Effects of feeding diets containing Morula (Sclerocarya birrea) kernel cake on bone development in commercial broiler chickens

F Manyeula, J C Moreki, L Baleseng¹, M Molapisi², L M Tabudi and T Khumoetsile

Department of Animal Sciences, Faculty of Animal and Veterinary Sciences, Botswana
jmoreki@buan.ac.bw
University of Agriculture and Natural Resources, P/Bag 0027, Sebele, Gaborone, Botswana
¹ Department of Agricultural Research, Ministry of Agriculture, Botswana, P/Bag 003, Sebele, Gaborone, Botswana
² Department of Food Science and Technology, Faculty of Agriculture, Botswana University of Agriculture and Natural Resources, P/Bag 0027, Sebele, Gaborone, Botswana

Abstract

The mineral bioavailability of birds fed diet containing Morula kernel cake (Mkc) tends to be lower compared to dietary soybean meal (SBM). This could negatively affect bone development and mineralisation in chickens reared on this alternative protein source. Therefore, this study evaluated the effects of partial replacement of SBM with Mkc in broiler chicken diets on bone development. A total of 160 two weeks old broiler chicks were allocated to 20 pens (experimental units). Four isonitrogenous and isocaloric commercial broiler diets containing 0, 4, 8, and 12% of Mkc were then randomly allocated to the pens. Broiler chickens were weighed at the beginning of the experiment (13097.75± 65.50 g) and on weekly basis thereafter. At the end of the experimental period (6 weeks), broiler chickens were humanely slaughtered for the determination of bone mineral composition and tibia traits. Results showed that there were neither linear nor quadratic effects of dietary levels of Mkc on tibia weight, width, tibia diameter proximal end, diameter distal end, density, index and ash. However, latency-to-lie test [y = 1 (±0) + 1 (±1.0)x_, R² = 1.0] and tibia length [y = 9.09 (±0.11) + 0.03 (±0.04) x_; R² = 0.30] increased linearly with dietary Mkc levels. There was a linear decrease in tibia Ca [y = 461.8 (±16.51) + -24.2 (±6.62)x_; R² = 0.96] and P [y = 236.17 (±17.4) -9.34 (±7.03)x_; R² = 0.82] contents in response to the incremental level of Mkc in the diets. No linear nor quadratic trends were observed for tibia Mg, Na, K, Fe, Mn, Cu and Zn in response to Mkc levels. Therefore, it was observed that higher inclusion levels of Mkc negatively affected bone Ca and P contents suggesting that dietary Mkc negatively impacts mineral bioavailability in broiler chickens. However, this reduction in mineral bioavailability did not compromise bone development. It was concluded that replacing SBM ingredients with Mkc in poultry diets up to 12 % inclusion levels does not adversely affect bone development in broiler chickens.

Keywords: anti-nutritional factors, bioavailability, bone mineralisation, tibia, soybean meal

Introduction

Marula or morula (Sclerocarya birrea) is a popular African wild tree that is distributed throughout Africa where the fruits are used for food, whereas leaves, stem bark and roots are used for traditional medicine (Mariod and Abdelwahab 2012; Tapiwa 2019). In addition, the leaves are browsed by livestock while wood is used to make a wide range of implements (Mojeremane and Tshwenyane 2004). Most farmers harvest fruits at ripe stage by picking them from the ground (Tapiwa, 2019) while some fruits are harvested directly from the tree. Fruits may be eaten fresh or are fermented to produce beer while the kernels are consumed or used for oil extraction (Mojeremane and Tshwenyane 2004) leaving the cake behind. Morula fruit juice has high ascorbic acid than orange juice (Mojeremane and Tshwenyane 2004) and contains sesquiterpene hydrocarbons, which are terpenes found in plants which have bacteriostatic properties (Mariod and Abdelwahab 2012). Other than the morula-based liqueur, known as ‘Amarula cream', which is produced, bottled and marketed across the world by Cape Distell Pty Ltd. in Stellenbosch (South Africa) there has been limited large-scale commercialization of morula products within Southern Africa (Wynberg et al 2002). Because of its multiple uses, morula tree is classified as a multipurpose tree that is important to smallholder farmers in arid and semi-arid areas (Mojeremane and Tshwenyane 2004; Tapiwa 2019). Morula tree, fruits, nuts and kernels are illustrated in Photos 1a to 1d.

Photo 1a. Morula tree at Mogonono extension	Photo 1b. Unripe Morula fruits area
Photo 1c. Morula nuts containing kernels	Photo 1d. Morula kernels

Although morula tree is grown in Central and East Africa, Asia, Europe, America and Australia (DAFF 2010), it is not cultivated commercially in Botswana but is planted in homesteads in the northern part of the country (Mojeremane and Tshwenyane 2004). A single tree can produce up to 500 kg of fruit per year. On average a tree can yield 30 kg fruits. The morula tree can be easily propagated by seeds, cuttings and grafting. The trees produce flowers from September to November and bear fruit from January to March (DAFF 2010). In addition, trees start to produce fruits at 3 to 5 years of age.

Conventional poultry diets are formulated with soybean meal (SBM) as the main plant protein source. However, for commercial chicken strains, the use of SBM as a protein source reduces profitability given the increasing price of this ingredient, which is imported as it is not produced in Botswana. Over the years, alternative protein sources such as oil cake by-products (e.g., canola cake, sunflower cake, cotton seed cake, etc.) have been evaluated and used in the formulation of low-cost diets. In Swaziland, Mthinyane and Mhlanga (2017) stated that S. birrea is a novel locally available low-cost alternative protein supplement for livestock and poultry.

Morula kernel cake is comparable to SBM in terms of protein quality but s it contains an array of secondary plant metabolites that may negatively affect some physiological functions in chickens (Khajali and Slominski 2012). Two such secondary plant compounds are phytic and oxalic acids, which are known to form insoluble complexes with protein and a range of minerals thereby reducing their bioavailability for poultry ( Khajali and Slominski 2012). Phytates have been reported to chelate divalent cations thereby reducing their availability (Catala-Gregori et al 2006). In addition to phytic and oxalic acid, Mkc also contains higher peroxides lipids and mycotoxin levels compared to SBM. Lipids peroxidation and mycotoxins in diets impacted unpalatable taste resulting in decreased feed intake (Mthinyane and Mhlanga 2017). Thus, the use of Mkc in poultry diets may result in lower mineral bioavailability, which could result in poor bone development. Bone ash content and bone breaking strength are commonly used to assess the bioavailability of calcium (Ca) and phosphorus (P) in chicken diets (Shaw et al 2010) since the mineral matrix contributing 60 to 70% of the bone is mainly composed of these two minerals (Rath et al 2000). Poor bone mineralisation increases the incidence of leg and bone weakness, lameness and other bone abnormalities (El-Husseiny et al 2012) resulting in high production losses and suboptimal birds’ welfare (Dibner et al 2007; Onyango et al 2003).

No studies have been conducted on effects of Mkc on bone characterisation and mineralisation in broilers in Botswana. As broiler chickens are marketed at 4-6 weeks of age, it is important that they have a strong skeletal system to support the increasing body mass for extended periods. Therefore, this study was conducted to determine the influence of graded levels of dietary Mkc on bone development in broiler chickens. It was hypothesised that inclusion of Mkc in commercial broiler diets negatively influences bone index, tibia characteristics and bone mineral composition.

Materials and methods

Study location and condition

This study was conducted at the Botswana University of Agriculture and Natural Resources (BUAN) Content Farm, Sebele, Gaborone (25.94°S, 24.58°E at altitude of 991 m) from April to July 2020. The ambient temperatures during the study time ranged from 12 °C to 25 °C.

Feed ingredients

All feed ingredients except Mkc were bought from Optifeeds (PTY) LTD, Gaborone, Botswana. The Mkc was purchased from DLG Natural (PTY) LTD in Gabane, Botswana. Both SBM and Mkc were pressed oil cakes.

Chemical analysis of the feedstuff

Prior to diet formulation, the chemical composition of Mkc (Table 1) and formulated experimental diets (Tables 2 to 3) were determined using the methods of AOAC International (AOAC, 2005) for Laboratory dry matter (DM; AOAC method no. 930.15). Nitrogen was determined by macro-Kjeldahl method (N; AOAC method no. 984.13) and was converted to crude protein by multiplying with the factor 6.25. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined using the ANKOM²⁰⁰⁰ Fibre analyser (ANKOM Technology, New York) (van Soest et al 1991

Dietary treatments and experimental design

Four isonitrogenous and isocaloric experimental diets in a mash form were formulated to meet the nutritional requirements of commercial broiler chickens (NRC 1994) by replacing soybean ingredients with graded levels of Mkc as follows: (1) Mkc0 = commercial broiler chicken\ diet without Mkc, (2) Mkc4 = a commercial chicken grower diet in which 4% of soybean ingredients were replaced with Mkc, (3) Mkc8 = a commercial broiler chicken diet in which 8% of soybean ingredients were replaced with Mkc, and (4) Mkc12 = a commercial broiler chicken diet in which 12% of soybean ingredients were replaced with Mkc. A total of 160 one day old broilers were purchased from Ross Breeders’ hatchery in Ramotswa (Botswana) and were reared up to 14 days. Average live weight of broilers at 14 days was 13097.75± 65.50 g) were purchased from Ross Breeders’ hatchery (Ramotswa, Botswana). Birds were allocated to 20 pens to which the four experimental diets were randomly allocated. Each dietary treatment had 5 replicate pens (1.00 mL × 1.00 mW× 1.00 mH) with each pen holding eight commercial broiler chickens as an experimental unit arranged in a completely randomised design (CRD).

Animal management

The rearing of broiler chickens throughout the experiment period was performed following the ethical guidelines of BUAN Animal Research Ethics Committee (AEC), accepted standards for the welfare and ethics of chickens (Ethics Number BUAN-2020-08). During brooding (day 1 to 14), the ambient temperature was kept at 32-33 ^oC and reduced by 1^oC for every three days until room temperature was attained. Broilers were fed commercial starter diet (day 1 to 14), grower diet (14 to 28 days) and finisher diet (day 28 to 35 days) which was purchased from Optifeeds (Pty) Ltd. (Gaborone, Botswana). A week before the start of the experiment, chicks were adapted to the experimental condition in the poultry house and reared in a 24 hours continuous light with average indoor humidity of 60% for 6 weeks. The pens were designed to meet space and welfare requirements of the commercial birds which were observed for any abnormalities regularly. Experimental diets and clean fresh water were offered ad libitum from 1 to 6 weeks of age. Birds were weighed at the beginning of the experiment and subsequently on weekly basis. At the end of the trial the broiler chickens were weighed to obtain the slaughter weight, then fasted for 12 hours to empty the digestive tract and subsequently humanely slaughtered for bone breaking strength, density and mineral composition analysis.

Slaughter procedures and measurements

Broiler chickens were electrically stunned, bled, defeathered and eviscerated. Both legs from each broiler chicken were excised at femorotibia articulation, defleshed and weighed. Tibia length (TL) and width (TW) were measured using digital Vernier calliper with an accuracy of 0.001 cm. Bone index was calculated as the ratio of bone weight to body weight. The tibia density (TD) was calculated as the proportion of tibia weight to tibia length (Seedox et al 1991).

Sheared tibiae pieces were collected and dried at 100 ^oC for 24 hours, cooled in a desiccator, and defatted by soaking in 200 mL anhydrous ether for 24 hours to remove moisture and lipids. The dried fat-free bones were weighed, then ashed in a muffle furnance overnight at 550 ^o C for about 6 hours (AOAC 2005, method number 924.05). Tibia ash was calculated as a proportion of tibia ash weight to tibia dry weight and reported as a percentage. Tibia ash was also used to analyse mineral contents (Ca, magnesium, sodium, iron, manganese, copper, sulphur and zinc) using ICP Mass Spectrometer. Phosphorus was determined calorimetrically using sodium phenol and ammonium molybdate plus ascorbic acid method (AOAC 2005, method number. 976.06). The ratio of Ca to P was calculated as the proportion of Ca to P mineral contents of tibia.

Statistical analysis

Data were evaluated for linear and quadratic effects using polynomial contrasts. Response surface regression analysis (SAS 2010) was applied to describe the responses of broiler chickens to inclusion levels of Mkc using the following quadratic model: y = ax² + bx + c, where y = response variable; a and b are the coefficients of the quadratic equation; c is intercept; and x is dietary Mkc (%). Data on mineral content and other characteristics of tibia were analysed using the general linear model procedures of SAS (2010) for a completely randomized experimental design with pen as the experimental unit.

Results

There were no significant (P > 0.05) linear and quadratic trends of all tibia traits with exception of Latency-to-lie test (LTL) and tibia length (Table 4). Latency-to-lie test linearly increased [y = 1 (±0) + 1 (±1.0) x_, R² = 1.0] and tibia length also increased linearly at y = 9.09 (±0.11) + 0.03 (±0.04)x_; R ² = 0.30 in response to incremental levels of dietary Mkc. Regarding tibia mineral contents, no linear or quadratic trends were observed for tibia Mg, Na, K, Ca:P, Fe, Mn, Cu and Zn contents with dietary Mkc levels (Table 5). However, there were linear decrease in tibia Ca (Figure 1 and P contents (Figure 2) with incremental levels of Mkc.

Figure 1. Effects of substitution of soybean products with graded levels of Morula kernel cake on calcium of the tibia of chicken	Figure 2. Effects of substitution of soybean products with graded levels of Morula kernel cake on phosphorus content of the tibia of chicken

Diets¹: Mkc0= commercial broiler chicken diet without Mkc; Mkc4 = a commercial broiler chicken diet in which 4% of soybean ingredients were replaced with CM; Mkc8 = a commercial broiler chicken diet in which 8% of soybean ingredients were replaced with Mkc; Mkc12 = a commercial broiler chicken diet in which 12% of soybean ingredients were replaced with Mkc

Discussion

Tibia characteristics are used as indicators of mineral adequacy in commercial broiler diets; if the mineral inadequacy is left unattended until maturity, the results will be seen in the bone (Bryden et al 2021). It is known that osteoporosis is not Ca deficiency per se, but may involve the inability of birds to sufficiently metabolize sufficient Ca because of inadequate dietary Ca, vitamin D3 or P supply (de Matos 2008). The presence of anti-nutritional factors (ANFs) in the ingredients that are used to formulate diet can lead to inability of birds to metabolize sufficient minerals (Khajali and Slominski 2012). In this study, dietary inclusion of Mkc did not influence any tibia characteristics suggesting that requirements for bone tissue maintenance were adequate. This is contrary to what was expected, since the presence of ANFs in Mkc such as fibre and tannin may decrease the absorption of minerals. Malebana (2018) reported the presence of higher ANFs such as oxalate, phytate, saponin and tannin in Mkc than in SBM. The current results are in line with those of Disetlhe et al (2017) who observed no effects of feeding oil cake canola-based diets on bone characteristics of broilers. The inclusion of Mkc in broiler diets to replace a maximum of 12% SBM in the present study did not affect tibia characteristics, despite the documented negative effects of ANFs in Mkc on bioavailability of minerals leading to avian osteoporosis and osteomalacia. Tibia length and latency-to-lie test increased with incremental levels of Mkc implying that Mkc inclusion promotes bone development. Latency-to-lie test is an indirect measure of bone development and strength in a broiler chicken with strong bone being able to stand for 600 seconds, whereas those with poor bone development might succumb to their weight and sit down within a short period of time (Disetlhe et al 2017). In this study, increasing the levels of Mkc in broiler diets increased the time of persistence implying that the amount of minerals increased with increasing Mkc levels in the diets. The minerals in the gut from the diets were assimilated and transferred to the blood and cells resulting in strong bone development (Ghost et al 2016).

The current study revealed that inclusion of Mkc above 12% resulted in lower concentration of tibia Ca and P. The reduced concentration of tibia Ca and P may be due to poor absorption of these minerals in the gastrointestinal tract (GIT). The ANFs in Mkc are reported to reduce mineral bioavailability (Mansoori et al 2015). This explains why broilers fed diet containing 12% Mkc had lower tibia Ca and P contents. Calcium and P are crucial for the formation and maintenance of leg bones (tibia, femur, metatarsus), which in turn have direct impact on the quality of poultry meat produced (Orban et al 1999), as well as, the welfare of the birds reared. In studies by Banaszkiewicz (2012) and Disetlhe et al (2017), tibia Ca and P contents were reported to be lower in broilers fed oil cake canola-containing diets compared to SBM-based diets. In this study, the concentration of tibia Mg, Na, K and Ca:P were found to be significantly similar across the treatment diets, indicating that inclusion of Mkc in broiler diets has no negative effect on bone formation. Magnesium promotes bone formation by activating osteoclasts (Toba et al 2000). Additionally, Mg participates in the processes that are essential for body homeostasis (Oliveira et al 2005). Potassium is the third most abundant mineral in the animal body, which combines with Na and Cl to form bone (Ahmad et al 2008).

Trace minerals are only required in small amounts and may contribute less to bone development (Hossain et al 2013). Zinc stimulates the synthesis of DNA in osteoblasts and increases Ca ion concentration and bone weight (Ma and Yamaguchi 2000). Lack of significant effects on the concentrations of Mn, Zn and Cu, as seen in tibia of broilers fed treatment diet, are known to stimulate bone growth and increase bone strength. Reports showed that these trace minerals are linked to the use of macro minerals during osteogenesis (Kwiecień et al 2016; Medeiros et al 1997).

Conclusion

• Higher inclusion levels of Mkc negatively affected bone Ca and P contents suggesting that dietary Mkc negatively impacts Ca and P bioavailability in broiler chickens which might affect osteogenesis.
• However, this reduction in Ca and P bioavailability appears insufficient to deleteriously affect osteogenesis. It was concluded that replacing 12% of soybean products in a commercial broiler diets with Mkc as a protein source does not compromise bone development in broiler chickens.

Acknowledgements

We are grateful to the Botswana University of Agriculture and Natural Resources laboratory for availing the laboratory for chemical analysis.

Conflict of interest

The authors declare that they have no competing interest.

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