Performance, Carcass Characteristics, Cardio-Pulmonary Morphometry, Gut-Morphology, Fatty Acid Prole and Humoral Immunity of Commercial Broiler Birds Fed Diet Supplemented with Blended Fish Protein

A 35-day trial was conducted to explore if different sources of dietary proteins at varying levels of metabolizable energy had any effect on the average daily feed intake (ADFI), average daily gain (ADG), feed eciency (FE), carcass characteristics, cardio-pulmonary morphometry, gut morphology, prole of unsaturated fatty acid (UFA), saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA) and humoral immunity of the broiler birds. Total 288 Ross-308 day old male broiler birds were randomly distributed in four dietary treatment groups in a 2×2 factorial arrangement (Two different sources of proteins, i.e., plant and animal with two different levels of metabolizable energy, i.e., low and high). Each treatment had nine replicates containing eight birds per pen. Results indicated that, the supplementation of blended sh protein substantially increased (p<0.001) the nal body weight and decreased (p<0.001) the ADFI at 1-14 d, 15-35 d and 1-35 d compared with plant protein. The ADG increased at 1-14 d (p<0.001), 15-35 d (p<0.05) and 1-35 d (p<0.05) and the FE improved (p<0.001) at 1-14 d, 15-35 d and 1-35 d. Similarly, the high energy diet signicantly increased (p<0.001) the nal body weight and decreased the ADFI at 1-14 d (p<0.001), 15-35 d (p<0.05) and 1-35 d (p<0.05) compared with low energy diet. The ADG increased (p<0.001) and the FE improved at 1-14 d, 15-35 d and 1-35 d. The animal protein increased the thigh weight (p<0.01), the neck weight (p<0.05) and the ratio (p<0.001) of ∑ UFA: ∑ SFA and ∑ PUFA: ∑ MUFA in the breast muscles of the broiler birds. The GLM identied no interaction effect (p>0.05) of energy and protein on the ADFI, ADG, FE, cardio-pulmonary morphometry and humoral immunity of the broiler birds against Newcastle and Infectious Bronchitis diseases. The RV:TV remained within standard range indicating no symptoms of cardiac dysfunctions. It was concluded that the shmeal supplemented high energy diet improved weight gain, feed eciency and meat quality of broiler by increasing ∑ ω-3 and ∑ ω-7 fatty acids as well as the ratio of ∑ ω-3: ∑ ω-6, ∑ UFA: ∑ SFA and ∑ PUFA: ∑ MUFA in the breast muscles of the broiler birds. uniform size abnormalities. feet incandescent bulbs. and birds from of 36 replicates were randomly selected for slaughter. gastrointestinal tract removed and separated into three intestinal segments, i.e., duodenum, jejunum and ileum. weight and diameter of duodenum taken distally gizzard to end pancreatic loop, jejunum taken distally pancreatic loop to Meckel's diverticulum, ileum taken Meckel's diverticulum to ileo-caecal junction. measurements triplicate and centrifuged at 3,000 g for 15 min, and the sera samples were -80 determination of antibody titers. Serum antibody titers were determined by means of the hemagglutination inhibition (HI) test and ELISA for and IBD respectively using ELISA test kit according to manufacturer instructions listed in the ELISA Kit (Symbiotic-USA). ω-3: ∑ ω-6, ∑ UFA: ∑ SFA and ∑ PUFA: ∑ MUFA in the breast muscles of the broiler birds.

The experiment was conducted in a two way 2 2 factorial arrangements (Two different sources of proteins, i.e., plant and animal with two levels of Metabolizable energy, e.g., low and high). Total 256 Ross-308 day old male broiler chicks were randomly distributed into eight dietary treatment groups with four replicates having 8 birds per pen ( Table 1). The chicks were purchased from Nahar Agro Ltd., Chattogram, Bangladesh. All chicks were examined for male, grade A, uniform size without abnormalities. Floor space for each bird was 0.17 square feet in brooding box and 1 square feet in the cage. The birds were exposed to continuous lighting for the 1st three days followed by 1-4 hours of dark for the remaining 1-4 weeks. The chicks were brooded at a temperature of 95°F, 90°F, 85°F and 80°F for the 1st, 2nd, 3rd and 4th weeks, respectively with the help of incandescent bulbs. Room temperature and humidity were measured by using wall mounted indoor analog thermo-hygrometer. Before arrival of the chicks, the shed was thoroughly cleaned and washed by using tap water with caustic soda. For disinfection, phenyl solution (1% v/v) was sprayed on the oor, corners and ceiling. Following spray, cleaning was done by using brush and clean water. Brooding boxes, rearing cages and pens were cleaned in the same manner. After cleaning and disinfection, the house was left empty one week for proper drying. After drying, all doors and windows were closed. The room was fumigated with single strength fumigant (Adding 40 ml formalin to 20 g KMnO 4 for 100 cubic feet area) and sealed for 24 hours. On the next day, lime was spread on the oor and around the shed. Footbath containing potassium permanganate (1% w/v) was kept at the entrance of the poultry shed and changed regularly. Feeders and drinkers were cleaned and washed with Timsen® solution (0.3% v/v) daily. All birds were vaccinated against Newcastle and Gumboro disease with both the primary and booster doses. After each vaccination, lemon juice as a vitamin-C supplement was supplied through drinking water to overcome the effect of stress due to vaccination.    Table 3 Initial live weight (ILW, g/bird), nal live weight (FLW, g/bird), average daily feed intake (ADFI, g/bird/d), average daily gain (ADG, g/bird/d) and feed e ciency (FE, ADFI/ADG) of the broiler birds fed diet supplemented with two different sources of dietary protein and two different levels of metabolizable energy

Experimental diets
Feed ingredients were purchased from Pahartali market, Chattogram, Bangladesh. During purchase, cleanliness and date of expiry was checked. Dry mash was provided to the birds throughout the whole experimental period. Four different types of rations were formulated. Each ration was two different types i.e., starter (0 to 14 days) and nisher (15 to 28 days). All rations were iso-caloric and iso-nitrogenous. Feed was prepared manually and supplied ad-libitum to the birds on round small feeder and waterer for 0-7 days. After 7th day, these feeders and waterers were replaced by medium linear feeders (2.21 ft X 0.25 ft) and round waterers. At 15th day, large linear feeder (3.5 ft X 0.38 ft) and round waterers (3 liter capacity) were provided for feeding and drinking of the birds.

Chemical analyses
From each treatment, 100 g of prepared mash feed was taken and preserved in an air tight bag to carry out the chemical analysis. Chemical Scienti c Equipment Company Ltd., 874-1 Wolgye 4-dong, Nowon-gu, Seoul, Korea). Gross energy (GE) of mixed diets was estimated by using the bomb calorimeter (Parr 6200 Calorimeter, Parr Instruments Co., USA).

Performance parameter
Mortality was recorded as occurred, while average daily feed intake (ADFI), average daily gain (ADG) and feed e ciency (FE) were recorded at fortnightly intervals. Carcass characteristics, hematological and biochemical parameters were recorded at 5th week. Weight gain was calculated by deducting initial body weight from the nal body weight of the birds. Feed intake was calculated by deducting leftover from the total feeds supplied to the birds. The FE was calculated dividing feed intake by weight gain.

Carcass characteristics
At day 35, three birds from each replicate were randomly selected and killed by severing the jugular vein and carotid artery. Once a bird was adequately bled out, it was scalded and feather was removed. After defeathering, the birds were eviscerated and the head and feet were removed as per standard technique (Jones, 1984). During evisceration process, abdominal fat, lung, liver, kidney, spleen, gizzard and proventriculus were excised separately and weighed. Dressed birds were weighed to obtain a dressed carcass weight.

Cardio-pulmonary morphometry
The heart was isolated from the carcass immediate after slaughter.

Gut morphology
At day-30, two birds from each of 36 replicates were randomly selected for slaughter. The gastrointestinal tract was removed and separated into the three intestinal segments, i.e., duodenum, jejunum and ileum. The length, weight and diameter of duodenum was taken distally from the gizzard to the end of the pancreatic loop, the jejunum was taken distally from the pancreatic loop to Meckel's diverticulum, and the ileum was taken from the Meckel's diverticulum to the ileo-caecal junction. All measurements were triplicate and averaged later on.

Fatty acid pro le
Lipids were extracted from the breast muscles (Pectoralis major and pectoralis minor) of the slaughtered birds by using a modi ed method (Folch et al., 1957). Fatty acid methyl esters were extracted by the gas chromatography using KOH/methanol. The Gas Chromatograph (Nexis, GC-2030, Shimadzu, Japan) was equipped with a robotic auto sampler (AOC 6000, Shimadzu, Japan), a hydrogen ame ionization detector The intercept of the regression model; The xed effect of the 'i th ' level of the factor 'α' (Sources of protein) on the value observed in Y ijk (i = plant, animal); β j = The xed effect of the 'j th ' level of the factor 'β' (Level of energy) on the value observed in Y ijk (j = low, high); (αβ) ij = The interaction effect of the of the 'i th ' level of the factor 'α' and the 'j th ' level of the factor 'β'; The observed effects of the variable under study at the 'i th ' level of the factor 'α' and the 'j th ' level of the factor 'β' for the 'k th ' repetition of measurements; ε ijk = The random sampling error due to 'i th ' level of the factor 'α' and 'j th ' level of the factor 'β' at the 'k th ' repetition of measurements.

Humoral immunity
The sources of dietary protein and the levels of metabolizable energy had no main or interaction effects (p > 0.05) on humoral immunity of the broiler birds against Newcastle and Infectious Bronchitis Diseases (Table 8).

Cost-bene t analysis
The high energy sh protein diets resulted maximum economic bene t compared with vegetable based low energy diets (Table 9).

Performance
In the present study, we observed decreased (P < 0.001) feed intake in the broiler birds due to supplementation of sh protein. The trend appears consistent with a previous study where bene ts of the inclusion of shmeal on feed intake were less evident during earlier stages but later on it was exhibited (Karimi, 2006). The reasoning could be the presence of biogenic amines (Barnes et al., 2001;Karimi, 2006), poor digestible protein content and objectionable odor of the sh meal (Halliwell and Chirico, 1993) which the chicks were unable to utilize at the earlier stages. The result, however, contradicts with the ndings of Oduguwa et al. (2021) who reported signi cantly (P ≤ 0.05) higher daily feed intake of the broiler birds supplied animal protein than those of the other treatments. In our study, we ensured normal appearance and smell of the sh meal when subjected to visual appraisal. The actual discrepancies, therefore, could be due to the variations of the levels of sh meal used, metabolizable energy and protein contents of the diets although incorporation of even 16.6% of the sun dried sh offal to the diets of growing RIR chicken did not affect feed intake in a previous study (Tera et al., 2011).
We further observed increased daily gain while birds were offered diets supplemented with sh protein compared with plant protein. The improved palatability of the sh protein, well balanced amino acid pro le, higher e ciency of utilization and improved feeding values of the diets supplemented with sh proteins might have contributed to the better weight gain of broiler during the starter and nisher period with the maximum bene t evident at 15-35 d (Memon et al., 2002;Metwally, 2004). It was also reported that this response was dose dependent although Karimi (2006) explained that the further increase of the levels of sh meal beyond optimum growth response was affected by sex and age of the birds (Negesse and Tera, 2010;Melesse et al., 2013). In another study, it was claimed that the shmeal could be used at different levels up to 8% in broiler diets without adversely affecting weight gain (Awoniyi et al., 2003). Santana-Delgado et al. (2008) further demonstrated that there were no differences in the weight gain of the broiler chicken providing diet with either soybean meal or shmeal. Similar results were reported elsewhere (Johnson et al., 1985).
We observed improved feed e ciency (FE) for the sh protein supplemented diets compared with the plant protein. The result is consistent with Hossain et al. (2012) who explained that the better protein digestibility of the sh protein forti ed diets as well as increased e ciency of utilization of energy available in sh proteins were responsible for improved FE (Ali et al., 2001;Alali et al., 2011). This response, however, may not always be consistent with all the sh by-products used for feed formulation since the sources of protein may vary in property, i.e., the protein quality, digestibility, biological value, amino acid pro le, physical and chemical properties (Sing and Panda, 1992). These characteristics of individual protein source may affect performance of the birds. However, several researchers reported that the major factor limiting the use of vegetable proteins in practical diet is the existence of naturally occurring deleterious factors including non-starch polysaccharides, tannins and trypsin inhibitors which have adverse effects on nutrient digestibility and absorption (Gatel, 1994;Smits and Annison, 1996). Saxena et al. (1962) reported that the unavailability of amino acids due to presence of anti-nutritional factors was considered a major factor in explaining the poor feed e ciency of the chicks fed diets containing soybean meal. Accordingly, despite being iso-caloric and iso-nitrogenous, better overall performance of the broiler birds in sh protein supplemented diets compared with soybean meal in our study is likely.

Carcass, cardio-pulmonary morphometry and gut morphology
We observed no changes in the carcass yield, breast meat yield, thigh yield, abdominal fat, liver weight, and heart weight for the sources of dietary protein and energy. It implies that the dietary sources of protein and energy did not alter the yield of carcass and the amount of its associated abdominal fat. Closely similar results were reported in the previous studies (Fanimo et al., 1996;Al-Marzooqi et al., 2010) where size of the liver, gizzard, heart and spleen did not differ signi cantly between the dietary treatments. The reasoning was that the adequate levels of essential amino acids in both the diets, particularly lysine and methionine used for protein accretion in body and a constant ME:CP ratio was maintained across all the dietary treatments (Si et al., 2001;Baker et al., 2002). Similarly, Hidalgo et al. (2004) also reported no differences in carcass yield, breast meat yield, and abdominal fat pad in broilers fed low CP diets with a constant ME:CP ratio. Dozier and Moran (2001) reported that feeding broiler birds with diets formulated to contain suboptimum levels of ME and CP impaired the amount and yield of carcass components. Contrastingly, Mikulec et al. (2003) reported that the addition of sh protein supplements enhanced the higher values for breast, thigh and drumstick.

Humoral immunity
In our study, we failed to elucidate the in uence of the sources of protein and the levels of metabolizable energy on humoral immunity of the broiler birds. Fish meal contains plasma ceruloplasmin which is an indicator of acute phase in ammation in chicken associated with production or release of in ammatory cytokine (Chamanza et al., 1999). Thus, the results of the present study suggested that the dietary sh meal probably contained anti-in ammatory factors. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of sh meal and sh oil can prevent cardiovascular diseases and altogether affect the vascular and haemo-static systems, the brain, retina and other body tissues (Stansby et al., 1965;Visentainer et al., 2000). Usually, materials of sea foods and sh meals contain greater amount of carnitine and taurine. Thus, it was hypothesized that the carnitine and taurine particularly rich in sh protein sources were concerned in the better immune performance observed in chicks fed diet containing sh meal during immune stimulation. Previous studies reported that the dietary carnitine We deserved more studies to clarify the physiological nature of the dietary supplemented carnitine and taurine as factors stimulating growth and immune response in sh meal. It was hypothesized that a dietary protein source with higher biological value will result a lower urea and uric acid concentrations in blood serum compared to those with lower biological values (Bandegan et al., 2010). Dietary supplementation with more than 3.3 g EPA + DHA/100 kg BW daily (and in some studies up to 20.7 g EPA + DHA/100 kg BW) has been reported to decrease tumour necrosis factor-a (TNF-a), interleukin (IL)-1, IL-2, IL-6 and interferon (IFN)-c by mononuclear cell (Early, 1993). Accordingly, it was reported that the sh meal high in n-3 fatty acids, when fed to poultry, can increase the n-3 content in the bird's esh, thus making this tissue another source of n-3 fatty acids in the human diets (Calder, 2001). Many researchers have demonstrated that the n-3 PUFA has anti-in ammatory properties that bene t the immune system, with particular effectiveness against asthma and rheumatic arthritis (Gerster, 1995;Calder, 2001). Since such direct effects against Newcastle and Infectious Bursal diseases are scant, therefore, similar humoral immune response from sh meal might be anticipated in our study based on ND and IBD titer values.

Fatty acid pro le
It was reported that the sh meal high in n-3 fatty acids when fed to poultry increased the ω-3 fatty acid contents in the muscle of the broiler birds which made those tissues another source of ω-3 fatty acids for the human diets (Early, 1993). Ponnampalam et al. (2002) further reported that the long-chain ω-3 fatty acid contents (20:5n-3, 22:5n-3 and 22:6n-3) in the longissimus thoracis muscle were substantially higher with sh meal. Accordingly, the sh oils available in the sh meal not only increase the long chain ω-3 fatty acids in the meat, but also improve the ω-6: ω-3 ratio (Phetteplace and Watkins, 1989). In the present study we obtained substantially high levels of total ω-3 fatty acids and the ratio of ∑ω-3:∑ω-6, ∑UFA:∑SFA and ∑PUFA:∑MUFA in the Pectoralis major muscle of the broiler birds fed diet supplemented with blended sh protein which are fully aligned with previous studies.

Conclusion
Fishmeal supplemented high energy diet improves weight gain, feed e ciency and meat quality of broiler by increasing ∑ω-3 and ∑ω-7 fatty acids as well as the ratio of ∑ω-3:∑ω-6, ∑UFA:∑SFA and ∑PUFA:∑MUFA in the breast muscles of the broiler birds.

Declarations Funding
The Ministry of Science and Technology, Bangladesh (Grant No. BS-285).

Con icts of interest/Competing interests
None.

Availability of data and materials
All the data used in the manuscript exclusively belongs to the mentioned authors.

Code availability
None.
Author's contribution Dr. Md. Emran Hossain conceived the study, procured research grant, led the research team, analyzed data, interpreted the results and nalized the draft. Dr. Nasima Akter conducted the animal trial, ran the immunization program, dressed the carcass and collected data. Dr. Sonnet Podder executed the cardio-pulmonary morphometry. Dr. Karabi Barua prepared the initial draft. Dr. Ahasanul Hoque provided additional insights. All authors read and approved the nal manuscript.

Consent for publication
Yes.

Consent to participate
Yes.