Among the non-nutritive additives available for lamb nutrition, direct-fed microbial (DFM) stands out for altering rumen fermentation and increasing animal productivity. This study was conducted to evaluate the effects of DFM and weaning systems on performance, mortality, and health of newborn lambs. A total of 60 newborn lambs were divided into 4 equal groups and assigned to one of 4 treatments: Control (C) without DFM and late weaning; T1, DFM and early weaning; T2, DFM and mid weaning; and T3, DFM and late weaning. Each lamb in the treated group received 3 doses of DFM (5 mL/lamb). The mortality was reduced by 80% compared to the control group. Lambs in the T2 and T3 with DFM groups had significantly (P < 0.05) higher body weight (BW) and body weight gain (BWG) than lambs in the C group. Glucose, creatinine, and urea nitrogen levels of T3 lambs were significantly (P < 0.05) higher in late weaning compared with the remining treatments. DFM supplementation and weaning system significantly (P < 0.01) reduced serum Zn concentration. These results suggest that the addition of DFM to the diet of newborn lambs and weaning at 60 days improves the overall performance and immunity of the lambs and consequently reduces the mortality rate.
Fattening lambs for sale or slaughter is the primary source of income for sheep farms. Farmers’ management practices are the main cause of the high newborn lamb mortality rate. Furthermore, the survival rate of newborn lambs is determined by health indicators in the first month after birth, which is the most important factor influencing sheep farm profitability . Productivity of ruminants can be increased by manipulating rumen microbiota, which has been studied extensively focusing on rumen health and animal performance . Following the ban on antibiotic use in ruminants and concerns about food safety, the feed industry has become more interested in direct-fed microbial (DFM) to improve animal performance . DFM is a mixture of mono- or mixed-cultures of live microorganisms as opposed to probiotics . DFM has a positive effect on the development of microbial flora in the gut by functioning in the intestine, they stimulate the protective functions of the digestive tract and act as a biotherapeutic and bioprotectant, which is also used to prevent intestinal and gastrointestinal infections [3,5]. DFM has been used in cattle , poultry , lambs , and calves . DFM has been shown to improve digestion, increase feed intake, reduce rumen disturbance, and increase animal productivity . Several attempts have been made to stimulate rumen development in ruminants for early weaning and to avoid gastrointestinal disturbances due to the transition feeding period . Weaning is a critical period in lambs because they only receive milk from their mothers and the microbial population begins to establish. The transition from a liquid to a solid diet causes stomach problems and diarrhea which reduces productivity and potentially lead to death . Early weaning occurs between 3.5 and 5.5 weeks of age, when the rumen is developing and thus in transition to ruminant, body growth and fat deposition are affected, which may reduce feed intake and growth rate, with high plasma non-ester fatty acid (NEFA) levels as an indicator of lipid mobilization . The weaning of lambs is usually accompanied by stress, which causes a change in body composition, resulting in fat loss or a sharp decrease in body fat accumulation, with an increase in the proportion of lean meat in both cases . Therefore, supplementing DFM as a rumen development stimulant with early weaning can improve sheep farm management practices and profitability. The study objective is to evaluate the performance and mortality rate of Najdi lambs at different stages of weaning at 30, 45, and 60 days of age after DFM administration.
2 Materials and methods
2.1 Animals and experimental design
An experiment was conducted at Al Khalediah farm, with 60 newborn Najdi lambs (body weight [BW] = 5.4 ± 0.10 kg) randomly divided into 4 groups. The experimental groups were randomly assigned to the following treatments: Control (C), without DFM administration and weaning at 60 days of age; T1, 3 DFM administration and weaning at 30 days of age; T2, 3 DFM administration and weaning at 45 days of age; and T3, 3 DFM administration and weaning at 60 days of age. Each lamb in the treated group received 3 doses of DFM (5 mL/lamb) at 5, 10, and 15 days postpartum. The DFM used as a treatment is a commercial gel containing eight naturally occurring microorganisms (Biostart Microbial Paste, Bio-Vet, USA). The eight species of microorganisms are Bacillus licheniformis, Bacillus subtilis, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus lactis, Pediococcus cerevisiae, and Saccharomyces cerevisiae. The total bacterial count is 2.00 billion CFU/mL. Most of the above bacteria were classified as lactic acid-producing bacteria .
2.2 Management of newborn lambs
In this study, a total of 60 multiparous ewes were selected before calving with an average weight of 71.39 ± 2.42 kg. At birth, the newborn navels were cut and treated with iodine spray. They were then allowed to suckle their mothers’ colostrum for 36 h. The lambs were vaccinated with 1 mL of Pasteurella spp. (Trivalent PasteurellaVaccin) subcutaneously and given selenium and vitamin E (oral dose of 3 mL). During the second week, the lambs began eating creep feed (small pellets, containing low fiber and high protein in addition to vitamins and salt), and received their second dose of selenium and vitamin E, and drank their mothers’ milk without restriction. After the sixth week, the lambs were vaccinated against Peste des petits ruminants (PPR) (Octavalent Enterotoxaemia Vaccine, 1 mL subcutaneously). After weaning, the lambs were fed every day with a total mixed ration (TMR) of alfalfa hay, Rhodes, and Concentra (crude protein content of 14%). Bodyweight was recorded at birth and then every two weeks until weaning. Mortality was recorded at the start of the experiment when DFM supplementation began receiving oral doses (5 mL) (at 5 days of age).
2.3 Samples collection and analysis
Blood samples from each lamb were obtained in 10 mL serum collection tubes (BD Vacutainer, USA) at days 1, 30, and 60 of the experimental phases, by puncture of the jugular vein. The serum was separated from the blood samples via centrifuging at 3,000 rpm for 10 min at 4°C and then stored in Eppendorf tubes. Subsequently, the samples were frozen at –20°C for their later analysis. Blood serum samples were analyzed for glucose, total protein, cholesterol, creatinine, urea nitrogen, and triglycerides by spectrophotometer (UDICHEM-310, Semi-Automated, United Diagnostics Industry, Dammam, KSA) using commercial kits (Randox Laboratories Limited, BT, UK). Cortisol levels were also analyzed in blood serum using ELISA (BioTek Instruments, VT, USA) and commercial kit (Human Gesellschaft Fur Biochemica und Diagnostica mbH, Germany). The blood concentrations of calcium, phosphorus, zinc, and copper serum were analyzed using UDICHEM-310 spectrophotometer (semi-automated, United Diagnostics Industry, Dammam, KSA) and commercial kits (Randox Laboratories Ltd, BT, UK).
2.4 Statistical analysis
Statistical analyses were performed using the Proc GLM procedure in SAS 9.4 (SAS Institute Inc. 9.4) for a Completely Randomized Design, according to the following model:
where Y ijk = measurement of the variables, μ = overall mean, DFM i = effect of the ith treatment (C and 3 treatments), WS j = effect of the jth weaning system (30, 45, and 60 days of age), and e ijk = residual error.
BW, body weight gain (BWG), average daily gain (ADG), biochemical parameters, and mineral concentrations of blood serum were dependent variables. Differences among treatment means were detected using Duncan test with P < 0.05 considered statistically significant unless otherwise noted.
3.1 Performance and mortality rate
The effects of DFM paste and weaning system on growth performance of newborn Najdi lambs are shown in Table 1. In this study, weaning system and DFM treatment had a significant (P < 0.05) effect on BW and BWG. In contrast, at mid and late weaning, lambs in T2 and T3 had significantly (P < 0.05) higher BW and BWG compared with lambs in T1 and the control group. In addition, ADG was significantly (P < 0.05) higher in lambs in T2 at mid weaning (45 days). While there was no statistically significant (P > 0.05) difference between C and T1 in BW, BWG, and ADG at different stages of weaning. It is noteworthy that the mortality rate in the early stage of lamb life and until weaning was reduced by 80% in this experiment in T2 and T3 groups (1 lamb was dead from 15 lambs – 6.67%), compared to the control group (5 lambs were dead from 15 lambs – 33.33%) (Figure 1).
|BW 1 day||5.68C ± 0.20||5.01C ± 0.11||5.67C ± 0.21||5.59C ± 0.31||0.09|
|BW 30 days||10.86B ± 0.73||10.29B ± 0.81||12.20B ± 0.55||11.24B ± 1.01||0.31|
|BW 45 days||14.63abAB ± 0.96||12.3cAB ± 0.96||16.53Aab ± 0.94||13.54bAB ± 1.30||0.02|
|BW 60 days||15.33bA ± 1.53||14.47cA ± 1.09||18.45aA ± 0.98||18.27aA ± 1.19||0.04|
|1–30 days||5.18C ± 0.58||5.28C ± 0.76||6.52C ± 0.49||5.66C ± 0.94||0.53|
|1–45 days||8.96abB ± 0.85||7.37cB ± 0.92||10.86aB ± 0.88||7.96bB ± 1.21||0.04|
|1–60 days||9.65A ± 1.38||9.47A ± 1.08||12.78A ± 0.91||12.69A ± 1.07||0.06|
|1–30 days||0.17 ± 0.02||0.18 ± 0.02||0.22 ± 0.02||0.19 ± 0.03||0.52|
|1–45 days||0.20ab ± 0.02||0.16c ± 0.02||0.24a ± 0.02||0.17b ± 0.03||0.04|
|1–60 days||0.16 ± 0.02||0.16 ± 0.02||0.21 ± 0.02||0.21 ± 0.02||0.06|
Data are presented as mean values ± SE (n = 15). A,B Mean values within a row with different subscript letters were significantly different (P < 0.05). a,bMean values within a column with different subscript letters were significantly different (P < 0.05). CControl (weaning at 60 days old). T1Weaning at 30 days old with 3hree doses of DFM (5, 10, and 15 days old). T2Weaning at 45 days old with 3 doses of DFM (5, 10, and 15 days old). T3Weaning at 60 days old with 3 doses of DFM (5, 10, and 15 days old). BW = body weight. BWG = body weight gain. ADG = average daily gain. d = day.
3.2 Metabolites in the blood
The metabolites concentration in the blood serum of Najdi lambs fed with DFM during different weaning stages (age 1, 30, and 60 days) are summarized in (Table 2). Serum concentrations of glucose, cholesterol, urea nitrogen, and triglycerides of the lambs were significantly (P < 0.05) affected by treatment and weaning system, with no significant (P > 0.05) difference in serum concentrations of total protein. DFM supplementation and late weaning (60 days) showed significantly (P < 0.05) higher concentrations of glucose, cholesterol, and triglycerides than blood serum of lambs in the C group. In addition, higher (P < 0.05) levels of glucose were found in the blood serum of lambs in the T2 group at mid weaning (45 days) and T3 at late weaning (60 days). However, the levels of cortisol, urea nitrogen, and triglycerides in the blood serum of lambs in the C group were significantly (P < 0.05) affected by the weaning system.
|1 day||93.87Aa ± 6.32||50.12 ± 9.22||63.73bA ± 8.66||61.17b ± 8.42||0.01|
|30 days||40.44B ± 7.68||35.23 ± 3.22||32.00C ± 3.27||45.12 ± 7.51||0.43|
|60 days||40.69B ± 3.65||49.31 ± 1.24||44.40B ± 3.04||52.64 ± 5.49||0.15|
|1 day||82.88 ± 5.73||74.40B ± 2.11||68.30B ± 9.12||84.55 ± 4.15||0.21|
|30 days||67.30b ± 6.93||92.90aA ± 6.21||97.24a A ± 6.52||59.58 ± 10.49||0.01|
|60 days||72.30a ± 5.13||39.56bC ± 5.53||50.20bC ± 4.81||73.38a ± 3.21||0.0002|
|Total protein (g/dL)|
|1 day||5.04 ± 0.73||4.75 ± 0.61||5.18 ± 0.56||6.39 ± 0.88||0.40|
|30 days||4.53 ± 0.25||5.50 ± 0.48||5.32 ± 0.38||5.74 ± 0.53||0.25|
|60 days||5.26 ± 0.21||4.33 ± 0.84||5.17 ± 0.48||6.28 ± 0.31||0.11|
|1 day||40.70 ± 5.17||45.88B ± 3.39||38.28B ± 2.66||47.62C ± 4.17||0.34|
|30 days||59.78 ± 8.36||69.16A ± 6.01||68.00A ± 5.16||79.45A ± 7.95||0.30|
|60 days||48.48b ± 5.03||66.50abA ± 4.84||28.46C ± 4.12||75.18aB ± 8.90||0.0002|
|1 day||0.75b ± 0.09||0.57c ± 0.06||0.58 ± 0.05||1.05aA ± 0.07||0.001|
|30 days||0.70 ± 0.05||0.71 ± 0.04||0.68 ± 0.04||0.56C ± 0.03||0.06|
|60 days||0.84a ± 0.05||0.51c ± 0.07||0.59b ± 0.10||0.82abB ± 0.04||0.01|
|Urea Nitrogen (mg/dL)|
|1 day||116.80aA ± 4.89||49.64bc ± 9.25||50.40b ± 6.79||39.50C ± 2.11||0.001|
|30 days||44.28C ± 4.28||44.92 ± 1.98||48.78 ± 2.62||43.72B ± 3.68||0.69|
|60 days||60.53bB ± 5.42||31.83d ± 4.48||49.35c ± 6.94||65.24aA ± 5.84||0.004|
|1 day||90.03bA ± 3.28||61.40dB ± 5.69||73.14cA ± 5.58||135.67aA ± 8.56||0.001|
|30 days||64.96B ± 9.80||75.96A ± 6.06||68.82B ± 5.14||53.4C ± 5.32||0.17|
|60 days||58.86aC ± 4.51||38.14C ± 3.05||36.94C ± 2.32||55.48abB ± 4.27||0.001|
Data are presented as mean values ± SE (n = 15). A,BMean values within a row with different subscript letters were significantly different (P < 0.05). a,bMean values within a column with different subscript letters were significantly different (P < 0.05). CControl (weaning at 60 days old). T1Weaning at 30 days old with 3 doses of DFM (5, 10, and 15 days old). T2Weaning at 45 days old with 3 doses of DFM (5, 10, and 15 days old). T3Weaning at 60 days old with 3 doses of DFM (5, 10, and 15 days old).
3.3 Mineral concentration
Mineral concentration in blood serum of lambs at late weaning showed higher concentration (P < 0.05) of calcium in groups C and T3 compared to concentration in blood serum of lambs in T1 and T2 groups (Table 3). On the other hand, phosphorus concentration was significantly (P < 0.05) decreased by the treatments (T1 and T3) and late weaning (60 days).
|30 days||9.91 ± 0.45||8.80 ± 1.51||11.02 ± 1.02||9.47 ± 0.88||0.51|
|45 days||8.66 ± 1.30||8.58 ± 0.66||9.29 ± 0.90||8.28 ± 0.48||0.87|
|60 days||10.74 ± 1.12||7.64 ± 0.91||7.17A ± 0.76||9.72 ± 0.68||0.04|
|30 days||6.60 ± 0.69||6.82 ± 1.03||6.50 ± 1.02||8.26 ± 0.57||0.45|
|45 days||7.13 ± 0.45||7.05 ± 0.11||7.07 ± 0.60||6.20 ± 0.26||0.33|
|60 days||6.40a ± 0.47||4.15A ± 0.29||5.05 ± 0.66||6.13 ± 0.27||0.01|
|30 days||132.70A ± 4.51||104.97 ± 6.08||93.94A ± 7.56||114.80A ± 4.59||0.002|
|45 days||242.17 ± 9.85||207.79 ± 9.43||139.28 ± 9.24||151.32 ± 7.10||0.001|
|60 days||167.89 ± 17.45||48.68A ± 3.25||102.14 ± 2.52||117.45 ± 9.74||0.001|
|30 days||201.19 ± 7.35||188.53 ± 22.02||175.95 ± 7.78||185.84 ± 11.46||0.63|
|45 days||190.82 ± 8.18||182.73 ± 11.71||184.38 ± 4.88||161.26 ± 10.25||0.16|
|60 days||166.34A ± 5.83||137.51 ± 7.43||140.41A ± 5.52||160.12 ± 5.84||0.01|
CControl (weaning at 60 days old). T1Weaning at 30 days old with 3 doses of DFM (5, 10, and 15 days old). T2Weaning at 45 days old with 3 doses of DFM (5, 10, and 15 days old). T3Weaning at 60 days old with 3 doses of DFM (5, 10, and 15 days old). SE = Standard error. Mean values followed by a common letter are not significantly different by the Duncan-test at the 95% level of significance.
Serum zinc concentration was significantly (P < 0.05) affected by the treatments and weaning system, with the C group having a higher zinc concentration (P < 0.05) at the different weaning periods (30, 45, and 60 days). Interestingly, the lowest serum zinc concentrations in T1 lambs (48.68 μg/dL) were found in the late weaning compared with the other treatments (Table 3). In addition, serum copper levels were significantly (P < 0.05) higher in groups C and T2 compared with the other treatments at late weaning (60 days).
4.1 Growth performance and mortality rate
The most important characteristic of a well-functioning digestive tract is a balanced bacterial microflora, which is disturbed when animals are exposed to sudden stress, such as weaning, high temperatures, castration, and feed changes . The most critical period is lamb weaning, which is associated with severe stress and results in change in body composition, manifested by fat loss or a sharp decrease in body fat accumulation with a corresponding increase in lean tissue . In the current study, differences in BW and BWG were observed between different groups. Improvement was seen in lambs with supplementation DFM in T2 and T3, particularly at late weaning. This can be returned to DFM to improve rumen microflora and get lambs sufficient milk with late weaning. A previous study found that consuming DFM before weaning increased final BW and daily weight gain . Similarly, probiotic administration was found to increase lamb BW . In comparison to the control group, DFM treatment had no significant effect on BW, feed intake, or conversion rate in growing lambs . Elghandour and colleagues  demonstrated rapid rumen development in DFM-supplemented newborn calves, as well as fermentation at 30 days of age as solid diet was increased. Furthermore, the levels of acetic acid and lactic acid in the rumen had a significant impact on gram-negative bacteria and, as a result, the uptake of some metabolites [10,21].
The percentage of mortality in this study was greatly reduced in T2 and T3, which was consistent with the findings of Alhidary and colleagues  and Kritas et al. , who reported lower mortality rates in lambs supplemented with DFM compared to the non-supplemented groups. In the perinatal period, lamb mortality rates reach up to 20.8% within 3 days of birth, 28.7% within the first 7 days, and approximately 8% within the next 8 days . The main cause of starvation in lambs that can lead to death was a lack of suckling milk at early weaning, 30 days, and a decrease in the development of rumen microflora. However, the decrease in mortality with late weaning, 60 days, demonstrated that the lamb was getting enough milk and that the rumen had developed the ability to digest diet. It is generally believed that feeding DFM stimulates the immune system, especially in young ruminants, resulting in greater susceptibility to microbial colonization by pathogens in the small intestinal tract [10,23] and improved health and productive yield through the production of cytokines by macrophages to fight infections .
4.2 Metabolites in the blood
In the current study, cortisol concentrations in the treated and control groups decreased at weaning compared to the perinatal period. On the contrary, weaning was associated with increased plasma cortisol levels, according to Vosooghi-Poostindoz et al. , and treatment with the probiotic mixture improved these levels. The serum glucose concentration significantly decreased in T1 and T2 lambs at late weaning compared to the other groups. The findings of this study were consistent with those reported by Antunović et al.  in growing lambs fed with a probiotic mixture. Feeding DMF and solid diets to calve at a young age may result in acidosis caused by lactic acid-producing bacteria, which has also been linked to lower rates of gluconeogenesis .
DFM supplementation affected serum cholesterol and triglyceride levels, resulting in a decrease in lambs in T1 and T2 compared to the other groups at late weaning. These finding were similar to those reported by Santillo et al.  and Moarrab et al. . These observations could be attributed to the activity of lactic acid bacteria (LAB), which reduced intestinal lipid absorption by deconjugating bile acids [30,31]. This explains why bacterial fermentation and inhibition of hydroxymethylglutaryl-CoA reductase, a protein that regulates cholesterol synthesis, results in higher fecal propionic acid concentrations and lower blood concentrations .
In this study, the late weaned lambs in T3 had a higher urea nitrogen concentration than the other treatments, while T1 had a lower concentration. This change could be related to acidosis, a condition in which there are significant changes in glutamine utilization and synthesis as a result of ammonia transfer from the rumen . The lower serum urea nitrogen in T1 lambs at late weaning may be due to some absorption of NH3 from the rumen in addition to full uptake of NH3 for glutamine synthesis . In addition, creatinine levels at late weaning were lower in T1 and T2 than in other treatments. Creatinine is produced in muscle, and its concentration is directly related to physical activity levels . The current study’s reduction in creatinine levels could be attributed to DFM supplementation. Furthermore, these metabolites provide information about the nutritional and metabolic status of the lambs in this experiment, which was healthy and disease-free.
4.3 Mineral concentration
Minerals exist as electrolytes in body tissues and fluids and play roles in osmotic pressure, acid-base balance, membrane permeability, and tissue irritability. Mineral deficiency generally has a negative impact on animal health and performance . Mineral concentration was generally influenced by DFM supplementation and weaning system in the current study. The impact of ruminant physiological stages on mineral absorption is poorly understood, and more research is needed to define the relationship between DFM and mineral intake . Rumen bacteria produce fermentation by-products such as short-chain fatty acids and lactic acid, which can contribute to pH reduction and, as a result, affect mineral solubility and uptake positively or negatively .
In this study, serum minerals (Ca, P, Zn, and Cu) decreased at late weaning in all treatments, but this was within normal limits and could be attributed to an increase in physiological demand. Low calcium and phosphorus concentrations could be the result of metabolic acidosis caused by high levels of lactic acid-producing bacteria as a result of DFM addition. Antunović and colleagues  found similar results in growing lambs fed with probiotics. This result contradicts the findings of Underwood and Suttle , who found a tendency for higher plasma phosphorus in chronic calcium deficiency. Parathyroid hormone (PTH) is involved in the reduction in blood calcium via the synthesis of 1,25-dihydroxycholecalciferol (1,25-(OH)2 D3) in the kidney, which affects calcium absorption from the intestine and kidney. Furthermore, metabolic acidosis causes increased excretion via the renal system .
The minerals zinc and copper are very important trace minerals that play an important role in the metabolism and health of animals, especially as components of many important enzymes . In the present study, zinc concentration in blood serum was significantly affected by DFM supplementation and weaning system. According to report by Herdt and Hoff , zinc levels in this study were normal (80–120 g/dL), with the exception of T1, which had the lowest levels (48.68 g/dL) when compared to other treatments. This could be due to the lambs’ physiological stage and increased mineral requirement. Because most ruminant diets are zinc deficient, a contribution of less than 45 mg/kg Zn in the diet is said to cause micromineral deficiency in cattle . Serum copper concentration at late weaning was significantly (P < 0.01) lower in lambs from groups C and T2 than in other groups in this study, despite the fact that all values were within the normal range (70–200 g/dL) according to Puls  and Herdt and Hoff .
DFM supplementation during the perinatal period and late weaning (60 days) improved their BW, average BW, and decreased mortality. The use of live bacteria as DFM to stimulate rumen development, as well as late weaning, allows lambs to get enough suckling milk; this practice is considered safe and profitable in sheep farming.
The authors sincerely acknowledge the Researchers Supporting Project for funding this work number (RSP-2021/232) at King Saud University, Riyadh, Saudi Arabia.
Funding information: The authors sincerely acknowledge the Researchers Supporting Project for funding this work number (RSP-2021/232) at King Saud University, Riyadh, Saudi Arabia.
Author contributions: Conceptualization: R.A.A. and A.R.A.; methodology: R.A.A. and A.A.A; validation: A.R.A., R.A.A., and A.M.M.; formal analysis: A.M.M.; investigation: R.A.A. and A.R.A.; data curation: A.R.A.; writing – original draft preparation: R.A.A. and A.R.A.; writing – review and editing: R.A.A., A.R.A., and A.M.M.; visualization: A.R.A., A.A.A., and M.Q.A.; supervision: A.R.A. and M.Q.A. All authors have read and agreed to the published version of the manuscript.
Conflict of interest: The authors declare that they have no conflict of interest.
Ethical approval: All experiments were conducted according to the Guidelines of the Institutional Animal Care and Use Committee of the Department of Animal Production, King Saud University.
Data availability statement: The authors confirm that the data supporting the findings of this study are available within the article.
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