Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 58 issue 5 (may 2024) : 797-806

Rumen Methanogens Diversity in Native Cattle under Different Feeding Regimen using 16s Metagenomic Techniques

G. Srinivasan1,*, Valli Chinnamani1, M. Chellapandian1, V. Leela1, T. Sathiamoorthy1
1Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 051, Tamil Nadu, India.
Cite article:- Srinivasan G., Chinnamani Valli, Chellapandian M., Leela V., Sathiamoorthy T. (2024). Rumen Methanogens Diversity in Native Cattle under Different Feeding Regimen using 16s Metagenomic Techniques . Indian Journal of Animal Research. 58(5): 797-806. doi: 10.18805/IJAR.B-5109.

ABSTRACT

Background: Pulikulam cattle is an indigenous cattle breed native to south India, Understanding the rumen microbiome is an important step to develop effective strategies to minimize their enteric methane emission. The rumen microbiome of this breed has not been documented.  Hence, a study was executed in Pulikulam cattle, fed different diets, to investigate the diversity of rumen methanogens using metagenomic techniques. 

Methods: Twenty recently calved Pulikulam cattle with mean body weight of 200 Kg were randomly distributed into four treatments, with five animals per treatment. The animals were fed their respective diets as per their treatment for 30 days, after which the rumen liquor was collected.  DNA from the samples were extracted and subjected for 16s rRNA gene amplification using region specific primers. 

Result: Methanogens belonging to five orders, four classes and eleven families were identified. Order Methanomassiliicoccales was not present in treatment group fed with high energy diet. The methanogens affiliated to the order Methanomicrobiales were predominant in treatment 1 (14%) and treatment 2 (16.30%). Order methanococcales (15%) was predominant in treatment 3 and order Methanosarcinales (18%) was predominant in treatment 4. Archaeal order belonging to Thermoplasmatales were also identified in Pulikulam cattle fed different dietary regimen except in treatment 3. Archaeal family Methanothermaceae belonging to the order Methanobacteriales was identified in Pulikulam cattle. Methanocorpusculum labreanum was the predominant species in all the treatment groups. Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus solfataricus, Sulfolobus sp. A20 and Sulfolobus tokodaii were identified in treatment 1, which had higher dietary crude protein. It was concluded that methanogen diversity varied under different feeding regimen and predominant methanogen is varied in Pulikulam breed of cattle.  M. labreanum was the predominant methanogen in Pulikulam cattle fed with different dietary regimen.

INTRODUCTION

Rising environmental temperature is a big threat to the whole world. The main reason for environmental temperature rise is emission of greenhouse gases. Methane is one of the important green house gas produced either naturally or by anthropogenic source. Cattle and buffaloes are accounted for 85 per cent of the annual enteric methane emission in India (Bhatta et al., 2019). As the country has 13 and 53 per cent of the world’s cattle and buffalo population, respectively (Government of India, 2019). Rumen methanogen community is very complex and depending on the substrate being fermented the methane emission varies. Majority of methanogens utilize carbon dioxide and formate while others use methylated compounds for producing methane. Till date, about 155 species of methanogens associated to six orders and twenty-nine genera have been isolated from different ecosystems (Holmes and Smith, 2016). However, only seven species of methanogens have been isolated from the rumen ecosystem (Janssen and Kirs, 2008). Isolated methanogens from the rumen represents less than 10 per cent of the archaeal community (Kim, 2012). Understanding of the rumen microbiome especially rumen archaea is important in order to develop effective strategies for reduction of enteric methane and thus contribute to decreased green house gas emission.
         
In India, majority of indigenous cattle are low yielders and are reared through grazing in wastelands or roadside. Pulikulam cattle is one such indigenous cattle breed of south India reared by nomadic grazing system, in a zero input system of management. Bulls of this cattle breed are reared for bull baiting sporting activity which is conducted during paddy harvest season in the state of Tamil Nadu, India. Currently the nomadic farmers show very little interest in rearing this breed and population statistics have shown a decline in their number. Conservation of this cattle breed will have a great impact on the socioeconomic status of the local farming community. Various measures are underway to ensure the conservation of this breed. One approach is gathering data on the unique rumen fermentation pattern of this breed that has made them resilient in the utilisation of limited nutrient resources that they obtain from the degraded pastures that they graze. The rumen microbiome of this breed has not been studied and this is the first kind of study in Pulikulam cattle breed.  Information on diversity of rumen microbes with special regard to methanogens in this breed of cattle will pave a way for their conservation. Hence, a study was carried out using 16s metagenomic techniques to explore the diversity of microbes emphasising on methanogens in the rumen of Pulikulam cattle fed different diets, under similar environmental conditions. 

MATERIALS AND METHODS

The study was carried out at Pulikulam Cattle Research Station (PCRS), Manamadurai, Tamil Nadu, India, which is a constituent unit of Tamil Nadu Veterinary and Animal Sciences University. The study period was between November to December, 2021. A total of 20 recently calved Pulikulam cattle having a mean body weight of 200 Kg, in first lactation, with average milk yield of 2 litres and milk fat percentage of 4 to 4.5, were selected, dewormed and randomly distributed to each one of the four treatment groups based on body weight, such that each treatment group had five animals. The details of experimental diets provided to the animals in their respective treatments is presented in Table 1. Pulikulam cattle breed have a small stature and have low milk yield, hence the dry matter, energy and protein requirement for diet formulation of this breed were based on the ICAR, (2013) recommendations. Three different energy and protein levels (20% above, same and 20% below) of ICAR were considered for this study. Treatment 1 was control diet followed by some farmers without standard level which was compared with treatment 2, 3 and 4 contains different energy protein levels of 100:80, 120:100 and 100:100 of ICAR, (2013) recommendations.
 

Table 1: Diet composition (DMB Kg/day) and calculated nutritive value of diets offered to animals of different treatment groups.


 
Rumen liquor collection
 
The animals in the various treatment groups were fed their respective diets for 30 days after which rumen liquor was collected from three animals per treatment. The collection was carried out early in the morning prior to feeding using a pedal operated vacuum pump (Bioplus®, Pedal suction apparatus BE-SU03, India).  During rumen liquor collection, the head of the animal was restrained and rumen liquor was collected by passing the tube using an oral speculum down the oesophagus into the rumen. The tube was gently pushed through the rumen mat to collect ruminal contents. Approximately 180 to 200 cm of the stomach tube was inside the cattle with the remaining portion of tube being outside of cattle, thus providing the flexibility to move the tube and extract ruminal fluid. Approximately 500 mL of initially sampled rumen liquor was discarded due to possible saliva contamination. After discarding the initial volume, an additional 1000 ml of rumen liquor was collected per animal and was used. The collected rumen liquor was transported using prewarmed thermos flask. The rumen liquor was filled up to the neck of the flask to maintain anaerobic condition. The collected rumen liquor was strained through four layer cheese cloth with continuous flushing of carbon dioxide to maintain strict anaerobic conditions and 39°C during transit.  In the laboratory the rumen liquor collected from each individual animal of the respective treatment groups were pooled and approximately 50 ml of rumen liquor from the pooled sample for each treatment was stored at -80! for DNA isolation and 16 s metagenomic study. DNA from the rumen liquor samples was extracted using Qiagen DNeasy Blood and tissue Kit. (Cat#69506). DNA from the samples were subjected for 16s rRNA gene amplification using region specific primers (16s rRNA primer) and PCR reaction was performed. Nanopore sequencing was performed on GridION X5 (Oxford Nanopore Technologies, Oxford, UK) using SpotON flow cell R9.4 (FLO-MIN106) in a 48 hours sequencing protocol.

RESULTS AND DISCUSSION

Comparative biodiversity of microbiome in different treatment groups
 
The estimated DNA concentration and nanopore read statistics in different treatment groups are presented in Table 2. The comparative microbiome diversity in different treatment groups were studied using Shannon index and rarefaction curve are presented in Fig 1 and 2 respectively, it reveals variation in microbiome community between treatment 1 and the other treatment groups. The results of this study indicate that there is diversity of methanogens in Pulikulam cattle and they vary under different dietary regimen. The rumen microbiota could be affected either by the geographical locations (Guan et al., 2008), host (Handerson et al., 2015; Guan et al., 2008; Roehe et al., 2016) or by the diet of the animal. Zhou et al., (2009) reported that methanogen composition varied between cattle herds of different feed efficiency. Difference in the rumen microbiota composition was observed between breeds fed on a similar diet (Paz et al., 2016). Handerson et al., (2015) reported that rumen microbiome varies depending upon the feed.
 

Table 2: Estimated DNA concentration and nanopore read statistics of different treatment groups.


 

Fig 1: Alpha diversity of ruminal microbiome in different treatment groups.


 

Fig 2: Rarefaction curve of ruminal microbiome in different treatment groups.


 
Effect of different dietary diets on methanogens community
 
A total of 125,442; 214613; 126123 and 126405 raw reads per sample were generated from treatment 1, treatment 2, treatment 3 and treatment 4 samples respectively. After removal of unclassified reads a total of 139, 428, 110 and 144 archaeal reads were retained for further analysis which is 0.1, 0.3, 0.08 and 0.1 per cent of total microbial reads in treatment1, treatment 2, treatment 3 and treatment 4 respectively. All filtered reads in the present study were affiliated to the rumen archaea. Total rumen archaeal phylum reads in different treatment groups is presented in Table 3. The ruminal archaea community in all the treatments were dominated by phylum Euryarchaeota. The archaea belonging to the other phylum Crenarchaeota and Thaumarchaeota were also present in all the treatments but the representation was lower than phylum Euryarchaeota. Phylum Candidatus Korarchaeota were present in treatment 2 and treatment 4 and phylum Candidatus Micrarchaeota was present only in treatment 2.
 

Table 3: Total number of rumen archaeal phylum reads in different treatment groups.


         
These results of this study concur to the previous reports (Abecia et al., 2014; Xue et al., 2020), where Crenarchaeota methanogens have acknowledged in the rumen.  Sulfolobales are physiologically and morphologically distinguished archaea, belonging to the phylum Crenarchaeota which occurs mainly in extreme thermo acidophilic ecosystems (Quehenberger et al., 2017). Dias et al., (2017) had reported that species Sulfolobus thuringiensis was in abundance they reported that S. thuringiensis was distributed up to 0.1 per cent of total archaea in dairy calves. Malik et al., (2021) also reported the presence of S. thuringiensis in livestock. However, the methanogenic capabilities of genera Sulfolobus and their influence to the ruminal methanogensis needs to be explored. Among the eight Sulfolobus species established in the literature, S. islandicus, S. solfataricus and S. acidocaldarius are best defined members of the genus. While S. islandicus is used as a model organism for comparative genomics and genetics (Reno et al., 2009) and for host virus interactions (Held and Whitaker, 2009). In this study all three Sulfolobus species were identified in the Pulikulam cattle.
         
Ruminal archaeal abundance percentage in different treatment groups at class level is presented in Table 4. Methanogens belonging to a total of four classes were identified in this study. Methanogens associated with the class Methanomicrobia were dominant in all the treatments. Methanogens associated with the class Thermoplasmata was absent in treatment 3.
 

Table 4: Ruminal archaeal abundance (%) in different treatment groups at class level.

    
 
Ruminal archaeal abundance percentage in different treatment groups at order and family level is presented in Table 5. Methanogens belonging to five orders were identified. The methanogens affiliated to the order Methanomicrobiales were predominant in treatment 1 (14 %) and treatment 2 (16.30 %).  Order methanococcales (15 %) was predominant in treatment 3 and order Methanos arcinales (18%) was predominant in treatment 4. Methonogens belong to Methanomassiliicoccales was distributed at lower frequency in treatment 1, 2 and 4 and not present in treatment 3. In treatment 1, Methanococcales was the second most abundant methoanogen (10%) followed by Methanobacteriales (8%) and Methanosarcinales (5%). In treatment 2, Methanobacteriales (11%) was the second most abundant methoanogen followed by Methanosarcinales (10.6%) and Methanococcales (7%). Methanomicrobiales (12%). In treatment 3 Methanosarcinales and Methanobacteriales abundance were present in equal amount. In treatment 4, Methanomicrobiales (9.6%) was the fourth abundant methonogen. Methanobacteriales and Methanococcales held second and third ranks in the abundance.
 

Table 5: Ruminal archaeal abundance (%) in different treatment groups at order and family level.


         
The results of this finding is similar to the finding of Sadan et al., (2019) in Vechur cattle, another native cattle breed of India. The authors had reported that Methanobactetriales, Methanomassiliicoccales and Methanomicrobiales were the predominant methanogen orders in rumen of Vechur cattle. Xue et al., (2016) reported that Methanobacteriales was the predominant archaea of rumen microbiota in the natural qrazing Yak in Sichuan. Other studies have reported an unprecedented greater abundance of Methanomicrobiales in cattle (Tajima et al., 2001; Shin et al., 2004) and buffaloes (Chaudhary and Sirohi, 2009; Singh et al., 2011; Singh et al., 2012). Previous studies have also reported that majority of the archaeal sequences retrieved from bovine rumens and cattle dung belonged to order Methanomicrobiales and Methanobacteriales (Rastogi et al., 2008; Tatsuoka et al., 2004). In contrast to the finding of the present study, Malik et al., (2021) reported that Methanomicrobiales proportion was less than 4 per cent of the total archaea in both cattle and buffaloes and the abundance frequency was consistent with the global data sets (Janssen and Kirs 2008; Handerson et al., 2015; Zhou et al., 2009). This could be due to the different DNA isolation methods, primer sets and animal diets. 
         
Methanomassiliicoccales is a novel group of archaea related to Thermoplasmatales (Horz et al., 2012). Methanomassiliicoccales are methylotrophic methanogens that utilize methanol and methylamines for producing methane (Lang et al., 2015; Sollinger et al., 2016).
         
In this study methanogen belonging to the order Methanomassiliicoccales was not present in treatment 3, diet of this treatment formulated with high energy contains more amount of maize than the other treatment group’s diets. A decrease in rumen pH due to high grain feeding is possibly a general cause for the stimulation or suppression of a particular type of methylotrophs in the rumen (Lana et al., 1998). The Methanomassiliicoccales may have specific properties that allowed their survival or inhibition during variable pH. The relative abundance of Methanomass iliicoccales affected by the diet rather than the host. Similarly, Xue et al., (2016) reported that methanogen Methanomass iliicoccales was absent in domesticated Yak maintained at China.
         
In this present study methanogens belonging to Methanosarcinales were present in all the treatment groups. Methanosarcinales grow on a broad range of substrates such as H2, CO2, methanol, methylamines and acetate. This is the only methanogen order capable of performing acetoclastic methanogenesis (Kendall and Boone, 2006). Malik et al., (2021) reported that methanogens belonging to Methanosarcinales were distributed at very small frequencies in both the cattle and buffaloes, their abundance was significantly greater in cattle compared to in buffaloes. Presence of Methanosarcinales in rumen was reported by (Patterson and Hespell, 1979; Jarvis et al., 2000) in both cattle and buffalo. VFA produced during digestion in the rumen are generally absorbed by the rumen epithelium and subsequently converted to animal proteins and therefore not available for utilization as a carbon source by Methanosarcinales residing in rumen. Therefore, abundance of  Methanosarcinales was minimal in rumen of cattle (Zinder, 1993). H2/CO2 is the only carbon source available in plenty for methanogens hence, hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales) that are capable of using H2/CO2 can multiply easily and their abundance were high in cattle rumen. Similarly, Denman et al., (2007) reported that the methanogens belonging to order Methanobacteriales was the major hydrogenotrophic methanogens in rumen of Brahman-crossbred (Bos indicus) steers.
         
In the present study methanogens belonging to 11 families were identified. Rumen archaeal composition at family level in different treatment groups are given in Fig 3, 4, 5 and 6. Families Methanocorpusculaceae, Methanomicro biaceae Methanoregulaceae Methanospirillaceae were affiliated to the order Methanomicrobiales. Methanoregulaceae distributed at lower frequency was present only in treatment 2 and treatment 3. Family Methanospirillaceae was present only in treatment 2 and treatment 4. Orders Methanosarcinales, Methanobacteriales and Methanococcales were characterized by two families of each. Methanothermaceae belonging to the order Methanobacteriales present only in treatment 4. Methanomassiliicoccaceae was absent in treatment 3.
 

Fig 3: Rumen archaeal composition at family level Treatment 1.


 

Fig 4: Rumen archaeal composition at family level in Treatment 2.


 

Fig 5: Rumen archaeal composition at family level in Treatment 3.


 

Fig 6: Rumen archaeal composition at family level in Treatment 4.


         
In this study at the species level methanogen M. labreanum belonging to order methanomicrobiales was the predominant species in all the treatment groups. The heat map comparison of different archaeal abundance at species level in different treatment groups is given in Fig 7. Methanococcus maripaludis, Methanococcus aeolicus, Methanobrevibacter millerae and Methanobrevibacter olleyae hold second, third and fourth predominant species in treatment 1. Abundance of species M. millerae and M. olleyae were similar. On the other hand, in treatment 2, M. labreanum, M. maripaludis, M. millerae and Methanosarcina mazei were the top four predominant species. Abundance of M. millerae and M. mazei were similar in this treatment. Abundance of M. olleyae was high compared to other treatments. M. labreanum, M. olleyae, M. maripaludis and Methanococcus voltae were the predominant species in treatment 3. Abundance of M. maripaludis and M. voltae were similar in this treatment. In treatment 4, M. labreanum was the predominant species followed by Methanococcus vannielii, M maripaludis and Methanobrevibacter smithii hold second and third position. Abundance of species, M maripaludis and M smithii were similar in this treatment. In the present study, species Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus solfataricus, Sulfolobus sp. A20 and Sulfolobus tokodaii were identified in treatment 1. Species Ferroplasma acidarmanus, Picrophilus torridus, Thermoplasma acidophilum, Thermoplasma volcanium, Thermoplasmatales archaeon BRNA1 species belonging to order Thermoplasmatales were also identified in the rumen of Pulikulam cattle.
 

Fig 7: The heat map comparison of different rumen archaeal abundance at species level in different treatment groups.


         
Similar to the finding of this study, in Murrah buffalo of India the majority of methanogens belonged to the genus methanomicrobium (Chaudhary and Sirohi, 2009). Shin et al., (2004) reported that 85 per cent of the total clones from the rumen of cattle belong to the order Methanomicrobiales, out of 104 clones 61 clones were resembling M. mobile.
         
In contrary, (Ozutsumi et al., 2012), Lwin et al., (2012) and Franzolin et al., (2012) have identified Methanobrevibacter as the dominant genus in Holstein cattle, water buffaloes and crossbred buffalo, respectively. In another study Jeyanathan et al., (2011) reported that in New Zealand Methanobrevibacter spp. were dominant in cattle. Leahy et al., (2013) also reported that in the cow rumen, Methanobrevibacter reported to be the dominant genus of the archaeal domain. In the present study Methanocella arvoryzae was identified only in treatment 2. Malik et al., (2021) reported that species M. arvoryzae, was identified in the buffalo rumen at a very low frequency.

CONCLUSION

From the study it is concluded that methanogen M. labreanum was the predominant species in Pulikulam cattle fed with different dietary regimen and diversity of methanogen varied in Pulikulam cattle fed with different diets. Methanogens belonging to archaeal family Methanothermaceae was identified in Pulikulam cattle and family Methanomassilii coccaceae was absent in rumen when high grain ration was fed to Pulikulam cattle. Since the predominant methanogen varies in this breed of cattle future studies are needed to explore the amount of enteric methane emission from the rumen of Pulikulam cattle under different feeding regimen and the effects of geographical location in the diversity of methanogen in this breed of cattle.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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