Effect of Effective Microorganisms treated Wheat Bran supplementation on intake, digestibility and milk yield and composition of lactating cows fed natural pasture hay as a basal diet

E T Tadesse, G A Minneeneh, Y Y Mummed, M Dejene and E Kebreab

African Center of Excellence for Climate-Smart Agriculture and Biodiversity Conservation, Haramaya University, P O Box 138, Dire Dawa, Ethiopia
eden.tesfaye27@gmail.com

Abstract

A study was conducted to evaluate the effect of different levels of effective microorganisms (EM)-treated wheat bran (EMWB) supplementation replacing concentrate mixture (EM) on intake and digestibility of nutrients and on milk yield, composition of lactating dairy cows. Twenty cross-bred lactating cows with an average milk yield of 8-10 L per day were assigned in a randomized complete block design (RCBD) to four treatment groups (EM0: ad libitum natural pasture hay (NPH) + 0.5 kg of concentrate mixture (CM)/liter of milk (control); EM33: ad libitum NPH + 66% CM + 33% CM replaced by EMWB; EM66: ad libitum NPH + 33% CM + 66% CM replaced by EMWB; EM100: ad libitum NPH + 100% CM replaced by EMWB). Cows were fed for 105 days 15 days for adaptation followed by a 90-day feeding trial. The crude protein (CP) contents were 6.43, 13.7 and 15.9%, and the NDF contents were 73.8, 46.3 and 38.7% for NPH, CM and EMWB, respectively. Supplementation of EM-treated wheat bran improved (p<0.001) the total DM and nutrient intake of cows in EM33, EM66, and EM100 compared with the control group. The apparent digestibility of most nutrients (DM, OM, CP and ADF) was greater for EM66 and EM100 than EM0. Milk yield was in the order of EM0 < EM33 < EM66 = EM100. Milk protein, SNF, and ash contents were lower for EM0 than for EM66 and EM100, while milk fat and lactose contents were unaffected by treatment. Therefore, it can be concluded that sole EMWB can be used as a supplement to lactating cows, replacing CM with better performance.

Key words: crude protein, milk protein, nutrient intake

Introduction

Among the livestock sectors production, dairy production plays an important role in Ethiopia, serving as a source of food and income. However, the productivity of dairy cattle in the country is low (Duguma et al 2012; FAO 2019), mainly due to feed shortages in both quantity and quality, both of which are subjected to seasonal variation in availability (Mengistu et al 2017; FAO 2019). Livestock feed in the country is derived mainly from unimproved natural pastures and crop residues (Tolera 2008). These roughages are rich in fiber and deficient in nitrogen, limiting ruminal fermentation and nutrient supply for microbial synthesis and to the animal (Santra and Karim 2003). Hence, manipulation of the digestion process through feed supplementation or use of probiotics is imperative not only to improve the utilization of these available feed resources and increase the productivity of ruminants but also to reduce methane production (Arowolo and Jianhua 2018).

Modern dairy farms target high milk production by utilizing feed composed of high concentrates to meet the metabolic demand of higher milk production. Such a feeding system is associated with metabolic dysfunction, such as rumen acidosis (Beauchemin et al 2008). Therefore, probiotic supplementation has been observed as a good alternative feed additive to prevent rumen acidosis, minimize the use of antibiotics by decreasing the load of pathogenic bacteria, improve dry matter intake and feed conversion efficiency, enhance nutrient utilization efficiency and production performance, stimulate and activate immune cells, reduce methane production, thereby minimizing energy loss, and generally promote growth and health performance as well as meat and milk production in ruminants (Chaucheyras-Durand et al 2012; Khan et al 2016; Arowolo and Jianhua 2018).

Effective microorganisms (EM) are a mixed culture of aerobic and anaerobic microbes living symbiotically with each other. These microorganisms are beneficial, natural, free-living, and safe. As a mixed culture of beneficial and naturally occurring microorganisms, EM contains selected species of microorganisms, including predominant populations of lactic acid bacteria and yeasts and smaller numbers of photosynthetic bacteria, actinomycetes, and other types of organisms (Higa 1994).

The use of EM in animal husbandry is widely accepted in many parts of the world. In a study conducted in Russia by Belookov et al (2019), EM was successfully used as a feed additive in cattle rations to increase the productivity and meat quality of calves. Similarly, studies conducted in Egypt by Yacout et al (2021) showed that the use of EM as a feed supplement improved digestibility in sheep. Likewise, several studies have been conducted in Ethiopia to investigate the effect of EM on enhancing the nutritive value of crop residues. For instance, Gulilat and Walelign (2017) and Asmare et al (2020) indicated that treating rice straw with EM improved intake, digestibility, and milk yield of lactating cows. Furthermore, studies have evaluated the effect of feeding EM-treated grass hay supplemented with niger seedcake on dry matter digestibility, average daily gain, and economic gain (Fikre et al 2019; Roba et al 2022). However, to date, most studies have focused on EM-treated roughages or supplementation of EM-treated wheat bran. The effects of a mixed ration containing different proportions of EM-treated wheat bran on intake, digestibility, and milk yield and composition have not been investigated. Therefore, the objective of this study was to evaluate the effect of different levels of effective microorganisms (EM)-treated wheat bran supplementation on the intake and digestibility of nutrients and on the milk yield and composition of lactating dairy cows.

Materials and methods

Study Site

The study was carried out at the Holetta Agricultural Research Center of the Ethiopian Institute of Agricultural Research (EIAR). The center is located at 9° 00’ N latitude and 38° 30’ E longitude, approximately 28 km west of Addis Ababa. The study area is situated at an altitude of 2400 meters above sea level and receives a mean annual rainfall of approximately 1444 mm. The mean minimum and maximum temperatures are 6 and 22 °C, respectively (Kitaw et al 2012).

Experimental animals and management

Twenty high-grade (75% blood level) cross-bred (Friesian x Boran) lactating cows with similar lactation performance (8-10 L of milk/day), the same stage of lactation (mid lactating, i.e., three months after calving) and body weight (380 – 420 kg) but different parities (two through five) were selected from the total dairy herd available at the station. All cows were weighed and drenched with broad-spectrum anthelminthic (2500 mg albendazole) before the start of the experiment.

Effective microorganisms -treated wheat bran preparation

An adequate quantity of activated EM packed in 20L plastic bottle was purchased from Weljeji PLC found in Bishoftu town, and molasses was purchased from the Ethiopian Sugar Corporation, Wenji branch. EM was diluted by mixing 1 litter of EM, 1 litter of molasses, and 18 litter of water at a ratio of 1:1:18. Then, 20 liters of diluted EM solution was poured gradually into 50 kg of wheat bran and mixed well. The mixture was put into a concrete hole that did not permit air entry to maintain anaerobic conditions and was protected from direct sunlight. Then, it was allowed to ferment for 21 days. After 21 days, EM-treated wheat bran was ready for use when it had a sweet fermented smell.

Experimental design and treatments

The twenty lactating cows were randomly assigned to the treatments in a complete randomized block design (CRBD). Cows were blocked based on parities and randomly allotted to one of the four dietary treatments, making five animals in each treatment. Cows in the control (EM0) were fed natural pasture hay ad libitum as a basal diet and 0.5 kg concentrate mixture per litter of milk produced per day. The major species of hay were Andropogon, Hyperrnenia, Cyperacea and Trifolium spp. The concentrate mixture was composed of 35% wheat bran, 20% maize, 21% rice bran, 3% molasses, 4% niger seedcake, 11% sunflower cake, 3% salt and 3% limestone. Cows in the 2^nd, 3^rd, and 4^th groups received the same basal diet but with 33, 66, and 100% of the concentrate mixture replaced by EM-treated wheat bran (treatments EM33, EM66 and EM100). Cows were fed for 105 days, of which 15 days were used for adaptation followed by 90 days for the feeding trial for measurements of intake, digestibility of nutrients, milk yield and composition and body weight change.

Chemical composition of experimental feed ingredients

Samples of natural pasture hay, wheat bran, EM-treated wheat bran, and concentrate mixture were analyzed for dry matter (DM), crude protein (CP), and total ash using procedures described by AOAC (1990). The organic matter (OM) content was calculated as 100 − ash content. The CP content was calculated by multiplying the nitrogen content by a factor of 6.25. The acid detergent fiber (ADF), acid detergent lignin (ADL) and neutral detergent fiber (NDF) of the samples were determined using the method of Van Soest and Robertson (1985).

The chemical composition of the experimental feeds is given in Table 1.

Feed intake and nutrient digestibility

Natural pasture hay was fed ad libitum and a concentrate mixture and/or EM-treated wheat bran supplemented daily to the lactating dairy cows was offered twice daily in two equal halves. Data on feed offered and feed refused were collected daily. Feed intake was determined for each animal as the difference between the quantity of feed offered and refused. Samples of feed offered and feed refusals of each cow were collected on a daily basis. The feed offered was bulked per feed type, and the refusal was bulked over the experimental period for each treatment.

An apparent digestibility trial was conducted after the feeding trial with the same animal used for the feeding trial. Daily total fecal output along with the daily feed offered and refusal were weighed and recorded for five consecutive days for each animal at the end of the experimental period. To minimize errors in feces collection, farm personnel were assigned around the clock to scoop feces into plastic buckets when the animals were defecating. Urinal contamination was minimized by frequent washing of the concrete floor with high-pressure running water using a plastic water hose. Individual cow feces were weighed every morning before 0800 hours and before feeds were given to the animals. The feces from each cow were thoroughly mixed, and a sample of 1% was taken and placed in a polyethylene bag. Composite samples of the daily collected fecal samples were mixed and stored in a deep freezer (-20) until the end of the collection period. At the end of the collection period, the pooled samples were thawed and mixed thoroughly and subsampled. The samples were oven dried at 65 for 72 hours, ground to pass through a 1-mm sieve, and stored in sample bottles at room temperature. Grabs of feed samples from each feed and refusal from each animal were collected each day to make a composite feed sample for each feed and refusal per treatment. The apparent digestibility of DM and nutrients was determined using the formula:

Milk yield and composition

The cows were milked twice a day at 0500 and 1600 hours, and milk yield was recorded individually for each animal throughout the experimental period. Aliquots (100 ml) of morning and evening milk were collected every two weeks to determine major milk components (milk fat, protein, lactose, and total solids). The sampling bottles were properly cleaned and sanitized before samples were taken to the Holetta Agricultural Research Center dairy laboratory. Milk composition (milk fat, protein, lactose, and total solids) was analyzed using a Lactoscan machine (Ultrasonic Milkanalyzer).

Body weight change

Body weights of the experimental cows were measured in the morning before feed and water were offered at the beginning and end of feeding trial using a digital scale. Body weight (BW) change was calculates as a difference of the final and initial BW.

Statistical analysis

All data from the feeding and digestibility trial and milk yield and compositions were analyzed using analysis of variance (ANOVA) following the general linear model (GLM) procedure of the SAS statistical program (SAS 2010). Means were separated using the least significant difference (LSD). The model used for analysis was Yijk = μ + αi + βj + εijk, where μ = overall mean; Yijk= observation of the jth block and the ith treatment; αi = effect of treatment i; βj = effect of block j; and εijk = experimental error.

Result

Dry matter and nutrient intake

Total dry matter and nutrient intakes for lactating crossbred cows fed natural pasture hay as a basal diet and different levels of concentrate mixture and/or EM-treated wheat bran as a supplement are presented in Table 2. Supplementation of EM-treated wheat bran improved (P<0.001) the total mean DM intake (DMI) and OM intake (OMI) of cows in EM33, EM66, and EM100 compared with the control group (EM0), but no difference was observed between EM33 and EM66. The mean DM and OM intake of cows supplemented with 100% EM-treated wheat bran (EM100) were higher (p<0.0001) than those of the other treatments. The CP intake increased (p<0.0001) with increasing levels of EM-treated wheat bran supplementation. NDF intake was unaffected by treatment (p>0.05), while effects on intakes of ADF and ADL did not follow a consistent trend.

Dry matter and nutrient digestibility

The apparent digestibility of dry matter and nutrients for the experimental animals are given in Table 3. The apparent digestibility of DM and all nutrients measured varied (p<0.05) among treatments. The mean digestibility of OM, CP, NDF, and ADF was highest (P<0.001) in cows fed natural pasture hay ad libitum supplemented with 100% CM replaced by EM-treated wheat bran (EM100) over the other treatments. The OM, CP and ADF digestibility values were also higher for EM66 than for EM0. Except for CP digestibility, all other digestibility parameters were similar for EM0 and EM33.

Milk yield and composition

There was difference (p<0.0001) among treatments in mean daily milk yield (Table 4). Daily milk was lowest for EM0, intermediate for EM33 and highest (p<0.001) for EM66 and EM100. Among the analyzed milk compositions, protein, solid not fat (SNF), and ash varied (p<0.05) among treatments (Table 4). Milk protein content was lower for EM0 than for EM66 and EM100, while the value for EM33 was similar to all other treatments.

The SNF was greater for EM66 and EM100 than for EM0 and EM33. The ash contents of milk were lower for EM0 than for EM66 and EM100. EM-treated wheat bran supplementation did not affect the lactose and fat contents of the milk (p>0.05).

Body weight change

The effect of supplementing different level of EM-treated wheat bran replacing concentrate mixture had no effect (p>0.05) on body weight of lactating cows under all treatments (Table 5).

Discussion

DM and nutrient intake

Animal dry matter and nutrient intake are inversely proportional to the concentration of cell wall components (NDF and ADF) and directly proportional to the dietary CP content (Bosa et al 2012; De Carvalho et al 2017). Therefore, higher dry matter and nutrient intakes were observed in cows supplemented with EM-treated wheat bran compared to the control (EM0), which could be the result of the difference in CP and fiber fraction of the EM-treated wheat bran and concentrate mixture used in this study. From the laboratory analysis results, it is observed that NDF and ADF of EM-treated wheat bran are lower than that of the concentrate mixture. In contrast, the CP content of EM-treated wheat bran was higher than that of the concentrate mixture used in the current study. In addition, the higher total DM and nutrient intake of cows in EM100 and EM66 may be related to the favorable rumen environment due to EM addition, such as microbial growth and production of fiber-degrading enzymes, which could have resulted in enhanced fermentation, rate of breakdown, and rate of digestion of the feed and resulted in a greater DM and nutrient intake (McDonald et al 2010).

One of the factors that affects feed intake and digestibility is the microbial density in the rumen. Effective fiber colonization of microbes in the rumen reduces the retention time of fiber components due to the production of fiber-degrading enzymes (Seo et al 2010). This result agrees with Gulilat and Walelign (2017) and Chalchissa and Arega (2018), who reported higher DM and nutrient intake in lactating dairy cows fed EM-treated rice and barley straw compared to untreated straws. Similarly, Roba et al (2022) reported higher DM and nutrient intake in a ram-fed ration containing EM-treated sugarcane bagasse and rice husk.

Apparent digestibility

Apparent digestibility of DM and nutrients was improved through EM-treated wheat bran supplementation in the current study. The improved DM and nutrient digestibility with EM-treated wheat bran supplementation may be associated with improved rumen fermentation efficiency and microbial biomass production, increased number and/or activity of rumen microbiota, and enhanced rumen microbial colonization and attachment to the digesta (Olafadehan 2013; Mahrous et al 2021). Similarly, Kitaw et al (2016) reported that the apparent DM and nutrient digestibility of lactating cows fed barley straw treated with EM were significantly improved compared with those of the control group. Roba et al (2022) and Fikre et al (2019) also reported improved digestibility of nutrients in rams fed rations containing EM-treated sugarcane bagasse, rice husk and EM-treated hay respectively.

Milk yield and composition

Milk yield and composition are the interaction of many elements within the cow and external environments (O’Connor 1994). The difference in milk yield of cows placed in different treatment groups in the current study might be related to differences in intake and digestibility of DM and nutrients. Cows in EM100 and EM66 had greater intake and digestibility than cows in EM33 and EM0. Increased feed intake and digestibility are positively correlated with the milk yield of cows (Mosavi et al 2012; Gulilat and Walelign 2017). The results of the current study were in agreement with those of Gulilat and Walelign (2017) and Asmare et al (2020), who reported that there was an increase in the milk yield of cows fed EM-treated rice straw. Similarly, Kitaw et al (2016) reported that feeding EM-treated barley straw to lactating cows improved milk yield and minimized the use of concentrate mixtures for lactating cows.

The high protein content of milk from cows supplemented with EM100 and EM66 could be due to the high CP intake and digestibility of cows grouped under EM100 and EM66. Diets have a considerable effect on protein and fat content of milk (Moran 2005). This finding is in line with the findings of Kitaw et al (2016) and Gulilat and Walelign (2017), who reported that the protein content of milk of cows fed EM-treated barely and rice straw was improved when compared with the control treatment. Similarly, the ash and solid-not-fat (SNF) content of milk showed an increase with EM-treated wheat bran supplementation. This might be due to intake and digestibility improvement by effective microorganisms that result in the supply of more nutrients in milk. It is believed that the SNF content can fall if the cow fed a low-energy diet (O’Connor 1994).

There was no difference (p>0.05) in the fat content of milk of cows among treatments, which might be related to the lack of differences in NDF intake of cows grouped under all treatments. It has been noted that cows in low roughage rations yield milk with lower fat content than cows fed a higher proportion of roughage diet (Moran 2005). There was also no difference (p>0.05) in the lactose content of milk among the treatment groups. This is expected, as the lactose content of milk remains unaffected by dietary changes (Moran 2005). Similarly, Kitaw et al (2016) reported that the lactose content of milk was not affected by feeding EM-treated barely straw.

Body weight change

There was no difference (p>0.05) in body weight of lactating cows among the treatment groups. This might be due to the lactation stage of the cows and improved milk yield. It is known that body weight is low but stable at the mid-lactation stage, and nutrient intake at this stage is partitioned toward milk production (Moran 2005). Similarly, the study of Gulilat and Walelign (2017) indicated that feeding EM-treated rice straw did not affect the body weight of lactating cows.

Conclusions

Supplementing EM-treated wheat bran to replace the concentrate mixture in lactating cows at 66 and 100% levels (EM66 and EM100) improved feed intake, apparent digestibility, milk yield, and protein and solid not fat content of milk compared to EM0. Therefore, it can be concluded that sole EM-treated wheat bran can be used as a supplement to lactating cows to replace concentrate mixtures with better performance.

Acknowledgments

The authors would like to acknowledge African Center of Excellence for Climate-Smart Agriculture and Biodiversity Conservation, Haramaya University and Ethiopian Agricultural Research Institute, Addis Ababa for funding this study.

References

AOAC 1990 Official methods of analysis, sixteenth ed. Association of Official Analytical Chemists, Arligton, Virgina, USA.

Arowolo M A and He J 2018 Use of probiotics and botanical extracts to improve ruminant production in the tropics. Journal of Animal Nutrition, 4, 241-249. https://doi.org/10.1016/j.aninu.2018.04.010

Asmare B, Bishaw F and Gezie T 2020On-farm evaluation the effective microbes and urea treated rice straw on performance of local dairy cows and farmers perception in Ethiopia. AgroLife Science Journal, 9, 35-44.

Beauchemin K A, Kreuzer M, O’Mara F and McAllister T A 2008 Nutritional management for enteric methane abatement: a review. Australian Journal of Experimental Agriculture, 48(2), 21-27. http://doi.org/10.1071/EA07199

Belookov A, Belookova O, Zhuravel V, Gritsenko S, Bobyleva I, Ermolova E, Ermolov S, Matrosova Y, Rebezov M and Ponomarev E 2019 Using of EM-technology (effective microorganism) for increasing the productivity of calves. International Journal of Engineering and Advanced Technology, 8 (4), 1058-1061.

Bosa R, Faturi C, Gildeli H, Vasconcelos R, Cardoso A M, Ramos A F O and de Azevedo J C 2012 Intake and apparent digestibility with different inclusion levels of coconut meal for sheep feeding. Acta Scientiarum Animal Science, 34 (1), 57–63. http://doi.org/10.4025/actascianimsci.v34i1.11936

Chalchissa G and Arega A 2018 Evaluation of effective microbe treated barely straw supplemented with bypass protein as intervention diet for crossbred dairy animal under small scale farmer’s condition. Journal of Biology, Agriculture and Healthcare, 8 (11), 74-78.

Chaucheyras-Durand F, Chevaux E, Martin C and Forano E 2012 Use of yeast probiotics in ruminants: effects and mechanisms of action on rumen pH, fiber degradation, and microbiota according to the diet. In: Probiotic in animals: pp 119-152. https://doi.org/10.5772/50192

De Carvalho G G P, Rebouças R A, Campos F S, Rufino L D A, Azevedo J A G and Cirne L G A 2017 Intake, digestibility, performance, and feeding behavior of lambs fed diets containing silages of different tropical forage species. Journal of Animal Feed Science and Technology, 228,140–148. https://doi.org/10.1016/j.anifeedsci.2017.04.006

Duguma B, Kechero Y and Janssens G P J 2012 Productive and reproductive performance of Zebu X Holstein-Friesian crossbred dairy cows in Jimma town, Oromia, Ethiopia. Global Veterinaria, 8, 67-72.

FAO 2019 Ethiopia. Availability and utilization of agroindustrial byproducts as animal feed. Rome. 64 pp.

Fikre T, Alemu B and Ayele S 2019 Effect of effective microorganism treated grass hay supplementation on feed intake, digestibility and growth performance of washera sheep fed natural grass hay as a basal diet. International Journal of Scientific and Research Publication, 9(12), 845–853. http://doi.org/10.29322/IJSRP.9.12.2019.p96109.

Gulilat L and Walelign E 2017 Evaluation of milk production performance of lactating Fogera cows fed with urea and effective microorganisms treated rice straw as basal diet. Journal of Agricultural Research and Development, 7, 0111-0119. http://dx.doi.org/10.18685/EJARD(7)2_EJARD-17-011

Higa T 1994 Effective microorganisms: a new dimension for nature farming. In: Proceeding of the Second International Conference on Kyusei Nature Farming. US Department of Agriculture, Washington, DC, USA, pp. 118-124.

Khan R U, Naz S, Dhama K, Karthik K, Tiwari R, Abdelrahman M M, Alhidary I A and Zahoor A 2016 Direct-fed microbial: beneficial applications, modes of action and prospects as a safe tool for enhancing ruminant production and safeguarding health. International Journal of Pharmacology, 12, 220-231. https://doi.org/10.3923/ijp.2016.220.231

Kitaw G, Daba T, Assefa G, Dejene M, Fekadu D and Kehaliw A 2012 EM Technology on Performance of Lactating Crossbred Cows and Barn Malodor Reduction. In proceeding of National Workshop on Effective Microorganisms in Ethiopia. Addis Ababa, Ethiopia, 20 March 2012. Institute of Agricultural Research Addis Ababa. P. 14-26.

Kitaw G, Kehaliw A, Feyissa F and Assefa G 2016 Evaluation of Activated Effective Microorganisms (EM-2) as Biological Crop Residue Treatment Option Targeted for Feeding Crossbred Dairy Cattle. Ethiopian Journal of Animal Production, 16, 17-35.

Mahrous A A, El-Tahan A A H, Hafez Y H, EI-Shora M A, Olafadehan O A and Hamdon H 2021 Effect of date palm (Phoenix dactylifera L.) leaves on the productive performance of growing lambs. Tropical Animal Health and Production, 53 (1), 1–8. https://doi.org/10.1007/s11250-020-02493-2

McDonald P, Edwards R A, Greenhalgh J F, Morgan G A, Sinclair L A and Wilkinson R G 2010 Evaluation of Foods: Digestibility, Animal Nutrition, Seventh ed. Pearson Educational Limited, Edinburgh, Great Britain.

Mengistu A, Kebede G, Feyissa F and Assefa G 2017 Review on Major Feed Resources in Ethiopia: Conditions, Challenges and Opportunities. Academic Research Journal of Agricultural Science and Research, 5 (3), 176-185. http://doi.org/10.14662/ARJASR2017.013

Moran J 2005 How the Rumen Works. In: Moran, J., Ed., Tropical Dairy Farming: Feeding Management for Small Holder Dairy Farmers in the Humid Tropics, Landlinks Press, Melbourne, 41-49.

Mosavi G H R, Fatahnia F, Alamouti M H R, Mehrabi A A and Kohi D H 2012 Effect of dietary starch source on milk production and composition of lactating Holstein cows. South African. Journal of Animal Science, 42, 201-209. https://doi.org/10.4314/sajas.v42i3.1

O’Connor C B 1994 Rural dairy technology. ILRI training manual No. 1. International Livestock Research Institute (ILRI), Addis Ababa, Ethiopia. 133p.

Olafadehan OA 2013 Feeding value of Pterocarpus erinaceus for growing goats. Animal Feed Science and Technology, 185 (1-2), 1–8. https://doi.org/10.1016/j.anifeedsci.2013.05.014

Roba R B, Letta M U, Aychiluhim T N and Minneeneh G A 2022 Intake, digestibility, growth performance and blood profile of rams fed sugarcane bagasse or rice husk treated with Trichoderma viride and effective microorganisms. Heliyon, 8, 1-8. https://doi.org/10.1016/j.heliyon.2022.e11958.

Santra A and Karim S A 2003 Rumen manipulation to improve animal productivity. Asian-Australian Journal of Animal Science, 16, 748-763. https://doi.org/10.5713/ajas.2003.748

SAS Institute 2010 SAS^®/ STAT Software Release 9.0. SAS Institute, Inc., Cary, NC.

Seo J K, Kim S W, Kim M H, Upadhaya S D, Kam D K and Ha J K 2010 Direct-fed microbials for ruminant animals. Asian-Australian Journal of Animal Science, 23, 1657-1667. http://doi.org/10.5713/ajas.2010.r.08.

Tolera A 2008 Feed resources and feeding management: A manual for feedlot operators and development workers. Ethiopia SPS-LMM - Texas Agricultural Experiment Station (TAES)/TAMU, Addis Ababa.

Van Soest P J and Robertson J B 1985 In: Analysis of Forages and Fibrous Foods a Laboratory Manual for Animal Science, 613. Cornell University, Ithaca, New York.

Yacout M H, Badawi E L AY, Khalel M S, Atia S E and Hassen A A M2021 Impact of effective microbes (em) bokashi supplementation on nutrients digestibility, rumen fermentation and gas volume production in diets of sheep. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Animal Science and Biotechnologies, 78(2), 66-75. https://doi.org/10.15835/buasvmcn-asb:2021.0009