Introduction
Rearing a healthy piglet with an adequate growth rates is essential for the success of swine production. However, during the first week of weaning, young piglets may face several stressors due to their immature immune system that are ensuing with high susceptibility to digestive diseases (Campbell et al., 2013). Besides, post-weaning diarrhea becomes the most prevalent disorder in piglets that mainly leads to mortality. Over the past decades antibiotic growth promoters (AGP) were widely used to overcome this circumstance. However, the overuse of AGP cause an antibacterial resistance in animals and leads to severe health issues in humans through food chain. Thus, the use of AGP in livestock feed has been strictly prohibited by many countries including South Korea since 2011 (Shanmugam et al., 2021). Consequently, this problem provoked animal nutritionist to find an effective alternative with innovative strategies. For instance, omega-3 fatty acids were found to enhance the immunity of weaned piglets (Qizhang et al., 2014) whereas pre- and probiotics were discovered to promote the gut microorganisms (Peera and James, 2013) and considered as effective additives for many years.
Prebiotics are defined as non-digestible food ingredients that have a beneficially effect on host by stimulating their growth performance (Yoon and Shin, 2017). Besides they can be used by host to stimulate the beneficial bacterial growth by stabilizing the intestinal microbiota (Roberfroid, 2000). The administration of oligosaccharides (OS) which comprise the major prebiotic group, has been reported to enhance the growth and feed efficacy in young ruminants ( et al., 2020). Also, prebiotic effect of lactulose has played a vital role in improving the growth performance of pigs at weaning age due to its palatability and become a readily digestible energy source to eases the transition from milk to solid feed (Zhao et al., 2021). On the other hand, Krueger et al. (2002) reported that the inclusion of lactulose (LAC) supplement has improved the intestinal microflora of sows. In addition, Cho and Kim (2014) addressed that LAC supplement had increased the body weight gain, apparent metabolizable nitrogen as well as reduced the ammonia and acetic acid gas emission in broilers. Furthermore, Schumann (2002) and Chen et al. (2011) reported that LAC supplement could be used as therapeutic for hepatic encephalopathy and to cure constipation in children. Though, above studies had addressed the positive effects of LAC on colonic metabolism in pigs, broilers, and humans to date, the application of 2 g·kg-1 LAC in weaning piglets’ diet is still scarce. Previously, Zhao et al. (2021) stated that piglets are born with high intestinal lactase activity which helps to fully digest dietary lactulose. In this study we used lactulose as a promising alternative and hypothesized that 1 g·kg-1 and/or 2 g·kg-1 lactulose supplementation would play a vital role in enhancing the growth performance, nutrient digestibility, bacterial counts, and reduce noxious odor emission in weaning pigs. Hence, the intention of this study was to determine the effect of lactulose on growth performance, nutrient digestibility, fecal microbial, and fecal noxious gas emission in weaning pigs.
Materials and methods
Ethical clearance
This experiment was conducted at “Dankook University experiment farm” located at Jeonui (Sejong, Korea). Prior to the trial, the research protocols were revised well and permitted (Permit No: DK-4-1755) by the Ethical Committee of Dankook University, Korea and all animals were humanely treated in according to the guidelines of Animal Care and Use Committee.
Trial design
Prior to the trial, all equipment and fostering room was disinfected. A total of 225, three weeks old crossed ([Yorkshire × Duroc] × Landrace) healthy weaning piglets with an initial body weight (BW) of 5.77 ± 1.22 kg were commercially procured from Gene-pig farm (Gongju, Korea). On the arrival day, all piglets were weighed individually and housed in an environmentally-controlled room. Ambient temperature of the piggery was setup at 30℃ at first, then gradually reduced up to 24℃ and 60% humidity was maintained throughout the experiment.
Diets and dietary schedules
This experiment had lasted for 42 days. The feeding program consist of 2 phases: Phase 1 [0 - 14 days] and Phase 2 [15 - 42 days]. Based on initial BW and sex piglets were randomly (complete block design) allocated into 1 of 3 dietary treatments with 15 replication and 5 pigs (2 female and 3 male) per pen, and the dietary treatments includes: CON, corn-soybean meal based basal diet without lactulose and basal diet incorporated with 1 g or 2 g lactulose per kg feed and designated as LAC1 and LAC2, respectively. The lactulose powder used in this study was commercially purchased from Easy Bio Co., Ltd. (Seoul, Korea) and added to piglets’ diet at the prescribed level. All diets were formulated according to the recommendation of NRC (2012) (Table 1) and the feed ingredients were finely mixed with DDK-801 feed mixer (Daedong Tech, Anyang, Korea) and provided for piglets at the same time intervals (14:00 - 15:00). Piglets had free access to clean water and feed until the end of the experiment.
Sampling and measurements
Individual piglets (BW) were weighed using a GL-6000S weigh machine (G-Tech Inc., LTD., Seoul, Korea) at beginning and end of each phase to determine the average daily gain (ADG), whereas the amount of feed intake and refusals (pen basis) were recoded to determine gain to feed ratio (G : F) and the average daily feed intake (ADFI).
Chromium oxide (0.3%) as an indigestible marker was added to the piglet’s diet on day 35 and provided for about one week until end of the experiment to measure the nutrient digestibility. The representative feed samples were collected using the sterilized plastic bags from each treatment groups right after mixing the marker. At the end of each phase, the fresh fecal samples were randomly collected from 30 piglets·treatment-1 (2 pigs - 1 gilt and 1 barrow·pen-1) by rectal palpation. Within half an hour the collected fecal samples were transported to laboratory and stored at -20℃ to prevent changes in nutrients. Before starting the analysis, fecal samples were placed in WOF-L800 convection oven dryer (Daehan Scientific Co., Ltd., Wonju, Korea) at 105℃ for 2 days. The samples were taken out from the dryer and grounded well (Willey mill, Swedesboro, USA) to pass through a 1-mm screen sieve. The nutrient digestibility of dry matter (DM), nitrogen (N), and gross energy (GE) were analyzed according to the procedures of AOAC (2000) Previously described by Thamaraikannan and Kim (2021). The chromium concentrations were determined using Ultraviolet (UV-1201) spectrophotometer (Shimadzu Co., Kyoto, Japan). The nitrogen content was determined using Tector KjeltecTM 8600 (FOSS Co., LTD., Hilleroed, Denmark) analyzer (Method 981.10). whereas, Parr 6400 oxygen bomb calorimeter (Moline, IL, USA) was used to determine GE. The calculation of digestibility was done based on the relative chromium concentration of feed and feces samples, using the following equation: ATTD (%) = 100 - [(NF/ND) × (CrD/CrF)] × 100], ATTD is the apparent total tract digestibility, whereas NF, ND, CrD, and CrF were nutrient concentration in the feces sample, nutrient concentration in the diet, chromium concentration in the diet, and chromium concentration in the feces sample, respectively.
At the end of each phase, fresh fecal samples were randomly collected from 30 piglets·treatment-1 (2 pigs·pen-1 - 1 gilt and 1 barrow) using micro-tubes and placed in sterile plastic bags. The samples were then placed in an insulated ice container and immediately taken to the research laboratory for microbial study. To confirm the existence of microbes, 1gm of fresh fecal sample was taken and diluted with 9 mL of 1% peptone broth and mixed well with a vortex mixer. Then 0.02% of peptone solution was poured into MacConkey agar plates and Lactobacilli medium III agar plates, respectively. The MacConkey agar plates were incubated at 37℃ for 1 day and Lactobacilli medium III agar plates were incubated at 39℃, for 2 days. Finally, Lactobacilli and E. coli colonies were enumerated immediately and log transformed for statistical analysis.
At the end of each phase, approximately 300 g of fecal samples were collected from 30 piglets·treatment-1 (2 pigs·pen-1 - 1 gilt and 1 barrow) and placed in a plastic box (2.6 L) with a small hole in the middle and sealed with plaster. The samples were fermented for seven days at 25℃, and 100 mL of sample was taken away from the headspace (approximately 2.0 cm) of the fecal sample for air circulation. Later the box was re-sealed and manually shaken for around 30 S to measure the crust formation on the surface. Finally, the concentrations of NH3 and H2S was determined within the scopes of 5.0 to 100.0 ppm and 2.0 to 20.0 ppm (No. 3La and 4LK, detector tube; Gastec Corp, Kanagawa, Japan).
Statistical analysis
All the data were analyzed as a completely randomized block design using the GLM procedure of SAS (version 9.2 SAS Institute, Cary, NC, USA). When significant differences were identified among treatment means and they were separated using T-test. Pens were considered as experimental units. Variability in the data was expressed as the SEM. p < 0.05 was considered to be statistically significant and p < 0.10 was considered as trends.
Results
The effects of increasing level of lactulose supplementation on the growth performance of weaning pigs is shown in Table 2. The piglets receiving lactulose supplement showed a higher body weight (p < 0.05) compared to those fed CON diet. Also, during phase 1, LAC1 and LAC 2 group pigs showed higher (p < 0.05) ADG and ADFI, whereas during Phase 2 ADG and G : F was significantly increased (p < 0.05) in pigs fed lactulose supplement compared to CON. Moreover, during the overall experimental period, BW, ADG and G : F were significantly (p < 0.05) increased with graded level of lactulose supplementation.
The effects of lactulose supplementation on the apparent total track nutrient digestibility of weaning pigs is presented in Table 3. The dietary inclusion of lactulose supplement has significantly (p < 0.05) increased the DM and N at phase 1, whereas during phase 2 the nitrogen digestibility and energy was significantly increased (p < 0.05) in pigs fed lactulose supplement compared to CON. Moreover, the fecal lactobacillus count was significantly increased in pigs receiving lactulose supplementation during phase 1 and 2. Whereas, E. coli count was significantly reduced (p > 0.05) in pigs fed lactulose diet during phase 2 (Table 4).
In addition, dietary lactulose supplementation has significantly decreased (p > 0.05) NH3 and H2S odor emission in weaning pig compared to CON during phase 2 (Table 5).
Discussion
Lactulose (LAC) is a kind of non-digestible oligosaccharide (NDO) (Schumann, 2002; Cho and Kim, 2014) and produced by isomerization of lactose by regrouping the glucose residue to a fructose molecule. Besides, piglets are born with high intestinal lactase activity that aids to easily digest the dietary lactulose (Zhao et al., 2012). The present study showed increased BW, ADG, ADFI, and G : F in weaning pigs fed lactulose (2 g·kg-1) supplement was consistent with the studies of O'Doherty et al. (2004) and Krueger et al. (2002) who found an increased growth performance in piglets fed lactulose supplement. Similarly, Fleige et al. (2007) reported that 30 g·kg-1 LAC supplementation has increased the growth performance of calves. However, Konstantinov et al. (2004) reported that 10 g·kg-1 of lactulose with other fermentable carbohydrates has no effect on the growth performance. Hence the main reason for increased body weight, daily gain, and G : F of weaning pigs in this study are mainly due to the palatability of lactulose supplement that observed through the increased digestibility and fecal microbiota.
In the earlier studies, Choct and Kocher (2000) and Montagne et al. (2003) had addressed the benefits of short-chain fatty acids in host organism that include the use of acetic acid in peripheral tissues, the use of propionic acid by the liver for gluconeogenesis, and the use of butyric acid in colon cells as the primary energy fuel that helps to increase the nutrient digestibility. Abrams et al. (2005) stated that prebiotics as Lactobacillus and Bifidobacterium can easily ferment by the gut microbes and aids to increase the mineral absorption. In this study, pigs fed graded level of LAC supplement has significantly increased the nutrient digestibility of DM, N, and E was correlated with O'Doherty et al. (2004) and Cho and Kim (2014) who observed an increased nutrient digestibility in weaning piglets and broilers, respectively. Furthermore, (Xu et al., 2002) stated that fructooligosaccharides (FOS) supplementation had stimulate the digestive enzyme activity (total protease, trypsin, and amylase) in the small intestine of growing pigs which corroborates with the current findings. Lactulose is an NDO (Natural disaccharide-octyl) that cannot be hydrolyzed by digestive enzymes but fermented to short chain fatty acids, such as acetate, propionate and butyrate in the lower gut (Klaschik et al., 2003). Previously, Cho and Kim (2014) have suggested that lactulose inclusion in the basal diet had reduce E. coli, and improve the Lactobacillus counts in the broiler. Similarly, Zhao et al. (2013) reported that inclusion of NDO in growing- finishing pigs’ diet has enhanced the growth of Lactobacillus and reduce E. coli. In our study, pigs fed lactulose supplement has significantly increased the population of beneficial Lactobacillus counts and suppress the E. coli concentration was agreed with Hossain et al. (2015) who observed a same finding in growing-finishing pigs. We believe that increased growth performance and digestibility in this study is mainly due to the presence of abundance lactobacillus bacteria in weaning pigs’ gut.
The high concentrations of hazard odor ammonia (NH3) and Hydrogen sulfide (H2S ) emission from the farm may cause adverse effect on animals and humans (Zhang and Kim, 2014). Such odors are highly correlated with nutrient utilization and the intestinal microbial ecosystem (Ferket et al., 2002). In this study, pigs fed LAC supplementation has significantly decreased the NH3 and H2S gas emission in weaning pigs was correlated with the literature of Zhao et al. (2013) who observed similar results in growing-finishing pigs fed fructo-oligosaccharides supplementation. Similarly, Cho and Kim (2014) reported that inclusion of lactulose supplementation in the diet of broilers has reduced NH3 and H2S gas emission. In 2011, Panesar and Kumari reported that lactulose supplement could be metabolized by LAB (lactic acid bacteria) and live in the hind gut to produce short chain fatty acids and carbon-dioxide that leads to a low pH in gut contents. Apart from this, Hoeksma et al. (1993) study showed that fecal pH has greatly affects NH3 emission thereby decrease NH3 and H2S emissions. The decreased NH3 and H2S odor release from weaning pigs fed LAC supplement is mainly because of increased nutrient digestibility and improved microbial ecology.
Conclusions
Our data revealed that administration of 1 g·kg-1 and/or 2 g·kg-1 LAC diet in weaning diet could be beneficial to improve the growth performance, nutrient digestibility of dry matter, nitrogen, and energy. Moreover, it plays a vital role in modulating the gut microbial as increase Lactobacillus population and decrease in E. coli counts. Moreover, the graded level of LAC supplementation has reduced NH3 and H2S toxic odor emissions from a swine farm. Therefore, we infer that LAC supplementation at a level of 1 g·kg-1 and/or 2 g·kg-1 would be optimal level of alternative feed additive to enhance the performance of weaning pigs.
