Effect of fibrolytic microbes and enzymes on nutrient and energy intake in crossbred calves

Introduction

In rural regions of our nation, raising livestock is a crucial economic activity. Livestock has long been considered one of humanity's oldest resources. It offers additional income to many families reliant on agriculture and to numerous landless households. Additionally, it supplies protein through milk. Livestock also contributes to income through draught power, organic manure, hides, and skins, providing a steady income throughout the year. To boost milk production and enhance farmers' earnings, a comprehensive strategy is necessary to advance the animal production sector. This involves the effective management of young animals with appropriate nutritional care and oversight (Anonymous, 2012) [3] . However, challenges such as rapid growth and high feed consumption can undermine the advantages and economic viability of these practices. India boasts a diverse agro-ecological landscape. The rising demand for feed and fodder, coupled with diminishing natural resources, presents new and varied challenges. The available feed resources, including dry roughages, green fodder, concentrates, and crop residues (straw and stovers), are insufficient and expensive to meet the nutritional needs of growing cattle and buffaloes. Walli et al. (2006) [36] suggested that the issue of feed scarcity could be addressed by adopting alternative feed technologies, potentially enhancing nutrient supply within the ruminant system. The capacity of young dairy calves to effectively process fibrous feeds significantly impacts nutrient intake, energy availability, and subsequent growth performance. In crossbred calves, the development of rumen functionality occurs gradually, and the early establishment of fibrolytic microbial populations is often inadequate to support optimal breakdown of plant cell wall components. Consequently, the intake and utilization of fiber-rich diets during the early growth phase may be limited, resulting in a reduced energy supply (Suárez et al., 2006) [33] .

In India, the poor quality of native grasses, crop residues, and agro-industrial by-products is a common issue in animal feed. Therefore, utilizing alternative feed resources derived from agro by-products is a practical approach in low-input systems (Dayani et al., 2012) [12] . Microorganisms have been isolated from the rumen, and through the use of fungi, bacteria, and enzymes, it is possible to enhance feed intake, growth, and feed efficiency in young animals. Numerous studies have documented improvements in fiber digestion and animal performance with the use of feed additives (Adesogan et al., 2007; Bala et al., 2009) [1,6] . Feed additives can enhance gut health in animals, potentially leading to a higher digestion rate and improved growth performance (Frizzo et al., 2010; Kawakami et al., 2010; Frizzo et al., 2011) [16,19,15] . The combination of direct-fed microbial (DFM) and exogenous fibrolytic enzyme (EFE) has shown significant positive effects on the growth performance and feed conversion efficiency of crossbred calves without negatively impacting their behavior (Kocyigit et al., 2016) [21] . However, some studies have reported no effect or even negative effects (Bowman et al., 2003, Vicini et al., 2003, Baloyi, 2008) [11,35,7] . In young calves, limited microbial activity and enzymatic capacity can hinder fiber breakdown. Nutritional strategies aimed at boosting rumen fibrolytic activity, such as supplementing with direct-fed microbes and exogenous fibrolytic enzymes, have been investigated to enhance nutrient and energy intake. Fibrolytic microbial additives are known to maintain rumen microbial balance and encourage the growth of cellulolytic bacteria, while exogenous enzymes may improve fiber hydrolysis through complementary or synergistic actions within the rumen environment (Beauchemin et al., 2003) [10] . Previous research has shown improvements in fiber digestibility and feed intake following such supplementation; however, responses can vary and are influenced by diet composition, enzyme characteristics, and animal age (Beauchemin & Holtshausen, 2010) [9] . Dietary supplementation with feed additives, especially the combined use of direct-fed microbes and exogenous fibrolytic enzymes, has been reported to enhance fiber digestibility, support gut health, and improve growth performance, feed efficiency, and nutrient utilization in ruminants (Adesogan et al., 2007; Dean et al., 2013; Lunagariya, 2019) [1,13,23] . Information on the combined effects of fibrolytic microbes and enzymes on nutrient and energy intake in crossbred calves, particularly under tropical feeding systems, remains limited. Therefore, the present study was undertaken to evaluate the influence of dietary supplementation of fibrolytic microbes and enzymes on nutrient and energy intake in crossbred calves. Many researchers have reported that yeast culture improves feed intake (Robinson and Erasmus, 2009; Ayad et al., 2013); feed conversion efficiency, growth rate (Kumar et al., 2015) [28,5] and nutrient digestibility (Segal et al., 2008) [29] in cost-effective ways (Hutjens, 2003) [17] . The positive effect of yeast on blood hematology results in improved animal health status (Agazzi et al., 2014) [2] . Dey et al. (2004) [14] ; Paul et al. (2004) [26] ; Tripathi et al. (2007) [34] used anaerobic fungi in ruminants and observed increased intake and digestibility of lignocellulosic feeds. Considering above facts in mind an experiment was planned to evaluate effect of fibrolytic microbes and enzymes on Nutrient and Energy Intake in Crossbred Calves.

Materials and Methods

The research took place over 140 days at the Livestock Research Station of Anand Agricultural University (AAU) in Anand. All experimental activities adhered to the ethical guidelines sanctioned by the Institutional Animal Ethics Committee of the College of Veterinary Science and Animal Husbandry, AAU, Anand (Approval No. 285/LPM/2018). The study involved 20 crossbred calves (Holstein Friesian × Kankrej), which were randomly divided into five dietary treatment groups. Each group consisted of one male and three female calves. At the beginning of the study, the calves had similar body weights (85.70±6.37 kg) and ages (167.55±21.70 days), ensuring consistency across the groups.

The feeding plan was designed to meet the nutrient requirements as recommended by ICAR (1998) [18] . Each calf was individually fed with green hybrid napier fodder (CO-3 variety), a concentrate mixture, and sorghum hay. The fibrolytic fungi, bacteria, and enzymes used for feed treatment were isolated and purified from animals housed at the same research facility by the Department of Animal Biotechnology, Veterinary College, AAU, Anand. The calves were allowed to exercise freely for two hours daily between 08:00 and 10:00, with clean drinking water available at all times. Additional water was provided twice daily at 12:00 and 14:00. Measured amounts of hybrid napier fodder (treated or untreated as per the treatment), concentrate mixture, and sorghum hay were offered at 10:00, 14:00, and 16:00, respectively. The control group received untreated hybrid napier fodder, while the treatment groups were given hybrid napier treated with the fibrolytic fungus Aspergillus spp. (1 × 10⁷ per g of feed), fibrolytic bacteria Escherichia spp. (1 × 10⁶ CFU per g of feed), xylanase enzyme (50 ml/kg fodder containing 1.2 IU/ml), or a combination of one-third dose of each additive for the Fungus Fed (FF), Bacteria Fed (BF), Enzyme Fed (EF), and Consortium Fed (CF) groups, respectively. For fodder treatment, 2 kg of hybrid napier was thoroughly mixed with the respective microbial or enzyme preparation about 20 hours before feeding. The treated fodder was spread evenly on a cemented floor, covered with a plastic sheet, and mixed twice during the incubation period before being offered to the calves. Moisture loss during treatment was accounted for when distributing the treated fodder equally among animals within each treatment group. Hybrid napier fodder and concentrate mixture were provided in amounts sufficient to minimize leftovers, while sorghum hay was given ad libitum to meet the remaining nutrient needs. Any leftover feed was collected and weighed the next morning. Feed allowances were adjusted every two weeks based on changes in body weight. Feed and fodder samples were collected weekly and analyzed for dry matter and crude protein content using standard analytical procedures outlined by AOAC (1995) [4] . Nutrient intakes such as CP, DCP, TDN, EE, NFE, CF, and related components were calculated based on dry matter intake. The experimental data were statistically analyzed using a Completely Randomized Design as described by Snedecor and Cochran (2014) [32] .

Results and Discussion

Nutrient and Energy Intake

The information regarding dry matter and nutrient intake is detailed in Table 2. There was no significant difference (p>0.05) in dry matter intake (kg/day, kg/100 kg BW, g/kg W 0.75 ) among Crossbred Calves fed with various Fibrolytic Microbes and Enzymes. Although the T 2 group exhibited a higher dry matter intake (kg/day, kg/100 kg BW, g/kg W 0.75 ) compared to the other group, it did not significantly impact the crossbred calves. The increased dry matter intake in the fungus-fed group might be attributed to the enhanced palatability of the feed. However, despite the rise in dry matter consumption with fungal feed, there was no variation in average daily gain. The enzyme-fed group showed a higher average daily growth than the other treatments, as the enzyme-containing dry matter intake was lower. The current findings align with Morsy et al. (2016) [25] , who found that daily feeding of 40 g of two commercial enzyme products did not significantly affect dry matter intake in Egyptian buffaloes. Lunagariya (2016) [24] also reported that the intake of dry matter and nutrients (crude protein, digestible crude protein, and total digestible nutrient) in Holstein crossbred cows was not significantly (p>0.05) affected by feeding exogenous fibrolytic enzymes supplemented TMR (240 mg/kg TMR) over a 140-day trial. Barbadikar (2012) [8] demonstrated that daily dry matter intake (3.63 vs. 3.69 kg), percentage dry matter intake (2.77 vs. 2.77 kg), DCP intake (313.51 vs. 320.51 g), and TDN intake (2.07 vs. 2.10 kg) in crossbred calves were not significantly different (P>0.05) between the control and fibrolytic enzymes (0.025%) group of crossbred calves. Kim et al. (2012) [20] indicated that feeding TMR with probiotics (Lactobacillus acidophilus: KCTC3140 and Saccharomyces cerevisiae: KACC30068) had no significant effect on daily feed intake in Hanwoo steers during the experiment. Dey et al. (2004) [14] also reported findings consistent with the present study, showing non-significantly/numerically lower dry matter intake (4.04±0.34 vs. 4.08±0.52kg/day), higher DCP intake (394.18±36.43 vs. 368.65±41.89 g/day), and higher TDN (2.46±0.22 vs. 2.16±0.31 kg/day) when feeding fungal culture (106 CFU/ml) at 160 ml/week to calves. Similarly, administering 250 ml of Neocallimastix sp. GR1 culture (≈106 tfu/ml) every fourth day to Murrah female buffalo calves resulted in similar dry matter intake (4.12±0.20 vs. 4.14±0.17 kg); however, DCP (295.39±14.86 vs. 275.13±9.68) and TDN (2.46±0.13 vs. 2.19±0.09) intake increased significantly in Murrah female buffalo calves (Sehgal et al., 2008) [29] over a 90-day period. Kumar et al. (2015) [22] also noted that feeding anaerobic fungal zoospores of Neocallimastix sp. GR-1, incorporated in wheat straw-based complete feed blocks (140×106 zoospores/block of 14 kg), did not significantly affect dry matter and DCP intake, whereas TDN intake was significantly (p<0.05) higher in 5-7 month old Murrah buffalo male calves, with values of 3.47±0.17 and 3.39±0.09 kg/day; 251.43±14.71 and 277.64±17.76 g/day; 2.14±1.55 and 2.25±1.43 kg/day, respectively, for the control and treated group. According to Sharma et al. (2018) [31] , in contrast to the current study, dry matter intake (1426.9 vs. 1402.9 g/day) of Murrah buffalo calves was improved (p<0.05) in the probiotics (Lactobacillus acidophilus) fermented milk feeding (200 ml daily with 108 CFU/ml) groups compared to the control. According to Shahzad et al. (2016) [30] , male Nili Ravi buffalo calves fed total mixed ration (TMR) containing 33% fungus-treated wheat straw (Arachniotus sp.) consumed more dry matter (2.95 kg/day) than control TMR or 67 and 100% fungus-treated wheat straw (2.88, 2.80, and 2.71 kg/day). Crossbred calves fed various feeds Fibrolytic Microbes and Enzymes did not significantly (p>0.05) differ in their intake of crude protein and digestible crude protein (kg/day, kg/100 kg BW, g/kg W0.75). In comparison to another group, the T 2 group consumed more crude protein (kg/day, g/kg W0.75) and digestible crude protein (kg/day), albeit this difference was not statistically significant in crossbred calves. The study's findings are consistent with those of Dey et al. (2004) [14] , who found that ruminal fungus had no discernible impact on the DCP intake of 13-month-old calves. For the control and fungal culture (Orpinomyces sp), the daily DCP consumption was 368.65±41.89 and 394.18±36.43 g/head, respectively. Sehgal et al. (2008) [29] reported 275.13±9.68 and 295.39±14.86 g/head/day in the control and fungus groups, respectively, for Murrah buffalo calves (8-11 months old) fed anaerobic fungus Neocallimastix sp. However, in buffalo calves (15 months old), Tripathi et al. (2007) [34] discovered a substantial impact on DCP intake from a complete feed mixture based on wheat straw, both with and without Orpinomyces sp. or Piromyces sp. culture administration. For the Control, Orpinomyces, and Piromyces groups, they reported DCP intake of 415+23.8, 464+24.2, and 453+10.9 g/head/day, respectively. Barbadikar (2012) [8] found that crossbred calves in T 1 [concentrate combination (60%) + wheat straw (40%)] and T 2 [concentrate mixture (60%) + wheat straw (40%) with fibrolytic enzymes @ 0.025%] consumed more DCP (313.51±16.33 and 320.51±12.23 g), respectively. The outcome indicated that fibrolytic enzyme had no discernible impact on crossbred calves' consumption of DCP. The ether extract, crude fibre, and nitrogen free extract intake (kg/day, kg/100 kg BW, g/kg W 0.75 ) were not significantly (p>0.05) differed in Crossbred Calves fed different feeding Fibrolytic Microbes and Enzymes. The energy intake includes total digestible nutrient intake (kg/day, kg/100 kg BW, g/kg W 0.75 ) and calculated metabolizable energy intake (Mcal/day, Mcal/100 kg BW, Kcal/kg W 0.75 ) were not significantly (p>0.05) differed in Crossbred Calves fed different feeding Fibrolytic Microbes and Enzymes. In line with present investigation, Dey et al. (2004) [14] discovered non-significant effect of ruminal fungus on TDN intake of 13 month old calves and the value were 2.16±0.31 and 2.46±0.22 kg, respectively for control and fungal cultures given (Orpinomyces sp). Higher TDN consumption levels of 2.07±0.11 and 2.10±0.08 kg/head/day were observed by Barbadikar (2012) [8] for T 1 [concentrate combination (60%) + wheat straw (40%)] and T 2 [concentrate mixture (60%) + wheat straw (40%) with fibrolytic enzymes @ 0.025%] in crossbred calves. This finding indicated that fibrolytic enzyme had no discernible impact on TDN intake in crossbred calves. According to Ramaswami et al. (2005) [27] , TDN consumption for 216day-old crossbred calves fed diets supplemented with or without L. acidophilus was 2.31±0.06 and 2.34±0.09 kilogram/100 kg body weight, respectively. The variations, however, were not statistically significant. The reports and the current study are comparable. In contrast with the present study Tripathi et al. (2007) [34] showed substantial effect of a complete feed mixture based on wheat straw without and with Orpinomyces sp. or Piromyces sp. culture administration in buffalo calves (15month age) on TDN consumption. For Group I (Control), Group II (Orpinomyces), and Group III (Piromyces), their calculated TDN intake was 2.5±0.1, 2.9±0.2, and 3.0±1.0 kg/head/day, respectively. According to Sehgal et al. (2008) [29] , the anaerobic fungus Neocallimastix sp. had a substantial impact on the TDN consumption (kg/head/day) of Murrah buffalo calves aged 8 to 11 months. The TDN intake values in the control and treatment groups were 2.19±0.09 and 2.46±0.13 kg, respectively. The value of TDN intake was higher than the present study.

Conclusion

The present study indicated that supplementation of fibrolytic microbes, exogenous fibrolytic enzymes, or their consortium did not significantly affect dry matter, nutrient, or energy intake in crossbred calves. Intake of CP, DCP, EE, CF, NFE, TDN, and metabolizable energy remained comparable among all treatment and control groups. Although the fungus-fed group showed a numerically higher dry matter intake, it did not result in improved growth performance. The enzyme-fed group exhibited better growth efficiency despite relatively lower dry matter intake, suggesting improved nutrient utilization. Overall, fibrolytic additives did not adversely influence feed intake. These findings support the safe use of fibrolytic microbes and enzymes in calf diets under tropical feeding systems.

Acknowledgement

AcknowledgementsWe really thank the College of Veterinary Science and Animal Husbandry, Anand Agriculture University, Anand, for its excellent assistance and cutting-edge facilities, which have been crucial to our academic and professional development.

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