Int J Biometeorol (2007) 52:97–107 DOI 10.1007/s00484-007-0097-4

ORIGINAL PAPER

Metabolic conditions of lactating Friesian cows during the hot season in the Po valley. 2. Blood minerals and acid-base chemistry Luigi Calamari & Fabio Abeni & Ferdinando Calegari & Luigi Stefanini

Received: 3 November 2006 / Revised: 12 March 2007 / Accepted: 22 March 2007 / Published online: 15 May 2007 # ISB 2007

Abstract In two consecutive summers, 21 and 18 cows respectively were monitored for acid-base chemistry and some blood minerals, to assess their variation according to the level of heat stress at different stages of lactation. During both years, the cows were monitored according to their lactation phase (early, mid-, and late) at the beginning of the summer. Climatic conditions were described through the temperature humidity index. Cows were monitored weekly for: breathing rate, rectal temperature, hemogas parameters and blood minerals (morning and afternoon collection). In the first year, two hotter periods were identified, with more severe conditions in the second one, when cows had rectal temperatures higher than 40°C. In the second year, only one hotter period was identified, with a heat stress comparable to that of the first period of the first year. The behaviour of rectal temperature, breathing rate and the parameters of the acid-base status indicated that the suffering of the cows was on the borderline between mild L. Calamari (*) Istituto di Zootecnica, Facoltà di Agraria, Università Cattolica del Sacro Cuore, Piacenza, Italy e-mail: [email protected] F. Abeni : L. Stefanini Azienda Sperimentale “V. Tadini”, Gariga di Podenzano, Piacenza, Italy F. Calegari Istituto di Genio Rurale, Facoltà di Agraria, Università Cattolica del Sacro Cuore, Piacenza, Italy Present address: F. Abeni CRA Istituto Sperimentale per la Zootecnia, Cremona, Italy

and high heat stress during the hotter periods only, according to the climatic conditions in the two years. During the hotter periods, the acid-base chemistry differed significantly with a reduction of HCO3 and an increase of Cl during the hotter hours of the day. The compensation mechanism for mild alkalosis during hotter hours maintained blood pH and the HCO3 returned to normal values during the night. Significant reductions were observed for Mg and Zn during the hotter periods. The cows in late lactation appeared to be less stressed by the hot climate. Keywords Heat stress . Dairy cows . Acid-base chemistry . Blood minerals

Introduction In Italy, persistent, intense heat and humidity characterize the summer months in the northern region, particularly in the Po Valley, from May through September. The Temperature Humidity Index (THI) is commonly used as an indicator of the degree of climatic stress on animals, where a THI of 72 and below is considered as no heat stress (cool), 73–77 as mild heat stress (HS), 78–88 as moderate and above 88 as severe (Fuquay 1981; Armstrong 1994). Heat stress occurs when any combination of environmental conditions, such as air temperature, relative humidity, air movement and solar radiation cause the effective temperature of the environment to be higher than the animal’s thermo-neutral zone or comfort zone (Bianca 1962). Prolonged exposure to high ambient temperatures has negative effects on the physiological balance of dairy cows, including acid-base alterations (West et al. 1991). The acidbase balance of animals depends on the intricate relationship between anions and cations in the blood. Under normal

98

conditions, acids and bases are added continuously to the body fluids as a result of either ingestion or production during cellular metabolism. To combat any changes in the normal acid-base balance, the body utilizes three basic mechanisms: chemical buffering, respiratory adjustment of blood carbonic acid and excretion of H+ or HCO3 by the kidneys (Houpt 1989). A cow subjected to hot climatic conditions can have acid-base disturbances resulting from respiratory alkalosis, subsequent renal compensation by increasing urinary excretion of bicarbonate and Na, and renal conservation of K (Collier et al. 1982). When the heat load increases, there is a subsequent increase in the respiratory frequency of cattle that increases evaporative heat loss from the upper respiratory passage. Panting tends to alter alveolar ventilation which subsequently alters blood pH, O2 and CO2. Carbon dioxide is eliminated faster than it is produced, pCO2 is lowered, and blood pH rises. Decreased pCO2 reduces renal tubular acid secretion and exaggerates the compensatory loss of alkali reserve in the urine. As CO2 is eliminated, H2CO3 is formed from H+ and HCO3 , so the pH increases and the concentration of HCO3 decreases. The net result of these processes is that pCO2 decreases, the pH increases, and the concentration of HCO3 decreases and is replaced by other buffers. There is no change in total buffer base. In order to restore the pH to normal, hyperventilation must be stopped, or the kidneys must eliminate HCO3 . The latter occurs because the low pCO2 and alkalosis reduce H+ and NH3 production by the kidneys. When H+ is not produced in sufficient amounts to capture all the filtered HCO3 , the latter spills into the urine (Robinson 1997). Heat stress is cyclical in nature, with cows generally being at peak stress by mid-afternoon and cooling somewhat in the evening and early morning hours. Cows may exhibit a respiratory alkalosis in the afternoon, but physiological responses may actually overcorrect, so that when cows become cool a metabolic acidosis occur. Cows in early lactation during hot weather exhibited a marked decline in plasma Na, K and Cl concentrations during the day, with concentration returning to normal at night (Maltz et al. 1994). Wide swings in the acidbase chemistry and blood electrolyte content of the cow occur within a short time span. (Sanchez et al. 1994). The aim of this paper was to verify, during two consecutive summers, the pattern of some blood parameters of the acid-base status of dairy cows as related to their phase of lactation during summer season in the Po Valley.

Materials and methods The study was carried out through two consecutive summers, from June to September, at the “Vittorio Tadini” Experimental Farm, near Piacenza; for housing and location details, see the companion paper (Abeni et al. 2007).

Int J Biometeorol (2007) 52:97–107

During the first year, 21 cows (9 primiparous and 12 pluriparous) were monitored, while during the second year the study involved 18 cows (5 primiparous and 13 pluriparous). The cows were of the Italian Friesian breed. They were chosen to be representative of three different phases of lactation at the beginning of the summer season: between 30 and 80 days of lactation (A), just before mid lactation (B), and in the second half of lactation (C); for details, see the companion paper (Abeni et al. 2007). The primiparous cows were homogenously distributed in the three lactation phases. Air temperature and humidity were recorded inside the barn, using a continuous recording system placed in the feeding lane. 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). The experimental period lasted 100 days each year. During those periods, blood was sampled weekly (on Wednesday) from the jugular vein, in the morning (0730 hours) and in the afternoon (1630 hours). Cows were locked by a self-locking system, with voluntary access to the feeding gate, avoiding any kind of stress. The afternoon sampling time was about 30 min after milking. Sampling was carried out with vacuum system tubes (Vacutainer) with Liheparin, avoiding air introduction in the tubes, which might alter hemogas parameters. Samples were then immediately stored in an ice bath until analyzed for hemogas and processed for plasma separation. Hemogas analysis was performed by a Gas-Analyzer (IL1306, Instrumentation Laboratory), within 90 min of sampling, on whole blood. Parameters directly measured were: pH, relative pressure of carbon dioxide (pCO2) and relative pressure of oxygen (pO2) (Calamari et al. 1995). Total bicarbonates (HCO3 ) were then calculated from the previously measured parameters (National Committee for Clinical Laboratory Standards 1982). Just after blood separation for hemogas analysis, packed cell volume (PCV) was determined by microhemocitometric centrifugation. The remaining blood was centrifuged at 3,500 g for 15 min to separate plasma, which was stored in four sub-samples at −20°C for subsequent analysis. The frozen samples were used to determine the following parameters: calcium; inorganic phosphorous; magnesium by a colorimetric reaction kit (blue of xilidile, Roche); zinc by a colorimetric kit (WAKO); sodium, potassium, and chlorine by potenziometric system with specific electrodes. In addition, glucose and total protein on plasma were measured as described in the companion paper (Abeni et al. 2007). During the first year, only the lactating phase A and B cows were controlled for blood parameters. Breathing rate (BR), rectal temperature (RT), feeds and diets were monitored as described in the companion paper (Abeni et al. 2007). The content of Na, K and Cl in feeds was

Int J Biometeorol (2007) 52:97–107

99

also measured by inductively coupled plasma optical emission spectrometry (ICP-OES Optima 2100 DV; Perkin Elmer, Shelton, Conn., USA). In defining the content of Na, K and Cl in the diet, the increase of Na and K requirements in animals under heat stress was also taken into account (NRC 2001). In each year, hotter periods were defined considering the minimum and the maximum daily THI: minimum daily THI near the lower values of the zone delimiting mild heat stress (72) and maximum daily THI higher than minimum values delimiting the high heat-stress zone (78), as indicated in Armstrong (1994). In the first year there were two hotter periods: the first week of July (Y1HP1) and the first 10 days of August (Y1HP2). In the second year there was one hotter period: the fourth week of July (Y2HP1). In addition, the occurrence of heat waves was detected according to Hahn et al. (1999); this analysis confirmed the previous one, classifying both the hotter periods of the first year and the hotter period of the second year as mild–moderate heat waves.

Data from each year were analyzed separately, using the MIXED PROC of the SAS (1996), including lactation phase (three levels), periods (three levels in the first year and two levels in the second year), interaction by lactation phase and period, day of control within period, interaction by lactation phase and day of control within period, random experimental error from cow nested within lactation phase and day of control. The description of the model is reported in the companion paper (Abeni et al. 2007). The differences between hotter periods (HP1 and HP2) with HP0 period in each year were estimated with pairs ttest. Correlation coefficients were calculated between all the recorded parameters, separately by year. Differences were considered significant when P<0.05; in addition, however, the presence of a trend was noted when P<0.10.

b 100.0

100.0

90.0

90.0

80.0

80.0

Breathing rate, n/min

Breathing rate, n/min

a

Statistical analysis

70.0 60.0 50.0 40.0

50.0 40.0

20.0

20.0

40.50 Rectal temperature, ˚C

40.50 Rectal temperature, ˚C

60.0

30.0

30.0

40.00 39.50 39.00 38.50 38.00 37.50 15/6

70.0

5/7

25/7

14/8

3/9

23/9

Day/Month

Fig. 1 Behaviour of breathing rate (BR) and rectal temperature (RT) measured in the afternoon (1530–1600 hours for BR and 1700– 1800 hours for RT) in dairy cows in early (A: , continuous line), mid- (B: , dashed line) and late lactation (C: , dotted line) in the

40.00 39.50 39.00 38.50 38.00 37.50 15/6

5/7

25/7

14/8

3/9

23/9

Day/Month

first (a) and second (b) year during the trial. The boxes highlight the hotter periods: Y1HP1 and Y1HP2 in the first year; Y2HP1 in the second year

100

Int J Biometeorol (2007) 52:97–107

Results

Microclimatic conditions

Feeds and diets

Microclimatic data are presented in the companion paper (Abeni et al. 2007).

Chemical and nutritive characteristics of the diets are reported in the companion paper (Abeni et al. 2007). In addition to these data, the content of electrolytes in the diet was 0.314, 1.617 and 0.389% DM respectively for Na, K and Cl in the first year; 0.311, 1.653 and 0.393% DM respectively for Na, K and Cl in the second year.

Rectal temperature (RT) and breathing rate (BR) Both parameters increased significantly during the hotter periods. During the first year, the mean value of RT was 39.18°C in Y1HP1 and in Y1HP2, when microclimatic

Table 1 Lsmeans of plasma electrolytes, and pH pCO2, pO2 and HCO3 in venous blood in in the hotter periods (Y1HP1 and Y1HP2 in the first year, Y2HP1 in the second year; characterized by minimum daily THI near 72 and maximum daily THI higher than 78) and in the other periods of summer (Y1HP0 in the first year and Y2HP0 in the second year) Year

1

Time of day

Morning

Afternoon

2

Morning

Afternoon

Parameter

Na K Cl pH pCO2 pO2 HCO3 Na K Cl pH pCO2 pO2 HCO3

Na K Cl pH pCO2 pO2 HCO3 Na K Cl pH pCO2 pO2 HCO3

Units

mmol/l mmol/l mmol/l mmHg mmHg mmol/l mmol/l mmol/l mmol/l mmHg mmHg mmol/l

mmol/l mmol/l mmol/l mmHg mmHg mmol/l mmol/l mmol/l mmol/l mmHg mmHg mmol/l

Period

Prob F

SE

HP0

HP1

HP2

LP

LP x P

Y1HP0 139.3 4.07 105.3 7.415 41.82 34.07 27.07 142.5 4.13 105.6 7.435 41.36 34.52 28.02

Y1HP1 139.3 4.11 104.8 7.419 42.12 32.62* 27.52 143.5 4.29* 105.7 7.430 41.34 30.27*** 27.75

Y1HP2 141.3*** 4.16 107.7*** 7.423 40.33* 33.35 26.59 141.4* 4.25 108.3*** 7.424* 38.55*** 36.65*** 25.53***

0.008 0.019 0.0005 NS 0.011 0.01 0.022 NS 0.019 0.005 NS NS 0.005 0.05

NS NS NS NS 0.079 NS 0.098 NS NS NS NS NS NS NS

0.250 0.040 0.300 0.004 0.314 0.587 0.257 0.340 0.051 0.330 0.004 0.320 0.670 0.270

Y2HP0 139.7 4.02 107.6 7.404 41.58 34.21 26.25 141.6 3.93 107.27 7.410 41.44 33.43 26.52

Y2HP1 139.8 3.95 107.4 7.409 40.78 32.94* 26.03 142.7* 4.04* 107.57 7.419* 38.69** 34.47* 25.27*

0.004 NS 0.099 0.062 NS NS NS 0.005 NS NS NS NS 0.018 NS

NS NS NS NS NS NS NS 0.077 NS NS NS NS NS NS

0.323 0.046 0.335 0.004 0.462 0.452 0.318 0.270 0.048 0.335 0.003 0.409 0.489 0.264

*P<0.05, **P<0.01, ***P<0.001, between hotter periods (HP1 and HP2) with HP0 period LP Lactation phase P Period (Y1HP0, Y1HP1 and Y1HP2 in the first year; Y2HP0 and Y2HP1 in the second year)

Int J Biometeorol (2007) 52:97–107

conditions were more adverse, the mean value of RT was 39.98°C (Fig. 1a). During the second year, RT reached values higher than 39°C only in Y2HP1. The RT pattern (Fig. 1b) was similar for the three phases of lactation. However, during Y2HP1, RT was higher (P<0.001) in midlactation (B phase: 39.76°C) and lower in late lactation (C phase: 39.03°C). A significant correlation between RT with maximum daily THI (r=0.65; P<0.001 in the first year and r=0.52; P<0.001 in the second year) was observed. Similar correlations were observed between RT with daily minimum temperature. A significant correlation between RT and BR (r=0.43, P<0.001 in the first year; r=0.70; P<0.001 in the second year) was noted. In the first year, BR reached the highest value in Y1HP1, whereas RT reached the highest value in Y1HP2 (Fig. 1a). The values of BR in the three phases of lactation confirm the results shown by rectal temperature. In the second year, the BR in Y2HP1 increased to a lower extent in late phase of lactation (85 n/min) than in the early and mid- ones (99.4 and 100 n/min, respectively; P<0.05). During the second year, the pattern of BR was very similar to those of RT. Electrolytes and acid-base balance In Table 1, the values of plasma electrolytes, blood pH, pCO2, pO2 and HCO3 observed in the samples collected in the morning and in the afternoon in the different periods of the 2 years are summarized. The main differences were observed in the afternoon data. During the first year, a significant increase (P<0.001) in plasma Cl was observed in Y1HP2. At the same time, a reduction in Y1HP2 of blood pCO2 (P<0.001) and HCO3 compared to Y1HP0 was observed. These reductions were confirmed in the second year. Blood pH increased, in the afternoon sampling, in Y1HP2 (P<0.05) and Y2HP1 (P < 0.05), but the increase was very slight (Fig. 2). The pattern of changes of Cl, pCO2 and HCO3 , measured in the blood collected in the afternoon, indicates an increase in the former and a decrease in the latter during the hotter periods, with a subsequent quick recovery (Fig. 2). Positive correlation was observed between afternoon blood pH with RT (P < 0.001) and with daily maximum THI (P<0.001) in the second year. Negative correlation was observed between afternoon blood pCO2 with RT (P<0.001), with BR (P<0.001) and with daily maximum THI (P<0.001) in the second year. The afternoon blood HCO3 was negatively correlated with RT (P<0.001 in the first year and P<0.01 in the second year), with BR (P<0.001 in the second year) and with daily maximum THI (P<0.001 in both years). In contrast, the blood HCO3 measured in the morning sampling was not correlated with RT, BR and with daily maximum THI. A

101

significant correlation was observed between Cl with pCO2 in the morning samples (r=−0.33 in the first year and −0.54 in the second year; P<0.001) and between Cl with HCO3 (r=−0.50 in the morning checks in the first year; r=−0.67 in the morning checks and r=−0.60 in the afternoon checks in the second year; P<0.001). Plasma K concentration increased, in the afternoon sampling, in Y1HP1 (P<0.05) and Y2HP1 (P<0.05). During Y1HP2, the values of plasma Na were higher than Y1HP0 in the morning (P<0.05) and were lower than Y1HP0 in the afternoon (P<0.05). Otherwise, plasma Na in the afternoon in Y2HP1 was higher than Y1HP0 (P<0.05). A significant correlation was observed between plasma Na and K with RT, BR and with daily maximum THI in both years, but the correlations were often negative and sometimes positive. Other blood parameters Variable differences for plasma Ca, P, Mg, and Zn, during the study were observed. Zn slightly decreased in both years during the hotter periods (P<0.05 in Y1HP1 vs Y1HP0 and in Y2HP1 vs Y2HP0). A significant increase of plasma Ca (P<0.001) in Y1HP2 was observed. A positive correlation between Ca with PCV was observed in the first (r=0.29; P<0.001) and second (r=0.39; P<0.001) year. During the second year, a reduction of plasma Mg (P<0.01) in Y2HP1 (Table 2) was noted. Negative correlation between plasma Mg, both in morning and afternoon blood sampling, with RT (P<0.001 in both years), with BR (P<0.001 in the second year) and with daily maximum THI (P < 0.001 in both years) were observed. Blood PCV in morning sampling was lower during the hotter periods of both years, compared to the other overall data. The differences were consistent in the afternoon, with lower values in the hotter period of the first year (0.275 vs 0.290 l/l in Y1HP2 vs Y1HP0; P<0.01) and second year (0.287 vs 0.300 l/l in Y2HP1 vs Y2HP0; P < 0.01). Negative correlation was observed between PCV, both in morning and afternoon blood sampling, with RT (P<0.01 in both years), with BR (P<0.01 in both years) and with daily maximum THI (P<0.01 in both years). Plasma glucose was significantly lower in the hotter periods in both years and the data are reported in the companion paper (Abeni et al. 2007). A negative correlation between plasma glucose with RT was observed in the first year (r=−0.38, P<0.001) and with daily maximum THI (r=−0.48, P<0.001 in the first year; r=−0.29, P< 0.001 in the second year). No significant differences were found in total protein concentration in the hotter periods compared to the other periods.

102

Int J Biometeorol (2007) 52:97–107

b 7.55

7.55

7.50

7.50

pH

pH

a

7.45

7.45

7.40

7.40

7.35

7.35

50.0 48.0

50.0

44.0

pCO2, mm Hg

pCO2, mm Hg

46.0

42.0 40.0 38.0 36.0 34.0 32.0

45.0 40.0 35.0 30.0

30.0 36.0

36.0

34.0

34.0 HCO3-, mmol/L

HCO3-, mmol/L

32.0 30.0 28.0 26.0 24.0

32.0 30.0 28.0 26.0 24.0 22.0

22.0

20.0

115 113 111 109 107 105 103 101 99 97 95 15/6

115 Cl, mmol/L

Cl, mmol/L

20.0

5/7

25/7

14/8

3/9

23/9

Day/Month

Fig. 2 Behaviour of blood pH, pCO2 and and plasma Cl measured in , dairy cows (bled in the afternoon at h 16.30) in early (A: continuous line), mid- (B: , dashed line) and late lactation (C: ,

110 105 100 95 15/6

5/7

25/7

14/8

3/9

23/9

Day/Month

dotted line) in the first (a) and second (b) year during the trial. The boxes highlight the hotter periods: Y1HP1 and Y1HP2 in the first year; Y2HP1 in the second year

Int J Biometeorol (2007) 52:97–107

103

Table 2 Lsmeans of PCV and plasma mineral in the hotter periods (Y1HP1 and Y1HP2 in the first year, Y2HP1 in the second year; characterized by minimum daily THI near 72 and maximum daily THI higher than 78) and in the other periods of summer (Y1HP0 in the first year and Y2HP0 in the second year) Year

Parameter

Units

Period

Prob F

SE

HP0

HP1

HP2

LP

LP x P

Y1HP2 0.288** 0.275* 2.60* 2.05* 1.03 14.00*

0.0001 0.015 0.045 0.0002 0.076 0.0004

NS NS NS NS NS NS

0.003 0.004 0.017 0.088 0.011 0.390

0.021 NS NS NS NS 0.006

NS NS NS NS NS NS

0.003 0.003 0.019 0.042 0.012 0.303

1

PCV (morning) PCV (afternoon) Ca (morning) P (morning) Mg (morning) Zn (morning)

l/l l/l mmol/l mmol/l mmol/l μmol/l

Y1HP0 0.301 0.29 2.55 1.90 1.02 14.97

Y1HP1 0.292** 0.281 2.53 1.80 1.00 13.63*

2

PCV (morning) PCV (afternoon) Ca (morning) P (morning) Mg (morning) Zn (morning)

l/l l/l mmol/l mmol/l mmol/l μmol/l

Y2HP0 0.300 0.300 2.54 1.74 1.12 14.26

Y2HP1 0.292* 0.287** 2.58 1.72 1.06** 13.37*

*P<0.05, **P<0.01, between hotter periods (HP1 and HP2) with HP0 period LP Lactation phase P Period (Y1HP0, Y1HP1 and Y1HP2 in the first year; Y2HP0 and Y2HP1 in the second year)

Discussion Rectal temperature and breathing rate The values of RT over the normal range of dairy cows (38.3–38.7°C) indicates heat stress and insufficient thermoregulation. This situation was observed in 8 of the 12 relieves during the first year and in 4 of the 11 relieves during the second year. At those times, the maximum daily THI was higher than the minimum values of the high heat stress zone (Armstrong 1994) and near the stress threshold of 25°C indicated by Hahn et al. (1999). The pattern of RT was closely related to those of minimum and maximum daily THI and was positively correlated. To maintain body temperature within the normal range (38.3–38.7°C), cattle need to increase heat loss and reduce the endogenous heat production. Several physiological changes indicate the response of dairy cows to heat stress, which is manifested by a number of overt reactions, including reduced feed intake, increased water intake and evaporative water losses from the body, increased BR, and maintenance requirements, as well as altered endocrine parameters. The sudden responses of cattle are an increase in water intake and BR, a reduction of feed intake and, subsequently, a reduction of metabolic rate. Breathing rate (BR) has long served as a gross indicator of heat load in cattle during hot weather, increasing when

animals need to maintain homeothermy by dissipating excess heat, as more benign avenues become inadequate. Hahn et al. (1999) indicates the value of 21°C as a threshold for increased BR. The BR is easy to check and it demonstrated a strong relationship with RT and environmental THI. The BR value increased more in the first hot period. This was probably due to the effective activation of the thermoregulatory system in cattle when exposed to high temperature, leading to a kind of acclimation and a less negative impact with subsequent THI rises. Cows in mid-lactation had the highest BR in hotter periods and they would appear to be the most stressed by the hot climate (Perera et al. 1986). These results confirm the observations on milk yield reported in the companion paper (Abeni et al. 2007). The reduction of PCV during the hotter periods may be due to an expanded plasma volume, to accommodate increased evaporative loss from skin. On the other hand, total protein did not decrease in hotter periods (Abeni et al. 2007 companion paper), suggesting that other factors were also involved in the reduction of PCV (Ronchi et al. 1999). The results confirm the close relationship of THI with BR and RT, indicating that during the trial the cows experienced mild heat stress and that their suffering was on the borderline between mild and high heat stress in the hotter periods alone.

104

Acid-base balance A cow subjected to hot climatic conditions can have acid-base disturbances resulting from respiratory alkalosis, subsequent renal compensation by increasing urinary excretion of bicarbonate and Na, and renal conservation of K (Collier et al. 1982). During extreme heat stress, cows exhibited a respiratory alkalosis with elevated blood pH and reduced concentrations of blood CO2 and bicarbonate (Schneider et al. 1984). In chamber experiments, cows subjected to heat stress had a higher blood pH during hot hours compared to those in a thermal neutral environment (Schneider et al. 1988). Heat stress is diurnally cyclical in nature, with cows generally being at peak stress by mid-afternoon and cooling somewhat in the evening and early morning hours. Cows may exhibit a respiratory alkalosis in the afternoon, but physiological responses may actually overcorrect, so that when cows become cool metabolic acidosis occurs. The reduction of pCO2 observed in the hotter periods was due to the faster elimination of CO2 caused by the increased BR. As CO2 is eliminated, H2CO3 is formed from H+ and HCO3 , so the pH increases and the concentration of HCO3 decreases and is replaced by other buffers. The increase in the HCO3 / H2CO3 ratio increases pH. In order to restore the pH toward its normal value, the kidneys must eliminate HCO3 . This occurs because the low pCO2 and the respiratory alkalosis reduce H+ and NH3 production by the kidney. When H+ is not produced in sufficient amounts to capture all the filtered HCO3 , the latter spills into the urine (Robinson 1997). The relative stability of blood pH during the trial and the limited, but significant, reduction of pCO2 and HCO3 in venous blood from afternoon sampling in the hotter periods confirm that heat stress was not severe and that the suffering of the cows was on the borderline between mild and high heat stress. Only blood HCO3 , measured in the afternoon, showed a significant and negative correlation with RT and with daily maximum THI in both years. On the contrary, the blood HCO3 measured in the morning sampling was not correlated with RT, BR and with daily maximum THI. These results suggest that heat stress was not severe and that the compensation mechanism for the mild alkalosis during hotter hours maintained blood pH and blood HCO3 returned to normal values during night. A higher excretion of HCO3 through urine, with an increase in urine pH, was also observed in mild heat stress (Bianca 1965). Our results seem to confirm that blood HCO3 , measured in the afternoon, after the hotter hours of the day, appears to be a sensitive parameter in the evaluation of mild heat stress. During heat stress, the increased excrection of HCO3 influences blood Cl. In many species, the collecting duct of nephron is capable of HCO3 secretion in response to alkalosis. Functional studies of the isolated perfused cortical collecting duct have demonstrated that the type B

Int J Biometeorol (2007) 52:97–107

intercalated cell contains an apical Cl/ HCO3 exchanger, with active H+ resorption and exchange of Cl in the tubule fluid for intracellular HCO3 (Verlander 1997). This mechanism can explain the negative relationship between plasma Cl and blood HCO3 levels. The higher values of plasma Cl during some hotter periods are related to the higher excretions of HCO3 in the urine. The negative correlation between Cl and pCO2 and between Cl and HCO3 confirm our previous results (Calamari et al. 1995). An increase in plasma Cl was also observed in calves in severe heat stress (Ronchi et al. 1995). On the other hand, a reduction of Cl was observed by Maltz et al. (1994) during the hotter hours of the day in severe heat stress. Potassium and sodium may be important, especially during heat stress, as major regulators of body water. Sodium is required at the kidney for K conservation and to balance bicarbonate excretion electrically (Schneider et al. 1986). El Nouty et al. (1980) noted that urinary Na output increased during heat stress. Potassium loss through skin secretions may be substantial during heat stress because the major inorganic constituents of bovine sweat are K2CO3 and KHCO3 (Schneider et al. 1988). K and Cl losses in sweat are significant (Maltz et al. 1994). Retention of Na, K and Cl, the main elements involved in sweat, was more efficient in summer than winter (Maltz and Silanikove 1996; Silanikove et al. 1997). These research papers concluded that the greater part of the elevated summer milkfree water balance (about 20 kg/day) is devoted to evaporative water loss. Irrespective of this fact, cows that calve in summer do not maintain plasma Na, K and Cl homeostasis and plasma volume (Maltz et al. 1994). A marked decline in plasma Na and K concentrations during the day in cows in early lactation during hot weather was observed (Maltz et al. 1994), returning to normal at night. Other authors (Ronchi et al. 1995), reported a reduction of concentration of K and Na in plasma and an increase of Cl, in calves exposed to very high temperature (THI=86). In our trial, plasma Na and K were not affected by THI, in accordance with the results of Schneider et al. (1988) in severe heat stress conditions. A significant correlation was observed between plasma Na and K with RT, BR and with daily maximum THI in both years, but the correlations were often negative and sometimes positive. These results suggest an implication and a relationship between these parameters with evaporative process, water intake and water reservoir (mainly in the gastrointestinal tract). Water loss from an animal, due to evaporative loss during heat stress, is a continuous process; in contrast, water intake is episodic (Kadzere et al. 2002). The fluctuations in drinking volumes and drinking pattern, not measured in our trial, challenge the physiological ability of the animal to maintain the gastrointestinal tract homeostasis of volume and solute, with substantial fluctuations in blood

Int J Biometeorol (2007) 52:97–107

plasma electrolyte concentrations and volume (Kadzere et al. 2002). Minerals Different responses were observed on plasma Ca during hotter periods, with an increase only in Y1HP2; this rise was not confirmed in the second year. An increase of plasma Ca was observed in Holstein cows during hot weather (Toharmat and Kume 1997) and in heat stressed Holstein and Jersey cows (Srikandakumar and Johnson 2004). A reduction of plasma Ca in a hot environment was observed in dairy cows (Kume et al. 1986; Sanchez et al. 1994) and in heifers (Ronchi et al. 1995). Plasma Ca might be affected by respiratory alkalosis, which could lead to a subtraction of Ca from plasma (Sanchez et al. 1994). The diluting effect of the rumen would likely reduce the degree to which passive calcium absorption would occur (NRC 2001). Nevertheless, active transport of calcium appears to be the major route for calcium absorption in mature ruminants and a careful homeostatic mechanism, regulating Ca absorption, resorption or by resorbing a larger portion of the Ca filtered across the renal glomerules, maintaining Ca constant in extracellular fluids (NRC 2001). Thus, the level of Ca in plasma is not related to the Ca intake (ASPA Commission 1999). The higher risk of an inflammatory situation (Bertoni 1998) could contribute to the Ca reduction as an effect of some cytokines (Ronchi et al. 1995; Bertoni 1998). Some cytokines could impair haematopoiesis with a reduction of PCV (Klasing 1988), contributing to explain the correlation observed between Ca with PCV. Phosphorus level in plasma is influenced by the amount of P supplied in the diet (ASPA Commission 1999). Passive absorption of P predominates when normal to large amounts of potentially absorbable phosphorus are consumed, and absorption is related directly to the amount in the lumen of the small intestine and to concentrations in blood plasma (Wasserman and Taylor 1976). Kume et al. (1986) observed a reduction of plasma P in cows kept in a hot environment, while an increase was observed by Ronchi et al. (1997). In hot conditions, lower feed intake reduces P supply and the lower blood flow in the digestive tract reduces the nutrient uptake (Bertoni 1998), both favorable to a reduction of plasma P. The slight and not significant variations observed in plasma P during the our trial seems to indicate that the absorption of P was not impaired in mild heat stress. A significant reduction of magnesium was observed in the hotter period in the second year alone. Plasma Mg is influenced by Mg intake (ASPA Commission 1999), so its reduction could be related to a decline in dry matter intake (DMI), not measured in our trial, often observed during heat

105

stress (Ronchi 1998). Magnesium is mainly, and perhaps only, absorbed in rumen and reticulum (Martens and Gabel 1986). Magnesium absorption from the rumen is dependent on the concentration of magnesium in solution in the rumen fluid (important to both the active and passive transport of magnesium across the ruminal wall) and the integrity of the magnesium transport mechanism (NRC 2001). During heat stress, the lower DMI often reported in literature (Ronchi 1998), associated with an increase in water content of digesta in the rumen (Silanikove 1992), could lead to inadequate magnesium in the ruminal fluid, reducing magnesium absorption. The active transport of magnesium across the rumen wall could be impaired in heat-stressed animals. Considering that the magnesium transport system is a sodium-linked active transport process (Martens and Gabel 1986), the Na reduction in plasma during heat stress, which was not, however, observed in our trial, could impair the active system transport. In addition, increased utilization of orally administered magnesium when administered with oral glucose has been reported, suggesting that glucose supplied the ruminal epithelium with a source of energy to power active transport of magnesium (Mayland 1988). The negative correlations between plasma glucose and rectal temperature could suggest an implication of energy balance on the reduction of plasma Mg. Finally, during heat stress blood flow to rumen epithelia is depressed (Hales et al. 1984) and this could also impair the active transport of magnesium across the rumen wall. Plasma Mg might be also related to the reduced activity of ALP observed in our trial and reported by Abeni et al. (2007 companion paper); in fact, this enzyme needs Mg for its activity (Ronchi et al. 1997). Plasma Zn reduction during heat stress was confirmed in both years. This observation agreed with data from Ronchi et al. (1997, 1999) and their suggestion that, in the absence of a significant increase in plasma ceruloplasmin and globulins, the low Zn concentration could not be attributed to a simple inflammatory response. Therefore, it is still difficult to explain plasma Zn reduction during heat stress and further research will be necessary.

Conclusions The behaviour of rectal temperature, breathing rate and the parameters of the acid-base status indicate that heat stress in dairy cattle was not severe during the trial. This was related to the climatic conditions, not particularly harsh in the two years studied, and to the open barn, which protected animals from solar radiation and took advantage of the natural ventilation. The suffering of the cows was on the borderline between mild and high heat stress only in the hotter periods. During these periods the compensation mechanism for the

106

mild alkalosis of the hotter hours, maintained blood pH and the HCO3 returned to the normal values during the night. The more sensitive parameters of acid-base chemistry to heat stress seem to be HCO3 and Cl; the former decreased and the latter increased in the hotter periods, when the animals suffered mild to high heat stress. Variable fluctuations of plasma Na and K during the hotter periods were observed, probably in relation to the hemodilution effect, whereas Mg and Zn often decreased significantly during hotter periods. These results suggest that even when heat stress is not severe the equations to calculate the requirements for some minerals have to be slightly modified, particularly for high milking cows and during early-mid lactation. Further research will be necessary to achieve better understanding of the mechanism behind the reduction of some blood minerals and the variations of some acid-base parameters when heat stress is not severe. Acknowledgment This research was conducted with a grant from the Emilia-Romagna region.

References Abeni F, Calamari L, Stefanini L (2007) Metabolic conditions of lactating Friesian dairy cows during the hot season in the Po valley. 1. Blood indicators of heat stress. Int J Biometeorol (in press) Armstrong DV (1994) Heat stress interaction with shade and cooling. J Dairy Sci 77:2044–2050 ASPA Commission (1999) Guida all’interpretazione dei profili metabolici. Università degli Studi di per uggia, Italy Bertoni G (1998) Effects of heat stress on endocrine-metabolic and reproductive of the dairy cows. Zoot Nutr Anim 24:273–282 Bianca W (1962) Relative importance of dry and wet bulb temperatures in causing heat stress in cattle. Nature 195:251 Bianca W (1965) Reviews of the progress of dairy science-section A. Physiology. Cattle in a hot environment. J Dairy Res 32:291–345 Calamari L, Lombardelli R, Calegari F, Molinari A, Abeni F (1995) Indagini preliminari sulle variazioni di pH, pCO2 e pO2 nel sangue di bovine Frisone durante il periodo estivo. Atti 11th Congr. Naz. ASPA, 71–72. DISPA, Udine Collier RJ, Beede DK, Thatcher WW, Israel LA, Wilcox CJ (1982) Influence of environment and its modification on dairy animal health and production. J Dairy Sci 65:2213–2227 El Nouty FD, Elbanna IM, Davis TP, Johnson HD (1980) Aldosterone and ADH response to heat and dehydration in cattle. J Appl Physiol 48:240–255 Fuquay JW (1981) Heat stress as it affects animal production. J Anim Sci 52:164–174 Hahn GL, Mader TL, Gaughan JB, Hu Q, Nienaber JA (1999) Heat waves and their impacts on feedlot cattle. In: Proceedings, 15th International Society Biometeorology Congress, September 1999, Sydney, Australia, pp 353–357 Hales JRS, Bell AW, Fawcett AA, King RB (1984) Redistribution of cardiac output and skin AVA activity in sheep during exercise and heat stress. J Thermal Biol 9:113–116 Houpt TR (1989) Water, electrolytes and acid-base balance. Dukes’ physiology of domestic animals, 10th edn. Cornell University press, Ihaca, NY, pp 486–506 Kadzere CT, Murphy MR, Silanikove N, Maltz E (2002) Heat stress in lactating dairy cows: a review. Liv Prod Sci 77:59–91

Int J Biometeorol (2007) 52:97–107 Klasing KC (1988) Nutritional aspect of leucocytic cytokines. J Nutr 118:1436–1446 Kume S, Kurihara M, Takahashi S, Shibata M, All T (1986) Effect of hot environmental temperature on major mineral balance in dairy cows. Jap J Zoot Sci 57:940–945 Ingraham RH, Stanley RW, Wagner WC (1979) Seasonal effects of tropical climate on shaded and nonshaded cows as measured by rectal temperature, adrenal cortex hormones, thyroid hormone, and milk production. Am J Vet Res 40:1792–1797 Maltz E, Silanikove N (1996) Kidney function and nitrogen balance of high-yielding dairy cows at the onset of lactation. J Dairy Sci 79:1621–1626 Maltz E, Silanikove N, Berman A, Shalit U (1994) Diurnal fluctuations in plasma ions and water intake of dairy cows as affected by lactation in warm weather. J Dairy Sci 77:2630–2639 Martens H, Gabel G (1986) Pathogenesis and prevention of grass tetany from the physiologic viewpoint. DTW Dtsch Tierarztl Wochen-schr 93:170–177 Mayland H (1988) Grass tetany. In: Church D (ed) The ruminant animal: digestive physiology and nutrition. Waveland Press, Prospect Heights, Ill National Committee for Clinical Laboratory Standards (1982) Tentative standard for definition of quantities and conventions related to blood pH and gas analysis. National Committee for Clinical Laboratory Standards 2:329–361 National Research Council (2001) Nutrient requirements of dairy cattle, 7th edn. National Academic Press, Washington DC Perera KS, Gwazdauskas FC, Pearson RE, Brumback TB Jr (1986) Effect of season and stage of lactation on performance of Holstein. J Dairy Sci 69:228–236 Robinson NE (1997) Acid-base homeostasis. In: Cunningam JG (ed) Textbook of veterinary physiology. Saunders, Philadephia, Pa., pp 621–633 Ronchi B (1998) Nutrition and feeling of dairy cattle during hot weather. Zoot Nutr Anim 24:283–293 Ronchi B, Bernabucci U, Lacetera NG, Nardone A, Bertoni G (1995) Effetti dello stress termico sullo stato metabolico di vitelle di razza Frisona. Zoot Nutr Anim 21:209–221 Ronchi B, Bernabucci U, Lacetera N, Nardone A (1997) Effetti dello stress termico sullo stato metabolico-nutrizionale di vacche Frisone in lattazione. Zoot Nutr Anim 23:3–15 Ronchi B, Bernabucci U, Lacetera N, Verini Supplizi A, Nardone A (1999) Distinct and common effects of heat stress and restricted feeding on metabolic status of Holstein heifers. Zoot Nutr Anim 25:11–20 Sanchez WK, McGuire MA, Beede DK (1994) Macromineral nutrition by heat stress interactions in dairy cattle: review and original researches. J Dairy Sci 77:2051–2079 SAS (1996) Statistical and analysis system. SAS Institute, Cary, N.C., USA Schneider PL, Beede DK, Wilcox CJ, Collier RJ (1984) Influence of dietary sodium and potassium bicarbonate and total potassium on heat-stressed lactating dairy cows. J Dairy Sci 67:2546–2553 Schneider PL, Beede DK, Wilcox CJ (1986) Responses of lactating cows to dietary sodium source and quantity and potassium quantity during heat stress. J Dairy Sci 69:99–110 Schneider PL, Beede DK, Wilcox CJ (1988) Nycterohemeral patterns of acid-base status, mineral concentrations and digestive function of lactating cows in natural or chamber heat stress environments. J Anim Sci 66:112–125 Silanikove N (1992) Effects of water scarsity and hot environment on appetite and digestion in ruminants: a review. Liv Prod Sci 30:175–194 Silanikove N, Maltz E, Valevi A, Shinder D (1997) Water, Na, K and Cl metabolism in high yielding dairy cows at the onset of lactation. J Dairy Sci 80:945–956 Srikandakumar A, Johnson EH (2004) Effect of heat stress on milk production, rectal temperature, respiratory rate and blood

Int J Biometeorol (2007) 52:97–107 chemistry in Holstein, Jersey and Australian Milking Zebu cows. Trop Anim Health Prod 36:685–692 Toharmat T, Kume S (1997) Effect of heat stress on minerals concentration in blood and colostrums of heifers around parturition. Asian Australasian J Anim Sci 10:298–303 Verlander JW (1997) Acid-base balance. In: Cunningam JG (ed) Textbook of veterinary physiology. Saunders, Philadephia, Pa., pp 546–554

107 Wasserman RH, Taylor AN (1976) Gastrointestinal absorption of calcium and phosphorus. In: Aubach GD (ed) Handbook of physiology, sect. 7: endocrinology, vol. VII, parathyroid gland. American Physiological Society, Washington, DC, pp 137–155 West JW, Mullinix BG, Sandifer TG (1991) Changing dietary electrolyte balance for dairy cows in cool and hot environments. J Dairy Sci 74:1662–1674