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IntroductionWith dwindling global freshwater supplies and increasing water stress, agriculture is coming under increasing pressure to reduce water use. Plant breeding requires high analytical capabilities. For this reason, near-infrared spectroscopy (NIRS) has been used to develop prediction equations for whole-plant samples, particularly for predicting dry matter digestibility, which has a major impact on the energy value of forage maize hybrids and is required for inclusion in the official French catalogue. Although the historical NIRS equations have long been used routinely in seed company breeding programmes, they do not predict all variables with the same accuracy. In addition, little is known about how accurate their predictions are under different water stress-environments.MethodsHere, we examined the effects of water stress and stress intensity on agronomic, biochemical, and NIRS predictive values in a set of 13 modern S0-S1 forage maize hybrids under four different environmental conditions resulting from the combination of a northern and southern location and two monitored water stress levels in the south.ResultsFirst, we compared the reliability of NIRS predictions for basic forage quality traits obtained using the historical NIRS predictive equations and the new equations we recently developed. We found that NIRS predicted values were affected to varying degrees by environmental conditions. We also showed that forage yield gradually decreased as a function of water stress, whereas both dry matter and cell wall digestibilities increased regardless of the intensity of water stress, with variability among the tested varieties decreasing under the most stressed conditions.DiscussionBy combining forage yield and dry matter digestibility, we were able to quantify digestible yield and identify varieties with different strategies for coping with water stress, raising the exciting possibility that important potential selection targets still exist. Finally, from a farmer’s perspective, we were able to show that late silage harvest has no effect on dry matter digestibility and that moderate water stress does not necessarily result in a loss of digestible yield.

Background Since the introduction of studies on maize silage digestibility at the end of the nineteenth century, protocols to estimate dry matter digestibility have not stopped evolving. Since the early 1980s, the protocol developed by Aufrère became a benchmark in many laboratories to estimate in vitro dry matter digestibility. In order to increase its throughput, to facilitate its execution and to decipher the impact of the different parameters of the protocol we decided to test the combination of 7 parameters in 21 different protocols. Results We thus tested the impact of (1) the presence or absence of pepsin in HCl solution, (2) the temperature of incubation during enzymatic hydrolysis, (3) the presence or absence of a gelatinization step, (4) washing/rinsing versus neutralization step, (5) the presence or absence of α-amyloglucosidase in enzymatic solution, (6) the duration of cellulase incubation, and (7) the concentration of the cellulase solution. The major result of our work highlighted that it was essential to carry out a gelatinization step to correctly estimate the in vitro dry matter digestibility of maize silage. Conclusions The proposed protocol in this paper is innovative, reliable, highthroughput and easy to implement in many laboratories to accurately quantity in vitro dry matter digestibility.