Age-Related Differences in Short-Term Transportation Stress Responses of Horses.

Transportation of horses on short journeys can lead to an increase in stress. There are known age-associated changes in immune and metabolic responses in horses; however, no research exists evaluating how age may influence these responses to transportation stress. Eleven mares within two age groups, aged (n = 5, 22 ± 1 year) or young (n = 6, 2 ± 1 year), were transported 1 hour and 20 minutes. Peripheral blood and saliva were collected before and after transportation at baseline (2 to 3 weeks prior to transportation), 24 hours pre-transport, 1 hour before loading, 15 minutes, 30 minutes, 1 to 3 hours, 24 hours and 8 days post-transport. Heart rates, rectal temperatures, under the tail temperatures, serum cortisol, plasma ACTH, serum insulin, salivary cortisol and salivary IL-6 were measured. Whole blood gene expression of the cytokines IL-1b, IL-2, IL-6, IL-10, IFNγ, and TNFα were determined through qPCR, and peripheral blood mononuclear cells were isolated, stimulated, and stained to determine IFNγ and TNFα production. Serum cortisol (P < .0001), salivary cortisol (P < .0001) and heart rate (P = .0002) increased in response to transportation with no age differences. Rectal (P = .03) and under the tail temperatures (P = .02) were increased in young versus aged horses. ACTH was higher in aged horses (P = .007) and post-transportation (P = .0001). Aged horses showed a greater increase in insulin compared with young horses (P < .0001). While age does not seem to impact cortisol responses to short-term transportation in horses, it did influence the post transportation insulin response to stress in aged horses.

The effects of cannabidiol on immune function and health parameters in senior horses.

Cannabidiol (CBD) has potential to reduce pain and inflammation in humans leading to the interest of use in equine. The purpose of this study was to determine the effects of CBD on immune function by measuring inflammatory cytokines and antibody responses to vaccination, as well as other health parameters in senior horses. Horses were orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days, from July 2021 to November 2021. Peripheral blood samples were collected on days 0, 30, 60, and 90 before administering treatments. On day 90 all horses were kept on treatment and vaccinated with an equine influenza vaccine and blood samples were collected post-vaccination on days 14 and 21. For all time points, plasma samples were analyzed for determination of CBD and metabolites, 7-OH CBD and 7-COOH CBD, using tandem mass spectrometry. For time points 0, 30, 60 and 90, blood samples were analyzed for CBC and chemistry. Additionally, peripheral blood mononuclear cells (PBMC) were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) then analyzed via flow cytometry. Real-time PCR (RT-PCR) analyzed both stimulated PBMCs and whole blood for cytokine gene expression. Inflammatory proteins C-reactive protein, interleukin 1 receptor agonist, and prostaglandin E2 were measured with equine-specific enzyme linked immunosorbent assays. Thyrotropin-releasing hormone stimulation test and oral sugar test were performed on all horses before and after the study to analyze metabolic function. Hemagglutination inhibition (HI) titers were measured for immune responses pre- and post-vaccination. All data were analyzed using either a paired t-test or a two-way repeated measures analysis of variance (significance P < 0.05). Plasma concentrations of CBD and metabolites were determined with 7-COOH CBD, the most significant metabolite, in CBD treated horses compared to control treated horses. A significant decrease was determined for whole blood inflammatory cytokine expression of IFN-γ at day 60, and for IL6 at day 60 and 90 for CBD-treated horses when compared to control horses. CBD did not significantly affect any other immune factors, HI titers, or health parameters. This study demonstrated that treatment with CBD reduced some inflammatory cytokine production with no negative side effects as measured by CBC or chemistry profiles. This study reveals the initial understanding of CBD in the horse, however more in-depth research is needed to fully understand its efficacy on the health of the horse.

Comparison of the host response to larvicidal and nonlarvicidal treatment of naturally acquired cyathostomin infections in horses.

This study aimed to collect information on local and systemic inflammatory responses, and goblet cell-associated components, following anthelmintic treatment with moxidectin and ivermectin in horses naturally infected with cyathostomin parasites. Thirty-six horses aged 2-5 years of age were randomly allocated to three groups. Group 1 received ivermectin/praziquantel (0.2 mg/kg), Group 2 received moxidectin/praziquantel (0.4 mg/kg) and Group 3 were untreated controls. Tissue samples from the Cecum, Dorsal and Ventral Colons were used for histopathological evaluation and preserved for RNA isolation and gene expression analysis. Whole blood was collected weekly for gene expression analysis as well. The control group had significantly higher inflammation associated with higher larval scores. The treatment groups displayed no differences in larval counts and inflammatory cell populations (p > .05). Mucosal larval counts were positively correlated with goblet cell hyperplasia scores (p = .047). The moxidectin-treated group had a significantly lower expression of IFN-γ (p < .05). The data suggest that removal of cyathostomins reduced the pro-inflammatory response associated with cyathostomin infections. Pro-inflammatory reactions associated with anthelmintic treatment were minimal, but lowest for moxidectin-treated horses. Results suggested that cecum, ventral and dorsal colons responded differently to cyathostomin larvae, which may have implications in the disease process.

Development and Evaluation of a Muscle Atrophy Scoring System (MASS) for Horses.

Loss of skeletal muscle mass likely compromises performance and welfare in horses and thus routine monitoring would be valuable. Currently available methods to assess muscle mass require expert knowledge and are often expensive. To provide a simple method, a muscle atrophy scoring system (MASS) was created and tested by three evaluators (raters) in 38 horses of varying age, breed, and health status. Inter-rater agreement on atrophy scores was in the good-to-excellent range for ratings of the neck (ICC = 0.62), back (ICC = 0.62) and hind (ICC = 0.76) regions but was poor for the abdominal region (ICC = 0.29). Due to this low agreement, the abdominal region was excluded from further analysis. Associations between muscle atrophy scores and age, pituitary pars intermedia dysfunction (PPID) status, and body composition indicators, including weight and estimated fat-free mass (FFM), were examined. Weight was inversely associated with neck, back and hind muscle atrophy scores (ß = -0.008, ß = -0.008, ß = -0.009, respectively; all P <0.001), but estimated FFM was not associated with muscle atrophy scores at any region (P >0.05). Age was positively related to neck (ß = 0.030, P <0.01), back (ß = 0.037, P <0.001) and hind (ß = 0.040, P <0.001) muscle atrophy scores. PPID-positive horses (n = 4) had higher muscle atrophy scores than PPID-negative horses (n = 23), even after adjusting for age (P <0.05). This data suggests that neck, back and hind region evaluations by individual raters likely have acceptable reliability. In addition, these findings support further evaluation of the potential benefits of the MASS to identify and monitor muscle atrophy in horses.

Cytokine and goblet cell gene expression in equine cyathostomin infection and larvicidal anthelmintic therapy.

AIMS: The role of the immune response to cyathostomin infections in horses remains unknown. Intestinal goblet cell hyperplasia has previously been noted as a component in cyathostomin infection; however, the function is unclear. The goal of this study was to evaluate the local and systemic gene expression to cyathostomin infections following larvicidal treatment and explore their relation to goblet cells. METHODS AND RESULTS: Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into three groups: fenbendazole-treated (10 mg/kg PO 5 days), moxidectin-treated (0.4 mg/kg PO once) and untreated control. Whole blood from all horses was collected weekly, and tissue samples from the large intestine collected during necropsy at 2 and 5 weeks post-treatment (WPT). Gene expression of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-22, IFN-γ, resistin-like molecule beta (RELM-ß), Mucin 2 (MUC2) and tumour necrosis factor (TNF)-α was measured using qRT-PCR. There were statistically significant linear correlations between luminal worm burdens and MUC2 (r = -.2358) and RELM-ß (r = -.2261). CONCLUSION: This suggests an active role of immune system post-treatment in parasite expulsion, specifically in goblet cells, and that the organs respond differently to treatment and the larvae themselves. This may have implications in the disease process and treatment.

Relationships of inflamm-aging with circulating nutrient levels, body composition, age, and pituitary pars intermedia dysfunction in a senior horse population.

Similarly to aged humans, senior horses (≥20 years) exhibit chronic low-grade inflammation systemically, known as inflamm-aging. Inflamm-aging in the senior horse has been characterized by increased circulating inflammatory cytokines as well as increased inflammatory cytokine production by lymphocytes and monocytes in response to a mitogen. Little is currently known regarding underlying causes of inflamm-aging. However, senior horses are also known to present with muscle wasting and often the endocrinopathy pituitary pars intermedia dysfunction (PPID). Despite the concurrence of these phenomena, the relationships inflamm-aging may have with measures of body composition and pituitary function in the horse remain unknown. Furthermore, nutrition has been a focus of research in an attempt to promote health span as well as life span in senior horses, with some nutrients, such as omega-3 fatty acids, having known anti-inflammatory effects. Thus, an exploratory study of a population of n = 42 similarly-managed senior horses was conducted to determine relationships between inflamm-aging and measures of circulating nutrients, body composition, age, and PPID. Serum was collected to determine vitamin, mineral, and fatty acid content. Peripheral blood mononuclear cells were also isolated to determine inflammatory cytokine production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following stimulation with a mitogen, as well as to determine gene expression of interleukin(IL)-1ß, IL-6, IL-10, IFN-γ, and TNF-α. Serum IL-6 and C-reactive protein were determined by enzyme-linked immunosorbent assay. Whole blood was collected for hematological and biochemical analysis. Body composition was evaluated via ultrasound and muscle scoring for all 42 horses as well as by deuterium oxide dilution for a subset of n = 10 horses. Pituitary function was evaluated by measuring basal adrenocorticotropin hormone concentrations as well as by thyrotropin releasing hormone stimulation testing (to determine PPID status). Results showed various relationships between inflammatory markers and the other variables measured. Most notably, docosadienoic acid (C22:2n6c), docosapentaenoic acid (C22:5n3c), and folate were positively associated with numerous inflammatory parameters (P ≤ 0.05). Although no relationships were found between inflamm-aging and PPID, being positive for PPID was negatively associated with vitamin B12 (P ≤ 0.01). No relationships between inflammation and body composition were found. Even within this senior horse population, age was associated with multiple parameters, particularly with numerous inflammatory cytokines and fatty acids. In summary, inflamm-aging exhibited relationships with various other parameters examined, particularly with certain fatty acids. This exploratory study provides insights into physiological changes associated with inflamm-aging in the senior horse.

Effects of Docosahexaenoic Acid-Rich Microalgae Supplementation on Metabolic and Inflammatory Parameters in Horses With Equine Metabolic Syndrome.

Much of the equine population is obese and therefore predisposed to the development of additional health concerns such as equine metabolic syndrome (EMS). However, pharmacologic treatments for EMS are limited. Omega-3 fatty acid supplementation is a therapeutic strategy in humans with metabolic dysfunction that improves insulin sensitivity and reduces inflammation, but the effects of omega-3 fatty acid supplementation in horses with EMS are unclear. Therefore, in this pilot study, 10 mixed-sex and mixed-breed horses with EMS were fed a docosahexaenoic acid (DHA)-rich microalgae containing 16 g DHA/horse/d or served as controls for 46 days. Inflammatory status was measured using serologic enzyme-linked immunosorbent assay and in peripheral blood mononuclear cells (PBMCs) using flow cytometry and reverse transcription polymerase chain reaction. Circulating fatty acids, triglyceride, leptin, and adiponectin concentrations were also determined. Insulin and glucose dynamics were assessed with oral sugar test (OST) and frequently sampled intravenous glucose tolerance testing. Postsupplementation, treated horses had an increase in many circulating fatty acids, including DHA (P < .001). Treated horses also had lower serum triglycerides postsupplementation (P = .02) and a trend (P = .07) for reduced PBMC tumor necrosis factor α. Interestingly, after 46 days, control horses had an increase in insulin responses to the OST (P = .01), whereas treated horses did not (P = .69). These pilot data indicate that DHA-rich microalgae supplementation alters circulating fatty acids, modulates metabolic parameters, and may reduce inflammation in horses with EMS.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

PROBLEM: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. METHODS: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. RESULTS: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1ß in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1ß expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. CONCLUSION: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

Effects of polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene on lymphocyte pro-inflammatory cytokine production of senior horses in vitro.

Senior horses (aged ≥ 20 years) exhibit increased chronic, low-grade inflammation systemically, termed inflamm-aging. Inflammation is associated with many afflictions common to the horse, including laminitis and osteoarthritis, which are commonly treated with the non-steroidal anti-inflammatory drugs (NSAIDs) flunixin meglumine and phenylbutazone. Although these NSAIDs are effective in treating acute inflammatory problems, long-term treatment with NSAIDs can result in negative side effects. Thus, bioactive polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene were investigated to determine their effectiveness as anti-inflammatory agents in vitro. Heparinized blood was collected via jugular venipuncture from senior horses (n = 6; mean age = 26 ± 2 years), and peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient. PBMC were then incubated 22 h at 37°C, 5% CO2 with multiple concentrations (320, 160, 80, 40, 20, 10 µM) of all five polyphenols (curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene), dissolved in DMSO to achieve the aforementioned concentrations. PBMC were stimulated the last 4h of the incubation period with phorbol 12-myristate 13-acetate (PMA)/ionomycin and Brefeldin A (BFA). A Vicell-XR counter evaluated cell viability following incubation. PBMC were stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) and analyzed via flow cytometry. Data was analyzed by one-way analysis of variance (ANOVA). Viability of PBMC incubated with various compound concentrations were compared with PBMC incubated with DMSO alone (positive control) to determine at what concentration each compound caused cytotoxicity. The highest concentration at which cell viability did not significantly differ from the positive control was: 20 µM for curcuminoids, 40 µM for hydroxypterostilbene, 80 µM for pterostilbene, and 160 µM for quercetin and resveratrol. Flunixin meglumine and phenylbutazone were then evaluated within this range of optimal concentrations for the polyphenol compounds (160, 80, 40, 20 µM) to compare the polyphenols to NSAIDs at equivalent concentrations. The highest concentration at which viability did not significantly differ from the positive control was: 40 µM for flunixin meglumine and 160 µM for phenylbutazone. All five polyphenols and flunixin meglumine significantly decreased lymphocyte production of IFN-γ, while only hydroxypterostilbene, pterostilbene, quercetin, and resveratrol significantly reduced lymphocyte production of TNF-α compared to the positive control (p < 0.05). Polyphenols performed similarly to or more effectively than common NSAIDs in reducing lymphocyte production of inflammatory cytokines of the senior horse in vitro. This study therefore supports the further investigation of polyphenols to determine whether they may be effective anti-inflammatory treatments for chronic inflammation in the horse.

Whole-body phenylalanine kinetics and skeletal muscle protein signaling in horses with pituitary pars intermedia dysfunction.

OBJECTIVE: To compare whole-body phenylalanine kinetics and the abundance of factors in signaling pathways associated with skeletal muscle protein synthesis and protein breakdown between horses with pituitary pars intermedia dysfunction (PPID) and age-matched control horses without PPID. ANIMALS: 12 aged horses (6 horses with PPID and 6 control horses; mean age, 25.0 and 25.7 years, respectively). PROCEDURES: Plasma glucose, insulin, and amino acids concentrations were determined before and 90 minutes after feeding. Gluteal muscle biopsy samples were obtained from horses 90 minutes after feeding, and the abundance and activation of factors involved in signaling pathways of muscle protein synthesis and breakdown were determined. The next day, horses received a priming dose and 2 hours of a constant rate infusion of (13)C sodium bicarbonate followed by a priming dose and 4 hours of a constant rate infusion of 1-(13)C phenylalanine IV; whole-body protein synthesis was determined. RESULTS: Plasma glucose and insulin concentrations were higher after feeding than they were before feeding for both groups of horses; however, no significant postprandial increase in plasma amino acids concentrations was detected for either group. Phenylalanine flux, oxidation, release from protein breakdown, and nonoxidative disposal were not significantly different between groups. No significant effect of PPID status was detected on the abundance or activation of positive or negative regulators of protein synthesis or positive regulators of protein breakdown. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggested that whole-body phenylalanine kinetics and the postprandial activation of signaling pathways that regulate protein synthesis and breakdown in muscles were not affected by PPID status alone in aged horses.

Effects of advanced age on whole-body protein synthesis and skeletal muscle mechanistic target of rapamycin signaling in horses.

OBJECTIVE: To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses. ANIMALS: Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses. PROCEDURES: Whole-body protein synthesis was measured with a 2-hour primed constant infusion of (13)C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-(13)C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis. RESULTS: Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples. CONCLUSIONS AND CLINICAL RELEVANCE: The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

The effect of an immunomodulator (parapoxvirus ovis) on cell-mediated immunity (CMI) in abruptly weaned foals.

The weaning process of foals involves a period of considerable stress which likely contributes to an increased risk of infectious disease in these young horses. Mechanisms responsible for this heightened risk of infection remain unknown, although likely due to compromised cell-mediated immunity. Parapoxvirus ovis (PPVO), an immmunomodulator, has been shown to limit the severity of infectious disease outbreaks among horses and has been shown to enhance CMI responses. Thus, an objective of this study was to investigate the effect of PPVO therapy on cell-mediated immune (CMI) responses of abruptly weaned foals. A group of foals (n=6) were given an intramuscular injection of PPVO on days -2, 0 (weaning) and 9. An additional group of foals (n=5) received the diluent only on the same days serving as controls. Peripheral blood samples were collected from all foals prior to weaning (day 0) and on days 1, 3, 5, 7, 9, 11, 14, and 21 after weaning. Whole blood samples were prepared to determine in vivo cytokine mRNA expression by reverse transcription and real-time PCR (RT-PCR). Peripheral blood mononuclear cells (PBMC) were isolated and stimulated to determine in vitro cytokine production by intracellular staining using flow cytometry and gene expression was measured by RT-PCR. Cytokines analyzed in this study were interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10). Regardless of PPVO treatment, foals undergoing the weaning process showed a significant decrease in both in vivo and in vitro cytokine (IFN-γ, TNF-α and IL-10) production. These results indicate that abrupt weaning significantly impacts CMI of the foal which may increase susceptibility to infectious agents.

The promoter region of interferon-gamma is hypermethylated in neonatal foals and its demethylation is associated with increased gene expression.

While born with a limited production, foals' interferon-gamma (IFN-γ) expression increases after birth. The underlying mechanisms remain unknown. DNA methylation is considered to be involved. Therefore, the DNA methylation status of the Ifng promoter in CD4(+) cells from neonatal foal was determined using a methylation-specific PCR (MSP), and its relevance to IFN-γ mRNA expression was estimated. The effect of environment on the DNA methylation was also evaluated by comparing ponies that were kept in a barn versus those on pasture. The DNA in the Ifng promoter was hypermethylated and its demethylation was correlated with an increase in IFN-γ mRNA expression and age. This age-associated demethylation was accelerated by barn-air exposure. In conclusion, IFN-γ expression in foals appears to be controlled by DNA methylation in the promoter region of Ifng. The age-associated demethylation of the DNA in foals may be induced by exposure to environmental antigens and their effect on lymphoproliferation.

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection.

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ(+) CD5(+)T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.

Effect of body condition, body weight and adiposity on inflammatory cytokine responses in old horses.

Advanced age is associated with a low-grade, systemic inflammatory response characterized by increased inflammatory cytokine production both in vitro and in vivo, termed inflamm-aging. It is also known that increased white adipose tissue, associated with obesity, leads to increased production of inflammatory cytokines. To date, it is unknown whether increased adiposity contributes to the age-related increased inflammatory status. Here we show that peripheral blood mononuclear cells (PBMC) from old horses compared to young horses have increased inflammatory cytokine production; moreover, fat old horses compared to thin old horses have even greater frequencies of lymphocytes and monocytes producing inflammatory cytokines. Therefore, we proposed that decreasing adiposity in old horses would reduce age-associated increases of inflammatory cytokines both in vitro and in vivo, and increasing adiposity in old horses would increase these measurements. To test this hypothesis further, eight old obese horses (20-28 year) were assigned to two consecutive treatments, dietary restriction (DR) during weeks 1-12 and increased dietary intake (DI) during weeks 13-30. Body weight, body condition score (BCS) and percent body fat were measured weekly. PBMC were stimulated in vitro and interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) production was measured by intracellular staining. Levels of nascent IFNgamma and TNFalpha mRNA expression were examined by RT-PCR. Serum concentrations of TNFalpha protein were also measured weekly. Reducing body weight and fat in old horses significantly reduced the percent of IFNgamma and TNFalpha positive lymphocytes and monocytes, and serum levels of TNFalpha protein. Further, when weight and fat increased in these old horses there was a significant increase in inflammatory cytokine production. Regression analysis also revealed significant relationships. These findings demonstrate that age-related obesity potentially plays a role in the dysregulation of inflammatory cytokine production by the immune system with age or inflamm-aging in the horse.