Research Article

Impact of Bentonite, Humic Acid or Zeolite Levels with Soybean, Sunflower or Cottonseed Meals as Dietary Protein Sources on In vitro Rumen Fermentation

Shorouk Aladdin Helmy1, Hossam Mahrous Ebeid2*, Mohamed Ahmed Hanafy1, Adel Eid Mohamed Mahmoud1, Reham Roshdy Ali El-tanany1

1Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt; 2Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, 12622, Giza, Egypt.

Received | February 21, 2022; Accepted | March 25, 2022; Published | June 01, 2022

*Correspondence | Hossam Mahrous Ebeid, Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, 12622, Giza, Egypt; Email :hm.abdel-aziz@nrc.sci.eg; hossamebaid55@gmail.com

Citation | Helmy SA, Ebeid HM, Hanafy MA, Mahmoud AEM, El-Tanany RR (2022). Impact of bentonite, humic acid or zeolite levels with soybean, sunflower or cottonseed meals as dietary protein sources on in vitro rumen fermentation. J. Anim. Health Prod. 10(2): 158-167.

DOI | http://dx.doi.org/10.17582/journal.jahp/2022/10.2.158.167

ISSN | 2308-2801

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

In intensive livestock production, protein degradation is the most single important factor affecting protein supply to the small intestine through the hydrolysis of microorganisms that count for 65% of the protein in the diet to organic acids, ammonia and CO2 in the rumen (McDonald et al., 2011). The ammonia produced with other peptides and free amino acids is used for microbial protein synthesis by microorganisms. However, the feeding on a high percentage of rumen degraded protein sources such as soybean, sunflower, and cottonseed meals or even non protein nitrogen (NPN) is causing an increasing problem in ammonia release in the rumen and causing ammonia toxicity in the blood that might lead to losing the animal (McDonald et al., 2011). In addition to aflatoxin and mycotoxins (poisons) problems produced by the fungi and moulds that grow on feed and affect animal health through lowering immunity and causing diseases that lead to diarrhea, liver, and kidney damage, weak bones, and reduced weight gain in animals (Jaynes et al., 2007).

In the mid of the 1960s, scientists of animal nutrition introduced clay as a natural source into livestock production diets as binding agents for feeding pellets and to promote the growth and health of the animals (Sulzberger et al., 2016). The actual use of clay minerals was directed towards the production of selective sorbents applicable to both human and veterinary medicine. However, previous reports showed that eating clay (including products such as, bentonite, zeolites, or humic acid) by animals in the wild, led to detoxification of the body, alleviation of gastrointestinal infections as against rumen disorders, reduced the absorption of aflatoxin from the gastrointestinal tract (Diaz et al., 2004; Kissell et al., 2013) and reduce ammonia release in the rumen (Montalvo et al., 2012; Sulzberger et al., 2016).

Bentonite known as Montmorillonite has an attraction to mono and divalent ions that bind proteins and nitrogenous compounds to reduce protein degradation and increase bypass protein from the rumen (Sulzberger et al., 2016; Trckova et al., 2004). On the other hand, humic acid exerts a protective action on the mucosa of the intestine as well as having antiphlogistic, adsorptive, antitoxic, and antimicrobial properties through improving the immune system. On the other hand, it plays a crucial role in reducing stress and enhancing liver function, as well as stimulating lymphocyte proliferation (Griban et al., 1991; EMEA, 1999; Kanana et al., 2019).

Most of the research on clay has been investigated by veterinarians and nutritionists. However the recommended application techniques in terms of doses, forms of addition, and types of animals along with other criteria still need more comprehensive research and experimentation. Therefore, clay’s effects on rumen fermentation and dry matter digestibility have varied among studies and it is necessary to conduct more research to enhance the performance. Moreover, the recommended levels of additives were not elucidated as well as the type of ration content. So, in this study we aim to evaluate the potential effects of different clay minerals such as bentonite, zeolite, or humic acid as feed additives in different levels added to different sources of protein (soybean, sunflower, and cottonseed meals) on the mitigation of ammonia release and modulation of rumen fermentation in vitro.

Materials and Methods

The in vitro experiment was conducted at the Dairy Sciences Department, National Research Centre, Egypt in cooperation with the Animal Production Department, Faculty of Agriculture, Cairo University, Giza, Egypt.

Clay minerals sources

Clays of bentonite and zeolites have been gotten from the Feeds Unit, Faculty of Agriculture, Cairo University, Giza, Egypt. The humic acid product was purchased from an Egyptian-Candian company for fertilizers commerce in Egypt and derived from humic lignite from the USA, containing 74% combined humic acids with 20% low molecular weight fulvic acid.

Experimental design

In vitro trials

Three protein sources (soybean meal, sunflower meal and cottonseed meal) and three clay minerals as additives (bentonite, zeolites, and humic acid) were used in this study. Each clay mineral was tested in four levels with three different mixed rations, for total of nine rations compared to three control ration groups (without addition) in a total of 12 groups for each protein source as illustrated in Table 1.

The effects of different clay mineral levels on degradable dry matter (dDM), rumen fermentation (pH and ammonia concentration), and total gas production (TGP) were studied using an in vitro batch culture system. The experimental ration mixture was used as a substrate in the ratio of 50:50 (R:C) and its chemical composition is shown in Table (2). Each treatment was tested in 6 replicates accompanied by blank vessels (no substrate). Around 400 mg of the substrate was added to the incubation vessels of 100 mL capacity. Each vessel was filled with 40 mL of the incubation medium, consisting of rumen inoculum and buffer a ratio of 1:4 (v/v). A buffer was prepared according to Szumacher-Strabel et al. (2004) as reported by Ebeid et al. (2020a and b). All vessels containing rations of substrate were incubated at 39°C for one day before fermentation started to be warm during the injection. Rumen fluid was collected from a slaughterhouse at 5:00 pm into preheated thermos vessels and transported to the laboratory, then squeezed through 4-layers of cheesecloth into a Schott Duran® bottle (L) with under flushing with CO2 in a water bath at 39°C where it was used as a source of inoculum. Forty mL of this rumen buffer inoculum was added to each vessel after which the headspace of each bottle was flushed with CO2, all vessels were immediately closed with rubber stoppers followed by sealing with an aluminum cap, and the vessels were incubated at 39°C.

Sampling and analyses

After 48 hours of incubation at 39°C, the TGP was measured by the displacement of a syringe glass (100 mL), which was connected to the serum flasks. After that, the bottles were transferred directly into the fridge to stop microbial activity and fermentation. Three vessels of six were transferred into crucibles (dried, and weighted previously), and oven dried at 70 °C until completely dry to measure the dDM. The other three vessels were filtered, and pH was measured immediately after the bottles were opened (HI 9024C, HANNA Instruments, Woonsocket, Rhode Island, USA), while ammonia samples (15 mL each) were collected and stored in a freezer (-20°C) for analysis. Nessler’s modified method was used to figure out how much ammonia was in the water (Szczechowiak et al., 2016).

Statistical analysis

Statistical analysis of data was carried out using the GLM procedure of SAS (2015) with three different clay minerals (humic acid, zeolite, and bentonite) in four different clay levels to three different protein sources in diets in a 3×4×3 factorial design. Differences between means of treatments for parameters (dDM, NH3-N, pH, TGP and TGP (g/kg DM) were separated by Duncan’s test (Duncan, 1955) with a P value < 0.05 to < 0.10.

The statistical model was:

Xicps= µ + αc + βps + (αβ)cps + eicps

Where: Xicps: observation icps. µ: overall mean. αc: effect of clay. βp: effect of protein source. (αβ)cp: interaction of two factors. eicps: experimental error

Results and Discussion

Ingredients and rations chemical composition

The ingredients and chemical compositions of the three rations (on DM basis) were prepared to be iso-nitrogenous (15.68, 15.65, and 15.64%) CP for soybean (R1), sunflower (R2), and cottonseed rations (R3), respectively as shown in Table (2). Also, the DM content was similar among rations, but OM, EE and CF were a little bit decreased in R3 compared to R1 and R2 which could be explained by the diffractions in chemical composition among the three protein sources. The percentage of wheat bran addition was 0.5 R3 lower than in R1 and R2.

Effect of humic acid addition on rumen fermentation kinetics

The effect of humic acid addition at three levels (0.5, 1, and 1.5%) on protein sources is shown in Table (3). Overall significant differences were observed in the amounts of dDM (g/kg DM), TGP (ml), and TGP (g/DM) for 3 protein sources (soybean, sunflower, and cottonseed meals) when compared with 3 levels of humic acid. Moreover, the ammonia concentrations and pH values did not show a significant difference between humic levels on soybean and sunflower rations, but a significant difference was observed in the cottonseed ration. Additionally, more than 1% of humic acid had an effect on dDM and rumen fermentation kinetics in rations containing soybean. Total GP (ml or g/DM) increased at 1% humic acid addition in rations containing sunflower and cottonseed meals, but dDM was significantly decreased. The ammonia nitrogen concentrations increased at 0.5% humic acid and decreased at 1%, while it had increased again in sunflower rations without any significant difference, but the inverse trend was significantly observed in cottonseed rations.

Previous research were reported that humic acids are the fraction of humic substances that are not soluble in water under more acidic conditions (pH < 2) but are soluble at higher pH values that could be induced by the high molecular weight acids and minerals (iron, manganese, copper, and zinc) (Weber, 2002). In a study conducted in vitro (Shi et al., 2001), the use of humic acid in livestock was applied directly to the beef cattle manures to reduce ammonia emissions and reducing cumulative ammonia emissions by 67.6%. Additionally, Ji et al. (2006) observed that ammonia emission from manure was reduced by 3 to 18% when animals fed on various humic dietary substances at rates of 0.5 and 1 % which is consistent with our findings. However, no differences were found for ruminant ammonia concentrations or ruminant pH with beef cattle (McMurphy et al., 2011).

Recently, El-Zaiat et al. (2018) found that adding drenched humic acid at a dose of 2 g/day per goat increased ruminal pH, while ammonia concentration was decreased, which is in agreement with our research. Moreover, (Degirmencioglu, 2012) reported that humic acid is not a single molecule, but a complex mixture of many different acids containing carboxyl and phenolate groups. The phenolate group has a negative effect on ammonia and protozoa counts in the rumen (Ebeid et al., 2020a and b). In spite of the actual mechanism of humic acid’s effect in animals being unclear or not yet understood, but Vaughan and Ord (1991) reported that humic substances inhibited urease activity and urease inhibition was higher in pH values. Other theories suggest that humate substances and humic binding capability may have the potential to alter rumen fermentation through slow NH3-N release, which could cause a gradual drop and recovery of pH over a longer period of time, and result in a better utilization of degradable intake protein by rumen microbes in the rumen (McMurphy et al., 2011). Therefore, the improvement of dDM in the current study may be related to humic acid, which is thought to have a beneficial impact on ruminal and intestinal microflora stabilization, ensuring enhanced feed efficiency.

 

Table 1: In vitro experimental treatments.

Protein source	Clay minerals sources levels (% from DM)
	Bentonite	Zeolite	Humic acid
Soybean meal	0.0, Control	0.0, Control	0.0, Control
	1.0	1.25	0.5
	2.0	2.5	1.0
	3.0	5.0	1.5
Sunflower meal	0.0, Control	0.0, Control	0.0, Control
	1.0	1.25	0.5
	2.0	2.5	1.0
	3.0	5.0	1.5
Cottonseed meal	0.0, Control	0.0, Control	0.0, Control
	1.0	1.25	0.5
	2.0	2.5	1.0
	3.0	5.0	1.5

 

Table 2: Ingredients and chemical composition of the total mixed ration in in vitro experiment

Item	Experimental rations*
	R1	R2	R3
Ingredients
Clover hay	50	50	50
Corn	25	25	25
Sunflower	0	9.6	0
Soybean	10	0	0
Wheat bran	13	13	12.5
Cotton seed meal	0	0	10
limestone	1.5	1.5	1.5
Salt	0.5	0.5	0.5
Urea	0	0.4	0.5
Total	100	100	100
Chemical composition (DM basis, %)
DM	89.08	88.88	89.00
OM	93.15	92.70	92.00
Ash	6.85	7.30	8.00
CP	15.68	15.65	15.64
EE	3.32	3.29	2.96
CF	22.41	23.61	21.16
NFE	51.74	50.15	52.24

*R1: Soybean ration; R2: Sunflower ration; R3: Cotton seed ration

 

Table 3: Effect of humic acid addition to the experimental rations on rumen fermentation kinetics

Protein source	Levels of humic	1dDM, %	NH3-N, mmol	pH	2TGP, ml	TGP, g/kg DM
Soybean	0.0, control	32.48ab	9.93	6.41	55.00a	151.29a
	0.5	37.80a	8.64	6.5	55.40a	153.64a
	1.0	31.65b	7.32	6.55	58.60a	162.34a
	1.5	28.91b	7.54	6.33	43.00b	117.87b
SEM		1.35	0.5	0.09	1.65	4.69
P value	Treatment	0.040	0.179	0.399	0.001	0.001
	Linear	0.050	0.069	0.658	0.003	0.003
	Quadratic	0.041	0.414	0.132	0.001	0.001
	Control vs. all	0.856	0.064	0.652	0.326	0.274
Sunflower	0.0, control	19.67bc	7.23	6.61	48.33ab	132.18ab
	0.5	23.82ab	9.3	6.51	40.00b	108.43b
	1.0	18.38c	6.56	6.45	58.00a	159.96a
	1.5	26.10a	8.02	6.44	51.60ab	142.87ab
SEM		1.17	0.49	0.05	2.61	7.30
P value	Treatment	0.015	0.171	0.676	0.099	0.077
	Linear	0.042	0.920	0.277	0.194	0.156
	Quadratic	0.220	0.718	0.654	0.836	0.793
	Control vs. all	0.070	0.476	0.271	0.790	0.756
Cottonseed	0.0, control	25.63ab	7.21b	6.52a	31.75b	88.05b
	0.5	28.79a	6.42b	6.35a	30.75b	84.49b
	1.0	16.83b	8.86a	6.47a	43.66a	121.53a
	1.5	33.07a	7.70ab	6.62a	26.00b	72.27b
SEM		2.49	0.37	0.05	1.77	4.98
P value	Treatment	0.058	0.036	0.313	0.0002	0.0003
	Linear	0.46	0.11	0.34	0.584	0.653
	Quadratic	0.080	0.680	0.136	0.001	0.001
	Control vs. all	0.862	0.415	0.748	0.424	0.446
P value
Protein source:		<.0001	0.294	0.672	<.0001	<.0001
SEM		0.950	0.373	0.048	1.44	3.99
Humic acid:		0.001	0.734	0.866	<.0001	<.0001
SEM		1.040	0.242	0.056	1.66	4.61
Protein*Treat.		0.007	0.015	0.287	0.004	0.002
SEM		1.910	0.779	0.097	2.87	7.94

Arithmetic mean in the same column within each ration with different letters differ (P<0.05).

1dDM: Degradable of dry matter; 2TGP: Total gas production

 

Table 4: Effect of zeolite addition to the experimental rations on rumen fermentation kinetics

Protein source	Zeolite level	1dDM,%	NH3-N, mmol	pH	TPG2, ml	TGP (g /kg DM)
Soybean	0.0, control	32.48	9.93	6.41	55.00a	151.29a
	1.25	34.11	8.5	6.58	56.40a	155.46a
	2.5	26.17	9.43	6.40	55.66a	152.24a
	5	25.91	8.78	6.36	45.33b	123.09b
SEM		1.638	0.306	0.048	1.26	12.79
P value	Treatment	0.151	0.330	0.447	0.007	0.020
	Linear	0.055	0.377	0.495	0.006	0.024
	Quadratic	0.729	0.535	0.323	0.008	0.126
	Control vs. all	0.276	0.157	0.733	0.275	0.196
Sunflower	0.0, control	19.67	7.23c	6.61a	48.33a	132.18a
	1.25	20.34	13.25a	6.44ab	37.80b	105.03b
	2.5	20.24	11.92b	6.28b	41.80ab	113.90ab
	5	18.8	8.425c	6.58a	43.00ab	118.29ab
SEM		0.458	0.818	0.048	1.59	4.29
P value	Treatment	0.750	<0.0001	0.027	0.148	0.185
	Linear	0.581	0.248	0.429	0.425	0.426
	Quadratic	0.349	<.0001	0.007	0.071	0.077
	Control vs. all	0.915	0.0001	0.048	0.066	0.075
Cottonseed	0.0, control	25.63a	7.21c	6.52	31.75	88.04
	1.25	18.52b	10.13a	6.45	30.5	84.56
	2.5	19.06b	9.30ab	6.38	33.0	90.6
	5	26.74a	8.11bc	6.50	31.33	84.25
SEM		1.472	0.432	0.054	0.839	5.577
P value	Treatment	0.013	0.009	0.853	0.770	0.867
	Linear	0.487	0.339	0.834	0.886	0.733
	Quadratic	0.002	0.003	0.455	0.910	0.630
	Control vs. all	0.033	0.007	0.609	0.947	0.652
P value
Protein source:		<.0001	0.0008	0.822	<.0001	0.003
SEM		0.868	0.227	0.048	1.09	7.77
Zeolite:		0.067	<.0001	0.212	0.065	0.132
SEM		0.987	0.271	0.056	1.36	10.93
Protein*Treat.		0.006	<.0001	0.399	0.017	0.044
SEM		1.814	0.704	0.097	2.24	17.96

Arithmetic mean in the same column within each ration with different letters differ (P<0.05).

1dDM: Degradable of dry matter; 2TGP: Total gas production

 

Table 5: Effect of bentonite addition to the experimental rations on rumen fermentation kinetics

Protein source	Bentonite level	1dDM, %	NH3-N, mmol	pH	2TPG, ml	TGP (g /kg DM)
Soybean	0.0, control	32.48	9.93ab	6.41	55.00ab	151.30a
	1	36.28	7.93b	6.56	57.33a	156.79a
	2	35.76	8.36b	6.33	46.25bc	126.90b
	3	30.70	11.34a	6.32	44.80c	124.24b
SEM		1.590	0.511	0.051	1.914	4.954
P value	Treatment	0.651	0.043	0.346	0.020	0.015
	Linear	0.739	0.174	0.318	0.009	0.007
	Quadratic	0.251	0.010	0.469	0.551	0.613
	Control vs. all	0.695	0.363	0.992	0.161	0.128
Sunflower	0.0, control	19.67b	7.23b	6.61	48.33a	132.18a
	1	23.01ab	12.83a	6.39	32.00b	88.96b
	2	22.15b	9.39b	6.62	31.00b	85.48b
	3	28.43a	10.35ab	6.67	25.00b	68.36b
SEM		1.165	0.816	0.041	2.427	6.632
P value	Treatment	0.060	0.033	0.054	0.0002	0.0003
	Linear	0.015	0.171	0.201	<.0001	<.0001
	Quadratic	0.420	0.043	0.063	0.043	0.065
	Control vs. all	0.038	0.017	0.473	<.0001	<.0001
Cottonseed	0.0, control	25.63b	7.21	6.52	31.75	88.04
	1	28.24b	7.39	6.37	29.75	82.3
	2	35.05a	8.29	6.36	31.66	86.55
	3	15.44c	8.18	6.39	33.75	92.86
SEM		2.713	0.357	0.051	0.783	2.145
P value	Treatment	0.002	0.733	0.729	0.438	0.463
	Linear	0.018	0.356	0.446	0.302	0.375
	Quadratic	0.001	0.868	0.448	0.221	0.191
	Control vs. all	0.720	0.475	0.288	0.988	0.878
P value
Protein source:		<.0001	0.003	0.026	<.0001	0.001
SEM		1.22	0.356	0.046	1.20	7.48
Bentonite:		0.028	0.049	0.730	<.0001	0.013
SEM		1.46	0.424	0.053	1.45	9.51
Protein*TRT		0.006	0.003	0.163	0.0002	0.037
SEM		2.69	0.780	0.093	2.51	15.64

Arithmetic mean in the same column within each ration with different letters differ (P<0.05).

1dDM: Degradable of dry matter; 2TGP: Total gas production.

 

Effect of zeolite addition on rumen fermentation kinetics

As shown in Table (4), adding of zeolite resulted in a significant linear and quadratic decrease for TGP (ml), and TGP (g/kg) with soybean ration at 5% level compared to 1.25, 2.5% zeolite, and control. The same trend was observed with dDM, NH3-N concentration, and pH value, but with no significant effect. However, the addition of zeolite to the sunflower ration affected pH value, TGP (ml), and TGP (g/kg) quadratically compared to the control treatment, especially at a 1.25% dose.

On the other hand, the NH3-N concentrations quadratically increased significantly at 1.25%, 2.5%, and 5% compared to control with the lowest concentration in rumen fluid solutions. Also, this trend was noticed with dDM%. Moreover, the ration containing cottonseed meal has increased significantly (P = 0.013) in terms of dDM at 5% zeolite compared to other zeolite levels (1.25 and 2.5%) but did not differ from control. Counterwise, 5% zeolite addition has a lower NH3-N concentration (P = 0.009) than the other zeolite levels (1.25 and 2.5%), but not less than control. The values of pH, TGP (ml), and TGP (g/kg DM) in the cottonseed rations were affected by the level of zeolite in the rumen fluid without significant differences among all levels and control. In general, the current results showed that rumen fermentation kinetics for zeolite addition, different types of protein sources and the interaction had a significant effect.

Previous results found that zeolite addition in an in vitro study increased the NH3-N concentration and ruminal pH (linear and quadratic effects, P<0.05) at 8 and 12 hours of incubation, but DM % was decreased insignificantly with zeolite added compared to control (Roque-Jiménez et al., 2018). This result agrees with our findings for NH3-N concentrations with sunflower and cottonseed rations, but is inconsistent with soybean rations which have reduced NH3 concentrations with the addition of zeolite.

Other experiments reported that adding zeolite to cows’ diets significantly reduced ruminal DM digestibility (Grabherr et al., 2009; Dschaak et al., 2010; Stojkovic et al., 2012). On the other hand, no change was observed for the addition of zeolites to pH, or ammonia content in the rumen (Bosi et al., 2002). Kardaya et al. (2012) found that ruminal pH decreased with the addition of zeolite, and indicated that zeolite was able to capture NH3 through its cation exchange capacity. However, their findings were in contrast with our observations, where ruminal pH was decreased, and ruminal NH3 increased in zeolite addition for sunflower and cottonseed rations, and these findings inconsistent with reported by Ghoneem et al. (2022).

It’s known that NH3-N concentration in the rumen is an indicator of the rate of ruminal N degradation. The present study results indicated that a higher inclusion of NH3-N can be obtained in rations involving urea (sunflower and cottonseed rations) with zeolite addition. This can be added safely with no adverse effects on ruminal pH level in a normal range of 6.5 to 7, where most of the ammonia would be present in the form of NH4+, as reported by (Abdoun et al., 2006), as a result of high ruminal NH3 and the rapid urea hydrolysis in the rumen (Roque-Jiménez et al., 2018). This suggestion is supported by our findings in the case of soybean rations without urea supplementation compared to others involving urea in diets, where ammonia was lower in soybean rations than others.

The conflict between previous results and present data is whether there is a positive, negative, or no effect of zeolite addition on rumen fermentation especially ruminal NH3 could be due to the type of protein rations, urea supplement or not, level of NDF in the rations, type of animal, roughage to concentrate ratio, etc.

Effect of bentonite addition on rumen fermentation kinetics

According to the results shown in Table (5), the type of protein source and bentonite level significantly affected rumen DM degradability, NH3-N concentrations, TGP (ml), and TGP (g/kg DM), while ruminal pH value was affected only by the type of protein source. No interaction effect was observed between bentonite additions and the protein sources. For soybean ration, bentonite quadratically increased (P = 0.01) NH3-N concentrations at 3% compared to other levels. On the other hand, both TGP (ml) and TGP (g/kg DM) values were linearly decreased with bentonite addition of 2 and 3%, but linearly increased with 0 to 1% addition. For rations of sunflowers, dietary addition of bentonite at 3% showed a significant increase in apparent rumen DM degradability and quadratically increased NH3-N concentrations at 1 and 3% of supplement levels compared to other levels.

Both of TGP (ml) and TGP (g/kg DM) were linearly decreased with bentonite addition. For rations of cottonseeds, NH3-N concentrations, ruminal pH values, TGP (ml), and TGP (g/kg DM) did not differ among levels of bentonite addition and control, but adding bentonite at 1 and 2% linearly increased the ruminal DM degradability, while adding bentonite at 3% quadratically decreased (P<0.001) the ruminal DM degradability in cottonseed rations.

An in vitro study by Jiang et al. (2020) found similar results between the adding of bentonite + aflatoxin B1 and control diet on NH3-N concentration and DM degradability and attributed it to the binding of aflatoxin B1 with bentonite that has a positive effect on rumen microflora activity. However, these findings are confounded with our results, where the response of bentonite addition was affected by the type of protein source as shown in Table 5. Moreover, ammonia concentration was reduced with the bentonite addition at 2 g/day in the rumen simulation technique (Rusitec) compared to control (Wallace and Newbold, 1991). Similarly, Hristov et al. (2003) observed that ammonia and soluble protein concentrations were reduced by bentonite addition. However, the ruminal pH values, nutrient digestibility and NH3–N concentrations were not affected by sodium bentonite addition (Chegeni et al., 2013). Interestingly, a greater increase of NH3-N concentrations were observed with sunflower and cottonseed meal treatments with the inclusion of urea than the soybean without urea inclusion. This was probably a result of the substantial amount of soluble protein introduced into the incubation media and this suggestion agrees with that reported by (Hristov et al., 2003). Further, Wallace and Newbold (1991) reported that bentonite effects have been attributed to adsorptive interactions between the bentonite and ruminal microorganisms or their growth substrates.

Therefore, different protein sources (soybean meal, sunflower, or cottonseeds) have different rates of protein degradation in the rumen while protein sources have large quantities of soluble nitrogen that can be converted into ammonia in the rumen. Other views, a basal diet of protein type, urea supplemented with or without the addition of bentonite levels may be partly due to different responses to rumen fermentation.

Conclusion

The addition of clay agents such as humic acid, zeolite and bentonite to different protein sources has different effects (positive, negative, and no effect) on ruminal fermentation (dDM, pH, reducing ammonia emission and TGP). Therefore, more studies are needed to select the best level of each clay with different protein sources in rations to study in vitro, which could reveal whether the discovered effects on decreased ammonia emission, modulating rumen fermentation and possibly, improving feed efficiency through increasing DM degradability in rumen have a positive response or not.

acknowledgements

The authors wish to thank Mr. Mohamed G. Embaby, Graduate student, Dept. Animal Science, Food and Nutrition Southern Illinois University, Carbondale, Illinois State, USA for his language revision and enhancing manuscript editing.

conflict of interest

All authors declare that there are no present or potential conflicts of interest among the authors or with other people and organizations that could inappropriately bias their work.

novelty statement

This relationship between clay minerals (bentonite, zeolite, and humic acid) as digestibility enhancers was rediscovered using different protein sources (soybean meal, sunflower meal, and cottonseed meal) via in vitro system. The data collected appeared the positive effects of clay minerals on rumen fermentation, especially gas production and reducing ammonia concentration.

authors contribution

AEM, HME and MAH contributed to the study conception and design. MAH contributed to supervision and validation. HME and SHAH prepared materials, investigation, collected data and formal analyses. HME prepared the first draft of the manuscript. MAH, AEM and RRAE revised the manuscript. All authors read and approved the final manuscript.

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