Chitosan (an N-acetyl-d-glucosamine polymer, CHI) is a natural, nontoxic, biodegradable biopolymer (Muzzarelli, 1993) derived from deacetylation of chitin, a major component of the shells of crustaceans and insects. The antimicrobial activity of chitosan has emerged as one of its most interesting properties (Matsuhashi & Kume, 1997), which has led to evaluation of its use in ruminant nutrition. Benefits observed seem to be caused by changes in ruminal fermentation, in particular by increased propionate proportion (Goiri et al., 2010) and decreased methane production (Goiri et al., 2009), which in turn lead to energetically more efficient fermentation patterns. Such shifts in the fermentation pattern could result in increased plasma glucose concentrations (Sauer et al., 1989) and as a consequence in improved productive performance or improved efficiency (McGuffey et al., 2001). The application of CHI as an animal feed additive to modify animal productive performance, however, is little studied to date. The objective of this study was, therefore, to evaluate the effects of CHI supplementation on lactational performance and plasma metabolites in dairy ewes.

The experiment was carried out in accordance with the European Council Directive 86/609/ECC (EC, 1986) for the protection of animals used for experimental and other scientific purposes.

Twenty-four blackfaced multiparous Latxa dairy ewes at early lactation from the Neiker-Tecnalia experiment station (42º51’N, 2º37’W) were blocked according to milk yield (1976 ± 276 mL), days in milk (16 ± 9 d postpartum), and BW (67.5 ± 8.5 kg). Ewes were group housed. Tall fescue (Festuca arundinacea) hay was group fed ad libitum in a feed bunk (0.5 m/ewe), water, and mineralized salt block. The quantity of offered fescue hay was based on morning bunk readings, and the amount of feed offered was adjusted daily to allow 10% refusals.

In each block ewes were randomly assigned to one of two experimental concentrate supplements that contained either 0 (control) or 1.2% CHI (Chitoclear; Deacetylation degree: > 95%; viscosity: < 500 mPa·s; Trades S.A., Barcelona) on a dry matter (DM) basis. Ingredients of both experimental concentrates are shown in Table 1. The experimental concentrates were offered in individual feeders in the milking parlour as two equal meals (420 g DM) during the morning and evening milkings.

The experimental period lasted for 25 d, of which the first 14 d were for treatment adaptation to allow the ruminal microflora to adjust to the supplemental chitosan, and the last 11 d for measurements and samplings.

Quantities of concentrate offered and refused were recorded 4 d/wk on an individual basis throughout the experiment. Individual fescue hay dry matter intake (DMI) was determined using the n-alkanes technique. Paper pellets containing the alkane C32 were prepared following the technique described by Mayes et al. (1986), with slight modifications. Twenty sheets of Albert 135 filter paper, 45 cm wide and 55 cm long, were placed in a stainless steel tray and dried at 105°C for 2 h in a forced-air oven. Meanwhile, 30 g of C32 were measured into a 1000-mL Pyrex screw-top flask and dissolved in 900 mL heptane by heating to 80°C and maintaining this temperature for 15 min. The rest of the process was conducted as described by Mayes et al. (1986). Pellets were made by introducing 1.5 g of shredded paper into an adapted reloading press. Average concentration of C32 in the dosed pellets was 85.2 ± 6.0 g/kg DM. Beginning on d 16 until the end of the experiment, a once-daily dose of paper pellet containing 128 mg of C32 alkane was given to each ewe with a dosing gun during the morning milking. Faecal grab samples were collected twice daily from each in the last 3 d of the experiment. The faecal samples from each ewe were kept at -20ºC until the end of the collection and bulked to a single sample per ewe for alkane analysis.

Weekly experimental concentrate and fescue hay samples were collected and composited by group. Half of the sample was dried at 60°C for 48 h for DM determination, and the other half was kept at -20ºC for chemical composition and alkane analysis.

Ewes were milked daily at 07:30 and 18:00 h, and milk yield was recorded individually four times weekly with an electronic meter (MM25 SG, DeLaval, Madrid). On d 16, 18, 23, and 25, a portion of milk from each ewe was stored with potassium bichromate (0.3 g/L) at 4ºC and analysed (ILL, Lekumberri, Spain) by near-infrared spectroscopy (Foss System 4000, Foss Electric, Hillerød, Denmark) for fat, crude protein, and lactose.

Blood samples (10 mL) were collected 2 h after morning milk feeding on d 19, 23, and 25 via jugular venipuncture into evacuated tubes containing potassium-EDTA. Once collected, blood samples were centrifuged (1,500 × g for 15 min at 4°C), and plasma was recovered and frozen at -20°C for glucose, non-esterified fatty acids (NEFA), and blood urea nitrogen (BUN) determinations.

Fescue hay, concentrates, and faeces were freeze-dried (Christ Alpha 1-4 LD Plus, Fisher Bioblock Scientific, Madrid) and ground to pass a 1-mm screen. Dry matter (method 934.01) and N (method 984.13) contents were determined following AOAC (1999). Nitrogen content was determined using the macro-Kjeldahl procedure on a Kjeltec Auto 1030 (Foss, Hillerød, Denmark). Neutral detergent fiber (NDF) was determined by the method of Van Soest et al. (1991) using an alpha amylase, but without sodium sulphite in the neutral detergent solution, and was expressed free of ash. Acid detergent fiber, expressed exclusive of residual ash, was determined by the method of Robertson & Van Soest (1981). Fat content was determined without hydrolysis by the automated soxhlet method (Selecta S.A., Barcelona) using hexane for 6 h as solvent. Ewes were weighed and body condition score (BCS) was determined on day 1 and 25.

The extraction of n-alkanes was carried out following the technique described by Mayes et al. (1986), with the modifications suggested by Vulich et al. (1995) and Olivan & Osoro (1999).

Plasma samples were analysed for glucose (glucose oxidase/peroxidase method; Karkalas, 1985), NEFA (NEFA-C kit; Wako Chemical, Richmond, VA, USA; Johnson & Peters, 1993), and BUN (liquid urea nitrogen reagent set, Pointe Scientific Inc., Canton, MI, USA).

Day milk production was adjusted to 6.5% fat-corrected milk (FCM) based on the following equation developed by Pulina & Nudda (2002):