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Article

The Effect of Humic Acid Supplementation on Selected Ruminal Fermentation Parameters and Protozoal Generic Distribution in Cows

by
Svetlana Malyugina
1,2,* and
Pavel Horky
1
1
Department of Animal Nutrition and Forage Production, Mendel University in Brno, Zemedelska 1665, 61300 Brno, Czech Republic
2
Agrovyzkum Rapotin Ltd., Vyzkumniku 863, 78813 Vikyrovice, Czech Republic
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(10), 1663; https://doi.org/10.3390/agriculture14101663
Submission received: 9 June 2024 / Revised: 16 September 2024 / Accepted: 17 September 2024 / Published: 24 September 2024
(This article belongs to the Section Farm Animal Production)
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Abstract

:
The objective of this study was to examine the effect of humic acid (HA) supplementation on the rumen fermentation and protozoal community in the rumen. For this purpose, four ruminally cannulated Simmental cows were randomly assigned in a replicated 4 × 4 Latin square design experiment to study the effect of HA dietary supplementation on feed utilization, rumen fermentation, and protozoal community for 84 days. The basal diet (BD) was composed of meadow hay (68.2% of dry matter [DM]), maize silage (17% of DM), and granulated feed mixture (14.9% of DM). There were four treatments, including the BD without additives (control diet, H0), the BD supplemented with 50 g HA/cow/day (H50 treatment), the BD supplemented with 100 g HA/cow/day (H100 treatment), and the BD supplemented with 200 g HA/cow/day (H200 treatment). HA supplementation did not affect the total or individual volatile fatty acid concentrations, the total protozoa, or the ruminal pH. However, HA at the dosage of 50 g/cow/day increased the NH3-N concentration and fecal nitrogen compared to the control (p < 0.001). HA supplementation also significantly impacted the abundance of individual protozoal genera in the rumen. The results of this study suggest that HA has potential in ruminants as a natural feed additive and may play a role in nitrogen metabolism and stabilizing the protozoal community without adverse effects on rumen fermentation.

1. Introduction

For many years, ruminant nutritionists and microbiologists have been interested in manipulating the rumen microbial ecosystem to improve animal production efficiency and reduce the use of growth promoters, hormones, and antibiotics in veterinary practice. In addition, there is a growing concern about developing effective strategies to mitigate greenhouse gas emissions from the livestock industry [1]. Recently, there have been numerous studies conducted using various feed additives (e.g., essential oils, tannin or algae extracts, fats) in order to modulate rumen fermentation, enhance microbial activity, and find an effective way to reduce enteric methane emissions in the rumen [2,3,4,5,6,7,8,9,10]. Among these feed additives, clay-derived humic substances have the potential to improve nutrient utilization, rumen fermentation, and animal performance. Humic and fulvic acids are the main extractable components of soil humates and are mainly used for soil fertility improvement [11]. It has been proposed that they can also be used as natural antibiotics, enhancing rumen fermentation, nutrient digestibility, and animal performance [12,13,14,15,16]. Humates have also been used for their anti-inflammatory, antiedematous, antibacterial, and antiviral effects in animals [17]. Experiments on applying humic acids (HAs) have mainly been conducted on monogastric animals.
The positive impact of HA supplementation (at the levels 0.1, 0.2, and 0.4%, and at the dosages 0, 1, and 2 mL/L, and 30 and 60 g/t feed) was demonstrated on the growth, performance, and immune status of broilers, and also on poultry production and egg quality [18,19,20]. Two studies conducted by Wang et al. [21,22] demonstrated that dietary HA improved the growth performance, health, and meat quality of pigs. Existing reports regarding the use of HA in ruminant diets have been inconclusive [23,24,25]. Milk yield increased after the administration of HA to goats’ diets [26]. Knolif et al. [13] evaluated the effect of dietary HA on feed utilization and milk production of lactating Friesian cows. They observed that HA supplementation (at the dosages 20 and 40 g/day/animal) increased nutrient digestibility, milk production, and milk efficiency, and enhanced milk nutritional quality parameters (e.g., increases in total conjugated linoleic acid (CLA) and unsaturated fatty acid [UFA] concentrations).
Sheng et al. [27] reported that HA caused a reduction in methane production in an in vitro study. In another in vitro study, the authors used RUSITEC to investigate the effect of HA on rumen fermentation. The inclusion of HA did not impact pH, volatile fatty acid (VFA) concentrations, or methane (CH4) or carbon dioxide (CO2) production [28]. In an in vivo study, Terry et al. [29] did not observe any antimethanogenic effect of HA supplementation in the diets of beef heifers. This discrepancy in the results could be attributed to the different dosages, chemical composition, and structure of HA used, as well as the length of the experiment and other factors related to animal species, type, and composition of the basal diets (BDs) to which HAs are added. Considering the above, more information is needed on this subject to overcome the amount of insufficient research or meaningful results.
Rumen protozoa play an important role in rumen metabolism and nutrition. They can benefit digestion and stabilize fermentation by regulating rumen pH and slowing down acid synthesis [30]. The significant contribution of protozoa to rumen carbohydrate metabolism [31,32], protein degradation, the regulation of bacterial N turnover, and the supply of soluble protein for microbial growth [33] have been reported in the literature. However, the scientific community still has not reached a consensus regarding the importance of protozoa to ruminant performance and production. Even less information is available on the impact of HA inclusion in ruminants’ diets on the ciliated protozoa community. Several studies associated HA supplementation with reduced a reduced number of protozoa and decreased ammonia production in the rumen, with concurrently increased VFA concentrations, which improved rumen fermentation [26,28,34,35]. The proposed mechanism of dietary HA is mainly their antimicrobial action, which reduces or eliminates ruminal protozoa [36]. However, since HA has antibacterial properties, it may also affect the bacteria population in the rumen and consequently decrease digestibility. This would make the results of HA administration deleterious. In addition, it is necessary to analyze the changes in the structure of the protozoal community and consider how protozoa (e.g., main protozoa orders and genera) differ in their metabolic activities in order to understand their true impact on feed efficiency. Considering the lack of information in this area, this study aimed to evaluate the effect of three dosages of HA supplementing cows’ diets and its impact on the selected rumen fermentation parameters, with an emphasis on an analysis of the ciliated protozoa community, which plays a significant role in maintaining the ruminal environment and rumen metabolism. We hypothesized that different dietary HA levels would affect the protozoal population at the genus level and improve fermentation parameters in the rumen.

2. Materials and Methods

2.1. Experimental Design and Animal Diets

This experiment was conducted to determine the effect of HA dietary inclusion on rumen fermentation parameters and ciliated protozoa community in the rumen. Four Simmental cows (age: 4 years ± 35 d; body weight [BW]: 560 ± 5.35 kg) surgically fitted with ruminal cannulas were used as experimental animals in a 4 × 4 Latin Square design (4 treatments and 4 periods). Cows were housed in the experimental facility at the Research Institution Agrovyzkum Rapotin (Rapotin, Czech Republic) in individual pens (6.7 m2) and were allowed access only to their individual ration (Figure 1b). Animals had unrestricted access to water, and their experimental diets were to meet their energy and nutrient requirements according to NRC [37] recommendations. The animal procedures were reviewed and approved by the Ministry of Agriculture (14608/2021-MZE-18134) of the Czech Republic; each experimental period lasted 21 days (7 days for adaptation to rations and 14 days for measurements of response variables). All animals were fed the BD consisting of meadow hay, maize silage, and granulated feed mixture (Table 1). The experimental diets were supplemented with the following HA levels: the control diet, 0% of dry matter (DM), H0; 0.3% of DM (4.2 g/kg DM), H50; 0.6% of DM (8.4 g/kg DM), H100; and 1.3% of DM (16.9 g/kg DM), H200. A commercial product HUMAK Natur AFM (Envi Product, Prague, Czech Republic) was used as a source of HAs.
The diets were fed as a total mixed ration (TMR), and HUMAK Natur AFM was mixed manually before each feeding. Animals were individually fed the diets once per day at 7 a.m. The cows were housed individually, had strict access to their ration only, and had free access to water. The proportions of feed ingredients and their chemical composition are shown in Table 1 and Table 2. The daily nutrient intake of cows fed the experimental diets H0, H50, H100, and H200 is represented in Table 3.

2.2. Sampling and Chemical Analyses of Feed Ingredients

Chopped meadow hay, corn silage, and granulated feed mixture were sampled to determine DM and nutrient composition. The chemical compositions of all diets (Table 2) were analyzed for DM, crude protein (CP), crude fat, crude fiber, and ash according to the EC Commission Regulation [39]. The acid detergent fiber (ADF) and neutral detergent fiber (NDF) concentrations were sequentially determined using an ANKOM A200 Fiber Analyzer (ANKOM Technology, Macedon, NY, USA) according to the methodology supplied by the company, which is based on the methods described by Van Soest et al. [40]. Starch content was determined by Ewers’ polarimetric method based on the partial acid hydrolysis of starch, followed by measurement of the optical rotation of the resulting solution according to STN EN ISO 10520 [41].

2.3. Sampling and Analyses of Rumen Fluid and Protozoa Identification

The ruminal fluid samples of each cow were collected three hours after the morning feeding (at 10:00 h) two times per week (on Mondays and Fridays) throughout the experiment. Samples were collected via the rumen cannula with a probe connected to a vacuum pump (Figure 1a) and sent to the laboratory for further analysis. After collection, rumen fluid samples were filtered through the synthetic cloth (119 μm, Uhelon 59 S, Silk & Progress) and subsampled for further chemical analyses and protozoa enumeration.
Tests of rumen fluid included the measurement of pH, the concentration of total nitrogen (Ntot), nitrogenous compounds (NCs) and ammonia (NH3-N), determination of the total number of protozoal ciliates (TPCs), the generic composition of the ciliated protozoa, the concentrations of VFAs, and the total amount of VFAs. The pH values were measured immediately after sample collection using a portable pH meter (EUTECH CyberScan PC510 pH/Conductivity Bench Meter, Fisher Scientific, Pardubice, Czech Republic). The ruminal fluid aliquots (20 mL) were stored at −20 °C for the subsequent analysis of VFAs, Ntot, NC, and NH3-N. Samples for rumen protozoa analysis were preserved in 1 mL of 10% formaldehyde solution, stained with Brilliant Green Dye, and allowed to stand overnight. The density of the rumen protozoa per mL was obtained using a Bürker counting chamber in a fluorescent optical microscope (INTRACO FL200, INTRACO MICRO, s.r.o., Tachlovice, Czech Republic) at a magnification of 40× according to the procedure described by Dehority [42].
The identification of the protozoa genera present in each sample was performed according to the phenotypical criteria described by Imai and Ogimoto [43], Baraka T.A. [44], and Dehority [42]. The concentrations of VFAs were determined by gas chromatography (Agilent 6820 Gas Chromatograph System; Agilent Technologies, Santa Clara, CA, USA) using the method described by Filipek and Dvorak [45]. The Kjeldahl method was used to determine the amount of total nitrogen, nitrogenous compounds, and ammonia, as described by Chen et al. [46].

2.4. Sampling and Analysis of Fecal Nitrogen

Fresh fecal samples were collected from each cow directly from the rectum using sterile gloves. During the experiment, samples were collected on the same days as rumen fluid samples. After collection, the samples of fresh feces were dried in an oven for a period of 48 h at a temperature of 65 °C and further analyzed for total Kjeldahl N by the method described by Dai and Karring [47].

2.5. Correlation Analysis

The correlation analysis was performed using STATISTIKA 14 (StatSoft CR, s.r.o., Prague, Czech Republic), and the Heatmap was created using Microsoft Excel software (ver. 2408). Pearson’s correlation coefficient was employed to calculate the strength of the associations between the variables (ruminal fermentation parameters and the protozoa community members at the genera level). The correlation coefficient (r) ranged from −1 to 1, with a positive correlation represented by r > 0 and negative correlation represented by r < 0.

2.6. Statistical Analysis

The software Statistica 14.0.0. (TIBCO Software Inc., Santa Clara, CA, USA) was used to perform the statistical analysis. Data were analyzed with a one-way analysis of variance (ANOVA). If statistical significance was observed, a post hoc comparison analysis via the post hoc Tukey’s test was performed. p < 0.05 was considered statistically significant, and the trend was 0.05 ≤ p < 0.1. The normality of the distribution of variables was tested using the Shapiro–Wilk test, and all variables were normally distributed. The statistical model for the analysis of variance for the 4 × 4 Latin square design was Yijk = μ + Ti + Pj + (T × P)ij + Ck + Eijk, where Yijk = response (dependent) variable; μ = general mean; Eijk = random error. Treatment (Ti) and period (Pj) were considered as fixed effects, and cow (Ck) as a random effect.
Protozoal community alpha diversity was estimated using Shannon and inverse Simpson diversity indices.
The Shannon–Wiener diversity index (H) was calculated using the following equation:
H = −∑[(pi) × log(pi)],
where
  • H = Shannon diversity index;
  • pi = proportion of individuals of i-th species in a whole community.
The Simpson’s diversity index was calculated using the following equation:
1/D = 1/[Σni × (ni − 1)/(N*(N − 1))],
where
  • n = number of individuals of each species;
  • N = total number of individuals of all species.

3. Results and Discussion

3.1. The Impact of HA Supplementation on the Ruminal Fermentation Variables

As shown in Table 4, the HA supplementation did not significantly affect the pH values (p = 0.064) or the total or individual VFA concentrations in the rumen (p > 0.05). In contrast, dietary HA significantly impacted N metabolism in the rumen. HA at the dosage of 50 g/day/animal increased the content of NC, total N, fecal N, and ruminal NH3-N concentrations.
Rumen pH, as well as other fermentation parameters, can be modulated by changing diet and using feed additives. During rumen fermentation, pH is considered a significant factor affecting microbial growth, enzyme activity, VFA concentration, and even methane production [48]. It is one of the most variable factors in the fermentation environment and usually reflects the concentration of acids produced by the rumen microorganisms [49]. In the current study, increasing doses of HA in the cows’ diet did not significantly affect ruminal pH nor the total or individual concentrations of VFAs (Table 4). In general, cows maintain rumen pH values between 5.5 and 7.5 through a complex acid–base regulation system depending on the type of diet provided [50]. The optimal ruminal pH for rumen bacteria proliferation ranges from 6.10 to 6.80 [51], and for the protozoal activity it is between 5 and 7.8 [49]. The protozoa ciliates tend to be more sensitive to pH changes compared to bacteria [49]. In this study, the ruminal pH values were within the physiological range (6.39–6.5) and were unaffected by HA administration (p > 0.05). The absence of changes in rumen pH can be caused by the increased buffering capacity of ruminal fluid after HA administration, which may lead to stabilizing rumen pH and the neutralization of VFAs. Buffer substances were proposed as a prevention against rumen acidosis due to maintaining more stable pH and acidity neutralization [52,53].
Our results are in accordance with those reported by Terry et al. [29], who also observed unaffected ruminal pH with HA supplementation at 1.5 g/d or 3.0 g/d using the rumen stimulation technique (RUSITEC) or with the in vitro experiment conducted by Sallam et al. [54]. In an in vivo study, Terry et al. [14] also did not observe any changes in pH after HA supplementation at the dosages 100, 200, and 300 mg/kg BW, which are comparable with the dosages used in our study. Similarly, Galip et al. [35] and McMurphy et al. [24] reported no changes in ruminal pH after HA supplementation. In contrast, El-Zaiat et al. [26] showed that adding drenched HA at a dose of 2 g/day per goat increased ruminal pH values. This study was conducted on dairy goats, and HA was administered directly to the rumen. The variation in results could potentially be explained by the different HA administration methods, animal species, their age, or health condition. In addition, such supplements may have various fermentation pattern responses under in vitro and in vivo conditions. The concentrations of the total or individual VFA were not affected by dietary HA (p > 0.05), which corresponds to the results obtained by Ikyume et al. [55], who observed unaffected total VFA as well as its proportions with high doses of HA under in vitro conditions. Likewise, other researchers reported no changes in the VFA profile after HA supplementation with HA butyrate, and total protozoa abundance in the rumen [14,56,57]. Sallam et al. [54], in an in vitro study, observed no changes in total or individual VFA concentrations after HA administration at the dosages 0, 2, 4, and 6 g/kg DM, as well as no changes in ruminal pH and total protozoa number. Interestingly, the in vivo experiment conducted within the same study resulted in a decrease in total protozoa and N-NH3 and an increase in butyrate in the ruminal fluid of Damascus goats supplemented with HA. This difference in results between in vitro and in vivo experiments partly explains discrepancies in the other studies that evaluated HA effect on rumen fermentation.
Ruminal NH3-N concentration is a crude predictor of the efficiency of dietary N conversion into microbial N [58]. In the rumen, ammonia is the product of ruminal proteolysis, which ruminal bacteria utilize as a source of N for growth [33]. In this study, we observed significant differences in ruminal ammonia concentrations among the diets (p = 0.001). HA dietary inclusion increased the ruminal ammonia N concentrations at the dosages 50 and 200 g/d/animal compared to the control (H0). In ruminants, excessive NH3–N produced due to high degradation or hydrolyses of urea and other N compounds is one of the main signs of low N efficiency and protein degradation [59]. Another possible reason for excessive ammonia production could be related to a non-synchronized diet and asynchronous availability of N and energy to the rumen, which leads to excessive ruminal proteolysis and deamination. The concept of a synchronous diet for ruminants was proposed as an effective nutritional management solution to improve the utilization of nitrogenous compounds, increase the efficiency of rumen microbial protein synthesis, and reduce excessive N excretion to urine and feces, which cause environmental pollution [60]. On the other hand, studies that evaluated the effect of a synchronous diet on rumen fermentation have been inconclusive [61,62,63]. Newbold and Rust [64] reported that the asynchrony between nitrogen and energy availabilities in the rumen has only short-term effects on bacterial growth and leads to the quick recovery of the microbial population in the rumen. Henning et al. [65] concluded that the pattern of energy supply is more important than the synchronization between N and the energy supply in the rumen.
However, in the present in vivo study, the ruminal NH3–N levels in all experimental groups were within a range (from 6.35 to 15.16 mg/dL) required for the normal function and growth of rumen microorganisms. The minimal ammonia concentration necessary for microbial growth was reported at the level of 5 mg/dL, and the optimum ammonia concentration ranges between 8,5 and 30 mg/dL [66]. According to Schwab et al. [67], the rumen microbiota requires 5–11 mmol/L ammonia to maximize microbial protein synthesis. In the present study, ammonia nitrogen ranged between 3.73 and 8.9 mmol/L (Table 1), which may indicate that experimental diets provided sufficient N and energy to meet the requirements of the rumen microorganisms. Odle and Schafer [68] conducted a study with beef cattle and showed optimal rumen ammonia of 125 mg/L for the degradation of barley and 61 mg/l for the degradation of maize. In soil, HA has been reported as a microbial growth promoter and consequently has been proposed that it may have a similar effect on the rumen bacteria, enhancing their activity and increasing rumen fermentation [28]. Nevertheless, the mechanism of action of HA on the microbial population has been studied primarily in soils, which makes it unclear how it acts in the rumen and how it may impact the rumen microbial ecosystem and fermentation.
Similar to our results, Terry et al. [29] reported increased ruminal ammonia concentration after HA administration at the dosage of 100 mg/kg body weight (BW). This dosage is comparable to treatment H100 used in the present study, considering that the concentration of HA in the additive was 65% compared to only 50.7% in the study of Terry et al. Corresponding with our results, the authors did not observe any changes in pH values or VFA concentrations (total and individual) after HA administration. In contrast, some authors reported a decrease in ruminal NH3-N with HA dietary inclusion [13,26,28]. They hypothesize that a reduction in NH3-N can indicate the improved efficiency of microbial protein synthesis due to a concurrent decrease in protozoa number and thus more ruminal by-pass CP. However, our results did not confirm this trend. In addition, it should be noted that protozoal genera have various metabolic functions and activities; thus, their relative abundance and diversity in relation to the bacterial community in the rumen should be investigated when evaluating ruminal fermentation and N metabolism. The impact of dietary HA on ciliate protozoa populations is discussed in Section 3.2.

3.2. Comparison of Protozoal Community in the Rumen among the Different Levels of HA Dietary Inclusion

The impact of HA supplementation on the rumen’s protozoal community is represented in Table 5, and the relative abundance of the protozoal genera in response to different HA levels is displayed in Figure 2. HA did not change the total protozoa in the rumen (p = 0.32). This result is in agreement with other studies reported by Terry et al. [14], Ikyume et al. [56], Sizmaz et al. [57], and Sallam et al. [54]. Terry et al. [29], in an in vivo study, reported increased total protozoa and N-NH3 with dietary HA at the dosage of 100 mg/kg BW (comparable with the H50 diet in our study) and a decrease in both at the dosage of 300 mg/kg BW (comparable with the H200 diet in our study). We did not observe any changes in the total protozoa number with H50 and H200, while the ammonia concentrations were significantly affected by the HA dosages of 50 and 100 g/d/animal.
Nevertheless, dietary HA significantly impacted the relative abundance of the majority of protozoal genera (p < 0.005) in the rumen with the exclusion of the genus Dasytricha spp., whose abundance showed no significant changes (p = 0.17) in all treatments.
Rumen ciliate protozoa have fascinated microbiologists since their discovery over 180 years ago due to their high importance for the health and productivity of their cattle hosts. They actively contribute to the digestion of animals’ diets using carbohydrate-active and other enzymes to break down carbohydrates that are otherwise indigestible by the host [69]. In addition, as a predator, they regulate the rumen bacteria population [70] and can reduce potential pathogens [71]. However, according to other authors, the presence of protozoa in the rumen can enhance the pathogenicity [72,73].
Undoubtedly, much more research is required to achieve comprehensive knowledge regarding the role of protozoa in the rumen and their contributions to feed digestion and fermentation.
Rumen protozoa have been morphologically categorized as Entodiniomorphids and Holotrichs, belonging to the Entodiniomorphida and Vestibuliferida (Holotrichs) orders, respectively [74]. In our study, we also evaluated the impact of HA supplementation on these two protozoal groups, which are characterized by different metabolic activities and functions in the rumen. Entodiniomorphids can modulate ruminal pH by (a) engulfing starch particles, thereby preventing their fermentation to lactate, and competing with amylolytic bacteria for starch [75,76], and (b) fermenting starch (at a slower rate) to VFA with lower dissociation and acidogenic potential than lactate; they are also able to take up some of the lactate and may prevent its accumulation in the rumen [77]. These factors stabilize ruminal pH and reduce the risk of acidosis. In this study, we observed a significant increase (p < 0.001) in this protozoal group in the animals supplemented with higher doses of HA supplementation (50 and 200 g/d/animal, respectively).
Interestingly, the concentration of the Holotrich protozoa significantly decreased with HA supplementation and only at the dosage of 50 g/d/animal. No existing research evaluated the impact of HA on the proportion of these protozoal groups. Since the presence of Entodiniomorphids and Holotrichs in the rumen plays an important role in maintaining the microbiome stability and in determining the fermentation pattern [32], we presume that HA supplementation supported the microbial stability in the rumen. Entodiniomorphids were reported to have higher predation activity compared to Holotrichs [74]. This is evidenced by Martin et al. [78], who observed an increase in N-NH3 in the presence of ciliates from the Entodiniomorphid order. Our results show a significant concomitant increase in Entodiniomorphid number (Table 5) and rumen ammonia (Table 4) with HA diets at the dosages H50 and H200 compared to the control. Belance et al. [79,80] demonstrated that Holotrich protozoa have much lower predation activity and, thus, a lower impact on rumen N-NH3 concentrations in comparison with Entodiniomorphids. Protozoa that predate upon bacteria regulate bacterial nitrogen turnover [58]. By engulfing microbes, ciliated protozoa (especially Entodiniomorphids) release oligopeptides and amino acids, which can be subsequently fermented to ammonia and VFAs by amino acid-fermenting bacteria. This bacteria, as well as hyper-ammonia-producing bacteria, were found to be in a mutualistic relationship with Entodinium caudatum [81]. In our study, dietary supplementation with H50, 100, and 200 led to an increase in Entodinium in the ruminal fluid. Although we did not evaluate the impact of dietary HA on bacterial populations, we can speculate that an increase in ammonia N in the rumen could result from an increase in protozoa species with higher predatory activity. Developing an understanding of the mutualistic and co-habitation relationship between rumen protozoa and bacteria represents a challenge for future research to achieve improved nitrogen efficiency in ruminants and the increased economic viability of ruminant procedures.
Numerous studies reported lowered or eliminated ruminal protozoal numbers with HA supplementation [23,26,28,34,35]. Our result did not show any effect on total protozoa or VFA concentrations, which could be explained by the different chemical compositions and HA dosages used in the studies.
The supplementation of HA impacted the relative abundance of the protozoal species in the rumen (Table 5, Figure 2). HA supplementation at the dosage of 50 g/day/animal reduced protozoal genera belonging to the Vestibuliferida (Holotrichs) order, such as Isotricha spp., Dasytricha spp., Charonina spp., and Buetschlia spp. (p < 0.001). The population of the genus Isotricha was significantly lower after the administration of the H50 diet compared to the control (H0), H100, and H200 diets (p = 0.003). In the two studies of Majewska et al. [82,83], HA supplementation of a sheep diet caused increased amylolytic activity in the rumen and thus increased the proportion of Isotricha and Entodinium ciliates, which mainly utilized starch and soluble sugars in the sheep rumen.
In another study [84], dietary HA (20 g/day) increased Entodinium ciliates without a significant effect on the amylolytic and cellulolytic activities in rumen fluid. In contrast, Galip et al. [35] did not observe a significant effect on the number of total ciliates and the genera Entodinium and Isotricha in the rumen when rams were fed 5 or 10 g/day HA. Our study also observed a significant increase (p < 0.001) in Entodinium spp. by dietary HA at the dosages of 50, 100, and 200 g/d/animal but a decrease in Isotricha spp. The differences in these results may have been caused by different HA dosages and animal species. In the mentioned studies, they used a much lower dosage of HA supplementation in animal diets compared to this study.
The population of Charonina was lower after the supplementation of HA at dosages of 50 and 200 g/day/animal compared to a diet with 0 g of HA. The presence of Buetschlia genera was also reduced in H50 and H200 diets compared to the control.
The effect of HA dietary inclusion on the protozoal populations of genera belonging to Holotrichs is remarkable, and further detailed investigation is needed. Generally, the Entodinium genera is one of the most abundant in the rumen, reaching up to 90% of the total protozoal population in high-concentrate diets [44]. In our study, Entodinium spp. was also the most abundant genera observed in all diets, ranging from 53 to 70% of the total protozoa.
Rumen protozoa markedly contribute to plant fiber digestion in the rumen, especially species that have high fibrinolytic enzyme activity, such as Epidinium, Ophryoscolex, and Polyplastron [23,85]. An increase in fibrinolytic activity can cause lower total tract NDF digestibility and reduce the production of enteric CH4 [86]. However, the relationship between the concentrations of the protozoa genera, fiber digestibility, and CH4 production in the rumen should be investigated further. In the present study, HA at the dosage of 200 g/day/animal reduced the number of Epidinium spp. compared to the control (p = 0.002). The H50 diet decreased the presence of Ophryoscolex spp. compared to the control. Ostracodinium spp. was reduced by adding HA at the dosage of 100 g (H100) compared to the H50 and H200 dosages. The available data concerning the effect of HA supplementation on the abundance of protozoan populations at the genera level are scarce. In the majority of research investigating dietary HA impact on ruminants, protozoal genera were not evaluated.
The strength of the linear correlations between the protozoa generic composition among diets and ruminal fermentation traits were analyzed using Pearson’s correlation coefficients and are visualized via Heatmap in Figure 3. Pearson’s correlations indicated significant associations among protozoa genera and between protozoa genera and some rumen fermentation traits. Within ciliate protozoa, positive correlations were observed between the abundance of Isotricha and Diplodinium (r  =  0.97; p  <  0.001), between Charonina and Buetschlia (r  =  0.4; p  =  0.03), between Buetschlia and Epidinium (r  =  0.49; p  <  0.007), and between the Epidinium genus and Ophryoscolex (r = 0.37; p  <  0.046), and strong negative correlations were noted between the genus Isotricha and Buetschlia, Charonina, and Epidinium (r  =  −0.56, r = −0.81 and r = −0.56, respectively; p = 0.01). Concerning interactions between protozoa and rumen fermentation variables, strong negative correlations were observed between the Epidinium genus with ruminal pH (r = −0.57; p = 0.001). At the same time, positive correlations occurred between the Entodinium and Diplodinium genera with ruminal ammonia concentrations (r = 0.44, r = 0.43; p = 0.01).
Dietary HA treatments did not have a strong impact on alpha diversity among protozoal genera in the rumen (Figure 4). However, a comparison of alpha diversity among diets shows that protozoal species richness was significantly reduced by the H50 diet compared to the control, and Simpson’s index confirms the same trend. Entodinium dominated the ciliate protozoa community (53–70%) in all dietary groups. Other predominant ciliate genera detected at an abundance above 5% in at least one of the diets were Dasytricha spp. (9–15.4%), Isotricha spp. (6–8.8%), and Diplodinium spp. (5–8.7%). The role of protozoa in ruminal fermentation and their contribution to animal metabolism and nutrition is still the subject of research and controversy. Further investigations are needed to understand their role in ruminal fermentation.

4. Conclusions

In conclusion, the dietary intake of HA significantly increased ruminal ammonia concentrations at the dosages of 50 and 200 g without significant effects on pH values or negative effects on total and individual VFA concentrations. The concentration of total protozoa was not affected by the inclusion of HA in the cows’ diet. However, the results of our study showed the potential of HA supplementation to alter rumen protozoal genera by increasing protozoal Entodiniomorphid concentrations, the protozoa responsible for stabilizing pH and regulating the bacterial populations in the rumen. In addition, Entodiniomorphid ciliates have higher predation activity; thus, an increase in their number can partially explain the increase in ammonia concentrations with HA administration. The number of protozoa associated with higher fibrinolytic activity in the rumen (Epidinium, Ophryoscolex) was decreased by HA administration, which may lead to lower fiber digestibility in the rumen. However, there is a lack of in vivo studies conducted on ruminants. Therefore, the impact of HA on ruminant metabolism, digestion, and health is not fully understood and needs to be investigated in further research. Furthermore, the effect of HA on the ciliate protozoa at the genus level should be examined to reveal their role in rumen N metabolism and fermentation, as well as their interaction with rumen bacteria populations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14101663/s1.

Author Contributions

Conceptualization, S.M.; methodology, S.M. and P.H.; investigation, S.M. and P.H.; formal analysis, P.H.; writing—original draft preparation, S.M.; and writing—review, and editing, P.H. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO1224 and by the TECHNOLOGY AGENCY OF CZECH REPUBLIC, grant number SS06020190.

Institutional Review Board Statement

The animal procedures were reviewed and approved by the Ministry of Agriculture (14608/2021-MZE-18134) of the Czech Republic.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

Author Svetlana Malyugina was employed by the company Agrovyzkum Rapotin Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. (a) Rumen fluid sampling; (b) the morning feeding during the experimental feeding schedule.
Figure 1. (a) Rumen fluid sampling; (b) the morning feeding during the experimental feeding schedule.
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Figure 2. The relative abundance of observed protozoal genera in the rumen of cows fed with four experimental diets (H0, H50, H100, H200).
Figure 2. The relative abundance of observed protozoal genera in the rumen of cows fed with four experimental diets (H0, H50, H100, H200).
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Figure 3. Heatmap based on Pearson correlation coefficients between the relative abundances of rumen protozoa genera and rumen fermentation parameters. Asterisks (* and **) indicate a significant correlation at p < 0.05 and 0.01. Red and green tiles indicate positive and negative correlations, respectively.
Figure 3. Heatmap based on Pearson correlation coefficients between the relative abundances of rumen protozoa genera and rumen fermentation parameters. Asterisks (* and **) indicate a significant correlation at p < 0.05 and 0.01. Red and green tiles indicate positive and negative correlations, respectively.
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Figure 4. Analysis of alpha diversity: richness (A) at the genus level (using the Shannon–Weiner index) and Simpson’s diversity index (B). Median values and interquartile ranges are indicated in the plots. Different superscript letters indicate the significant differences between the treatment groups (p < 0.05).
Figure 4. Analysis of alpha diversity: richness (A) at the genus level (using the Shannon–Weiner index) and Simpson’s diversity index (B). Median values and interquartile ranges are indicated in the plots. Different superscript letters indicate the significant differences between the treatment groups (p < 0.05).
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Table 1. Ingredient composition of the experimental diets.
Table 1. Ingredient composition of the experimental diets.
Diets 1
H0H50H100H200
Ingredients, % of dry matter
Meadow hay71.371.070.870.4
Maize silage2120.920.820.7
Granulated feed mixture (GFM) 27.77.77.77.7
Supplement HUMAK Natur AFM 300.30.61.3
1 Diets: control diet without HUMAK Natur AFM supplement (H0); diet contains 50 g of HUMAK Natur AFM (H50); diet contains 100 g of HUMAK Natur AFM (H100); diet contains 200 g of HUMAK Natur AFM (H200). 2 Granulated feed mixture BIOSTAN (BIOKRON, s.r.o., Blucina, Czech Republic; quantity per kg of product): barley (15%), oat mill feed (65%), wheat (15%), malt flower (10%), sunflower expellers (5%), and extracted soybean meal (3%). 3 Supplement HUMAK Natur AFM: humic acids (65%), fulvic acids (5%), Ca (42,278 mg/kg), Mg (5111 mg/kg), Fe (19,046 mg/kg), Cu (15 mg/kg), Zn (37 mg/kg), Mn (142 mg/kg), Co (1.24 mg/kg), Se (1.67 mg/kg), V (42.1 mg/kg), and Mo (2.7 mg/kg).
Table 2. Chemical composition of feed ingredients.
Table 2. Chemical composition of feed ingredients.
Ingredients
Nutrient Composition,
% of Dry Matter 		Meadow HayMaize SilageGFMHUMAK
Dry matter, %90.6393.7291.5875.64
Crude protein, % 4.877.4313.372.72
Starch, %0.0035.4138.824.7
Ash, %66.13.87.631.3
Crude fiber, %32.515.45.626.68
Fat, %1.573.12.680.07
Neutral detergent fiber (NDF), %35.137.1819.60.22
NEV *, MJ38.0912.6511.41-
PDIE, g571.8157.9161.5-
PDIN, g569.4109.2163.2-
NEV—net energy value for fattening ruminants; PDIE—protein digested in the small intestine depending on rumen-fermented organic matter; PDIN—protein digested in the small intestine depending on rumen degraded protein. * NEV (net energy for fattening) was calculated according to the NEV equations for ruminant feedstuffs according to Sommer et al. [38].
Table 3. Daily nutrient intake of dry matter, nutrients, and energy by cows on experimental diets.
Table 3. Daily nutrient intake of dry matter, nutrients, and energy by cows on experimental diets.
Diets 1
H0H50H100H200
Nutrient intake
DM, kg/d/animal11.81311.85911.90611.999
CP, g/d/animal10131014.641016.311019.65
CF, g/d/animal262.29265.7269.1275.9
Starch, g/d/animal1605.361622.561639.761674.16
NEV *, MJ59.78---
PDIE, g891.2---
PDIN, g841.8---
1 Diets: control diet without HUMAK Natur AFM supplement (H0); diet contains 50 g of HUMAK Natur AFM (H50); diet contains 100 g of HUMAK Natur AFM (H100); diet contains 200 g of HUMAK Natur AFM (H200). DM—dry matter; CP—crude protein; CF—crude fiber; NEV—net energy value for fattening ruminants; PDIE—protein digested in the small intestine depending on rumen-fermented organic matter; PDIN—protein digested in the small intestine depending on rumen-degraded protein. * NEV (net energy for fattening) was calculated according to the NEV equations for ruminant feedstuffs according to Sommer et al. [38].
Table 4. Fermentation parameters in the rumen of cows fed total mixed rations (TMR) with various HA supplementation levels.
Table 4. Fermentation parameters in the rumen of cows fed total mixed rations (TMR) with various HA supplementation levels.
ItemH0H50H100H200SEMp-Value
pH6.396.286.386.51.90.064
Nitrogenous compounds (g/kg)3.42 a5.04 b3.36 ac4.49 abc1.870.007
Total nitrogen (g/kg)0.54 a0.79 b0.55 a0.69 a0.030.022
NH3-N(mg/dL) 16.35 a15.16 b7.27 ac9.55 c0.470.001
Fecal nitrogen (%)9.06 a12.46 b7.99 a9.57 a0.21<0.001
VFAtot (mmol/L)102.9998.6399.6697.271.520.54
Acetate (mmol/L)79.9874.6975.4174.881.160.25
Propionate (mmol/L)14.4714.5915.7112.060.440.06
Butyrate (mmol/L)8.529.1710.488.960.640.4
Acetate/Propionate5.675.284.856.310.180.77
a,b,c Means with different superscript letters in the same row differ from each other (p < 0.05). SEM, standard error of the mean. 1 NH3-N (mmol/L): H0 (3.73); H50 (8.9); H150 (4.27); H200 (5.55).
Table 5. The effect of humic acid supplementation on the ciliate protozoa (total count, abundance of the major groups, and at the genera level).
Table 5. The effect of humic acid supplementation on the ciliate protozoa (total count, abundance of the major groups, and at the genera level).
ItemH0H50H100H200SEMp-Value
Total protozoa count (104/mL)26.0727.0425.0224.260.540.32
Holotrich protozoa 29.8 a18.1 b28.4 a24.8 a0.77<0.001
Entodiniomorphid protozoa 61.4 a76.5 b66.7 ad69.3 cd0.83<0.001
Isotricha6.17 a4.06 b7.5 ac8.8 ad0.440.003
Dasytricha15.479.713.910.90.720.17
Charonina5.7 ac3.7 b4.2 abc3.8 b0.20.001
Buetschlia2.42 a0.58 b2.71 ac1.65 ad0.1<0.001
Entodinium53.1 a70.1 b61.6 cd62.5 cd0.8<0.001
Diplodinium8.69 a5.13 b5.25 bc5.77 bd0.24<0.001
Epidinium1.89 a1.23 ac1.39 abc0.75 bc0.10.002
Ophryoscolex2.73 a1.57 b2.28 ab2.19 ab0.130.01
Ostracodinium3.18 a3.67 a1.45 b3.52 a0.1<0.001
a,b,c,d Means with different superscript letters in the same row differ from each other (p < 0.05). SEM, standard error of the mean.
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Malyugina, S.; Horky, P. The Effect of Humic Acid Supplementation on Selected Ruminal Fermentation Parameters and Protozoal Generic Distribution in Cows. Agriculture 2024, 14, 1663. https://doi.org/10.3390/agriculture14101663

AMA Style

Malyugina S, Horky P. The Effect of Humic Acid Supplementation on Selected Ruminal Fermentation Parameters and Protozoal Generic Distribution in Cows. Agriculture. 2024; 14(10):1663. https://doi.org/10.3390/agriculture14101663

Chicago/Turabian Style

Malyugina, Svetlana, and Pavel Horky. 2024. "The Effect of Humic Acid Supplementation on Selected Ruminal Fermentation Parameters and Protozoal Generic Distribution in Cows" Agriculture 14, no. 10: 1663. https://doi.org/10.3390/agriculture14101663

APA Style

Malyugina, S., & Horky, P. (2024). The Effect of Humic Acid Supplementation on Selected Ruminal Fermentation Parameters and Protozoal Generic Distribution in Cows. Agriculture, 14(10), 1663. https://doi.org/10.3390/agriculture14101663

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