Stress at weaning can negatively impact piglet immunity and gut health, impairing growth and feed efficiency and often resulting in diarrhoea(Reference Campbell, Crenshaw and Polo1). Along with the stress of weaning, passive immunity of the piglets is also reduced at this time, while active immunity is not fully developed. This makes weaned pigs more prone to disease(Reference Blecha, Pollmann and Nichols2), in particular post-weaning (pw) diarrhoea which can be caused by enterotoxigenic Escherichia coli (Reference Amezcua, Friendship and Dewey3) or other pathogens(Reference Ruiz, Bersano and Carvalho4). To reduce the incidence of these pathogens and the occurrence of pw diarrhoea and to prevent the weaning-associated growth check, in-feed antibiotic and/or zinc oxide treatments are frequently used(Reference Pluske5). However, in-feed antibiotic growth promoters were banned in the European Union in 2006, and a ban on the preventive use of antibiotics in groups of animals and via medicated feed will enter into force in the European Union in 2022. In the same year, the use of pharmacological levels of zinc oxide will also be banned. As a result, alternative treatments, such as probiotics, will be of increased importance in the future. Probiotics not only control pathogens, but they can also improve pig growth and feed efficiency(Reference Pluske5,Reference Gaggìa, Mattarelli and Biavati6) .
Bacteria from Bacillus spp. are commonly used as probiotics in pig production(Reference Poulsen, Jonge and Nielsen7–Reference Alexopoulos, Georgoulakis and Tzivara9). Species from this genus form spores, which increases their resistance to hostile conditions such as those encountered in the gastrointestinal tract and during feed manufacture(Reference Nicholson, Munakata and Horneck10,Reference Setlow11) . In addition, the vegetative cells of Bacillus spp. produce extracellular enzymes, which can increase nutrient availability in the diet and improve digestibility(Reference Leser, Knarreborg and Worm12), and Bacillus are well known for the production of antimicrobials(Reference Hong, Duc and Cutting13–Reference Guo, Li and Lu15). On this basis, many studies which administered spores of Bacillus spp. to weaned pigs found improved growth performance and feed conversion(Reference Davis, Parrott and Brown16–Reference Alexopoulos, Karagiannidis and Kritas18), while the incidence of pw diarrhoea was also reduced in some cases(Reference Kyriakis, Tsiloyiannis and Vlemmas19). Nevertheless, commencing the administration of Bacillus spores to pigs pw may not be the most effective strategy. First, it may be too late, as evidence suggests that early-life gut microbiota interventions are more effective(Reference Alexopoulos, Georgoulakis and Tzivara20–Reference Barba-Vidal, Martín-Orúe and Castillejos25). Second, the spores may not germinate in the gastrointestinal tract(Reference Prieto, O’Sullivan and Tan26), and last, which may/may not be related to lack of germination, Bacillus administered as spores does not usually persist for more than 1 week after ceasing administration(Reference Leser, Knarreborg and Worm12).
A cheaper and potentially more effective alternative to probiotic supplementation of pw diets is the inclusion of Bacillus spores in the diet of gestating and/or lactating sows. Vertical transmission of the probiotic from sows to their offspring can then occur between birth and weaning(Reference Taras, Vahjen and Simon27,Reference Taras, Vahjen and Macha28) , although this is sometimes limited(Reference Michiels, Possemiers and Degroote29). Maternal administration can also benefit the sow, minimising weight loss during lactation and improving reproductive performance and milk quality(Reference Alexopoulos, Karagiannidis and Kritas18,Reference Alexopoulos, Georgoulakis and Tzivara20,Reference Stamati, Alexopoulos and Siochu30,Reference Hayakawa, Masuda and Kurosawa31) . These maternal benefits sometimes increase the number of piglets weaned per sow(Reference Stamati, Alexopoulos and Siochu30), although some studies did not find any significant effects on sow productivity(Reference Baker, Davis and Spencer22,Reference Link, Reichel and Kyzeková24,Reference Taras, Vahjen and Simon27,Reference Taras, Vahjen and Macha28) . Probiotic administration to sows also leads to improved weight gain and feed efficiency in the offspring pw(Reference Alexopoulos, Karagiannidis and Kritas18,Reference Alexopoulos, Georgoulakis and Tzivara20,Reference Baker, Davis and Spencer22,Reference Taras, Vahjen and Macha28) . However, the mechanisms by which maternal probiotic supplementation benefits offspring growth are not fully understood. Probiotic administration stimulates the immune system of sows, which confers passive immunity to offspring through colostrum and milk(Reference Inatomi, Amatatsu and Romero-Pérez32). Stimulation of the immune system of the piglets may even start before the piglets are born, as piglets become immunocompetent in utero and their active immunity depends on maternal antibody levels(Reference Salmon33). Furthermore, the faecal bacterial community of the sows, including any administered probiotic and/or probiotic-modulated taxa, can be transferred to their litter through the intake of maternal faeces(Reference Taras, Vahjen and Macha28).
However, most studies that administer probiotics to gestating/lactating sows do not follow the growth of offspring beyond the weaner stage, as they are usually focused on the incidence of pw diarrhoea(Reference Poulsen, Jonge and Nielsen7,Reference Alexopoulos, Karagiannidis and Kritas18,Reference Alexopoulos, Georgoulakis and Tzivara20,Reference Link, Reichel and Kyzeková24,Reference Taras, Vahjen and Simon27–Reference Michiels, Possemiers and Degroote29,Reference Hayakawa, Masuda and Kurosawa31) . The aim of the present study was therefore to evaluate the efficacy of a novel Bacillus altitudinis probiotic delivered as spores to sows and/or their offspring on sow health, reproductive performance and colostrum quality, as well as on lifetime growth and health and carcass characteristics of the offspring.
Materials and methods
Ethical approval
Ethical approval for this study was granted by the Teagasc Animal Ethics Committee (approval no. TAEC148/2017), and the project was authorised by the Health Products Regulatory Authority (project authorisation no. AE19132/P066). The experiment was conducted in accordance with Irish legislation (SI no. 543/2012) and the European Union Directive 2010/63/EU for animal experimentation.
Experimental design and diets
A total of twenty-four sows (Large White × Landrace; Hermitage Genetics) were selected on day (D) 100 of gestation and blocked by parity, body weight (BW) and back fat (BF) depth, following which they were individually housed and randomly assigned to one of two experimental treatment groups as follows: (1) control (CON, n 12), fed with a standard gestation diet from D100 of gestation to farrowing, followed by a standard lactation diet for 26 d until litters were weaned; and (2) probiotic (PRO, n 12), fed the standard gestation/lactation diet supplemented with B. altitudinis WIT588 spores (about 4 × 109 spores daily from D100 of gestation to farrowing and about 1·2 × 1010 spores daily during lactation for 26 d until weaning of litters, administered as outlined below). Cross-fostering of piglets was performed between 24 and 48 h postpartum to equalise litter size (14 piglets/litter) if necessary, but only within the same treatment group.
At weaning (at D26 (sem 1·5) of age), a total of 144 piglets from these sows (n 72/sow treatment) were selected across all litters, blocked by sow treatment, sex, BW and litter origin and randomly assigned to dietary treatments. Offspring from each of the two sow treatments were assigned as same sex pairs of pigs to either a CON (no probiotic) or PRO (probiotic-supplemented) treatment for 28 d pw, resulting in four treatment groups (n 36 piglets/treatment) as follows: (1) piglets weaned from CON sows, fed a CON diet (CON/CON); (2) piglets weaned from CON sows, fed a probiotic-supplemented diet (CON/PRO); (3) piglets weaned from PRO sows, fed a CON diet (PRO/CON) and (4) piglets weaned from PRO sows, fed a probiotic-supplemented diet (PRO/PRO). Probiotic supplementation consisted of about 1 × 109 colony forming units (CFU) of B. altitudinis WIT588 spores administered daily, as outlined below. Probiotic supplementation was ceased at D28 pw, but pigs were monitored until the end of the finisher period (about D127 pw). A starter/link diet was fed for the first 28 d pw, followed by a weaner diet until D55 pw, and thereafter a finisher diet was fed until slaughter at D127 pw.
The ingredient composition and nutrient content of all sow and offspring diets are shown in Table 1. The diets were manufactured in the Teagasc feed mill (Moorepark) and were formulated to meet or exceed National Research Council recommendations (NRC, 2012)(34) for pigs at the relevant stage of the production cycle. All starter/link diets were formulated with 10·74 MJ/kg net energy and 14·0 g/kg standardised ileal digestible lysine using the same ingredients. Similarly, the weaner diet was formulated with 10·55 MJ/kg net energy and 11·49 g/kg standardised ileal digestible lysine. The finisher diet was formulated with 9·80 MJ/kg net energy and 9·97 g/kg standardised ileal digestible lysine. All diets were fed in 3 mm pellet form. Sows were fed 2·7 kg/d of feed up to the day of farrowing and thereafter were provided with ad libitum access to feed from a trough using a computerised feed delivery system (DryExact Pro; Big Dutchman). Water was available on an ad libitum basis to sows during gestation and lactation from a single-bite drinker in the feed trough and to suckling piglets from a bowl in the farrowing pen. Suckling piglets were offered creep feed in pelleted form from D12 of age to weaning. At all stages pw, pigs were provided with ad libitum access to feed from a 30 cm wide stainless-steel feeder (O’Donovan Engineering) and to water from one nipple-in-bowl drinker (BALP). Representative samples were taken from all diets and analysed for DM, ash, crude protein, total oil, crude fibre and neutral-detergent fibre by Sciantec Analytical Services Limited.
Composition of experimental diets
(on an air-dry basis; kg/tonne unless otherwise stated)
* Lactoflo 70 contains 70 % lactose, 11·5 % protein, 0·5 % oil, 7·5 % ash and 0·5 % fibre (Volac).
† Premix provided per kg of complete diet: Cu, 15 mg; Fe, 70 mg; Mn, 62 mg; Zn, 80 mg; I, 0·6 mg; Se, 0·2 mg; vitamin A, 344 μg; vitamin D3, 25 μg; vitamin E, 100 mg; vitamin K, 2 mg; vitamin B12, 15 μg; riboflavin, 5 mg; nicotinic acid, 12 mg; pantothenic acid, 10 mg; choline chloride, 500 mg; biotin, 200 mg; folic acid, 5 g; vitamin B1, 2 mg; vitamin B6, 3 mg.
‡ Premix provided per kg of complete diet: Cu, 155 mg; Fe, 90 mg; Mn, 47 mg; Zn, 120 mg; I, 0·6 mg; Se, 0·3 mg; vitamin A, 2064 μg; vitamin D3, 25 μg; vitamin E, 100 mg; vitamin K, 4 mg; vitamin B12, 15 μg; riboflavin, 2 mg; nicotinic acid, 12 mg; pantothenic acid, 10 mg; choline chloride, 250 mg; vitamin B1, 2 mg; vitamin B6, 3 mg; Endox, 60 g.
§ Premix provided per kg of complete diet: Cu, 15 mg; Fe, 24 mg; Mn, 31 mg; Zn, 80 mg; I, 0·3 mg; Se, 0·2 mg; vitamin A, 688 μg; vitamin D3, 12·5 μg; vitamin E, 40 mg; vitamin K, 4 mg; vitamin B12, 15 μg; riboflavin, 2 mg; nicotinic acid, 12 mg; pantothenic acid, 10 mg; vitamin B1, 2 mg; vitamin B6, 3 mg.
|| The diet contained 500 phytase units (FYT) per kg feed from RONOZYME HiPhos (DSM).
¶ Calculated from tabulated ingredient values(Reference Sauvant35).
Preparation and administration of probiotic spores
B. altitudinis WIT588 is a rifampicin resistant variant of a seaweed-derived isolate (WIT572; NCIMB 43558) characterised, both in vitro and in vivo as a probiotic for pigs, used to facilitate enumeration in the porcine gastrointestinal tract(Reference Prieto, O’Sullivan and Tan26,Reference Prieto, O’Sullivan and Tan36,Reference Prieto, O’Sullivan and Tan37) . The strain was first referred to as B. pumilus on the basis of sequencing of the gyrB and pyrE genes(Reference Prieto, O’Sullivan and Tan26), but has since been identified as B. altitudinis on the basis of whole genome sequencing (unpublished results). The B. altitudinis WIT588 spore suspension used in the current feeding trial was prepared according to the nutrient exhaustion method described by Prieto et al. (2014)(Reference Prieto, O’Sullivan and Tan37), and the spores were suspended in sterile water. The concentration was then determined using a haemocytometer and adjusted to about 109 spores/ml. Aliquots of this spore suspension were stored at –20°C until use. Probiotic spores were administered once daily in the morning to the respective treatment groups. The doses used for sows and weaned pigs, as outlined above, were calculated based on data from previous experiments and doses used for comparable commercially available probiotics. The amount of spore suspension required each day was thawed overnight at 4°C. On the morning of administration, spore suspensions were diluted in distilled water to the required dose and top-dressed onto the feed in a final volume of 4 ml for gestating sows and weaned pigs and 12 ml for lactating sows. The same volume of sterile water was top-dressed onto the feed of CON pigs not administered probiotic.
Animal housing and management
PRO sows were housed separately from CON sows, with two farrowing rooms for PRO sows, each with seven pens per room, and one room for CON sows with fourteen pens per room. Farrowing pens (2·5 m × 1·8 m) had a farrowing crate on a partially slatted floor with a heated floor pad for piglets. The temperature of the farrowing rooms was maintained at about 24°C at farrowing and gradually reduced to 21°C by D7 of lactation. Each room was illuminated by daylight and artificial light. The temperature inside the building was automatically controlled. Ventilation was via punched ceiling ventilation with air exhausted via a variable speed fan linked to a thermostat and controlled automatically via a controller (135-L2 Pro climate computer; Big Dutchman) outside each room.
At weaning, piglets were housed in same sex pairs in seventy-two pens (n 2 pigs/pen) across four rooms. Each room contained twenty-four pens (1·2 m × 0·9 m), with treatments distributed equally across rooms. Pens were fully slatted with plastic flooring (Faroex). Empty pens were left between treatments to minimise probiotic cross-contamination, and strict hygiene procedures were followed. Pigs were penned as pairs for the first 7 d pw. A total of forty pigs (n 10/treatment; one pig from each of ten pen pair replicates per treatment) were killed by captive bolt stunning followed by exsanguination on D8 pw to facilitate sampling of digesta and intestinal tissue. To coincide with this, one pig from each of the remaining pairs of pigs was also removed from the trial at this time and the remaining piglets (n 72) were individually penned until slaughter at D127 pw. The temperature of the weaner rooms was maintained at 28°C for the first 7 d pw, gradually reduced to 22°C by D28 pw and maintained at 22°C until D56 pw. Temperature and ventilation were controlled by a hot air heating system and an exhaust fan drawing air from under slat level connected to a controller (Stienen PCS 8400; Stienen BV). At D56 pw, pigs were moved to one of four finisher rooms, each with eighteen pens/room, where they were individually penned in fully slatted pens (1·81 m × 1·18 m) until the end of the experimental period (D127 pw). Pigs were kept in the same order as in the weaner rooms but without the empty pen between treatments. Finisher rooms were ventilated with fans and air inlets controlled by a Stienen PCS 8200 controller (Stienen BV). Air temperature was maintained at 20–22°C. Sows and piglets were observed closely at least twice daily. Any pig showing signs of ill health was treated as appropriate, and this was recorded. All veterinary treatments were recorded, including identity of pig, symptom, medication used and dosage.
Data recording and sampling
During sampling and weighing of sows and offspring, strict hygienic measures were taken to prevent cross-contamination between treatments. CON pigs not receiving probiotic were handled first, followed by PRO treatment groups. Gloves were changed between pigs, and fresh disposable overalls were worn by all personnel prior to commencing sampling of each treatment group. All equipment, such as weighing scales and the cradle used for collection of blood samples, were disinfected thoroughly with 1 % Virkon® after use to prevent cross-contamination at subsequent weighings/samplings. In both CON and PRO farrowing rooms and beside both PRO and CON pens within the weaner rooms, settle plates containing agar medium selective for the probiotic strain (see below) were exposed for 30 min at faecal sampling time points and incubated with the faecal sampling plates as outlined below in order to check for the presence of the probiotic strain in the air.
Sow body weight and back fat thickness
Feed intake of sows was recorded daily between D100 of gestation and D28 of lactation. BW and BF were recorded at the start of the experiment (D100 of gestation), on the expected farrowing date (D114 of gestation) and again at weaning of litters (about D26 of lactation). Sow BW was recorded using an electronic sow scales (EziWeigh 7i; O’Donovan Engineering). Sow BF was measured using a digital BF indicator (Renco LEAN-MEATER; Renco Corporation) by placing the probe of the digital indicator on the back of the sow at the level of the second last rib, 6·5 cm from the side of the backbone. A reading was taken from the right and left side of the sow’s back, and the average of both readings was recorded.
Colostrum and milk sampling
Colostrum samples (n 12 sows/treatment) were collected by manual milking of the first four teats immediately distal to the sow’s head on one side of the udder within 12 h of farrowing. On D14 of lactation, milk samples were collected from sows (n 12 sows/treatment) in the same way but this time following administration of a 1 ml (10 IU) intramuscular injection of oxytocin (Eurovet 247 Animal Health) to induce milk let-down. Samples for compositional analysis were stored at −20°C until analysis and samples for immunoglobulin analysis were stored at –80°C.
Litter data at birth and pre-weaning piglet growth performance
Reproductive parameters were recorded per litter, that is number of piglets (total born, born alive, stillborn). The weight and sex of each piglet were recorded at birth, and each piglet was tagged for identification purposes. Thereafter, piglets were individually weighed at birth (D0), D14 and D26 postpartum, and these data were used to determine pre-weaning piglet average daily gain (ADG). Piglet mortality between birth and weaning was also recorded.
Post-weaning growth performance, faecal scoring and carcass measurements
Growth performance of piglets was measured by weighing pigs individually and monitoring individual feed intake in order to calculate ADG, average daily feed intake and feed conversion ratio (FCR). Feed disappearance was recorded weekly, and pigs were individually weighed at weaning (D0 pw), D14 pw, the changeover to weaner feed (D28 pw), changeover to finisher feed (D56 pw), D105 pw and immediately before slaughter (D127 pw). Pigs were fasted for 12 h prior to pre-slaughter weighing.
The incidence of pw diarrhoea was assessed by daily faecal consistency scoring between weaning and D28 pw. The scoring system used was as follows: 0 for dry pelleted faeces; 1 for soft faeces with shape; 2 for very soft or viscous liquid faeces (mild diarrhoea) and 3 for severe diarrhoea with or without blood.
Pigs were slaughtered at about 123·5 (sem 1·38) kg live weight by CO2 stunning followed by exsanguination. Carcass weight was estimated by multiplying the weight of the hot eviscerated carcass 45 min after slaughter by 0·98. Kill-out percentage was calculated as carcass weight/live weight at slaughter. BF thickness and muscle depth measured at 6 cm from the edge of the split back at the level of the third and fourth last rib were determined using a Hennessy Grading Probe (Hennessy and Chong). Lean meat content was estimated according to the following formula: Estimated lean meat content (%) = 60·3 – 0·847x + 0·147y where x = fat depth (mm); y = muscle depth (mm)(38).
Faecal sampling
Faecal samples were collected from sows (n 24) directly from the rectum using gentle digital stimulation on D100 and D115 of gestation, about D13 of lactation and at weaning of litters (about D26 of lactation). Pre-weaning, rectal swabs were taken from offspring on about D13 of lactation (n 12 pig replicates per treatment), and faecal samples were obtained by digital rectal stimulation at weaning (n 10 pig replicates per treatment), D27 pw and D56 pw (n 10 pig replicates per treatment). Faeces were collected into sterile containers and, together with swabs, were put on ice and stored at 4°C until analysis for the administered probiotic strain (within 12 h), as outlined below.
Blood sampling
Blood samples were taken from sows (n 24) by anterior vena cava/jugular venepuncture on D100 and D114 of gestation and at weaning of litters (about D26 of lactation). Piglets (n 10 pig replicates per treatment) were blood sampled by anterior vena cava/jugular venepuncture on D0 pw, D28 pw and D57 pw. Blood samples were also collected from piglets killed at D8 pw (n 10 pig replicates per treatment) at exsanguination. In all cases, about 1–2 ml of whole blood was collected in a Vacutainer® tube containing EDTA (Becton-Dickson Ltd) (except at kill when the volume was about 9 ml) and immediately inverted a number of times to prevent clotting. Samples were kept at room temperature, and haematological analysis was performed within 6 h, as outlined below.
Intestinal sampling
After euthanasia of piglets on D8 pw (n 10 pig replicates per treatment), the entire intestinal tract was immediately removed. Digesta samples from the ileum (15 cm proximal to the ileo-caecal junction), caecum (terminal tip) and rectum were collected aseptically into sterile containers, put on ice and stored at 4°C until analysis for the administered probiotic strain (within 12 h), as outlined below. Samples (about 2 cm) of tissue were excised from the duodenum (15 cm distal to the pyloric junction), jejunum (1·5 m distal to the pyloric junction) and the ileum (15 cm proximal to the ileo-caecal junction). Tissue samples were rinsed in PBS immediately post-harvest and placed in No-Tox, an alcohol/aldehyde fixative (Scientific Device Lab) on a shaker for 48 h prior to histological analysis, as outlined below.
Analysis of sow colostrum and milk
Compositional analysis of sow colostrum and milk
Colostrum and milk samples were defrosted at room temperature. When fully thawed, samples were mixed by inverting several times to disrupt settled solids and mixed well. The volume of each sample was recorded prior to decanting into 50 ml tubes on ice. Sterile water was added to bring the volume up to 40 ml. Tubes were mixed thoroughly and kept on ice. Each sample was analysed in duplicate for total solids, lactose, fat, protein, true protein and casein B content by near-infrared absorption using a Bentley Dairyspec FT (Bentley Instruments, Inc.). Data were recorded as % (g/100 g), taking the dilution factor into account.
IgA and IgG quantification in colostrum
IgA and IgG concentrations in colostrum were determined using ELISA kits (Pig IgA and IgG ELISA Kits; Bethyl Laboratories Inc.). First, 200 μl of colostrum was diluted 1:2 with PBS (1×, pH 7·4) and centrifuged at 10 000 rpm for 20 min at 4°C. The fat was then removed, and the supernatant was collected and diluted 1:100 000 and 1:500 000 with 1× Dilution Buffer B (Bethyl Laboratories Inc.) for IgA and IgG analyses, respectively. The rest of the analysis was performed according to the manufacturer’s protocol. All colostrum samples were analysed in duplicate. Absorbance was measured at 450 nm using a plate reader (ELx808 Absorbance Microplate Reader; BioTek). The IgA and IgG concentrations in the colostrum were obtained by reading absorbance values from standard curves prepared using standard solutions containing 1000·0, 333·3, 111·1, 37·0, 12·3, 4·1 and 1·4 ng/ml of IgA and 500·0, 250·0, 125·0, 62·5, 31·3, 15·6 and 7·8 ng/ml of IgG.
Small intestinal histology
Duodenal, jejunal and ileal tissue samples were removed from the No-Tox fixative and dehydrated through a graded alcohol series, cleared with xylene and embedded in paraffin wax. Tissue samples were sliced into 5 μm sections using a microtome (Leica RM2135), mounted on microscope slides and stained with haematoxylin–eosin for the determination of gross morphological parameters of intestinal structure (villus height and width and crypt depth and width). For each pig, ten villi and ten crypts were measured on five fields of view, where villi were attached to the lumen, and the means were utilised for statistical analysis. The goblet cell number was determined by periodic acid-Schiff staining. Positively stained periodic acid-Schiff cells were enumerated on ten villi/sample, and the means were utilised for statistical analysis.
Microbiological analysis of faecal and digesta samples
Faecal and digesta samples and rectal swabs were homogenised and subsequently diluted in maximum recovery diluent (Merck) as described by Gardiner et al. (2004)(Reference Gardiner, Casey and Casey39). Appropriate dilutions were spread-plated in duplicate on brain heart infusion agar containing 3·5 % NaCl, 200 μg/ml rifampicin (Sigma-Aldrich) and 50 U/ml nystatin (Sigma-Aldrich) in order to enumerate the administered probiotic strain. Plates were incubated aerobically for 2 d at 37°C, the colonies were counted and the counts were averaged and presented as log10 CFU/g of the original sample or log10 CFU/swab.
Haematological analysis of blood samples
Haematological analysis was performed on whole blood using an Abbot Cell-Dyn 3700 analyser (GMI-Inc.). The following parameters were measured: leucocyte number, lymphocyte number and percentage, monocyte number and percentage, granulocyte number and percentage, eosinophil number and percentage, basophil number and percentage, erythrocyte number, Hb, mean corpuscular volume, mean corpuscular Hb, platelets and packed cell volume.
Statistical analysis
Power calculations were performed to determine the minimum number of observations required to detect effect sizes, using a statistical power of 80 %, an α level at 5 % and standard deviation of variables of interest from seven previously published studies. The power calculation indicated that twelve sows per treatment were required to see a difference of 2·5 mm in BF depth, ten piglets were required to see a 2 log10 CFU/g difference in selected microbial counts between treatments and that eighteen piglets were required to see a 1·5 log10 CFU/g difference in microbial counts between treatments.
The experiment was a 2 × 2 factorial arrangement, with the factors being maternal treatment (control or probiotic supplementation) and pw treatment (control or probiotic supplementation). All data were analysed using the MIXED procedure in SAS® 9.4 (SAS Institute Inc.), unless otherwise stated. The model included maternal treatment and pw treatment as fixed effects and their interaction. Where required, data were analysed as a repeated measure with sampling day as the repeated variable and the appropriate covariance structure, as indicated by the model fit statistics, was fitted to the data. Simple main effects were obtained using the ‘slice’ option in SAS.
The sow/litter was the experimental unit for sow performance, sow haematology, sow probiotic count data, colostrum and milk composition and colostrum IgA and IgG. The individual pig was the experimental unit for analysis of pre-weaning and pw pig growth performance, carcass characteristics, haematology, small intestinal morphology and probiotic count data. The normality of scaled residuals was investigated using the Shapiro–Wilk and Kolmogorov–Smirnov tests within the UNIVARIATE procedure of SAS. Differences in least square means were investigated using the t test after Tukey adjustment for multiple comparisons. df were estimated using Satterthwaite adjustment.
For sow performance, litter size and pre-weaning mortality data, block was included as a random effect. The initial value (D100 of gestation) was included as a covariate in the analysis when significant in the model. Pre-weaning performance was analysed as repeated measures, including sex (male, female) as a fixed effect and block as a random effect. Birth weight was included as a covariate when significant in the model. pw performance was analysed as repeated measures, including sex (male, female) as a fixed effect and weaning weight as a covariate, when significant in the model. For carcass characteristics, sex (male, female) was included as a fixed effect and BW at weaning was included as a covariate when significant in the model. Counts of B. altitudinis WIT588 were analysed as repeated measurements. For the faecal counts of B. altitudinis WIT588 in the sows, block was included as a random effect. For the faecal counts of B. altitudinis WIT588 in the post-weaned piglets, the count at weaning was included as a covariate in the analysis, when significant. Haematological parameters were analysed including the initial value (D100 of gestation for sows or D0 pw for the offspring) as a covariate in the analysis when significant in the model. In addition, block was included as a random effect for the haematological values of sows. The haematological parameters that were not normally distributed were further analysed to find the best fitting distribution using the GLIMMIX procedure in SAS, using a gamma distribution. For these variables, the ilink function was used to back-transform the data to the original scale. The small intestinal morphology data were analysed using sex (male, female) as a fixed effect.
The results are presented in the text and tables as the least square means together with the pooled standard errors of the mean. Differences between treatments were considered significant for P ≤ 0·05, while 0·05 < P ≤ 0·10 was considered as a tendency.
Results
Sow reproductive performance and tissue mobilisation
The effect of supplementing sow diets with B. altitudinis WIT588 spores from D100 of gestation to weaning (D26 of lactation) on sow weight, BF depth, feed intake and reproductive performance is presented in online Supplementary Table S1. There was no treatment × day interaction for any of the variables of interest. Sows from the CON group were heavier than those in the PRO group at weaning (257·0 v. 248·7 (sem 2·71) kg; P = 0·03). However, gestation length (114·8 v. 114·6 (sem 0·33) d), total born per litter (14·62 v. 15·49 (sem 1·253)), live born per litter (13·50 v. 13·97 (sem 1·170)), percentage of piglets live born per litter (93·3 v. 90·8 (sem 3·25) %), stillbirths per litter (1·15 v. 1·51 (sem 0·592)) and the numbers of piglets suckling per litter at 48 h postpartum (14·3 v. 14·2 (sem 0·40)) were not affected by sow treatment (P > 0·1). Although not significant, there was a numerical reduction in pre-weaning mortality (15·6 v. 10·1 (sem 2·82) %; P = 0·18) when the probiotic was fed and because of this a numerical increase in the number of piglets weaned per litter (11·8 v. 12·6 (sem 0·55); P = 0·29) in response to probiotic supplementation of sows.
Recovery of Bacillus altitudinis WIT588 from the faeces of sows and their litters during lactation
Faecal counts of the administered probiotic (B. altitudinis WIT588) from the faeces of sows during gestation and lactation and from their offspring during lactation are shown in Table 2. Prior to commencing probiotic treatment (D100 of gestation), B. altitudinis WIT588 was not detected in the faeces of either CON or PRO sows. There was a treatment × day interaction for faecal counts of B. altitudinis WIT588 in sows. Counts of B. altitudinis WIT588 increased over time in PRO sows from D100 of gestation until D13 of lactation, declining slightly on D26 of lactation (P < 0·001). Faecal counts of B. altitudinis WIT588 were higher in PRO than in CON sows at all time points during probiotic administration (D115 of gestation, and D13 and D26 of lactation; P < 0·001), as the administered probiotic was essentially undetectable in CON sows. Although not administered the probiotic themselves, most of the offspring from PRO sows shed B. altitudinis WIT588 by D13 of age. There was a treatment × day interaction for faecal counts of B. altitudinis WIT588 in the offspring of PRO sows, with probiotic counts increasing over time (P < 0·001). However, counts are not comparable, as the D13 count is presented as CFU/swab and the D26 count as CFU/g faeces. Similar effects were observed in the offspring as in the sows, in that piglets born to PRO sows had higher faecal counts of B. altitudinis WIT588 at D13 and D26 of age than piglets born to CON sows (P < 0·001), again due to lack of probiotic detection in the offspring from CON sows.
Effect of supplementing sow diets with Bacillus altitudinis WIT588 spores from day (D) 100 of gestation to weaning (D26 of lactation) on faecal counts (log10 CFU/g) of sows and their piglets
(Least square mean values with their pooled standard errors of the mean (SEM)).
* Mean values were significantly different between treatments when P ≤ 0·05.
† CON: non-probiotic-supplemented sows.
‡ PRO: probiotic-supplemented sows.
§ The limit of detection of the assay for B. altitudinis WIT588 was 1000 CFU/g faeces or /swab. Values below the limit of detection were recorded as 3·00 log10 CFU/g faeces or /swab.
|| Counts are from rectal swabs and are presented as log10 CFU/swab.
¶ A rectal swab was taken from three pigs in the probiotic treatment group due to insufficient faecal sample. Probiotic was detected in these animals, but the counts were excluded from the statistical analysis.
Haematological parameters of sows during gestation and lactation
The full results for all haematological parameters measured in sows are presented in online Supplementary Table S2. Only results for haematological parameters where there were significant treatment differences are reported in Table 3. There was a tendency for a treatment × day interaction for mean corpuscular Hb concentration (P = 0·09), which decreased on D114 of gestation in CON sows, increasing again at weaning (D26 of lactation). The only treatment difference found for blood cell counts was for basophils. Overall, PRO sows had a higher basophil count than CON sows (P < 0·01). This was also found on D114 of gestation (P = 0·04), and a tendency for this effect was found on the day of weaning (D26; P = 0·07). Similar results were found for the overall percentage of basophils, where PRO sows had higher levels than CON sows (P = 0·001). This was also found on D114 of gestation (P = 0·05) and on the day of weaning (D26; P < 0·01). Regarding the other parameters measured, treatment differences were also observed for mean corpuscular volume and mean corpuscular Hb. Overall, CON sows had higher mean corpuscular volume than PRO sows (P < 0·001), and this was also found on D114 of gestation (P = 0·001) and on the day of weaning (D26; P < 0·01). Overall, CON sows had greater mean corpuscular Hb levels than PRO sows (P = 0·001), and this was also found on D114 of gestation (P = 0·01) and at weaning (D26; P = 0·001). In addition, the mean corpuscular Hb concentration was higher for PRO sows than for CON sows on D114 of gestation (P = 0·04).
Effect of supplementing sow diets with Bacillus altitudinis WIT588 spores from day (D) 100 of gestation to weaning (D26 of lactation) on haematological parameters of sows
(Least square mean values with their pooled standard errors of the mean (SEM)).
G100, D100 of gestation; G114, D114 of gestation; W26, weaning (D26 of lactation).
* Mean values were significantly different between treatments when P ≤ 0·05; Mean values tended to be different between treatments when 0·05 ≤ P ≤ 0·10.
† CON: non-probiotic-supplemented sows.
‡ PRO: probiotic-supplemented sows.
§ Percentages are based on the differential count of leucocytes.
Colostrum and milk composition
The effect of supplementing sow diets with B. altitudinis WIT588 spores from D100 of gestation to weaning of litters (D26 of lactation) on the composition of sow colostrum and milk is shown in Table 4. Colostrum composition was impacted by maternal treatment for all of the parameters measured, with the exception of fat percentage (P = 0·75) and IgA and IgG concentrations (P = 0·46 and P = 0·34, respectively). The colostrum from PRO sows had a higher percentage of total solids (P = 0·02), protein (P = 0·04), true protein (P = 0·05) and casein B (P = 0·05) and had less lactose (P = 0·01) than the colostrum from CON sows. However, milk composition was not affected by sow treatment (Table 4).
Effect of supplementing sow diets with Bacillus altitudinis WIT588 spores from day (D) 100 of gestation to weaning (D26 of lactation) on the composition of sow colostrum and milk
(Least square mean values with their pooled standard errors of the mean (SEM)).
* Mean values were significantly different between treatments when P ≤ 0·05.
† CON: non-probiotic-supplemented sows.
‡ PRO: probiotic-supplemented sows.
§ Milk was sampled 14 d postpartum.
Pre-weaning and post-weaning pig growth performance
Pig weights and ADG while suckling the sow were not affected by treatment (online Supplementary Table S3; P > 0·05). Birth weight averaged 1·47 (sem 0·029) kg and weaning weight averaged 7·27 (sem 0·168) kg for piglets from both treatments.
The effects of B. altitudinis WIT588 spore supplementation to sow and piglet diets on pw growth and carcass characteristics are shown in Table 5. No maternal treatment × pw treatment × day interaction was found. A maternal treatment × pw treatment interaction was found for BW on D127 pw (P = 0·05) with a tendency for the same on D105 pw (P = 0·07) and overall (P = 0·09). On D105 pw, PRO/PRO pigs tended to be heavier than CON/PRO pigs and on D127 pw, PRO/PRO pigs were heavier than pigs born to CON sows. At D105 pw, BW was 91·7 and 95·2 (sem 0·98) kg (P = 0·01), while at D127 pw, it was 121·0 and 124·5 (sem 0·97) kg (P = 0·01) for pigs born to CON and PRO sows, respectively. Overall, pigs born to PRO sows were heavier than pigs born to CON sows (P = 0·01). ADG from D0 to D127 pw was 890 and 922 (sem 10·9) g/d (P = 0·04) for pigs born to CON and PRO sows, respectively. Overall, pigs born to PRO sows had higher ADG than pigs born to CON sows (P = 0·04). A maternal treatment × pw treatment interaction was found for FCR from D0 to D14 pw (P < 0·001), where PRO/CON pigs had better FCR than CON/PRO pigs. During this period (D0–D14 pw), pigs born to PRO sows had better FCR than those born to CON sows (1·28 v. 1·45 (sem 0·030) g/g; P < 0·001). A maternal treatment effect for FCR was also observed for the overall period (P = 0·02). A pw treatment effect was observed from D0 to D14 pw, where CON pigs had better FCR than PRO pigs (1·30 v. 1·43 (sem 0·030) g/g; P < 0·01). A tendency for a pw treatment effect was also observed from D57 to D105 pw and during the entire pw period (D0–127 pw), but this time with PRO pigs having a better FCR than CON pigs (2·21 v. 2·13 (sem 0·032) g/g (P = 0·06) and 2·07 v. 2·04 (sem 0·014) g/g (P = 0·07), respectively).
Effect of Bacillus altitudinis WIT588 spore supplementation to sow and piglet diets on post-weaning growth and carcass characteristics
(Least square mean values with their pooled standard errors of the mean (SEM)).
ADG, average daily gain; ADFI, average daily feed intake.
a,bMean values within a row with unlike superscript letters were significantly different (P ≤ 0·05).
A,BMean values within a row with unlike superscript letters tended to be different (0·05 ≤ P ≤ 0·10).
† CON/CON, non-probiotic-supplemented sow/non-probiotic-supplemented piglet.
‡ CON/PRO, non-probiotic-supplemented sow/probiotic-supplemented piglet.
§ PRO/CON, probiotic-supplemented sow/non-probiotic-supplemented piglet.
|| PRO/PRO, probiotic-supplemented sow/probiotic-supplemented piglet.
¶ Mortality: Due to polyserositis and septicaemia (Streptococcus suis infection).
** Off trial: Pigs were removed from the trial due to lameness (PRO/CON, n 1), pneumonia (CON/CON, n 1 and CON/PRO, n 1), bloody diarrhoea (CON/CON, n 1 and PRO/CON, n 1) and abdominal hernia (CON/PRO, n 1).
†† Day 0 pw is the day of weaning.
There was no maternal treatment × pw treatment interaction for carcass weight or carcass quality parameters (P > 0·05). Carcass weight and kill-out percentage were 90·9 and 94·4 (sem 1·22) kg (P = 0·05) and 75·0 and 75·9 (sem 0·187) % (P < 0·01) for pigs born to CON and PRO sows, respectively. There was no effect of pw treatment on carcass weight or carcass quality parameters (P > 0·05).
Recovery of Bacillus altitudinis WIT588 from the faeces and intestinal digesta of pigs post-weaning
Counts of the administered B. altitudinis probiotic in the faeces and ileal, caecal and rectal digesta of the offspring pw are shown in Table 6. No maternal treatment × pw treatment × day interaction was found. A maternal treatment × pw treatment interaction was found at D27 pw (P < 0·001), and a tendency for this effect was also found at weaning (P = 0·08). At weaning, B. altitudinis WIT588 counts tended to be higher in the faeces of PRO/CON than PRO/PRO piglets. A maternal treatment effect was observed at weaning, where piglets born to PRO sows had higher B. altitudinis WIT588 counts than those born to CON sows (4·70 v. 3·00 (sem 0·088) log10 CFU/g faeces; P < 0·001), due to lack of detection in the latter. At D8 pw, pw treatment affected counts in the intestinal digesta. B. altitudinis WIT588 counts were higher in the ileal, caecal and rectal digesta of PRO compared with CON piglets (P < 0·001), as the administered strain was undetectable in the latter. B. altitudinis WIT588 counts were also higher in the faeces of PRO v. CON piglets on D27 pw (5·93 v. 3·00 (sem 0·021) log10 CFU/g faeces; P < 0·001), and there was a tendency for this effect at weaning (3·96 v. 3·74 (sem 0·088) log10 CFU/g faeces; P = 0·08).
Effect of Bacillus altitudinis WIT588 spore supplementation to sow and piglet diets on ileal, caecal and rectal digesta counts (log10 CFU/g)* of piglets euthanised on day (D) 8 post-weaning and on faecal counts at D0, D27 and D56 post-weaning
(Least square mean values with their pooled standard errors of the mean (SEM)).
a,bMean values within a row with unlike superscript letters were significantly different (P ≤ 0·05).
A,B,CMean values within a row with unlike superscript letters tended to be different (0·05 ≤ P ≤ 0·10).
† CON/CON, non-probiotic-supplemented sow/non-probiotic-supplemented piglet.
‡ CON/PRO, non-probiotic-supplemented sow/probiotic-supplemented piglet.
§ PRO/CON, probiotic-supplemented sow/non-probiotic-supplemented piglet.
|| PRO/PRO, probiotic-supplemented sow/probiotic-supplemented piglet.
¶ The limit of detection of the assay for B. altitudinis WIT588 was 1000 CFU/g faeces. Values below the limit of detection were recorded as 3.00 log10 CFU/g faeces.
Faecal scoring of pigs post-weaning
Statistical analysis of the probiotic effect on pw diarrhoea prevalence could not be conducted, as the occurrence of faecal consistency scores higher than 0 was rare. Out of 504 faecal consistency scores given to each one of the four treatments up to D28 pw, a score of 1 (soft faeces with shape) was given 45 times to the CON/CON treatment group, 28 times to the CON/PRO treatment group, 38 times to the PRO/CON treatment group and 27 times to the PRO/PRO treatment group. No scores higher than 1 were given at any time to any animal.
Haematological parameters of pigs post-weaning
The effects of B. altitudinis WIT588 supplementation to sow and piglet diets on the haematological parameters of pigs pw are shown in Table 7. No maternal treatment × pw treatment × day interactions were found for any of the parameters measured, except for mean corpuscular volume (P = 0·08) and mean corpuscular Hb (P = 0·09) which tended to decrease with increasing age in the pigs.
Effect of Bacillus altitudinis WIT588 spore supplementation to sow and piglet diets on haematological parameters of piglets at weaning and days 8, 28 and 57 post-weaning
(Least square mean values with their pooled standard errors of the mean (SEM)).
a,bMean values within a row with unlike superscript letters were significantly different (P ≤ 0·05).
A,BMean values within a row with unlike superscript letters tended to be different (0·05 ≤ P ≤ 0·10).
† CON/CON, non-probiotic-supplemented sow/non-probiotic-supplemented piglet.
‡ CON/PRO, non-probiotic-supplemented sow/probiotic-supplemented piglet.
§ PRO/CON, probiotic-supplemented sow/non-probiotic-supplemented piglet.
|| PRO/PRO, probiotic-supplemented sow/probiotic-supplemented piglet.
¶ Day 0 pw is the day of weaning.
** Percentages are based on the differential count of leucocytes.
Pigs on the pw PRO treatment had higher leucocyte counts on D57 pw than CON pigs (14·62 v. 11·68 (sem 0·962) × 103 cells/µl; P = 0·04). There was a tendency for a maternal treatment × pw treatment interaction for the total lymphocyte count on D57 pw (P = 0·10). An effect of pw treatment was found for the total number of lymphocytes and lymphocyte percentage at D57 pw, where PRO pigs had a higher lymphocyte count and percentage than CON pigs (10·97 v. 7·29 (sem 1·145) × 103 cells/µl (P = 0·03) and 68·03 v. 59·33 (sem 2·954) % (P = 0·04), respectively). Similarly, the overall lymphocyte count and lymphocyte percentage tended to be higher in PRO compared with CON pigs (10·61 v. 8·42 (sem 0·822) × 103 cells/µl (P = 0·06) and 68·95 v. 61·11 (sem 2·135) % (P = 0·01), respectively).
A maternal treatment × pw treatment interaction was found on D8 pw for monocyte count (P < 0·01), with counts lower in the CON/CON group than in the PRO/CON group. Likewise, a tendency for a maternal treatment × pw treatment interaction was also found for the percentage of monocytes on D8 pw (P = 0·09), with piglets from the CON/CON group having a lower percentage than their PRO/CON counterparts. This led to offspring from PRO sows having a higher monocyte percentage than pigs born to CON sows at D8 pw (6·65 v. 4·76 (sem 0·667) %; P = 0·05). In addition, pigs on the pw probiotic treatment had a lower percentage of monocytes than CON pigs on D57 pw (7·95 v. 10·65 (sem 0·873) %; P = 0·03) and overall (6·36 v. 8·28 (sem 0·631) %; P = 0·04).
A maternal treatment × pw treatment interaction was observed at weaning for the neutrophil count (P = 0·05), where pigs from the CON/PRO group had a higher count than PRO/PRO pigs. A tendency for a pw treatment effect was observed overall for the neutrophil percentage, where probiotic-supplemented pigs had a lower percentage of neutrophils than CON pigs (21·90 v. 26·90 (sem 1·877) %; P = 0·07).
There was a maternal treatment × pw treatment interaction for both the eosinophil count (P = 0·01) and percentage (P = 0·001) on D57 pw, with pigs from the PRO/CON group having a higher eosinophil count and percentage than pigs from the CON/PRO and PRO/PRO groups. A pw treatment effect was also observed, with probiotic-supplemented pigs having lower eosinophil counts than CON pigs on D8 pw (0·11 v. 0·16 (sem 0·017) × 103 cells/µl; P = 0·03), D57 pw (0·15 v. 0·22 (sem 0·019) × 103 cells/µl; P < 0·01) and overall (0·15 v. 0·19 (sem 0·014) × 103 cells/µl; P = 0·050). Similarly, probiotic-supplemented pigs had a lower eosinophil percentage than CON pigs on D57 pw (0·95 v. 1·89 (sem 0·140) %; P < 0·001) and overall (0·97 v. 1·47 (sem 0·102) %; P = 0·001).
A maternal treatment × pw treatment interaction was found for basophil count (P = 0·001) and percentage (P = 0·02) on D8 pw, with CON/CON pigs having a lower basophil count and percentage than pigs from the CON/PRO and PRO/CON groups. In addition, pigs born to CON sows had a lower basophil count than those born to PRO sows at weaning (0·07 v. 0·12 (sem 0·012) × 103 cells/µl; P = 0·05) and D8 pw (0·04 v. 0·06 (sem 0·006) × 103 cells/µl; P = 0·02). This led to offspring from CON sows having a lower basophil percentage than those from PRO sows at weaning (0·58 v. 1·16 (sem 0·108) %; P = 0·01) and D8 pw (0·37 v. 0·55 (sem 0·058) %; P = 0·03). An effect of pw treatment was also observed for basophil percentage overall, where probiotic-supplemented pigs had a lower percentage than CON pigs (1·56 v. 2·07 (sem 0·179) %; P = 0·05).
At weaning, tendencies for a maternal treatment effect were observed for erythrocyte count (7·82 v. 6·98 (sem 0·318) × 106 cells/µl; P = 0·07), Hb (15·08 v. 13·64 (sem 0·594) g/dl; P = 0·10) and haematocrit (0·50 v. 0·45 (sem 0·018) l/l; P = 0·05), with offspring from CON sows having higher levels than those from PRO sows. A tendency for a maternal treatment × pw treatment interaction was observed for mean corpuscular Hb at weaning (P = 0·10), D57 pw (P = 0·08) and overall (P = 0·07), and for mean corpuscular Hb concentration overall (P = 0·06). On D8 pw, PRO-supplemented pigs tended to have a higher mean corpuscular Hb concentration than CON pigs (28·88 v. 28·43 (sem 0·186) g/dl; P = 0·10).
Regarding platelet counts, a significant maternal effect was found on D8 pw, with the offspring from CON sows having a lower platelet count than those from PRO sows (224·25 v. 332·28 (sem 22·892) × 103 cells/µl; P < 0·01).
Intestinal morphology of piglets post-weaning
There was no maternal treatment × pw treatment interaction (P > 0·05) for any of the intestinal morphological parameters investigated (online Supplementary Table S4). In addition, there was little effect of pw treatment, except for an increase in villous height:crypt depth ratio in the jejunum (1·9 to 2·1 (sem 0·06); P = 0·03) and an increase in villous area in the ileum (36 786 to 42 443 (sem 1724·3) µm2; P = 0·03) in response to feeding the probiotic pw. For this reason, only the main effects of maternal treatment are presented in Table 8.
Effect of supplementing sow diets with Bacillus altitudinis WIT588 spores from day (D) 100 of gestation to weaning (D26 of lactation) on small intestinal morphology of piglets at D8 post-weaning
(Least square mean values with their pooled standard errors of the mean (SEM)).
CON, non-probiotic-supplemented sows; PRO, probiotic-supplemented sows; VH:CD ratio, villous height:crypt depth ratio.
* Mean values were significantly different between treatments when P ≤ 0·05; Mean values tended to be different between treatments when 0·05 ≤ P ≤ 0·10.
Pigs born to PRO sows had longer villi (P < 0·01), greater villous area (P < 0·01), deeper crypts (P = 0·04) and a tendency for greater crypt area (P = 0·06) in the duodenum than pigs born to CON sows (Fig. 1). The offspring from PRO sows also had deeper crypts (P = 0·04) and a greater crypt area (P < 0·01) in the jejunum than those from CON sows. Ileal villous height (P = 0·06) and area (P = 0·10) tended to be greater in pigs born to PRO sows than in the offspring from CON sows.
Intestinal morphology of duodenum sections taken on day 8 post-weaning from piglets born to sows receiving the Bacillus altitudinis WT588-supplemented diet (a) or a control diet (b). The black line shows the villous height measurement. Box plots show the significant effects of the maternal treatment on the crypt depth (c) and villous height (d) of the duodenum of the offspring. Significant differences between treatments are indicated as ** (P ≤ 0·01) and * (0·01 < P ≤ 0·05).
Discussion
This study assessed the effect of supplementing B. altitudinis WIT588 spores to transition and lactating sows and/or their offspring on the growth and health of sows and their offspring. While a number of probiotic supplementation studies with a similar design have been published, piglet growth has rarely been determined after the early pw period(Reference Alexopoulos, Karagiannidis and Kritas18,Reference Alexopoulos, Georgoulakis and Tzivara20,Reference Link, Reichel and Kyzeková24,Reference Taras, Vahjen and Simon27–Reference Michiels, Possemiers and Degroote29) . The novelty of this study lies in the fact that the offspring of probiotic-supplemented sows were followed from birth to slaughter. To our knowledge, this is the first study to date that conclusively demonstrates lifetime growth benefits in the offspring of probiotic-supplemented sows.
Maternal probiotic supplementation improved FCR of offspring during the first 14 d pw. Improved FCR early pw is considered a good indicator of improved intestinal health at this critical period(Reference Le Bon, Davies and Glynn40). This was corroborated in the present study when increased villous height was found at D8 pw in the small intestine of pigs born to probiotic-supplemented sows. This indicates increased absorptive capacity which may account for the increased lifetime growth in these animals. In fact, improved FCR early pw has previously been shown to correlate well with increased lifetime growth(Reference Saintilan, Brossard and Vautier41). This held true in the current study. Incremental increases in growth in offspring due to maternal probiotic supplementation were observed, with the initial increases in pig live weight at D14, D28 and D56 pw not being statistically significant. It was only in the late finishing period (D105 and 127 pw) when increased live weight in pigs in response to feeding probiotic to the sows became significant. The improvement in live weight at the end of the finishing period resulted in a 3·5 kg increase in carcass weight in offspring from probiotic-supplemented v. control sows.
Interestingly, there was no additive effect of pw supplementation of the offspring from probiotic-supplemented sows, nor was there any benefits of probiotic supplementation of weaned pigs alone. This agrees with the findings from a previous study from our group in which growth benefits in weaned pigs supplemented with this strain were only found when compared with a medicated diet containing apramycin and pharmacological levels of zinc oxide, and not when compared with the negative control(Reference Prieto, O’Sullivan and Tan26). The lack of effect in weaned pigs may be due to the fact that commencing supplementation to pigs pw might be too late to see an effect, as it is understood that there is a critical window early in life during which gut microbiota modulation is more impactful(Reference Stokes42). Probiotic supplementation of sow diets offers an effective means of early-life (prior to weaning) probiotic administration, as litters do not consume appreciable amounts of creep feed until about D14 of age and oral dosing of individual piglets prior to this is not feasible on a commercial pig unit.
In the present study, B. altitudinis WIT588 was detected as early as D13 of age in suckling piglets born to sows fed this probiotic strain, even though the probiotic had not been administered to the piglets themselves. This demonstrates probiotic transfer from sows to offspring. Although the use of Bacillus strains as probiotics in pig production is well documented, whether administered to weaned piglets(Reference Luise, Bertocchi and Motta8,Reference Alexopoulos, Georgoulakis and Tzivara9) or to gestating sows and their offspring(Reference Alexopoulos, Karagiannidis and Kritas18,Reference Alexopoulos, Georgoulakis and Tzivara20,Reference Link, Reichel and Kyzeková24,Reference Taras, Vahjen and Simon27–Reference Michiels, Possemiers and Degroote29) , few studies have reported probiotic transmission from the sow to the piglet(Reference Taras, Vahjen and Simon27,Reference Taras, Vahjen and Macha28) . Although the mechanisms by which the probiotic is vertically transmitted in the present study are not fully understood, it is most likely via the faecal-oral route(Reference Aviles-Rosa, Rakhshandeh and McGlone43). In fact, we hypothesise that Bacillus spores excreted in the sow’s faeces germinate in the farrowing house environment and, due to the relatively high gastric pH in suckling piglets(Reference Kidder and Manners44), survive gastric transit as vegetative cells in the piglets leading to early colonisation of the gut. This early colonisation may also help to explain why beneficial effects are observed in these animals and not in piglets to which the probiotic spores are administered pw, as it appears from our previous work that the spores do not germinate in the gut(Reference Prieto, O’Sullivan and Tan26). Another mechanism by which the probiotic could be vertically transmitted to the piglets is that the spores might be transferred to the piglets in dust from the sow feed or indeed via direct contact with the feed, hence bypassing faecal transplantation from the mother. However, this potential mechanism leaves little opportunity for the spores to germinate outside the pig and become metabolically active and so is not considered by the authors to be as important as faecal-oral transfer.
Similar to the lack of persistence found in weaned piglets, which no longer shed B. altitudinis WIT588 1 month after ceasing probiotic administration, this early colonisation in suckling piglets was also transient. This is evidenced by the fact that B. altitudinis WIT588 was not detected in the intestinal digesta of piglets from the PRO/CON group on D8 pw, that is 1 week after contact with the probiotic-supplemented mothers had ceased. This lack of persistence post-administration is not uncommon with probiotics(Reference Leser, Knarreborg and Worm12). In addition, this early colonisation in suckling piglets was not at as high a level or as consistent as when the probiotic was directly administered to weaned piglets. Not all of the piglets born to probiotic-supplemented sows shed B. altitudinis WIT588 at both time points prior to weaning, and some of those that shed the probiotic at D13 were no longer doing so at D26. However, the probiotic was recovered from all of the piglets at some point prior to weaning, and the differences in shedding may be due to variations in the level of probiotic to which the piglets were exposed and also to variations in gastric pH(Reference Kidder and Manners44) or coprophagic behaviour(Reference Sansom and Gleed45).
One possible mechanism by which the probiotic strain improved lifetime growth of the progeny of the sows to which it was administered is via modulation of colostrum composition. Although all of the measured colostrum and milk compositional values fell within reference ranges(Reference Hurley46), the colostrum from probiotic-fed sows had a higher protein content than that from control sows, indicating that it was of higher nutritional value(Reference Aumaitre and Seve47). In previous studies, protein, together with fat content, of milk was also increased as a result of Bacillus supplementation of sows(Reference Alexopoulos, Georgoulakis and Tzivara9,Reference Stamati, Alexopoulos and Siochu30) , although others reported only an increase in fat content(Reference Alexopoulos, Karagiannidis and Kritas18). The higher protein content of the colostrum from the probiotic-supplemented sows in the current study may have resulted from increased mobilisation of the sows’ body reserves as these sows were lighter than control sows on the weaning day and lost more weight (numerically) during the lactation period. However, probiotic-supplemented sows also had to produce more milk during lactation, as they suckled more piglets to weaning. Furthermore, we do not know the exact mechanism by which probiotic supplementation increased colostrum protein content. Another avenue that we explored was that higher concentrations of Ig in the colostrum of probiotic-supplemented sows would confer increased immune protection to offspring, thereby helping to explain the observed growth benefits, the numerical reduction in pre-weaning mortality and the improved intestinal morphology were found in piglets born to probiotic-supplemented sows. However, maternal probiotic supplementation did not have a significant effect on the concentrations of IgA or IgG in the colostrum.
Interestingly, some of the haematological parameters measured in sows indicate a possible inflammatory response after the first 2 weeks of probiotic treatment (D114 of gestation) which persisted throughout the suckling period. Basophil counts in probiotic-supplemented sows were higher than those in control sows, although all values were within reference ranges, except the basophil percentage at weaning (the upper limit is 2·0 %, and the value in probiotic-supplemented sows was 2·32 %)(Reference Weiss and Wardrop48). Probiotic-supplemented sows also had lower mean corpuscular volume and less mean corpuscular Hb than control sows from farrowing to weaning, but values were within the normal ranges, being indicative of subtle anaemia or possible inflammation. This possible immune modulation in the sow could have affected the pigs in utero (despite swine placenta being epitheliochorial), which may also help to explain the improved gut health early pw and the subsequent growth benefits. It has previously been reported that Bacillus spores can trigger immune responses in the gut(Reference Gialdroni-Grassi and Grassi49,Reference Caruso, Flamminio and Folghera50) , which may protect against external pathogens. However, specific immune assays in intestinal cells are required in order to further investigate the probiotic-mediated immunomodulation hypothesised in the current study.
It is interesting to note that some of the haematological effects found in the sows were mirrored in the offspring. For example, piglets born to probiotic-fed sows had higher basophil counts and percentages than the offspring from control sows on the day of weaning and at D8 pw. This may have been caused by an in utero effect, or it could be indicative of immune stimulation during the early stages of suckling due to early-life probiotic exposure. Nonetheless, this effect diminished after D8 pw and was not observed thereafter. Furthermore, there was no effect of pw treatment with the probiotic on basophil levels; however, piglets that were never exposed to B. altitudinis WIT588 had the lowest counts. Other significant differences of note were the effects on leucocyte populations found due to probiotic administration pw. These included elevated total leucocyte and lymphocyte counts and reduced monocyte and eosinophil levels, albeit all were within reference values(51). Interestingly, all were observed 2 months pw (D57 pw). However, it is difficult to explain these differences because at this stage, the piglets were no longer shedding B. altitudinis WIT588. The effects may however be residual. In any case, these pw treatment-related haematological effects did not translate into improved growth, highlighting the fact that maternal supplementation is the preferred route of administration to pigs for this probiotic strain.
Conclusions
The data presented in this study indicate that B. altitudinis WIT588 dietary supplementation to sows during late gestation and lactation is more beneficial than pw administration to piglets. Piglets born to sows supplemented with the probiotic displayed faecal shedding of the administered strain while suckling. This vertical transmission is rarely reported for other probiotics and demonstrates that maternal supplementation is an effective means of early-life probiotic administration. Maternal treatment improved feed efficiency in the early pw period in progeny and increased live weight at the end of the finishing period, which resulted in increased carcass weight at target slaughter age. Possible mechanisms of action are improved colostrum quality in sows, maternal immunomodulation, which was mirrored to a certain extent in the offspring, and increased small intestinal absorptive capacity in offspring early pw. However, further analyses are needed to elucidate the mechanism(s) of action, including immune assays. In summary, the novelty of this study lies in the fact that the offspring of probiotic-supplemented sows were followed from birth to slaughter. The lifetime growth benefits observed offer considerable economic advantages for commercial pig producers in search of alternatives to in-feed antibiotics and pharmacological levels of zinc oxide. Work is ongoing to develop a product containing spray/freeze-dried spores to facilitate formulation of the probiotic strain into commercial pig diets.
Acknowledgements
We thank Tomas Ryan, Florence Viard, Eoghan Gubbins-Cloney and the farm staff in the Pig Development Department at Teagasc Moorepark for assistance with the pig study. We also acknowledge Renz Ybanez, Thomas Byrne and Emily Dempsey (Waterford Institute of Technology) and David Clarke (Teagasc) for technical assistance, Prof Joseph Cassidy (University College Dublin) for assistance with interpretation of histology slides and Jim O’Donovan (Department of Agriculture Food and the Marine) for haematology analysis.
The project leading to these results was co-funded by Enterprise Ireland and the European Regional Development Fund (ERDF) under Ireland’s European Structural and Investment Funds Programme 2014–2020 (grant number CF-2015-0361). Thomas Byrne who provided technical assistance was funded by a Students into Work grant from the Society for Applied Microbiology.
G. G., P. L. and A. M. have a patent ‘An isolated Bacillus altitudinis strain and its use as a probiotic’ pending.
G. E. G. and P. G. L. conceived the study and, together with A. M. and S. R., designed the experiment. P. G. L. and G. E. G. directed the study. S. R. and P. G. L. conducted the animal experiment. G. E. G., A. M. and R. H. performed laboratory analyses together with J. P., who also interpreted the haematology data. D. C.-P., M. A. B., S. R. and P. G. L. statistically analysed the data. D. C.-P., G. E. G. and P. G. L. interpreted the data and drafted and revised the manuscript. All authors read and approved the final version of the manuscript.
Supplementary material
For supplementary material referred to in this article, please visit https://doi.org/10.1017/S0007114521001203
