Determination of Nutritional Value and Methane Production Potential of Phacelia tanacetifolia in Different Stages of Growth

Backround: This study was carried out to determine the nutritional value and methane production potential of Phacelia tanacetifolia plant in different growth stages in April-May year in the ecological conditions of the province of Adıyaman in Turkey. Methods: Phacelia ( Phacelia tanacetifolia ) plant samples were collected with four replications in four different stages, pre-flowering, early flowering, mid-flowering and end of flowering (seeding). The nutrients of the feed samples were determined. The gas production was measured after 24 hours of incubation of the forage with rumen fluid. Result: The effect of harvest time on chemical composition was highly significant. In vitro gas production was lowest in stage I. and highest in II. The methane (CH 4 ) lowest production values were found in the 1 st stage, whereas the highest value was observed in the 4 th stage (seed-binding stage), where the difference was statistically significant. ME was 7.49, 9.54, 8.90 and 8.61 MJ/kg DM respectively and the difference between the stages was statistically significant. It was concluded that Phacelia tanacetifolia , which is known as an important nectar source in the world and in Turkey. It is appropriate to harvest the phacelia plants in the second stage, i.e ., at early flowering for higher ME contents and OMD.


INTRODUCTION
In ruminant nutrition, feed costs account for 60-70% of production costs. In order to ensure the sustainability of livestock production and to obtain high quality and economical products, rations for ruminants must contain significant amounts of coarse forage. Turkish meadows and pastures are of great importance for coarse forage production in the country. Livestock production in Turkey depends on natural pastures. In recent years, indoor livestock production has gained importance. The productivity of meadows and pastures, which provide a large part of the fodder needs of Turkish livestock, has declined due to centuries of irresponsible use and high livestock numbers. For this reason, part of the agricultural land should be reserved for growing fodder crops as part of crop rotations. A plant suitable for this purpose is phacelia (Phacelia tanacetifolia). Phacelia is also known as a bee plant, as it is one of the rare plants that can meet roughage needs and provided a source of pollen and nectar for bees in early spring. Studies conducted in Turkey and other countries have shown that Phacelia can be grown both as a forage crop and as an important nectar source. As a forage crop, Phacelia can be fed to animals in the form of green and dry grass and for this purpose, it is beneficial to harvest it preferabily during the 50% flowering stagestage. Since it is used as a source of nectar during the flowering stagestage, it loses this characteristic towards the end of the flowering stagestage.
Although phacelia loses quality, harvesting the plant at the end of the flowering stagestage provides the opportunity to use the same area as bee pasture and forage crop (Sağlamtimur et al., 1989). Climate change and global warming are known to be a major problem that can endanger the life of living beings in the world (Sağlam et al., 2008). Greenhouse gases (CH 4 , N 2 O), which come from agriculture and livestock in all countries, cause global warming. Methane gas released to nature from dairy farms in the United States accounts for about 71% of methane emissions from agricultural activities and 19% of total methane emissions (Steinfeld et al., 2006;). Manure from ruminants accounts for 16.4% of annual methane gas production. This accounts for about 2.9% of all greenhouse gases causing global warming (Johnson et al., 1991). In studies to reduce methane emissions in ruminants, remarkable results have been obtained by using plant-based feed additives (Wallace, 2004). Essential oils of plants such as onion, ginger, clove, fennel and garlic have been found to reduce methane production under in vitro conditions (Kamra et al., 2006).

Determination of Nutritional Value and Methane Production Potential of Phacelia tanacetifolia in Different Stages of Growth
In this study, in addition to the nutritional value of grass samples collected during certain growing seasons of the plant Phacelia tanacetifolia, its effect on methane gas causing global warming and energy losses, especially in ruminants, was investigated.

MATERIALS AND METHODS
The forage material used in this study was obtained from Phacelia plants by mowing at 15-day intervals in April-May 2016 in different regions under the ecological conditions of Adıyaman Province, Turkey and at the site of the Provincial Directorate of Food, Agriculture and Livestock of Adıyaman (37.77 latitude and 38.26 longitude). Plant samples of Phacelia were collected with four replicates at four different times, pre-flowering, early flowering, mid flowering and end of flowering (seeding) and the dry matter content was determined by taking the samples to the laboratory. A sufficient amount of rumen fluid was collected from yearling lambs slaughtered in slaughter houses and placed in a previously prepared hot water bath, with the addition of CO 2 to ensure that anaerobic conditions prevailed. The dried leaves of the Phacelia plant were ground so that they pass through a 1-mm sieve and prepared for chemical analysis. Crude nutrients of the forage samples used in the study (dry matter (DM), crude protein (CP), crude fat (CF), crude ash (CA)) were determined according to the standards given by AOAC (1990). Neutral detergent fiber (NDF) and acid detergent fiber (ADF), which are the cell wall components of the forage, were determined according to the method described (Van Soest et al., 1991;Van Soest, 1994). The condensed tannin content of Phacelia leaves was determined according to the method described by . The organic matter digestion degree (OMDD) and metabolic energy (ME) values of Phacelia leaves were calculated using an equation described previously by . In this study, the method described by Makkar et al. was used to calculate gas production (Menke et al., 1979). This method is based on the calculation of the amount of gas formed after 24 hours of incubation of the forage with rumen fluid. The methane concentration (%) of the gasses formed after 24 hours of incubation was determined using an infrared gas analyzer (Sensor Europe GmbH, Erkrath, Germany) (Goel et al., 2008). ME (MJ/kg DM) and OMD of phacelia plants were estimated using the equation of Menke and Steingass (1988) as follows: GP= 24 h of net gas production (mL 200 mg -1 ). CP = Crude protein (%). EE = Ether extract (%). CA = Ash content (%). The methane content of the gas produced after 24-h incubation was determined using an infrared methane analyzer (Sensor Europe GmbH, Erkrath, Germany) (Goel et al., 2008), using the following equation: Methane production (mL)= Total gas production (mL) percentage methane (%) ....3 Statistical analyses of the data obtained in the study were performed using the one-way analysis of variance method (ANOVA) in the SPSS 9.0 (2003) programme. To determine the significance of differences between means, Duncan's comparison test was used.

RESULTS AND DISCUSSION
The values obtained from the chemical analyses of the Phacelia plant harvested in different growth stages are shown in Table 1.
W hen the effects of harvest timing on the chemical components of Phacelia were evaluated (Table 1), it was found that the effects on all constituents except CT (condensed tannin) were highly significant (p<0.001). As the vegetative process of Phacelia progressed, the amount of dry matter increased due to development and maturation. The reason for the stageic increase in dry matter was the maturation of the plant and the associated increase in the amounts of cellulosic materials (ADF and NDF) in the cell wall. As the harvest stage progressed, an increase in hay

Determination of Nutritional Value and Methane Production Potential of Phacelia tanacetifolia in Different Stages of Growth
yield was observed. While the percentage of dry matter in the first stage was 32.62%, in the last stage it was 54.58%. The dry matter content of the samples were 32.62, 37.77 43.17 and 54.58% for the first, second, third and fourth stages, respectively and the difference between the stagestages was statistically significant (p<0.001). An increase in dry matter content was observed with the delay in harvesting stage and the differences between stages were highly significant. This result is consistent with the results of previous studies (Soya et al., 1999;Kamalak et al., 2014;Ayaşan et al., 2020a;Ayaşan et al., 2020b). When examining the influence of harvest stage on CA %, the results for the first and second stages were statistically similar and there was no significant difference. The differences between the third and fourth stages were found to be highly significant (p<0.001). W hile the ash content in the first stage was 11.18%, it was 10.61% in the second stage. The CA rates in the third and fourth stage were 9.60 and 8.66%, respectively. The CA contents of Phacelia in all stages were higher than those reported in a previous study for alfalfa, vetch, pea, cloverleaf and dried rapeseed grasses (Canbolat and Kara, 2013). In their study with two different Phacelia varieties, researchers (Geren et al. 2007) generally reported similar average CA ratios as in this study. In other studies of quinoa and wild sainfoin in plants (Kaplan et al., 2017, Kamalak et al., 2014, the finding that CA content decreases as the plant matures was comparable to the data obtained in this study. In this study, it was found that the CP amounts (DM%) of the different stages of the Phacelia plant varied from 14.76 to 19.79%. The crude protein content for the first, second, third and fourth stages were 19.79, 18.86, 15.71 and 14.76%, respectively. The highest crude protein content was 19.79% in the first stage and the lowest CP was 14.76% in the fourth stage. The difference between all stages in terms of CP ratios was significant (p<0.001). The results of chemical analysis of Phacelia showed that CP content decreased with the growth and maturity of the plant, which is consistent with the results of some previous studies (Soya et al., 1999;Kamalak et al., 2014;Kaplan et al., 2017;Ayaşan et al., 2021). In another study (Geren et al., 2007) on two different Phacelia cultivars, it was observed that the mean values of the ratio CP were lower than the values obtained for all four stages in this study. Similar to this study, other researchers Kamalak et al. (2014) and Ayaþan et al. (2021) also reported that there was a negative correlation between an increase in dry matter and a decrease in crude protein content.
The CF amounts (DM%) in the four different growing stages of Phacelia plant were studied, the crude fat content was 2.80, 2.04, 1.94 and 1.42%, respectively and the difference in these CF ratios between all stages was significant (p<0.001). Canbolat and Kara (2013) reported higher crude fat contents in alfalfa, vetch, pea, clover and canola than found in the study for Phacelia. In their study with wild clover grass, Kamalak et al. (2014) found that the ratio CF decreased with maturity. These results were similar to those of the present study. W hile the stageic increases in the rates CA, CP and CF were similar in our study and the ratios of these chemical compounds were high in the fistage, the ratios of these constituents decreased as the Phacelia plant developed and matured. The ADF and NDF contents are among the factors that directly affect the quality of the forage. The increase in ADF and NDF contents, which are cell wall components, as a function of advancing harvest stage proved to be highly significant (p<0.001). Similar results were obtained by other researchers. It was found that as harvest time progressed, poorly digestible materials such as ADF and NDF increased and corresponding lignification occurred (Cassida et al., 2000;Kamalak et al., 2014;Kaplan et al., 2017, Ayaşan et. al. 2020aAyaşan et al., 2021). Lower ADF ratios than in our study were observed in alfalfa, vetch, pea, bird's-foot trefoil and colza dried grasses, but the NDF ratios observed in alfalfa, vetch and pea in the same study were similar to those in our study (Canbolat and Kara, 2013).
Gas production, methane production, metabolic energy and degree of organic matter digestion of Phacelia in different time stages are shown in Table 2. As shown in the table, the differences between the data of different stages were highly significant (p<0.001).
The highest value of daily in vitro gas production (GP) of Phacelia was recorded at the beginning of flowering, in the second stage, with 45.68 ml. The second highest value was obtained in the samples taken in the middle of the p *** *** *** *** *** GP: Gas production in 24 hours (mL), CH 4 : Methane production (mL/%), ME: Metabolic energy, OMDD: Organic matter digestibility (%), abc: Difference between groups in the same column is statistically significant. p: Level of significance, ***p<0.001.

Determination of Nutritional Value and Methane Production Potential of Phacelia tanacetifolia in Different Stages of Growth
flowering stage (42.08 ml), corresponding to the third stage. The difference in daily in vitro gas production between the studied stages proved to be highly significant (p<0.001). The daily gas production of Phacelia ranged from 30.56 mL to 45.68 mL in all four stages. A previous study by Canbolat and Kara (2013) found higher daily gas production levels in alfalfa, vetch, pea, bird's-foot trefoil and colza dried grasses in comparison to the levels found in our study. Other studies on quinoa and wild trefoil (Kaplan et al. 2017) and Kamalak et al. (2014) showed that in vitro gas production decreased with increasing maturity. In our study, these values increased in a general trend. The daily gas production values of Phacelia plant for the preflowering stage in this study were very low compared to the values reported for quinoa and wild trefoil (Kaplan et al., 2017;Kamalak et al., 2014Ayaþan et al. 2020bAyaşan et al., 2021). In this study, CH 4 concentrations for the first, second, third and fourth stages were 0.93, 5.33, 4.74 and 5.38 mL, respectively, while CH 4 ratios were 3.05, 11.66, 11.28 and 12.85%, respectively. The highest CH 4 concentration (5.38 mL) and CH 4 ratio (12.85%) were obtained in the samples from the second stage. The lowest CH 4 concentration (0.93 mL) and CH 4 ratio (3.05%) were found in the samples from the first stage. In a study on quinoa and wild trefoil (Kaplan et al. 2017;Kamalak et al. 2014), the amount (mL) and percentage (%) of CH 4 were found to decrease with maturity. However, in previous study, these values generally increased. It was found that the amount (mL) and percentage (%) of CH 4 were higher in quinoa and wild trefoil Kaplan et al. (2017), Kamalak et al. (2014) compared to Phacelia. In addition to, Gautam et al. (2018) reported that the addition of 10% deoiled ajwain (Trachyspermum ammi) meal (DOAM) to the concentrate in the mixture resulted in lower methane production. Osita et al. (2019) conducted a study to determine the effects of adding yeast (Saccharomyces cerevisiae) to his diet on methane production. Based on the results, it was suggested adding Saccharomyces Cerevisiae to the legume diet to improve methane emission. Jafari et al. (2020) investigated the effect of bamboo leaf (BL) on rumen methane gas production in vitro. They reported that methane gas production (mL/250 mg DM) decreased at a decreasing rate with higher BL levels. Moreover, Murillo-Ortiz et al (2018) reported that net gas production decreased linearly when alfalfa hay was substituted, while methane and CO 2 production decreased linearly with the addition of water hyncinth. The results showed that W H has emerged as a promising alternative to reduce methane production in ruminants. In addtion, Kaur et al. (2017) in vitro analysis revealed that the net gas production was the lowest in P. minor seeds (216.37 L/kg DM/24 h). Methane production (L/kg DM/24 h) from P. minor seeds (43.03) were lower than wheat (54.33) and barley (57.35). Dey et al. (2022) investigated the in vitro fermentation model of the stovets obtained from three different new sorghum (Sorghum bicolor L.) cultivars in buffalo. The fermentation pattern of brown midrib sorghum stovers reported higher total gas production than normal and sweet sorghum stovers. Apart from all these research studies mentioned above, Sarkar et al (2018) showed significant increase in total gas (mL/g DM) between different diets, CH 4 (%, mL/24h and mL/100 mg DDM) and NH 3 -N (mg/dL) on supplementation of Aegle leaves if any of the diets. The values of CH4 (%, mL/24h and mL/100 mg DDM) and NH 3 -N (mg/dL) on supplementation of Aegle leaves if any of the diets were found to be not significant.
The ME (metabolic energy) amounts (DM%) of Phacelia plant for the first, second, third and fourth stages were 7. 49, 9.54, 8.90 and 8.61 MJ/kg DM, respectively. On the other hand, in a previous study (Canbolat and Kara 2013) higher metabolic energy contents were found in alfalfa, vetch, pea, bird's-foot trefoil and colza dried grasses. Another study by Kamalak et al. (2014), conducted on wild trefoil, found that the amount of metabolic energy decreased with increasing maturity. However, in our study, this parameter varied with advancing maturity with different tendency.
In study, the OMD (%) of Phacelia plant for the first, second, third and fourth stages were 51.68, 64.95, 60.51 and 58.48%, respectively. The lowest value was found in the pre-flowering stage, while the highest value was obtained at early flowering stage. In a previous study, it was found that the organic matter digestibility of alfalfa, vetch, pea, bird's-foot trefoil and colza dried rapeseed grass were higher than the values obtained in our study with Phacelia. In addition, other studies on quinoa and wild trefoil (Kaplan et al. 2017;Kamalak et al. 2014) found that ripening reduced organic matter digestibility. In contrast, this study found that ripening generally increased digestion.

CONCLUSION
According to the results of this study, it is appropriate to harvest the Phacelia plant, which is known as an important source of nectar in the world and Turkey, in the pre-flowering stage in terms of GP, crude ash, crude protein, crude fat, CH 4 amount and CH 4 ratio. Due to the high amounts of ME and OMD, it is appropriate to harvest in the second stage, i.e., at early flowering. Nevertheless, it is important to investigate the effects of harvest stage of Phacelia on feed value and consumption of the crop by animals with new in vitro and in vivo studies.