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EFFECTS OF ENTODINIUM CAUDATUM MONOCULTURE INOCULATION ON IN VITRO FERMENTATION, METHANE PRODUCTION AND PREVENTION OF SUB-ACUTE RUMINAL ACIDOSIS.

Byline: N. Gulsen, H. D. Arik, M. S. Alatas and M. N. Tahir

Keywords, Acidosis, protozoa, gas production technique, methane production, volatile fatty acids.

INTRODUCTION

Intensive feeding with high concentrate levels, highly fermentable forages, and insufficient dietary coarse and physically effective fiber can lead to the observation of various disorders related to digestion and metabolism by increasing ruminal acid production and lowering pH than optimum (Plaizier et al., 2009; Ingvartsen, 2006). Among these disorders, sub acute ruminal acidosis (SARA) is the most common and observed in 11-19% of the animals in early lactation and 20-26% in the mid lactation animals (Oetzel et al., 1999; Garrett et al., 1997). In their extensive review on possible consequences of SARA, Plaizier et al. (2009) have listed the following: feed intake depression, reduced fiber digestion, milk fat depression, diarrhea and laminitis. Additionally, grain based SARA is also responsible for increased release of endotoxin and subsequently systemic inflammation (Qumar et al., 2017; Plaizier et al., 2012).

It has been suggested that manipulation of rumen fermentation either by enhancing the speed of desirable processes such as degradation of fibre or protein or reducing the speed of those which are inefficient such as methane production or harmful such as rapid production and accumulation of volatile fatty acids (VFA), can have beneficial effects on both the rumen and the animal (DiLorenzo, 2011). The most popular understanding developed for the mitigation of SARA is maintaining rumen pH above 5.5 (Plaizier et al., 2009) which can be achieved by feeding management to avoid a rapid buildup of VFA, or alternatively fast removing/clearance of VFA from the rumen, thereby reducing VFA accumulation (Henning et al., 2010; Beauchemin et al., 2003).

Despite that the rumen ciliate protozoa make a substantial portion of rumen biomass, little is known on their specific development conditions, inter-community interactions, and effects on rumen fermentation due to their complex morphology (Newbold et al., 2015; Zhang et al., 2007). One of the consequences of SARA is the fluctuation of bacterial and protozoal community in the rumen and protozoa are more sensitive to decrease in pH. But, Entodinium genus is resistant to low pH, and present approximately 90-99% in the protozoal community in high grain fed animals (Nagaraja and Titgemeyer, 2007).

Ciliated protozoa play important role in regulating the fermentation by utilizing the sugars and starch, which they sequesters from the bacteria. Entodinomorphid mostly utilize starch and delay the process of fermentation and helps in the clearance of lactic acid from the rumen pool, helping in stabilization of ruminal pH (Nagaraja and Titgemeyer, 2007; Dehority, 2005). Moreover, presence of ciliated protozoa causes the reduction of bacterial population which helps in slowing the fermentation rate, leading to more stable ruminal pH (Willaim and Coleman, 1997; Nagaraja et al., 1992).

As mentioned earlier that during SARA microbial community is disturbed and especially, ciliated protozoa. So, in order to facilitate adaptation to high grain feeding, or in nutritional disorder where the rumen environment such as pH is impaired, protozoa can be supplemented as an inoculum, which could be helpful in restoring the rumen ecosystem by regulating the fermentation pattern. The objective of present study was to determine the effect of protozoa monocultures inoculation on in vitro fermentation of corn and wheat in defaunated buffered rumen fluid.

MATERIALS AND METHODS

Care of cannulated animals and production of E. caudatum monocultures: The experimental protocol was approved by the Selcuk University Ethical Committee on Animal Experimentation ((#2012/048; Konya, Turkey) and study was conducted in March-July, 2015. The Entodinium (E.) caudatum cultures used in the present study were prepared from rumen fluid collected from two rumen cannulated Holstein heifers with 450 +- 20 kg live weight. Dry matter consumption was adjusted during collection of rumen fluid for in vitro testing to meet animal maintenance requirements (NRC, 2001). The nutrient composition of diet and in vitro ingredients is presented in table-1. Animals were offered alfalfa hay in two equal meals at 0800 h and 1700 h for at least 7 days as adjustment period prior to rumen fluid collection to better determine the effect of E. caudatum cultures on acidity.

The rumen fluid was collected through a suction tube attached to a mechanical vacuum pump (#3T PVC Sample Tube, Bar Diamond, Parma, ID, USA) through the cannula, and subsequent defaunated. For defaunation, 50 mL volumetric falcon tubes were filled with rumen fluid and centrifuged twice at 1000 x g for 5 minutes. (Allegra 64R, Beckman Coulter, USA). After the last centrifugation, the supernatant was discarded and the pellet layer remaining in the bottom of the tube was aspirated.

The E. caudatum cultures were frozen in two-stage freezing protocol (Ice Cube 14S, Sy-Lab, Austria) in a 2 mL cryotube with a computer-controlled freezer, and then stored at -196AdegC (Nsabimana et al., 2003). Frozen E. caudatum cultures were re-suspended for 5 minutes at 39AdegC to check viability. These cultures were grown in an incubator in 250 mL culture flasks at 39AdegC in anaerobic medium using anaerobic dilution fluid (Dehority, 2005). For this purpose, the amount of the culture broth was divided into two parts every three days and Medium M (table-2) was added to this half culture broth (Dehority, 1998). The protozoa were daily offered a substrate comprising 1.5% wheat and 1.0% alfalfa (Dehority, 1998), both ground to pass a screen of 0.425 mm (Retsch, SM100 Comfort, Germany).

Establishment of acidosis in the medium and detection of E. caudatum monoculture activity by gas production technique: The rumen fluid was collected just before morning feeding from the same rumen cannulated animals as used for preparing E. caudatum monocultures, transported to the gas in vitro laboratory in insulated flasks and drained through double layer of cheesecloth under continuous supply of CO2. The rumen fluid was defaunated using the procedure described earlier, and the supernatant was examined with a 10 x 10 lens on a light microscope and protozoa control was performed.

In vitro media were prepared according to the method of Menke and Steingass (1988). The gas production recordings were carried out in triplicates and the process was repeated on three occasions. Samples of 30 g/L of wheat and corn each were added separately to 100 mL pressure-resistant pyrex fermentation bottles (three bar pressure resistant) for 24 h at 39AdegC. To these bottles, 15 mL of defaunated buffered rumen fluid with a rumen fluid to buffer ratio of 1:2 (v/v) were added (Menke and Steingass, 1988). The fermentation bottles had previously been flushed with CO2 and remained under the continuous supply of CO2 during the whole experiment. The bottles were placed in a shaking water bath (40 rpm), and connected to an automated monitoring system for recording of gas production.

The prepared E. caudatum monoculture was charged to a separation funnel and expected to sediment during 1 h at 39 AdegC in the incubator. One-tenth of the sediment was collected and counted (Dehority, 1984). One and two mL of culture sediments with an average of 5.5x104 E. caudatum per mL were added to the fermentation bottles by an automatic pipette. A total of 18 fermentation bottles including three replicates per treatment, two controls without E. caudatum and four blanks without substrate were included at each occasion and were incubated at 39AdegC for 24 h. The gas pressures (digital manometer; Keller Leo 1, Switzerland, 0.2% sensitivity), E. caudatum culture sediments and medium pH values (HI 8314, Hanna Instruments, Portugal) were recorded every 4 h during the whole incubation period, and cumulative gas productions were calculated from individual recordings (Lopez et al., 2007).

All other parameters were recorded at 8, 16 and 24 hrs of incubation.

Chemical analyses: Dry matter, crude protein, crude fat and crude ash of feeds were determined according to methods described in AOAC (1984). The neutral detergent fibre and acid detergent fibre levels of the feeds were determined using Ankom 200(tm) Fiber Analyzer (ANKOM Technology, Macedon, NY) according to the method reported by Goering and Van Soest (1970). The volatile fatty acid (VFA) concentrations were determined using 80/120 Carbopack B-DA/ 4% Carbowax 20M as column (Supelco, 1998). Concentration of methane was calculated stochiometrically (Ramin and Huntanen, 2013) while those of lactate (Kimberley and Taylor 1996) and ammonia (Weatherburn 1967) were determined proximately.

Statistical analyses: The data were analyzed according to General Linear Model with Repeated Measures statement using statistical package for social sciences (SPSSA(r); IBM, 1989-2015, SPSS Statistics, Release 16.0)) software and significance was declared when P<0.05. The following model was used:

Yijkl = u + Gi + Cj + Tk + (T x G)ik + (T x C)jk + (G x C)ij + (T x G x C)ijk + E ijk

Yijk = dependent variable, u = population mean, Gi = ith grain type, Cj = jth E. caudatum number; Gi and Cj represent the between-subject factors, Tk = kth incubation time; represents within-subject factor and Eijk = error term. The factors within parentheses represent interactions.

Table-1: Chemical composition of Alfalfa, Corn and Wheat used in the experiment (g/kg DM).

Ingredients###DM###CP###EE###CA###NDF###ADF

Alfalfa hay###926.8###159.4###19.7###104.7###465.9###343.8

Corn###912.7###86.3###40.6###18.3###134.8###40.4

Wheat###921.2###115.5###17.0###18.7###202.1###36.5

Table-2. Medium M components.

Components###mL/L

Mineral mixture M1###500.0

Sodium acetate, 1.5%###50.0

Rumen fluid (1000 x g supernatatnt)2###100.0

Sodium bicarbonate, 6%###83.3

Distilled water###260.0

Cysteine HCl, 3%###06.7

Cardon dioxide###continuous supply

Table-3: Effects of E. caudatum inoculation and substrate source on parameters of rumen fermentation in vitro.

Items###Inoculation###pH###NH3 mMol/L1###CGP, mL2###Methane, mL

###Blank3###5.40###5.10###47.28###5.95

Wheat###1###5.47###5.25###48.28###6.66

###2###5.43###5.02###49.09###5.49

###Blank###5.27###1.19###51.08###5.56

Corn###1###5.38###1.51###53.73###6.10

###2###5.43###1.56###54.08###6.04

Inoculum###Control4###5.40a###3.14###49.18###5.76b

###1###5.43b###3.38###51.00###6.38a

###2###5.43b###3.30###51.88###5.77b

SEM###0.02###0.15###1.15###0.13

Probability###------------------- P = -------------------

G*###0.039###0.001###0.001###0.586

C**###0.100###0.803###0.002###0.061

T***###0.001###0.001###0.005###0.074

G x C****###0.318###0.467###0.450###0.080

TxG###0.001###0.077###0.069###0.513

TxC###0.248###0.670###0.814###0.492

TxGxC###0.441###0.148###0.101###0.055

Table-4: Effects of E. caudatum inoculation and substrate source on volatile fatty acids and lactate levels in vitro.

Items###Inoculation###Total VFA1###Acetic acid###Propionic###Butyric acid###Lactate

###mol/100 mol###acid mol/100###mol/100 mol###mmol/100

###mol###mol

###Blank2###1.04###51.64###29.54###14.46###1.60

Wheat###1###1.15###51.51###28.41###14.56###1.61

###2###0.96###50.58###29.17###15.74###1.61

###Blank###1.04###50.55###31.23###14.46###1.60

Corn###1###1.10###51.58###29.79###14.56###1.60

###2###1.13###48.56###31.39###15.94###1.49

Inoculation###Control3###1.04###51.10###30.39###14.48###1.60

###1###1.13###51.04###29.10###15.42###1.60

###2###1.05###49.57###30.28###15.84###1.54

SEM###0.02###0.47###0.30###0.33###0.15

Probability###------------------- P = -------------------

G*###0.413###0.457###0.001###0.460###0.086

C**###0.170###0.307###0.069###0.282###0.196

T***###0.011###0.078###0.001###0.792###0.001

G x C***###0.076###0.371###0.785###0.482###0.397

TxG###0.067###0.072###0.001###0.925###0.138

Tx C###0.734###0.500###0.151###0.957###0.942

TxGxC###0.130###0.534###0.330###0.803###0.97

RESULTS

Figure-1 showed viability levels depending on the incubation period of E. caudatum. The number of protozoan tended to be lower (G x C, P <0.058) in the wheat medium (6.38 vs. 6.66 x 104/mL), as compared to the medium containing corn and this state continued until the end of incubation due to the progression of incubation times (G x C x T interactions, P <0.009). Protozoa survived during the incubation. The protozoa consumed the substrate during the first 8 h of incubation and then became immotile.

The type of substrate changed the pH and ammonia levels (P0.05). Overall protozoa inoculum tended (P=0.10) to increase the pH as compared to control. Corn substrate and E. caudatum inoculation increased (P0.05) but E. caudatum inoculation and a G x C interaction tended (P0.05) the proportional levels of acetic and butyric acids in total VFA, as well as total VFA or lactate production (table-4). The molar proportions of propionic acid were affected by the substrate source (P<0.01), where corn as a substrate increased the molar proportion of propionic acid as compared to wheat, while wheat tended (P<0.10) to increase the lactate proportion as compared to corn. The E. caudatum inoculation tended (P<0.10) to decrease the molar proportion of propionic acid, while a substrate by inoculums interaction tended to increase total VFA.

DISCUSSIONS

The number of protozoa in the rumen fluid was in the range of 1.51 x 105-5.3 x 105/mL (Williams and Coleman, 1997; Dehority 1984), of which about 85% were Entodinium spp. (Baah et al., 2007). Entodinium-type rumen protozoa resist SARA environment and maintain their presence at 3.2 x 104/mL especially in high grain diets (Hristov et al., 2004). In this study, it was also seen that the number of E. caudatum inoculated in vitro was higher than the number of protozoa persist during in vivo SARA conditions, indicating that the inoculums was sufficient to test the protozoa activity. The results of present study and those of Umucallilar et al. (2012) pointed out that corn was more effective in increasing the number of E. caudatum or maintaining the viability in SARA conditions as compared to wheat.

Franzolin and Dehority (2010) reported that decreasing the pH of rumen caused strong negative impact on ciliates protozoa viability. During SARA, the number of protozoa decreases due to the lower pH but does not disappear totally and as the environment becomes suitable, these protozoa reappear back (Nagaraja and Titgemeyer, 2007). According to study conducted by Pourazad et al. (2016), the rumen fermentation process in order to establish the acidosis pH level takes at least 10-11 h. From the present study it can be speculated that fluctuation in protozoal population after 8 h might be the result of the lower pH at this interval as a result of substrate fermentation, which eventually recovered back after 16 h when the environment was hospitable. The average pH in this study was 5.39, but the number of protozoa and activity decreased after the first 20 h of incubation. Oetzel (1999) reported as low as 5.0-5.5 rumen pH during SARA and the pH values obtained in this study were within this range.

Protozoa are competing for substrate with amylolytic bacteria, and protozoa ferment starch more slowly than amylolytic bacteria (Owens et al., 1998; Nagaraja et al., 1992), resulting in reduced rumen starch digestibility and pushing starch digestion to the intestines (Mendoza et al., 1993).

In this study, E. caudatum inoculations did not affect the total VFA and molar ratios of individual VFA in total VFA. In their meta-analysis including limited studies on the effects of defaunation on in vivo performance of ruminants, Newbold et al. (2015) found that the removal of rumen ciliates slightly reduced VFA concentrations. More interestingly, their data suggested that defaunation substantially decreased butyrate, slightly increased acetate and had no effect on propionate molar proportions. Qin et al. (2012) conducted an experiment on the possible effects of defaunation on fermentation patterns using corn, rye and wheat grains as substrates. They reported that defaunation significantly increased the molar concentrations of propionate on the expense of acetate and butyrate. In the present study, the protozoa slightly decreased the molar proportion of the propionate, that might be seen as the only effect on VFA production.

The high starch diets like corn and wheat favors the production of propionate, but E. caudatum are known to efficiently ingest and store starch granules and can delay the fermentation process. It is therefore assumed that E. caudatum might have reduced starch availability for bacterial fermentation, hence resulted in low propionate production. Brossard et al. (2004) suggested that protozoa transformed the starch into more butyric acid. Contrary to this view, protozoans have been suggested to play an important role in preventing lactate accumulation and controlling pH as they use lactate (Dehority, 2005). In a study conducted by Hayirli et al. (2014) where lactic acid was added to in vitro medium, it was reported that E. caudatum selectively consumed lactic acid and maintained a concentration of <1.2 mM.

It was interesting that the metabolites produced by E. caudatum in the fermentation medium do not change ammonia production regardless of substrate source. Increasing effects of ammonia production of protozoa were determined in some studies (Williams and Coleman, 1997), while in others reduced ammonia production levels were recorded (Qin et al., 2012). In this study, the use of any protein source as substrate and the low protein content of corn and wheat may not have changed the production of ammonia.

In the present study, doubling the E. caudatum inoculation levels increased cumulative gas production. There is not enough information about the gas production property of E. caudatum. However, a study conducted using E. caudatum reported that it increased gas production (Zeitz et al., 2013). In the current study, the amount of methane as molar ratio in the total gas also tended to increase. Methanogens associated with ciliates are responsible for 9-25% of the methanogenesis of the rumen fluid (Moss et al., 2000). Pei et al. (2010) reported that rumen defaunation of protozoa caused a 30-45% reduction of methane emissions. While in the present study methane production increased with protozoa inoculation, which is opposite to defaunation and confirms the finding of literature.

Conclusion: Entodinium caudatum monoculture inoculation did not reduce the chances of SARA in wheat or corn based in vitro media owing to poor relationship between protozoa number and VFA levels in the rumen.

Acknowledgements: The authors wish to thank Faculty of Veterinary Medicine, University of Selcuk, Konya, Turkey for their financial support for the research presented in this paper.

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