Spirulina platensis Algae Enhances Endogenous Antioxidant Status, Modulates Hemato-Biochemical Parameters, and Improves Semen Quality of Growing Ram Lambs

Animal reproduction is a crucial issue because of its impact on the overall benefits of the herd. Management of animal reproduction is the base of the achievement of good economic and productive performances for maximizing animal longevity (Maquivar et al., 2021). Fertility is one of the most crucial concerns in the flock for its impact on the productivity and economic aspects of the animal industry (Van Metre et al., 2012). Selection and acquisition of males, and well-managed tools of males at mating and pre- and post-breeding season, are representing challenges for using males either in natural or artificial insemination (Beltman, 2013). In sheep flock, healthy status, and the nutritional and environmental factors affect the productivity of ram lambs or rams (Maquivar et al., 2021).

Male infertility is caused by the harmful effects of oxidative stress which causes damage to the germ cells and sperm function development (Correia et al., 2017). The antioxidant defense system can protect the body cells against the deleterious effects of reactive oxygen species (ROS) produced by exogenous agents, so increasing ROS generation over the ability of the intracellular antioxidant defense system causes the oxidative stress (Cocuzza et al., 2008).

Spirulina platensis (Arthrospira platensis), a blue-green microalga, family Oscillatoriaceae, is considered a source of proteins and vitamins (Somchit et al., 2014), and has properties as antioxidant, anticancer, anti-apoptotic, anti-inflammatory, antiviral, immunomodulatory (Somchit et al., 2014), anti-apoptotic, antiviral, and immunomodulatory (Wu et al., 2016). Also, it contains 50–70% proteins, all essential amino acids, vitamins, minerals (Zn, Mg, and Se), phycocyanin, tocopherols, phenolics, carotenoids, and fatty acids, especially γ-linolenic acid (Gargouri, et al., 2019), and carotenoids which have impacts on fertility in many species (Zhang et al., 2020). In diet of different species of fishes, SPL was added as an alternative natural carotenoid source (Mantog, 2012).

To our knowledge, there are few research reports on the effects of dietary Spirulina on small ruminant productivity. Therefore, the current research aimed to detect the impact of dietary incorporation of Spirulina platensis alga on growth, hemato-biochemical parameters, antioxidant status, and semen quality of crossbred growing rams.

This current study was conducted within the framework of scientific and research cooperation between Animal Production Research Institute (APRI), Agricultural Research Center, Egypt and Faculty of Veterinary Medicine Kafrelsheikh University, Egypt.

Clinically normal twenty-four growing rams taken from the flock of Sakha Animal Production Research Station, Egypt were served as experimental animals, At the beginning of the experiment, animals have 32.93±0.48 kg body weight and aged 10 months. The experimental animals were in good health status, clinically free of external and internal parasites, and normal external genitalia. Animals were housed in a hygienic pen. Animal management and handling during the whole experiment and blood samples were collected by an expert veterinarian.

The experimental animals (n=24) were randomly distributed to 3 groups, 8/group. Animals in the 1st group were fed on corn silage and concentrate without any additives (G1, control), while G2 and G3 were fed the same diet with SPL at levels of 1 and 5 g/kg concentrate feed mixture CFM), respectively, from 10 to 15 months of age. The CFM was a mixture of wheat bran (40%), ground yellow corn (35%) undecorticated cottonseed meal (19%), molasses (3%), limestone (2%) and common salt (1%). The feeds were offered to animals to cover their requirements according to NRC (2007). The amounts of feeds were adjusted based on body weight changes and the physiological state while water was provided ad libitum.

Throughout the experimental period from 10 to 15 months of age, animals were individually weighed (bi-weekly) electronically by digital balance, then the scrotal circumference of each animal was measured at the median region of both testes using a tape.

At the beginning of the experimental period (10 months of age), semen ejaculates were collected once/week from all animals in each group up to 15 months of age, then the averages of semen characteristics were monthly calculated. Semen was collected by an artificial vagina of rams. Evaluation of semen included ejaculate volume, and sperm initial motility (Melrose and Laing, 1970), live sperm (Eosin and Nigrosin stain), and abnormal sperm percentages. Neubeuer hemocytoometer was used for determining sperm count microscopically, while total sperm count was computed by multiplying sperm concentration by ejaculate volume. Seminal plasma was obtained by centrifugation of semen samples at 700 g for 20 min, then stored at -20 oC until analysis.

At the terminal of the experimental period (15 months of age), blood samples were taken from the jugular vein of all animals in each group into two types of test tubs. The 1st type, blood samples were collected into test tubs containing EDTA, as an anticoagulant, for hematological parameters. The 2nd samples were taken in plain centrifuge tubes without anticoagulants. Blood serum was obtained after clotting (2-3 h) and centrifugation (3000 g for 15 min) and stored (-20 °C) for further analysis of biochemicals, hormones, lipid peroxidation, and antioxidant markers.

In the 1st blood samples (the whole blood), blood samples expose to determining the count of red (RCBs) and white (WBCs) blood cells, packed cell volume (PCV), and hemoglobin concentration (Hb). Also, erythrocytic indices including means of corpuscular volume (MCV), corpuscular hemoglobin (MCH), and corpuscular hemoglobin concentration (MCHC) as well as differential counts of WBCs (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) were measured by using automated blood cells counter with an Auto Hematology Analyzer (Sysmex F-800, Japan) according to (Buttarello, 2004).

In the serum of the 2nd blood samples, total protein and albumin concentrations (Henry et al., 1974) were estimated, while globulin was calculated. Serum triglycerides and total cholesterol was determined according to Richmond (1973), high density lipoproteins (HDL) according to (Abell et al., 1952), while low density lipoproteins (LDL), and very LDL (VLDL) concentrations were calculated according to an equation (Friedwald et al., 1972). Serum urea and creatinine were assayed according Henry et al. (1974) and Fabiny and Einghausen (1971), respectively. The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was measured as described by Reitman and Frankel (1957). Serum biochemical determinations were performed by spectrophotometer and chemical kits (Bio diagnostics, Cairo, Egypt).

Lipid peroxidation in terms of malondialdehyde level (Ohkawa et al., 1979) and activity of antioxidant enzymes such as superoxide dismutase (Owens and Belcher, 1965), catalase (Sinha, 1972), and glutathione peroxidase (Paglia and Valentine, 1967) were determined in blood serum and the seminal plasma using chemical kits (Biodiagnostics, Cairo, Egypt).

The enzyme-linked immunosorbent assay (ELISA) was used to determine serum and seminal plasma testosterone and serum thyroid hormones (triiodothyronine, T3, and tetraiodothyronine, T4) by using commercial kits (DRG Diagnostics GmbH, Marburg, Germany following the manufacturer’s instructions (DRG Diagnostics).

The results were tabulated as the means±standard error. One-way ANOVA was used to study the effect of treatment on all parameters studied, except for semen variables. Factorial design (3 treatments x 6 ages) was used to study the effect of three treatments, 6 ages, and their interaction on all semen variables. A computer program (SAS, 2009) was used for the statistical analysis. The significant differences in means of treatment or age were tested using Multiple Range test of Duncan (1955). All means were set at a level of P < 0.05.

Results of growth changes (Figure 1) revealed a significant improving effect of Spirulina (SPL) supplementation in a dose-response manner on the live body weight of animals starting at 13 months of age, reflecting higher total weight gains in G3 than in G1 and G2 at 13, 14, and 15 months of age.

The differences in the scrotal circumference of animals in the experimental groups were not significant at all ages (Table 1).

Table 1: Average scrotal circumferences (cm) of animals fed the experimental diets.

Table 2: Hematological findings of animals in the experimental groups at the end of the experimental period.

a and b: Means within the same row with different superscripts are significantly different (P<0.05). RBCs: Red blood cells count. Hb: Hemoglobin. PCV: Packed cell volume. MCV: Mean corpuscular volume. MCH: Mean corpuscular hemoglobin. MCHC: Mean corpuscular hemoglobin concentration. WBCs: White blood cells count.

Table 3: Serum biochemical analysis of animals in different experimental groups at the end of the experimental period.

a, b, and c: Means within the same row with different superscripts are significantly different (P<0.05). A/G ratio: Albumin/globulin ratio. TG: Triglycerides. TC: Total cholesterol. HDL: High-density lipoprotein. Low-density lipoprotein. V-LDL: Very low-density lipoprotein. AST: Aspartate aminotransferase. ALT: Alanine aminotransferase.

The count of RBCs and WBCs, Hb concentration, and PCV% were significantly enhanced in G2 and G3 compared with G1. However, RBCs indices including MCV, MCH, and MCHC in addition to the count of neutrophils, lymphocytes, monocytes, eosinophils, and basophils were not affected significantly by treatments (Table 2).

Results in Table 3 declared that SPL administration significantly increased the concentration of serum total protein and HDL-cholesterol, and significantly decreased the concentration of total cholesterol, LDL, v-LDL, and urea as well as the activity of AST and ALT in blood serum of animals as compared to control. However, triglyceride concentration significantly increased only in G3 as compared to other groups. The differences in albumin, globulin, and creatinine concentrations were not significant. Despite these results, SPL exhibited the best effect on serum biochemical and enzyme activity in G3 as compared to G2.

Dietary inclusion of both levels of SPL revealed a significant increase in the overall mean of ejaculate volume (EV),

Table 4: Semen variables of ram lambs as affected by SPL treatment.

a, b, and c: Means within the same column with different superscripts are significantly different at (P<0.05). EV: Ejaculate volume. SM: Sperm motility. SL: Sperm livability. SCC: Sperm cell count. TSO: Total sperm output.

Table 5: Semen variables of ram lambs as affected by animal age.

a, b, f: Means within the same row with different superscripts are significantly different (P<0.05). EV: Ejaculate volume. SM: Sperm motility. SL: Sperm livability. SCC: Sperm cell count. TSO: Total sperm output

Table 6: Lipid peroxidation biomarker (MDA) and antioxidant enzyme activity in blood serum and seminal plasma of animals in different experimental groups at the end of the experimental period.

, ba and c: Means within the same row with different superscripts are significantly different (P<0.05). MDA: Malonaldehyde. GSH: Reduced glutathione. SOD: Superoxide dismutase. CAT: Catalase. GPx: Glutathione peroxidase.

sperm motility (SM), sperm livability (SL), and sperm cell concentration (SCC). However, the overall mean of all semen characteristics was significantly improved in G3 as compared to G2 and G1, in terms of increasing EV, SM, SL, SCC, and total sperm output (TSO), and decreasing abnormal sperm (AS, Table 4).

As affected by age, the overall mean of EV, SM, SL, SCC, and TSOD significantly increased, while AS significantly decreased by age advancing from 10 up to 15 months of age (Table 5).

Analysis of variance revealed that the effect of interaction between group x age was not significant on all semen characteristics, indicating the superiority of animals in G3, followed by those in G2 in comparison with controls at all ages studied (G1, Figure 2).

Results illustrated in Figure 3 showed that SPL treatment significantly affected testosterone concentration in blood serum, while testosterone concentration in the seminal plasma was not affected by SPL treatment. SPL at both levels (G2 and G3) significantly increased serum testosterone as compared to the control.

Dietary SPL supplementation showed significant enhancement in lipid peroxidation and antioxidant enzymatic activities in blood serum and seminal plasma of animals. The level of malondialdehyde (MDA) was significantly reduced, while the activity of GSH, SOD, CAT, and Gpx significantly increased in the serum and seminal plasma of animals in G2 and G3 compared with G1. The highest level of SPL in G3 (1 g/kg) showed significantly more benefits on MDA level and antioxidant enzyme activity in the seminal plasma (Table 6).

Dietary incorporation of SPL (1 and 5 g/kg) revealed a remarkable and significant enhancement in serum concentrations of triiodothyronine (T3) and thyroxine (T4) compared with the control (Table 7).

SPL was incorporated in the diets of fish, shrimp, and poultry as a protein and vitamin complement to aqua feeds (Anvara and Nowruzi, 2021). The reports on the effects of dietary SPL on small ruminant performance are rare, so the goal of this study was to detect the impact of dietary incorporation of SPL alga on growth, hemato-biochemical parameters, antioxidant status, and semen quality of crossbred rams. In the current study, we hypothesized that dietary inclusion of SPL, with its potent antioxidative properties, could have beneficial effects on the semen quality of animals. The obtained results indicated that final body weight gain significantly increased only for animals fed a diet supplemented with 5 g SPL/kg (G3) for 5 months, as a feeding period (10-15 month of age), in comparison with the controls. The positive impacts of SPL administration on weight gain were reported in Santa Inês ram lambs (Bezerra et al., 2010), growing rabbits (Hassanein et al., 2014), and even in the broilers (Kaoud, 2012). Also, SPL supplementation improved weight gains in both dams and offspring (Khalifa et al., 2016). Moreover, SPL exhibited an important role in boosting body weight and carcass yield in lambs (El-Sabagh et al., 2014), rabbits (Dalle Zotte et al., 2014), and poultry (Evans et al., 2015). The positive effect of SPL on health and production (El-Sabagh et al., 2014) may be attributed to the high nutritional value of SPL. Also, SPL may stimulate extracellular enzymes the release of via the microbiota in the gut to improve weight gain in growing lambs (Lamminen et al., 2019). In ruminants, SPL could reduce degradation of protein resulting in alterations in diversity of bacteria and elevating the synthesis of microbial protein synthesis in the rumen (Panjaitan et al., 2014). Also, SPL reduced the retention time of microbial crude

Table 7: Thyroid hormones in blood serum of animals in different experimental groups at the end of the experimental period.

a and b Means within the same row with different superscripts are significantly different (P<0.05). T3: Triiodothyronine. T4: Tetraiodothyronine (thyroxin).

protein in the rumen (Quigley and Poppi, 2009). About 20% of dietary SPL escape from the degradation in the rumen being available within the abomasum for the direct absorption (Quigley and Poppi, 2009; Panjaitan et al., 2010; Zhang et al., 2010). SPL is a rich source of calcium, iron, beta carotene, and protein with abundant in micro- and macro-nutrients, as well as having strong nutritional properties (Soni et al., 2019). SPL reacts as a usable food sustaining favorable intestinal microflora. It is well known that the testicular size is concerning animal live body weight. In our study, there is an association of increased live body weight of animals in SPL-groups (not significant in G2 and significant in G3), with a tendency of increase in scrotal circumference (testicular size) as compared to controls, but these increases were not significant.

Concerning the hematological findings, we detected that erythrogram and leukogram variables significantly increased in animals fed both SPL levels compared with the controls. The improving the hematological variables in our study may be due to the rich content of SPL from crude protein, vitamin B12, Fe, and essential macro- and micro-elements (El-Deeb et al., 2023). Our results are in accordance with the results of El-Sabagh et al. (2014), who indicated that SPL supplementation increased the total leukocytic count of fattening lambs. SPL increased leucocytes chickens (Qureshi et al., 1996) and fish (Watanuki et al., 2006). Increased WBCs production might be related to phycocyanin and polysaccharides contents in SPL supplemented to animals (Zhang et al., 2001). These findings may be in parallel with improving the immune response through measuring WBCs. Watanuki et al. (2006) found that SPL enhanced animal immunity and antibody production in mice. Also, SPL was found to strengthen the immune response by stimulating function of macrophages (monocytes), phagocytes (monocytes and neutrophils), and immunoglobins (Grzanna et al., 2006). Therefore, SPL is considered as a natural antioxidant and immune stimulant for human and animal without adverse effects in comparison with the synthetic antioxidant (Wu et al., 2016; Liang et al., 2020).

In our study, serum biochemical observations revealed normal hepatic synthetic function through enhanced serum total protein, with normal albumin concentration, and non-significant changes in serum hepatic injury biomarkers (AST and ALT) in both SPL groups. Also, SPL had a significant impact on kidney function via a reduction in urea and creatinine concentrations in blood serum, but SPL at a level of 5 g/kg showed the best results. SPL protects rats against nephrotoxicity by reducing concentration of urea and creatinine. Dietary SPL administration has the ability to decrease cytokine tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 to protect liver and kidney functions (Abdel-Daim et al., 2016). SPL contains phenolic antioxidants and C-phycocyanin targeting Nrf2 and NF-κB pathways (Wu et al., 2016) and heptadecanewhich decrease activity of NF-κB (Kim et al., 2013; Finamore et al., 2017). These findings proved the ability of SPL to protect hepatic-renal sfunction. Moreover, containing SPL blue pigment phycocyanin reduces the hepatic toxicity induced by paracetamol-free radicals (Bhat and Madyastha, 2001). Presence of SOD, β-carotene, Se, or vitamins in SPL have immune-stimulant properties (Mokhbatly et al., 2020; Latif et al., 2021). Improving kidney function by SPL in the current paper may be attributed to triterpenes and flavonoids in SPL as an antioxidant (Sharoud, 2015). In addition, positive effects of SPL on lipid profile were in dose-response pattern. Similar results were reported in sheep (Liang et al., 2020) and cows (Lamminen et al., 2019), whereas SPL decreased concentration of total lipid, total cholesterol, and triglycerides and raised HDL. SPL has anti-lipidemic and anti-hyperglycemia actions by decreasing glucose, cholesterol, and triglycerides, and increasing insulin resistance (El-Sayed et al., 2018). The hypolipidaemic activity of SPL may be due to β-carotene (Seo et al., 2004) and linolenic acid (Morise et al., 2004) in SPL; both affect synthesis of the cholesterol. In this context, SPL reduced cholesterol in rabbits fed a diet containing high cholesterol level (Colla et al., 2008). Phycocyanin in SPL likes to bile acids in the jejunum, and reduced absorption of cholesterol (Nagaoka et al., 2005).

According to the obtained results, dietary administration of SPL significantly improved semen quality in terms of increasing EV, SM, SL, SCC, and TSO, and decreasing AS, particularly, in ejaculates of animals in fed 5 g SPL/kg diet as compared to those fed 1 g/kg or the controls. Oral SPL administration improved the mercury-induced toxicity in the reproductive system of male rats (El-Desoky et al., 2013). In accordance with our results, a significant increase in sperm motility and the count was found after treatment with SPL in rats (El-Desoky et al., 2013; Afkhami-Ardakani et al., 2018). It is of interest to note that improving ejaculate volume and semen quality by SPL is associated with increased weight gain testosterone level significantly in blood serum and a tendency of increase in the seminal plasma. The impact of SPL on the reproduction may be due by regulating the reproductive hormones (Khalifa et al., 2016) or/and by inhibiting the oxidative stress via decreasing ROS, and raising antioxidant enzyme capacity (Wu et al., 2016). Concerning the antioxidant status of dietary SPL administration, our results showed that SPL incorporation enhanced the endogenous antioxidant status through a remarkable enhancement in enzyme activities of GSH, SOD, CAT, and GPx, and decreased lipid peroxidation by reducing MDA levels in a dose-manure pattern in both blood serum and the seminal plasma compared with the unsupplied control group. As such, we detected an improvement in lipid peroxidation marker by decreasing the MDA level and increasing the activity of antioxidant enzymes (GSH, SOD, CAT, and GPx) in blood serum and seminal plasma in SPL treatment groups, but the highest level of SPL showed more impact on the seminal plasma than in blood serum. In accordance with the obtained results, SPL has antioxidants contents such as phycocyanin, polysaccharides, α-tocopherol and ß-carotene with potent antioxidant capacities (Riss et al., 2007), and SOD which reduce ROS (Belay, 2002). As proven in our study, Wu et al. (2016) mentioned that SPL treatment increased GSH, CAT, and SOD activities and decreased level of MDA which indicate the antioxidant, immunological, and anti-inflammatory activities of SPL (Liang et al., 2020). Generally, Ibrahim and Abdel-Daim (2015) reported non-toxic, bio-available, and potent antioxidant capacity of SPL. In chemoprevention, phycocyanin and Se derived from SPL can exert potent antioxidant capacity (Chen and Wong, 2008) to reduce the lipid peroxidation (Mokhbatly, 2020). Also, C-phycocyanin and carotenoids in may help in improving animal reproduction (Niu et al., 2017).

Moreover, we observed additional benefits of SPL on serum concentration of T3 and T4. In association with the amelioration in the level of thyroid hormones by SPL treatment, there was an in attenuation of oxidative stress in the thyroid tissues by decreasing caspase-3 activity and DNA damage (Ebrahim, 2020). The contents in SPL may also eliminate inflammatory gene expression in splenocytes by inhibition of the Toll-like receptor 4 signaling pathways. Moreover, increasing γ-linolenic acid in SPL (Ronda and Lele, 2008) can decrease LPS-induced inflammatory gene expression in monocytes and macrophages (Chang et al., 2010). Organic extract of SP represses TNF-α expression and secretion in macrophages (Pham et al., 2016). Spirulina displays high antioxidant and anti-inflammatory activities with numerous neutralizing and oxidative effects on free radicals and heavy metals (Wu et al., 2016).

We can conclude that the Spirulina platensis algae can be used safely as a potentially healthy feed for growing ram lambs as dietary incorporation at a level of 5 g/kg diet. It can improve weight gain, hematology, liver and kidney function, lipid profile, semen characteristics, and lipid peroxidation in blood serum and seminal plasma of growing ram lambs from 10 up to 15 months of age.

The effect of dietary incorporation of Spirulina platensis (SPL) alga during the pre-pubertal stage (15-16 month of age) on improving the growth, health, antioxidant status, and semen quality of ram lambs raised for natural mating or artificial insemination.

Substantial contributions to conception and design DHA, MME-M, AEA, and RAA-W. Acquisition of data ZIE, HKZ, and AEA. Analysis and interpretation of data DHA, MME-M, AAE-B, RAAW and AEA. Statistical analyses RAAW, AAE-B, HKZ and AEA. Drafting of the manuscript DHA, AEA, MME-M and ZIE. Critically revising the manuscript for important intellectual content, AEA, DHA, MME-M and AAE-B. Final approval of the manuscript for publication (all authors).

The experimental procedures were carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.

The authors have declared no conflicts of interest.