DISCUSSION
Values of the daytime THI recorded during SU indicated conditions capable of inducing a moderate heat stress (Armstrong, 1994). Values of RT and RR of SU cows confirmed conditions of moderate stress (Johnson, 1987).
Even if the possibility that the differences found in the oxidative status of spring and summer cows were also partially dependent on other seasonal factors cannot be excluded, previous studies that referred to other species (Bellò-Klein et al., 2000) indicated that seasonal alterations of the oxidative status depend more on weather conditions such as temperature and humidity. For this reason, and for lack of information on the effects of other seasonal factors on the oxidative status of dairy cows, this discussion will provide a possible interpretation of our results in light of literature on the effects of heat stress on oxidative status.
In the present study, we found that plasma markers of the oxidative status in moderately heat-stressed cows did not differ from those observed in SP cows. Unfortunately, previous studies on heat stress and oxidative status of dairy cows were carried out only in midlactating animals (Harmon et al., 1997; Trout et al., 1998; Calamari et al., 1999). Harmon et al. (1997) reported the reduction of antioxidant activity of plasma in midlactating cows exposed to a chamber temperature of 29.5°C, and when THI reached danger level for livestock during summer. Calamari et al. (1999) found a reduction of plasma lipid soluble antioxidants (vitamin E and ß-carotene), and an increase of plasma TBARS in moderately heat-stressed, midlactation cows during summer. Trout et al. (1998) reported no effects of short exposure to a hot environment on the concentration of lipid soluble antioxidants (
-tocopherol, ß-carotene, retinol, and retinyl palmitate) or on the concentration of MDA in muscle. The different physiological status of cows between previous studies and our own might be responsible for the discrepancy between our results and those of said previous studies.
In contrast, we found significant differences in the oxidative status of SU and SP cows by observing erythrocyte markers. In particular, SU cows showed higher erythrocyte SOD, GSH-Px-E, SH, and TBARS, which would indicate some effects of moderate hot summer weather on the oxidative status of transition dairy cows.
The role of intracellular SOD is to scavenge the superoxide (•O2-) that is produced by a number of reaction mechanisms, including several enzyme systems, as a part of normal cellular functions (Fee et al., 1975). In particular, the oxidation or autooxidation of hemoglobin (Hb-Fe2+ into Hb-Fe3+) into the erythrocytes results in the continuous formation of •O2- (Hebbel and Easton, 1989). In this study, the higher erythrocyte SOD activity found in SU cows was probably a response to the higher •O2- generation. SOD catalyzes the dismutation of •O2- into oxygen and hydrogen peroxide (H2O2), and it is an important antioxidant defense mechanism in aerobic organisms, although too much SOD may sometimes be deleterious (Halliwell and Chirico, 1993). In fact, the dismutation of •O2- results in a rise in H2O2. Since SOD activity increases H2O2 production, protection from reactive oxygen would only be conferred by a coordinate increase of catalase and GSH-Px-E activities (Clemens and Waller, 1987; Frei, 1994; Kehrer and Smith, 1994). In support of this conjecture, GSH-Px-E was found to be increased in SU cows. The decomposition of H2O2 or its interaction with •O2- would generate hydroxyl radicals (OH•) (Hochstein and Jain, 1981). Hydroxyl radicals can attack all biological molecules, including membrane lipids, and can result in initiation of lipid peroxidation (Halliwell and Chirico, 1993).
The resulting lipid-centered free radicals rearrange and react with molecular oxygen to form lipid hydroperoxides (Trevisan et al., 2001). Hydrolysis of lipid hydroperoxide leads to a complex mixture of small acyl compounds aldehydes, alcohols, and hydrocarbons (Armstrong and Browne, 1994). One of the commonly applied assays to estimate lipid peroxidation is the thiobarbituric acid test. Thiobarbituric acid reacts with MDA to form a fluorescent adduct. However, thiobarbituric acid is not specific for MDA, and the test is considered to be a good general indicator of oxidative stress rather than a marker of lipid peroxidation (Armstrong and Browne, 1994). The increment in erythrocyte TBARS indicates that the balance between the oxidants and the antioxidants favors the former.
The method we used to assess thiol content is unable to distinguish the GSH from the other free thiols. However, it represents a fast and easy tool to observe the response at cytosolic level (Kusmic et al., 2000) and provides a good estimation of total intracellular GSH content, since GSH represents about 95% of the intracellular thiols (van den Berg et al., 1992). Glutathione plays an important role in protecting cells against oxidative stress and toxic agents (Zollner et al., 1991; Cnubben et al., 2001), and it is a substrate for GSH-Px-E (Sies, 1991). The increase of cellular GSH and total intracellular -SH groups would indicate a higher exposure of the erythrocytes to the risk of oxidative stress (Yu, 1994; Ohtsuka et al., 1997; John et al., 2001). John et al. (2001) reported that the increase of -SH groups in oxidative-stressed erythrocytes could be due to the decrease of glutathione-S-transferase activity of erythrocytes. The same authors suggested that the lower conjugation of -SH groups due to the decrease of glutathione-S-transferase activity may cause toxic conditions in the erythrocytes.
We speculate that the coordinate increase of erythrocyte SOD, GSH-Px-E, and SH is an indirect compensatory response of cells to increased oxidant challenge during heat stress. In the present study, the concentrations of TBARS were significantly higher in erythrocytes of heat-stressed cows which, in parallel with increased erythrocyte enzymes activity and SH concentration, supported the hypothesis that heat stress represents an oxidative challenge for erythrocytes and causes alterations in the antioxidant status of these cells.
The combination of moderate heat stress conditions and the elevated antioxidant capacity of plasma (Frei et al., 1998) might be the reason for the absence of differences between seasons in plasma oxidative markers found in our study. In contrast, the high polyunsaturated fatty acid content of erythrocyte membrane, and the continuous exposure to high concentration of oxygen and iron in hemoglobin are the factors that make erythrocytes very sensitive to the oxidative injury (Clemens and Waller, 1987) and make erythrocytes an appropriate model to study oxidative stress (Kusmic et al., 2000; Alicigüzel et al., 2001).
In a recent study on hyperthyroid patients, Alicigüzel et al. (2001) hypothesized the hypermetabolic effects of thyroid hormones as the cause of erythrocyte oxidative stress. In our instance we can exclude the hyperthyroidism and the hypermetabolism as the causes, since it is well known that thyroid hormones and metabolism are decreased when dairy cows are heat-stressed (Webster, 1991; Nardone et al., 1997). Under heat stress, the increase of oxygen pressure of blood due to the increased RR (unpublished data) might be the cause of alteration of oxidative status. As reported before, the high polyunsaturated fatty acid content of erythrocyte membrane and the presence of high concentration of iron in hemoglobin may result in oxygen toxicity via generation of free radicals (DiGiuseppi et al., 1984) that could in turn cause alteration in erythrocyte antioxidant defense systems.
Noble et al. (1976) observed a reduction of the proportion of linoleic acid (C18:2) in the cholesteryl ester fraction from cattle exposed to hot environments. Kuiper et al. (1971) cited by Noble et al. (1976), found a reduction of C18:2 concentration in the erythrocyte membrane from heat-stressed hamsters. Linoleic acid is one of the principal polyunsaturated fatty acids of cell membranes, which is sensitive to lipid peroxidation process (Zollner et al., 1991). Utoh and Harasaki (1992) reported an increase of membrane fragility in erythrocytes exposed to high in vitro temperatures. Oxidative stress can lead to increase in TBARS (Halliwell and Chirico, 1993), and the cytotoxic effects of TBARS are well known (Zollner et al., 1991). In particular, TBARS can induce a reduction of membrane fluidity (Chen and Yu, 1994), and increase erythrocyte membrane fragility (Spicket et al., 1998).
Data from our study that show an alteration of erythrocyte oxidative status with the increase of TBARS in heat-stressed cows might explain the reduction of C18:2 and the increase of erythrocyte membrane fragility as a consequence of exposure to hot condition.
Variation of plasma and erythrocyte oxidants and antioxidants during the transition period observed in the present study revealed an alteration of the oxidative status after calving. These results confirm our previous (Ronchi et al., 2000) and others' (Miller et al., 1993; Formigoni et al., 1997) findings that clearly indicated an imbalance of the oxidative status during the early lactation phase in Holstein cows. It can be concluded that a hot environment is responsible for oxidative stress, which worsens the alteration of oxidative status commonly observed in transition dairy cows.
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