In-feed Resin Acids: Bioactive Compounds From Coniferous Trees Supporting Gut Health in Farm Animals

Pos te r s and A b s tr ac t s

PROGRES ® – A RESIN ACID BASED FEED INGREDIENT

Progres® is a patented, natural resin acids product, which improves gut integrity and performance of animals. It’s refined from a side-stream of cellulose industry. Resin acids originate from rosin – a substance, which protects coniferous trees. Progres® was developed in Finland, where rosin has been used throughout the centuries for wound healing and is known for its anti-inflammatory properties.

AB Vista acquired Progres® in 2023.

In collaboration with top of the art research institutions around the world, various performance benefits and the absolutely unique mode of action of in-feed resin acids have been profoundly researched. We believe that the well documented performance benefits of resin acids for farm animals result from its impact on the intestinal epithelium. This is a crucially important tissue for high-performing farm animals. Even subclinical inflammation impairs the epithelial barrier function and reduces the capacity for nutrient absorption.

Intestinal epithelium is composed by a layer of cells, which are attached to the collagen-rich basal membrane. Inflammation induces the expression of specific matrix metalloproteinase (MMP) enzymes, which degrade collagen and other structural molecules of the extracellular matrix. This process damages epithelial integrity and leads to the “leaky gut syndrome”.

Research has demonstrated that diet-derived resin acids inhibit the expression and collagenolytic activity of inflammation-associated MMPs. Resin acids also reduce the number of inflammatory T-lymphocytes in the small-intestinal tissue. By these mechanisms, resin acids reduce the impact of inflammation to the epithelial integrity. Resin acids also beneficially modulate intestinal microbiota, favoring butyrate producers and lactobacilli, and reducing the growth of Gram-positive pathogens.

Resin acids have been studied in different forms, including Progres® (TOFA), which is a plant derived oil containing 8–10% of resin acids. Selected results of this long-lasting research have been presented in numerous international symposiums and also published as scientific articles.

This collection includes posters with the related abstracts and abstracts submitted for oral presentations (sections by species). In the Appendixes, you will find an annotated list of scientific articles and a list of publications dealing with resin acids/Progres® in the alphabetic order.

Learn more at www.abvista.com

Broiler chicken posters

Kettunen, H., Simao, F., Vienola, K., Vartiainen, S., Apajalahti, J. and Vuorenmaa, J. (2022) Effects of in-feed resin acid concentrate on growth performance and ileal gene expression of broiler chickens. Abstract id-87 at: 7th International Conference on Poultry Intestinal Health (ICPIH2022), Cartagena De Indias, Colombia, 30 March – 1 April, 2022.

Kettunen, H., Vuorenmaa, J., Valkonen E. and Alleno C. (2022) Dietary tall oil fatty acids improved egg shell quality and reduced the proportion of dirty eggs in laying hens with or without salt stress challenge. Abstract id-80 at: 7th International Conference on Poultry Intestinal Health (ICPIH2022), Cartagena De Indias, Colombia, 30 March – 1 April, 2022.

Kettunen, H., Vuorenmaa, J., Vienola, K., Valkonen, E., Apajalahti, J. and Vartiainen, S. (2019) Resin acid composition reduces small-intestinal expression of collagen-degrading matrix metalloproteinases in broilers. At: 6th International Conference on Poultry Intestinal Health (IHSIG 2019), Rome, Italy 3–5 April 2019.

Kolpe, A., Goossens, E., Van Immerseel, F., Antonissen, G., De Gussem, M., Dedeurwaerder, A., Ducatelle, R., Vuorenmaa, J., Hasan, S., Valkonen, E. and Kettunen, H. (2019) In-feed resin acids improve small-intestinal mucosal characteristics of broiler chickens during dysbiosis challenge. At: 3rd International Symposium on Alternatives to Antibiotics (ATA), December 16–18, 2019, Bangkok, Thailand.

Valkonen, E., Apajalahti, J., Kettunen, H., Vienola, K., Vuorenmaa, J. and Raatikainen, K. (2019) In-feed resin acids do not accumulate in broiler breast muscle or adipose tissue At: 2 2nd European Symposium in Poultry Nutrition (ESPN 2019), 10–13 June, Gdańsk, Poland.

Vuorenmaa, J., Goossens, E., Kettunen, H., Aguirre, M., Valkonen, E., Ducatelle, R., Hasan, S. and Van Immerseel, F. (2019) Natural resin acid composition supports the connective tissue of small intestinal epithelium and brings consistent performance benefits to broilers. At: World’s Poultry Science Association, Bangladesh Branch, 11th International Poultry Seminar, 5–6 March 2019, Dhaka, Bangladesh.

Vuorenmaa, J., Valkonen, E., Apajalahti, J., Kettunen, H. and Vienola, K. (2019) Beneficial performance effect of tall oil fatty acids is likely based on the effects of resin acids. At: 22nd European Symposium in Poultry Nutrition (ESPN 2019), 10–13 June, Gdańsk, Poland.

Valkonen, E., Picart, C., Launay, C., Kettunen, H. and Vuorenmaa, J. (2021) Resin acid containing tall oil fatty acid product improved broiler performance. At: World Poultry Congress 2021 - Electronic poster session.

Valkonen, E., Sanson, G., Kettunen, H. and Vuorenmaa, J. (2021) Resin acid containing tall oil fatty acid reduced mortality and oocyst shedding in broilers challenged with Eimeria. At: World Poultry Congress 2021 - Electronic poster session.

Broiler chicken presentations

Kettunen, H., Vartiainen, S., Vienola, K., Apajalahti, J. and Vuorenmaa, J. (2021) Immunomodulatory and performance-enhancing effects of dietary resin acids in broiler chickens. At: 72nd annual meeting of European Federation of Animal Science (EAAP) August 30 – September 3, 2021, Davos, Switzerland

Kettunen, H., Vuorenmaa, J., Valkonen, E. and Apajalahti, J. (2014) A resin acid -based product improved performance of broiler chicks in a necrotic enteritis model using a commercialtype dietary formula. At: The XIVth European Poultry Conference (EPC) 2014, Stavanger, Norway.

Kolpe, A., Aguirre, M., Vuorenmaa, J., Valkonen, E., Kettunen, H., Callens, C., Haesebrouck, F., Ducatelle, R., Van Immerseel, F. and Goossens, E. (2019) Resin acids reduce expression of matrix metalloproteinases in vitro and in the ileal mucosa of broilers. At: 6th International Conference on Poultry Intestinal Health (IHSIG 2019), Rome, Italy 3–5 April 2019.

Valkonen, E., Kettunen H., Kivinen J. and Vuorenmaa, J. (2018) Performance enhancing effect of a natural resin acid composition in broiler chickens under a variety of challenge conditions. At: The 15th European Poultry Conference (EPC) 2018, Dubrovnik, Croatia.

Vuorenmaa, J., Valkonen, E. and Kettunen, H. (2017) Dietary resin acid composition as a performance enhancer for broiler chickens. At: 5th International Conference on Poultry Intestinal Health, 11–12 October 2017, Bangkok, Thailand.

Turkey posters

Kettunen, H., Apajalahti, J., Valkonen, E., Rinttilä, T., Grönberg, H. and Vuorenmaa, J. (2014) A novel, resin-based dietary ingredient reduces the risk of necrotic enteritis in turkeys. At: The XIVth European Poultry Conference (EPC) 2014, Stavanger, Norway.

Lipiński, K., Vuorenmaa, J., Antoszkiewicz, Z., Kaliniewicz, J., Mazur M. and Makowski, Z. (2017) The efficiency of resin acid composition in turkeys nutrition. At: The 21st European Symposium on Poultry Nutrition (ESPN), Salou, Spain.

Pig posters

Hasan, S., Junnikkala, S., Peltoniemi, O. and Oliviero, C. (2017) Dietary supplementation of tall oil fatty acid and resin acid in farrowing sows can affect the colostrum quality.

At: The 9th European Symposium of Porcine Health Management (ESPHM) 2017, Prague, Czech Republic.

Hasan, S., Kamyzek, M., Vuorenmaa, J., Valkonen, E. and Kettunen, H. (2019) Dietary resin acid supplementation improves the performance of sows and piglets.

At: 3rd International Symposium on Alternatives to Antibiotics (ATA) December 16–18, 2019, Bangkok, Thailand.

Kettunen, H., Hasan, S., Suokanto, M., Valkama, S., Daka, J., Vuorenmaa, J., and Heikkilä, T. (2022) Raising weanling piglets without medical doses of zinc: an example from Finland. Abstract in: ZeroZincSummit2022, Copenhagen, Denmark, 22–23 June, 2022.

Middelkoop, A., Tichelaar, R., Guan, X., Kettunen, H., Vuorenmaa, J., Rydal, M.P., Brøndsted, L., Gambino, M., Nielsen, J. P. and Molist, F. (2022). Effect of dietary resin acids and hydrolysed yeast in piglets challenged with F4-Enterotoxigenic Escherichia coli. Abstract in: ZeroZincSummit2022, Copenhagen, Denmark, 22–23 June, 2022.

Yun, J., Hasan, S., Saha, S., Oliveiro, C. and Peltoniemi, O. (2018) Effects of supplementing prepartum sow diets with organic acids on neonatal piglet mortality. At: 10 th European Symposium on Porcine Health Management, 9–11 May 2018 Barcelona, Spain.

Pig presentations

Guan, X., Kettunen, H., Santos, R., Vuorenmaa, J. and Molist F. (2021) Effect of dietary resin acid and zinc oxide in weaned piglets subjected to an immune challenge. At: 72nd annual meeting of European Federation of Animal Science (EAAP) August 30 –September 3, 2021, Davos, Switzerland

Hasan, S., Saha, S., Yun, J., Peltoniemi, O. and Oliviero, C. (2018) Dietary supplementation with tall oil fatty acid and resin acid increases sow colostrum IgG and piglets’ colostrum intake in free farrowing pen. At: The 10 th European Symposium of Porcine Health Management (ESPHM) 2018, Barcelona, Spain.

Hasan, S., Sani, S., Junnikkala, S., Orro, T., Peltoniemi, O. and Oliviero, C. (2018) Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum IgG, piglet colostrum intake and modulates sow gut microbiota. At: 14th International Symposium on Digestive Physiology of Pigs, 21–24 August 2018, Brisbane, Australia.

Ruminant posters

Kettunen, H., Frantzi, M., Vuorenmaa, J., Breves, G. and Eger, M. (2019) Resin acid composition increases propionate and butyrate production in a Rumen Simulation Technique (RUSITEC) model. At: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019.

Kairenius, P., Mäntysaari, P., Lidauer P., Franco M., Frantzi, M., Kettunen H. and Rinne, M. (2019) The effects of in-feed resin acid inclusion on milk production responses of dairy cows. At: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019.

Qin, N., Kairenius, P., Lidauer, P., Franco, M., Niku, M., Junnikkala, S. and Kettunen, H. (2019) The effects of in-feed resin acid composition on the colostrum composition and immunity of dairy cows. At: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019.

Qin, N., Niku, M., Junnikkala S., Vuorenmaa, J. and Kettunen, H. Biomarkers of inflammation and gut permeability in dairy cows with or without dietary resin acids Abstract no 21.29 at the European Federation of Animal Science (EAAP) virtual meeting 1–4 December 2020. Presented as a recorded oral presentation.

Aquaculture posters

Kettunen, H., Vuorenmaa, J., Jintasataporn, O. (2023) Effects of dietary tall oil fatty acids with resin acids on the performance and immunity of juvenile White shrimp (Litopenaeus vannamei) with and without an intentional Vibrio parahemolyticus challenge. At: Aquaculture Europe 2023, Vienna, Austria, 18–21 September 2023.

Kettunen, H., Vuorenmaa, J., Jintasataporn, O. (2023) Effect of dietary tall oil fatty acids (TOFA) on performance, immunological status, and pathogen (Aeromonas spp.) load of juvenile Asian seabass (Lates calcarifer). At: Aquaculture Europe 2023, Vienna, Austria, 18–21 September 2023.

In vitro posters

Roy, K., Lyhs, U. and Pedersen, K. (2015) Inhibition studies of natural resin acids to Clostridium perfringens and Escherichia coli O149. At: The 1st International Conference on Necrotic Enteritis in Poultry 2015, Copenhagen, Denmark. Abstract only, poster not available.

Abstract

Effects of in-feed resin acid concentrate on growth performance and ileal gene expression of broiler chickens

H. Kettunen1, F. Simao2 , K. Vienola 3 , S. Vartiainen3 , J. Apajalahti 3 , J. Vuorenmaa1

1Hankkija Oy, Hyvinkää, Finland

2Genevia Technologies Ltd, Tampere, Finland, 3Alimetrics Ltd, Espoo, Finland

The Problem

Amending broiler chicken diets with coniferous resin acids has in previous experiments resulted in better growth performance, improved gut microbial composition, and reduced small-intestinal collagen degradation by matrix metalloproteinases (MMPs). The results indicate that resin acids have beneficial effects on intestinal homeostasis. The present broiler chicken study was conducted to identify genes and genetic pathways of ileal mucosa which are affected by dietary resin acid concentrate (RAC).

How we investigated or researched the problem

Male Ross 308 hatchlings were randomised into 20 pens, 14 birds/pen, and two diet groups (10 pens/ group): 1) control wheat-soy -based diet, and 2) the same diet amended with 176 g/ton of RAC (40% resin acids + 60% wheat flour; Hankkija Oy, Finland). Bird performance and feed intake were measured for the starter (d1-21) and grower (d21-35) periods and over the whole study. Two birds/pen were sampled for gut mucosal tissue on d21. Total RNA was extracted from 6 control group samples and 6 RAC-group samples, and sequenced using an Illumina instrument. Performance data was analysed with independent sample’s T-test using SPSS software. The sequencing data was analysed with principal component analysis (PCA), differential expression gene (DEG analysis, and Gene set enrichment analysis (GSEA) using R software.

Results

Compared to control birds, RAC-fed birds were heavier on d21 (+8.6%) and on d35 (+5.7%), they consumed more feed for d1-21 (+6.5%) and had an improved feed conversion ratio for d1-21 (-6 points; p<0.05 for all). For all 12 samples, DEG analysis showed no differences between treatments. After removal of two highly divergent samples from the control group, identified by the PCA analysis, the DEG analysis revealed 27 genes with different expression between the treatments (p<0.05). The GSEA analysis which shows suppressed and activated biological processes, recognized 10 activated and 10 suppressed processes in RAC-group, compared to control group. One of the significantly suppressed processes was ‘Extracellular matrix organization’, which includes the MMP2 protein coding gene. In contrast, processes related to ‘Intracellular transport’ were activated. Specific pathways related to inflammation were not altered by the dietary RAC amendment.

Implications / Conclusions

The present study confirmed earlier findings on the performance-enhancing effects of RAC. The study indicated that RAC affects biological processes related to extracellular matrix, including expression of MMP2, thus supporting previous research which has indicated less MMP activity in ileal mucosa with in-feed resin acids. Due to removal of 2 outliers from a group of 6 control birds, the sequencing results should be considered tentative. More research is needed to draw strong conclusions on the effect of dietary RAC on the gene expression of small-intestinal mucosa.

Effects of in-feed resin acid concentrate on growth performance and ileal gene expression of broiler chickens

H. KETTUNEN1, F. SIMAO2, K. VIENOLA 3, S. VARTIAINEN3, J. APAJALAHTI3, J. VUORENMAA1

INTRODUCTION AND TARGET

Amending broiler chicken diets with coniferous resin acids has in previous experiments resulted in

• better growth performance,

• improved gut microbial composition,

• reduced extracellular matrix degradation in small-intestinal mucosa by matrix metalloproteinases (MMPs). Here we aimed to identify ileal mucosal genes and genetic pathways which are affected by dietary resin acid concentrate (RAC).

RESULTS

RAC improved bird performance (Table 1).

The gene sequencing results are presented in Figure 1.

DEF analysis: RAC affected the expression of 27 genes (p < 0.05)

GSEA analysis:

RAC significantly suppressed 10 biological pathways (BPs) and activated 10 BPs (p < 0.05)

SUPPRESSED

Extracellular matrix organization

Anatomical structure morphogenesis

Multicellular organism development

Developmental process

System development

Multicellular organismal process

ACTIVATED

Intracellular transport

Steroid metabolic process

Steroid biosynthetic process

Glutamate metabolic process

Endosomal transport

Fatty acid catabolic process

MMP2-gene, previously identified being suppressed by resin acids, is included in the Extracellular matrix organization BP.

CONCLUSIONS

• In line with earlier studies, RAC improved the performance of broiler chickens.

• In ileal mucosa, RAC affected biological processes related to extracellular matrix, including expression of MMP2.

• The result supports previous findings suggesting less MMP activity in ileal mucosa of RAC-fed birds.

• These gene expression results should be considered tentative, and no strong conclusions can be drawn.

MATERIALS AND METHODS

Trial outline

Male Ross 308 hatchlings were randomised into 20 pens, 14 birds/pen, and two diet groups (10 pens/group). Bird performance and feed intake were measured for the starter (d1-21) and grower (d21-35) periods and over the whole study. Two birds/pen were sampled for gut mucosal tissue on d21.

Dietary treatments

• Control: Wheat-soy –diet without amendments;

• RAC: Wheat-soy –diet + 176 g/ton of RAC (40% resin acids + 60% wheat flour; Hankkija Oy, Finland).

Analysis

• Performance data was analysed with independent sample’s T-test using SPSS software.

• Total ileal RNA was extracted from 6 birds/tr. and sequenced using Illumina instrument.

• Sequencing data was analysed with principal component analysis (PCA), differential expression gene (DEG) analysis, and Gene set enrichment analysis (GSEA) using R software.

• Two control birds were removed because the PCA analysis identified them as highly divergent, leaving N=4 for the control group.

1Hankkija Oy, Hyvinkää, Finland, 2Genevia Technologies Ltd, Tampere, Finland, 3Alimetrics Ltd, Espoo, Finland

Figure 1. The gene sequencing results

Table 1. Performance results

Abstract

Dietary tall oil fatty acids improved eggshell quality and reduced the proportion of dirty eggs in laying hens with or without salt stress challenge

H.

Kettunen1, J. Vuorenmaa1, E. Valkonen1, C. Alleno2

1Hankkija Oy, Hyvinkää, Finland

2 Zootests, Ploufragan, France

The Problem

Gastrointestinal problems may negatively affect egg yield and quality in laying hens. Too high dietary salt irritates gut mucosa and increases the proportion of dirty eggs. A salt stress challenge model would be useful in investigating the effects of feed additives on laying hens. In-feed resin acids improve gut barrier functions in chicken by reducing inflammation-associated collagen breakdown in small-intestinal mucosa. Here we studied whether dietary tall oil fatty acid (TOFA) with resin acids affects laying performance and egg quality of hens with or without salt stress challenge.

How we investigated or researched the problem

Rhode Island layers were randomized into 4 treatments with 320 hens/tr. and 20 hens/cage: T1) Normal Salt (NS) feed (Na 1.7 g/kg) without amendment; T2) NS feed with TOFA at 0.7 g/kg (Hankkija Oy, Finland; 90% free fatty acids and 9% resin acids); T3) High Salt (HS) feed (Na 2.6 g/kg) without amendment; T4) HS feed with TOFA at 0.7 g/kg. The diets were fed for 42 days starting at 47 weeks of age. The comparability of the replicates was ensured by a pre-trial follow-up for weeks 42-46. Mortality, feed consumption, egg production and dirty eggs (%) were recorded for weeks 42-46 and for weeks 47-53, and bodyweight at weeks 42 and 53. Egg size and feed consumption were evaluated once every 2 weeks. Egg quality traits (individual egg weight, shell strength and Haugh Units) were measured at weeks 44 and 53, and fecal dry matter and N content at week 49. Data was analysed by ANOVA followed by LSD tests on SAS software.

Results

The salt stress treatment or TOFA supplementation did not affect laying rate, egg weight, feed intake, FCR, body weight, or mortality. The salt stress did not affect egg shell quality or proportion of dirty eggs. The fecal N was higher for the NS feed (1.08%) than for HS feed (0.95%; p = 0.002). Fecal dry matter differed between treatments (p < 0.001), being 23.7%, 21.6%, 21.1% and 21.8% for T1-T4, respectively. Egg shell quality was improved by TOFA, with static stiffness 190.3 N/mm for TOFA vs. 184.4 N/mm for nonsupplemented (p = 0.004), and fracture force 43.9 N for TOFA vs. 42.3 N for nonsupplemented (p = 0.002), but there were no effects on Haugh Unit. Proportion of dirty eggs tended to decrease with TOFA, compared to the nonsupplemented group (1.52% vs. 1.81%, respectively; p = 0.053).

Implications / Conclusions

High salt treatment used in the present trial induced few changes, so the salt stress challenge model needs further development. Dietary TOFA improved egg shell quality and tended to reduce the proportion of dirty eggs, which suggest improved intestinal condition in TOFA-fed birds. This is the first controlled experiment to confirm these effects, which have been seen also in farm-level tests. The study suggests that TOFA may be useful in improving egg shell quality and reducing the negative effects of dysbiosis on the proportion of dirty eggs in laying hens.

Dietary tall oil fatty acids improved eggshell quality and reduced the proportion of dirty eggs in laying hens with or without salt stress challenge

H. KETTUNEN1*, J. VUORENMAA1, E. VALKONEN1, C. ALLENO2

1Hankkija Oy, Hyvinkää, Finland, 2Zootests, Ploufragan, France *hannele.kettunen@hankkija.fi

INTRODUCTION AND TARGET

• In-feed tall oil fatty acids (TOFA) is known to improve performance and gut integrity of meat poultry.

• Egg yield and quality are negatively affected by intestinal problems. High-NaCl diet, “salt stress”, could act as a controlled dysbiosis model for laying hens.

• We studied the effect of dietary TOFA on the laying performance and egg quality of hens with or without salt stress challenge.

RESULTS

• TOFA significantly improved eggshell strength parameters (Figure 1) and tended to reduce the proportion of dirty eggs (Figure 2).

• Bird performance was not affected by the treatments.

• Fecal N was the only parameter affected by the high salt treatment. It was higher for the NS feed (1.08%) than for HS feed (0.95%; p = 0.002).

• Fecal dry matter differed between treatments (p < 0.001), being 23.7%, 21.6%, 21.1% and 21.8% for T1-T4, respectively.

CONCLUSIONS

• TOFA improved eggshell quality and tended to reduce the proportion of dirty eggs.

• Results suggest improved intestinal condition in TOFA-fed birds.

• High salt treatment induced few changes. The salt stress model needs further development.

MATERIALS AND METHODS

Trial outline

• Rhode Island layers were randomized into 4 treatments with 320 hens/tr., and 20 hens/cage.

• Diets were fed for 42 days starting at 47 wks of age.

• The comparability of the replicates was ensured by a pre-trial followup for weeks 42-46.

Dietary treatments

• T1 - Normal Salt (NS) feed (Na 1.7 g/kg) without amendment;

• T2 - NS feed with TOFA at 0.7 g/kg (Progres®, Hankkija Oy, Finland; 90% free fatty acids and 9% resin acids);

• T3 - High Salt (HS) feed (Na 2.6 g/kg) without amendment;

• T4 - HS feed with TOFA at 0.7 g/kg.

Measurements and statistics

• Performance: bird body weight, feed intake, mortality, egg production, egg size, proportion of dirty eggs;

• Egg quality traits: individual egg weight, shell strength and Haugh Units;

• Fecal dry matter and N content;

• Data analysis: ANOVA followed by LSD tests, SAS software.

Figure 1. Eggshell quality: static stiffness (A) and fracture force (B)

Figure 2. Proportion of dirty eggs, % on

Abstract

Resin acid composition reduces small-intestinal expression of collagen-degrading matrix metalloproteinases in broilers

Hannele Kettunen1, Juhani Vuorenmaa1, Kirsi Vienola 2 , Eija Valkonen1, Juha Apajalahti 2 , Suvi Vartiainen2

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 Alimetrics Ltd, Koskelontie 19, FIN-02920 Espoo, Finland

The Problem

Collagen is an important structural component of the intestinal basal lamina and submucosa of vertebrates. Intestinal inflammation increases the mucosal presence of matrix metalloproteinases (MMPs) which degrade collagen and weaken the gut barrier function. Natural resin acid composition (RAC) of the coniferous trees Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) has previously been shown to improve broiler performance and reduce foot pad lesions, an inflammatory condition of broilers. Here we investigated the hypothesis that in-feed RAC would reduce the gene expression of matrix metalloproteinases in the small intestinal epithelium of broiler chickens.

How we investigated or researched the problem

Day-old Ross 308 broilers were allocated in 22 pens and fed with 0 (Control) or 265 g/ton of RAC in a commercial-type diet, without any intentional challenge factors. One chick per pen was sacrificed on day 14 and sampled for jejunal and ileal tissue. The tissue samples were immersed in RNAlater solution (Sigma), and subsequently analysed for the expression of MMP-2, MMP-7 and MMP-9 by TaqMan™ Gene Expression Assays (Thermo Scientific). The gene expression was normalised against the housekeeping genes Tata-box binding protein and RNA Polymerase II subunit B.

Results

The expression of MMPs varied depending on the intestinal segment: MMP-2 had the highest expression in jejunum, while the expression of MMP-7 and MMP-9 was high in ileum. In-feed RAC significantly decreased the expression of those MMPs that were the most abundant in the respective gut segment. For jejunum, the MMP-2 expression in the RAC-fed group was 64.2% of that of control birds, while the expression of MMP-7 and -9 was similar in both treatments. For ileum, the expression of MMP-7 and -9 were 25.1% and 53.1% of those of the control group, while no effect on MMP-2 expression was observed.

Implications / Conclusions

In conclusion, in-feed RAC inhibited the small-intestinal expression of three collagenolytic MMPs in broiler chickens. The finding offers novel possibilities for supporting the integrity of intestinal mucosa during inflammation or under other stress factors

INTRODUCTION

Resin acid composition reduces smallintestinal expression of collagendegrading matrix metalloproteinases in broilers

H. KETTUNEN1, J. VUORENMAA1, K. VIENOLA2, E. VALKONEN1, J. APAJALAHTI2, S. VARTIAINEN2

1Hankkija Ltd, FIN-05800 Hyvinkää, Finland, 2 Alimetrics Ltd, FIN-02920 Espoo, Finland

• Collagen is a key contributor to mucosal integrity

• In-feed coniferous resin acid composition (RAC) inhibited collagenolytic matrix metalloproteinases (MMPs) in broiler ileumREF

• Inflammation increases collagenolytic activity of MMPs and thus compromises mucosal integrity

• We studied the effect of in-feed resin acids on the expression of MMPs 2, 7 and 9 in the ileal and jejunal tissue of broilers

RESULTS

A. In-feed RAC significantly suppressed the expression of MMP-2 in jejunum and MMP-7 in ileum. The expression of MMP-9 was low in both sites, and only numerically reduced by in-feed resin acids.

B The expression of MMPs varied between tissues. The highest expression was for MMP-2 in jejunum and MMP-7 in ileum

MATERIALS AND METHODS

CONCLUSIONS

The results suggest that in-feed resin acids support the integrity of small-intestinal mucosa by reducing the expression of collagenolytic MMPs.

The effect may be especially beneficial during intestinal inflammation.

Three collagenolytic MMPs

• MMP-2, gelatinase A

• MMP-7, matrilysin-1

• MMP-9, gelatinase B

All of them have been linked to the degradation of extracellular matrix during intestinal inflammation.

Abstract

In-feed resin acids improve small-intestinal mucosal characteristics of broiler chickens during dysbiosis challenge

A. Kolpe1*, E. Goossens1, F. Van Immerseel1, G. Antonissen1, M. De Gussem2 , A. Dedeurwaerder2 , R. Ducatelle1, J. Vuorenmaa 3 , S. Hasan3 , E. Valkonen3 & H. Kettunen3 .

1Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.

2 Poulpharm, Prins Albertlaan 112, 8870, Izegem, Belgium

3 Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

In-feed coniferous resin acids had previously shown to reduce both duodenal inflammatory T cell infiltration and small intestinal matrix metalloproteinase (MMP) activity towards collagen type I and type IV in the ileum of non-challenged broiler chickens, indicating a protective effect of resin acids on intestinal barrier integrity by preservation of the basal membrane and the extracellular matrix. Here we fed coniferous resin acid composition (40% natural resin acids in wheat flour) to study whether resin acids affect mucosal histology and collagen-degrading activity in a diet-induced challenge model, in which increased MMP expression and collagen breakdown were previously shown. Male Ross 308 hatchlings were randomly divided into three dietary treatments: 1) non-challenged control (NCC), 2) challenged control (CC), and 3) CC+ RAC at 250 g/ton (RAC); 10 replicate pens/treatment and 28 chicks/ pen. The CC diet was produced by inclusion of 7.5% rye (replacing an equal amount of wheat) for all dietary phases (starter d1-13, grower d14-26, finisher d27-39). One bird/pen were sampled for duodenal, jejunal and ileal tissue on d26. Duodenal samples were processed by routine histology into haematoxylinstained slides and measured by light microscopy for the length of villi and the depth of crypts. Homogenized tissue samples from all intestinal areas were measured for the relative activities of collagen type I and type IV degradation by EnzChek™ kit (Thermo-Fisher Diagnostics, USA).

Bird performance, as measured by daily weight gain and feed conversion, was significantly lower for CC than NCC (p < 0.05). Feed conversion was better for RAC than CC for the starter period (p < 0.05), but otherwise performance was similar for CC and RAC treatments. Mortality was unaffected by the treatments. The relative ileal activity of collagen type I and type IV degradation was higher for CC than NCC (by +38% and +86%, respectively; p < 0.05 for both). In RAC, the activity of collagen degradation was similar to NCC for both collagen types. In duodenum and jejunum, the collagen-degrading activity was unaffected by the treatments. The challenge significantly shortened duodenal villi and reduced the depth of crypts, thus thinning the entire mucosal surface (NCC vs. CC p < 0.05 for both variables). In RAC, the length of villi and depth of crypts were similar to NCC and differed significantly from CC (RAC vs. DCC p < 0.05 for both variables). In conclusion, the diet-induced challenge increased relative collagen-degrading activity in the ileum, and caused alterations in histological parameters. In-feed resin acids prevented these diet- induced changes and maintained mucosal characteristics similar to those in birds without a dietary challenge. Thus, dietary amendment by coniferous resin acids may partly protect intestinal mucosa of broiler chickens during intestinal disturbances.

IN-FEED RESIN ACIDS IMPROVE SMALL-INTESTINAL MUCOSAL CHARACTERISTICS OF BROILER CHICKENS DURING DYSBIOSIS CHALLENGE

A.

KOLPE1*, E. GOOSSENS1, F. VAN IMMERSEEL1, G. ANTONISSEN1, M. DE GUSSEM2, A. DEDEURWAERDER2, R. DUCATELLE1, J. VUORENMAA3, S. HASAN3, E. VALKONEN3 & H. KETTUNEN3

1Department of Pathology, Bacteriology and Avian Diseases, Ghent University, 9820 Merelbeke, Belgium. 2Poulpharm, 8870, Izegem, Belgium, 3Hankkija Ltd, FIN-05801 Hyvinkää, Finland.

INTRODUCTION

In-feed coniferous resin acids had previously shown to reduce both duodenal inflammatory T cell infiltration and small intestinal matrix metalloproteinase (MMP) activity towards collagen type I and type IV in the ileum of non-challenged broiler chickensREF This indicates a protective effect of resin acids on intestinal barrier integrity by preservation of the basal membrane and the extracellular matrix.

Here we studied whether resin acids affect mucosal histology and collagen-degrading activity in dysbiosis-challenged broiler chickens.

MATERIAL AND METHODS

Male Ross 308

• 3 treatments

• 10 pens/tr.

• 28 chicks/pen

Dietary phases:

• Starter d1-13

• Grower d14-26

• Finisher d27-39

Performance for each pen and phase

• Feed intake

• Daily weight gain

• Feed conversion

• Mortality

DIETARY TREATMENTS

NCC Non-challenged control diet

CC Dysbiosis-challenged control diet containing 7.5% rye

RAC

CC + coniferous resin acids (40%) in wheat flour at 250 g/ton

Day 26: Sampling one bird/pen for tissue from duodenum, jejunum and ileum. Duodenal samples were processed by routine histology into haematoxylin-stained slides and measured by light microscopy for the length of villi and the depth of crypts. Homogenized tissue samples from all intestinal areas were measured for the relative activities of collagen type I and type IV degradation by EnzChek™ kit (Thermo-Fisher Diagnostics, USA).

PERFORMANCE RESULTS

• The challenge impaired bird performance, as measured by weight gain and FCR

• RAC improved FCR in the starter period compared to CC (p < 0.05)

• Mortality was unaffected by the dietary treatments

SUMMARY AND CONCLUSIONS

• Dysbiosis challenge increased relative collagen-degrading activity in the ileum, and altered gut histology

• Mucosal characteristics in RAC-group remained similar to those in nonchallenged birds

• Dietary resin acids may partly protect intestinal mucosa of broiler chickens during dysbiosis by preservation of the basal membrane and the extracellular matrix

RESULTS

The relative ileal activity of collagen type I and type IV degradation was higher for CC than NCC (Table 1). In RAC, the activity of collagen degradation was similar to NCC. In duodenum and jejunum, the collagen-degrading activity was unaffected by the treatments.

TABLE 1. Relative ileal activity of collagen degradation; mean ± SE, N = 10; * difference to NCC, p < 0.05

Relative breakdown (RLU) of Diet Collagen type I Collagen type IV

NCC 67.39 ± 5.07 21.36 ± 2.85

CC 93.12 ± 4.43* 39.83 ± 3.19*

RAC 68.77 ± 5.87 21.81 ± 2.71

Histology: CC but not RAC decreased the length of duodenal villi (Fig. 1). The depth of crypts was reduced by CC and RAC, compared with NCC (p <0.05; data not shown).

FIGURE 1. Effect of the diets on the length of duodenal villi (N= 10, mean ± SE); *p < 0.05

In-feed resin acids do not accumulate in broiler breast muscle or adipose tissue

E. Valkonen1, J. Apajalahti 2 , H. Kettunen1, K. Vienola 2 , J. Vuorenmaa1, K. Raatikainen2

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 Alimetrics Ltd, Koskelontie 19, FIN-02920 Espoo, Finland

Tall oil -based, natural resin acid composition (RAC) of coniferous trees has in previous trials shown positive influence on broiler performance and gut mucosal integrity, reduced the incidence of foot pad lesions, and favoured the ileal presence of lactobacilli. Here we investigated the concentration of resin acids in the gastrointestinal tract and tissues of RAC-fed broilers. Ross 308 broilers were fed with 0 (Control) or 3000 mg/kg of RAC, corresponding to 265 mg/kg of resin acids in feed, and being a 4-fold overdose compared with commercial dosing. Titanium dioxide was added to the diets as a digestibility marker. Faeces were collected on day 34. On day 35, six birds from six pens per diet were sacrificed and sampled for several tissues and intestinal contents. Samples of feeds and birds were analysed by gas chromatography for abietic (AA) and dehydroabietic (DHAA) acids.

No resin acids were detected in the control birds’ samples. Of the total resin acids introduced to the diets, about 70% was found in jejunal contents, and about 60% in ileum and faeces of RAC-fed birds. The concentration of AA in blood plasma, jejunal tissue and bile of RAC-fed birds was on average 0.31, 2.1 and 44 mg/kg, respectively. The average concentration of AA in breast meat of six birds was 0.02 mg/kg. Resin acids were not detected in adipose tissue. In every sample the concentration of DHAA was lower than that of AA. The ratio of DHAA to AA was relatively constant between the samples. In conclusion, some of the diet-derived resin acids were absorbed in the small intestine and re-introduced into the gut via bile. From the consumer safety perspective it is notable that even a 4-fold overdose of RAC resulted in negligible resin acid levels in muscle and adipose tissues.

INTRODUCTION

In-feed resin acids do not accumulate in broiler breast muscle or adipose tissue

E. VALKONEN1*, J. APAJALAHTI2, H. KETTUNEN1, K. VIENOLA2, J. VUORENMAA1, K. RAATIKAINEN2

1 Hankkija Ltd, FIN-05800 Hyvinkää, Finland, 2 Alimetrics Ltd, FIN-02920 Espoo, Finland

Tall oil -based resin acids (RAC) contains 9 % of resin acids and 90 % of free fatty acids, mainly linoleic and oleic acids. Previously, in-feed RAC has improved broiler performance1,2 and gut mucosal integrity3, reduced foot pad lesions2, and favored the ileal presence of lactobacilli1. From a consumer safety perspective it is important to know whether a feed additive is present in edible parts of the animal. We investigated the fate of resin acids of RAC-fed broiler chickens.

Abietic acid

Dehydroabietic acid

Resin acids, the protection mechanism of coniferous trees, are diterpene carboxylic acids. The two most common resin acids of Norway spruce and Scots pine are abietic acid and dehydroabietic acid.

CONCLUSIONS

Resin acids do not accumulate in breast muscle, liver or adipose tissue of broilers even when fed at four times the commercial dosage. Resin acids are partly absorbed from the intestine and re-circulated into gut lumen via bile in a conjugated format. Eventually, most of them are voided via feces.

RESULTS

• No resin acids were detected in the control birds’ samples

• Of the total resin acids introduced to the diets, about 70 % was found in jejunal contents, and about 60 % in ileum and faeces of RAC-fed birds.

• The concentration of DHAA was lower than that of AA in all samples

• The concentration of AA in bile was 44 mg/kg, suggesting that it is absorbed but voided via bile

• Resin acids did not accumulate in edible parts of broilers

MATERIALS AND METHODS

Trial outline

• 330 male Ross 308 broilers

• 11 replicate pens/treatment

• Ad.lib. feeding on days 0–35

• Wheat-soy -based diet

• Titanium dioxide as a digestibility marker

Dietary treatments

➤ Control (no amendments)

➤ RAC 3000 mg/kg, giving 265 mg/kg of resin acids

Here RAC was used at 4x the commercial dose.

Sampling, on day 35, one bird/pen

• Jejunal and ileal tissue and digesta

• Blood plasma, breast muscle, adipose tissue, liver and bile

• Two samples were pooled to get 6 replicate pools

Feces were sampled on day 34.

Samples were analysed by gas chromatography for abietic (AA) and dehydroabietic (DHAA) acids.

REFERENCES:; 1Kettunen et al. 2017, 2Vienola et al. 2018; 3Aguirre et al. 2019

Adipose tissue 2,5 2,0 1,5 1,0 0,5 0

Breast muscle Blood plasma Abietic acid, mg/kg

H HO

H O

CH3 H3C CH3 HO

H O

CH3

Abstract

Natural resin acid composition supports the connective tissue of small intestinal epithelium and brings consistent performance benefits to broilers

J. Vuorenmaa1, E. Goossens2 , H. Kettunen1, M. Aguirre Morales2 , E. Valkonen1, R. Ducatelle2 , S. Hasan1, F. Van Immerseel 2

1Hankkija Ltd, Peltokuumolantie 4, FIN-05800 Hyvinkää, Finland

2 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, University of Ghent, Salisburylaan 133, B-9820 Merelbeke, Belgium

Introduction

Coniferous trees of the northern hemisphere secrete viscous rosin on the site of mechanical injury, in order to prevent the entry of pathogenic yeasts and bacteria. Diterpene, non-volatile resin acids of rosin are known for wound healing, anti-inflammatory and antimicrobial effects in Finnish folk medicine as well as in recent biomedical literature (Jokinen and Sipponen, 2016). Environmental challenges such as parasites, pathogenic microbes or heat stress may damage the structure and function of intestinal epithelium of animals. Such effects may induce inflammatory processes in the intestinal epithelium, thus reducing the efficacy of digestion and feed conversion. Integrity of mucosal connective tissue, consisting by large of collagen fibers, is vital for the gastrointestinal functions and stress-resistance of poultry. A natural resin acid composition (RAC) of Pinus sylvestris and Picea abies has been shown to reduce the in vitro growth of pathogenic strains of Clostridium perfringens and Staphylococcus aureus (Roy et al., 2017), and to benefit the performance of non-challenged and necrotic enteritis -challenged broiler chickens (Kettunen et al., 2017 and Vienola et al., 2018). Here we studied RAC for its effects on the collagen and gelatin breakdown in broiler ileum and for its effects on broiler performance.

Material and methods

Groups of ten broiler hatchlings were fed a commercial-type diet with or without RAC at 2.0 g/kg for 21 days. They were then euthanized and collected for ileal tissue. Gelatinase activity and collagen type I and IV activities were measured from ileal protein lysates using EnzChek® Gelatinase/Collagenase Assay Kit (Thermo Fisher). Diet of broiler chickens was amended with RAC at 0.500 – 0.750 g/kg diet in thirteen institutional experiments in Europe and the USA, using various feeding and management systems, but without any intentional challenge. Five of those experiments included intentional challenges such as high stocking density, low temperature, coccidiosis vaccine, coccidiosis and/or necrotic enteritis (NE). The trial sites and challenge types are given in Figure 1.

Results and discussion

Gelatinase activity and collagen type I and IV activities in ileal protein lysates of broiler chickens were significantly lower for the RAC-amended than control diet group (p < 0.05). The result suggests that ileal collagenolytic activity was suppressed by RAC in broilers of normal health status and thus implies that natural resin acids may support the integrity of intestinal tissue of broilers. The effect of RAC on the daily weight gain and feed conversion ratio of broiler chickens in the 13 institutional trials is given in Figure 1. In 80% of the challenge studies and 69% of all studies, resin acids improved feed conversion, bird growth and/or mortality (p < 0.05). It is noteworthy that RAC did not prevent the growth of pathogens or formation of NE-induced lesions in challenge trials, excluding direct medicinal or anti-pathogenic actions.

In conclusion, a unique, direct anti-collagenolytic action of resin acids on intestinal epithelium of broiler ileum was revealed, but the causal link between this mode-of-action and the performance-enhancing effect remains to be verified.

Broiler chicken posters

INTRODUCTION

Natural resin acid composition supports the connective tissue of small intestinal epithelium and brings consistent performance benefits to broilers

J. VUORENMAA1*, E. GOOSSENS2, H. KETTUNEN1, M. AGUIRRE2, E. VALKONEN1, R. DUCATELLE2, S. HASAN1, F. VAN IMMERSEEL2

1 Hankkija Ltd, FIN-05800 Hyvinkää, Finland

2 Faculty of Veterinary Medicine, University of Ghent, B-9820 Merelbeke, Belgium

Collagen has a key role in the integrity of intestinal epithelium. Inflammation often leads to collagen degradation and thus impairs gut barrier function. We studied the effects of a natural resin acid composition (RAC) of Pinus sylvestris and Picea abies on the collagenase and gelatinase activity in broiler ileum, and on the performance of broilers in 13 institutional experiments.

RESULTS

EXPERIMENT 1.

RAC significantly decreased ileal enzymatic activity towards gelatin and collagen types I and IV; Figure 1)

EXPERIMENT 2

RAC significantly improved broiler performance in 9 out of 13 trials and in 4 out of 5 challenge trials (~80%; Figure 2)

MATERIALS AND METHODS

EXPERIMENT 1.

– RAC in diet at 0 or 2.0 g/kg for 21 days.

– Broilers sampled for ileal tissue (N=10)

– Gelatinase activity and collagenase type I and IV activities were measured from ileal protein lysates with EnzChek® Gelatinase/ Collagenase Assay Kit

EXPERIMENT 2.

– 13 institutional experiments in Europe and the USA

– RAC in diet at 0 or 0.50 – 0.75 g/kg

– Various feeding and management systems

– Including five challenge trials such as high stocking density, low temperature, coccidiosis vaccine, coccidiosis and/or necrotic enteritis.

– Trial sites and challenge types are given in Figure 2.

CONCLUSIONS

In-feed RAC reduced ileal collagen-degrading activity, which might support the mucosal integrity. Furthermore, RAC has constantly improved broiler performance especially in challenging conditions.

Trial sites and

1.-2.

FIGURE 2. Effect of resin acid composition (RAC) on broiler performance in 13 institutional trials. Upper panel: difference in weight gain (g/day); lower panel: difference in feed conversion ratio (kg/kg). Statistically significant differences (p < 0.05) between the control and RAC treatments of each trial are denoted by an asterisk (*).

challenge types:

Alimetrics Ltd, Finland; 3.-5. Devenish Nutrition Research Farm, UK; 6.-7. University of Warmia and Mazury, Poland; 8. Agrifirm Innovation Center, The Netherlands; 9. Alimetrics Ltd, Finland, necrotic enteritis (NE) challenge; 10. Schothorst Feed Research, The Netherlands, NE challenge; 11. InVivo, France, high stocking density and low temperature challenge; 12. Aviforum Research Farm, Switzerland, coccidiosis vaccine and low temperature challenge; 13. Colorado Quality Research, USA, NE challenge.

FIGURE 1. Breakdown of gelatin (A) and collagen types I and IV (B and C, respectively) by ileal enzymes of broilers with or without in-feed RAC (N=10, *p < 0.05, *p< 0.01)

Beneficial performance effect of tall oil fatty acids is likely based on the effects of resin acids

J. Vuorenmaa1, E. Valkonen1, J. Apajalahti

2

, H. Kettunen1, K. Vienola

2

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 Alimetrics Ltd, Koskelontie 19, FIN-02920 Espoo, Finland

Tall oil fatty acid -based natural resin acid composition (RAC) originates from coniferous trees and contains free fatty acids (90%) and resin acids (9%). In previous trials RAC has consistently improved animal performance. The mode of action of resin acids on broilers has been investigated, and a direct anti-inflammatory effect on gut epithelium has been suggested. We hypothesized that resin acids are the main bioactive component in RAC, and that concentrated resin acids (CRA) without free fatty acids would have the same effect on broiler performance as RAC. We conducted a 35-day performance trial with 540 male Ross 308 broilers. The birds were housed in 36 pens, 15 birds per pen. The pens were randomly allocated into three dietary treatments to give twelve pen replicates. The commercial-type, wheat-soy -based experimental diets were supplemented either with 750 g/ton of RAC or with 71 g/ton of CRA to yield an equal resin acid content (70 mg/kg feed) for both diets. A non-supplemented diet served as a control treatment (CTR).

At 35 days, both experimental resin acid treatments significantly outperformed the control treatment in body weight gain (CTR: 2467 g, RAC: 2610 g, CRA: 2601 g; P<0.05). Moreover, the mortality-corrected feed conversion ratio was improved by resin acids until 21 days of age (CTR: 1.42, RAC: 1.37, CRA: 1.39; P<0.05). The supplements did not affect feed intake or daily mortality. Thus, similar performance effects were produced by both supplements with the same in-feed resin acid content, whether the resin acids were added as RAC or as CRA. In conclusion, the experiment supported our hypothesis suggesting that resin acids are the most important performance-improving components of RAC.

Beneficial performance effect of tall oil fatty acids is likely based on

the effects of resin acids

J. VUORENMAA1*, E. VALKONEN1, J. APAJALAHTI2, H. KETTUNEN1, K. VIENOLA2

1 Hankkija Ltd, FIN-05800 Hyvinkää, Finland, 2 Alimetrics Ltd, FIN-02920 Espoo, Finland

CONCLUSIONS

Broiler performance was equally improved by RAC and CRA supplements with the same in-feed resin acid content. The result suggests that resin acids are the most important performance-improving components of RAC.

INTRODUCTION

Tall oil fatty acid -based natural resin acid composition (RAC) contains 90 % of free fatty acids and 9 % of resin acids. According to previous research, RAC improves broiler performance. We investigated whether the presence of tall-oil -derived fatty acids are needed for the performance benefits of RAC, by amending broiler diets with either RAC or pure concentrated resin acids (CRA).

RESULTS

Both RAC and CRA significantly increased body weight gain (BWG) at all measured periods (p < 0.05; Figure 1) Mortalitycorrected feed conversion ratio (FCR) was significantly improved for the first 21 days (p < 0.05; Figure 2).

The supplements did not affect feed intake or daily mortality. The effects of the two resin acid supplements was similar in all time periods.

• 35-day performance trial

• 36 pens, 15 birds per pen

• Male Ross 308 broilers

• Wheat-soy -based, pelleted, commercial-type feeds without antibiotics or coccidiostats

Dietary treatments

➤ Control (no amendments)

➤ RAC at 750 g/tn, giving 70 g/ton of resin acids

➤ CRA at 71 g/ton, giving 70 g/ton of resin acids

All diets contained an equal amount of fatty acids.

Measurements

• BWG, Feed intake and Mortality-corrected FCR for d0-14, d0-21, d0-28 and d0-35

• Daily mortality

ANTI-INFLAMMATORY EFFECTS: Collagen-degrading activity Matrix metalloproteinases

FAVORABLE MICROBIAL MODULATION

Gram + pathogens

Lactobacilli

Butyrate producers REFs4,5,6

Imroved animal performance

REFERENCES: 1Aguirre et al. 2019; 2Kolpe et al. 2019; 3Kettunen et al. 2019, 4Kettunen et al. 2015; 5Roy et al. 2018, 6 Vienola et al. 2018; 7Kettunen et al. 2017, 8 Valkonen et al. 2018, 9 Vuorenmaa et al. 2019

Figure 1. Per cent difference in BWG between control and resin acid -treated birds.

Figure 2. Mortality-corrected FCR.

Abstract

Resin acid containing tall oil fatty acid product improved broiler performance

Eija Valkonen1, Cécile Picart 2 , Claire Launay2 , Hannele Kettunen1, and Juhani Vuorenmaa1

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 ADM Animal Nutrition – Neovia, Talhouet, FR-56250 Saint Nollf, France

Tall oil fatty acid, originating from coniferous trees is comprised of free long-chain fatty acids (90%) and bio-active resin acids (8.5%). In previous trials tall oil fatty acid has consistently shown positive effects on animal performance.

An experiment was conducted with sodium salt of tall oil fatty acid (STOFA) to evaluate its effects on broiler health and performance under challenging housing conditions, and with or without in-feed coccidiostat (maduramicin). The experiment had 3×2 factorial design with three levels of STOFA; 0, 1.0, and 2.0 g/kg feed (equating 0, 0.5, and 0.1 g of tall oil fatty acid/kg feed), and two levels of coccidiostat (maduramicin); 0 and 5 mg/kg feed. A total of 7680 Ross 308 mixed sex one-day-old chicken were randomly allocated into 6 experimental treatments with 8 replicate pens in each treatment. In each pen maximal allowed animal density was maintained along the trial with extra fencing. The house temperature was reduced by 3 centigrade lower than the recommendation after day 2. The broilers were weighed at days 14, 20, 28 and 34. Feed intake and FCR were calculated for each period and for the entire trial period. The results were subjected to two-way ANOVA for mean effects and interaction effects with R software (R i386 3.6.1). Post-hoc comparisons were made if the differences were statistically significant (p<0.05).

Diet significantly affected the final body weight of the broilers. There was a significant interaction between the effect of coccidiostat and the effect of STOFA; STOFA improved birds body weight more in coccidiostat free diet group than in group with in-feed coccidiostat (p<0.05). Birds on coccidiostat-free STOFA diets reached similar average body weight as birds on coccidiostat diets, while birds without STOFA and in-feed coccidiostat had the lowest body weight. Dietary treatments had no statistically significant effects on feed conversion ratio. Based on the current trial showing significantly improved final body weight, sodium salt of tall oil fatty acid with bio-active resin acids supports broiler growth performance. The effect was more pronounced in broilers on coccidiostat-free diet.

1Hankkija

Resin acid containing tall oil fatty acid product improved broiler performance

An experiment was conducted with sodium salt of tall oil fatty acid (STOFA) to evaluate its effects on broiler health and performance under challenging housing conditions, and with or without in-feed coccidiostat (maduramicin).

Conclusions

In current trial, sodium salt of tall oil fatty acid with bioactive resin acids supported broiler growth performance, showing significantly improved final body weight. The effect was more pronounced in broilers on coccidiostatfree diet.

R Results

• Diet significantly affected the final body weight of the broilers

• There was a significant interaction between the effect of coccidiostat and the effect of STOFA; STOFA improved birds body weight more in coccidiostat free diet group than in group with infeed coccidiostat (p<0.05).

Tall oil fatty acid, originates from coniferous trees, and is comprised of free long-chain fatty acids (90%) and bio-active resin acids (8.5%). In previous trials tall oil fatty acid has consistently shown positive effects on animal performance.

• Birds on coccidiostat-free STOFA diets reached similar average body weight as birds on coccidiostat diets

• Birds without STOFA and in-feed coccidiostat had the lowest body weight

• Dietary treatments had no statistically significant effects on feed conversion ratio

Ctrl

STOFA 1 g/kg

STOFA 2 g/kg

T Trial outline

• A total of 7680 Ross 308 mixed sex one-day-old chicken

• 8 replicate pens/treatment

• Maximal allowed animal density

• Reduced house temperature; 3 centigrade lower than the recommendation after day 2

• Wheat-soy-maize -based diet without enzymes

• Ad.lib. feeding on days 0-34

• 3×2 factorial design with

• 3 levels of STOFA; none, 1.0, and 2.0 g/kg feed (equating none, 0.5, and 0.1 g of tall oil fatty acid/kg feed, respectively)

• 2 levels of coccidiostat (maduramicin); none or 5 mg/kg feed

• The broilers were weighed at days 14, 20, 28 and 34

• Feed intake and FCR were calculated for each period and for the entire trial

Eija Valkonen1, Cécile Picart2, Claire Launay2, Hannele Kettunen1, and Juhani Vuorenmaa1

Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland; 2ADM Animal Nutrition – Neovia, Talhouet, FR-56250 Saint Nollf, France

Abstract

Resin acid containing tall oil fatty acid reduced mortality and oocyst shedding in broilers challenged with Eimeria

Eija Valkonen1, Gorm Sanson2 , Hannele Kettunen1, and Juhani Vuorenmaa1

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 Felleskjøpet Fôrutvikling AS, Nedre Ila 20, NO-7018 Trondheim, Norway

Resin acids have well documented anti-inflammatory and tissue healing effects. An experiment was conducted to evaluate the effect of resin acid composition (RAC), containing bioactive resin acids and free fatty acids from coniferous trees, on broiler health and performance under an Eimeria challenge. A total of 880 Ross 308 one-day-old chicken were housed in a trial facility. The birds received one of the 2 dietary treatments during the 30 days experiment: control (CTRL), or RAC supplemented diet (1.5 g RAC/ kg of feed) (RAC). There were in total 11 pens in each treatment; 3 pens of females, 3 pens of males and 5 pens with 50% of each sex. Each pen housed 80 chicken. The pens were divided in 3 rooms, sexes and dietary treatments were balanced within the rooms. On day 16 all chicks were challenged with a 10-fold dose (0,25 ccm/chick) of Eimeria vaccine (Huveguard, Huvepharma, Belgium). Growth, feed intake, feed conversion ratio (FCR) were recorded per pen, and shedding of Eimeria oocysts (OPG) at days 7, 14, 20, 21, 22, 23 and 28 per dietary treatment. The results were subjected to ANOVA or when response variables were not normally distributed, to two-sided Χ2-test with R software.

In both treatments there were no oocysts found in excreta samples taken at day 7, 14 or 20. At d21, total oocyst counts were high (> 600 000 OPG) in both treatments, but 10% lower in treatment RAC than in CTRL. Furthermore, RAC gave better reduction of OPG from d21 to d23 compared to CTRL (–86% vs. –60%), and from d21 to d28 (–50% vs. –37%). The birds receiving RAC diet had a lower mortality in comparison to CTRL (2-sided Χ2-test, p<0.05). There were no statistically significant differences between the diets in growth or FCR.

In current trial, RAC gave protection against the Eimeria challenge when compared to the control, denoted by the lower mortality and improved reduction of OPG. RAC seems to be a useful tool in supporting gut health and performance in coccidiostat-free broiler production.

Resin acid containing tall oil fatty acid reduced mortality and oocyst shedding in broilers challenged with Eimeria

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2Felleskjøpet Fôrutvikling AS, Nedre Ila 20, NO-7018 Trondheim, Norway

An experiment was conducted to evaluate the effect of resin acid composition (RAC, Progres® Liquid, Hankkija Ltd, Finland), containing bioactive resin acids and free fatty acids from coniferous trees, on broiler health and performance under an Eimeria challenge.

C Conclusions

In current trial, RAC gave protection against Eimeria challenge denoted by the lower mortality and improved reduction of OPG. RAC seems to be a useful tool in supporting gut health and performance in coccidiostat-free broiler production.

• There were no oocysts found in excreta samples taken before the challenge (at day 7, 14) or four days post challenge at day 20

• At day 21, total oocyst counts were high (> 600 000 OPG) in both treatments, but 10% lower with RAC than with Ctrl diet

Reduction of total Eimeria oocyst count from day 21

• Birds receiving RAC diet had lower mortality in comparison to Ctrl (2-sided Χ2-test, p<0.05)

• RAC gave better reduction of OPG from day 21 to day 23 compared to Ctrl (-86% vs. -60%), and from day 21 to day 28 (50% vs. -37%)

Trial outline

• 1760 Ross 308 broilers

• 11 replicate pens/treatment

• Ad.lib. feeding on days 0-30

• Wheat-soy -based diet

• Dietary treatments

1. Control (Ctrl)

2. Resin acid composition 1500 mg/kg feed (RAC)

• Challenge:

• On day 16 chicks were challenged with a 10-fold dose (0.25 ccm/chick) of Eimeria vaccine (Huveguard, Huvepharma, Belgium)

• Shedding of Eimeria oocysts (OPG) assessed at days 7, 14, 20, 21, 22, 23 and 28 per dietary treatment.

Eija Valkonen1, Gorm Sanson2, Hannele Kettunen1, and Juhani Vuorenmaa1

Abstract

Immunomodulatory and performance-enhancing effects of dietary resin acids in broiler chickens

H. Kettunen*, S. Vartiainen†, K. Vienola†, J. Apajalahti†, J. Vuorenmaa*

* Hankkija Ltd, FI-05800 Hyvinkää, Finland

†Alimetrics Ltd, FI-02920 Espoo, Finland

In-feed coniferous resin acids have been reported to improve the performance of broiler chickens and to reduce the inflammation-associated collagen degradation in intestinal epithelium. Here we studied the effect of dietary a resin acid concentrate (RAC) on the growth performance of broiler chickens and on the expression of ten immune response -related genes in their intestine. On day 1, 280 male Ross 308 hatchlings were allocated into 20 pens, 14 chicks/pen. The two dietary treatments, both with 10 replicates, included a wheat-soy -based control diet (CON) and the same diet amended with 176 g/ton of RAC (40% resin acids + 60% wheat flour; Hankkija Ltd, Finland).

The birds were weighed on days 1, 21 and 35. Feed intake and feed conversion ratio (FCR) were determined for periods d1-21, d21-35, and d1-35. On d21, gut mucosal tissue samples were collected from 2 birds/pen. The samples were stored in RNA-preserving solution and analysed for the expression of nuclear factor (NF)-κB subunits 1 and 2, NF-κB inhibitor α, matrix metalloprotein (MMP)-2, MMP-7, MMP-9, interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α and peroxisome proliferator activated receptor-ϒ relative to two housekeeping genes: RNA polymerase II subunit B and Tatabox binding protein. Independent sample’s T-test and Pearson’s correlation analysis were used for data analysis.

Compared with CON, the body weight of RAC-fed birds was higher by 8.6% on d21 and by 5.7% on d35 (p<0.05 for both). Feed intake was increased by RAC for d1-35 (+7%, p<0.05). RAC improved FCR (-6 points, p<0.05) for d1-21, and tended to improve FCR also for d21-35 and 0-35 (p<0.1 for both). The expression of the immune modulator NF-κB subunit 2 was significantly higher in the RAC than CON group, but the other genes showed a similar expression in CON and RAC groups. Bird body weight correlated negatively with the expression of the three inflammation-associated MMP-genes. The results support earlier findings on the performance-enhancing effects of in-feed resin acids in broiler chickens, and suggest a possible immunomodulatory activity through the NF-κB subunit 2 gene.

A resin acid -based product improved performance of broiler chicks in a necrotic enteritis model using a commercial-type dietary formula

H. Kettunen1, J. Vuorenmaa 2 , E. Valkonen2 , J. Apajalahti1

1Alimetrics Ltd, Espoo, Finland

2 Hankkija Oy, Hyvinkää, Finland

In broiler chick models of necrotic enteritis (NE), the disease is often induced by a sudden change to a high-protein diet, concomitant with a coccidial and/or Clostridium perfringens (C.p.) challenge. As atypical diets may limit the applicability of the results, we developed a NE model which uses a simple wheat-soy based broiler starter formula throughout the trial. The effect of a resin acid composition (RAC; Progres®, Hankkija Oy) on bird performance was tested with the model. In previous studies, RAC has been found to inhibit the growth of C.p. in vitro. RAC was added in the diet at 0, 0.1, 0.5, 1 and 3 kg/tn. 48 male Ross 508 hatchlings in eight replicate pens were allocated to each dietary group. NE was induced by an oral inoculum of Eimeria maxima on day 11 and a fresh culture of C.p. on day 14. The C.p. strain used originates from a field case of NE.

In the first 14 days, RAC improved body weight gain dose-dependently. Compared to the control group, the birds in the diets with RAC at 0.5 and 1 kg/tn were 11% heavier on day 11 (p < 0.05). RAC at all levels improved feed conversion efficiency (p < 0.01 for 0.1, 1 and 3 kg/tn treatments). On the final day (day 17) the RAC-treated birds were numerically heavier than the control birds. RAC at 0.3% decreased mortality to NE. The results indicated that RAC may improve broiler chick performance and partially protect against NE.

Broiler chicken presentations

Abstract

Resin acids reduce expression of matrix metalloproteinases in vitro and in the ileal mucosa of broilers

Annasaheb Kolpe1, Marisol Aguirre1, Juhani Vuorenmaa 2 , Eija Valkonen2 , Hannele Kettunen2 , Chana Callens1, Freddy Haesebrouck1, Richard Ducatelle1, Filip Van Immerseel1, Evy Goossens1

1Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2 Hankkija Ltd, Peltokuumolantie 4, 05800 Hyvinkää, Finland

The Problem

The chicken gut is constantly exposed to harmful microorganisms and feed toxins which endanger the integrity of the intestinal wall. Protecting and strengthening of intestinal mucosal integrity is a key target for feed additives that aim to promote intestinal health in broilers. Rosin of coniferous trees contains resin acids, which are known for wound healing, anti-inflammatory and antimicrobial effects, all properties that might support broiler intestinal health. Recently, dietary inclusion of resin-based products has been shown to improve broiler performance. However, the mode of action is still largely unknown.

How we investigated or researched the problem

In order to understand the mode of action of resin acids, we studied the effect of resin acid supplementation of broiler feed on a wide range of broiler intestinal health parameters, including matrix metalloproteinase (MMP) expression in the intestinal wall. Ross 308 broilers were fed a diet supplemented with coniferous resin acids for 22 days, after which the effect on both the intestinal microbiota as well as on the activity of host collagenases was assessed. Further, we used a cell culturebased model of A549 cells, which express high levels of matrix metalloproteinase-7 (MMP-7). The A549 cells were treated with pure resin acids for 24h, followed by fixation with 4% paraformaldehyde, staining of cells with MMP-7 specific antibody and cell imaging by fluorescence microscopy.

Results

Dietary inclusion of resin acids induced minor effects on the intestinal microbiota. However, resin acidssupplementation reduced both duodenal inflammatory T-cell infiltration and ileal MMP activity towards collagen type I and type IV. The microscopic analysis of resin acids treated A549 cells showed a significant reduction in MMP-7 protein expression.

Implications / Conclusions

Reduced expression of MMP-7 in A549 cells and lower breakdown of collagen type I and IV in ileal tissue indicate a protective effect of resin acids on intestinal barrier integrity by preservation of the basal membrane and the extracellular matrix. MMP proteins are involved in the breakdown of extracellular matrix in tissue remodeling. Secretion of specific MMPs is important for facilitating wound healing or tissue repair during inflammatory intestinal diseases. MMP inhibitors can potentially be used in therapies that are involved in inhibition of tissue degradation and remodeling. Our results suggest that resin acids could be used as a feed additive in protecting integrity of the intestinal wall and potentially promoting the intestinal health of the poultry.

Performance enhancing effect of a natural resin acid composition in broiler chickens under a variety of challenge conditions

E. Valkonen1, H. Kettunen2 , J. Kivinen1, J. Vuorenmaa1

1Hankkija Oy, Hyvinkää, Finland; 2 Sciandics, Tervakoski, Finland

Coniferous trees secrete viscous rosin at the site of injury to inhibit invasion of pathogenic microbes. Rosin contains a mixture of diterpene carboxylic acids collectively referred to as resin acids, according to species and geographical origin. The antimicrobial and anti-inflammatory potential of resin acids is well documented and utilized e.g. in rosin salves that accelerate wound healing. The natural resin acid mixture of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) consists mainly of abietic, dehydroabietic and pimaric acids, and is found in tall oil fatty acids (TOFA).

Supporting the gastrointestinal health and performance of broiler chickens is important especially in antibiotic-free feeding. In our previous research, a patented and standardized resin acid composition (RAC; Progres®, Hankkija Ltd), sustainably produced from tall oil, inhibited the growth of several pathogenic Gram-positive bacterial cultures, including Clostridium perfringens and Staphylococcus aureus. Here we studied the potential of RAC as a performance enhancer for broiler chickens in different environmental conditions and feeding regimens, and also with and without intentional challenge. Eight feeding trials without intentional challenge were run in four European research institutes, using local feeding and management systems. RAC at 0.500-0.750 g/kg feed significantly (p < 0.05) improved feed conversion ratio in four trials and the daily growth also in four tr ials, compared with nonsupplemented control treatments. In total, statistical improvement in performance was measured in five out of eight experiments (62.5%). Intentional challenge procedures, including Eimeria maxima, Clostridium perfringens, high stocking density, low temperature and/or coccidiosis vaccine, were implemented in additional five broiler experiments in European and US research institutes. Significant (p < 0.05) improvement of feed conversion, bird growth and/or mortality was reported in four out of five (80%) challenge trials. Improved foot pad health was observed in both trials in which it was measured. In trials with Clostridium perfringens -challenge, RAC did not prevent the onset of necrotic enteritis nor did it reduce the formation of lesions or decrease the pathogen load. However, RAC improved the performance of challenged broiler chickens more often than unchallenged birds.

Antimicrobial effects of resin acids unlikely fully explain the improved bird performance in our trials. Future research has been directed to reveal whether modulation of intestinal microbiota, reduced inflammatory processes in the gut epithelium, or other physiological functions are involved in the modeof-action of dietary resin acid supplementation.

Abstract

Dietary resin acid composition as a performance enhancer for broiler chickens

J. Vuorenmaa1, E. Valkonen1 and H. Kettunen2

1Hankkija Ltd /Suomen Rehu., Hyvinkää Finland

2 Sciandics Ltd., Tervakoski Finland

The Problem

Broiler performance depends on the health and proper function of gastrointestinal system. Coccidiosis, necrotic enteritis (NE) and dysbacteriosis are common performance-decreasing problems. Resin acids from coniferous trees have antimicrobial and anti-inflammatory properties which make them potential performance enhancers for broilers.

How we investigated or researched the problem

We evaluated the effects of a resin acid composition (RAC) on broiler performance in 3 trials. The RAC (Progres®, Hankkija Ltd/Suomen Rehu, Finland) consisted of 5.0–8.5% of resin acids, 88.5–93.0% free fatty acids and 2–3% neutral components. In trial 1, male broilers (n=192) received diets with RAC at 0, 0.5, 1.0, or 3.0 g/kg from 0 to 17 d of age. All birds received Eimeria maxima (d 11) and Clostridium perfringens (d 14) challenge. In trial 2, male broilers (n=510) received diets with RAC at 0, 1, 2, or 3 g/kg from 0 to 35 d of age. A non-challenged, non-supplemented group, served as a control. The other birds got Eimeria maxima (d 9) and C. perfringens (d 14) challenge. In trial 3, unsexed broilers (n=1400) were reared from 0 to 42 d of age on 1 of 4 diets from a 2x2 factorial design with 2 coccidiostats (chemical (CC) or ionophore (IC)) and 2 RAC doses (0 and 0.5 g/kg).

Results

RAC dose-dependently improved body weight gain (BWG) in trials 1 and 2. In trial 1, clinical coccidiosis was evident and RAC tended to reduce mortality. In trial 3, RAC with CC improved BWG (3.3%) and feed conversion ratio (5.7%). With IC RAC tended to improve the same variables.

Implications / Conclusions

Dietary RAC improved broiler performance in NE challenge trials and under a natural challenge of a commercial-type setting where broilers received in-feed anticoccidials.

Broiler chicken presentations

A novel, resin-based dietary ingredient reduced the risk of necrotic enteritis in turkeys

H. Kettunen1, J. Apajalahti1, E. Valkonen2 , T. Rinttilä1, H. Grönberg1, J. Vuorenmaa 2

1Alimetrics Ltd, Espoo, Finland

2 Hankkija Oy, Hyvinkää, Finland

The prevalence of necrotic enteritis (NE) in Finnish turkey flocks has raised significantly in the recent years. Gastrointestinal presence of the Gram-positive bacterium Clostridium perfringens (C.p.) is a prerequisite for the onset of NE outbreak. Here we measured the capacity of a natural resin acid composition (RAC, Progres®, Hankkija Oy) to inhibit the growth of C.p. in vitro and in vivo in the small intestine of turkey poults. The growth of a pure culture of C.p. in vitro was measured as gas production and optical density of the growth medium during the 8-hour incubation. RAC in the growth medium at or above 0.001% prevented the growth of C.p. completely, while 0.0005% RAC caused partial growth inhibition. Commercial starter diet for turkey poults was amended with 0, 0.6, or 3 kg/ton of RAC. Three replicate pens of ten turkey poults were fed with each diet for 28 days. RAC tended to reduce the 28-day feed conversion ratio. Three poults per pen were analysed for the presence of C.p. in ileal contents by quantitative real-time PCR. The frequency of poults with detectable levels of C.p. in ileal contents was 78%, 33%, and 44% for the 0, 0.6, and 3 kg/ton RAC, respectively. These data suggest that RAC acts against C.p. both in vitro and in the small intestine of turkey poults. Consequently, the dietary RAC amendment has a potential to reduce the risk of NE in commercial turkey farms.

A novel, resin-based dietary ingredient reduces the risk of necrotic enteritis in turkeys

Kettunen H1, Apajalahti J 1, Valkonen E 2, Rinttilä T 1, Grönberg H 1, Vuorenmaa J 2

1 Alimetrics Ltd, Koskelontie 19B, FI-02920 Espoo, Finland

2 Hankkija Ltd, Peltokuumolantie 4, FI-05800 Hyvinkää, Finland

Background

Conclusions

In the present study, a natural resin-based product (RAC; Progres ®) effectively inhibited the growth of Clostridium perfringens (Cp) in vitro. Moreover, dietary RAC decreased the frequency of Cp in the small intestinal contents of turkey poults. These results suggest that dietary RAC has potential to reduce the risk of necrotic enteritis in commercial turkey farms.

Necrotic enteritis (NE) has a major economic impact on poultry industry worldwide. The primary causative agent of NE is the Gram-positive bacterium Clostridium perfringens (Cp).

Resin of coniferous trees has been used as a traditional medicine for centuries in the Nordic countries. Resin contains resin acids which have antimicrobial properties especially against Gram-positive bacteria. Here we measured the capacity of a novel resin-based product to inhibit the growth of Cp in vitro and in the small intestine of turkey poults.

Results

Experiment 1. In vitro growth of Cp

• RAC significantly inhibited the growth of Cp at or above the concentration of 5 mg/l (Figures 1 and 2)

Experiment 2. 28-day in vivo trial with turkey poults

• RAC at 0.6 and 3 kg/tn tended to improve FCR (data not shown)

• Dietary RAC at 0.6 and 3 kg/tn decreased the frequency of ileal digesta samples with detectable levels of Cp (Figure 3)

Material and methods

Experiment 1. Pure culture of Cp was grown anaerobically at 37 °C in TSY medium for 8 hours. RAC was added at seven concentrations. Optical density of the medium was measured at 3, 6 and 8 hours, and the total gas produced in the vials was measured at 8 hours. These two variables indicated the growth of Cp.

Experiment 2. Commercial starter diet for turkey poults without coccidiostat was amended with 0, 0.6, or 3 kg/tn of RAC. Three replicate pens of ten turkey poults were fed with each diet for 28 days. The body weight gain (BWG), feed intake and feed conversion ratio (FCR) were measured. Three poults per pen were analysed for the density of Cp (alpha-toxin-encoding phospholipase C -gene copies/g) in ileal contents by quantitative real-time PCR.

Figure 1. Production of gas by Cp during the 8-hour fermentation (mean ± SE, N = 4). RAC at or above 5 mg/l significantly inhibited the metabolism of Cp (p < 0.001).

Figure 2. Optical density of the simulation vessels at the 6 and 8 hours time points (mean ± SE, N = 4). RAC at or above 5 mg/l significantly inhibited the growth of Cp at both time points (p < 0.001).

Figure 3. Frequency (%) of ileal digesta samples of turkeys with detectable levels of Cp (N = 9). Dietary RAC decreased the frequency of samples with Cp density above the detection limit of 1*10 4 plc gene copies/g digesta fresh weight.

The efficiency of resin acid composition in turkeys nutrition

K. Lipiński1, J. Vuorenmaa 2 , Z. Antoszkiewicz1, J. Kaliniewicz1, M. Mazur1 and Z. Makowski1

1University of Warmia and Mazury, Department of Animal Nutrition and Feed Science, Oczapowskiego 5, 10–718 Olsztyn, Poland

2 Hankkija Ltd, Peltokuumolantie 4, 05800 Hyvinkaa, Finland

A 105-day experiment was conducted on 600 BIG 6 female turkeys to determine the effect of Progres® (natural resin acids of Scots pine and Norway spruce, RAs) on the growth performance of birds, the incidence of footpad dermatitis (FPD), litter quality, carcass quality, structure and function of the gastrointestinal tract – GIT (pH, viscosity, short-chain fatty acids; SCFAs). The experiment involved 600 turkeys divided into three groups in ten replications in each. The birds from each subgroup (20) were placed in a separate pen. The birds received a basal diet without RAs (T1 ), or diets supplemented with RAs at 0.5 kg/t (starter 1 and 2, finisher), and at 1.0 kg/t (grower 1 and 2) (T2), or diets supplemented with RAs at 0.75 kg/t (starter 1 and 2, finisher), and at 1.5 kg/t (grower 1 and 2) (T3).

The inclusion of the Progres® RA preparation in turkey diets tended to increase the body weights of birds by 1.6% (0.5–1.0 kg/t RAs) and 2.1 % (0.75–1.5 kg/t RAs); P≤0.10. Dietary supplementation with RAs significantly improved the values of feed conversion ratio (FCR; P <0.01) and European Efficiency Index (EEI; P<0.05). The addition of RAs to the diets reduced the incidence of FPD at 9 and 15 weeks of age (P<0.0 1) and improved litter quality (P<0.01). The inclusion of the analyzed preparation in turkey diets had no effect on carcass quality, meat quality and most parameters of GIT structure and function.

The experiment suggests that Progres® (RAs) preparation improves the performance and well-being of turkeys.

THE EFFICIENCY OF RESIN ACID COMPOSITION IN TURKEYS NUTRITION

K. Lipiński1, J. Vuorenmaa2, Z. Antoszkiewicz1, J. Kaliniewicz1, M. Mazur1, Z. Makowski1

1Department of Animal Nutrition and Feed Science, University of Warmia and Mazury in Olsztyn Oczapowskiego 5, 10-718 Olsztyn, Poland

2Hankkija Ltd, Peltokuumolantie 4, FI-05800 Hyvinkää, Finland

INTRODUCTION

Resin is a substance that coniferous trees secrete at the sites of mechanical injury to prevent the invasion of pathogenic bacteria and fungi and to deter herbivorous animals Resin contains compounds such as abietic, dehydroabietic, neoabietic, isopimaric, levopimaric and palustric acids, which are a diverse group of hydrophobic diterpene carboxylic acids with antimicrobial, antifungal and antiparasitic activity Gram-positive bacteria are especially sensitive to resin acids Traditional medicine in Scandinavia has used the antimicrobial properties of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) resin for centuries, and novel applications, like resin-salves for the treatment of severe wounds, have been developed in recent years

Different superscripts in same row are significant or trending (A/B: P ≤ 0.01; a/b: P ≤ 0.05; x/y 0.05 <P≤ 0.10).

* European Efficiency Index, ** FPD score, 0-2 scale, FPD classes: 0=no lesions; 1=mild lesions; 2=severe lesion, FPD score =(number lesion class 1x 0.5 + number lesion class 2 x 2) x 100)/10 *** litter score, 0-4 scale, litter classes: 0= completely dry and flaky; 1= dry but not easy to move foot; 2=leaves imprint of foot and will form a ball if compacted, but ball does not stay together well, 3= sticks to boots and sticks readily in a ball if compacted, 4= sticks to boots once the cap or compacted crust is broken. Litter quality=(number places 0 x 100 + number places 1 x 67 + number places

RESULTS

OBJECTIVE

The aim of the study was to determine the effect of application of Progres (Resin acids) preparation in diets for turkeys on the growth performance of birds, incidence of footpad dermatitis (FPD), litter quality, carcass quality, structure and function of the gastrointestinal tract – GIT (pH, viscosity, short-chain fatty acids - SCFAs)

MATERIAL AND METHODS

A 105-day experiment was conducted on 600 BIG 6 female turkeys to determine the effect of Progres (natural resin acids of Scots pine and Norway spruce, RAs) on the growth performance of birds, incidence of footpad dermatitis (FPD), litter quality, carcass quality, structure and function of the gastrointestinal tract – GIT (pH, viscosity, short-chain fatty acids - SCFAs) (3 treatments, 10 replications, 20 birds per replication) The birds received a basal diet without RAs (T1), or diets supplemented with RAs at 0 5 kg/t (starter 1 and 2, finisher), and at 1 0 kg/t (grower 1 and 2) (T2), or diets supplemented with RAs at 0 75 kg/t (starter 1 and 2, finisher), and at 1 5 kg/t (grower 1 and 2) (T3)

At 105 days of age (end of fattening), the highest body weight was found in birds of group T3 (Progres – higher level) In comparison with birds from the T1 group, the difference was approx 0 20 kg (9 60 vs 9 40 kg) The mean body weight showed a nearly significant trend in favor of Progres turkeys (P=0 069) The resin acids applied in the diets for turkeys improved the feed conversion ratio (FCR) The differences observed were statistically significant between T1 and T3 group (higher level of Progres) Analyses of the results from the entire fattening period indicate that the application of the Progres preparation in the diets improved FCR (2 23, 2 20 vs 2 28, P≤0 01) The analysis of the European Efficiency Index demonstrated that the use of the study feed additive had a positive impact on the value of this index (P>0 05) Throughout the entire period there was no difference in the mortality rates between groups

FPD scores were significantly different between Resin acid groups (T2 and T3) and the control group at weeks 9 and 15 (P≤0 01) In the control group, the footpad dermatitis score was higher than in the groups fed with Progres There was also a strong positive effect of RAs addition on litter quality (P<0 01)

The use of the study of Progres preparation in diets had a positive impact on the breast muscle yield, although the differences were not statistically significant Analysis of chemical composition of breast muscle meat did not demonstrate a significant impact of the applied diet on the study indicators

In the experiment, an assessment of the structure of individual sections of the gastrointestinal tract was performed The applied nutrition did not have any effect on the weight of the crop, the proventriculus, the gizzard or the small intestine, but the weight of the ceca was varied A higher weight of the ceca was in the T1 group, the observed differences in comparison with the T2 group were statistically significant (3 60 vs 3 14 g/kg BW, P<0 05) The weight and pH of the chyme in the crop, the proventriculus and the gizzard did not vary between the individual experimental groups Turkeys fed diets with Resin acids were characterized by a lower weight of the digesta in the small intestine (P<0 01) and in the ceca (P 0 064) The applied diets increased the pH of the digesta in the small intestine (P<0 01)

The viscosity of the chyme of the small intestine and the ceca were not varied among the experimental groups

CONCLUSIONS

 The analysis of the FCR demonstrated that supplementation with the studied feed additive significantly improved this indicator (P<0.01).

 The use of a resin acid preparation in diets for turkeys affected an increase in the total body weight by 1.6% (Progres RA 0.5-1.0 kg/t) and by 2.1% (Progres RA 0.75-1.5 kg/t).

 The analysis of the European Efficiency Index demonstrated that the use of a resin acid preparation resulted in an improvement of this index (P<0.05).

 The use of Progres RA preparation in diets did not affect the survival of turkeys.

 The addition of resin acids in the diets decreased footpad lesions at 9 and 15 weeks (P<0.01) and increased litter quality (P<0.01).

 The inclusion of the Resin acids in turkey diets had no effect on carcass quality, meat quality or most parameters of GIT structure and function.

Table 1 Effect of resin acid supplementation on growth performance, FPD score and litter quality

Abstract

Dietary supplementation of tall oil fatty acid and resin acid in farrowing sows can affect the colostrum quality

Hasan SMK.1, Junnikkala S. 2 , Peltoniemi O.1, Oliviero C.1

University of Helsinki, Faculty of Veterinary Medicine

1Department of Production Animal Medicine

2 Department of Veterinary Biosciences

Background & objectives

Colostrum plays an essential role in piglet survival and growth, providing the piglets with a source for both immunoglobulin (mainly IgG) and energy. The neonatal piglets lack IgG, which makes them dependent on colostrum as the sole source of antibody. The aim of this study was to examine whether tall oil fatty acid and resin acid (RA) derived as a co-product in pulp production, added to a late pregnancy diet affected colostrum composition and yield (CY) in sows.

Materials & Methods

44 sows were randomly allocated to two groups as follows: a negative control diet (n= 21) and the same diet supplemented with 5 ml RA/day (n=23) during the last 1 week of pregnancy to whole lactation period. The RA used was Progres® (Hankkija Oy/Suomen Rehu, Hyvinkää, Finland). Within the first 2 hours from the beginning of farrowing, a 10 ml colostrum sample was obtained to check for nutritional composition (protein, fat, lactose, dry matter, with FITR analysis), and immunoglobulin content (IgA, IgM and IgG with ELISA analysis). All piglets were individually weighted at birth and 24 hours later in order to calculate CY.

Results

Colostrum content of protein, lactose, fat and dry matter did not significantly differ between the two groups. While RA fed sows had higher level of IgG (86.37 ± 5.27 mg/ml vs 70.94 ± 5.49 mg/ml) in colostrum (p < 0.05), IgA and IgM levels in colostrum remain same in two groups. There was also no changes in the CY of the sows in two groups.

Discussion & Conclusions

In conclusion, adding RA to late pregnancy diet in sows did not affect the CY and protein, fat and lactose content in colostrum, but contributed to higher IgG content. Therefore RA added to sow diet seems to increase colostrum IgG and ensure the better survival of neonate piglets.

FACULTY OF VETERINARY MEDICINE

Dietary supplementation of tall oil fatty acid and resin acid in farrowing sows can affect the colostrum quality

INTRODUCTION

Tall oil fatty acid (TOFA) is the non-aqueous layer of tall oil, typically comprises 90% fatty acids and 10% resin acids Fractional distillation of crude tall oil, obtained as a by-product of the Kraft process of wood pulp manufacture, produces distilled tall oil, further refinement of distilled tall oil produces TOFA. TOFA has been used in animal feed as an energy source because of its inexpensive source of fatty acids TOFA modulated the microbial population in the digestive system of animal and changes the microbial metabolism and improve feed conversion ratio and gut immunity The aims of this study was to examine whether TOFA added to a late pregnancy diet affected colostrum composition and yield (CY) in sows

RESULTS

MATERIALS AND METHODS

91 sows from two different herds were randomly allocated to two groups as follows: a negative control diet (n= 44) and the same diet supplemented with 5 g TOFA/kg (n=47) during the last week of pregnancy The TOFA used was Progres® (Hankkija Oy/Suomen Rehu, Hyvinkää, Finland) Within the first 2 hours from the beginning of farrowing, a 20 ml colostrum sample was obtained to check for nutritional composition (protein, fat, lactose, dry matter, with Fourier transform infrared spectroscopy - FTIR), and immunoglobulin content (IgA, IgM and IgG with ELISA analysis) All piglets were individually weighted at birth and 24 hours later in order to calculate CY

Colostrum content of protein, lactose and dry matter did not differ between the two groups, while TOFA fed sows had tendency in lower level of fat in colostrum (4 17 % vs 4 73 %; p = 0 09; Fig 1) Colostrum Yield and IgA, IgM levels in colostrum did not differ between the two groups (Fig 2 and 3) Colostrum IgG was 68.34 mg/ml in the control group and 81 71 mg/ml in the TOFA fed group (p < 0 05; Fig 2)

CONCLUSION

In conclusion, adding TOFA to late pregnancy diet in sows did not affect the CY, protein, fat, lactose and DM content in colostrum, but contributed to higher IgG content Therefore TOFA added to sow’ late pregnancy diet seems to increase colostrum IgG, which can promote better survival of neonate piglets.

Shah Hasan1, Sami Junnikkala2, Olli Peltoniemi1 and Claudio Oliviero1

1Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Finland

2Department of Veterinary Bioscience, Faculty of Veterinary Medicine, University of Helsinki, Finland

Shah.hasan@helsinki.fi

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Figure 1. Colostrum composition analysed with FTIR. Asterisk indicates tendency (P = 0.09).

Figure 2. Colostrum immunoglobulin content, analysed with ELISA. Asterisk indicates significant differences (P<0.05).

Figure 3. Colostrum yield in the two study groups.

COLOSTRUM COMPOSITION (%)

COLOSTRUM YIELD (G)

Progres Control

Dietary resin acid supplementation improves the performance of sows and piglets

S. Hasan1, M. Kamyzeck 2 , J.Vuorenmaa1, E. Valkonen1, H. Kettunen1

1Hankkija Ltd, Peltokuumolantie 4, FI-05801 Hyvinkää, Finland

2 Pawlowice Instytut Zootechniki, Zakład Doświadczalny Pawłowice, ul. Mielżyńskich 14, 64-122 Pawłowice, Poland

Coniferous resin acids have anti-inflammatory and antimicrobial effects. We have previously shown that dietary resin acids improve the reproductive performance of sows and increased the production and immunoglobulin G -content of colostrum. Here we studied the effect of a tall oil -based resin acid composition (RAC) with 8.5% resin acids to the performance sows and their offspring.

Fifty-six Polish synthetic line 990 sows were allocated to two dietary treatments. The standard lactation feed was amended with 0 kg/tn (Control; C) and 1.0 kg/tn (Treated; T) of RAC from 2 wk before to 4 wk after farrowing. The parity number, backfat thickness at start and end of lactation, course and length of parturition, number of total born, born alive and stillbirth piglets, number and weight of piglets and weight of litter at weaning were recorded for the sow. The piglets were weaned at 4 wk of age. From both diet groups, 80 castrated male and 80 female weanlings were housed in groups of eight and allocated to C and T dietary treatments, to reach a total 320 piglets in the following four treatments: CC, CT, TC, TT. The piglets were weighed at 0, 2 and 6 wk after weaning, and rec-orded for daily feed intake, feed conversion ratio (FCR) and mortality. For statistical evaluation of the performance data, ANOVA was used.

Dietary RAC decreased the course of parturition, the number of stillborn piglets, and piglet mortality before wean-ing, increased piglet weight at birth, and resulted in an average of 0.96 more piglets per litter (p<0.05). For 2 wk post-weaning, piglet average daily gain (ADG) was lower for the CC than other dietary groups (p<0.05). ADG re-mained significantly lower in CC than CT group for 6 wk post-weaning. Average daily feed intake was higher in pig-lets of the CT group than in TC group (p<0.05). FCR was better in piglets of TT group than in piglets of the TC group. In conclusion, the performance of sows and piglets was improved by dietary RAC supplementation at lactation and nursery respectively.

INTRODUCTION

DIETARY RESIN ACID SUPPLEMENTATION IMPROVES THE PERFORMANCE OF SOWS AND PIGLETS

S. HASAN1*, M. KAMYZECK2, J. VUORENMAA1, E. VALKONEN1 & H. KETTUNEN1

1Hankkija Ltd, FIN-05801 Hyvinkää, Finland, 2Pawlowice Instytut Zootechniki, Zakład Dos´wiadczalny Pawłowice, ul. Mielzyn ´ skich 14, 64-122 Pawłowice, Poland

Dietary inclusion of coniferous resin acids improves the reproductive performance of sows and increases the production and immunoglobulin G -content of colostrumREF. Here we studied the effect of a tall oil -based resin acid composition (RAC; Progres®; Hankkija Ltd) with 8.5% resin acids on the performance sows and their offspring (Figure 1).

PHASE 1: Sows

CONTROL, C

PHASE 2: Piglets

CONCLUSIONS

The performance of both sows and piglets was improved by dietary RAC supplementation.

PHASE 1: SOWS

MATERIALS AND METHODS

TREATED, T

1. Trial setup

PHASE 2: Piglets

MATERIALS AND METHODS

From both sow diet groups, 80 female + 80 castrated male weanlings were housed in groups of eight and allocated to C and T dietary treatments: CC, CT, TC, TT. The piglets were weighed at 0, 2 and 6 wk after weaning, and recorded for daily feed intake (FI), feed conversion ratio (FCR) and mortality. Data was evaluated using ANOVA.

RESULTS, see Table 2

Other results

• Average daily feed intake was higher in piglets of the CT group than in TC group (p < 0.05)

• FCR was better in piglets of TT group than in piglets of the TC group

REF Hasan et al., Animal. 2018 D ec 27:1-8

The standard lactation feed of 56 Polish synthetic line 990 sows was amended with 0 kg/tn (Control; C) and 1.0 kg/tn (Treated; T) of RAC from 2 wk before to 4 wk after farrowing. The parity number, backfat thickness at start and end of lactation, length of gestation and parturition, number of total born, born alive and stillborn piglets, number and weight of piglets and weight of litter at weaning were recorded for the sows.

RESULTS

Dietary amendment with RAC improved sow performance (Table 1), resulting in an average of 0.96 more piglets per sow than in control group (p < 0.05).

TABLE 1. Sow performance

TABLE 1. Sow performance

Sow feeding group

FIGURE

Abstract

Raising weanling piglets without medical doses of zinc: an example from Finland

H. Kettunen1, S. Hasan1, M. Suokanto1, S. Valkama1, J. Daka1, J. Vuorenmaa1, T. Heikkilä 2

1Hankkija Oy, Hyvinkää, Finland; 2 Heikkilä Farm, Rusko, Finland,

Background and objectives

Conditions in the nursery and optimized feeding are critical factors in avoiding post-weaning diarrhea (PWD) when raising piglets without medical doses of zinc in diet. Especially at 3-6 days post-weaning, drought and too low ambient temperature lead to increased feed intake and a higher PWD risk. Here we provide an example of technologies involved in a successful withdrawal of medical zinc from piglet feeds in a commercial sow-piglet operation (Heikkilä Farm, Larjava, Rusko, Finland), in which the smallest 40% of weaned piglets from 3500 sows stay at the home farm. The use of medical doses of zinc in piglet diets was stopped in January 2016.

Technologies

Piglet management is based on top quality nursery conditions, optimization of feeding, and all-in-all-out concept. The piglets (DanBred genetics, Denmark) arrive to the nursery at the age of 29-31 days and weight of 6.2 kg. They are housed in compartments of 16 pens (x5m2) with 70% solid flooring and 30% slatted flooring. The floors are heated with two floor heating units, one for the floor under canopies and the other for the rest of the floor. The canopies are fully automated, equipped with heat lamps (Veng Systems, Denmark), and programmed with different settings for winter and summer periods. The air ventilation system (Fancom BV, The Netherlands) is precise and fitted with air inlets towards canopy roofs, in order to avoid air flow over manure. The slurry cooling system (Alpha Innotec, Germany) is very efficient (winter time ~50W/manure m2). Excellent environmental condition for piglets is necessary also because tails are not docked Finland. Every pen is equipped with plastic chew toys (Bite-Rite, Ikadan, Denmark) and a straw automat.

Feeding

Good feeding hygiene is invaluable in zinc-free feeding. The liquid feeding system has 40 mm main pipes and 32 mm pipes to the pens. A 2.5 m feeding trough allows adequate space for 12-15 piglets/pen. The Weda Active Cleaning System (WEDA Dammann & Westerkamp GmbH, Germany) for automatic cleaning of the pipes was developed in this farm. There are five feeding phases for piglets from 6 to 32 kg. Trough emptying time is measured by sensors to adjust feed quantity. During the 4-day transition from prestarter to starter diets (days 3-6 post-weaning), feed intake is softly reduced by altering the taste of the feed, in order to reduce PWD risk. The other dietary transitions take place gradually over 7 days. Feed is optimized for reduced protein and supplemented with synthetic amino acids, to ensure maximal amino acid digestibility. Calcium levels are kept low to reduce the buffering capacity of diets. The feeds (Hankkija Oy, Finland) are acidified with a blend of organic acids, including supplementation by lactic acid (Pekoni Lakta-Happo, Finlactic Oy, Finland) at the farm. Intestinal functions of piglets are supported by two special products: yeast hydrolysate Progut® and resin acid based Progres® (both from Hankkija Oy, Finland).

Results and conclusions

The technology and feeding has worked very well and there has been no need to re-start using medical zinc. The health and condition of piglets is consistently very good in this nursery, with the following growth performance indicators: 6-32 kg, ADG 480-490 g, FCR 1.55-1.65 kg/kg. The health problems, when present, usually originate from the pre-weaning period. The system has been running in the present form for six years, and it has proven that it is fully possible to maintain good piglet performance without medical doses of zinc in the diet. Investing in top quality technology has paid back in good piglet performance in this farm.

Pig posters

Raising weanling piglets without medical doses of zinc: an example from Finland

H. KETTUNEN1, S. HASAN1, M. SUOKANTO1, S. VALKAMA1, J. DAKA1, J. VUORENMAA1, T. HEIKKILÄ 2

Hankkija

INTRODUCTION

Risk for post-weaning diarrhea (PWD) is highest at 3–6 days postweaning, with drought and too low ambient temperature as critical risk factors.

Heikkilä Farm (Rusko, Finland) successfully stopped using medical doses of zinc in piglet feeds in January 2016.

At Heikkilä, the smallest 40% of weaned piglets from 3500 sows stay at the home farm. As everywhere in Finland, the tails are not docked.

Piglet management is based on top quality nursery conditions, optimization of feeding, and all-in-all-out concept.

TECHNOLOGIES

• The piglets (DanBred genetics, Denmark) arrive to the nursery at the age of 29 –31 days and weight of 6.2 kg.

• Housing in compartments of 16 pens (x 5m2) with 70% solid flooring and 30% slatted flooring.

• One floor heating unit under canopies and another for the rest of the floor.

• Fully automated canopies with heat lamps (Veng Systems, Denmark), and with different settings for winter and summer periods.

• Air ventilation system (Fancom BV, The Netherlands) is fitted with air inlets towards canopy roofs, avoiding air flow over manure.

• Efficient slurry cooling system (Alpha Innotec, Germany).

• Plastic chew toys (Bite-Rite, Ikadan, Denmark) and a straw dispenser in every pen.

• The liquid feeding system has 40 mm main pipes and 32 mm pipes to the pens. A 2.5 m feeding trough allows adequate space for 12–15 piglets/pen.

• The Weda Active Cleaning System WACS (WEDA Dammann & Westerkamp GmbH, Germany) for automatic cleaning of the pipes was developed at this farm.

CONCLUSIONS

The technology and feeding has worked very well and there has been no need to re-start using high doses of zinc in piglet feeds.

The health and condition of piglets are consistently very good, with the following growth performance indicators: 6–32 kg, ADG 480–490 g, FCR 1.55–1.65 kg/kg.

The health problems, when present, usually originate from the preweaning period.

Investing in top quality technology has paid back in good piglet performance at Heikkilä farm.

FEEDING

• Five feeding phases for piglets from 6 to 32 kg.

• During the 4-day transition from prestarter to starter diets (days 3 – 6 post-weaning), feed intake is softly reduced by altering the taste of the feed, in order to decrease PWD risk.

• Feed is optimized for reduced protein and supplemented with synthetic amino acids, for maximal amino acid digestibility.

• Calcium levels are kept low to reduce the buffering capacity of diets.

• Two special products for supporting intestinal functions: yeast hydrolysate Progut® and resin acid -based Progres® (Hankkija Oy, Finland).

• The feeds (Hankkija Oy, Finland) are acidified with a blend of organic acids, including supplementation by lactic acid (Pekoni Lakta-Happo, Finlactic Oy, Finland) at the farm.

• Trough emptying time is measured by sensors to adjust feed quantity.

Oy, Hyvinkää, Finland, 2Heikkilä Farm, Rusko, Finland

Abstract

Effect of dietary resin acids and hydrolysed yeast in piglets challenged with F4-Enterotoxigenic Escherichia coli

Anouschka Middelkoop1, Ramon Tichelaar1, Xiaonan Guan1, Hannele Kettunen2 , Juhani Vuorenmaa 2 , Martin Peter Rydal 3 , Lone Brøndsted3 , Michela Gambino3 , Jens Peter Nielsen3 , and Francesc Molist1

1Schothorst Feed Research, Lelystad, The Netherlands

2 Hankkija Oy, Hyvinkää, Finland

3 University of Copenhagen, Frederiksberg, Denmark

Background and Objectives

Enterotoxigenic Escherichia coli (ETEC) that expresses F4 fimbriae is a major cause of post-weaning diarrhoea. Adding tall oil fatty acids (TOFA) to the diet improves the intestinal barrier function and Saccharomyces cerevisiae yeast hydrolysate (YH) has a high activity against ETEC in vitro. We studied the effect of dietary amendment with TOFA consisting of 9% resin acids and/or YH in an F4-ETEC challenge with piglets.

Material and Methods

From weaning (30 days old, ~7.8 kg), piglets received: a control diet (crude protein level of 20%) + colistin via drinking water as positive control (T1: PC), a control diet as negative control (T2: NC), TOFA at 1.0 kg/ ton feed (T3), YH at 1.5 kg/ton feed (T4), or TOFA+YH at 1.0+1.5 kg/ton (T5). On D10 post-weaning each piglet (n=10/treatment) was orally administered an F4-ETEC at ~5.9x109 CFU/piglet. Faecal consistency was scored and faeces collected for ETEC qPCR and/or myeloperoxidase quantification on D0, 8, 11-15, 18, 20 and 22 post-weaning.

Results

Treatments affected faecal consistency (P<0.0001), with improved scores for T1, T3 and T5 versus T2 and T4. Also a shorter duration of diarrhoea after F4-ETEC inoculation was seen for T1, T3 and T5 versus T2 (P<0.05). The proportion of pigs shedding F4-ETEC over time is visualised in Figure 1 for each treatment group. Myeloperoxidase concentrations were not affected by treatment (P=0.26). Piglet performance was equal in all groups between D0-22 post-weaning. Between D8-15, average daily gain was 452b, 295a, 379ab, 387ab and 273a g/piglet/d (P=0.09) and the feed conversion ratio was 1.02a, 1.37bc , 1.22b, 1.19ab, and 1.44 c for T1-T5, respectively (P=0.02).

Conclusion and Discussion

TOFA and/or YH in the weaner diet increases piglet resilience to F4-ETEC in the post-weaning period. Against expectations, no cumulative effect of dietary TOFA+YH was observed.

Figure 1. Proportion of piglets shedding the inoculated F4-ETEC in their faeces over time

Effect of dietary resin acids and hydrolysed yeast in piglets challenged with F4-enterotoxigenic Escherichia coli

Anouschka Middelkoop1, Ramon Tichelaar1, Xiaonan Guan1, Hannele Kettunen2, Juhani Vuorenmaa2, Martin Peter Rydal3, Lone Brøndsted3, Michela Gambino3, Jens Peter Nielsen3, and Francesc Molist1 1SchothorstFeedResearch,Lelystad,TheNetherlands ; 2HankkijaOy,Hyvinkää,Finland ; 3UniversityofCopenhagen,Frederiksberg,Denmark

Contact: amiddelkoop@schothorst.nl

Introduction

PROGRES® contains tall oil fatty acids that can lower inflammation PROGUT® is a Saccharomyces cerevisiae yeast hydrolysate, of which its cell wall contains macromolecules that can bind to F4-ETEC fimbriae We hypothesized that PROGRES® and PROGUT® may inhibit F4-ETEC proliferation and diarrhoea in weaned piglets

Objectives

To test the ability of PROGRES® and PROGUT®, in combination and alone, to reduce proliferation of F4-ETEC and post-weaning diarrhoea in piglets challenged with F4-ETEC after weaning

Results

Methods

• 50 piglets, 7 8 kg BW, weaned at 29 7 days of age, mixed sex

• 5 9x109 CFU F4-ETEC inoculation in all piglets at d10 post-weaning

• Zootechnical performance (d0, 8, 15, 22), faecal consistency (score

≤4: diarrhoea, d0, 8, 11-15, 18, 20, 22), faecal F4-ETEC shedding (qPCR; d8, 11-15, 18, 20, 22) and faecal MPO (d0, 8, 15, 22)

• 5 treatments with 10 piglets/treatment, and 5 piglets/pen, d0-22:

COLISTIN Control diet + colistin via drinking water

CONTROL Control diet: barley, wheat, soybean meal, maize-based

PROGRES Control diet + 1.0 g/kg PROGRES®

PROGUT Control diet + 1.5 g/kg PROGUT®

COMBI Control diet + 1.0 g/kg PROGRES® + 1.5 g/kg PROGUT®

PROGRES, PROGUT and their COMBI reduce F4-ETEC shedding PROGRES and COMBI improve post-weaning faecal consistency

COLISTIN CONTROL PROGRES PROGUT COMBI

*

PROGRES and COMBI shorten the diarrhoeic period after F4-ETEC inoculation

Treatment Days with diarrhoea

COLISTIN 0.80±0.36a

CONTROL 2.97±0.77b

PROGRES 1.20±0.55a

PROGUT 2.00±0.60ab

COMBI 1.10±0.38a

P-value 0.06

PROGRES® and PROGUT® , in combination and alone, make weaned piglets more resilient to enterotoxigenic F4-positive Escherichia coli and represent alternatives for managing post-weaning diarrhoea

PROGRES and PROGUT maintain zootechnical performance after F4-ETEC challenge

Effects of supplementing prepartum sow diet with organic acids on neonatal piglet mortality

J. Yun, S. Hasan, S. Saha, C. Oliviero, O. Peltoniemi.

University of Helsinki, Helsinki, Finland

The primary goal of the present study was to examine the effect of supplemental organic acids to the late gestation diet on neonatal piglet mortality. An additional goal was to study the effect of the loosehoused farrowing system on neonatal piglet mortality. A total of 60 sows were moved to farrowing units 7 days before the expected parturition date. The sows and their offspring were allocated to a factorial design with two factors, diet [CON (normal sow diet) vs. ORG (normal sow diet supplementing tall oil fatty and resin acids)] and housing [CRATE (crate size: 225 × 65 × 65, pen size: 325 × 250) vs. FREE (crate size: 225 × 159 × 191, pen size: 325 × 250)].

The live-born piglet mortality rate was remarkably higher in FREE than in CRATE (11.8% ± 1.9 vs. 3.3% ± 1.8, P < 0.01). This was due to the higher rate of crushed piglets seen in FREE compared to CRATE (11.5% ± 1.8 vs. 2.4 ± 1.8, P < 0.001). In FREE, the sows with ORG diet had a lower rate of crushed piglets (5.6% ± 2.5 vs. 17.5% ± 2.6, P < 0.01), and thus the lower live-born piglet mortality rate (6.0% ± 2.5 vs. 17.5% ± 2.7, P < 0.05) than the sows with CON diet, whereas among sows in CRATE, the live-born piglet mortality rate was not affected by different diets (1.3% ± 2.6 for CON vs. 5.3% ± 2.6 for ORG).

Consequently, these data indicate that supplemental tall oil fatty and resin acids to the diet of the prepartum sow could reduce mortality rates of neonatal piglets in loosed-housed system within 24 h postpartum.

Poster not available.

Effect of dietary resin acid and zinc oxide in weaned piglets subjected to an immune challenge

X. Guan*, H. Kettunen1†, R. Santos*, J. Vuorenmaa†, F. Molist*

*Schothorst Feed Research B.V., 8200 AM Lelystad, The Netherlands; †Hankkija Ltd, FI-05800 Hyvinkää, Finland

Resin acids derived from coniferous trees are phytochemicals with antimicrobial, antifungal and antiparasitic activities. This study aimed to compare the effect of dietary resin acid concentrates (RAC) with therapeutic level of ZnO on immune responses of weaned piglets subjected to a systemic challenge with E. Coli lipopoly-saccharide (LPS).

Forty-eight 26 day old piglets entered a completely randomized design with four treatments, including a non-challenged control (phosphate-buffered saline, PBS), a challenged control (LPS) and 2 chal-lenged groups with dietary treatments of 2500 mg ZnO (LPS + ZnO) and 2000 mg/kg of RAC (LPS+RAC). Piglets were fed ad libitum for 21 days (d) and subjected to LPS or PBS injections on d 7 and 21. Blood samples were taken at 1.5 hour (h) and 3 h after each infection. Weekly growth performance and rectal temperature at various times were recorded. Rectal temperature reflected activation of the immune system by LPS. There was no treatment effect on overall growth performance. Serum immune biomarkers including Interleukin (IL)-1β IL-4, IL-6, IL-8, IL-10, IL-12, Tumor necrosis factor (TNF)-α, Interferon (IFN)-α and IFN-γ were meas-ured. Data were analyzed by two-way ANOVA using time and treatment as factors.

No treatment effects on IL-1β, IFN- α, IFN-γ and IL-4 were observed. There was an interaction between treatment and time (P<0.001) at the both challenges (i.e. d 7 and d 21) for IL-10 and TNF-α. For IL-10, piglets received PBS showed low level at 1.5 h and 3h, piglets from LPS and LPS+ZnO groups showed higher levels at 1.5 h but decreased at 3 h, and piglets from LPS+RAC showed the highest level at 1.5 h and 3 h. For TNF-α, piglets from LPS+ZnO and LPS+RAC groups showed the highest level at 1.5 h, followed by piglets from LPS and piglets from PBS showed the lowest level. All piglets showed low level of TNF-α at 3 h. Treatment effect were found on IL-6 and IL-8 on d 7 and d 21 (P<0.001), piglets from LPS+RAC showed the highest levels of IL-6 and IL-8, followed by piglets from LPS+ZnO, and piglets from PBS and LPS groups showed the lowest level.

These results indicate that RAC compared to ZnO and control, may possibly boost the immune response of piglets without penalizing growth performance during a LPS challenge.

Dietary supplementation with tall oil fatty acid (TOFA) and resin acid (RA) in different farrowing housing at late pregnancy influences sow colostrum quality and piglets colostrum uptake

Shah Hasan, Sani Saha, Jinhyeon Yun, Olli Peltoniemi and Claudio Oliviero University of Helsinki, Faculty of Veterinary Medicine

Colostrum is essential for piglet survival and growth, providing the piglets with a source for immunoglobulin and energy. The aim of this study was to examine whether TOFA and RA (PRO) added to a late pregnancy diet affect colostrum composition, colostrum yield (CY, g) and colostrum intake (g) in different farrowing housing.

One week before farrowing, 60 sows were assigned to a 2×2 factorial [housing (CRATE,PEN), diet (PRO, CON)] experiment. Diet supplemented daily with 5 ml/sow of PRO or control basic sow diet (CON) during the last week of pregnancy. At the beginning of farrowing, 20 ml colostrum were collected to check nutritional composition (protein, fat, lactose, dry matter)), and IgG content (mg/ml, ELISA). Piglets were weighted individually at birth and 24h later to calculate CY.

The CY in the PEN sows was higher than the CRATE sows (4949.3 ± 184.0 vs. 4528.5 ± 162.5, P=0.06) and ensured higher colostrum intake for the piglets (342 ± 6.8 vs. 319 ± 6.9, P=0.01). Sows fed with PRO diet had higher colostrum IgG content than CON fed sows (113.8± 4.9mg/ml vs. 97.5 ± 5.1mg/ml, P<0.05). Considering the effect of housing sows fed with PRO diet had a tendency to higher colostrum IgG content (119.1 ± 6.3 vs. 103.8 ± 7.9 and 108.9 ± 7.3 vs. 92.1 ± 6.6, P=0.06, PEN and CRATE respectively) and significantly higher colostrum uptake in PEN sows (355 ± 8.9 vs. 325 ± 10.4, P<0.05). A linear regression model predicts 300 g of more colostrum when PRO is used, an additional 300 g when housing in PEN and 1.1 g less colostrum every minute more of farrowing duration (P<0.05).

In conclusion, as shown previously, this study confirmed the positive correlation between TOFA, IgG level in colostrum, and CY, considering also the interaction due to the housing effect.

Abstract

Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum IgG, piglet colostrum intake and modulates sow gut microbiota

Hasan, S., Sani, S., Junnikkala, S., Orro, T., Peltoniemi, O., Oliviero, C. University of Helsinki, Faculty of Veterinary Medicine

Tall oil fatty acid (TOFA) and resin acid (RA) commonly termed resin acid-enriched composition (RAC) can modulate the microbial population in the gut, changes metabolism, and improve the feed conversion ratio. We investigated the effects of dietary supplementation of RAC on sow colostrum yield, colostrum composition and gut microbiota.

The experiment was conducted in three trials in three respective herds. Sows were allocated either a control diet or a control diet supplemented with 5g RAC/day/sow during the last week of pregnancy. In one herd, faecal microbiota populations of sows at farrowing were assessed using 16S rRNA gene sequencing. Colostrum samples were examined for nutritional composition, acute phase proteins (APP) and immunoglobulin (Ig) content. All piglets were individually weighed at birth and 24 hours later in order to calculate colostrum yield (CY), and later at three to four weeks to calculate average daily gain (ADG).

The RAC-fed sows had significantly higher IgG levels (P < 0.05) in all three herds but treatment did not influence colostrum IgA and IgM concentration. Protein, lactose and fat content of colostrum did not significantly differ between sows of the two diet groups (P > 0.05), but RAC fed sows had higher levels of colostrum serum amyloid A (SAA). CY was significantly higher in RAC-fed sows in herds 2 and 3 with heavier piglets between 3 and 4 weeks of age (P < 0.05), but not in herd 1 (P > 0.05). RAC supplementation significantly increased some beneficial and fermentative bacteria (Romboutsia and Clostridium sensu stricto) than the control diet group (P < 0.01) while some opportunistic pathogens (Barnesiella, Sporobacter, Intestinimonas and Campylobacter), including Proteobacteria, were suppressed. Therefore, RAC added to the sow diet at late pregnancy increases colostrum IgG, colostrum availability for neonate piglets, and seems to promote better maternal intestinal microbial sources.

Abstract

Resin acid composition increases propionate and butyrate production in a Rumen Simulation Technique (RUSITEC) model

Hannele Kettunen1, Milla Frantzi1, Juhani Vuorenmaa1, Gerhard Breves2 , Melanie Eger2

1Hankkija Ltd, Peltokuumolantie 4, FIN-05801 Hyvinkää, Finland

2 Institute for Physiology and Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany

Tall oil fatty acid -based resin acid composition (TOFA) with 9% coniferous resin acids is a known microbial modulator and performance enhancer for chickens and pigs, but the effects of resin acids on rumen fermenta-tion are incompletely understood. Here we studied the effects of TOFA on the ruminal fermentation of dairy cows in a 16-day Rumen Simulation Technique (RUSITEC) experiment.

Six 750-ml RUSITEC fermentation vessels were loaded with substrate bags containing 6 g hay and 4 g concen-trate feed, and seeded with fresh bovine rumen fluid. After an equilibration phase of eight days, either TOFA (330 µl/day) or ethanol (550 µl/day) was added to the fluid of the fermenters during the daily exchange of the substrate bags. Ethanol was used as a solvent for another treatment not included here. Earlier studies suggest no effects on the used level of ethanol. On days 8-16, the vessels were measured for redox potential, pH, NH3-N and short chain fatty acid (SCFA) concentrations, gas volume and percentages of CH4 and CO2. Organic matter degradation was analysed from substrate bags of days 9 - 11, and 13-15. On days 8, 12 and 16, the ves-sels were sampled for 30 ml of fluid and the substrate bags for liquid- and solid-associated microbes, respec-tively. Microbial DNA was extracted and subjected to microbial population analysis by Illumina MiSeq 16S rRNA amplicon sequencing.

The pH and the redox values stayed at the physiological range of 6.6 to 6.8 and -270 mV to -290 mV, respec-tively, with no treatment effects. The total SCFA production was similar in both treatments, but TOFA signif-icantly increased the production rates and the molar proportions of propionate and butyrate, and decreased those of isovalerate. The molar proportion of acetate decreased significantly by TOFA. Degradation of organic matter and the concentrations of NH3-N, gas volume and percentages of CH4 and CO2 were not affected by TOFA. The treatments showed no differences in relative abundances of microbial phyla at the individual time-points of either the liquid or solid phase. Analysis for the operational taxonomic units (OTUs) revealed only a few transient changes as a response to TOFA.

In conclusion, TOFA shifted the fermentation towards propionate and butyrate production but did not have other major impacts on fermentation in this model. Increased ruminal propionate production has been linked to increased capacity of milk production. Studies with lactating dairy cows are needed to verify such an effect for TOFA.

Resin

acid composition increases propionate and butyrate production in a Rumen

Simulation Technique (RUSITEC) model

H. KETTUNEN 1, M. FRANTZI1, J. VUORENMAA1, G. BREVES 2, M. EGER2

1Hankkija Ltd, FIN-05801 Hyvinkää, Finland, 2Institute for Physiology and Cell Biology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany

INTRODUCTION

Increased ruminal propionate production has been linked to a higher capacity for milk production by lactating dairy cows. Tall oil fatty acid -based resin acid composition (TOFA; Progres®, Hankkija Ltd) is a known microbial modulator and performance enhancer in monogastric farm animals. Our hypothesis was that TOFA would favorably modulate ruminal bacterial populations and enhance propionate production. We studied the effects of TOFA in a Rumen Simulation Technique (RUSITEC) experiment.

CONCLUSIONS

As hypothesized, TOFA shifted the fermentation towards the production of propionate. However, TOFA did not have other major impacts on fermentation in this model. Studies with lactating dairy cows are needed to evaluate the effects of TOFA on milk production.

RESULTS

• Increased by TOFA: Production rates and molar proportions of propionate and butyrate (p<0.01; Figures 1A and 1B)

• Decreased by TOFA: Molar proportion of acetate (p<0.05; Figure 1C), production rate and molar proportion of isovalerate (p<0.01; data not shown)

• Not affected by TOFA: pH, redox values, organic matter digestibility, total production rates of short chain fatty acids, NH3-N, gas volume, percentages of CH4 and CO2, relative abundances of microbial phyla of the liquid or solid phase

• Analysis for the operational taxonomic units revealed only a few transient changes as a response to TOFA.

MATERIALS AND METHODS

Trial outline

• Six 750 ml RUSITEC fermentation vessels, loaded with fresh bovine rumen content (liquid and solid)

• Daily substrate: 6 g hay + 4 g concentrate feed

• Equilibration phase of 7 days, control period of 1 day

• Experimental period of 8 days, during which TOFA (330 µl) or control (ethanol 550 µl) was added to vessels during the daily exchange of the substrate bags. This amount of ethanol is known not to affect the fermentation.

Measurements

• Days 8-16: redox potential, pH, NH3-N and, gas volume and percentages of CH4 and CO2

• Days 9-11 and 13-15: organic matter degradation

• Days 8, 12 and 16: liquid- and solid-associated microbes. Microbial DNA was extracted and subjected to bacterial population analysis by Illumina MiSeq 16S rRNA amplicon sequencing (V34 region).

Figure 1. Molar proportion of propionate (A), butyrate (B) and acetate (C) of all short chain fatty acids in fermentation vessels during the control period (CP; day 8) and the experimental period (Days 9-16). Treatments: Control (Con) and tall oil fatty acids (TOFA). Significance: * p<0.05; ** p<0.01; *** p<0.001.

Abstract

The effects of in-feed resin acid inclusion on milk production responses of dairy cows

Kairenius

P.1,

Mäntysaari,

P.1,

Lidauer P.1, Franco M.1, Frantzi, M. 2 , Kettunen H. 2 & Rinne, M.1

1Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland

2 Hankkija Ltd, FI-05800 Hyvinkää, Finland

There is increasing interest in supporting the healthy microbiota within the digestive tract and the immune system of livestock with natural feed ingredients to improve the efficiency of food production and reduce the need for antimicrobial medication. The objective of this study was to investigate the effects of in-feed resin acids on milk production of dairy cows during early lactation.

Thirty-six Nordic Red cows were used in a continuous feeding trial that started 3 weeks prepartum and lasted for 10 weeks during lactation. The cows were housed in a loose-house system with individual feed intake measurements. The diet consisted of grass silage fed ad libitum and 12 kg of pelleted concentrate from automated feeders. The cows were assigned to three dietary treatments: Control with basal concentrate (CON), basal concentrate supplemented with tall oil fatty acids (TOFA; 9 % resin acids) at 7 g/cow/day or basal concentrate supplemented with resin acid concentrate (RAC; 37.5% resin acids) at 1.68 g/cow/day. Feed intake and milk production were measured continuously and milk samples were collected at weeks 2, 3, 6 and 10.

The proportion of concentrate averaged over all diets was 0.40 of total dry matter (DM) intake. The average energy corrected milk (ECM) production of the cows on diets CON, TOFA and RAC were 43.0, 43.3 and 43.5 kg/day and respective total DM intakes 23.4, 23.7 and 23.7 kg/day resulting in no differences in feed efficiency presented as kg ECM production per kg DM intake (1.88, 1.87 and 1.86). Milk fat concentration was 46.3, 47.9 and 44.7 g/kg while that of milk protein 39.7, 39.2 and 39.1 g/kg for diets CON, TOFA and RAC, respectively. The effect of time was statistically significant for all reported performance parameters (P<0.01), but the effect of diet and diet×time interaction were not statistically significant (P>0.05), although numerically ECM production on diets TOFA and RAC were numerically 0.29 and 0.51 kg/day higher during the 10-week observation period. It seemed that both supplements resulted in faster and higher development of milk production during the first weeks of lactation as e.g. during weeks two and three, the milk production on RAC diet was on average 2.07 kg/day higher than that on the control diet (P=0.116). Sensory analysis of milk taste and smell revealed no differences between the dietary treatments.

Further studies on rumen fermentation and blood analyses will provide clearer insight into the mode of action of resin acids.

The effects of in-feed resin acid inclusion on milk production responses of dairy cows

P. KAIRENIUS 1, P. MÄNTYSAARI1, P. LIDAUER1, M. FRANCO1, M. FRANTZI2, H. KETTUNEN2 AND M. RINNE1

1Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland

2Hankkija Ltd, FI-05800 Hyvinkää, Finland

CONCLUSIONS

In-feed resin acid supplementation resulted in faster and higher milk production peak during the first weeks of lactation. Sensory analysis of milk revealed no differences between treatments. Further studies on rumen fermentation and blood analyses will provide a clearer insight into the mode of action of resin acids.

INTRODUCTION

Resin acids, the natural compounds secreted by coniferous trees, have been shown to have antibacterial, antifungal and anti-inflammatory effects. The objective of this study was to investigate the effects of in-feed resin acids on milk production of dairy cows during early lactation.

MATERIALS AND METHODS

Feeding trial: from 3 weeks prior to the predicted parturition to 10 weeks postpartum

RESULTS

CON, Basal diet based on grass silage and concentrate

TOFA , Basal diet + 7 g/cow/day tall oil fatty acids (9 % resin acids, Progres®, Hankkija Ltd, Finland)

RAC , Basal diet + 1.68 g/cow/day resin acids concentrate (37.5 % resin acids; Forchem Ltd)

• The proportion of concentrate during lactation averaged over all diets was 0.40 of dry matter (DM) intake (P>0.10).

• The average energy corrected milk (ECM) production of the cows on diets CON, TOFA and RAC were 43.2, 43.5 and 43.6 kg/day and respective total DM intakes 23.9, 24.3 and 23.9 kg/day (P>0.05).

• No differences were found in feed efficiency presented as kg ECM production per kg DM intake (1.82, 1.80 and 1.84).

• Milk fat concentration was 46.3, 47.9 and 44.7 g/kg while that of milk protein 39.7, 39.2 and 39.0 g/kg for diets CON, TOFA and RAC, respectively.

• The proceeding lactation time was statistically significant for all reported performance parameters (P<0.01), but diet and diet×time interaction were not significant (P>0.05), although ECM production on diets TOFA and RAC were numerically 0.29 and 0.40 kg/day higher during the 10-week period.

0.63 g/cow/day resin acids

The cows were loose-housed with individual feed intake measurements. The diet consisted of grass silage ad libitum and the amount of pelleted concentrate given from automated feeders was gradually increased from 3.5 kg at parturition to 12 kg per day.

Figure 1. During lactation weeks 2 and 3, the milk production on RAC diet was on average 2.08 kg/day higher than on CON diet (P<0.075).

Abstract

The effects of in-feed resin acid composition on the colostrum composition and immunity of dairy cows

Qin N.1,3 , Karenius P. 2 , Lidauer P. 2 , Franco M. 2 , Niku M.1, Junnikkala S.1, and Kettunen H. 3

1Department of Veterinary Biosciences, University of Helsinki, 00014 Helsinki, Finland

2 Natural Resources Institute Finland (Luke), 31600 Jokioinen, Finland

3 Hankkija Ltd, FI-05800 Hyvinkää, Finland

Dairy cows prevalently undergo a subacute systemic inflammation during the transition period. A slow resolution of postpartum inflammation may predispose dairy cows to metabolic disorders and divert the nutrients that could be used for production and normal physiological functions. In-feed coniferous resin acids in sow diets have been reported to increase immunoglobulin G (IgG) concentration in colostrum and to improve piglet performance. The objective of this study was to investigate the effects of in-feed resin acids on colostrum composition of dairy cows during early and mid-lactation.

Thirty-six Nordic Red cows were assigned to a basal diet based on grass silage and concentrate (CON) and two treatment diets supplemented with the same level of resin acids, from 3 wk prior to the predicted parturition to 10 wk postpartum. The two treatment diets were the basal diet supplemented either with tall oil fatty acids (TOFA; 9 % resin acids) at 7 g/cow/day or with resin acid concentrate (RAC; 37.5% resin acids) at 1.68 g/cow/day. The first colostrum was measured with Brix refractometer and representative samples were collected for the analysis of fat, protein, lactose, urea, somatic cell count, and IgG.

The RAC group had a higher colostrum urea concentration (8.6 mg/ml) compared with the CON (6.2 mg/ ml; P < 0.05) and TOFA group (6.3 mg/ml; P < 0.05), which may reflect the modification of rumen microbiota or ruminal nitrogen metabolism by the resin acids supplement. Colostrum lactose content of the RAC group (3.0 g/kg) was lower compared with the TOFA group (3.4 g/kg; P < 0.05) and tended to be lower compared with the CON group (3.3 g/kg; P < 0.10). The decreased colostrum lactose content may reflect the decrease of blood glucose level in the RAC group, which may have possibly resulted from changes in ruminal fermentation. In contrast, the diet had no effect on colostrum fat, protein, and dry matter contents, the weight of first milking colostrum, and somatic cell count in colostrum. Moreover, colostrum IgG concentration (56.1 mg/ml for CON, 54.2 mg/ml for TOFA, and 57.6 mg/ml for RAC) and Brix value were not affected by the resin acids supplement.

In conclusion, the changes in colostrum composition reflect the potential influence of resin acids supplement on rumen microbial fermentation. Further studies on rumen microbiota will provide clearer insight into the effect of resin acids.

The effects of in-feed resin acid composition on the colostrum composition and immunity of dairy cows

N. QIN1,3, P. KAIRENIUS2, P. LIDAUER2, M. FRANCO2, M. NIKU3, S. JUNNIKKALA3, AND H. KETTUNEN1

1 Hankkija Ltd, FI-05800 Hyvinkää, Finland

2 Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland

3 Department of Veterinary Biosciences, University of Helsinki, FI-00014 Helsinki, Finland

CONCLUSIONS

In-feed resin acid concentrate increased urea concentration and decreased lactose concentration in the colostrum of dairy cows. Further studies on rumen microbiota may provide more comprehensive interpretation about the effects of resin acid supplementation.

MATERIALS AND METHODS

INTRODUCTION

Resin acids, the natural compounds secreted by coniferous trees, have been shown to have antibacterial, antifungal and anti-inflammatory effects. The dietary supplement of resin acids to sows has been found to increase immunoglobulin G (IgG) concentration in colostrum and to improve piglet performance. This poster presents the first study involving resin acids in the feed of dairy cows, in which we investigated the effects of resin acids on the colostrum composition.

Feeding trial: from 3 weeks prior to the predicted parturition to 10 weeks postpartum

Nordic Red x 36

RESULTS

x 12 x 12 x 12

CON, Basal diet based on grass silage and concentrate

TOFA , Basal diet + 7 g/cow/day tall oil fatty acids (9 % resin acids, Progres®, Hankkija Ltd, Finland)

RAC , Basal diet + 1.68 g/cow/day resin acids concentrate (37.5 % resin acids; Forchem Ltd)

• The lower colostrum lactose content in the RAC group compared with the other groups may be attributed to the changes in blood glucose level or glucose partitioning before parturition.

• The RAC group had a higher colostrum urea concentration compared with the CON and TOFA groups, which may reflect the increase of ammonia derived from ruminal breakdown of protein and thus the changes in ruminal microbial fermentation

• Colostrum fat, protein, and dry matter contents, the weight of first milking colostrum, and somatic cell count in colostrum were not affected by the supplementation.

• Colostrum IgG concentration was similar in all the groups (56.1±14.9 mg/ml for CON, 54.2±18.2 mg/ml for TOFA, and 57.6±16.8 mg/ml for RAC; P > 0.10).

• The TOFA and RAC diets had different effects on colostrum composition. This may come from the different composition of the two supplement, since the TOFA diet contains other fatty acids from coniferous tree.

0.63 g/cow/day resin acids

FUTURE STUDIES

• Rumen and gut microbiota

• Blood biomarkers of inflammation

Biomarkers of inflammation and gut permeability in dairy cows with or without dietary resin acids

Qin N.1, Niku M.1, Junnikkala S.1, Vuorenmaa, J. 2 and Kettunen H. 2

1Department of Veterinary Biosciences, University of Helsinki, 00014 Helsinki, Finland

2 Hankkija Oy, FI-05800 Hyvinkää, Finland

During the periparturient period and early lactation, dairy cows are prone to low-grade systemic inflammation which decreases their productive capacity. Inflammatory processes impair intestinal barrier functions and lead to increased gut permeability. The present study investigated the effects of coniferous resin acids on biomarkers of intestinal permeability and systemic inflammation in dairy cows during the first 10 weeks of lactation.

Thirty-six Nordic Red cows were assigned to a control diet (CON), resin acid concentrate -supplemented diet (RAC), and a tall oil fatty acid -supplemented diet (TOFA) by a randomized block design from three weeks prior to the predicted parturition until ten weeks postpartum. In both treatments, the daily dose of resin acids was adjusted to 0.65 g per cow. The cows were sampled for blood from the tail vein at wk 2, 3, 6, and 10 postpartum. The following parameters were analysed from blood plasma using commercial ELISA kits: interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), intestinal fatty acid-binding protein (I-FABP), lipopolysaccharide-binding protein (LBP), and serum amyloid A (SAA). The log-2 transformed data were analysed with repeated-measures ANOVA.

The effect of time was statistically significant for all studied parameters, likely reflecting natural changes in systemic inflammation in dairy cows during the first weeks after calving. Neither of the treatments showed significant effects on IL-1β, TNF-α, LBP, or SAA. The gastrointestinal barrier function biomarker I-FABP tended to be decreased by RAC (p < 0.1) but was not affected by TOFA. The two proinflammatory cytokines IL-6 and IL-8, as well as the anti-inflammatory cytokine IL-10 were slightly elevated by TOFA (p < 0.05) but none of them was affected by RAC. The results suggest different mechanism of action for RAC and TOFA. The used level of TOFA resulted in mild immunomodulating effects. The potential of RAC to positively influence intestinal permeability of lactating dairy cows needs to be verified in future experiments.

Effects of dietary tall oil fatty acids with resin acids on the performance and immunity of juvenile White shrimp (Litopenaeus vannamei ) with and without an intentional Vibrio parahemolyticus challenge

H. Kettunen1, J. Vuorenmaa1, O. Jintasataporn2

1Hankkija Oy, FI-05801 Hyvinkää, Finland; 2 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand

Introduction

Tall oil fatty acids with 9% natural coniferous resin acids (TOFA) is used as a feed ingredient for poultry and swine for improved production performance and beneficial effects on intestinal condition, but its potential for aquatic species has remained unexplored. The present experiment studied the effect of dietary TOFA on performance, immunity and disease resistance of white shrimp (Litopenaus vannamei) in an experimental model which included an 8-week period without intentional challenge factor, followed by a 1-week period with Vibrio parahemolyticus (V.p.) challenge.

Materials and methods

The experiment was carried out in 30 aquariums with 120 liters of 15 ppt saline water. Juvenile white shrimp were stocked at 200 shrimp/m3, 25 individual/aquarium. The water in each aquarium was aerated to maintain DO >5 mg/l, and 20% of water was changed every 3 days. Commercial-type shrimp feed, based on soybean meal, wheat flour, fishmeal, poultry meal and corn gluten was amended with TOFA (Progres®, AB Vista, UK) at 0, 0.5, and 1.0 kg/ton for the dietary treatments T1-T3, respectively. Feed was applied to the shrimp 3 times/day at 3-5% of body weight. Uneaten feed was siphoned out 1 hour after feeding, dried in hot air and weighed. Shrimp weight, weight gain, feed intake and feed conversion ratio (FCR) were recorded every 2 weeks and survival rate every 4 weeks. At the 8-wk time point, 6 shrimp/tr. were sampled for haemolymph, hepatopancreas and intestinal contents. The experiment continued by allocating 3 replicate aquariums/tr. into a 7-day challenge period which started by a subcutaneous injection of the virulent EMS strain of V.p. (6.9*107 cfu/ml) to each shrimp. The survival rate was monitored daily. On day 7 post-challenge, the shrimps were sampled as described earlier. Haemolymph samples were analysed for parameters relating to immunity and oxidative status. Density of Vibrio spp. in samples was determined by plate culturing. The study was conducted in a completely randomize design. Data was analysed with one-way ANOVA, followed by Duncan’s Multiple Range Test, using p<0.05 as a limit for statistical significance.

Results

Results are summarized in Table 1. On day 1, the mean shrimp weight was 2.46 g, with no difference between treatments. Compared to T1 (control) treatment, shrimp weight, weight gain and FCR were improved by T2 and T3 from the 4-wk time point onwards (p <0.05). Post-challenge survival rate was increased from 32.5% in the control group to 55.0 and 65.0% in T2 and T3, respectively (p <0.05). Compared to T1, the haemocyte count, phenoloxidase activity, lysozyme activity, superoxide dismutase activity and glutathione concentration in haemolymph were all significantly higher, while the density of Vibrio spp. in tissues was lower for T2 and T3 both before and after the challenge. Haemolymph protein was increased by TOFA only post-challenge.

Discussion and Conclusions

In the present study, dietary TOFA at 0.5 and 1.0 kg/ton improved the performance and survival rate of juvenile white shrimp, suggesting a better production potential for TOFA-fed shrimps, in comparison to

Aquaculture abstracts

the control group. The decreased Vibrio load in the tissues and the parameters analysed from haemolymph indicate that TOFA positively affected several immunological and antioxidative functions and the disease resistance of the shrimp. Previously, in-feed resin acids have been shown to reduce intestinal inflammatory processes (Aguirre et al. 2019) and to positively modulate gut microbiota (Vienola et al., 2018) in chicken. In conclusion, TOFA may become a dietary strategy for supporting the performance, immunity and disease resistance of white shrimp.

Table 1. Effect of the treatments on parameters measured at the 8-week time point (pre- challenge) and 7 days post-challenge, with statistical evaluation by ANOVA and Duncan’s Multiple Range Test. Item

1 TOFA 0 kg/ton, 2 TOFA 0.5 kg/ton, 3 TOFA 1.0 kg/ton, 4 feed conversion ratio, 5 Vibrio parahemolyticus, 6 colony forming units, a, b, c Different letters in the same row indicate significant differences between treatments (p<0.05).

References

Aguirre, M., Vuorenmaa, J., Kettunen, H., Valkonen, E., Callens, C., Haesebrouck, F., Ducatelle, R., Van Immerseel, F., Goossens, E., 2019. In-feed resin acids reduce matrix metalloproteinase activity in the ileal mucosa of healthy broilers without inducing major effects on the gut microbiota. Veterinary Research 50: 15.

Vienola, K., Jurgens, G., Vuorenmaa, J., Apajalahti, J., 2018. Tall oil fatty acid inclusion in the diet improves performance and increases ileal density of lactobacilli in broiler chickens. British Poultry Science. 59: 349–355.

Aquaculture posters

Effects of dietary tall oil fatty acids with resin acids on the performance and immunity of juvenile white shrimp (Litopenaeus vannamei) with and without an intentional Vibrio parahemolyticus challenge

INTRODUCTION

H. Kettunen1, J. Vuorenmaa1, O. Jintasataporn2

1Hankkija Oy, Hyvinkää, Finland; 2Kasetsart University, Bangkok, Thailand

❑ Tall oil fatty acids (TOFA) is a feed material with 9% natural resin acids of coniferous trees Resin acids have antimicrobial and anti-inflammatory properties

❑ TOFA is used in poultry and swine diets for improved production performance and intestinal condition, but it has not been studied in aquatic species

❑ Vibrio parahemolyticus (V p ) is a common pathogen of white shrimp (Litopenaeus vannamei) in Thailand

❑ The aim: to study the effect of dietary TOFA at 0 5 and 1 0 kg/ton on the performance and immunity of white shrimp

❑ The protocol included an 8-week period without a challenge factor, followed by a 7-day V p challenge

CONCLUSIONS

Dietary TOFA significantly improved growth, survival and immunity of white shrimp, and reduced Vibrio load in the tissues TOFA may become a dietary strategy for supporting the performance and disease resistance of white shrimp

RESULTS AND DISCUSSION

Dietary TOFA significantly improved shrimp performance (data in the abstract), and survival rate after the challenge (Figure 1), compared to the control treatment Vibrio count in the tissue samples was significantly decreased by TOFA, especially post-challenge (Figure 2) Many physiological and immunity-related parameters were increased by both TOFA dosages (Table 1) These results suggest improved immunity, disease resistance and performance of juvenile white shrimp with dietary TOFA amendment

MATERIALS AND METHODS

Dietary treatments and study outline

T1: TOFA at 0 kg/ton (Control)

T2: TOFA at 0.5 kg/ton

T3: TOFA at 1.0 kg/ton

• Test material: TOFA (Progres®, AB Vista, UK).

• 30 aquariums with 120 liters of 15 ppt saline water, at Kasetsart University, Thailand.

• 25 juvenile white shrimp/aquarium.

• Feed was applied 3x/day, 3-5% of body weight. Uneaten feed was removed, dried, and weighed.

• 6 shrimp/tr. were sampled for haemolymph, hepatopancreas and intestinal contents at 8 wk.

Challenge period

• 3 replicate aquariums/treatment.

• Vibrio parahemolyticus challenge by a subcutaneous injection of the virulent EMS strain (6.9*107 cfu/ml) to each shrimp.

• Survival rate was monitored daily.

• Sampling for haemolymph, hepatopancreas and intestinal contents on day 7 post-challenge.

Data analysis

• Completely randomized design.

• One-way ANOVA, followed by Duncan’s Multiple Range Test.

• p<0.05 as a limit for statistical significance.

Table 1. Effects of the dietary treatments on parameters measured from haemolymph samples at the 8-week time point (prechallenge) and 7 days post-challenge.

Figure 1. Shrimp survival rate, %

Figure 2. Vibrio count in the tissue samples, (log10 cfu/ml), 7 days post-challenge

Abstract

Effect of dietary tall oil fatty acids (TOFA) on performance, immunological status, and pathogen ( Aeromonas spp.) load of juvenile Asian seabass (Lates calcarifer)

H. Kettunen1, J. Vuorenmaa1, O. Jintasataporn2

1Hankkija Oy, FI-05801 Hyvinkää, Finland; 2 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand

Introduction

Tall oil fatty acids with 9% natural coniferous resin acids (TOFA) is a novel feed ingredient which improves the production performance and intestinal microbiota of farm animals (e.g. Vienola et al., 2018, Uddin et al., 2021). Resin acids are anti-inflammatory and antibacterial secondary metabolites of coniferous trees (San Feliciano et al., 1993). The present study investigated the effects of dietary TOFA on the performance, immunological status and load of the pathogenic bacterium Aeromonas spp. in the intestinal tract and liver of juvenile Asian seabass (Lates calcarifer) in Thailand.

Materials and methods

Juvenile Asian sea bass were allocated into 20 freshwater cages of 2 m3, 15 fish/cage, at a site with a known background challenge of Aeromonas histolytica. The water was aerated to maintain DO >5 mg/l. Feed was of commercial-type, with main ingredients of fishmeal, poultry meal, soybean meal and tapioca, and it was amended with 0 (Control), 0.35, 0.7, or 1.0 kg/ton of TOFA (Progres®, AB Vista, UK) for treatments T1-T4, respectively. The fish were fed 3 times/day at 3-5% of body weight for 16 weeks. Fish weight, weight gain, feed intake and feed conversion ratio (FCR) were determined every 2 wk, and the survival rate every 4 wk. Two fish/replicate were sampled for blood at 8- and 16-wk time points for determining the red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin and haematocrit, and the concentrations of total serum protein and immunoglobulin M (IgM). The same fish were sampled for liver and intestinal contents for determining the number of Aeromonas spp. by plate culturing, using Aeromonas-selective media. The study design was completely randomized. Data was analysed with one-way ANOVA, followed by Duncan’s Multiple Range Test, using P<0.05 as a limit for statistical significance.

Results

Mean weight of the fish was 54.6 g, 111.8 g, and 170.0 g for wks 0, 2, and 4, respectively, without a significant difference between treatments. Dietary TOFA supplementation dose-dependently improved fish weight and weight gain from the 6-wk time point onwards (p<0.05). Table 1 presents fish weight on wks 8 and 16, and FCR for wks 0-8 and 0-16. Feed intake and fish survival rate was not affected by the treatments. At the end of the study, the survival rate was 89.3%, 92.0%, 93.3% and 96.0% for T1-T4, respectively. The treatments significantly affected FCR for the following time periods: wks 0-6, 0-8, 0-10, and 0-12 (p <0.001), with the best performance in T4.

Parameters analysed from blood and tissue samples are reported in Table 1. The TOFA supplementation significantly increased haematocrit (wk 8) and IgM (wks 8 and 16), and decreased Aeromonas spp density in the liver and intestinal tract (wk 16).

Discussion and conclusions

In the present study, supplementing the diet of juvenile Asian seabass with TOFA promoted the growth performance, feed conversion and immunity of the fish, and at the same time improved their ability to control the growth of Aeromonas spp. in the liver and intestinal tract. Increased haematocrit and the trend towards higher serum protein levels in TOFA-supplemented groups may indicate positive effects on

Aquaculture abstracts

the physiological status of the fish. The effects were dose-dependent, and the treatment with TOFA at 1.0 kg/ton gave the highest responses. The results were in line with previous observations from studies with poultry and swine, in which dietary TOFA had positive effects on performance, gastrointestinal microbiota and disease resistance of animals (Vienola et al., 2018; Uddin et al., 2021). In conclusion, TOFA may function as a dietary strategy to improve the production performance and disease resistance of Asian seabass.

1 TOFA 0 kg/ton, 2 TOFA 0.35 kg/ton, 3 TOFA 0.7 kg/ton, 4 TOFA 1.0 kg/ton, 5 feed conversion ratio, 6 red blood cell, 7 white blood cell, 8 colony forming units, a, b, c Different letters in the same row indicate significant differences between treatments (p<0.05).

References

San Feliciano A., Gordaliza M., Salinero M.A., Del Corral J.M., 1993. Abietane acids; sources, biological activities, and therapeutic uses. Planta Medica, 59: 485–490.

Uddin, M.K., Hasan, S., Mahmud, M.R., Peltoniemi, O., Oliviero, C., 2021. In-feed supplementation of resin acid-enriched composition modulates gut microbiota, improves growth performance, and reduces post-weaning diarrhea and gut inflammation in piglets. Animals 11: 2511.

Vienola, K., Jurgens, G., Vuorenmaa, J., Apajalahti, J., 2018. Tall oil fatty acid inclusion in the diet improves performance and increases ileal density of lactobacilli in broiler chickens. British Poultry Science. 59: 349–355.

Table 1. Effect of the treatments on parameters measured at 8 and 16 weeks, with statistical evaluation by ANOVA and Duncan’s Multiple Range Test.

Abstract

Inhibition studies of natural resin acids to Clostridium perfringens and Escherichia coli O149

Krisna Roy, Ulrike Lyhs and Karl Pedersen

National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Copenhagen, Denmark

Introduction

As feed antibiotics are no longer allowed in Europe and some other countries, natural means to control the proliferation of pathogenic bacteria in the intestinal lumen of animals are needed. Natural resin acids, for example, abietic acid, dehydroabietic acid, isopimaric acid and palustric acid are such natural products which have antimicrobial properties. However, there is limited data available in the literature on the effects of resin acids on pathogens. In the present study, we aimed to evaluate the inhibitory activity of a resin-based product at different concentrations on intestinal bacterial pathogens.

Materials and methods

The targeted product was Progres® (Suomen Rehu) containing 8% resin acids. Clostridium perfringens isolated from chickens, turkeys and pigs, respectively, and Escherichia coli O149 from pigs were tested. Growth of the pathogens was tested at 0.01%, 0.1% and 0.5% concentrations of the product. Inhibitory bioactivity of the product was examined following optical density (OD600nm) measurements on growing cultures, by a 10-fold broth dilution method (DM), and by using an agar diffusion method (ADM). The OD method was followed only in one strain of E. coli O149 with readings at 2, 4, 8, and 24 hours. The DM was applied to three strains of each of the bacteria. Samples were taken after 2, 5, 8, and 24 h incubation, where after 10-fold dilutions were made and plated onto blood agar plates. Counts were expressed as colony forming unit per ml (cfu/ml). The ADM was run on one strain of each of the bacteria, where zones of inhibition (mm) were measured against the aforementioned product concentrations. Subsequently, all these strains and seven additional strains of Cl. perfringens (total ten Cl. perfringens strains: five from pigs, four from chickens and one from turkey) were tested with ADM against 0.5%, 1% and 5%.

Results

In general OD measurements were difficult to interpret due to a considerable contribution of the test product to the turbidity of the cultures. Therefore c.f.u. measurements were considered more accurate. In DM, no Cl. perfringens was found at any concentration of the product, indicating an efficient inhibition of Cl. perfringens. At 0.1% and 0.5% of the product, there was apparently lower cfu/ml of two strains of E. coli O149 compared to the corresponding controls (difference from control was ≥4.40 x 108 cfu/ml), but E. coli was considerably less inhibited than Cl. perfringens. In ADM, zone of inhibition (ZI) was evolved around the product-concentration of 0.5% (ZI: 8 to 10 mm), 1% (8.5 to 12.0 mm), and 5% (9.0 to 19.5 mm) when performed on all ten strains of Cl. perfringens. No strain of E. coli O149 was inhibited by the product at any concentration in ADM, and not at 0.01% in DM.

Conclusion

Cl. perfringens was inhibited even at lower concentration of the product containing resin acids, but there seemed to be some strain variation. E. coli O149 was only inhibited by high concentrations.

APP. 1. ANNOTATED LIST OF SCIENTIFIC ARTICLES

Resin acids in farm animal nutrition: the peer-reviewed studies

Discovering the potential of coniferous resin acids in farm animal nutrition and developing the tall oil fatty acid -based Progres® product has been a research-driven process of thirteen years. This process was started by the Finnish company Hankkija FFI. Its efficient, top-quality research network revealed the mechanism-of-action of coniferous resin acids in farm animals and to proved the commercial value of Progres® in animal feeding.

AB Vista, which acquired Progres® in 2023, will go on with the product's development. This collection presents the research carried out until 2022. This far, resin acid research program has produced 14 peer-reviewed scientific papers and 33 congress publications.

The peer-reviewed research articles, introduced here by Dr. Hannele Kettunen, reveal the evolution of knowledge on the value of resin acids in farm animal nutrition.

Year 2015

1. Kettunen, H., Rinttilä, T., Grönberg, H., Valkonen, E. and Apajalahti, J. (2015) Natural resin acid-enriched composition as a modulator of intestinal microbiota and performance enhancer in broiler chicken. Journal of Applied Animal Nutrition 3: 1–9.

Even before the research project of Hankkija, the efficacy of resin acids against some Gram-positive pathogenic bacteria had been scientifically proven. However, there was no information on the effect of resin acids on farm animal -specific strains of these pathogens. The series of Progres®- publications started with a paper including in vitro -results on the efficacy of Progres® against Clostridium perfringens, the causative agent of necrotic enteritis (NE) in poultry. It also included results of a NE-challenge trial with broiler chickens indicating better body weight and reduced mortality in Progres®-amended diet groups. The study was conducted at Alimetrics Research Ltd., the invaluable partner of Hankkija in very many R&D-projects over almost two decades.

Year 2017

2. Kettunen, H., van Eerden, E., Lipiński, K., Rinttilä, T., Valkonen, E. and Vuorenmaa, J. (2017) Dietary resin acid composition as a performance enhancer for broiler chickens. Journal of Applied Animal Nutrition 5: 1–8.

Quite soon it became evident that Progres® especially well supports the performance of broiler chickens which live in challenging conditions. Different challenge factors, such as temperature, stocking density, or Eimeria -inoculation with or without Clostridium perfringens, were tested. The second Progres®-article described two broiler studies, one conducted in Poland and the second in The Netherlands. At the University of Warmia and Mazury in Poland, Progres® was compared against the chemical coccidiostat diclazuril and the ionophoric coccidiostat salinomycin in conditions which represent the challenge level in commercial broiler houses. Progres® improved the body weight gain by 3.3% and feed conversion ratio by 5.7% with diclazuril, and improved footpad lesion scores with salinomycin. The study was conducted by professor Lipiński, with whom Hankkija has collaborated for a long time. The second experiment was conducted at Schothorst Feed Research, another very important research partner for Hankkija. The experiment re-enforced previous findings by proving the performance-enhancing capacity of Progres® in NE-challenged broiler chickens.

3. Roy, K., Lyhs, U., Vuorenmaa, J. and Pedersen, K. (2018) In vitro inhibition studies of natural resin acids to Clostridium perfringens, Staphylococcus aureus and Escherichia coli O149. Journal of Applied Animal Nutrition 5: 1–5.

The effect of Progres® on the growth of three pathogenic bacteria, Clostridium perfringens, Staphylococcus aureus and Escherichia coli O149, was studied in vitro at the National Veterinary Institute of Denmark. The bacterial strains originated from chicken, turkey, pig, or cattle. The study highlighted the efficacy of Progres® against Clostridium perfringens and Staphylococcus aureus. In contrast, E. coli as a Gram-negative bacterium was proven to be relatively resistant against Progres®.

4. Vienola, K., Jurgens, G., Vuorenmaa, J. and Apajalahti, J. (2018) Tall oil fatty acid inclusion in the diet improves performance and increases ileal density of lactobacilli in broiler chickens. British Poultry Science. 59: 349–355.

The second Progres® -article from Alimetrics Research Ltd focussed on the in vivo -effects of the product in two 35-day broiler trials. In both trials, Progres® improved body weight gain at days 14, 21 and 35, and feed conversion efficiency during the first 21 days. Progres® significantly increased ileal lactobacilli density on day 14, which also correlated positively with broiler body weight on day 14. It was also shown in vitro that C. perfringens was strongly inhibited by Progres® at 10 mg/l while common lactobacilli were resistant to >250 mg/l. Thus, the beneficial Gram-positive lactobacilli tolerate much higher concentrations of Progres® than pathogenic clostridia. Even more noteworthy was that Progres® also supported the growth of lactobacilli in the ileum of live birds.

5. Hasan, S., Saha, S., Junnikkala, S., Orro, T., Peltoniemi, O. and Oliviero, C. (2018) Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum IgG content, piglet colostrum intake and modulates sow gut microbiota. Animal 13: 1599-1606.

The first swine-specific Progres® article was published on 2018 by Dr. Shah Hasan from the University of Helsinki, Finland. The study investigating the effects of Progres® on sow colostrum yield and quality, and on the pre-weaning performance of the offspring was conducted in three trials in three respective herds: two in Finland and one in The Netherlands. Sows were fed either a control diet or the same diet supplemented with 5 g Progres®/day /sow during the last week of gestation. Progres® significantly increased the IgG levels of colostrum in all three herds, and also increased the quantity of colostrum in one herd. At weaning, piglets from Progres®-fed sows were heavier in two herds. In sow feces, Progres® significantly increased some beneficial and fermentative bacteria while some opportunistic pathogens were suppressed. Thus, the publication proved that Progres® amendment to sow diets benefits the performance of sows and piglets.

Year 2019

6. Aguirre, M., Vuorenmaa, J., Kettunen, H., Valkonen, E., Callens, C., Haesebrouck, F., Ducatelle, R., Van Immerseel, F. and Goosens, E. (2019) In-feed resin acids reduce matrix metalloproteinase activity in the ileal mucosa of healthy broilers without inducing major effects on the gut microbiota. Veterinary Research 50: 15.

The mechanism-of-action of Progres® really started to unravel with the beginning of the research collaboration with professors Filip van Immerseel and Richard Ducatelle from the University of Ghent, Belgium. Their team, including Dr. Marisol Aguirre, was able to demonstrate that resin acids ingested by broiler chickens reduce the inflammation-associated collagen breakdown in the intestinal epithelium. The collagen degradation during inflammation is caused by the increased expression and activity of specific matrix metalloproteinase enzymes, of which MMP-7 is the most important. In the experiment, Ross 308 broilers were fed a diet supplemented with coniferous resin acids for 22 days before multiple samples from intestine were collected. In-feed resin acids did not alter the morphology of the small-intestine, and

only minor effects on the intestinal microbiota were observed. However, the resin acids reduced the MMP activity towards collagen type I and type IV in jejunum and ileum, and lowered the density of inflammatory T-cells in duodenum. These hugely beneficial effects reduce the negative effects of proinflammatory challenge to the gut, regardless of the type of challenge. Progres® is the first and only natural feed ingredient with this proven mechanism-of-action.

Year 2020

7. Apajalahti, J., Vienola K., Raatikainen K., Kettunen, H. and Vuorenmaa, J. (2020) Distribution, metabolism, and recovery of resin acids in the intestine and tissues of broiler chickens in a feeding trial with tall oil fatty acid-supplemented diets. Frontiers in Veterinary Science 7: 437. As resin acids had not been fed to farm animals before the invention of Progres®, it was necessary to study their intestinal uptake and body distribution in farm animals. This research was conducted at Alimetrics Research Ltd, and it was published by Dr. Apajalahti. A 35-day broiler trial was conducted, with Progres® in feed at 0, 750 and 3,000 g/t, the highest dose being a 3-4 -fold overdose compared to commercial dietary levels. The diets also included an indigestible marker to quantify resin acid uptake in different intestinal compartments. From digesta, both free and conjugated resin acids were quantified. At least 75% of resin acids ingested were absorbed in duodenum and jejunum. Approximately 45% of ingested resin acids were conjugated in bile, and re-entered the digestive tract in conjugated form. Recovery of resin acids in excreta was 45% and 70% when TOFA was fed at 750 and 3,000 g/t, respectively. Only trace amounts of resin acids were found in jejunal tissue, blood, liver, bile, breast muscle, and abdominal fat. The concentrations of resin acids in the edible parts of broiler chickens remained so low that feeding resin acids to broiler chickens brings no concerns from the consumer safety perspective. Thus, most of the resin acids in Progres® are absorbed from the proximal small intestine, are sent back to the gut lumen via bile in a conjugated form, and voided via feces. The gastrointestinal microbiota and/or the host may modify part of the resin acids, since a maximum of 70% of feed-derived resin acids were found in the feces.

8. Lipiński, K., Vuorenmaa, J., Mazur-Kuśnirek, M. and Antoszkiewicz, Z. (2020) Effect of resin acid composition on growth performance, footpad dermatitis, slaughter value, and gastrointestinal tract development in turkeys. Journal of Applied Poultry Research 30: 100112. The benefits of Progres® to turkeys was studied at the University of Warmia and Mazury in Poland by professor Lipiński and his team. A total of 600 BIG 6 turkeys were allocated to the 105-day feeding trial with three dietary treatments, 10 replicates, and 20 birds per replicate. Progres® significantly improved feed conversion ratio and the European Efficiency Index, reduced the incidence of footpad lesions and improved litter quality, but had no effects on carcass quality or meat quality, and on most parameters of the GIT structure and function.

Year 2021

9. Uddin, M.K., Hasan, S., Mahmud, M.R., Peltoniemi, O. and Oliviero, C. (2021) In-feed supplementation of resin acid-enriched composition modulates gut microbiota, improves growth performance, and reduces post-weaning diarrhea and gut inflammation in piglets. Animals 11: 2511.

Led by professors Olli Peltoniemi and Claudio Oliviero from the Veterinary Faculty of the University of Helsinki, the PhD student Karim Uddin examined the effect of Progres® on sow and piglet performance and gut health. First, 40 sows were divided into Control and Progres® diet groups six weeks before the expected parturition. The Progres® dosing for sows was 1.5 kg/ton. From both groups, the piglets were divided into Control and Progres® groups, with Progres® dose of 1.0 kg/ton. At weaning, the piglets from all groups were divided into Control and Progres® groups, again with Progres®-dose of 1.0 kg/ton. The trial ended when the piglets were seven weeks of age. The results showed that Progres® supplementation to sows and piglets favorably modulated gut microbiota, reduced inflammatory

biomarkers, improves growth performance of post-weaning piglets, and very efficiently prevented post-weaning diarrhoea and piglet mortality. The best piglet performance was in the group where Progres® was fed first to sows and then to piglets during both the pre-weaning and weaning periods. The authors concluded that Progres® supplementation represents a potential strategy to improve piglet growth performance, and is a natural alternative to antibiotics for preventing post-weaning diarrhoea.

10. Guan, X., Santos, R., Kettunen, H., Vuorenmaa, J. and Molist, F. (2021) Effect of resin acid and zinc oxide on immune status of weaned piglets challenged with E. coli lipopolysaccharide. Frontiers in Veterinary Science 8:761742. doi: 10.3389/fvets.2021.761742

The banning of high dosages of zinc oxide (ZnO) in piglet diets in Europe led to a surge of research efforts to find effective alternatives to medical ZnO. From Schothorst Feed Research, Dr. Francesc Molist and Dr. Xiaonan Guan took the task to compare dietary resin acids to a high dose of ZnO in piglets. They evaluated the effect of ZnO at 2,500 mg/kg diet and resin acid concentrate (RAC) at 200 mg/kg diet on the immune defense of weaned piglets. The trial involved a challenge with lipopolysaccharide (LPS), which is a pro-inflammatory molecule originating from the outer membrane of Gram-negative bacteria, in this case from E. coli. In the experiment, piglets were challenged at days 7 and 21 post-weaning. They were sampled for blood at 1.5 and 3.0 h after each challenge to determine serum levels of pro- and anti-inflammatory cytokines. Blood samples were also taken on day 14 when no challenge was given. The results showed that on day 14 without the challenge, neither ZnO nor RAC stimulated cytokine secretion. At days 7 and 21, both ZnO and RAC increased the serum levels of several cytokines after the LPS challenge. The anti-inflammatory cytokine IL-10 was much more elevated in the RAC-group than in the ZnO-group. The authors concluded that in-feed resin acids appear as a candidate to replace the medical zinc in piglet diets.

11. Lipiński, K., Vuorenmaa, J., Mazur- Kuśnirek, M., Sartowska-Żygowska, K., Kettunen, H. (2021) Dietary resin acid concentrate improved performance of broiler chickens and litter quality in three experiments. Animals 11: 3045.

In the three broiler trials conducted and published by Prof. Lipiński at the University of Warmia and Mazury in Poland, broiler chicken diets were amended with a resin acid concentrate (RAC) product at 125–250 g/ton. Bird weight gain increased, and the efficiency of broiler production was improved. Furthermore, the litter material of pens stayed drier in RAC groups, which indicates better intestinal condition of the birds. The results suggest that the resin acid concentrate improves bird performance and the efficiency of broiler production.

12. Kairenius, P., Qin, N., Tapio, I., Mäntysaari, P., Franco, M., Lidauer, P., Stefański, T., Lidauer, M.H., Junnikkala, S., Niku, M., Kettunen, H. and Rinne, M. (2021) The effects of dietary resin acid inclusion on productive, physiological and rumen microbiome responses of dairy cows during early lactation. Livestock Science 255: 104798.

Time around calving is a critical period in the life of a dairy cow, as it involves metabolic stress and carries a high risk of inflammatory conditions. Progres® has been shown to alleviate inflammation in poultry and swine, so it was of interest to test its effects on dairy cows around parturition. Two product forms were tested: the regular Progres® Liquid (7 grams/cow/day) and a resin acid concentrate (RAC; 1.7 g/cow/day). The experiment was conducted at the Natural Resources Institute Finland, which is another trusted research partner of Hankkija. The experiment started three weeks before calving and ended after ten weeks of lactation. The organic matter and neutral detergent fibre digestibility of was higher with Progres®, and Progres® also resulted in mild immunomodulatory effects. It is noteworthy that RAC significantly reduced the number of cows with sickness or inflammation by 55% and 38%, respectively, and reduced the number of cow-days with sickness or inflammation by 44% and 36%, respectively. The onset of milking seemed to be faster with both test materials than in the control group, but the overall lactation during the 10 weeks was similar in all groups.

13. Bayat, A.R., Vilkki, J., Razzaghi, A., Leskinen, H., Kettunen, H., Khurana, R., Brand, T. and Ahvenjärvi, S. (2021) Evaluating the effects of high-oil rapeseed cake or natural additives on methane emissions and performance of dairy cows. Journal of Dairy Science 2021 Nov 16:S0022-0302(21)01014-6.

The second Progres® trial with dairy cows, also conducted at the Natural Resources Institute Finland, studied the ability of rapeseed cake and some natural additives to reduce methane emissions. Progres® (15 g/cow/day) was tested in combination with Hankkija’s another feed innovation, the yeast hydolysate Progut® Extra (30 g/cow/day). The rapeseed cake diet was very successful in reducing the methane emissions in this experiment, while the tested additives showed no effect on methane emissions. Year 2022

14. Kettunen, H., Bento, H., Lewis, E., Remington, S., Matthews, A., Hendrix, S., and Vuorenmaa, J. (2022) Tolerability of graded levels of tall oil fatty acids as a nutritional additive for broiler chickens: a 45-day target animal safety study. British Poultry Science. DOI: 10.1080/00071668.2022.2106776.

The benefits of Progres® on the performance and well-being have been proven over the years for many farm animal species. However, entry to the North American markets can only open after a specific Target Animal Safety (TAS) study. The TAS-protocol was first applied to broiler chickens. The study was conducted at the Colorado Quality Research Inc. in the USA. The feed of Cobb 500 broiler was amended with Progres® at 0, 1, 3 and 5 kg/ton, and fed to the birds for 42 days. The birds were weighed individually on days 0, 16, 31, and 45. Bird performance was determined for starter, grower and finisher phases, and over the whole study. On day 42, every bird was sampled for blood for haematology and blood chemistry measurements, and two birds per pen were subjected to gross pathological examination and sampling of several tissues for histopathology. Bird performance was typical for Cobb 500 -broilers and similar for all diet groups. Progres® had no effect on clinical chemistry or histopathological parameters. The authors concluded that feeding Progres® to broiler chickens for 45 days had no negative effects, and that the product is well tolerated by the chickens up to the level of 5.0 kg/ton of feed.

APP. 2 LIST OF PUBLICATIONS

Published peer-reviewed articles:

Aguirre, M., Vuorenmaa, J., Kettunen, H., Valkonen, E., Callens, C., Haesebrouck, F., Ducatelle, R., Van Immerseel, F. and Goosens, E. (2019) In-feed resin acids reduce matrix metalloproteinase activity in the ileal mucosa of healthy broilers without inducing major effects on the gut microbiota. Veterinary Research 50: 15.

Apajalahti, J., Vienola K., Raatikainen K., Kettunen H. and Vuorenmaa, J. (2020) Distribution, metabolism, and recovery of resin acids in the intestine and tissues of broiler chickens in a feeding trial with tall oil fatty acid-supplemented diets. Frontiers in Veterinary Science 7: 437.

Bayat, A.R., Vilkki, J., Razzaghi, A., Leskinen, H., Kettunen, H., Khurana, R., Brand, T. and Ahvenjärvi, S. (2021) Evaluating the effects of high-oil rapeseed cake or natural additives on methane emissions and performance of dairy cows. Journal of Dairy Science 2021 Nov 16:S0022-0302(21)01014-6.

Guan, X., Santos, R., Kettunen, H., Vuorenmaa, J. and Molist, F. (2021) Effect of resin acid and zinc oxide on immune status of weaned piglets challenged with E . coli l ipopolysaccharide. Frontiers in Veterinary Science 8:761742. doi: 10.3389/fvets.2021.761742

Hasan, S., Saha, S., Junnikkala, S., Orro, T., Peltoniemi, O. and Oliviero, C. (2018) Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum IgG content, piglet colostrum intake and modulates sow gut microbiota. Animal 13: 1599-1606.

Kairenius, P., Qin, N., Tapio, I., Mäntysaari, P., Franco, M., Lidauer, P., Stefański, T., Lidauer, M.H., Junnikkala, S., Niku, M., Kettunen, H. and Rinne, M. (2021) The effects of dietary resin acid inclusion on productive, physiological and rumen microbiome responses of dairy cows during early lactation. Livestock Science 255: 104798.

Kettunen, H., Bento, M. H. L., Lewis, E. A., Remington, S., Matthews, A., Hendrix, S. and Vuorenmaa, J. (2022) Tolerability of graded levels of tall oil fatty acids as a nutritional additive for broiler chickens: a 45-day target animal safety study. British Poultry Science, DOI:10.1080/00071668.2022.2106776.

Kettunen, H., van Eerden, E., Lipiński, K., Rinttilä, T., Valkonen, E. and Vuorenmaa, J. (2017) Dietary resin acid composition as a performance enhancer for broiler chickens. Journal of Applied Animal Nutrition 5: 1–8.

Kettunen, H., , Rinttilä, T., Grönberg, H., Valkonen, E. and Apajalahti, J. (2015) Natural resin acid-enriched composition as a modulator of intestinal microbiota and performance enhancer in broiler chicken. Journal of Applied Animal Nutrition 3: 1–9.

Lipiński, K., Vuorenmaa, J., Mazur-Kuśnirek, M. and Antoszkiewicz, Z. (2020) Effect of resin acid composition on growth performance, footpad dermatitis, slaughter value, and gastrointestinal tract development in turkeys. Journal of Applied Poultry Research 30: 100112.

Lipiński, K., Vuorenmaa, J., Mazur- Kuśnirek, M., Sartowska-Żygowska, K. and Kettunen, H. (2021) Dietary resin acid concentrate improved performance of broiler chickens and litter quality in three experiments. Animals 11: 3045.

Roy, K., Lyhs, U., Vuorenmaa, J. and Pedersen, K. (2018) In vitro inhibition studies of natural resin acids to Clostridium perfringens, Staphylococcus aureus and Escherichia coli O149. Journal of Applied Animal Nutrition 5: 1–5.

Uddin, M.K., Hasan, S., Mahmud, M.R., Peltoniemi, O. and Oliviero, C. (2021) In-feed supplementation of resin acid-enriched composition modulates gut microbiota, improves growth performance, and reduces post-weaning diarrhea and gut inflammation in piglets. Animals 11: 2511.

Vienola, K., Jurgens, G., Vuorenmaa, J. and Apajalahti, J. (2018) Tall oil fatty acid inclusion in the diet improves performance and increases ileal density of lactobacilli in broiler chickens. British Poultry Science. 59: 349–355.

Congress abstracts

Guan, X., Kettunen, H., Santos, R., Vuorenmaa, J. and Molist F. (2021) Effect of dietary resin acid and zinc oxide in weaned piglets subjected to an immune challenge. Abstract in: 72nd annual meeting of European Federation of Animal Science (EAAP) August 30 – September 3, 2021, Davos, Switzerland (Presentation, Abstract)

Hasan, S., Junnikkala, S., Peltoniemi, O. and Oliviero, C. (2017) Dietary supplementation of tall oil fatty acid and resin acid in farrowing sows can affect the colostrum quality. At: The 9 th European Symposium of Porcine Health Management (ESPHM) 2017, Prague, Czech Republic. (Poster)

Hasan, S., Kamyzek, M., Vuorenmaa, J., Valkonen, E. and Kettunen, H. (2019) Dietary resin acid supplementation improves the performance of sows and piglets. At: 3rd International Symposium on Alternatives to Antibiotics (ATA) December 16–18, 2019, Bangkok, Thailand. (Poster)

Hasan, S., Saha, S., Yun, J., Peltoniemi, O. and Oliviero, C. (2018) Dietary supplementation with tall oil fatty acid and resin acid increases sow colostrum IgG and piglets’ colostrum intake in free farrowing pen. At: The 10 th European Symposium of Porcine Health Management (ESPHM) 2018, Barcelona, Spain. (Presentation, Abstract)

Hasan, S., Sani, S., Junnikkala, S., Orro, T., Peltoniemi, O. and Oliviero, C. (2018) Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum IgG, piglet colostrum intake and modulates sow gut microbiota. At: 14 th International Symposium on Digestive Physiology of Pigs, 21–24 August 2018, Brisbane, Australia. (Presentation, Abstract)

Kairenius, P., Mäntysaari, P., Lidauer P., Franco M., Frantzi, M., Kettunen H. and Rinne, M. (2019)

The effects of in-feed resin acid inclusion on milk production responses of dairy cows. At: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019. (Poster)

Kettunen, H., Apajalahti, J., Valkonen, E., Rinttilä, T., Grönberg, H. and Vuorenmaa, J. (2014) A novel, resinbased dietary ingredient reduces the risk of necrotic enteritis in turkeys. At: The XIVth European Poultry Conference (EPC) 2014, Stavanger, Norway. (Poster)

Kettunen, H., Frantzi, M., Vuorenmaa, J., Breves, G. and Eger, M. (2019) Resin acid composition increases propionate and butyrate production in a Rumen Simulation Technique (RUSITEC) model. At: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019. (Poster)

Kettunen, H., Hasan, S., Suokanto, M., Valkama, S., Daka, J., Vuorenmaa, J. and Heikkilä, T. (2022) Raising weanling piglets without medical doses of zinc: an example from Finland. Abstract in: ZeroZincSummit2022, Copenhagen, Denmark, 22-23 June, 2022. (Poster)

Kettunen, H., Hasan, S., Uddin, K., Oliviero, C., Molist, F., Guan, X., Santos, R. and Vuorenmaa, J. (2022) Dietary resin acids improved performance of sows and piglets and acted as an immune modulator for piglets. Abstract in: ZeroZincSummit2022. Copenhagen, Denmark, 22–23 June, 2022. (Presentation, Abstract)

Kettunen, H., Hayat, Z., Hasan, S., Vuorenmaa and J., Khan, M.Z.U. (2022) Effects of dietary tall oil fatty acids on broiler performance and intestinal immunology. Abstract in: Proceedings of 73rd Annual Meeting of the European Federation of Animal Science, September 5–9, 2022, Porto, Portugal (Presentation, Abstract)

Kettunen, H., Simao, F., Vienola, K., Vartiainen, S., Apajalahti, J. and Vuorenmaa, J. 2022. Effects of in-feed resin acid concentrate on growth performance and ileal gene expression of broiler chickens. Abstract id-87 at: 7th International Conference on Poultry Intestinal Health (ICPIH2022), Cartagena De Indias, Colombia, 30 March – 1 April, 2022. (Poster)

Kettunen, H., Vartiainen, S., Vienola, K., Apajalahti, J. and Vuorenmaa, J. (2021) Immunomodulatory and performance-enhancing effects of dietary resin acids in broiler chickens. Abstract in: 72nd annual meeting of European Federation of Animal Science (EAAP) August 30 – September 3, 2021, Davos, Switzerland (Presentation, Abstract)

Kettunen, H., Vuorenmaa and J., Jintasataporn, O. (2022) Dietary tall oil fatty acids with resin acids improve the performance of seabass and white shrimp. Abstract in: Proceedings of 73rd Annual Meeting of the European Federation of Animal Science, September 5–9, 2022, Porto, Portugal (Presentation, Abstract)

Kettunen, H., Vuorenmaa, J., Valkonen E., Alleno C. 2022. Dietary tall oil fatty acids improved egg shell quality and reduced the proportion of dirty eggs in laying hens with or without salt stress challenge. Abstract id-80 at: 7th International Conference on Poultry Intestinal Health (ICPIH2022), Cartagena De Indias, Colombia, 30 March – 1 April, 2022. (Poster)

Kettunen, H., Vuorenmaa, J., Valkonen, E. and Apajalahti, J. (2014) A resin acid -based product improved performance of broiler chicks in a necrotic enteritis model using a commercial-type dietary formula. At: The XIVth European Poultry Conference (EPC) 2014, Stavanger, Norway. (Presentation, Abstract)

Kettunen, H., Vuorenmaa, J., Vienola, K., Valkonen, E., Apajalahti, J. and Vartiainen, S. (2019) Resin acid composition reduces small-intestinal expression of collagen-degrading matrix metalloproteinases in broilers. At: 6 th International Conference on Poultry Intestinal Health (IHSIG 2019), Rome, Italy 3 – 5 April 2019. (Poster)

Kolpe, A., Aguirre, M., Vuorenmaa, J., Valkonen, E., Kettunen, H., Callens, C., Haesebrouck, F., Ducatelle, R., Van Immerseel, F. and Goossens, E. (2019) Resin acids reduce expression of matrix metalloproteinases in vitro and in the ileal mucosa of broilers. At: 6 th International Conference on Poultry Intestinal Health (IHSIG 2019), Rome, Italy 3 – 5 April 2019. (Presentation, Abstract)

Kolpe, A., Goossens, E., Van Immerseel, F., Antonissen, G., De Gussem, M., Dedeurwaerder, A., Ducatelle, R., Vuorenmaa, J., Hasan, S., Valkonen, E. and Kettunen, H. (2019) In-feed resin acids improve smallintestinal mucosal characteristics of broiler chickens during dysbiosis challenge. At: 3rd International

Lipiński, K., Vuorenmaa, J., Antoszkiewicz, Z., Kaliniewicz, J., Mazur M. and Makowski, Z. (2017) Effect of resin acid composition on growth performance, footpad dermatitis, slaughter value and gastrointestinal tract development in turkeys. At: The 21st European Symposium on Poultry Nutrition (ESPN), Salou, Spain. (Poster)

Middelkoop, A., Tichelaar, R., Guan, X., Kettunen, H., Vuorenmaa, J., Rydal, M.P., Brøndsted, L., Gambino, M., Nielsen, J. P. and Molist, F. (2022). Effect of dietary resin acids and hydrolysed yeast in piglets challenged with F4-Enterotoxigenic Escherichia coli. Abstract in: ZeroZincSummit2022, Copenhagen, Denmark, 22 – 23 June, 2022.

Qin, N., Karenius, P., Lidauer, P., Franco, M., Niku, M., Junnikkala, S. and Kettunen, H. (2019) The effects of in-feed resin acid composition on the colostrum composition and immunity of dairy cows. Abstract in: XIIIth International Symposium on Ruminant Physiology (ISRP 2019), Leipzig, Germany, 3–6 September 2019. (Poster)

Qin, N., Niku, M., Junnikkala S., Vuorenmaa, J. and Kettunen, H. Biomarkers of inflammation and gut permeability in dairy cows with or without dietary resin acids Abstract no 21.29 in: The European Federation of Animal Science (EAAP) virtual meeting 1–4 December 2020. (Poster)

Roy, K., Lyhs, U. and Pedersen, K. (2015) Inhibition studies of natural resin acids to Clostridium perfringens and Escherichia coli O149. At: The 1st International Conference on Necrotic Enteritis in Poultry 2015, Copenhagen, Denmark. (Poster)

Valkonen, E. (2018) Performance enhancing effect of a natural resin acid composition in broiler chickens under a variety of challenge conditions. At: The 15th European Poultry Conference (EPC) 2018, Dubrovnik, Croatia. (Presentation, Abstract).

Valkonen, E., Apajalahti, J., Kettunen, H., Vienola, K., Vuorenmaa, J. and Raatikainen, K. (2019) In-feed resin acids do not accumulate in broiler breast muscle or adipose tissue At: 22nd European Symposium in Poultry Nutrition (ESPN 2019), 10–13 June, Gdańsk, Poland. (Poster)

Valkonen, E., Picart, C., Launay, C., Kettunen, H., Vuorenmaa, J. (2021) Resin acid containing tall oil fatty acid product improved broiler performance. At: World Poultry Congress 2021 – Electronic poster session. (Poster)

Valkonen, E., Sanson, G., Kettunen, H. and Vuorenmaa, J. (2021) Resin acid containing tall oil fatty acid reduced mortality and oocyst shedding in broilers challenged with Eimeria. At: World Poultry Congress 2021 – Electronic poster session. (Poster)

Valkonen, E., Sarker, N., Hasan, S., Kettunen, H. and Vuorenmaa, J. (2019) Effects of resin acid composition on broiler performance in comparison to ionophoric in-feed coccidiostat. At: World’s Poultry Science Association, Bangladesh Branch, 11th International Poultry Seminar, 5–6 March 2019, Dhaka, Bangladesh. (Presentation, Abstract)

Vuorenmaa, J., Goossens, E., Kettunen, H., Aguirre, M., Valkonen, E., Ducatelle, R., Hasan, S. and Van Immerseel, F. (2019) Natural resin acid composition supports the connective tissue of small intestinal epithelium and brings consistent performance benefits to broilers. At: World’s Poultry Science Association, Bangladesh Branch, 11th International Poultry Seminar, 5–6 March 2019, Dhaka, Bangladesh. (Poster)

Vuorenmaa, J., Valkonen, E., Apajalahti, J., Kettunen, H. and Vienola, (2019) Beneficial performance effect of tall oil fatty acids is likely based on the effects of resin acids. At: 22nd European Symposium in Poultry Nutrition (ESPN 2019), 10–13 June, Gdańsk, Poland. (Poster)

Vuorenmaa, J., Valkonen, E. and Kettunen, H. (2017) Dietary resin acid composition as a performance enhancer for broiler chickens. At: 5th International Conference on Poultry Intestinal Health, 11–12 October 2017, Bangkok, Thailand. (Presentation, Abstract)

Yun, J., Hasan, S., Saha, S., Oliveiro, C. and Peltoniemi, O. (2018) Effects of supplementing prepartum sow diets with organic acids on neonatal piglet mortality. At: 10 th European Symposium on Porcine Health Management, 9–11 May 2018 Barcelona, Spain. (Poster)