In vitro Gas production and rumen fermentation for rations containing increasing levels of Panicum maximum cv. Mombasa with or without Spirulina

In vitro Gas production and rumen fermentation for rations containing increasing levels of Panicum maximum cv. 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Mombasa with or without Spirulina mohamed meteab, Mahmoud Mohamed Khorshed, Abeer Mohamed Abd El-halim El-Essawy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3946950/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jan, 2025 Read the published version in Tropical Animal Health and Production → Version 1 posted 5 You are reading this latest preprint version Abstract This study aimed to evaluate the impact of replacing alfalfa hay with Panicum maximum hay, with or without ascending levels of Spirulina supplementation on in-vitro gas production and fermentation parameters. Isocaloric and isonitrogenous experimental diets were formulated to contain 40% roughage + 60 % concentrate, ascending replacement with Panicum maximum hay at levels 0, 25%, 50%, 75%, and 100% take place in rations R1, R2, R3, R4, and R5, respectively. Each ration supplemented with incremental levels 0, 0.5, 1.5, 2, 2.5, and 3kg spirulina per Ton of feed. Results indicated that the potential of gas production after 24 h was linearly increased by increasing the level of Spirulina. The control group (R1) recorded the highest values of gas production, while it was the lowest at (R5) (p<.0001). In vitro degradability of dry and organic matter was negatively affected by the increment of Panicum maximum and Spirulina levels and vice versa, until 2 kg/ton of Spirulina (p<.0001). The addition of Spirulina significantly (p<.0001) increased total volatile fatty acids (TVFA) and N-NH3 concentration, until 2Kg/ton, while the addition of Panicum maximum hay significantly increased N-NH3 concentration, until it reached at (R4). As a result, the best mixing ratio between Alfalfa and Panicum maximum hays was recorded at R2. In conclusion, the substitution of alfalfa hay with 25% Panicum maximum hay (R2) and incorporation of Spirulina as feed additive (2Kg/ton) could offer a good solution in dry periods to improve the quality of Panicum maximum and can be used to maximize degradability and ruminal fermentation parameters. Panicum maximum cv. Mombasa Alfalfa Spirulina in vitro gas production rumen fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Water scarcity and soil salinity are major constraints for forage crop production in Egyptian deserts. Cultivating plants adapted to these conditions can increase the potential yield of high-quality forage crops. Desert areas in Egypt suffer from a shortage of fodder, especially in the summer, because of water shortage and soil salinity. Feed shortage remains one of the main constraints to developing animal production in Africa. One of the alternative solutions to improve livestock feeding, and thus productivity, could be the cultivation of forages that can tolerate salinity and water shortages. So, Panicum maximum presents one of the greatest potential productions of dry matter in subtropical and tropical environments. The use of Panicum maximum is a possible alternative for use as a source of energy, due to its high yield as well as seed propagation (Jank et al. ,2013). Panicum maximum, which is like other tropical grasses, showed a rapid decline in the crude protein with age, quality can be improved by mixing it with other rich legumes. Alfalfa ( Medicago sativa L .) is a perennial legume widely cultivated to provide high-quality forage in the form of hay, silage, and to a lesser extent as a grazing crop, but Alfalfa has higher water requirement compared to other crops (Li et al., 2023 ). So, intercropping Panicum maximum with forage legumes improves the quality of the fodder (Alasa et al., 2014 ), reduces water consumption, and reduces the feed cost for small breeders, particularly in desert areas. Replacing alfalfa hay with increasing amounts of Panicum hay may cause a decrease in the mixture's nutritive value. However, supplementing the rations with a digestibility enhancer can improve efficiency. Algae are gaining interest as alternative sources of micronutrients due to their rich functional metabolite content including polysaccharides, proteins, peptides, amino acids, lipids, polyphenols, and minerals (Brown et al., 2014 ). Han and McCormick ( 2014 ) suggested that most algae are considered high protein supplements with high-fat content, soluble carbohydrates, macro and micro minerals, and polyunsaturated fatty acids (PUFA) while having low fiber content. Thus, they are suitable for use as feed additives with health-improving characteristics for livestock. Also, this supplement contains pigments, antioxidants, provitamins, vitamins, growth factors, and all the essential nutrients. It can help to improve the health and quality of animal products, as reported by Kotrbáček et al. ( 2015 ). Spirulina platensis ( Arthrospira sp .) is a natural antioxidant and immune stimulant that is safe for both humans and animals. It is also more cost-effective than synthetic products and has fewer side effects (Abdel-Daim et al., 2013 ). Holman et al. ( 2012 ) have reported that dietary supplementation with Spirulina can have a positive impact on animal health and productivity. However, still limited research on the use of Spirulina platensis as a feed additive in ruminant feeding was reported. The present in vitro experiment aimed to evaluate the impact of gradually replacing alfalfa hay with Panicum maximum hay, with or without ascending level of Spirulina extract supplementation, on in vitro gas production and fermentation parameters. Materials and Methods This research was conducted in the Animal and Poultry Nutrition Department labs, Desert Research Center and Animal Nutrition Research lab, Animal Production Department Faculty of Agriculture, Ain Shams University. Forage Materials and additives: Alfalfa hay ( Medicago sativa ) was harvested near to flowering, and 1 meter height from Maryout Research Station. Panicum maximum cv. Mombasa , was harvested before it could flower and had also grown to a higher 1meter from Siwa Research Station farm. Both stations are affiliated to the Desert Research Center (DRC), Ministry of Agriculture and Land Reclamation, Cairo, Egypt. The collected plants were sun-dried for six days until hays were obtained. Alfalfa hay ( Medicago sativa ) and Panicum hays were finely crushed and preserved in plastic bags to prevent the absorption of moisture for further proximate analysis and in vitro experiments. Spirulina extract with 100% purity in powder form was purchased from the Algal Biotechnology Unit in Dokki, Giza, Egypt. E XPERIMENTAL PROCEDURES : 1. Preparation of experimental rations and treatments: An experiment (In vitro) was conducted using an isocaloric and isonitrogenous rations, consisting of a roughage-to-concentrate ratio of 40–60%, respectively. The control ration contained 100% alfalfa hay as roughage portion while ascending replacement with Panicum maximum hay (P) at level 25%, 50%, 75% and 100% take place in rations R2, R3, R4 and R5, respectively. The ration formulation and chemical compositions of the total ration are presented in Table (1). Additionally, Spirulina extract was added to the experimental rations in ascending levels of 0 (control), 1, 1.5, 2, 2.5, and 3 Kg/ton feed. 2. Rations Formulation 500g of each fodder sample was manually chopped using a machete into particles (2–5 cm) and dried at 60 o C until constant weight in a ventilated drying oven (Qallenhamk OVE25010G). After drying, samples were crushed using a hammer mill to pass through sieve size 1 mm, and then preserved in plastic sachets. Five rations × six levels of Spirulina extract (i.e., 30 treatments) were formulated in this study. 3. Analytical Methods 3.1. Chemical Analysis Proximate chemical analyses were conducted on the experimental samples for crude protein (CP), crude fiber (CF), ether extract (EE), and total Ash as per the procedures laid down by the Association of Official Analytical Chemists AOAC ( 1990 ). neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to sequential procedures of Van Soest et al. ( 1991 ) using Ankom 200 (Ankom Technology Corp., Fairport, NY) filter bag technique. 3.2. Fatty acids analysis: Gas- Liquid Chromatography (GLC) analysis of saponifiable fraction: The extracted fatty acids of each plant and the standards were converted to the corresponding methyl esters using an ethereal solution of diazomethane (Farag et al., 1980 ). The methyl esters of the fatty acids were analyzed with Agilent – 8890 gas – chromatographic apparatus. The fraction of fatty acids methyl esters was conducted using (GLC) column. Peak identification was performed by comparing the relative retention time of each compound with those of standard materials. The relative proportions of each compound were estimated as the ratio of the partial areas to the total area as mentioned by Fryer et al., ( 1960 ), Nelson et al., ( 1969 ), and Farag et al., ( 1980 ). The following conditions were used: Apparatus: Agilent – 8890 GC System. Column: HP-5 Capillary Tubing (15 m x 0.25 mm) packed with fused silica with Film Thickness 0.25µm coated with (5%-Phenyl)-methylpolysiloxane. Column temp.: 300°C. Injector temp.: 250°C. Detector temp.: 250°C. Rate of temp.: 3°C / minute Attenuation: 32 x 10 − 2 Chart speed: 2 mm/minute. Initial temp.: 100°C. Final temp.: 240°C. Initial time: 1 minute. Final time: 20.8 minutes. 3.3. Phytochemical screening Qualitative phytochemical screening was conducted on the alcoholic extracts of alfalfa hay, Panicum maximum hay, and Spirulina extract. The presence of total tannins, saponins, and total phenols was determined using methods described by Makkar et al., ( 1993 ), Tava and Avato, ( 2006 ) (Table 3). Quantitative estimation for total tannins was carried out by gravimetric method (Makkar et al. ,1993), saponins and total phenols (Tava and Avato, 2006 ) (Table 3). 4. In vitro gas production experiment: An in vitro batch culture technique was applied as described by Szumacher-Strabel et al. ( 2002 ). The experiment was carried out in triplicates for each treatment. About 500 ± 3 mg of feed (according to the experimental design) was weighed into 120 ml incubation vessels using an electric balance (KERN 770). At least 3 blanks were included, and Alfalfa hay was used in triplicate as the standard. The rumen fluid was obtained from adult sheep immediately after slaughter at Al-Marg slaughterhouse. The collected rumen fluid was mixed and squeezed through a 4-layer cheesecloth into a bottle (2L) with an O2-free headspace and maintained in an insulated container containing warm water 39 o c, then immediately transported to the laboratory. Buffer solution was made up of 9.8 g NaHCO 3 , 2.44 g Na 2 HPO 4 , 0.57 g KCl, 0.47 g NaCl, 0.12 g MgSO 4 .7H 2 O, and 0.16 g CaCl 2 .2H 2 O per liter of distilled water. It's important to note that CaCl2 must be added only after all the other components have completely dissolved. During the warming and reducing step, urea is added to the buffer at a rate of 1.0 gm/liter. Rumen fluid was mixed with buffer solution in a ratio of 1:4 (v/v) to use as a source of inoculum. Each vessel was filled with 50 ml of the incubation medium and dispensed anaerobically before being closed. The samples were then incubated at 39°C for 24 hours. Finally, the vessels are randomly distributed in the rack in the incubator and the tubes are swirled at least twice daily. 4.1. Total gas production Volumes of gas produced were measured after 24 h using a 100 ml glass syringe. To calculate the accurate volume of gas produced, the following formula was used: GP (ml/sample) = V24 - GP0, where V24 represents the volume of gas produced after 24 hours of incubation, and GP0 represents the volume of gas produced by the blank after 24 hours of incubation. 4.2. Evaluation of in Vitro Degradability of Dry Matter (DMD), Neutral detergent fiber (NDFD) and Acid detergent fiber (ADFD) After 24 hours of incubation, the gas production and pH value were recorded. Then, the filtration process was performed on each of the 120 ml vessels using a filter bag (F57 Ankom). Ammonia and total volatile fatty acids (TVFs) concentrations were determined in the liquid part. After the filtration process the filter bags were dried at 105° C for 3 h in an oven (Qallenhamk OVE25010G) to estimate residual DM, NDF and ADF. The DMD, NDFD and ADFD were calculated as the difference between the weight of the incubated substrate and the weight of non-degraded residue at the end of incubation, according to the following formula (Van Soest et al., 1991 ): IVD (%) =((R-P))/ R ×100 Where R = Weigh the sample inside. P = The true weight of the out sample. 4.3. In vitro rumen fermentation end-products The pH of rumen liquor was immediately recorded using pH meter (Gallen Kamp pH Stick pH K-120 – B). Rumen liquor samples were analyzed to determine ammonia concentration, (NH 3 ) by Nessler’s method modified by Szumacher- Strabel et al. (2002) and total volatile fatty acids (TVF’s) by steam distillation according to Warner ( 1964 ). 4.4. Calculated parameters: After 24 hour - incubation, the gases produced and corrected by gases of the blank tubes were used to calculate the: In vitro digestibility of organic matter, using the following regression equation (Lemoufouet et al., 2019 ): OMD (%) = 14.88 + 0.889 GP + 0.45CP + 0.065 Ash. The content of the Metabolizable energy (ME) was calculated according to the following equation (Lemoufouet et al., 2019 ): ME (MJ/kg DM) = 2.20 + 0.136GP + 0.057CP. Short chain fatty acids (SCFA) were calculated as described by Getachew et al. , (2000): SCFA= (0.0222GP)-0.00425. Where, GP = quantity of gas produced for 200 mg DM of sample after 24 hours of incubation, CP = crude proteins. Statistical Analyses The data were statistically analyzed using the statistical analysis system SAS software (V.9.3, SAS Inst. In., Cary, NC, USA,2011) Separation among means was carried out according to Duncan’s multiple-range test (Duncan, 1955 ). The collected data were subjected to the analysis of variance with two ways with interaction analysis model according to the General Linear Model. The statistical model was as follows: Y ij = µ + α I + βj + (α β) I J + E ij Where, Y ij = Observation on the ration subjected to factors I and J; µ = general average; α I = effect of the type of Hay I; βj = effect of the Algae J; eij = residual error on the ruminal liquid subjected to factors I and J; (α β) I J = effect of the interaction between factors I and J. Results Nutrient Degradability and gas production after 24 hours of incubation The data presented in Table 4 and Fig. (1A, 1B, 1C) show the effect of substituting alfalfa hay with increasing levels of panicum maximum hay on in vitro DM, NDF, and ADF degradability. The data showed a significant (p < .0001) decrease in DMD with the ascending substitution of alfalfa hay with Panicum maximum hay, with no significant difference between the control ratio (100% alfalfa hay, R1) and the ration containing 75% alfalfa hay + 25% Panicum hay (R2), and both treatments were higher than the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Also, the ration contained 50% alfalfa hay + 50% Panicum hay (R3) recorded higher in vitro DM, NDF, and ADF degradability than the ration contained 100% Panicum (R5). Concerning the effect of spirulina supplementation levels, the data presented in Table (4) and Fig. (2A, 2B, 2C) demonstrate that the degradation of DM, NDF, and ADF increase as the concentration of spirulina supplementation increases from 0.5 kg/ton to 2.0 kg/ton. However, the degradation of DM, NDF, and ADF remain unchanged at 3 kg/ton, showing similar values as the control (un-supplemented) group. The data of Table (4) and Fig. (3A, 3B, 3C) show that there is a significant (p < .0001) decrease in gas production parameters (GP per g DM, g OM, and g DDM) as the amount of Panicum maximum hay substituted for alfalfa hay. The control ratio (R1), which contained 100% alfalfa hay as roughage portion, recorded higher gas production parameters compared to the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Both rations containing 25% and 50% Panicum hay as a substitute for alfalfa hay (R2 and R3) were higher than the ration containing 75% and 100% Panicum, with no significant differences between R2 and R3. Concerning to effect of the spirulina supplementation levels, the results displayed in Table (4) and Fig. (4A, 4B, 4C) reveal increase (p < .0001) in gas production parameters (GP per g DM, g OM, and g DDM) as the concentration of spirulina supplementation increase. The data indicates that the rations supplemented with 3 kg spirulina per ton of feed had the highest (p < .0001) gas production per g DM and g OM compared to the other supplemented rations (2.5, 2, 1.5, 1, 0.5, and 0 kg spirulina per ton of feed). The rations supplemented with 2.5 and 2 kg spirulina per ton of feed also showed higher gas production per g DM and g OM compared to the control rations (not supplemented). However, all the rations supplemented with 3, 2.5, and 2 kg spirulina per ton of feed recorded higher gas production per g DMD compared to the rations supplemented with 1.5, 1, 0.5, and zero kg spirulina per ton of feed, with no significant differences among them. Fermentation and Calculated parameters after 24 hours The data of Table 5 and Fig. (5A, 5B, 5C) showed the impact of substituting alfalfa hay with Panicum hay on in vitro serum parameters. The in vitro serum pH values of the different experimental rations were within the normal range, and no abnormal values were observed Fig. (5 A). However, the data indicated a decrease in pH value with an increase in the replacement of alfalfa hay with Panicum hay. The highest pH value was recorded for the ration containing 25% Panicum and 75% alfalfa hay (R2), and it decreased to the lowest pH value for the rations containing 100% Panicum (R5), with no significant differences between control ration (R1) and R5. The data presented in Table (5) and Fig. (5B) showed no significant differences among the rations containing 25%, 50%, and 75% Panicum as a substitute for alfalfa hay in ammonia concentration after 24 h of in vitro fermentation, and all three rations had higher ammonia concentrations than the control ration (R1) and the ration containing 100% Panicum (R5) and the lowest significant ammonia concentration was recorded for R5. The data presented in Table (5) and Fig. (5C) showed a significant decrease in TVFA's with the ascending substitution of alfalfa hay with Panicum hay. The control ratio (R1) was higher than the values for R2, R3, R4, and R5. Regarding the impact of ascending levels of spirulina supplementation on in vitro serum pH value, ammonia concentration and volatile fatty acid (TVFA) concentration, the data of Table (5) and Fig. (6A) showed significantly (P < .0001) increase in pH value as the level of spirulina supplementation increased up to 1kg spirulina per ton of feed. There were no significant differences among spirulina supplementation levels of 1, 1.5, and 2 Kg/ton of feed, but the pH value decreased with 2.5 and 3 kg spirulina supplementation per ton of feed. The data of ammonia concentration in Table (5) and Fig. (6B) showed that it increased with an increase in spirulina supplementation until 2 Kg/ton, and then decreased again to a lower concentration at 3 kg/ton of feed. As for the volatile fatty acid’s concentration, the data in Table (5) and Fig. (6c) showed that control ration recorded the highest TVFAs concentration compared to all levels of supplementation. However, there was a significant increase from 0.5 kg up to 2 kg/ton of feed, followed by decrease until 3kg spirulina supplementation per ton of feed. The data of Table (5) and Fig. (7A, 7B, 7C) show that there is a significant (p < .0001) decreased in organic matter degradability (OMD) Fig. (7A), ME content Fig. (7B) and SCHFA Fig. (7C) as the amount of Panicum maximum hay substituted for alfalfa hay increase. The control ratio (R1), which contains 100% alfalfa hay as roughage portion, recorded higher calculated parameters as OMD, ME, and SCHFA compared to the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Both rations contained 25% and 50% Panicum hay as a substitute for alfalfa hay (R2 and R3) were higher OMD, ME, and SCHFA than the ration containing 75% and 100% Panicum, with no significant differences between R2 and R3. Supplementing the experimental rations with ascending levels of spirulina showed a positive relationship between the concentration of spirulina and the calculated parameters (OMD, ME and SCFA) until the highest values (40.51,7.16 and 0.64, respectively), which were produced with the highest concentration of spirulina 3 kg/ton, Table (5) and Fig. (8A, 8B, 8C). Discussion Nutrient Degradability and gas production after 24 hours of incubation Although the rations were formulated to be isocaloric and isonitrogenous, the decrease in degradability with the ascending substitution of alfalfa hay with Panicum maximum hay, this may be due to 1) the increase in CF, NDF, ADF and Ash content as substituting rate increased parallel with the decrease in OM contents (table 1), 2) the higher content of tannins and total phenols for Panicum hay compared to alfalfa hay (table 3). In this connection, Cilliers and Van der Merwe, ( 1993 ) concluded that the decrease in Panicum digestibility is associated with a decrease in N content and an increase in NDF, ADF, and ADL contents. Indeed Zhong et al. ( 2021 ) reported that dry matter degradation increased in feedstuff with low levels of lignin and NDF because high levels of lignin may resist fiber degrading microorganisms’ activity in the rumen. A similar trend was observed by Ramirez et al. ( 2009 ). Also, Yasmin et al., ( 2008 ) found that the presence of secondary metabolites or anti-nutritional factors (ANFs) in animal diets affects nutrient digestion and absorption negatively where the studied panicum maximum contained high levels of tannins (table 3). Anti-nutritional factors (ANFs) such as tannins, glucosides, flavonoids, alkaloids, terpenoids, cyanides, coumarin, nitrate, oxalate, and organic acids have different effects on animal performance. High levels of ANFs in an animal’s diet prevent the growth of microbes and fungi in the rumen, thus affecting the rate of nutrient digestion and absorption (Acamovic and Brooke 2005). The higher gas production parameters observed in the control ratio may be due to the fact that alfalfa is a legume that contains higher CP% and lower contents of both fiber fractions NDF, ADF, and Ash compared to Panicum maximum hay (Table 1). Although the rations were formulated to be isocaloric and isonitrogenous, there is a positive relationship between Dry Matter, NDF, and ADF Degradability (Table 1) and gas production. This relationship has been confirmed by Blummel et al. ( 1999 ), who reported that in-vitro gas production is an indirect measurement of positive changes in dry matter degradability. In addition, Blummel and Orskov ( 1993 ) observed a positive correlation between DM disappearance and gas production. Blummel et al. ( 1999 ) reported that gas production is closely related to SCFA production, which will be discussed later (Table 5). Concerning to effect of the spirulina supplementation levels on increased degradability, this may be due to several factors, including the high nutrient density of Spirulina and the stimulation of extracellular enzyme secretion by gut microflora (Tovar-Ramírez et al., 2002 ). Spirulina also contains vitamins, minerals, essential fatty acids, amino acids, and other nutrients that promote faster growth (Costa et al., 2016 ). Additionally, Spirulina reduces rumen protein degradation and alters bacterial community composition, leading to an increase in the efficiency of rumen microbial crude protein production in steers (Panjaitan et al., 2010 ). The increase in gas production with spirulina supplementation levels may be due to two reasons. Firstly, the degradability rates increase as spirulina supplementation increased (Table 4), which activate the microflora in the rumen, leading to an increment in the production of microbial protein and an increase in fiber degradability (Costa et al., 2016 ). Secondly, spirulina contains all essential amino acids and has a great impact on digestibility (Lafarga et al., 2020 ; Chia et al., 2019 ). Fermentation and Calculated parameters after 24 hours The level of ammonia in rumen liquor is an indicator of nutritional conditions, as many types of rumen micro-organisms use ammonia as a source of nitrogen. Mixing Alfalfa and Panicum maximum showed improved pH and NH3 than other treatments containing either of them alone. This may be attributed to complementarity between Alfalfa and Panicum maximum, leading to an improvement in the quality of the fodder, as confirmed by Alasa et al, ( 2014 ) when concluded that intercropping grasses (Panicum maximum) with forage legumes ( lablab purpureus ) improve the quality of the fodder. Additionally, there was an increase in degradability rate and gas production as substituting levels increased (Table 4). Elevated values of ammonia were associated with an increased percentage of Panicum in rations (R2 and R5). This may be attributed to an increased in CP content of the concentrate ration portion through increasing protein sources, especially Soya meal (highly degradable protein source, Table 1) to compensate for the low CP content of Panicum to get iso protein rations. This is in line with Jahan et al. ( 2018 ), who reported that many nitrogenous substances in the high concentration diet may be the possible reason for increasing NH3-N concentration. Nousiainen et al. ( 2009 ) also supported this explanation, where they found that increased concentrate protein feeding improved whole-diet digestibility in cows in a curvilinear manner because of positive effects on the ruminal environment for fiber digestion, but the magnitude of the effect was not very large. Regarding the volatile fatty acids, carbohydrates are fermented by a variety of bacteria in the rumen and transformed into volatile fatty acids (VFA) by the corresponding enzymes (Wang et al. 2020 ). The data showed a significant decrease in TVFA with the ascending substitution of alfalfa hay with Panicum hay. The control ratio (R1) was higher than the values for R2, R3, R4, and R5. This may be due to the low content of fiber fractions (NDF and ADF) and Ash in alfalfa hay compared to Panicum hay, with comparable contents of carbohydrates (NFC contents) in both alfalfa and Panicum hays (Table 1). Additionally, there was an increase in the degradability of DM, NDF and ADF as well as gas production (Table 4), as well as an increase in OMD, ME, and SCFA (Table 5). All these results improve the fermentation process, leading to an increment of VFA. The inclusion of 2 kg/ton spirulina resulted in a significant (P < .0001) increase in pH value and ammonia concentration, without affecting the value of TVFA concentration. These results may be due to the high nutrient density of Spirulina and the stimulation of extracellular enzyme secretion by gut microflora (Tovar-Ramírez et al., 2002 ). Spirulina also contains vitamins, minerals, essential fatty acids, amino acids, and other nutrients that promote faster growth (Costa et al., 2016 ). Similar observations were reported by Panjaitan et al., ( 2010 ) feeding Spirulina platensis as a supplement along with low CP containing guinea grass (Panicum maximum) hay improved efficiency of microbial protein production in cattle. Panjaitan et al. ( 2015 ) increased microbial protein synthesis and rumen ammonia-N in a quadratic fashion with increasing Spirulina inclusion in the diet. The differences between this study and our findings may be due to the differences in the experimental condition, ration composition and levels of supplementation as well as type of animals. It’s clear that there is an inverse relationship between the ratio of Panicum maximum in ration, and the calculated parameters (OMD, ME and SCFA). This may be attributed to 1) decrease CP and EE contents in rations with panicum hay (table1), this observation was in line with the reported work of Blummel and Orskov ( 1993 ) reported that, there is a positive correlation between the calculated metabolizable energy from in vitro gas production together with CP and EE contents as well as ME value of conventional feeds measured in vivo. 2) Decrease OM contents and increased of CF, NDF and ADF in rations with panicum hay (table1), The low OMD and ME obtained for the level of panicum in the treatments increased might probably connected with the presence of high fiber (Blummel and Orskov, 1993 ) especially in treatment R5 (panicum 100%). 3) Relationship between the SCFA and gas production (table 4), similar observation was reported by Getachew et al . (2000) the SCFA estimated from in vitro gas production, has been widely used to evaluate the energy value of several classes of feed. Blummel and Orskov ( 1993 ) who suggested that gas production from different classes of feeds incubated in- vitro in buffered rumen fluid was closely related to the production of SCFA which was based on carbohydrate fermentation. The present findings indicated that all in-vitro parameters are affected by the concentration of panicum in ration. Supplementing the experimental rations with ascending levels of spirulina showed a positive relationship between the concentration of spirulina and the calculated parameters (OMD, ME and SCFA) until the highest values (40.51,7.16 and 0.64, respectively), which were produced with the highest concentration of spirulina 3 kg/ton, Table (5) and Fig. (8A, 8B, 8C). Referring to increased values of calculated ME with increasing spirulina concentration proved that Spirulina specifically 3 kg/ton has a good potential to enhance energy content of roughage feedstuffs. Moreover, as the level of SCFA, which is an indicator of the energy content of the ration, its production increased with increasing spirulina concentration in ration. This is due to the fact that Spirulina species, known as cyanobacteria, contain the essential fatty acids, linoleic acid (LA, 18:2 delta-9,12) (table 2) and gamma-linolenic acid (GLA, 18:3 delta-6,9,12) (Gupta et al., 2008 ), high quality proteins, carbohydrates, vitamins (B1, B2, tocopherols), minerals (sodium, potassium, calcium, magnesium, phosphorus, iron), carotenes (especially beta-carotene), chlorophyll a, phycocyanin, and some phenolic acids (Gupta et al., 2008 ). Consequently, rations associated with spirulina makes it possible to raise digestion rates, the energy supply and mineral elements to the microbes present in the rumen fluid, thus improve their growth and activity, and thus, increase the rates of degradability of OMD, ME and SCFA. Conclusion Replacing alfalfa hay with ascending level of Panicum hay may lead to ascending decrease in the mixture nutritive value. The best mixing of Alfalfa hay with Panicum maximum cv. Mombasa was 75% alfalfa hay with 25% Panicum maximum cv. Mombasa (R2). Incorporation of Spirulina as feed additive (2Kg/ton) could offer a good solution to improve the quality of Panicum maximum and alfalfa hay, can be used to maximize degradability and ruminal fermentation parameters. Declarations Acknowledgements The authors acknowledge the role of the Desert Research Center and Faculty of Agriculture, Ain Shams University, in supporting the research. Author contributions Conceptualization: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by (Meteab, M.I), (Abeer, M. EL-Essawy) and (El-Bordeny N. E). Investigation and methodology were performed by (Khorshed, M.M), (Abeer, M. EL-Essawy), (Nassar, M.S) and (El-Bordeny N. E). The first draft of the manuscript was written by (Meteab, M.I) and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Open-access funding is provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Data availability : The datasets generated and analyzed during the current study will be provided upon reasonable request from the corresponding author. Ethics approval Experimental procedures were conducted per the Animal Ethics Committee guidelines of the animal and Poultry Production Division of the Desert Research Center (Egypt), (approval No. 2022–0179). Consent for publication Not applicable. Competing interests: The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3946950","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276332519,"identity":"9aa4b292-1a5c-40cf-baa9-c9eea33c2075","order_by":0,"name":"mohamed 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12:22:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":224914,"visible":true,"origin":"","legend":"","description":"","filename":"TablesTROPD2400259.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3946950/v1/538c37a7a70ae8416d40bcf1.pdf"}],"financialInterests":"","formattedTitle":"In vitro Gas production and rumen fermentation for rations containing increasing levels of Panicum maximum cv. Mombasa with or without Spirulina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWater scarcity and soil salinity are major constraints for forage crop production in Egyptian deserts. Cultivating plants adapted to these conditions can increase the potential yield of high-quality forage crops. Desert areas in Egypt suffer from a shortage of fodder, especially in the summer, because of water shortage and soil salinity. Feed shortage remains one of the main constraints to developing animal production in Africa. One of the alternative solutions to improve livestock feeding, and thus productivity, could be the cultivation of forages that can tolerate salinity and water shortages. So, Panicum maximum presents one of the greatest potential productions of dry matter in subtropical and tropical environments. The use of Panicum maximum is a possible alternative for use as a source of energy, due to its high yield as well as seed propagation (Jank \u003cem\u003eet al.\u003c/em\u003e,2013). Panicum maximum, which is like other tropical grasses, showed a rapid decline in the crude protein with age, quality can be improved by mixing it with other rich legumes. Alfalfa (\u003cem\u003eMedicago sativa L\u003c/em\u003e.) is a perennial legume widely cultivated to provide high-quality forage in the form of hay, silage, and to a lesser extent as a grazing crop, but Alfalfa has higher water requirement compared to other crops (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). So, intercropping Panicum maximum with forage legumes improves the quality of the fodder (Alasa et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), reduces water consumption, and reduces the feed cost for small breeders, particularly in desert areas. Replacing alfalfa hay with increasing amounts of Panicum hay may cause a decrease in the mixture's nutritive value. However, supplementing the rations with a digestibility enhancer can improve efficiency. Algae are gaining interest as alternative sources of micronutrients due to their rich functional metabolite content including polysaccharides, proteins, peptides, amino acids, lipids, polyphenols, and minerals (Brown et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Han and McCormick (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) suggested that most algae are considered high protein supplements with high-fat content, soluble carbohydrates, macro and micro minerals, and polyunsaturated fatty acids (PUFA) while having low fiber content. Thus, they are suitable for use as feed additives with health-improving characteristics for livestock. Also, this supplement contains pigments, antioxidants, provitamins, vitamins, growth factors, and all the essential nutrients. It can help to improve the health and quality of animal products, as reported by Kotrb\u0026aacute;ček et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Spirulina platensis (\u003cem\u003eArthrospira sp\u003c/em\u003e.) is a natural antioxidant and immune stimulant that is safe for both humans and animals. It is also more cost-effective than synthetic products and has fewer side effects (Abdel-Daim et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Holman et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) have reported that dietary supplementation with Spirulina can have a positive impact on animal health and productivity. However, still limited research on the use of Spirulina platensis as a feed additive in ruminant feeding was reported. The present in vitro experiment aimed to evaluate the impact of gradually replacing alfalfa hay with Panicum maximum hay, with or without ascending level of Spirulina extract supplementation, on in vitro gas production and fermentation parameters.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis research was conducted in the Animal and Poultry Nutrition Department labs, Desert Research Center and Animal Nutrition Research lab, Animal Production Department Faculty of Agriculture, Ain Shams University.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eForage Materials and additives:\u003c/h2\u003e \u003cp\u003eAlfalfa hay (\u003cem\u003eMedicago sativa\u003c/em\u003e) was harvested near to flowering, and 1 meter height from Maryout Research Station.\u003c/p\u003e \u003cp\u003ePanicum \u003cem\u003emaximum cv. Mombasa\u003c/em\u003e, was harvested before it could flower and had also grown to a higher 1meter from Siwa Research Station farm. Both stations are affiliated to the Desert Research Center (DRC), Ministry of Agriculture and Land Reclamation, Cairo, Egypt. The collected plants were sun-dried for six days until hays were obtained.\u003c/p\u003e \u003cp\u003eAlfalfa hay (\u003cem\u003eMedicago sativa\u003c/em\u003e) and Panicum hays were finely crushed and preserved in plastic bags to prevent the absorption of moisture for further proximate analysis and in vitro experiments. Spirulina extract with 100% purity in powder form was purchased from the Algal Biotechnology Unit in Dokki, Giza, Egypt.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE XPERIMENTAL PROCEDURES\u003c/b\u003e:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1. Preparation of experimental rations and treatments:\u003c/h2\u003e \u003cp\u003eAn experiment (In vitro) was conducted using an isocaloric and isonitrogenous rations, consisting of a roughage-to-concentrate ratio of 40\u0026ndash;60%, respectively. The control ration contained 100% alfalfa hay as roughage portion while ascending replacement with Panicum maximum hay (P) at level 25%, 50%, 75% and 100% take place in rations R2, R3, R4 and R5, respectively. The ration formulation and chemical compositions of the total ration are presented in Table\u0026nbsp;(1). Additionally, Spirulina extract was added to the experimental rations in ascending levels of 0 (control), 1, 1.5, 2, 2.5, and 3 Kg/ton feed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2. Rations Formulation\u003c/h2\u003e \u003cp\u003e500g of each fodder sample was manually chopped using a machete into particles (2\u0026ndash;5 cm) and dried at 60\u003csup\u003eo\u003c/sup\u003eC until constant weight in a ventilated drying oven (Qallenhamk OVE25010G). After drying, samples were crushed using a hammer mill to pass through sieve size 1 mm, and then preserved in plastic sachets. Five rations \u0026times; six levels of Spirulina extract (i.e., 30 treatments) were formulated in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3. Analytical Methods\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e3.1. Chemical Analysis\u003c/h2\u003e \u003cp\u003eProximate chemical analyses were conducted on the experimental samples for crude protein (CP), crude fiber (CF), ether extract (EE), and total Ash as per the procedures laid down by the Association of Official Analytical Chemists AOAC (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to sequential procedures of Van Soest et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) using Ankom\u003csup\u003e200\u003c/sup\u003e (Ankom Technology Corp., Fairport, NY) filter bag technique.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Fatty acids analysis:\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eGas- Liquid Chromatography (GLC) analysis of saponifiable fraction:\u003c/h2\u003e \u003cp\u003eThe extracted fatty acids of each plant and the standards were converted to the corresponding methyl esters using an ethereal solution of diazomethane (Farag et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). The methyl esters of the fatty acids were analyzed with Agilent \u0026ndash; 8890 gas \u0026ndash; chromatographic apparatus. The fraction of fatty acids methyl esters was conducted using (GLC) column. Peak identification was performed by comparing the relative retention time of each compound with those of standard materials. The relative proportions of each compound were estimated as the ratio of the partial areas to the total area as mentioned by Fryer et al., (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1960\u003c/span\u003e), Nelson et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1969\u003c/span\u003e), and Farag et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1980\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe following conditions were used:\u003c/h2\u003e \u003cp\u003eApparatus: Agilent \u0026ndash; 8890 GC System.\u003c/p\u003e \u003cp\u003eColumn: HP-5 Capillary Tubing (15 m x 0.25 mm) packed with fused silica with Film Thickness 0.25\u0026micro;m coated with (5%-Phenyl)-methylpolysiloxane.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn temp.: 300\u0026deg;C.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInjector temp.: 250\u0026deg;C.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetector temp.: 250\u0026deg;C.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRate of temp.: 3\u0026deg;C / minute\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttenuation: 32 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChart speed: 2 mm/minute.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial temp.: 100\u0026deg;C.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal temp.: 240\u0026deg;C.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial time: 1 minute.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFinal time: 20.8 minutes.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Phytochemical screening\u003c/h2\u003e \u003cp\u003eQualitative phytochemical screening was conducted on the alcoholic extracts of alfalfa hay, Panicum maximum hay, and Spirulina extract. The presence of total tannins, saponins, and total phenols was determined using methods described by Makkar et al., (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), Tava and Avato, (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) (Table\u0026nbsp;3). Quantitative estimation for total tannins was carried out by gravimetric method (Makkar \u003cem\u003eet al.\u003c/em\u003e,1993), saponins and total phenols (Tava and Avato, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) (Table\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4. In vitro gas production experiment:\u003c/h2\u003e \u003cp\u003eAn in vitro batch culture technique was applied as described by Szumacher-Strabel et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The experiment was carried out in triplicates for each treatment. About 500\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mg of feed (according to the experimental design) was weighed into 120 ml incubation vessels using an electric balance (KERN 770). At least 3 blanks were included, and Alfalfa hay was used in triplicate as the standard.\u003c/p\u003e \u003cp\u003eThe rumen fluid was obtained from adult sheep immediately after slaughter at Al-Marg slaughterhouse. The collected rumen fluid was mixed and squeezed through a 4-layer cheesecloth into a bottle (2L) with an O2-free headspace and maintained in an insulated container containing warm water 39\u003csup\u003eo\u003c/sup\u003ec, then immediately transported to the laboratory.\u003c/p\u003e \u003cp\u003eBuffer solution was made up of 9.8 g NaHCO\u003csub\u003e3\u003c/sub\u003e, 2.44 g Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.57 g KCl, 0.47 g NaCl, 0.12 g MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, and 0.16 g CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO per liter of distilled water. It's important to note that CaCl2 must be added only after all the other components have completely dissolved. During the warming and reducing step, urea is added to the buffer at a rate of 1.0 gm/liter. Rumen fluid was mixed with buffer solution in a ratio of 1:4 (v/v) to use as a source of inoculum. Each vessel was filled with 50 ml of the incubation medium and dispensed anaerobically before being closed. The samples were then incubated at 39\u0026deg;C for 24 hours. Finally, the vessels are randomly distributed in the rack in the incubator and the tubes are swirled at least twice daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e4.1. Total gas production\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eVolumes of gas produced were measured after 24 h using a 100 ml glass syringe. To calculate the accurate volume of gas produced, the following formula was used: GP (ml/sample)\u0026thinsp;=\u0026thinsp;V24 - GP0, where V24 represents the volume of gas produced after 24 hours of incubation, and GP0 represents the volume of gas produced by the blank after 24 hours of incubation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2. Evaluation of in Vitro Degradability of Dry Matter (DMD), Neutral detergent fiber (NDFD) and Acid detergent fiber (ADFD)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 24 hours of incubation, the gas production and pH value were recorded. Then, the filtration process was performed on each of the 120 ml vessels using a filter bag (F57 Ankom). Ammonia and total volatile fatty acids (TVFs) concentrations were determined in the liquid part. After the filtration process the filter bags were dried at 105\u0026deg; C for 3 h in an oven (Qallenhamk OVE25010G) to estimate residual DM, NDF and ADF. The DMD, NDFD and ADFD were calculated as the difference between the weight of the incubated substrate and the weight of non-degraded residue at the end of incubation, according to the following formula (Van Soest et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1991\u003c/span\u003e): IVD (%) =((R-P))/ R \u0026times;100\u003c/p\u003e \u003cp\u003eWhere R\u0026thinsp;=\u0026thinsp;Weigh the sample inside. P\u0026thinsp;=\u0026thinsp;The true weight of the out sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3. In vitro rumen fermentation end-products\u003c/h2\u003e \u003cp\u003eThe pH of rumen liquor was immediately recorded using pH meter (Gallen Kamp pH Stick pH K-120 \u0026ndash; B). Rumen liquor samples were analyzed to determine ammonia concentration, (NH\u003csub\u003e3\u003c/sub\u003e) by Nessler\u0026rsquo;s method modified by Szumacher- Strabel \u003cem\u003eet al.\u003c/em\u003e (2002) and total volatile fatty acids (TVF\u0026rsquo;s) by steam distillation according to Warner (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1964\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Calculated parameters:\u003c/h2\u003e \u003cp\u003eAfter 24 hour - incubation, the gases produced and corrected by gases of the blank tubes were used to calculate the:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIn vitro digestibility of organic matter, using the following regression equation (Lemoufouet et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e): OMD (%)\u0026thinsp;=\u0026thinsp;14.88\u0026thinsp;+\u0026thinsp;0.889 GP\u0026thinsp;+\u0026thinsp;0.45CP\u0026thinsp;+\u0026thinsp;0.065 Ash.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe content of the Metabolizable energy (ME) was calculated according to the following equation (Lemoufouet et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e): ME (MJ/kg DM)\u0026thinsp;=\u0026thinsp;2.20\u0026thinsp;+\u0026thinsp;0.136GP\u0026thinsp;+\u0026thinsp;0.057CP.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eShort chain fatty acids (SCFA) were calculated as described by Getachew \u003cem\u003eet al.\u003c/em\u003e, (2000): SCFA= (0.0222GP)-0.00425.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eWhere, GP\u0026thinsp;=\u0026thinsp;quantity of gas produced for 200 mg DM of sample after 24 hours of incubation, CP\u0026thinsp;=\u0026thinsp;crude proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eThe data were statistically analyzed using the statistical analysis system SAS software (V.9.3, SAS Inst. In., Cary, NC, USA,2011) Separation among means was carried out according to Duncan\u0026rsquo;s multiple-range test (Duncan, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1955\u003c/span\u003e). The collected data were subjected to the analysis of variance with two ways with interaction analysis model according to the General Linear Model.\u003c/p\u003e \u003cp\u003eThe statistical model was as follows:\u003c/p\u003e \u003cp\u003eY ij\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;α I\u0026thinsp;+\u0026thinsp;βj + (α β) I J\u0026thinsp;+\u0026thinsp;E ij\u003c/p\u003e \u003cp\u003eWhere, Y ij\u0026thinsp;=\u0026thinsp;Observation on the ration subjected to factors I and J; \u0026micro;\u0026thinsp;=\u0026thinsp;general average; α I\u0026thinsp;=\u0026thinsp;effect of the type of Hay I; βj\u0026thinsp;=\u0026thinsp;effect of the Algae J; eij\u0026thinsp;=\u0026thinsp;residual error on the ruminal liquid subjected to factors I and J; (α β) I J\u0026thinsp;=\u0026thinsp;effect of the interaction between factors I and J.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNutrient Degradability and gas production after 24 hours of incubation\u003c/h2\u003e \u003cp\u003eThe data presented in Table\u0026nbsp;4 and Fig.\u0026nbsp;(1A, 1B, 1C) show the effect of substituting alfalfa hay with increasing levels of panicum maximum hay on in vitro DM, NDF, and ADF degradability. The data showed a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;.0001) decrease in DMD with the ascending substitution of alfalfa hay with Panicum maximum hay, with no significant difference between the control ratio (100% alfalfa hay, R1) and the ration containing 75% alfalfa hay\u0026thinsp;+\u0026thinsp;25% Panicum hay (R2), and both treatments were higher than the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Also, the ration contained 50% alfalfa hay\u0026thinsp;+\u0026thinsp;50% Panicum hay (R3) recorded higher in vitro DM, NDF, and ADF degradability than the ration contained 100% Panicum (R5). Concerning the effect of spirulina supplementation levels, the data presented in Table\u0026nbsp;(4) and Fig.\u0026nbsp;(2A, 2B, 2C) demonstrate that the degradation of DM, NDF, and ADF increase as the concentration of spirulina supplementation increases from 0.5 kg/ton to 2.0 kg/ton. However, the degradation of DM, NDF, and ADF remain unchanged at 3 kg/ton, showing similar values as the control (un-supplemented) group.\u003c/p\u003e \u003cp\u003eThe data of Table\u0026nbsp;(4) and Fig.\u0026nbsp;(3A, 3B, 3C) show that there is a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;.0001) decrease in gas production parameters (GP per g DM, g OM, and g DDM) as the amount of Panicum maximum hay substituted for alfalfa hay. The control ratio (R1), which contained 100% alfalfa hay as roughage portion, recorded higher gas production parameters compared to the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Both rations containing 25% and 50% Panicum hay as a substitute for alfalfa hay (R2 and R3) were higher than the ration containing 75% and 100% Panicum, with no significant differences between R2 and R3. Concerning to effect of the spirulina supplementation levels, the results displayed in Table\u0026nbsp;(4) and Fig.\u0026nbsp;(4A, 4B, 4C) reveal increase (p\u0026thinsp;\u0026lt;\u0026thinsp;.0001) in gas production parameters (GP per g DM, g OM, and g DDM) as the concentration of spirulina supplementation increase. The data indicates that the rations supplemented with 3 kg spirulina per ton of feed had the highest (p\u0026thinsp;\u0026lt;\u0026thinsp;.0001) gas production per g DM and g OM compared to the other supplemented rations (2.5, 2, 1.5, 1, 0.5, and 0 kg spirulina per ton of feed). The rations supplemented with 2.5 and 2 kg spirulina per ton of feed also showed higher gas production per g DM and g OM compared to the control rations (not supplemented). However, all the rations supplemented with 3, 2.5, and 2 kg spirulina per ton of feed recorded higher gas production per g DMD compared to the rations supplemented with 1.5, 1, 0.5, and zero kg spirulina per ton of feed, with no significant differences among them.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFermentation and Calculated parameters after 24 hours\u003c/h2\u003e \u003cp\u003eThe data of Table\u0026nbsp;5 and Fig.\u0026nbsp;(5A, 5B, 5C) showed the impact of substituting alfalfa hay with Panicum hay on in vitro serum parameters. The in vitro serum pH values of the different experimental rations were within the normal range, and no abnormal values were observed Fig.\u0026nbsp;(5 A). However, the data indicated a decrease in pH value with an increase in the replacement of alfalfa hay with Panicum hay. The highest pH value was recorded for the ration containing 25% Panicum and 75% alfalfa hay (R2), and it decreased to the lowest pH value for the rations containing 100% Panicum (R5), with no significant differences between control ration (R1) and R5. The data presented in Table\u0026nbsp;(5) and Fig.\u0026nbsp;(5B) showed no significant differences among the rations containing 25%, 50%, and 75% Panicum as a substitute for alfalfa hay in ammonia concentration after 24 h of in vitro fermentation, and all three rations had higher ammonia concentrations than the control ration (R1) and the ration containing 100% Panicum (R5) and the lowest significant ammonia concentration was recorded for R5. The data presented in Table\u0026nbsp;(5) and Fig.\u0026nbsp;(5C) showed a significant decrease in TVFA's with the ascending substitution of alfalfa hay with Panicum hay. The control ratio (R1) was higher than the values for R2, R3, R4, and R5. Regarding the impact of ascending levels of spirulina supplementation on in vitro serum pH value, ammonia concentration and volatile fatty acid (TVFA) concentration, the data of Table\u0026nbsp;(5) and Fig.\u0026nbsp;(6A) showed significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;.0001) increase in pH value as the level of spirulina supplementation increased up to 1kg spirulina per ton of feed. There were no significant differences among spirulina supplementation levels of 1, 1.5, and 2 Kg/ton of feed, but the pH value decreased with 2.5 and 3 kg spirulina supplementation per ton of feed. The data of ammonia concentration in Table\u0026nbsp;(5) and Fig.\u0026nbsp;(6B) showed that it increased with an increase in spirulina supplementation until 2 Kg/ton, and then decreased again to a lower concentration at 3 kg/ton of feed. As for the volatile fatty acid\u0026rsquo;s concentration, the data in Table\u0026nbsp;(5) and Fig.\u0026nbsp;(6c) showed that control ration recorded the highest TVFAs concentration compared to all levels of supplementation. However, there was a significant increase from 0.5 kg up to 2 kg/ton of feed, followed by decrease until 3kg spirulina supplementation per ton of feed.\u003c/p\u003e \u003cp\u003eThe data of Table\u0026nbsp;(5) and Fig.\u0026nbsp;(7A, 7B, 7C) show that there is a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;.0001) decreased in organic matter degradability (OMD) Fig.\u0026nbsp;(7A), ME content Fig.\u0026nbsp;(7B) and SCHFA Fig.\u0026nbsp;(7C) as the amount of Panicum maximum hay substituted for alfalfa hay increase. The control ratio (R1), which contains 100% alfalfa hay as roughage portion, recorded higher calculated parameters as OMD, ME, and SCHFA compared to the values for other substituting levels (50%, 75%, and 100% for R3, R4, and R5, respectively). Both rations contained 25% and 50% Panicum hay as a substitute for alfalfa hay (R2 and R3) were higher OMD, ME, and SCHFA than the ration containing 75% and 100% Panicum, with no significant differences between R2 and R3. Supplementing the experimental rations with ascending levels of spirulina showed a positive relationship between the concentration of spirulina and the calculated parameters (OMD, ME and SCFA) until the highest values (40.51,7.16 and 0.64, respectively), which were produced with the highest concentration of spirulina 3 kg/ton, Table\u0026nbsp;(5) and Fig.\u0026nbsp;(8A, 8B, 8C).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNutrient Degradability and gas production after 24 hours of incubation\u003c/h2\u003e \u003cp\u003eAlthough the rations were formulated to be isocaloric and isonitrogenous, the decrease in degradability with the ascending substitution of alfalfa hay with Panicum maximum hay, this may be due to 1) the increase in CF, NDF, ADF and Ash content as substituting rate increased parallel with the decrease in OM contents (table 1), 2) the higher content of tannins and total phenols for Panicum hay compared to alfalfa hay (table 3). In this connection, Cilliers and Van der Merwe, (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) concluded that the decrease in Panicum digestibility is associated with a decrease in N content and an increase in NDF, ADF, and ADL contents. Indeed Zhong et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that dry matter degradation increased in feedstuff with low levels of lignin and NDF because high levels of lignin may resist fiber degrading microorganisms\u0026rsquo; activity in the rumen. A similar trend was observed by Ramirez et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Also, Yasmin et al., (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that the presence of secondary metabolites or anti-nutritional factors (ANFs) in animal diets affects nutrient digestion and absorption negatively where the studied panicum maximum contained high levels of tannins (table 3). Anti-nutritional factors (ANFs) such as tannins, glucosides, flavonoids, alkaloids, terpenoids, cyanides, coumarin, nitrate, oxalate, and organic acids have different effects on animal performance. High levels of ANFs in an animal\u0026rsquo;s diet prevent the growth of microbes and fungi in the rumen, thus affecting the rate of nutrient digestion and absorption (Acamovic and Brooke 2005). The higher gas production parameters observed in the control ratio may be due to the fact that alfalfa is a legume that contains higher CP% and lower contents of both fiber fractions NDF, ADF, and Ash compared to Panicum maximum hay (Table\u0026nbsp;1). Although the rations were formulated to be isocaloric and isonitrogenous, there is a positive relationship between Dry Matter, NDF, and ADF Degradability (Table\u0026nbsp;1) and gas production. This relationship has been confirmed by Blummel et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), who reported that in-vitro gas production is an indirect measurement of positive changes in dry matter degradability. In addition, Blummel and Orskov (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) observed a positive correlation between DM disappearance and gas production. Blummel et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) reported that gas production is closely related to SCFA production, which will be discussed later (Table\u0026nbsp;5). Concerning to effect of the spirulina supplementation levels on increased degradability, this may be due to several factors, including the high nutrient density of Spirulina and the stimulation of extracellular enzyme secretion by gut microflora (Tovar-Ram\u0026iacute;rez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Spirulina also contains vitamins, minerals, essential fatty acids, amino acids, and other nutrients that promote faster growth (Costa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, Spirulina reduces rumen protein degradation and alters bacterial community composition, leading to an increase in the efficiency of rumen microbial crude protein production in steers (Panjaitan et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The increase in gas production with spirulina supplementation levels may be due to two reasons. Firstly, the degradability rates increase as spirulina supplementation increased (Table\u0026nbsp;4), which activate the microflora in the rumen, leading to an increment in the production of microbial protein and an increase in fiber degradability (Costa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Secondly, spirulina contains all essential amino acids and has a great impact on digestibility (Lafarga et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFermentation and Calculated parameters after 24 hours\u003c/h2\u003e \u003cp\u003eThe level of ammonia in rumen liquor is an indicator of nutritional conditions, as many types of rumen micro-organisms use ammonia as a source of nitrogen. Mixing Alfalfa and Panicum maximum showed improved pH and NH3 than other treatments containing either of them alone. This may be attributed to complementarity between Alfalfa and Panicum maximum, leading to an improvement in the quality of the fodder, as confirmed by Alasa et al, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) when concluded that intercropping grasses (Panicum maximum) with forage legumes (\u003cem\u003elablab purpureus\u003c/em\u003e) improve the quality of the fodder. Additionally, there was an increase in degradability rate and gas production as substituting levels increased (Table\u0026nbsp;4). Elevated values of ammonia were associated with an increased percentage of Panicum in rations (R2 and R5). This may be attributed to an increased in CP content of the concentrate ration portion through increasing protein sources, especially Soya meal (highly degradable protein source, Table\u0026nbsp;1) to compensate for the low CP content of Panicum to get iso protein rations. This is in line with Jahan et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who reported that many nitrogenous substances in the high concentration diet may be the possible reason for increasing NH3-N concentration. Nousiainen et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) also supported this explanation, where they found that increased concentrate protein feeding improved whole-diet digestibility in cows in a curvilinear manner because of positive effects on the ruminal environment for fiber digestion, but the magnitude of the effect was not very large. Regarding the volatile fatty acids, carbohydrates are fermented by a variety of bacteria in the rumen and transformed into volatile fatty acids (VFA) by the corresponding enzymes (Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The data showed a significant decrease in TVFA with the ascending substitution of alfalfa hay with Panicum hay. The control ratio (R1) was higher than the values for R2, R3, R4, and R5. This may be due to the low content of fiber fractions (NDF and ADF) and Ash in alfalfa hay compared to Panicum hay, with comparable contents of carbohydrates (NFC contents) in both alfalfa and Panicum hays (Table\u0026nbsp;1). Additionally, there was an increase in the degradability of DM, NDF and ADF as well as gas production (Table\u0026nbsp;4), as well as an increase in OMD, ME, and SCFA (Table\u0026nbsp;5). All these results improve the fermentation process, leading to an increment of VFA. The inclusion of 2 kg/ton spirulina resulted in a significant (P\u0026thinsp;\u0026lt;\u0026thinsp;.0001) increase in pH value and ammonia concentration, without affecting the value of TVFA concentration. These results may be due to the high nutrient density of Spirulina and the stimulation of extracellular enzyme secretion by gut microflora (Tovar-Ram\u0026iacute;rez et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Spirulina also contains vitamins, minerals, essential fatty acids, amino acids, and other nutrients that promote faster growth (Costa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similar observations were reported by Panjaitan et al., (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) feeding Spirulina platensis as a supplement along with low CP containing guinea grass (Panicum maximum) hay improved efficiency of microbial protein production in cattle. Panjaitan et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) increased microbial protein synthesis and rumen ammonia-N in a quadratic fashion with increasing Spirulina inclusion in the diet. The differences between this study and our findings may be due to the differences in the experimental condition, ration composition and levels of supplementation as well as type of animals.\u003c/p\u003e \u003cp\u003eIt\u0026rsquo;s clear that there is an inverse relationship between the ratio of Panicum maximum in ration, and the calculated parameters (OMD, ME and SCFA). This may be attributed to 1) decrease CP and EE contents in rations with panicum hay (table1), this observation was in line with the reported work of Blummel and Orskov (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) reported that, there is a positive correlation between the calculated metabolizable energy from in vitro gas production together with CP and EE contents as well as ME value of conventional feeds measured in vivo. 2) Decrease OM contents and increased of CF, NDF and ADF in rations with panicum hay (table1), The low OMD and ME obtained for the level of panicum in the treatments increased might probably connected with the presence of high fiber (Blummel and Orskov, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) especially in treatment R5 (panicum 100%). 3) Relationship between the SCFA and gas production (table 4), similar observation was reported by Getachew \u003cem\u003eet al\u003c/em\u003e. (2000) the SCFA estimated from in vitro gas production, has been widely used to evaluate the energy value of several classes of feed. Blummel and Orskov (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) who suggested that gas production from different classes of feeds incubated in- vitro in buffered rumen fluid was closely related to the production of SCFA which was based on carbohydrate fermentation. The present findings indicated that all in-vitro parameters are affected by the concentration of panicum in ration. Supplementing the experimental rations with ascending levels of spirulina showed a positive relationship between the concentration of spirulina and the calculated parameters (OMD, ME and SCFA) until the highest values (40.51,7.16 and 0.64, respectively), which were produced with the highest concentration of spirulina 3 kg/ton, Table\u0026nbsp;(5) and Fig.\u0026nbsp;(8A, 8B, 8C). Referring to increased values of calculated ME with increasing spirulina concentration proved that Spirulina specifically 3 kg/ton has a good potential to enhance energy content of roughage feedstuffs. Moreover, as the level of SCFA, which is an indicator of the energy content of the ration, its production increased with increasing spirulina concentration in ration. This is due to the fact that Spirulina species, known as cyanobacteria, contain the essential fatty acids, linoleic acid (LA, 18:2 delta-9,12) (table 2) and gamma-linolenic acid (GLA, 18:3 delta-6,9,12) (Gupta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), high quality proteins, carbohydrates, vitamins (B1, B2, tocopherols), minerals (sodium, potassium, calcium, magnesium, phosphorus, iron), carotenes (especially beta-carotene), chlorophyll a, phycocyanin, and some phenolic acids (Gupta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Consequently, rations associated with spirulina makes it possible to raise digestion rates, the energy supply and mineral elements to the microbes present in the rumen fluid, thus improve their growth and activity, and thus, increase the rates of degradability of OMD, ME and SCFA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eReplacing alfalfa hay with ascending level of Panicum hay may lead to ascending decrease in the mixture nutritive value. The best mixing of Alfalfa hay with Panicum maximum \u003cem\u003ecv. Mombasa\u003c/em\u003e was 75% alfalfa hay with 25% Panicum maximum \u003cem\u003ecv. Mombasa\u003c/em\u003e (R2). Incorporation of Spirulina as feed additive (2Kg/ton) could offer a good solution to improve the quality of Panicum maximum and alfalfa hay, can be used to maximize degradability and ruminal fermentation parameters.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the role of the Desert Research Center and Faculty of Agriculture, Ain Shams University, in supporting the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions Conceptualization:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by (Meteab, M.I), (Abeer, M. EL-Essawy) and (El-Bordeny N. E). Investigation and methodology were performed by (Khorshed, M.M), (Abeer, M. EL-Essawy), (Nassar, M.S) and (El-Bordeny N. E). \u0026nbsp;The first draft of the manuscript was written by (Meteab, M.I) and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Open-access funding is provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study will be provided upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental procedures were conducted per the Animal Ethics Committee guidelines of the animal and Poultry Production Division of the Desert Research Center (Egypt), (approval No. 2022\u0026ndash;0179).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Daim, M.M., Abuzead, S.M.M. and Halawa, S.M., 2013. 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Agriculture, 11 (10), 1015. https://doi.org/10.3390/agriculture11101015.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Panicum maximum cv. Mombasa, Alfalfa, Spirulina, in vitro, gas production, rumen fermentation","lastPublishedDoi":"10.21203/rs.3.rs-3946950/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3946950/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to evaluate the impact of replacing alfalfa hay with Panicum maximum hay, with or without ascending levels of Spirulina supplementation on in-vitro gas production and fermentation parameters. Isocaloric and isonitrogenous experimental diets were formulated to contain 40% roughage + 60 % concentrate, ascending replacement with Panicum maximum hay at levels 0, 25%, 50%, 75%, and 100% take place in rations R1, R2, R3, R4, and R5, respectively. Each ration supplemented with incremental levels 0, 0.5, 1.5, 2, 2.5, and 3kg spirulina per Ton of feed. Results indicated that the potential of gas production after 24 h was linearly increased by increasing the level of Spirulina. The control group (R1) recorded the highest values of gas production, while it was the lowest at (R5) (p\u0026lt;.0001). In vitro degradability of dry and organic matter was negatively affected by the increment of Panicum maximum and Spirulina levels and vice versa, until 2 kg/ton of Spirulina (p\u0026lt;.0001). The addition of Spirulina significantly (p\u0026lt;.0001) increased total volatile fatty acids (TVFA) and N-NH3 concentration, until 2Kg/ton, while the addition of Panicum maximum hay significantly increased N-NH3 concentration, until it reached at (R4). As a result, the best mixing ratio between Alfalfa and Panicum maximum hays was recorded at R2. In conclusion, the substitution of alfalfa hay with 25% Panicum maximum hay (R2) and incorporation of Spirulina as feed additive (2Kg/ton) could offer a good solution in dry periods to improve the quality of Panicum maximum and can be used to maximize degradability and ruminal fermentation parameters.\u003c/p\u003e","manuscriptTitle":"In vitro Gas production and rumen fermentation for rations containing increasing levels of Panicum maximum cv. Mombasa with or without Spirulina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 12:22:22","doi":"10.21203/rs.3.rs-3946950/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision with re-assessment","date":"2024-05-16T03:14:26+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-11T14:13:07+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-04T18:46:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-29T08:01:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2024-02-28T04:27:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"81e6cbb6-0278-462e-8ed0-248b688429b4","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T16:00:28+00:00","versionOfRecord":{"articleIdentity":"rs-3946950","link":"https://doi.org/10.1007/s11250-024-04262-x","journal":{"identity":"tropical-animal-health-and-production","isVorOnly":false,"title":"Tropical Animal Health and Production"},"publishedOn":"2025-01-15 15:57:12","publishedOnDateReadable":"January 15th, 2025"},"versionCreatedAt":"2024-03-06 12:22:22","video":"","vorDoi":"10.1007/s11250-024-04262-x","vorDoiUrl":"https://doi.org/10.1007/s11250-024-04262-x","workflowStages":[]},"version":"v1","identity":"rs-3946950","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3946950","identity":"rs-3946950","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}