Blood Serum Components and Serum Protein Test of Hybro-PG Broilers of Different Ages

Blood serum samples of HYBRO PG broilers were analyzed, with 30 samples collected from 21-day-old broilers (G1), 30 from 35-day-old birds (G2), and 30 from 42-day-old birds (G3), with the aim of establishing normal values of some blood serum parameters. The activities of the enzymes gamma-glutamyl-transferase (GGT), aspartate aminotransferase (AST), creatine kinase (CK), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), serum levels of total calcium, calcium ion, phosphorus, sodium, potassium, magnesium, chlorides, creatinine, uric acid, triglycerides, cholesterol, total protein, albumin, total and indirect and direct bilirubin, and electrophoretic profile of serum proteins in acrylamide (SDS-PAGE) and agarose gel were determined. There was no influence of age on total bilirubin and albumin levels. All the other evaluated parameters presented differences in at least one age group. Protein electrophoretic profile also changed as a function of age. The obtained results can be considered as normal for the studied ages, and therefore be used as references for the interpretation of laboratory exams of broilers of this genetic line in the evaluated ages.


INTRODUCTION
In the last few years, the poultry industry has shown extraordinary growth in Brazil, which is currently one of the main global producers and exporters of broilers.
When an animal population grows, there is often a proportional increase in disease incidence.Diseases present such a wide variety of symptoms that the physical exam is not sufficient to provide a diagnosis.
Blood components may be influenced by physiological factors, such as age and species, and by pathological factors (Szabo et al., 2005;Lloyd & Gibson, 2006).The loss of cell membrane integrity caused by hypoxia or traumatism causes enzymes to leak to the extracellular fluid, where they are measured, allowing to determine the degree of cell or tissue lesions.These analyses are discussed in several studies with domestic animals (Jain, 2000), but only a few specifically refer to broilers (Kaneko et al., 1997).
The evaluation of the levels of total protein and its fractions supply the information required to interpret the occurrence of dehydration, infections, immune diseases, and inflammatory responses.
The determination of blood component values using laboratory exams is an important procedure to aid the diagnosis of several diseases and dysfunctions, as they provide reliable results, and may also give inputs for research studies on nutrition, physiology, and pathology (Bounous et al., 2000).
This study aimed at determining the levels of blood serum components of broilers of different ages, including serum protein test in acrylamide gel (SDS-PAGE), in order to provide useful data for the diagnosis and prognosis of broiler diseases.

MATERIAL AND METHODS
Ninety blood serum samples were collected from HYBRO-PG broilers, being 30 samples collected at 21 days of age (G1), 30 colleted at 35 days of age (G2), and 30 collected at 42 days of age (G3).Broilers were fed a corn and soybean meal-based diet, which supplied the nutritional requirements recommended by Rostagno (2000).The ages chosen for blood collection coincided with diet change (21 and 35 days of age), and with market age (42 days of age.
Blood samples were collected by puncture of the jugular vein in 21-day-old broilers, and axillary vein in 31-and 42-day-old broilers, using 5-ml plastic syringes, and 25 X 7 mm needles.
Serum components were expressed as means and standard deviation, which were submitted to statistical analysis.Means were compared by the test of Tukey after sample homogeneity was verified (Zar, 1999).Significance was considered at 5% probability level.Serum protein test results were expressed as lower and upper limits.

RESULTS AND DISCUSSION
Table 1 shows the means and standard deviations of the serum activities of the enzymes AST, ALP, GGT, LDH, and CK of healthy broilers of different ages: 21 days of age (G1), 35 days of age (G2), and 42 days of age (G3).Serum activity of all enzymes changed as a function of bird age.AST serum activity progressively increased with age, and LDH and CK activities were higher at older ages, which is consistent with the observations of other studies (Sandhu et al., 1998;Agawane & Lonkar, 2004).This is possibly due to the increase of liver metabolism and to the significant muscle development that usually happens during this period, as observed by Szabo et al. (2005) in turkeys of commercial strains.ALP serum activity, as opposed to AST, LDH, and CK, was higher at 21 days of age due to a higher bone development, as compared to older ages.The activity of GGT was not different between extreme ages (G1 and G3), but it was higher at 35 days of age, a phase when there is rapid body development due to higher feed intake and feed gain (Almeida et al., 2006), with a concurrent significant increase of liver metabolism.The differences detected among groups relative to serum enzymes are probably due to physiological changes that are normal in these different ages.Nevertheless, birds were not tested for mycotoxins, which may also influence these parameters and it is frequently present in farms.Table 2 shows the means and standard deviations of mineral serum levels.There were changes in the levels of Ca, P, Mg, Na, and K, with the highest values obtained at 35 days of age.Chlorine serum level was the lowest in G2.Calcium and phosphorus level at 21 days of age were similar to those observed in the control group in the study of Oguz et al. (2000), whereas sodium and potassium levels at 42 days of age were different from those analyzed in healthy broilers by Oguz et al. (2002).Harr (2002) stressed that blood mineral results levels may be different as a function of the methodology applied, and breed and genetic line may also influence these parameters (Gyenis et al., 2006).The higher mineral levels observed in the sera of 35-day-old birds are probably due to the higher nutrient supply (Rajman et al., 2006), and to the more intense metabolism that commonly occurs at this age (Almeida et al., 2006).Therefore, the obtained results suggest that the different blood mineral components usually vary with age due to physiological changes that happen at each development phase.Serum levels of creatinine, uric acid, triglycerides, cholesterol, total protein, albumin, as well as total, direct and indirect bilirubin levels are presented in Table 3.It is observed that age did not influence total bilirubin and albumin levels, but all other components changed as a function of age.Creatinine level at 35 days of age was higher than in the other age groups, and no difference was detected between G1 and G3.The level of this catabolite directly related to muscle volume and activity, and therefore its lower blood levels in old and young chickens (Rajman et al., 2006;Agawane & Lonkar 2004;Sandhu et al., 1998).Uric acid level was higher in 21-day-old birds, and there was no difference between G2 and G3. the highest dietary protein level is supplied in the starter phase of production, and a study (Szabo et al., 2005) found a direct relation between the amount of ingested protein and uric acid serum level.Rajman et al. (2006) found higher uric acid levels in broiler breeders during the rearing phase, at 30 and 44 days of age, as compared to older breeders.
Triglycerides and total cholesterol present a similar pattern of variation as a function of age, with higher values in G1 and G3 birds, as observed in other studies (Sandhu et al., 1998;Oguz et al., 2002).The higher values of these components in G1 correspond to their low mobilization by tissues and to their intense synthesis The evaluation of total protein serum levels did not reveal any differences between G2 and G3; however, at 21 days of age, its level was lower, due to the high requirement for growth.These data are compatible with those presented above for uric acid level, main catabolite of protein metabolism, which presented the highest serum levels at 21 days of age.Direct and indirect bilirubin levels were different in the three studied ages, and their variation is probably linked to variation in liver metabolism as a function of age.
Table 4 shows the results of the serum protein test obtained in acrylamide gel SDS-PAGE).Electrophoretic fractioning performed by this technique allowed the identification of 24 proteins, out of which 11 were identified by their name, including acute-phase proteins and immunoglobulins, whereas the remaining 13 were identified on molecular weight basis.by the liver (Szabo et al., 2005).At 42 days of age, the increase of these values is related to the higher dietary energy supply (Rajman et al., 2006).The lower triglycerides and cholesterol levels at 35 days of age are due to the bird's high energy requirement at this stage caused by high body development (Almeida et al., 2006).

Table 2 -
Means and standard deviations of total calcium, calcium ion, phosphorus, magnesium, sodium, potassium, and chlorine of broilers at 21 days of age (G1), 35 days of age (G2), and 42 days of age (G3).

Table 3 -
Means and standard deviations of serum levels of creatinine, triglycerides, uric acid, cholesterol, total protein, albumin, total bilirubin, direct bilirubin, indirect bilirubin of broilers at 21 days of age (G1), 35 days of age (G2), and 42 days of age (G3).

Table 4 -
Molecular weight (MW; Kilodalton, Kd) and lower limit (LL), mean (X) and upper limit (UL) of serum levels (mg/dL) of proteins in the serum protein test obtained in acrylamide gel (SDS-PAGE) of broilers at 21 days of age (G1), 35 days of age (G2), and 42 days of age (G3).
* Protein identified only by molecular weight.There are presently no purified proteins to nominally identify these fractions.