Effect of Nitrate, Two Oils and Their Combinations Added To Two Different Forage: Concentrate Ratios On Some Rumen Parameters and Protozoa Population.

This study aimed to determine the effects of nitrate, and two oils (O) (soybean and hazelnut oils) alone or in combination, on in vitro methane (CH 4 ) production, volatile fatty acid (VFA) and ammonia (NH 3 ) concentrations, pH, and protozoa population. For that, 2 x 2 x 3 factorial design with 2 two different forage:concentrate (F:C) ratios (40F:60C, and 60F:40C), 2 sources of nitrogen (sodium nitrate (NO −3 : 45.94 g/kg DM) or urea (16.45 g/kg DM for control group) and 2 oils (hazelnut oil (HO: 36.58 g/kg DM) or soybean oil (SO: 36.58 g/kg DM)). For every forage: concentrate (F:C) ratio, six (6) treatment groups were formulated: F:C + Urea, F:C + NO −3 , F:C + Urea + HO, F:C + Urea + SO, F:C + NO −3 + HO, F:C + NO −3 + SO. For every F:C ratio while NO −3 (p<0.01), oils (p<0.01), and NO −3 + oils (SO, HO) (p<0.01) decreased CH 4 content, protozoa population, ammonia (NH 3 ) concentration, acetic acid, total VFA, acetic acid: propionic acid ratio and pH, they increased butyric acid and propionic acid concentrations. Furthermore, CH 4 production (12.44 vs 9.09 ml), ammonia (8.23 vs 7.37 mmol/l), propionic acid (19.14 vs 17.93 mmol/l), butyric acid (15.50 vs 14.50 mmol/l), total VFA (86.46 vs 85.66 mmol/l), protozoa population (32.16 vs 26.96 x10 4 ml.) were high in early lactation period. In both lactation periods, SO, and NO −3 + SO decreased acetic acid concentration, protozoa population, and thus CH 4 production. Stabilwax-DA, 260°C. of to about of ruminal liquid uid the maintained -20°C and centrifuged at 10000 rpm at + 4°C.


Introduction
Methane (CH 4 ), a product of ruminal microbial fermentation, is a major contributor to global warming (IPCC, 2014). One of the main actors in CH 4 production are ruminants. The amount of CH 4 produced in the rumen is an indicator for estimating environmental impacts and energy costs in the animal production sector (Auffret et al., 2018). Archaea are responsible for microbial fermentation in anaerobic rumen environment (Yang et al., 2017). For this reason, the decrease of the population of archaea is related to CH 4 mitigation in the rumen.
Usingnitrate as a feed additive in ruminant nutrition, is one of best strategies to reduce enteric CH 4 emissions (Hristov et al., 2013). In the rumen, nitrate uses free hydrogen for the production of ammonia to the detriment of CH 4 production. Thus, CH 4 production decreases (van Zijderveld et al., 2011). Some studies show a decrease of enteric methanogenesis, microbial growth (Ungerfeld and Kohn, 2006), inhibition of total gas volume and CH 4 emission (Guyader et al., 2016), an increase of ammonia (NH 3 ) concentration in the rumen (Shari et al., 2018) due to the use of nitrate in the ration. Another CH 4 mitigation way is the use of lipids in the diet. In recent years, the use of lipids as a feed additive has been adopted as an alternative to mitigate CH 4 in the rumen (Boadi et al., 2004;Martin et al., 2010). Some researchers have reported that the alfalfa plant (because of its high nutritional value) positively affects the levels of digestion and absorption of nutrients, leading to increased productivity levels in ruminants (Paterson et al., 1982;Hunt et al., 1985;Brandt and Klopfenstein, 1986a;Leng 1990; Ørskov et al., 1999). On the other hand, some studies reported a low ber content for alfalfa (28-30%) (

Determination of CH 4 production
Infrared CH 4 analyzer (Sensor Europe GmbH, Erkrath, Germany model) was used to determine the CH 4 production in the rations used in present study (Goel et al., 2008). After 24 hours, the gas accumulated in the injectors was taken to the CH 4 analyzer by means of a special tube (using plastic injectors) and CH 4 production (ml) was determined as a percentage of total gas CH 4 production (ml) = Total gas production (ml) x % CH 4 2.3. Determination of NH 3 concentration in the rumen uid.
For the determination of NH 3 concentration, 5 ml of rumen uid was taken from the syringes after 48 hours. As in the protein analyses, a distillation was performed. Then the titration was done and the volume of HCl (0.1) was noted. Due to following formula, the amount of NH 3 was determined. 14: Molar masses of nitrogen.

pH and VFA analysis in rumen uid
In our study, 5 ml of rumen uid was added to 2 wheaton asks before incubation and 4 drops of H 2 SO 4 were added to determine the content of VFA in the rumen uid to be used in the study. The rumen uids thus prepared were kept at room temperature until the analyzes

Determination of Protozoa Population
After the mixing 0.6 g methyl green, 8 g sodium chloride (NaCl) and 100 ml 37% formaldehyde solution for staining the protozoa, the volume was increased to 1000 ml with distilled water. One mililiter of rumen inoculum from the fermenter was mixed with 1 ml of methyl- Yijkl = µ +α i + β j + λ k + (αβ)ij + (βλ) jk + (αλ) ik + (αβλ) ijk + e ijkl Where Y ijkl : i th application subject to j th feed variety (CH 4 production, etc.) k th observation value of the sample (gas production, etc.).
µ: mean population, α i : Effect of i th ration βj: Effect of the j th additive λ: Effect of the k th oil addition (αβ) ij : Interaction of i th ration and j th additive effect (βλ) jk : Effect of interaction between j th inci additive with k th vegetable oil type (αλ) ik : Effect of interaction between i th ration with k th vegetable oil type (αβλ) ijk : Effect of interaction between i th ration, j th additive with k th vegetable oil type eijk: shows a random error.
Duncan Multiple Comparison test was used to compare the means if the differences between the applications or feed types were statistically signi cant. SPSS 22.0 statistical package program licensed by Ondokuz Mayıs University was used for statistical analysis.

Methane (CH 4 ) production
In current study, F:C ratio (p=0.002), feed additive (FA) (p<0.001), oils addition (O) (p<0.001), F:C ratio x FA interaction (p<0.001), FA x O interaction (p=0.007), F:C ratio x O interaction (p<0.005), and F:C ratio x FA x O interaction (p=0.004) were found to affect CH 4 production (Table 3). After 24 hours of fermentation, the high-concentrate (40F:60C) content rations led to higher CH 4 productions. In 40F:60C and 60F:40C, NO − 3 supplementation (compared to urea supplementation) and NO − 3 + O supplementation (compared to urea + O supplementation) decreased CH 4 production. This combined effect of NO − 3 + oils supplementation on CH 4 production was more evident with a high forage content (60F:40C) compared to high concentrate content (40F:60C). However, in 40F:60C and 60F:40C, SO supplementation led to lower CH 4 production compared to HO supplementation).    In current study, the effect of NO − 3 supplementation on CH 4 production in both forage:concentrate ratios was different. In this study the increase of forage level (60F:40C) led to a decrease of CH 4 production. An interaction was found between ration type (roughage/concentrate ratio) and CH 4 reducing agents (such as nitrate) in cattle (Alvarez-Hess et al., 2019). A high CH 4 production found in a high concentrate level, is consistent with some studies (Hristov et  In our study, it was found that NO − 3 + O added rations decreased CH 4 production in both forage:concentrate ratios (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 CH 4 (Pal et al., 2014). This is consistent with our results. In our study, while a high CH 4 production was observed in 40F:60C (12.44 ml), a lower CH 4 production (9.09 ml) was recorded in low a high forage level (Table 3). This result is related to the increase in saponin and condensed tannins level and their effects on CH 4 production in a high forage level.
In our study, oils used were rich in PUFA (SO) and MUFA (HO). As expected, SO with a high content of PUFA decreased CH 4 production at a higher level than HO. This nding is consistent with studies reporting that the mitigation effect of fats on CH 4 production is related to In both forage:concentrate ratios the higher negative effect of SO (rich in PUFA) in the reduction of CH 4 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 axseed and rapeseed rich in PUFA on CH 4 mitigation was found by some researchers (Chung et

NH 3 concentration
In the current study, rumen NH 3 values determined for rations used in both forage:concentrate ratios are above the recommended minimum NH 3 concentration (4.39 to 7.32 mmol/l) (Satter and Slyter, 1974), which is considered su cient for maximum microbial growth rates. The high NH 3  In our study, the forage:concentrate ratios associated with NO − 3 supplementation decreased NH 3 (p<0.001). NO − 3 is converted to nitrite, which has a toxic effect on rumen bacteria, and therefore NO − 3 addition reduces NH 3 concentration at a high level compared to urea addition (control group). Nitrate alters the fermentation pro le and decreases the NH 3 production. However, the conversion rate of NO − 3 to NH 3 in rumen is slower than urea to NH 3 .
Various studies investigated the effect of O (rich in MUFA or PUFA) supplementation on NH 3 concentration. While in some studies oil supplementation had no effect (Jalc et al., 2005) on NH 3  In our study, a combined effect of NO − 3 + O supplementation link to the forage:concentrate ratios decreased NH 3 concentration (p<0.05). However, combined effect of NO − 3 + SO (compared to NO − 3 + HO) was more evident on NH 3 concentration in the both forage:concentrate ratios. In the same time, the biohydrogenation (due to O supplementation) and hydrogen sink reaction (due to NO − 3 supplementation) were happened to use the free hydrogen in rumen. Like that, NH 3 production decreased because of lack of hydrogen. Previously, combined  Table 3). The pH difference in this study is due to forage/concentrate ratio. In this study while a high concentrate level decreased pH, a high forage level increased pH. Although, it was found that NO − 3 addition link to forage:concentrate ratios decreased pH values (p<0.05). This nding is in agreement with some studies ( supplementation indicates that microorganisms were not accustomed to digesting nitrate. It suggested that NO − 3 supplementation caused a dramatic change in rumen conditions. A decreased in pH due to the high concentrate level can be associated to a high starch content which creates an environment to inhibit nitrate and nitrite metabolism. This means that a high concentrate level provided su cient energy for the microorganisms to convert nitrate to nitrite and then nitrite to NH 3 . For this reason, NH 3 concentration was high in the high concentrate level (Table 3) In the present study, oil addition associated to forage:concentrate ratios decreased pH values. A decrease in ruminal pH, AA concentration, and CH 4 production observed due to oil addition in our study can be associated with the degree of unsaturation of oils used (SO and HO). 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 level 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 forage level, is associated with the increase in ber content (in this case NDF and ADF). Depending on an increase in brous content of ration, ruminal hydrogen concentration used in the production of AA and CH 4 increased. Our results are consistent with some studies (Kljak et  The increase in PA concentration due to NO − 3 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 NH 3 concentration, AA and AA: PA ratio decreased in the rumen. But, a decrease in BA (due to NO − 3 supplementation) was caused by the rapid reduction of NO − 3 (to nitrite then ammonia) which use up the electrons needed for the production of BA. However, a high forage level decreased BA concentration. This was due to the combined effect of tannin and NO In this study, the decrease in AA due to NO − 3 supplementation can be explained by the use of free hydrogen for production of NH 3 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 NO − 3 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 NO − 3 + O on VFA and AA: PA ratio changed according to the source of fatty acids (MUFA and PUFA). For that, NO − 3 + SO (rich in PUFA) associated with forage: ratios decreased AA concentration but it increased PA and BA concentrations. This result can be explained by the simultaneous effect of NO − 3 (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 NO − 3 + SO on AA, CH 4 , NH 3 , TVFA and AA:PA ratio. However, while NO − 3 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, CH 4 , NH 3  In the present study, NO − 3 added rations associated with forage:concentrate ratios decreased PP. Otherwise, nitrite which come from a transformation of nitrate, inhibits rumen protozoa population and thus CH 4 production. This is consistent with ndings of Iwamoto et al.

Conclusion
In this study, a high forage level (because of a presence of saponins and condansed tannins in high level) decreased CH 4 , AA, NH 3 , TVFA, PP, and AA:PA ratio. While the 60F:40C associated to NO − 3, O alone or in combination decreased CH 4 , AA, TVFA, PP, and AA:PA ratio, it increased PA, BA. This study shows that NO − 3 and O (HO and SO) affect CH 4 production, protozoa population, NH 3 and VFA concentrations. The combination of NO − 3 and O (HO and SO) reduced acetic acid, protozoa population (thus CH 4 ), and increased propionic acid and butyric acid more than individual use of nitrate and oils. Our study showed that a combined effect of nitrate and oils can be considered more avantageous to reduce methane production and protozoa population without a negatif effect on PA and BA concentrattions.
The fermentation properties of rations supplemented with nitrate or oils have a potential to improve rumen fermentation. It has been found that the degree of unsaturated fat alone or in combination with NO − 3 decreases CH 4 production and increases VFA.

Declarations
Funding This study was supported by Ondokuz Mayıs University as PYO. ZRT.1904.19.010 Scienti c Research Project.

Con it of interests
The authors declare that they have no con ict of interest. GARIPOĞLU. All authors read and approved the nal manuscript.