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Pesquisa Veterinária Brasileira

Print version ISSN 0100-736X

Pesq. Vet. Bras. vol.33 no.6 Rio de Janeiro June 2013 



Effects of environmental modification on mastitis occurrence and hormonal changes in Holstein cows


Efeito da modificação ambiental sobre a ocorrência de mastite e alterações hormonais de vacas Holandesas



Juliana R.P. ArcaroI; Soraia V. MatarazzoII,*; Claudia R. PozziI; Irineu Arcaro JuniorI; Luciandra M. de ToledoI; Elizabeth O. CostaIII; Mariana S. de MirandaI

ICentro APTA Bovinos de Leite, Instituto de Zootecnia (IZ), Rua Heitor Penteado 56, Nova Odessa, SP 13460-000, Brazil
IIDepartamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado Km 16, Ilhéus, BA 45662-900, Brazil
IIIDepartamento de Medicina Veterinária Preventiva, Universidade de São Paulo (USP), Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil




The purpose of this research was to evaluate the effects of evaporative cooling in freestall on mastitis occurrence, milk production, and composition, as well as cortisol, T3 (triiodothyronine), and T4 (thyroxin) levels in lactating dairy cows. Twenty-eight multiparous cows averaging 70 ± 10 day postpartum were used in four treatments from January to March 2003. The treatments were: Day (cooling from 7:00 a.m. to 7:00 p.m.); Night (cooling from 7:00 p.m. to 7:00 a.m.); 24-hour (cooling 24-hour); and Control (no cooling). Wired cup test was used for clinical mastitis diagnosis, and the California Mastitis Test (CMT) was used to identify subclinical mastitis. Blood and milk samples were taken weekly for microbiological and hormonal analyses. The cortisol levels were higher than normal values in all treatment groups, suggesting stress conditions, but T3 and T4 levels remained normal in all groups. The occurrence of subclinical mastitis was lower in Day and Night groups than in Control and 24-hour groups. Regarding the microbiological analyses, in all groups the isolation of Corynebacterium sp. from milk samples increased while negative coagulase staphylococci (CNS) declined as etiological agents of subclinical mastitis. However, in Day and 24-hour groups, coagulase positive staphylococci (CPS) increased mainly Staphylococcus aureus (49.8% and 47.7% respectively). The Night group showed a decrease in subclinical mastitis occurrences. Our data indicate that all animals subjected to treatments presented high levels of cortisol, indicating a stress condition. The Night treatment presented a reduction in microbial isolation, suggesting a reduced susceptibility to mastitis.

Index terms: Intramammary infection, mastitis, evaporative cooling, cortisol, thyroid hormones, dairy


O trabalho teve como objetivo avaliar a eficiência do sistema de resfriamento adiabático evaporativo, acionado em diferentes horários, em instalação do tipo freestall e seus reflexos sobre a ocorrência de mastite, produção e composição do leite e respostas hormonais de vacas em lactação. Foram utilizadas 28 vacas em lactação (70±10 dias), multíparas, das raças Holandesa Preta e Branca e Pardo Suíça, com produção média diária de 23±2,3 kg leite/dia. O período experimental de 56 dias teve início em 20 de janeiro de 2003. Os tratamentos foram: Controle (sem resfriamento); Dia (resfriamento 7 as 19 h); Noite (resfriamento 19 às 7 h) e 24 horas (resfriamento durante 24 h). A temperatura de bulbo seco (TBS), umidade relativa do ar (UR) e a temperatura de globo negro (TGN) foram mensuradas ao longo das 24 horas. A ordenha foi realizada às 7 h e 19 h. Amostragens semanais de leite e sangue foram realizadas para análise da composição do leite (gordura, proteína, lactose e contagem de células somáticas) e determinações hormonais de cortisol, tiroxina (T4) e triiodotironina (T3). Para avaliação da ocorrência de mastite clínica e subclínica foram feitos exames semanais de TAMIS (caneca de fundo preto) e California Mastitis Test (CMT). Foram colhidas amostras de leite de todos os quartos para identificação microbiológica dos agentes causais da mastite. O tratamento Dia diminuiu (P<0,05) a temperatura do freestall em 5,3°C às 12h e em 3,5°C às 14h em relação ao grupo Controle.  A umidade relativa esteve elevada (P<0,05) às 7h no tratamento Noite e às 12h, 14h e 21h no tratamento Dia. Os maiores valores de ITU foram registrados no tratamento Noite às 12h, 14h e 21h. Não foram observadas diferenças entre os tratamentos (P>0,05) para a produção e composição do leite. Nos animais do tratamento Os níveis de cortisol mostraram-se acima (P<0,05) dos níveis normais em todos os tratamentos. Já os teores de T3 e T4 estiveram dentro da faixa de normalidade. Na fase pré-experimental a maior frequência de isolamento bacteriano foi para Staphylococcus coagulase negativa. No tratamento noite e dia, houve uma diminuição na proporção de casos positivos de mastite subclínica da fase pré-experimental em relação à última semana da fase experimental. Na última semana da fase experimental houve uma diminuição de Staphylococcus coagulase negativa e aumento da ocorrência de Corynebacterium sp.

Termos de indexação: Infecção intramamária, mastite, resfriamento evaporativo, hormônios tireoidianos, vacas leiteiras.




Environmental conditions such as high temperature, humidity, and solar radiation compromise the ability of the lactating dairy cow to dissipate heat, resulting in heat stress. Also, climate variables are associated with increases in incidence of mastitis due to enhance animals' susceptibility to infections (Hammami et al. 2013).

Cooling methods should be used to alleviate the thermal stress in these conditions (Renaudeau et al. 2010). Environmental modifications for dairy facilities are generally provided by evaporative cooling such as misting or sprinklers associated to fans (Collier at al. 2006). Keister et al. (2002) verified a lower incidence of mastitis in Jersey cows housed in a freestall with a misting system compared to animals in a non-cooled freestall during the summer period in Arizona.

In general, plasma cortisol levels are increased in dairy cows during short periods of exposure to thermal stress. On the other hand, extended periods of exposure reduce these levels (Du Preez 2000). In addition, basal serum thyroxine (T4) and triiodothyronine (T3) concentrations decline under thermal stress. The modification of thyroid activity is accompanied by a decrease in metabolic rate, food ingestion, growth, and milk production.

The purpose of this study was to evaluate the effects of a misting system in a freestall on mastitis occurrence, milk production, and composition, as well as cortisol, T3 and T4 levels in dairy cows.



The study was conducted at the Centro APTA Bovinos de Leite do Instituto de Zootecnia, located in Nova Odessa, São Paulo, Brazil at the coordinates 22°42' S and 47°18' W. The region has a subtropical climate with a hot and rainy summer. All procedures were approved by the Animal Care and Use Committee of the CAPTA Bovinos de Leite Nova Odessa, SP, Brazil.

The trial was carried out from January to March 2003. Twenty-eight multiparous cows averaging 70±10 days postpartum and 23±2.3 kg milk/day. The treatments were randomly assigned to the cows. The freestall were divided into four sections and then 1 of 4 treatments was installed: Day (cooling from 7:00 a.m. to 7:00 p.m.); Night (cooling from 7:00 p.m. to 7:00 a.m.); 24-hour (cooling 24-hour); and Control (no cooling). The treatments were activated by an automatic controller. The misting system (0.3 L/h) was installed using PVC pipes with valves within a 2.8-m distance. The fans (2.5m/s) were placed about 2.5 m above the freestall bedding. The misting cycle included 5 min with 1 min of water spraying and 4 min turned off. Microclimate parameters' dry bulb temperature (DBT) and air relative humidity (RH) inside the facility were measured every 60 min over a period of 24 h using a data logger. The temperature and humidity index (THI) was calculated using the equation described previously by McDowell & Johnson (1971).

Weekly blood samples were collected from the coccygeal vein of the cows. The plasma was separated and stored at -80°C. The concentration of hormones was determined by a solid phase radioimmunoassay (RIA) using a commercial kit Coat-A-Count DPC® (DPC, Los Angeles, CA).

The animals were milked twice daily (7:00 a.m. and 7:00 p.m.). Milk samples from both milking were collected weekly to determine fat, protein, lactose, total solids (Bentley2000® Instruments Inc.), and somatic cell counts (Somacount® 300).

Wired cup tests were used to diagnose clinical mastitis and California Mastitis Tests (CMT) were used to identify subclinical mastitis. Milk samples from all mammary glands were aseptically collected daily for six consecutive days in the pre-experimental phase and during the last week of the trial. The samples were cultivated in sheep blood agar, incubated under aerobic conditions at 37°C for 72 h with readings after 24, 48 and 72 h. The samples were also plated on MacConkey's agar, where they were incubated at 37°C for 48 h. The microbiological techniques used for the identification of bacteria complied with the guidelines described by Murray et al. (1999). Staphylococci sp. was identified based on colony morphology, mannitol salt agar, and tests of coagulase, urea, maltose, trehalose, xylose, lactose, sucrose, and raffinose. Corynebacterium sp. was identified based on catalase, urea, maltose, lactose, and sucrose tests (Costa et al. 1986). Streptococcus uberis, Streptococcus agalactiae, and Streptococcus dysgalactiae were identified based on catalase, esculin, and CAMP tests.

Milk yield, milk composition and, hormones were analyzed by repeated measures using PROC MIXED (SAS 2001) with a heterogeneous compound symmetry covariance structure. Parity number was adopted as a covariate for the adjustment of milk production. For meteorological variables, days were used as the observation unit. The model contained treatments, time of day and treatment by time of day interaction. The data were subjected to an analysis of variance, using the PROC MIXED for repeated measures (SAS 2001) with a heterogeneous toeplitz covariance structure. Differences were considered significant when P<0.05. The difference between the incidence of mastitis in the mammary glands evaluated in the pre-experimental phase and in the last week was performed using the Instat Graphpad Sofware with chi-square test (χ2).



The environmental conditions recorded from 7:00 a.m. to 9:00 p.m. during the experimental period are shown in Table 1. The highest dry bulb temperatures (DBT) were recorded from 12:00 a.m. to 5:00 p.m. and were correlated with the lowest absolute values of relative air humidity (RH). Day treatment decreased the DBT by 5.3°C at midday and 3.5°C at 2:00 p.m. in relation to the Control group. The relative humidity was increased at 7:00 a.m. in Night treatment (96.5%) and at midday (70.0%), 2:00 a.m. (64.8%), and 9:00 p.m. (90.0%) in Day treatment. The highest values established for the THI were registered in the Night treatment at midday (79.3), at 2:00 p.m. (80.5), and at 5:00 p.m. (79.4). The 24-hour treatment presented the higher THI at 9:00 p.m (77.7).



Cortisol concentration of Control treatment (1.1µg/dL) was increased in relation to Night (0.6 µg/dL) and 24-hour (0.7µg/dL) treatment. No differences (P>0.05) were found in the levels of triiodothyronine and thyroxin among treatments. Mean values of treatments were 55.4 µg/mL and 2.9 ng/dL to T3 and T4 respectively (Table 2).



No differences in milk production and composition (P>0.05) were observed (Table 3). In the pre-experimental phase, 3.5% of the animals and 1.8 % of the quarters had clinical mastitis. In the last week of the experimental phase, the number of animals and quarters with clinical mastitis remained the same when compared with the pre-experimental phase. The frequency of subclinical mastitis in the pre-experimental phase in relation to the number of animals and mammary quarters was 78.5 and 65.1% respectively. There was a decrease in the percentage of animals and mammary quarters affected with subclinical mastitis with 67.8 and 40.1% in the last week of the experimental phase (Table 4).





The percentages of quarter milk samples without microbial isolates at the pre-experimental phase in the Control, Day, Night, and 24-hour treatments were 45.9%, 40.0%, 37.2%, and 37.4%, respectively (Fig.1). Control treatment showed a decrease (P < 0.05) in the negative microbial isolates in the last week of experimental phase (45.9 vs. 37.7%). On the other hand, Day (40 vs. 51.2%) and Night (37.2 vs. 50.2%) treatments presented an increase in the quarter without microbial isolates. No significant difference (P>0.05) in the quarter without microbial isolates was observed in the 24-hour treatment (37.4% vs. 43.2%).



Table 5 shows the somatic cell count and bacterial isolations of the pre-experimental phase and the last week of the experimental phase. Day (5.9 vs 0.0%) and Night (6.7 vs. 0.0%) treatments decreased (P<0.05) the frequency of Staphylococcus sp. isolates in the last week of experimental phase. Coagulase-positive staphylococci (CPS), Staphylococcus aureus, and Corynebacterium spp. isolates were increase in the Day (36.2, 18.8, and 35.1%) and 24-hour treatments (45.3, 21.6, and 42.2%) in the last week of the experimental phase. There was no difference (P>0.05) for somatic cell counts between the pre-experimental and the last week of the experimental phase.




The effects of heat stress on dairy cows can be alleviated using a heat abatement system such as shades, fans, misters, and sprinklers. In our trial, Day treatment was efficient to reduce the freestall temperature during the hottest hours (12:00 p.m. to 2:00 p.m.). The temperature was 5.3°C lower at midday and 3.5°C lower at 2:00 p.m. in relation to the Control group. These results are in agreement with Smith et al. (2006) who reported a decrease of 3.1°C in dry bulb temperature using evaporative cooling in the North Mississippi, an area characterized by high temperature and humidity.

The main objective of the misting system is to decrease the air temperature. However, the system efficiency is highly related with relative humidity (RH). High environmental temperature and RH (higher than 70%) may limit the performance of the confined cows because of a reduction in heat dissipation. This restriction of thermal changes will provide an increase in body temperature due to the storage of heat (Davis 2001). RH values higher than 70% were found in all treatments at 7:00 a.m. and at 9 p.m. in the Day, Night, and 24-hour treatments. The high RH observed in these situations is probably associated with the large volume of steam (from breathing) and urine produced by the animals that cannot be removed from the building. Thus, the air changes should be improved to move the warm and humid air away from the facilities.

According to Hahn (1985), THI values of 70 or less are considered comfortable, 71–78 mild stress, 79–83 moderate, and values greater than 83 as severe stress with lactating cows being unable to maintain their thermoregulatory mechanisms or normal body temperature. The highest values found for the THI in the Night treatment suggested that it is required to provide an effective cooling system during the hottest hours of the day in order to help cows dissipate more heat into the environment.

Cortisol concentrations were high in all treatment groups. According to Smith (1993), the normal levels for bovine cortisol are around 0.35g/dL. There is evidence that cortisol, together with other hormones, influences the metabolism of the mammary gland. On the other hand, T3 and T4 levels were within the normal ranges, which in cows are 41-170 ng/dl for T3 and 3.6-8.9mg/ml for T4. These results are similar to those observed by Avendaño-Reyes et al. (2010) who found lower concentrations of T3 and T4 in the months when the temperature and the air humidity were higher, although they remained within the normal range.

In the pre-experimental phase, 3.5% of the animals presented clinical mastitis being this percentage higher than the 3.0% that is accepted internationally (Reneau 1993). To know the clinical mastitis levels allows us to make inferences on subclinical mastitis rates. Pearson et al. (1971) verified a proportion of 1:14 between the cases of clinical mastitis for subclinical mastitis. In relation to mammary quarters, it was expected that this proportion would be higher due the cows present more than one quarter with mastitis. In our results, were found the proportion of 1:22 (animals) and 1:36 (mammary quarters) cases of clinical mastitis for subclinical mastitis. The increase of mammary quarters with negative CMT can be attributed to the spontaneous cure that results from the efficiency between udder defense mechanisms and the inflammatory process.

The increase in the quarter without microbial isolates in Day (40 vs. 51.2%) and Night (37.2 vs. 50.2%) treatments can be attributed to animal resistance. Heat stress promotes acute and chronic changes in plasma cortisol levels, which when increased leads to the suppression of immune response and a decrease in the number of circulating leukocytes (Granner 1994). Cortisol levels were higher in the Control treatment, which can partly explain the great number of positive cases in the experimental phase.

The increase in the number of CPS isolates, especially Staphylococcus aureus, in the Day and 24-hour treatments could be due to the decrease in animal resistance. According to Sutra (1990), some Staphylococcus aureus strains cause depression of the immune system and a consequently weak cellular response to infection in the mammary gland. Consistent with this, an increase in the number of Corynebacterium spp. isolates was observed in the quarter milk samples in all treatments.

The contagious mastitis occurs in different forms that depend on the microorganism involved, pathogenicity, and the ability to prevent tissue invasion. The host response is related to the resistance of the mammary gland (Blood & Radostits 1991). On the other hand, heat stress increases susceptibility to infection by decreasing animal resistance, or increasing exposure to pathogenic agents by promoting conditions that enhance their growth and proliferation in the environment (Whitaker et al. 2004).



Our data indicate that all the animals subjected to treatments presented high levels of cortisol, indicating a stress condition.

The Night treatment showed a reduction in microbial isolation, suggesting a reduced susceptibility to mastitis. In contrast, the animals subjected to the 24-hour treatment had the highest occurrence of Staphphylococcus aureus and therefore, the worst results.

Acknowledgment.- The study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Process nº 01/11093-2).



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Received on February 25, 2013.
Accepted for publication on April 25, 2013.



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