- Mastitis is a major endemic disease in dairy cows resulting in significant economic losses for the dairy industry. The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that is able to bind and be activated by natural (e.g., fatty acids) and synthetic (e.g. thiazolidinedione) compounds. PPARγ plays important roles in adipocyte differentiation, inflammation, and re-epithelialization in monogastric. In ruminants, PPARγ may play a role in milk fat synthesis. The aim of this study was to assess the role of PPARγ in host response to mammary infection and milk fat synthesis in ruminants. Our hypothesis is that activation of PPARγ improves the host response to mastitis and increases milk fat yield. By using a synthetic PPARγ agonist in dairy goats in combination with intramammary infection to induce subclinical mastitis, the objectives of the present experiments were to test if activation of PPARγ improves 1) the response to mastitis and 2) milk fat production. To achieve our objectives we performed two in vivo experiments (Experiments 1 and 2).
In Experiment 1, 24 Saanen lactating goats with a low body condition score and getting a low-energy diet without vitamin supplementation received a daily intrajugular injection of either 8 mg of 2,4-thiazolidinedione (TZD) per kg of BW or saline (as a control) and, after a week of TZD injection, an intramammary infusion (IMI) of either Streptococcus uberis to induce subclinical mastitis or saline used as a control (6 goats/group). Milk yield and components, body weight, rectal temperature, leukocyte phagocytosis, blood metabolic and inflammation parameters plus insulin, adipocyte size by histology, and expression by RT-qPCR of PPARγ target genes in adipose tissue obtained through biopsy and in mammary epithelial cells (MEC) isolated from milk were assessed. In MEC, expression of CCL2 and IL8 was also measured. Data were analyzed by GLIMMIX of SAS with Mastitis, TZD, and Time and all interactions as main effects and goat as random effect. Statistical significance and tendencies were declared at P < 0.05 and 0.05 ≤ P ≤ 0.10, respectively. The induction of mastitis was successful achieved as indicated by >5-fold increase of milk somatic cells count (SCC) in goats receiving Strep. uberis and by 30% decrease of % polymorphonuclear leukocytes in blood. The SCC in milk were overall lower in TZD-treated goats. Mastitis induction but not TZD decreased milk yield and production of milk fat. Goats receiving Strep. uberis had increased concentrations of glucose, triglycerides, and non-esterified fatty acids (NEFA) in blood after IMI. NEFA was not affected in TZD goats, which did not receive Strep. uberis. Inflammatory markers increased in blood of all goats but the increase of haptoglobin was overall lower in TZD treated goats. Indicators of liver activity, including albumin, paraoxonase, and cholesterol, overall decreased after IMI but cholesterol did not decrease in TZD-treated goats. The bactericidal myeloperoxidase was higher
in TZD-treated goats after mastitis. Insulin sensitivity was not affected by TZD or mastitis. Adipocytes size increased over time and was higher in TZD goats not receiving Strep. uberis. Subclinical mastitis increased expression of CCL2 and prevented a decrease in expression of IL8. MEC from TZD-treated goats tended to have higher expression of PPARG, FASN and SCD1 after 3 weeks of TZD treatment. Neither mastitis nor TZD affected the expression of genes in adipose tissue. Overall the data of Experiment 1 indicated that the subclinical mastitis model was successfully achieved. The treatment with TZD decreased somatic cells in milk, improved the response of liver, decreased the severity of inflammation, and increased the killing capacity of neutrophils after IMI. The data suggested a more lipogenic adipose tissue in TZD-treated goats but also some active, although minor, nutrigenomic effect of TZD on MEC that may have counteracted the competition of lipid substrates between mammary and adipose tissue. Blood metabolic data suggested that goats responded to Strep. uberis intramammary infusion similar to dairy cows in negative energy balance. Data obtained from Experiment 1 indicated that TZD aids with mastitis response. TZD had some effect on milk fat synthesis but, overall, had a smaller-than-expected nutrigenomic effect probably also due to the low body condition and low energy in the diet of the goats. Thus, the effect of PPARγ on milk fat synthesis is still unclear.
The rationale to perform Experiment 2 stemmed from the possibility that the limited nutrigenomic response observed in Experiment 1 was due to a potential dietary deficiency. Subsequent in vitro work demonstrated that TZD is a strong activator of PPAR but only in the presence of 9-cis-retinoic acid, a metabolite of vitamin A and the activation of PPARγ obligate heterodimer Retinoic-X-Receptor (RXR). Therefore, we hypothesized that continuous activation of PPARγ by TZD in dairy goats supplemented with adequate amount of vitamin A improves the inflammatory response to subclinical mastitis. In order to test this hypothesis we used 12 Saanen
multiparous goats in early lactation. Goats received a diet that met the NRC requirements, including vitamin A, and a daily injection of 8 mg TZD per kg of BW (n=6) or saline (n=6; CTRL). Following 14 days of treatment, all goats received an IMI of Strep. uberis to induce subclinical mastitis in the right half of the udder with the left half used as control. Metabolic, inflammation, and oxidative-status profiling in blood including 20 parameters was performed. Milk yield, SCC, rectal temperature and leukocytes phagocytosis were measured. Expression of several PPARγ target genes and genes involved in inflammation was measured in MEC, macrophages isolated from milk, and liver tissue. Data were analyzed by GLIMMIX of SAS with treatment (TRT) and Time and TRTxTime interaction as main effects and goat as random effect. For milk and SCC, mammary half was also included in the main effect (including interactions). Statistical significance and tendencies were declared at P < 0.05 and 0.05 ≤ P ≤ 0.10, respectively. Milk yield decreased after IMI but the decrease was larger in TZD-treated goats. SCC increased after IMI but was not affected by TZD administration. Milk fat decreased after IMI in all halves except in the untreated half of TZD-treated goats. In blood within 2 days from IMI, ceruloplasmin, haptoglobin, and glucose were increased while Zn was decreased. These data confirmed successful induction of sub-clinical mastitis and a status of slight inflammation after IMI. None of the parameters in blood was affected by TZD with the exception of a lower bilirubin concentration and a tendency for higher haptoglobin in TZD vs. CTRL after IMI, indicating a more robust response of the liver to inflammation. The stronger inflammation was also supported by a tendency for higher reactive oxygen metabolites in TZD vs. CTRL group after IMI. We also detected a tendency for a higher globulin in TZD vs. CTRL indicating a better adaptive immune system. Leukocyte phagocytosis was strongly reduced by TZD treatment. None of the genes measured were affected by TZD in liver. In milk macrophages
and MEC, expression of inflammatory genes was higher compared to control in halves receiving Strep. uberis, whereas no effect of TZD was observed with the exception of a lower SCD1 in TZD-treated goats compared to CTRL. We conclude that, contrary to our hypothesis, in goats receiving NRC recommended amount of vitamin A, TZD had a minor effect on the response to mastitis with a likely better liver response, but a lower phagocytosis and minor effect on expression of genes.
Considering both in vivo experiments, we can conclude that TZD has an important effect on inflammatory response in dairy goats receiving low energy diet without vitamins supplementation but the effect disappears for the most part in goats receiving adequate feeding, including vitamin A. Furthermore, the lack of effects on expression of PPARγ target genes does not support TZD being a strong PPARγ agonist in dairy goats.