This study was undertaken to investigate MSC as an alternative to the economically and environmentally unsustainable conventional SBM protein source for broiler chicken diets. In keeping with previous studies [23, 24], our results found MSC to have a similar CP content as SBM. Considering its amino acid composition also mimicking that of SBM, except for lysine [22, 23], MSC offers great promise to replace SBM in poultry diets in Southern Africa and elsewhere. Interestingly also, local marula oil-extracting factories have improved their efficiency of oil extraction from marula kernels as evidenced by relatively low residual oil content in MSC used in this study compared to previous MSC products (289.6–343.5 g/kg DM) [23, 24]. With more improvements in oil extraction efficiency, iso-energetic MSC-containing broiler and other animal diets can henceforth be formulated with greater ease and the feed product is expected to have less problems with fungal and hence mycotoxin infestation as observed previously [23]. Of interest also is the relatively low fibre content in MSC used in this study in comparison to values observed in previous studies [27, 35]. The observed low fibre content of MSC renders the feed product even more ideal for use in diets of broiler chickens and other non-ruminants that are unable to utilize high fibre-containing diets [36, 37]. Further, our study showed MSC to be richer in ash, an indicator of the mineral content, compared to previous MSC products (48.5–54.3 g/kg DM: [23, 27, 35]). Furthermore, the concentration of CTs observed in MSC used in this study is higher than that reported by Malebana et al. [22] yet within the nor-mal range that is considered safe for the feed product to be used in broiler diets without induction of adverse effects on bird growth performance [38]. Indeed, previous studies have shown that inclusion of up to 3% tannins in broiler diets improves gut health and digestive performance [39, 40].
As part of the investigation of the nutritive value of MSC for broiler chickens, this study tested effects of incremental dietary inclusion levels of the marula by-product on broiler growth performance and mortality during the whole production cycle from d1 to 42 (i.e. starter, grower, and finisher phases). The observed increase in FI from 0 to 15%, as well as BWG and FCE from 0 to 10%, of dietary MSC inclusion level beyond which they decreased, corroborates observations in pig studies by Hlongwana et al. [35], Mabena et al. [27] and Thabethe et al. [28] but contradicts those of Mazizi et al. [41] in Japanese quails. In a previous study, the decrease in performance parameters with increasing inclusion levels of MSC was suspected to be induced by extensive lipid peroxidation and mycotoxin infestation of MSC [23]. Indeed, these researchers found high lipid peroxidation and low concentrations of mycotoxins deoxynivalenol (DON) and T-2 toxin in MSC.
Notwithstanding, there is a possibility that the observed decrease in performance at high (15 to 20%) dietary MSC inclusion levels may also be related to the high oleic acid content in the residual oil-rich MSC. Indeed, dietary oleic acid was previously shown to decrease food intake through induction of satiety in mice [42] and to decrease food energy intakes in humans [43] through its eliciting of production of oleoylethanolamide [44], which is known to decrease food intake [45–47] through a mechanism involving the histaminergic system [48]. Oleic acid also has capacity to regulate body weight in animals as was demonstrated through a decrease in this parameter in rats fed a diet supplemented with 10% olive oil [49], a rich (70–80%) source of oleic acid. Mechanistically, this might occur through high MUFA diets exhibiting greater rates of oxidation leading to decreased body weight [50]. Otherwise, another putative mechanism might involve oleic acid inducement of a laxative (cathartic) effect as has been demonstrated in rats [51]. Hence, these effects of oleic acid may explain the decreased FI, BWG and FCE in broiler chickens fed high dietary levels of MSC in this study. These mechanisms may also explain the observed significant decrease in slaughter weight, HCW and CCW in these broilers when fed high (15 to 20%) dietary inclusion levels of MSC.
Otherwise, considering the lack of effect of dietary MSC on the weights and lengths of all internal organs, it would therefore appear that the marula by-product does not contain detrimental antinutritional factors and is thus safe to incorporate at 5 to 10% inclusion levels in broiler diets. This notion is further supported by the lack of effect of dietary MSC on mortality in this study. Generally, the feeding of alternative plant-derived feedstuffs or their extracts with high levels of antinutritional factors and fibre is associated with in-creased size and length of the digestive system [52, 53]. Further, the observation of lack of effects of dietary MSC on all performance parameters even at as high inclusion levels of the marula by-product as 20% in week 6 suggests age-dependent attainment of adaptation to consumption of the novel alternative protein source by birds. This was reflected in the significant diet x time (week) interaction for weekly BWG. Indeed, the digestive system of broiler chickens undergoes major anatomical and physiological changes as the birds grow with increases in size and length as well as ability to secrete digestive enzymes, alongside improved digestive ability, as they grow older [54]. If the MSC contained any antinutritional substances, their adverse impact appears to have decreased as the age of birds advanced, similarly to previous observations [55, 56].
The lack of significant effects of dietary MSC inclusion on most haemato-biochemical parameters of broiler chickens in this study is indicative of reasonable biosafety of the marula by-product in relation to the health of the birds. Indeed, the kernels of marula fruits are a safe and delicious source of nutrition generally indulged upon by millions of mainly rural people in numerous countries in Africa without any health perturbations. Generally, they are consumed as a snack [57], incorporated into porridge and boiled meat as flavour enhancers [58] or their extracted oil used for meat preservation [59–61]. In poultry nutrition, their dietary consumption in the form of MSC has also elicited no deleterious effects in Japanese quails [41]. Hence, their deleterious effects on broiler chicken white blood cells including lymphocytes at dietary inclusion levels beyond 10% and 5%, respectively, as observed in this study, was unexpected. Notwithstanding, many studies have reported inhibitory effects of oleic acid in its neat form [62–64] or as dietary olive oil [65, 66] or cashew kernel oil [67] on lymphocytes and their proliferation in different tissues including blood. Also, consumption of an oleic acid-rich Mediterranean diet decreased the number of leukocytes and platelets in human subjects [68]. The mechanisms underlying these deleterious effects of oleic acid on leukocytes including lymphocytes seem to involve the MUFA-induced cellular oxidative stress, mitochondrial depolarization [68–71] and apoptosis [72, 73]. This was the first study to investigate the full repertoire of haemato-biochemical parameters including immuno-physiological biomarkers in broilers. Hence, it remains to be seen in future studies whether diets supplemented with high levels of MSC would induce similar deleterious effects on leukocytes in other breeds of chicken. Also, there is a need for elucidation of molecular mechanisms underlying the observed MSC-induced perturbations in immunological parameters of broilers and other breeds of chicken.
The safety of MSC as broiler chicken feed at low (0 to 15%), and its apparent toxicity at high (20%), dietary inclusion levels was clearly embodied in SDMA responses. Correlated well with renal function, SDMA is a biomarker of acute kidney injury [74] and has previously been measured in broiler and quail studies of alternative protein sources and phytogenic feed additives [75, 76]. Considering the linearly decreasing serum SDMA concentrations in broilers fed diets with 0 to 15% MSC, it is evident that MSC was safe to use at these relatively low dietary inclusion levels but induced kidney injury in the chickens when it was included at a higher (20%) level, mirroring the observed decremental responses in performance and immunological parameters of birds fed diets with high dietary inclusion levels of the marula by-product. As mentioned above, it is argued that the high oleic acid content of MSC particularly at 20% inclusion level of the marula by-product induced cellular oxidative stress [69–71] and possibly apoptosis as well [72, 73] in the chickens. Lending support to this contention are previous observations of elevated SDMA levels in disease conditions involving oxidative stress including diabetes mellitus, atherosclerosis, inflammation, apoptosis, and compromised immune function [77]. In fact, some studies have postulated that SDMA itself may be an inducer of oxidative stress by elevating reactive oxygen species in monocytes [78] whilst enhancing NADPH-oxidase through endothelial Toll-like receptor-2 activation [79]. Further, literature evidence reporting abrogative effects of administration of antioxidants including epigal-locatechin-3-gallate, melatonin, N-acetylcysteine, vitamin E, and others on kidney injury, measured as asymmetric dimethylarginine (ADMA), a structural isomer of SDMA [77], further reinforces the contention of oleic acid having induced oxidative stress in broilers at high dietary MSC inclusion levels. Unfortunately, our data could not be compared with literature values as this was the first study to investigate SDMA responses to dietary MSC supplementation in broilers. In future studies, there is a need for investigation of biomarkers of and mechanisms underlying oxidative stress in birds fed high MSC-containing diets.
Another intriguing finding in the current study was the linear increase in broiler se-rum cholesterol responses to incremental dietary inclusion levels of MSC. Whilst our se-rum cholesterol values were about 1.8 times lower than plasma cholesterol ones previously observed in quails [80], there are currently no comparable literature serum cholesterol responses to dietary MSC supplementation in broilers. However, it is evident that the observed increase in the chicken serum cholesterol concentrations with increasing dietary MSC inclusion levels in the current study is again associated with oleic acid. Indeed, a previous study showed consumption of oleic acid-rich olive oil to increase blood plasma and adipose tissue concentrations of high-density lipoprotein cholesterol (HDL-C) [49]. Apparently, the MUFA has unique ability to selectively increase the levels of the health-beneficial blood HDL-C whilst decreasing those of its cardiovascular disease (CVD)-associated low-density lipoprotein cholesterol (LDL-C) counterpart [49, 81, 82] resulting to its attenuation of CVD risk in hypercholesterolemic patients [83, 84]. Hence, it will be necessary to measure concentrations of both HDL-C and LDL-C in MSC-fed broilers in future studies in order to discern which exactly between the two cholesterol species is responsible for the observed elevation in serum cholesterol levels of birds fed incremental marula by-product-containing diets. Also, the observed dietary MSC-associated in-crease in broiler serum cholesterol suggests a need for investigation of underlying molecular mechanisms in terms of cholesterol biosynthesis in future studies.