Effect of supplementing hydrolysable tannins extract on milk proteins and productive performance of early lactating Nili Ravi buffaloes

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Effect of supplementing hydrolysable tannins extract on milk proteins and productive performance of early lactating Nili Ravi buffaloes

https://doi.org/10.21203/rs.3.rs-1603755/v1

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Supplementation of tannin extract with proper dosage can increase the protein precipitation, decreasing the ruminal protein losses, ensuring the availability of nutrient and their utilization in the body. Nine early lactating Nili Ravi buffaloes were selected and divided into 3 groups. Buffaloes were randomly assigned to 1of 3 treatments in a 3× 3 Latin square design. Treatments were 0, 15, and 30 g/d per animal supplemental levels of tannin extract. The period length of each treatment was 21 days. All the buffaloes were fed similar basal diet and tannin extract was mixed in concentrate. Dry matter intake and milk protein content increased linearly with increasing tannin supplementation. Nitrogen intake, apparent digestibility of nitrogen, and milk nitrogen efficiency were similar among treatments. Blood urea nitrogen and milk urea nitrogen decreased with tannin supplementation indicating reduced protein degradation. Manure scoring increased but feed efficiency decreased linearly with increasing level of tannin supplemental level. Overall, it is observed that tannin supplementation decreases blood and milk urea nitrogen and increases milk protein content and tends to increase dry matter intake.

Buffalo

Milk nitrogen efficiency

Milk urea nitrogen

Nitrogen digestibility

Milk production

Amino acids are the building block of proteins present in animal’s feed. Proteins are long chains of amino acids (Lopez and Mohiuddin, 2020). Proteins are most expensive components of dairy animal’s feed (Onsongo et al. 2018). Dairy animals require amino acids present in small intestine for milk production as well as tissue growth (Estes, 2017). Ruminant have less capability to utilize whole nitrogen. Therefore nitrogen loss in the form of ammonia occurs in the rumen (Nasiru et al. 2014). Lactating cows use up to 25% of ingested nitrogen to produce milk. The rest is excreted through feces and urine in the environment that also causes environmental pollution (Herremans et al. 2020).

Excess ammonia is absorbed through the villi of rumen. It goes to liver by hepatic portal veins and its breakdown occurs in the liver. It converted to urea in liver by urea cycle. Urea is finally excreted from kidneys through urine (Bach et al. 2005). However, in ruminants, urea is also recycled to rumen, small and large intestine. It can enter the rumen in saliva and be excreted from kidneys or secreted in sweat or milk (Getahun et al. 2019).

Conversion of ammonia to urea is the only way to avoid ammonia toxicity in blood. Elimination of urea is an energy dependent process as animals use energy to excrete the extra urea through urine. Excess protein consumption leads to high level of ammonia as well as urea in the blood and it has bad impact on milk yield, reproduction, embryo viability, immune function, and in the environment. In order to reduce the nitrogen loss in the rumen, the proteins are needed to be protected from extreme degradation in rumen so that amino acids are absorbed from lower gut for synthesis of proteins and gluconeogenesis in liver (Walli, 2005).

Tannins decreases urinary nitrogen loss and increases fecal nitrogen loss in ruminants (Powell et al. 2009). Balanced concentrations of tannins in ruminant diets can minimize the nitrogen loss and increase flow of amino acids towards intestines due to forming complexes with feed proteins and thus protecting them from degradation in the rumen and increase protein supply (Min et al. 2003). Tannins are water-soluble polyphenols having higher molecular weight with multiple phenolic hydroxyl group that form complexes with proteins, amino acids and metal ions present in rumen. Tannin forms tannin-protein complex through hydrogen bonding which is stable at 5–7 pH of rumen and intestine but dissociated at very low pH of abomasum as pH of abomasum is 2–3. Tannins bypass the protein from rumen to small intestine which leads to increased digestibility and absorption ultimately improving the production performance of animals (Makkar, 2003). To determine the effect of tannin supplementation in buffaloes, dose-response studies are required that are not done previously in buffalo as per author’s knowledge. In the light of these observations, our objective in this study was to investigate the effect of different levels of tannin supplementation on Milk yield, milk composition, plasma metabolites, apparent nitrogen digestibility and tannin estimation in feces.

Animal

The experiment was conducted at Buffalo Research Institute, Livestock and Dairy Development Department, Punjab, Pakistan, in September 2019. Nine (9) early lactating Nili-Ravi buffaloes with average weight of 550 ± 22 kg were taken and 70 ± 5 days in milk (dry matter intake) were used. One week before start of experiment, the average daily milk yield of buffaloes was 12 ± 5.0 kg/day. All animals were administered ivermectin and levamisole for deworming before starting research trial. The buffaloes were individually tied in a semi-open shed and were provided free access to water.

Experimental design, treatments, and feedings

Nine (9) buffaloes were randomly divided into 3 groups. Each group had 3 animals. Animals were randomly assigned to 1 of 3 treatments. Treatments were 0, 15, and 30 g/d per animal supplemental levels of tannin extract in a 3 × 3 Latin square design. Supplemented hydrolysable tannin extract with concentration of 100gm (VEGANOK Pharmaceuticals®, Italy) was purchased from market and mixed in feed with prescribed doses. The total duration of experiment was 63 days and the duration of each period was 21 days, where the first 7 days were used for dietary adaptation. The diets were formulated using CPM-Dairy 3.0.10 based on Cornell Net Carbohydrate and Protein System (CNCPS) 5.0 (Fox et al. 2004). All diets were isonitrogenous iso-energetic and provided 1.48 Mcal of NE_L. The ingredient composition and nutritive values of diets are presented in Table 1. Briefly, the diet was composed of (total mixed ration) TMR having corn silage (50%), concentrate (45%), and wheat straw (5%). Buffaloes were offered 17 kg of dry matter (DM) per day. This was achieved by feeding ad libitum diet 7 days before the start of experiment and estimating the individual buffalo dry matter (DM) intake. The diets were offered once in the morning at 0900 h, whereas buffaloes were milked twice at 0500 and 1500 h.

Table 1

Ingredient, chemical, and nutrient compositions of the basal diet
Item	Basal diet
DM (%)	51.6
Ingredient (% of DM)
Corn silage	50.0
Wheat straw	5.00
Corn grain	10.0
Canola meal	2.40
Corn gluten 30%	7.90
Wheat bran	13.0
Sugarcane molasses	5.00
Rapeseed meal	6.00
Mineral premix	0.50
Sodium bicarbonate	0.20
Nutrient composition (% of DM)
Crude protein	14.0
Neutral detergent fiber	43.3
Acid detergent fiber	23.1
Ether extract	2.98
Ash	7.86
Nonstructural carbohydrates	31.8
Nutritive values
RUP^a (% of CP)	35.5
RDP^b (% of CP)	64.5
NE_L^c (Mcal/kg of DM)	1.48
^aRUP: Rumen undegradable protein predicted using CNCPS evaluation
^bRDP: Rumen degradable protein predicted using CNCPS evaluation
^cNE_L: Net energy for lactation predicted using CNCPS evaluation

Measurement, sample collection, and preparation

Diet offered as well as refused was weighed daily. Three samples of each feedstuff were collected in each period for evaluating the dry matter (DM) and for further laboratory analysis. Diet Samples were analyzed for dry matter (DM), crude protein (CP), nutrient detergent fibers (NDF), acid detergent fibers (ADF), ether extract (EE), and ash following official methods of AOAC (2005). Milk yield of buffaloes was recorded twice a day (morning and evening). Milk samples were collected and analyzed (morning and evening) twice a week in the first 2 weeks and every day in the last week of every period for milk composition using Ultrasonic milk analyzer Milk tester Master (Milkotester LTD, Belovo, Bulgaria). Ultrasonic milk analyzer was used for analysis of milk protein, fats, and lactose concentration. Blood samples of all the buffaloes were collected on the last 3rd day of each period from jugular vein in heparinized syringes and centrifuged immediately. Plasma was separated, aliquoted, and stored at − 20°C for the analysis of urea, by spectrophotometer using enzymatic kits (Fluitest Urea, Analyticon Biotechnologies AG, Lichtenfels, Germany at university diagnostic lab, Ravi campus Pattoki). Urease converts urea into ammonia and carbon dioxide through enzymatic hydrolysis. Reaction of ammonia with 2-oxoglutarate and NADH yields glutamate in presence of glutamate dehydrogenase. Milk urea nitrogen (MUN) was estimated using a colorimetric p- Dimethyl amino benzaldehyde methods define by AOAC, (2000).

On 19d (3rd last day) of each period, faecal samples from each buffalo were taken. In the first period, total fecal output of buffaloes was determined through total collection method, whereas in the subsequent 2 periods only fecal samples were collected through grab method. These 2 different methods of fecal sample collection were used to obtain average results. Fecal grab samples were taken every 12h in d 18–19 of each period (4 samples/ buff/period) in 24 hours. A composite sample (50g) from each individual sample (4) was made. A fifty gram of wet sample was dried at 55° C for 48 hours and was ground to pass 1-mm screen size to determine N (Leonardi et al. 2003). Daily fecal scoring (1–5) was done according to the method described by Sterrett et al. (2014). The total tannin estimation was done by modified method of Makkar (2003). Buffaloes were weighed on the last day of each period.

Statistical analyses

Collected data were analyzed mixed procedure of SAS (SAS Institute Inc. 1996). Analysis of tannins was done according to the method described by Makkar HPS (2003). Treatments, periods, and buffaloes were the main effects. Treatments were compared in linear and quadratic polynomial contrasts. A linear component indicates that there is a significant difference between first and the last level. Quadratic component implies the lack of fit of intermediary level. Significance level was set at P ≤ 0.05 and the tendency was set at 0.05 < P ≤ 0.10.

Intake, milk production, and milk composition

Dry matter intake (DMI), milk yield and its composition data are presented in Table 2. Despite slight numeral linear increase, there was no significant difference in DMI. Milk yield did not differ significantly, although numerically a light linear decrease was observed. There was no treatment mean difference for milk composition and component yield, however a significant (P < 0.05) numerical linear increment was observed on protein content percent (Table 2). Average for milk protein yield was 435 g/d, for milk fat yield was 751 g/d, average milk lactose yield was 489 g/d, and solid not fat (SNF) average yield was 1033 g/d. All milk contents and yield were unaffected by tannin administration.

Table 2

Effects of different doses of tannin extract supplementation on milk yield and milk composition of Nili Ravi buffaloes
Item^a	Treatments^b			RMSE^c	P-value
	0	15	30		Treat	L	Q
DMI (kg/day)	16.5	16.6	16.7	0.03	0.18	0.06	0.93
Milk yield (kg/day)	12.5	12.5	12.5	0.07	0.96	0.79	0.95
Protein
Yield (g/day)	425	437	443	14.0	0.60	0.33	0.83
Content (%)	3.43	3.45	3.67	0.05	0.06	0.03	0.28
Fat
Yield (g/day)	750	768	735	35.1	0.76	0.73	0.51
Content (%)	6.03	5.83	6.05	0.141	0.77	0.96	0.48
Lactose
Yield (g/day)	483	507	477	13.0	0.16	0.75	0.11
Content (%)	3.88	4.00	3.95	0.073	0.38	0.40	0.26
^aDMI: dry matter intake
^bTreatments were three different supplemental levels of tannin extract (0, 15, and 30 g/d)
^cRMSE: root mean square error

Body weight and feed efficiencies

Body weight and feed efficiency are presented in Tables 3 and 4. No effect was observed on body weight of buffaloes. The initial body weight (IBW) increased numerically in quadratic manner which was not significant and there was not difference in treatment for final body weight (FBW). Feed efficiency of control group was higher than those treated with 30 g (P < 0.05), however there was no significant difference between group with feed efficiency of 15 g and both control (0 g) and 30 g groups.

Table 3

Effects of increase in tannin supplementation on nitrogen intake, digestibility and efficiency in buffaloes
Item^a	Treatments^b			RMSE^c	P-value
	0	15	30		Treat	L	Q
N intake (g/d)	328	334	328	2.11	0.40	0.95	0.17
N digestibilty (%)	62.8	62.0	62.5	0.90	0.78	0.78	0.51
Neff (%)	19.6	20.1	20.2	0.61	0.71	0.45	0.73
^aN = Nitrogen; N Dig = Nitrogen Digestibility; Neff = Nitrogen Efficiency
^bTreatments were three different supplemental levels of tannin extract (0, 15, and 30 g/d)
^cRMSE, root mean square error

Table 4

Feed efficiency and body weight of Nili Ravi buffaloes in response to different levels of tannin supplementation
Item^a	Treatments^b			RMSE^c	P-value
	0	15	30		Treat	L	Q
FE	0.75	0.74	0.73	0.01	0.03	0.01	0.54
IBW (Kg)	529	574	557	37.84	0.52	0.47	0.38
FBW (Kg)	551	551	551	15.16	1.00	0.98	0.99
^aFE = Feed Efficiency; IBW = Initial Body Weight; FBW = Final Body Weight
^bTreatments were three different supplemental levels of tannin extract (0, 15, and 30 g/d)
^cRMSE: root mean square error

Blood urea nitrogen milk urea nitrogen, and manure scoring

The effect of effect of tannin supplementation on blood urea nitrogen (BUN), milk urea nitrogen (MUN), nitrogen digestibility and manure scoring in Nili Ravi buffaloes (Table 5). Mean BUN was significant (P < 0.05) with tannin addition in basal diet from 0 g to 30 g. The MUN decreased (P < 0.01) while manure scoring increased and both effects were linearly with tannin addition as shown in Table 5.

Table 5

Blood and milk urea nitrogen and manure scoring of Nili Ravi buffaloes in response to different levels of tannin supplementation
Item^a	Treatments^b			RMSE^c	P-value
	0	15	30		Treat	L	Q
BUN (mg/dl)	19.53	18.80	18.16	0.37	0.02	< 0.01	0.90
MUN (mg/dl)	13.66	12.66	11.88	0.42	< 0.01	< 0.01	0.79
Manure scoring	2.58	2.63	2.93	0.58	< 0.01	< 0.01	0.04
^aBUN: blood urea nitrogen; MUN = milk urea nitrogen
^bTreatments were three different supplemental levels of tannin extract (0, 15, and 30 g/d)
^cRMSE: root mean square error

Tannin in Feces

The traceable tannins were nearly absent in buffaloes feces. In control group it was 0.13% ± 0.006 while in group with feed efficiency of 15g and 30g, it was 0.26% ± 0.007 and 0.343% ± 0.005 of dry matter intake (DMI) respectively. Nearly no extractable total tannins were present in the feces.

The influence of tannins on DMI in ruminants are controversial. In this study, the DMI was almost unaltered in different experimental groups. It did not exert any negative impact on DMI in early lactating buffaloes. Similar to our findings, many authors reported no effect on DMI when supplemented with tannins (Sliwinski et al. 2004; Benchaar et al. 2008; Mezzomo et al. 2011; Alves et al. 2017). Contrary to our findings, (Carulla et al. 2005) studies found increase in dry matter intake in sheep when fed Acacia mearnsii. In contrast, there existed decrease in DMI in few studies due to tannin supplementation. A linear decrease was reported in DMI with increasing level of tannin (45–1.80% of DM) in dairy cows when fed with the mixture of quebracho-chestnut tannin extracts (Aguerre et al. 2016). Normal level of tannins in various fruits and leaves is up to 2–5% (Haslam, 2007). Addition of quebracho tannin at 3% of diet DM decreased DMI of lactating cows (Dschaak et al. 2011). Intra-ruminal administration of tannins at 3g/kg DM in ewes also reduced DMI when fed with alfalfa hay (Hervas et al. 2003).

The contradictory findings are related to ruminant’s specie, rumen micro flora differences, animal stage, tannin type, feeding type and their concentration (Bueno et al. 2020). Use of tannin as a supplement is dose dependent. Dosage determine the effect of tannin. In the current study, there was no substantial increase or decrease in milk yield of buffaloes. Buffaloes from different treatment groups maintained their production. Similar to our findings, several authors found no effect of tannin addition on decrease or increase in milk production (Aguerre et al. 2016; Liu et al. 2013; Dscchaak et al. 2011; Benchaar et al. 2008; Siliwinski et al. 2004). Contrariwise, many others reported enhancement in milk production in cows, goats and in ewes (Woodward et al. 1999; Rouissi et al. 2006; Penning et al. 1988; Wang et al. 1996). The major cause for more milk production in their trial was attributed to the provision of increase protein supply to lower gut (Patra and Saxena 2011). Protection of protein in rumen resulted into more milk production. In some studies, harmful impacts of tannin addition on milk production existed. Dairy cows showed lowered milk production from 3.6–9.7% when fed acacia mearnsii as a source of condensed tannins (Grainger et al. 2009). In ewes, reduction in milk yield was recorded when supplemented with acacia cyanophylla foliage as a source of tannin (Maamouri et al. 2011). According to current study, milk contents remained unchanged except proteins which increased linearly with addition of tannin in the diet. Our results are in agreement with Wang et al. (1996) in dairy cows and woodward et al. (2001) who reported increased milk protein contents in dairy sheep. Bhatta et al. (2001) found significant increase of protein content in milk. It was concluded that tannins has a positive effect on milk protein content at low concentration. Aguerre et al. (2016) found substantial increase in proteins contents of milk when mixture of chestnut-quebracho tannin was offered in diet at 0.45% of DM. Others found no change in milk composition of tannin administration on milk proteins, milk fat, and lactose contents of milk (Benchaar et al. 2008; Dschaak et al. 2011, Liu et al. 2013).

The BUN and MUN decreased significantly with tannin addition in buffalo’s diet. These findings are consistent with Aguerre et al. (2016) who noticed a gradual decrease in BUN from 15.4mg/dl to 13.5mg/dl and MUN from 14 to 12.9 mg/dl when a mixture of chestnut- quebracho tannin was added in dairy ration. This reflects lowered rumen degradation of proteins. Dschaak et al. (2011) also observed decrease in milk urea nitrogen from 14.8 to 12.3 mg/dl without loss of milk proteins. Jolazadeh et al. (2015) found a linear decrease in blood urea nitrogen (BUN) in Holstein bulls. Barajas et al. (2011) also observed bulls that were supplemented with tannin extract had 1.48mg/dl lesser BUN level than the control group that were not supplemented with tannin. Hence it leads to less breakdown of protein in rumen and reduction in NH3-N production in the rumen which resulted in decrease in MUN concentration. Others reported unaltered milk composition while supplementing tannin in diet. (Benchaar et al. 2008; Liu et al. 2013).

The protein concentration in buffalo diet was same across the trial. Nitrogen digestibility was not affected by administration of tannin in buffalo’s diet. Nitrogen digestibility remained almost similar among different treatment groups. Our results were consistent with other studies that had also reported unaltered nutrient digestibility when tannin was added to buffalo’s diet. Our results were similar to the work conducted by Dschaak et al. (2011) who observed that digestibility of CP, DM, OM, NDF, and ADF remained unaltered by adding CT in basal feed. Likewise, Benchaar et al. (2008) also observed no change in digestibility of nutrients (CP, ADF and OM) when 0.64% of DM CTE was added in feed. Similarly, Baah et al. (2007) noted that there were no significant changes in different nutrient when supplemented with quebracho CTE at 0.6% DM in Jersey heifers.

In contrast, digestibility %DM of nutrients declined linearly with increasing levels of dietary tannin (Aguerre et al. 2016). Others also reported lower nutrient digestibility when quebracho was added to the diet (Dawson et al. 1999; Ahnert et al. 2015). The strong binding affinity of tannins for protein relative to other substrates is demonstrated by reduction in nitrogen apparent digestibility. In addition, a decreased digestibility of dietary feed fractions, especially of fiber, will likely slow digesta clearance from the rumen, resulting in a DMI reduction. Beauchemin et al. (2007) supplemented with condensed tannin in beef cattle and concluded that there was gradual decline in digestibility of different nutrients. In this study, tannin extracts has saved dietary protein from excessive breakdown in rumen as confirmed by the low value BUN, and MUN concentrations which likely resulted in an increase of feed protein flow to the intestine.

Nearly no traceable tannins were present in the faeces of buffalo. This indicates the removal of tannins from feed when it passed through the digestive tract. In the present study, the extractable tannins present in the feed disappeared in the gastrointestinal tract. These values are in agreement with that of Degen et al. (1995). Alternatively, Robbins et a1. (1991) described that 75% of ingested quebracho tannins were present in the faeces. Hagerman and Robbins (1993) proposed that difference in these results could be due to lower binding of quebracho tannins to proteins. The quebracho tannin has a branched chain structure which is compact and it is not very accessible for binding to macromolecules.

It has been observed in current study that tannin extracts has prevented dietary protein from excessive breakdown and loss in rumen which is confirmed by presence of low value of BUN, and MUN level. More level of feed protein in rumen ultimately go to intestine where it is absorbed in the form of amino acids in the blood. Consequently, it leads to more milk production.

Ethical approval Entire experiment was conducted in accordance with regulations by the ethical committee for animal welfare at University of Veterinary and Animal Sciences, Lahore, Pakistan.

Conflict of Interest The authors declare that they have no conflicts of interest.

Funding This research work was partially funded by Hilton Pharma Pvt. Ltd. Pakistan.

Data availability Dataset used and analyzed for the current study are available from the corresponding author on reasonable request.

Authors’ contribution M. R. and M. Q. S. designed the research. M. R. and B. A. collected the data. M. R., S. A. and M. U. A. conducted the data analysis. M. R. and U. A. wrote the manuscript. T. N. P., S., and S. A. supervised and edited the manuscript. All the authors read and approved the final manuscript.

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Effect of supplementing hydrolysable tannins extract on milk proteins and productive performance of early lactating Nili Ravi buffaloes

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