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Plant Based High Energy Mash Diets Supplemented With Nahco3, L-arginine+ Vitamin-c and Vegetable Oils Are Effective Against Tachycardia and Polycythemia in Broiler Chicken

The study aimed to investigate if plant based high energy mash diets supplemented with NaHCO3, L-arginine + vitamin-C and vegetable oils were effective against tachycardia and polycythemia in the commercial broiler chicken. Total 256Ross-308 day old male broiler chicks were randomly distributed into eight dietary treatment groups in a three way 2 3 factorial arrangements (Three factors, i.e., NaHCO 3, L-arginine + vitamin-C and vegetable oil each with two levels, e.g., 0 and 0.1% for NaHCO 3 and L-arginine + vitamin-C; 3 and 4% of vegetable oil supplemented with basal diet). Iso-caloric and iso-nitrogenous diets were formulated and supplied ad libitum. The average daily feed intake (ADFI), average daily gain (ADG), feed eciency (FE), carcass characteristics, cardio-pulmonary morphometry, total protein (TP), hemoglobin (Hb), triiodothyronine (T 3 ), incidence of tachycardia and polycythemia were examined up to 35 d. Supplementation of NaHCO 3 decreased (p<0.001) the ADFI at 1-14 d, 15-35 d, 1-35 d, improved (p<0.01) the FE at 1-14 d and increased (p<0.05) the serum TP. Dietary L-arginine + vitamin-C decreased (p<0.01) the heart rate without affecting the performance parameters, carcass characteristics and hemato-biochemical indices. Supplementation of vegetable oil decreased (p<0.01) the ADFI at 1-14d, 15-35 d, 1-35 d, increased (p<0.01) the ADG at 1-14d, improved (p<0.001) the FE at 1-14 d and increased (p<0.05) the heart rate, Hb and PCV. Further, NaHCO 3, L-arginine + vitamin-C and vegetable oil synergistically interacted to decrease the left and right ventricular weight, RV:TV and increased the T 3 without affecting overall performance, carcass characteristics and hemato-biochemical indices. It was concluded that, plant based high energy mash diets are not susceptible to tachycardia and polycythemia. Addition of NaHCO 3, L-arginine + vitamin-C ameliorate the propensity of tachycardia and polycythemia without deteriorating performance, carcass characteristics and hemato-biochemical indices of the commercial broiler birds in a dose dependent manner.


Introduction
Tachycardia in broiler is a patho-physiological interplay between the lungs and the heart initiated by the terminal consequences of the excessively elevated blood pressure within the pulmonary circulation (Wideman et al. 2013; Khajali and Wideman 2016). A broiler chick initially weighing around 40 g at hatch is likely to attain more than 4000 g in 8 weeks. This elevated growth of almost 100-fold in just 8 weeks cannot be sustained devoid of similar remarkable increases in the functional capability of the heart and lungs (Wideman et al., 2013). The cardiac output increases 100 fold in 8 weeks post hatch, ranging from 8ml/min for a 40 g chick to around 800 ml/min for a broiler weighing 4 kg (Wideman 1999;Wideman et al. 2013). Furthermore, in the study of Decuypere et al. (2000) on the muscle bre typology it was evident that the choice for increased breast meat yield did not result in a proportionate increase in the heart, blood and lung weight due to the relative independence of glycolytic white muscle to the requirement for their oxygen. Due to these discrepancies, pulmonary vascular capacity in broiler birds is restricted and only a bit able to cope with continuous increases in the cardiac yield (Wideman and Bottje, 1993;Wideman, 2000). Therefore, modern broiler birds are prone to initially tachycardia and later on to the progressive development of pulmonary hypertension syndrome (PHS).
The PHS stimulates the red blood cells racing too rapidly through the pulmonary vasculature. Thus, the RBCs cannot achieve full blood-gas equilibrium because of a short transit time at the gas exchange surfaces leading to incomplete diffusive exchange of O 2 and CO 2 (Henry and Fedde, 1970;Powell et al., 1985). This inadequate residence time, thus, causes blood exiting the lungs and to enter the systemic ow with a lower than the usual partial pressure of O 2 leading to hypoxemia. In the systemic ow, hypoxemia evokes extensive arteriolar dilatation to raise the blood ow and reinstate ample O 2 delivery to the organs as well as tissues Wideman 2000; Wideman and Tackett 2000; Ruiz-Feria and Wideman 2001). The systemic arteriolar vasodilatation lets blood to way out to the large arteries more quickly accompanied by increment in the rate at which venous blood comes back to the right ventricle. The rise in venous return and the commencement of systemic arteriolar hypotension automatically stimulate the heart to increase the cardiac output leading to tachycardia. Additionally, persistent hypoxemia stimulates hematopoietin and markedly increases the hematocrit (Burton et al., 1971;Julien et al., 1985).
Increased hematocrit results polycythemia which boosts up blood thickness and is the most important reasons of the increased resistance to the blood-ow that consequences in PHS (Snyder, 1971; Penney et al., 1988). High energy pellet diets provoke PHS by stimulating lipolysis and being de cient in protein and thereby arginine as well. Plant proteins, by virtue, are more susceptible to be de cient in critical amino acids, i.e., lysine, methionine and arginine. Consequently, we warned that plant based high energy mash diet may prone to reduce the performance of the birds. We therefore, aimed to justify if plant based high energy mash diet supplemented with L-arginine + vitamin-C and sodium bicarbonate was effective against tachycardia and polycythemia in commercial broiler birds.

Study design, animals and housing
The experiment was conducted in a three way 2 3 factorial arrangements (Three dietary factors, i.e., NaHCO 3, L-arginine + vitamin-C and vegetable oil each with two levels, e.g., NaHCO 3, 0 and 0.1%; L-arginine + vitamin-C, 0 and 0.1%; Vegetable oil, 3 and 4% of the basal diet). 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. 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.

Chemical analysis
Chemical analyses of the experimental diets were carried out in triplicate for dry matter (DM), crude protein (CP), crude ber (CF), nitrogen free extracts (NFE), ether extracts (EE) and total ash (TA) in the animal nutrition postgraduate laboratory, Chattogram Veterinary and Animal Sciences University, Chattogram as per standard procedure (AOAC, 2019

Performance parameter
Mortality was recorded as occurred, while average daily feed intake (ADFI), average daily gain (ADG), feed e ciency (FE) were recorded fortnightly. 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 two birds from each replicate were randomly selected and killed by severing the jugular vein and carotid artery. Once a bird was adequately bleed out, it was scalded and defeathered. 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.  (αβγ)ijk = The interaction effect of the of the 'i th ' level of the factor 'α', the 'j th ' level of the factor 'β', and 'k th ' level of the factor 'γ'; Yijk = The observed value of the variable under study for the 'n th ' repetition of the combination of the 'i th ' level of factor 'α', the 'j th ' level of the factor 'β', and the 'k th ' level of the factor 'γ'; εijk = The random sampling error due to 'i th ' level of the factor 'α', 'j th ' level of the factor 'β', and 'k th ' level of the factor 'γ'.  (Table 4). Table 4 Initial live weight (ILW, g/bird/d), nal live weight (FLW, g/bird/d), 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 diets supplemented with various levels of NaHCO 3, Larginine + vitamin-C and vegetable oils

Carcass characteristics
Main as well as interaction effects of supplementing NaHCO 3 , L-arginine + vitamin-C and vegetable oil appeared nil (p > 0.05) on the carcass characteristics of the experimental broiler birds (Table 5).
Under critical episodes of progressive tachycardia, the pulmonary arterial pressure accelerates the rate of blood ow which then enters the systemic ow with a lower than usual partial pressure of O 2 and a higher partial pressure of CO 2 resulting hypoxemia (Peacock et al. 1990;Reeves et al. 1991;Julian and Mirsalimi 1992;Roush et al. 1997).

Performance
Our study indicated that, the addition of L-arginine + vitamin-C in the diet of the broiler chicken signi cantly improved feed intake from 3-5 weeks. Similar result was reported by Al-Daraji and Salih (2012). Supplementation of L-arginine + vitamin-C in the mash feed resulted better weight gain during last two weeks of rearing which is in line with the ndings of other studies (Njoku 1986; Al-Daraji and Salih 2012). Signi cantly higher weight gain might have been due to increased feed intake. It is well established that, vitamin-C reduces the extents of oxidative heat stress e ciently (Masad 2012; Dalia et al. 2018) which is the underlying cause of increased feed intake and weight gain (Njoku, 1986) in the broiler birds in our study. Further, these two additives improve the immune status of the birds which could have helped in better performance of the commercial broiler birds used in current study (Masad 2012; Dalia et al. 2018). Similarly, L-arginine, an indispensable amino acid for poultry as well as human which improves the release of growth hormone and muscle growth that could have contributed to the better feed intake and daily weight gain in our study (Stevens et al. 2000).
In the present study, incorporation of vegetable oil in the diet of boiler birds signi cantly improved average daily gain and FCR during the initial period. These ndings coincide the results of Attia et al. (2020) although differ from the observation of Ayed et al. (2015). One of the objectives of the current study was to see the interaction effect of NaHCO 3 , L-arginine + vitamin-C and vegetable oil in accordance with the performance of broiler chicken. Interaction effect of NaHCO 3 and L-arginine + vitamin-C showed signi cant rise in feed intake in the last two weeks of the trial. As stated above, effect of reducing heat stress due to addition of additives present in the diet could be one of the reasons behind the improved feed intake (Masad 2012; Dalia et al. 2018). Accordingly, at the same time, NaHCO 3 on diet apparently had association with the increasing feed intake and the reason appeared to be due to the bicarbonate ion which was associated with an increased water intake (Balnave and Gorman, 1993). Similar interaction effects were seen in the birds fed diet containing combination of L-arginine + vitamin-C and vegetable oil. As mentioned earlier, vitamin-C in the test diets helped to reduce the environmental stress (Masad 2012; Dalia et al. 2018). Further, the vegetable oils in the diet met the increasing demand of energy (Attia et al., 2020), especially during the nisher stage. These effects might be the reason of rise in energy intake in the broiler birds fed diets containing both the L-arginine + vitamin-C and the vegetable oil.
From the result of our study, it is obvious that, average feed intake of the birds fed NaHCO 3 supplemented diet throughout the trial increased substantially which agrees with the previous ndings (Arp et al., 1984;Roussan et al., 2008;Osman et al., 2015). The increased feed intake, however, might be a result of reduced heat stress in broilers caused by NaHCO 3 (Roussan et al., 2008).
In our study, NaHCO 3 also improved the average daily gain of birds in the last two weeks which is aligned with the previous Here, in our study, liver weight was found lower in broiler birds fed experimental diets containing L-arginine + vitamin-C that further coincides with the ndings of Susantoputro et al. (2014). The interaction effects of test ingredients on carcass merit were also found nonsigni cant between diets.

Cardio-pulmonary morphometry
Dietary supplementation of 130% arginine of the requirement improved the intestinal morphology and performance and decreased the cold induced ascitic mortality in broiler chickens (Abdulkarimi et al., 2019). In a previous study, additional arginine administered in ovo or in the feed reduced the vulnerability of broilers to pulmonary hypertension (Saki et al., 2013). Additionally, broiler chickens reared at high altitude and predisposed to pulmonary hypertension and ascites were partly explained by arginine supplementation (Khajali et al., 2011). Supplemental arginine improved the pulmonary vascular performance of hypoxic broiler birds and its outcomes were further enhanced by the addition of the vitamin-C. Arginine and antioxidant vitamins might have taken part in synergistic functions to improve nitric oxide bioavailability as potent natural vasodilator and lessen oxidative damage, thus increasing cardiopulmonary performance (Bautista-Ortega and Ruiz-Feria, 2010).
It was further reported that, arginine or guanidinoacetic acid supplementation of diets did not affect gross response of birds under standard temperature, but addition of arginine to the diet signi cantly reduced the incidence of cold stress on performance, gut development and ascites syndrome (Kodambashi et al. 2017). In fact, L-arginine is a substrate for nitric oxide, which acts as a potent endogenous pulmonary vasodilator that substantially reduces the right ventricle: total ventricle ratio (Wideman et al. 1995). Consistent with these points, addition of L-arginine and thereby, reduced susceptibility of the birds against tachycardia and polycythemia evident in our study is likely. Rostami et al. (2016) reported that, the birds receiving ax oil had signi cantly higher serum concentration of nitric oxide. The right-to-total ventricle weight ratio (RV/TV) and death from pulmonary hypertension were signi cantly (p < 0.05) declined in birds that fed on ax oil. It was argued that, n-3 fatty acids could signi cantly lessen RV:TV and PHS death in birds by escalating circulatory level of nitric oxide and suppressing hepatic lipogenesis. In another study, administration of ax oil reduced blood viscosity, right ventricular hypertrophy, hematocrit and hemoglobin content and improved erythrocyte deformability by increasing the quantity of unsaturated fatty acids in the erythrocyte membranes and thereby decreased ascites induced mortality (Walton et al., 1999(Walton et al., , 2001. With reference to these points, addition of vegetable oil in the present study might have contributed additional n-3 fatty acids responsible for improving RV:TV and thereby no evidence of tachycardia in the experimental birds fed test diets in our study. Supplementation of vitamin C substantially improved cellular integrity and reduced incidence of mortality by ascites in a previous study (Roch et al., 2000). Similarly, addition of vitamin C reduced the possibility of thick-walled peripheral vessels in the lungs and thereby, the incidence of ascites (Xiang et al., 2002). Vitamin-C reduces muscularization of the pulmonary arterioles by scavenging oxygen-derived free radicals thus lower the number of thick-walled peripheral vessels to decrease the resistance to blood ow in the pulmonary vessels of broilers (Hassanzadeh Ladmakhi et al., 1997). The levels of α-tocopherol and γ-tocopherol were decreased in the mitochondria of an ascitic broiler, suggesting inadequate reactive oxygen scavenger in the primary site of energy transduction (Cawthorn et al., 2001). It could therefore be inferred that, addition of L-arginine + vitamin-C in the form of feed additive e ciently stabilized RV:TV, prevented tachycardia and polycythemia in the experimental birds used in our study.

Hemato-biochemical indices
A decline in blood pH lowers the oxygen a nity of haemoglobin, while increased blood pH increases oxygen a nity to increase haemoglobin saturation in the lungs (Isaacks et al., 1986). Thus, increased blood pH can improve the loading capacity of oxygen by haemoglobin in the lungs due to the Bohr effect. Accordingly, feeding bicarbonate supplemented diets result in a decrease in pulmonary hypertension (Barer et al. 1966; Balnave and Gorman 1993;Squires and Julian 2001). Perhaps, this is the reason behind the absence of tachycardia and thereby polycythemia in the experimental birds used in the present study.
The majority of commercial meat birds are fed crumbled or pelleted diets to attain utmost growth and feed e ciency. Feeding mash diet reduces growth rate. Broiler birds that consumed pellet feed had frequently been shown to have higher incidence of tachycardia than the birds that consumed the same diet in mash form (Shlosberg et al., 1992; Bölükbasi et al., 2005). Unlike pellets, mash diets are not subjected to steam conditioning prior to pelleting. Thus, mash diets are less palatable and have reduced microbial and enzymatic digestibility. Consequently, mash diets have fur less susceptibility for tachycardia and polycythemia in broiler birds as evident in our study.
In ascitic birds, Dahiya et al. (2001) reported a signi cant rise in haemoglobin, packed cell volume, total erythrocyte count, serum alanine aminotransferase, asparate aminotransferase, alkaline phosphates and phosphorus and marked fall in serum total proteins, albumin globulin ratio (A:G), calcium and sodium although erythrocyte indices, i.e., mean corpuscular volume, and mean corpuscular haemoglobin concentration remained unchanged. Consistent results were reported elsewhere indicating signi cant Additionally, the HDL cholesterol signi cantly decreased re ecting progressive cardiomyopathy and subsequently cardiac failure in previous studies. Since direct evidences are scant, relying upon above reports, it can indirectly be inferred that the birds susceptible for tachycardia and polycythemia will reveal similar symptoms. Interestingly, all above hemato-biochemical parameters as well as heart beat persisted within standard range (Bounous and Stedman, 2000) exhibiting no unusual changes thereby least possibility of tachycardia or polycythemia either in control or test groups in our study.

Conclusion
Plant based high energy mash diets are not susceptible to tachycardia and polycythemia. Addition of NaHCO 3, L-arginine + Vitamin-C ameliorate propensity of tachycardia and polycythemia without deteriorating performance, carcass characteristics and hemato-biochemical indices of the commercial broiler birds in a dose dependent manner.

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