we do not rule out the possibility that some endemic bacterial species may correlate with aging in certain regions or under certain conditions, it is difficult to use the canine gut microbiota as a sensitive marker of chronological aging.\u003c/p\u003e \u003cp\u003eIn summary, we found that elevated plasma total protein is a relatively sensitive indicator of chronological aging in healthy small-sized dogs. We also found that NAD⁺ levels and microbiome composition, which have been reported as aging markers in other species, have very low sensitivity as markers of chronological aging, regardless of dog size. Limitations of this study include its single-center design and the limited breeds included. To generalize the aging-related indices to all small dogs, studies should be conducted at multiple institutions and include small dog breeds other than toy poodles. Additionally, the relationship between the aging indices identified in this study and the decline in body function with aging needs to be examined in a longitudinal study over a longer time span. Such further efforts will lead to the development of methods for better understanding and preventing aging in dogs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by RIKEN Aging Project (to M.Y), Japan Society for the Promotion of Science (JSPS) KAKENHI (20K11553 to T.K.I.), and funding by Meiji Holdings Co., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by RIKEN Aging Project (to M.Y), Japan Society for the Promotion of Science (JSPS) KAKENHI (20K11553 to T.K.I.), and funding by Meiji Holdings Co., Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e16S rDNA sequencing data is available under accession number DRA019236 (under project number PRJDB18730) from the DDBJ DRA database (https://www.ddbj.nig.ac.jp/dra/index-e.html). Other data that support the findings of this study are available from the corresponding authors, T.K.I and S.O., upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.K.I conceived and S.O. and T.K.I. conceptualized the study. S.O. and T.K.I. designed the experiments and analyzed the data. S.O. developed figures. S.O. performed blood biochemistry and antibody titer analysis. S.O. and S.H. performed blood NAD⁺\u0026nbsp;measurement. E.M. performed cardio echography. E.M. performed the frailty index measurement. Y.H. performed microbiome sequencing. K.H. performed statistical analysis for microbiome data. M.M. and M.Y. provided intellectual input and essential resources for project completion. S.O. and T.K.I. prepared the manuscript with input from co-authors.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol was approved by the Laboratory Animal Committee of Meiji Holdings Corporation (Application No. 2020_1000_0034) and was conducted in compliance with experimental facility guidelines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.O., Y.H., S.H., K.H., and M.M are employees of Meiji Holdings Co., Ltd.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBergstrom A et al Origins and genetic legacy of prehistoric dogs. 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PLoS ONE 15:e0234712. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0234712\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0234712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeluso A, Damgaard MV, Mori MAS, Treebak JT (2021) Age-Dependent Decline of NAD(+)-Universal Truth or Confounded Consensus? \u003cem\u003eNutrients\u003c/em\u003e, vol. 14, no. 1, Dec 27 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/nu14010101\u003c/span\u003e\u003cspan address=\"10.3390/nu14010101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plasma protein, aging, dogs, NAD⁺, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-5341224/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5341224/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough extensive research has examined aging markers in larger dog breeds, little is known about small breeds. This study assesses the relevance of aging biomarkers examined in larger breeds and other biological species, focusing on toy poodles (N\u0026thinsp;=\u0026thinsp;40) as a model of small breeds and retrievers (N\u0026thinsp;=\u0026thinsp;17) serving as a large-sized reference. Healthy individuals with no significant health declines for up to a year post-data collection were studied for age-related changes in various parameters, excluding disease factors. Our cross-sectional analysis identified significant correlations between age and increases in plasma protein concentration and amylase levels across both breeds, with breed-specific age-related declines in vaccine responses to various viruses observed only in toy poodles. Longitudinal analysis over one year confirmed a significant temporal increase in plasma protein in toy poodles, with a similar, albeit non-significant, trend in retrievers. Unlike in other species, NAD⁺ levels and fecal microbiota showed no age-related changes. Additionally, the previously reported frailty index correlated with age in retrievers but not in toy poodles. Notably, including deceased individuals during the study strengthened correlations. These results suggest plasma protein increase as a chronological aging factor in toy poodles and enhance our understanding of aging in healthy small dog breeds.\u003c/p\u003e","manuscriptTitle":"Plasma protein increase as a chronological aging factor in healthy toy poodles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 19:32:18","doi":"10.21203/rs.3.rs-5341224/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"097306fb-da41-4063-8e00-557eec88d165","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-04T06:53:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-06 19:32:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5341224","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5341224","identity":"rs-5341224","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}