Title: Optimizing dietary rumen-degradable starch to rumen-degradable protein ratio improves lactation performance and nitrogen utilization efficiency in mid-lactating Holstein dairy cows

The dietary rumen-degradable starch (RDS) to rumen-degradable protein (RDP) ratio, denoted as the RDS-to-RDP ratio (SPR), has been proven to enhance in vitro rumen fermentation. However, the effects of dietary SPR in vivo remain largely unexplored. This study was conducted to investigate the effect of dietary SPR on lactation performance, nutrient digestibility, rumen fermentation patterns, blood indicators, and nitrogen (N) partitioning in mid-lactating Holstein cows. Seventy-two Holstein dairy cows were randomly assigned to three groups (24 head/group), balanced for (mean ± standard deviation) days in milk (116 ± 21.5), parity (2.1 ± 0.8), milk production (42 ± 2.1 kg/d), and body weight (705 ± 52.5 kg). The cows were fed diets with low (2.1, control), medium (2.3), or high (2.5) SPR, formulated to be isoenergetic, isonitrogenous, and iso-starch. The study consisted of a one-week adaptation phase followed by an eight-week experimental period. The results indicated that the high SPR group had a lower dry matter intake compared to the other groups ( p  < 0.05). A quadratic increase in milk yield and feed efficiency was observed with increasing dietary SPR ( p  < 0.05), peaking in the medium SPR group. The medium SPR group exhibited a lower milk somatic cell count and a higher blood total antioxidant capacity compared to other groups ( p  < 0.05). With increasing dietary SPR, there was a quadratic improvement ( p  < 0.05) in the total tract apparent digestibility of crude protein, ether extract, starch, neutral detergent fiber, and acid detergent fiber. Although no treatment effect was observed in rumen pH, the rumen total volatile fatty acids concentration and microbial crude protein synthesis increased quadratically ( p  < 0.05) as dietary SPR increased. The molar proportion of propionate linearly increased ( p  = 0.01), while branched-chain volatile fatty acids linearly decreased ( p  = 0.01) with increasing dietary SPR. The low SPR group (control) exhibited higher concentration of milk urea N, rumen ammonia N, and blood urea N than other groups ( p  < 0.05). Despite a linear decrease ( p  < 0.05) in the proportion of urinary N to N intake, increasing dietary SPR led to a quadratic increase ( p  = 0.01) in N utilization efficiency and a quadratic decrease ( p  < 0.05) in the proportion of fecal N to N intake. In conclusion, optimizing dietary SPR has the potential to enhance lactation performance and N utilization efficiency. Based on our findings, a medium dietary SPR (with SPR = 2.3) is recommended for mid-lactating Holstein dairy cows. Nevertheless, further research on rumen microbial composition and metabolites is warranted to elucidate the underlying mechanisms of the observed effects.