Effect of Pre-Treating Dietary Green Seaweed with Proteolytic and Fibrolytic Enzymes on Physiological and Meat Quality Parameters of Broiler Chickens

The use of seaweeds as nutraceuticals in chicken diets is limited by high fibre levels and low protein digestibility. Therefore, we tested the effect of pre-treating dietary seaweed (Ulva sp.) with a combination of protease and fibrolytic enzymes on physiological and meat quality parameters of Cobb 500 broilers. Five dietary treatments were formulated by including untreated (T1); fibrolytic (12 g/kg) enzyme-treated (T2); fibrolytic (12 g/kg) and protease (5 g/kg) enzyme-treated (T3); fibrolytic (12 g/kg) and protease (10 g/kg) enzyme-treated (T4); fibrolytic (12 g/kg) and protease (15 g/kg) enzyme-treated (T5) seaweed (35 g/kg) in a standard broiler diet. Three hundred, two-week-old chicks (239.3 ± 8.57 g live weight) were evenly distributed to 30 replicate pens to which the diets were then randomly allocated. Birds fed diet T1 had the highest feed intake (1144.5 g/bird). Neither linear nor quadratic trends were recorded for growth performance and carcass traits in response to protease pre-treatment levels. Gizzard weight linearly increased, while symmetric dimethylarginine, calcium, meat pH24, and hue angle24 quadratically responded to protease levels. Diet T1 promoted the lowest serum phosphorus levels (3.37 mmol/L). In conclusion, pre-treatment of seaweed with a combination of protease and fibrolytic enzymes did not improve diet utilization, physiological parameters, and meat quality in broilers.


Introduction
The poultry industry significantly contributes to agri-food value chains around the world [1]. Chicken meat production has been increasing worldwide [2], driven by a growing human population, which is projected to breach the 9 billion mark by 2050 [3]. Over the years, poultry producers have relied heavily on high-performing conventional chicken strains to meet the increasing demand for animal protein. However, these chicken strains have high nutrient requirements resulting in high feeding costs and lower profit margins. In addition, intensive production systems predispose these chickens to a variety of stressors leading to a high incidence of diseases. Consequently, antibiotic growth promoters (AGP) have traditionally been used to control infections and enhance feed utilization efficiency [4]. However, the emergence of antimicrobial resistance as well as the threats posed by antibiotic residues to consumer health has seen AGPs being banned in some countries [4,5]. However, due to a lack of effective alternatives, the use of AGPs has continued in low-income countries that are burdened by high incidences of infectious disease outbreaks. A possible solution is the use of readily available plant-based feed additives with nutraceutical properties as AGP alternatives in poultry production.

Study Site and Ingredient Sources
The feeding trial was conducted at the North-West University Experimental Farm (26 • 41 36" S, 27 • 05 35" E) in South Africa from October to November 2020. The green seaweed (Ulva sp.) was harvested as described by Nhlane et al. [11] from an abalone farm (Western Cape, South Africa). The seaweed was oven-dried (60 • C) then milled (2 mm, Retsch grinder, 42781 Haan, Germany) to produce seaweed powder (SWP). The SWP was chemically analyzed prior to diet formulation and the chemical composition is reported in Nhlane et al. [11]. The fibrolytic enzyme mixture (Viscozyme ® L) was purchased from Sigma-Aldrich (Modderfontein, South Africa), and is composed of cellulase, hemicellulose, xylanase, β-glucanase, and arabanase enzymes. The Viscozyme ® L is completely miscible with water and has an enzyme activity of 100 fungal beta-glucanase per gram and a density of 1.2 g/mL. The protease mono-enzyme, as well as the other feed ingredients, were purchased from Nutroteq (PTY) LTD (Pretoria, South Africa). The protease has an enzyme activity of 600,000 U/g.

Feeding Trial and Broiler Management
Three hundred Cobb 500 chicks (male, one day old) were bought from Super Birds farm (North West, South Africa). The chicks were weighed and randomly placed in 30 replicate pens (experimental units) holding 10 birds each. The pens measured 3.5 m × 1.0 m × 1.85 m (L × B × H) with sunflower husks as bedding. The chicks were given a stress pack for the first 3 days and reared on a commercial starter mash diet until day 10 of age. At day 11, the birds were adapted to the experimental diets until day 13 of age. The house temperature was maintained at 35 • C using infrared electric bulbs placed in the brooder to ensure constant supply of heat for the first two weeks. Measurements commenced from day 14 to day 28 for the grower phase and from day 29 to day 42 for the finisher phase. The birds had unlimited access to clean fresh water and feed for the entire duration of the feeding trial.
Amount of feed consumed was determined every morning by subtracting the amount of refused feed from the amount offered. The initial body weights (239.3 ± 8.57 g live weight) of the birds were measured at 2 weeks of the age. Thereafter, the birds were weighed weekly to determine average weekly weight gain (ABWG). The ratio of weight gain to feed consumption was used as a measure of feed conversion efficiency (FCE).

Blood Collection and Analysis
At day 40 of age, 4 mL of blood samples were collected early in the morning before feeding from 12 birds randomly selected per dietary treatment. The blood samples were collected from the branchial vein using 23 gauge disposable needles and 5 mL syringes. After each collection, the samples (2 mL) were immediately transferred into labelled serum and whole blood tubes. The automated LaserCyte Hematology and the Vet Test Chemistry Analyzer machines (IDEXX Laboratories SA (PTY) LTD, Gauteng, South Africa) were used to determine hematological and serum biochemical parameters, respectively.

Slaughter, Internal Organs, and Carcass and Meat Quality Traits
At day 42 of age, feed was withheld for 13 h before the birds were weighed to determine final body weight (FBW). The birds were then taken to a commercial abattoir (Rooigrond, North West, South Africa) where they were electrically stunned and slaughtered by cutting the jugular vein [12]. After bleeding, the carcasses were plucked and eviscerated to determine visceral organ weights (liver, gizzard, proventriculus, spleen, duodenum, jejunum, ileum, and caecum), carcass weights and carcass cuts (wing, breast, drumstick, and thigh), as well as meat quality parameters as previously described by Kumanda et al. [14].
Breast meat pH and color coordinates (L* = lightness, a* = redness and b* = yellowness) were measured as described by Matshogo et al. [12] and Kumanda et al. [14]. The color coordinates a* and b* were used to calculate hue angle and chroma values [19]. Water-binding capacity (WBC) of breast meat samples was determined following the filter-paper press method by Grau & Hamm [20]. Breast meat drip loss and cooking loss were determined as described by Honikel et al. [21]. Shear force values (N) for raw breast meat samples were determined [22] as described by Matshogo et al. [12].

Statistical Analysis
Data were first tested for normality using the NORMAL option in the Procedure Univariate statement and for homogeneity of variances using Levene's test. Except for T1 data, linear and quadratic coefficients for physiological and meat quality data were evaluated using response surface regression analysis [23]. Weekly measured data were analyzed using the repeated measures analysis option in the general linear models (GLM) procedure of SAS version 9.4 [23] to determine the interactive effect of time (in weeks) and diet. Overall growth performance, hemato-biochemical parameters, and meat quality data were analyzed using one-way ANOVA (PROC GLM; [23]), where diet was the only variable. Significance was considered at p < 0.05 for all statistical tests and the least squares means were compared by using the probability of difference option in SAS.

Growth Performance and Hemato-Biochemical Parameters
Repeated measures analysis indicated that there were no significant week × diet interaction effects on average weekly body weight gain (p = 0.946) and average weekly FCE (p = 0.124). However, an interaction effect was observed on average weekly feed intake (p = 0.018). Table 3 shows that there were no significant linear and quadratic effects for average weekly feed intake. However, significant dietary effects were observed in week 6 of age, where birds fed diet T1 had a higher (p < 0.05) feed intake (1144.5 g/bird) than those fed diets T2, T3, and T5, whose feed intake values did not differ. Table 3. Effect of diets containing seaweed pre-treated with fibrolytic and protease enzymes on average weekly feed intake (g/bird) in Cobb 500 chickens. In a row, means with similar superscripts do not differ (p > 0.05). 1 Diets: T1 = standard grower or finisher diet containing 35 g/kg untreated seaweed; T2 = standard grower or finisher diet containing 35 g/kg seaweed pre-treated with 12 g/kg fibrolytic enzymes; T3 = standard grower or finisher diet containing 35 g/kg seaweed pre-treated with 12 g/kg fibrolytic enzymes and 5 g/kg protease mono-enzyme; T4 = standard grower or finisher diet containing 35 g/kg seaweed pre-treated with 12 g/kg fibrolytic enzymes and 10 g/kg protease mono-enzyme; T5 = standard grower or finisher diet containing 35 g/kg seaweed pre-treated with 12 g/kg fibrolytic enzymes and 15 g/kg protease mono-enzyme. 2 SEM = standard error of the mean.

Growth Performance and Hemato-Biochemical Parameters
This study investigated the effectiveness of pre-treating dietary seaweed with a combination of a proteolytic enzyme and fibrolytic multi-enzymes as a strategy to improve physiological parameters and meat quality of broiler chickens. Repeated measures analysis revealed a significant diet and week interaction effect on feed intake only, which indicates that the ranking of dietary treatments in terms of feed intake changed as the birds grew older. The results revealed that pre-treatment of green seaweed powder with the exogenous enzymes did not improve growth performance of the birds. Matshogo et al. [12] found that the inclusion of untreated green seaweed at 35 g/kg in Cobb 500 broiler diets compromised the performance and feed efficiency of the birds. This was reportedly due to high levels of indigestible non-starch polysaccharides such as cellulose, hemicellulose, xylans, and ulvans in the seaweed [8]. Moreover, high levels of condensed tannins in green seaweeds could reduce the digestibility of protein by forming tannin-protein complexes that are indigestible by endogenous digestive enzymes [24]. Indeed, the use of untreated green seaweed (Ulva lactuca) at a rate of 30 g/kg in broiler diets did not improve body weight gain or feed conversion ratio of the birds [25]. It was, therefore, expected that pre-treatment of the seaweed with both the protease mono-enzyme and cellulolytic multi-enzymes would improve the utilization of the diets resulting in improved digestibility and growth performance. The lack of improvement in the feed value of substrates treated with exogenous fibrolytic and protease enzymes has similarly been reported by Mnisi et al. [26] and Mnisi and Mlambo [27] in Japanese quail fed canola-containing diets. Likewise, Sayyazadeh et al. [28] reported no significant effects on body weight and feed efficiency of chickens fed cereal-based diets supplemented with exogenous enzymes. In contrast, several studies have reported positive results when exogenous enzymes were used in poultry diets [29]. Indeed, feed intake could be fully compensated by the effect of enzyme treatment on feed efficiency so that the birds can meet their nutritional requirement by consuming a smaller amount of feed. The lack of improvement upon pre-treatment of seaweed could be due to other factors (pH, viscosity, etc.) in the gastrointestinal tract of the birds that play a crucial role in the effectiveness of enzyme activity on the animal. The benefits of supplementation with enzymes are more evident during the early stages of the life of birds because of different physiological needs throughout the life of the chicken [30]. The combination of amylase, xylanase, and protease has been reported to enhance nutrient digestibility and chicken performance [31]. These inconsistent reports could be attributed to the various types of feed substrates used in these studies as well as different application methods, enzyme activities, and treatment levels [26]. The lack of differences between the control treatment group (T1) and treatment group (T4) on feed intake of six-week-old broilers further confirms the inefficacy of the enzymes to improve the utility of the seaweeds.
Hematological profiles have been used as indicators of dietary responses in farm animals [32]. The results from this study revealed that feeding enzyme-treated seaweed had no impact on the hematological parameters of the birds, and all the observed values were within the normal range reported for healthy chickens [12,14]. The lack of adverse effects indicates that the dietary treatments did not compromise the health of broiler chickens. Additionally, serum biochemical indices give useful information on the health and nutritional status of animals consuming non-conventional feed ingredients [32,33]. The findings from this study showed that seaweeds treated with exogenous enzyme had no negative effects on the blood parameters of broiler chickens. However, the observed quadratic responses of calcium, phosphorus, and symmetric dimethylarginine to protease treatment levels in this study might be attributed high levels of fiber, sterols, and other bioactive compounds in seaweeds [34]. According to Slominski [16] and Shalash et al. [35] supplementing broiler diets with a combination of xylanase, amylase glucanase, and/or protease did not improve the performance of the birds. The response to a multi-enzyme pre-treatment of substrate depends on various factors, such as age of the birds, genetic strain, chemical composition of the diet, and enzyme dose [36].

Carcass Characteristics, Visceral Organs and Meat Quality Attributes
The effect of supplementing green seaweed with exogenous enzymes on the carcass characteristics, visceral organs, and meat quality of broiler chickens is less reported. From this study, combining proteolytic and fibrolytic enzymes did not result in an improvement in carcass traits and visceral organs. Our findings corroborate Mohammed et al. [37], who observed no effect of the exogenous enzyme supplementation on the carcass weight, abdominal fat, and breast meat weight of broiler chickens. Previous reports on the effects of exogenous enzyme treatments on meat quality and organ weight in broilers have been inconsistent. While Symeon et al. [38] reported improvements in meat quality when exogenous enzymes were used, Zakaria et al. [15] did not observe such improvements. An underdeveloped gizzard restricts the broiler chicken's ability to efficiently digest large feed particles [39]. In the current study, dietary treatments had significant effects on gizzard weights with heavier gizzards in birds reared on seaweed that was treated with both the fibrolytic and protease enzymes compared to untreated seaweed. This finding was not expected because diets rich in fiber have been reported to cause an increase in gizzard size. The consumption of fibrous diets is expected to induce changes in visceral organ sizes as an adaptation mechanism [40]. However, the relative weights of livers, spleen, proventriculus, and intestines were not affected by the diets.
The current study showed that feeding seaweed treated with fibrolytic and protease enzymes had no effect on the carcass characteristics of broiler chickens. Similarly, Fischer et al. [41] found that a multi-enzyme complex did not improve nutrient digestibility and growth performance in broiler chickens. The color of the meat is the most important indicator when consumers buy meat products and is the major factor that affects consumer acceptance of the meat [42]. In this study, no dietary influences were observed on color indicators, which shows that enzyme-treatment of seaweed had no influence on the appearance of the meat. Meat pH is influenced by glycogen levels in meat muscle before slaughter and the extent to which glycogen is converted to lactic acid after slaughter [43]; this explains the drop in meat pH values measured 24 h post-slaughter. Likewise, Hossain et al. [44] reported that meat pH is a direct indicator of the consistency of the muscle acid content. It is not clear why meat pH and hue angle measured 24 h post-mortem quadratically responded to protease enzyme levels; this signifies a need for more research to understand the effect of exogenous enzymes on meat quality attributes. Nonetheless, the observed pH values were in line with the normal pH values reported for broiler meat [45]. Pre-treating seaweed with the combination of fibrolytic and proteolytic enzymes did not improve feed intake, physiological parameters, or meat quality characteristics, which suggests that other feed additives should be evaluated to improve the feed value of dietary seaweed in broiler chicken diets.

Conclusions
Pre-treatment of dietary green seaweed powder with a combination of a protease mono-enzyme and fibrolytic multi-enzymes did not improve feed intake, physiological, or meat quality parameters in Cobb 500 broiler chickens. In addition, an optimum protease treatment level could not be determined using the growth performance data, indicating a need to investigate levels beyond 15 g/kg of the protease mono-enzyme to generate nonlinear responses. Future studies should be designed to investigate the effect of seaweed extracts, instead of the meal, on nutrient digestibility, growth performance, and meat quality parameters of broiler chickens. Funding: The financial assistance from the National Research Foundation (ZA) received by the first author (NRF grant number: 118224) is hereby acknowledged. We are also grateful to the North-West University PhD bursary for contributing financially in this study.
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Production Research Ethics Committee of the North-West University, South Africa (protocol code: NWU-00356-19-A5 and date of approval: 04 July 2019).
Informed Consent Statement: Not applicable.