Effects and mechanism of the timing of alfalfa hay supplementation on rumen development of Hu lambs

The present study investigated the effects of alfalfa hay supplementation at different time points on rumen development in pre-weaning lambs and its underlying mechanisms. Thirty-six 7-day-old lambs (3.88 ± 0.92 kg) were randomly allocated to two feeding treatments, with 18 lambs in each group. Three lambs were housed together in the same pen as a single unit. After 7 days of adaptation to the milk replacer (MR), one group received MR and starter pellets, along with chopped alfalfa starting at 14 d of age (EAF), and the other group received the same diet but with chopped alfalfa introduced at 42 d of age (LAF). All lambs had ad libitum access to starter pellets and chopped alfalfa. At 42, 56, and 70 d of age, six lambs were randomly selected from each treatment group for slaughter. Rumen contents and epithelial tissues were collected for bacterial 16S rRNA sequencing and transcriptome analysis, respectively. At 42 d of age, the results indicated that the body weight (P<0.05), average daily gain (P<0.05), total dry matter intake (P<0.05), rumen papillae length (P=0.04), and rumen muscle layer thickness (P=0.01) were higher in the EAF compared with LAF group. Nevertheless, the α-amylase activity (P=0.04) and stratum corneum thickness (P<0.01) were lower in the EAF compared with LAF group. However, no statistically significant difference in growth performance, rumen fermentation parameters and tissue morphology indicators at 56 and 70 d of age between EAF and LAF groups (P>0.05). The EAF group exhibited lower abundances of Lachnospiraceae_NK3A20 and Muribaculaceae compared to the LAF group at 42 d of age (P<0.05). The transcriptomic results showed that early supplementation with alfalfa upregulates the expression of solute carrier family 2 like 14 (BCL2L14) and downregulates the expression levels of keratin 2 (KRT2) and stearoyl-CoA desaturase (SCD) in 42-day-old lambs. In conclusion, supplementing alfalfa at 14 d of age is beneficial to rumen development. However, this advantage gradually diminishes as the lambs grow older. The mechanism underlying the promotion of rumen development may be related to the expression of KRT2 and BCL2L14.