**3. Results**

**2.1. Measured variables**

110 Forage Groups

described by Jasaitis et al. [10].

*2.1.2. Variables measured in the silage*

pearance of DM, and (v) NH3-N content.

those jars were used to measure CO<sup>2</sup>

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.

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

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

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* disap-

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

remained 5d uncovered and was used to measure the temperature at 08:00, 14:00 and 20:00 h.

production following Crossno et al. [11]; the third one

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

beginning, half and end of the process of filling the containers.

The average pH of the fresh forage was 6.9 ± 0.3 and its buffering capacity was equivalent to 339 ± 50 meq 10−<sup>3</sup> lactic acid, with no effect of the combinations of experimental phases, harvesting hours, wilting times and DM content of the forage at the time of ensiling.

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

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

#### **3.2. Contents of crude protein, neutral detergent fiber and alcohol-soluble carbohydrates in forage to be ensiled**

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


**Table 1.** Dry matter content (DM) of alfalfa and orchard grass herbage under two harvest hours, freshly cut and after one or 2 h of wilting during three experimental phases (LS-means).

> freshly cut forage and the reduction in content due to ensiling (**Figure 1**). There were significant differences between the treatments in phase two because of the wilting time factor, where the treatments without wilting maintained an ASC of 1.2%, with one-hour of wilting was 0.8%

> **Table 3.** Means pH and ammoniacal nitrogen of silages alfalfa and orchard grass cut at two different hours, with or

**pH N-NH<sup>3</sup>**

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

Hour at cutting Inoculant Phase 1 Phase 2 Phase 3 Phase 1 Phase 2 Phase 3 8:00 0 4.7b 4.9<sup>a</sup> 4.8<sup>a</sup> 11<sup>a</sup> 16<sup>a</sup> 15b 8:00 1 4.8<sup>a</sup> 4.9<sup>a</sup> 4.9<sup>a</sup> 11<sup>a</sup> 14<sup>a</sup> 18<sup>a</sup> 14:00 0 4.8<sup>a</sup> 4.6b 4.6b 11<sup>a</sup> 14<sup>a</sup> 13c 14:00 1 4.4c 4.6b 4.7b 9<sup>a</sup> 14<sup>a</sup> 12c

 **(% of total N)**

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The CP content of silages was not affected (p > 0.05) by the treatments, it was on average 18% in phase one, 15% in phase two and 16% in phase three. The differences in CP of silage with respect to freshly cut forage were small, in phase one the protein of the silage was 0.5% lower,

in phase two the decrease was 1.7% while in phase three contents were similar.

**Figure 1.** Relationship between the content of alcohol-soluble carbohydrates in the original forage and its

and with 2 h of wilting 1.1%.

corresponding silage.

**3.5. Nutritional composition of silages**

abc Means that do not share any literal within columns are different (p ≤ 0.05).

without addition of bacterial inoculant, in three distinct experimental phases.


**Table 2.** LS-means and standard error (SE) of alcohol soluble carbohydrates (ASC) and NDF alfalfa and orchard grass forage before ensiling.

#### **3.3. Temperature, pH and ammoniacal nitrogen of silages**

The average temperature at the opening of the silos was 21 ± 0.6°C in phase one, 20 ± 0.7°C in phase two and 18 ± 1.1°C in phase three, not affected by treatments. In phase one, the silage pH of forage harvested at 14:00 and with inoculant was lower (p < 0.05) than that of forage harvested at the same time but without inoculant and those of forage harvested at 08:00 (**Table 3**). The NH3-N content was not affected (p > 0.05) by treatments in phases one and two. In phase three the NH3-N content of forage harvested at 14:00 and with inoculant was lower (p < 0.05) than that of forage harvested at the same time but without inoculant and those of forage harvested at 08:00 (**Table 3**).

#### **3.4. Alcohol-soluble carbohydrates in silages**

The ASC in freshly cut forage was reduced by ensiling and hence was lower in the correspondent silages, in such a way that there was a linear relationship (p < 0.01) between ASC in


**Table 3.** Means pH and ammoniacal nitrogen of silages alfalfa and orchard grass cut at two different hours, with or without addition of bacterial inoculant, in three distinct experimental phases.

freshly cut forage and the reduction in content due to ensiling (**Figure 1**). There were significant differences between the treatments in phase two because of the wilting time factor, where the treatments without wilting maintained an ASC of 1.2%, with one-hour of wilting was 0.8% and with 2 h of wilting 1.1%.

#### **3.5. Nutritional composition of silages**

**3.3. Temperature, pH and ammoniacal nitrogen of silages**

abcd Means that do not share any literal within columns are different (p ≤ 0.05).

Hour at cutting Freshly cut forage DM(%)

112 Forage Groups

or 2 h of wilting during three experimental phases (LS-means).

forage harvested at 08:00 (**Table 3**).

forage before ensiling.

**3.4. Alcohol-soluble carbohydrates in silages**

The average temperature at the opening of the silos was 21 ± 0.6°C in phase one, 20 ± 0.7°C in phase two and 18 ± 1.1°C in phase three, not affected by treatments. In phase one, the silage pH of forage harvested at 14:00 and with inoculant was lower (p < 0.05) than that of forage harvested at the same time but without inoculant and those of forage harvested at 08:00 (**Table 3**). The NH3-N content was not affected (p > 0.05) by treatments in phases one and two. In phase three the NH3-N content of forage harvested at 14:00 and with inoculant was lower (p < 0.05) than that of forage harvested at the same time but without inoculant and those of

**Table 2.** LS-means and standard error (SE) of alcohol soluble carbohydrates (ASC) and NDF alfalfa and orchard grass

**ASC (%) NDF (%)**

**Phase 1 Phase 2 Phase 3**

Hour at cutting Wilting time (h) Phase 1 Phase 2 Phase 3 Phase 1 Phase 2 Phase 3 8:00 0 3.8<sup>a</sup> 3.4b 4.3b 57<sup>d</sup> 75<sup>a</sup> 69b 8:00 1 3.5<sup>a</sup> 3.1b 4.0b 63b 75<sup>a</sup> 73<sup>a</sup> 8:00 2 3.3<sup>a</sup> 3.3b 4.3b 68<sup>a</sup> 75<sup>a</sup> 66c 14:00 0 4.3<sup>a</sup> 3.9<sup>a</sup> 4.7<sup>a</sup> 60c 73b 67bc 14:00 1 5.1<sup>a</sup> 4.5<sup>a</sup> 4.5<sup>a</sup> 61bc 71c 67bc 14:00 2 3.8<sup>a</sup> 4.2<sup>a</sup> 4.9<sup>a</sup> 57<sup>d</sup> 71c 63<sup>d</sup> SE of means 0.8 0.2 0.2 2.6 1.9 2.1

**Table 1.** Dry matter content (DM) of alfalfa and orchard grass herbage under two harvest hours, freshly cut and after one

8:00 23 ± 1.1b 17 ± 0.7b 20 ± 1.1<sup>a</sup> 14:00 29 ± 1.1<sup>a</sup> 21 ± 0.7<sup>a</sup> 22 ± 1.1<sup>a</sup>

8:00 27 ± 2ª 20 ± 2b 19 ± 1b 14:00 27 ± 2ª 24 ± 2ª 26 ± 1ª ab Means with different literal within columns (of freshly cut or after wilting) are different (p ≤ 0.05).

Post-wilting DM (%)

The ASC in freshly cut forage was reduced by ensiling and hence was lower in the correspondent silages, in such a way that there was a linear relationship (p < 0.01) between ASC in The CP content of silages was not affected (p > 0.05) by the treatments, it was on average 18% in phase one, 15% in phase two and 16% in phase three. The differences in CP of silage with respect to freshly cut forage were small, in phase one the protein of the silage was 0.5% lower, in phase two the decrease was 1.7% while in phase three contents were similar.

**Figure 1.** Relationship between the content of alcohol-soluble carbohydrates in the original forage and its corresponding silage.

In all three phases the NDF content decreased 10% in silage with respect to that of freshly cut forage. In phases one and three the NDF content in the silage was not affected by the treatments (p > 0.05). In phase two the hour of cutting x wilting time interaction led to differences (p < 0.05); while silage from forage harvested at 8:00 with one-hour wilting and at 14:00 with 2 h wilting had the highest contents, those from forage harvested at 8:00 with 2 h wilting and at 14:00 with one-hour wilting had the lowest contents (**Table 4**).

#### **3.6. Fermentation and ruminal in vitro disappearance of DM**

The volume of gas produced by in vitro silage fermentation was not affected by the treatments in phase one (p > 0.05); however, in phase two the volume of gas produced by the fermentation of silage from forage harvested at 08:00 h was lower (p < 0.05) than the volume reached with forage harvested at 14:00 (**Table 5**). In phase three, the gas volumes were affected (p < 0.05) by the interaction of wilting time x inoculant; treatments without wilting and with 2 h of wilting with inoculant application reached higher gas volumes than treatments without inoculant. In the case of treatments with one-hour of wilting, the upper value of gas volume was achieved in treatments without inoculant. The *in vitro* disappearance of DM did not show significant differences between treatments of phases one and two; on the other hand, in phase three the percentage of disappearance was lower (p < 0.05) in silages from forage harvested at 08:00 h (**Table 5**).

rational interpretation was feasible. In phases two and three no effect (P > 0.05) of treatments on this variable was detected. Differences among means of phases one, two and three (0.17,

**Table 5.** Means of volume of gas produced by fermentation and ruminal in vitro disappearance of DM of silage from

**Hour at cutting Phase 1 Phase 2 Phase 3**

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

08:00 218<sup>a</sup> 193b 227a 14:00 223<sup>a</sup> 209<sup>a</sup> 238<sup>a</sup>

Ruminal *in vitro* disappearance of DM (%) 08:00 60<sup>a</sup> 49<sup>a</sup> 50b 14:00 58<sup>a</sup> 50<sup>a</sup> 54<sup>a</sup>

alfalfa and orchard forage harvested at contrasting times of the day in three experimental phases.

Gas volume (ml g−<sup>1</sup> DM)

ab Means that do not share any literal within columns are different (p ≤ 0.05).

interaction Hour at cutting x Inoculant (**Table 6**) in the three phases implied that inoculation was not effective to control pH rise in silage from forage cut at 08:00 but it was on silage from forage cut at 14:00 (p < 0.05). The interaction Inoculant x wilting time (**Table 6**) in phase one

Hour at cutting Inoculant Phase 1 Phase 2 Phase 3 08:00 0 0.18<sup>d</sup> 0.86<sup>a</sup> 0.18<sup>a</sup> 08:00 1 0.63<sup>a</sup> 0.95<sup>a</sup> 0.42<sup>a</sup> 14:00 0 0.37b 0.58b −0.002b 14:00 1 0.25c 0.32b 0.15<sup>a</sup> Inoculant Wilting time Phase 1 Phase 2 Phase 3 0 0 0.33<sup>d</sup> 0.91<sup>a</sup> 0.12<sup>a</sup> 0 1 0.10<sup>e</sup> 0.77<sup>a</sup> 0.05<sup>a</sup> 0 2 0.40c 0.49<sup>a</sup> 0.09<sup>a</sup> 1 0 0.04<sup>f</sup> 0.72<sup>a</sup> 0.30<sup>a</sup> 1 1 0.84<sup>a</sup> 0.65<sup>a</sup> 0.57<sup>a</sup> 1 2 0.45b 0.52<sup>a</sup> 0.00<sup>a</sup> abcde Means that do not share any literal within columns of each interaction are different (p ≤ 0.05).

**Table 6.** Means of rate of change of pH during aerobic deterioration of alfalfa and orchard grass silages.

**Rate of change of pH units d−<sup>1</sup>**

production rate, no effects of treatments were detected in any

in phases one, two and three, respectively. In the rate of change of pH, the

production were 9, 9.4 and 19.8 milli moles of

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−0.27, 0.03°C d−<sup>1</sup>

CO<sup>2</sup> g−<sup>1</sup>

Concerning the variable CO<sup>2</sup>

DM d−<sup>1</sup>

) were also odd.

of the three phases(P > 0.05); the rates of CO<sup>2</sup>

#### **3.7. Aerobic stability of silages**

The measurement of the rate of change of temperature did not produce clear results. In phase one the interactions hour at cutting x inoculant (P < 0.05) on the rate of change of temperature resulted in silage from forage harvested at 8:00 with one-hour wilting and at 14:00 with 2 h wilting the highest rates were found, while in silages from forage harvested at 8:00 with 2 h wilting and at 14:00 with one-hour wilting the lowest rates were detected. In the same first phase the results of the wilting time x inoculant interaction were of such a nature that no


**Table 4.** Means of NDF content of silage from alfalfa and orchard grass forage harvested at two distinct times of the day and subjected to different times of wilting during three experimental phases.


In all three phases the NDF content decreased 10% in silage with respect to that of freshly cut forage. In phases one and three the NDF content in the silage was not affected by the treatments (p > 0.05). In phase two the hour of cutting x wilting time interaction led to differences (p < 0.05); while silage from forage harvested at 8:00 with one-hour wilting and at 14:00 with 2 h wilting had the highest contents, those from forage harvested at 8:00 with 2 h wilting and

The volume of gas produced by in vitro silage fermentation was not affected by the treatments in phase one (p > 0.05); however, in phase two the volume of gas produced by the fermentation of silage from forage harvested at 08:00 h was lower (p < 0.05) than the volume reached with forage harvested at 14:00 (**Table 5**). In phase three, the gas volumes were affected (p < 0.05) by the interaction of wilting time x inoculant; treatments without wilting and with 2 h of wilting with inoculant application reached higher gas volumes than treatments without inoculant. In the case of treatments with one-hour of wilting, the upper value of gas volume was achieved in treatments without inoculant. The *in vitro* disappearance of DM did not show significant differences between treatments of phases one and two; on the other hand, in phase three the percentage of disappearance was lower (p < 0.05) in silages from forage harvested

The measurement of the rate of change of temperature did not produce clear results. In phase one the interactions hour at cutting x inoculant (P < 0.05) on the rate of change of temperature resulted in silage from forage harvested at 8:00 with one-hour wilting and at 14:00 with 2 h wilting the highest rates were found, while in silages from forage harvested at 8:00 with 2 h wilting and at 14:00 with one-hour wilting the lowest rates were detected. In the same first phase the results of the wilting time x inoculant interaction were of such a nature that no

**Phase 1 Phase 2 Phase 3**

at 14:00 with one-hour wilting had the lowest contents (**Table 4**).

**3.6. Fermentation and ruminal in vitro disappearance of DM**

**Hour at cutting Wilting (h) NDF (% of DM)**

abcde Means that do not share any literal within columns are different (p ≤ 0.05).

and subjected to different times of wilting during three experimental phases.

8:00 0 47<sup>a</sup> 59b 63<sup>a</sup> 8:00 1 48<sup>a</sup> 61<sup>a</sup> 64<sup>a</sup> 8:00 2 45<sup>a</sup> 54<sup>e</sup> 60<sup>a</sup> 14:00 0 48<sup>a</sup> 57c 62<sup>a</sup> 14:00 1 50<sup>a</sup> 56<sup>d</sup> 65<sup>a</sup> 14:00 2 51<sup>a</sup> 61<sup>a</sup> 59<sup>a</sup>

**Table 4.** Means of NDF content of silage from alfalfa and orchard grass forage harvested at two distinct times of the day

at 08:00 h (**Table 5**).

114 Forage Groups

**3.7. Aerobic stability of silages**

**Table 5.** Means of volume of gas produced by fermentation and ruminal in vitro disappearance of DM of silage from alfalfa and orchard forage harvested at contrasting times of the day in three experimental phases.

rational interpretation was feasible. In phases two and three no effect (P > 0.05) of treatments on this variable was detected. Differences among means of phases one, two and three (0.17, −0.27, 0.03°C d−<sup>1</sup> ) were also odd.

Concerning the variable CO<sup>2</sup> production rate, no effects of treatments were detected in any of the three phases(P > 0.05); the rates of CO<sup>2</sup> production were 9, 9.4 and 19.8 milli moles of CO<sup>2</sup> g−<sup>1</sup> DM d−<sup>1</sup> in phases one, two and three, respectively. In the rate of change of pH, the interaction Hour at cutting x Inoculant (**Table 6**) in the three phases implied that inoculation was not effective to control pH rise in silage from forage cut at 08:00 but it was on silage from forage cut at 14:00 (p < 0.05). The interaction Inoculant x wilting time (**Table 6**) in phase one


abcde Means that do not share any literal within columns of each interaction are different (p ≤ 0.05).

**Table 6.** Means of rate of change of pH during aerobic deterioration of alfalfa and orchard grass silages.

without inoculant, one-hour wilting precluded the rise of pH, but two-hour wilting did not (p < 0.05), while with the addition of inoculant, wilting led to rises in pH (p < 0.05), in phases two and three there was no effect of this interaction (p > 0.05).

**3.8. Results of principal component analysis**

**4.1. Properties of forage to be ensiled**

weather conditions during wilting [4].

alfalfa forage for 2 and 4 h.

the original forage [3].

temperature.

**4.2. Characteristics of silages**

**4. Discussion**

Three classes of silages were identified; Class 1, Class 2 and Class 3 comprising 39%, 53% and

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

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The pH of forage to be ensiled was not affected by treatments and ranged between 6.5 y 7.0 in the scope of results quoted by Coblentz and Muck [4], but somewhat higher than the range between 6.1 and 6.2 reported by Santos and Kung [17]. The results attained in terms of buffering capacity with no effect of treatments, differ from those reported by Zheng et al. [18] who found that wilting resulted in an on average reduction in 5% of the buffering capacity of alfalfa forage; but, the effect of wilting on the buffering capacity is highly dependent on the

As expected from results reported by Tremblay et al. [3] DM content of forage cut in the afternoon was higher than that of forage cut in the morning, which was coupled with higher humidity and lower ambient temperature, solar radiation and wind speed in the morning hours, leading to lower evapotranspiration which concurs with Owens et al. [19]. During morning hours low evapotranspiration is the probable cause of the lack of effect of wilting on DM content of forage to be ensiled; conversely, in forage harvested at 14:00 in phases two and

The content of ASC of fresh forage in phase one did not show significant differences, while in phases two and three forage treatments harvested at 14:00 had higher concentrations of ASC, which was to be expected [3]. On the other hand, the wilting time did not affect the concentration of ASC; on the contrary, Zheng et al. [18] found reductions of 8 and 17% when wilting

The NDF content of forage harvested at 14:00 was lower than that of forage harvested at 08:00, probably due to the accumulation of photosynthetic products in the cell content [19]. This represents advantages in terms of the nutritional composition and fermentative characteristics of

Silage temperatures (in the range between 18 and 21°C) were adequate, Borreani and Tabacco [20] report that in well preserved silage the temperature should be close to the ambient

Silage pH is one of the main factors that influences the degree of proteolysis [21] in silages; the results attained (**Table 3**) were not conclusive since treatments effects differed between phases and short-term weather variables are a feasible explanation for these differences. The pH values fluctuated between 4.6 and 4.9; analogously, in alfalfa silage with *L. buchneri* inoculant

three there was a linear increase in DM content as the wilting time increased.

8% of silages (**Figure 2**). Characteristics of those classes are described in **Table 7**.

**Figure 2.** Classes of alfalfa and orchard grass silages harvested at 08:00 or 14:00, submitted to 0, 1 or 2 h of wilting and with or without addition of bacterial inoculant.


**Table 7.** Attributes of three classes of alfalfa and orchard grass silages harvested at 08:00 or 14:00, wilted 0, 1 or 2 h and with or without addition of bacterial inoculant.

#### **3.8. Results of principal component analysis**

Three classes of silages were identified; Class 1, Class 2 and Class 3 comprising 39%, 53% and 8% of silages (**Figure 2**). Characteristics of those classes are described in **Table 7**.
