Growth performance and health status of weaned piglets fed diets including catfish (Pangasius hypophthalmus) by-product protein hydrolysate and β-glucans

Nguyen Thi Thuy and Nguyen Cong Ha¹

Department of Animal Sciences, College of Agriculture and Applied Biology, CanTho University, 3/2 Street, Ninh Kieu District, Can Tho City.
nthithuycn@ctu.edu.vn
¹ Department of food Technology, College of Agriculture and Applied Biology, CanTho University, 3/2 street, Ninh Kieu District, Can Tho City.

Abstract

An experiment was conducted to evaluate the effects of inclusion of Catfish (Pangasius hypophthalmus) by-product protein hydrolysate (CPH) and β-glucan produced from brewer’s yeast cell (BG) in diets on the performance and health status of weaned piglets. A total of 48 crossbred castrated (Yorkshire x Landrace) male piglets (8.0 ± 1.5 kg/piglet) at weaning were allocated to four treatment groups in a randomized complete block design. In each group, the piglets were divided randomly into 4 pens (replicates) with 3 piglets per pen. The control diet contained marine fishmeal (FM) as the sole protein supplement (FMD), and the experimental diets consisted of three different diets, in which 100% crude protein (CP) from FM in the FMD was replaced by the CP from CPH (CPHD) without any β-glucan product, and with commercial β-glucan (CPHCB) or β-glucan produced from brewer’s yeast cell (CPHBG) at 0.1%.

Average daily gain (ADG) was highest in CPHBG; the lowest was in FMD. Feed consumption was lowest in CPHD and highest in FMD. Feed conversion ratio (FCR) was poorest in FMD and similar in the other three diets. Piglets fed diets with β-glucan from brewer’s yeast cell product and commercial β-glucan were less affected by diarrhoea and had less E.coli in feces than piglets fed the FMD diet. The cost/gain was lowest in piglets fed CPHBG and highest in FMD. In conclusion, it was best to combine the catfish by-product protein hydrolysate and β-glucan from brewer’s yeast cell in diets for weaned piglets, resulting in improved average daily gain and feed conversion ratio, and reduced diarrhoea incidence, fecal score and E.coli in feces.

Key words: diarrhoea incidence, E coli, fecal score

Introduction

In the Mekong Delta of Viet Nam, pig production is the dominant and rapidly developing form of livestock production. However, conventional protein supplements - marine fish meal and soya bean meal - have become relatively expensive. Therefore, it is important to find alternative protein sources. Total production of Pangasius hypophthalmus (called Tra catfish) in Vietnam reached 1.17 million tonnes in 2013 (Nguyen Phuoc Minh 2014). So the by-product of Tra catfish fillet processing is a potentially valuable alternative protein source. The increase in catfish production means an increase in its by-products (head, bone, scrap meat and skin) which accounts for 62-67% of whole catfish (Nguyen Thi Thuy et al 2007). Traditionally, these by-products are ground fresh, boiled, and the oil extracted. The residue after oil extraction is dried to produce catfish by-product meal. Recently, some research has been done on hydrolyzing the protein in catfish by-product into protein hydrolyzate by addition of enzymes, then drying and grinding to produce a powder product (Dang Minh Hien et al,2015). Nguyen Cong Ha et al (2015) showed that the optimal conditions for protein hydrolysis were by addition of the enzyme bromelain at pH 6.5 and at 55^oC for 120 minutes. The major components after hydrolysis of this by-product are peptides with varying molecular weight.

β-glucan is now being researched for use in the diets for pigs as this component is easily absorbed, and improves health especially of young pigs. β-glucan has no nutritional function in the animal digestive tract and thus functions as a non-food energy supply. β-glucan in animal feed particularly for pigs improves the immune system and is thus an alternative to use of antibiotics. Investigations on the extraction and purification of b-glucan from spent yeast for humans have been carried by Nguyen Cong Ha and Tran Thanh Doi (2010). However, there are no studies on processing and use of b-glucan from yeast protein after the brewing process as feed for livestock in Vietnam.

The period from weaning (at 28 days) to 2 months is a critical time for pigs in order to maintain good health and growth and to reduce diarrhoea incidence. Therefore, supplementation of feed with powdered catfish protein hydrolysate and β-glucan produced from beer processing by-products merits investigation.

Materials and methods

Animals and experimental design

Forty eight crossbred (Yorkshire x Landrace) castrated male piglets at weaning, 26-28 days of age, from 10 litters (5-6 male piglets/litter), whose body weights were on average 8.0 ± 1.5 kg (mean ± SD) were used. The piglets were allocated randomly into four groups of twelve, balanced for initial body weight and litter origin. In each group, the piglets were divided randomly into four pens (replicates), three piglets/pen and each group was fed one of four diets for 5 weeks. The pens had slatted floors with no litter, and each pen was equipped with a feeder and nipple drinker. Four experimental diets (Table 2) were fed according to a randomized complete block design with pen as the experimental unit, and blocks based on initial body weight.

Figure 1. Piglets in the first week and at the end of the experiment

Feeds and supplements

The diets were formulated to contain around 19.0% CP and 12.8-13.0 MJ/kg of metabolisable energy (ME). Rice bran, broken rice, maize meal and soya bean meal were used as basal ingredients to provide 60% of the total CP of the diets. The remainder of the CP was provided by marine fish meal alone in the basal diet (FMD); in the other diets 100% of the CP from FM was replaced by the CP of the CPH (CPHD) without any β-glucan product, with commercial β-glucan (CPHCB) or β-glucan produced from brewer’s yeast cell (CPHBG) at 0.1%.of the diet. All diets were supplemented with a standard mixture of vitamins and minerals, and were fed ad libitum. The rations were given in four meals per day at 08:00h, 11:00h, 14:00h and 17:00h. The refusals were collected the following morning before the first meal. Samples were taken and stored at -18^oC for DM analysis. Feed intake was calculated for each week. Feed conversion ratio (FCR) was calculated as feed intake divided by weight gain.

Preparation of catfish by-product protein hydrolysate (CPH) and β-glucan

The catfish by-product was collected from a catfish fillet processing factory in the industrial zone in Can Tho city, and transported to the catfish by-product meal factory. The production of CPH was carried out as described by Dang Minh Hien et al. (2015). Catfish by-product (head, bone and skin) was ground, boiled and pressed to get a soluble product. This was then centrifuged and extracted to get a protein solution. Hydrolysis was carried out by adjusting pH and temperature to the optimal condition; 7.5 and 55^oC, respectively. The enzyme papain was added to the protein solution according to the ratio 1 mg E/2.5 g catfish by-product. The protein hydrolyzate was finally transported to a dryer with the previous solid matter and dried to produce the final product (CPH). The powder CPH was mixed each week with the basal ingredients and fed to the piglets.

In order to produce powdered semi-purified β-glucan, spent brewer’s yeast cell was broken down in a high pressure homogenizer (Manton Gaulin 15M). After that it was subjected to two treatment environments: acid (acetic acid 0.5N) and alkaline (NaOH 0.5N). First, spent brewer’s yeast cell (18% DM) was diluted to 15% to standardize for the production line later. It was incubated at 50^oC,at pH 5 for 24 h with stirring to make the cell easier for disruption. Then it was heated at 80^oC for 15 minutes and cooled to room temperature (26-30^oC). The treated cell wall was centrifuged at 3565g for 10 minutes at room temperature, the supernatant was released and the cell wall of the yeast (CW) collected. This was diluted to 15% DM with distilled water before being homogenized at 300 atm with 10 passes until most of the cell was broken. To semi-purify the β-glucan, the homogenized cell wall (HCW) to was purified in an alkaline environment with the ratio of 1:2 (V_HCW/V_{NaOH 0,5N}) at 85^oC for 2 h. The HCW was then centrifuged at 3565g for 10 minutes at room temperature, to remove the supernatant and the residue then washed out by distilled water. This stage was repeated three times until neutralization. Next, the residue was purified again in acid environment to remove the rest of the protein with the ratio of 1:2 (V_{Paste glucan}/V_{Acid acetic 0.5N}) at 75^oC for 1 hour. It was then centrifuged at 3565g for 10 minutes at room temperature, the supernatant was released and the residue washed out by distilled water (3 times repeated) to collect the semi-purified β-glucan. In order to process β-glucan into powder form, the mass of semi-purified β-glucan was diluted by distilled water with the ratio 1:2 before transfer to a spray dryer (LABPLANT CE, SD-05, England) to dry the product. The inlet temperature of the dryer was fixed at 185^oC, with the outlet temperature at 85^oC. The final powdered semi-purified β-glucan ( purification >80% and 13.6% moisture) was packaged and preserved at room temperature.

Diarrhoea score and E. coli in feces

The health status of the piglets during the first 2 weeks (period 1) and then 3 weeks continuously (period 2) after weaning was assessed by fecal consistency scoring using a four-grade system, where 0 corresponded to firm and dry (normal); 1 to pasty; 2 to thick and fluid; and 3 to watery (Cupere et al 1992). Scoring was performed twice daily by two independent individuals and the incidence of diarrhoea (%) was calculated as the sum of the total number of diarrhoeal piglets over the period divided by the number of piglet days in the period multiplied by 100. The "fecal score" was calculated as the sum of the diarrhoea score over the period divided by the number of piglet days in the period.

The infestation of E.coli (CFU/g) in feces was determined by colony counting. Homogenous samples were implanted in appropriate agar environment containing lactose, and then incubated at 44⁰C for 24 h. The number of characteristic colonies having the shape of coliforms was counted and confirmed as E.coli by IMViC (Indol, Methyl Red, Voges Proskauer and Citrate) (Tran Linh Thuoc, 2006). The quantity of E.coli (CFU/g) was calculated as:

(CFU/g) = N/( n₁vf₁+ … + n_ivf_i) * R

N: The total number of colonies counted;
f₁ : dilution at each plate

n_i :  The number of plates in each dilution  
R : the positive rate

v : The volume (ml) of dilution to grow in each plate

Chemical analysis

The chemical composition of feed offered and refusals was determined by AOAC (1990). Dry matter (DM) was measured by drying the fresh samples at 105 ^oC. Crude protein was determined by the Kjeldahl method. Total ash was the residue after ashing the samples at 550^oC and organic matter (OM) was calculated by difference. The ether extract (EE) was determined by Soxhlet extraction.

Statistical analysis

The data were analysed using the General Linear Model (GLM) of Minitab Statistical Software Version 16.0. Tukey’s pair – wise comparisons were used to determine the differences between treatment means at P<0.05. The statistical model used was as follows:

 Y_ij = µ + α_i + β_j + e_ij

Where: Y_ij is growth performance or faecal score; µ is overall mean averaged over all treatments and all possible blocks; α_i is effect of treatment i; β_j is effect of block j; e_ij is random error associated with assigned to treatment i in block j.

Results

The DM, CP and EE contents were different between FM and CPH (Table 1). The experimental diets contained relatively the same protein and calculated ME content . However, the EE content in FMD was lower than that in the other treatments.There were no significant differences in DM, Ash, CF, NFE among the diets. Replacing 100% CP of the FM in the basal diet by CP of the CPH caused a significant increase in EE intakes compared with the .control diet.

The results in Table 3 shown that ADFI was reduced in CPH diets compared with FM diet. The ADG was also highest in CPHCB, followed by CPHBG, CPHD and lowest in FMD. The FCR was improved in CPHD, CPHCB and CPHBG compared with FMD. None of the piglets had a fecal score of 3 (watery faeces) during the first 2 weeks nor in weeks 3-5. However, some piglets in all the treatment groups showed symptoms of diarrhoea (Table 4). In period 2, the incidence of diarrhoea as well as the fecal score was reduced compared with the first period. Overall in the five weeks, the piglets fed FMD diet had higher diarrhoea incidence and fecal score than piglets fed the CPHD, CPHCB and CPHBG diets. Quantity of E.coli in feces was also lower in CPHCB and CPHBG compared with that in CPHD and FMD.

Figure 2. Growth curves of the piglets

Discussion

In this study, the CP content of the catfish by-product protein hydrolysate (CPH) was relatively low, as the by-products used to produce the CPH constituted head, bone and skin thus making the CP in the CPH lower than that from the FM. Recent research has been on the utilization of catfish by-products to produce protein hydrolysate using alcalase enzyme (Nguyen Phuoc Minh 2014) or bromelain enzyme (Nguyen Cong Ha et al 2015). This is another way to process and preserve catfish by-products, which traditionally is often used in the production of catfish meal. Most of the protein hydrolysate from catfish by-products undergoes complete hydrolysis and forms low molecular weight peptides and amino acid mixtures (Nguyen Cong Ha et al 2015). The EE content in CPH is higher than that in FM, which is a typical characteristic of catfish by-product in the Mekong Delta (Nguyen Thi Thuy et al 2007).

The inclusion of CPH in the diets improved ADG and FCR of the piglets. It may be because the protein hydrolysate can be used to improve or modify the physicochemical and functional properties such as solubility, fat absorption or sensory properties of proteins without losing its nutritional value (Nguyen Phuoc Minh 2014). The results from this study indicated that the CPH was a high quality product and gave a better response than the FM. Theoretically, the addition of CPH to weaned piglet diets can provide partially predigested proteins and give the young animal a head start in the digestive process. Protein hydrolysate consists mainly of low molecular weight peptides which can be absorbed rapidly and improve growth performance of piglets (Wu 1998). This is due to the fact that fish protein hydrolysate is known to be a good and easily digestible protein supplement (Hevroy et al 2005). Furthermore, the explanation for the positive effects of fish protein hydrolysate on animal performance may be the high content of short peptides and free amino acids which are palatable and more readily absorbed than intact protein without preceding digestion by pancreatic proteases (Gilbert et al 2008). On the other ha is the effect of higher levels of fat in the diets with CPH; catfish oil contains a high proportion of unsaturated fatty acids (Nguyen Thi Thuy et al 2007), which can improve the efficiency of energy utilization in pigs. This could explain the improvement of FCR in CPHD compared with FMD.

This study showed that the ADG and FCR of piglets fed CPHCB and CPHBG were better than in piglets fed CPH or FM diets. It may be because the addition of β-glucan to the diet increases the operational efficiency of macrophages and heterophils (Lowry et al 2005). The study by Dritz et al (1995) showed that supplementing piglets with 0.025% beta-glucan in the diets increased growth performance. Hahn et al (2006) found that supplementing β-glucan at 0.01 to 0.04% in diets of piglets during 5 weeks post weaning resulted in a linear increase in nutrient digestibility. A similar trend emerged from research of Schoenherr et al (1994) who reported that β-glucan supplementation improved growth performance and feed efficiency in nursery pigs. Theoretically, the inclusion of β-glucan in diets may balance the intestinal flora of piglets, create a healthy condition, strengthen resistance of pigs to the growth of harmful microorganisms and avoid stress during weaning. The combination of protein hydrolysates and β-glucan in the diets improved feed conversion, stimulated digestion, prevented intestinal diseases, and also stimulated the natural immune system (Hiss and Sauerwein 2003).

In this study, the diets with commercial and locally-produced β-glucan gave similar results, so it can be concluded that the β-glucan produced from by-products of beer processing is comparable with the commercial product.

Weaning piglets often suffer from post-weaning diarrhea. In the pathogenesis of diarrhea, enteropathogenic E. coli strains play a major role. In this trial, the diarrhea incidence and E.coli in levels in feces of pigs fed CPH diets, especially with β-glucan supplementation, were very low in comparison with FMD. The lowest diarrhea scores and E.coli levels were in piglets fed CPHGB; the highest were in piglets fed FMD over the 5 weeks post weaning period.

It is hypothesized that dietary supplementation of hydrolysed proteins would increase the availability of amino acids for the gut wall and therefore result in an improved small intestinal integrity and growth performance of piglets after weaning. However, research from Vente- Spreeuwenberg et al (2004) found that dietary supplementation of protein hydrolysate did not affect villus architecture during the 1^st week post weaning, but it enhanced growth performance during the 2^nd week.

The use of highly digestible and palatable protein sources is important in diets for young pigs, in order to stimulate feed intake and improve the growth performance post weaning. The challenge of dealing with diarrhea problems after weaning has been focused on minimizing the level of dietary crude protein and the amount of undigested crude protein.

β-glucan has the capacity to activate the immune system, thereby enhancing the defense barriers and thus providing protection against an otherwise severe or lethal infection (Vetvicka et al 2002). In our research, piglets receiving feed supplemented with β-glucan for 2 weeks after weaning showed a decreased susceptibility to E.coli, suggesting that β-glucan could be an alternative for antibiotics for prevention of post-weaning infections with E.coli (Stuyven et al 2009).

However, studies in which β-glucan has been tested to protect piglets against an infection are few up to now. A study of Dritz et al (1995) shows that administration of β-glucan originating from Saccharomyces cerevisiae in the feed resulted in an increased growth performance of the piglets. In fact, the β-glucan used in this study was produced from brewer’s yeast cells from the beer processing by-product and this also originated from yeast.

The present study was undertaken to compare the two different sources of β-glucan for their protective effects against an infection in newly-weaned piglets. The results showed that piglets fed the locally-produced β-glucan were less susceptible to colonization with E. coli and diarrhea than that those fed commercial β-glucan.

Escherichia coliis an important cause of diarrhea in neonatal, suckling and newly weaned piglets. After weaning, piglets completely stop receiving antibodies from their mother, because during the neonatal and suckling period, the piglets can be passively protected by antibodies present in milk (Osek et al 1995). A supplement of β-glucan in the diets of piglets in the early weaning is therefore important.

In this study, the reduced bacterial excretion and diarrhea suggested a correspondingly reduced colonization of the small intestine with E.coli due to β-glucan supplementation. This means that the use of β-glucan as a substrate could induce an increase in population of lactobacillus spp. In the current study, dietary β-glucan decreased the numbers of E. coli in feces. It is a also reported that β-glucan might prevent the adhesion of E.coli in the small intestine.

Conclusions

• Combining catfish by-product protein hydrolysate and β-glucan in diets for weaned piglets improved growth rate and feed conversion, reduced diarrhea incidence and fecal score and reduced E.coli in feces.
  
  
• β-glucan produced from by-products of beer processing is compatable with commercial β-glucan in terms of piglet performance and health.
  
  
• Replacing fish meal by catfish by-product protein hydrolysate may be considered an alternative protein source in diets for pigs in the Mekong Delta of Viet Nam.

Acknowledgements

This research was funded by Viet Nam National Foundation for Science and Technology Development (Nafosted) under grant number 106-NN.05-2013.68. Sincere gratitude goes to the pig farm in An Giang Province for carrying out the experiment.

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Received 18 August 2016; Accepted 10 October 2016; Published 1 November 2016

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