Citation: Xu, Z.; Heuschele, D.J.; Lamb, J.F.S.; Jung, H.-J.G.; Samac, D.A. Improved Forage Quality in Alfalfa (Medicago sativa L.) via Selection for Increased Stem Fiber Digestibility. Agronomy 2023, 13, 770. https://doi.org/10.3390/ agronomy13030770 Academic Editor: Photini V. Mylona Received: 3 February 2023 Accepted: 6 March 2023 Published: 7 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). agronomy Article Improved Forage Quality in Alfalfa (Medicago sativa L.) via Selection for Increased Stem Fiber Digestibility Zhanyou Xu * ,† , Deborah J. Heuschele † , JoAnn F. S. Lamb, Hans-Joachim G. Jung and Deborah A. Samac USDA, Agricultural Research Service, Plant Science Research Unit, 1991 Upper Buford Circle, St. Paul, MN 55108, USA * Correspondence: zhanyou.xu@usda.gov † These authors contributed equally to this work. Abstract: The low digestibility of fiber in alfalfa (Medicago sativa L.) limits dry matter intake and energy availability in ruminant animal production systems. Previously, alfalfa plants were identified for low or high rapid (16 h) and low or high potential (96 h) in vitro neutral detergent fiber digestibility (IVNDFD) of plant stems. Here, two cycles of bidirectional selection for 16 h and 96 h IVNDFD were carried out. The resulting populations were evaluated for total herbage, percentage of stems to total biomass, IVNDFD, neutral detergent fiber (NDF), and acid detergent lignin as a proportion of NDF (ADL/NDF) at three maturity stages. Within these populations, 96 h IVNDFD was highly heritable (h 2 = 0.71), while 16 h IVNDFD had lower heritability (h 2 = 0.46). Selection for high IVNDFD reduced NDF and ADL/NDF in plant stems at the late flowering and green pod maturity stages and reduced seasonal variability in stem digestibility but did not alter the percentage of stems. Stability analyses across 12 harvest environments found that selection for high IVNDFD had little effect on environmental stability of the trait compared to the unselected population. Thus, selection for stem IVNDFD was a highly effective strategy for developing alfalfa populations with improved nutritional quality without changing the percentage of stems to total biomass. Keywords: alfalfa; forage quality; lignin; in vitro neutral detergent fiber digestibility; IVNDFD 1. Introduction In the United States, alfalfa (Medicago sativa L.) is the third largest field crop produced, with an annual estimated value of over USD 10 billion [1]. Primarily used in animal feeds, it is also valued for its diverse environmental services [2] such as carbon sequestration and mitigation of nitrogen leaching from surface water and tile drainage. Alfalfa herbage is an excellent source of nutrients for milk production in dairy cattle and increasing muscle mass in beef cattle [3–5]. Alfalfa herbage is comprised of a protein-rich, highly digestible leaf fraction and a less digestible carbohydrate-rich stem fraction [6]. The stem fraction is higher in indigestible fiber concentration than the leaf fraction, and as the stems mature, the fiber concentra- tion increases within stems while the fiber in leaves remains low and fairly constant [7]. Because stems make up approximately 46 to 60% of the dry matter in alfalfa herbage [8], increasing the digestibility of alfalfa stems would impact the dairy market substantially through increased animal productivity, reduced feed costs, and reduced animal waste [4,9]. Additionally, increasing the available energy of the stem at later maturity stages would also increase dry matter yield [10,11]. Thus, it has been recognized that alfalfa breeding should focus on improving stem digestibility to improve the overall quality of alfalfa forage [12]. In forage quality analysis, plant cell walls (CWs) are typically characterized by the detergent fiber fractions: neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL). Generally, forages that limit feed intake have greater NDF concentrations and more highly lignified CWs [13]. Because alfalfa stems contain more Agronomy 2023, 13, 770. https://doi.org/10.3390/agronomy13030770 https://www.mdpi.com/journal/agronomy