The objective of this study was to compare how maturity affects leaf:stem ratio (L:S), fiber composition [NDF, indigestible NDF (iNDF), acid detergent lignin (ADL) and in situ total-tract NDF digestibility (ISTTNDFD)] of 4 alfalfa varieties. Two conventional alfalfa varieties developed for high digestibility (C1, C2), a transgenic reduced lignin (RL) variety (GMO) and a RL alfalfa developed by conventional breeding (CB) were compared. First cutting alfalfa at the University of Wisconsin Arlington research station (43°18′9.47″ N, 89°20′43.32″ W) were sampled twice weekly from early vegetative to full flower from May 4 to June 19, 2017. Samples were harvested 7 cm above soil level by hand. Each of the 4 fields (each approximately 8ha) were split into 3 units for replication. Samples were immediately dried at 60°C for 48 h. Leaves were manually stripped from dried samples, and L:S determined. Stems were ground to 1 mm by Wiley mill and weighed (0.5 g) into Ankom F-57 bags. Duplicates were placed into rumens of 2 lactating dairy cows for 24, 30, 48, and 240 h (iNDF). Calculation of ISTTNDFD was according to Lopes et al. (2015). Stem ADL analysis were in triplicate with Ankom bags that contained ADF residue soaked in 72% H₂SO₄ for 3 h. Data were analyzed by split plot design in PROC MIXED in SAS, with variety, day (D), and their interaction as fixed effects. Field section was a random effect and D a repeated measure. Both RL varieties had higher L:S than conventional. The GMO stems had a lower ADL than the other stems. The RL stems had higher ISTTNDFD compared with C2 stems. Table 1. Leaf:stem ratio and fiber analysis of stems of 2 conventional alfalfa varieties (C1, C2) and 2 reduced lignin alfalfas (GMO and CB)

Variable	Alfalfa variety				SEM	P - value
C1	C2	GMO	CB	Variety	D	Variety×D
L:S ratio, DM	0.57b	0.59b	0.64a	0.65a	0.01	<0.01	<0.01	<0.01
Stem composition
ADL, % of DM	7.61b	7.95a	6.74c	7.42b	0.08	<0.01	<0.01	<0.01
NDF, % of DM	46.0	48.4	45.7	45.8	0.8	0.06	<0.01	0.24
iNDF, % of NDF	45.6ab	48.8a	41.3b	45.2ab	1.6	<0.04	<0.01	0.12
ISTTNDFD, % of NDF	39.5ab	37.3b	41.1a	41.5a	1.0	<0.01	<0.01	0.04

^a–cMeans within a row with different superscripts differ (P < 0.05).