CD3+ cells in the lymphoid system of broilers

Hatching time is characterized by a series of physiological events, such as the maturation of the gastrointestinal tract (physical and functional growth due to increased villi length, enterocyte polarization, and enzyme activities) and development of the immune system (maturation of primary and secondary lymphoid organs, establishment of passive immunity) (Willemsen et al., 2010).

The management practices adopted by the poultry industry during the early stages of birds´ life, can pose additional challenges to the development of broilers. Factors such as genetics, breeder age, nutritional conditions, egg size/weight, and the delayed access to feed and water, directly affect the early development and can impact performance at the end. Within one same hatcher, differences can exist in the hatch time among the eggs, from 36 to 48 hours, which conforms the hatch window (Vieira et al., 2005). After standard pull times, hatchery operative factors including sexing, vaccination, farm delivery, etc., can extend the fasting period and result in chick weight losses of up to 10% (Cançado and Baião, 2002). Chicks fasted this way can fall in dehydration, ketosis, damages in the intestinal mucosa, cell apoptosis, and damages in the breast muscles (Yamauchi et al., 1996), poor yolk sac utilization (Maiorka et al., 2006), and delayed immune system development (Friedman et al., 2003), in addition to stress-associated immunodepression (Handy et al., 1991). Despite that in the literature vast information exists about post-hatch fasting on bird development, only a few papers exist about the fate of early hatching chicks that remain in the hatcher for extended periods of time.

The purpose of this study was to evaluate the presence of CD3+ cells in the lymphoid tissue of chicks from eggs of two different weights, hatching at different times within one same hatcher.

One thousand one hundred and fifty two (1,152) eggs laid by 38-week-old Cobb^® breeders were used. Prior to setting, these eggs were stored for two days at 15˚C and 75% relative humidity, and then weighed, and both the mean and the standard deviation were estimated (65.4 ± 4.6 g). Eggs were classified as either light or heavy. All eggs were identified and incubated at controlled temperature and humidity (37.8˚C and 60%, respectively) in Casp CMg 125R^® setters to day 19. At transfer to the hatcher, groups of 5 light and 5 heavy eggs were scattered in specific compartments within the experimental trays, in a uniform, random manner.*

The chicks were distributed using a 2 x 3 factorial design, with 2 egg weights (light and heavy), and 3 incubation lengths (<472 hours; 472 - 488 hours; and 488 - 504 hours of incubation), i.e., chicks staying in the hatcher for more than 32 hours after hatch (hatch window [HW]  +32 h), between 32 and 16 h residence time in the hatcher after hatch (32 h HW), and between 16 and 0 hours residence time after hatch (16 h HW).
For each HW and for each weight, 20 chicks were identified and left inside the hatcher until pull, in accordance with the regular procedures in the hatchery.

At standard pull time, the chicks were killed and posted. Thymus and spleen samples were taken for immunohistochemistry in order to enumerate CD3+ cells. Twenty (20) fields of both thymus and spleen (100X) were counted. Results were subjected to analysis of variance, and whenever differences were found (P<0.05), factors were broken down and the means were compared using Tukey´s test, at the 5 % error probability.

Figure 1. Photo micrograph of thymus (A) and spleen (B) for the identification of CD3+ cells by immunohistochemistry (40X).

The numbers of CD3+ in the thymus had an influence on HW (P<0.05) since longer time periods of residence inside the hatcher after hatch (+ 32 h HW), resulted in lesser numbers of these cells in such organ. In the spleen, a HW x egg weight interaction was seen, with an effect on CD3+ cell counts (P<0.05) (Tables 1 and 2).

Table 1. Mean and standard deviation in CD3+ cell numbers by immunohistochemistry in both the thymus (100X) and the spleen (100X) in birds subjected to 3 different hatch Windows from eggs of two different weights, at regular pull time

Table 2. Interaction between hatch window (+ 32, 32 and 16 h HW) and egg weights (light and heavy) on the number of CD3+ cells in the spleen, at standard pull time

The primary lymphoid organs, such as the thymus and the bursa of Fabricius, start to develop during embryo life. The thymus, the site of development and maturation of T cells, has its own CD3, CD4 and CD8 marker cells during the embryo stage (Mast and Goddeeris, 1999). After hatch, waves of migration of these cells occur towards secondary lymphoid organs, such as the spleen, which is still immature at hatch. This transport from the thymus to other organs may explain the findings in this study, since a decrease in the number of CD3+ cells in the thymus of birds with longer HWs was seen.

When studying the body population of CD3+ cells, Mast and Goddeeris (1999) explained that the spleen provides the ideal microenvironment for the interaction of lymphoid and non-lymphoid cells, and it is mostly responsible for storing and transporting lymphocytes to the blood stream and tissues. In this study, interaction was seen between egg weight and HW on CD3+ cell numbers in the spleen. The animals from light eggs, with a >32 hour HW showed a longer proportion of CD3+ cells in the spleen as compared to those chicks from heavy eggs that stayed in the hatcher for the same period. We can suggest that the chicks from heavy eggs and hatch earlier, transport more CD3+ cells from the spleen to other tissues than those from light eggs. The animals hatching in the 32 - 16 hours prior to standard pull time (32 h HW) showed no difference in the amounts of CD3+ cells in the spleen regarding egg weight, thus showing that in this period no difference exists in the transport of these cells to other tissues between chicks from either light or heavy eggs. In the animals that hatched near pull time (16 h HW) from light weight eggs, showed a higher number of CD3+ than those from heavy eggs, which once again suggests a higher efficiency in the transport of these cells from the spleen to other tissues.

According to Handy et al. (1991) the birds that stayed in the hatcher for a period longer than 12 hours after hatch, are subjected to stress factors, since they produce more body heat and the excess temperature in the hatcher, so that they respond with a higher physiological release of corticosterone. The long presence of this hormone, can cause lymphoid atrophy in the thymus, the bursa of Fabricius, and the spleen (apoptosis), as well as suppression on the cellular immune response (Rogausch et al., 1999). Nevertheless, the microscopic evaluation of thymus and spleen from the birds in this experiment, showed no evidence of apoptosis, which does not mean that this phenomenon could not be seen in periods longer than those studied herein.

The birds that hatch early and stay for longer periods in the hatcher, show lesser numbers of CD3+ cells in the thymus and spleen.
Fasting in the hatcher does not seem to have a negative effect on the development of the organs evaluated; nevertheless, further studies are granted on the tissue development of animals hatching at different time periods, and subjected to different conditions prior to and after hatch, such as stressors, inadequate temperatures, fasting, and field challenges.

Cançado SV & Baião NC. 2002. Effects of fasting period between hatching and housing andaddition of oil to the feed on performance of broiler chicks and digestibility of the ration.Arquivo Brasileiro de Medicina Veterinária e Zootecnia 54:630-635.

Friedman A, Bar-Shira E, Sklan D. 2003. Ontogeny of gut associated immune competence in the chick. World´sPoultry Science Journal 59:209-219.

Handy AMM, Henken AM, Vander Hel W. 1991. Effects of incubation humidity and hatching time on heat tolerance of neonatal chicks. Growth performance after heat exposure. Poultry Science 70:1507-1515.

Maiorka A, Dahlke F, Morgulis MSFA. 2006. Adaptações pós-eclosão em frangos. Ciência Rural 36:701-708.

Mast J & Goddeeris BM. 1999. Development of immunocompetence of broiler chickens. Veterinary Immunology and Immunopathology 70:245-256.

Rogausch H, del Rey A, Oertel J, Besedovsky HO. 1999. Norepinephrine stimulates lymphoid cell mobilization from the perfused rat spleen via-b-adrenergic receptors. American Journal of Physiology 276:R724-R730.

Vieira SL, Almeida JG, Lima AR, Conde ORA, Olmos AR. 2005. Hatching distribution of eggs varying in weight and breeder age. Brazilian Journal of Poultry Science 7:73-78

Willemsen H, Debonne M, Swennen N, Everaert N, Careghi C, Han H, Bruggeman V, Tona K, Decuypere E. 2010. Delay in feed access and spread of hatch: importance of early nutrition. World´s Poultry Science Journal 66:177-188.

Yamauchi K, Kamisoyama H, Isshiki Y. 1996. Effects of fasting and refeeding on structures of the intestinal villi and epithelial cells in White Leghorn hens. British Poultry Science 37:909-921.