The poultry industry is one of the most developed and relevant economic sectors in Ecuador. In recent years, production has increased by 80%1. Despite the competitiveness of the sector, factors such as intensive production and high exposure to microbial agents contribute to stress in broiler chickens, making the animals more prone to immunosuppression and to diseases, especially infectious ones2,3,4. The consequences of infectious diseases are mainly reflected in lower production, particularly in terms of animal mortality and growth rate5. For this reason, it is important to apply different strategies to prevent and control the illnesses.
The most commonly employed strategies are biosecurity programs, vaccination and medication schedules and making the most of genetic resistance to diseases6. However, there are other safe, effective and inexpensive alternatives including the utilization of food additives with immunomodulatory capacities, defined as substances that stimulate, suppress or modulate the elements of the immune system7-10. These products have certain advantages in comparison with other alternatives, specifically, their direct influence on the gastrointestinal immune system and a higher acceptance amongst consumers in contrast to antibiotics4,11,12.
The spray-dried animal plasma is a commercial by-product rich in functional and digestible proteins, growth factors and biologically active peptides13-16. It is reported that the inclusion of spray-dried plasma in the diet of pigs, cows, turkeys and broiler chickens improves the animals’ performance, especially those reared in non-sanitary environments. Other studies showed that cattle and pigs immunologically challenged with enteric bacteria and fed with spray-dried animal plasma were less susceptible to developing a disease related to the challenge14,17-20. Although, the action mechanism has been studied, the way in which the product accomplishes those results is unclear and the studies were carried out mainly in pigs17,20. The immunoglobulin G and the glycoproteins of the plasma interact with antigens to decrease the overstimulation of the immune system, preventing energy distribution being inclined towards other non-productive activities. Furthermore, the product maintains the integrity of the intestinal mucosa, enhances its morphology and helps the digestive enzymatic activity17,19,21.
Shrimp hydrolysate, obtained from by-products of the aquaculture industry, contains proteins, lipids, carotenoids, chitin and chitosan22,23. The principal studies focussing on its effect on animals have concentrated on the field of aquafarming. It has been demonstrated that amino acids and small peptides of the hydrolysate are responsible for the improvement of animal performance, due to the easy and fast absorption and digestion of those components from an early age24. It has been reported that peptides in fish and chitin and chitosan in other animals, enhance the activity of some elements of the immune system such as macrophages and lysozyme23-25. Thereby, the shrimp hydrolysate favors protection against infectious diseases. However, in investigations with Pagrus major in the presence of a challenge, no significant effect was demonstrated on the prevention of the Edwardsielosis disease24.
The aim of this study was Z to evaluate the immunomodulatory effect of spray-dried bovine plasma and shrimp hydrolysate in components of the immune system and its influence on zootechnical parameters in broiler chickens.
MATERIALS AND METHODS
Bird management: Day-old male broiler chickens (Cobb500), unimmunized against Avian pneumovirus, were obtained from a commercial hatchery (Grupo Oro, Ecuador), raised for 42 days in a controlled temperature under 180 m² experimental shed at 2500 m.a.s.l. Poultry gas heaters kept the temperature at 31°C during the first week and decreased by 2°C each week until it reached 20°C during the last week. Cross-ventilation and light required was achieved behandling curtains and conventional light. The animals were reared in floor pens with 75% reused and untreated litter (broiler breeders 41-week-old) and 25% new litter (new rice hulls). Water was offered ad libitum and mash feed was provided according to the feed intake restriction to avoid ascites. This restricted feed intake consisted of around 20% less food than the ration recommended by the broiler’s genetic house (Cobb, 2015). All diets were formulated using the Brill® Formulation program to fulfill all the nutrient requirements according to the poultry breed, for the initial (0-14 days), growth (15-21 days) and final (22-42 days) phases (Table 1).
Experimental design and diets: The feeding trial used a randomized complete block design with 5 groups, each with 5 replicates and each replicate with 60 broilers. It involved the evaluation of 2 dietary additives, commercial spray-dried bovine plasma (PROLECHON, LICAN) and commercial shrimp hydrolysate (Actipal HP1-s1, Aquativ), in two different dosing strategies: S1 (5, 2.5 and 1.5%) and S2 (2.5, 1.5 and 0.5%) in initial, growth and final phases respectively, compared with a control diet composed primarily of corn and soybean meal. All assessed treatments are shown in Table 2.
||Ingredients and nutritional composition of experimental diets
aVitamin and mineral premix contained the following ingredients kg-1, Vitamin A: 7.3MIU, Vitamin D3: 3.7MIU, Vitamin E: 36.6KIU, Vitamin K: 2.3 g, Thiamin: 2.3 g, Riboflavin: 5.5 g, Pyridoxine: 2.7 g, Niacin: 38 g, Folic
acid: 1.3 g, Cyanocobalamine: 10.7 mg, Pantothenic acid: 10 g, Biotin: 86.7 mg, Cu: 9 g, I: 0.67 g, Fe: 33.5g, Mn: 60 g, Se: 0.22 g, Zn: 60 g and csp: 1 kg. bSpray-dried bovine plasma (Prolechon®). cShrimphydrolysate
(HP1S1®). SDBPS1: Spray-dried bovine in dosing strategy 1, SDBPS2: Spray-dried bovine in dosing strategy 2, SHS1: Shrimp hydrolysate in dosing strategy 1 and SHS2: Shrimp hydrolysate in dosing strategy 2
|| Treatments in the study
Sampling and measurements
Blood parameters: On day 1, 2 mL blood samples from the jugular vein of 25 birds were collected in EDTA tubes and the same blood samples from 20 birds were collected in tubes without anticoagulant, in line with Abdel-Fattah et al.26.
On days 7 and 14, 20 broilers per treatment (4 per repetition) with the same weight were selected for blood extraction (from the jugular vein). The samples were divided into tubes with or without anticoagulant. Over the subsequent weeks, until the 6th week, the same procedure was executed but the samples were taken from the wing vein.
Five blood samples per treatment collected into EDTA tubes were used to count blood cells (lymphocytes, monocytes, heterophils and heterophils: lymphocytes ratio) under a microscope, according to Wang et al.27.
The samples in tubes without anticoagulant were stored at a tilted angle at 4°C for 2 days. Then the plasma was harvested and stored at -18°C for subsequent analysis of antibody titers of APV by indirect ELISA. The analysis was performed using the commercial IDEXX APV Ab test kit according to the manufacturer’s recommendations (IDEXX, Maine, USA).
Immune organ indices: Every 7th day, 5 birds per treatment, previously used in the blood sampling, were selected at random and weighed. They were stunned by low electrical voltage, slaughtered and bled. The spleen, thymus and bursa of Fabricius were immediately removed and weighed. The relative weights of each lymphoid organ and the ratio between the different lymphoid organ weights were determined.
Performance parameters: On a daily basis, we recorded the mortality, culling and feed intake for each repetition. Also, the birds with 12 h of fasting were weighed weekly. The recollected data was used to calculate the body weight gain, feed intake and feed conversion ratio for the whole period of rearing and for each of the 3 development phases (initial, growth and final). Additionally, a weekly survival rate was determined.
Statistical analysis: The normality and homogeneity of the studied variables were analyzed using the Shapiro Wilk test.
When necessary, before analysis, the data were transformed to log or square root. An appropriate statistical analysis of variance was applied for a randomized complete block design (two-way ANOVA) with the pens’ positions within the experimental shed as the blocking criterion. A Tukey test was used for the determination of significant differences amongst treatments. p<0.05 was considered statistically significant.
The data that did not adjust to the model assumptions of normality and homogeneity of variance were analyzed using the Kruskal Wallis test and Dunn Bonferroni test in order to determine significant differences between treatments. Mortality was assessed via the Log Rank test in order to compare survival amongst treatments. All the statistical analyses were performed with SPSS 23.0.
Blood parameters: As shown in Table 3, the APV antibody titers of birds in the control group and all treatments decreased pronouncedly until day 14. The highest antibody titers during the decrease, precisely on days 7 and 21, were found in the SDBP S2 group. The result of this treatment for both days was significantly different in relation to the control group (p<0.05). In addition, on day 7, the antibody titers of the SH S1 were significantly different to the control (p<0.05).
The effects of SDBP and SH on the studied hematological parameters of broilers are presented in Table 4 and 5. In certain periods of the experiment, the measured parameters were influenced by the immunomodulators. Between 1-7 days, SDBP S2 showed a significant increase (p<0.05) in lymphocytes and a significant decrease (p<0.05) in heterophils and H:L ratio. Over the same period of time, the monocytes decreased significantly in SH S2. Between 7-14 days, SDBP S2 manifested a significant decrease and SH S2 a significant increment (p<0.05) in lymphocytes. At day 42, the lymphocytes, heterophils and H:L ratio presented significant differences (p<0.05) in SDBP S1, SDBP S2 and SH S1respect to the control. Specifically, from day 28 to day 42 those 3 parameters showed significant increases in the treatments mentioned above, with the exception of SH S1 in lymphocytes and SDBP S2 in H:L, in which they decreased significantly.
|Table 3:||Antibody titers for APV of each treatment
|Values are geometric means. NS: No significance at p<0.05|
|Table 4:||Percentages of lymphocytes, monocytes and heterophils in each treatment
|Values are means. Mean values with different letters in the same column differ significantly at p<0.05. NS: no significance at p<0.05|
|Table 5:|| Heterophils:lymphocytes ratio in each treatment
|Values are means. Mean values with different letters in the same column differ significantly at p<0.05. NS: No significance at p<0.05|
Immune organ indices: The relative lymphoid organ weights and the ratio between the different lymphoid organ weights were not affected by any dietary supplementations (p<0.05), with the exception of the relative weight of the thymus in SDBP S1 on day 35, where it showed a significant increment (p<0.05) as compared to the control (Table 6 and 7).
Performance parameters: The effects of SDBP and SH on the performance parameters of broiler chickens are presented in Table 8. During the initial phase, the feed intake was significantly higher (p<0.05) by 1.42 % in broilers fed with SH S2 as compared to the control group. Contrarily, the feed intake in broilers fed with SDBP S1 and SH S1 was significantly lower (p<0.05). The largest difference was found in SH S1 with a decrease of 2.21%. Additionally, for weight gain, significant differences were not found between the experimental groups and the control (p<0.05). Although, the SDBP S2 diet group manifested a higher weight gain than the control, it was not significantly different.
|Table 6:||Lymphoid organ relative weights (g g1) in each treatment
|Values are means. Mean values with different letters in the same column differ significantly at p<0.05. NS: No significance at p<0.05|
||Lymphoid organ weights ratio (g g1) in each treatment
|Values are means. Mean values with different letters in the same column differ significantly at p<0.05. NS: No significance at p<0.05
The experimental diets did not affect (p<0.05) the feed conversion ratio but the SDBP S2 group showed the lowest value on this parameter among the other groups.
In the growth phase and over the whole period of rearing, the feed intake was significantly lower (p<0.05) in groups fed the SH S1 and SDBP S1 diets compared with the control and SDBP S2 treatment. The largest difference was found in SH S1 with a decrease of 1.99% during the growth phase and a decrease of 1.49% over the whole period of rearing, as compared to the control. Although, the SDBP S2 group did not present a significant difference (p<0.05), its feed intake value was the highest of all the groups. Additionally, the broilers fed with SH S1 showed a significantly lower (p<0.05) weight gain in comparison with the control. No significant differences in the feed conversion ratio were found (p<0.05) in comparison with the control. Even though the SDBP S1 group had the lowest feed conversion ratio during the growth phase and SDBP S2 over the whole period of rearing. The only significant difference (p<0.05) for this parameter was found between treatments, SDBP S1 and SH S1.
|Table 8:|| Body weight gain, feed intake and feed conversion ratio of each treatment during all phases and over the whole period
|Values are means. Mean values with different letters in the same column differ significantly at p<0.05. NS: No significance at p<0.05|
Kaplan-Meier survival curves that show the effect of the treatments on the survival percentage of broilers
In the final phase, broiler chickens fed a SH S1 diet showed a significantly lower (p<0.05) feed intake by 1.09%, as compared to the control and SDBP S2. Furthermore, the weight gain and the feed conversion ratio was significantly higher and lower (p<0.05), respectively, in broilers fed with SDBP S2.
The survival ratio was not significantly dependent on the experimental diets (Log Rank p<0.05). However, as shown in Fig. 1, the group of animals fed with SDBP S2 presented a higher survival percentage during the first three weeks of rearing. At the end of the experiment, the broilers fed with SH S2 diet had the lowest survival ratio and the control group the highest.
It is well established that without vaccination, the initial antibody titers and maternal antibodies decrease in the first 7-10 days of the chick's life until the titers decline below the protection level between 15-20 days28-30. In this study, that tendency was evident for the APV antibody titers in the broiler chicken groups studied, due to the absence of a vaccination program for the virus. However, the maternal antibody titers in the first week for the groups of broilers fed with SDBP S2 and SH S1 had less of a significant decrease than the other groups, which might demonstrate a higher stability of the antibodies, probably as a result of the action of the immunomodulators. Selegean et al.31 found a similar effect when studying the influence of a polysaccharide extract from Pleurotus ostreatus against the infectious bursal disease virus in unvaccinated broilers31.
The measurement of immune organ weight is a well-known method for determining the chicken's immune status32. Here, the inclusion of SDBP and SH in the diet of the broilers had no significant effect on the relative lymphoid organ weights and the ratios between the different lymphoid organ weights. These results are in accordance with the findings of Lei and Kim32 and Zhou et al.33 who studied natural supplements in broilers with a comparable composition to SDBP and SH (supplements such as egg powder rich in immunoglobulin G similar to the composition of SDBP and chitooligosaccharides that are the result from the hydrolysis of chitosan, the main component of SH).
Even though the ratios between the different lymphoid organ weights showed no significant differences, the values of the bursa: spleen ratio were proof that the experimental flock presented adequate immunocompetence, because according to Perozo-Marin et al.34, a value greater than 2 for the ratio bursa:spleen is a sign of the immune system’s competence.
The impact of environmental, nutritional and pathological stresses are commonly assessed through changes in hematological parameters35. In this study, the differences found in the percentages of immune cells during the whole period of rearing in comparison to the daily established percentages for Cobb broilers36, the reversal of the normal tendency of the number of lymphocytes and heterophils on days 7 and 28 and the increment of the H:L ratio on the same days are signals of the presence of an inflammatory process. It is estimated that the stimulation of this kind of process is the result of an adaptation to stress. In this case, the stress is an immune challenge originating from rearing the animals in reused litter37. Adamu et al.38 demonstrated that in broilers, reused and untreated litter generates a higher white cell count than reused litter treated with a disinfectant.
Considering the hematological parameters in the different study groups, statistical differences were found on days 7 and 42 for lymphocytes, heterophils and H:L and on day 14 for lymphocytes and monocytes. These significant differences indicate that in the first week, SDBP S2 generated better results in terms of percentages of white cells in the presence of a challenge. The same happened in the sixth week, not only with SDBP S2 but also with SDBP S1 and SH S1. McWilliams39 determined that a constant level of lymphocytes under conditions of stress, in this case a smaller decrease of such cells, is an indicator that the birds are less susceptible to the presence of a stressor. However, our results differ from what Jamroz et al.40 obtained. These authors did not find significant differences in the percentage of lymphocytes and monocytes when evaluating the effect of porcine plasma on the elements of the immune system of broilers reared under controlled conditions.
Both the inclusion of SDBP and SH with the lowest concentration in the whole cycle and in the initial phase, respectively, showed a significant increase in the feed intake, as seen in the results of Okoye et al.41 In the initial phase of rearing broilers, they found a significant increment of feed intake when shrimp waste meal was added to their diet. Additionally, Henn et al.42 determined that a diet including spray-dried porcine plasma in the same animals increased their feed intake only in the final phase. However, Campbell et al.13 and Mahata et al.43 disagree with these results. They found that the inclusion of both products, separately, in broilers' diets didn't significantly affect their feed intake.
The reason for the increment of feed intake is not totally elucidated. Pierce et al.44 studied animal plasma in pigs and stated that the specific mechanism that influences this parameter is not clear. Various authors attribute the increase to different factors such as bird management, feed organoleptic characteristics and the environment45-48. In this study, there is not enough evidence to determine the reason for the increment. The management, environment and feed organoleptic characteristics were similar in the control and in all the treatments.
SDBP S2 treatment showed better values for body weight gain and feed conversion ratio in the whole cycle of rearing and in all the development phases, with the exception of the growth phase. In the latter, the control had the best body weight gain and SDBP S1 had the best feed conversion ratio. SH S1 was the least effective treatment in both parameters during all the experimental time period.
Previous studies have described that including animal plasma in the diet of different animals (poultry, pigs, calves etc.) improves their performance, especially in non-sanitary or challenging environments where its action is enhanced13,49. Campbell et al.19 confirmed these results when studying the effect of porcine plasma on the performance of broilers. They observed that the immunomodulator improved the body weight gain and feed conversion ratio, which agrees with what was obtained in our experiment. In the aforementioned study, the authors postulated that this improvement is related to the effect of the immunomodulator on the level of stimulation of the immune system19. Stimulating the immune system activates processes that require energy consumption, so the availability of energy is reduced for productive functions such as growth50. Torrallardona17 concluded that activating the immune system increases proinflammatory cytokines that inhibit food consumption and animal growth.
Other studies stated that the use of animal plasma improves animal health and hence performance due to its composition17. Immunoglobulins provide antimicrobial protection and reduce the activation of the intestinal immune system in pigs and rats17. Glycoproteins prevent antigenic binding at the moment in which pathogens enter the body17. Moreover, bovine plasma enhances intestinal health and morphology, keeping pathogens from crossing the intestinal barrier17,51. Therefore, the overstimulation of the immune system is minimized and energy expenditure is decreased19,42.
Shrimp hydrolysate has been described as having an important effect on body weight gain and feed conversion ratio in broilers43. These authors observed that despite the significant differences in these parameters, this product, used up to a certain concentration, generates an effect in animal performance similar to the control treatment; these results correspond with our findings. However, Bui et al.24 observed a significant improvement in animal growth, proving the effect of the shrimp hydrolysate on the performance of the red sea bream. The authors attributed this improvement to the nature of the hydrolysate. The peptides of low molecular weight in composition are easily assimilated, allowing for an improvement in the absorption of protein.
Therefore, the effect of the bovine plasma in animal performance is demonstrated by the way in which it reinforces animal immunity and intestinal morphology. While the effect of shrimp hydrolysate is more linked to its ability to enhance the absorption of the intestine. It should be noted that bovine plasma generates better results in the presence of an immune challenge, specifically in the case of this research, the presence of reused litter49. It appears that the presence of these challenging conditions does not modify the effect of shrimp hydrolysate on animal performance. The results obtained in this study do not differ from those reviewed in the literature in the absence of an immunological challenge.
Even though not all the comparisons between S1 and S2 of both immunomodulators were significantly different in all phases of rearing, the treatments with lower concentration showed better results for all the performance parameters. Other authors have found different results when studying various concentrations of spray-dried bovine plasma in broilers. Campbell et al.13 arrived at the conclusion that better performance results are reached with a higher concentration of bovine plasma (1.25%). Their work was performed under an adequate adjustment of the diet in order to maintain a similar amino acid content in all treatments13. A high concentration of animal plasma can generate nutritional imbalance, especially of amino acids and therefore decrease the productive responses17. In all probability, a protein imbalance of the diet in the initial phase of our study occurred when working with the high concentration of bovine plasma. The performance variables might have been affected by the amino acid imbalance. The animal, in order to adequately fulfil physiological functions and manifest suitable performance, has to be fed with a diet adapted to the ideal protein concept to reach a nutritional balance42-53.
Campbell et al.19 studied the effect of porcine plasma on the performance of broilers and observed an increase in animal growth only in the initial phase. It is suggested that the antibody portion of the animal plasma compensates for the immature state of GALT and enhances protection against pathogens to avoid the suppression of the performance. These results do not agree with our research. However, it is known that when porcine or bovine plasma is included in animals’ diets from early stages, the development of the immune and intestinal systems improves and the performance enhances in the subsequent phases of rearing19.
Our experiment proved that the addition of SH and SDBP in the diet of broilers does not improve their survival. Other authors found the same results when studying the effect of spray-dried bovine and porcine plasma and flour made with shrimp by-products in broilers and the effect of SH in tilapia fish7,42,46,54.
The studies in which different concentrations of shrimp hydrolysate or shrimp waste meal were used in the diet of broilers have suggested that up to a certain concentration, the effect on animal performance is comparable to that of the control group. At a higher concentration, a decrease in performance parameters is observed43,54,55. Similar results were found in our experimental work, where the strategy of lower concentration (S2) yielded better performance. However, it is noted that the aforementioned investigations do not coincide with the maximum concentration at which the product can be used (8-25%). Mahata et al.43 established that it is possible to use the immunomodulator up to a concentration of 8%. Ingweye et al.54 and Okonkwo et al.55 recommended applying the shrimp waste meal up to a concentration of 5 and 25%, respectively, considering that the product was used as a macro-ingredient in the diet, different from the hydrolysate that is a micro-ingredient. According to Leal et al.46, the different results in these investigations could be attributed to the variation in the products’ composition, which depends on the method in which they were obtained and their conditions. The high amount of fiber, ash and chitin in shrimp meal influences the digestibility and absorption of nutrients because they can become a physical barrier between digestive enzymes and lipids or proteins, thus hindering the shrimp meal’s proper use43,46.
The immunomodulatory effect of bovine plasma and shrimp hydrolysate was more evident on the percentage of white cells studied than on the rest of the immunologicals parameters analyzed, especially when there was evidence of stress caused by an immune challenge. During the initial weeks, SDBP S2 showed better immune cell percentages than the other groups and during the final weeks, the treatments with the best percentages were SDBP S2, SDBP S1 and SH S2. The addition of shrimp hydrolysate to the diet of broilers does not generate a positive effect on animal performance. The lower concentration (S2) acts in a similar way to the control and the higher concentration (S1) shows a negative effect on performance. Bovine plasma in a lower dosing strategy (S2) produces a better performance throughout the fattening cycle as compared to the control. The mortality of the flock was not affected by the inclusion of either of the two products.
Authors would like to thank Integracion Avicola Oro S.A. for funding and Biolaboratory Immunological for technical support.