Artificial rearing of lambs using milk replacer (MR) is widely applied in sheep husbandry because of insufficient milk secretion of ewes, mastitis, postpartum paralysis and other reasons (Belanche et al., 2019; McCoard et al., 2020; Arshad et al., 2021; Mialon et al., 2021). Artificial feeding to make up for the lack of breast milk can increase the survival rate of lambs, shorten the breeding cycle of ewes, and improve animal welfare. In China, Hu sheep constitute an important livestock for lamb production because of their excellent prolificacy, rapid growth, and ability to adapt to poor-quality feeds and diverse environments (Yue, 1996; He et al., 2019). However, multiparous ewes also easily lead to insufficient breastmilk for lambs. Artificial rearing is important for the utilization of surplus lambs from dairy sheep systems, and lambs from meat sheep operations that are not able to be naturally reared to generate additional revenue from meat production (McCoard et al., 2020). Therefore, the rational utilization of MR significant to improve the survival rate and growth performance of lambs.

MR is an artificial milk produced by replacing milk protein with non-milk protein based on the nutritional standards of breast milk (Li et al., 2021). The nutritional components and physical form of MR are similar to breast milk, and its quality is not easily affected by the external environment (Toral et al., 2015). Evidence suggests that feeding lambs with MR could accelerate growth performance, has long-term benefits for sheep health (Zhang et al., 2019), enhance immunity, reduce the stress reaction caused by sudden changes of diets (Amdi et al., 2021), and affect post-weaning starter intake and average daily gain (Chapman et al., 2016). The beneficial effects of MR administration in early-weaning lambs also function via regulating their rumen microbiota (Bhatt et al., 2009).

However, high levels of MR might be detrimental to rumen development in early lambs and affect their rumen microbiota. The rumen of a newborn ruminant is not completely developed and does not contain a fully established microflora (Ekiz et al., 2016); early lambs rumen function is similar to monogastric animals (Longenbach and Heinrichs, 1998). The period from birth to 2 months of age is a critical stage of rumen development, representing a time window to regulate microbial colonization in lambs, and the feeding strategy in the first few weeks of life has been reported to affect rumen development (Carballo et al., 2019). Although little research has been conducted on the effects of MR feeding level on the rumen development, excessive intake of MR might reduce the intake of starter feed, which is essential for rumen development. Reports suggested that feeding high levels of MR could reduce post-weaning starter digestibility, especially for NDF (neutral detergent fiber) and ADF (acid detergent fiber; Terré et al., 2006; Hill et al., 2010). Low digestibility in young ruminants fed large quantities of MR is likely to be associated with suboptimal development of the rumen (Hill et al., 2016). These changes might be closely related to the rumen microbiota. Furman et al. proposed that both deterministic effects, driven by age and diet, and stochastic effects, driven by early colonization events, shape the composition of the rumen microbiome throughout life (Furman et al., 2020). MR is usually fed quantitatively in artificially reared lambs, and the feeding level varies greatly in different farms. Therefore, it is important to clarify the effect of the MR level on the rumen microbiota and its relationship with lamb development, and to balance rumen development and microbiota colonization using an appropriate feeding MR level. In recent years, several studies have examined the length of MR feeding period on rumen fermentation and microbial diversity (Carballo et al., 2019; Zhang et al., 2019; Mao et al., 2021), but relatively few have been directed at assessing how MR feeding level can affect rumen development. The appropriate MR level for lambs and the effect of feeding MR on rumen development and microbial colonization remains unclear.

Thus, in the present study, we hypothesized that feeding early lambs with intensive MR would affect rumen function and the rumen microbiota through changes in feed structure and intake, thereby affecting performance. We analyzed the growth performance and starter intake of lambs fed with different levels of MR, and evaluated the development and functionality of the rumen via ruminal fiber degradation rates, rumen weight, fermentation parameters, enzyme activities and rumen histomorphology. Furthermore, we employed 16S rRNA sequencing to explore the effect of the MR feeding level on the rumen microbiota and its relationship with rumen function and development. A detailed understanding of the regulation of early rumen development (function, morphology, and colonization) could provide the basis for the rational use of early nutritional regulation strategies to improve the productivity and health of lambs.

To obtain a higher price for lambs and more income from a flock, it is essential to have the minimum loss of lambs and optimum growth during the neonatal phase (Poonia et al., 2015). Meanwhile, appropriate feeding strategies to promote rumen development and microbiota establishment might be beneficial to the performance of lambs in the later period. Despite the accumulated knowledge regarding nutritional regulatory strategies during the early life of ruminants, the effects of the MR feeding level on rumen development, microbial colonization, and the regulatory mechanisms of host-microbial interactions in pre-ruminants are largely unknown.

Up to 3 weeks old, the growth performance of lambs was significantly improved by the higher level of MR feeding, although the low-level feeding group had a higher starter feed intake. The intake of solid feed in lambs before 3 weeks old is low, and the digestion and utilization efficiency of plant-based feed was much lower than that of MR (Li et al., 2022). Thus, at this stage, milk or MR provides most of the nutrients needed for growth and development, and the better bioavailability of protein and energy along with minerals, enzymes, and growth factors results in increased weight gains (Lee et al., 2009). Studies have also shown that high levels of MR can increase the ADG of lambs before weaning (Zhang et al., 2019). After the were 4 weeks old, with the rapid improvement of starter intake and digestive function, the effect of the MR feeding level on daily gain was no longer significant, indicating that the development of digestive function and the increase of starter intake are very important for the transition from liquid milk to plant feed in young lambs.

It should be noted that although high levels of MR feeding improved the growth performance of early lambs, it had adverse effects on rumen development. In the present study, the rumen weight and papillae length at 49 days old was significantly lower in the high feeding level group, and the lower intake of starter was the main factor limiting rumen development. Studies have shown that feeding large volumes of milk delays solid feed intake, which might compromise rumen development before and during weaning (Jasper and Weary, 2002). An adequate starter intake can stimulate the development of the rumen, which is necessary to establish the rumen microbiota (Khan et al., 2016). In early lambs, MR bypasses the rumen to the abomasum (Zhang et al., 2019), therefore, the solid feed that enters the rumen is the decisive factor for rumen fermentation parameters. Interestingly, we did not find any differences in the main rumen fermentation parameters, enzyme activities, and fiber degradation rates between the groups in the present study. Considering that the two groups of lambs have the same diet composition, these results indicated that the composition of the starter diet was more important for rumen fermentation function than the starter intake.

Interestingly, we found that the MR feeding level had a profound influence on the rumen microbiota, which was reflected in the α-diversity, β-diversity, and taxa abundance. In this study, despite the increasing of starter intake, the Shannon and Chao1 indices of the rumen microbiota decreased in L group. Although highly diverse microbiota is generally considered beneficial for host health and is regarded as a sign of a mature gut (Konopka, 2009; Le Chatelier et al., 2013), many studies have indicated that early starter intake decreased the ruminal bacterial diversity (Meale et al., 2016; Wang et al., 2016; Li et al., 2020). These findings were consistent with the results obtained from this study. We found that many bacteria genera and species disappeared in L group. The main reason may be that the increased starter intake promotes the establishment of predominant microflora and the depletion of transient bacterial species and genera. With the increase of plant-based feed intake, carbohydrate degrading bacteria occupied the widest niche, which suppressed the colonization of foreign flora, and most of the ‘disappeared’ bacteria are aerobic bacteria or bacteria without fermentation function (Wang et al., 2016). Besides, on average, the phyla Proteobacteria, Firmicutes, and Bacteroidetes were predominant in all samples. These findings agreed with previous studies on the ruminal microbiota (Morgavi et al., 2015). Proteobacteria are a large group of bacteria that ferment carbohydrates to ethanol, playing an important role in rumen metabolism, such as the formation and fermentation of biofilms (Zeng et al., 2017). Firmicutes represent the core bacterial component that is predominant within the rumen, mainly comprising diverse fibrolytic and cellulolytic bacterial genera. Bacteroidetes express relatively large numbers of genes encoding carbohydrate-active enzymes; thus, promoting the breakdown of structural polysaccharides in the rumen and also fermenting amino acids into acetate (Zhang Y. K. et al., 2021). Notably, the high MR feeding level decreased the relative abundance of Fibrobacteres, which were reported as major bacterial degraders of lignocellulosic material in the herbivore gut (Ransom-Jones et al., 2012). High MR feeding level decreased the starter intake, and the reduced available substrates may be the main reason for the reduction of lignocellulosic degrading bacteria. Besides, the high MR feeding level increased the relative abundance of Synergistetes, Euryarchaeota, and Verrucomicrobia, which have been reported to be associated with inflammation in the gastrointestinal epithelium (Forterre et al., 2014; Sichert et al., 2020; McCracken and Nathalia Garcia, 2021), suggesting that excessive feeding of MR might have adverse effects on gastrointestinal health by altering the microbiota.

In particular, we found that the MR feeding level affected the abundance of many carbohydrate-degrading bacteria. The abundance of Lachnospiraceae_NK3A20_group, Succiniclasticum, Ruminococcus_1, Ruminococcaceae_UCG-014, and Moryella decreased with the increasing MR level. Lachnospiraceae ferment diverse plant polysaccharides to short-chain fatty acids and alcohols (Boutard et al., 2014). Succiniclasticum, which ferment succinate quantitatively to propionate, were isolated from a high dilution of rumen ingesta obtained from a dairy cow fed a production diet containing grass silage as the main roughage source (Van Gylswyk, 1995). Ruminococcus_1 plays an important role in plant fiber degradation (Devillard et al., 2004). Moryella has been reported to be associated with cellulose or its metabolites (Tian et al., 2019). A key deterministic factor framing the process of microbial succession is diet, especially because the abundance of certain bacteria changes in response to a fiber-based diet (Furman et al., 2020). The pre-ruminant rumen microbiota is highly active and ready to ferment a solid diet from the first week of life (Malmuthuge et al., 2019), and the increased abundance of certain important fiber degrading bacteria might be an adaptive mechanism of the rumen microbiota in response to increased solid diet intake. Typically, under ad libitum feed intake, as the starter intake increases, the rate of digesta passage from the rumen increases and nutrient digestibility decreases (Bourquin et al., 1990). In this study, the starter intake was significantly lower in the high MR feeding level group; however, the rumen degradation rates of NDF and ADF did not increased, which mighty be related to the decreased abundance of these carbohydrate-degrading bacteria.

We further analyzed the relationship between rumen fermentation parameters and bacterial abundance, and found that many bacterial genera with significant differences, such as Ruminococcaceae_UCG-014 and Moryella, correlated significantly and positively with rumen cellulase activity, and Succinivibrio correlated positively with rumen ADF degradation rate and the total VFA concentration. Considering the small sample size and the vast differences in microbiome composition between the different groups, we cannot infer specific links through correlation due to large fluctuations in the microbiome between groups. This would likely yield inaccurate correlations due to strong habitat filtering (Berry and Widder, 2014) and the heteroscedasticity of the data. Although the correlation relationship does not necessarily indicate a direct causal effect, the observed multiple significant correlations between relative abundances of microbial taxa and rumen fermentation parameters provide some insight into potential host-microbiotic interactions in the rumen, suggesting that changes in the microbiota, especially the abundance of carbohydrate-degrading bacteria, may be the mechanisms for the rumen to adapt to changes in food structure and starter intake and can affect rumen function. There has been a study indicating that VFAs produced by the early microbiome were associated with the rumen tissue metabolism and the development of the epithelium with the host transcriptome and microRNAome (Malmuthuge et al., 2019). However, further studies are needed to determine the long-term consequences of the adverse effects of a high MR feeding level on rumen weight and the microbiota of lambs. A suitable balance between promoting rumen development and enhancing weight gain in early stage needs to be determined by selecting appropriate levels of MR feeding.

Increasing the feeding level of MR from 2 to 4% of the body weight of lambs significantly increased the weight gain, but decreased the intake of starter and had adverse effects on rumen development. A high MR feeding level increased the rumen microbial diversity, but decreased the abundance of many rumen carbohydrate catabolizing bacteria. This study provides new insights into the regulation of early rumen development (function, morphology, and microbial colonization) of lambs. The results suggests that the adverse effect of excessive MR feeding on rumen development and the microbiota should not be ignored; and a suitable balance between promoting rumen development and enhancing weight gain in the early stage needs to be identified by selecting appropriate levels of MR feeding.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA890401.

The animal study was reviewed and approved by the Gansu Agricultural University’s Academic Committee and the National Natural Science Foundation of China (Approval no. 31760682) according to guidelines established by the Biological Studies Animal Care and Use Committee of Gansu Province.

YH, GW, and QZ: methodology, investigation, and writing-original draft. ZC, XZ, XW, and DZ: validation and visualization. CL: funding acquisition. WW: supervision and project administration. PC, ZM, and CL: writing-review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China under grant no. 31760682 and Discipline Team Project of Gansu Agricultural University under grant no. GAU-XKTD-2022-20.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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