**2. Material and methods**

**1. Introduction**

108 Forage Groups

enzymes [5].

silage fermentation [4].

which will be producing CO<sup>2</sup>

decay of the silage [7].

responding silages.

rapid decline of pH within the ensiled forage [2].

Alfalfa (*Medicago sativa* L.) grasslands are dominant in irrigated temperate Mexico; however, their yield in the dry autumn-winter season is 50% lower than during the rainy springsummer [1]. Conservation of forage surpluses is therefore necessary; but, due to summer rains, haymaking is risky and hence silage making becomes the best option. Nonetheless, ensiling alfalfa forage is difficult because of its low content of water-soluble carbohydrates and high buffering capacity, which delay the lactic acid production and therefore hinder a

Technical alternatives for ensiling alfalfa forage are to harvest in afternoon hours [3], wilting [4] and the use of lactic acid bacteria (LAB) homo- and heterofermentative inoculants with

Cutting in the afternoon and wilting increased dry matter (DM) and alcohol soluble carbohydrates (ASC) concentration in alfalfa, which led to silage with improved conservation attributes such as lower pH, greater concentrations of lactate, lower concentrations of volatile fatty acids and NH3–N [3]. However, the effect of wilting largely depends on weather conditions, since events of rain during wilting, together with poor drying conditions (low potential evapotranspiration) may lead to considerable rise in pH and losses of sugars, causing worse

The use of inoculants consisting of homofermentative bacteria hastened the drop of pH during the fermentation but did not improve the aerobic stability of the silage [6]. Such result is consistent with [7], who reported that inoculants based on homofermentative lactic acid bacteria did not improve aerobic stability in about two thirds of the cases; on the contrary, inoculant based on the heterolactic acid bacterium *Lactobacillus buchneri* increases the aerobic

Aerobic stability is defined as the length of time that silage remains cool and does not spoil after it is exposed to air [7]. Since silage making relies on keeping anaerobic conditions, once the silo is open in the feeding phase and hence the silage is exposed to oxygen, it becomes liable to oxidation by the coming into action of dormant aerobic bacteria, yeasts and molds,

The fermentation and storage phases are dominated by anaerobic processes; as a result, silage contains yeasts, molds and some aerobic bacteria which are dormant under these anaerobic conditions. Introduction of oxygen, by deterioration of the sealing or opening the silo for the feeding phase, activates these aerobic microorganisms whose respiration consumes valuable nutrients, producing carbon dioxide and water with loss of dry matter and nutrients and

Based on the above stated, this study was aimed at assessing during the rainy season, in three different months (different short-term weather conditions) the effect of hour at harvest, wilting and use of a mixed inoculant on quality properties of freshly cut forage from a mixed alfalfa and orchard grass (*Dactylis glomerata* L.) grassland dominated by alfalfa, and the cor-

[8], leading to rises in temperature and pH [7].

stability of silages mainly due to a rise in the concentration of acetic acid.

The field work was carried out in three silage phases between June and September 2011 at Chapingo University, State of Mexico, Mexico, 19° 29 'N, 98° 54 'W and 2240 meters above sea level, under temperate sub-humid climate with summer rains.

Forage from a 2-year old alfalfa-orchard grass mixed grassland (0.37ha) was used, harvested after 42 days of regrowth, yielding on average 2480 kg DM/ha per harvest. Such mixed grasslands are regularly used within an irrigated grassland and forage crops rotation for dairy production under grazing; during the first 2 years of these grasslands, alfalfa is dominant (more than 70% of dry matter of harvested herbage). As silos, plastic containers with a capacity of 200 L, 52 cm in diameter and 95 cm in height were used, with lid and strap for air tight sealing.

The experiment comprised three phases (phase 1: June–July, phase 2: August, phase 3: September) in which 12 treatments were evaluated with two replicates in each phase. The treatments resulted from the combination of 2 × 2 × 3 complete factorial arrangement of two cutting schedules (08:00 and 14:00 h), two levels of LAB inoculant (0 and 5 g/t forage) and three wilting times (0, 1 and 2 h). During each day of operation, two containers were filled (one with forage cut at 08:00 h and the other with forage cut at 14:00 h), following a random order.

Forage was cut with a scythe, according to treatments it was wilted on the field, thereafter gathered and carried 300 m to the ensiling facility where it was cut into 3 cm (on average) particles using a Mapusa ® (Pudong, Shanghai, China) mincer. During mincing the forage was covered with a polyethylene film to avoid dehydration and contamination.

The inoculant used was BIOTAL PLUS II® (Lallemand Animal Nutrition, Milwaukee, Wisconsin) that contained viable cells of the LAB *Pediococcus pentosaceus* 12,455 (homofermentative) and *Propionibacterium freudenreichii* R2453 (heterofermentative) and specific enzymes, which were expected to enhance fiber hydrolysis during ensiling [5]. The inoculant was applied following instructions; hence, it was expected to supply 100,000 CFU/gram of forage.

For the compaction of the forage a structure was designed consisting of two vertical concrete cylinders of 0.25 m in diameter and 2.8 m in height, separated 1.15 m and joined at the top by a steel crossbar of 7.62 cm diameter and 1.25 m long. A hook with a pulley was placed on the crossbar, which was used to compact forage, operating with a rope to vertically move a cement piston with a diameter of 49 cm, height 25 cm and weight of 53 kg. The controlled displacement of the piston inside the containers was stabilized by placing them inside a metal ring 40 cm high that had hinges to open it when entering or removing each container.

The forage to be ensiled was placed within the containers in layers of 5 kg extended with pitchfork and the inoculant was applied with atomizer; two operators used the rope and the pulley to raise and drop the piston 60 times on each forage layer; when the section to be filled with the container was of less height than the piston, a metal sheet tube 50 cm high and 50 cm in diameter was used as a guide for the falling piston. Once each container was filled, the lid was placed and sealed with adhesive tape and secured with the strap. The containers were weighed on a Trutest® (Auckland, New Zealand) scale to verify that density was within target ranges (resulting in 608 ± 31 kg m−<sup>3</sup> ) and thereafter placed outdoors for 60 days, after which they were opened for sampling.

#### **2.1. Measured variables**

With a Davis Instruments Vantage Pro2® (Hayward, California) meteorological station, temperature, rainfall and humidity data were recorded at the time of forage harvesting, wilting and ensiling; these data were used to unravel the effect of weather variables on silage properties. There were no clear differences among phases with on average 0.14 mm rainfall in the morning hours and much less in the afternoon hours (0.04 mm); on the contrary, evapotranspiration was low in the morning hours (on average 0.21mm) and higher in the afternoon hours (on average 2.54 mm). The containers were opened 60 days after ensiling, a silage top layer of 30 cm was removed, the temperature was measured, and samples were taken for determinations of pH, DM content, aerobic deterioration and chemical composition.

The CP content of the silage was estimated by the Kjeldahl method [12], while to estimate its

Ensiling Alfalfa (*Medicago sativa* L.) and Orchard Grass (*Dactylis glomerata* L.) Forage Harvested…

The ruminal fermentation of silage samples was estimated using the gas production technique [14] with three replicates per sample. The gas pressure generated by the fermentation

key and hypodermic needle, the measurements were made with intervals of one-hour in the first 24 h and then every 4 h, after each reading the pressure was made equal to zero. The total gas production was estimated and once the incubation period was over, the residue was filtered and dried, which was considered as the residual DM. The in vitro disappearance of

 was quantified by ultra violet light chromatography with a visible ultraviolet light spectrophotometer (UV/VIS Lambda 35, Perkin Elmer, Waltham, Massachusetts) at 630 nm [15]. Data were analyzed using a generalized linear model (GLM procedure 16 SAS 9.0®) which corresponded to the complete factorial arrangement and hence with effects of cutting schedules, LAB inoculant, wilting times, double interactions, phases and replicates within phases, the Tukey procedure (α = 0.05) was used for comparisons of LS-means [16]. Additionally, a Principal Component Analysis was carried out with Microsoft Excel® using XLSTAT® (Addinsoft, New York, NY).

The average pH of the fresh forage was 6.9 ± 0.3 and its buffering capacity was equivalent

The DM content of forage wilted for 2 h did not differ (p < 0.05) from that of the forage wilted for 1 h. The forage cut at 14:00. had a higher DM content than that cut at 08:00 h in Phase 1 (p = 0.0029) and in Phase 2 (p = 0.0065), but not in Phase 3 (p = 0.3348) (**Table 1**). No differences (p > 0.05) were found in DM content between wilting 1 or 2 h. The post-wilting DM content was not affected by wilting in phase one; but, in phases two and three the forage after 1 or 2 h of wilting, cut at 2:00 PM had a higher DM content than that cut at 08:00 h under the same

Hour of cutting and wilting time did not affect (p > 0.05) CP content in the three experimental phases; contents were on average 18.5 ± 1.2, 16.7 ± 0.7 y 16.0 ± 0.6. In phase one the content of ASC was not affected (p > 0.05) by hour of cutting or wilting time, while in phases two and

harvesting hours, wilting times and DM content of the forage at the time of ensiling.

**3.2. Contents of crude protein, neutral detergent fiber and alcohol-soluble** 

three it was higher (p < 0.05) in forage harvested at 14:00 (**Table 2**).

**3.1. Dry matter content of forage at cutting and post-wilting**

lactic acid, with no effect of the combinations of experimental phases,


DM (DM*iv*D) was calculated as the difference between initial DM and residual DM.

, equipped with a three-phase

http://dx.doi.org/10.5772/intechopen.81509

111

NDF content, the Van Soest method described by 13 Sosa [13] was used.

was measured with a manometer with a scale of 0–1 kg cm−<sup>2</sup>

For the estimation of the NH3

NH3

**3. Results**

to 339 ± 50 meq 10−<sup>3</sup>

wilting time (p < 0.05) (**Table 1**).

**carbohydrates in forage to be ensiled**

#### *2.1.1. Morphological characterization and quality indicators of the forage before ensiling*

In the forage to be ensiled samples were taken to measure botanical composition, and DM content, temperature, pH, ASC (alcohol soluble carbohydrates), and buffering capacity.

Botanical and morphological composition of forage was estimated by means of hand separation. The DM content was estimated by drying at 65°C to constant weight, this variable was estimated in freshly cut forage or after wilting according to treatments, and additionally at the beginning, half and end of the process of filling the containers.

Temperature and pH measurements were made with a portable Orion 3-Star® meter (Thermo Fisher Scientific Inc., Chelmsford, Massachusetts). The temperature was measured at the beginning, middle and end of the container filling process. For the pH measurement the samples of approximately 50 g of fresh forage were taken in the field, they were frozen and subsequently the pH was measured in the laboratory. The determination of ASC was carried out in 40–50 mg of sample previously ground using the method of Dubois et al. [9]. The buffer capacity of the fresh forage was determined in 2.5 g fresh samples according to the method described by Jasaitis et al. [10].

#### *2.1.2. Variables measured in the silage*

The measurements made in the silage comprised the following variables: (i) temperature, pH and ASC content, such as in fresh forage, (ii) aerobic deterioration, (iii) crude protein content (CP) and neutral detergent fiber (NDF), (iv) rate of ruminal fermentation and *in vitro* disappearance of DM, and (v) NH3-N content.

At the opening of each silo, the temperature of the silage was measured at five points of the surface layer, and five points at a depth of 30 cm; 100 g samples were taken, which were refrigerated for pH measurement in the laboratory 4 h later. Likewise, samples were taken to which the ASC content was determined with the same procedure used in fresh forage samples.

The aerobic deterioration of the silage was estimated with measurements of temperature and CO2 production. Three 300 g silage samples were placed in 1 L transparent glass jars. Two of those jars were used to measure CO<sup>2</sup> production following Crossno et al. [11]; the third one remained 5d uncovered and was used to measure the temperature at 08:00, 14:00 and 20:00 h.

The CP content of the silage was estimated by the Kjeldahl method [12], while to estimate its NDF content, the Van Soest method described by 13 Sosa [13] was used.

The ruminal fermentation of silage samples was estimated using the gas production technique [14] with three replicates per sample. The gas pressure generated by the fermentation was measured with a manometer with a scale of 0–1 kg cm−<sup>2</sup> , equipped with a three-phase key and hypodermic needle, the measurements were made with intervals of one-hour in the first 24 h and then every 4 h, after each reading the pressure was made equal to zero. The total gas production was estimated and once the incubation period was over, the residue was filtered and dried, which was considered as the residual DM. The in vitro disappearance of DM (DM*iv*D) was calculated as the difference between initial DM and residual DM.

For the estimation of the NH3 -N content, metaphosphoric acid was added to the samples and NH3 was quantified by ultra violet light chromatography with a visible ultraviolet light spectrophotometer (UV/VIS Lambda 35, Perkin Elmer, Waltham, Massachusetts) at 630 nm [15].

Data were analyzed using a generalized linear model (GLM procedure 16 SAS 9.0®) which corresponded to the complete factorial arrangement and hence with effects of cutting schedules, LAB inoculant, wilting times, double interactions, phases and replicates within phases, the Tukey procedure (α = 0.05) was used for comparisons of LS-means [16]. Additionally, a Principal Component Analysis was carried out with Microsoft Excel® using XLSTAT® (Addinsoft, New York, NY).
