Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 04/06/2025 Aceptado:19/08/2025 Publicado: 24/09/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 8 Revista Cienﬁca, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35715 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Eﬀects of exogenous ﬁbrolyc enzymes supplementaon on growth performance and ruminal fermentaon in pre-weaning Simmental calves Efectos de la suplementación con enzimas ﬁbrolícas exógenas sobre el rendimiento del crecimiento y la fermentación ruminal en terneros Simmental predestete Tuncay Tufan* 1 , Cavit Arslan 2 , Oktay Kaplan 3 , Mehmet Sarı 4 , Mehmet Irmak 1 ¹ Department of Animal Nutrion and Nutrional Disease, Faculty of Veterinary Medicine, Siirt University, Siirt, 56100, Turkey. ² Department of Animal Nutrion and Nutrional Disease, Faculty of Veterinary Medicine, Selçuk University, Konya, 42250, Turkey. ³ Department of Animal Nutrion and Nutrional Disease, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, 21200, Turkey. ⁴ Department of Animal Science, Faculty of Agriculture, Kırşehir Ahi Evran University, Kırşehir, 40200 Turkey. *Corresponding author: tuncaytufan@siirt.edu.tr ABSTRACT Early adaptaon of newborn calves’ forestomaches to concentrates and roughage is crucial for cost-eﬀecveness. Therefore, the use of addives that will facilitate early adaptaon to feed and posively impact forestomach development is crucial. This study aimed to determine the eﬀects of exogenous ﬁbrolyc enzyme supplementaon on growth performance and rumen fermentaon in pre-weaning calves. Eighteen Simmental male calves of the same age (4 days) were randomly assigned to 3 groups and supplemented with exogenous ﬁbrolyc enzyme addive, 0 (Control), 2 g.d - 1 , or 4 g,d -1 for 84 days treatments included with exogenous ﬁbrolyc enzyme addive, 0 (Control), 2 g.d-1 or 4 g.d -1 . The exogenous ﬁbrolyc enzyme supplementaon to calves signiﬁcantly improved feed conversion rao (P<0.01). Ruminal pH and ammonia nitrogen (NH₃-N concentraons were not aﬀected by exogenous ﬁbrolyc enzyme supplementaon on days 42 and 84 of the study (P>0.05). Ruminal concentraons of acec acid, propionic acid, and butyric acid were not aﬀected by exogenous ﬁbrolyc enzyme supplementaon on day 42 of the study (P>0.05). The propionic acid concentraon was higher in both exogenous ﬁbrolyc enzyme -supplemented groups than in the Control group on the 84th day of the study (P<0.01). The butyric acid concentraon at 2 g.d -1 exogenous ﬁbrolyc enzyme supplemented group was higher than the other groups on the 84th day of the study (P<0.001). The acec acid to propionic acid rao was higher in the Control and 2 g.d -1 exogenous ﬁbrolyc enzyme -supplemented groups than in the 4 g.d -1 exogenous ﬁbrolyc enzyme-supplemented group on the 42nd day of the study (P<0.05). The acec acid to propionic acid rao was higher in the Control group than in the exogenous ﬁbrolyc enzyme -supplemented groups on day 84 of the study (P<0.01). The results indicated that 2 or 4 g.d - 1 exogenous ﬁbrolyc enzyme supplementaon had a beer feed conversion rao and ruminal propionic concentraon in pre-weaning calves. Key words: Calf; exogenous ﬁbrolyc enzyme; growth; ruminal fermentaon RESUMEN La adaptación temprana de los terneros recién nacidos a los concentrados y al forraje es crucial para la rentabilidad. Por lo tanto, el uso de adivos que faciliten la adaptación temprana al alimento e inﬂuyan posivamente en el desarrollo del ternero es crucial. Este estudio tuvo como objevo determinar los efectos de la suplementación con enzima ﬁbrolíca exógena sobre el rendimiento del crecimiento y la fermentación ruminal en terneros antes del destete. Dieciocho terneros machos de raza Simmental, de la misma edad (4 días), fueron asignados aleatoriamente a tres grupos y suplementados con enzima ﬁbrolíca exógena a dosis de 0 (Control), 2 g.d - 1 o 4 g.d -1 durante 84 días. La suplementación con enzima ﬁbrolíca exógena a terneros mejoró signiﬁcavamente la tasa de conversión alimencia (P < 0,01). El pH ruminal y las concentraciones de nitrógeno amoniacal (NH₃-N) no se vieron afectados por la suplementación con enzima ﬁbrolíca exógena en los días 42 y 84 (P>0,05). Las concentraciones de ácido acéco, ácido propiónico y ácido burico no se vieron alteradas por la suplementación con enzima ﬁbrolíca exógena en el día 42 (P>0,05). En el día 84, la concentración de ácido propiónico fue superior en ambos grupos suplementados con enzima ﬁbrolíca exógena en comparación con el grupo Control (P<0,01). La concentración de ácido butírico fue mayor en el grupo suplementado con 2 g.d-1 de enzima ﬁbrolíca exógena en comparación con los otros grupos (P<0,001). La relación ácido acético: ácido propiónico fue más alta en los grupos Control y 2 g.d -1 que en el grupo de 4 g.d -1 en el día 42 (P<0,5). La relación ácido acético / ácido propiónico fue mayor en el grupo control que en los grupos suplementados con enzima ﬁbrolíca exógena al día 84 del estudio (P<0,01). En conclusión, la suplementación con 2 o 4 g.d -1 de enzima ﬁbrolíca exógena mejoró la tasa de conversión alimencia y aumentó la concentración ruminal de ácido propiónico en terneros en etapa de pre-destete. Palabras clave: Ternero; enzimas ﬁbrolícas exógenas; crecimiento; fermentación ruminal

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Newborn calves are considered pre-ruminant because their anterior stomachs are not developed at this stage [1]. Pre- ruminant calves are fed liquid feeds such as milk or milk replacer unl their reculo-rumen is fully developed anatomically, physiologically and microbially [2]. When calves are fed both liquid (such as whole milk and milk replacer) and dry feed, their rumen is fully developed between 12 and 16 weeks [3 , 4 , 5]. However, the content of liquid feeds is expensive. For economical calf feeding, in addion to liquid feeds, they should be fed dry feeds [such as calf starter feed (CS) and quality roughages] during the pre-weaning period to smulate the development of the foregut stomach and establish rumen microorganisms [2 , 3 , 4 , 5]. In this way, the calves’ rumen can reach full maturity as soon as possible. Microbial enzymes secreted by rumen microorganisms constute an important part of feed digeson in ruminants [6]. The rumen of newborn calves is unable to digest dry feed, especially plant cell walls, due to the negligible acvity of enzymes that degrade starch and cell wall polysaccharides, which becomes evident as the rumen microbial community develops [7 ,8]. The Exogenous ﬁbrolyc enzymes (EFE) are powerful probiocs produced primarily through bacterial and fungal fermentaon [9 , 10]. The EFE can break down the complex structure of cellulose into soluble carbohydrates [10]. Few studies have been conducted to add EFE to improve growth performance and increase ruminal fermentaon in young calves during the pre-weaning period [10 , 11 , 12 , 13 , 14]. The EFE speciﬁcally designed for ruminants contains cellulase and xylanase acvies that may enhance ﬁber digeson in the rumen and improve feed eﬃciency [15]. EFE’s precise mode of acon in ruminant diets has not been fully understood. However, supplementaon of EFE had a marked eﬀect on increasing the ruminal microorganism populaon as well as increasing total tract digesbility, enhanced microbial protein synthesis [16], and provided the energy and nutrients required for ruminal microbial growth [13]. Colombao et al. [17] detected that EFE enhances the fermentaon of cellulose by combining pre- and post-incubaon eﬀects. The EFE could also enhance the aachment and improve access to the cell wall components (such as crude ﬁber, NDF and ADF) by rumen microorganisms and thus increase the rumen’s digeson rate [18]. Previous studies found that live weight gain, FCR [13], and growth performance [14] improved with EFE supplementaon in pre-weaned calves fed with whole milk, CS, and alfalfa hay. Contrary to those studies, there have been studies shown that EFE [12 , 14] or EFE plus probioc supplementaon unchanged the growth performance in pre-weaned calves [19 , 20]. Recent studies indicated that ruminal total volale fay acid (VFA), acec acid and butyric acid concentraon increased [13 , 14], NH 3 -N concentraon and pH decreased, and acec acid to propionic acid rao unchanged in calves fed with whole milk, CS and alfalfa hay in the pre-weaning period [14]. There are limited studies on the eﬀects of EFE supplementaon on growth performance and ruminal fermentaon of calves in the pre- weaning period, especially on calves fed with whole milk, CS, and roughages [5], 21]. In this study, it is hypothesized that supplementaon of 2 or 4 g.day -1 of EFE to pre-weaning calves fed whole milk, CS, and dried grass hay will improve growth performance, increase ruminal fermentaon, and increase the ruminal microorganism populaon compared to calves not receiving EFE supplementaon. This study was carried out to determine the eﬀect of 2 or 4 g.day -1 EFE supplementaon to calves fed with whole milk plus calf starter feed and dried grass hay on growth performance and ruminal fermentaon in the pre-weaning period. MATERIALS AND METHODS Animals, management and treatments This study was carried out at Kaas University Veterinary Faculty Research and Applicaon Farm (Kars, Turkey) by guidelines of the Dollvet A.S. Animal Experiments Local Ethics Commiee (Date 07.03.2014, No: 2014/17). Newly born calves (Bos taurus) were fed 4 L of colostrum for the ﬁrst 3 days (d) of life, weighed (TEM Scale, 70X110 cm 500 kg, Türkiye), and transferred to individual pens (2.5 x 3 m). Eighteen, 4-d-old male Simmental calves (40.36 ± 1.73 kg of LW) were randomly assigned (n= six calves per treatment) to three diets including (I) no supplementaon of EFE (Control), (II) 2 g.d -1 orally EFE supplementaon (EFE-2), and (III) 4 g.d -1 orally EFE supplementaon (EFE-4). Calves were given 4 L of whole milk per day between days 1-14 of the study, 5 L of whole milk per day between days 15-77 and 2.5 L of whole milk per d between days 78-84. In addion to whole milk, all calves were fed the CS prepared according to NRC [7] and chopped 2-3 cm dried pasture grass hay (GH: The pasture grass used in this study, “gramineae, Leguminoseae, and other plant families”, constute 64.2%, 22.8%, and 13.0% of the pasture grass populaon, respecvely [22]) as ad libitum in separate feeders during the study. The chemical composion of the whole milk, CS and GH used in this study are presented in TABLE I. Commercial EFE used in this study (ForagezymeTM, Global Nutritech, USA) were supplemented to the calves 2 or 4 g.d -1 aﬅer dissolving in 50 mL of dislled water in the EFE-2 and EFE-4 groups respecvely, at 09:00 h every morning with the help of a syringe without needles. The calves in the Control group received only 50 mL of dislled water. According to the manufacturer company, the EFE used in this study contains 1,000,000 CU.kg -1 cellulase and 1500,000 XU.kg -1 xylanase. Fresh and clean drinking water was available ad libitum during the study. The calves were housed in individual pens in a closed barn for 1 to 6 weeks of the study and under the shed in the open air for 7 to 12 weeks. 2 of 8

Enzymes supplementaon on growth performance and ruminal fermentaon Simmental / Tufan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE I Chemical composion of the diets, (g.kg -1 DM) Item Milk Calf Starter Grass Hay Ingredient composion Corn grain, ground 250.0 Barley grain, ground 200.0 Wheat grain, ground 100.0 Soybean meal 205.0 Sunﬂower meal 60.0 Wheat bran 40.0 Corn bran 100.0 Vegetable oil 17.0 Salt 4.0 Limestone 20.0 Vitamin-mineral premix 1 4.0 Chemical composion, g.kg -1 DM) Dry maer 132.0 901.0 927.6 Metabolic energy, MJ.kg -1 12.05 Crude protein 35.5 185.5 98.1 Crude ash 75.0 100.4 90.3 Ether extract 42.0 36.0 25.4 Crude ﬁbre 65.2 360.7 1 Contained: 250 000 IU vitamin A, 50 000 IU vitamin D, 1 500 IU vitamin E, 2.25 g Mn, 20 g Mg, 8 g Zn, 1.25 g Fe, 3 g S, 15 mg Co, 1.25 g Cu, 58 mg I and, 10 mg Se per kg premix. 2 Calculated from NRC [7] Growth performance Each calf’s live weight (LW) was recorded at the start and every 14 d before morning feeding. The average daily gain (ADG) was calculated by dividing the LW diﬀerence over 14 d by 14. Calves were weighed using a scale (TEM Scale, 70X110 cm 500 kg, Türkiye) with a precision of 100 g. Daily intake and refusal of whole milk, CS, and GH were recorded to calculate dry maer intake (DMI). Total DMI (milk, CS, and GH) was assessed on a biweekly basis. CS and GH samples were dried at 60°C (NÜVE, KD400, Turkey) and ground, and nutrient analyses were performed (Dry maer, metabolic energy, crude protein, crude ash, ether extract, crude ﬁbre). [22]. The feed conversion rao (FCR) was calculated on a biweekly basis. Rumen fermentaon parameters Rumen ﬂuid samples were collected from all calves 3 h aﬅer morning feeding on d 42 and 84 using a ruminal tube with a vacuum pump. Immediately aﬅer taking the rumen ﬂuid, the pH was measured (Thermo, Orion 3 Star, Germany) and the samples were ﬁltered through four layers of cheesecloth. For VFA analysis, 10 mL of ﬂuid was mixed with 2 mL of 250 g.L -1 meta-phosphoric acid. An addional 10 mL was taken for NH₃-N analysis. All samples were stored (Arçelik, 5194-NFY, Türkiye) at -20 °C unl analysis. Chemical analyses Whole milk, CS, and GH samples were analysed for dry maer, ash, crude ﬁber (CF: in CS and GH), crude protein (CP: 6.25xN), and ether extract (EE) according to AOAC [23]. Before analyses, ruminal ﬂuid samples were thawed at room temperature and clariﬁed by centrifuging (Nüve, NF1200, Türkiye) (10,000 x g for 20 min). The clariﬁed supernatant was analysed for NH 3 -N concentraons using a Modiﬁed Kjeldahl Method according to AOAC [23]. Ruminal AA, PA, and BA concentraons were measured in a Gas Chromatography device (Agilent Technologies, 6850N, USA) according to the method reported by Erwin et al. [24]. Stascal analysis This study created a research trial design according to the Resource Equality Method [25 , 26 ,[27]. Data were subjected to analyses of variance. Duncan’s mulple-range test assessed the signiﬁcance of the diﬀerences among the groups [28]. Two sample T-tests were performed to compare two independent groups for the 42nd and 84th-d comparisons of ruminal pH, NH 3 -N, and VFAs. SPSS 16.0 [SPSS for Windows, Version 16.0. Chicago, USA, SPSS Inc] package program was used for stascal evaluaon. Results were given mean ± standard error of means. Means were considered signiﬁcantly diﬀerent at P < 0.05. RESULTS AND DISCUSSION Growth performance The LW and ADG results from the groups for bi-week intervals are presented in TABLE II. The LW of the calves at the inial and throughout the study were similar among the groups. The ADG of the calves did not diﬀer at 1-2, 3-4, 5-6, 9-10, 11-12 weeks, 3 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico and overall the study (1-12 weeks), but signiﬁcantly higher in the EFE-2 and EFE-4 groups than the Control group at 7-8 weeks (P<0.001). TABLE II Eﬀect of the EFE supplementaon on LW and ADG at pre-weaning period in Simmental calves Weeks Control EFE-2 EFE-4 P value ------------------------ Live weight, kg --------------------- Inial 39.57 ± 2.83 41.30 ± 2.26 40.20 ± 1.63 0.865 2 48.82 ± 3.31 51.62 ± 3.05 49.63 ± 2.03 0.778 4 60.63 ± 4.20 62.83 ± 3.67 60.73 ± 1.96 0.877 6 72.47 ± 4.84 75.67 ± 4.12 71.38 ± 2.55 0.737 8 82.52 ± 5.07 88.17 ± 4.96 84.95 ± 2.49 0.661 10 95.32 ± 5.47 102.53 ± 5.26 98.73 ± 2.57 0.557 12 110.20 ± 5.74 119.02 ± 5.41 113.83 ± 2.70 0.448 -------------------- Average daily gain, kg ---------------- 1-2 0.661 ± 0.07 0.737 ± 0.09 0.674 ± 0.06 0.733 3-4 0.844 ± 0.08 0.801 ± 0.05 0.793 ± 0.04 0.804 5-6 0.846 ± 0.07 0.917 ± 0.06 0.761 ± 0.06 0.267 7-8 0.718 ± 0.03 b 0.893 ± 0.05 a 0.969 ± 0.03 a 0.001 9-10 0.914 ± 0.04 1.026 ± 0.03 0.985 ± 0.04 0.091 11-12 1.063 ± 0.05 1.178 ± 0.02 1.079 ± 0.03 0.070 1-12 0.841 ± 0.04 0.925 ± 0.04 0.877 ± 0.02 0.276 ab Means with diﬀerent leers in the same line are signiﬁcantly diﬀerent from each other, Average daily gain, EFE-2: Exogenous ﬁbrolyc enzymes (2g.d -1 ), EFE-4: Exogenous ﬁbrolyc enzymes (4g.d -1 ), Live weight The ﬁnal LW of the calves was higher at 8.00 and 3.29 % in the 2 and 4 g.d -1 EFE-supplemented calves, respecvely, when compared to control calves. The numerical increase in the LW of calves in both EFE groups suggests that 2 or 4 g.d -1 EFE posively aﬀects the LW of calves under faening condions but does not show parallelly by increasing the amount of EFE. These results agree with Winders et al. [29] and Beauchemin et al. [30], who reported a 7 and 9 % increase in LW of bison calves and steers fed with ﬁbrolyc enzymes added to dry forages compared to the Control. Ghorbani et al. [12] stated that the addion of EFE or EFE plus probiocs to the pre-weaning calf diets unchanged the LW of calves [19 , 20]. Ti and Tabbaa [31] found that adding diﬀerent amounts of EFE to the diet signiﬁcantly increased the living weight in male calves compared to females. In the present study, supplementaon of 2 or 4 g.d -1 EFE did not aﬀect the ADG of calves during the bi-weekly and overall period, except for the 7-8 weeks (TABLE II). However, supplementaon of 2 or 4 g.d -1 EFE induced a numerical enhancement of ADG (84 g.d -1 and 35 g.d -1 more LW gain, respecvely) compared to the Control group. Similar results were reported by feeding pre-weaning calves with EFE Ghorbani et al. [12] or EFE plus probioc [19 , 20]. McAllister et al. [33] observed that ADG was quadracally related to the EFE doses, but the increase in ADG in this study did not jusfy this paern. Similarly, it was observed that supplementaon of enzyme plus probioc from 7 d to 6 months of age did not signiﬁcantly increase the ADG but resulted in a numerical enhancement in calves [11]. Contrarily, Liu et al. [13] reported that EFE supplementaon to the raon signiﬁcantly increased the ADG of pre-weaning calves fed with whole milk, CS and alfalfa hay. Previous studies also found that ADG increased with the diﬀerent amounts of EFE supplementaon in buﬀalo calves [33]. Cruywagen and Goosen [34] indicated that adding 5 and 10 mg.kg -1 EFE to lamb raons signiﬁcantly increased the ADG, whereas 1 mg.kg -1 did not cause any change. The average CS, GH, and Total dry maer intake (TDMI) of the groups were not diﬀerent at bi-week intervals or overall (TABLE III). It was noced that all calves drank the oﬀered milk throughout the study. TABLE III Eﬀect of the EFE supplementaon on CS, GH and TDMI at pre- weaning period in Simmental calves, kg.d -1 in DM basis Weeks Feed Control EFE-2 EFE-4 P Value 1-2 CS 0.276 ± 0.04 0.257 ± 0.06 0.249 ± 0.05 0.924 GH 0.029 ± 0.04 0.046 ± 0.10 0.047 ± 0.02 0.439 TDMI 0.833 ± 0.04 0.833 ± 0.07 0.825 ± 0.04 0.991 3-4 CS 0.622 ± 0.06 0.607 ± 0.07 0.612 ± 0.05 0.984 GH 0.097 ± 0.02 0.145 ± 0.03 0.109 ± 0.03 0.408 TDMI 1.379 ± 0.08 1.412 ± 0.09 1.382 ± 0.03 0.933 5-6 CS 1.110 ± 0.13 1.107 ± 0.10 1.024 ± 0.09 0.812 GH 0.162 ± 0.03 0.187 ± 0.03 0.129 ± 0.02 0.299 TDMI 1.932 ± 0.14 1.955 ± 0.12 1.813 ± 0.08 0.651 7-8 CS 1.379 ± 0.17 1.467 ± 0.12 1.447 ± 0.09 0.876 GH 0.181 ± 0.01 0.218 ± 0.04 0.160 ± 0.01 0.234 TDMI 2.220 ± 0.16 2.346 ± 0.15 2.267 ± 0.08 0.805 9-10 CS 1.986 ± 0.15 1.936 ± 0.15 1.985 ± 0.09 0.956 GH 0.282 ± 0.03 0.267 ± 0.03 0.250 ± 0.02 0.757 TDMI 2.928 ± 0.17 2.864 ± 0.18 2.895 ± 0.08 0.954 11-12 CS 2.992 ± 0.15 3.010 ± 0.16 3.056 ± 0.04 0.937 GH 0.352 ± 0.02 0.366 ± 0.03 0.340 ± 0.02 0.759 TDMI 3.840 ± 0.16 3.872 ± 0.18 3.891 ± 0.05 0.968 1-12 CS 1.394 ± 0.10 1.253 ± 0.08 1.252 ± 0.04 0.353 GH 0.184 ± 0.02 0.206 ± 0.03 0.173 ± 0.01 0.518 TDMI 2.189 ± 0.11 2.214 ± 0.12 2.179 ± 0.04 0.967 CS: Calf starter, DM: Dry maer, EFE-2: Exogenous ﬁbrolyc enzymes (2 g.d -1 ), EFE-4: Ex- ogenous ﬁbrolyc enzymes (4 g.d -1 ), GH: pasture grass hay, TDMI: Total dry maer intake This study determined that supplementaon of 2 or 4 g.d -1 EFE did not aﬀect CS, HG, and TDMI during the bi-weekly and overall study period (TABLE III). Similarly, two studies conducted in calves reported that diﬀerent levels of EFE supplementaon did not aﬀect feed intake [12 , 13]. Beauchemin et al. [35] reported that the eﬀects of EFE on dry maer intake diﬀer among enzyme products; therefore, all enzyme mixtures may not increase feed intake. 4 of 8

Enzymes supplementaon on growth performance and ruminal fermentaon Simmental / Tufan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The FCR at 7-8 weeks, as well as the overall FCR for the study period (1-12 weeks), was signiﬁcantly higher in the EFE-2 and EFE-4 groups compared to the Control group (P<0.01). No diﬀerences were observed during the other bi-weekly intervals of the study (TABLE IV). TABLE IV Eﬀect of the EFE supplementaon on FCR rao at pre-weaning period in Simmental calves, kg feed.kg -1 ADG Weeks Control EFE-2 EFE-4 P value 1-2 1.26 ± 0.09 1.13 ± 0.05 1.22 ± 0.05 0.309 3-4 1.63 ± 0.21 1.76 ± 0.09 1.74 ± 0.07 0.673 5-6 2.29 ± 0.10 2.13 ± 0.05 2.38 ± 0.10 0.123 7-8 3.10 ± 0.18 a 2.63 ± 0.12 b 2.34 ± 0.09 b 0.005 9-10 3.20 ± 0.09 2.79 ± 0.12 2.94 ± 0.13 0.066 11-12 3.61 ± 0.10 3.29 ± 0.13 3.61 ± 0.12 0.103 1-12 2.52 ± 0.04 a 2.29 ± 0.04 b 2.37 ± 0.05 b 0.004 ab Means with diﬀerent leers in the same line are signiﬁcantly diﬀerent, ADG: Average daily gain, EFE-2: Exogenous ﬁbrolyc enzymes (2 g.d -1 ), EFE-4: Exogenous ﬁbrolyc enzymes (4 g.d -1 ), FCR: Feed conversion rao, In the present study, it was observed that supplementaon of 2 or 4 g.d -1 EFE improved the FCR in 7-8 weeks and the overall study period compared to Control in calves (TABLE IV). Beer FCR in the 2 or 4 g.d -1 EFE-supplemented groups is associated with unchanged TDMI in all groups (TABLE III), and numerically higher ADG in both ﬁbrolic enzyme groups (TABLE II). Colombao et al. [17] conducted a study that reported that exogenous enzymes can synergiscally enhance the hydrolyc potenal of endogenous microbial enzymes in the rumen. This improvement leads to more eﬀecve digeson of dietary ﬁber in the rumen. Supplementaon of EFE could compensate for calves’ limited endogenous enzymes and promote nutrient digeson in the rumen and intesnal tract, which improved FCR. Similarly, Liu et al. [13] and Tirado-González et al. [36] reported that adding ﬁbrolic enzymes to calf raons improves FCR. Contrarily, many previous studies have reported that the addion of ﬁbrolyc enzyme [12] or ﬁbrolyc enzyme plus probioc to calf raon did not aﬀect FCR [19 , 20]. The diﬀerences in growth performance in this study compared to other studies may be aributable to several factors, including diﬀerences in animal species such as calves, buﬀalo calves, or lambs, diﬀerences in age of animals used in the study, and animal housing and locaon. Addionally, the enzyme composions and dosages applied, along with the types and proporons of concentrate and roughage provided, likely contributed to the observed discrepancies. Ruminal fermentaon parameters Rumen ﬂuid pH and NH₃-N concentraons were not signiﬁcantly diﬀerent among the groups on either the 42nd or the 84th d of the study (TABLE V). The rumen’s pH level indicates the rumen’s internal environment is inﬂuenced by various factors, such as dietary composion, rumen circulaon rate, and saliva producon. Changes in pH levels dynamically aﬀect microbial acvity, digeson, and absorpon of nutrients [10]. In this study, supplementaon of 2 and 4 g.d -1 EFE did not inﬂuence the rumen ﬂuid pH (ranged between 5.88 - 6.03) on the study’s 42nd and 84th d (TABLE V). The rumen content in young calves generally has a low pH [37]. The measured pH values in this study are based on ﬁndings, which reported that the pH value of calves’ rumen should be under 6 unl the 10th week of age. The unchanged ruminal pH might be related to similar CS, GH, and TDMI among the groups (TABLE III). Krause and Oetzel [38], suggested that dry maer intake signiﬁcantly determines ruminal pH. Similarly, diﬀerent amounts of EFE supplementaon in the raon for beef cale [15 , 32 , 39 ,[40] and lamb (Ovis aries) [41] did not inﬂuence ruminal pH. In contrast to these studies’ results, Wang et al. [14] observed that supplementaon of EFE decreased the ruminal pH of calves that were fed whole milk, CS, and alfalfa hay in the pre-weaning period. There were also no signiﬁcant within-group diﬀerences between the 42nd and 84th d, except for the EFE-2 group, in which the NH₃-N concentraon was signiﬁcantly higher on d 42 than on day 84 (P<0.05). In the present study, there were no stascal diﬀerences among the groups in ruminal NH 3 -N concentraon at the 42nd and 84th d of the study (TABLE V). The possible reason for this unchanged NH 3 -N concentraon among the groups, 2 or 4 g.d -1 EFE supplementaon, could be unchanged ruminal crude protein degradability and amino acid deaminaon in pre-weaning calves. Similarly, it was reported that the supplementaon of ﬁbrolyc enzyme in raon did not change the ruminal NH 3 -N of beef cale [39] and lamb [41]. In contrast to these studies’ results, Wang et al. [14] determined that ruminal NH 3 -N concentraon decreased in calves fed with EFE during the pre-weaning period. TABLE V Eﬀect of EFE supplementaon on ruminal pH, NH 3 -N, and VFAs during the pre-weaning period in Simmental calves Days Control EFE-2 EFE-4 P value ---------------------------- pH ----------------------------- 42 5.91 ± 0.11 5.92 ± 0.09 5.88 ± 0.12 0.951 84 6.03 ± 0.14 6.02 ± 0.18 5.99 ± 0.17 0.977 P value 0.489 0.653 0.603 ------------- Ammonia nitrogen, mmol.L -1 -------------- 42 186.00 ± 9.51 218.00 ± 1.65 A 200.00 ± 7.52 0.192 84 190.67 ± 9.04 173.33 ± 7.84 B 191.33 ± 15.47 0.461 P value 0.729 0.034 0.625 --------------- Acec acid, mol.100 mol -1 --------------- 42 70.40 ± 1.98 71.64 ± 0.92 66.45 ± 1.39 0.065 84 69.54 ± 1.42 74.80 ± 1.42 70.09 ± 1.69 0.052 P value 0.733 0.091 0.127 ------------- Propionic acid, mol.100 mol -1 ------------ 42 16.27 ± 0.81 a 16.88 ± 0.33 18.18 ± 0.77 0.157 84 10.51 ± 1.48 b 16.98 ± 1.40 a 17.74 ± 0.65 a 0.002 P value 0.007 0.947 0.672 ------------- Butyric acid, mol.100 mol -1 --------------- 42 5.92 ± 0.36 a 6.90 ± 0.54 a 5.85 ± 0.28 a 0.163 5 of 8

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 84 1.95 ± 0.11 b 3.52 ± 0.13 ab 1.99 ± 0.09 b 0.000 P value 0.000 0.000 0.000 ---- Acec acid / Propionic acid, mol.100 mol -1 --- 42 4.33 ± 0.14 ab 4.24 ± 0.07 a 3.66 ± 0.18 b 0.007 84 6.62 ± 0.93 a 4.41 ± 0.41 b 3.95 ± 0.22 b 0.004 P value 0.012 0.457 0.316 ab Means with diﬀerent leers in the same line are signiﬁcantly diﬀerent. EFE-2: Exogenous ﬁbrolyc enzymes (2 g.d -1 ), EFE-4: Exogenous ﬁbrolyc enzymes (4 g.d -1 ), NH₃-N: Ammonia nitrogen, Volale fay acid Ruminal AA, PA, and BA concentraons were not diﬀerent among the groups on the 42nd d of the study (TABLE V). AA concentraons were also not diﬀerent among the groups on the 84th day of the study. The PA concentraon in the EFE-2 and EFE-4 groups was higher than the Control group on the 84th d of the study (P<0.01). PA concentraon at the 42nd d of the study was higher than at 84th d in the Control group (P<0.01). The BA concentraon in the EFE-2 group was higher than the Control and EFE-4 groups on the 84th d of the study (P<0.001). The BA concentraon in all groups was higher at the 42nd d of the study than at the 84th d (P<0.001). AA to the PA rao in the Control and the EFE-2 groups was higher than the EFE-4 group on the 42nd d of the study (P<0.01). It was also higher in the Control group than in the EFE-2 and EFE-4 groups on the 84th d of the study (P<0.01). AA to PA rao at the 84th d of the study was higher than the 42nd d in the Control group (P<0.05). The non-stascally signiﬁcant increase in AA concentraon on the 84th d of the study suggests that the supplementaon of 2 and 4 g.d -1 EFE had a posive eﬀect on crude ﬁbre digeson. Because the amount of AA increases when cellulose digeson is increased. The signiﬁcant increase of ruminal PA and BA concentraon in the EFE-2 and EFE-4 groups might be associated with the numerically higher CS and GH intake last six weeks of the study (7 to 12 weeks) when compared to the ﬁrst six weeks of the study (1 to 6 weeks). The increased ruminal PA and BA concentraons in both EFE-supplemented groups suggested that nutrient digeson in the rumen increased. On the other hand, increased PA and BA concentraon on the 84th d of study may explain numerically increased ADG in both enzyme groups. These two fay acids have long been considered chemical smuli for rumen development and may supply more incredible energy than acetate, which could improve animal performance [42]. The higher ruminal VFA concentraon with EFE supplementaon might be associated with increased microbial abundance and enzyme acvity [43]. Moreover, the synergisc eﬀect between supplemented EFE and endogenous enzymes might have improved the colonizaon and digeson of ruminal microbes to feed parcles, therefore improving ruminal fermentaon. The increased ruminal AA, PA, and BA concentraons in this study follow previous study’ results [40]. Similarly, it was determined that ruminal AA and BA concentraons increased by adding ﬁbrolyc enzymes to calf diets [13 , 14]. In contrast, a study in beef cale [40] found that EFE supplementaon did not aﬀect ruminal VFA concentraon. The AA to PA rao was higher in the Control and EFE-2 groups than the EFE-4 groups on the 42nd day of the study; it was lower for both EFE groups than the Control group on the 84th day of the study. Wang et al. [14] found that the addion of EFE to calves diet in the pre-weaning period did not change the AA to PA rao. In general, the diﬀerences in ruminal pH value, NH 3 -N, and VFA concentraon between this study and other studies might depend on the diﬀerence in roughage and concentrate feed, animal species, age of the animals, and the type and dose of ﬁbrolyc enzymes. CONCLUSION Supplementaon of 2 or 4 g.d -1 EFE did not aﬀect LW, daily gain, and feed intake but signiﬁcantly improved FCR in pre- weaning calves. Supplementaon of 2 or 4 g.d -1 EFE did not alter the ruminal pH, NH 3 -N, and AA concentraon on the 42nd and the 84th d of the study but increased the PA concentraon. Addionally, 2 g.d -1 EFE increased the butyric acid concentraon on the 84th d of the study. Due to the improved FCR and posive eﬀect on rumen VFAs concentraon, supplementaon of 2 or 4 g.d -1 EFE would beneﬁt calves fed with calf starter and grass hay and whole milk in the pre-weaning period. ACKNOWLEDGEMENTS I would like to thank Kaas University Scienﬁc Research Support Coordinatorship for their contribuon (No: 2014-VF- 39). In this research, “Global Nutritech, USA” has supplied “ForagezymeTM,” an enzyme used as a feed addive. Conﬂicts of interest None of the authors have any ﬁnancial or personal conﬂicts of interest that could inappropriately inﬂuence or bias the con- tent of the arcle. BIBLIOGRAPHIC REFERENCE [1] Diao Q, Zhang R, Fu T. Review of Strategies to Promote Rumen Development in Calves. Animals. [Internet]. 2019; 9(8):490. doi: hps://doi.org/p5ks [2] Dias J, Marcondes MI, Noronha MF, Resende RT, Machado FS, Mantovani HC, Dill-McFarland KA, Suen G. Eﬀect of Pre-weaning Diet on the Ruminal Archaeal, Bacterial, and Fungal Communies of Dairy Calves. Front Microbiol. [Internet]. 2017; 8:1553. doi: hps://doi.org/p5kt [3] Chen D, Zhong G, Su HEW, Rahman MA, Chen K, Tang J, Li F. 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