Research efforts pertaining to identifying superior thermo-tolerant indigenous breeds are gaining significance as the approach may help to sustain livestock production in the changing climate scenario. The present study being first of its kind, is one such attempt in trying to establish thermo-tolerance in three different indigenous goat breeds based on changes associated with rumen microbiome. The results from the study revealed that the animals were exposed to extremely severe heat stress as reflected by the weather variables and THI. Further, the panting score also revealed that the animals experienced the heat stress. In addition, heat stress was found to influence rumen microbiome at various levels such as phyla, class and genera.
The weather variables and THI indicated that the animals in the shed were at comfort condition while the animals exposed to outside environment were subjected to extremely severe heat stress. As per McDowell (1972) THI model any values above 78 is considered extremely severe heat stress and the average fortnightly THI values of 86.5, 86.3 and 86.8 in outside environment reflected that the heat stress group animals were exposed to extremely severe heat stress throughout the study duration. Similar observations using the same THI was also established by other researchers (Shaji et al. 2016; Aleena et al. 2019). Likewise the panting score established among the heat stress groups reflected the differences in the level of heat stress being experienced across the breeds. Gaughan (2003) established that panting score has the potential to be used in the assessment of the welfare status of animals. Although all the three breeds were experiencing severe heat stress as reflected by their panting score but the magnitude of this effect was less in Salem Black breed as compared to both Osmanabadi and Malabari breeds. The panting score in the heat stress groups of Osmanabadi and Malabari breeds were comparable. Similar observations of validating the magnitude of heat stress experienced by goats based on panting score changes was established by Darcan et al. (2007) and Fioni (2014). In a recent report Reddy et al. (2019) has demonstrated that panting score could be a reliable easy to use methodology to assess thermal resilience capacity of goats.
Firmicutes, Proteobacteria and Bacteroidetes were among the most abundant phyla in all the breeds irrespective of heat stress, which was in line with the findings reported by previous studies revealed by rumen metagenomics in goats goats (Wang et al. 2016; Do et al. 2017). Furthermore, Firmicutes and Bacteroidetes were found to be the dominant bacterial phyla in rumen fluid of buffalo and Jersey cows; indicating that these phyla could be the dominant core microbiome in ruminant species (Henderson et al. 2015). Similar prevalence of dominant phyla Firmicutes, Proteobacteria and Bacteroidetes were also reported by Zhong et al. (2019) in Boer goats subjected to heat stress. Firmicutes are primarily composed of gram positive low GC content bacteria (Boone et al. 2001). Previous studies have indicated that the abundance of Firmicutes is related to the feed utilization efficiency and found to be higher in gut of efficient feed utilizing animals (Myer et al. 2015). Firmicutes are the largest bacterial phylum and their higher abundance may increase carbohydrate fermentation in the rumen (Fernando et al. 2010). Thus, higher abundance of Firmicutes in Malabari and Salem Black breed control groups in the present study indicates that these breeds are more efficient in breaking down of complex lignocellulosic feeds than Osmanabadi. The phylum, Firmicutes, includes a majority of butyrate- producing bacteria (Li, 2015), which has the least abundance in Salem Black heat stressed goats while highest in Osmanabadi heat stressed goats. However, the phylum Bacteroidetes is known as good degraders of complex polysaccharides present in the plant cell wall than Firmicutes (Khafipour et al. 2016). This phylum has the highest abundance in Salem Black goats subjected to heat stress than the other two breeds. Thus the decreased Firmicutes and higher Bacteroides abundance in Salem Black goats could indicate the better adaptation of this breed to heat stress when compared to the other two breeds of goat. Further, it is an established fact that Firmicutes phylum also has high abundance of known H producer species (Stewart et al. 1997). Therefore, the lower Firmicutes phylum in the heat stress groups of Salem Black breed establishes the advantage of this breed over other two breeds in terms of contributing low H in the rumen environment. Nearly similar alteration of rumen microbiome at class level was noticed in heat stressed Osmanabadi, Malabari and Salem Black goats when compared to their control. This could be attributed to the indigenous nature of these breeds.
Previous studies have shown that Bacteroidetes possesses a strong ability to degrade protein and polysaccharides (Huo et al. 2014; Pitta et al. 2016). Moreover, Bacteroidetes are considered net H (hydrogen) utilizers (Stewart et al. 1997). As evident from the present study, the Bacteroidetes was reported to be the third abundant phylum, with highest abundance in Salem Black breed heat stress group. Hence, this breed could possess better protein and polysaccharide degrading ability in addition to increased utilization of H. Thus, Salem Black breed could have the dual advantage of increased production and low methane emission potential.
Güllert et al. (2016) suggested that low ruminal Firmicutes to Bacteroidetes ratio would increase the capacity of biomass degradation as the Bacteroidetes phylum had higher glycoside hydrolase enzymes. When comparing the three goat breeds subjected to heat stress, this ratio was least for Salem Black goats and highest for Osmanabadi goats which further add on to the potential superiority of Salem Black goats to adapt to heat stress. Though the relative abundance of several other phyla varied as a consequence of heat stress, there is paucity of information on their functional role in the rumen.
Generally, the genus Prevotella was found to degrade nonstructural carbohydrates and protein (Belanche et al. 2012; Thompson et al. 2015). Further, this genus also involves in amino acid metabolism, nucleotide metabolism, energy metabolism, and glycan biosynthesis (Hook et al. 2011). However, the present study indicated no significant change in this genus in control and heat stress groups across all the breeds with a slightly higher prevalence in Salem Black breed as compared to other breeds indicating the better feed degradation and nutrient utilizing ability by this breed.
The Butyrivibrio genus was known to modulate secretion of hemicelluloses-degrading enzymes thereby proposed to play an important role by contributing polysaccharide degradation in rumen (Dunne et al. 2012). The significantly increased abundance of this particular genus in Salem Black breed heat stress groups signifies the better digestion efficiency of this breed over other breeds. Ruminococcus is one of the most predominant fibrolytic bacteria in the rumen (Hespell et al. 1997) and their concentration was expected to increase for the breakdown of generally increased cellulose and hemicellulose content in ruminant feed (Biddle et al. 2013). In the present study, the abundance of Ruminicoccus was found to decrease in heat stress group of Salem Black breed as compared to control, indicating the sensitivity of this genus to heat stress in this particular breed.
Bifidobacteria are known to be the beneficial gut bacteria, as they act as probiotic and helps maintain healthy internal environment of the host (Song et al. 2012). In the present study, we did not observe any reduction in beneficial rumen bacteria (probiotic) like Bifidobacteria in control and heat stress groups, respectively across all breeds. This could be attributed to the indigenous nature of all three breeds. However, the overall increased concentration of this particular genus in Salem Black breed indicates the better adaptability of this breed for heat stress.
Butyrivibrio group is known to be a fibrolytic bacterium (Fernando et al. 2010) and are the most active bacterial species involved in the biohydrogenation of C18 unsaturated FA (Durmic et al. 2008). The increased concentration of Butyrivibrio in all heat stress groups across the breeds indicates that these breeds are much tolerant and well adapted to degrade unsaturated fatty acids efficiently even in heat stress condition. However, the evident increase in concentration of this particular genus in Salem Black heat stress group over other breeds establishes the better adaptability of this breed to heat stress challenges.
Additionally, an evident increase in Methanobrevibacter, having a significant role in contributing towards methane production was observed in heat stress groups of both Osmanabadi and Salem Black goats as compared to Malabari breed. However, this particular result needs to be validated by measuring methane production in addition to specifically targeting metagenomics analysis for 16s archeal microbial population to have a clear picture of this association. This is because as per the results of the previous studies, it has been proved Salem Black breed to be well adapted and also maintain its production during heat stress when compared to Osmanabadi and Malabari goats (Aleena et al. 2018; Pragna et al. 2018). Further, Denman et al. (2015) reported that abundance of Prevotella was stimulated when methanogenesis (which is a hydrogen-consuming process) was inhibited. However, the present study turned out to be a deviation of this previous finding (Denman et al. 2015); we reported a higher abundance of Methanobrevibacter as well as slight increase in Prevotella in heat stress groups of Osmanabadi and Salem Black breed.
The very less number of significantly abundant microbes, due to heat stress, at all taxonomic levels in the Salem Black goat indicates the sub-threshold level of heat stress in this breed to induce changes in the microbial composition unlike Osmanabadi and Malabari. These findings again establish the immense adaptive potential of Salem Black breed to keep intact the rumen microbial composition probably to maintain the digestive functions.
At each taxonomic (phyla, class and genera) level, the number of OTUs in common between the control and heat stress groups was higher in Salem Black breed as compared to both Osmanabadi and Malabari breeds indicating the lesser impact of heat stress on the microbial diversity in Salem Black breed. Further, the number of unique OTUs in the Salem Black heat group was higher as compared to both Osmanabadi and Malabari heat groups. This shows that the level of heat stress in the study was of lower threshold to induce higher impact on rumen microbial population in Salem Black breed. These findings points towards the superior ability of Salem Black breed over the other two breeds to maintain rumen microbial population to elicit important biological functions.
Both level 01 and level 02 KEGG analysis as well as COG analysis revealed that heat stress did not influence the abundance of predicted functional rumen metagenome pathways in Osmanabadi and Malabari breeds. However in Salem Black breed, the heat stress group showed less abundance of all the rumen metagenome pathways. This indicates that the magnitude of heat stress did not elicit stress response to the level to influence predicted functional metagenomics pathways in Salem Black breed. The probable reason for the reduced abundance of all these pathways in heat stressed Salem Black breed could be attributed to the sub-threshold level of the heat stress experienced by these animals. Further, this hypothesis was justified by the lower level of panting score recorded in the heat stress group animals of this breed in contrast to other two breeds. Generally, the metabolic activities are expected to be higher in breeds wherein there is more requirement of energy to support life sustaining activities to cope with heat stress. Since the heat stress level was sub-threshold in Salem Black breed, the metabolic and other cellular pathways associated with adaptive mechanisms are of less abundance in this breed. In a similar study conducted on the Salem Black breed, lower levels of expression was established for cellular heat stress markers such as HSP70 and HSP90 and the authors attributed this to the sub-threshold level of the heat stress experienced by this breed (Madhusoodhan et al. 2020). One should never overlook the fact that the THI established in this study falls in the category of extremely severe heat stress. Further, the higher THI induced significantly increased levels of stress response in heat stress groups of both Osmanabadi and Malabari breed as evident from the higher magnitude of panting scores recorded in these breeds. This indicates that the higher threshold stress level in Osmanabadi and Malabari breed was of sub-threshold level in Salem Black breed reflecting the inherent ability of this particular breed to cope with heat stress of higher magnitude without altering much of its adaptive pathways.
The morphometric characteristics of ruminal papillae in terms of height and width has significant role in adaptation of animals to heat stress challenges (Yan-fen et al. 2013). These anatomical structures in the rumen have implications in determining the digestion efficiency of animals during heat stress in tropical countries. In our study it was observed among the three breeds, the Salem Black breed showed lesser degree of changes in ruminal mucosa than the other two breeds indicating the better genetic potential of this breed in adapting to adverse environment. These findings in the histological changes in the rumen among the breeds further supports the superior genetic potential of Salem Black breed to counter heat stress challenges. Further, studies are required though to document in details the heat stress associated changes in the rumen histology.