Effects of Herbal-Plant Supplementation During Pregnancy on Ewes’ Blood Parameters and Lambs’ Birth Weight

The ban set on antibiotics use to reduce energy loss and enhance productivity in livestock industry forced those concerned with animal production to search for safer and better alternatives [1,2]. In recent years, herbal plants have gained much attention as alternative growth promoters to antibiotic. Various medicinal plants and their extracts have been used as feed additives due to their various health-promoting effects such as being anti-oxidative, antimicrobial, anti-inflammatory, and growth-promoting [3-5]. Previous studies have reported that medicinal plants and their extracts may be included in ruminant diets to improve nutrient digestibility, rumen fermentation, immune function, milk production, and composition [6,7].

Nigella sativa is a small aromatic black seed slightly smaller than the sesame seed. It is commonly known as black seed and belongs to the botanical family of the Ranunculaceae, which grows in countries bordering the Mediterranean basin and other places of similar climates [8-10]. This herb has long been used in traditional medicine due to its amazing pharmacological properties, and it also enjoys a rich historical and religious background [11]. Nigella sativa seeds contain many active components includingthymoquinone, thymohydroquinone, dithymoquinone, nigellone, melanthin, nigilline nigelamine, damascenone, p-cymene, and pinene, in addition to a large number of minerals (e.g., calcium, phosphorus, potassium, magnesium, iron, cobalt, and zinc) and vitamins such as A, B, C, D and E [12-14]. Seeds of Nigella sativa represent a major source of fixed and essential oils, proteins, saponins, polyphenols, alkaloids, and flavonoids. Previous reports suggest that thymoquinone (an alkaloid), nigellone (a carbonyl polymer of thymoquinone) and fixed oils are the most active component ingredients in Nigella sativa [8-15]. Nigella sativa seeds have many beneficial properties as antitumor, antidiabetic, anti-asthmatic antihypertensive, antioxidative, antimicrobial (antibacterial, antifungal and anthelminthic), anti-inflammatory, analgesic, antipyretic, diuretic, carminative, laxative, antimicrobial and antineoplastic activity [9,16,17].

Zingiber officinale is a rhizome (ginger root) that is widely used as a spice and a herbal remedy for a large number of diseases for many years [18,19]. Zingiber officinale contains several active compounds such as gingerol, gingerdiol, and gingerdione [19]. These compounds possess strong antioxidant, immunomodulatory, anti- cancer, anti-inflammatory, anti-apoptotic, anti-hyperglycemic, anti-lipidemic, antifungal, and anti-stress activities [5,20,21].

The objective of the present study was to investigate the effect of dietary supplementation of Nigella sativa seeds and Zingiber officinale powder on blood hematological parameters, biochemical parameters, and oxidative stress markers in pregnant ewes as well as lambs birth weight. We hypothesized that ewes supplemented by herbal plants will exhibit better blood parameters and oxidative status compared with the control counterparts.

Animals and managements

All procedures were approved by The Ethics of Animal Experiments Committee, Damanhour University. The current study was carried out at the experimental farm of the Faculty of Agriculture, Damanhour University located in Al-Bostan. Fifteen pregnant crossbred (Rahmany × Balady) ewes, aged 3-5 years old and weighing an average of 48.5 ± 2.03 Kg, were divided into three groups; one control and two experimental groups. Ewes fed like those in our previous report [7]. Briefly, ewes in all groups were fed a basal diet comprising wheat straw and concentrate feed mixture of 55% yellow corn grain, 20% wheat bran, 12.5% cotton seed meal, 10% soy bean meal, 1% sodium chloride, 1.4% lime stone, and 0.1% avimix mineral mixture. Each 1 kg of avimix mineral mixture (AGRIVET) consisted of manganese sulphate (16.66 g), zinc sulphate (20 g), iron sulphate (10 g), potassium iodide (0.83 g), cobalt chloride (0.17 g), sodium selinite (0.066 g), and calcium carbonate (952.27 g). While the control group fed the basal diet only, the 2 experimental groups received 5 g/h/d of either Nigella sativa seeds or Zingiber officinale powder. Concentrate feed mixture was offered two times a day at a rate of 2.5% of animal weight while wheat straw and water were offered ad libitum. We analyzed wheat straw, concentrated feed mixture, Nigella sativa seeds and Zingiber officinale powder for moisture, ash, crude protein, ether extract, crude fiber and nitrogen free extract according to the Official Methods of AOAC [22]. Cell wall constituents were estimated according to the method described by Van Soest [23]. Non fibrous carbohydrates were calculated according to Calsamiglia [24]. (Table 1) presents data resulting from these analyses.

Table 1: Chemical composition and cell wall constituents of concentrate feed mixture, wheat straw, Nigella sativa seeds, and Zingiber officinale fine powder (on dry matter basic) used in this experiment.

Sampling and measurements

Blood analysis

Blood samples were collected biweekly until parturition from each group (in the morning before feeding). Samples were obtained from the jugular vein in two clean tubes, one containing EDTA disodium salt as an anticoagulant for hematological parameters while the other tube contained no anticoagulant since blood biochemical parameters were estimated from the serum. Samples were immedi ately kept in an ice tank. Blood samples were centrifuged at (3500 rpm) for 15 minutes to separate the serum, which was stored at (-20 0C) until analysis.

The concentrations of total protein, glucose, total cholesterol, triglyceride, total antioxidant capacity, and malondialdehyde in serum were analyzed by assay kits according to the manufacturer’s instructions (Biodiagnostic Company, Giza, Egypt). While hematological parameters such as red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelets, and white blood cells (WBCs) were determined in fresh blood through an automatic blood analyzer (EXIGO VET, Boul Medical AB, Spanga, Sweden).

Statistical analysis

Data were statistically analyzed using One-Way Analysis of Variance (ANOVA) test ‘Duncan’s Multiple Range Test’ with alpha at the significance level of 0.05. Data are presented as means with standard error. The collected data were performed using PASW statistic 18.0 (SPSS, Inc., Chicago, IL, USA).

Table 2: Effect of Nigella sativa seeds and Zingiber officinale powder supplementation on blood hematological parameters of pregnant ewes.

Ewes fed Nigella sativa seeds had significantly lower WBCs count compared with ewes fed Zingiber officinale powder or basal diet. Meanwhile, there were no significant changes in the percentages of Hb, PCV, MCV, and MCHC, as well as counts of RBCs and platelets (Table 2). Findings from former studies are mixed. Consistent with our results, Abd El-Halim [25] reported significantly (P ≤ 0.05) lower total WBCs count in growing lambs fed Nigella sativa oil (NSO) than the control. Meanwhile RBCs count transiently decreased at 2 wk, but it significantly increased (P ≤ 0.05) after 6 wk. They found no significant differences in the percentages of PCV, MCV, MCH, and MCHC among NSO animals throughout their experiment [25]. On the other hand, Hendawy. [7] reported that counts of RBCs and WBCs significantly increased in lactating ewes fed Zingiber officinale powder compared with lactating ewes fed basal diet or Nigella sativa seeds. In contrast, MCH was higher in both the Nigella sativa seeds and control groups than the Zingiber officinale powder group (P < 0.05). While, there were no significant changes in the percentages of Hb, PCV, MCV, MCHC, and platelets. Unlike our findings, EL-Ghousein [26] reported no changes in RBCs, Hb, and WBCs in ewes fed 10 g/h/d Nigella sativa seed. Still the observed variation in these parameters can be due to the variation of preparation, doses, duration of supplementation, animal species, sex, age, and factors that affect their absorption from the gut and their bioavailability.

The concentration of serum total protein and glucose increased (P < 0.05) in animals treated with medicinal plants compared with the control animals. On the contrary, serum levels of total cholesterol and triglyceride were lower in the treatment groups compared with the control group (Figure 1A and 1B). Enhancement of serum protein expressed animals treated with Nigella sativa and Zingiber officinale is congruent with reports of several previous studies. In accordance, Saleh [27] reported that supplementing lactating ewes with Nigella sativa seeds increased (P < 0.05) serum protein compared with ewes fed basal diet. In the same line, Allak [28] reported that Ossimi rams fed Nigella sativa seeds or Zingiber officinale had significantly (P ≤ 0.05) higher serum protein compared with control animals. In agreement, Khattab [29] confirmed that buffalo supplemented with Nigella sativa seed oil exhibited increased protein anabolism due to higher protein digestibility, which resulted in higher blood plasma total protein—the main contributor to increased tissue protein biosynthesis. In the same regard, Habeeb and El-Tarabany [30] recorded that Nigella sativa additive to the diet of Zaraibi kids significantly increased plasma total proteins and globulin. In contrast, Ibrhim [31] reported no changes in total protein level of lactating Awassi ewes supplemented with Zingiber officinale extract by 2 ml/2 liter per day in drinking water. It is likely that herbal plants increase serum protein by creating favorable rumen conditions that promote microbial activity toward the best utilization of ruminal ammonia and the most efficient conversion of dietary substrates into microbial protein—feed the microbiome and let the microbiome feed the animal [6].

The reductions in total cholesterol and triglyceride levels observed following ewe’s treatment with Nigella sativa seeds and Zingiber officinale powder in the present experiment (Figure 1C and 1D) are consistent with finding reported by many other studies. Consistent with our findings, Allak [28] noted that Ossimi rams fed Nigella sativa seeds and Zingiber officinale herbs had significantly (P ≤ 0.05) lower cholesterol and triglyceride levels compared with control animals. In the same line, Saleh reported decreased triglyceride and total cholesterol (P < 0.05) in lactating ewes supplemented with Nigella sativa seeds compared with ewes on basal diet [27]. Alike, Khattab et al. reported decreased cholesterol level in buffalo fed NSO [29]. Similar effects of Nigella sativa were obtained in growing Zaraibi goats [30]. On the contrary, Retnani [32] reported no change in blood glucose and triglyceride levels of lambs fed diet containing 10% or 20% Nigella sativa. The cholesterol lowering effect of both Nigella sativa seeds and Zingiber officinale may be attributable to their high contents of unsaturated fatty acids [29].

The present research revealed significant drop in serum concentration of malondialdehyde in pregnant ewes fed medicinal plants-supplemented diets compared with their control counterparts. Moreover, Nigella sativa seeds and Zingiber officinale treatments increased total antioxidant capacity; however, that increase was insignificant (Figure 2A and 2B). In accordance, El-Far [5] reported that supplementing sheep with Nigella sativa seeds and Zingiber officinale powder significantly decreased malondialdehyde and significantly increased serum total antioxidant capacity and blood antioxidant enzyme e.g., catalase, glutathione peroxidase, glutathione S-transferase and total superoxide dismutase (P < 0.05). Such enzymes rapidly convert the corrosive superoxide (O2 •−) to less dangerous H2O2, which is further degraded to water [33,34]. Another study revealed no effect of herbal blend additives on levels of malondialdehyde and total antioxidant capacity in plasma or rumen fluid [31]. Altogether, Nigella sativa seeds and Zingiber officinale powder seem to be efficient in counteracting oxidative stress via enhancement of the production of internal antioxidants.

We recorded an increase in lambs birth weight of ewes supplemented with herbal plants-rich diet; nonetheless, that increase was not significant (Figure 2C). Consistent with this report, Dudkoa et al. indicated that enriching sheep meal with a blend of essential oil in O. vulgare and Citrus spp. caused no significant difference lambs’ body weight at birth [35]. On the other side, Sahoo et al. reported that supplementing Black Bengal goats with polyherbal immunomodulator (immu-21) during the last month of pregnancy resulted in several positive effects: significantly higher birth weights, absence of kid mortality, and heightened concentrations of blood protein and colostrum immunoglobulin. The researchers concluded that polyherbal immunomodulator supplementation to goats during late pregnancy and to kids during early growth periods is much more beneficial than solo administration at either stage [36].

Effects of herbal plants on lamb birth weight and growth in the first few weeks of life speak for their multiple effects on mother ewes—increased protein synthesis, immune enhancement, and improved redox state.

In conclusion, results of this study highlight a positive effect of herbal-plant dietary supplementation to pregnant ewes on some blood parameters as well as markers of oxidative stress. Late gestation and early post parturition represent a time of great physiological stress, which is characterized by increased oxidative stress. Therefore, it is recommended to supplement ewes during these stages with Nigella sativa seeds and Zingiber officinale powder to increase the activity of their antioxidant system. It will be beneficial if several large sample size studies were performed as replication of the current study on different farm animals to check the effect of herbal plants.

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