Hematological Profiles In Heat Stressed Lactating Dairy Cows Biology Essay

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This study was conducted to determine the effects of either dietary selenium (Se) source or dose on a range of dairy cow metabolic and hematological profiles and their subsequent relationship with oxidative status and environmental temperature. Forty lactating cows, offered the same basal diet, were blocked by days in milk, milk yield and parity and then randomly allocated to 1 of 5 dietary treatments: negative control (CTRL; 0.098 mg Se kg− 1 DM), two levels of Se yeast (SY) supplementation (0.31 and 0.50 mg total Se kg− 1 DM), and two levels of sodium selenite (SS) supplementation (0.31 and 0.50 mg total Se kg− 1 DM). Whole blood samples were taken from all animals at the start of the study (23 March) and after 28, 56, 84, 112, 126, and 140 d. Whole blood samples were analyzed for total Se, glutathione peroxidase (GPX-1) and a range of hematological parameters. Plasma was analyzed for total Se, glutathione peroxidase (GPX-3), metabolites related to energy and protein metabolism, concentration of minerals, enzyme activities, positive acute phase proteins and oxidative status markers. Glutathione peroxidase activity and total Se in whole blood and plasma were greater (P<0.001) in Se supplemented cows than CTRL. The temperature humidity index (THI) values indicate that during the trial cows experienced a slight-mild heat stress. A negative effect of THI on plasma glucose, non-esterified fatty acids (NEFA), thiol groups, plasma Na and K, and leukocyte count was observed. Conversely, a positive effect of THI on aspartate aminotransferase (AST) activity and GPX-3 activity was observed. Lower values (P<0.05) of thiobarbituric acid reactive substances (TBARS) during the hotter period were observed in SY supplemented animals when compared with CTRL and SS. Furthermore, plasma total antioxidants were lower (P<0.05) in SY supplemented animals when compared with SS during the hotter period. Plasma reactive oxygen metabolites were also numerically lower in SY when compared to SS. These results could be interpreted as an improvement in the preventive antioxidant systems of cows fed Se yeast.

Key words: dairy cow, selenium, heat stress, metabolic conditions, hematological profile.

1. Introduction

It is well known that high environmental temperatures negatively affect production and reproduction in dairy cows (Jordan, 2003; West, 2003). Similar observations in dairy cows have been reported for temperate areas such as Italy (Calamari and Mariani, 1998; Bernabucci et al., 2002).

Heat stress has been reported to induce the production of oxygen derived free radicals, resulting in some of the deleterious effects associated with heat stress (Loven, 1988). Nevertheless, studies on the effects of heat stress on markers of oxidative status in cattle are contradictory. Harmon et al. (1997) reported a reduction in antioxidant activity of plasma, while Calamari et al. (1999) observed only weak negative effects on some plasma markers of oxidative status in mid-lactating Holstein cows. Lakritz et al. (2002) observed a decrease in the reduced glutathione content with a commensurate increase in oxidized glutathione content in whole blood of adult cows. Trout et al. (1998) reported no effects of heat stress on plasma concentrations of vitamin E and β-carotene and on muscle thiobarbituric acid reactive substances. Bernabucci et al. (2002) observed that season did not modify plasma oxidative markers, although some erythrocyte markers of oxidative status indicated oxidative stress in transition dairy cows during the summer when compared to those during spring.

Free radicals compromise cellular function by removing electrons from a variety of molecules. As a result protein crosslinking, DNA damage, and lipid peroxidation (Dargel, 1992), and disruption of normal metabolism and physiology (Trevisan et al., 2001) can occur. Deficiencies of natural protective substances or excess exposure to stimulators of free radical production may result in oxidative stress. When the production of free radicals is faster than their neutralization by antioxidative mechanisms, oxidative stress is induced (Surai, 2006). These conditions can contribute to the onset of health disorders in cattle (Miller et al., 1993).

Natural and synthetic antioxidants in the feed as well as optimal levels of minerals, principally Se, help to maintain efficient levels of endogenous antioxidants in tissues. Selenium protects tissues against oxidative stress (Surai, 2006), as it is a component of the glutathione peroxidase (GPX) enzyme (Rotruck et al., 1973), which destroys free radicals in the cytoplasm (MacPherson, 1994). Selenium has been shown to reduce health disorders and enhance the immune system (Hernken et al., 1998)

A multitude of data is available on the effects of Se supplementation in heat stressed animals (Rochet and Mazzia, 2008), but little data exists on the effects of Se yeast compared with selenite. Thatcher (2006) reported an increase in immunocompetence at parturition, an improvement in uterine health and second service pregnancy rate during the summer months in cows fed Se yeast prior to calving. Increased neutrophil function, improved immuno-responsiveness and uterine health, and increased second service pregnancy rate during summer in a selenium-deficient environment were observed by Silvestre et al. (2007) in cows supplemented Se-yeast in Florida.

The aim of the current study was to evaluate the effects of feeding diets containing either no Se supplementation, or two Se sources (Se yeast and sodium selenite) at two different doses (0.31 and 0.50 mg total Se kg DM-1) to mid lactation dairy cows and the subsequent effects on metabolic and hematological profiles and their relationships with oxidative status and environmental temperature.

2. Materials and methods

2.1. Animal and management conditions

The research protocol and the animal care were in accordance with the EC Council Directive guidelines for animals used for experimental and other scientific purposes (European Community, 1986).

The Italian Friesian dairy cows involved in this study were raised in a free stall barn at the experimental farm "Vittorio Tadini", located near Piacenza (45°01'N, 9°40'E; altitude 68 m asl) (Italy). Cows were raised in pens holding 12-16 cows each, consisting of a resting area with cubicles and, outside, an unshaded hard court paddock. In each pen fresh potable water was available ad libitum. Description of housing and husbandry was reported previously (Calamari et al., 2010). Furthermore, each pen was equipped with 2 axial flow fans (0.75 kW; 90 cm diameter; 22500 m3 h-1 maximum airflow rate) installed along the feed driveway. Fans were mounted at a height of approximately 2.5 m and angled downward at about 10 degrees from vertical. The variable speed fans were thermostatically controlled and were switched on at 23° C and reached maximum flowrate at 27° C. Sprinklers (delivery rate of 4 L min-1 and a pressure of 150-200 kPa) spaced at 150 cm intervals were placed perpendicular to the air flow of the fans along the feed alley. The sprinklers were thermostatically controlled to 27°C in an uneven way: 50 seconds of showering and ventilation followed by 5 minutes of ventilation alone.

Feeding management and diet composition were also reported previously (Calamari et al., 2010). Samples of forages and concentrate mixes were collected twice monthly, whilst TMR samples were collected weekly. All samples were pooled monthly for the analyses.

2.2. Experimental design

The study was conducted on forty cows that received the same basal diet (dietary concentration of 0.10 mg total Se kg DM-1) that differed in only Se source (Se yeast or sodium selenite) or dose (0.31 or 0.50 mg total Se kg DM-1). The period of Se supplementation lasted 140 d (from March 23, 2007 to August 10, 2007). Cows were blocked by age, milk yield and day in milk (DIM) and randomly allocated to one of 5 dietary treatments (8 cows per treatment): negative control (CTRL, background Se only), Se yeast supplementation [Sel-Plex® Se yeast (Saccharomyces cerevisiae CNCM I-3060) containing 63% SeMet (Alltech, Nicholasville, KY)] to achieve either 0.31 and 0.50 mg of total Se kg DM-1 (SY03 and SY05, respectively), or sodium selenite supplementation to achieve either 0.31 and 0.50 mg of total Se kg DM-1(SS03 and SS05, respectively) (Table 1). Mean calving number and mean DIM of cows of each treatment were reported previously (Calamari et al., 2010). Total mixed rations, which contained the Se supplements, were prepared fresh daily and offered to the cows of each treatment. The Se supplement was added to the mixing wagon using corn meal as a carrier to obtain the correct concentration of Se for each dietary treatment.

2.3. Measurements and sampling

2.3.1. Microclimatic conditions.

Temperature and relative humidity of the inside barn were recorded daily during the study period using 2 electronic probes (Gemini Data Logger, UK) connected to a data logger programmed to record every 10 min. Mean daily temperature and humidity and daily minimum and maximum temperature and humidity were calculated from temperature and relative humidity data recorded throughout the trial. Data were used to compute a composite climatic welfare index, the Temperature Humidity Index (THI), according to the formula of Kelly and Bond, as reported by Ingraham et al. (1979). Mean daily THI (AVG THI), daily minimum THI (MIN THI), daily maximum THI (MAX THI), and the average THI of the week before each blood sampling (WK THI) were calculated throughout the trial and heat stress was estimated according to Armstrong (1994). In addition, the occurrence of heat waves was detected according to Hahn et al. (1999).

2.3.2. Blood sampling.

Blood samples on all cows were obtained before feeding at the start of the trial (23 March T0) and after 28 (April: T28), 56 (May: T56), 84 (18 June: T84), 112 (11 July: T112), 126 (25 July: T126) and 140 d (8 August: T140) of Se supplementation. At each sampling point, 3 blood samples were collected by venepuncture from the jugular vein: two 10-mL Li-heparin treated tubes (Vacuette, containing 18 IU of Li-heparin mL-1, Kremsmünster, Austria) and one 3-mL K3EDTA treated tubes (Venojet, containing 2.1 g of K3EDTA ml-1, Terumo, Leuven, Belgium). Blood samples were immediately placed into an ice-bath before processing.

2.4. Laboratory analyses

2.4.1. Selenium content in feeds, total Se and glutathione peroxidase in whole blood.

Selenium content of the specific premixes and composite feedstuffs was determined on mineralized samples using ICP-MS (Inductively Coupled Plasma Mass Spectrometry, Elan 6100, Perkin Elmer, Norwood, MA). The first Li-heparin treated tube of whole blood was used to measure total Se and glutathione peroxidase (GPX-1) activity as described by Calamari et al. (2010).

2.4.2. Plasma metabolic profile and Se content.

The second Li-heparin treated tube was centrifuged (3500 x g for 15 min at 10°C) and plasma separated and immediately stored at -20°C until analysis. Plasma metabolites were analyzed at 37°C by an automated clinical analyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA). Analysis for glucose, urea, calcium, inorganic phosphorus, magnesium, total protein, albumin, total bilirubin, and creatinine were conducted using commercial kits (Instrumentation Laboratory, Lexington, MA), also zinc (Wako Chemicals GmbH, Neuss, Germany). Enzymatic analysis for total cholesterol and triglycerides were conducted using commercial kits (Instrumentation Laboratory, Lexington, MA). A potentiometric system, with specific electrodes, was employed to determine Na, K, and Cl. Plasma thiol groups (SHp) were analyzed using commercial kits (Diacron International, Grosseto, Italy), as described by Bernabucci et al. (2005). Total plasma antioxidants (TA) were analyzed employing a commercial kit (Oxy-Adsorbent test, Diacron International, Grosseto, Italy), according to Trotti et al. (2001). The Oxy-Adsorbent test assesses the antioxidant power of the plasma barrier by measuring the ability of the barrier to oppose the massive oxidant action of hypochlorous acid (HClO). Results are expressed as micromoles of HClO mL-1 remaining after the reaction. Total plasma reactive oxygen metabolites (ROM) were measured using a commercial kit (d-ROMs test, Diacron International, Grosseto, Italy), according to Bernabucci et al. (2005). Results are expressed as milligrams of hydrogen peroxide per 100 mL of plasma. This reagent kit measures not only ROM existing in the matrix, but also the species developing during the Fenton reaction (Oriani et al., 2001). The thiobarbituric acid-reacting substances (TBARS) were measured with fluorimetric method according to Yagi (1976). Plasma glutathione peroxidase (GPX-3) was determined according to the method of Paglia and Valentine (1967) using a commercial kit (Ransel kit, Randox, UK). Kinetic analysis was adopted to determine activity of glutamate dehydrogenase (GDH, EC 1.4.1.2) using a commercial kit (Randox Laboratories, Antrim, UK), and alkaline phosphatase (AP, EC 3.1.3.1), aspartate aminotransferase (AST, EC 2.6.1.1), -glutamyltransferase (GGT, EC 2.3.2.2), L-lactate dehydrogenase (LDH, EC 1.1.1.27), alanine aminotransferase (ALT, EC 2.6.1.2), creatine kinase (CK, EC 2.7.3.2) determinations were also conducted using commercial kits (Instrumentation Laboratory, Lexington, MA). Total bilirubin concentration in plasma was determined using commercial kits (Instrumentation Laboratory, Lexington, MA). Ceruloplasmin (Cp) and haptoglobin were determined with reagents prepared according to the method reported by Bertoni et al. (1998). Total Se content in plasma was analyzed as described by Calamari et al. (2010).

2.4.3. Hematological Profile.

The K3EDTA treated tube of whole blood was used to measure the hematological profile using a Cell-Dyn 3700 hematology analyzer (Abbott Diagnostici, Roma, Italy). The measurements were: total red blood cells number (RBC; M L-1); hemoglobin (HGB; g dL-1); hematocrit (HCT; %); mean corpuscular volume (MCV; fL); mean corpuscular hemoglobin (MCH; pg); mean corpuscular hemoglobin concentration (MCHC; g dL-1); width of RBC volume distribution (RDW; %); total white blood cells number (WBC; K L-1); neutrophils (NEU; K L-1 and % on WBC); lymphocytes (LYM; K L-1 and % on WBC); monocytes (MON; K L-1 and % on WBC); eosinophils (EOS; K L-1 and % on WBC); basophils (BAS; K L-1 and % on WBC); total platelet number (PLT; K L-1); mean platelet volume (MPV; fL).

2.5. Statistical analysis

Results were analyzed using the MIXED models procedure (SAS Inst. Inc., Cary, NC) according to Littell et al. (1998). Sources of variation included treatment effect (5 levels), time, treatment x time interaction and the continuous random effect of THI (WK THI). The random variable was cow within treatment. Pre-experimental data were used for covariate adjustment. Each variable analyzed was subjected to 3 covariance structures: Autoregressive Order, Compound Symmetry, and Spatial Power (Littell et al., 1998). Using the lowest Akaike's information criterion and Schwarz Bayesian criterion, the Spatial Power was the covariance structure that fitted the model best. Results are presented in tables as least square means and standard error of the mean (SEM). If a significant effect of dietary treatment or THI was detected, comparisons were made between any treatment means during the hotter period (T112, T126, and T142) as follows: 1) CTRL vs. other treatments (SY03, SY05, SS03, and SS05) to evaluate the effects of Se supplementation; 2) Se yeast supplementation (SY03 and SY05) vs. selenite supplementation (SS03 and SS05) to evaluate source effect; 3) low level (SY03 and SS03) vs. high level of Se supplementation (SY05 and SS05) to evaluate the dose effect.

Correlation coefficients between the measured variables were also calculated first for the global dataset and then separately within CTR + SS03 + SS05 and within CTR + SY03 + SY05. Results were presented as significant when differences between values differ for P<0.05; a trend was considered when P<0.10.

3. Results

3.1. diet characteristics and Se content

The concentration of total Se in the negative control diet was 0.098 ± 0.027 mg Se kg DM-1 and was similar to estimated values before the beginning of the study (Table 1). Chemical and nutritive characteristics of the basal diet used in this study, and the Se content of the TMR of each treatment have already been reported by Calamari et al. (2010). Results on Se status (total blood Se, total plasma Se, and GPX-1 activity) obtained in this study have also been reported by Calamari et al. (2010).

Overall mean values of milk yield have already been reported by Calamari et al. (2010), and neither source nor dose effects were observed. At the start of the study (T0), average daily milk yield was 34.91 ± 1.71 kg d− 1 and dropped to 28.14 ± 1.71 kg d− 1 at the end of the study (T140), with an average rate of decline of about 4.43% per month. The rate of decline showed high variability through the experimental period. Greater rates of decline were observed between T56 and T84 (10.23% per month) and between T112 and T140 (9.94% per month).

3.2. Microclimatic conditions

The course of daily minimum and maximum THI during the experimental period is shown in Figure 1. The period during which the cows experienced daily MAX THI above 72 was from end of April to early August. Cows were exposed to daily AVG THI greater than 72 from early July to the end of July, during which time mean daily THI was 72.23 ± 2.69. Then, in the second half of July cows were exposed to daily AVG THI above 72 (73.59 ± 0.89) and to average daily maximum THI above 78 (79.09 ± 1.24).

3.3. Energy, protein and mineral metabolism.

Plasma metabolites related to energy and protein metabolism are shown in Table 2. During the hotter period Se supplementation affected plasma concentrations of NEFA and BHBA, with lower NEFA and greater BHBA concentrations in CTRL animals when compared to those supplemented with Se. No significant effects were observed for total cholesterol. During the hotter period a source effect was evident for NEFA only, concentrations being higher in SY supplemented animals when compared to those supplemented with SS (Table 2). A negative effect of THI on glucose and NEFA and a positive effect on urea was observed.

Among plasma minerals only inorganic Na was affected by Se supplementation during the hotter period (Table 2). A dose effect during was observed for inorganic P, but the difference was very slight (1.93 vs. 1.77 mmol L− 1 in cows fed 0.3 vs. 0.5 mg total Se kg− 1 DM, respectively). A negative effect of THI on plasma Na and K concentration was observed; conversely, the other minerals were unaffected.

3.4. Acute-phase proteins, oxidative status and enzymes activities

Selenium supplementation did not affect plasma acute phase protein concentrations, and no THI effect was observed.

Reactive oxygen metabolites (ROMs) were not affected by Se supplementation (Table 3). Reactive oxygen metabolites were positively correlated with +APP, and mainly with Cp (r = 0.97; P<0.001), haptoglobin (r = 0.52; P<0.001), and also globulin (r = 0.67; P<0.001). Reactive oxygen metabolites were correlated with SHp (r = -0.65; P<0.001) only in SS supplemented cows. Conversely, ROMs were correlated with GPX-3 (r = 0.62; P<0.001) and TBARS (r = 0.42; P<0.001) in both SS and SY supplemented animals.

Both source (P<0.05) and dose (P<0.01) effects on TA concentration were observed during the hotter period (Figure 2), with lower values in cows supplemented with Se yeast when compared to those receiving selenite supplements (SY03 vs. SS03 and SS05 vs. SY05) and in cows supplemented with higher doses of Se (SS05 vs. SS03 and SY05 vs. SY03). Total antioxidants showed a small, positive correlation with positive acute phase protein (+APP). However, a significant correlation was observed in SY supplemented cows (with Cp: r = 0.35; P<0.01; and also with globulin: r = 0.30; P<0.01). Furthermore, in these animals, a positive correlation with ROM (r = 0.37; P<0 0.01), and a small negative correlation with Se status (total blood Se: r = -0.24; P<0.05; total plasma Se: r = -0.24; P<0.05) was observed. A small negative correlation was observed between TA and GPX-1 (r = -0.22; P<0.05) regardless of treatment or season.

A small dose effect (P=0.051) was observed for SHp during the hotter period, with lower values in cows supplemented with 0.31 when compared to those supplemented with 0.50 mg total Se kg− 1 DM (Table 3). A negative effect of THI on SHp was observed. Thiol groups were mainly correlated with albumin (r = 0.68; P<0.001). In SS supplemented groups SHp was correlated with +APP (with Cp: r = -0.66; P<0.001; haptoglobin: r = -0.55; P<0.001; and also with globulin: r = -0.65; P<0.001) and negatively correlated with total plasma Se (r = -0.40; P<0.001) and GPX-3 (r = -0.69; P<0.001). However these same observations were absent from SY supplemented animals.

During the hotter period a source effect was observed for TBARS, with lower values in SY supplemented cows (P<0.001) compared with those supplemented with SS (Figure 3). Small correlations were observed between TBARS and +APP regardless of treatment or season. Conversely, only in SY supplemented cows, correlations between TBARS and indicators of Se status were observed (total blood Se: r = 0.34; P<0.01; total plasma Se: r = 0.27; P<0.05; GPX-1: r = 0.40; P<0.001).

Among enzymes activities only AST, LDH and plasma glutathione peroxidase (GPX-3) activities were affected by THI (Table 3), with a positive (AST and GPX-3) and negative (LDH) relationship. During the hotter period LDH activity was affected by Se source and dose (P<0.05), with lower values in cows supplemented with Se yeast when compared with animals supplemented with selenite, and with higher values in cows supplemented with the highest levels of Se supplementation when compared to those receiving diets supplemented with the lowest levels of Se supplementation. Plasma glutathione peroxidase (GPX-3) was greater in supplemented cows (P<0.05), however, there was no significant difference between the two Se sources or doses.

3.5. Hematological response

In general Se supplementation did not affect WBC or leukocyte populations during the hotter period, the exception being lymphocytes (Table 4). A source effect was noted during the hotter period on leukocytes count, with lower values of LYM (P<0.05) and MON (P<0.05) in SY supplemented animals when compared with SS supplemented cows. However, these differences were not apparent when each population was expressed as a percentage of WBC (Table 4). A negative effect of THI on WBC count and on the percentage of LYM was observed. The NEU and LYM, both as number and as a percentage of WBC, were significantly correlated (P<0.001) with SHp (r = -0.61, and r = 0.5261, for NEU and LYM percentage of WBC, respectively). Among the hematological measurements and Se status, only the basophils were correlated with total Se of blood and plasma (r = 0.36, and r = 0.38, respectively; P<0.001).

Selenium supplementation did not affect RBC during the trial, whereas a significant source effect during the hotter period was observed for RDW and PLT (Table 5).

4. Discussion

Cows of each treatment supplemented with Se showed greater overall mean values (P<0.05) of total Se in blood, plasma, and milk when compared with CTRL animals, as highlighted by Calamari et al. (2010). Animals fed the unsupplemented diet (CTRL) had mean blood Se concentrations of 163.5 ng g− 1 (150.6 to 179.1 ng g− 1) and mean plasma Se concentrations of 72.2 ng g− 1 (65.5 to 82 ng g-1), indicating marginally adequate Se status, if we consider that at least 100 μg L− 1 in whole blood is required to achieve optimal immune capacity and optimal fertility (Stowe and Herdt, 1992). From Silvestre et al., (2007), it can be concluded that beneficial effects of Se supplementation occur only when the animals are Se deficient. In our study, as previously reported by Calamari et al. (2010), mean whole blood Se concentrations of CTRL cows (164.3 ± 8.72 ng g-1) were in the range of 150.6 to 179.1 ng g-1, therefore easily meeting the recommendations of Gerloff (1992).

Heat stress in dairy cows is considered to be negligible when temperature humidity index (THI) values are lower than 72. Index values higher than 72, 78 and 88 reflect the potential for mild, high and severe levels of heat stress, respectively (Armstrong, 1994). During spring, the mean daily THI was below the upper critical value of 72 established for dairy cows. Values of daytime THI recorded during the hotter period were above the upper critical value of 72, indicating conditions capable of inducing moderate heat stress (Armstrong, 1994). Based on these index values, the potential existed in this study for dairy cows to suffer high heat stress for 22 d. These conditions were observed in July and August. During these hotter periods, the daily minimum THI reached values near the minimum value of the zone delimiting mild heat stress, representing mild-moderate heat waves (Hahn et al., 1999). In this period, according to Hahn et al. (1999), two heat waves (a period of at least 3 consecutive days during which there were less than 10 recovery hours with THI below 72) occurred. The first wave was recorded between 16 and 21 of July, with 19 hours where the THI was greater than 79. This five day period was classified as slight-mild heat stress. The second wave was recorded between 27 and 29 of July, with 12 hours where the THI was greater than 79, and could be classified as slight heat stress.

Cows in the current study were exposed to a THI above 72 and even over 79 for long periods, but the potential for heat stress was mild. The values of all the blood metabolites and enzymatic activities were found to be within the normal reference intervals (Bertoni et al., 2000). Measured energy and mineral metabolism parameters seem to confirm that heat stress occurred during the hotter period. The negative effect of THI on plasma glucose observed in the current study agrees with results obtained in heat stressed cows (Ronchi et al., 1997; Abeni et al., 2007). This difference can be explained by different factors: decreased energy intake, as consequence of the reduction in dry matter intake; increased cost for thermoregulation; and negative effect of the heat on gluconeogenesis, as an endocrine acclimation to hot conditions. The negative relationship between THI and NEFA (Table 2) was a further confirmation of the metabolic adaptations to the heat stress condition during the hotter period (Ronchi et al., 1999; Abeni et al., 2007), suggesting increased use of NEFA as fuel in hepatic and peripheral tissues during heat stress (Ronchi et al., 1999). The different pattern of DMI during the night time and daytime in the summer season could also explain the reduction of NEFA and also the increase of BHB in plasma from cows bled before the morning meal in hot season (Ronchi, 1998). In fact there is a meal effect, with a reduction in NEFA and increase in BHB after a meal (ASPA Commission, 1999). Concerning the mineral metabolism parameters, the negative effect of THI on plasma Na and K concentration observed in the current study agrees with the results obtained by Ronchi et al. (1995) in calves exposed to very high temperatures.

During severe heat stress the reduction in intestinal blood flow can elicit an inflammatory response (Lambert, 2009), resulting in the promotion of the synthesis of +APP, and as a consequence the reduction of several essential proteins (including albumins) called negative acute phase proteins (-APP). In the current study the APP were not affected by THI, confirming that cows were not exposed to severe heat stress. Furthermore, these APP were not affected by Se supplementation.

Among the plasma markers of oxidative status, the negative relationship between THI and SHp could be a consequence of increased plasma protein oxidation (Sandre et al., 2004). Increases in AST activity indicate a slight impairment of tissues, and also this effect could be a consequence of an oxidative effect. However, the values of LDH activity, and the activity of other enzymes related to tissue impairment, falls within range and are very close to values observed by Juniper et al. (2008). Therefore, these results seem to indicate that a general impairment of tissues was not evident. Among the other markers of oxidative status, ROM were not affected by THI, nevertheless the positive effect of THI on GPX-3 could be interpreted as an up-regulation in stress conditions. Indeed, GPX is well regulated and its increased activity can be considered an additional protective mechanism in stress conditions (Surai, 2006).

The plasma TBARS concentrations in the current study were greater than the values obtained with the fluorimetric method (Mudron et al., 2007) and lower than values observed by Bernabucci et al. (2002) in early lactating cows. The TBARS were not affected by THI, nevertheless, during the hotter period significant differences were observed. The lower TBARS values in SY cows compared with SS cows could be related to lower oxygen-free radical values, considering that lipids are a possible target for the latter, in the process of lipid peroxidation and production of lipoperoxides (increasing TBARS) and impaired membrane integrity. Cytotoxic aldehydes (e.g., malonaldehyde) that remain after termination of lipid peroxidation provide the basis for the TBARS test in body fluids. (Miller et al., 1993).

Oxidative stress resulting from increased production of free radicals and reactive oxygen species, and/or a decrease in antioxidant defense, leads to damage of biological macromolecules and disruption of normal metabolism and physiology (Trevisan et al., 2001). In the current study no source effect was observed on whole blood GPX (Calamari et al., 2010) and on plasma GPX. Also the SHp's were not affected by Se source. However, different relationships between SHp and indicators of Se status were observed between animals supplemented with SY when compared to those supplemented with SS. Furthermore, differences were also observed in the relationship between SHp and ROM in SY when compared to SS supplemented animals. Different hypothesis could be formulated to explain these different results obtained with SS and SY.

Selenite is capable of promoting radical formation and oxidative stress through its reaction with reduced glutathione (Surai, 2006), inactivating critical thiol-containing enzymes by oxidation and producing ROM. It has been observed that selenite generates ROM and causes cellular damage in the presence of sulfhydril compounds (Surai, 2006). Conversely, selenomethionine and Se-methylselenocysteine are not oxidizing agents (Drake, 2006). In most experiments SeMet was not able to produce ROM when added into an incubation medium in combination with reduced glutathione (Surai, 2006). These findings contribute to explain the negative correlation between SHp and total plasma Se observed only in cows supplemented SS. Furthermore, these findings also contribute to explain the negative correlation between ROM and SHp observed only in cows supplemented SS.

The inactivation of critical thiol-containing enzymes by oxidation reduces the activity of the first group of endogenous antioxidants (including GPX). The maintenance of glutathione reductase activity (Mahmoud et al., 2003) enhances and facilitates the ability to reduce oxidized glutathione (GSSG to 2 GSH). A thiol redox system, consisting of the glutathione and thioredoxin systems, is believed to be the major player of the redox status of the cells (Surai, 2006). Thioredoxin reductase, a selenoenzyme (Surai, 2006), can reduce not only thioredoxin, but also oxidized glutathione (Surai 2006). Rousseau et al. (2006) observed higher blood oxidized glutathione and lower SHp in exercised elderly subjects when compared with exercised young subjects. In the current study, blood oxidized glutathione was not measured, nevertheless, the different relationships between SHp and with the indicators of Se status and plasma ROM seem to indicate a less pronounced impairment of the second group of the endogenous antioxidants (SHp) in SY rather than in SS supplemented animals. This consequently results in the impairment of the first group of endogenous antioxidants, including GPX.

Castillo et al. (2006) suggested that decreased serum TA is not necessarily an undesirable condition when the production of reactive species (which would be reflected in malonaldehyde values, as observed in this study in SY cows) decreases. The lower plasma TA in cows fed SY in the current study could be interpreted as an improvement in their preventive antioxidant systems in terms of the free radical formation and chain-breaking antioxidants. Despite GPX-1 activities not differing between SY and SS supplemented animals (Calamari et al., 2010), the differences observed between the markers of oxidative status with Se status seem to indicate that SY improves the prevention of free radical formation, with a consequent reduction in plasma TA. Because Se (glutathione and thioredoxin systems) is also involved in the line of defense consisting of chain-breaking antioxidants (Surai, 2006), it could be hypothesized that the reduction in plasma TA could be a consequence of an improvement in the chain-breaking antioxidants not detected with the analysis of TA in plasma (i.e., intracellular systems as phospholipid hydroperoxide GPX or GPX-4 and thioredoxin system). Furthermore, these reduced systems decrease oxidized glutathione, and then improve the activity of GPX. However, further studies are needed to confirm these hypotheses.

The values for hematological profiles were found to be within normal reference intervals for dairy cows in the environment of this study (Archetti and Ravarotto, 2002). The negative effect of THI on circulating WBC and of each leukocyte population agrees with the results of Paape et al. (1973), when a regular alternation between hot days and cooler nights take place. The lower values of PLT in SY groups during the hotter period did not agree with the results of Sakr et al (2007) on the relationship between PLT count and plasma Se. In fact, they reported a positive correlation between these two features, but in the current trial the SY groups, characterized by low PLT during the hotter period, are the same with higher plasma Se (Calamari et al., 2010).

5. Conclusions

The temperatures and humidity recorded during this trial indicated that cows in this study may have suffered slight-mild heat stress during the hotter period, as highlighted by microclimatic condition as well as by the animal response (i.e. changes in plasma glucose, NEFA and electrolytes). The low Se supplementation in CTRL cows, as well as the total Se in whole blood and plasma, indicate a marginally adequate Se status in these animals. This was confirmed by the lack of important effects on hematological profiles, generally more responsive when Se is deficient. Oxidative stress, with greater plasma TBARS, during the hotter period was observed in CTRL animals compared to those observed in cow fed SY. Furthermore, during the hotter period, cows fed SY, when compared to those fed SS have shown lower plasma TBARS and plasma total antioxidant, as well as numerically lower plasma ROM. These results could be interpreted as an improvement in the preventive antioxidant systems in terms of the prevention system of free radical formation and chain breaking antioxidants in cows fed Se yeast, with a lower lipid peroxidation during hotter periods, when animals are subjected more to oxidative stress.

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