| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Dipartimento di Produzioni Animali, Università della Tuscia, 01100, Viterbo, Italy
Corresponding author: N. Lacetera; e-mail: nicgio{at}unitus.it.
 |
ABSTRACT |
|---|
 TOP ABSTRACT REFERENCES |
|---|
was depressed at concentrations of NEFA of 2, 1, 0.5, 0.25, and 0.125 mmol/L. Increases of plasma NEFA might contribute to explain the higher incidence of infections observed in cows suffering from energy deficit.
Key Words: heifer • nonesterified fatty acid • lymphocyte function
Abbreviation key: BrdU = 5-bromo-2'-deoxyuridine, ConA = concanavalin A, ECM = enriched culture medium, IFN- = interferon-, PBMC = peripheral blood mononuclear cells, PHA = phytohemagglutinin, PWM = pokeweed mitogen
Results of in vitro studies performed in the bovine to verify whether high concentrations of ketones contribute to the high incidence of infections in ketotic cows are conflicting (Targowski and Klucinski, 1983; Franklin et al., 1991; Suriyasathaporn et al., 1999). We reported immunodepression in ketotic ewes and negative relationships between immune functions and plasma NEFA or BHBA (Lacetera et al., 2001). We also found that the addition of NEFA to culture media altered sheep lymphocyte functions (Lacetera et al., 2002). The effect of NEFA on lymphocyte functions has not been studied before in cows.
The objective of the current in vitro study was to verify the effects of various concentrations of NEFA on DNA synthesis, IgM and interferon- (IFN-) secretion of mitogen-stimulated peripheral blood mononuclear cells (PBMC) of cows.
Nine healthy, midpregnant Holstein heifers of approximately the same age and BW were used. Blood samples were collected via jugular venipuncture, using evacuated glass tubes coated with sodium heparin. An aliquot of blood was centrifuged to separate plasma and to determine concentrations of plasma glucose (Instrumentation Laboratory, Lexington, USA) and NEFA (Wako Fine Chemical Industries, Inc., Dallas, TX).
A NEFA mixture was made that contained a combination of C16:0 (30%), C16:1 (5%), C18:0 (15%), C18:1 (45%), and C18:2 (5%). This mixture was prepared as described previously (Lacetera et al., 2002) by using the method of Strang et al. (1998). Briefly, a stock solution of NEFA (88.8 mM) was prepared by dissolving fatty acids in 0.1 N KOH at a molar ratio of 1:1.13 at 60°C. The NEFA-KOH solution, still at 60°C, was neutralized by the addition of 1 N HCl and diluted with sterile redistilled water. That solution was diluted in RPMI-1640, adjusted to pH 7.4, and sterilized by passage through a filter. The solution was added to cell cultures in RPMI-1640 to final concentrations of 0.0625, 0.125, 0.25, 0.5, 1, and 2 mmol/L of cell suspension. All reagents were purchased from Sigma (Milan, Italy). Concentrations of NEFA were designed to mimic those in healthy cows (0.0625, 0.125, 0.25, and 0.5 mmol/L) and ketotic cows (1 mmol/L). The highest concentration of NEFA (2 mmol/L) has instead to be considered beyond physiologic levels.
The DNA synthesis was evaluated as described (Lacetera et al., 2001). After isolation, PBMC were resuspended at a concentration of 1 x 106 cells/mL of RPMI-1640 enriched culture medium (ECM). Concentrations of NEFA and glucose in the ECM were measured and were equal to 0.006 and 9.87 mmol/L, respectively. Triplicate cultures were assayed, by using 96-well tissue-culture plates. Each well contained 1 x 105 PBMC in 100 µL of ECM. An optimal concentration of phytohemagglutinin (PHA, 2.5 µg/mL), pokeweed mitogen (PWM, 1 µg/mL) or concanavalin A (ConA, 2.5 µg/mL) (Sigma) was added to plates. Plates were incubated in an atmosphere of 95% air and 5% CO2 for 48 h at 39°C. We then added to wells 100 µM of 5-bromo-2'-deoxyuridine (BrdU) in 10 µL of RPMI-1640, and plates were incubated for an additional 18 h. The DNA synthesis was quantitated by an ELISA assay (Lacetera et al., 2002). The assay was performed with a commercial kit (APB, Milan, Italy) that is based on measurement of BrdU incorporated during DNA synthesis. Values for DNA synthesis were expressed as the optical density for test wells minus the optical density for control wells that did not contain BrdU. The intraassay coefficient of variation was 7.3%. Secretion of IgM was established by growing PWM-stimulated cells (1 x 106 cells/well) under the same conditions described above. Concentration of PWM was 0.2 µg/mL. Cells were cultured in duplicate for 12 d in 24-well tissue-culture plates. At the end of the incubation, supernatants were collected and stored at -20°C until analyzed. The IgM released in culture medium were quantitated by a capture ELISA (Lacetera et al., 2002). Plates were coated with a rabbit-purified antibody against bovine IgM. The IgM were detected by use of a phosphatase-conjugated sheep antibovine IgM polyclonal antibody. Antibodies were purchased from Bethyl Laboratories (Montgomery, TX). Antibody concentrations (ng/mL) were calculated using a computer program (Fulvio Montauti, Pisa, Italy). The 50% absorbance values of serial dilutions of supernatant were interpolated onto the linear portion of a standard curve obtained by using purified bovine IgM (VMRD, Pullman, WA). Interassay coefficient of variation was 11.4%; intraassay coefficient of variation was 2.4%. Secretion of IFN- was established by growing ConA-stimulated cells (1 x 106 cells/well) under the same conditions described above. Cells were cultured in duplicate for 72 h in 24-well tissue-culture plates. At the end of incubation, supernatants were collected and stored at -20°C until analyzed. The IFN- released in culture medium was quantitated by a capture ELISA, which was carried out by use of a commercial kit (CSL, Victoria, Australia). The IFN- concentration (ng/mL) was established by using the same computer program utilized for IgM quantification. In this case, the standard curve was obtained by using bovine rIFN-, which was kindly provided by Stephen Jones (CSL, Victoria, Australia). The interassay coefficient of variation was 9.6%; the intraassay coefficient of variation was 5.11%.
Data were reported as mean ± SEM. Data were analyzed by a one-way ANOVA (SAS, 1996), and effects were considered to be significant at a value of P < 0.05.
Values of plasma glucose and NEFA in the 9 heifers indicated a physiologic state (ranging from 3.40 and 3.64 mmol/L for glucose, and from 0.222 and 0.296 mmol/L for NEFA).
The DNA synthesis was significantly diminished at concentrations of NEFA of 2, 1, or 0.5 mmol/L (Figure 1
). The IgM secretion was inhibited at concentrations of NEFA of 2, 1, 0.5, and 0.25 mmol/L (Figure 2). Secretion of IFN- was significantly depressed at concentrations of NEFA of 1, 0.5, 0.25, and 0.125 mmol/L (Figure 3). The IFN- secreted from PBMC not stimulated or cultured in the presence of 2 mmol/L of NEFA was not detectable.
|
|
|
Received for publication September 24, 2003. Accepted for publication November 3, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  K. M. Moyes, T. Larsen, N. C. Friggens, J. K. Drackley, and K. L. Ingvartsen Identification of potential markers in blood for the development of subclinical and clinical mastitis in dairy cattle at parturition and during early lactation J Dairy Sci, November 1, 2009; 92(11): 5419 - 5428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hiss, C. Weinkauf, S. Hachenberg, and H. Sauerwein Short communication: Relationship between metabolic status and the milk concentrations of haptoglobin and lactoferrin in dairy cows during early lactation J Dairy Sci, September 1, 2009; 92(9): 4439 - 4443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. F. Duffield, A. R. Rabiee, and I. J. Lean A Meta-Analysis of the Impact of Monensin in Lactating Dairy Cattle. Part 3. Health and Reproduction J Dairy Sci, June 1, 2008; 91(6): 2328 - 2341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. M. van Knegsel, G. de Vries Reilingh, S. Meulenberg, H. van den Brand, J. Dijkstra, B. Kemp, and H. K. Parmentier Natural Antibodies Related to Energy Balance in Early Lactation Dairy Cows J Dairy Sci, December 1, 2007; 90(12): 5490 - 5498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Burvenich, D. D. Bannerman, J. D. Lippolis, L. Peelman, B. J. Nonnecke, M. E. Kehrli Jr., and M. J. Paape Cumulative Physiological Events Influence the Inflammatory Response of the Bovine Udder to Escherichia coli Infections During the Transition Period J Dairy Sci, June 1, 2007; 90(13_suppl): E39 - E54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Scalia, N. Lacetera, U. Bernabucci, K. Demeyere, L. Duchateau, and C. Burvenich In Vitro Effects of Nonesterified Fatty Acids on Bovine Neutrophils Oxidative Burst and Viability J Dairy Sci, January 1, 2006; 89(1): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Lacetera, D. Scalia, U. Bernabucci, B. Ronchi, D. Pirazzi, and A. Nardone Lymphocyte Functions in Overconditioned Cows Around Parturition J Dairy Sci, June 1, 2005; 88(6): 2010 - 2016. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
