Effects of protein nutrition levels on milk yield, composition, amino acid profiles and plasma metabolites of indigenous lactating buffaloes

Abstract

Article Info AGRICULTURE AND NATURAL RESOURCES The dietary crude protein (CP) supply was optimized for indigenous lactating buffaloes in Bangladesh. Twelve buffaloes (age 4 yr) with an average daily milk yield of 2.33-3.03 kg/d and average ± SD body weight of 390 ± 10 kg were divided into three groups (each with four animals) that were randomly assigned to three levels of CP in the concentrate feed mixture: 14.22%, 15.62%, and 17.81% (dry matter basis) in a completely randomized design. The total duration of the study was 100 d with an initial 10 d of adjustment. The diets were formulated to ensure the same energy level. The dietary protein level showed no effect on dry matter intake (p > 0.05). The nitrogen intake of buffaloes increased linearly (p = 0.01) with increasing CP levels. The milk yield tended to increase in a quadratic fashion. No effect was observed (p > 0.05) on the milk protein, fat and lactose contents. The level of plasma urea nitrogen increased (p < 0.05) when the dietary CP levels were raised, whereas the glucose, protein, albumin, globulin, calcium and triacylglycerol levels remained unchanged (p > 0.05). The nitrogen efficiency of lactating buffaloes increased (p < 0.05) by optimizing dietary protein nutrition accurately to each animal's requirements. The milk amino acid composition was unaffected (p > 0.05) by the dietary CP supplies. In conclusion, the 15.62% CP level resulted in higher milk production (p < 0.05) and high nitrogen efficiency (p < 0.05) in the indigenous lactating buffaloes under the study conditions.

Full text

Agr. Nat. Resour. 55 (2021) 935–944
Article history:
Received 29 March 2021
Revised 30 October 2021
Accepted 4 November 2021
Available online 20 December 2021
Keywords:
Amino acid prole,
Blood metabolites,
Crude protein,
Dairy buffaloes,
Milk production and quality
Research article
Effects of protein nutrition levels on milk yield, composition, amino acid
proles and plasma metabolites of indigenous lactating buffaloes
Md. Zakirul Islama,†, S.M. Rajiur Rahmana,†,*, Md. Nurul Islama, Md. Mehedi Hasan Khandakara,
Mohammad Shohel Rana Siddikia, Nathu Ram Sarkerb, Watcharawit Meenongyaic, Md. Harun-ur-Rashida
a Department of Dairy Science, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh
b Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh
c Faculty of Natural Resources and Agro-Industry, Kasetsart University, Chalermphrakiat Sakon Nakhon Province Campus,
Sakon Nakhon- 47000, Thailand
† Equal contribution.
* Corresponding author.
E-mail address: smrajiurrahman@yahoo.com (S.M.R. Rahman)
AbstractArticle Info
AGRICULTURE AND
NATURAL RESOURCES
The dietary crude protein (CP) supply was optimized for indigenous lactating buffaloes
in Bangladesh. Twelve buffaloes (age 4 yr) with an average daily milk yield of 2.33–3.03
kg/d and average ± SD body weight of 390 ± 10 kg were divided into three groups
(each with four animals) that were randomly assigned to three levels of CP in
the concentrate feed mixture: 14.22%, 15.62%, and 17.81% (dry matter basis) in
a completely randomized design. The total duration of the study was 100 d with an
initial 10 d of adjustment. The diets were formulated to ensure the same energy level.
The dietary protein level showed no effect on dry matter intake (p > 0.05). The nitrogen
intake of buffaloes increased linearly (p = 0.01) with increasing CP levels. The milk
yield tended to increase in a quadratic fashion. No effect was observed (p > 0.05) on
the milk protein, fat and lactose contents. The level of plasma urea nitrogen increased
(p < 0.05) when the dietary CP levels were raised, whereas the glucose, protein, albumin,
globulin, calcium and triacylglycerol levels remained unchanged (p > 0.05). The nitrogen
efficiency of lactating buffaloes increased (p < 0.05) by optimizing dietary protein
nutrition accurately to each animal’s requirements. The milk amino acid composition
was unaffected (p > 0.05) by the dietary CP supplies. In conclusion, the 15.62% CP level
resulted in higher milk production (p < 0.05) and high nitrogen efciency (p < 0.05)
in the indigenous lactating buffaloes under the study conditions.
online 2452-316X print 2468-1458/Copyright © 2021. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/),
production and hosting by Kasetsart University of Research and Development Institute on behalf of Kasetsart University.
https://doi.org/10.34044/j.anres.2021.55.6.04
Journal homepage: http://anres.kasetsart.org

936 Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
Introduction
Buffaloes inhabit and breed throughout Asia, Europe
and South America over a wide range of geographical,
environmental and agronomic settings and they are second
only to cows in their milk production globally (Eldahshan
et al., 2020). The greatest expense in dairy farming is feed,
which accounts for more than 70% of the total cost in developing
countries (Singh et al., 2003). It is more common for indigenous
buffaloes to grow slowly, delay puberty and produce less milk
due to inadequate protein consumption (Pasha, 2013; Habib
et al., 2020; Mohd Azmi et al., 2021). Nitrogen is one of the
main elements in the formulation of rations for ruminants,
resulting in greater attention being paid to excretion by
ruminants due to growing environmental concerns regarding
the nitrogen and phosphorus released (Islam et al., 2002).
Generally, excess dietary protein is not needed by ruminants
and it is converted into ammonia (Abbasi et al., 2018). It has
been established that the feed and feeding strategies in ruminant
animals may further significantly influence the microbial
population of the rumen (Huws et al., 2018) and the composition
of the ruminal gut microbiota is signicantly associated with
feed efficiency (Guan et al., 2008; Carberry et al., 2012).
Bevans et al. (2005) reported that alterations in the structure
of the ruminant gut microbiota could substantially improve
the feed intake, efciency and body weight gain of ruminants.
In addition, buffaloes can utilize feed more proficiently
where the feed is supplied in lower quantity or quality or
both (Chanthakhoun et al., 2012). Such feed efciency can be
explained by the buffaloes having a different rumen microbial
ecology with a higher population of cellulolytic bacteria and
fungal zoospores, a lower protozoal population and a greater
capacity to recycle nitrogen to the rumen (Wanapat, 2000;
Wanapat and Cherdthong, 2009; Cherdthong et al., 2010;
Chanthakhoun et al., 2012). However, a good understanding of
the protein requirement for dairy buffaloes is essential to ensure
a proper supply of the assorted amino acid required for milk
production and cow maintenance while minimizing the costs,
and importantly, reducing unnecessary excretion of surplus
nitrogen by circumventing any overfeeding of protein (Mohd
Azmi et al., 2021). Better feeding and management could
improve buffalo fertility and ensure a high milk yield of good
quality (Qureshi et al., 2007; Delno et al., 2021) and, likewise,
in dairy cows (Islam et al., 2020). The higher supply of crude
protein for dairy buffaloes in pasture over a sufcient period
improved the nutritional status of the milk yield (Hayashi
et al., 2005). The amount of milk protein could be increased
from 0.05% to 0.15% by manipulating the diet based on
the energy and protein contents (Santos, 2002). Increasing
the amount of dietary protein for a constant dietary energy
level had little effect on milk protein synthesis (Broderick,
2003). However, the abundance and effective use of nutrients
in the rumen is crucial for their effective use by the animal
host (Franzolin and Alves, 2010). Therefore, the promotion
of a suitable level of efficiency of nitrogen metabolism
by the animal via a healthy diet and the utilization of protein
and amino acids is an effective way to minimize nitrogen
losses while ensuring adequate levels to meet the actual
physiological requirements of the animal (Steinfeld
and Wassenaar, 2007).
Most small-scale buffalo farmers in tropical countries
like Bangladesh are not aware of the benets of better animal
feeding; in general, farmers feed their animals with locally
available crop wastes, roughage and a tiny quantity of
concentrates (Rahman et al., 2019a, 2019b). Therefore, most
of the buffaloes typically yield less milk and farmers are not
aware of the potential from providing concentrate in the diet.
Despite this, a few farmers fed their animals a small amount
of concentrate feed without being aware of the specic crude
protein (CP) requirements. Accordingly, the current experiment
was designed to optimize the protein nutrition levels with the
least quantity of concentrate supply possible by considering
these factors. In addition, information is scanty regarding
the effect of feeding the buffaloes various dietary protein
levels particularly on the milk amino acid proles. Therefore,
dose-response experiments, which have not been done
previously to the authors’ knowledge, are required to determine
the optimum protein supply of indigenous lactating buffaloes.
Thus, this study was undertaken to investigate the effect of
different protein levels in the diet of lactating buffaloes and to
evaluate the milk yield, components, amino acid composition
and blood metabolites.
Materials and Methods
Animals, feed types and management
The Buffalo Research Farm, Animal Production Research
Division, Bangladesh Livestock Research Institute, Savar,
Dhaka, Bangladesh, was the site of the experiment. Twelve,
lactating indigenous buffaloes aged 4 yr and having average
daily milk yields from 2.33 kg/d to 3.03 kg/d, and mean ± SD
body weight (BW) of 390 ± 10 kg were randomly assigned
to receive one of three CP levels (14.22%, 15.62% or 17.81%)

937
Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
in the concentrate mixture (Table 1) in a completely randomized
design experiment. The duration of the study was 100 d and
the rst 10 d were assigned for adjustment. There were three
groups with four buffaloes in each. The buffaloes were kept
in individual pens where water and mineral blocks were
provided ad libitum. All buffaloes were supplied ad libitum
with Napier silage (fodder cut at age 35 d, chopped into
2–3 cm lengths, compacted in a concrete silo pit and stored
for 60 d), while additional concentrate was fed at 3.5 g/kg
of BW. All buffaloes were fed their allocated diets during
the period of the experiment, and concentrate was fed to
them in two equal amounts at 0700 hours and 1630 hours.
The daily dry matter intake (DMI) was determined by
weighing any residual Napier silage feed before the morning
feeding. The BW of each buffalo was measured at the beginning
and the end of each period of 30 d. The dried concentrate
mixture and Napier silage samples (offered and leftover)
were ground in a MAC® WILLEY grinder to pass through
a 1-mm sieve and pooled, with the proximate composition
of the samples determined using the method of Association
of Official Analytical Chemists (2005). All animals were
cared for during milk and blood sampling according to
the protocol of Animal Welfare and Experimentation
Ethics Committee, Bangladesh Livestock Research Institute,
Savar, Dhaka.
Feed TDN, ME, FCM and N efciency calculation
Total digestible nutrient (TDN) was estimated according
to the following equations: TDN for silage (% dry matter
(DM)) = − 17.2649 + (1.2120 × %CP) + (0.8352 × %NFE)
+ (2.4637 × %EE) + (0.4475 × %CF); TDN for concentrate
(%DM) = 40.3227 + (0.5398 × %CP) + (0.4448 × %NFE) +
(1.4218 × %EE) − (0.7007 × %CF) as well as metabolizable
energy (ME) values were estimated according to Kearl
(1982); ME (MJ/kg DM) = [− 0.45 + (0.04453 × %TDN)]
× 4.184. Fat corrected milk (FCM) was measured using
software of Progressive Dairy Solutions. Inc. 120 S Sierra Ave,
Oakdale, CA 95361. N efciency was calculated as nitrogen in
milk/nitrogen in CP according to Naveed-ul-Haque et al.
(2018).
Milk and blood analysis
Once a week, individual samples of milk were collected
from two successive milking events and used to analyze
milk components (total solids, solids-not-fat, fat, protein,
lactose, ash content). The milk compositional parameters
were analyzed using an automated milk analyzer (Lactoscan
SP, MILKOTONIC Ltd., Bulgaria). At intervals of 30 d,
blood samples (about 10 mL) were taken from the jugular
Table 1 Ingredients and chemical composition of concentrates and Napier silage (NS) by treatment
Item Treatment
14.22% CP 15.62% CP 17.81% CP NS
Wheat bran (kg) 50.50 48.00 47.00 –
Soybean meal (kg) 1.00 6.00 10.00 –
Broken maize (kg) 45.50 43.00 40.00 –
DCP (kg) 2.00 2.00 2.00 –
Salt (kg) 1.00 1.00 1.00 –
Total (kg) 100.00 100.00 100.00 –
Chemical composition of
the diet (%)
TDN 69.68 69.74 69.70 51.03
DM 88.03 87.92 88.05 17.12
CP 14.22 15.62 17.81 7.68
NDF 24.40 24.28 24.36 85.61
ADF 6.68 9.72 10.18 45.33
Ash 5.07 4.73 5.79 10.22
‡ME (MJ/kg DM) 11.00 11.01 11.00 8.37
CP = crude protein; NS = Napier silage; DCP = di-calcium phosphate; TDN = total digestible nutrient; DM = dry matter; NDF = neutral detergent ber;
ADF = acid detergent ber; ME = metabolizable energy

938 Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
vein throughout the 90 d experimental period at 3 hr post
offering of the concentrate mixture in the morning.
Blood was collected into tubes containing 12 mg of
ethylenediaminetetraacetic acid and the plasma was separated
using centrifugation at 500×g at 4°C for 10 min and stored
at -20°C until analysis. Various blood biochemical parameters
were assessed using diagnostic kits (Span Diagnostics Ltd.,
Surat, India). The GOD–POD method was followed for glucose
(Ambade et al., 1998), the cresolphthalein complexone method
(Ranjan et al., 2012) for calcium, the modified Biuret and
BCG dye-binding method (Ranjan et al., 2012) for total
protein, albumin, globulin and the CHOD–PAP method
for total cholesterol (Deeg and Ziegenhorn, 1983).
Determination of milk amino acids
For the buffalo milk samples, the composition of the amino
acid (AA) was determined using the method of Raq et al.
(2016). A sample (1.5 mL) of milk was used as the starting
point for sample preparation. Following that, using 10 mL
of H2SO4 (6 mol/L), milk samples were mixed thoroughly
and hydrolyzed in a sealed glass apparatus at 110°C for
24 hr under a constant nitrogen flow. After centrifugation,
the supernatant was moved to a 5 mL centrifuge tube and diluted
with 0.02 mol/L H2SO4 to a final volume of 5 mL. Before
operating the amino acid analyzer (Model: L-8900 Amino
Acid Analyzer, Hitachi, Japan) for AA analysis, a 0.22 mm
syringe lter was used to lter the previous mixture.
Statistical analysis
The data were analyzed using one-way analysis of variance.
A general linear model for repeated measures procedure was
used to analyze the blood prole data, considering treatment
as the between-subjects main effect and sampling period as
the within-subject factor. The significance level was set at
p < 0.05. Duncan’s multiple range test was used to ascertain
the differences between means. All analyses were facilitated
by the SPSS statistical program version 17.0 (SPSS Inc.;
Chicago, IL, USA).
Result and Discussion
Feed intake, milk yield, and quality
Milk production and composition, dry matter intake
(DMI), feed and nitrogen efciencies and body weight data
are provided in Table 2. The average DMI levels during
the 90 d experimental period were not signicantly different.
This was supported by experiments on lactating buffaloes
where differing protein supplies had no impact on DMI (Bovera
et al., 2002; Bartocci et al., 2006; Terramoccia et al., 2012).
Actual herd management records have shown that the variation
in DMI of dairy cows in response to altered dietary protein or
AA supply varies with the experimental period (Naveed-ul-
Haque et al., 2018). It was possible that the length of treatment
in the current study was sufcient to expect complete DMI
Table 2 Effects of crude protein supplies on dry matter intake, milk yield, milk composition, feed and nitrogen efciencies and body weight of the
lactating buffaloes
Item 14.22% CP 15.62% CP 17.81% CP p-value
DMI (kg/d) 12.0±0.33 12.2±0.56 12.1±0.68 0.44
N intake (g/d) 200±4.23c209±5.33b216±6.87a0.01
Milk yield (kg/d) 3.47±1.02b3.82±0.74a3.27±0.48b0.02
FCM (kg/d) 5.66±1.05b6.36±1.12a5.40±1.22b0.03
Protein (%) 3.64±0.45 3.65±0.53 3.66±0.26 0.43
Fat (%) 7.34±1.87 7.55±0.92 7.46±0.95 0.30
Lactose (%) 5.20±0.73 5.22±0.41 5.23±1.00 0.57
MY:DMI 0.29±0.01 0.31±0.03 0.27±0.03 0.41
FCM:DMI 0.47±0.02 0.52±0.02 0.45±0.11 0.11
†N efciency 24.0±1.67a20.1±1.83b19.0±2.08c0.01
Body weight (kg) 398±3.45c400±3.98b405±2.49a0.02
CP = crude protein; DMI = dry matter intake; MY = milk yield; FCM = fat corrected milk
Mean ± SD values in a row superscripted with different lowercase letters differ signicantly (p < 0.05).

939
Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
responses. The total N intake increased linearly (p < 0.05)
which was in agreement with Naveed-ul-Haque et al. (2018).
The average daily milk production was 9.2% higher for
the 15.62% CP (3.82 kg) than for the 14.22% CP group
(3.47 kg) and 14.4% higher than for the 17.81% CP group
(3.27 kg) (p < 0.05). Milk yield increased in a quadratic
fashion with increasing CP supplies (p < 0.05). Increased
dietary CP supplies were predicted to increase milk production
in the current research. However, the results showed that the
protein nutrition increased milk yields quadratically, which was
in agreement with Naveed-ul-Haque et al. (2018). Additionally,
in a study on lactating buffaloes, increasing the dietary CP
levels from 9.3% to 12.2% (DM basis) increased the energy-
corrected milk yield by 7% (Campanile et al., 1998). Likewise,
several studies on lactating dairy cows have shown an increase
in milk yield by increasing the dietary protein supplies (Vérité
and Delaby, 2000; Brun-Lafleur et al., 2010). However,
when coupled with the high-energy diets, increasing protein
supplies had a higher impact on milk production (Brun-Laeur
et al., 2010), suggesting that supplies of protein and energy
interact with each other. This effect has also been shown with
lactating buffaloes, whereas increasing the amount of dietary
CP to 17.90% from 15.50% enhanced milk production by 9%
(Bartocci and Terramoccia, 2010). However, there was no
effect of the treatments on the protein, fat or lactose contents
of the milk (p > 0.05). In this regard, Santillo et al. (2016)
reported that different dietary protein levels in dairy buffalo
had no effect on fat and lactose but did affect the protein
content of milk. Similarly, in the current study, there was no
signicant effect on feed efciency (fat-corrected milk/DMI).
Likewise, the dietary protein levels in the diets of growing
buffaloes had no signicant effect on feed efciency (Singh
et al., 2015). Increasing the dietary CP level linearly decreased
the dietary N efciency for milk (p < 0.01). According to the
ndings for lactating buffalo studies, the nitrogen efciency
rate ranges from 18 to 20%, accordingly in our investigation
the rate averaged 21%, which is compatible with the ndings
of Bartocci et al. (2006), Terramoccia et al. (2012), and
Naveed-ul-Haque et al. (2018). Nonetheless, in lactation,
the N responses of milking buffaloes were lower than those of
Holstein cows, which ranged from 26% to 28% (Recktenwald
et al., 2014). In contrast, there was an effect on the body weight
gain of buffaloes (p < 0.05) by changing the dietary protein
levels. A higher protein level was associated with a small
increase in body weight gain (p < 0.05), which was consistent
with Dung et al. (2013) and Naveed-ul-Haque et al. (2018).
Blood biochemical prole
The plasma urea N (PUN), glucose, protein, albumin, globulin,
calcium, and triglycerides levels increased progressively
with advancement of the lactation stage (p < 0.05) but there
was no significant difference in the dietary crude protein
levels except for PUN (p < 0.05), as shown in Table 3.
The PUN level increased linearly (p < 0.05) along with the rise
in various periodic intervals by feeding dietary crude protein.
Naveed-ul-Haque et al. (2018) and Singh et al. (2015) reported
more or less similar findings to the current investigation
regarding dietary protein supply. Additionally, Naveed-ul-
Haque et al. (2018) found a signicant effect of increasing CP
levels on PUN but Singh et al. (2015) reported dietary protein
levels did not affect plasma metabolites. However, a higher
PUN level was observed following increased dietary CP,
perhaps because of the increased dietary N contents. Jordan et
al. (1983) used 23% and 12% CP in the diet of high-production
dairy cows and reported a 3.5 times higher PUN for a diet
containing 23% CP than for the diet having 12% CP. According
to Neglia et al. (2014) and Patra et al. (2020), ruminal NH3-N
concentrations increased from 0.8 mg/100 mL to 56.1 mg/100
mL when the dietary CP was elevated from 8% to 24%.
The surplus ammonia that forms in the reticulo-rumen is
absorbed either in the reticulo-rumen or the lower gastrointestinal
tract, and then transported to the liver, where it is converted
into urea. However, a higher level of blood biochemical
proles with the advancement of lactation trial was explained
by Ranjan et al. (2012) as a result of a positive energy balance
on account of better nourishment of the animals with advancing
stage of lactation. The lower N efficiencies in lactating
buffaloes compared to cows could be explained by the higher
PUN observed in the current investigation (22.22 mg/dL)
and supported by the investigation by Bartocci et al. (2006),
who reported 39 mg/dL compared to values in lactating cows
of 15.6 mg/dL (Roseler et al., 1993) and 24 mg/dL (Colmenero
and Broderick, 2006). In addition, with the N intake, PUN
increased linearly as was reported in experiments with lactating
dairy cows (Colmenero and Broderick, 2006), demonstrating
the ineffective use of dietary protein. Furthermore, Rafsanjanny
et al. (2019) reported a higher level (28 mg/dL) of blood urea
concentration in crossbred lactating cows. A similar pattern
was observed with the albumin concentration. Remarkably,
with the PUN observed in the current study (17.52–22.22
mg/dL), optimizing the protein nutrition for lactating buffaloes
provided useful information to improve N efciency and to
formulate economic diets.

940 Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
Table 3 Blood biochemical prole of lactating buffaloes fed different level of crude protein
Item 14.22% CP 15.62% CP 17.81% CP p-value
Treatment Period
PUN (mg/dL)
0 d 14.0±2.56 15.6±2.78 18.2±3.11
0.03 0.01
30 d 16.0±2.77 18.1±2.98 20.0±2.12
60 d 19.0±3.00 21.3±3.13 24.1±3.39
90 d 21.1±2.56 24.8±2.53 26.6±2.50
Overall mean 17.52±3.14c19.95±3.98b22.22±3.82a
Glucose (mg/dL)
0 d 49.01±1.15 49.32±1.12 50.93±1.79
0.22 0.02
30 d 51.20±1.21 52.96±1.80 52.60±1.24
60 d 52.11±1.32 53.55±1.59 53.11±1.55
90 d 52.29±1.50 54.18±2.00 53.97±2.06
Overall mean 51.15±1.25 52.50±1.63 52.65±1.52
Protein (g/dL)
0 d 7.55±0.031 8.92±0.11 8.63±0.64
0.42 0.00
30 d 7.96±0.051 8.96±0.32 8.91±0.91
60 d 8.51±0.60 9.32±0.30 9.79±0.84
90 d 9.10±0.42 9.76±0.45 10.12±0.27
Overall mean 8.28±0.67 9.24±0.40 9.36±0.70
Albumin (g/dL)
0 d 2.79±0.21 2.60±0.45 2.60±0.20
0.13 0.01
30 d 3.12±0.49 2.90±0.35 2.97±0.46
60 d 3.70±0.28 3.21±0.32 3.18±0.52
90 d 3.90±0.47 3.69±0.32 3.56±0.24
Overall mean 3.37±0.52 3.10±0.46 3.07±0.40
Globulin (g/dL)
0 d 4.97±0.44 5.19±0.54 4.93±0.72
0.10 0.02
30 d 5.22±0.44 5.93±0.48 5.33±0.48
60 d 5.91±0.51 6.13±0.64 6.00±0.34
90 d 6.21±0.47 6.50±0.12 6.62±0.24
Overall mean 5.57±0.58 5.93±0.55 5.72±0.75
Calcium (mg/dL)
0 d 5.00±0.68 5.00±0.46 4.96±0.98
0.08 0.04
30 d 5.65±0.52 5.13±0.99 5.00±0.85
60 d 5.95±0.52 6.50±0.19 6.55±0.52
90 d 6.23±0.62 7.00±0.92 7.26±0.71
Overall mean 5.70±0.53 5.92±1.00 5.94±1.15
TG (mg/dL)
0 d 175±3.21 176±1.89 178±1.93
0.89 0.56
30 d 176±2.54 180±2.22 181±1.99
60 d 177±0.98 180±2.30 182±1.48
90 d 181±1.25 182±1.79 184±1.27
Overall mean 177.25±2.63 179.5±2.52 181.25±2.50
CP = crude protein; PUN = plasma urea nitrogen; TG = triglycerides. Mean ± SD values in a row superscripted with different lowercase letters
differ signicantly (p < 0.05).

941
Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
Milk amino acid prole
The quality of protein is largely determined by the amino
acid composition. Amino acids are the building blocks of
proteins and have a vital role in the human body (Wolfe et al.,
2016). The milk amino acid composition data are presented
in Table 4. There were no significant differences for the milk
amino acid composition in the three dietary groups of buffaloes.
Haque et al. (2012) stated that the dietary crude protein levels
did not affect the amino acid composition of buffalo milk.
In the current study, it was clear that each buffalo milk sample
was rich in essential amino acids and the dominant amino acids
were glutamic acid (0.98 g/100 g milk), followed by proline,
lysine, leucine and asparagine (Table 4). Raq et al. (2016) studied
amino acids profiling of milk from different animal species
and stated that among the non-essential amino acids, glutamic
acid was in the highest concentrations in both casein and whey
proteins. However, in the current investigation, the ratios of
essential and non-essential amino acids in response to the shift
in the dietary protein levels were a bit higher numerically in the
17.81% CP group. Lysine and threonine are the scarcest amino
acids in a wide range of protein resources; however, essential
being catabolic-sensitive and necessary for protein synthesis
(Raq et al., 2016). Notably, differences in amino acid proles
in dietary proteins influence their utilization in the body. For
example, compared to soy protein, milk proteins elicited a greater
increase in branched-chain amino acids (BCAAs) concentrations
(26%) in peripheral tissues (Bos et al., 2000; Fouillet et al.,
2002). Furthermore, BCAAs play a significant role in weight
control by regulating glucose homeostasis and lipid metabolism
(Raq et al., 2016). Furthermore, sulfur-containing amino acids,
such as methionine and cysteine, boost the immune system via
intracellular glutathione conversion, functioning as antioxidants
(Hall et al., 2003). However, among different buffalo breeds,
the milk amino acid composition was significantly different
Zhou et al. (2018), though there has been a lack of scientific
evidence regarding the amino acid composition of buffalo
milk resulting from changing dietary protein levels.
Table 4 Amino acid composition of milk (g/100 g of milk) from indigenous lactating buffaloes
Amino acid type 14.22% CP 15.62% CP 17.81% CP p-value
Essential amino acid
Methionine 0.12±0.01 0.13±0.02 0.13±0.01 0.21
Valine 0.22±0.03 0.23±0.03 0.23±0.04 0.31
Lysine 0.45±0.04 0.45±0.02 0.45±0.02 0.50
Isoleucine 0.19±0.04 0.19±0.04 0.20±0.03 0.32
Phenylalanine 0.28±0.02 0.28±0.12 0.28±0.01 0.52
Leucine 0.39±0.10 0.40±0.05 0.40±0.02 0.13
Threonine 0.22±0.02 0.22±0.10 0.22±0.03 0.50
Non- Essential amino acid
Asparagine 0.36±0.03 0.36±0.01 0.36±0.01 0.31
Serine 0.25±0.05 0.24±0.05 0.24±0.05 0.49
Glutamic acid 0.96±0.12 0.98±0.11 0.99±0.12 0.31
Proline 0.51±0.01 0.51±0.03 0.51±0.11 0.39
Glycine 0.10±0.01 0.10±0.00 0.10±0.01 0.21
Alanine 0.16±0.04 0.16±0.03 0.16±0.03 0.11
Cysteine 0.02±0.01 0.02±0.00 0.02±0.00 0.17
Tyrosine 0.18±0.01 0.18±0.01 0.18±0.04 0.12
Histidine 0.12±0.03 0.12±0.01 0.12±0.03 0.15
Arginine 0.13±0.02 0.13±0.04 0.13±0.01 0.08
EAA 1.87±0.12 1.9±0.25 1.98±0.14 0.05
NEAA 2.79±0.01 2.8±0.17 2.81±0.23 0.05
TAA 4.66±0.55 4.7±0.52 4.79±0.48 0.05
EAA/NEAA (%) 67±1.56 68±1.12 70±1.83 0.51
CP = crude protein; EAA = essential amino acid; NEAA = non- essential amino acid; TAA = total amino acid.
Mean ± SD values in a row superscripted with different lowercase letters differ signicantly (p < 0.05).

942 Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
The current study inferred that supplementing concentrate
with 15.62% CP enhanced milk production and nitrogen
efficiency in indigenous lactating buffaloes, while having
no negative impact on DMI. The PUN also progressively
increased with the level of dietary CP, whereas other blood
metabolites and the amino acid composition of milk were not
signicantly inuenced. In this regard, a strategic supply of
CP can be recommended, especially for protein nutrition in
the diet of indigenous lactating buffaloes while incorporating
a limited supply of concentrate into the diet formulation.
The current study would be benecial for dairy processing
industries to develop nutritional and functional milk-based
innovative products for vulnerable parts of the population
based on the buffalo milk amino acid profile. However,
more research is needed to determine the relationship between
the energy and protein levels in indigenous lactating buffaloes
in Bangladesh and elsewhere in the world.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgments
The authors are grateful to the Bangladesh Academy
of Science, Dhaka, Bangladesh for financial support for
this project. The Department of Dairy Science, Bangladesh
Agricultural University assisted with samples analysis and the
Bangladesh Livestock Research Institute conducted the trial
at the Buffalo Research Farm, Animal Production Research
Division.
References
Abbasi, I.H.R., Abbasi, F., Abd El-Hack, M.E., Abdel-Latif, M.A.,
Soomro, R.N., Hayat, K., Cao, Y. 2018. Critical analysis of excessive
utilization of crude protein in ruminants ration: impact on
environmental ecosystem and opportunities of supplementation
of limiting amino acids—a review. Environ. Sci. Pollut. Res. 25:
181–190. doi.org/10.1007/s11356-017-0555-4
Ambade, V.N., Sharma, Y.V., Somani, B.L. 1998. Methods for estimation
of blood glucose: A comparative evaluation. Med. J. Armed Forces
India. 54: 131–133. doi.org/10.1016/S0377-1237(17)30502-6
Association of Official Analytical Chemists. 2005. Official Methods
of Analysis of AOAC International, 18th ed. Association of Ofcial
Analytical Chemists, Washington, DC, USA.
Bartocci, S., Terramoccia, S. 2010. Variations in the production,
qualitative characteristics and coagulation parameters of the milk of
the riverine buffalo determined by the energy/protein content of the
diet. Asian-Australas. J. Anim. Sci. 23: 1166–1173. doi.org/10.5713/
ajas.2010.80578
Bartocci, S., Terramoccia, S., Tripaldi, C. 2006. The utilisation of a high
level energy/protein diet for lactating Mediterranean buffaloes: Intake
capacity and effects on quanti-qualitative milk parameters. Livest. Sci.
99: 211–219. doi.org/10.1016/j.livprodsci.2005.06.015
Bevans, D.W., Beauchemin, K.A., Schwartzkopf-Genswein, K.S.,
McKinnon, J.J., McAllister, T.A. 2005. Effect of rapid or gradual
grain adaptation on subacute acidosis and feed intake by feedlot cattle.
J. Anim. Sci. 83: 1116–1132. doi.org/10.2527/2005.8351116x
Bos, C., Gaudichon, C., Tome, D. 2000. Nutritional and physiological
criteria in the assessment of milk protein quality for humans.
J. Am. Coll. Nutr. 19: 191–205. doi.org/10.1080/07315724.2000.
10718068
Bovera, F., Calabrò, S., Cutrignelli, M.I., Di Lella, T. 2002. Effect of
dietary energy and protein contents on buffalo milk yield and quality
during advanced lactation period. Asian-Australas. J. Anim. Sci. 15:
675–681. doi.org/10.5713/ajas.2002.675
Broderick, G.A. 2003. Effects of varying dietary protein and energy levels
on the production of lactating dairy cows. J. Dairy Sci. 86: 1370–1381.
doi.org/10.3168/jds.S0022-0302(03)73721-7
Brun-Laeur, L., Delaby, L., Husson, F., Faverdin, P. 2010. Predicting
energy × protein interaction on milk yield and milk composition
in dairy cows. J. Dairy Sci. 93: 4128–4143. doi.org/10.3168/jds.
2009-2669
Campanile, G., de Filippo, C., Di Palo, R., Taccone, W., Zicarelli, L.
1998. Inuence of dietary protein on urea levels in blood and milk
of buffalo cows. Livest. Prod. Sci. 55: 135–143. doi.org/10.1016/
S0301-6226(98)00123-7
Carberry, C.A., Kenny, D.A., Han, S., McCabe, M.S., Waters, S.M. 2012.
Effect of phenotypic residual feed intake and dietary forage content
on the rumen microbial community of beef cattle. Appl. Environ.
Microbiol. 78: 4949–4958. doi.org/10.1128/AEM.07759-11
Chanthakhoun, V., Wanapat, M., Kongmun, P., Cherdthong, A. 2012.
Comparison of ruminal fermentation characteristics and microbial
population in swamp buffalo and cattle. Livest. Sci. 143: 172–176. doi.
org/10.1016/j.livsci.2011.09.009
Cherdthong, A., Wanapat, M., Kongmun, P., Pilajun, R., Khejornsart, P.
2010. Rumen fermentation, microbial protein synthesis and cellulolytic
bacterial population of swamp buffaloes as affected by roughage to
concentrate ratio. J. Anim. Vet. Adv. 9: 1667–1675. doi.org/10.3923/
javaa.2010.1667.1675
Colmenero, J.J.O., Broderick, G.A. 2006. Effect of dietary crude protein
concentration on milk production and nitrogen utilization in lactating
dairy cows. J. Dairy Sci. 89: 1704–1712. doi.org/10.3168/jds.S0022-
0302(06)72238-X
Deeg, R., Ziegenhorn, J. 1983. Kinetic enzymic method for automated
determination of total cholesterol in serum. Clin. Chem. 29:
1798–1802. doi.org/10.1093/clinchem/29.10.1798
Delfino, N.C., Silva, R.D.G.E., Alba, H.D.R., Oliveira, M.X.D.S.,
Carvalho, G.G.P.D., Araújo, M.L.G.M.L.D., Freitas Júnior, J.E.D.
2021. Milk yield and composition, blood, and urinary parameters of
Murrah buffaloes in different maturity stages during the transition
period and early lactation. J. Appl. Anim. Res. 49: 247–256. doi.org/
10.1080/09712119.2021.1940188

943
Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
Dung, D.V., Ba, N.X., Van, N.H., Cuong, V.C., Yao, W. 2013. Practice on
improving fattening local cattle production in Vietnam by increasing
crude protein level in concentrate and concentrate level. Trop. Anim.
Health Prod. 45: 1619–1626. doi.org/10.1007/s11250-013-0407-2
Eldahshan, E., Saddick, E., Selim, S. 2020. Lactation performance, milk
fatty acid composition, and blood lipid prole of lactating buffaloes
in response to dietary soybean and linseed oils. Trop. Anim. Sci. J. 43:
254–262. doi.org/10.5398/tasj.2020.43.3.254
Fouillet, H., Mariotti, F., Gaudichon, C., Bos, C., Tome, D. 2002. Peripheral
and splanchnic metabolism of dietary nitrogen are differently affected
by the protein source in humans as assessed by compartmental
modeling. J. Nutr. 132: 125–133. doi.org/10.1093/jn/132.1.125
Franzolin, R., Alves, T.C. 2010. The ruminal physiology in buffalo
compared with cattle. In: Proceedings of the 9th World Buffalo
Congress. Buenos Aires, Argentina, pp. 104–111.
Guan, L.L., Nkrumah, J.D., Basarab, J.A., Moore, S.S. 2008. Linkage
of microbial ecology to phenotype: Correlation of rumen microbial
ecology to cattle’s feed efciency. FEMS Microbiol. Lett. 288: 85–91.
Habib, M.R., Islam, M.Z., Bari, M.S., Islam, M.Z., Sarker, M.A.H., Rashid,
M.H., Islam, M.A. 2020. Effect of concentrate supplementation during
transition period on production and reproduction of indigenous buffalo.
Bangladesh J. Anim. Sci. 49: 83–90. doi.org/10.3329/bjas.v49i1.49385
Hall, W.L., Millward, D.J., Long, S.J., Morgan, L.M. 2003. Casein
and whey exert different effects on plasma amino acid profiles,
gastrointestinal hormone secretion and appetite. Br. J. Nutr. 89:
239–248. doi.org/10.1079/BJN2002760
Haque, M.N., Rulquin, H., Andrade, A., Faverdin, P., Peyraud, J.L.,
Lemosquet, S. 2012. Milk protein synthesis in response to the
provision of an “ideal” amino acid prole at 2 levels of metabolizable
protein supply in dairy cows. J. Dairy Sci. 95: 5876–5887. doi.
org/10.3168/jds.2011-5230
Hayashi, Y., Maharjan, K.L., Kumagai, H. 2005. Feeding traits, nutritional
status and milk production of dairy cattle and buffalo in small-scale
farms in Terai, Nepal. Asian-Australas. J. Anim. Sci. 19: 189–197.
doi.org/10.5713/ajas.2006.189
Huws, S.A., Creevey, C.J., Oyama, L.B., Mizrahi, I., Denman, S.E.,
Popova, M., Morgavi, D.P. 2018. Addressing global ruminant
agricultural challenges through understanding the rumen microbiome:
past, present, and future. Front. Microbiol. 9: 2161. doi.org/10.3389/
fmicb.2018.02161
Islam, M.R., Ishida, M., Ando, S., Nishida, T. 2002. In situ dry matter,
nitrogen and phosphorous disappearance of different feeds for
ruminants. Asian-Australas. J. Anim. Sci. 15: 793–799. doi.org/
10.5713/ajas.2002.793
Islam, M.Z., Islam, M.Z., Barman, K.K., Bari, M.S., Habib, M.R.,
Rashid, M.H., Islam, M.A. 2020. Concentrate supplementation impact
on the performances of extended transitional crossbred Zebu cow.
J. Bangladesh Agril. Univ. 18: 117–123. doi.org/10.5455/JBAU.94751
Jordan, E.R., Chapman, T.E., Holtan, D.W., Swanson, L.V. 1983.
Relationship of dietary crude protein to composition of uterine
secretions and blood in high-producing postpartum dairy cows.
J. Dairy Sci. 66: 1854–1862. doi.org/10.3168/jds.S0022-
0302(83)82023-2
Kearl, L. 1982. Nutrient Requirement of Ruminant in Developing
Countries. Ph.D. thesis, Utah State University. Logan, UT, USA.
Mohd Azmi, A.F., Ahmad, H., Mohd Nor, N., et al. 2021. the impact of
feed supplementations on Asian buffaloes: A review. Animals 11: 2033.
doi.org/10.3390/ani11072033
Naveed-ul-Haque, M., Akhtar, M.U., Munnawar, R., Anwar, S., Khalique,
A., Tipu, M.A., Ahmad, F., Shahid, M.Q. 2018. Effects of increasing
dietary protein supplies on milk yield, milk composition, and
nitrogen use efciency in lactating buffalo. Trop. Anim. Health Prod.
50: 1125–1130. doi.org/10.1007/s11250-018-1539-1
Neglia, G., Balestrieri, A., Gasparrini, B., et al. 2014. Nitrogen and
phosphorus utilisation and excretion in dairy buffalo intensive
breeding. Ital. J. Anim. Sci. 13: 3362. doi.org/10.4081/ijas.2014.3362
Pasha, T.N. 2013. Prospect of nutrition and feeding for sustainable buffalo
production. Buffalo Bull. 32: 91–110.
Patra, A.K., Pal, K., Lalhriatpuii, M. 2020. Prediction of nitrogen excretion
in buffalo production systems using dietary and animal variables.
Agric. Syst. 182: 102845. doi.org/10.1016/j.agsy.2020.102845
Qureshi, M.S., Khan, S., Ahmad, N. 2007. Pregnancy depresses milk yield
in dairy buffaloes. Ital. J. Anim. Sci. 6: 1290–1293. doi.org/10.4081/
ijas.2007.s2.1290
Raq, S., Huma, N., Pasha, I., Sameen, A., Mukhtar, O., Khan, M.I. 2016.
Chemical composition, nitrogen fractions and amino acids prole of
milk from different animal species. Asian-Australas. J. Anim. Sci. 29:
1022–1028. doi.org/10.5713/ajas.15.0452
Rafsanjanny, H., Sarker, M.A.H., Islam, M.Z., Islam, K.M.S., Haque,
M.R., Islam, M.N., Siddiki, M.S.R. 2019. Blood prole of lactating
crossbred cows rearing at Bangladesh Agricultural University Dairy
Farm. Bangladesh J. Anim. Sci. 48: 139–144. doi.org/10.3329/
bjas.v48i2.46768
Rahman, S.R., Islam, M.N., Harun-ur-Rashid, M., Sarker, N.R., Siddiki,
M.S.R., Islam, M.Z., Islam, M.A. 2019a. Buffalo milk yield, quality,
and marketing in different agro-climatic districts of Bangladesh.
J. Buffalo Sci. 8: 62–67. doi.org/10.6000/1927-520X.2019.08.03.2
Rahman, S.R., Islam, M.N., Harun-ur-Rashid, M., Siddiki, M.S.R.,
Islam, M.A. 2019b. Dairy buffalo production under intensive system
in semi-arid area of Bangladesh. Buffalo Bull. 38: 83–98.
Ranjan, A., Sahoo, B., Singh, V.K., Srivastava, S., Singh, S.P., Pattanaik,
A.K. 2012. Effect of bypass fat supplementation on productive
performance and blood biochemical profile in lactating Murrah
(Bubalus bubalis) buffaloes. Trop. Anim. Health Prod. 44: 1615–1621.
doi.org/10.1007/s11250-012-0115-3
Recktenwald, E., Ross, D., Fessenden, S., Wall, C., van Amburgh,
M. 2014. Urea-N recycling in lactating dairy cows fed diets with
2 different levels of dietary crude protein and starch with or without
monensin. J. Dairy Sci. 97: 1611–1622. doi.org/10.3168/jds.2013-7162
Roseler, D.K., Ferguson, J.D., Sniffen, C.J., Herrema, J. 1993. Dietary
protein degradability effects on plasma and milk urea nitrogen and
milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76: 525–534.
doi.org/10.3168/jds.S0022-0302(93)77372-5
Santillo, A., Caroprese, M., Marino, R.O., Sevi, A., Albenzio, M. 2016.
Quality of buffalo milk as affected by dietary protein level and axseed
supplementation. J. Dairy Sci. 99: 7725–7732. doi.org/10.3168/jds.
2016-11209
Santos, J.E.P. 2002. Feeding for milk composition. In: Proceedings of the
6th International Congress on Bovine Medicine. Spanish Association of
Specialists in Bovine Medicine, Santiago de Compostela, Spain, pp. 163–172.

944 Md.Z. Islam et al. / Agr. Nat. Resour. 55 (2021) 935–944
Singh, S., Kushwaha, B.P., Maity, S.B., Singh, K.K., Das, N. 2015. Effect
of dietary protein on intake, nutrients utilization, nitrogen balance,
blood metabolites, growth and puberty in growing Bhadawari buffalo
(Bubalus bubalis) heifers. Trop. Anim. Health Prod. 47: 213–220. doi.org/
10.1007/s11250-014-0713-3
Singh, J., Singh, B., Wadhwa, M., Bakshi, M.P.S. 2003. Effect of level
of feeding on the performance of crossbred cows during pre-and
post-partum periods. Asian-Australas. J. Anim. Sci. 16: 1749–1754.
doi.org/10.5713/ajas.2003.1749
Steinfeld, H., Wassenaar, T. 2007. The role of livestock production in
carbon and nitrogen cycles. Annu. Rev. Environ. Resour. 32: 271–294.
doi.org/10.1146/annurev.energy.32.041806.143508
Terramoccia, S., Bartocci, A., Giovanni, S.D., Bartocci, S. 2012.
The influence of dietary characteristics on the milk quantity and
quality of riverine buffaloes: Estimate of the energy/protein
requirements, for a medium-high production, in the rst ninety days of
lactation. Asian-Australas. J. Anim. Sci. 25: 335–340. doi.org/10.5713/
ajas.2011.11220
Vérité, R., Delaby, L. 2000. Relation between nutrition, performances
and nitrogen excretion in dairy cows. Ann. Zootech. 49: 217–230.
doi.org/10.1051/animres:2000101
Wanapat, M. 2000. Rumen manipulation to increase the efficient use
local feed resources and productivity of ruminants in the tropics.
Asian-Australas. J. Anim. Sci. 13: 59–67.
Wanapat, M., Cherdthong, A. 2009. Use of real-time PCR technique
in studying rumen cellulolytic bacteria population as affected by
level of roughage in swamp buffalo. Curr. Microbiol. 58: 294–299.
doi.org/10.1007/s00284-008-9322-6
Wolfe, R.R., Rutherfurd, S.M., Kim, I.Y., Moughan, P.J. 2016. Protein
quality as determined by the digestible indispensable amino acid
score: Evaluation of factors underlying the calculation. Nutr. Rev. 74:
584–599. doi.org/10.1093/nutrit/nuw022
Zhou, L., Tang, Q., Iqbal, M.W., et al. 2018. A comparison of milk protein,
fat, lactose, total solids and amino acid profiles of three different
buffalo breeds in Guangxi, China. Ital. J. Anim. Sci. 17: 873–878.
doi.org/10.1080/1828051X.2018.1443288