This study was undertaken to investigate the impact of artificial rearing of lambs (i.e separation from the dam and milk replacer feeding) on rumen and lower gut microbiota colonization in the first month after birth as well as possible post-weaning effects on GIT development.
Performance And Sanitary Status Of Mothered And Artificially-reared Lambs
No difference in lambs’ weight and ADG was observed between groups before weaning or on the whole experiment. Several previous works reported impaired early growth rate in lambs submitted to artificial rearing compared with mothering (12,22). On the contrary, in the present study, the post-weaning ADG was significantly higher for the ART group, and appeared more variable in the MOT group, also after weaning. This higher variability in MOT lambs might result from the stress due to separation of lamb and dam at weaning. The composition of milk replacer varies from one supplier to another and could explain differences observed between studies, as well as the mode of suckling which differed also between studies (commercial buckets vs in-house system used here). Our results suggest that the milk replacer and the suckling system we used allowed to cover well the lambs’ needs and avoided competition for teat allocation. One hypothesis to explain the higher ADG measured in the ART group after weaning could be that although MOT and ART lambs had access to solid feed at the same age, MOT lambs were not prone to rapidly shift from dam’s milk to feed. ART lambs were likely to perceive digestive discomfort due to high milk replacer intake and thus tended to start consuming starch-rich pellets earlier in age before weaning. Indeed, a more degraded sanitary status was observed in ART lambs compared to MOT animals, at weeks 1 and 5. This was mainly due to dirtier perianal areas, indicating altered digestive transit. Previous work on lambs also showed minor diarrheal events from weeks 2 to 5 with artificial rearing, leading to lower growth rates during the 1st month but the animals reached similar weaning weights than mothered lambs (22), as we observed in the present study. The dirtiness of the rear end observed on ART lambs in the present work was associated to a disturbed microbiota in their feces, and detection of higher relative proportions of Cryptosporidium taxa. Cryptosporidium is an ubiquitous protozoan parasite that infects a broad range of vertebrate hosts, including sheep (25). Note that total parasite taxa and Eimeria, a well-known coccidian parasite in lambs (26), were also found in higher proportions in ART than in MOT rumens. In addition to a lower sanitary score in the ART lambs, the IgG concentrations in blood serum measured on this group were lower than that measured in the MOT group in the first days of life, although the mean IgG concentrations were rather comparable to published literature (from 15.7 to 65 g/l) (22,27). In addition, 3 lambs from the ART group had a serum IgG level lower than 10 g/L, indicating failure of passive transfer (FPT). These results reflect a lower immunocompetence in the ART group, probably due to the limited intake of colostrum during the 12h lambs were kept with their mother. Colostrum ingestion is of paramount importance for the neonate and FPT is associated with an increased neonatal mortality (28). Delaying colostrum feeding within 12 h of life has been shown to decrease the passive transfer of IgG in calves (29). In agreement with these observations, lower serum IgG has been often observed in ruminants fed milk replacer (18). In addition to bioactive proteins, colostrum includes oligosaccharides which probably provide adequate substrates to the pioneer gut bacteria (30). Limited colostrum intake may then also delay the bacterial colonization of the intestine, possibly leaving the newborn vulnerable to infections during the preweaning period.
The Physiology Of The Lamb Git Post-weaning Was Not Affected By The Pre-weaning Rearing Mode
To investigate possible long-term effects of the newborn rearing mode on the lamb physiology post-weaning, the GIT anatomy and cellular activity were evaluated in the two groups. No impact of the rearing mode was observed at day 64 on the GIT anatomy as assessed by measuring the relative weight of organs, rumen wall thickness and papillae dimensions. We also quantified the expression of a selection of genes coding for proteins involved in epithelium integrity, metabolism or immune response in the rumen and colon mucosa, but no significant difference could be observed between the ART and MOT lambs. This suggests that after 64 days, the epithelium metabolism and activity is similar whatever the rearing system, even though it could have been different earlier in life. Indeed, the rumen metabolism of pre-weaning lambs has been shown to be mainly stimulated by SCFA (31,32) and should be then influenced by the activity of the microbiota. In the pre-weaning period, the rumen epithelium undergoes morphological development as well as cellular molecular adaptation of nutrients absorption and metabolism (31,32). It was shown that introduction of solid feed to lambs impacts the rumen wall metabolism at 42 days of age (31), hay facilitated establishment of immune function, while concentrate starter enhanced nutrient transport and metabolism. Also in calves, a highly active early microbiome was shown to regulate the rumen development at the cellular level (33). Anyway, our results suggest that after 64 days of age, the lamb pre-weaning rearing mode had no more impact on the rumen and intestinal epithelium activity, even though significant differences in the rumen microbiota can still be observed between the two groups at day 64 (i.e F. succinogenes population level).
Finally, our data showed differential expression of certain genes in the rumen vs the colon epithelium of lambs from both ART and MOT groups and bring additional information to the few papers comparing gene expression in these two GIT segments in sheep. Among the differential genes identified here, Claudin1, HMGSC2, HMGSL and MCT1 were more expressed in the rumen wall while IGF-1, IL-10 and TLR4, participating to intestinal homeostasis, epithelial regeneration, and immunity, appeared more expressed in the colon epithelium. Expression of genes involved in innate immune functions has demonstrated variations according to GIT segment in bovines (34). The expression of TLR4 (Toll like receptor 4, known to recognize lipopolysaccharide of Gram-negative bacteria) was previously found higher in the colon epithelium than in the rumen mucosa both in calves and cows (34,35), according to the fact that the colon plays a more important role in the GIT immune system than the rumen. Claudin1 is one of the main tight-junction Claudin proteins which mediate adhesive functions between epithelial cells. Tight junctions play a key role in maintaining the polarity of epithelial cells, regulating the permeability of the epithelial barrier and preventing the translocation of LPS and other toxins (36). HMGSC2 and HMGSL encode the 3-hydroxy-3-methylglutaryl-CoA synthase isoform 2 and a 3-hydroxy-3-methylglutaryl-CoA lyase, respectively, these two proteins being involved in the ketogenesis pathway. It is known that, while the neonate rumen epithelium is not ketogenic, the ketogenesis pathway is expressed in the sheep rumen mucosa from 42 days of age (37). MCT1 protein, a monocarboxylate transporter 1 which mediates transport of SCFA across the GIT epithelium, is recognized as highly distributed in the forestomach and large intestine of sheep (38). More precisely, MCT1 facilitates the efflux of SCFA, lactate and ketone bodies across the basolateral membranes of epithelial cells toward the blood side (38), and its gene expression has been already demonstrated in the ovine rumen epithelium (39). Our results agree well with MCT1 playing a significant role in the transport of short chain fatty acids and their metabolites in the rumen epithelium of sheep.
The rearing mode has a strong impact on the rumen and intestinal microbiota of pre-weaned lambs
The present study highlights huge modifications in the rumen and lower gut microbiota colonization depending on the mode of rearing before weaning. Artificial rearing led to lower richness and slower maturation of the rumen microbiota, with delayed establishment of functional fibrolytic populations and protozoa, leading to lower rumen fermentative activities, particularly after weaning.
Indeed, in MOT lambs we clearly observed an evolution of bacterial communities with age, both in rumen and feces, which was not seen in ART lamb microbiota. The rumen and distal gut microbiota of MOT animals diversified and matured with age, according to known stages of GIT colonization in sheep and bovines: Proteobacteria, including facultative anaerobic bacteria, constitute the major phylum during the first days of life, and they consume the oxygen present allowing the gradual colonization of strictly anaerobic bacteria and fungi in the first two weeks (5,7,8,40–43). From 14 days to weaning, the rumen microbiota grows in complexity, becoming dominated by Bacteroidetes and Firmicutes as a result of a gradual increased in solid feed intake (5,21,40). In the lower gut, as reflected by monitoring the fecal microbiota during the first weeks of age, Bifidobacterium, Enterobacteriaceae, Streptococcus and Lactobacillus taxa decreased while strictly anaerobic bacteria increased and fibrolytic bacteria such as Ruminococcus flavefaciens and Fibrobacter sp. become detectable only around weaning (44). Such successions were observed here in MOT rumen and feces, as shown by alpha and beta diversity, microbiota analysis and qPCR quantification, but not in the rumen and feces of ART animals. It has been previously observed that bacterial diversity was higher in mothered lambs compared with lambs fed milk replacer and separated from their dam (19,20,22).
The ART rumen and feces microbiota revealed a lactic profile with high proportions of Streptococcaceae and Lactobacillaceae, respectively, compared with MOT microbial communities. This was confirmed by qPCR quantifications which showed higher proportions of lactic acid bacteria and Bifidobacterium in the feces of pre-weaned ART lambs. These families/genera are generally associated with primarily milk feeding diet, as it has been shown in humans that milk oligosaccharides and glycans can act as prebiotics stimulating the growth of Bifidobacterium and Lactobacillus spp. (45). Streptococcaceae may have been promoted by the concentrate intake as it contained quite high levels in starch and sugars. However, it was not possible to measure individually the solid feed intake so the difference in bacterial composition between groups cannot be directly attributed to difference in feed intake. Their higher persistence in ART lamb intestine may reflect the delay in maturation of the rumen and hindgut microbiota and impaired establishment of the fibrolytic communities. Indeed, the cellulolytic bacteria R. albus and F. succinogenes were already well established at day 7 in the MOT lamb rumens, but, although R. albus was also quantified at high levels in ART rumens, F. succinogenes establishment was clearly delayed in ART animals. This species was found in lower proportions in the artificially reared lambs, even after weaning. Strong delay in establishment of F. succinogenes was previously detected in calves or lambs separated from their dam (5,10,46). A decrease in the Fibrobacteres phylum was also observed at parturition on ewes (47), indicating that Fibrobacter may be quite sensitive to changes in the ecological conditions of the rumen. F. succinogenes is known as an important fibrolytic microorganism, particularly efficient in degrading recalcitrant cellulosic substrates such as straw (48,49) and also very sensitive to oxygen.
Another remarkable finding is the much lower relative proportion of Proteobacteria in ART newborn lambs compared with MOT animals, both in rumen (at day 7) and feces (at day 14), the major phylum in ART samples at these ages being Firmicutes. In MOT lambs, the Proteobacteria sequences are in majority affiliated to Neisseriaceae in the rumen and to Enterobacteriaceae including Escherichia as dominant genus in the feces. Neisseriaceae are known to colonize the rumen mucosal surface and belong to the core rumen epithelial bacteria of several ruminant species (50,51). They were found abundant and active at this site in a metatranscriptome study of the bovine rumen which revealed high expression of genes involved in nitrogen metabolism and of genes related to coping with oxidative stress conditions (52). Thus, members of this family are assumed to participate to oxygen scavenging, an important function of the rumen epimural microbiota, and could then be very effective in oxygen removal from the MOT rumens, providing conditions suitable to subsequent colonization by strictly anaerobic rumen microbes.
Because Escherichia was a dominant genus in the feces of MOT lambs, we sought the presence of E. coli virulence genes in these samples. No difference between MOT and ART lambs’ feces was detected, except for 2 genes with opposite detection frequencies. Despite a strong increase of Enterobacteriaceae relative abundance observed in MOT lambs’ feces at d14, it was not associated with an increase in virulence potential of the colibiota, in accordance with the sanitary status results of these animals. This high proportion of Enterobacteriaceae and Escherichia/Shigella observed could be beneficial to the animal, as already observed in piglets for example (53,54). Indeed, the commensal E. coli can fill an ecological niche no more available for pathogenic E. coli colonization, providing a protective effect to the mothered lambs.
Among other differential bacterial taxa in the rumen of the two lamb groups, Anaeroplasmataceae and Acidobacteriaceae were much more abundant in MOT rumens. These two families have been correlated to ADG in sheep and associated to the succinate to propionate pathway in goat, respectively (55,56). They may then be involved in propionate synthesis which favors gluconeogenesis and consequently meat production (57).
Regarding the Eukaryota community, artificially reared lambs had no rumen ciliate protozoa at weaning, while natural rearing promoted an early colonization of the rumen by a diverse protozoal population. These results confirm previous findings on lambs and goat kids (10,22,23). The MOT lamb rumen protozoa belonged to many different ciliate taxa, with Entodinium, Eudiplodinium and Polyplastron as dominant genera. It is known that the establishment of ciliate protozoa in the rumen requires the preliminary establishment of a diverse bacteria community (3) so the higher bacterial diversity observed in the rumen of MOT lambs certainly triggered better ecological conditions for ciliate protozoa settlement. Both lamb groups showed a diverse anaerobic fungi community from the Neocallimastigomicota phylum, including monocentric and polycentric taxa, with no difference in composition in the rumens and feces of both groups although colonization by anaerobic fungi was reported to be perturbed during artificial rearing of young ruminants (10,23). The ability of anaerobic fungi to form resistant spores may allow them to retain good viability, even in a less anaerobic environment (58). However, regarding other fungi phyla, MOT feces showed higher relative proportions of Cryptomycota and Chytridiomycota while several yeast genera from the Saccharomycetales order had higher relative abundance in ART rumens and feces. Yeasts such as Candida spp. have been found in the sheep farm environment and animal feed, as well as in ewe’s milk and teat surface or ruminant digestive tract (59,60), but the fungal diversity within farms and according to farming practices has not been studied much. It is then hard to make assumptions about differences found in ART and MOT lambs about these communities.
Finally, the dysbiosis observed in the ART rumen and hindgut microbiota reveals impaired establishments of functionally important microbes, which may be due to several factors. Nutritional factors such as limited colostrum feeding or quality of milk replacer may be important in the artificial rearing management, but other stresses such as the separation with the dam and other adult ruminants may also affect the health and wellbeing of the lamb (12).