Experimental forage

An irrigated and well-established 4 ha lucerne plot (cvar Aragón) in the second year of growth, at La Alfranca farm, Zaragoza, Spain (41°40′N, 0°53′E; 199 m asl) was cut six times a year according to traditional lucerne management in the area (Fanlo et al. Reference Fanlo, Chocarro, Lloveras, Ferran, Serra, Salvia, Muñoz, Andueza, Delgado, Lloveras, Gonzalez-Rodriguez, Vazquez-Yañez, Piñeiro, Santamaria, Olea and Poblaciones2006). The growth periods used were the second (May), fourth (July) and sixth (September) cuts, chosen to represent a wide range of climatic and daylight conditions. For all growth cycles, the plot was cut at mid-bloom (0·50 of plants flowering). Cut dates, climate data and proportions of leaves (proportion of dry weight) are given in Table 1. Proportion of leaves was estimated from a near infrared spectroscopy (NIRS) calibration equation based on 109 samples (standard error of validation=2·7; R ²=0·91) and expressed as a coefficient (Andueza et al. Reference Andueza, Delgado and Muñoz2009).

Table 1.

Hay growth conditions, proportion of leaves and climate conditions of forages cut at sunrise (AM) or at sunset (PM). Accumulated temperature (HDD) was calculated as the sum of positive average daily temperatures from the cutting date

* Av. temp.: average temperature of the growth cycle.

Paired cuts per growth period were taken at AM and at PM, giving six experimental forages. On the date of cutting, half of the plot was cut before 08:00 h using a mower conditioner leaving stubble of 50 mm, and the other half-plot was cut later the same day, after 20:00 h. The forages were sun-dried in natural conditions until reaching a minimum of 800 g dry matter (DM)/kg of fresh forage. Each pair of forages was baled the same day, and two bales (400 kg) per treatment were immediately transported to CITA Research Centre in Zaragoza (Spain) (41°44′N, 0°48′E) and stored for animal utilization.

Animals and experimental procedures

The study was conducted indoors at the CITA farm. All animals were handled by specialist personnel trained in animal care and welfare according to the European Union Directive No. 609/1986 under agreement No. A63 565.

Rumen degradation of DM and N

Ruminal degradation characteristics were determined using three 4-year-old wethers (Fleichchaff×Rasa Aragonesa), averaging 62 kg live weight, fitted with a rumen cannula and adapted for 30 days to a diet composed of 0·70 lucerne hay and 0·30 concentrate (barley plus mineral–vitamin complement). The diet was divided into two equal meals given at 08:00 h and 16:00 h.

Thermally sealed nylon bags of 70×110 mm inside capacity and 46 μm pore size were used. Approximately 3 g (air-dry basis) of forage samples were placed in the bags. Forage samples were formed by thoroughly mixing subsamples from the six periods of digestibility phase. DM and N degradability were determined using the in situ technique (Ørskov & McDonald Reference Ørskov and McDonald1979). Bags containing forages chopped to a length of 20 mm were incubated in the animal's rumen, with two replicates per wether and per incubation time. The bags were left in the rumen for 2, 4, 8, 16, 24 and 48 h before being washed, oven-dried at 60°C and stored at –20°C until analysis. Before analysis, the bags were thawed and washed in a washing machine (3×10 min) with cold water until the rinse water ran clear. Duplicate samples of a common lucerne hay used as a ‘standard’ were incubated for 8 h in the rumen of each sheep to detect any changes in the level of rumen degradation activity during the experiment. DM and N disappearance data obtained for each animal was fitted to the equation proposed by Ørskov & McDonald (Reference Ørskov and McDonald1979). The model is described by

where A is the soluble fraction, B is the insoluble potentially degradable fraction, A+B is the degradable fraction and C is the fractional degradation rate. The values for rumen degradation kinetics were fitted to this equation using the NLIN procedure of the SAS statistical package (SAS 1998). Effective degradability of the DM (EDDM) and N (EDN) in each animal was calculated using the equation:

where ED represents effective degradability and k is the rumen outflow rate. ED was calculated for a k of 0·06/h (Vérité & Peyraud Reference Vérité, Peyraud and Jarrige1989).

Digestibility and voluntary intake

Twelve 4-year-old wether sheep (mean live weight: 45 kg) of Rasa Aragonesa breed were used in a 6×6 Latin square design. The digestibility phase was carried out in metabolism cages for 15 days, during which the first 10 days were devoted to adaptation to the diet and the last 5 days to data collection (daily offering and daily total faeces excretion). The diet offered consisted of forage chopped into 50–70 mm lengths and offered at maintenance level (40 g DM/kg BW0·75) twice a day, at 08:00 h and 16:00 h. After weighing, the faeces of each wether were subsampled (0·20 of each animal's daily output); subsamples from 5 days were then combined to form one sample per animal and per period. Finally, a faeces sample was taken (according to the weight excreted by each wether) from each of the six animals for laboratory analyses.

After determination of digestibility, the voluntary intake of hays was determined using sheep wethers in individual pens over 23 days, during which the first 15 days were devoted to adaptation to the diet and the last 8 days were used to measure the quantities offered and refused by the animals. The animals received hay chopped into 50–70 mm lengths and offered ad libitum, allowing a maximum refusal of 0·10. The forage was offered twice a day, at 08:00 h and 16:00 h.

Forage offered and refusals were sampled daily for analysis, giving one forage sample for both the digestibility phase and intake phase. During the experimental period, animals had free access to water and vitamin–mineral blocks (manufacturer-specified composition: 2 g Mg/kg, 3 g Fe/kg, 0·81 g Zn/kg, 0·05 g I/kg, 0·018 g Co/kg, 0·01 g Se/kg, 985 g NaCl/kg and 9 g moisture/kg).

Before starting the experiment, animals were fed a common lucerne hay (chemical composition: 98 g ash/kg; crude protein (CP), 190 g/kg; neutral detergent fibre (NDF), 410 g/kg; acid detergent fibre (ADF), 235 g/kg and acid detergent lignin (ADL), 52 g/kg) for a 14-day period and then randomly assigned to one of the experimental forages (two animals per experimental forage).

Samples and laboratory analysis

All samples were oven-dried at 60°C for 48 h and ground in a Fritsch pulverisette mill through a 1 mm screen.

Calculations

Dry matter digestibility (DMD), OMD, neutral detergent fibre digestibility (NDFD), acid detergent fibre digestibility (ADFD) and digestible organic matter intake (DOMI) were calculated from in vivo results and chemical composition data. DMI and DOMI were expressed in g/kg BW0·75.

Statistical analysis

Data on the chemical composition of the forages and the rumen degradation trial were analysed as repeated measurements, with diurnal cut and growth period as repeated measures. Fixed effects included growth period and diurnal cut. Animal in the rumen degradation trial and replicate in the chemical composition data were included as random effects. Data for digestibility measures, DMI and DOMI were analysed as a Latin square. The statistical model included animal, period, forage and residual error. Fixed effects included period and forage, with animal as the random effect. Orthogonal contrasts were established to compare the differences between AM and PM hay in each growth period. All analyses were performed using the MIXED procedure of the SAS statistical package (SAS 1998) Significance was taken as P<0·05.