Animal Science

Effect of three collections of Tithonia diversifolia on the ruminal microbial population of cattle

0000-0001-8639-4693Juana Galindo^{1  *}  , 0000-0003-3415-6241Niurca González¹  , 0000-0002-1690-1140T. Ruiz¹  , 0000-0002-2641-1815Magaly Herrera¹  , Onidia Moreira¹  , Aned Capó¹  , H. Díaz¹ 

¹Instituto de Ciencia Animal, Carretera Central, km 47 ½, San José de las Lajas, Mayabeque

Abstract

In order to determine the effect of three collections of Tithonia diversifolia (mv-12, mv-14 and mv-17), collected in the eastern region of Cuba, on the ruminal microbial population of cattle, an experiment under in vitro conditions was developed. The control treatments with Cynodon nlemfuensis (star grass) were compared: star grass + mv-12; star grass + mv-14 and star grass + mv-17. The three collections were included at a rate of 20 % of the dry matter. The crude protein content was 19-23 % of the dry matter. With the materials mv-14 and mv-17 higher populations of cellulolytic bacteria were obtained, at three and six hours after starting fermentation with respect to that obtained with star grass. The three collections doubled the population of proteolytic bacteria three hours after starting fermentation, while at six hours only mv-12 produced effects in this bacterial group. The methane values were 37.4, 32.6, 33.2 and 32.33 g/kg of digested organic matter for the control, and the treatments mv-12, mv-14 and mv-17, respectively. Although they did not differ between plant materials, mv-12, mv-14 and mv-17 produced less methane than the star grass treatment only (P = 0.0477). It is concluded that the treatments mv-12, mv-14 and mv-17 caused modifications in the ruminal ecosystem, by increasing the population of total cellulolytic organisms and proteolytic bacteria; in addition to reducing the protozoan population. The methane estimated from crude protein concentration also decreased.

Key words: cellulolytic bacteria; proteolytic; methane; SCFAs; protozoa

Ruminants have a consortium of microorganisms in their rumen-reticulum able to fermenting feed, mainly fibrous ones. Currently, efforts are made to diversify the forage supply and silvopastoral systems are promoted with T. diversifolia (Gallego et al. 2017, ^{Galindo et al. 2018} and ^{Rivera et al. 2021}). The latter constitutes an alternative for feeding dairy cattle.

T. diversifolia (Hemsl.) Gray is commonly called sunflower, tree marigold, tithonia, among others. It is an herbaceous plant, from the Compositae (Asteracea) family, native to Central America and naturalized in Cuba. It has characteristics that give it a high potential for animal production, among which is its tolerance to poor soils, resistance to water stress and cut, with an approximate production of 55 t DM/ha/year (Gallego et al. 2014). It improves the environment and adapts to various climatic conditions (^{González-Castillo et al. 2014}).

Among its nutritional characteristics, the protein content, soluble carbohydrates and the level of tannins highlighted, which help to improve the nutritional balance, in terms of the energy and protein contribution in the diet of dairy cattle (Gallego-Castro et al. 2017, ^{Mahecha et al. 2017} and ^{Arguello et al. 2020}).

The protein content of the plant varies from 14.0 to 36.6 %. Animals intake the whole plant, preferably leaves and flowers (^{Maina et al. 2012}), which contributes to the ruminal balance and increases the efficiency for the transformation of ammonia into microbial protein, which would imply lower energy costs due to losses of ammonia, methane and ruminal CO₂, an aspect that reduces possible environmental contamination.

^{Mahecha et al. (2007)} evaluated the effect of the inclusion of tithonia forage as a partial replacement of the concentrated feed in Holstein crossed cows by Cebu, without finding differences in the production and quality of milk, for which they recommend its use as a strategic option for cattle production. ^{Gallego (2016)} showed that a silvopastoral system of tithonia, associated with Pennisetum clandestinum (kikuyo), increased milk production and, importantly, its quality, by improving the passage of long-chain fatty acids, which serve as precursors for some of the fatty acids and reduce cell counts in milk, which improves udder health. ^{García-López (2016)} obtained increases in production, when used a silvopastoral system with tithonia for milk production.

^{González et al. (2014)} defined tithonia as a plant with high protein value, which have a level of soluble carbohydrates higher than other forages. Its tannin content is not high, and it does not significantly affect ruminal activity. This can have an important effect on the productive performance of the animals, it is to be expected that ruminal health will improves and a great economic and environmental effect will be achieved.

Studies related to the action of tithonia in the ruminal ecology of cattle in Cuba were conducted with ecotypes collected in the western region of the country. However, the effect of the materials collected at different sites in the eastern zone, with regard to the ecology of the rumen, is not known.

The objective of this study was to determine the effect of three tithonia materials (mv-12, mv-14 and mv-17), collected in the eastern region of Cuba, on the ruminal microbial population, under in vitro conditions.

Materials and Methods

Research location. The study was performed at the Instituto de Ciencia Animal, located in San José de las Lajas municipality, Mayabeque province, at 92 m a.s.l. and 22(53’North latitude and 82(02’ West longitude. The soil of this region is fersialitic, undulated, with 4.84 % organic matter, 0.26 total nitrogen, 40.59 p.p.m. of phosphorus, 4.60 of calcium, 0.46 of magnesium and pH of 6.34. It has desiccation and is clayey and deep on limestone (^{Hernández et al. 2015}).

The tithonia plant materials were collected in the eastern part of Cuba (Camagüey, Las Tunas and Granma) and were planted at Miguel Sistach Naya Pastures and Forages Experimental Station, which belongs to the Institute itself.

Preparation of tithonia samples for in vitro studies. The star grass was used as base substrate of fermentation. For the preparation of these substrates, leaves with their petioles were collected. The material was dried in an oven at 60^oC for 48 h. The fraction that was sampled was 1 kg. Subsequently, it was milled in a mill until reaching a 1 mm particle size. The dried milled samples were stored in amber glass flasks, at room temperature, until their later use in the experiments. A total of 100 g were quickly transferred to Unidad Central de Laboratorios (UCELAB) to determine its bromatological composition, according to ^{AOAC (2016)}.

Procedure for the experiment preparation. The microbiological studies were conducted in the rumen microbiology laboratory, for which the ^{Theodorou et al. (1994)} technique was used.

Treatments. Each treatment consisted of a mixture of 80 % star grass and 20 % tithonia. The inclusion percentage of tithonia was selected from previous studies by ^{Galindo et al. (2012)}.

The treatments consisted of star grass (control treatment, without tithonia); star grass + tithonia mv-12; star grass + tithonia mv-14 and star grass + tithonia mv-17.

Ruminal fluid donor animals. For the development of the experiments, a battery made up of four Holstein-Cebu crossbred cows was used, which for microbiological purposes were considered donor animals. These animals were kept in stabling conditions and intake low quality fibrous diets and 2 kg of commercial concentrate. They were fistulated in the dorsal sac of rumen, where a simple cannula was inserted. Ruminal fluid was extracted in fasting animals through the cannula, with the help of a vacuum pump. This was kept in thermos with hermetic closure to guarantee the temperature conditions (39 ºC) and anaerobiosis during the transfer to the laboratory. The samples were taken to the rumen microbiology laboratory.

The ruminal content of all sampled donor animals was mixed and integrated into a single sample that was filtered through muslin. A small portion of ^{Menke and Steingass (1988)} buffer solution was added to the resulting solid and stirred for a few seconds in a domestic blender to loosen the microorganisms adhering to the fiber. The filtrate from this portion was incorporated into the liquid fraction. During all the time, the ruminal fluid was kept under a CO₂ atmosphere.

The experimental units consisted of 100 mL glass bottles, which contained 0.5 g of the feed to be evaluated. A total of 50 mL of a mixture of rumen fluid and buffer solution from ^{Menke and Steingass (1988)} was added to each bottle, in a 1:3 (v/v) ratio. Subsequently, each one was sealed with a butyl and graphed stopper. Bottles without substrate were included as control to correct the effect of the ruminal liquid on the volumes of produced gas. All bottles were randomly placed in a 39 °C temperature controlled bath.

The composition of the buffer solution was as follows. 5.7 g of Na₂HPO₄; 6.2 g of KH₂PO₄; 0.6 g of MgSO₄ ^.7 H₂O; 13.2 g CaCl₂ ^. 2 H₂O; 10 g of MnCl₂ ^. 4 H₂O; 1 g of CaCl₂ ^.6H₂O; 0.8 g FeCl₂ ^. 2 H₂O, 35 g of NaHCO₃ and 4 g of NH₄HCO₃. The procedure was carried out in a CO₂ atmosphere, in order to guarantee strict anaerobic conditions.

Culture of microorganisms from the rumen. The ^{Hungate (1950)} culture technique was used in rolled tubes and under strict anaerobic conditions.

The culture of total viable, cellulolytic and proteolytic bacteria was carried out in the culture media of ^{Caldwell and Bryant (1966)}. In the case of proteolytic bacteria, 10 % skim milk was added, according to Galindo et al. (1988). For the determination of the fungal population, the culture medium of ^{Joblin (1981)} was used.

For the inoculations (10 %) three dilutions were used, and each one of them was replicated three times. The results were expressed in colony-forming units (CFU) for bacteria and in thallus-forming units (TFU) for fungi per milliliter of rumen fluid (mL) at the determined dilution.

Rumen protozoa count. To perform the protozoa counts, the ruminal fluid samples were preserved in a 10 % formaldehyde solution. The protozoa were counted directly under the light microscope in a Neubauer chamber, after staining them with a gentian violet solution at 0.01 % in glacial acetic acid. For its reading under the light microscope, a 1:1 (v/v) dilution was used. Similarly, the counts were expressed as cells per milliliter of rumen fluid (mL) at the determined dilution.

Determination of the chemical composition of plant materials. The analysis of the chemical composition of tithonia plant materials, evaluated in each of the treatments, was carried out according to the techniques described by the ^{AOAC (2016)} and they are showed in each case. The fibrous fractions were determined by the method of ^{Goering and van Soest (1970)}. The chemical composition of the evaluated tithonia collections is shown in table 1.

Table 1 Chemical composition of some studied tithonia plant materials, % DM 

Collections	CP	NDF	ADF	Cellulose	Lignin	Ash
mv-12	23.39	50.1	44.93	28.23	11.87	16.88
mv-14	21.71	48.89	42.89	26.64	10.97	14.36
mv-17	21.01	48.23	44.27	30.63	10.95	17.83

Determination of final fermentation products. The NH₃ concentration was determined according to the technique described by ^{Conway (1957)}. The concentration of total and individual short-chain fatty acids (SCFAs) was recorded by gas chromatography.

pH measurement. It was determined by reading in a digital pH meter, Sartorius brand.

Calculation of the stoichiometric balance of ruminal fermentation. The BALANCE - RUMETANO program was used to estimate the stoichiometric balance of ruminal fermentation and the contribution to animal metabolism (^{Stuart 2015}).

Experimental design and statistical processing. A completely random design with factorial arrangement was applied. The factors were the treatments and fermentation hours (four treatments and three fermentation hours). For the processing of the results, the multivariate analysis of variance was used. In case of finding interaction between the treatments and the sampling times, a split plot model was applied, where the treatments were the main plot, and the sampling times the subplot. If there were not interaction, a linear model was used for the effects of the treatments and the sampling hours. ^{Duncan (1955)} test was applied for P<0.05, when necessary. The INFOSTAT statistical program, proposed by ^{Balzarini et al. (2001)} was used.

Statistical treatment of microorganism counts. The normality and homogeneity of the information obtained from the experimental results was determined. The counts of viable microorganisms were transformed according to Log N, to guarantee normal conditions in the growth curve. For the analysis, the formula $(\mathrm{K}+\mathrm{N}).{10}^{\mathrm{x}}$ was applied, where:

K	- is the constant that represents the logarithm of the dilution to which the microorganism was inoculated.
N	- is the logarithm of the colony count, determined as CFU/mL, TFU/mL, or cells/mL.
10	- is the base of logarithms.
X	- is the dilution at which the inoculation was performed.

Results and Discussion

There was no significant interaction between the treatments and the hours after the fermentation start for the microbial populations of total viable bacteria and ruminal cellulolytic fungi (0, 3 and 6 h after fermentation). Table 2 shows the results.

Table 2 Effect of tithonia plant material on the population of total viable bacteria and cellulolytic fungi 

	Plant material	Star grass	mv-12	mv-14	mv-17	SE (±) Signif.
Indicators
Total bacteria, 10^11CFU/mL		10.00^a	6.11^b	11.59^a	6.78^b	1.09 P=0.0012
Cellulolytic fungi, 10^5 TFU/mL		6.98^b	12.00^a	10.41^a	9.07^ab	0.99 P=0.0049

CFU: colony forming units

TFU: thallus forming units

a, b Different means in the same row differ to P < 0.05 (^{Duncan 1955})

The mv-12 and mv-17 propitiated fewer populations of total viable bacteria (P=0.0012), while the mv-14 keep the same populations as the star grass control. The effect of tithonia on the total population of rumen bacteria was highly variable in the different experiments and plant materials evaluated. ^{Galindo et al. (2018)} attributed it to the defaunanting effect produced by this plant, because of ecological relations of predation exerted by protozoa on total bacteria.

The mv-12 and mv-14 were able to increase the populations of cellulolytic fungi in the rumen (P=0.0049) with respect to the control, while the mv-17 showed intermediate fungal populations between the star grass control and the other two plant materials of tithonia evaluated.

The increase in the populations of cellulolytic fungi in the rumen recorded in this experiment is of great importance, since although these microbial groups are numerically smaller than cellulolytic bacteria, their extracellular enzymes are able to degrading, largely, the cellulosic materials contained in vegetables. It is estimated that approximately 58 % of ruminal cellulolysis is due to the presence of the mentioned microorganisms, which are capable of adhering, colonizing and degrading cellulosic materials and even modifying the structure of lignin.

In this research, the effect of the fermentation time in the presence of cellulolytic fungi of the rumen was showed (table 3). At 3 and 6 h after the start of fermentation, the fungal populations were highly significant (P<0.001) with respect to those registered at zero hour (before the start of fermentation). The populations of total viable bacteria did not show differences between the sampling hours that were evaluated.

Table 3 Effect of fermentation time on the population of total viable bacteria and fungi 

	Time, h	0	3	6	SE (±) Signif.
Indicators
Fungi, 10^5TFU/mL		5.92^b	10.70^a	12.23^a	0.86 P<0.0001
Total viable bacteria, 10^11CFU/mL		7.83	10.14	7.89	0.94 P=0.1495

CFU: colony forming units, TFU: thallus forming units

a b Different means in the same row differ to P < 0.05 (^{Duncan 1955})

The evaluation of the population dynamics of the cellulolytic bacteria of the rumen with the inclusion of tithonia plant materials is shown in figure 1. With the inclusion of mv-12, mv-14 and mv-17 in the diet, higher populations of cellulolytic bacteria were achieved at 3 and 6 h after the fermentation start with respect to that obtained with star grass.

Figure 1 Effect of tithonia plant material and fermentation time on the ruminal cellulolytic bacteria population, 10⁶ CFU/mL, SE ± 3.11, P = 0.0267 

The treatment that included mv-12 showed similar cellulolytic bacteria populations at 3 and 6 h. While, the inclusion of plant materials 14 and 17 highlighted for its variability. The mv-17 stood out for producing high populations of cellulolytic bacteria, at 3 and 6 h after the start of fermentation. These results are similar to those obtained by ^{Ruiz et al. (2017)} with materials of T. diversifolia, collected in the western region of Cuba.

The results related to the presence of cellulolytic bacteria and fungi, when mv-12, mv-14 and mv-17 were used coincide with those obtained by López et al. (2019), who found higher ruminal degradability values of NDF and ADF with the referred tithonia plant materials.

The different plant materials of tithonia showed their effect on the ruminal protozoa population. There was interaction (P <0.0001) between the treatments and the fermentation times in this indicator (figure 2). As can be seen, 3 and 6 h after the begining of the fermentation, all the plant materials showed superior protozoa population than the control (star grass) and the higher were at 6 h in mv-14 and mv-17.

Figure 2 Effect of tithonia plant material and fermentation time on ruminal protozoa population, 10⁵ cells/mL, SE ± 0.55 P < 0.0001 

The effect of different tithonia collections on the protozoa population has been studied in previous experiments by ^{Galindo et al. (2012)} and ^{Galindo et al. (2018)}, in which decreases in its population were showed. Among the advantages of protozoa reduction or defaunation are: increase in the population of cellulolytic microorganisms, stabilization of rumen pH, decrease in free ammonia, reduction of methanogenesis, and increase in the efficiency of digestive use of different diets, mainly fibrous ones.

The obtained results coincide with compilation reports by Hristov (2013), in which it is asserted that the most outstanding contribution of the reduction of protozoa in rumen is that it improves energy metabolism and reduces losses due to methane production, which is an environmental pollutant. This effect was showed in this research. In this regard, ^{Leng (2014)} reported that defaunation reduces the enteric emission of CH₄, due to the flow of microbial cells from the rumen and the reduction in the acetate/ propionate ratio, events that are considered electron sinks.

Table 4 shows the population of proteolytic bacteria and the pH of the rumen. In both indicators of ruminal fermentation, there were interaction between treatments and fermentation times. Regarding the population of proteolytic bacteria, the high values of these groups highlighted at 6 h, when mv-12, mv-14 and mv-17 are used, which does not differ from that obtained with mv-12 and mv -14, at 3 h of fermentation.

Table 4 Effect of tithonia plant material and fermentation time on the population of proteolytic bacteria and rumen pH 

Indicators	mv	Time, h
		0	3	6	SE (±) Signif.
Proteolytic bacteria, 10^6CFU/mL	Star grass	6.94^d	5.08^d	3.39^d	6.51 P=0.0006
	mv-12	16.78^bcd	36.78^ab	34.11^abc
	mv-14	15.33^cd	36.22^ab	37.11^ab
	mv- 17	6.33^d	15.67^cd	42.67^a
pH	Star grass	6.39^c	6.44^c	6.14^d	0.07 P<0.0390
	mv-12	6.73^ab	6.68^ab	6.66^b
	mv-14	6.84^ab	6.79^ab	6.88^a
	mv-17	6.69^ab	6.78^ab	6.81^ab

a, b, c, d Different means in the same row differ to P < 0.05 (^{Duncan 1955})

It is important to highlight that mv-17showed 2.47 and 2.72 times more proteolytic bacteria at 3 and 6 h after the start of fermentation, respectively. In the mv-12 and mv-14 they were 2.19 and 2.36 times, respectively, more numerous at 3 h with respect to the population found before starting the fermentation. At 6 h there was no increase.

The results found in the populations of proteolytic bacteria are related to the high CP content of these materials and, specifically, to the protein solubility. The largest populations of bacteria that degrade proteins in rumen are conditioned by factors such as their high protein content and their solubility. This produces the elimination of the terminal amino group of them, higher release of ammonia and, consequently, pH increase.

In this research, with all the tithonia plant materials and times evaluated, the rumen pH was higher with respect to the star grass, which confirms the hypothesis previously exposed (table 4).

Due to the presence of the different plant materials of tithonia, there was no effect on the molar percent of ruminal acetic acid and propionic acid, determined as percent of the total concentration of SCFAs in the rumen (table 5). In all cases, the fermentation pattern was acetic. ^{Ramírez et al. (2014)} reported that ruminal microbial activity produces varied concentrations of SCFAs, which are absorbed through the ruminal epithelium and are used as an energy source. Other products of the fermentation process, such as carbon dioxide (CO₂) and hydrogen (H₂), are not used by the ruminant, but serve as a substrate for a particular community of microorganisms belonging to Archaea domain, the methanogens. These microorganisms produce methane (CH₄) as a metabolic strategy to obtain the energy necessary for their growth.

Table 5 Effect of tithonia plant materials on SCFAs concentration in rumen and some elements of the stoichiometric balance of fermentation 

	Treatment	Control	mv-12	mv-14	mv-17	SE (±) Signif.
Indicator
Acetic acid, %		69.03	62.32	67.08	67.49	1.65 P=0.0581
Propionic acid, %		16.41	17.41	16.30	14.72	0.62 P=0.0503
CH4, g/kg digested OM		37.4^b	32.6^a	33.2^a	32.33^a	2.53 P=0.0477
Acetic/propionic		4.22^ab	3.58^b	4.13 ^ab	4.77^a	0.23 P=0.0200

In this experiment it was not possible to determine the methane concentration, reasons that led to its estimation from the equation of ^{Sauvant et al. (2011)}, who considered that the methane production that takes place in the rumen is related to the crude protein content of the intake feed. In this regard, it is reported that the CH₄ content, determined in grams /kg of digested OM = 40.1 - (0.32 × CP), % DM.

The methane values were 37.4, 32.6, 33.2 and 32.33 g/kg of digested OM for star grass and T. diversifolia plant materials mv-12, mv-14 and mv-17, respectively. The different plant materials did not differ from each other for this indicator, but produced less methane than the star grass (P=0.0477).

Results similar to those of this research were reported by ^{Delgado et al. (2011)} and Pérez-Can et al. (2020), when foliages from different tropical plants were evaluated. The authors found that leucaena and tithonia were the plants that produced the least methane (mL/gDM) compared to ten others, and showed that the response is associated with the higher content of condensed tannins and saponins that act on methanogens and protozoa, in addition to they have the ability to increase the molar ratio of propionic acid.

The acetic/propionic ratio (table 5) varied erratically between the treatments, without differences between the plant materials and the star grass control. However, it is clear that mv-12 and mv-17 differed from each other (P=0.0200).

From these results, it is concluded that the mv-12, mv-14 and mv-17, obtained in the eastern region of Cuba, led to modifications in the ruminal ecosystem, by increasing the population of total cellulolytic organisms and proteolytic bacteria, in addition to reducing the population of protozoa and the methane estimated from the concentration in CP.

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Received: October 19, 2021; Accepted: December 06, 2021

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Conflict of interest: The authors declare that there are no conflicts of interests among them

Author´s contribution: Juana Galindo: Conceptualization, Investigation, Writing – original draft. Niurca González: Conceptualization, Investigation. Tomás Ruiz: Conceptualization, Investigation. Magaly Herrera: Metodology, Formal analysis. Onidia Moreira: Investigation. Aned Capó: Investigation. Humberto Díaz: Investigation