In Vitro and In Vivo Assessment of Dietary Supplementation of Both Natural or Nano-Zeolite in Goat Diets: Effects on Ruminal Fermentation and Nutrients Digestibility

Simple Summary Increasing fibrous feed digestibility while reducing methane (CH4) emission through manipulating rumen fermentation patterns to improve animal performance is the most critical challenge in the animal nutrition field. Nanotechnology has revolutionized the commercial application of nano-sized minerals in medicine, engineering, information, environmental technology, pigments, food, electronics appliances, biological and pharmaceutical applications, and many more. Therefore, animal nutrition scientists also resorted to using minerals and clays such as zeolite with different forms in feeding animals and evaluate this additive in animal performance. The natural zeolite clay is known for its high cation exchange capacity and adsorption characteristics that can modify ruminal fluid viscosity and binding capacity with ammonia (NH3-N). After evaluating the addition of zeolite in vivo and in vitro, results indicated that zeolite (natural and nano forms) maintained rumen pH, increased protozoa numbers, and improved propionate production. Medium supplementation level of the natural form of zeolite at 20 g/kg dry matter (DM) was the most efficient dose in reducing CH4 production, while the zeolite nano-form supplemented at 0.4 g/kg DM was the most effective dose in improving the organic matter (OM) degradability and reducing the NH3-N concentration compared to the control. Abstract This study aimed to evaluate in vitro and in vivo dietary supplementation with different levels of natural or nano-zeolite forms on rumen fermentation patterns and nutrient digestibility. In the in vitro experiment, a basal diet (50% concentrate: 50% forage) was incubated without additives (control) and with natural zeolite (10, 20, 30 g/kg DM) or nano-zeolite (0.2, 0.3, 0.4, 0.5, 1.0 g/kg DM) for 24 h to assess their effect on ruminal fermentation, feed degradability, and gas and methane production using a semi-automatic system of in vitro gas production (GP). The most effective doses obtained from the in vitro experiment were evaluated in vivo using 30 Barki goats (26 ± 0.9 SE kg body weight). Goats were allocated into three dietary treatments (n = 10/treatment) as follows: control (basal diet without any supplementations), natural zeolite (20 g/kg DM diet), and nano-zeolite (0.40 g/kg DM diet). The in vitro results revealed that only the nano-zeolite supplementation form quadratically (p = 0.004) increased GP, and the level of 0.5 g/kg DM had the highest GP value compared to the control. Both zeolite forms affected the CH4 production, linear, and quadratic reductions (p < 0.05) in CH4 (mL/g DM), consistent with linear increases in truly degraded organic matter (TDOM) (p = 0.09), and propionate molar proportions (p = 0.007) were observed by nano zeolite treatment, while the natural form of zeolite resulted in a linear CH4 reduction consistent with a linear decrease (p = 0.004) in NH3-N, linear increases in TDOM (p = 0.09), and propionate molar proportions (p = 0.004). Results of the in vivo experiment demonstrated that the nutrient digestibility was similar among all treatments. Nano zeolite enhanced (p < 0.05) the total short-chain fatty acids and butyrate concentrations, while both zeolite forms decreased (p < 0.001) NH3-N compared to the control. These results suggested that both zeolite supplementation forms favorably modified the rumen fermentation in different patterns.


Introduction
The application of feed additives in ruminant rations is one solution to improve the animal's performance via manipulating ruminal fermentation patterns and improving nutrients utilization. Microbial fermentation of the dietary organic matter results in loss of gross energy and nitrogen. Enteric CH 4 emission in ruminants represents a loss of up to 15% of gross energy of feeds; also, 75-85% of the nitrogen consumed by ruminants is excreted in the feces and urine [1]. Therefore, enhancing fibrous feed digestibility, reducing CH 4 emission, and nitrogen excretion by ruminants have to improve their performance [2].
Natural zeolite clay is composited of crystalline aluminosilicates and characterized by a high cation exchange capacity, high sorbent property that can modify ruminal fluid viscosity and binding capacity with NH 3 -N; therefore, it has been extensively used as a potential feed additive [3]. It also can capture ammonium ions, reducing the rate of their release and absorption from the rumen wall, and act as adsorbents for mycotoxins [4]. Besides, clinoptilolite of zeolite can enhance microbial ruminal fermentation by regulating ruminal pH to act as a pH-buffering agent [5].
The literature reported that zeolite supplementation levels had been examined ranging from 1% to 9% of DM of ruminant diets [6][7][8]. Dietary supplementation with zeolite clay exhibited positive effects on nutrients digestion and growth performance of sheep [9]. Furthermore, zeolite positively affected animal health status and performance due to its characteristic sorbent properties that modify the ruminal environment [10,11]. Nanoclays and other nano-particles have been shown to specifically absorb mycotoxins through the gastrointestinal tract of ruminants [12]. Nanotechnology is one of the most promising applications of the twenty-first century. It can create new materials with unique properties, which change the physical and chemical characteristics of the molecules/element to have the potential to revolutionize agriculture sectors and has given birth to the new area of agro-nanotechnology, particularly in livestock production. Size reduction of materials to the nano range can increase their adsorption, absorption, and cation exchange capacity [13]. Comparative research studies of nano and natural zeolite supplementations on rumen fermentation patterns and nutrient digestibility are limited. Therefore, we hypothesized that the effects of nano zeolite on ruminal microbial activity might differ from its natural form. Therefore, the objective of this study was to investigate the in vitro dose-response effects of natural and nano-zeolite supplementations on ruminal antimethanogenic activity, fermentation end-products, and nutrient degradation. The most effective doses of both zeolite forms were evaluated in vivo for ruminal fermentation characteristics and nutrient digestibility.

Materials and Methods
The study was carried out at the Advanced Laboratory of Animal Nutrition and experimental farm Faculty of Agriculture, Alexandria University and Laboratory of Livestock Research Department of Arid Land Cultivation Research Institute, the City of Scientific Research and Technological Applications, Alexandria. All procedures following protocols were approved and authorized by the Institutional Animal Care and Use Committee of the Alexandria University (ALEXU-IACUC/08-19-05-14-2-22).

Experimental Feed Additives
Natural zeolite was commercially purchased from A & O trading company, Giza, Egypt. Zeolite is composed of a microporous arrangement of silica and alumina tetrahedra (Clinoptilolite) with general formula (Ca, K 2 , Na 2 , Mg) 4  Gray-green The nano-zeolite powder was prepared mechanically by a high-energy planetary ball mill (Retsch PM, Germany) [14]. The mechanical route was performed in a period of 6 h with a reverse rotation speed of 300 rpm and vial rotation speed of 600 rpm with a ball to powder ratio of 9:1 mass/mass. The particle size of the obtained nano zeolite was measured by N 5 submicron particle size analyzer (BECKMAN COULTER, Brea, CA, USA), with a range of 3 nm-5 µm of particle size.
To detect the distribution size and shape of zeolite nano-particles, the scanning electron microscope (SEM; Jeol JSM-6360 LA, 3-1-2 Musashino, Akishima, Tokyo, Japan) and transmission electron microscope (TEM; JEOL JEM-2100, 3-1-2 Musashino, Akishima, Tokyo, Japan) were used to provide three-dimensional images, which are very useful for understanding the morphological characters of the tested nanoparticles [15]. The sample was coated with gold to improve the imaging of the sample. The SEM was operated at a vacuum of the order of 10, and the accelerating voltage of the microscope was kept in the range of 10-20 kV (Figure 1). The TEM nano-particles' shape and size were prepared by dropping approximately 10-15 µL of a dilute sample of ZnO-NPs on the top of the carbon-coated copper grid and left in the hood to dry (Figure 2). The particle size mean was 60.2 nm of the nano zeolite. To identify the functional groups of the prepared nano-zeolite form, the Fourier Transform Infra-Red Spectroscopy (FTIR) analysis was performed using an infrared spectrometer (Shimadzu FTIR-8400S, Nakagyo-ku, Kyoto, Japan) by employing the KBr pellet technique [16], as shown in Figure 3.
The surface charge of the nano-zeolite was measured by zeta potential analysis using a Malvern ZETASIZER Nano series (Malvern, Worcestershire, England, United Kingdom) [17], under the following circumstances: temperature (°C) 25.0, count Rate (kcps) 347.4, measurement position (mm) 2.00, and attenuator 7.00. The zeta potential of the prepared nano-zeolite was −5.85 (mv), zeta deviation and conductivity were 63.8 (mV) and 0.00165 (mS/cm), respectively, as presented in Figure 4.   To identify the functional groups of the prepared nano-zeolite form, the Fourier Transform Infra-Red Spectroscopy (FTIR) analysis was performed using an infrared spectrometer (Shimadzu FTIR-8400S, Nakagyo-ku, Kyoto, Japan) by employing the KBr pellet technique [16], as shown in Figure 3.
The surface charge of the nano-zeolite was measured by zeta potential analysis using a Malvern ZETASIZER Nano series (Malvern, Worcestershire, England, United Kingdom) [17], under the following circumstances: temperature (°C) 25.0, count Rate (kcps) 347.4, measurement position (mm) 2.00, and attenuator 7.00. The zeta potential of the prepared nano-zeolite was −5.85 (mv), zeta deviation and conductivity were 63.8 (mV) and 0.00165 (mS/cm), respectively, as presented in Figure 4. To identify the functional groups of the prepared nano-zeolite form, the Fourier Transform Infra-Red Spectroscopy (FTIR) analysis was performed using an infrared spectrometer (Shimadzu FTIR-8400S, Nakagyo-ku, Kyoto, Japan) by employing the KBr pellet technique [16], as shown in Figure 3.

Basal Diet
The experimental basal diet (used in the in vitro and in vivo experiments) consisted of (g/kg DM) 500 g concentrate and 500 g berseem hay (Trifolium alexandrinum) formulated as a total mixed ration (TMR) diet to meet the nutrient requirements of lactating goats [18]. The AOAC [19] analytical procedures were used for dry matter (DM), organic matter (OM), crude protein (CP as 6.25 × N; by Kjeldahl technique), and ether extract (EE). Cell wall ingredients (neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin contents (ADL)) were determined sequentially by an Ankom 200 fiber analyzer unit (ANKOM Technology Corporation, Macedon, NY, USA) and expressed exclusive of residual ash as described by Van Soest et al. [20]. Concentrations of hemicellulose were calculated as NDF-ADF, and cellulose as ADF-ADL.
The major ingredients and chemical composition of the experimental diet are presented in Table 2.

Item
Basal Diet (g/kg Dry Matter) Ingredients Berseem clover hay 500 The surface charge of the nano-zeolite was measured by zeta potential analysis using a Malvern ZETASIZER Nano series (Malvern, Worcestershire, England, United Kingdom) [17], under the following circumstances: temperature ( • C) 25.0, count Rate (kcps) 347.4, measurement position (mm) 2.00, and attenuator 7.00. The zeta potential of the prepared nano-zeolite was −5.85 (mv), zeta deviation and conductivity were 63.8 (mV) and 0.00165 (mS/cm), respectively, as presented in Figure 4.

Basal Diet
The experimental basal diet (used in the in vitro and in vivo experiments) consisted of (g/kg DM) 500 g concentrate and 500 g berseem hay (Trifolium alexandrinum) formulated as a total mixed ration (TMR) diet to meet the nutrient requirements of lactating goats [18]. The AOAC [19] analytical procedures were used for dry matter (DM), organic matter (OM), crude protein (CP as 6.25 × N; by Kjeldahl technique), and ether extract (EE). Cell wall ingredients (neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin contents (ADL)) were determined sequentially by an Ankom 200 fiber analyzer unit (ANKOM Technology Corporation, Macedon, NY, USA) and expressed exclusive of residual ash as described by Van Soest et al. [20]. Concentrations of hemicellulose were calculated as NDF-ADF, and cellulose as ADF-ADL.
The major ingredients and chemical composition of the experimental diet are presented in Table 2.

Item
Basal Diet (g/kg Dry Matter) Ingredients

Basal Diet
The experimental basal diet (used in the in vitro and in vivo experiments) consisted of (g/kg DM) 500 g concentrate and 500 g berseem hay (Trifolium alexandrinum) formulated as a total mixed ration (TMR) diet to meet the nutrient requirements of lactating goats [18]. The AOAC [19] analytical procedures were used for dry matter (DM), organic matter (OM), crude protein (CP as 6.25 × N; by Kjeldahl technique), and ether extract (EE). Cell wall ingredients (neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin contents (ADL)) were determined sequentially by an Ankom 200 fiber analyzer unit (ANKOM Technology Corporation, Macedon, NY, USA) and expressed exclusive of residual ash as described by Van Soest et al. [20]. Concentrations of hemicellulose were calculated as NDF-ADF, and cellulose as ADF-ADL.
The major ingredients and chemical composition of the experimental diet are presented in Table 2.

The In Vitro Experiment
The experimental treatments consisted of control (basal diet without supplementation), five supplemental doses of the nano-zeolite (0.2, 0.3, 0.4, 0.5, and 1 g/kg DM basal diet), and three doses of the natural zeolite (10, 20, and 30 g/kg DM), and were evaluated in vitro.

Gas Production Procedure
Method of the semi-automatic system of GP equipped with pressure transducer and a data logger (Pressure Press Data GN200, Sao Paulo, Brazil) as described by Bueno et al. [21] and adapted by Soltan et al. [22] was used to evaluate the dose-response effects of the experimental supplementations.
Rumen contents were collected freshly from adult fasted slaughtered of three Egyptian buffalo steers at the slaughterhouse of Faculty of Agriculture Alexandria University. The slaughtered animals were fed ad libitum a diet consisting of 50:50 commercial concentrate mixture: clover hay (Trifolium alexandrinum L.) and had free access to fresh water. Rumen contents were collected and kept separately in pre-warmed containers (39 • C) under anaerobic conditions. To prepare the rumen inocula (n = 3), the rumen content of each animal was blended for 10 s, squeezed through three layers of cheesecloth, and kept in a water bath (39 • C) under CO 2 until inoculation took place. The different ruminal inocula were used to prevent the unusual effects of rumen environmental conditions [23,24].
Four analytical repetitions (4 bottles/inoculum/treatment) were used; two for the fermentation parameters and protozoal count, and the other two were for the determination of truly degraded organic matter (TDOM). Similarly, blank bottles (rumen fluid and buffer solution), and internal standard bottles (rumen inoculum, buffer solution, and clover hay) were prepared to correct for the sensitivity induced by the inocula [24,25]. Samples (0.5 g) of the experimental supplemented diets were weighed into numbered bottles and were incubated with 45 mL of diluted rumen fluid (15 mL mixed rumen fluid + 30 mL of Menkes buffered medium) in 120 mL incubation bottles [24,25]. Bottles were then sealed immediately with 20 mm butyl septum stoppers (Bellco Glass Inc., Vineland, NJ, USA), mixed, and incubated in a forced-air oven (FLAC STF-N 52 Lt, Treviglio, Italy) at 39 • C for 24 h. The gas head-space pressure of all bottles was recorded at 3, 6, 9, 12, 24 h incubation using a pressure transducer and a data logger (Pressure Press Data GN200, Piracicaba, Sao Paulo, Brazil). The pressure of GP in all bottles at each measuring time was converted into volumes to calculate the total accumulative gas produced through 24 h [22].
For CH 4 determination through 24 h, one mL of gas of the bottle head-space was sampled by a syringe (med Dawliaico, Assiut, Egypt) at each gas pressure measuring time and accumulated in a 5 mL vacutainer tubes (BD Vacutainer ® Tubes, Franklin Lakes, NJ, USA). Methane concentration was determined using a gas chromatograph (Model 7890, Agilent Technologies, Inc., CO 80537, Santa Clara, CA, USA); the separation conditions were in detail described by Soltan et al. [22]. The amounts of CH 4 produced were calculated according to Longo et al. [26]. Net values of both GP and CH 4 were corrected for the corresponding blank values.

Rumen Degradability
At the end of the incubation, all bottles were put in cold water (4 • C) to stop the microbial fermentation process. Determination of TDOM was carried out according to Blümmel et al. [27] by immediate addition of neutral detergent solution (70 mL) without heat-stable α-amylase and incubated in a forced-air oven at 105 • C for 3 h. The remains were filtered in clean pre-weighed crucibles, washed with hot water, and dried at 105 • C for 16 h, and allowed to be burned at 550 • C for 4 h. The TDOM values were calculated from the difference between the amounts of the incubated OM and those remaining nondegraded. The portioning factor (PF) was calculated as the ratio of TDOM (mg) and gas volume (mL) [27].

Rumen Fermentation Characteristics
Rumen pH was determined using a pH meter (GLP 21 model; CRISON, Barcelona, Spain) in all fermentation bottles. Protozoal count was microscopically determined and differentiated by Digital Zoom Video microscope (LCD 3D, GiPPON; Wanchai, Hong Kong) following the procedure described by Dehority et al. [28].

Animals and Experimental Design
Based on the in vitro assay results, the most effective level of both natural and nanozeolite was selected to evaluate their responses on apparent nutrients digestibility. Thirty female non-lactating Barki goats were randomly divided into three dietary treatments (n = 10/treatment) according to initial body weight (26 ± 0.9 kg SE bodyweight) as follows: control (the same control basal diet that was used in the in vitro experiment), natural zeolite (20 g/kg DM), and nano-zeolite (0.40 g/kg DM). Animals were fed their experimental diets ad libitum. Zeolite supplementation was orally administrated to ensure the complete dose was received.
Goats were fed twice daily at 08:00 and 16:00 and allowed free access to fresh water throughout the experimental period. Animals were adapted to the experimental diets for 15 days, followed by 7 days as a collection period.

Rumen Fermentation Parameters
Samples of rumen fluid (~30 mL) were collected using an esophageal probe 3 h after the morning feeding. The first 15 mL of the ruminal sample was discarded to avoid saliva contamination; all samples were then strained through three layers of cheesecloth and immediately subjected to ruminal pH using the same portable digital pH meter that was used in the in vitro assay. Ruminal individual SCFAs, total protozoa numbers, and NH 3 -N concentration were analyzed as described previously in the in vitro experiment.

Apparent Nutrients Digestibility
Fresh fecal samples (~40 g each) were obtained daily from each goat at 09:00 and 17:00, about 1 h post-feeding. Apparent nutrient digestibility was determined in which acid-insoluble fiber was used as an internal marker based on the relative concentrations of these nutrients in the feed and feces [20]. These samples were pooled per goat and stored at −20 • C for later analysis. At the end of this period, all the fecal samples were dried in a forced-air oven at 60 • C for 72 h, ground to pass through a 1 mm screen, and chemically analyzed for DM, OM, EE, NDF, and ADF as described previously.

Statistical Analyses
All results were analyzed using the general linear model procedure (PROC GLM) procedure of SAS [31]. The in vitro gas production experiment was performed in one run for all treatments. The analytical replicates were averaged before statistical analysis, with each inoculum being the statistical replicate; thus, the statistical number of replications of treatments (n = 3) are the true replications. Orthogonal contrast statements were designed to test the linear and quadratic responses of each dependent variable to the increasing concentrations of nano or natural zeolite. The results of in vivo experiment were subjected to analysis of variance using the following statistical model as Yi = µ + Ti + ei, where Yi = observations mean, µ = overall mean, Tj = treatment effect, and ei = residual error. Differences between the treatments were considered significant at (p < 0.05), and trends were accepted if (p < 0.10). Tukey's procedure for multiple comparisons was used to detect differences among means of the in vivo experiment.

In Vitro Experiment
The effects of different levels of natural and nano-zeolite forms on ruminal GP, CH 4 , TDOM, and partitioning factors are presented in Table 3. The GP increased quadratically (p = 0.004) with increasing doses of nano-zeolite supplementations, while the natural zeolite did not affect the GP values. Linear reductions (p < 0.05) in CH 4 production (related to the incubated DM and TDOM) consistent with tended increases (p = 0.09) in TDOM were observed by both zeolite form supplementations. The most significant CH 4 reductions (49 and 15%) were achieved by supplementations of 20 g/kg DM natural zeolite, and 0.4 g/DM kg nano zeolite, respectively, compared to the control. Neither nano nor the natural form of zeolite supplementation affected the partitioning factor. Table 3. Supplementation effects of natural and nano-zeolite forms on ruminal gas production (GP), methane, truly degraded organic matter (TDOM), and partitioning factor through 24 h incubation period (in vitro experiment).

GP (mL/g DM Incubated)
Methane  The in vitro effects of natural and nano-zeolite forms on rumen protozoal count are presented in Table 4. Increases in the total protozoal count consistent with increases in Diplodinium sp. and Epidinium sp. were observed by nano zeolite (quadratic effect; p < 0.05) and natural zeolite (linear effect, p < 0.01) supplementations. Only natural zeolite supplementation increased linearly (p = 0.001) and quadratically (p = 0.02) the Eudiplodinium sp., while no effects were observed by nano zeolite treatments. Similarly, Isotricha sp. tended to be increased (linearly, p = 0.09, and quadratically p = 0.05) with the increasing levels of the natural zeolite, while neither Entodinium nor Ophryscolex sp. was affected by both zeolite supplementations. Quadratic increases (p < 0.05) in total SCFAs concentrations and acetate molar proportions by the natural zeolite, while no effects were observed by the nano zeolite form. Both zeolite forms linearly enhanced (p < 0.05) and tended to increase quadratically (p < 0.001) propionate to molar proportions. Ratio of C2:C3 declined linearly (p = 0.01) by nano-zeolite, and quadratic (p = 0.02) by natural zeolite supplementation. The ruminal pH was not affected by dietary levels of nano or natural zeolite, while only natural zeolite linearly decreased (p = 0.004) the NH 3 -N concentration (Table 5). Contrast 1 = effects of control (0 supplementation g/kg DM) compared with nano zeolite supplementations, and Contrast 2 = effects of control (0 supplementation g/kg DM) compared with natural zeolite supplementations. C2:C3 = acetate to propionate ratio. SEM: standard error of the mean.

In Vivo Experiment
The effects of zeolite type supplementation on ruminal fermentation characteristics and protozoal count are shown in Table 6. Nano-zeolite increased total SCFAs (p = 0.021) and butyrate (p = 0.001) concentrations compared to other treatments, while it decreased (p = 0.03) valeric molar proportion compared with the natural form of zeolite. Goats fed natural zeolite had an increase (p = 0.05) in ruminal pH compared with goats fed the control diet, while no differences were observed between both zeolite forms on ruminal pH.
Both natural and nano-zeolite forms declined (p < 0.001) NH 3 -N concentration compared with the control. Moreover, both nano and natural zeolite increased (p < 0.001) ruminal Isotrica sp. populations compared with the control, while no differences were detected among the experimental treatments on the other protozoal populations. Table 6. Supplementation effects of natural or nano-zeolite on goat rumen fermentation parameters and protozoal count 3 h post-feeding (in vivo experiment). The digestibility coefficients of DM, OM, CP, and EE are shown in Table 7. Natural and nano zeolite supplemented diets did not affect the DMI and nutrients digestibility. Table 7. Supplementation effects of natural or nano-zeolite on dry matter intake (DMI) and apparent nutrients digestibility of goats (in vivo experiment).

Discussion
Both TEM and SEM images of the experimental nano zeolite indicated that the mechanical grinding of the natural zeolite reduced their particle size and successfully presented in the nano-scale. The zeta potential of nano zeolite was negative charges that favorably enhance the affinity of palygorskite with cationic matters, e.g., cationic dyes, and then enhance the adsorption capacity. Results of the FTIR of the nano form of zeolite indicated the high efficiency of the performed nano-particles; 20 well-defined peaks of zeolite functional groups were observed, and 12 of them were found in higher frequencies at 2126-4009, while only four peaks appeared in the lower frequency range (from 466 to 789). These physico-chemical properties of the zeolite nano-form may result in different effects in rumen fermentation compared to its natural form. Increases in GP values caused by nanozeolite addition may indicate the higher efficiency of nano-zeolite to improve the ruminal microbial fermentation than the natural zeolite; this can be due to the large surface areas, large capacity for cation exchange, and high activities caused by the size-quantization effect [32]. Rumen CH 4 production is strongly related to microbial fermentation extent; therefore, enhancements in GP and nutrient degradability can increase rumen CH 4 emission [33]. Thus, such increases in the total GP caused by the nano zeolite may partly explain the low efficiency of the nano-zeolite to reduce CH 4 production compared to the normal form. Reductions in CH 4 production caused by nano or natural forms confirmed the anti-methanogenic activity of zeolite in this study. Zeolite may act as an alkalinizer and has a high capacity for H+ exchange at different pH ranges [34,35]. Therefore, zeolite can reduce CH 4 emission by affecting rumen H+ exchange capacity and can also affect all the end fermentation characteristics.
Most common CH 4 inhibitors may adversely affect the ruminal nutrient degradability and/or microbial fermentation at doses that achieve desirable CH 4 reduction [22]. In the in vitro study, CH 4 reduction consistent with increases in TDOM and GP caused by nano zeolite supplementations may indicate that both zeolite types might benefit the alteration of ruminal fermentation pattern towards less CH 4 production without adverse effect on feed degradability. This can be due to the catalytic activity of zeolite nano-particles which can increase some digestive fiber enzymes (such as amylase, α-amylase) to improve OM degradability [36]. Moreover, the literature reported that enhancing the rumen nutrient degradability is a typical action of zeolite through the buffering effect and maintaining the ruminal pH from the rapid decrease [36]. In the current study, both zeolite forms enhanced the TDOM in vitro and the pH in vivo, while no differences were detected in the nutrient digestibility in vivo. The reasons for this phenomenon are not clear, but it seems that the activity of zeolite is more efficient in the rumen than in the post-ruminal digestive tract parts. The current results are in line with Galindo et al. [37], who reported that zeolite could provide favorable conditions for the increase of cellulolytic rumen bacteria and subsequently increase the ruminal degradable organic matter. The lacking effects of zeolite supplementation on apparent nutrient digestibility are consistent with what reported by Câmara et al. [38], as total tract DM digestion was unaffected when zeolite supplemented at levels of 30-50 g zeolite/kg of dietary DM, while McCollum et al. [39] observed enhancements in ruminal digestion of OM and starch with supplementation of 25 g zeolite/kg of finishing diet.
Reduction in CH 4 can be achieved indirectly by decreasing protozoal abundance [22], but results of the current study indicated that CH 4 reduction was consistent with increases in the total protozoal count, which is mainly related to the significant increases in Diplodinium sp., the highest number of protozoal species naturally found in the typical rumen conditions of ruminants. This may partly explain the high TDOM caused by both zeolite types, as Diplodinium sp. is known for the high efficiency for cellulose degradation, and consequently, H+ abundance [33][34][35][36][37][38][39][40]. Therefore, the current results indicated that the CH 4 reduction was a result of the high rumen H+ exchange capacity of zeolite.
According to some previous studies [40,41], ruminal pH stability provides more favorable environmental conditions for more microbial proliferation. In the present study, the observed increase in total protozoal abundance may be due to the practical stability of rumen pH within the normal range associated with the available energy (as SCFAs production) and nitrogen (as adequate NH 3 -N concentration) for more microbial protein synthesis. This explanation agrees with Dschaak et al. [42], who reported that the great affinity of zeolites for holding water and osmotically active cations could enhance ruminal microbial fermentation and osmotic activity that can regulate pH in the rumen by buffering against hydrogen ions of organic acids. Our results also confirmed that rumen pH plays an important role in the survival of rumen -ciliated protozoa [40,41].
The SCFAs patterns of both zeolite forms declared the ability of nano zeolite to modify the microbial fermentation activity differently from its natural form. The in vitro experiment revealed that natural zeolite quadratically enhanced acetate concentration; consequently, the total SCFAs (as acetate is the main contributor of total SCFAs), while these were not caused by the nano form of zeolite. Additionally, the nano form of zeolite enhanced butyric concentration in the in vivo experiment compared with the natural zeolite form. These differences may confirm our suggested hypothesis that performing the nano form of the zeolite may affect their efficiency as feed additive differently from its natural form.
Results of the in vitro assay showed that both zeolite forms enhanced propionate molar proportions concentration. These results, alongside decreases in acetate to a propionate ratio, might be due to shifting SCFAs production pattern from acetate toward more propionate production, which may explain that the fermentation process occurred in a more efficient manner where more hydrogen ion (H + ) may be used by ruminal microbes to synthesize SCFAs (propionate) rather than CH 4 . Additionally, differences in fermentation patterns were observed by the in vivo and in vitro experiments using the same experimental dose. It seems that the time of collection of the ruminal samples (3 h post-feeding) of the in vivo assay, rather than the nutritive buffering solution used in the in vitro assays, may affect the obtained results.
Both zeolite forms decreased NH 3 -N concentration in the in vivo assay, while it occurred only by the natural form in the in vitro experiment. Zeolite, as a cation exchanger, is capable of exchanging and holding the ammonium ion before its release by the sodium ion (Na + ) present in the saliva that was entering the rumen [43][44][45]. In this regard, zeolite additive could exhibit a higher potential to sink hydrogen through its cation exchange capacity, which might be another possible explanation for zeolite-buffering properties. Lower ruminal NH 3 -N concentration with the addition of nano-zeolite indicated that zeolite was able to capture NH 3 through the character of cation exchange capacity [46].
The modulation of rumen fermentation patterns that occurred by both zeolite forms may be nutritionally advantageous for lactating and growing ruminants through enhancing ruminal OM degradability and propionate production [47].

Conclusions
The nano transformation of the natural zeolite positively affected the physico-chemical properties of the natural zeolite. Zeolite, whether in its natural or nano-form, was able to maintain rumen pH while reducing NH3-N concentration and affecting CH4 production without adverse effects on the apparent nutrient digestibility. Zeolites as clay minerals play a role in improving the rumen environment and fermentation end-products because of their buffering role. In both experiments, nano zeolite modified the SCFAs pattern differently from the natural zeolite. These results may suggest that the consideration of zeolite as a modifier of rumen fermentation was not only dose-dependent but also particle-size-dependent.

Conflicts of Interest:
The authors declare no conflict of interest.