Effects of lactulose supplementation on growth performance, nutrient digestibility, blood profiles, faecal microbial shedding, faecal score and faecal noxious gas emission in weanling pigs

Abstract

A total of 120 weanling pigs [(Yorkshire × Landrace) × Duroc, weaned at day 21 after birth] with an average body weight (BW) of 7.95 ± 1.22 kg were used in 6-week experiment to evaluate the effects of lactulose supplementation on growth performance, nutrient digestibility, blood profiles, faecal microbial shedding, faecal score and faecal noxious gas emission. Pigs were randomly allotted to one of four experimental diets according to initial BW. There were six replicate pens per treatment with five pigs per pen. Dietary treatments were: NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose. The experiment included two phases (days 1–14 and days 15–42). Pigs fed PC and L1 diets had greater (P < 0.05) average daily gain (ADG) and gain/feed (G/F) than those fed NC diet during days 1–14. The average daily feed intake (ADFI) was improved (P < 0.05) by PC and L1 treatments compared with NC treatment during days 15–42. During days 1–42, ADG and ADFI were higher (P < 0.05) in PC and L1 than that in NC. Pigs fed L1 diet increased (P < 0.05) the apparent total tract digestibility (ATTD) of energy compared with those fed NC diet at week 2, and the ATTD of N in PC and L1 treatments was increased (P < 0.05) compared with NC treatment. The concentration of faecal Lactobacillus was increased (P < 0.05), and E. coli counts were decreased (P < 0.05) by L1 treatment compared with NC treatment. Pigs fed L1 and L2 diets had lower (P < 0.05) faecal score compared with those fed NC diet. Dietary supplementation of 0.1% lactulose decreased (P < 0.05) the faecal NH₃ emission compared with NC treatment. In conclusion, dietary supplementation with 0.1% lactulose improved the growth performance, ATTD of energy and N, and concentration of faecal Lactobacillus, and decreased E. coli counts and excreta NH₃ emission in weaning pigs.

Keywords:

• faecal microbial
• growth
• lactulose
• weanling pig

1. Introduction

Antibiotic supplementation has been well accepted to improve growth and efficiency in swine (Hahn et al. 2006). However, recently ban on the use of antibiotic leads to a growing interest in alternative to antibiotics growth promoters such as non-digestible oligosaccharides (NDO), which could reach the colon intact, where they are selectively fermented by bacteria supposed to have beneficial effects (Banner et al. 2004).

Lactulose is claimed as a kind of synthetic NDO (Schumann 2002; Martín-Peláez et al. 2010), which is produced by isomerization of lactose due to regrouping the glucose residue to a fructose molecule (Kochetkov & Bochkov 1967). A series of studies have reported beneficial effects of lactulose by improving the humoral immunity of humans (Liao et al. 1994; Schumann 2002; Foster et al. 2010; Chen et al. 2011). Sutton and Patterson (1996) confirmed the effect of lactulose on reducing fermentative diarrhoea by reducing E. coli during the pig rearing period. Furthermore, Konstantinov et al. (2004) reported that lactulose elicits a prebiotic effect (increased counts of Bifidobacteria and Lactobacillus) in pigs, which may reduce the faecal noxious gas emission due to a more balanced intestinal microbiota ecosystem (Wang et al. 2009; Yan & Kim 2013; Cho & Kim 2014), and has been proposed as a feed additive (1%) to prevent salmonellosis in fattening pigs (Wiemer 1999). However, lactulose supplementation research on the performance of weaning pigs is limited.

Therefore, the aim of the present study was to evaluate the effects of lactulose supplementation on growth performance, nutrient digestibility, blood profiles, faecal microbial shedding, faecal score and faecal noxious gas emission in weaning pigs.

2. Materials and methods

2.1. Animals and experimental design

A total of 120 weanling pigs [(Yorkshire × Landrace) × Duroc, weaned at day 21 after birth] with an average body weight (BW) of 7.95 ± 1.22 kg were used in a 6-week experiment. Pigs were randomly allotted to one of four experimental diets according to initial BW. There were six replicate pens per treatment with five pigs per pen. All pigs were housed in an environmentally controlled room, where provided 0.26 × 0.53 m² for each pig. Each pen was equipped with a one-sided, stainless steel self-feeder and a nipple drinker that allowed access to feed and water ad libitum. Dietary treatments were: NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose. The experiment included two phases: days 1–14 and days 15–42. All nutrients in diets were formulated to meet or exceed the recommendation of NRC (2012) for weanling pigs and fed in a mash form (Table 1). The Animal Care and Use Committee of Dankook University approved the experimental procedures used in this study.

Table 1. Feed compositions of control diet (as-fed basis).

Item	Phase 1 (days 1–14)	Phase 2 (days 15–42)
Ingredient (%)
Extruded corn	44.49	61.97
Soybean meal (48% CP)	21.20	27.80
Fish meal (66% CP, Brazil)	3.50	–
Soy oil	2.55	1.05
Lactose	8.30	–
Whey	10.00	5.00
MCP	–	–
DCP	1.5	1.5
Sugar	3.00	–
Plasma powder (AP 920)	3.00	–
L-Lys HCl (78%)	0.39	0.46
DL-Met (50%)	0.30	0.24
L-Thr (89%)	0.19	0.20
Choline chloride (25%)	0.10	0.10
Vitamin premix^a	0.10	0.10
Trace mineral premix^b	0.20	0.20
Limestone	0.98	1.13
Salt	0.20	0.25
Total	100.00	100.00
Calculated nutritional content
ME (kcal/kg)	3540	3410
CP (%)	20.00	19.00
Lys (%)	1.50	1.35
Met (%)	0.62	0.53
Met + Cys (%)	0.97	0.84
Ca (%)	0.95	0.90
Total P (%)	0.75	0.70
Avail P (%)	0.55	0.43
Crude fat (%)	5.02	3.98
Crude fibre (%)	1.87	2.45

^aProvided per kg of complete diet: 11,025 IU vitamin A; 1103 IU vitamin D₃; 44 IU vitamin E; 4.4 mg vitamin K; 8.3 mg riboflavin; 50 mg niacin; 4 mg thiamine; 29 mg d-pantothenic; 166 mg choline; 33 μgvitamin B₁₂.

^bProvided per kg of complete diet: 12 mg Cu (as CuSO₄•5H₂O); 85 mg Zn (as ZnSO₄); 8 mg Mn (as MnO₂); 0.28 mg I (as KI); 0.15 mgSe (as Na₂SeO₃•5H₂O).

2.2. Sampling and measurements

Individual pig BW and feed disappearance were recorded to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed efficiency (G/F). All pigs were fed diets mixed with 0.20% chromium oxide (Cr₂O₃) as an indigestible marker during days 8–14 and days 36–42, fresh faecal samples were collected from two pigs per pen (days 12–14 and days 40–42) via rectal massage. Representative samples were stored in a freezer at –20°C until analyzed (Fenton & Fenton 1979). Before chemical analysis, the faecal samples were thawed and dried at 60°C for 72 h, after which they were finely ground to a size that could pass through a 1-mm screen. The procedures utilized for the determination of dry matter (DM), nitrogen (N) and energy (E) digestibility were conducted in accordance with the methods established by the AOAC (1995). Chromium levels were determined via UV absorption spectrophotometry (Shimadzu, UV-1201, Kyoto, Japan). Nitrogen was determined by a Kjectec 2300 Nitrogen Analyzer (Foss Tecator AB, Hoeganaes, Sweden). Gross energy was analyzed by oxygen bomb calorimeter (Parr 1600 Instrument Co., Moline, IL, USA).

For the blood profiles, two pigs per pen were randomly selected and blood samples were collected via anterior vena cava puncture on days 14 and 42. At the time of collection, blood samples were collected into both non-heparinized tubes and vacuum tubes containing K₃EDTA (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) to obtain serum and whole blood, respectively. After collection, serum samples were centrifuged (3000×g) for 15 min at 4°C. The white blood cells (WBC), red blood cells (RBC), lymphocyte counts and haptoglobin concentration in the whole blood were determined using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA).

Faecal samples were collected directly via massaging the rectum from two pigs per pen and then pooled and placed on ice for transportation to the lab. One gram of the composite faecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and then homogenized. Viable counts of bacteria in the faecal samples were then conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and Lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the E. coli and Lactobacillus, respectively. The Lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. The E. coli and Lactobacillus colonies were counted immediately after removal from the incubator.

Faecal scores were determined at 08:00 and 20:00 using the following faecal scoring system: 1 hard, dry pellet; 2 firm, formed stool; 3 soft, moist stool that retains shape; 4 soft, unformed stool that assumes shape of container; 5 watery liquid that can be poured (Hill et al. 2000). Faecal samples were collected at days 14 and 42, and then placed in aluminium foil cups. The aluminium foil cups were weighed and placed in a drying oven at 100°C for 24 h and then reweighed to calculate moisture loss.

Faeces were collected on day 42 to determine faecal noxious gas emission according to the method described by Cho et al. (2008). A mount of 150 g fresh faeces and 150 g urine mixture samples were stored in 2.6 L plastic boxes for replicates. The samples were fermented for 5 days at room temperature (28°C). After the fermentation period, a Gastec (model GV-100) gas sampling pump was utilized for gas detection. The adhesive plasters were punctured, and 100 ml of headspace air was sampled approximately 2.0 cm above the faeces surface.

2.3. Statistical analysis

All data were subjected to the statistical analysis as a randomized complete block design using the General Linear Model (GLM) procedures of SAS (1996) (SAS Inst. Inc., Cary, NC, USA), and the pen was used as the experimental unit. Before carrying out statistical analysis of the microbial counts, logarithmic conversion of the data was performed. Differences among treatment means were determined using the Duncan's multiple range test with p < 0.05 indicating significance.

3. Results

3.1. Growth performance

Pigs fed PC and L1 diets had greater (P < 0.05) ADG and G/F than those fed the NC diet during days 1–14 (Table 2). The ADFI was improved (P < 0.05) in PC and L1 compared with that in NC during days 15–42. During the whole experimental period, pigs fed PC and L1 diets showed greater (P < 0.05) ADG and ADFI than those fed NC diet.

Table 2. The effects of lactulose on growth performance in weanling pigs.^.

Items	NC	PC	L1	L2	SE
Phase 1 (days 1–14)
ADG (g)	340^b	371^a	373^a	358^ab	7
ADFI (g)	534	545	535	533	7
G:F	0.637^b	0.681^a	0.697^a	0.672^ab	0.013
Phase 2 (days 15–42)
ADG	562	588	589	570	10
ADFI	991^b	1051^a	1050^a	1022^ab	19
G:F	0.567	0.559	0.561	0.554	0.014
Overall (days 1–42)
ADG	488^b	516^a	517^a	499^ab	8
ADFI	839^b	882^a	878^a	859^ab	12
G:F	0.582	0.585	0.589	0.572	0.011

Note: Means in the same row with different superscripts differ (p < 0.05).

SE, standard error; NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose.

3.2. Apparent total tract nutrient digestibility (ATTD) and faecal moisture

There was no dietary effect on the ATTD of DM and N at week 2, as well as the ATTD of DM and energy at week 6 (Table 3). However, pigs fed L1 diet increased (P < 0.05) the ATTD of energy compared with those fed NC diet at week 2, and the ATTD of N in PC and L1 diets was increased (P < 0.05) compared with NC diet. Faecal moisture was not affected by dietary treatments at both weeks 2 and 6.

Table 3. The effects of lactulose on digestibility and faecal moisture content in weanling pigs.^.

Items (%)	NC	PC	L1	L2	SE
Digestibility
2 weeks
DM	84.68	85.43	85.90	86.24	1.01
N	83.21	86.11	85.93	83.90	1.16
E	82.73^b	85.76^ab	86.59^a	85.74^ab	1.03
6 weeks
DM	83.13	84.08	84.05	84.01	1.05
N	81.77^b	85.35^a	85.28^a	82.59^ab	1.08
E	82.21	85.13	85.11	84.55	1.00
Faecal moisture content
2 weeks	70.47	70.11	68.99	69.54	1.74
6 weeks	70.61	70.41	69.56	68.95	0.76

Note: Means in the same row with different superscripts differ (p < 0.05).

SE, standard error; NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose.

3.3. Blood profiles

The RBC, WBC, haptoglobin concentrations and lymphocyte counts were not affected (P > 0.05) by dietary treatments (Table 4).

Table 4. The effects of lactulose on blood profiles in weanling pigs.

Items	NC	PC	L1	L2	SE
Lymphocyte (%)
2 weeks	39.43	43.63	45.43	44.86	4.27
6 weeks	62.40	58.44	59.64	65.89	3.98
RBC (10^6/µl)
2 weeks	6.09	6.04	6.13	6.03	0.14
6 weeks	6.61	6.57	6.67	6.93	0.21
WBC (10^3/µl)
2 weeks	19.33	21.44	20.63	20.97	2.47
6 weeks	19.44	15.56	15.27	17.17	1.57
Haptoglobin (mg/dL)
2 weeks	12.44	12.21	11.94	12.04	0.75
6 weeks	10.63	11.24	11.77	10.90	1.23

SE, standard error; NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose.

3.4. Faecal microflora, faecal scores and faecal noxious gas emission

The concentration of faecal Lactobacillus was increased (P < 0.05) while the population of E. coli was decreased (P < 0.05) by L1 treatment compared with NC treatment (Table 5). Pigs fed L1 and L2 diets had lower (P < 0.05) faecal score compared with those fed NC diet. L1 treatment decreased (P < 0.05) the faecal NH₃ emission compared with NC treatment, but no effect on faecal total mercaptans, H₂S and acetic acid emission was observed among treatments.

Table 5. The effects of lactulose on faecal microflora and noxious gas emission in weanling pigs.

Items	NC	PC	L1	L2	SE
Faecal microflora (log10 cfu/g)
Lactobacillus	7.40^b	7.47^ab	7.60^a	7.48^ab	0.04
E. coli	6.43^a	6.36^ab	6.21^b	6.28^ab	0.06
Faecal score^a	3.23^a	3.19^ab	3.14^b	3.13^b	0.03
Faecal noxious gas emission (ppm)
NH3	28.13^a	23.50^ab	19.48^b	23.53^ab	3.00
Total mercaptans	4.35	4.05	3.95	3.95	0.12
H2S	3.98	3.88	3.58	3.55	0.23
Acetic acid	2.78	2.68	2.63	2.63	0.65

Note: Means in the same row with different superscripts differ (p < 0.05).

SE, standard error; NC, basal diet; PC, NC + 0.05% tiamulin; L1, NC + 0.1% lactulose; L2, NC + 0.2% lactulose.

^aFaecal scores were determined at 08:00 and 20:00 using the following faecal scoring system: 1 hard, dry pellet; 2 firm, formed stool; 3 soft, moist stool that retains shape; 4 soft, unformed stool that assumes shape of container; 5 watery liquid that can be poured.

4. Discussion

Growth performance was improved by antibiotic supplementation, which was in agreement with previous findings (Jin et al. 2008; Shen et al. 2009; Choi et al. 2011).The increased overall ADG may be associated with the increased ADG during the phase 1. However, we did not found consistent effects of antibiotic administration, which may be explained by the age of pigs (Hahn et al. 2006).

Lactulose inclusion in the diet improved ADG and ADFI. In agreement with our results, Krueger et al. (2002) noted that lactulose supplementation in diets for periparturient sows and weaning pigs improves daily weight gains. Estrada et al. (2001) proposed that dietary fructo-oligosaccharide (FOS) supplementation benefited growth performance of weaning pigs. In contrast, Zhao et al. (2011) and Mikkelsen et al. (2003) reported that FOS does not affect growth performance of weanling pigs. However, Waldroup et al. (1993) reported that broiler fed 0.375% FOS yielded consistent improvement in growth rate and feed efficiency.

In our study, pigs fed 0.1% lactulose-supplemented diet showed improved energy and nitrogen digestibility at weeks 2 and 6, respectively, which may have been responsible for the increased ADG by 0.1% lactulose inclusion in the diet. Burr et al. (2008) demonstrated that supplementation of prebiotics had significantly increased protein digestibility compared with the basal diet in fish. Lee et al. (2009) also demonstrated that supplemental synbiotics from anaerobic microflora (probiotics from yeast, mould, bacteria) increased DM and crude protein digestibility in early-weaning pigs. Limited reports are available to compare the effects of lactulose on nutrient digestibility with others; thus, we could only compare our results with those reported in FOS studies. Mountzouris et al. (2006) demonstrated that FOS did not affect nutrient digestibility in growing pigs at levels of 0.68%, 1.35% or 0.1%. However, Zhao et al. (2011, 2013) proposed that dietary FOS supplementation at 0.1% has a substantial positive effect on nutrient digestibility. The increased energy and nitrogen digestibility observed in the current study may have been due to the enhanced intestinal structure by lactulose supplementation as mentioned previously by Shim (2005). Thus, the potential mechanism of the nutrient digestibility improving effect remains to be clarified.

One report showed that most dietary lactulose is absorbed; however, a slight amount (0.25–2%) of dietary lactulose could be absorbed into the cardiovascular system and cause an immune response (Schumann 2002). An in vitro experiment demonstrated that lactulose decreased TNF-α level produced by an endotoxin produced by monocytes (Liehr & Heine 1981). However, no report has investigated the effect of lactulose on blood parameters. We suggest that the environment we provided may have had no negative effect on the pigs and less of a response on the blood parameters to dietary lactulose.

Post-weaning diarrhoea is one of the critical problems faced by the hog industry, and lactulose is a potential diarrhoea inhibitor (Kleesen et al. 2001; Loh et al. 2013). Lactulose acts as an effective prebiotics in (non-typhoid) Salmonella carriers (Schumann 2002). Another possible factor that could result in diarrhoea is imbalanced gut microflora (Lactobacillus and E. coli). The clinical response to lactulose is dependent on its breakdown to organic acids by bacteria and the consequent lowering of colonic pH, so a relationship might be expected between the effects of lactulose and faecal microflora (Vince et al. 1974). In agreement with our study, Haenel et al. (1958) found that E.coli and Streptococcus counts decrease. Conn and Floch (1970) found that lactulose treatment results in a moderate increase in the mean number of Lactobacillus. Salminen and Salminen (1997) also reported that lactulose promoted the growth of lactic acid bacteria. The faecal score was reduced by 0.1% lactulose inclusion in the diet, which may be clear evidence of an increased Lactobacillus count, which led to a beneficial gut environment. However, faecal moisture content was not affected by the dietary treatments, which may have been caused by the subjective faecal score evaluation method.

Lactulose is metabolized by bacteria that live in the hind gut. These bacteria produce short chain fatty acids and carbon dioxide (Panesar & Kumari 2011), which leads to a low pH in gut contents. Sommer and Husted (1995) reported that slurry pH is of great importance for ammonia emission. Because the effect of pH on ammonia is very strong, a minor change in pH can have a large effect. Therefore, ammonia emission was reduced by 0.1% lactulose. It is well accepted that volatile sulphides include a wide range of sulphur-containing compounds produced through both in vivo fermentation in the hindgut and in vitro anaerobic fermentation of a manure slurry during storage (Kadota & Ishida 1972). In a related study, FOS was not effective for decreasing the production and excretion of volatile sulphides in growing pigs (Rideout et al. 2004). Our results were consistent with this report. Volatile sulphide emission may be related to differences in the post-absorptive urinary loss of inorganic sulphur-containing compounds and the time period allowed for in vitro anaerobic fermentation during manure storage. The volatile fatty acid content we reported was the acetic acid concentration. Lactulose is metabolized by bacterial flora in the colon to short chain fatty acids, which acidify colonic content (Patil et al. 1987). However, similar to our study, Rideout et al. (2004) found that decreased faecal pH does not occur in response to the increase in faecal acetic acid content when pigs are fed FOS. This may be because lactulose fermentation mainly affected other short chain fatty acids but not acetic acid.

5. Conclusion

Taken together, our results suggest that dietary supplementation with 0.1% lactulose could benefit growth rate and feed intake of weanling pigs and improve the gut environment.

Funding

This work was supported by the research grant of Chungbuk National University in 2014.

Additional information

Funding

Funding: This work was supported by the research grant of Chungbuk National University in 2014.