Influence of Sex, Age, and Fasting on Blood Parameters and Body, Bursa, Spleen and Yolk Sac Weights of Broiler Chicks

The effects of water and feed fasting for 24, 48 and 72 hours posthatching on blood parameters (mean corpuscular volume, MCV; red blood-cell, RBC; hematocrit, HCT; hemoglobin, HGB; plasma glucose, CGP; plasma total protein, PP, and differential leukocytes count), and on body, liver, spleen, bursa, and yolk sac weights were analyzed. Erythrogram data were obtained with a blood cell counter. Total plasma protein and plasma glucose were determined by using the Bradford method (1976) and a glucose PAP liquiform kit (Labtest, cat. n. 84), respectively. Specific leukocyte counts were carried out on blood smears stained with Rosenfeld solution. According to the obtained data, water and feed post-hatching fasting reduced MCV values, which also were lower in males than that in females. Fasting for 48 hours promoted an increase in PP, while fasting for 72 hours reduced HCT. Chicks submitted to fasting presented lower body weights as compared to fed chicks, but their liver weight did not increase between 48 and 72 hours of age. Fasting decreased spleen weight, but bursa and yolk sac weight were not affected. Data showed that female and male chicks react in a similar way to post-hatching fasting, which affects body weight, liver and spleen weight, and HCT and PP values. Moreover, 72 hours of fasting affected more intensely HCT and MCV values.


INTRODUCTION
The first post-hatching week is an essential period for broiler development and corresponds to approximately 17% of its growth period before slaughter (Lilburn, 1998).During this period, most of the energy and proteins (amino acids) are utilized for the development and morphological and functional maturation of the organic systems, especially of the mid-gut, and overall body growth of the young chicks (Maiorka, 2002).
Water and food fasting reduces intestinal digestive and absorptive area as a result of slower development of the intestinal villi and of an increase in cell extrusion rate (Yamauchi et al., 1996;Gomide et al., 2004).Body weight also decreases in 5 to 10% because of water loss by respiratory evaporative process (Baião & Cançado, 1998), which can affect bird maximal growth potential.
After placement, the chick intestinal mucosa is exposed to a diversified microflora, to which the body reacts by activating its immune system.Therefore, it is important that, at placement, young chicks do not present changes in the hematological profile that can affect the quality of their immune reaction to new intestinal microbiota or make them susceptible to enteric diseases.
In the present study, the effects of post-hatching water and feed fasting on blood parameters and body, spleen, liver, and yolk sac weights of the male and female chicks were analyzed.

MATERIALS AND METHODS
Approximately 200 fertile eggs (57.5±2.1g)from 29week-old broiler breeders (Cobb500 ® ) were obtained from a commercial hatchery, and incubated at 37.8ºC and 60ºC humidity in two forced-air rotating incubators (IP120, Premium Ecologica) with automatic control of egg turning (1 turn every 2 hours) and temperature.Immediately after hatching chicks were sorted by sex (using sex-linked feather phenotype), and male and female chicks were divided into 4 groups of 15 birds per sex, with two groups fed water and feed (2,800 kcal/Kg and 22% protein) ad libitum, and two groups submitted to water and feed fasting.All groups were reared in commercial cages (Premium Ecologica, 50cm x 90cm), containing two 40W light bulbs (one green and another blue).Twenty four, 48 and 72 hours after the beginning of the experimental treatments, birds were weighed and 6 birds from each treatment were randomly selected (3 birds per replicate) and slaughtered after blood collection.
Data for the erythrogram were followed a 2 (fed ad libitum with water and feed, or submitted to water and feed fasting) x 2 (female and male) x 3 (24, 48 and 72 hours of treatment) factorial arrangement in a completely randomized experimental design.Body and organ weight data followed a 2 (fed ad libitum with water and feed or submitted to water and feed fasting) x 2 (female and male) x 2 (48 and 72 hours of treatment) factorial arrangement in a completely randomized experimental design.
Blood samples were collected from wing vein, placed in plastic vials containing EDTA (15µl/ml blood), and put on ice, and submitted to the lab in order to determine hematocrit, red blood cell count, hemoglobin, mean corpuscular volume, white blood cells count, and plasma glucose and protein.
Immediately after blood collection, liver, spleen, bursa of Fabricius, and yolk sac were removed and weighed.
For plasma protein (g/dL) and glucose (mg/dL) determination, blood samples were centrifuged at 1,500 x g at 4ºC for 15 min.Plasma samples were placed into plastic vials and stored at -20ºC until analysis.Total plasma protein was determined using the Bradford method (1976), whereas plasma glucose was determined using a glucose PAP liquiform kit (Labtest,cat. n. 84).Two readings per bird were made for glucose and total protein in 505nm and 595nm, respectively.
Specific leukocyte counts were carried out in blood smears stained with Rosenfeld solution.Monocyte, lymphocyte, heterophil, eosinophil, and basophil counts were determined by counting the number of each leukocyte type in 100 analyzed cells.Data are expressed as estimated percentages of the 100 analyzed cells (Gonzáles et al., 2003).
Data were submitted to analysis of variance, and significant treatment means were separated by Tukey's test (p<0.05),using the SAS software package (2002).

RESULTS AND DISCUSSION
In the present study, body weight was significantly (p<0.05)influenced by sex, age, and treatment.Body weight was higher in females than in males, in 72-hourold chicks than in 48-hour-old chicks, and in chicks that received water and feed than in fasted chicks (Table 1).However, there was a significant (p<0.05)interaction between age and treatment, showing that chicks submitted to fasting did not increase in body weight from 48 to 72 hours of age (Table 3).Liver weight was not affected by sex, but it was significantly (p<0.05)influenced by age and treatment, being higher in 72 h-old than in 48 h-old chicks, as well as being in fed as compared to fasted chicks.In addition, significant interaction (p<0.05) was observed between age and treatment (Table 3): liver weight of fasted chicks did not increase from 48 to 72 hours, and these chicks reached 72 h of age with lower liver weight as compared to fed chicks.Sex and age did not affect (p>0.05)spleen weight, but treatments had a significant (p<0.05) on this parameter (Table 1).Spleen was heavier in fed than in fasted chicks.No significant (p>0.05)effect of sex, age, or treatment was observed on yolk sac weight (Table 1), and there were no significant interactions among sex, age, and treatment for this parameter.
These data show that chicks submitted to posthatching fasting for 48 and 72 hours did not gain weight, and did not increase liver and spleen weights, as observed in fed chicks after hatching.In addition, fasted chick body weight was 12% and 40% lower, and liver weight 15.4% and 52.3% lower as compared to fed chicks at 48 and 72 hours post-hatching, respectively.The post-hatching period is characterized by rapid body growth and by morphological and When dehydrated, the body tries to maintain its homeostatic balance by ingesting water and by reducing water excretion volume or using interstitial and intracellular fluids (Bruno & Macari, 2002).However, animals submitted to fasting, and that are already dehydrated, utilize tissue substances for metabolic energy production, which can also be used by the animals to maintain homeostasis.In the present study, young chicks were submitted to water and feed fasting.Therefore, it is possible that the utilization of interstitial and intracellular fluids, as well as of metabolic energy, is involved in the absence of weight gain and the lack of increase in liver and spleen weights shown by fasted chicks.functional differentiation of the organic systems, which utilize a large part of the ingested energy and proteins (Noy & Sklan, 1999).It is well-known that yolk is the largest source of nutrients for chick during their first days of life outside the egg.Therefore, it would be expected that yolk nutrients were utilized more quickly by fasted chicks than by fed chicks, allowing young chicks to maintain their physiological functions.However, similarly to the observations of Baião & Cançado (1998), and Maiorka et al. (2003), no difference in yolk sac weight due to fasting was observed in the present study, indicating that feed ingestion after hatching is essential for chick body growth and organ development.Negative effects of the post-hatching food and water deprivation on liver weight were also reported by Donaldson & Christensen (1991) and Maiorka (2002).Bursa weight results are shown in Table 1.No significant (p>0.05)effect of sex, age, or treatment was observed on this parameter.No significant (p>0.05)interaction among sex, age and treatment was detected.The bursa presents considerable development after hatching, particularly up to 8 weeks of age (Verma et al., 1999).Our results seem to indicate that the post-hatching bursa development may begin after 72 h of hatching, which could explain the absence of effects of fasting on this organ during the experimental period.The absence of differences between males and females in bursa weight show that both sexes present a similar pattern of early bursal development.
Tables 2 and 3 show erythrogram results.No significant effect of sex or treatment was observed on RBC and HCT, but there was significant (p<0.05)effect of age on both parameters.RBC and HCT values were higher in 72 h-old than in 24 or 48 h-old chicks.There was a significant (p<0.05)interaction between age and treatment on RBC and HCT, indicating that chicks submitted to fasting did not increase the values of these parameters between 48 and 72 h of age.These chicks reached 72 h of age with lower HCT values than fed chicks .There were significant (p>0.05)effects of sex and treatment on MCV values, but not of age (Tables 2 and 3).Female chicks presented higher MCV values than male chicks, and fed chicks had higher values than fasted chicks.In addition, there was a significant (p<0.05)interaction between age and treatment (Tab. 3): fed chicks presented an increase in MCV values between 48 and 72 h of age, which was not observed in fasted chicks, and at 72 h, had higher MCV values as compared to fasted chicks.No significant (p>0.05)effects of sex, age, or treatment were observed on HGB values, nor any significant interaction among them (Tables 2 and 3).
According to those data, when chicks were submitted to water and feed fasting for 72 hours, there was no increase in RBC, HCT, or MCV values between 48 and 72 hours of age, as reported in the fed chicks, and in addition presented lower HCT and MCV values at 72 hours of age, but with no simultaneous reduction in HGB values.Lower MCV values were also observed by Maxwell et al. (1990) in Gallus domesticus submitted to feed restriction starting on the fourth week of life.However, in contrast with our data, these authors reported an increase in RBC and reduction in HGB values in chicks submitted to feed restriction.Therefore, our data suggest that the intensity of the erythrocytic response elicited in an animal depends on the degree of food deprivation to which this animal is submitted.Considering that the fasted chicks did not receive either water nor feed, our results indicate that the simultaneous occurrence of HCT reduction and HGB increase in these birds was caused by the reduction in the of MCV values as a consequence of water fasting.In addition, they suggest that prolonged fasting induced microcytic anemia in the fasted chicks.Dehydration produces changes in blood parameters, particularly in HCT values, which are abnormally increased when the animal is not able to maintain homeostasis (Swenson, 1996).In the present study, the young chicks that were submitted to fasting maintained MCV and HCT values constant during the entire experimental period (72 hours), indicating that they maintained their homeostatic equilibrium under prolonged water and feed fasting, probably using interstitial and intracellular fluids (Bruno & Macari, 2002).It is well known that, with an isometric increase in size, volume increases faster than surface area (Woods, 1999); the opposite is also true.This seems to explain the occurrence of higher MCV values without an increase in the HGB values in the female chicks; however, this but was not observed in males.
Plasma protein (PP) values observed in the present study show significant (p<0.05)effects of age and treatment on this parameter (Tables 2 and 3).PP values were higher in chicks submitted to fasting than in fed chicks, and in 48 h-old than in 24 and 72 h-old chicks.However, the significant (p<0,05) interaction between age and treatment showed that fasted chicks did not present higher PP values at 48 h as compared to 24 and 72 h of age, and that they presented higher PP values than fed chicks at 48 h of age.This result is consistent with previous reports of Warris et al. (1992) that chicks submitted to fasting for 48 hours presented higher PP values as compared to chicks that received water and feed.This increase in the PP values may have been caused by an increase in protein breakdown promoted by the body to maintain its physiological functions during fasting using metabolic energy (Katanbaf et al., 1988).On the other hand, chicks submitted to prolonged fasting (72 hours) presented PP values similar to those obtained by fed chicks at the same age (our data).This result is very interesting and suggests that protein depletion could be used by fasted chicks to obtain energy and/or metabolic water.
Plasma glucose (PG) values were significantly (p<0.05)influenced by sex, age, and treatment (Table 2).PG values were higher in female than in male chicks, in 48 h-old than in 24 and 72 h-old chicks, and in fed described above, our study also found a reduction in monocyte, lymphocyte, and thrombocyte counts in the fasted chicks.In adult broilers and turkeys submitted to feed restriction, Maxwell et al. (1991) and Maxwell et al. (1992) reported only a reduction in thrombocyte and lymphocyte counts, respectively.These differences and similarities between our results and those of other authors show that bird immune reactions change with the intensity and the type of stress to which they are exposed, and that reduction in the monocyte count occurs under severe stressful conditions (in this case, prolonged water and feed fasting).Our data support the results obtained by Gross & Siegel (1986), who also reported an increase in H/L ratios in chicks submitted to food fasting.However, our results are different from those reported by Hocking et al. (1993) and De Jong et al. (2002), who did not observe changes in the H/L ratios in adult broilers submitted to food restriction.These differences between our data and those obtained by other researchers in birds submitted to stress promoted by fasting are related to fasting duration and type, as well as to bird age during fasting.
There were no effects of sex on differential leucocytes counts.In both sexes, heterophils, lymphocytes, and eosinophils were the most numerous leukocytes in the blood, corresponding to approximately 38, 30, and 15-20% of the total number of immune cells, respectively.Lymphocytes and heterophils were also the most frequent cells in the blood of layers, broilers, and turkeys (Bounous & Steedman, 2000).According to our results, there was an increase in the eosinophil and basophil counts according to bird's age .After hatching, birds are exposed to a high diversity of microorganisms that constitute the natural intestinal microbiote and that stimulate their immune system (Spencer & Garcia, 1995).This exposure to new agents appears to explain the increase in leukocyte counts after hatching observed in the present study.
Similarly, as verified in the present study, Maxwell et al. (1991Maxwell et al. ( , 1992) also observed an increase in basophil, heterophil, and eosinophil counts, and H/L ratios in broilers and turkeys submitted to feed restriction, respectively.We observed a reduction in thrombocyte, lymphocyte, and monocyte counts, while a reduction in thrombocyte counts was found only in broilers (Maxwell et al., 1991) and a reduction in thrombocyte counts only in turkeys (Maxwell et al., 1992).Gross & Siegel (1986) also reported an increase in H/L ratio of chicks submitted to fasting.Alternatively, Hocking et al. (1993) and De Jong et al. (2002) did not find any changes in H/ L ratio of broilers caused byfeed restriction.These data suggest that the type and the intensity of immune reaction depend on stress type, fasting degree, stress duration and age in which the birds are submitted to stress, and that reduction in monocyte counts are caused by severe stress caused by prolonged fasting.

CONCLUSION
Post-hatching water and feed fasting for a period longer than 24 hours markedly affects organ growth, blood parameters, and immune system of broilers of both sexes.Water and feed must be offered immediately after hatching to prevent detrimental changes in the physiological functions of broilers.
a-b, A-B -Comparisons among ages (columns) and between treatments (rows).Means followed by different letters in the same column and row are significantly different (P<0.05) by Tukey's test.

Table 1 -
Effects of sex (female or male), age (48 or 72 hours), and treatment (fed with water and feed, water and feed fasted) on body weight, and liver, spleen, bursa, and yolk sac weights in newly-hatched broiler chicks.
a-b -Comparisons between sexes (columns), among ages (columns), and between treatments (columns).Means followed by different letters in the same column are significantly different (P<0.05) by Tukey's test.

Table 5 -
Effects of sex (female or male), age (24, 48, or 72 hours) and treatment (fed with water and feed, or water and feed fasted) on differential leukocyte counts and H/L ratios in newly-hatched broiler chicks.Comparisons between sexes, among ages and between treatments (columns).Means followed by different letters in the same column are significantly different (P<0.05) by Tukey's test.