Impacts of algae supplements (Arthrospira & Chlorella) on growth, nutrient variables, intestinal efficacy, and antioxidants in New Zealand white rabbits

An 8-week trial to examine the impacts of Arthrospira platensis and Chlorella vulgaris on the growth, nutrient aspects, intestinal efficacy, and antioxidants of 75 New Zealand white male rabbits (initial body weight = 665.93 ± 15.18 g). Herein the study was designed in one-way ANOVA to compare the effects of the two algae species with two levels of supplementations in the feeds of New Zealand white rabbits. The rabbits were divided into five groups (n = 15/group), where the first group was allocated as the control group (Ctrl) while the second and third groups received A. platensis at 300 or 500 mg/kg diet (Ap300 or Ap500). The fourth and fifth groups fed C. vulgaris at 300 or 500 mg/kg diet (Ch300 or Ch500). The basal diet rabbits exhibited the lowest values of weight, lipase, protease, and the highest feed conversion ratio, which improved noticeably with algae addition, particularly with Ap500, Ch300, and Ch500. All tested groups showed normal intestinal structure. Amylase potency, hematological indicators, and serum biochemistry revealed non-significant variation except for a higher serum total protein and lower total cholesterol in algal groups. The best GPx existed in groups fed algal diets, while favorable SOD and CAT efficiency occurred at the higher level of Arthrospira and both levels of Chlorella. In conclusion, incorporating Arthrospira or Chlorella in the diet of New Zealand white rabbits improved performance, nutrient utilization, intestinal efficacy, and antioxidants. Arthrospira (Ap500) and Chlorella (Ch300 or Ch500) have almost the same beneficial effect on rabbit performance.

). The rabbits in the reference group (Ctrl) were fed a basal diet with no additions (Table 2), whereas the remainder of the groups were provided a basal diet with 300 or 500 mg of Arthrospira platensis (Ap300 or Ap500) or Chlorella vulgaris (Ch300 or Ch500).
Performance variables. The weight of the rabbits at the start and conclusion of the trial and the amount of feed consumed were recorded as follows: Weight gain, g/ rabbit = W T − W 0  Sampling procedure. After 8 weeks of feeding, 5 rabbits/group were allocated for blood collection and slaughter. Blood samples were drawn without anesthesia from the lateral saphenous superficial vein of the back leg after wetting the fur with alcohol using a 1 ml syringe with heparin for hematological measurements or without anticoagulants to separate the serum. Heparin-treated blood was employed for hematocrit (Ht) quantification using microhematocrit tubes and rotary centrifugation (13,000 rpm for 5 min) 36 . Non-heparinized blood was centrifuged [3000 rpm undercooling (4 °C) for 10 min] to harvest serum. Hematological and biochemical blood indices were measured using CBC Micros ABX, France automatic analyzer with P500 kinetic & Quality control Diatron Q.C kits according to package guidelines. The liver and small intestine were separated on an ice layer, cleaned with regular saline solution (0.90%; pH 7.5), and subjected directly to the determination of hepatic antioxidants, intestinal structure, and digestive enzyme activities.
Intestinal enzymes and histology assessment. Parts of the collected intestine (duodenum) were finely homogenized in freezing iced NaCl (0.86%) using VEVOR, FSH-2A device, and centrifuged at 8000 rpm for 5 min, 4 °C. The filtrate was employed for the colorimetric detection of amylase and lipase at A 714 and A 540 7 . Protease potency was measured using a non-specific protease vigor methodology utilizing casein 37 . For histological evaluation, samples (duodenum, jejunum, ileum) were fixed in a neutral buffered (10% formalin solution) for 72 h, dehydrated in rising grades of ethanol (60-100%), cleared in xylene, embedded in paraffin wax (24 h), and then sectioned with Rotary Microtome 2145, Leica Microsystems at a 3-5 μm in thickness.
Hepatic antioxidants. Liver samples (5 rabbits/treatment) were finely homogenized in cold iced potassium phosphate buffer (pH 7.4, 10% w/v) using VEVOR, FSH-2A device, and centrifuged at 4 °C, 12,000 rpm for 10 min. The filtrate was employed for the colorimetric detection (Jenway UV-Vis spectrophotometer 7415, Staffordshire, UK) of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) at 550, 280, 412 nm using Biodiagnostic and research reagents, Dokki, Giza, Egypt. Statistical analysis. The study was designed in one-way ANOVA to compare the effects of the two algae species with two levels of supplementations in the feeds of New Zealand white rabbits. The rabbits were divided into five groups. The first group received a diet without either A. platensis or C. vulgaris (Control group, Ctrl). Conversely, the second group was given a diet with 300 mg/kg of A. platensis (Ap300), the third group a diet with 500 mg/kg of A. platensis (Ap500), the fourth group a diet with 300 mg/kg of C. vulgaris (Ch300), and the fifth group a diet with 500 mg/kg of C. vulgaris (Ch500). The data was examined using the IBM® SPSS® Inc., IL, USA program (IBM SPSS Statistics Ver. 26.0). The Shapiro-Wilk and Levene tests were employed to verify variance normality and homogeneity. The outcomes of the one-way ANOVA and Duncan's post hoc test were presented as a mean of three replicates with standard errors.

Results
Chemical composition of the Algal supplements. Arthrospira platensis dry biomass comprises 56.4 ± 3.3, 6.6 ± 0.6, and 26.2 ± 0.98% of protein, lipids, and carbohydrates, compared to Chlorella vulgaris's proportions of 43.6 ± 2.4, 20.19 ± 1.2, and 23.8 ± 0.94%. A total of 25 active chemical compounds were characterized in the extracts of both algae. The identified chemical products with their retention time and % peak area of both extracts were shown in Table 3. The chromatograms of both extracts were shown in Fig. 3.
Performance variables. Table 4 shows the growth and nutrient efficiency of New Zealand white rabbits fed experimental diets for 8 weeks. Rabbits fed the basal diet exhibited the lowest final weights and weight gains and the highest feed conversion ratio, which improved noticeably with algae addition, particularly with Ap500, Ch300, and Ch500. Feed intake did not change with treatments except for the high level of Chlorella vulgaris (Ch500), which showed the lowest FI value.
Intestinal efficiency. Figure 4 shows the intestinal structure of New Zealand white rabbits fed experimental diets for 8 weeks. All rabbit groups showed intact and normal intestinal structures with no pathological alterations such as degeneration, necrosis, hemolysis, edema, congestion, hemorrhages, and hypertrophy. The efficiency of intestinal enzymes is shown in Table 4. A remarkable enhancement in the efficiency of lipase and protease occurred in algal groups compared to the control, while the efficiency of amylase did not change between the experimental groups.
Blood health. Table 5 exhibits the blood profile of New Zealand white rabbits after 8 weeks of feeding trial.
Hematological indicators comprising hematocrit (Ht), hemoglobin (Hb), red blood cells (RBCs), and white blood cells (WBCs) showed non-significant variation with dietary treatments. Similarly, serum biochemistry displayed no alteration in glucose, triglyceride, alanine transaminase (ALT), and aspartate transaminase, while a significant alteration occurred in total protein and total cholesterol. Rabbits treated with Arthrospira (Ap300 and Ap500) and Chlorella (Ch300 and Ch500) exhibited higher total protein and lower total cholesterol compared with the reference group. The lowest level (P < 0.05) of cholesterol was found in the blood of rabbits given a high level of Arthrospira (Ap500) and both levels of Chlorella (Ch300 and Ch500). Figure 5 displays the hepatic superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in New Zealand white rabbits after 8 weeks of feeding trial. Rabbits fed the basal diet exhibited the poorest antioxidant potency (SOD, CAT, and GPx). The best GPx existed in all groups fed algal diets, while the favored SOD and CAT efficacy appeared at higher Arthrospira (Ap500) and both levels of Chlorella (Ch300 and Ch500).

Discussion
Stimulating the maximum production of animals necessitates specific procedures to ensure quantity, quality, and animal health 7 . Nutraceuticals feed additives in the animal production business as natural substitutes for antibiotics have risen in importance 6 . Rabbit production is an appropriate agricultural investment because of its low production costs, superior fertility, short generation intervals, and ability to utilize a range of forages 1 .
Results of growth variables showed that rabbits fed the basal diet exhibited the poorest performance, which improved noticeably with algae addition, particularly with Ap500, Ch300, and Ch500 (Table 4). The high nutritional value of algae may be one of the reasons for the enhanced performance in animals fed with algae supplements. In this sense, Mahmoud et al. 18 found that soybean substitution by A. platensis at levels of 20, 40, and 60% in rabbit feed did not show any negative outcomes and maintained indicators of growth, health, and meat quality. Furthermore, Seyidoglu et al. 26 found an enhancement in the immune system of growing rabbits with A. platensis-diets. In contrast, Gerencsér et al. 38 assumed that Arthrospira (5%) and thyme (3%), either alone or in combination, did not substantially alter the growth or health of growing rabbits. Chlorella has been suggested to boost the performance and health of animals 17,30,39 . In a previous study, Hassanein et al. 40 compared the influence of Arthrospira (Spirulina) platensis and Chlorella vulgaris at levels of 0.75 and 1.5 g/kg diet on growing New Zealand white rabbits and concluded that both levels of A. platensis improved the growth and reduced liver enzyme, cholesterol and total lipids contents in serum in comparison with Chlorella vulgaris supplements. Moreover, An et al. 39  www.nature.com/scientificreports/ improved growth, blood cell counts, and declined total lipids in serum. According to Abdelnour et al. 30 , adding 1.0 g of Chlorella vulgaris to the diet of growing New Zealand white rabbits could boost their immunological and antioxidant health, as well as reduce blood lipid accumulation. Despite these valuable results and to the authors' best knowledge, so far there are no planned studies that have compared the potentials of Arthrospira and Chlorella on intestinal histology, digestive enzyme potency, and hepatic antioxidants of New Zealand white rabbits. Consequently, the current trial was designed to cover these parameters. Likewise, the improvement in weight with algae supplements may be linked to a change in the feed conversion ratio (↓ FCR). The detected reduction in FCR may be linked with the amended intestinal efficiency (Table 4), particularly digestive enzymes (lipase and protease). Several studies have demonstrated that adding algal biomass www.nature.com/scientificreports/ or extracts improves growth and nutrient use. In Arthrospira impacts regard, Alazab et al. 20 found that adding Spirulina platensis (SP) to the diet of growing rabbits at a level of 0.6 g/kg diet resulted in considerably better growth performance parameters and enhanced feed conversion ratio in comparison to those provided the low level (0.3 g/kg diet) or those fed a basal diet. Moreover, Aladaileh et al. 21 highlighted that exogenous supplementation of SP enhanced the growth traits of rabbits subjected to Pb. In addition, Peiretti and Meineri 22,23 demonstrated that rabbits receiving Arthrospira at a level of 10% exhibited higher feed consumption. Regarding the effect of Chlorella, Sikiru et al. 31 noted that dietary implementation of the Chlorella vulgaris amidst 200 and 500 mg/kg diet considerably raised the rabbits' weights without substantial alteration in feed intakes, but substantially enhanced feed to gain ratio. In another study by Sikiru et al. 32 on New Zealand white rabbits, a significant positive boost in the final body weight and feed intake with the addition of Chlorella vulgaris. In contrast to the findings of the current study, no alteration was observed in the growth aspects with the dietary incorporation of Arthrospira (Spirulina) [22][23][24]38 or Chlorella 30 and this may be due to the different conditions of the experiment. Blood status is a precise sign of the welfare and health status of animals, hence are direct reflectors of stressors and external stimuli 41 . Hematological indicators and serum biochemistry showed non-significant variation except for serum total protein and total cholesterol (Table 5). Rabbits treated with Arthrospira and Chlorella had higher total protein (TP) and lower total cholesterol than the reference group. The higher levels of TP in rabbits fed algae may suggest an improvement in rabbit health. In this context, Hassan et al. 19 reported an enrichment in plasma total protein in rabbits provided a diet enhanced with Zn-Se-rich Spirulina compared to the reference group. A similar improvement in glycoprotein appeared with Chlorella treatment 33 . The hypocholesterolemic effect of algae could explain the lower cholesterol levels associated with supplementation. In line with the present results, Cheong et al. 27 suggested that spirulina consumption can reduce hypercholesterolemic atherosclerosis by lowering total serum cholesterol in New Zealand White rabbits. Also, Hassan et al. 19 found low levels of total cholesterol, LDL-and VLDL-cholesterol in Se-rich Spirulina and Zn-Se-rich Spirulina groups of New Zealand White male rabbits. Similar impacts on cholesterol were reported with Chlorella incorporation. In this regard, Abdelnour et al. 30 found a reduction in serum VLDL in the Chlorella-treated groups relative to those in the control group.
The oxidative state of the animal is positively related to its immunity and wellbeing 42 . Oxidative stress is caused by an imbalance in the generation and clearance of reactive oxygen species (ROS) 43 . Several enzymes in the oxidative system, such as SOD, CAT, and GPx aid in the elimination of ROS and the maintenance of cell homeostasis 44 . In the present trial, algae dietary application mediates a substantial rise in SOD, CAT, and GPx activities. This may be due to the unique compositions of Arthrospira and Chlorella that are rich in effective compounds with an antioxidant impact, e.g., minerals, vitamins, β carotene, β-glucan, linolenic acid, tocopherols, phycocyanin, flavonoids, and phenols. Similar interpretations were reported for New Zealand White rabbits fed Arthrospira by Hassan et al. 19 or Chlorella by Abdelnour et al. 30 . Several studies have found enhanced antioxidant enzymes in rabbits fed Arthrospira 21,28,29 and Chlorella 30,32 .
The content of active chemicals in feed additives is mostly responsible for their beneficial effects. The overall results indicated that including Chlorella surpassed Arthrospira additives in New Zealand white rabbit feeds. These results indicated superior improvements in the growth performance, feed efficiency, and intestinal and blood health of New Zealand white rabbits fed Chlorella. These observations could be associated with the content of Chlorella and its effect on intestinal health and body immunity in rabbits. The GC-MS analysis of the crude extracts of the two algae showed the presence of 25 chemical substances with known favorable bioactivity on rabbits [19][20][21]31 and humans 45 . It is challenging to explain the effects of algae dietary supplements at the level of a single ingredient because algal extracts contain a significant number of active compounds and the best strategy is to classify them into major categories. Arthrospira exceeded Chlorella in its content of ketone, cholesterol, and terpenes, but Chlorella surpassed Arthrospira in its content of esters, fatty compounds, and hydrocarbon. Both extracts contain the majority of the active chemicals, albeit in different amounts which amply explain the convergence of the impacts supporting the performance and wellbeing of New Zealand white rabbits. In this context, phytol is a diterpene compound found in almost all crude extracts of the used algae and known for its anticancer and antioxidative properties 46 . The hydrocarbon pentadecane and the fatty acid Pentanoic acid, 4-methyl-are known for their antimicrobial activity 47 and antitumor activity 48 , as well as a growth promoter 49 .

Conclusions
The present study sheds light on the potential of algae feed additives (Arthrospira platensis VS Chlorella vulgaris) on the performance and wellbeing of New Zealand white rabbits. Incorporating Arthrospira platensis at 500 mg/kg diet or Chlorella vulgaris at levels of 300 and 500 mg/kg diet improved growth, nutrient aspects, intestinal enzyme efficiency, blood health, and antioxidants of New Zealand white rabbits. It will be vital for rabbit production in the future to monitor molecular responses to external feeds and/or supplements and to concentrate on obtaining a precise nutritional formula for algal biomass in rabbit feeding without compromising performance or health.   www.nature.com/scientificreports/ Intestinal structure (duodenum, jejunum, ileum, H&E = 40 X) of New Zealand white rabbits fed experimental diets for 8 weeks. Ctrl = the control group; Ap300 and Ap500 = Arthrospira platensis inclusion levels at 300 and 500 mg/kg; Ch300 and Ch500 = Chlorella vulgaris inclusion levels at 300 and 500 mg/kg.  www.nature.com/scientificreports/