Nutrient intake and blood profile of Nellore calves supplemented with cellulolytic fungi from rumen

ABSTRACT This study evaluates the effects of cellulolytic fungi from rumen on the nutrient intake and blood profile of Nellore calves. A diet was formulated with 56.79% lignified hay of Urochloa brizantha and 43.21% of concentrate. Sixteen Nellore calves were evaluated in completely randomized blocks using two calf groups: four males and four females. The first group was supplemented with a culture medium containing a mixture of Aspergillus terreus (VN 15 isolate) and Trichoderma longibrachiatum (VN 20 isolate). The second group received the sterile culture medium only. Ether extract ingestion was higher in the control calves; however, the supplemented calves showed a tendency to improve feed efficiency. No significant differences were detected in the blood profiles or serum biochemistry between the calf groups. Further research analyzing different dosages, adjusting the diet protein levels, and increasing the proportion of roughage could better elucidate the interference of these fungi on the performance and blood parameters of weaned Nellore calves.


Introduction
The use of tropical pasture alone during the dry season does not permit the sustainable productivity of cattle.However, the inclusion of probiotics or microbial additives represents a new alternative for improving the performance of these animals (Cholewińska et al. 2020;Soltan and Patra 2021).Additionally, after weaning, stress and health risks are high for calves because their immunity is reduced, they are also more susceptible to diseases related to alterations in the digestive tract during this period (Timmerman et al. 2005).
The microorganisms frequently used in feed are yeasts, filamentous fungi, and bacteria, which can colonize and multiply in the rumen and improve the degradation of plant fibres (Khan et al. 2016).These microorganisms may favour homeostasis in the rumen environment by reducing pathogenic microorganisms through competition or production of antimicrobial substances (Rode et al. 2001).
Anaerobic cellulolytic fungi from the rumen may produce enzymes with fibrolytic activity, which can increase the availability of amino acids and soluble carbohydrates (Gruninger et al. 2014).The action of these fungi favours the growth of rumen microorganisms and, consequently, the productivity of the ruminants.However, their use on an industrial scale is difficult given that these fungi are difficult to grow under laboratory conditions (Gruninger et al. 2014).
Anaerobic facultative fungi can be detected in the rumen ecosystem at concentrations of approximately 1 × 10 4 colonyforming Units (CFU)/mL in cattle raised on tropical pastures during the dry season (Abrão et al. 2014;Almeida et al. 2014).These microorganisms produce expressive activity of cellulases that may improve the digestibility of tropical forage (Abrão et al. 2017).In a previous study, beef cattle fed on lignified tropical pastures presented with Aspergillus spp.and Trichoderma spp. in the rumen (Abrão et al. 2014), both of which have shown potential for ruminant supplementation and biotechnological industries (Abrão et al. 2018).
Therefore, supplementing with fungi from the rumen may represent an alternative during the dry season, thereby contributing to improvements in cellulolytic microbiota in the digestive tract of ruminants.However, the effects of this supplementation on performance are not known, despite its influence on the nutrient intake capacity and blood parameters of cattle.
In addition, hematological analyses have contributed to the assessment of the health status and better performance of cattle, favouring an accurate diagnosis of possible diseases (Knowles et al. 2000), which could provide information on the food safety of the inclusion of these fungi in young cattle.The objective in this study was to analyze the effects of the inclusion of cellulolytic fungi on the nutrient intake and blood profile of weaned Nellore calves.

Study design
The experiment was carried out in the city of Montes Claros, North of Minas Gerais state, Brazil (646 m of altitude, 16°4 2 ′ 16 ′′ latitude south and 43°49 ′ 13 ′′ longitude west).The climate in this region is of the AW type (according to the Köppen classification), which is characterized by a long dry season in winter and a rainy season in summer.The average annual temperature is 24.2°C and the average annual rainfall is approximately 1,050 mm, with a drought period between April and October (INMET 2020).
In this study, we evaluated 16 weaned Nellore calves (pure of origin (PO)), approximately eight months old, in a randomized block design with two groups of four males and four females.The initial body weight (IBW) of these calves was 284.5 kg ± 38 kg.The diet was formulated according to the National Research Council (NRC 2016), for an average daily gain (ADG) of 400 grams (g) provided daily at 8:00 am and 3:00 pm (Table 1).To allow consumption at will, the quantities of food offered were adjusted daily according to leftovers and maintained at 5%.Water was provided ad libitum.The calves were housed individually, and all procedures adopted in this study were approved by the Ethics Committee on the Use of Animals of the Federal University of Minas Gerais (CEUA/UFMG) under number 209/2018.

Selected fungi evaluated
Two selected isolates of mycelial fungi were obtained from the rumens of Nellore steers raised on U. decumbens pastures supplemented with minerals and urea (Abrão et al. 2014).These fungi were identified by sequencing ribosomal DNA (rDNA), as reported by Altschul et al. (1997) and Abrão et al. (2014).The DNA sequences were analyzed using BLASTn v. 2.215 of BLAST 2.0 (Altschul et al. 1997).The species was considered an isolate with a similarity of 99% or more, and the sequences were deposited in the GenBank, with Aspergillus terreus [KF781532] and Trichoderma longibrachiatum [KF781535].

Experimental groups
In this study, the calves were divided into two groups of four males and four females for block analysis.One group of supplemented calves (SCs) with a mixture of T. longibrachiatum (isolated VN20) and A. terreus (isolated VN15) was compared to the control calves (CCs) group receiving only the culture medium.
The experiment was conducted for 70 days, with 15 days of adaptation to the diet and pens, anthelmintic treatment (albendazole 15 mg/kg subcutaneously), and vaccination against clostridial disease, followed by 55 days for the administration of the experimental diets.The calves were confined to individual pens, covered in a trough area, of 1.5 m (width) x 3.0 m (length), and located side by side.
The culture medium was composed of 2% U. decumbens hay, 2.5 g starch, 2.5 g soybean and 2.5 g dextrose in one litre of distilled water.Before the first feeding, the SCs were supplemented daily with 160 ml of culture medium containing 2.0 × 10 8 CFU/mL of T. longibrachiatum and 4.4 × 10 8 CFU/mL of A. terreus mixed with 200 g of concentrate.The CCs received 160 ml of sterile culture medium in the concentrate.

Sample collection
During the experimental period, the calves were weighed every 15 days using a mechanical balance (FIZIOLA®.model 3106000.São Paulo.Brazil).To minimize differences in filling between calves, the initial and final weights were followed by fasting from solids for approximately 16 h prior to feeding.The feed efficiency was calculated as the ratio of weight gain per day to dry matter ingestion per day.
Samples of the diets and feeds were analyzed for dry matter content at 105°C, ether extract, mineral matter, and crude protein according to the Association of Official Analytical Chemists (AOAC 1975), neutral detergent fibre (NDF), and acid detergent fibre (ADF), according to the methodology reported by Van Soes et al. (1991).Total carbohydrate (TCH) and nonfibrous carbohydrate (NFC) contents were obtained using the equations described by Sniffen et al. (1992).

Analysis of blood parameters
After the experimental period, eight mL of blood was collected via jugular venipuncture in tubes containing EDTA (BD Vacutai-ner® EDTA K 2 ) to measure blood parameters.The animals were immobilized in a containment chute and collections were performed between 7 and 11 am.

Statistical analysis
Each calf was used as an experimental unit.The statistical model used was as follows: in which Y ijk = average observation of the dependent variable in each plot, measured in the i th treatment class, at the j th block, and the k th replication; µ = effect of the overall average; T i = fixed effect of treatment classes, for i = 1and 2; b j = random block effect, for j = 1 and 2; and E ijk = the random error of the plot associated with i th and j th block, and the k th replication.
The initial live weight (ILW in kg) and final live weights (FLW in kg) were used to calculate the mean metabolic live weight (MLW).Daily weight gain (DWG) was analyzed from the slope coefficient of the straight line resulting from the regression of individual measurements of live weight without fasting as a function of time, using the REG procedure of SAS (2004).
The performance variables, nutrient consumption, and blood parameterswhich were normally distributed using the Shapiro-Wilk testwere subjected to variance analysis using the PROC GLM from SAS (2004).The means were compared using Student's t-test or Wilcoxon test at 5% probability.Additionally, P values between 0.05 and 0.1 were considered tending be significantly different.

Results
The inclusion of a microbial additive in the calves' diet did not change (P > 0.05) animal performance or nutrient intake, except for ether extract, which was higher than that of the control calves.The supplemented calves tended to have improved FE (Table 2, P = 0.07).
The concentrations of erythrocytes and other cells in the hemogram did not differ significantly (P > 0.05) and were within the reference ranges, except for MCV and MCHC (Table 3).Means of MCV in the control (37.2 ± 2.7 µ3) and treated (39.7 ± 4.7 µ3) groups were lower than the lower limit of normality.Conversely, the averages of MCHC (43.6 ± 2.0% to control calves) and (42.0 ± 2.5% to supplemented calves), extrapolated the normal range of 30-36% (Kaneko et al. 2008).
No significant differences were detected in the serum biochemical profiles of animals supplemented with an additive containing autochthonous anaerobic fungi (Table 4).Serum variables were within the reference ranges for cattle (Kaneko et al. 2008) except for ALT, glucose, albumin, globulin, and urea levels.Urea, ALT, and albumin levels were not within the minimum limits of normality (Meyer and Harvey 1998;Kaneko et al. 2008).However, glucose and globulin levels exceeded the reference values (Kaneko et al. 2008).

Discussion
In this study, the feed efficiency of the supplemented calves increased by 19.7%, with lower CEE levels in these animals; however, these FE did not promote a significant improvement in performance for the WG of these animals, which could be explained by the fact that the supplied diet consisted of 43.2% of the total dry matter consumed.This proportion of concentrate could underestimate the cellulolytic action of microorganisms because the presence of excess volatile fatty acids (VFA) can limit the growth of A. terreus, as reported by Goularte et al. (2011).The A. terreus strain evaluated in this study did not produce toxins and tolerated different concentrations of VFAs.However, its growth was reduced when acetic, propionic, and butyric acids were used at a ratio of 50:40:10 ( Abrão et al. 2018).
Another study that evaluated the effect of Saccharomyces cerevisiae or Aspergillus niger on fermented Napier grass mixed with fresh cassava root in beef calves (Piamphon et al. 2017), observed that dry matter intake was similar among the evaluated calf  groups.However, inoculation with the two fungi improved crude protein intake and nutrient digestibility and did not change blood biochemistry, blood enzymes, or hematological parameters in growing beef cattle.The mechanism of action of these fungi and their fermentation products, which optimize animal performance, is complex, and the effects vary according to the strains used, administered doses, and individual factors of the animals, such as physiological status and stress (Chiquette 1995).
In the present study, the calves had an average age of eight months, which may have influenced the effects of the evaluated fungi.The effects of fungal inclusion may be more evident in younger animals, as reported by Adeyemi et al. (2019).
In a study on newly weaned calves, the inclusion of S. cerevisiae in the diet increased FLW (P = 0.001) and DWP (P = 0.04), and did not influence DMI (P > 0.05), indicating a trend towards improvement in FE and immune response (Chiquette 1995).The DGW results observed in this study corroborated those reported by Riddell et al. (2010), who observed that supplementing calves with a probiotic containing Bacillus spp.did not result in changes in weight gain.
When analyzing the blood parameters of the evaluated calves, the analysis of the hemoglobin content of the animals supplemented in this study is similar to the results described by Jones and Allison (2007) for Holstein Friesian x Kankrej calves supplemented with Aspergillus spp.(9.07 ± 0.08 g/dL).Kapadiya et al. (2019) did not observe any changes in this parameter in Brahman calves crossed with a native Thai breed.
The calves evaluated in this study showed concentrations of erythrocytes, hemoglobin, and HCM within physiological parameters, indicating that hematopoiesis was not compromised in animals supplemented with fungal isolates.In a study on the supplementation of Enterococcus faecium and Saccharomyces cerevisiae in weaned Jersey calves, Bet Flores et al. (2019) also did not find hematological changes in supplemented animals.
The glucose concentrations observed in both groups evaluated in this study were extrapolated to reference values (Kaneko et al. 2008) and does not resemble what is suggested by the NRC (2016) which states that, with advancing age, the absorption capacity of glucose by the small intestine decreases due to the development of ruminal functions, an increase in the microbial population, and the production of short-chain fatty acids that become a source of energy for the calves.However, the parameters established for weaned Nelore calves raised under tropical conditions are not yet available in the literature and should be established and investigated in future studies.Piamphon et al. (2017) observed that plasma glucose levels in dairy calves decreased significantly after weaning because of the switch from glycolytic metabolism to glycogenic metabolism, which is typical of cattle becoming functional ruminants.
Elevated globulin levels, as observed in both groups of calves evaluated in this study, may suggest infectious diseases, recent vaccinations, or alterations in protein metabolism, although the latter is more forceful in terms of inflammatory processes (Hayashi et al. 2006).The steers were vaccinated and dewormed prior to the experimental period.Additionally, the calves showed no clinical signs of infectious diseases.Therefore, it is believed that animals may have responded adequately to the vaccines used, indicating adequate functioning of the immune system.
In this study, the albumin concentrations in the calves did not meet the minimum limits of normality (Kaneko et al. 2008).Albumin can reflect the protein content in the diet, although changes are slow, owing to its half-life of approximately 20 days (González 2009).Hypoalbuminemia can alter the metabolism and concentration of other substances given that albumin acts as a transporter of free fatty acids (Hayashi et al. 2006).The albumin and urea concentrations in this study corroborated the pattern of dietary protein deficiency described by González et al. (2000).The albumin and globulin values observed in this study support a negative correlation between these proteins.The elevation of globulin inhibits the synthesis of albumin in the liver to maintain the total protein level and, consequently, the osmotic pressure in the blood (González 2009).
The plasma urea concentration observed in this study does not obey the minimum limits established by Kaneko et al. (2008) and differed from those reported by González (2009) when they evaluated beef cattle on native pasture in southern Brazil (24.7 ± 1.7 mg /dL).In this study, the diet contained the amount of protein recommended for young cattle according to the NRC (2016).However, calves with rigorous genetic selection to improve weight gain were evaluated, which may have increased their requirement for this nutrient.Thus, a higher level of protein in the diet should be evaluated in future studies, which would allow for better expression of these fungi to improve the performance of weaned calves.Adequate levels of plasma urea indicate a correct balance between proteins and fermentable carbohydrates because this synchronicity between nutrients can increase the concentration of urea in the blood and its excretion in milk and urine (Kaneko et al. 2008).

Conclusions
The addition of a microbial additive containing cellulolytic fungi to the diet improved feed efficiency and reduced the ingestion of ether extract during the experimental period; however, it did not significantly alter the productive performance or blood parameters of Nelore calves.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Table 1 .
Nutritional composition of the diet to evaluated calves (g/kg of dry matter).

Table 2 .
Analysis of variance for performance and nutrient intake of Nellore calves supplemented or not with microbial additive.

Table 3 .
Mean concentrations and standard error of the mean of erythrogram, leukogram, and platelet count of Nellore calves supplemented or not with microbial additive.

Table 4 .
Mean concentrations and standard error of the mean of the serum biochemical profile of Nellore calves supplemented or not with microbial additive.Supplementation with facultative anaerobic fungi: Aspergillus terreus (VN 15 isolate) and Trichoderma longibrachiatum (VN 20 isolate)