Dietary supplementation of low levels of unprocessed Mucuna pruriens utilis seed meal induces mild antinutritional entero-physio-metabolic perturbations without compromising performance and meat quality in finisher broilers

The rapidly growing world human population accentuates the need for improved production especially of protein-rich food. Broiler meat production offers opportunity to ensure security of this food. However, the production of modern broilers is not only limited by high feed costs due to dietary use of expensive energy and protein sources but also their meat possesses undesirable quality attributes. This study thus examined the effect of dietary Mucuna pruriens utilis seed meal (MSM) on growth performance, blood profile, carcass traits, and meat quality in finisher broiler chickens. In a completely randomised design (CRD), 320 21-day-old chicks were randomly allocated to 32 pens in which they were allotted 4 dietary treatments with 0, 2.5, 5, and 10% MSM, each with 8 replicate pens of 10 birds, for 28 days. Growth performance, carcass characteristics, internal organs, haemato-biochemistry, and meat quality were measured. Results showed that dietary MSM did not affect (P > 0.05) broiler performance, weights, and lengths of carcass cuts and internal organs, haematology, and meat quality. The only exception was MSM-induced increase in duodenal weight (linear, P < 0.05) and serum phosphorus (quadratic, P = 0.05) in contrast to a decrease in procalcitonin (quadratic, P < 0.01) and serum levels of total protein (linear, P < 0.05; and quadratic, P < 0.01), albumin (quadratic, P < 0.05), and bilirubin (quadratic, P = 0.001). Therefore, MSM could be supplemented up to 10% without compromising performance, carcass traits, internal organs, haemato-biochemistry, and meat quality in finisher broiler diets.


Introduction
The world human population continues to grow rapidly and is envisaged to reach between 9.4 and 10.1 billion by 2050 (United Nations, 2019).This concomitantly increases the demand for food and thus accentuates the need for strategies to improve production especially of protein-rich food to eradicate both food insecurity and malnutrition particularly in low-and middle-income countries of Africa (Ayodele et al., 2021;OECD/FAO, 2021).Considering its fastest global growth among all agricultural subsectors (Research and Market, 2019), its perception as being relatively nutritionally valuable with a preponderance of health-desirable unsaturated fatty acids (Barroeta, 2007;Cavani et al., 2009), as well as its affordability to most consumers (Valceschini, 2006), broiler meat is the most strategic type of protein-rich food for which development interventions are required to enhance its capacity and efficiency of production.
Notwithstanding, the production of broiler chickens is limited by high feed costs.These represent about 65-75% of total variable costs (Panda et al., 2014), of which about 95% are ascribable to energy and protein (plant and animal derived) sources (Mallick et al., 2020).To decrease these costs, replacement of expensive energy and protein ingredients with cheaper and locally abundant alternative feedstuffs, among other options, has been the most prudent strategy to sustain broiler production (El-Deek et al., 2020).Particularly, the focus of replacement has been directed at maize and soya bean meal (SBM), the major dietary plant-derived protein sources commonly used in broiler diets (Khalil et al., 2023).Aside exorbitant feed costs, modern fast-growing broiler strains produce meat with undesirable quality attributes imputable to intensive genetic selection and breeding programmes over the past decades.They deposit excessive abdominal fat (Tumova et al., 2021), and produce meat with high fat content and low pH (Kocer et al., 2018).The pH of meat is an important quality characteristic, the decline of which is associated with paler and softer meat with higher drip loss.Drip loss is visually unattractive to consumers and can result to excessive cooking losses and cooked meat dryness (Marchewka et al., 2023).Therefore, there is a need for strategies to improve not only production but also the quality of broiler meat to ultimately improve its appeal among consumers.One of the alternative feedstuffs with nutritional attributes that qualify it for partial replacement of maize and soya bean meal in broiler diets, as well as bioactive compounds with great potential to enhance desirable quality properties of the modern birds' meat, is MSM.
Notwithstanding, due to its endowment with L-DOPA, dietary intake of MSM induces weight loss and decreases feed intake and feed conversion efficiency in non-ruminants including broilers (Del Carmen et al., 2002;Flores et al., 2002) especially when included at high levels (≥ 15%) (Vadivel and Pugalenthi, 2010a).For this reason, it may be prudent to include MSM at low levels (≤ 10%) in broiler diets as it has recently been recommended (Ayodele et al., 2021) as a strategy to impart its anabolic effects on broiler meat as it was shown in mice (Imbs and Schwartz, 2011) whilst minimising the likelihood of its toxicity.Therefore, the objective of this study was to investigate the growth performance, carcass characteristics, internal organs, haemato-biochemistry, and meat quality in finisher broilers fed low-graded levels of unprocessed MSM as a partial replacement for maize and SBM as well as a phytogenic feed additive.

Study site and ethical consideration
The protocols used in this study were approved by the North-West University (NWU) Animal Production Sciences Research Ethics Committee (NWU-00814-31-AS).The study was conducted using broiler facilities at the NWU Experimental (Molelwane) farm (coordinates: 25° 40.459′ S, 26° 10.563′ E) situated in the Mahikeng Local Municipality, North-West Province, South Africa.

Sourcing of M. pruriens utilis seeds and other ingredients, diet formulation, and chemical analysis
Mucuna seeds were purchased from TWK (Piet Retief, RSA), whilst all other dietary ingredients were procured from Optifeeds (PTY) LTD., Lichtenburg, RSA.The experimental diets were formulated at SimpleGrow Agric Services (Pty) Ltd. (Irene, Gauteng, South Africa) to produce 5 iso-caloric and iso-nitrogenous (18% CP) diets with maize, SBM, and full fat soya bean being partially substituted with incremental inclusion levels of MSM (0, 2.5, 5, and 10%) to meet nutritional requirements of finisher broilers (d22-49) (Table 1) (NRC, 2001).Then, 100 g samples of MSM and experimental diets were milled (1 mm screen) and analysed for DM (930.15),CP (954.01),EE (920.39),ash (942.05), and CF (AOAC, 2005).Their DM was determined by ovendrying (105 °C) 1 g of sample in pre-weighed crucibles for 12 h followed by cooling in a desiccator and weighing.It was then calculated as a difference between initial sample weight and moisture weight.CP was determined following the Kjeldahl method and multiplying nitrogen values by 6.25.EE was determined by ether extraction of crude fat using an automated Soxhlet Fat analyser ANKOMXT15 extractor following the operator's manual (ANKOM Technology, Macedon, NY, USA).Ash was determined by calcinating a dry 1 g sample in the muffle furnace (550 °C) (Nabertherm GmbH, Germany) for 6 h.CF was analysed using the ANKOM2000 Fibre Analyzer with 0.255 N crude fibre acid solution and 0.313 N crude fibre base solution.Also, NDF, ADF, and ADL were analysed following protocols of Van Soest et al. (1991).Both NDF and ADF were analysed using the automated ANKOMDELTA fibre analyser (ANKOM Technology, Macedon, NY, USA), whilst ADL was determined by immersing ADF residue bags for 3 h in 72% (wt/wt) sulphuric acid.

Study design and broiler management
Day-old Cobb 500 male broiler chicks purchased from Eagle Pride Hatchery were initially ad libitum fed a commercial starter diet from d1 to 21, after which they were introduced to experimental finisher diets from d22 to 49.They were supplied with StressPack with electrolytes and vitamins for 48 h.In a CRD, 320 21-day-old chicks were randomly allocated to 32 pens (1.8 m high × 1.5 m long × 1.5 m wide each) in which they were allotted 4 dietary treatments (0, 2.5, 5, and 10% MSM), each with 8 replicate pens of 10 birds, for 28 days.The pen was the experimental unit.Fresh feed and water were ad libitum supplied throughout the duration of the trial.The feeding trial was carried out in a deep-litter system in a broiler house wherein temperature and ventilation were controlled by opening curtains during the day (0800 h to 1700 h).Daily house temperature was maintained in the range of 18 to 21 °C and humidity at 40 to 60%, with a multi-metre device used to monitor them.

Growth performance
Average feed intake (AFI) was calculated by subtracting the weight of leftover feed from the weight of feed offered and dividing the difference by the total number of birds per pen.Body weight was initially measured on d21 and thereafter weekly by weighing all the birds in each pen using a weighing balance.Body weights were used to calculate average body weight gain (ABWG) according to the equation: where t0 = initial time (days); T = final time, W (T) = final body weight (g), and W (t0) = initial body weight (g).Weekly feed conversion efficiency (FCE) was calculated as ABWG divided by AFI.

Carcass characteristics and internal organs
Following 12 h of feed withdrawal, 4 birds were randomly selected from each replicate pen on d50, electrically stunned, bled, and de-feathered.Hot carcass weight (HCW) was measured by weighing eviscerated carcasses within 45 min after slaughter, whilst cold carcass weight (CCW) was measured after chilling the carcasses at 4 °C for 24 h.The weight and length of various carcass cuts (breast, wing, drumstick and thigh) and internal organs (gizzard, proventriculus, spleen, liver, duodenum, jejunum, ileum, caecum and colon) were measured using a digital scale and tape.

Meat quality
Breast muscle pH and temperature were measured 45 min post-slaughter and 24 h after chilling (4 °C) using a corning Model 4 pH-temperature metre (Corning Glass Works, Medfirld, MA), whilst meat colour (lightness L*, redness a*, and yellowness b*) was measured within 45 min and 24 h after euthanasia using a colorimeter (Minolta, Tokyo, Japan).Shear force was determined using a Meullent-Owens razor shear blade whilst drip loss, water holding capacity, and cooking loss were measured according to Cheng et al. (2019).

Haemato-biochemistry
On d 50, blood was collected from 2 birds per pen (16 per treatment) from the wing vein using a 21-gauge needle.It was placed into purple-top EDTA-coated vacutainer tubes and analysed for haematology using an automated IDEXX LaserCyte Hematology Analyzer (IDEXX Laboratories (Pty) Ltd., Johannesburg, South Africa), whilst blood collected into red top Vacuette® Serum Clot Activator tubes without EDTA (Greiner Bio-One, GmbH, Frickenhausen, Germany) was analysed for biochemistry using an automated IDEXX Vet Test Chemistry Analyzer (IDEXX Laboratories (Pty) Ltd., Johannesburg, South Africa).

Statistical analysis
All data were tested for normality using Levene test (Levene, 1960) and homogeneity of variance using Shapiro Wilkinson test (Shapiro and Wilk, 1965).Then, normally distributed data including weekly ADWG, FI, FCE, weights, and lengths of carcass cuts and internal organs, haemato-biochemistry, and meat quality were analysed for linear and quadratic effects by employing polynomial contrasts.The optimum dietary inclusion level of MSM was estimated using response surface regression analysis (SAS, 2002(SAS, -2012 n.d.) n.d.) according to the quadratic model: y = ax 2 + bx + c; where y = response variable, a and b = coefficients of the quadratic equation, c = intercept, x = MSM level (%), and − b/2a = x value for optimal response.Also, weekly ADWG, FI, and FCE data were analysed as repeated measures to determine the diet by week interaction effect, whilst the rest of data (overall ADWG, FI, and FCE; weights and lengths of carcass cuts and internal organs; haemato-biochemistry; and meat quality) were analysed using the general linear models procedure of SAS (2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012) in a CRD, with diet being the only main factor.Least square means were compared using the probability of difference option and differences among them deemed significant at P ≤ 0.05.
Notwithstanding, a previous study showed decreased blood phosphorus and alkaline phosphatase (hypophosphataemia) in rats following consumption of unprocessed MSM-containing (10%) diets (Huisden et al., 2019).The reason for the differences in animal responses to dietary MSM between the current study and that of Huisden et al. (2019) is unknown.Hence, it would be of interest to further investigate blood (serum) phosphorus and other minerals in broilers fed MSM-supplemented diets in future.
Notwithstanding, our data also demonstrated deleterious effects of dietary MSM on entero-physio-metabolic processes particularly when included at 5 and 10% levels, with most of these effects elicited in blood serum components.In this regard, the observed increase in the weight of the duodenum with incremental dietary levels of MSM (Table 3) points to the presence of toxic phytochemicals most likely L-DOPA in the legume seed meal, if not the high dietary fibre content (Agyekum et al., 2012) (Table 1).
Osei and Dei (1998) observed similar hypertrophic effects on the intestine in broilers fed high (15%) MSM-containing diets.Also, other studies observed increased lengths of the duodenum and other intestinal segments due to consumption of diets supplemented with MSM (Oloruntola et al., 2018) and tannin-rich leguminous leaf meals (Miya et al., 2020), in addition to dietary fibre stimulation (Wu et al., 2004) in broilers.However, it is interesting that the relative change in duodenal weight did not influence carcass weights, implying that the observed enteric weight change was merely an adaptation mechanism.
The observation of quadratic decreases in procalcitonin and serum total protein, albumin, and bilirubin appears to be congruent with the observed increase in the weight of the duodenum and suggests MSM-induced enteric and liver toxicity particularly at 10% inclusion level.Produced by liver neuroendocrine cells and macrophages (Matzaraki et al., 2007), increased blood procalcitonin is indicative of liver injury (Rule et al., 2015), and this appears to have been more intense at 10% MSM inclusion level.Also, serum total protein (composed mainly of albumin, globulins, and other proteins) is ordinarily measured as a biomarker of the body's nutritional status and liver function, with its levels usually decreased in liver dysfunction-associated conditions such as ageing and malnutrition (Furruqh et al., 2004;Sabatino et al., 2017).Constituting 65% of serum total proteins, albumin is a biomarker for malnutrition and is responsible for the transportation, among other substances, of unconjugated bilirubin (Tian et al., 2014).Decreased serum bilirubin levels, on the other hand, occur in physiological conditions associated with oxidative stress and inflammation (Salomone et al., 2013;Tian et al., 2018).It would therefore appear that dietary consumption of high (10%) L-DOPA-infested MSM induced hepatotoxicity that led to protein undernutrition alongside compromised production of albumin, hence poor transportation and decreased serum levels, of bilirubin in broilers.Indeed, there is a positive correlation between procalcitonin and total bilirubin level (Qu et al., 2016) as well as serum total protein and bilirubin ( Jonathan et al., 2021).Otherwise, the observed concentrations of blood procalcitonin are similar to those (0.40 to 0.49%) of Matshogo et al. (2021) whilst serum total protein values are within normal ranges (25.0 to 45.0 g/L) for broiler chickens (Harr et al. 2002) and serum bilirubin values are slightly lower but comparable with those in the literature (3.2 to 5.0 μmol/L) (Egbu et al., 2022).

Conclusion
Our results showed that dietary MSM feeding did not affect growth performance, carcass characteristics, internal organs, as well as haemato-biochemical and meat quality characteristics.Dietary MSM increased duodenum weight and serum phosphorus and decreased procalcitonin and serum levels of total protein, albumin, and bilirubin.The MSM could therefore be supplemented up to 10% without compromising growth performance, carcass traits, internal organs, haemato-biochemistry, and meat quality in finisher broiler diets.

Table 1
Ingredient, chemical, and amino acid composition (%) of experimental finisher diets

Table 2
The effect of dietary MSM inclusion on growth performance parameters of Cobb 500 broilers ADWG, average daily body weight gain; ADFI, average daily feed intake; FCE, feed conversion efficiency; MSM, mucuna seed meal; SEM, standard error of the mean based on a pooled estimate of variation (n = 80) CCW , cold carcass weight; HCW, hot carcass weight; MSM, mucuna seed meal; SEM, standard error of the mean based on a pooled estimate of variation (n = 80)

Table 4
The effect of dietary MSM inclusion on haematology of Cobb 500 broilers MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume; MSM, mucuna seed meal; MPV, mean platelet volume; PDW, platelet distribution width; RCDW, red cell distribution width; WBC, white blood cells; SEM, standard error of the mean based on a pooled estimate of variation (n = 80)

Table 6
The effect of dietary MSM inclusion on meat quality parameters of Cobb 500 broilers 24 h refers to meat quality parameters 24 h after slaughter (i.e., on the cold carcass); 45 min refers to meat quality parameters 45 min after slaughter; MSM, mucuna seed meal; SEM, standard error of the mean based on a pooled estimate of variation (n = 80)

Table 7
The effect of dietary MSM inclusion on meat quality parameters of Cobb 500 broilers MSM, mucuna seed meal; WHC, water holding capacity; SEM, standard error of the mean based on a pooled estimate of variation (n = 80)