Experimental Design, Animals, and Feeding
The experiment was carried out from April until the end of August in a commercial dairy herd located in central Italy, approximately 50 km south of Rome (13°00' North, 42°72' East). Twenty Holstein cows were utilized. Eleven cows gave birth during spring (SP cows), in a period from April 13 to May 12. The remaining 9 cows gave birth during summer (SU cows), in a period between June 27 and August 26. Spring cows and SU cows were similar for parity (2.6 ± 2.1 and 2.8 ± 2.2 for SP and SU cows, respectively). During both SP and SU, diets for dry cows and lactating cows consisted of a base-ration fed as a TMR given daily at 0930, and offered ad libitum to achieve 5 to 10% refusals. The close-up diet was offered during the last 10 d before the expected calving. All diets were kept constant for the entire experimental period (Table 1
).
Measurements and SamplingsTemperature and relative humidity of the air were measured and recorded at 1-h intervals using electronic recorder stations (Data-Hog recorders, Skye Instruments Ltd., Llandrindod Wells, UK). Temperature-humidity index (
THI) was calculated according to the formula reported by
Ingraham et al. (1979). The animals were monitored during the last 3 wk of pregnancy and the first 35 d of lactation. Rectal temperatures (
RT) and respiration rates (
RR) were registered at -21, -3, +1, +15, and +35 d from calving at 1500 h. On the same days, blood samples were taken from the jugular vein using Vacutainer tubes containing lithium heparin as an anticlotting agent and put in ice.
Laboratory Analyses
Within 2 h of the bleedings, packed cell volume (PCV) was determined by microhemocytometer, and blood samples were centrifuged at 3500 rpm for 10 min at 4°C and plasma was thus separated. Erythrocytes were obtained by centrifuging 0.5 ml of blood at 3500 rpm for 10 min. Erythrocytes were then washed four times with 3 ml of 0.9% NaCl solution, by centrifuging for 10 min at 2200 x g. After the final wash, the red blood cells were lysed by hypotonic shock using 2.0 ml of cold redistilled water. The hemolysate was mixed and left at 4°C for 15 min. The plasma and the lysate of erythrocytes were then analyzed.
Plasma reactive oxygen metabolites (ROM) were measured by the analytical method patented by Diacron (Cesarone et al., 1999). The results of the analyses were expressed in arbitrary units (a.u.). The value of 1 a.u. corresponds to a concentration of 0.08 mg/dl of hydrogen peroxide.
Erythrocyte glutathione peroxidase (GSH-Px-E) and plasma glutathione peroxidase (GSH-Px-P) activities were determined by a kinetic method with a commercial kit (RANSEL by Randox Laboratories, UK), and were expressed in international units per milliliter of PCV and international units per milliliter, respectively.
To determine superoxide dismutase activity in erythrocytes (SOD), the lysate was diluted 50-fold with 0.01 mmol/L phosphate buffer, pH 7.0, and then SOD was measured by a kinetic method using a commercial kit (RANSOD, by Randox Laboratories), and results were expressed in international units per milliliter.
Intracellular thiol contents of lysates of erythrocytes (SH) (Kusmic et al., 2000) and plasma thiol groups (SHp) were determined by titration with 5,5-dithiobis-2-nitrobenzoic acid by use of commercial kit (Diacron, Italy), and were expressed in micromoles per milliliter of PCV and in micromoles per liter, respectively.
Thiobarbituric acid reactive substances (TBARS) in erythrocyte lysates and plasma were measured by the fluorimetric method of Maseki et al. (1981) and were expressed in nanomoles malondialdehyde (MDA) equivalents per milliliter of lysate.
Statistical Analysis
Data were analyzed as repeated measures using the GLM procedure of SAS (SAS, 1996) by multifactorial models with interactions including season, cows within season, days from calving, and the physiological phase (pregnancy or lactation) as main effects, and an error term. To isolate the variation due to cow from variation due to season, the error term used to test for a season effect was cow within season. Least squares means were separated with the PDIFF procedure of SAS (SAS, 1996). Significance was declared at P
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