REGISTRO DOI: 10.5281/zenodo.12639583
José Evandro de Moraes1,2, Thiago Bernardino2,3*, Mariane Marton2, Luana Alves2, Patricia Tatemoto2, Beatrice Morrone Lima2, Anna Cristina de Oliveira Souza2, Ana Carolina Peroni Gomes1, Júlia Mayumi Fujii Ferrazo1,Sadala Mehesen Cruz Tfaile1, Gerson Barreto Mourão4, Carla Cachoni Pizzolante1, Adroaldo Jose Zanella2
ABSTRACT
Housing densities of laying hens kept in cages in limited space results in an unfavorable and stressful environment. 750 lying hens from 1 day old until 17 weeks of age received the same management and vaccination protocol. In the production phase, since 23 weeks old, the animals were submitted to different housing densities and their vaccine response were evaluated. A complete block design was used and the treatments were five housing densities in the cages (321.43; 375; 450; 562.5 and 750 cm²/bird) and six replications, totaling 150 experimental plots. Blood samples were collected at the beginning of the experiment (control sampling) and at 34, and 44 weeks of age, summing 325 samples. The titers of vaccine antibodies against the Newcastle virus (NDV) and Gumboro (IBD) diseases were evaluated through the ELISA. The results were analyzed using analysis of variance and the means between treatments were compare using the Tukey-Kramer test, as a post hoc, with 5% of probability. There was no significant effect (p > 0.05) of housing densities on the analyzed variables. Serum antibody titers ranged from 11635 to 6266 for Newcastle and Gumboro, respectively, values that exceed the expected cutoff minimum titer line, which was 2515 for Newcastle and 1877 for Gumboro.
Keywords: antibodies,Gumboro disease, housing densities, immunity, Newcastle disease, vaccine responses
INTRODUCTION
Stress is a physiological response of the organism and can unbalance the homeostasis of birds, which seeks to provide the body with conditions to respond and adapt to these changes. Animals subjected to stress trigger several physiological mechanisms to activate the immune system in order to reestablish the balance of the organism’s functions (Klasing & Korver, 1997).
Stress metabolism is a mechanism that has a high cost for the animal, once the energy that should be used for maintenance and production, such as laying rate, weight gain, feed consumption and egg production, is diverted to mobilize the organism in the combating stressors or the invading agent (Guahyba, 2000).
Studies indicate that in many infectious diseases, the main pathological aspects are not related to a direct action of the aggressor agent, but to an abnormal immune response, which include activities of the pathogen itself, poor welfare, among other stressors that can determine immunosuppression and the increase of diseases (Jeurissen et al., 2002, 1994; Quinteiro Filho, 2009; Vieira et al., 2015).
Stressors are stimuli that trigger a stress response, such as temperature variations (cold and heat), transport, mixing of lots, housing density, space in the feeder and drinker, among others can alter nutritional use (Çetin et al., 2010; Jahanian & Mirfendereski, 2015; Lieboldt, 2015; Lieboldt et al., 2016; Yang et al., 2016) and the immune response to vaccines applied to birds (da Silva Guahyba, 2000; Salak-Johnson & McGlone, 2007). Prolonged exposure to these factors can result in a state of chronic stress, reflecting in losses in productive performance and immune response, possibly increasing disease morbidity and mortality rates in the squad (Elrom, 2000; Mazzuco, 2006; Vieira et al., 2015).
The immune response relies on innate or cellular immunity and adaptive or humoral immunity, divided into two systems: primary lymphoid (bursa of Fabricius and Thymus) and secondary lymphoid (spleen and mucosa-associated lymphoid tissues – Harder’s gland, intestine, bronchi, Peyer’s patches, Meckel’s diverticulum, cecal and pineal tonsils) (Guidotti, 2011; Olah et al., 2014).
The humoral immunity of birds consists of three different types of antibodies or immunoglobulins: IgA, which is the main antibody present in the mucous membranes; IgY, which corresponds to 75% of the total serum immunoglobulins and matches to mammalian IgG (Abbas et al., 2015); and IgM, which is functionally and structurally similar to mammals IgM (Tizard, 2018). Variations in their levels directly affect the ability of birds to protect against pathogens.
Immune deficiencies, whether of innate immunity (phagocytic cell dysfunction and complement system deficiency) or adaptive immunity (deficiency in antibody production or deficiency in T and B cell function), are strongly associated with increased susceptibility to infections, because of poor or undesirable welfare conditions (Davison, 2014; Hoerr, 2010; Jeurissen et al., 2002, 1994; Vainio & Imhof, 1995; Weinstock et al., 1989).
Susceptibility to infection in birds involves traits of resistance, which refers to the ability of a variety of anatomical, physiological and immune systems to eliminate pathogens, and persistence of production, which refers to the ability to maintain their productivity (eg. growth, feed efficiency, egg production) during the health challenge (Klasing & Korver, 1997; Rutz et al., 2002). Some diseases have the ability to immunosuppress birds without stimulating the production of corticosteroids, including Gumboro disease and Newcastle disease (da Silva Guahyba, 2000).
Newcastle disease is an avian pneumoencephalitis or nervous respiratory disorder, is highly contagious, it is caused by viruses and can affect the respiratory, nervous and digestive systems of any age birds (Flores, 2012, 2007), causing great economic losses (Paulillo & Doreto Jr, 2006; Zanetti et al., 2005). This is a zoonosis, compromising human health, especially people who work with birds (farms, slaughterhouses, and laboratories), attributing an occupational character and, therefore, it is part of the list of notifiable diseases of the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH) (2022) and mandatory notification. The occurrence of this disease in the country leads to the immediate suspension of exports of poultry products, with serious consequences for international trade (Oliveira Júnior et al., 2005; Santana et al., 2008).
Infectious Bursa Disease or Gumboro Disease is one of the main immunosuppressive diseases in birds and its presence in poultry flocks is responsible for economic losses related to immunosuppression, poor performance and mortality (Kneipp, 2000; Sharma et al., 2000). The main target organ of the virus is the Bursa of Fabricius and, for this reason, the disease has immunosuppressive effects, presenting considerable mortality and high morbidity (Alam et al., 2002; Allan et al., 1972; Faragher, 1974; Müller et al., 2003), in addition to causing susceptibility to secondary infections (Eterradossi and Saif, 2013). Animals that had the bursa removed are resistant to this infection (Ivanyi & Morris, 1976; Käufer & Weiss, 1980), which explains the resistance of adult birds to the virus when this organ atrophies physiologically (Tessari et al., 2000; Tessari & Cardoso, 2011). This disease is not a zoonosis and is not a public health concern (Eterradossi & Saif, 2013).
A biosecurity measure for the control and prevention of Newcastle and Gumboro diseases adopted by farms is vaccination (Baxendale & Lutticken, 1982). According to Oliveira et al. (2007), after vaccination, the results can be evaluated by the indirect Enzyme Linked Immunosorbent Assay – ELISA or the hemoagglutination inhibition test (HI). Current vaccines technologies aim to reduce the effect of immunosuppression and accelerate steps so the immune response against the disease happen faster, dynamic and effectively. The effectiveness of vaccination programs and information about the virus circulation in the environment can be obtained through the study of immunity profiles through a serological analysis, which evaluates the sanitary conditions of the flocks (Hoerr, 2010; Romero et al., 1989).
The indirect ELISA can be used through specific commercial kits to process a large number of serum samples to evaluate the immune response after vaccination programs (Santos & Silva, 2000). Furthermore, this technique is increasingly being applied in programs for serological monitoring of different viral diseases in the poultry industry (Richtzenhain et al., 1993).
In light of the foregoing, the present study aimed to compare the levels of specific antibodies against Newcastle disease virus and Gumboro disease virus in light laying hens reared at five densities of housing in cages of the conventional rearing system, in order to understand the immunological behavior of laying hens exposed to the stress of rearing in cages.
MATERIALS AND METHODS
Animals and housing
The laying hens were housed in the poultry sector of the Institute of Animal Science in Nova Odessa – SP, in a masonry shed measuring 10 meters wide x 60 meters long and 2.80 meters high. The shed had ceramic tile roof, a side opening protect with one-inch diameter mesh against the entry of other birds and animals and two fans inside.
The cages arranged in a battery system measuring 50 cm in front x 0.45 cm in depth x 0.40 cm in height, installed suspended one meter from the floor, arranged in four rows and distributed in two service aisles. Each cage was separated by PVC partitions and had in the frontal part feeders of the “gutter” type and inside nipple drinkers, which were considered the experimental plots, and the housing densities (stocking rates) according to the experimental treatments.
It was used 750 commercial laying hens (Gallus gallus domesticus) from five different genetic lineages. The birds were reared in pens until 17 weeks of age and submitted to the same handling and feeding conditions, receiving feed and water ad libitum. The feed were prepared to meet the nutritional needs of each strain, according to the breeding manuals of the strains, following the recommendations of (Rostagno et al., 2011), for light commercial layers.
Until puberty period, the birds were vaccinated according to the current Brazilian laying hens practices, being immunized against viral diseases – Infectious Bronchitis (H120), Newcastle (LaSota), Gumboro (mild and strong), Pneumovirus, avian Yaws and Encephalomyelitis – and bacterial diseases – Coryza Aquosa, Mycoplasmosis and Samonella enteritidis. They were vaccinated on the 7th, 14th, 21st, 28th, 35th, 42nd, 56th, 63rd, 90th and 110th-115th days, by ocular, intramuscular (chest and thigh) and wing membrane, as shown in table 1.
Table 1. Vaccination program for laying hens until puberty period.
| Baby chick stage | ||
| Age (days) | Vaccine | Application route |
| 1 | – Marek HVT + Rispens | hatchery |
| 7 | – Bronchitis (H120) (1st dose) – Newcastle (LaSota) (1st dose) – Gumboro – mild (1st dose) – Salmonella enteritidis (1st dose) | Eyepiece |
| 14 | – Pneumovirus (swollen head) (1st dose) – Gumboro – mild (2nd dose) | Eyepiece |
| 21 | – Gumboro – strong (1st dose) | Eyepiece |
| 28 | – Bronchitis (H120) (2nd dose) – Newcastle (LaSota) (2nd dose) – Gumboro – strong (2nd dose) | Water |
| Teenage bird stage | ||
| 35 | – Watery coryza (Single Dose) – Mycoplasma (1st dose) | Intramuscular (thigh) Eyepiece |
| 42 | – Avian Yaws (Single Dose) – Encephalomyelitis (Single Dose) | Wing membrane |
| 56 | – Bronchitis (H120) (3rd dose) – Newcastle (LaSota) (3rd dose) – Salmonella enteritidis (2nd dose) | Water |
| 63 | – Pneumovirus (2nd dose) – Escherichia colli (Single Dose) | Water |
| 90 | – Mycoplasma (2nd dose) | Eyepiece |
| 110-115 | – Triple oily (SHS) (Newcastle, Bronchitis (H120) e Gumboro) – Pneumovirus (3rd dose) | Intramuscular (chest) |
Egg production and housing
At 20 weeks of age, body weight and egg production were standardized, discarding birds with low or high body weight and after that the animals were transferred from the floor pens to the cages and distributed in the experimental plots, being submitted to an adaptation period to the ambient conditions of the shed and to the experimental diets.
750 laying hens of five genetic lines were used from 23 weeks old at the beginning of the first experimental period to 43 weeks old at the end of the fifth experimental period. At 23 weeks old the birds were distributed using a complete randomized block design, characterized by the concatenation of the genetic lines with a column of cages; the treatments were five densities or stocking rates in the cage (321.43; 375.00; 450.00; 562.50 and 750 cm2/bird) with six replications, totaling 150 experimental plots.
Temperature and relative humidity were recorded daily throughout the pre-experimental and experimental period using a digital thermo-hygrometer, scale between -10 and 90ºC, with an error limit of ± 1ºC.
Blood sampling
To analyze the effects of housing density on the welfare of laying hens, vaccine antibodies were measured by collecting 2 mL of blood from three samples per treatment, for each of the laying hens’ genetic lines. In total, three collections were performed along of the experiment: 25 samples before the allocation in the treatments (control or basal collection), 150 samples in the middle of the experiment (birds with 34 weeks old) and 150 samples at 140 days of the experiment (birds with 44 weeks old), making a total of three blood samples in duplicate from the five treatments over five periods of 28 days each, totaling 325 samples. The birds collected were randomly chosen within the treatments, with each animal participating in only one blood collection during the entire experimental period, which means that no laying hens was collected more than once.
During the collections, it was used gauze and 97°GL alcohol for local antisepsis, 10 mL syringes, 14/7 needles, 2 mL microtubes, supports for microtubes with a capacity of 300 samples, conventional ice for immediate cooling of the samples after collection, Styrofoam box, disposable gloves, pen to identify the microtubes and lamps to facilitate the visualization of the blood vessel.
Antibody measurement
The titers of vaccine antibodies were analyzed in test samples from the blood serums in bigger quantity for Gumboro, Newcastle and Salmonella enteritidis antigens, but the last one did not reach satisfactory levels for the study in the present experiment.
The samples for quantification of anti-Gumboro and Newcastle vaccine antibodies were analyzed using the Enzyme Linked Immunosorbent Assay test – ELISA, which is based on antigen-antibody reactions detectable through enzymatic reactions.
The ELISA tests were performed in the laboratory of the Department of Preventive Veterinary Medicine and Animal Health (VPS/USP) in Pirassununga – SP with commercial ELISA kits for the Gumboro (IBD) and Newcastle (NDV) tests (BioChek LTD, United Kingdom), both specific for chicken serum. The ambient temperature of the laboratory was kept controlled at 23°C during all analyzes, because the variation of the ambient temperature can interfere with the ELISA results.
Statistical Analysis
The titrations obtained in all phases of the experiment were organized and evaluated with the aid of the SAS program and, when appropriate, the Tukey-Kramer test was applied as a post hoc for the comparison of means (p<0.05), to obtain of the results that will be discussed in this research.
RESULTS AND DISCUSSION
Mean titers of vaccine antibodies against the virus of Newcastle and Gumboro diseases in the control (baseline) treatment are shown in Table 2.
Table 2. Mean titers of vaccine antibodies against the virus of Newcastle (NDV) and Gumboro (IBD) diseases, in the control (baseline) collection of light laying hens at 21 weeks of age, which underwent immunization using vaccines associated in the laboratory, measured by the hemoagglutination inhibition test (HI) (log10).
| Means of vaccine antibody titers | ||
| Period | Newcastle | Gumboro |
| 1 | 11635 | 6266 |
| SEM1 | 314.8 | 452.8 |
| CV2 (%) | 0.1353 | 0.3613 |
1. SEM: standard error mean; 2. CV: Coefficient of variation
The serological evaluation of the light laying hens showed titers ranging from 11635 to 6266 and the coefficient of variation, which measures the uniformity of antibody titers detected, was 0.13 and 0.36% for Newcastle and Gumboro, respectively (Table 1). We observed that the values found greatly exceed the expected cutoff minimum titer line, which was 2515 and 1877 for Newcastle and Gumboro, respectively (Opengart, 2003; Ristow, 2004).
The laying hens belonging to the control group showed high titers. Thus, the detection of antibodies against Newcastle and Gumboro in birds was directly related to vaccination (Jorge et al., 1998), suggesting that the vaccination reached the expected objective, since they undergo an intensive program of vaccination during the first 17 weeks of life, aiming to protect them (Meszaros et al., 1992; Ribeiro & dos Santos, 2009; Romero Cullerés et al., 2017).
The results obtained in this study confirm those already found by (Bacallao et al., 1998). The authors argue that young laying hens, when vaccinated via the eye and drinking water, did not show significant differences in antibody titer during all periods evaluated, justifying that the LaSota lutenogenic strain is successful by the routes of application of vaccines used.
The mean titers of vaccine antibodies against the virus of Newcastle and Gumboro disease in the second and third collections are shown in Table 3. The housing densities studied did not influence (p>0.05) the mean titers of vaccine antibodies against the virus of Newcastle or Gumboro disease. We can see that the values found greatly exceed the line of the minimum expected security, which was 2515 and 1877 of the cutoff point (cutoff) for Newcastle and Gumboro, respectively.
The increase in the level of vaccine response may be related to the optimal efficiency of the vaccination performed in the present study. The titration results obtained were satisfactorily above the expected cut-off point, indicating that the densities studied did not interfere with the immune response of the laying hens. This can probably be explained by the vaccination of healthy birds, appropriate vaccines for the pathogenic viruses studied, correct dosage used for each bird and the route of administration used for live vaccines, in addition to good administration practices such as the preparation and maintenance of the vaccine solution and the birds.
Table 3. Mean titers of Newcastle (NDV) and Gumboro (IBD) vaccine antibodies, of light laying hens at 34 and 44 weeks old, housed in five densities and that underwent immunization using associated vaccines in the laboratory, measured by the hemoagglutination inhibition test (HI) (log10), in the second and third collection period.
| Mean titers of Newcastle vaccine antibodies | ||||||||||
| Densities | ||||||||||
| Housing density (cm2/bird) | ||||||||||
| Period | 750 cm2 (3 birds) | 562.5 cm2 (4 birds) | 450 cm2 (5 birds) | 375 cm2 (6 birds) | 321.4 cm2 (7 birds) | P3 Value | ||||
| 2 | 11469 (a) | 10849 (a) | 10751 (a) | 11244 (a) | 11351 (a) | P(1)>.8898 | ||||
| SEM2 | 612.61 | 660.17 | 574.33 | 612.79 | 612.65 | |||||
| 3 | 11227 (a) | 11401 (a) | 11399 (a) | 11438 (a) | 11660 (a) | P(1)>.9870 | ||||
| SEM2 | 554.24 | 554.21 | 554.20 | 554.22 | 554.22 | |||||
| Mean titers of Gumboro vaccine antibodies | ||||||||||
| Densities | ||||||||||
| Housing density (cm2/bird) | ||||||||||
| Period | 750 cm2 (3 birds) | 562.5cm2 (4 birds) | 450cm2 (5 birds) | 375cm2 (6 birds) | 321.4cm2 (7 birds) | P3 Value | ||||
| 2 | 7115 (a) | 6953 (a) | 7392 (a) | 8120 (a) | 7907 (a) | P(1)>.3248 | ||||
| SEM2 | 469.06 | 504.22 | 440.21 | 469.03 | 469.04 | |||||
| 3 | 7799 (a) | 7619 (a) | 7450 (a) | 8583 (a) | 8272 (a) | P(1)>.3692 | ||||
| SEM2 | 451.42 | 451.42 | 451.42 | 451.42 | 451.42 | |||||
1. Means followed by different letters, on the same line, differ statistically from each other by the Tukey-Kramer test at 5%; 2. SEM: standard error mean; 3. P: Probability.
Different housing densities did not reduce the vaccine responses and did not impair the birds’ immunity, as observed by the high titers obtained from vaccine antibodies against Newcastle and Gumboro (Table 3).
Some studies relate chronic stress with a decrease in the body’s defenses, but there are no data regarding changes in the humoral immune response in birds. As in the case of this study, housing densities probably did not expose the birds to reinfection, which, associated with housing stress and the presence of other pathogens, could compromise the birds’ immunity development, which did not occur.
The vaccination of the birds was carried out until 17 weeks old and the presence of an adequate amount of antibodies titers suggest that it was well done. Therefore, it is possible that the levels of vaccine immunoglobulins were not affected by the stress that the birds may have suffered at the densities studied.
According to (Dohms & Saif, 1984), immunosuppression is a temporary or permanent state of dysfunction of the immune response, which results from damage to the immune system that leads to a decreased response in the levels of antibodies against other microorganisms and leads to an increase in susceptibility to illnesses. One of the first scientists to demonstrate the link between stress and weakening of the immune system was Frenchman Louis Pasteur, in the late 19th century, when he observed that chickens exposed to stressful conditions were more susceptible to bacterial infections than non-stressed chickens (Amâncio et al., 2010). The authors explain that stress is related to the release of hormones, which alter various aspects of physiology and have a modulating effect on the body’s defenses.
Maternal antibodies can be transferred from the breeding bird to the embryo’s circulatory flow through the yolk and play an important role in the immunocompetence of chicks to pathogens (Ribeiro & dos Santos, 2009). This transfer occurs through specific receptors present on the surface of the membrane of the embryo sac and allows a selective transport of IgY present in maternal blood (Chalghoumi et al., 2009). These antibodies, mostly IgY, can interfere with live vaccine immunity to the same strain (Lopez, 2006) and may disappear between 10 to 20 days after hatching (Tizard, 2018). Therefore, to maintain optimal antibody levels, it is important to follow the complete vaccination program.
According to Canal & Vaz (2008), the ideal vaccine must be easy to administer, have an affordable acquisition cost, have stability of the product during storage and after inoculation in the organism, suitability for mass vaccination programs and the ability to stimulate adequate and lasting immunity. The objectives of vaccination are: to prevent infection; prevent the clinical manifestation of the disease and its consequences; attenuate the clinical disease and its consequences, reducing the severity and intensity of the symptoms; immunize breeding animals, avoiding vertical infection; guarantee passive immunity to the fetus; prevent pathogen excretion and infection of new animals, reducing environmental contamination; eradicate the agent from the population, establishing the immunity of the herd (Barbosa, 2014).
In Brazil, vaccines against Newcastle disease are produced by a live lutenogenic virus (they present very mild clinical signs), being represented by the LaSota and B1 strains (Nunes et al., 2002; Orsi, 2010) and the best way to administering this live vaccine is through spray or eye drops, because these stimulate local and humoral immunity (Alexander, 2001). Vaccines with this character are available on the market and provide protection to the avian species for which they have been formulated, but they are less effective in protecting against infection and normally only reduce the amount of virus shed by the infected bird. In laying hens, vaccination is recommended in the first week of age by the eye drops method, as maternal antibodies offer 75% protection on the first day of life of the chicks and these antibodies can increase to 90% protection when there is vaccination. According to (Bacallao et al., 1998), immunization with the lutenogenic strain LaSota presents an efficient immune response when performed via the ocular routes and drinking water. A revaccination is required 2 to 3 weeks after the first dose and a third dose 4 to 6 weeks (Borne & Comte, 2003), as occurred in our research.
Contrary to our results obtained for Gumboro, Van Den Berg (2000) evaluating vaccination programs against this disease for broilers, concluded that vaccination was not effective in protecting birds, even for the classical strains. Therefore, virus-induced immunosuppression continued to result in secondary infections, poor development and condemnation of carcasses at the slaughterhouse. The results obtained differ from those (Alexander, 2001), when he states that stressor factors, such as changes in the environment and management, interfere with the immune response of vaccinated birds and not only those related to the application of the vaccine. They also differ from the results obtained by (Paulillo & Doreto Jr, 2006), who state that the immune response is unlikely in old birds with falling antibody levels and a mature immune system, and also from those obtained by (Jorge et al., 1998), who state that immunity in older birds, over one year old, would be contained by pre-existing immunity, without causing perceptible immune reinforcement by the hemagglutination inhibition test (HI).
According to Besedovsky (1996), at the same time that the conventional system densifies a certain number of birds in a certain space, allowing an increase in production, it also favors the incidence of diseases, because, induced alterations in the activities of the central nervous system, caused by stress, modify the functions of the immune system causing a greater susceptibility to opportunistic diseases.
Studies indicate that laying hens housed at a density of 300 cm2/bird, much lower than the 750 cm2/bird currently recommended in the European Union, have a higher incidence of diseases and mortality and have metabolic changes that can significantly alter the immune response (Laganá et al., 2007; Pavan et al., 2005; Rech et al., 2010). There are also studies demonstrating the existing relationships between the immune, nervous and endocrine systems in farm animals subjected to stress (Jeurissen et al., 2002; Quinteiro Filho, 2009).
Koenen et al., (2002) concluded that broilers had higher IgM titers, while in laying hens, IgY titers were higher. In addition, they observed that the cellular immune response in laying hens has higher rates and correlate that heavier chickens have lower titers when compared to lighter chickens, which have higher and longer-lasting titers of antibodies against the antigens studied.
The greatest benefit related to the use of vaccines in laying hens is the protection of the immune system functionality and the induction of a response by antibodies against the crucial components of the vaccine program that aims at a more efficient egg production (Alexander et al., 2004; Rautenschlein et al., 2013).
The biosecurity of birds can be monitored by several techniques, making the vaccination program more efficient and safe (Barbosa, 2014; Bernardino et al., 2004) and antibody titers to Newcastle disease can be used as an indicator of humoral immunity (Bernardino & Leffer, 2009; Molnár et al., 2011; Nunes, 2008).
CONCLUSION
The different housing densities studied did not modify the immunological response of laying hens, since the titers of vaccine antibodies against Gumboro and Newcastle Diseases obtained through ELISA tests were satisfactorily above the cut-off levels.
ACKNOWLEDGMENTS
To FAPESP, Process: 2014/22559-2 for the financial funding; to the Breeding Companies Hendrix Genetics, Mercoaves e HY-Line for the donation of the chicks; Granja Kakimoto and Agropecuária Peeters S/A; to Ceze Feed for the feed mixtures.
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1 Instituto de Zootecnia/ APTA/ SAA – Rua Heitor Penteado, 56, Centro, Nova Odessa-SP, Brasil, CEP. 13.380-011
2 Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Medicina Veterinária Preventiva e Saúde Animal, Pirassununga – SP, Brazil
3 Universidade Santo Amaro, Programa de Pós-graduação em Saúde Única
4 Departament of Animal Science, Universidade de São Paulo, Piracicaba, Brazil
*Corresponding author: evandro.moraes@sp.gov.br
evandromoraes0906@gmail.com