Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 19/09/2025 Aceptado: 06/12/2025 Publicado: 21/12/2025 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 7 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI https://doi.org/10.52973/rcfcv-e361781 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Substuon of alfalfa hay with Rumex pulcher L. and its eﬀect on in vitro gas producon and ruminal fermentability Sustución de heno de alfalfa por Rumex pulcher L. y su efecto sobre la producción de gas in vitro y fermentación ruminal Mehtap Güney 1 * , Çağrı Kale 2 ¹ Van Yuzuncu Yil University, Department of Animal Science, Faculty of Agriculture, 65090, Van, Türkiye ² Department of Animal Nutrion and Nutrional Diseases, Faculty of Veterinary Medicine, Van Yuzuncu Yil University, 65090, Van, Türkiye. *Correspondence author: mguney@yyu.edu.tr ABSTRACT In this study, the eﬀects of Rumex pulcher L. forage added to alfalfa hay as an alternave roughage source in ruminants at diﬀerent mixing raos (0, 25, 50 and 100%) on gas and methane producon, in vitro digesbility properes and rumen fermentaon parameters were determined by in vitro gas producon technique. Experimental groups were formed of control (C: 100 % alfalfa hay), Rumex pulcher L. forage 1 (75 % alfalfa hay + 25 % Rumex pulcher L. forage), Rumex pulcher L. forage 2 (50 % alfalfa hay + 50 % Rumex pulcher L. forage) and Rumex pulcher L. forage 3 (100 % Rumex pulcher L. forage). Each experimental group was designed with 5 replicates. Rumex pulcher L. forage was signiﬁcantly higher (P < 0.001) crude protein and lower neutral detergent ﬁber and acid detergent ﬁber content than alfalfa hay. While gas and methane producon did not change in all treatments, Rumex pulcher L. forage 3 had the highest organic maer digesbility compared to the other treatment groups. As the rates of Rumex pulcher L. forage increased, an increase was observed in the amount of microbial protein synthesis parameters. Rumen parameters except acec acid were diﬀerent among all treatments. Rumex pulcher L. forage can be included in ruminant raons in order to reduce feed costs, due to its superior properes compared to alfalfa hay in terms of nutrient composion, in vitro digesbility and microbial protein synthesis. Furthermore, it is important to conduct further studies on this species, which grows unculvated in nature, and explore it as an alternave forage source. Keywords: Alternave roughage; in vitro gas and methane; rumen parameters RESUMEN En este estudio, se determinaron los efectos del forraje de Rumex pulcher L. añadido al heno de alfalfa como fuente alternava de forraje en rumiantes, en diferentes proporciones de mezcla (0, 25, 50 y 100 %), sobre la producción de gas y metano, las propiedades de digesbilidad in vitro y los parámetros de fermentación ruminal, mediante la técnica de producción de gas in vitro. Se conformaron los siguientes grupos experimentales: control (C: 100 % heno de alfalfa), forraje de Rumex pulcher L. 1 (75 % heno de alfalfa + 25 % forraje de Rumex pulcher L.), forraje de Rumex pulcher L. 2 (50 % heno de alfalfa + 50 % forraje de Rumex pulcher L.) y forraje de Rumex pulcher L. 3 (100 % forraje de Rumex pulcher L.). Cada grupo experimental se diseñó con 5 repeciones. El forraje de Rumex pulcher L. presentó un contenido signiﬁcavamente mayor (P < 0,001) de proteína cruda y menor de ﬁbra detergente neutra y ﬁbra detergente ácida que el heno de alfalfa. Si bien la producción de gas y metano no varió en ninguno de los tratamientos, el forraje de Rumex pulcher L. 3 mostró la mayor digesbilidad de la materia orgánica en comparación con los demás grupos. A medida que aumentaron las tasas de forraje de Rumex pulcher L., se observó un incremento en los parámetros de síntesis de proteína microbiana. Los parámetros ruminales, a excepción del ácido acéco, presentaron diferencias entre los tratamientos. La forraje de Rumex pulcher L. puede incorporarse a las raciones de rumiantes para reducir los costos de alimentación, debido a sus propiedades superiores en comparación con el heno de alfalfa en cuanto a composición nutricional, digesbilidad in vitro y síntesis de proteína microbiana. Además, es importante realizar estudios adicionales sobre esta especie, que crece de forma silvestre, y explorarla como fuente alternava de forraje. Palabras clave: Forraje alternavo; gas y metano in vitro; parámetros ruminales

Alternave roughage for ruminants / Güney and Kale UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Today, problems such as decreasing agricultural land, climate change and environmental polluon have led to diﬃcules in the producon and supply of feed resources for ruminants. It is inevitable that serious losses will occur because of the decrease in biodiversity due to these eﬀects in animal and plant producon. Studies examining the genec characteriscs of diﬀerent species and creang condions where they can beer adapt to climac condions have become more important today [1]. In addion, new feed sources require more sustainable feed producon. For this reason, non-tradional feed sources in ruminant nutrion have been increasing in recent years. Indeed, in livestock producon areas, some naturally occurring and non- culvated forages have the potenal to parally or completely replace tradional forages in ruminant diets [2]. Therefore, these feeds, which can be an alternave for ruminants, can also be a soluon to the roughage problem and contribute to the development of low-cost diets [3]. Rumex species, one of the alternave roughages for ruminants, have been used as medicine for humans from past to present, and studies on a wide variety of biological acvies of this species have been reported. It has also been reported that some Rumex species are eﬀecve against many fungi and bacteria [4]. Rumex acetosella L., which is both commercially culvated and collected from the wild in Türkiye, has been reported to have an average phenolic substance content and anoxidant capacity. However, it is stated that the bioavailability of these anoxidave properes is quite high [5]. In a study invesgang the alternave forage properes of Rumex acetosella species, it was compared with alfalfa hay (AH) and sainfoin. The researchers reported that Rumex acetosella hay had a higher crude protein content and lower plant cell wall elements, and therefore had a higher digesbility value compared to the tradionally used alfalfa (Medicago sava) hay and sainfoin (Onobrychis viciifolia) in animal nutrion. In addion, it was reported that the hay in queson is good quality roughage and can also be used as protein feed due to its high protein content [6]. It has been reported that this herb grows naturally in many provinces from the east to the west of Türkiye [6]. Researches stated that Rumex pulcher forage (RPH) has a strong anoxidant acvity, all parts of this grass are hairy, its stems are square- shaped, the undersides of the nodes are not swollen, the leaf edges are toothed and it can grow upright between 10 and 60 cm [7]. Rumex pulcher L., a species belonging to the Polygonaceae family, has a high nutrional content and high degradaon potenal in the rumen, as well as having the eﬀect of reducing greenhouse gas producon in the rumen, and therefore it has been reported that this forage can be used as an alternave in ruminant nutrion [8]. Apart from the above study, no study was found examining the eﬀects of RPH on ruminant animals. The fact that this forage, which grows freely in nature, can be used in ruminants may be a soluon to some extent in terms of roughage producon. The objecve of this study was to evaluate the substuon of AH by RPH on chemical composion, gas and methane producon, in vitro digesbility and ruminal fermentaon parameters. MATERIALS AND METHODS This study was conducted in accordance with the animal research procedures outlined in the instuonal commiee on animal use (case number 2024/10-10). In this study, AH and RPH were obtained from Van province in Türkiye. The experimental groups were formed as control (C: 100 % AH), RPH1 (75 % AH + 25 % RPH), RPH2 (50 % AH + 50 % RPH) and RPH3 (100 % RPH) by supplemenng increasing amounts of RPH on dry maer (DM) basis to AH, which is tradionally used in animal nutrion. Each experimental group was designed with 5 replicaons for rumen fermentaon parameters and with 4 replicaons for microbial protein synthesis. A total of 45 (9 blank) (Bos taurus) gas producon syringes (Model: Fortuna, 100 mL:1, 40 mm capillary tube, boro, amber grad, Poulten & Graf GmbH Wertheim/Germany) were used. In the study, AH and RPH were ground in a feed mill with a 1 mm sieve diameter (Retsch GmbH 5657 Haan, West- Germany) and then the mixing raos were determined. Rumen ﬂuid was obtained from Simmental cale (Bos taurus) of 600 ± 25 kg live weight, belonging to a farm fed a roughage based raon and brought to a slaughterhouse in Van. Rumen ﬂuid was quickly brought to the laboratory in a thermos containing 39 ± 1 °C water, maintaining anaerobic conditions and filtered through a 4-layer cheese cloth. Then, it was mixed with arﬁcial saliva in a rao of 1/2 (rumen ﬂuid/arﬁcial saliva) under anaerobic condions. Chemical composion (DM, ash, CP, EE, ADF, NDF) Dry maer (DM) and ash analyses of roughages and their mixtures were determined according to the Associaon of Oﬃcial Analycal Chemists (methods 934.01 and 942.05, respecvely) [9]. Crude protein (CP) analysis was determined by Kjeldahl steam disller (method 984.13) [9], ether extract (EE) analysis was measured using an ANKOM XT15 device (Ankom®, Macedon, USA) [10], neutral detergent ﬁber (NDF and acid detergent ﬁber (ADF) analyses were analyzed using an ANKOM A220 device (Ankom, Macedon, USA) as described by Van Soest et al. [11]. İn vitro gas, methane producon and digesbility assay The study was conducted to determine the eﬀect of AH and RPH mixtures on 24-h in vitro ruminal gas producon [12]. The treatment groups (200 ± 1mg) were incubated with 30 mL of rumen ﬂuid + arﬁcial saliva mixture in 100 mL in vitro gas producon syringes in a custom-made water bath at 39 ± 1 °C for 24 hours (h). For methane measurement, the gas formed in glass syringes was taken with a three-way plasc syringe system and injected into a computer-aided methane gas measuring device (Sensors Europe Analysentechnik GmbH, Erkrath, Germany) and the methane gas value (%) was read on the computer. By using 24-hours gas producon (GP) and chemical composion parameters, metabolizable energy (ME) content and organic maer digesbility (OMD) of feed treatments were determined by the calculaon [12]. OMD (%) = 14.88 + 0.889GP + 0.45CP + 0.0651 ash ME (Mj / kg DM) = 2.20 + 0.136GP + 0.057 CP 2 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In vitro microbial protein synthesis parameters The treatment groups (500 ± 1 mg) were incubated with 40 mL of rumen ﬂuid + arﬁcial saliva mixture in 100 mL in vitro gas producon syringes in a custom-made water-bath at 39 ± 1 °C for 24 h. At the end of the incubaon, the gas was removed from the syringes and the material in the syringes for microbial protein analysis was transferred to 250 mL beakers, 70 mL of NDF soluon was added and boiled for 1 h. Aﬅer boiling, the material in the beakers was vacuum ﬁltered through Gooch crucibles and dried in an oven (Nuve, FN 500, Turkey) at 105 ± 1 °C for 12 h. True digesbility (TD), microbial protein producon (MP), microbial protein synthesis eﬃciency (MPSE) and paron factor (PF) were calculated using the formulas of Blummel et al. [13]. TDDM (mg) = incubated DM – remained DM, TD (%) = (TDDM / incubated DM) X100 MP (mg /g DM) = TDDM – (GP X 2,2 mg/ml), MPSE = ((TDDM – (2.2 x GP))/TDDM) X100 TF = TDDM/GP Rumen fermentaon parameters In this study, pH measurement was made by taking the content transferred to 250 mL boles in in vitro gas producon syringes at the end of incubaon with a digital pH meter (Thermo, Orion Star A11, USA; 0.01) immediately. Then, ammonia nitrogen analysis was performed in rumen ﬂuids ﬁltered with 4 layers of cheesecloth according to the Kjeldahl method. For this, rumen ﬂuids were rotated at 4500 G for 5 min. 5 mL of rumen ﬂuid was placed in glass tubes connected to a Kjeldahl disller (Tecnal® TE 036/1) and water and sodium hydroxide were added, and the samples were dislled in a boric acid soluon (30 g/L). The soluons obtained from the disllaon (approximately 100 mL) were trated with sulfuric acid (H 2 SO 4 ; N/70) [14]. Rumen ﬂuid samples were stored at -18 °C for VFAs (Acec acid, Propionic acid, Butyric acid) by adding 2 mL of 1/1 HCl (absolute) to a 10 mL tube immediately aﬅer incubaon [15]. Aﬅer approximately 15 days, the samples were removed from the deep freezer (Arçelik, 2172 JEI, Türkiye) and thawed in the refrigerator overnight (Arçelik, 570431, Türkiye). Then, samples were centrifuged at 12000 G for 10 min. The supernatant obtained was sampled into 2 mL vials, and high performance liquid chromatography (HPLC, Thermo Scienﬁc/Agilent, 1100, USA) condions were adjusted with 300 x 7.8mm HPX-87H_ column as mobile phase 0.015 N H 2 SO 4 +0.0034 M EDTA, ﬂow rate 0.7 mL/min, column temperature 45 ºC, detector PDA-DAD and 50 µL injecon volume [16]. Stascal analysis Stascal analyses of the study were performed using a linear model according to a completely randomized trial design, and diﬀerences at P < 0.05 were considered stascally signiﬁcant. Diﬀerences between means were determined using the Duncan Mulple comparison test, and all analyses were performed in SAS 9.4 [17]. RESULTS AND DISCUSSION The nutrient composions of RPH and AH used in the study are in TABLE I. Diﬀerences between roughages were found to be signiﬁcant in terms of all parameters analysed. TABLE I Chemical composion of Rumex pulcher L and Alfalfa hay % Rumex pulcher L Alfalfa hay P-value Items x̄ ± S x̄ x̄ ± S x̄ DM 92.37 ± 0.05b 92.74 ± 0.03a 0.004 OM 79.57 ± 007b 85.77 ± 0.02a < .0001 Ash 12.80 ± 0.02a 6.97 ± 0.01b < .0001 CP 27.81 ± 0.19a 15.60 ± 0.17b < .0001 EE 4.50 ± 0.14a 3.33 ± 0.04b 0.001 NDF 13.66 ± 0.27b 35.78 ± 0.55a 0.000 ADF 9.84 ± 0.03b 26.51 ± 0.11a 0.000 DM: Dry maer; OM: organic maer; CP: Crude protein; EE: Ether extract; NDF: Neutral detergent ﬁber; ADF: Acid detergent ﬁber There are many naturally growing plant species in nature. These plant species play a very important role in the protecon of natural diversity. In the literature searches, we did not ﬁnd any studies other than one on the use of RPH in ruminants. Therefore, the results were compared with similar species of this forage. The DM and OM content of RPH was lower than AH. The most striking thing was that the OM content of RPH was considerably higher than AH. This result was due to the higher ash content of RPH (12.80 %) compared to AH (6.97 %) (TABLE I). The sum of the mineral substances in the plant, and the mineral substances in the soil contaminated with the plant gives the ash content [18]. It is also stated that the diﬀerence in the ash content varies depending on the type of forage, ferlizaon and irrigaon, soil structure, climate, harvest me, and the environmental condions in which forage is grown [19]. In this study, it is thought that such high ash content is related to the plant species. In the study, the CP content of RPH (27.81 %) was determined to be higher than AH (15.60 %) (TABLE I). It is stated that RPH has CP levels similar to AH with high nitrogen ﬁxaon and that AH has the highest CP content among legume forage [20]. The study shows that the CP content of RPH is considerably higher than AH. In addion, it is reported that the CP content in forages may vary depending on the species, vegetaon, climate and nitrogen level of the soil [21]. The CP level obtained from RPH in the current study is similar to the values obtained from RPH [22]. Due to this very high CP content obtained from RPH, it is thought that it can be recommended for use in the nutrion of farm animals and can also be seen as a protein source feed material. It was observed that the NDF and ADF contents of RPH (13.66-9.84 %) were signiﬁcantly lower than AH (35.78-26.51 %), respecvely (TABLE I). 3 of 7

Alternave roughage for ruminants / Güney and Kale UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico It has been reported that ADF is an indicator of the digesbility of the plant and NDF is an indicator of the intake by animals, and that the ADF rate in feeds should be 30 % and below, and the NDF rate should be 40 % and below [23]. In the present study, the values obtained from the NDF and ADF results were found to be lower than the values obtained from RPH [6]. It is reported that the low ﬁber content of RPH may be related to the physiological state of the plant (early vegetaon) [24]. İn vitro gas producon, CH 4 producon, OMD and ME values of RPH added to AH at diﬀerent levels are in TABLE II. It was observed that adding RPH to AH at diﬀerent rates did not change the total gas, methane values (%, mL), but changed the OMD and ME values, and the highest rate was obtained in the group using RPH3 (TABLE II). TABLE II Eﬀect of using diﬀerent rates of Rumex pulcher L. hay instead of alfalfa hay on in vitro gas producon The rao of experimental feeds N Control RPH1 RPH2 RPH3 P-value Items x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ Total gas* 5 146.16 ± 1.44 135.08 ± 0.98 136.62 ± 0.71 147.10 ± 0.37 0.162 Methane, % 5 11.66 ± 0.47 11.54 ± 0.25 11.53 ± 0.21 11.32 ± 0.27 0.895 Methane, mL 5 11.83 ± 0.56 11.95 ± 0.23 11.99 ± 0.28 11.74 ± 0.04 0.949 OMD, % 5 52.32±0.59b 50.72±0.34c 52.22±0.44b 56.54±0.33a <.0001 ME** 5 7.97±0.20b 7.84±0.13b 8.12±0.10b 8.80±0.05a 0.001 OMD: Organic maer digesbility; ME: Metabolizable energy; a,b Diﬀerent superscript leers in the same row represent signiﬁcant diﬀerence, x̄ ±S x̄: mean±standart error mean; *mL/g DM; ** Mj/kg DM Methane and carbon dioxide gas, which are formed because of the fermentaon of nutrients in ruminants, are one of the important sources of greenhouse gas emissions. It is also considered an important problem as the cause of climate change worldwide. Using RPH at diﬀerent rates instead of AH did not change the in vitro gas producon (mL/g DM), methane (%) and methane (mL) producon (TABLE II). In a study comparing RPH with some weeds with forage potenal, it was stated that in vitro gas producon was lower, feed value was higher, and it could be used in ruminant animals due to its digesbility and greenhouse gas reducing eﬀects [8]. A other study from various non-tradional plants, including Rumex forage, reported that forages with high CP and low ﬁbre content had a reducing eﬀect on methanogens and protozoa in the rumen in ruminants [25], thereby reducing greenhouse gas emissions [24 , 26]. It has been reported that methane producon, as well as gas producon, is related to the amount of fermentable carbohydrates [27]. In current study, it was expected that gas and methane producon would decrease as the rates of RPH increased, but the results were not as expected. This situaon showed that the use of RPH instead of AH did not inhibit ruminal microbial acvity, connued and was a good nutrient source for rumen microorganisms, while it had no eﬀect on methanogens in the rumen. It stated that in addion to measuring gas producon in the in vitro gas producon technique, determining the degree of digeson allows more informaon to be obtained about the feeds [27]. In addion to it is important to determine energy and digesble nutrients, while determining the diﬀerences between feeds [28]. In the study, OMD and ME levels were observed to reach the highest value when 100 % RPH was used (TABLE II). The higher OMD and ME levels obtained when 100 % RP was used may be due to the lower plant cell wall structure of RPH compared to AH. Furthermore, low cell wall coverage is thought to be a result of hydrolysis into simple sugars, and the higher OMD of RPH compared to AH may be due to the solubility of its metabolizable energy. It was stated that oak nuts supplementaon to AH increased OMD and ME levels in vitro [29]. The in vitro digesbility and ME values obtained from the study were found to be similar to the results obtained from Rumex acetocella [6 ,[22]. In the study, the true digeson degree and microbial protein synthesis parameters are in TABLE III. When the table was examined, the diﬀerence between all treatments examined was signiﬁcant. As the AH rates decreased, an increase was determined in MP, PF and TD, with the highest rates observed in RPH3. Protein degradaon in the rumen is the result of microbial acvity, and varies depending on the type of proteins, ruminal diluon rate, ruminal pH, fermented substrate and dominant species of rumen ﬂora [30]. Moreover, protein and carbohydrates in the composion of feeds aﬀect microbial protein yield in rumen [31]. In the study, MP values increased as RPH rates increased. While the lowest in vitro MP amount was obtained from 100 % AH with 172.63 %, the highest value was reached in 100 % RPH with 242.48 %. The degree of true digesbility increased in accordance with the MP values. The highest value of 88.16 % was obtained from 100 % RPH (TABLE III). It is thought that the increase in protein degradaon in the rumen due to the higher CP content of RPH compared to AH increases MP, and low NDF and ADF content increased TD values due to increased soluble carbohydrates in the rumen. With these properes, RPH can be considered as an alternave forage source. 4 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico It is reported that the addion of 1 unit of oak nuts to the raon results in an increase of 0.6477 in microbial protein producon, 0.4105 in microbial protein synthesis eﬃciency, and 0.262 in the paroning factor [32]. In another study, it was stated that oak nuts (Quercus spp.) supplementaon added to alfalfa hay at diﬀerent levels reduced the amount of protein destroyed in the rumen and increased bypass proteins, in vitro organic maer digesbility and metabolizable energy values as a result of 24-h in vitro incubaon [29]. It is reported that caramba hay has higher TD raos than vetch (vicia) and alfalfa hay [21]. TABLE III Eﬀect of using diﬀerent rates of Rumex pulcher L. hay instead of alfalfa hay on in vitro microbial proteins synthesis parameters and TD The rao of experimental feeds N Control RPH1 RPH2 RPH3 p x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ MP 5 172.63 ± 3.33c 178.15 ± 4.08c 201.27 ± 10.88b 242.48 ± 4.61a < .0001 MPSE 5 55.03 ± 0.93ab 53.06 ± 1.25b 54.16 ± 2.22b 59.00 ± 0.75a 0.044 PF 5 1.91 ± 0.02c 1.96 ± 0.02c 2.10 ± 0.06b 2.33 ± 0.03a < .0001 TD, % 5 67.04 ± 0.18d 72.13 ± 0.41c 79.61 ± 1.01b 88.16 ± 0.88a < .0001 MP: Microbial protein; MPSE: Microbial protein synthesis eﬃciency;; PF: Paroning factor; TD: True digesbility;; a,b,c Diﬀerent superscript leers in the same row represent signiﬁcant diﬀerence, x̄ ±S x̄: mean±standart error mean It reported that using Rumex asotocella hay instead of AH in lamb diets decreased in vitro methane producon while increasing MP, MPSE, PF and TD [22]. The researchers also stated that there was a negave relaonship between methane producon and microbial protein synthesis parameters. However, in the current study, while the amount of methane did not decrease, an increase in PF, MP, and TD was observed. These results revealed that rumen fermentaon did not suppress methane-producing bacteria and hence fermentaon. Moreover, it can be said that microbial protein producon increased posively with the increase in energy and protein in roughage. It is stated that the digeson rate and feed consumpon of forages with high PF values are high [13]. It has also been reported that the PF values of feeds are the most important element in determining the microbial protein synthesis eﬃciency, and that feeds with high PF values also have high microbial protein synthesis eﬃciency [33]. In the study, RPH3 has the highest PF value, microbial protein producon and TD are also high. İn vitro rumen fermentaon parameters of the roughage mixtures used in the study were determined and are in TABLE IV. Diﬀerences between treatment groups were signiﬁcant for all parameters examined except AA concentraon. TABLE IV Eﬀect of using diﬀerent rates of Rumex pulcher L. hay instead of alfalfa hay on rumen fermentaon parameters The rao of experimental feeds N Control RPH1 RPH2 RPH3 P Title x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ x̄ ± S x̄ pH 5 6.35 ± 0.007d 6.41 ± 0.007c 6.45 ± 0.004b 6.52 ± 0.007a < .0001 NH 3 , mL/100 5 29.49 ± 0.38d 35.74 ± 0.21c 39.22 ± 0.56b 42.09 ± 0.63a < .0001 AA, mmol/L 5 92.56 ± 1.13 90.13 ± 1.01 94.16 ± 2.64 95.43 ± 0.43 0.1261 PA, mmol/L 5 22.18 ± 0.61a 21.93 ± 0.24a 22.32 ± 0.56a 19.99 ± 0.49b 0.0142 BA, mmol/L 5 14.06 ± 0.60b 19.96 ± 1.10a 14.27 ± 0.49b 12.91 ± 0.20b < .0001 NH 3 : ammonia; AA: Acec acid; PA: Propionic acid; BA: Butyric acid; a,b,c,d Values within a column with diﬀerent superscripts diﬀer signiﬁcantly The eﬀect of using RPH on rumen pH values was stascally signiﬁcant (TABLE IV). It is seen that rumen pH values increased as RPH rates increased. The highest pH value was reached in the experimental group where RPH3 was used. All pH values in the study varied between 6.35 and 6.52 and were found to be within acceptable limits for rumen fermentaon [34]. It is reported that rumen pH did not change signiﬁcantly with increases in protein, energy and rumen degradable protein levels and was determined in the range of 6.88-7.22 [35]. In this study, ammonia nitrogen levels also increased signiﬁcantly in parallel with the increase in RPH rates. It is determined that increasing pH and ammonia rates are related to increased fermentaon because of RPH degradaon. In this study, ammonia nitrogen levels were considerably higher in RPH compared to AH. Therefore, while ammonia nitrogen levels were 29.49 mL/100 mL in control, also increased as RP rao increased and reached 42.09 with RPH3. It is seen as a natural result that high ammonia nitrogen producon in RPH supports MP producon. Increased MP producon has been reported to increase ammonia nitrogen ulizaon and eﬃciency of ﬁbre 5 of 7

Alternave roughage for ruminants / Güney and Kale UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico digeson, thus ensuring opmum ulizaon of the diet oﬀered to the animal [36]. In fact, in present study, OMD and TD was also the highest in the RPH treatment group, and it increased with the increase in RPH rate. It can be said that the degradaon of RPH in the rumen is high, and the microbial acvity occurring in the rumen degrades the protein source in RPH into ammonia. It is stated that a low rumen degradable protein level may reduce ruminal ammonia nitrogen levels, DM intake, and microbial protein synthesis [37]. Total VFA is a product of rumen microbial acvity resulng from the digeson of the energy source in the feed [38]. It is stated that diﬀerences in rumen degradable protein levels may alter VFA producon. In addion to the amount and composion of roughage in the diet aﬀects the metabolism of cellulose degradaon and VFA producon [39]. In the study, in vitro rumen AA rates did not change with diﬀerent rates of RPH contribuon to AH. However, PA levels were higher in the treatment group with RPH1 than in the other groups. The reason for the higher PA in this treatment group is unknown, but it is thought that the synergisc eﬀect of the roughage may have increased PA levels. CONCLUSIONS AND IMPLICATIONS In the treatment groups prepared from RPH mixtures at diﬀerent rates instead of AH, 100% RPH provided a signiﬁcant increase in in vitro digesbility, MP, and TD compared to AH and other mixtures. RPH grown freely in nature can be considered an oponal roughage source due to its high digesbility, rich nutrient composion, crude protein content, and low cell wall elements. 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