Effect of adding biochar in diets containing probiotics on in vitro fermentation variables, health indicators, rectum bacteria, and blood enzymes of Holstein calves

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.