The DM (38.2 vs. 38.4%), NDF (27.8 vs. 26.4%), and ADF (19.7 vs. 20.1%) concentrations of PL before ensiling were close to those reported by Kazemi and Mokhtarpour (2021). The primary purpose of silage preparation with higher quality is to minimize DM losses and maintain the maximum aerobic stability and nutritive value using different additives or new methods. Some of the quality of silages can be evaluated by chemical analyses. The treatment of the present silages with or without additives compared to before ensiling affected some chemical composition (DM, CP, Ash, and NFC) after 60-day ensiling. The range of DM of the prepared silages was between 29.1% and 39.1%, which was within an ideal range reported by Ergün et al. (2001) for different silages. In line with other reports (Li et al. 2014, 2019; Wang et al. 2019), we found an increase in DM content of PL ensiled with additives compared to the control silage. This increase can be attributed to the growth of lactic acid-producing bacteria, which reduces the pH of the silo environment, then inhibits the growth of harmful anaerobic bacteria, reduces the consumption of nutrients by these microorganisms, then preserves nutrients, and finally result in the increase of DM content of silages containing additives (yogurt and molasses). In line with the present study, Baytok et al. (2005) reported an increase in DM content of corn silage when molasses was added to the silages. To make good quality silage, it is necessary to produce silage from wilted material that contains DM between 32 to 38% (Knotek 1997). Also, it is reported that the DM content of the primary substrate for ensiling should be above 35% to ensure successful fermentation (Đorđević et al. 2001). Therefore, according to the above reports, PL had an ideal DM content (about 38.2%) for ensiling. An increase in CP content of PL after ensiling compared to before ensiling could be attributed to the concentration effect due to the loss of organic carbon during fermentation or the combination of proteolysis inhibition and concentration effect (He et al. 2018). Also, the increase in CP content of the control silage compared to before ensiling was not only associated with increased protein supply but was also associated with a stable rate of proteolysis during ensiling, as evidenced by close percentages of DM loss due to ensiling. However, the primary mechanism underlying such findings remained ambiguous. We found that the PL ensiled with additives had lower ADF, EE, and ADF contents compared with the PL before ensiling. Consistently, other studies have reported that molasses or other additives can decrease the fiber fractions (NDF and ADF) of silages (Li et al. 2014; Babaeinasab et al. 2015).
We found an increase of minerals (exception sodium) in the control silage compared to PL before ensiling and also a decrease in minerals content (calcium, phosphorus, potassium, magnesium, manganese, iron, and zinc) between silages containing additives or no additives. The decrease in the amounts of minerals after ensiling of PL can be attributed to the use of organic matters by microorganisms, which has ultimately led to an increase in minerals.
Fermentation quality of silage
The rate of pH reduction is considered as an essential indicator for reflecting microbial activity and silage fermentation (Ni et al. 2017). In this study, all silages containing additives showed lower pH values than the control silage. In general, lactic acid because of its stronger acid characteristics (pKa 3.86) compared to acetic (pKa 4.76) is the final goal of the end product of fermentation in the silage (Muck 2010). High concentrations of lactic acid lead to a rapid decrease in the pH of silage, which reduces the activity of harmful microorganisms and the production of butyric acid. A decrease in pH of ensiled PL with additives can be related to their higher lactic and acetic acids production compared to the control silage. The pH value (3.98–4.06, Table 3) of the ensiled PL with additives suggests that these silages underwent a proper fermentation process. Some researchers suggested a pH range of 3.8–4.2 as indicative of good fermentation for silage of tropical grass (Rabelo et al. 2019). Kleinschmit and Kung (2006) reported that microbial inoculants decreased the pH of the silo environment. A higher concentration of ammonia nitrogen in the ensiled PL with additives can be attributed to excessive protein breakdown caused by a slow drop in pH (Gandra et al. 2016). Higher butyric acid in the control silage can be related to yeast activity (Gandra et al. 2016). As we found a high level of butyric acid (0.66% of DM) in the control silage, Kung and Shaver (2001) reported that a high level of butyric acid (> 0.5% of DM) indicates the clostridial fermentation, which is one of the unsuitable fermentations. It is also reported that silages containing high levels of butyric acid are usually low in nutritive value and have higher ADF and NDF levels because many of the soluble nutrients have been degraded. In this regards, we also found that control silage had high level of butyric acid as well as higher NDF and ADF rather than other prepared silages. In line with our results, the addition of whey as a natural source of bacteria improved the fermentation characteristics of alfalfa silage (Mariotti et al. 2020). The present additives increased lactic acid and acetic acid production as well as ammonia nitrogen in the silo environment simultaneously, which its reason is unknown. This simultaneous increase may be due to the different nature of pomegranate leaves. It is reported that if the concentration of ammonia nitrogen is less than 10% of total nitrogen, fermentation quality is ideal, as in our study, the concentration of ammonia nitrogen was less than this value (McDonald et al. 2010). Although two applied additives (yogurt and molasses) increased ammonia nitrogen in silages, the amount of ammonia nitrogen in these silages was normal (below 10% of total nitrogen). When the pH value is high enough to limit the activity of proteolytic bacteria, the proteins in the fresh material are preserved (Man and Wiktorsson 2002). So, although the different CP contents of the PL silages in our study cannot prove the efficiency of additives in protein preservation, however, the proteolytic activity of microorganisms inside the PL silages seems to be negligible. In line with the present study, Steg and Meer (1985) reported that the addition of molasses to silage increased acetic and lactic acids compared to the control silage. They suggested that the use of molasses stimulated the growth of hetero-fermentative instead of homo-fermentative lactic acid bacteria due to the specific source of water-soluble carbohydrates in molasses, i.e., sucrose (Steg and Meer 1985). Similar to our results, the addition of molasses to corn silage increased lactic acid and ammonia nitrogen compared to the control group (without additive) (Baytok et al. 2005).
Gas production and ruminal fermentation parameters
Due to its ease of implementation and low cost, the gas production technique can easily be used to evaluate feedstuffs and forages. Gas production reflects the produced short-chain fatty acids in gas production techniques and provides essential information on the ruminal fermentation of phytochemicals and animal feed (Makkar 2005). We found PL before ensiling or ensiled PL with two additives had higher bgas and 12, 24, 48, and 72 h gas production rather than the control silage. The increased gas production can be attributed to their higher NFC content rather than the control silage. When a feedstuff or forage is incubated in vitro, the carbohydrates are fermented to the short-chain fatty acids, gases (mainly CO2 and CH4), and microbial cells (Getachew et al. 1998). In the present study, higher (12.5%) potential gas production (35 vs. 30.6 ml/200 mg DM) for PL before ensiling was observed than that reported by Kazemi and Mokhtarpour (2021).
A strong correlation between DMD, OMD, and bgas with TVFA was reported by Kazemi and Valizadeh (2019) and Kazemi (2019). So, higher TVFA in PL before ensiling or ensiled PL with 4% yogurt or molasses can be attributed to more DMD, OMD, and bgas. One of the most critical applications of the gas production technique is determining the digestibility and energy value of animal feed (Krishnamoorthy et al. 2005). We found PL before ensiling or PL ensiled with two additives had higher metabolism energy rather than the control silage. This increase can be attributed to their higher 24 h gas production. In line with our results, the addition of 1% of a commercial yogurt containing Lactobacillus plantarum, L. bulgaricus, L. casei, L. acidophilus, and L. bifidus as a natural source of bacteria increased the in vitro DMD and OMD of sugarcane silage (Reyes-Gutiérrez et al. 2018). One of the most critical be attributed to higher content indicators of feed evaluation is determining its digestibility, which can affect feed intake and largely relies on chemical compounds, especially the fibrous and structural parts of the plant (Chabot et al. 2008). So, PL before ensiling or PL containing two additives in terms of DMD and OMD were superior to the control silage. Ammonia nitrogen is an index for ruminal proteolysis. Some of the increase in ammonia nitrogen can be attributed to the higher content of CP in PL after ensiling.
Although high ammonia nitrogen concentration indicates high levels of ruminal degradable protein in the diet, lower concentrations of ammonia nitrogen may occur in similar conditions when the CP has a lower degradability coefficient or CP quality is low (Minson 1990). So, another part of increasing levels of ammonia nitrogen in PL after ensiling can be related to its higher ruminal degradable protein.
Moharrery (2007) reported that the buffering system of ruminants is controlled by three essential mechanisms including, (1) the dietary additive buffers, (2) the buffering capacity of the feed consumed, and (3) the salivary buffer system (Moharrery 2007). The buffering capacity of some protein sources and leguminous fodders has been reported to be higher than 85 mEq × 10–3 (Montanez-Valdez et al. 2013), which is consistent with our study. In this study, the highest acid and also acid–base buffering capacity in PL ensiled 4% molasses indicated more acid is needed to change in pH of the water-soluble plant sample and high control of this plant in ruminal pH balance. Initial pH and titratable acidity have been reported to be the most critical determinants of rumen fluid pH. In the present study, the highest titratable acidity was observed for PL after ensiling (21.67 mEq × 10–3), indicating high resistance to acidification. By evaluating the pH and buffering capacity of the ration, we can predict the need for buffers to control and maintain rumen pH (Bujňák et al. 2011). All experimental silages had acidic pH and therefore, their consumption could lead to rumen pH reduction. It is reported that the amount and composition of minerals in the ash have a particular buffering effect on the plant’s initial pH (Levic et al. 2005). Due to the different ash content of the present plant species (5.33–8.60%), their buffering capacity was also different.
In summary, ensiling of PL alone resulted in the loss of some nutrients, reduction of some minerals, reduction of gas production potential, and reduction of dry and organic matter digestibility, as well as reduction of TVFA production in the culture medium. Molasses and yogurt improved the fermentation characteristics of the silage environment, increased nutrient digestibility, and improved nutritional indicators and buffering parameters. In general, it is recommended that PL is ensiled with yogurt or molasses. Higher levels of yogurt or molasses (4%) than lower levels (2%) are recommended to add to the silo environment. In objective observations, we found that the silage containing 4% molasses had a better appearance quality and smell than other silages.
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