4.1. CH4 production
A low in vitro CH4 production recorded in 60R:40C (high roughage) may be associated with the presence of high levels of secondary metabolites (saponin and condensed tannin) in Medicago sativa used as a roughage in this study (Kozlowska et al., 2020). Previously, Castro-Montoya et al. (2012) and recently Kozlowska et al. (2020) found that the plantes rich in saponin (quillaja plant, alfalfa or Medicago sativa) used in high level in diet (high roughage for example) decreased CH4 production compared to high concentrate. Likewise, in some studies, saponin has been found to reduce CH4 production (Morgavi et al., 2012; Jayanegara et al., 2014; Chen et al., 2019). A low in vitro CH4 production found in high roughage and high roughage can be explained by low NDF and high CP of all of diets used in this study (Table 2). However a lowest in vitro CH4 production found in a high roughage (60R:40C) compare to the high concentrate (40R:60C) is due to the presence of saponin and condensed tannins (in high level, Table 2) and the combined effect of secondary metabolites (Saponin and Condensed tannins), NO3− and oils. While some researchers found that a low condensed tannin (CT < 0.001%) content in alfalfa hay decreased CH4 production, some researchers reported a decrease in CH4 production due to the low NDF and a high crude protein content and a presence of secondary metabolites in alfalfa (Cheok et al., 2014; Rira et al., 2015; Moate et al., 2017; Szumacher -Strabel et al., 2019). These reports are consistent with our findings.
In current study, the effect of NO3− supplementation on in vitro CH4 production in the high concentrate and high roughage was different. In this study the increase of roughage level (60R:40C) led to a decrease of in vitro CH4 production. This can be explained by the presence of secondary metabolites (Saponin and Condensed tannin) and the effect of nitrate which act to reduce in vitro CH4 production. Previously an interaction was found between ration type (roughage/concentrate ratio) and CH4 reducing agents (such as nitrate) in cattle (Alvarez-Hess et al., 2019). This result is consistent with our findings.
In vitro CH4 production was high when a concentrate ratio was high in the diet and was gradually decrease with the high roughage ration in the diet. This could be due to that roughage (Medicago sativa) content a high secondary metabolites (Saponin and condensed tannin) which could explain the lower in vitro CH4 production when roughage fraction was maximun (60%) in the diets. A high in vitro CH4 production found in a high concentrate level, is consistent with some studies (Hristov et al., 2015; 2017; Moate et al., 2017; 2019; Olomonchi et al., 2022). In this study, a relationship between in vitro CH4 production, dietary starch rate and digestion can be established. This is in line with previous findings (Herrera-Saldana et al., 1990; McAllister et al., 1996; Alvarez-Hess et al., 2019).
In our study, it was found that NO3− + O added rations decreased in vitro CH4 production in the high concentrate (40R:60C) and high roughage (60R:40C) (p < 0.001). Some researchers reported that a combined effect of oils (rich MUFA or PUFA) and NO− 3 is an effective method to reduce CH4 production in rumen (Leng and Preston, 2010; Yang et al., 2016). It has been determined that a lower effect of NO3− on in vitro CH4 production is associated to NO3− and nitrite reducing microorganisms (Guo et al., 2009). Nitrate acts as a hydrogen acceptor. There are studies showing that nitrate has a significant inhibitory effect on CH4 production (El-Zaiat et al., 2014; Olijhoek et al., 2016). In the current study, the reduction of nitrate to nitrite and then to NH3 reduces H ions concentration required for the conversion of the CO2 to CH4 compound in the rumen and thus in vitro CH4 production decreases. Previously some studies reported the same result (Zhou et al., 2012; Liu et al., 2017). In addition, it has been determined that nitrite has a toxic effect on methanogens (Božic et al., 2009; Zhou et al., 2011). However, in this study the reducing effect of NO3− on CH4 is more evident in rations with a high roughage which is rich in condensed tannins or saponins. Similar results have been reported from an experiment by Pal et al. (2014).
In our study, while a high in vitro CH4 production was observed in 40R:60C (12.44 ml), a lower CH4 production (9.09 ml) was recorded in low a high roughage level (Table 3). This result is related to the increase in saponin and condensed tannins level and their effects on CH4 production in a high roughage level.
In our study, oils used were rich in PUFA (SO) and MUFA (HO). As expected, SO with a high PUFA content decreased in vitro CH4 production at a higher level than HO. This finding is consistent with studies reporting that the mitigation effect of oils on CH4 production is related to degree of unsaturation (Rodrigues et al., 2017; Vargas et al., 2017).
In high concentrate and high roughage, the higher negative effect of SO (rich in PUFA) in vitro CH4 production (compared to the effect of HO) is associated with a high presence of α-linolenic acid (C18:3 cis-9, cis-12, cis-15) and linoleic acid (C18:2 cis-9, cis-12) in SO. Previously, the effect of oils such as flaxseed and rapeseed rich in PUFA on CH4 mitigation was found by some researchers (Chung et al., 2011; Benchaar et al., 2015; Veneman et al., 2015). As a matter of fact, a lowering effect of oils rich in MUFA (oleic acid (C18:1)) on CH4 mitigation was found (Dong et al., 1997). Although, in the high concentrate and high roughage, HO decreased in vitro CH4 production. Likewise, in some studies canola oil (22% linoleic acid, 11% linolenic acid, and 54% oleic acid) caused a reduction of CH4 production (Dohme et al., 2000; Beauchemin and McGinn, 2015)). It was found that oil (rich in MUFA or PUFA) reduced the cellulolytic bacteria population, methanogenic bacteria, and then CH4 production (Freitas et al., 2018; Nur Atikah et al., 2018).
In present study, the decrease in CH4 production due to oils addition can be associated with the decrease in protozoa population. In the high concentrate and high roughage, it was determined that NO3− + O supplementation caused a higher decrease in CH4 production compared to the use of O and NO3− separately. This result is consistent with previous studies (Duthie et al., 2017; Villar et al., 2019). Likewise, Guyader et al. (2015) and Veneman et al. (2015) reported that CH4 production decreases when nitrate and flaxseed oil (high in MUFA) are added to ration.
4.2. NH3 concentration
In the current study, rumen NH3 values determined for rations used in high concentrate and high roughage are above the recommended minimum NH3 concentration (4.39 to 7.32 mmol/l) (Satter and Slyter, 1974), which is considered sufficient for maximum microbial growth rates. The high NH3 concentration found in the high concentrate (40R:60C) might be related to the high number of proteolytic bacteria in rumen. Because, proteolytic bacteria increase ruminal NH3 concentration by accelerating protein degradation in rumen. While this finding is in agreement with some studies (Kljak et al., 2017; Liu et al., 2019), it disagreed with other studies (Jadhav et al., 2017; Liu et al., 2018).
A high roughage (60R:40C) decreased NH3 concentration in present study. This finding can be associated with a high level of alfalfa (rich in saponins), which increased saponin level in ration. Saponin decreased or inhibited NH3 production. Our results were consistent with some previous studies (Belanche et al., 2016; Jadhav et al., 2018).
In our study, 40R:60C x NO3− and 60R:40C x NO3− treatment groups decreased NH3 (p < 0.001). NO3− is converted to nitrite, which has a toxic effect on rumen bacteria, and therefore NO3− addition reduces NH3 concentration at a high level compared to urea addition (control group). Nitrate alters the fermentation profile and decreases the NH3 production. However, the conversion rate of NO3− to NH3 in rumen is slower than urea to NH3. This can explained the high NH3 concentrattions found in control groups (urea supplementation) compared to other treatment groups.
Various studies investigated the effect of O (rich in MUFA or PUFA) supplementation on NH3 concentration. While in some studies oil supplementation had no effect (Jalc et al., 2005) on NH3 concentration, in some studies oil supplementation increased (Jalc et al., 2002) or decreased (Szumacher-Strabel et al., 2009; Doreau et al., 2017) NH3 concentration.
In this study, oils rich in PUFA (SO) or in MUFA (HO) associated with the R:C ratios (40R:60C, and 60R:40C) decreased NH3 concentration (p < 0.001). This can be explained by the presence of linolenic acid (SO) and oleic acid (HO). But the effect of SO (rich in PUFA) was more evident. In fact, the biohydrogenation of linoleic acid consumes more hydrogen (compare to biohydrogenation of oleic acid). Thus, in our study, the lack of hydrogen causes the decrease in NH3 production. Previously, while Bayat et al. (2017), and Kubelkova et al. (2018) found that flaxseed oil (rich in PUFA) compared to rapeseed oil (rich in MUFA) decreased the rumen pH and NH3 concentration at a high level, some researchers reported that Moringa oleifera oil rich in MUFA (oleic acid (74.99%), stearic acid (2.09%), linolenic acid (1.75%), and linoleic acid (1.27%)) increased rumen protected (by-pass) protein and decreased NH3 concentration (Gassenschmidt et al., 1995; Belewu et al., 2014).
In our study, a combined effect of NO3− + O supplementation link to the high concentrate or high rougahge decreased NH3 concentration (p < 0.05). However, combined effect of NO3− + SO (compared to NO3− + HO) was more evident on NH3 concentration in high concentrate or high roughage. In the same time, the biohydrogenation (due to O supplementation) and hydrogen sink reaction (due to NO3− supplementation) were happened to use the free hydrogen in rumen. Like that, NH3 production decreased because of lack of hydrogen. Previously, combined effect of NO3− + O supplementation was reported in some studies (Veneman et al., 2015 (NO3− + linseed oil supplementation); Villar et al., 2019 (NO3− + canola oil supplementation)).
4.3. pH, VFA, and AA: PA ratio
In the present study, pH values of rations used, are determined from the fluids remaining in the injectors after 48 hours of incubation. The pH values vary between 5.99 and 6.25 (Table 3). The pH difference in this study is due to R:C ratio. In this study while a high concentrate decreased pH, a high roughage increased pH. Firstly, this could be to that a high roughage (60R:40C) contained more NDF, ADF and cellulose than a high concentrate (40R:60C). Secondly, a decreased in pH due to the high concentrate can be associated to a high starch content which creates an environment to inhibit nitrate and nitrite metabolism. This means that a high concentrate provided sufficient energy for the microorganisms to convert nitrate to nitrite and then nitrite to NH3. For this reason, NH3 concentration was high in the high concentrate (Table 3).
Although, it was found that NO3− addition link to R:C ratios decreased pH values (p < 0.05). This finding is in agreement with some studies (Li et al., 2012; Villar et al., 2019). Likewise, rumen pH values found in our study are consistent with the value reported by Latham et al. (2016). A decline in pH observed due to NO3− supplementation indicates that microorganisms were not accustomed to digesting nitrate. It suggested that NO3− supplementation caused a dramatic change in rumen conditions.
In the present study, oil addition associated to R:C ratios decreased pH values. A decrease in ruminal pH, AA concentration, and CH4 production observed due to oil addition in our study can be associated with the degree of unsaturation of oils used (SO and HO). Some researchers have reported that oils addition reduces ruminal pH, AA concentration, and CH4 production with oils (rich in MUFA or in PUFA) supplementation (Wu et al., 2016; Majewska et al., 2017; Alvarez-Hess et al., 2019). However, it was found that SO (compared to HO) decreased significantly pH, AA concentration, and CH4 production (Compared to HO). This can be associated to the high level of linolenic acid in SO. This result is consistent with some studies (Russell and Wilson, 1996; Mertens, 1997). By the way, NO3− + O supplementation combine with R:C ratios decreased more pH, AA concentration, and CH4 production. This is associated to the biohydrogenation of unsaturated fatty acids (PUFA, and MUFA) provided by oil (SO, and HO), and hydrogen sink reaction (due to NO3− supplementation) which occurred in the same time.
In this experiment, TVFA, individual concentration of VFA (AA, PA, BA, and AA: PA ratio) were affected by R:C ratios, NO3−, O, R:C ratios x NO3−, R:C ratios x O, NO3− x O.
It was determined that a high concentrate decreased AA concentration, and AA: PP ratio (p < 0.001). An increase in PA, BA, TVFA concentration found in the high concentrate can be explained by the lowering pH due to the increase in lactic acid content derived from the high easily fermentable carbohydrates content of rations used, and an increase in carbohydrate fermentation. An increase in BA concentration can be also associated with the increase in ammonia concentration which inhibited bacterial growth and promotes a fermentation for BA production in this study. As it is known, VFA are produced as a result of microbial fermentation of carbohydrates in the rumen. However, the increase in AA, TVFA, and AA:PA ratio found in the high roughage, is associated with the increase in fiber content (in this case NDF and ADF). Depending on an increase in fibrous content of ration, ruminal hydrogen concentration used in the production of AA and in vitro CH4 increased. Our results are consistent with some studies (Kljak et al., 2017; Moate et al., 2017; Alende et al., 2019).
The increase in PA concentration due to NO3− addition can be explained by the competition between the mechanism of PA production and nitrate (for ammonia production). In other words, propionic acid producing bacteria population (Selenomonas ruminantium, Propionibacterium and Tessaracoccus) increased and they used free H ions present in the rumen to produce propionic acid. For this reason, hydrogen required for nitrate reduction (nitrite then ammonia) decreased. Consequently, PA concentration increased and NH3 concentration, AA and AA: PA ratio decreased in the rumen. But, a decrease in BA (due to NO3− supplementation) was caused by the rapid reduction of NO3− (to nitrite then ammonia) which use up the electrons needed for the production of BA. However, a high roughage level decreased BA concentration. This was due to the combined effect of tannin and NO3−. Our results were consistent with some studies (van Zijderveld et al., 2011; Adejoro and Hassen, 2017; Wang et al., 2018).
In this study, the decrease in AA due to NO3− supplementation can be explained by the use of free hydrogen for production of NH3 and PA. Like this, hydrogen concentration required for the production of AA decreased. One of the possible reasons for the reduction in the concentration of AA due to the combined effect of NO3− and the two types of oil (MUFA or PUFA) is the use of free hydrogens for the production of PA and BA.
In our study, the effect of NO3− + O on VFA and AA: PA ratio changed according to the source of fatty acids (MUFA and PUFA). For that, NO3− + SO (rich in PUFA) associated with R:C ratios decreased AA concentration but it increased PA and BA concentrations. This result can be explained by the simultaneous effect of NO3− (hydrogen sinks) and the biohydrogenation of PUFA which consume more free hydrogen than the biohydrogenation of MUFA. A high concentrate level increased more the combined effect of NO3− + SO on AA, CH4, NH3, TVFA and AA:PA ratio. However, while NO3− stimulated the population of propionic acid-producing bacteria, the unsaturated fatty acids (PUFA and MUFA) in SO and HO used free hydrogens for biohydrogenation. Thus, the production of AA, CH4, NH3, TVFA and AA:PA ratio decreased. Our findings are in conformity with those found by Popova et al. (2017) and Villar et al. (2019). Our results showed that AA and CH4 were more decreased due to the combined effect of NO3− + SO which can be explained by biohydrogenation of PUFA and NO3− mechanism (transformation of NO3− to nitrite then to NH3) for obtaining PA having different and associative mechanism for using available hydrogen. Use of NO3− and O in the same time in the ration led to reduction in ruminal hydrogen concentration.
4.4. Protozoa population (PP)
In the current study, a high concentrate level increased the number of PP compared to the high roughage (p < 0.001). Previously, it was shown that a high concentrate can increase (Franzolin and Dehority, 1996; Lengowski et al., 2016) or decrease (Gozho et al., 2005; Khafipour et al., 2009; Hook et al., 2011) protozoa population.
However, the increase in roughage (rich in secondary metabolites: saponins and tannins) content of ration link to NO3− addition caused a decrease in the protozoa number. This can be explained by the combine effect of NO3− and saponins which acted negatively on protozoa population. Our findings are consistent with those of Lin et al. (2013).
In the present study, NO3− added rations associated with R:C ratios decreased PP. Otherwise, nitrite which come from a transformation of nitrate, inhibits rumen protozoa population and thus in vitro CH4 production. This is consistent with findings of Iwamoto et al. (2001).
Furthermore, in our study, there is a parallelism between NH3 concentration and PP in a high roughage level, and this finding was reported by some studies (Hu et al., 2005; Liu et al., 2018).
In our study, use of NO3− alone or in combination with HO (rich in MUFA: oleic acid) and SO (rich in linolenic acid and linoleic acid) reduced protozoa population. However, the combined effect of NO3− + SO decreased protozoa population more than individual use of NO3− and O. This can be explained by the simultaneous mitigation effect of NO3− and PUFA (linolenic acid and linoleic acid) on PP. Previously it was demonstrated that NO3− alone (Sar et al., 2005; Asanuma et al., 2015) or in combination with linseed oil (Veneman et al., 2015) or canola oil (Villar et al., 2019) decreased PP. While some authors notified a toxic effect of NO3− and lipids on protozoa PP (Morgavi et al., 2010), other researchers reported no significant effect on PP (Guyader et al., 2016).