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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Effect of Feeding Nano and Neem Coated Urea on Nutrient Intake, Digestibility and Blood Chemistry of Lactating Crossbred Cow

M
Mamata Joysowal1,*
T
Tapas K. Dutta2
B
Bibeka N. Saikia3
A
Anupam Chatterjee2
G
Gunaram Saikia1
M
Mousumi Hazorika3
L
Lakhyajyoti Borah1
1Department of Animal Nutrition, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
2Division of Animal Nutrition, Eastern Regional Station, National Dairy Research Institute, Karnal-132 001, Haryana, India.
3Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.

ABSTRACT

Background: With diminishing communal grazing lands, cost-effective protein supplementation for ruminant livestock is crucial. Utilizing nano-formed urea and a novel nano-formed nitrogen source, in conjunction with balanced energy sources, is anticipated to decrease the feeding costs for ruminant livestock. Nano-urea alone or with neem-coated urea supplementation may be used as good economic source of nitrogen, if found suitable.

Methods: Two doses of Nano-urea (160 µL Nu) and combined with neem-coated urea (160 µL Nu+1% Ncu) were selected for lactation trial based upon in vitro rumen fermentation system.

Result: Addition of Nano-urea and combination of Nano-urea and neem-coated urea enhanced voluntary intake of different nutrients and increased DCP value in crossbred dairy cows. TDN intake pattern was similar among all treatment groups. However, DCP intake was greater (p<0.001) and  increased blood urea nitrogen  in T1 and T2 compared to T0 groups. However, Feeding nano-urea and combination of nano-urea + neem-coated urea marginally benefitted in terms of feeding economics for milk production.

INTRODUCTION

Nano-Urea as a source of non-protein nitrogen may be a potential strategy to reduce the overall emission intensity of nitrogenous compound through milk production system of dairy animals, since nano-urea is prepared with nano-technology. Soybean meal (SBM), groundnut cake (GNC), mustard cake, tiloil (sesame) cake has long been used as a prominent source of crude protein for ruminants (Koukolová et al., 2020). However, with its increasing price, the use of these protein sources results in ultimately higher cost of production. Therefore, the use of urea as a protein (NPN source) replacement is attractive in ruminant diets because of its low cost compared with other protein feeds such as soyabean meal with high rumen degradability (Wanapat, 2009; Xin et al., 2010; Muralidharan et al., 2016). Driven by the gradual loss of community pastureland in India, progressive farmers are increasingly adopting intensive dairy farming systems. This shift necessitates a focus on cost-effective agribusiness, where a primary challenge lies in optimizing rumen function. Specifically, farmers must synchronize the availability of energy and nitrogen from feed to maximize nutrient utilization, thereby overcoming the traditionally high costs associated with protein nutrition for ruminant livestock (Alipour et al., 2020). As a non-protein nitrogen source, urea is a popular, low cost and easily obtained protein supplement (Wahyono et al., 2022). Slow-release urea (SRU) is a coated non-protein nitrogen (NPN) source for ruminant nutrition (Salami et al. 2020; Manju et al., 2022). These products are usually available for feeding to all ruminant species (cattle, buffalo, sheep and goat).Rumen bacteria can convert NPN sources into true protein (Simoni et al., 2023). Therefore, use of urea (NPN compound) is cost effective as ruminant animal can convert non protein nitrogen into true protein that can be utilized by host animal for production purpose. 

MATERIALS AND METHODS

The research  was conducted during the period of  August, 2023 to December, 2024 at the experimental facility within the laboratories of animal nutrition department, Instructional  livestock cattle farm ILF©, college of veterinary science which is located in Khanapara,  Assam, India. Eighteen crossbred (Jersey × Holstein Friesian) cow (weight between 350 kg to 400kg  were equally divided into 3 dietary treatment groups denoted as T0, T1 and T2. Animals were distributed to each square based on parity and lactation stage. Live weights were measured using electronic/electrical balance. Prior to commencement of the experiment, the animal underwent deworming. Intake parameters  was recorded at the fortnight basis of each experimental period. The animals were randomly allocated to receive one of the experimental diets which are TMRs based diet i.e. Concentrate: Green roughage: Dry roughage (40:45:15), where T0 (control; only TMR diet); T1 (addition of 160 µL Nano urea on the diet DM basis in the TMR based diet); T2 (addition of 160 µL Nano urea+1%neem coated urea in the TMR based diet DM basis). Nano urea product produced by IFFCO which is available in the local market. Diets were formulated to meet the nutrient requirement of cows according to NRC (2001) recommendations. Amounts of feed offered and orts of each cow were weighted daily and were restricted to 5 to 10% of intake as-fed basis.
       
Throughout the 120days of duration of the experiment, the  animal in each group adhered to their respective feeding schedules and had access to their allocated feed.So, there was continuous monitoring of  growth performance and nutrient intake of the experimental animal were using established protocols. Throughout the experiment, experimental animals received consistent access to clean drinking water and their daily feed consumption was precisely measured using a double pan balance. Bi-weekly, between 8:00 and 9:00 AM, the weight of each animal was recorded to monitor growth. For the estimates of digestibility of the diets total 6day collection period was fixed. This analysis aimed to estimate the intake, digestibility and availability of various nutrients, as per the procedures laid out in AOAC, (2012). Analysis of minerals were done using atomic absorption spectroscopy (AAS). Blood samples were collected from the jugular vein of all experimental cows at 0, 60 and 120 days of feeding under aseptic conditions. Some relevant blood metabolites (Protein, albumin, globulin, glucose, blood urea nitrogen) estimated by commercially available kit.
 
Statistical analysis
 
Data related to the chemical and mineral composition of different samples were statistically analyzed by using one way analysis of variance (ANOVA). The intake of nutrients during digestion trial, blood parameters were analyzed by a mixed ANOVA with repeated measures, where treatment was a fixed factor (between subject effect), time period (within-subject) was a random factor having subject (cow) nested within it and their (period treatment) interaction was evaluated. A computerized IBM SPSS 26.0 software program was used for ANOVA. Tukey’s HSD test was used to measure the differences of means.

RESULTS AND DISCUSSION

Chemical composition and mineral profile of experimental diet 
 
The chemical and mineral compositions of various feed components including maize grain, wheat bran, mustard oil cake, soya bean cake, rice polish, paddy straw and maize fodder are provided in Table 1. Mustard oil cake had comparatively  higher protein content than soyabean cake (32.99 vs. 26.65%).Samples of total mixed ration collected from study area had maximum CF (13.95%) and AIA (2.58%). Wheat bran had the maximum NDF and hemicelluloses content while soyabean oil  cake contained highest ADF, cellulose and lignin levels. Maize grain contained least ADF, cellulose and lignin among all the concentrates. The crude protein content of mustard cakes and soyabean oil  cakes observed in the present study were well within the range reported by earlier workers (Chowdhury et al., 2010 and Kumar et al., 2002). The proximate compositions and fibre fractions of feedstuffs analyzed in the present study are also within the normal range (Yadav et al., 2018). The diverse chemical compositions and fiber fractions observed in feedstuffs can be attributed to variations in oilseed varieties and agronomic factors, including fertilizer application, harvest timing and field drying practices.The DM, OM, CP, EE, total ash, T-CHO, NFC, NDF, ADF, hemicelluloses, cellulose and lignin content of concentrate mixture and green fodders were within the normal range as reported by Ranjhan, (1991) for Indian feeds and fodders. The results on macro and micro mineral profile of concentrate feeds collected from dairy farms are presented in Table 2, respectively. Compounded cattle feeds exhibited the optimum concentrations of major minerals (Ca, P, Mg) and micro minerals (Cu, Zn, Mn, Fe, Co) compared to other concentrate feeds as per Ranjhan, (1991).

Table 1: Chemical composition on (%DM basis) and mineral contents of different feed ingredients used for the preparation of total mixed ration.



Table 2: Chemical composition and mineral profile of total mixed ration diets used during experimental period.


 
Evaluation of chemical and mineral composition in different TMR based feeds
 
Table 2 outlines the chemical and mineral composition of the rations. While DM, OM, EE, TCHO, ADF, hemicellulose and lignin were consistent among the three TMRs, NDF and cellulose were higher in T1 and T2 compared to T0. However, three TMRs had a similar concentration of Ca and P. TMR T1 and TMR T2 contained greater amounts of Mg, Cu, Zn, Mn, Co and Fe compared to T0, which may be due to addition of nano urea may influence the bioavailability of some of major and trace minerals.
       
Compounded cattle feeds provided an optimal balance of essential minerals, showing the highest concentrations of major minerals (Ca, P, Mg) and ideal levels of micro minerals (Cu, Zn, Mn, Fe, Co) among all concentrate feeds (Yadav et al., 2018). Of all the concentrates, maize grain demonstrated the lowest concentrations of several macro and micro minerals, specifically Mg, Cu, Zn, Mn and Co.
 
Intake, apparent digestibility and nutritive value of feeds
 
Table 3 summerizes intake performance of  digestion trial. Although total DM intake was significantly (p<0.001) lower in T2 than T0 and T1, but DMI per 100 kg BW and g/kg W0.75 was statistically similar among three treatment groups. Table 3 also depicts the value of DMI (g)/kg W0.75 was 101.88, 105.69 and 98.21 g in crossbred dairy cows under T0, T1 and T2 groups, respectively. However, diets containing Nano-urea and combination of Nano-urea and neem-coated urea resulted in increased (p<0.001) CP intake in T1 and T2 compared to T0. CPI (g)/kg W0.75 was estimated 10.45, 13.61 and 13.83 g in T0, T1 and T2 treatments, respectively. In contrast to our findings, Salami et al. (2020) reported no significant effect on overall dry matter intake (DMI) and crude protein intake (CPI) in beef cattle fed slow-release urea. Similarly, neutral detergent fiber (NDF) intake and apparent dry matter digestibility remained consistent across all treatment groups. Consistent with our findings, Salami et al. (2021) reported a significant decrease (P<0.05) in dry matter intake (DMI, -500 g/d) in slow-release urea treated groups compared to controls. These results can be explained by the increased ruminally available nitrogen, following a rise in ammonia nitrogen concentration. This optimal nitrogen availability likely stimulated microbial growth, leading to an apparent increase in dietary intake (Wahyono et al., 2022). Conversely, McGuire et al. (2013) and Xu et al. (2019) stated that dry matter intake (DMI) and crude protein intake (CPI) are higher in sheep who receive urea supplementation than  non-supplemented animals. Different results were shown by Kozloski et al. (2007) and Currier et al. (2004), who reported that supplemental urea to ruminants consuming low-quality roughage does not affect an animal’s dry matter intake (DMI). Despite consistent dry matter intake (DMI), we hypothesize that urea supplementation altered the intake of other nutrient components. Therefore, it can be enumerated that nano urea likely created the most favorable conditions for rumen function compared to other treatments in the present study. We did not find any report in relation to the utilization of nano-urea in ruminant species. Based on the current observations it may be interpreted that feeding nano-urea has resulted increased feed intake pattern compared to control (T0) and combination of both (Nu + Unc). Probably, neem-coated urea did not produce any tangible impact on the intake performance in lactating crossbred cows.

Table 3: Nutrients intake and nutrients digestibility during digestion trial in lactating cows.


       
These variations in nutrient intake, observed with urea supplementation in sheep, may stem from several factors, including differences in crude protein (CP) concentration and the quality of the basal diets (Wang et al., 2015) differences in the chemical and/or physical characteristics of the forage/feedstuff (Sano et al., 2011) differences in the slow-fast degradable carbohydrate and/or forage-concentrate composition of the basal diets (Zhao et al., 2007; Galina et al., 2007).The digestibility’s of organic matter (OM) (p<0.001) and crude protein (CP) (p<0.001) were notably higher in T1 and T2 compared to control T0 (Table 3). Despite supplementation with Nano urea and a combination of Nano-urea + neem-coated urea, the digestibility coefficients of dry matter, ether extract, nitrogen-free extract, NDF and ADF remained unchanged.These outcomes indicated that rumen degradable nature of Nano and neem coated urea may enhance nitrogen availability in the rumen in the treatment groups compare to the control groups. Comparing our findings with earlier workers is difficult, because no published reports are available on the utilization of Nano-urea in ruminant diets.However, Cherdthong et al. (2011) found that the urea and slow-release urea (urea-calcium sulfate and urea-calcium chloride) did not influence dry matter intake. However, improved CP and OM digestibility was found in the Nano urea and Nu +Ncu added groups, which also supported by Phillip et al. (2023); Alizadeh et al. (2019); Sevim et al. (2019). However, the effect of urea and non-protein nitrogen sources on digestibility depends on their concentration, physical structure and adaptation period. Increased digestibility of optigen and coated urea could be due to these treatments supplied enough nutrients for microorganisms in the rumen. Earlier it was reported that SRU in rations had no effect on DM, NDF and ADF digestibility (Xin et al., 2010). With some exceptions, feeding FGU and SRU did not affect nutrient intake and digestibility on lactating dairy cow (Simoni et al., 2023). 
 
Availability of nutrients and nutritive value of TMR feeds
 
Data pertaining to nutrients intake in all the treatment groups (T0, T1 and T2) was presented in the Table 4. TDN intake pattern was similar among all treatment groups. However, DCP intake was greater (p<0.001) in T1 and T2 compared to T0 control. The results demonstrated that DCP percentage was significantly higher (p<0.001) in treatment groups (T1 and T2) compared to control T0. Digestible NDF value of TMR feeds remained similar in the groups, indicating that dairy animals likely adjusted their feed intake to maintain a consistent digestible NDF intake in all treatment groups. Phillip et al. (2023) reported that there was no significant (p<0.05) differences found for total digestible nutrients (TDN) value among tested groups; Acetic acid addition to urea-treated rice straw in dairy ewes resulted in a significant (p<0.05) increase in digestible crude protein (DCP). This enhancement, coupled with the fulfillment of daily dry matter (DM), total digestible nutrients (TDN) and DCP intake recommendations outlined by NRC (2001), which specifies an energy requirement of 1.67 Mcal/kg for dairy cows, demonstrates effective nutritional management.

Table 4: Availability of different nutrients in lactating cows fed with different TMR based rations.



Blood chemistry of the experimental animal
 
Plasma total protein concentration found significant in terms of treatment effect (P=0.032) and period effect (p<0.001) and no significant difference found for interaction between treatment and period (T×P). Notably, we did not find any difference among treatment groups in relation to plasma albumin, globulin and albumin: globulin ratio (Table 5). The periodic effect was highly significant (p<0.001) for globulin, but the interaction (T×P) effect was statistically similar for albumin, globulin and albumin/globulin ratio. Moreover, plasma glucose concentration increased significantly (p<0.001) with different period of time. The BUN (mg/dL) value was significant (P=0.002) increased in T2 Groups compare to the other. Periodic effect was also higher (p<0.04) for this parameter. Moreover, significant (p=0.027) interaction effect was also found for treatment and period. But, for bilirubin, there was no significant (p<0.05) difference found   for treatment effect (T), period effect (P) and interaction between treatment and period effect (T×P).

Table 5: Blood metabolites in animals fed with different TMRs.


       
However, our findings are in close agreement with the finding of Alizadeh et al. (2019) who investigated the blood glucose concentration in plasma increased by soya bean meal replacement with slow-release urea in the diet. Similarly, Huntington et al. (2006) suggested that feeding urea increases plasma glucose concentration. Increased in glucose concentration in the T2(Nu+Ncu) treatment might be due to greater DM and CP intake per unit body weight and OM digestibility compared to control diet (T0). However, current study supported the observation of  Xin et al. (2010); Mazinani et al. (2021); Abyane et al. (2020). Consistent with our findings, Gonçalves et al. (2015) also demonstrated that blood urea nitrogen concentrations were elevated in lactating dairy cows receiving slow-release urea and soybean meal compared to regular urea.Therefore, it could be enumerated that higher BUN value (mg/dL) concentration might be due to rapid hydrolysis of urea in the rumen leads to high ammonia levels. This excess ammonia can be absorbed into the bloodstream and potentially contribute to negative health effects. The rumen itself can recycle some of this ammonia back into urea, but significant amounts may still be excreted in the urine.

CONCLUSION

Addition of Nano urea and neem coated urea in TMR based diet enhanced voluntary intake of different nutrients and increased DCP value of the TMR feeds in crossbred dairy cows. At the feeding rates used in these trials, our findings suggest there is no improvement in animal performance when a ruminal nitrogen met with Nano urea and slow release urea. However current studies demonstrated measurable effects of Nano-urea, these effects were minimal in lactating crossbred cows. Further research is warranted to validate these findings across different ruminant species (cattle, buffalo, goats and sheep) at various physiological stages (growth, pregnancy, lactation).

ACKNOWLEDGEMENT

The authors are thankful to the Dean and Director of Postgraduate Studies, Faculty of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, for providing financial support in the experiment.
 
Author contribution
 
Mamata Joysowal: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Original draft writing Tapas K. Dutta: Conceptualization, Funding acquisition, Project administration, Writing – review and editing, Formal analysis. Bibeka N. Saikia: Writing- review and editing, Software. AD: Writing-review and editing, Software. Anupam Chatterjee: Funding acquisition, Resources, Supervision. AM: Conceptualization, Software. SR: Funding acquisition, Resources, Supervision. All authors read and approved the final manuscript.
 
Data availability
 
All data generated or analyzed during this study are included in this published article.
 
Declarations
 
Ethics approval
 
All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Ethics Committee (IAEC), The Institute research granted approval for the experiment with reference no.770/GO/Re/S/03/CPCSEA/FVSc/AAU/IAEC/23-24/1065.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interests.

REFERENCES


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    Full Research Article

    Effect of Feeding Nano and Neem Coated Urea on Nutrient Intake, Digestibility and Blood Chemistry of Lactating Crossbred Cow

    M
    Mamata Joysowal1,*
    T
    Tapas K. Dutta2
    B
    Bibeka N. Saikia3
    A
    Anupam Chatterjee2
    G
    Gunaram Saikia1
    M
    Mousumi Hazorika3
    L
    Lakhyajyoti Borah1
    1Department of Animal Nutrition, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
    2Division of Animal Nutrition, Eastern Regional Station, National Dairy Research Institute, Karnal-132 001, Haryana, India.
    3Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.

    ABSTRACT

    Background: With diminishing communal grazing lands, cost-effective protein supplementation for ruminant livestock is crucial. Utilizing nano-formed urea and a novel nano-formed nitrogen source, in conjunction with balanced energy sources, is anticipated to decrease the feeding costs for ruminant livestock. Nano-urea alone or with neem-coated urea supplementation may be used as good economic source of nitrogen, if found suitable.

    Methods: Two doses of Nano-urea (160 µL Nu) and combined with neem-coated urea (160 µL Nu+1% Ncu) were selected for lactation trial based upon in vitro rumen fermentation system.

    Result: Addition of Nano-urea and combination of Nano-urea and neem-coated urea enhanced voluntary intake of different nutrients and increased DCP value in crossbred dairy cows. TDN intake pattern was similar among all treatment groups. However, DCP intake was greater (p<0.001) and  increased blood urea nitrogen  in T1 and T2 compared to T0 groups. However, Feeding nano-urea and combination of nano-urea + neem-coated urea marginally benefitted in terms of feeding economics for milk production.

    INTRODUCTION

    Nano-Urea as a source of non-protein nitrogen may be a potential strategy to reduce the overall emission intensity of nitrogenous compound through milk production system of dairy animals, since nano-urea is prepared with nano-technology. Soybean meal (SBM), groundnut cake (GNC), mustard cake, tiloil (sesame) cake has long been used as a prominent source of crude protein for ruminants (Koukolová et al., 2020). However, with its increasing price, the use of these protein sources results in ultimately higher cost of production. Therefore, the use of urea as a protein (NPN source) replacement is attractive in ruminant diets because of its low cost compared with other protein feeds such as soyabean meal with high rumen degradability (Wanapat, 2009; Xin et al., 2010; Muralidharan et al., 2016). Driven by the gradual loss of community pastureland in India, progressive farmers are increasingly adopting intensive dairy farming systems. This shift necessitates a focus on cost-effective agribusiness, where a primary challenge lies in optimizing rumen function. Specifically, farmers must synchronize the availability of energy and nitrogen from feed to maximize nutrient utilization, thereby overcoming the traditionally high costs associated with protein nutrition for ruminant livestock (Alipour et al., 2020). As a non-protein nitrogen source, urea is a popular, low cost and easily obtained protein supplement (Wahyono et al., 2022). Slow-release urea (SRU) is a coated non-protein nitrogen (NPN) source for ruminant nutrition (Salami et al. 2020; Manju et al., 2022). These products are usually available for feeding to all ruminant species (cattle, buffalo, sheep and goat).Rumen bacteria can convert NPN sources into true protein (Simoni et al., 2023). Therefore, use of urea (NPN compound) is cost effective as ruminant animal can convert non protein nitrogen into true protein that can be utilized by host animal for production purpose. 

    MATERIALS AND METHODS

    The research  was conducted during the period of  August, 2023 to December, 2024 at the experimental facility within the laboratories of animal nutrition department, Instructional  livestock cattle farm ILF©, college of veterinary science which is located in Khanapara,  Assam, India. Eighteen crossbred (Jersey × Holstein Friesian) cow (weight between 350 kg to 400kg  were equally divided into 3 dietary treatment groups denoted as T0, T1 and T2. Animals were distributed to each square based on parity and lactation stage. Live weights were measured using electronic/electrical balance. Prior to commencement of the experiment, the animal underwent deworming. Intake parameters  was recorded at the fortnight basis of each experimental period. The animals were randomly allocated to receive one of the experimental diets which are TMRs based diet i.e. Concentrate: Green roughage: Dry roughage (40:45:15), where T0 (control; only TMR diet); T1 (addition of 160 µL Nano urea on the diet DM basis in the TMR based diet); T2 (addition of 160 µL Nano urea+1%neem coated urea in the TMR based diet DM basis). Nano urea product produced by IFFCO which is available in the local market. Diets were formulated to meet the nutrient requirement of cows according to NRC (2001) recommendations. Amounts of feed offered and orts of each cow were weighted daily and were restricted to 5 to 10% of intake as-fed basis.
           
    Throughout the 120days of duration of the experiment, the  animal in each group adhered to their respective feeding schedules and had access to their allocated feed.So, there was continuous monitoring of  growth performance and nutrient intake of the experimental animal were using established protocols. Throughout the experiment, experimental animals received consistent access to clean drinking water and their daily feed consumption was precisely measured using a double pan balance. Bi-weekly, between 8:00 and 9:00 AM, the weight of each animal was recorded to monitor growth. For the estimates of digestibility of the diets total 6day collection period was fixed. This analysis aimed to estimate the intake, digestibility and availability of various nutrients, as per the procedures laid out in AOAC, (2012). Analysis of minerals were done using atomic absorption spectroscopy (AAS). Blood samples were collected from the jugular vein of all experimental cows at 0, 60 and 120 days of feeding under aseptic conditions. Some relevant blood metabolites (Protein, albumin, globulin, glucose, blood urea nitrogen) estimated by commercially available kit.
     
    Statistical analysis
     
    Data related to the chemical and mineral composition of different samples were statistically analyzed by using one way analysis of variance (ANOVA). The intake of nutrients during digestion trial, blood parameters were analyzed by a mixed ANOVA with repeated measures, where treatment was a fixed factor (between subject effect), time period (within-subject) was a random factor having subject (cow) nested within it and their (period treatment) interaction was evaluated. A computerized IBM SPSS 26.0 software program was used for ANOVA. Tukey’s HSD test was used to measure the differences of means.

    RESULTS AND DISCUSSION

    Chemical composition and mineral profile of experimental diet 
     
    The chemical and mineral compositions of various feed components including maize grain, wheat bran, mustard oil cake, soya bean cake, rice polish, paddy straw and maize fodder are provided in Table 1. Mustard oil cake had comparatively  higher protein content than soyabean cake (32.99 vs. 26.65%).Samples of total mixed ration collected from study area had maximum CF (13.95%) and AIA (2.58%). Wheat bran had the maximum NDF and hemicelluloses content while soyabean oil  cake contained highest ADF, cellulose and lignin levels. Maize grain contained least ADF, cellulose and lignin among all the concentrates. The crude protein content of mustard cakes and soyabean oil  cakes observed in the present study were well within the range reported by earlier workers (Chowdhury et al., 2010 and Kumar et al., 2002). The proximate compositions and fibre fractions of feedstuffs analyzed in the present study are also within the normal range (Yadav et al., 2018). The diverse chemical compositions and fiber fractions observed in feedstuffs can be attributed to variations in oilseed varieties and agronomic factors, including fertilizer application, harvest timing and field drying practices.The DM, OM, CP, EE, total ash, T-CHO, NFC, NDF, ADF, hemicelluloses, cellulose and lignin content of concentrate mixture and green fodders were within the normal range as reported by Ranjhan, (1991) for Indian feeds and fodders. The results on macro and micro mineral profile of concentrate feeds collected from dairy farms are presented in Table 2, respectively. Compounded cattle feeds exhibited the optimum concentrations of major minerals (Ca, P, Mg) and micro minerals (Cu, Zn, Mn, Fe, Co) compared to other concentrate feeds as per Ranjhan, (1991).

    Table 1: Chemical composition on (%DM basis) and mineral contents of different feed ingredients used for the preparation of total mixed ration.



    Table 2: Chemical composition and mineral profile of total mixed ration diets used during experimental period.


     
    Evaluation of chemical and mineral composition in different TMR based feeds
     
    Table 2 outlines the chemical and mineral composition of the rations. While DM, OM, EE, TCHO, ADF, hemicellulose and lignin were consistent among the three TMRs, NDF and cellulose were higher in T1 and T2 compared to T0. However, three TMRs had a similar concentration of Ca and P. TMR T1 and TMR T2 contained greater amounts of Mg, Cu, Zn, Mn, Co and Fe compared to T0, which may be due to addition of nano urea may influence the bioavailability of some of major and trace minerals.
           
    Compounded cattle feeds provided an optimal balance of essential minerals, showing the highest concentrations of major minerals (Ca, P, Mg) and ideal levels of micro minerals (Cu, Zn, Mn, Fe, Co) among all concentrate feeds (Yadav et al., 2018). Of all the concentrates, maize grain demonstrated the lowest concentrations of several macro and micro minerals, specifically Mg, Cu, Zn, Mn and Co.
     
    Intake, apparent digestibility and nutritive value of feeds
     
    Table 3 summerizes intake performance of  digestion trial. Although total DM intake was significantly (p<0.001) lower in T2 than T0 and T1, but DMI per 100 kg BW and g/kg W0.75 was statistically similar among three treatment groups. Table 3 also depicts the value of DMI (g)/kg W0.75 was 101.88, 105.69 and 98.21 g in crossbred dairy cows under T0, T1 and T2 groups, respectively. However, diets containing Nano-urea and combination of Nano-urea and neem-coated urea resulted in increased (p<0.001) CP intake in T1 and T2 compared to T0. CPI (g)/kg W0.75 was estimated 10.45, 13.61 and 13.83 g in T0, T1 and T2 treatments, respectively. In contrast to our findings, Salami et al. (2020) reported no significant effect on overall dry matter intake (DMI) and crude protein intake (CPI) in beef cattle fed slow-release urea. Similarly, neutral detergent fiber (NDF) intake and apparent dry matter digestibility remained consistent across all treatment groups. Consistent with our findings, Salami et al. (2021) reported a significant decrease (P<0.05) in dry matter intake (DMI, -500 g/d) in slow-release urea treated groups compared to controls. These results can be explained by the increased ruminally available nitrogen, following a rise in ammonia nitrogen concentration. This optimal nitrogen availability likely stimulated microbial growth, leading to an apparent increase in dietary intake (Wahyono et al., 2022). Conversely, McGuire et al. (2013) and Xu et al. (2019) stated that dry matter intake (DMI) and crude protein intake (CPI) are higher in sheep who receive urea supplementation than  non-supplemented animals. Different results were shown by Kozloski et al. (2007) and Currier et al. (2004), who reported that supplemental urea to ruminants consuming low-quality roughage does not affect an animal’s dry matter intake (DMI). Despite consistent dry matter intake (DMI), we hypothesize that urea supplementation altered the intake of other nutrient components. Therefore, it can be enumerated that nano urea likely created the most favorable conditions for rumen function compared to other treatments in the present study. We did not find any report in relation to the utilization of nano-urea in ruminant species. Based on the current observations it may be interpreted that feeding nano-urea has resulted increased feed intake pattern compared to control (T0) and combination of both (Nu + Unc). Probably, neem-coated urea did not produce any tangible impact on the intake performance in lactating crossbred cows.

    Table 3: Nutrients intake and nutrients digestibility during digestion trial in lactating cows.


           
    These variations in nutrient intake, observed with urea supplementation in sheep, may stem from several factors, including differences in crude protein (CP) concentration and the quality of the basal diets (Wang et al., 2015) differences in the chemical and/or physical characteristics of the forage/feedstuff (Sano et al., 2011) differences in the slow-fast degradable carbohydrate and/or forage-concentrate composition of the basal diets (Zhao et al., 2007; Galina et al., 2007).The digestibility’s of organic matter (OM) (p<0.001) and crude protein (CP) (p<0.001) were notably higher in T1 and T2 compared to control T0 (Table 3). Despite supplementation with Nano urea and a combination of Nano-urea + neem-coated urea, the digestibility coefficients of dry matter, ether extract, nitrogen-free extract, NDF and ADF remained unchanged.These outcomes indicated that rumen degradable nature of Nano and neem coated urea may enhance nitrogen availability in the rumen in the treatment groups compare to the control groups. Comparing our findings with earlier workers is difficult, because no published reports are available on the utilization of Nano-urea in ruminant diets.However, Cherdthong et al. (2011) found that the urea and slow-release urea (urea-calcium sulfate and urea-calcium chloride) did not influence dry matter intake. However, improved CP and OM digestibility was found in the Nano urea and Nu +Ncu added groups, which also supported by Phillip et al. (2023); Alizadeh et al. (2019); Sevim et al. (2019). However, the effect of urea and non-protein nitrogen sources on digestibility depends on their concentration, physical structure and adaptation period. Increased digestibility of optigen and coated urea could be due to these treatments supplied enough nutrients for microorganisms in the rumen. Earlier it was reported that SRU in rations had no effect on DM, NDF and ADF digestibility (Xin et al., 2010). With some exceptions, feeding FGU and SRU did not affect nutrient intake and digestibility on lactating dairy cow (Simoni et al., 2023). 
     
    Availability of nutrients and nutritive value of TMR feeds
     
    Data pertaining to nutrients intake in all the treatment groups (T0, T1 and T2) was presented in the Table 4. TDN intake pattern was similar among all treatment groups. However, DCP intake was greater (p<0.001) in T1 and T2 compared to T0 control. The results demonstrated that DCP percentage was significantly higher (p<0.001) in treatment groups (T1 and T2) compared to control T0. Digestible NDF value of TMR feeds remained similar in the groups, indicating that dairy animals likely adjusted their feed intake to maintain a consistent digestible NDF intake in all treatment groups. Phillip et al. (2023) reported that there was no significant (p<0.05) differences found for total digestible nutrients (TDN) value among tested groups; Acetic acid addition to urea-treated rice straw in dairy ewes resulted in a significant (p<0.05) increase in digestible crude protein (DCP). This enhancement, coupled with the fulfillment of daily dry matter (DM), total digestible nutrients (TDN) and DCP intake recommendations outlined by NRC (2001), which specifies an energy requirement of 1.67 Mcal/kg for dairy cows, demonstrates effective nutritional management.

    Table 4: Availability of different nutrients in lactating cows fed with different TMR based rations.



    Blood chemistry of the experimental animal
     
    Plasma total protein concentration found significant in terms of treatment effect (P=0.032) and period effect (p<0.001) and no significant difference found for interaction between treatment and period (T×P). Notably, we did not find any difference among treatment groups in relation to plasma albumin, globulin and albumin: globulin ratio (Table 5). The periodic effect was highly significant (p<0.001) for globulin, but the interaction (T×P) effect was statistically similar for albumin, globulin and albumin/globulin ratio. Moreover, plasma glucose concentration increased significantly (p<0.001) with different period of time. The BUN (mg/dL) value was significant (P=0.002) increased in T2 Groups compare to the other. Periodic effect was also higher (p<0.04) for this parameter. Moreover, significant (p=0.027) interaction effect was also found for treatment and period. But, for bilirubin, there was no significant (p<0.05) difference found   for treatment effect (T), period effect (P) and interaction between treatment and period effect (T×P).

    Table 5: Blood metabolites in animals fed with different TMRs.


           
    However, our findings are in close agreement with the finding of Alizadeh et al. (2019) who investigated the blood glucose concentration in plasma increased by soya bean meal replacement with slow-release urea in the diet. Similarly, Huntington et al. (2006) suggested that feeding urea increases plasma glucose concentration. Increased in glucose concentration in the T2(Nu+Ncu) treatment might be due to greater DM and CP intake per unit body weight and OM digestibility compared to control diet (T0). However, current study supported the observation of  Xin et al. (2010); Mazinani et al. (2021); Abyane et al. (2020). Consistent with our findings, Gonçalves et al. (2015) also demonstrated that blood urea nitrogen concentrations were elevated in lactating dairy cows receiving slow-release urea and soybean meal compared to regular urea.Therefore, it could be enumerated that higher BUN value (mg/dL) concentration might be due to rapid hydrolysis of urea in the rumen leads to high ammonia levels. This excess ammonia can be absorbed into the bloodstream and potentially contribute to negative health effects. The rumen itself can recycle some of this ammonia back into urea, but significant amounts may still be excreted in the urine.

    CONCLUSION

    Addition of Nano urea and neem coated urea in TMR based diet enhanced voluntary intake of different nutrients and increased DCP value of the TMR feeds in crossbred dairy cows. At the feeding rates used in these trials, our findings suggest there is no improvement in animal performance when a ruminal nitrogen met with Nano urea and slow release urea. However current studies demonstrated measurable effects of Nano-urea, these effects were minimal in lactating crossbred cows. Further research is warranted to validate these findings across different ruminant species (cattle, buffalo, goats and sheep) at various physiological stages (growth, pregnancy, lactation).

    ACKNOWLEDGEMENT

    The authors are thankful to the Dean and Director of Postgraduate Studies, Faculty of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, for providing financial support in the experiment.
     
    Author contribution
     
    Mamata Joysowal: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Original draft writing Tapas K. Dutta: Conceptualization, Funding acquisition, Project administration, Writing – review and editing, Formal analysis. Bibeka N. Saikia: Writing- review and editing, Software. AD: Writing-review and editing, Software. Anupam Chatterjee: Funding acquisition, Resources, Supervision. AM: Conceptualization, Software. SR: Funding acquisition, Resources, Supervision. All authors read and approved the final manuscript.
     
    Data availability
     
    All data generated or analyzed during this study are included in this published article.
     
    Declarations
     
    Ethics approval
     
    All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Ethics Committee (IAEC), The Institute research granted approval for the experiment with reference no.770/GO/Re/S/03/CPCSEA/FVSc/AAU/IAEC/23-24/1065.

    CONFLICT OF INTEREST

    The authors declare that they have no conflict of interests.

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