Influence of particle size and size distribution of sorghum and field pea on digestibility and growth performance of pigs

Influence of particle size and size distribution of sorghum and field pea on digestibility and growth performance of pigs Giang T Nguyen Master of Agricultural Sciences A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 Queensland Alliance for Agriculture and Food Innovation Abstract Cereals have higher starch and lower protein contents than legumes/pulses Field pea, a legume/pulse, is lower in anti-nutritional factors than other legumes/pulses, and is a desirable protein supplement in cereal-based feeds Grains (cereals and legumes/pulses) are milled to reduce particle size and improve feed efficiency, and while hammer-mills are used more in feed manufacture, disc-mills have different milling mechanisms and are increasing in importance Mill differences manifest in particle size and particle size distribution, and mills can yield narrow, broad and skewed distributions The dependence of in-vitro and in-vivo digestion on particle size distributions has not been thoroughly studied, and maximising digestion and energy delivery in pigs, demand defining a grain optimum particle size range Particle size characteristics influence residence time distributions in the gastrointestinal tract (GIT) with nutritional consequences, and understanding the mixing patterns in the GIT provides useful information on this Using sorghum and field pea, this project specifically (i) investigated the influence of milling techniques on particle size characteristics, and how these affected in-vitro digestion of starch and protein; (ii) determined how particle size characteristics affected growth performance of weaner pigs; (iii) modelled residence time distributions in the GIT of grower pigs cannulated at the terminal ileum, and investigated how this modelling revealed mixing in the GIT, which in turn was related to digestion patterns in the pigs; (iv) probed how feed ingredients influenced grain digestion; and (v) examined the status of starch in ileal digesta Feed mills in Australia and Vietnam were surveyed, revealing differences in particle size characteristics of mash diets within and between grains and mills Also, diets for weaner- and growerpigs varied in particle size characteristics In-vitro starch and protein digestions of the milled sorghum and field pea significantly (p ≤ 0.05) depended on particle size (Dgw), and particle size distribution (Sgw) significantly (p ≤ 0.05) affected the grain water absorption and solubility indices Using a pH drop method for protein digestion, and glucose release for starch digestion, irrespective of the year (2012 - 2014), the rate of protein digestion in the milled grains was much faster than the rate of starch digestion, with implications for nutrient asynchrony Generally, pig feed ingredients significantly (p ≤ 0.05) affected apparent enzyme diffusion coefficients in the milled grains, and the effects on the field pea were generally different from that on the sorghum There were also differences between the effects on starch and protein in both grains i Growth performance in weaner pigs was investigated with mash diets containing milled sorghum of particle sizes 0.4 - 0.8 mm, and milled field pea of particle sizes 0.6 - 0.8 mm over a 21-day period The weaner-pigs gained weight (average daily gain) during the feeding period, and neither mill type (disc- or hammer-mill) nor grain particle size significantly (p > 0.05) affected the feed conversion ratio (feed intake:weight gain) The particle sizes obtained were inferred to be within the optimum for the grains for maximum feed efficiency In-vitro digestion parameters reasonably (p ≤ 0.2) correlated with the growth performance of the weaner pigs The sorghum and field pea were disc-milled to make mash diets fed to cannulated grower pigs, with titanium dioxide as an indigestible marker Ileal digesta samples, collected through a T-cannula from - 720 after feeding, generated apparent multi-peak residence time distributions irrespective of the treatments, and gravitational sedimentation mainly in the stomach was proposed to be responsible The distributions were modelled using a compartmental approach, with the stomach and small intestine respectively as mixing and displacement compartments The model revealed pronounced mixing in the stomach, and gastric mean residence time ranged from - 20 hr, while a range of - hr was obtained for the small intestine However, there were no particle size effects on the ileal starch digestibility (95 - 98%), and the particle size characteristics (from light microscopy) of the ileal digesta samples were essentially the same (Dgw-ileal = 0.17 ± 0.03; Sgw-ileal = 0.12 ± 0.03), and independent of the diet particle size characteristics The residence time distribution parameters correlated (r2 > 0.800, p ≤ 0.30) positively with Dgw-diet and negatively with Sgw-diet Confocal microscopy revealed starch granules in, and separated from intact cells, irrespective of the digesta collection time Using in-vitro and in-vivo approaches, the present study showed how grains, mill types and particle size characteristics influenced animal performance with insights in apparent enzyme diffusion coefficients, flow profiles in the GIT and the status of starch during passage through the GIT This information is valuable in food and feed processing on grain milling, and in controlling milled grain particle sizes The range (0.4 - 0.9 mm) of particle sizes studied for the field pea and sorghum was within the optimum for the grains, in terms of weaner pig growth performance and grower pig ileal starch digestibility ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis iii Publications during candidature Peer-reviewed paper Nguyen, G T., Gidley, M J and Sopade, P A (2015) Dependence of in-vitro starch and protein digestions on particle size of field peas (Pisum sativum L.) LWT-Food Science and Technology, 63, 541-549 Nguyen G T., Bryden, W L., Gidley M J and Sopade P A (2015) Pig feed ingredients affect enzyme diffusion coefficients Animal Production Science, 55, 1537 Nguyen G.T., Bryden, W L., Gidley M.J and Sopade P.A (2015) Variation in particle sizes of commercial pig feeds in Vietnam Animal Production Science, 55, 1565 Nguyen G T., Collins, C., Henman, D., Diffey, S., Tredrea, A M., Black, J L., Gidley M J and Sopade P A (2015) Growth performance of weaner pigs fed diets containing grains milled to different particle sizes I Sorghum Animal Production Science, 55, 1566 Nguyen G T., Collins, C., Henman, D., Diffey, S., Tredrea, A M., Black, J L., Gidley M J and Sopade P A (2015) Growth performance of weaner pigs fed diets containing grains milled to different particle sizes II Field pea Animal Production Science, 55, 1567 Conference proceedings Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) A survey of particle size and particle size variability of milled grains available for use in Australian pig feeds Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 55 Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) Design and evaluation of a manual sieving device for monitoring particle size in feed manufacture Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 56 iv Nguyen, G T., Bryden, W L, Gidley, M J., and Sopade, P A (2013) Particle size and particle size dispersion drive hydration of grains: Field peas (Pisum sativum L.) as a case study (2013) Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 120 Nguyen, G T., Bryden, W L, Gidley, M J., and Sopade, P A (2013) In-vitro starch and protein digestion in field peas (Pisum sativum L.) reveal particle size dependence Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 121 Conference abstracts Nguyen G T., Gidley M J and Sopade P A (2013) Kinetics of protein and starch digestion in field peas Poster 63rd Annual Conference of the Australian Cereal Chemistry Conference (ACCC), 25 28 August 2013, Fremantle, Perth, Australia Nguyen G T., Zhang, J, Bozec, M., Gidley, M J and Sopade, P A (2014) Can Fourier Transform Infra-Red spectroscopy contribute to the understanding of protein digestion? Abstract Summer School 2014 of the Australian Institute of Food Science Technology Food Science (AIFST), - February 2014, The University of Queensland, Brisbane, Australia v Publications included in this thesis Incorporated as Chapter Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) A survey of particle size and particle size variability of milled grains available for use in Australian pig feeds Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 55 Contributor Statement of contribution Giang Tuyet Nguyen (Candidate) Designed study (40%) Conducted experiment (40%) Analysed data (80%) Wrote first draft of paper (100%) Finalised paper (70%) Wayne Bryden Reviewed paper (5%) Mike Gidley Designed study (10%) Reviewed paper (20%) Anthony Edwards Designed study (10%) Conducted experiment (20%) Reviewed paper (10%) Sara Willis Designed study (10%) Conducted experiment (10%) Reviewed paper (10%) John Black Designed study (5%) Reviewed paper (10%) Rob Wilson Designed study (5%) Conducted experiment (10%) Reviewed paper (10%) Peter Sopade Designed study (20%) Conducted experiment (20%) Analysed data (20%) Reviewed paper (35%) vi Finalised paper (30%) Nguyen G.T., Bryden, W L., Gidley M.J and Sopade P.A (2015) Variation in particle sizes of commercial pig feeds in Vietnam Animal Production Science, 55, 1565 Contributor Statement of contribution Giang Tuyet Nguyen (Candidate) Designed study (50%) Conducted experiment (100%) Analysed data (80%) Wrote first draft of paper (100%) Finalised paper (70%) Wayne Bryden Reviewed paper (20%) Mike Gidley Designed study (20%) Reviewed paper (30%) Peter Sopade Designed study (30%) Analysed data (20%) Reviewed paper (50%) Finalised paper (30%) Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) Design and evaluation of a manual sieving device for monitoring particle size in feed manufacture Manipulating Pig Production 14th - Proceedings of the Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 56 Contributor Statement of contribution Giang Tuyet Nguyen (Candidate) Designed study (70%) Conducted experiment (40%) Analysed data (80%) Wrote first draft of paper (100%) Finalised paper (70%) Wayne Bryden Reviewed paper (5%) Mike Gidley Reviewed paper (20%) Anthony Edwards Conducted experiment (20%) vii Reviewed paper (10%) Sara Willis Conducted experiment (10%) Reviewed paper (10%) John Black Reviewed paper (10%) Rob Wilson Conducted experiment (10%) Reviewed paper (10%) Peter Sopade Designed study (30%) Conducted experiment (20%) Analysed data (20%) Reviewed paper (35%) Finalised paper (30%) Incorporated as Chapter Nguyen, G T., Gidley, M J and Sopade, P A (2015) Dependence of in-vitro starch and protein digestions on particle size of field peas (Pisum sativum L.) LWT-Food Science and Technology, 63, 541-549 Contributor Statement of contribution Giang Tuyet Nguyen (Candidate) Designed study (60%) Conducted experiment (100%) Analysed data (80%) Wrote first draft of paper (100%) Finalised paper (70%) Mike Gidley Designed study (10%) Reviewed paper (40%) Peter Sopade Designed study (30%) Analysed data (20%) Reviewed paper (60%) Finalised paper (30%) viii Incorporated as Chapter Nguyen G T., Collins, C., Henman, D., Diffey, S., Tredrea, A M., Black, J L., Gidley M J and Sopade P A (2015) Growth performance of weaner pigs fed diets containing grains milled to different particle sizes II Field pea Animal Production Science, 55, 1567 Contributor Statement of contribution Giang Tuyet Nguyen (Candidate) Designed study (30%) Conducted experiment (50%) Analysed data (40%) Wrote first draft of paper (100%) Finalised paper (70%) Cherie Collins Designed study (20%) Conducted experiment (30%) Reviewed paper (10%) Dave Henman Designed study (5%) Reviewed paper (10%) Simon Diffey Designed study (10%) Analysed data (40%) Reviewed paper (10%) Annette Tredrea Designed study (5%) John Black Designed study (5%) Reviewed paper (10%) Mike Gidley Designed study (5%) Reviewed paper (10%) Peter Sopade Designed study (20%) Conducted experiment (20%) Analysed data (20%) Reviewed paper (50%) Finalised paper (30%) ix protease, xylanase or amylase can increase voluntary feed intake in male weaners The combination of all three enzymes, however, produced no improvement in nutrient intake or growth, and it is postulated that there are interactions between some enzyme combinations, especially at high dose rates Sorghum also may be very responsive to phytase, as the grain contains the highest level of phytic acid (0.24% phytic phosphorus) out of all cereal grains There is a need to assess the individual and combinations of feed additives that will optimise nutrient intake Previous studies have shown that the in-vitro starch and protein digestion rates of milled grain particles increase with the inverse square of the particle size What is not known is how particle size influences starch and protein digestion 2.2 State the aims of the project To determine ileal and faecal digestibility of dry matter, gross energy, nitrogen and amino acids of 53 (32 + 21) diets containing different cereal grains To determine the ileal and faecal digestibility of dry matter, gross energy, nitrogen and amino acids of eight diets containing two different sorgham grains and enzyme combinations To determine the critical particle size for optimal digestibility of starch and protein for peas and sorgum 2.3 Briefly outline what will happen to the animals to be used in the amended protocols It is important to note that this section is a summary only Expanded detail of procedures on animals is required in the Project Description Seven batches of 18 grower pigs are acclimatised for two weeks to pens in the MSLE Animal House facility Surgery is undertaken, under general anaethesia, to implant an ileal cannula to facilitate the collection of samples from the small intestines The pigs are fed a series of ten different grain diets formulations over five successive weeks For the last two days of the week the pigs are put into metabolic cages to facilitate the collection of faecal and ileal samples Samples are collected to be tested for their fermentablility and starch profiles, which will be an indicator of the quality of the carbohydrate (energy) remaining undigested Feeds previously tested in other batches are included, to allow comparisons to be made between batches To determine the critical particle size for milled sorgum and peas four grades of milling (fine, medium, coarse, and very coarse) will be fed of each feed To determine the progression in the digestion samples from the ileum will be collected over a 12 hour period A marker (Titanium Oxide) will be added to the feeds to determine the rate of passage JUSTIFICATION FOR THE USE OF ANIMALS 2.4 Explain the significance and the potential benefit of the proposed project The project is designed to assist in the assessment of the nutient values of the wide variety of grains that are available for feeding pigs At present the alternative options involve laboratory tests on the grain that delay the decisions on ration selection and feed trials that are expensive and require the use of many animals in experimental units The assessment of grains using NIR Spectrometry could provide a quick and accurate assesment of the energy values In the longer term this would mean fewer animals used for feeding trials For commercial pig enterprises it would mean better selection of more suitable feeds that would produce more consistent growth and development of growing pigs and therefore better match the market requirements This would maximise the use of animals in production 2.5 Will the degree of pain or distress experienced by animals be the same as outlined in the original project Pigs generally adapt well to the confined housing used in this project Page - - Date: 26 August 2014 12:08:31 The major pain and distress would be associated with the surgical procedure i.e the insertion of the ileal cannula This is carried out under general anaesthesia The post- surgical healing and resolution is usually uneventful and with few complications Once the tissue of the surgical wound resolves around the cannula there is little discomfort evidenced There is minimal distress involved in the handling required for the collection of samples and the maintenance of the cannula The killing of the pigs will be by an overdose of barbiturate (Lethabarb), after sedation to minimise distress 2.6 Does this project duplicate work that has been carried out previously? If so, please explain why it is necessary to duplicate the work No 2.7 Have any of the animals been the subject of a previous research or teaching activity? If yes, provide AEC Register Number/s of the other project/s, describe what was done to the animals previously, and justify their use in this project No REPLACEMENT 3.1 ALTERNATIVES Have alternatives that totally or partially replace the use of animals been incorporated into this project? If no, identify potential alternatives and explain why they are unsuitable for use in this project If yes, please describe what alternatives are to be used in this project Yes - The experimental design incorporates an incomplete block design that minimised animal use The same animals will be used to test five different feed samples This will minimise the number of replicates required whilst still providing high statistical power The technique of ileal cannulation and ileal sampling is an alternative to feeding trials that would require many more animals and not provide the same accuracy REDUCTION ANIMALS REQUESTED APPROVED NUMBER OF ANIMALS Type/Species Breed/Strain Pigs Commercial grower 25-50kg Sex Age M 12 weeks AMENDED NUMBER OF ANIMALS REQUESTED Type/Species Breed/Strain Sex Pigs Commercial grower 25-50kg M Age 12 weeks Total Number 127 Total Number 145 Source Other - Commercial Pig Supplier, Aussie Pride Pork, Shepparton Source Other - Commercial Pig Supplier, Aussie Pride Pork, Shepparton NOTE: refer to section 6.2 for details of genetically modified and/or cloned animals JUSTIFICATION FOR NUMBER OF ANIMALS REQUESTED Page - - Date: 26 August 2014 12:08:31 5.1 Provide an explanation for the number of animals being requested This explanation should be based on statistical analysis and/or other considerations in the experimental design (e.g., multiple time points, quantity of tissue required) Where appropriate, present the numbers in table form The proposed design (incomplete block design) requires seven week-long periods for each of five batches of pigs Each batch comprises 14 pigs, although only 10 of these will be included in the feeding trials (total pigs= batches x 14 pigs = 70 pigs) In total this provides 350 (= week-long periods x batches x 10 experimental animals) individual test periods There are up to 70 individual test feeds and each is replicated five times (70 x = 350) This means that each batch of 14 pigs will be brought in and ten of those will be cannulated and used for the experiment The others will be available if a cannula fails Ten "experimental" animals are required to produce an experimental statistical power greater than 80% and four "spares" are estimated to replace animals that cannot be used e.g due to cannula breakage during the course of an experiment This happens occasionally because they are made of flexible nylon and some may become damaged due to movement or rubbing If a spare is not available, the entire batch-experiment would have to be repeated Experience from previous experiments indicates that although the incidence of cannula failure is low, four spare animals are sufficient to maximise the likelihood of a successful completion of the conduct of an experiment of this size Assuming the ten continue through the process, they will be sequentially tested on different feeds over seven week-long feeding and collecting periods (7 x 10 = 70 week-long periods) As there are 70 different feeds to test, this means that each batch of 10 pigs will have a collection period for each There will be four more batches that will replicate the testing of the 70 feeds This means that each feed is tested by five different animals The design is called an incomplete block design because each pig in a batch is not feed that same feeds as the others in the batch Batches and comprised of 18 pigs (15 to be used for collections and the other three for back-up) This format means that 10 feeds can be fed six or eight and the number of week-long runs per batch is (5 x 15 = 75 individual feedings in the batch) This means that feeds can be tested and more efficiently and accurately The batch of 15 pigs collected means that 10 test feeds are fed to seven or eight different pigs (accumulating to 75 feedings - some runs have more than one pig fed some of the feeds) This allows high power to compare the differences between feeds, but also effects due to pigs, week of collection and batch The requested extra three batches for this amendment will follow the format of the batches of 18 pigs as outlined in the paragraph above This could mean up to 30 feeds 10 feeds x batches) could be fed, but to improve the repeatability of the testing, of the 10 feeds in each batch will be feeds previously tested (7 x = 21 new feeds tested) For the particle-size experiment a batch of 18 pigs is requested As previously stated three pigs are used as "spares" to replace pigs that may develop broken cannulae Five pigs will be used for continuation of the grain digestion program (allowing the inclusion of three further grains to be tested) For the particle-size experiment eight feeds (four milling-grades of the two feeds, sorgum and peas) will be tested in five replicates (8 x = 40) to ten pigs over four periods (10 x = 40) This has been calculated to produce a power of greater than 80% (P,0.05) 5.2 To reduce animal use, would the animals or their tissues be suitable for use in another project at the end of your experiment/s? Identify the suitable project, if known Yes - After the five weeks of collection one batch of these pigs would be available for use by the project 1413113.1 Oral enzymes to treat carbohydrate intolerance - proof of concept in a ileal cannulated pig model REFINEMENT 6.1 USE OF ANIMALS Justify your choice of animal (species/strain/sex/age) Page - - Date: 26 August 2014 12:08:31 The data obtained from these experiments will be unique to commercial strains of growing pigs These are the target animals for the results that will be obtained 6.2 Genetic Modification or Cloning of Animals This project does not involve the use or production of genetically modified animals This project does not involve the use or production of cloned animals 6.3 Transport 6.3.1 Will animals need to be transported from the source location/s to the location where they will be held for this project? Yes - The pigs will be purchased from Aussie Pride Pork, a commercial pigger near Shepparton, Victoria and will be transported to Parkville Campus Animal House (total distance 217 kms or hours 40 mins) using a covered and well ventilated transport vehicle (a truck with a specifically designed crate for transporting animals) Transport will not occur on days when the expected ambient temperature is not between 0oC and 30oC 6.3.2 Where will procedures be performed? If animals need to be transported from where they are housed to where the procedures are carried out, provide details of transport and acclimatisation procedures Cannulation of these animals will be conducted in the purpose-built surgery in the MSLE Animal House Facility All other procedures will occur in the specifically designed pig pens in the facility 6.4 Location & Housing 6.4.1 Where will animals be housed? Type/Species Pigs 6.4.2 Breed/Strain Commercial grower 25-50kg Housing Land & Environment Animal Facility - Parkville Will any animals be housed outdoors? If, contrary to the needs of the species, no shelter is provided, justify the lack of shelter No 6.4.3 What type of housing will be used? Describe any special housing requirements Pigs will be housed in a controlled environment with constant temperature of 21 oC and relative humidity at 50% For the bulk of the experiment the animals are housed in individual pens with solid sides to prevent cannula damage and to allow free movement of the pigs The pens are raised off the ground level with flooring (see attached photo) of hard plastic mesh with adequate space for faeces and urine to pass through The pens will provide 1.2 m2 per pig This is above the industry minimum space requirement of 0.44 m2 for 41-60kg growers Pigs will be fed twice daily and have constant water supply via a nipple drinker Every week, the pigs are transferred to metabolism cages (see attached photo) for days at a time for collection of digesta before being returned to the individual pens The metabolism cages are 560mm wide and metre long Page - - Date: 26 August 2014 12:08:31 6.4.4 Will any animals be housed individually? If yes, explain why, for how long and how the impact of social isolation will be minimised Yes - Individual pens are necessary to prevent other pigs tearing at the ileal cannulas and to ensure individual diets are consumed by the designated pigs Direct contact with neighbouring pigs is possible through parts of the pen Pigs will be provided with novel toys to play with while in pens Chains will be provided in the meatabolic cages for play PROJECT DESCRIPTION Pre-surgery For two weeks after arrival in the facility the pigs (from 25 kg liveweight) will be housed individually and fed a standard grower diet ad libitum Water will be supplied ad libitum via nipple drinkers Individual pens will be washed twice weekly They will be monitored twice daily For the week pre-surgery and the week after, Ranitidine Hydrochloride 1000mg/g (UlcerguardTM) will be fed prophylatically to help prevent gastric ulceration It will be added to the feed at a rate of 6mg per kg bodyweight Surgery The pigs will be fasted for 24 hours surgery, and water will be withheld for to hours before The pigs will be fitted with simple T-piece cannulas about 150 mm anterior to the ileo-caecal valve as described by van Barneveld (1993) with the addition that skin barriers (Stomahesive® System with 70mm flange; Bristol-Myers Squibb, Princeton, NJ, 08543-4000 USA), developed for use around stoma in human ileostomy patients, will be incorporated between the flange of the cannula and the skin to promote healing of the wound and to prevent any leakage around the cannula The basic surgical procedure involves the following: The pigs will be fasted for 24 hours prior to surgery, and water will be withheld for to hours before Pigs are pre-medicated with a single approximately mL intramuscular injection in to the rump consisting of Ketamine hydrochloride and Xylazine via a 19 Gauge 4cm long needle When sedated, the right flank is shaved and the pig is washed The pig is transferred to the surgery table and an IV catheter (18G 1¼ inch TERUMO, cat no SR+OX1832C) is inserted into an ear vein Intravenous 5% sodium thiamylal is administered via the catheter to induce deep anaesthesia The pig is then intubated with a 7mm endotracheal tube, attached to a gaseous anaesthetic machine and placed in lateral recumbency An intravenous drip containing 10% Aminolite in saline is introduced via the ear vein catheter at the rate of 10mL/kg/hour Anaesthesia is maintained with 2-4% isofluorane The flank is surgically prepared by scrubbing with povidone iodine, sprayed with alcohol and sprayed with iodine The area is covered with a sterile surgical drape and autoclaved instruments are prepared The surgeons are gowned, hands scrubbed and sterile latex gloves are worn A 8-10cm posterior-anterior incision is made in line with the ilial crest of the hip and the tip of the last rib Muscles are dissected by blunt dissection The terminal ileum is located and externalised and irrigated with sterile saline Approximately 10 cm anterior to the ileao-caecal junction a three cm incision is made in the intestine and the cannula inserted The cannula is a nylon impermiable tube that links the internal ileal cavity to the exterior A flange holds the ileal surface firmly against the abdominal wall It is secured via a double purse string suture using Perma hand silk 3-0 braided with a tapercut needle The site of externalisation of the cannula is irrigated with Triplopen and the cannula returned to the peritoneal cavity A second incision is made approximately 10 cm anterio-ventrally from the first using a surgical cork borer and blunt dissection The cannula neck is externalised through the second incision and secured with a flange The peritoneal cavity is irrigated with Triplopen in saline Muscle layers below the original incision are closed using interrupted mattress sutures using coated Vicryl Rapide 2-0 with a tapercut needle Isofluorane levels are scaled back to zero and oxygen administration maintained The skin layer is secured using Monosyn 3-0 with straight cutting needle The pig is administered doses of long-acting antibiotic, analgesic, and vitamin K The incisions are sprayed with Chloromide The pig is returned to a recovery pen and observed until a swallowing reflex is evident The endotracheal tube is then removed and observation continued until the pig is able to stand in the pen Electrolytes are provided in the drinking water until food is made available 24 hours after surgery Post-surgical pain is Page - - Date: 26 August 2014 12:08:31 minimised by the administration of Finadyne (approximately mL via a 19 Gauge needle) and Buprenorphine approximately 2mL initially given following sedation and then every hours for days Post-surgery Following surgery, pigs will be housed individually in solid-sided pens (1.5 x 2m) and fed a commercial grower diet (0.7 g available lysine MJ-1 DE; 14.0 MJ DE kg-1) for a recovery period of seven days Immediately post-surgery, pigs will be adequately monitored to ensure full recovery from the administered anaesthetics Water will be provided ad libitum via nipple drinkers The area surrounding the cannulas will be washed with Iovone daily They respond well to daily care and maintenance of their cannulas and can be easily touched and worked with After the surgery rectal temperatures, pulse rates and respiratory rates and checking the surgical site for wound healing and cannula integrity is done twice a day until the termination of the experiment Feeding The pigs will be allocated to each experimental diet based on an incomplete block design The experimental diets are formulated to incorporate grain types and varieties that have previously been used for commercial pig diets Daily feeding rates will be adjusted to three times maintenance (3 x (0.5 MJ DE kg-1 body weight0.75)/diet DE) Daily rations will be halved and fed at 12 h intervals and water was provided ad libitum via nipple drinkers Collections Following the day recovery period, experimental diets will be introduced Diets will be fed for days, with a day period of diet adaption, with collections of ileal digesta made on days and Ileal digesta will be collected and immediately frozen to prevent further digestion of the sample To facilitate collections, pigs will be transferred into metabolism cages using a transfer cage and hydraulic lift Pigs enter and leave this transfer cage after minimal training Following collection of digesta, diets will be re-allocated and the procedure repeated until all pigs have received the five allocated diets Following each collection samples will be bulked, mixed, subsampled, and freeze-dried prior to chemical analysis While the pigs are housed in the metabolism cages for continuous collection of digesta, random subsamples of faeces voided during this two day period will be collected, bulked and stored at -20° for determination of DE At the end of each collection period, samples of digesta and faeces will be thawed, subsampled, freeze-dried and ground prior to chemical analyses Administered substances Parnell Ketamine Injection (Ketamine hydrochloride 100 mg/ml): 10-15 mg/kg intramuscularly Xylaze Injection (Xylazine base 20 mg/ml): 0.5-1 mg/kg intramuscularly Pentothal Injection (Sodium thiamylal mg/vial): 5% solution, 0.25 ml/kg Isofluorane: 2-4% vapour inhalation Triplopen (Fortified benethamine penicillin for injection, 000 000 units): 1.5-2 ml Terramycin L/A or similar (Oxytetracycline 200 mg/ml): ml/10 kg Chloromide (Chloroxylenol 3g, O-phenylphenol 1g, Cetrimide 10g, gentian violet 600 mg, N-octylbicycloheptene dicarboximide 6g, di-N-propylisocinchomeronate g, pyrethrins 375 mg): Spray-on Iovone (Polyvinylpyrrolidone iodine 10% w/v) Finadyne solution (Flunixin meglumine 50 mg/ml): ml/45 kg Vitamin K injection: 0.2 ml/45 kg Amino-lite injection (Amino acids, dextrose, electrolytes and vitamins): ml/kg Buprenorphine (33 micrograms/mL) 0.02 mg/kg intramuscularly The Particle-size Experiment will use the identical procedures for transport, acclimatisation, surgery, and recovery During the collection stage eight feeds will be fed (four grades of milling: fine, medium, coarse, and very coarse of two Page - - Date: 26 August 2014 12:08:31 diets: sorgum and field pea based) will be fed with five replications (8 x = 40) to ten pigs over four nine day periods (10 x = 10) On the seventh day of each feeding the pigs will be transferred to metabolic cages On the nineth day the pigs will be feed at 7.30am with the addition of a marker (Titanium Oxide) The collections from the ileum then occur at the times: 8:00, 8:30, 9:00, 9:30, 10:00, 10:30, 11:00, 12:00, 13:00, 14:00, 15:00, 16:00, 18:00, 20:00, 20:30 during the collection day Normally collections occur over an eight hour period We foresee no issues with extending the time of collection PERSONNEL PROJECT SUPERVISOR Name Cakebread, Mr Peter Qualifications Bachelors Degree, University of Melbourne Department Agriculture And Food Systems Species Pigs Pigs Pigs Technique /Procedure Times Performed Training Required Assessing health and welfare needs Ileal cannula surgery more than 20 No Killing with Lethabarb injection more than 20 No Yes Training Provided by Expertise of Trainer Dr Robert van Barneveld He has performed cannulation surgery for over 18 years on more than 700 animals and has perfected the technique to a point that post-operative loss is less than 0.5% OTHER INVESTIGATOR Name Bland, Dr Ian Qualifications PhD, University of Aberdeen, Masters (Coursework & Research), University of Aberdeen, Bachelors Degree (Honours), University of Leeds, Post Graduate Certificate, The Open University Department Agriculture And Food Systems Species Technique /Procedure Times Performed Training Required Pigs Sample collection No Pigs Monitoring health and welfare more than 20 more than 20 Name Qualifications Department Species Pigs Name Qualifications Department Species Pigs Training Provided by Expertise of Trainer No Van Barneveld, Robert B.Agr.Sci (Hons), PhD Dr van Barneveld is a specialist nutritionist with specific expertise in feed evaluation He has been undertaking research with cannulated pigs since 1990, having performed cannulation surgery on more than 700 animals and has perfected the technique to a point that post-operative loss is less than 0.5% BECAN Consulting Group Pty Ltd, Springwood Technique /Procedure Times Performed Training Required Cannulation surgery and animal care more than 20 No Training Provided by Expertise of Trainer Dunshea, Prof Frank Bachelors Degree (Honours), La Trobe University, Doctorate (Research), La Trobe University Agriculture And Food Systems Technique /Procedure Times Performed Training Required Assessing growth and more than No Page - 10 - Training Provided by Expertise of Trainer Date: 26 August 2014 12:08:31 Pigs Name Qualifications Department Species Pigs Pigs Pigs Name Qualifications Department Species Pigs Name Qualifications Department Species development of growing pigs Ration formulation 20 more than 20 No Bittner, Mr Evan Masters (Coursework & Research), University of Melbourne, Bachelors Degree (Honours), University of Melbourne Agriculture And Food Systems Technique /Procedure Times Performed Training Required Training Provided by Expertise of Trainer Monitoring health and feeding Administering anaesthetic induction agents Collecting ileal samples more than 20 less than No Yes Peter Cakebread Practicing vet for over 25 years to 20 Yes Maree Cox Research Assistant with over 20 years experience with pig procedures Training Provided by Expertise of Trainer Eason, Paul Department of Primary Industries Technique /Procedure Times Performed Training Required Administering anaesthetic induction agent more than 20 No Tuyet Giang, Ms Nguyen Centre for Nutrition and Food Sciences, The University of Queensland Technique /Procedure Times Performed Training Required Training Provided by Expertise of Trainer Pigs Collecting illeal samples to 20 Yes Maree Cox Pigs Monitoring health and welfare of pigs less than Yes Maree Cox Over 20 years experience with experimental procedures in pigs Collecting ileal samples for past three years Over 20 years experience with experimental procedures in pigs Collecting ileal samples for past three years ANIMAL FACILITY MANAGER Name Cox, Ms Maree Qualifications Other (Qualification), Victoria University of Technology Department Agriculture And Food Systems Species Pigs Pigs Pigs Pigs Technique /Procedure Times Performed Training Required Management and husbandry procedures Collecting samples more than 20 more than 20 more than 20 more than 20 No Killing with Lethabarb injection Monitoring health and welfare Page - 11 - Training Provided by Expertise of Trainer No No No Date: 26 August 2014 12:08:31 OTHER Name Qualifications Department Species Pigs 9.1 Chauhan, Mr Surinder Singh Melbourne School Of Land And Environment Technique /Procedure Times Performed Training Required Monitoring health and feeding more than 20 No Training Provided by Expertise of Trainer MONITORING Day-to-day monitoring during the project Who will monitor the animals on weekdays? Mr Evan Bittner, Ms Maree Cox Who will monitor the animals after hours (incl Weekends and holidays)? Mr Evan Bittner, Mr Peter Cakebread, Mr Surinder Singh Chauhan, Ms Maree Cox 9.2 Day-to-day monitoring during the project: What specific signs will be monitored and how frequently? Attach a copy of the monitoring checklist you will use to record these observations In the pens the pigs are monitored twice daily for food and water intake, external and behavioural signs of health Animals in the metabolism cages are monitored four times a day (see attached Monitoring Sheet) 9.3 Monitoring during and after procedures/interventions: What specific signs will be monitored and how frequently? Attach a copy of the monitoring checklist you will use to record these observations Experimental animals are monitored constantly during surgery for depth of anaesthesia (heart rate response to stimuli, blink reflex and toe pinch reflexes) and vital signs (using a heart rate monitor and checking respiratory rate, gum colour and capillary refill reflex) They are monitored constantly immediately post-surgery until they have attained sternal recumbency (sitting up) for heart rate, respiratory rate, rectal temperature, gum colour and capillary refill reflex After an initial recovery period, they are monitored hourly by an animal attendant until such time that they have demonstrated a full recovery from the administered anaesthetic and are walking freely around the pen After the surgery rectal temperatures, pulse rates and respiratory rates and checking the surgical site for wound healing and cannula integrity is done twice a day until the termination of the experiment 9.4 What clinical, behavioural or other signs will be used to indicate that intervention is needed to alleviate an animal's pain or suffering? What action will be taken if these indicators are reached? During the time in the pens or the metabolism cages the monitoring signs listed above are recorded If outside normal signs or values Dr Peter Cakebread will be consulted and appropriate veterinary action will be taken In the post-surgical period specific signs or events requiring intervention are: 1.When a cannula breaks or is dislodged In this instance the pig is in no pain (or pain lasts for only a short period) and it could continue to live quite happily However, as the pig cannot be sold, and due to the fact that digesta will continue to leak from the cannula site until it heals over, the pig is euthanased Criteria: Broken cannula 2.Intestinal torsion This is rare if post-operative care is adequate but is possible with any kind of gut surgery The pig will not respond to medication for potential infection, will not eat, will not stand comfortably, there is no evidence of digesta flow through the cannula, and the extremities are cold and blue In this rare instance, the pig Page - 12 - Date: 26 August 2014 12:08:31 must be killed immediately as it will be in pain 3.Experimental completion All animals are killed at the facility In all cases, the decision to kill an animal is made by Dr Peter Cakebread 9.5 Who is responsible for the management of emergencies? Mr Peter Cakebread, Ms Maree Cox 10 10.1 FATE OF ANIMALS What will be the maximum period of time that an individual animal or group of animals will be used in this project? weeks acclimatisation + week post-surgery + x week test-feeds and collections = weeks 10.2 What will happen to the animals at the completion of the project? All pigs will be killed Pigs could be restored at the completion of the experiment, but this would require an additional surgical period As they are production animals with human consumption as an end-point, they are euthansed at the completion of the experiment rather than subjected to an additional surgical event 10.3 If the animals are to be killed, how will this be done and by whom? Include information about agents, dose rates, method and route of administration and experience of personnel The pigs will be killed by Lethabarb injection This will be performed by either Dr Peter Cakebread or Ms Maree Cox They have both had considerable experience in the technique The pigs are pre-medicated with a single approximately 16 mL intramuscular injection, divided in two, in to the rump muscle on both sides, consisting of Ketamine hydrochloride (10mg/kg) and Xylazine (1mg/kg) IM via a 19G 4cm long needle When heavily sedated, an IV catheter (18G 1.25 inch Terumo) is inserted into an ear vein Approximately 30 mL intravenous pentobarbital (Lethabarb), 1mL or 325mg/2kg body weight, is administered via the catheter to cause death They will be checked in the subsequent minutes to ensure there is a cessation of reflexes, heart beat and breathing 10.4 What will be the method of disposal of dead animals? They are disposed of via the animal tissue disposal system (incineration) provided by the Faculty of Veterinary Science 11 ADDITIONAL DETAILS 11.1 Risk Management 11.1.1 Does the research involve procedures or agents that might pose a health risk to other animals and/or personnel? If Yes, please explain the risk and describe what precautions will be taken Yes - Handling pigs of this age is relatively free of risks (e.g from bites), but good husbandry procedures minimise these All personel will be experienced and well-trained in safe handling procedures 11.1.2 Is the acquisition, holding, or use of the animals subject to any permit, law or regulation of the State or Commonwealth (e.g., OGTR, protected native or imported)? If yes, please specify permit numbers Page - 13 - Date: 26 August 2014 12:08:31 No 11.2 Glossary of Scientific Terms Scientific Term NIR 11.3 Lay Description Near Infra-Red Spectroscopy: a laboratory technique for assessing energy content of materials Attachments The following attachments should accompany this application: 11.4 Category Description Miscellaneous Monitoring checklist proforma Photos of facilities Daily Monitoring Sheet Attached Via Themis Yes Yes Hard Copy Only No No Funding and Contracts 11.4.1 Identify the principal source of funding for this project (internal/external/commercial or private) External funding agency 11.4.2 Is this project Commercial in Confidence? No 11.4.3 Is this project covered by a research contract? Under Negotation 12 GENETIC MODIFICATION OR CLONING OF ANIMALS None Specified 13 AUTHORISATIONS The Research Office has verified that the following nominated personnel have signed off on this project application online through THEMIS on the given date/s: Project Supervisor Investigator Investigator Investigator Animal Facility Manager Cakebread, Mr Peter Dunshea, Prof Frank Bland, Dr Ian Bittner, Mr Evan Cox, Ms Maree 02 June 2014 02 June 2014 02 June 2014 02 June 2014 02 June 2014 The Project Supervisor has acknowledged that all trainers listed on this application have the relevant expertise and that they have accepted responsibility to train their nominated researcher/s to be competent in the necessary procedures Page - 14 - Date: 26 August 2014 12:08:31 Appendix 7.4 The calibration curve of titanium used for marker analysis 1000 Absorbance 800 600 400 y = 90.8x + 4.3 r² = 0.991 200 0 TiO2 content (mg) 286 10 Appendix 7.5 Samples of milled field pea and sorghum grains used in the cannulation experiment Fine field pea Medium field pea Coarse field pea Very coarse field pea Fine sorghum Medium sorghum Coarse sorghum Very coarse sorghum 287 Appendix 7.6 Relationship between residence time parameters and particle size characteristics of the milled field pea grain 288 Appendix 7.7 Relationship between residence time parameters and particle size characteristics of the milled sorghum grain 289 ... field pea Physical properties and chemical composition of the field pea Particle size and particle size distribution of the milled field pea Hydration properties of the milled field pea Regression... protein digestion, and the square of the particle size of the field pea and sorghum samples The relationship between the rates of digestion of starch and protein of the field pea and sorghum samples... Summary of particle size effects on digestibility and growth performance of pigs as well as feed conversion ratio Effect of particle size uniformity on nutrient digestibility and growth performances