Effect of Dietary Supplementation of a Phyto-supplement on Carcass Characteristics of Broiler Chickens

Poultry is one of the lucrative food industries worldwide. The demand for poultry meat is in an upward trend in developed and developing countries and to meet the demand, most of the commercial poultry is being produced under intensive systems. Under field conditions, sustaining and meeting the high feed efficiency of poultry is highly challenging as the intensive production system exposes them to various infections (Carrasco et al., 2019). Under such circumstances, subtherapeutic doses of antibiotics are being used as growth promoters (AGPs) in poultry to reduce sub-clinical infections, improve feed conversion efficiency, growth and economic returns (Murugesan et al., 2015; Suresh et al., 2018). However, concern exists with the use of AGPs due to the potential link between AGPs use and the surge in emergence, amplification and transmission of antibiotic-resistance in bacteria including zoonotic pathogens (Lillehoj et al., 2018; Durairajan et al., 2021). In this context, plant sources are being explored as growth promoters (Alloui et al., 2014). Phytoproducts refers to whole plants, herbs, various parts of the plants and their products, plant extracts and essential oils of plants/parts of plants. In the recent past, phytoproducts have gained much attention as feed supplements in poultry production (Abudabos et al., 2016; Singh et al., 2020; Mech et al., 2021). The phytoproducts exhibit multiple pharmacological properties such as potent antimicrobial, antioxidant, anti-inflammatory effects etc (Manafi, 2015). Some phytoproducts were advantageous in poultry in terms of better production performance and overall health of broilers (Paraskeuas et al., 2017). Owing to these facts, researchers across the globe are focusing on the possibility of using phytoproducts in poultry.
       
In India, the plants namely Cynodon dactylon (Family: Poaceae), Piper betle (Family: Piperaceae), Aerva lanata (Family: Amaranthaceae) and Piper nigrum (Family: Piperaceae) have been commonly used in traditional systems of medicine. C. dactylon is a perennial grass that contains an array of phytochemicals like ethyl α-d-glucopyranoside, linoleic acid, ethyl ester, thymol, and phytol (Jananie et al., 2011). Recently, Da Silva et al., (2019) have reported similar carcass characteristics and meat quality in sheep fed alfalfa and C.dactylon hay. The plant Piper betle Linn is an evergreen vine cultivated in Asian countries. The leaves (called ‘betel leaf’) are commonly consumed as mouth freshener after meals. Supplementation of P. betle leaf meal enhanced nutrient digestibility, body weight gain and improved the growth performance of broiler chickens (Basit et al., 2020). The leaves of A. lanata are a rich source of flavonoids (Oso et al., 2019) and a number of studies have recorded improved carcass characteristics in broilers fed dietary flavonoids (Prihambodo et al., 2021). The seeds of P.nigrum (black pepper) contains the phytochemical piperine as a major constituent. Black pepper has been shown to improve performance and health status of broiler chickens when incorporated in broiler diets (Abou-Elkhair et al., 2014). Recently we reported that dietary supplementation of a novel phyto-supplement (PS) containing A. lanata, P. betle, C. dactylon and P. nigrum improves growth, nutrient digestibility and intestinal morphology in broilers and could be a potential alternative to AGPs (Oso et al., 2019). The chicken meat is marketed as a whole as well as cut-up into parts due to the different cooking and taste choices. So, the carcass yield and high-quality cut-up parts are more important for the producers, processors and consumers (Marapana, 2016). Recently the study by Hafsa and Ibrahim (2018) has reported improved dressing percentage and weight of various organs in broilers fed diets with included polyphenols. Therefore, the current study was designed to investigate the carcass traits and organ weights of broilers fed diet supplemented with a phyto-supplement prepared from A. lanata, P. betle, C. dactylon and P. nigrum.

Preparation of phyto-supplement
 
The phyto-supplement used in the present study was prepared from A. lanata, C. dactylon, P. betle and P. nigrum. The aerial parts of C. dactylon and A. lanata used in the present study were purchased from a commercial source (Jeyam Herbal Ltd, Madurai, India). The P. nigrum seeds were procured from herbal merchants from Bangalore, Karnataka and ground to fine power before use. Freshly harvested P. betle leaves were procured from a farm and cleaned and cut into small pieces before use. To prepare the phyto-supplement, equal quantities of all the four products were weighed (1:1:1:1 ratio) and the three products except betel leaves were kept separately. The pre-weighed P. betle leaves were mixed with distilled water, ground into a fine semisolid paste, allowed to settle overnight and was filtered using muslin cloth. The filtrate thus obtained was mixed with the other three products (already weighed), mixed thoroughly and dried at 35^oC to a constant weight. The resultant product (phyto-supplement) was stored at 4^oC in an air-tight container until use.
 
Broiler experiment: Design and protocol
 
The broiler experiment was conducted at the ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka during the year 2018. The animal care, use and experimental protocol were approved by the institutional animal ethics committee. For the present study, one hundred and ninety-two numbers (192) of day old, commercial (VenCobb) broiler chicks were procured.  The chicks were weighed and randomly distributed to four experimental groups and each group was separated into 6 replicates, with 8 birds per replicate. The experimental birds were housed in battery cages in a well-ventilated building. A 24-hour lighting was ensured with electric bulbs. On day 1, the temperature inside the cages were maintained at 32oC and reduced gradually. From 4^th week, the day and night temperatures spanned between 30-34^oC and 23-28^oC respectively and the relative humidity was 40-60%. Each unit of cage had individual waterer, feeders and dropping trays and the birds were maintained under standard management conditions. Feed and water were made available to all the birds throughout the experimental period. The birds were subjected to four dietary treatments: Positive control (basal diet + antibiotic, chlortetracycline @355 g/metric ton of feed), negative control (0% PS: Basal diet only), 1% PS (basal diet +1% PS) and 2% PS (basal diet +2% PS). The feeding regime was divided into three phases: starter diet from day 0 to day 14, grower diet from day 15 to day 21 and finisher diet from day 22 to day 42 (Table 1). The birds were fed as per National Research Council (NRC, 1994).
 

 
Sampling, carcass and organ characteristics
 
At the end of the experimental period of 42 days, two birds from each replicate (12 birds per treatment) were randomly chosen and processed to evaluate the carcass characteristics. The birds were sacrificed, feathers were removed and dressed, carcass weight (without head, feet and viscera) and weight of retail cuts (drumstick, thigh, breast and back) were measured. Dressing percentage was calculated from dressed carcass weight as a percentage of the body live weight. The weights and relative weights (expressed as percentage of body weights) of gizzard, lung, liver, heart, pancreas, kidney, spleen, thymus and bursa were measured.
 
Statistical analysis
 
The data were first analysed statistically using the GLM procedures of SAS statistical software (SAS Institute, 2000) to examine the treatment effect. A completely randomized design was considered for ANOVA and all the treatments were compared with the Tukey’s test and the effects were considered significant at P<0.05. The “contrast” option of the GLM procedure was employed to determine the effect of different levels of phyto-supplement. Further, orthogonal polynomial contrasts were also employed to determine the Linear (L) and Quadratic (Q) responses to different levels of phyto-supplement used for the treatments.

Carcass and organ characteristics
 
The dietary supplementation of phytogenic feed additives is being explored as a strategy to improve carcass characteristics in broilers (Jamroz et al., 2003; Mech et al., 2021). The effect of dietary supplementation with phyto-supplement on dressing percentage and retail cut parts is shown in (Table 2).
 

       
Broilers of antibiotic group and 1% PS group showed the highest (P=0.0205) and negative control group recorded the lowest dressing percentage. Orthogonal effect of PS levels revealed a linear increase (P=0.042) in dressed weight and a quadratic improvement (P=0.024) in dressing percentage as supplemental levels of PS increased from 0 to 1%, but reduced with 2% PS. Dietary treatment had no effect on weight of drumstick, thigh and breast meat. Dressing percentage is the proportion of live weight yielded after animals have been eviscerated. Singh et al., (2009) reported improved dressing percentages in birds fed with polyherbal growth promoter when compared to control group. Nasir and Grashorn (2010) also reported improved carcass yield following dietary inclusion of 1% Nigella sativa seed powder. A similar increment in dressing percentage obtained for antibiotic group and broilers fed diet supplemented with 1% PS suggested that PS included at this level could efficiently replace in-feed antibiotic without compromising dressing percentage. However, a reduction in dressed weight and dressing percentage obtained for the negative control group is expected since carcass traits are a reflection of growth response (Oso et al., 2019).
       
The effect of dietary supplementation with the phyto-supplement on the weight and relative weights of organs of broilers are presented in (Table 3).
 

       
The weight and relative weight of gizzard, heart, pancreas, spleen, thymus and bursa were not affected by dietary treatments. Orthogonal effect of PS levels revealed a quadratic reduction in relative weight of gizzard (P=0.008) as supplemental levels of PS increased from 0 to 1%, but showed increase in relative weight with 2% PS. Broilers fed diet supplemented with 1 and 2% PS exhibited higher (P<0.0001) weight and relative weight of lung than other treatments. The relative weight of lung increased linearly as the supplemental levels of PS increased. Broilers fed diet supplemented with 1 and 2% PS exhibited lower weight (P=0.0032) and relative weight (P=0.0002) of liver when compared with the antibiotic group. Orthogonal effect of PS levels showed a quadratic reduction (P=0.022) in the relative weight of liver as supplemental levels of PS increased from 0 to 1%, but recorded higher relative weight with 2% PS. The relative weight of heart also increased quadratically (P=0.044) with 1% PS supplementation, but reduced with 2% PS supplementation. Broilers on negative control diet recorded the highest weight (P = 0.0002) and relative weight (P<0.0001) of kidney. Contrast analysis also revealed a linear and quadratic reduction in weight and relative weight of kidney as PS levels increased from 0 to 1%.
       
As a part of the digestive tract, gizzard plays a key role in utilization of dietary nutrients. The relative weight of gizzard has been reported to increase in birds supplemented phytogenics like neem leaf extract (Maidala et al., 2016) while no changes were observed in birds supplemented with lemon peel extract or orange peel extract or Curcuma xanthorrhiza (Akbarian et al., 2013). It is worthy to notice in this study that the relative weight of gizzard was increased in a quadratic manner in response to the increase of PS supplementation from 1 to 2%, suggesting improved gizzard function and nutrient utilization (Choct, 2009). An increase in gizzard weight results in an increased secretion of hydrochloric acid by the proventriculus which in turn reduces the entry of pathogenic bacteria into the digestive tract  and eventually the immune stress on broilers (Engberg et al., 2002; Engberg et al., 2004). Phytochemicals are known to attenuate cardiovascular diseases in rats and humans (Peluso, 2006). Therefore, the higher weight and relative weight of heart obtained for broilers fed with 1% PS may be linked with the constituent phytochemical compounds present in the formulated phyto-supplement. The quadratic increase in the relative weight of the heart following 1% PS supplementation observed in the current study may be beneficial since low relative heart weight is associated with high prevalence of metabolic disorders such as sudden death syndrome and ascites in broilers (Havenstein et al., 2003).  This is because, heart with low weight have reduced capacity to supply oxygen to body tissues, resulting in the development of several metabolic disorders (Gaya et al., 2007). Moghadam et al., (2005) suggested that selection for a larger ratio of heart weight to live body weight could overcome this problem in chicken. Therefore, the increased relative weight of heart following 1% PS supplementation may also be characterized for weight gain selection (Havenstein et al., 1994). The protective potential of medicinal plants and phytochemicals against lung diseases are well researched and established in animals (Jasemi et al., 2020). Hence, it is possible that the phytochemicals present in PS exerted control on growth of the lungs resulting in higher lung weight observed in these birds. Under commercial broiler production systems, the inability of lungs to grow in pace with the rapid growth rate of the body affects the health and welfare of the birds (Stevenson, 2003). Therefore, the improved lung weights observed with PS supplementation would be helpful to improve the health of birds.
       
Our study demonstrated that supplementation of 1 or 2% of PS in the diet reduced the weight and relative weight of liver as compared to broilers fed antibiotic. This result is similar to Amad et al., (2011) who reported that the application of a mixture of thymol and anethole essential oils reduced the relative weight of liver in broiler chickens. In chicken, increase in liver weight is linked with high abdominal fat content and possible incidence of metabolic disorders such as fatty liver diseases and sudden mortality (Skrivan et al., 2000). The observed results of the study suggests that supplementation of PS reduces the susceptibility of broilers to hepatic hyperplasia and hypertrophy (Pearson et al., 1983). The highest weight of kidneys observed in broilers of negative control group indicates the possible malfunctioning of kidneys (Selvaraj et al., 1998).

In conclusion, supplementation with the phyto-supplement improved dressing percentage and weight of organs in a dose-dependent manner with the best response at 1% PS level. Further dietary supplementation with 1% PS increased relative weight of heart and weight and relative weight of liver and showed no adverse effect carcass characteristics. Thus, supplementation of PS would be advantageous to producers, processors and customers.

The funding for the research provided to the first author by the Department of Science and Technology, Government of India, under the Visiting Fellowship Grant of the ‘CV Raman International Fellowship for African Researchers’ vide letter No. DST/INT/CVRF/2016 and the additional fund provided by host institute (ICAR- National Institute of Animal Nutrition and Physiology, Bangalore, India) towards this research is also acknowledged.