Replacement of Panicum maximum for Alfalfa and its effect on nutritional

Introduction

Gas production techniques have been extensively applied to evaluate the nutritional value of ruminant feeds, particularly in estimating fermentation characteristics of forages and mixed diets (Getachew et al., 2000) [12] . This method allows simultaneous assessment of multiple feedstuffs by measuring gas volume at different incubation times, reflecting the microbial degradation of organic matter and production of volatile fatty acids (VFAs) as well as metabolic gases such as methane (CH₄) and carbon dioxide (CO₂) (Amanzougarene & Fondevila, 2020) [2] . The rate and volume of gas production are closely linked to feed chemical composition and physical structure, with rapidly fermentable carbohydrates promoting higher gas and VFA output, whereas protein contributes partially through ammonia and CO₂ interactions, and fat contributes minimally to gas formation.Volatile fatty acids, including acetate (C2), propionate (C3), and butyrate (C4), are the primary energy source for ruminants, accounting for up to 75% of ME, with acetate representing approximately 70%, propionate 20%, and butyrate 10% (Dijkstra et al., 2005; Eom et al., 2018) [7,8] . Methane is predominantly produced in the rumen (87-93% of total methane) through three main pathways: hydrogenotrophic, methylotrophic, and acetoclastic, under anaerobic conditions where microbial populations utilise hydrogen, carbon dioxide, and one-carbon compounds to generate CH₄ (Kebreab, 2006) [17] . Methane emission is influenced by dietary composition, microbial activity, volatile fatty acid ratios, and the efficiency of fermentation; diets high in acetate and butyrate increase CH₄, while propionate reduces it (Brask et al., Replacing forages like alfalfa with alternative grasses such as Panicum maximum may alter fermentation dynamics, gas production patterns, and microbial protein synthesis, providing opportunities to optimise ruminant nutrition and reduce methane emissions. This study aims to evaluate the effects of gradually replacing increasing proportions of Panicum maximum instead of alfalfa with two levels of concentrated feed to roughage on gas production, volatile fatty acids, methane emission, metabolisable and net energy, and microbial protein synthesis in ruminants, establishing the link between fermentation activity and key nutritional parameters.

Material and Methods

In this experiment, samples of Panicum maxima and AF plants grown in the Jablah area, Babylon province, were used. The samples were collected from various locations in the field, and the crops were harvested at a height of 5 cm above the soil surface to avoid contamination. The collection of Panicum maxima and AF samples was carried out during the seventh cut. Chemical analyses of the forage samples were conducted in the laboratories of the College of Veterinary Medicine and the College of Agriculture at Al-Qasim Green University, in addition to in vitro digestion trials performed at the laboratories of Razi University. As for the raw materials that make up the concentrated feed, they were prepared from one of the feed manufacturing plants in Al-Mahaweel District, affiliated with Babil Governorate, and the ingredients included (wheat bran, barley, corn, and soybean meal), in addition to some ingredients from the local market, such as (table salt, sodium bicarbonate, slow-release urea, and a vitamin mineral supplement).

Preparation of Experimental Rations

After preparing the concentrate feed from the mentioned raw materials, it was mixed with different proportions of PM and AF. In the first five treatments, the proportion of concentrate feed was 60% and roughage 40%, while in the remaining treatments (T 6 , T 7 , T 8 , T 9 , T 10 ) the proportion of concentrate feed was 70% and roughage 30%, as follows:  T 1: 60% concentrate, 40% roughage (100% AF, 0%

Estimation of Methane Production (CH₄)

Total gas production was measured following Menke (1988) [22] using TMR with graded replacement of Alfalfa by Panicum maximum (100, 75, 50, 25, 0%) combined with concentrate. Samples (200 mg) were incubated in 100 mL syringes with 20 mL artificial saliva and 10 mL filtered rumen fluid from freshly slaughtered sheep. CO₂ was added to ensure anaerobic conditions. Syringes were incubated in a water bath at 39 °C, and gas production was measured at 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours. Four replicates per treatment plus four blanks were used. pH was measured, and 4 mL of 4% NaOH was added to two syringes to determine methane (Fievez, 2005). IVOMD (%) and SCFA (mmol/100 mL) were calculated after 96 h incubation using Menke (1988) [22] equations. Gas components were estimated using Orskov and McDonald (1979) [26] :

IVOMD (%) = 14.88 + 0.889GV + 0.45 × CP SCFA = 0.00425 + (0.0222 × Gas 24) P represents the amount of gas produced at time t, a is the gas produced from the rapidly fermentable fraction, and b is the gas produced from the slowly fermentable fraction. C denotes the fermentation rate and gas production of fraction b, and t is the time divided by Euler's number e (approximately 2.718). SCFA: Short-chain fatty acids (mmol/100 mL). Artificial saliva was prepared from: KCl 0.77 g, Na₂HPO₄ 2.77 g, NaHCO₃ 9.8 g, MgCl₂•7H₂O 2.16 g, CaCl₂•2H₂O 16 g, NaCl 0.47 g, dissolved in 1 L distilled water.

Measurement of Metabolizable Energy (ME)

Metabolizable energy (ME, MJ/kg DM) was calculated using the following equation according to Menke (1988) [22]: ME = 2.20 + 0.136GP + 0.057CP + 0.0029EE ME: Metabolizable energy (MJ/kg dry matter), GP: Gas volume produced after 96 hours of incubation (mL/200 mg dry matter), CP: Crude protein (g/kg dry matter), EE: Ether extract (%) Net Energy (NE) NE = 0.54 + 0.096 GAS + 0.0038CP + 0.000173EE

Microbial Protein (MP)

The amount of microbial protein produced from the incubated samples was calculated using the equation (Makkar and Becker, 2010):

MCP: Microbial protein produced (mg/g of dry matter), GP: Gas volume produced over 96 hours (mL/200 mg of dry matter)

Statistical Analysis

Data were analyzed using SAS software (2018) in a 2×5 factorial completely randomized design. Analysis of variance (ANOVA) was performed, and means were compared using Tukey's test. The statistical model used was Yijk = μ + Ai + Bj + (A*B)ij + eijk. where: Yijk = observed value at the ith level of factor A, jth level of factor B, and kth replication. μ = overall mean. Ai = effect of factor A at level i (i = 1, 2). Bj = effect of factor B at level j (j = 1, 2, 3, 4, 5). (A*B) ij = interaction effect between factors A and B at levels i and j. eijk = random error, assumed normally distributed with mean zero and constant variance.

Results and Discussion

Effect of Concentrate-to-Forage Ratio on Nutritional Value

Table (1) shows the effect of different concentrate-to-forage ratios on several nutritional parameters. The 30:70 treatment showed a significant superiority (p<0.01) in all measured traits compared to the 40:60 treatment. ME increased to 11.22 MJ/kg DM, NE to 6.87 MJ/kg DM, and microbial protein to 144.93 mg/100 mL. In addition, short-chain fatty acid (SCFA) concentration rose to 1.31 mmol/100 mL, and CH₄ reached 11.32 mL/200 mg DM.

This improvement is attributed to the higher content of rapidly fermentable carbohydrates and lower content of slowly fermentable fibers in the diet, which enhanced the rate of organic matter degradation and increased volatile fatty acid production, providing a favorable environment for microbial fermentation in the rumen. This also contributed to higher microbial protein synthesis and energy derived from fermentation. The increase in CH₄ is associated with the greater release of hydrogen, which methanogenic microbes utilize to produce methane. These results are consistent with the findings of Saini et al. (2012) [31] , Nagadi (2019) [25] , and Ramos et al. (2021) [28] , who reported increases in ME, NE microbial protein, and SCFA concentration in diets with higher concentrate content than forage. Similarly, Suharti et al. (2011) [32] and Kumar et al. (2013) [18] observed a significant effect of increased concentrate on total volatile fatty acids. Conversely, these findings differ from Iqbal et al. (2008) [15] and Nagadi (2019) [25] , who reported a decrease in methane (CH₄) production with higher concentrate levels and Panicum maximum inclusion.

Effect of Gradually Replacing Alfalfa with Panicum maximum on Nutritional Value

The results in Table (2) show the effect of gradually replacing Alfalfa with Panicum maximum on nutritional parameters (energy and protein). Significant increases (p<0.05) in ME were observed in treatments T 1 , T 2 , T 3 , and T 4 , with values of 11.07, 10.97, 11.07, and 10.96 MJ/kg DM, respectively, compared to T 5 , which recorded 10.79 MJ/kg DM. The lower value in T 5 is attributed to the slower fermentation of PM and insufficient synchronization between energy and nitrogen availability. These results align with Hassan et al. (2022) [14] , who reported increased ME in diets containing a concentrate mixture with 100% AF, and with Meteab et al. (2025) [23] , who observed higher ME in diets with 100% AF compared to other Panicum maximum replacement levels (50%, 75%, and 100%). NE also showed significant superiority (p<0.05) in T 1 , T 2 , T 3 , and T 4 , with values of 6.77, 6.70, 6.76, and 6.69 MJ/kg DM, respectively, compared to T 5 (6.57 MJ/kg DM), due to higher digestibility of organic matter and increased volatile fatty acid production. Regarding microbial protein (MP), treatments T 1 and T 3 were significantly superior (p<0.05), reaching 142.89 and 143.04 mg/100 mL, respectively, compared to T 5 (139.57 mg/100 mL). The superiority of T 3 is attributed to the combination of rapidly fermentable energy from jet hay and sustained energy from Panicum, providing a temporal gradient that allowed rumen microbes to use nitrogen more efficiently for microbial protein synthesis. The superiority of T 1 is linked to greater microbial diversity and complementary interactions among species. These results are consistent with Hassan et al. (2022) [14] , who reported that replacing 25-50% of clover with Panicum maximum optimized rumen fermentation and microbial protein production, and with Uzegbu and Ukonu (2022) [33] , who observed increased microbial protein with Panicum supplementation. For SCFA, treatments T 1 -T 4 were significantly higher (p<0.05), recording 1.30, 1.28, 1.29, and 1.28 mmol/100 mL, respectively, compared to T 5 (1.24 mmol/100 mL). This is attributed to a more favorable rumen environment and the availability of degradable nitrogen, enhancing organic matter fermentation and gas production. These results agree with Abdelrahman et al. (2017) [1] , Ma et al. (2014, 2015) [19, 20] , who showed that including clover in lamb diets increases volatile fatty acids, particularly acetate and propionate, improving rumen fermentation efficiency and providing a rapid energy source compared to high-fiber hay. Regarding methane (CH₄) production, treatments T 1 -T 4 recorded significantly higher values (p<0.05) of 11.21, 11.04, 11.13, and 11.06 mL/200 mg DM, respectively, compared to T 5 (10.70 mL/200 mg DM). This increase is attributed to the availability of rapidly fermentable carbohydrates in T 1 -T 4 due to clover inclusion (100-25%), which increased hydrogen release utilized by methanogenic microbes for CH₄. In contrast, T 5 contained 0% jet hay and 100% Panicum maximum. These findings align with Meteab et al. (2025) [23] , who reported decreased gas production with increased replacement of clover hay by graded levels of Panicum maximum. They differ from Phesatcha et al. (2020) [27] , Kang et al. (2016) [16] , and Anantasook and Wanapat (2012) [3] , who reported reduced calculated CH₄ production with higher concentrate levels in the diet.

Effect of the Interaction between Concentrate-to-Forage Ratio and Panicum Maximum Replacement for Alfalfa on Nutritional Value

The results in Table (3) indicate that the interaction between concentrate-to-forage ratio and the replacement level of Panicum Maximum for Alfalfa had a significant effect on nutritional parameters (energy and protein). Treatment T 6 showed a significant superiority (p<0.05) in ME, reaching 11.40 MJ/kg DM, compared to T 1 , T 2 , T 3 , T 4 , T 5 , and T 10 , which recorded lower values of 10.74, 10.73, 10.93, 10.76, 10.48, and 11.10 MJ/kg DM, respectively. This superiority is attributed to the higher content of rapidly fermentable starch at the 70% concentrate level, combined with increased degradable nitrogen, which enhanced microbial mass and efficiency, thereby increasing volatile fatty acid and total gas production. These results differ from Fuller et al. (2020) [11] , who reported that increases in ME are directly proportional to decreases in feed concentration. T 6 also exhibited significant superiority in NE, reaching 7.00 MJ/kg DM, compared to other treatments (6.54, 6.53, 6.66, 6.55, 6.35, 6.78). This is due to the abundance of rapidly soluble carbohydrates at the higher concentrate level, providing a quick energy source, while the forage component contributed degradable nitrogen in the rumen, creating optimal synchronization between energy and protein. This enhanced microbial growth and efficiency, accelerating fermentation and energy production. Microbial protein (MP) was highest in T 6 , at 147.16 mg/100 mL, compared to 135.69-143.45 mg/100 mL in other treatments. This improvement is attributed to the ideal synchronization between energy and rumen-degradable protein, allowing optimal nitrogen utilization without losses as ammonia, thus promoting microbial growth and protein synthesis. These findings are consistent with Ramos et al. (2009) [29] , Broderick (2001) [6] , Guo et al. (2025) [13] , and Mohammadzadeh et al. (2021) [24] , who showed that increasing concentrate and clover levels or rumendegradable starch improved microbial protein synthesis and rumen nitrogen utilization efficiency. SCFA were significantly higher in T 6 (1.35 mmol/100 mL) compared to 1.19-1.29 mmol/100 mL in other treatments. This is associated with high levels of rapidly fermentable starch at the 70% concentrate level, combined with highly digestible forage, which stimulated rumen microbes to accelerate fermentation and total gas production. These results agree with Kumar et al. (2013) [18] , Phesatcha et al. (2020) [27] , and Hassan et al. (2022) [14] , who observed increased SCFA production with higher concentrate diets and inclusion of clover. Finally, methane (CH₄) production was significantly higher in T 6 (11.59 mL/200 mg DM) compared to 10.28-11.12 mL/200 mg DM in other treatments. This is attributed to increased total fermentation from higher volatile fatty acids, hydrogen release, and microbial proliferation, especially methanogens, which enhanced CH₄ despite relative shifts to propionate. These results are consistent with Eun et al. (2004) [9] and Hassan et al. (2022) [14] , who reported higher CH₄ with high (70%) concentrate diets. Asikin et al. (2018) also noted that adequate neutral detergent fiber (NDF) in forage supports digestion and increases gas production over incubation time, aligning with the observed rise in methane due to improved microbial fermentation efficiency. However, these findings differ from other studies (Meteab et al., 2025; Van Wyngaard et al., 2018) [23,34] . Values with different letters indicate significant differences (p<0.05).

Conclusion

The inclusion of PM at 100% of the roughage portion, under concentrate-to-forage ratios of 60% concentrate with 40% roughage and 70% concentrate with 30% roughage, led to a deterioration in metabolizable energy (ME), net energy (NE), microbial protein (MP), short-chain fatty acids (SCFA), and methane gas (CH 4 ). In contrast, higher concentrate levels (70% concentrate and 30% roughage)

References

1. Abdelrahman MM, Alhidary I, Alyemni AH, Khan RU, Bello ARS, Al-Saiady MY, et al. Effect of alfalfa hay on rumen fermentation patterns and serum biochemical profile of growing Naemi lambs with ad libitum access to total mixed rations. Pakistan J Zool. 2017;49(4):1519-1522.
2. Amanzougarene Z, Fondevila M. Fitting of the in vitro gas production technique to the study of high concentrate diets. Animals. 2020;10(10):1935.
3. Anantasook N, Wanapat M. Influence of rain tree pod meal supplementation on rice straw-based diets using in vitro gas fermentation technique. Asian-Australas J Anim Sci. 2012;25(3):325-331.
4. Asikin N, Jayanegara A, Ridla M, Samsudin AA. The potential of tropical grass as a feed in ruminant by using an in vitro gas production. In: MATEC Web of Conferences. EDP Sciences; 2018. p. 197:06005.
5. Brask M, Weisbjerg MR, Hellwing ALF, Bannink A, Lund P. Methane production and diurnal variation measured in dairy cows and predicted from fermentation pattern and nutrient or carbon flow. Animal. 2015;9(11):1795-1806.
6. Broderick GA. Maximizing utilization of alfalfa protein: The example of the lactating dairy cow. In: Delgado L, Lloveras J, editors. Quality in lucerne and medics for animal production. 2001. p. 183-192.
7. Dijkstra J, Forbes JM, France J. Quantitative aspects of ruminant digestion and metabolism. Wallingford: CABI; 2005. p. xi + 734.
8. Eom SY, Lee YS, Lee SG, Seo MN, Choi BS, Kim YD, Park JD. Lead, mercury, and cadmium exposure in the Korean general population. J Korean Med Sci. 2018;33(2):e93.
9. Eun JS, Fellner V, Gumpertz ML. Methane production by mixed ruminal cultures incubated in dual-flow fermentors. J Dairy Sci. 2004;87(1):112-121.
10. Fievez V, Oudshoorn BO. Estimation of direct and indirect gas production in syringes: a tool to estimate short chain fatty acid production that requires minimal laboratory facilities. Anim Feed Sci Technol. 2005;123124:197-210.
11. Fuller AL, Wickersham TA, Sawyer JE, Freetly HC, Brown-Brandl TM, Hales KE. The effects of the forage-to-concentrate ratio on the conversion of digestible energy to metabolizable energy in growing beef steers. J Anim Sci. 2020;98(8):skaa231. doi: 10.1093/jas/skaa231.
12. Getachew G, Makkar HPS, Becker K. Effect of polyethylene glycol on in vitro degradability of nitrogen and microbial protein synthesis from tannin-rich browse and herbaceous legumes. Br J Nutr. 2000;84(1):73-83.
13. Guo W, Liu Y, Na M, Zhang Y, Na R. Effects of rumen-degradable starch levels on in vitro rumen fermentation and microbial protein synthesis in alfalfa silage. Fermentation. 2025;11(2):106. doi: 10.3390/fermentation11020106.
14. Hassan AA, Sayed HA, Yacout MH, El-Badawi AA, Shwerab AM. Evaluation of feeding Panicum maximum forage for ruminant. Egyptian J Nutr Feeds. 2022;25(3):291-301.
15. Iqbal MF, Cheng YF, Zhu WY, Zeshan B. Mitigation of ruminant methane production: current strategies, constraints and future options. World J Microbiol Biotechnol. 2008;24(12):2747-2755.
16. Kang S, Wanapat M, Phesatcha K, Norrapoke T, Foiklang S, Ampapon T, et al. Using krabok (Irvingia malayana) seed oil and Flemingia macrophylla leaf meal as a rumen enhancer in an in vitro gas production system. Anim Prod Sci. 2016;57(2):327-333.
17. Kebreab E, Clark K, Wagner-Riddle C, France J. Methane and nitrous oxide emissions from Canadian animal agriculture: a review. Can J Anim Sci. 2006;86(2):135-157.
18. Kumar S, Dagar SS, Sirohi SK, Upadhyay RC, Puniya AK. Microbial profiles, in vitro gas production and dry matter digestibility based on various ratios of roughage to concentrate. Ann Microbiol. 2013;63(2):541-545.
19. Ma T, Deng KD, Tu Y, Zhang NF, Jiang CG, Liu J, et al. Effect of dietary forage-to-concentrate ratios on urinary excretion of purine derivatives and microbial nitrogen yields in the rumen of Dorper crossbred sheep. Livest Sci. 2014;160:37-44.
20. Ma T, Tu Y, Zhang NF, Deng KD, Diao QY. Effect of ratio of non-fibrous carbohydrates to neutral detergent fiber and protein structure on intake, digestibility, rumen fermentation, and nitrogen metabolism in lambs. Asian-Australas J Anim Sci. 2015;28(10):1419-1427.
21. Makkar HP, Becker K. Are Jatropha curcas phorbol esters degraded by rumen microbes? J Sci Food Agric. 2010;90(9):1562-1565.
22. Menke KA. Estimation of the energy of the energetic feed value obtained from chemical analysis and in vitro gas production rumen fluid. Anim Res. 1988;37:207222.
23. Meteab MI, Khorshed MM, El-Essawy AM, Nassar MS, El-Bordeny NE. in vitro gas production and rumen fermentation for diets containing increasing levels of Panicum maximum cv. Mombasa with or without spirulina. Trop Anim Health Prod. 2025;57(1):25-34.
24. Mohammadzadeh M, Kazemi-Bonchenari M, HosseinYazdi M, Mirzaei M. Forage source (alfalfa hay vs wheat straw) and rumen undegradable to degradable protein ratio: effects on growth performance, microbial protein yield, digestibility, blood metabolites, and behavior of Holstein dairy calves. Span J Agric Res. 2021;19(3):e0610.
25. Nagadi SA. in vitro gas production, methane emission and rumen fermentation characteristics with increasing roughage to concentrate ratios. Meteorol Environ Arid Land Agric Sci. 2019;28:27-36.
26. Orskov ER, McDonald I. The evaluation of protein degradability from incubation measurements weighed according to rates of passage. J Agric Sci. 1979;92:499503.
27. Phesatcha K, Phesatcha B, Wanapat M, Cherdthong A. Roughage to concentrate ratio and Saccharomyces cerevisiae inclusion could modulate feed digestion and in vitro ruminal fermentation. Vet Sci. 2020;7(4):151. doi: 10.3390/vetsci7040151.
28. Ramos SC, Jeong CD, Mamuad LL, Kim SH, Kang SH, Kim ET, et al. Diet transition from high-forage to highconcentrate alters rumen bacterial community composition, epithelial transcriptomes and ruminal fermentation parameters in dairy cows. Animals. 2021;11(3):838.
29. Ramos S, Tejido ML, Martínez ME, Ranilla MJ, Carro MD. Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage. J Anim Sci. 2009;87(9):2924-2934.
30. Reichardt N, Duncan SH, Young P, Belenguer A, McWilliam Leitch C, Scott KP, et al. Phylogenetic distribution of three pathways for propionate production within the human gut microbiota. ISME J. 2014;8(6):1323-1335.
31. Saini JK, Hundal JS, Wadhwa M, Bakshi MPS. Effect of roughage to concentrate ratio in the diet on the rumen environment and nutrient utilization in goat and sheep. Indian J Anim Nutr. 2012;29(4):333-338.
32. Suharti S, Astuti DA, Wina E, Toharmat T. Rumen microbial population in the in vitro fermentation of different ratios of forage and concentrate in the presence of whole lerak (Sapindus rarak) fruit extract. Asian-Australas J Anim Sci. 2011;24(8):1086-1091.
33. Uzegbu OH, Ukonu EC. Evaluation of the potential of Panicum maximum forage on gut microbial population of pigs. Niger J Anim Prod. 2022;49:2089-2093.
34. Van Wyngaard JDV, Meeske R, Erasmus LJ. Effect of concentrate feeding level on methane emissions, production performance and rumen fermentation of Jersey cows grazing ryegrass pasture during spring. Anim Feed Sci Technol. 2018;241:121-132.
35. Williams SRO, Hannah MC, Jacobs JL, Wales WJ, Moate PJ. Volatile fatty acids in ruminal fluid can be used to predict methane yield of dairy cows. Animals. 2019;9(12):1006.