y. rouzbehan

Probiotics accelerate and improve rumen development, stability, and balance of beneficial microbes in the digestive system and decrease microflora disruption, which will increase the activity of desirable enzymes. This action of probiotics increases digestibility, feed efficiency, livestock performance, and general defense including antioxidant power. However, the response of different animals to the specific probiotics is not uniform and similar. Therefore, it is necessary to provide factors that improve the conditions for the establishment and functioning of microbial additives in the rumen or to use compounds that have a synergistic effect with probiotics to receive a uniform and reassuring response from the herd. One of the potentially useful materials to achieve this goal is biochar. In the present study, it was hypothesized that if biochar (as a favorable habitat for microorganisms) is added to probiotic-containing diets, the conditions for establishing these microbial products in the fermentation environment may be improved and probiotics can act more efficiently. Moreover, a probable synergy between probiotics and biochar may increase the efficiency of these additives compared to their separate usage. However, there is no special information available in this regard, especially about the effect of including biochar in probiotic-containing diets on different microbial populations and rumen hydrolytic enzymes. Therefore, this study aimed to investigate the potential of biochar in enhancing the effectiveness of the probiotic sources (Bacilli and/or Lactobacilli) on microbial populations, hydrolytic enzyme activity, digestibility, antioxidant capacity, and fermentation products in the sheep rumen, in vitro.
The experimental treatments were: 1. A basal diet without probiotics and biochar (control), 2. Basal diet containing probiotic Bacilli (B. coagulans, B. subtilis, and B. licheniformis, at the ratio of 2×1011, 5×109, and 5×109 CFU/g, respectively), 3. A basal diet containing probiotic Lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium, at the ratio of 2×109, 2×1010, and 2×1010 CFU/g, respectively), 4. A basal diet containing biochar, 5. A basal diet containing Bacilli-biochar, 6. A basal diet containing Lactobacilli-biochar, and 7. A basal diet containing Bacilli-Lactobacilli-biochar. Diet digestibility was determined by the two-stage Tilley and Terry method. In addition, the 24 and 72-h in vitro gas production techniques were conducted. At the end of each incubation, cellulolytic and proteolytic bacteria, as well as protozoa population, enzymatic activity (carboxymethyl cellulose, microcrystalline cellulose, filter paper degrading, and α-amylase), truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), methane release, antioxidant capacity, pH, ammonia-N (NH3-N), and volatile fatty acids (VFA) were determined by the standard methods. All in vitro tests were done in three replicates and two batches (runs) in different weeks. Data were analyzed in a completely randomized design using the GLM procedure of SAS.
Separate inclusion of probiotic Bacilli and biochar in the diet increased the cellulolytic bacteria population and activity of fibrolytic enzymes compared to the control (P<0.05), but these variables were not affected by Lactobacilli. The highest values of these variables were observed with the inclusion of biochar in probiotic Bacilli-containing diets (i.e., Bacilli-biochar and Bacilli-Lactobacilli-biochar treatments). These results were due to probiotics' ability to provide superior growth conditions for useful ruminal microbes, as well as the positive influence of the biochar structure, as a desirable habitat, on establishing, attaching, and developing rumen microorganisms. The protozoa population was not affected by biochar, but it was decreased (P<0.05) in the probiotic-containing diets (Bacilli and Lactobacilli treatments without or with biochar) in comparison to the control. The number of proteolytic bacteria and protease activity were not affected by the experimental treatments. Alpha-amylase activity in 24-h incubation in Basilli and Lactobacilli treatments was higher than the control (P<0.05) but was not affected by biochar. The activity of this enzyme in 72-incubation was lower in the control than in probiotic or biochar treatments (P<0.05). The alpha-amylase activity was the highest in the Lactobacilli-biochar and Bacilli-Lactobacilli-biochar groups. Separate inclusion of probiotics and biochar in the diet increased the diet digestibility, degraded substrate, and microbial biomass production (P<0.05). The maximum values of these parameters were detected in the probiotics-biochar diets. The reason for these increases can be related to the improvement of the cellulolytic bacteria population, alpha-amylase, and fibrolytic enzyme activity in the probiotic or biochar groups. The 24-h total antioxidant activity was not affected by the treatments. In 72-h incubation, the probiotics did not affect this variable, but a tendency to increase in antioxidant capacity was observed in diets containing biochar (without or with probiotics). The improving effect of biochar on the antioxidant power could be owing to its activity in trapping pollutants, poisons, and adverse factors in the incubation medium. Methane release, NH3-N, and acetate to propionate ratio were decreased, but total VFA was increased by separate use of the probiotics or biochar in the diet, compared to the control (P<0.05). More importantly, the inclusion of biochar in the diets containing the probiotic (i.e., Bacilli-biochar, Lactobacilli-biochar, and Bacilli-Lactobacilli-biochar) resulted in the greatest total VFA and the lowest NH3-N and methane release (P<0.05). One reason for the increased methane and ammonia was the lower protozoa population in the additives groups. The methane decline may also be related to the decreasing effect of the additives on methanogens, and their increasing effect on methanotrophs. Moreover, the improved bacterial biomass production could be considered as another reason for the decreased ammonia; i.e., more ammonia was assimilated into the bacterial protein. The increased VFA production was due to the higher digestibility and degraded substrate in the probiotic and/or biochar groups. Regarding the valuable characteristics of probiotics and biochar and their probable synergistic effects, their simultaneous application resulted in the highest improvement of fermentation variables.
Separate use of probiotics Bacilli and Lactobacilli or biochar in the diet improved in vitro ruminal microbial and enzymatic activity and reduced energy (as methane) and nitrogen (as ammonia) losses. More importantly, the addition of biochar to the probiotics-containing diets was a suitable strategy for enhancing the effectiveness of the probiotic sources on digestibility and microbial biomass and reduction of methane and ammonia. However, these results need to be confirmed by further experiments. The experimental treatments were: 1- basal diet without probiotic and biochar (control), 2 - basal diet containing probiotic Bacilli (B. coagulans, B. subtilis, and B. licheniformis, at the ratio of 2×1011, 5×109, and 5×109 CFU/g, respectively), 3- basal diet containing probiotic Lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium, at the ratio of 2×109, 2×1010 and 2×1010 CFU/g, respectively), 4- basal diet containing biochar, 5- basal diet containing Bacilli-biochar, 6- basal diet containing Lactobacilli-biochar, and 7- basal diet containing Bacilli-Lactobacilli-biochar. Diet digestibility was determined by the two-stage Tilley and Terry method. In addition, the 24 and 72-h in vitro gas production techniques were conducted. At the end of each incubation, cellulolytic and proteolytic bacteria, as well as protozoa population, enzymatic activity (carboxymethyl cellulose, microcrystalline cellulose, filter paper degrading, and α-amylase), truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), methane release, antioxidant capacity, pH, ammonia-N (NH3-N) and volatile fatty acids (VFA) were determined by the standard methods. All in vitro tests were done in three replicates and two batches (runs) in different weeks. Data were analyzed in a completely randomized design using the PROC GLM of SAS. Separate including probiotic Bacilli and biochar in the diet increased the cellulolytic bacteria population and activity of fibrolytic enzymes compared to the control (P<0.05), but these variables were not affected by Lactobacilli. The highest values of these variables were observed with the inclusion of biochar in probiotic Bacilli-containing diets (i.e., Bacilli-biochar and Bacilli-Lactobacilli-biochar treatments). These results were due to probiotics' ability to provide superior growth conditions for useful ruminal microbes, as well as the positive influence of the biochar structure, as a desirable habitat, on establishing, attaching, and developing rumen microorganisms. The protozoa population was not affected by biochar, but it was decreased (P<0.05) in the probiotics-containing diets (Bacilli and Lactobacilli treatments without or with biochar) in comparison to the control. The number of proteolytic bacteria and protease activity were not affected by the experimental treatments. Alpha-amylase activity in 24-h incubation in Basilli and Lactobacilli treatments was higher than the control (P<0.05) but was not affected by biochar. The activity of this enzyme in 72-incubation was lower in the control than in probiotic or biochar treatments (P<0.05). The alpha-amylase activity was the highest in Lactobacilli-biochar and Bacilli-Lactobacilli-biochar groups. Separate inclusion of probiotics and biochar in the diet increased the diet digestibility, degraded substrate, and microbial biomass production (P<0.05). The maximum values of these parameters were detected in the probiotics-biochar diets. The reason for these increases can be related to the improvement of the cellulolytic bacteria population, alpha-amylase, and fibrolytic enzyme activity in the probiotic or biochar groups. The 24-h total antioxidant activity was not affected by the treatments. In 72-hour incubation, the probiotics had no effect on this variable, but a tendency to increase in antioxidant capacity was observed in diets containing biochar (without or with probiotics). The improving effect of biochar on the antioxidant power could be owing to its activity in trapping pollutants, poisons, and adverse factors in the incubation medium. Methane release, NH3-N, and acetate to propionate ratio were decreased, but total VFA was increased by separate use of the probiotics or biochar in the diet, compared to the control (P<0.05). More importantly, the inclusion of biochar in the diets containing the probiotic (i.e., Bacilli-biochar, Lactobacilli-biochar, and Bacilli-Lactobacilli-biochar) resulted in the greatest total VFA and the lowest NH3-N and methane release (P<0.05). One reason for the increased methane and ammonia was the lower protozoa population in the additives groups. The methane decline may also be related to the decreasing effect of the additives on methanogens, and their increasing effect on methanotrophs. Moreover, the improved bacterial biomass production could be considered as another reason for the decreased ammonia; i.e., more ammonia was assimilated into the bacterial protein. The increased VFA production was due to the higher digestibility and degraded substrate in the probiotic and/or biochar groups. Regarding the valuable characteristics of probiotics and biochar and their probable synergistic effects, their simultaneous application resulted in the highest improvement of fermentation variables. Separate use of probiotics Bacilli and Lactobacilli or biochar in the diet improved in vitro ruminal microbial and enzymatic activity and reduced energy (as methane) and nitrogen (as ammonia) losses. More importantly, the addition of biochar to the probiotics-containing diets was a suitable strategy for enhancing the effectiveness of the probiotic sources on digestibility and microbial biomass and reduction of methane and ammonia. However, these results need to be confirmed by further experiments.

To be successful in rearing newborn calves, it is necessary to maximize feed efficiency and health status. One way is to use feed additives such as probiotics. Probiotics reduce the harmful microflora of the digestive tract (such as coliforms) and metabolic and infectious diseases and improve beneficial microbes, the defense system, nutrient absorption, feed consumption, and animal growth. However, the animal response to probiotics is not uniform. Therefore, if the conditions for the activity of probiotics in the digestive system are improved, it may be possible to increase the effectiveness of these microbial additives and achieve a more uniform and reproducible response. For this purpose, it may be possible to use biochar in diets containing probiotics. Biochar can absorb organic substances and gases, bind toxins, and provide a favorable environment for useful microorganisms, increasing fermentation efficiency and livestock performance. However, few findings are available on the effect of biochar, Saccharomyces boulardii (yeast), and different commercial lactobacilli mixtures in young calves. We hypothesized if biochar is included in diets containing probiotics, it could probably provide better conditions for the probiotic activity in the digestive system and thus improve the response of the calves to these additives. Therefore, this research investigated the effect of adding biochar (pomegranate and plum woods) in diets containing lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium mixture) and yeast (S. boulardii) on in vitro fermentation, microbial population, methane release, antioxidant capacity, as well as health indicators, rectum bacteria and blood enzymes of the pre- and post-weaning calves.
The experimental treatments were: 1. Basal diet without probiotics and biochar (control), 2. Diet + lactobacilli mixture, 3. Diet + S. boulardii, 4. Diet + biochar, 5. Diet +lactobacilli-biochar, and 6. Diet + S. Boulardii-biochar. The in vitro experiment was conducted with three replicates and two different runs. Digestibility coefficients were determined using Tilley and Terry method. A gas production technique was used to assess truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), cellulolytic bacteria, total protozoa, methane release, and antioxidant capacity. The in vivo experiment was done using 60 newborn Holstein calves (six treatments and 10 replicates) in a randomized complete block design. The start of the experiment was at the age of 10 d, weaning at 75 d, and the end of the experiment at 100 d. The daily intake and growth of the animals were recorded. In pre- and post-weaning calves, the rectum bacteria (coliforms, lactobacillus, and total aerobics), urinary and fecal pH, health score criteria (including scores of feces, nose, eye, ear, cough, temperature, navel, and joint), and total average health score were determined using standard methods. Moreover, the blood serum enzymes (alkaline phosphatase, Gamma-glutamyl transferase, aspartate aminotransferase, alanine transaminase, and lactate dehydrogenase) were assessed. Data were analyzed using the MIXED procedure of SAS.
Separate inclusion of the lactobacilli mixture, S. boulardii, and biochar in the diet improved in vitro digestibility, TDS, cellulolytic bacteria population, and antioxidant capacity compared to the control group (P<0.05) with maximum improvements when probiotic-biochar mixtures were used. The MBP and PF were improved by including the additives in the diet. Probiotics and probiotic-biochar mixtures decreased protozoa (P<0.05). Different additives decreased methane production, and the least methane was observed in diets containing lactobacilli-biochar and yeast-biochar (P<0.05). Probiotics provide better conditions for the growth and activity of appropriate microorganisms. In addition, the highly porous structure and high surface area of biochar increased the establishment, attachment, and growth of useful microbes. These properties improved cellulolytic bacteria, organic matter degradation, and MBP. The improved antioxidant status could be due to the effect of probiotics in eliminating oxidant compounds in the digestive system, and the activity of biochar in absorbing toxins and unfavorable factors in the fermentation environment. As a result, the simultaneous use of probiotics and biochar improved in vitro fermentation variables more effectively. The reduction of methane can be due to the decrease of protozoa and methanogens, and the increase of methanotrophs. The results of the second experiment (in vivo) showed the probiotics and biochar reduced the rectum coliforms and improved (reduced) the fecal score and average health score of pre- and post-weaning calves so that the greatest improvement was seen in probiotic-biochar treatments (P<0.05). Cough and body temperature scores also tended to improve by feeding additives. Alanine phosphatase, gamma-glutamyl transferase, aspartate aminotransferase, and alanine transferase were not affected by the treatments. However, lactate dehydrogenase in the pre-weaning period was lower in the additive treatments compared to the control group (P<0.05), and the minimum value was observed in probiotic-biochar groups. The use of probiotics can improve the rumen development, ruminal and intestinal population of useful microbes, and prevent diarrhea, which results in enhancing digestion, passage rate, feed consumption, and animal performance. Moreover, adding biochar enhances digestibility and microbial growth, binds toxins, reduces energy loss, and induces a suitable environment for beneficial microbes, resulting in better animal performance. The reduction of coliforms may be due to the binding of probiotics to the digestive system wall, nutrient competition, preventing harmful microbes, improving digesta flow, and changing the pH of the digestive tract. Moreover, biochar provides a suitable growth environment for beneficial microbes, removes toxins and unwanted substances from the digestion environment, reduces viscosity, and probably makes the digestive system unsuitable for harmful species including coliforms.
Separate inclusion of the lactobacilli mixture, S. boulardii, and biochar in the diet improved in vitro fermentation variables and the health status of pre and post-weaning Holstein calves, without negative effects on blood enzymes. The best in vitro and in vivo responses were observed when probiotics and biochar were used simultaneously. Therefore, the addition of biochar (1% of DM) to diets containing probiotics (lactobacilli and yeast; 2 g/d) can be recommended as a strategy to enhance the effectiveness of the probiotics in calves and to reduce environmental pollution due to the decreased methane emissions.

Pastures provide an important part of ruminant fodder needs, and farmers store some pasture plants to use in winter-feeding of livestock. Therefore, having enough information about the nutritional value of such plants can help to optimize their consumption in animal feeding. Completing the information on the good-quality plants adapted to different environments will help to use and develop these valuable resources in a better way. In addition, it may be possible to improve rumen fermentation by aromatic pasture plants. Adiantum capillus-veneris (Adiantum) and Salvia officinalis L. (Salvia) are valuable genetic resources in pastures that are also used in livestock feeding. Due to the presence of plant secondary metabolites, they may improve rumen fermentation. However, there is little information on the nutritional value of Adiantum. Some information is available on the chemical composition of Salvia species (especially the leaves) and the effect of its essential oil on the rumen, but data on the use of the whole plant as forage are limited. Therefore, this study aimed to assess the nutritional value of Adiantum and Salvia compared to alfalfa, and the effect of including different levels of them in the diet on in vitro ruminal fermentation variables and digestibility.
The experimental pasture plants (Adiantum and Salvia) were obtained in July, after flowering, from the pasture of Safrin village located in the rural district of Rajaeedasht in western Alamut (Qazvin, Iran). In experiment I, the chemical composition of the fodders was determined by the standard methods, considering alfalfa as the control forage. In experiment II, the effect of different dietary levels of the fodders was assessed using in vitro gas production technique with five treatments including 1. A diet without the rangeland plants (control diet), 2. A diet containing 15% of Adiantum, 3. A diet containing 30% of Adiantum, 4. A diet containing 15% of Salvia, and 5. A diet containing 30% of Salvia (on a DM basis). The in vitro digestibility, fermentation variables, microbial biomass production (MBP), total antioxidant power (TAP), protozoa numbers, and methane production were determined. The gas test was performed in three replicates and two runs, and data were analyzed using the GLM procedure of SAS.
According to the results of experiment I, Adiantum and Salvia had higher ash compared to alfalfa (P<0.05). The crude protein and digestibility of Adiantum were lower than Salvia and alfalfa (P<0.05). The lower digestibility of Adiantum could be due to its higher lignin and ash and the lower crude protein concentration. Therefore, when consuming Adiantum in diets of high-productive ruminants, it is necessary to pay more attention to the use of energy supplements. Compared to alfalfa, the in vitro fermentation of Adiantum and Salvia resulted in higher in vitro ruminal MBP and TAP and lower ammonia-N and protozoa population (P<0.05), due to the presence of plant secondary metabolites such as phenolics, tannins, flavonoids, anthocyanins, and terpenes. The results of experiment II showed that the inclusion of Adiantum and Salvia in the diet had no significant (P>0.05) effect on in vitro gas production, organic matter digestibility, and metabolizable energy. Dietary use of Salvia, however, enhanced truly degraded substrate (P<0.05). Sometimes, high amounts of secondary metabolites in plants may have negative effects on the rumen but the amounts of metabolites in the pasture plants used in this study were not so high as to hurt rumen microbes and digestion. Dietary inclusion of Adiantum and Salvia improved in vitro TAP and decreased protozoa population, methane production, and acetate to propionate ratio (P<0.05). The lowest methane production was observed for the diets containing Salvia. The positive effect of the rangeland plants on in vitro TAP was because both plants contain significant amounts of various secondary metabolites (such as phenolic compounds, flavonoids, and terpenes) that are known to be favorable and high-capacity antioxidants. These plant metabolites can remove free radicals, bind transition metals (such as free iron), remove active oxygen from the environment, induce antioxidant enzymes, and reduce and moderate the conditions of oxidative stress and destruction. The reduction of methane release using Adiantum and Salvia in the diet was probably related to the fact that plant secondary metabolites destruct the protozoa or interfere with their metabolic pathways. In addition, they may inhibit methanogenic and hydrogen-consuming bacteria or hydrogen-producing Gram-positive bacteria. The dietary addition of Adiantum and Salvia improved in vitro ruminal MBP and decreased the ammonia-N concentration (P<0.05) compared to the control diet. The result could be due to the reduction of the protozoa population, causing a decrease in nitrogen recycling in the rumen and a decrease in bacteria predation. The formation of phenolic-protein complexes can also be another possible reason for reducing protein breakdown and ammonia concentration. Moreover, as rumen protozoa are reduced, the bacteria will have fewer natural predators and their population (reflected in the higher MBP) will increase, improving the degradation of feed ingredients. The inclusion of Adiantum and Salvia in the diet did not affect in vitro pH, total volatile fatty acids, and partitioning factor (P>0.05).
It is possible to include whole plants of Adiantum and Salvia in the diet, up to 30% DM, without adverse effects on digestibility. It also improves diet efficiency by increasing in vitro ruminal MBP and TAP and reducing methane and ammonia production.

Sheep adapted to consume tannins rich feeds such as oak leaf (OL) appear to develop defensive mechanisms by their ruminal bacteria against these polyphenols. The capabilities of ruminal isolated tannins resistant bacteria from these animals to ferment a tanniniferous feed (i.e., pistachio hulls, (PH) which were incubated with rumen fluid from Holstein dairy cows was assessed. Six g positive cocci were isolated from the rumen of sheep and the 16s rRNA gene sequences showed them to be closely related to Streptococcus gallolyticus. In three runs of in vitro gas production (GP), the effect of two of the isolates incubated with bufferedruminal fluid of Holstein cow and PH was evaluated. The GP was recorded from 1 to 96 h of incubation. Incubating either of the isolates with PH caused a significantly higher in vitro gas production, estimated parameters, in vitro organic matter disappearance, metabolisable energy and volatile fatty acids than those without any isolate. The improvement in the ruminal parameters when either of the isolates was used suggested the possible presence of isolated tannins-resistant bacteria (Streptococcus gallolyticus sp.),however, in vivo studies must be conducted to confirm the in vitro results.

The nutritive value of the annual alfalfa (Medicago rigidulla, Medicago polymorpha and Medicago scutellata) species harvested at flowering stage was assessed by chemical composition, in vitro dry matter and nitrogen digestibility, in sacco dry matter and nitrogen degradation (0, 8, 12, 24, 48 and 72 h) and palatability (short-term intake rate, STIR) methods. Mean values of the chemical analysis results (%) for M. rigidulla, M. polymorpha and M. scutellata respectively were as follows: OM 85.1, 86.1 and 86.9 CP 25.1, 23.8 and 15.6 NDF 23.2, 24.8 and 30.0 ADF 18.3, 19.9 and 24.0 ADIN 0.36, 0.11 and 0.22 calcium 1.4, 1.3 and 1.2 phosphorus 0.23, 0.28 and 0.24 potassium 1.5, 1.5 and 1.4. The digestibilities of the DM and OM for M. rigidulla were 0.82 and 0.79 M. polymorpha 0.83.5 and 0.80 M. scutellata 0.75 and 0.69, respectively. The degradabilities of DM and CP at outflow rate of 0.05 for M. rigidulla were 0.72 and 0.55 M. polymorpha 0.71 and 0.57 M. scutellata 0.63 and 0.58, respectively. Finally, the palatabilities (using short-term intake rate method) for M. rigidulla, M. polymorpha and M. scutellata species were 13.6, 12.8 and 11.3 (g DM/min) respectively. According to the methods used, the descending ranking order (high to low) of these species on the basis of their nutritional value was M.rigidulla, M.polymorpha and M.scutellata.