Influence of energy level and nicotinic acid supplementation on apoptosis of blood leukocytes of periparturient dairy cows

- Functional apoptosis assay compared with gene expression analysis in blood leukocytes.
- Measured apoptotic variables are significantly influenced by parturition.
- PBMC apoptosis is more susceptible to the changes around parturition than PMN.
- Apoptosis in blood leukocytes correlates to energy variables.

The periparturient period of dairy cows is accompanied by an immunosuppression that leaves the animal more susceptible to infections and metabolic disorders. Non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) which peak shortly after parturition due to lipolysis are known to impair immune cell functions. Niacin with its well-known anti-lipolytic effect may have the ability to ameliorate this situation. Additionally, niacin shows also anti-inflammatory effects that may be beneficial to the immune status of the cow. To address this 29 multiparous and 18 primiparous German Holstein cows were subjected to four different feeding groups. They were fed either a ration with a high concentrate proportion of 60% (HC), or a low concentrate proportion of 30% (LC). After parturition both concentrate levels were reduced to 30% and increased again to 50% either within 16 days (LC-group) or within 24 days (HC-group). Half of the animals received either 24 g per day of nicotinic acid from 42 days prepartum until 24 days postpartum (LC-NA, HC-NA) or no supplement (LC-CON, HC-CON). Apoptosis in polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) was examined with an Annexin V and propidium iodide (PI) based fluorescence flow cytometry assay and distinguished into early apoptotic (Annexin V positive and PI negative) and late apoptotic (Annexin V and PI positive) cells. Additionally, the pro-apoptotic gene BAX, the effector caspase CASP3, and the anti-apoptotic genes BCL2 and BCL-xL, as well as the NFκB subunit RELA were quantified by real-time PCR in blood leukocytes. All variables showed time dependencies that were mainly related to parturition (p < 0.01). Early apoptotic PBMC were significantly affected by concentrate level showing higher numbers of apoptotic cells in the HC groups (p = 0.029). PBMC were characterized by a more pronounced apoptosis than PMN and seemed to be more susceptible to the changes that occur around parturition. The genes BAX and CASP3 were positively correlated (0.631) and their peak preceded the apoptotic peak around parturition in the blood leukocytes. The LC animals showed a decrease in BCL2 expression before parturition, whereas the HC animals showed a continuous increase in BCL2 mRNA abundance (p = 0.059). RELA correlated stronger with the pro-apoptotic genes (0.715 and 0.650 with BAX and CASP3 respectively) and its expression was higher in primiparous than in multiparous cows (p = 0.011). Nicotinic acid supplementation did show some influence in increasing numbers of early apoptotic PMN and late apoptotic PBMC between 42 and 100 DIM.