The objective of the study was to evaluate the relationships between body condition and lymphocyte functions in periparturient dairy cows. Thirty days before expected calving, 21 Holstein cows were categorized as thin (n = 6), medium (n = 8), or overconditioned (n = 7) based on body condition score (BCS). Blood samples were collected on 21, 14, 7, and 3 d before calving and on d 3, 7, 14, 21, 28, and 35 after parturition. An aliquot of blood was used to determine plasma nonesterified fatty acids (NEFA) and glucose. At 14 and 7 d before, and 14 and 35 d after calving, a second aliquot of blood was used to assess peripheral blood mononuclear cell (PBMC) functions: DNA synthesis, immunoglobulin (Ig) M, and interferon-gamma (IFN-gamma) secretion after mitogen stimulation. During the experiment, all 21 cows showed a decline in BCS. Overconditioned cows lost significantly more BCS than thin cows. After calving, overconditioned cows had higher plasma NEFA compared with thin and medium cows. Conversely, plasma glucose never differed between the 3 categories of cows. Regardless of BCS, DNA synthesis and IgM secretions were significantly lower in PBMC isolated on 7 d before calving compared with those recorded 14 and 35 d after parturition. Conversely, PBMC from the 21 cows did not show any change of IFN-gamma secretion during the experimental period. Taking into consideration the BCS categories, PBMC isolated from overconditioned cows presented lower IgM secretion compared with thin cows on d 14 and 35 after calving. Furthermore, PBMC isolated from overconditioned cows secreted less IFN-gamma compared with thin and medium cows on d 7 before calving. The DNA synthesis of PBMC stimulated with the 3 mitogens did not differ between the 3 categories of cows. In conclusion, immunodepression occurring in cows around calving would be particularly evident in overconditioned cows.

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625 mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2 mM) was associated with a dramatic increase of phagocytosisassociated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the rela-

This in vitro study was performed to assess the effects of various concentrations of nonesterified fatty acids (NEFA) on lymphocyte function of heifers. Nine Holstein heifers were studied. Peripheral blood mononuclear cells were incubated with various concentrations of NEFA (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mmol/L). The mixture of NEFA was represented by C16:0 (30%), C16:1 (5%), C18:0 (15%), C18:1 (45%), and C18:2 (5%). The DNA synthesis was diminished at concentrations of NEFA of 2, 1, and 0.5 mmol/L. The IgM secretion was inhibited at concentrations of NEFA of 2, 1, 0.5, and 0.25 mmol/L. Secretion of IFN-gamma 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.

This study was undertaken to assess whether peripheral blood mononuclear cells (PBMC) isolated from Brown Swiss (Br) and Holstein (Ho) cows and stimulated with concanavalin A differ in response to chronic exposure to incubation temperatures simulating conditions of hyperthermia. Five multiparous Br and 5 Ho cows were utilized as blood donors. Peripheral blood mononuclear cells were subjected for 65 h to each of 5 treatments (T). Cells were exposed to 39 degrees C continuously (T39) and three 13-h cycles at 40 (T40), 41 (T41), 42 (T42) or 43 degrees C (T43), respectively, which were interspersed with two 13-h cycles at 39 degrees C. Treatment T39 was adopted to mimic normothermia; T40, T41, T42, and T43 mimicked conditions of more severe hyperthermia alternating with normothermia. Measures evaluated at the end of the incubation period were proliferative response (DNA synthesis), intracellular reactive oxygen species (ROS) concentrations, and mRNA abundance of the 72-kDa heat-shock protein (Hsp72). In Br cows, DNA synthesis began to decline when PBMC were repeatedly exposed to 41 degrees C (-22%), whereas DNA synthesis in cells isolated from Ho cows did not begin to decline until 42 degrees C (-40%). Furthermore, under T41 and T42, DNA synthesis from Br cows was lower than in Ho(-24 and -54%, respectively). In both breeds, increased incubation temperatures caused a reduction of intracellular ROS (from -39.6 and -69.7%). Increase in incubation temperatures enhanced Hsp72 mRNA levels only in PBMC isolated from Br cows. The Hsp72 mRNA in Br cows increased significantly under T41 and T43 compared with T39. In both breeds, DNA synthesis was positively and negatively correlated with intracellular ROS and Hsp72 mRNA abundance, respectively (r = 0.85 and r = -0.70, respectively). Results indicated that PBMC from Br cows are less tolerant to chronic heat exposure than those from Ho cows, and that the lower tolerance is associated with higher expression of Hsp72, suggesting that the same level of hyperthermia may be associated with a differential decline of immune function in the 2 breeds.

This study was carried out to ascertain the effects of intense high environmental temperatures (HET) on lymphocyte functions in periparturient dairy cows. The study was undertaken from the beginning of March through the end of July 2003 in a commercial dairy unit located approximately 40 km north of Rome. Thirty-four Holstein cows were utilised in the study. Twenty-two of these cows gave birth in spring (SP cows), from 28 March to 30 April. The remaining 12 cows gave birth in summer (SU cows), between 15 June and 2 July. The two groups of cows were balanced for parity and were fed the same rations. Blood samples were taken 4, 3, 2 and 1 week before calving, and 1, 2 and 4 weeks after calving, in order to evaluate peripheral blood mononuclear cell (PBMC) function in vitro, and to determine plasma cortisol concentrations. After isolation, the PBMC were stimulated with mitogens and their response in terms of DNA synthesis and IgM secretion was measured. During spring, either the day (9-20 h) or the night (21-8 h) temperature humidity index (THI) was below the upper critical THI (72) established for dairy cows. During summer, the mean daily THI values were 79.5+/-2.9 during the day and 70.1+/-4.7 during the night. Furthermore, during summer, three heat waves (a period of at least 3 consecutive days during which there were less than 10 recovery hours) occurred. Recovery hours were intended hours with a THI below 72. The first heat wave lasted 5 days, the second 6 days, and the third 15 days. Compared to the SP cows, over the entire periparturient period the extent of DNA synthesis and IgM secretion levels were lower (P ranging from <0.01 to 0.0001) and higher (P<0.01) respectively, in the SU cows. Before calving, the SU cows also presented higher (P<0.01) concentrations of plasma cortisol compared to the SP cows. This study indicates that the effects of HET on the immune response depend on the specific immune function under consideration, and that neuroendocrinal changes due to HET may play a role in the perturbation of immune functions.