Effect of Dietary
Supplementation with Betaine on Growth Performance and Blood Biochemical
Profile of Nili-Ravi Buffalo Calves
Zakir Hussain1†, Zahid Iqbal2*†, Nabila Roohi1† and Sirzamin Khan3†
1Department of Zoology, University of the Punjab, Lahore 54590,
Pakistan
2Department of Animal Production, Riphah
College of Veterinary Sciences, Riphah International University, Lahore 54000,
Pakistan
3Department of Poultry Sciences, Agriculture
University, Peshawar 25120, Pakistan
*For correspondence: zahidzizi@gmail.com
†Contributed equally to this
work and are co-first authors
Received 03 August 2021; Accepted 14 October 2021;
Published 15 December 2021
Abstract
This study aimed to
determine the effect of betaine supplementation on production performance and
blood biochemical parameters in calves. Sixty Nili-Ravi calves were randomly
divided into four groups: the control group, which received a total mixed
ration (TMR) without betaine and three experimental groups that received TMR
blended with 0.2, 0.4 and 0.6% betaine for 8 weeks. Feed and water intake was
recorded daily and production performance parameters were recorded on weekly
basis. Blood samples were collected weekly and at the end of the experiment.
The results showed that feeding betaine to calves increased dry matter intake,
body weight gain and average daily gain (P
< 0.001). Dietary addition of 0.6% betaine improved feed conversion
ratio (P < 0.01). Plasma cortisol,
superoxide dismutase, and malondialdehyde levels were positively affected (P < 0.05) by betaine supplementation
as compared to the control group; however, it caused HSP70 levels to decrease (P < 0.05). The results indicated that
supplementing betaine to the diet of Nili-Ravi
calves enhanced growth performance and improved their antioxidant
status. © 2021 Friends Science Publishers
Keywords: Nili-Ravi
calves; Betaine; Growth; Blood biochemical
Introduction
The great potential
of converting low-valued fibrous feedstuff into useful degradable products
marked the buffalo (Bubalus bubalis) as a significant contributor to
global meat and milk production (Terramoccia et al. 2000). For sustainable
buffalo production, calf rearing has been considered an important segment in
the livestock sector. Several serious
concerns, linked to the initial stage of calf rearing impacting the growth and
post maturity performances, are still needed to be dealt with. Besides
management and health concerns, the dietary formulations of calves seek substantial attention for better
production. In the last decades,
several dietary supplements have been extensively evaluated to improve calf
health, production, and growth performance (Kaufhold
et al. 2000). Among
different feed additives, the Betaine (BET) supplementation showed a positive impact on the
growth, reproductive, and production performances of different animals and
birds (Wang et
al. 2019; Alhotan et al. 2021;
Zhong et al. 2021). The
remarkable influence of BET supplementation on the reduction of heat stress (Hall et al.
2016) could notably improve the buffalo performance which is generally
highly affected in summer due to its black body coat. In addition, betaine
improves the digestibility of nutrients by maintaining stomach pH, physiological
and nutritional functions (Eklund et al. 2005). The purified
betaine is highly absorbable in the intestine and has a synergistic effect on
nutrient uptakes, improves digestibility of carotenoid, fat, lysine, protein
and methionine (Kettunen et al. 2001) which enhance body weight (BW) in different
animals (Nezamidoust et al. 2012; Wang et al.
2019).
In simple stomach animals, the
role of BET is well established in improving cell integrity and function. As a
methyl donor, it supplies the methyl group which is an integral part of many
intermediate substances required for the synthesis of protein and energy
metabolic pathways (Wang et al. 2019). In ruminants, supplemental BET plays its role
in two ways (Hall et al. 2016). Firstly, it is degraded in the rumen to a
variable extent and utilized by the rumen microbes resulting in enhanced
fermentation activity and increased production of short-chain volatile fatty
acids which are a major source of energy for ruminants (Wang et al. 2020a).
Under normal conditions, the acetic acid is the ultimate end product of BET but
under stressed and hyperosmotic conditions the end product of BET degradation
is propionate which is a glycogenic precursor and an energy source. Table 1:
Ingredient composition of total mixed rations (TMR) used in the
post-weaning experiment on Nili -Ravi calves
Ingredients |
Inclusion % |
Ground maize |
37.0 |
Wheat straw |
15.0 |
Maize Gluten 30% |
13.0 |
Rice polish |
11.0 |
Molasses |
5.0 |
Soybean meal |
16.50 |
Mineral premix |
1.0 |
Salt |
0.5 |
Lime |
1.0 |
Nutrient composition |
|
DM% |
88.5 |
CP |
16.0 |
ME (M. cal/kg) |
2.70 |
Fat (%) |
2.5 |
NDF (%) |
26.0 |
ADF (%) |
15.4 |
Ash (%) |
6.91 |
Ca (%) |
1.18 |
P (%) |
0.59 |
Secondly, some BET which escapes rumen degradation has a role
similar to that seen in simple stomach animals. Studies have been carried out
on the use of BET in different farm animals like transition and lactating cows,
rams, goats and bulls under normal and stressed conditions with variable
results on DMI, milk yield and composition, weight gain, feed conversion
efficiency and carcase characters. Relatively, the dietary effect of BET on the
production of post-weaning Buffalo calves has scarcely been deliberated.
Therefore, the current study was designed to evaluate the effects of dietary
supplementation with BET on the growth performance and blood biochemical
profile of Nili-Ravi buffalo calves under hot summer environmental conditions
of Pakistan.
Materials and Methods
Experimental design, animals, housing and treatments
The experiment was conducted at
Government Dairy Farm Jhelum, Punjab, Pakistan from July to September 2018. Sixty healthy female
buffalo calves having an average BW 71.69 ± 5 kg at weaning were blocked by BW
and allocated to one of the four treatment groups each having fifteen animals,
in a randomized complete block design. The animals were previously vaccinated
and dewormed as per Farm's policy. Strict bio-security measures were observed
with a close watch on health. The calves were fed a basal total mixed ration
(TMR) without BET (CON) or with BET as feed additive at the rate of 0.2, 0.4
and 0.6% of the ration dry matter for eight weeks excluding one week of
adaptation. Feed grade BET product 'Betafin®' (DuPont Animal Nutrition, USA)
was procured from 'UM Enterprises Pakistan' in a single consignment and
thoroughly mixed at the time of feed preparation for each treatment at the farm
feed milling unit. The ingredient composition and nutrient profile of TMR are
given in Table 1. All the calves were individually housed in cages under shed
and overhead fans were used for ventilation. A measured quantity of drinking
water was made available round the clock in steel buckets. TMR was offered once
daily in the morning to ensure a minimum 5% refusal. Temperature and humidity
were recorded thrice a day in the morning, afternoon and evening with the help
of a digital Temperature Humidity meter (Onway Technology, China).
Body weights and
sampling
The amount of feed and water offered and refused were recorded daily to
calculate feed and water intake. The previous day's orts were collected and
weighed in the subsequent morning before offering a fresh feed. The dry matter
content of TMR was determined fortnightly. All the calves were weighed
individually at the start and on weekly basis until the end of the experiment.
The weighments were carried out before feeding using a locally manufactured
digital weighing scale (Wazan Tech®). Bodyweight gain was calculated
by subtracting the initial weight from the final weight at the end of each
week. Average daily gain (ADG) by each calf during the experimental period was
calculated by dividing the total weight gained by the number of days. Feed
conversion ratio (FCR) was calculated by dividing average daily dry matter intake
(DMI) by the ADG (Abd-Allah and Daghash 2019).
Blood sampling and
analyses
Blood samples from the calves were collected via the jugular
vein using anticoagulant coated (sodium-heparinized) and plain vacutainer tubes
(10 mL) on weekly basis. The samples were centrifuged for 15 min at 3000 rpm
and the plasma and serum samples harvested were stored at -20°C until analysis.
Plasma concentrations of glucose were determined calorimetrically using
commercial kits (Alfawassermann, Milano, Italy) as per the manufacturer’s
instructions. Serum electrolytes and enzymes were measured photo-metrically
with an automatic biochemical analyzer (Daytona®, Randox) at
Combined Military Hospital Laboratory Jhelum Cantonment using various
commercial kits according to procedures described by previous researchers (Ježek et al.
2010; Wang et al. 2010a, 2020b).
Creatinine level was estimated using an automatic creatinine analyzer (Beckman
Synchron LX20 with Synchron LX Creatinine Reagent Kit). The serum cortisol
concentration level was determined using a commercial Bovine ELISA test kit
(Stress Xpress® Cortisone CLIA Kit). Malondialdehyde (MDA) and
superoxide dismutase (SOD) were assayed using a commercial kit (Wang et al.
2010a, 2020b).
Statistical analysis
Data collected were analyzed in
a randomized complete block design using the software SPSS 20 version (SPSS 2011). The significant association of parameters between Table 2: Effect of betaine supplementation on growth
performance of Nili-Ravi calves (Mean ± SEM)
Parameters |
BET 0 (Control) |
BET 2 |
BET 4 |
BET 6 |
Dry matter intake (kg/day) |
3.60 ± 0.023c |
4.28 ± 0.025 b |
4.32 ± 0.035b |
4.49 ± 0.023a |
Water intake (Litres/day) |
11.72 ± 0.136c |
12.40 ± 0.108b |
12.84 ± 0.045a |
12.98 ± 0.087a |
Weight gain (kg) |
22.80 ± 0.5787c |
26.43 ± 0.511b |
27.64 ± 0.868b |
31.84 ± 0.201a |
ADG (kg/day) |
0.40 ± 0.009c |
0.47 ± 0.008b |
0.49 ± 0.015b |
0.56 ± 0.004a |
FCR |
9.04 ± 0.24b |
9.00 ± 0.13b |
8.85 ± 0.062b |
7.90 ± 0.07a |
Different superscripts (a, b, c) within the same row indicate significant association (P < 0.01) between two different groups. ADG= Average daily gain, FCR=Feed to gain ratio
Table 3: Effect of betaine
supplementation on serum enzymes and blood chemical parameters of Nili-Ravi calves (Mean ± SEM)
Parameters |
BET 0 (Control) |
BET 2 |
BET 4 |
BET 6 |
Glucose (mg/dL) |
5.791 ± 0.055a |
5.524 ± 0.066a |
5.589 ± 0.046b |
5.564 ± 0.047b |
Creatinine (mg/dL) |
11.221 ± 0.166c |
12.042 ± 0.104a |
11.995 ± 0.120b |
12.237 ± 0.130b |
Cortisone (mg/dL) |
13.017 ± 0.210 |
13.106 ± 0.468 |
13.422 ± 0.178 |
13.803 ± 0.299 |
Sodium (mg/dL) |
136.085 ± 0.836a |
128.618 ± 0.975b |
129.616 ± 1.176b |
125.643 ± 0.295c |
MDA (nM)
|
1.35 ± 0.42c |
1.45 ± 0.20b |
2.06 ± 0.74a |
2.08 ± 0.74a |
SOD (U/L) |
13.07 ± 0.23b |
13.91 ± 0.20b |
14.88 ± 1.87a |
15.08 ± 0.87a |
Calcium (mg/dL) |
3.394 ± 0.032 |
3.433 ± 0.094 |
3.622 ± 0.149 |
4.104 ± 0.249 |
Potassium (mg/dL) |
11.983 ± 0.126 |
11.890 ± 0.029 |
11.970 ± 0.060 |
11.719 ± 0.074 |
Different superscripts (a, b, c) within the same row indicate
significant association (P < 0.05) between two
different groups. MDA= Malondialdehyde, SOD= Superoxide dismutase
four groups was estimated with the implementation of the One-way
analysis of variance (ANOVA) method. Verification of significant differences
was also done using Tukey’s test with a significance level of ≤ 0.05.
Results
In
this experiment BET supplementation positively (P < 0.05) affected the DMI, water intake, growth performance and FCR in buffalo calves
(Table 2). Feed conversion ratio (Average daily DMI to average daily
gain) was significantly better for BET 6 as compared to Control, BET 2 and
BET4. Mean values of various blood chemical parameters and serum enzymes as
influenced by the treatments have been presented in Table 3. Serum creatinine
concentration remained significantly higher in BET supplemented groups as
compared to control.
Discussion
The
results of this experiment are in agreement with those of Lakhani et al.
(2020) who indicated that dietary supplementation with BET increased BW
gain, DMI and FCR in crossbred cow heifers. A study carried out under the same
tropical summer conditions by Deshpande et al. (2020), described a
similar growth-promoting effect of BET in Buffalo heifers in the shape of
higher (P ≤ 0.05) ADG and DMI as compared
with control. The findings are in line with several other studies. A 2x2
factorial study conducted by Wang et al. (2020a) reported 25.2%
higher (P ≤ 0.01) ADG and 16.25% lower FCR
by feeding BET supplemented diet to Angus bulls
without the addition of rumen-protected folic acid. DiGiacomo et al. (2014) also found a trend (P ≤ 0.05) of higher carcase weight in feedlot steers with dietary
addition of BET during summer. Another research by Bock et al. (2004) reported
increased ADG and dressing percentage of steers. In contrast, a previous study
reported that dietary supplementation with 2 g/kg BET did not affect relative
growth parameters, especially weight gain and average daily gain of 6-month-old
calves, however; certain carcase characters like marbling score showed
improvement (Loest et al. 2002). The reason for this difference in performance
could be related to the complete degradation of supplemental BET by the rumen
microbes in later study restricting the availability of methyl group for the
animal (Loest et al. 2002; Nezamidoust et al. 2012). The inability
of BET to affect homocysteine remethylation in the said study might also be
attributed to the non-availability of a vitamin co-factor like pyridoxine
and/or folic acid (Lambert et al. 2004). Improved animal growth rate in current
and other studies with BET supplementation could partially be attributed to the
increased nutrient digestibility, enhanced microbial fermentation, more
production of volatile fatty acids and maintenance of mammalian cell integrity under stressful
conditions (Wang et al. 2010b; Hall et al.
2016). Another idea that has been advanced to explain the mechanism by
which betaine supplementation improves body performance is that BET improves
intestinal function by enhancing the digestive enzymes, ameliorating intestinal
morphology, and enriching intestinal microbes (Wang
et al. 2018). Feed conversion
ratio in this experiment was also improved by the increasing level of BET in
the diet which is also consistent with the findings of Chand et al. (2017) for
broilers and Yu et al. (2004) for pigs. The possible reason can be due
to the accumulation of BET residues in muscle cells and an increase in water
retention capacity in muscles (Matthews et al. 2001). In our
study, improvement in growth performance can also be correlated with the
phenomenon that BET helps in energy and protein metabolism (Metris et al.
2014), improves fiber and other nutrient digestibility, and thus
improves growth (Liu et al. 2021). Higher water intake in BET fed animals is also
in agreement with Hall et al. (2016). Higher DMI as noticed in our experiment
substantiate the earlier findings of Shankhpal et al. (2018) and Deshpande et al.
(2020) who also reported higher DMI which positively correlated with ADG
in BET supplemented animals. However, the same is in partial agreement with Hall et al.
(2016), who reported a higher DMI in BET supplemented lactating Holstein
cows under thermo-neutral environmental conditions but a lower DMI in BET
supplemented groups under heat stress. The reason could be species difference
and lactation stress in the later study.
Blood biochemical indices are
considered a key indicator of animal health status. In the present study, most
of the blood biochemical parameters were non-significantly affected by BET
addition in buffalo calves' diet (Table 3). Among serum enzymes, creatinine
showed significant influences because of dietary supplementation with BET.
Likewise, a previous study also concluded that BET has the potential to play a
vital role in the synthesis of metabolic substances like creatinine and
cortisone by transmethylation reaction converting methyl group into choline and
methionine (Ratriyanto et al. 2009). Plasma glucose levels increased significantly
with dietary BET at the rate of 0.2% as compared to control and other treatment
groups. The results of the present study are in line with the findings of Abd-Allah and Daghash (2019) who observed an
increase in glucose level in Ossimi rams treated with BET supplemented diet. The
increase in glucose level might be attributed to the elevation of thyroid
hormones including T3 and T4. During the circulation in the body, T4 is mainly
transformed into T3 (Zhang et al. 2014). Circulating T3 is an indicator of metabolic
and nutritional status because of the involvement of this hormone in the
production of glucose as a result of protein synthesis and lipid metabolism (McDonald 1980). The administration of T3
hormones has been known to increases glucogenesis, glycogenolysis and
absorption of glucose from the intestinal tract. In contrast, a lack of
residual effect of the BET feeding was noted in the plasma glucose
concentration of sheep by DiGiacomo (2011).
Malondialdehyde and SOD activity was higher (P < 0.05) in the
treatment groups as compared to the control group. These results of the present
study are in agreement with the findings of (Zhang
et al. 2014). Betaine has an
antioxidant capacity, which enabled it to scavenge free radicals and protect
cells from loss in rats (Lu et al. 2008). Results of the current study indicate a
reduction in the reactive oxygen species and free radicals, along with an
improvement in the antioxidant capacity. Besides growth performance and serum
enzymes, the BET supplementation showed a negative impact on the sodium and
potassium concentration, however; calcium concentration was positively
increased in treatment groups. These findings are in agreement with the
findings of Khattak et al. (2012) who observed a decrease in sodium and
potassium concentration in betaine treated group as compared to the control.
Betaine supplementation has an influence on sodium concentration by enlarging
villi and increasing the thickness of crypts (Lee
et al. 2019).
Conclusion
This experiment has
shown that supplementation of BET to the diet of Nili-Ravi calves enhanced growth performance and improved their
antioxidant capacity.
Acknowledgements
The authors are
thankful to Military Farms Jhelum authorities, for permitting and facilitating
us to conduct this research at their farm.
Author Contributions
All authors contributed to the study
conception and design as well as manuscript writing. Material preparation, data
collection and analysis were performed by Zakir Hussain. All authors read and
approved the final manuscript for submission.
Conflict
of Interest
The authors declare that they have no
conflict of interest.
Data
Availability
Data used in this study can be made available
on a serious request to the authors.
Ethics
Approval
All applicable international, national and
institutional guidelines for care and use of animals were followed. The
experimental protocol was approved by the Animal
Care and Use Committee, University of the Punjab, Lahore.
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