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Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses

Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses † † †,1, ‡ † † K. K. Fikes, J. A. Coverdale, J. L. Leatherwood, J. M. Campbell, T. H. Welsh, Jr., C. J. Hartz, † † ‖, †, M. Goehring, A. A. Millican, A. N. Bradbery, and T. A. Wickersham † ‡ Department of Animal Science, Texas A&M University, College Station, TX 77843; APC, LLC, Ankeny, IA 50021, USA; and Department of Animal and Range Sciences, Montana State University, Bozeman, MT 59717, USA ABSTRACT:  Twenty-seven mature Quarter IL-1β. Data were analyzed using PROC MIXED horses were used in a randomized design to de- of SAS. A trend towards treatment × time inter- termine the effects of bioactive protein sup- action was observed in ROM of the knee at the plementation on gait kinematics and systemic walk (P  =  0.10), due to the increasing ROM for inflammatory markers in a 34-d trial. Treatments 40BP and 80BP as time increased and decreasing consisted of oral doses of 230 g/d of pelleted sup- ROM for CON. A  treatment × time interaction plements containing 0  g (CON; n  =  9), 40  g of was observed (P < 0.01) for hock ROM at a walk bioactive protein (40BP; n = 9; LIFELINE, APC, resulting from CON and 80BP decreasing from LLC, Ankeny, IA), and 80 g of bioactive protein day 14 to 28 with 40BP increasing, while from day (80BP; n  =  9) daily. Horses were fed a commer- 28 to 34 ROM at a walk decreased for 40BP and in- cial concentrate at 0.5% BW (as-fed) and received creased for 80BP. The main effect of treatment on ad libitum coastal bermudagrass (Cynodon dac- hock ROM at the walk was quadratic (P  <  0.01) tylon) hay daily. On day 33, horses consistent in and characterized by higher ROM values for exercise (CON, n  =  6; 40BP, n  =  8; 80BP, n  =  7) 40BP compared to CON or 80BP. Dietary treat- participated in a trailering and riding challenge. ment lengthened (P  =  0.04) SL of the hind limb Kinematic gait analysis was performed on day 0 at the walk for 40BP and 80BP compared to CON for use as a covariate, and on day 14, 28, and 34 to on both days 14 and 28. A significant treatment × allow for the determination of potential time and time interaction was observed in the expression dosage effects. Video footage was collected and of IL-1β (P  <  0.01) and can be explained by analyzed using gait analysis software (EquineTec, lower concentrations of IL-1β for 80BP on day Monroe, GA) for the determination of stride 34 compared to the other treatments, with 40BP length (SL) and range of motion (ROM). Blood being intermediate and CON being the highest. was collected via jugular venipuncture on days 0, Increased articular ROM with decreased expres- 14, 28, and 34 for determination of systemic ex- sion of IL-1β may indicate potential anti-inflam- pression of tumor necrosis factor (TNF)-α and matory effects of 80 g/d of bioactive proteins. Key words: bioactive proteins, equine, gait kinematics, inflammation © The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2021.5:1-10 doi: 10.1093/tas/txab017 Corresponding author: leatherwood@tamu.edu Received June 16, 2020. Accepted February 4, 2021. 1 Fikes et al. INTRODUCTION in horses on the relationship between blood chem- istry parameters, serum inflammatory biomarkers, The performance potential of equine athletes and performance gait kinematics. Therefore, the is dependent upon the ability of articular joints objective of this study was to determine the effect to maximize the range of motion to create long, of dietary BP supplementation on gait kinematics fluid strides. Data gathered through gait kinematic and systemic inflammatory markers in horses over software are closely associated with local and sys- time and following an extensive transport and ex- temic inflammatory responses which when elevated ercise challenge. We hypothesized that dietary BP may the limit range of motion, stride length, and will reduce basal inflammation and the inflamma- overall comfort of movement for the performance tory response to a transport and exercise challenge horse. Furthermore, excessive inflammation results resulting in improved gait kinematics and overall in long-term damage to the joint and subsequent performance potential. osteoarthritis, which is the greatest single economic loss to the equine industry (Frisbie et  al., 2006). MATERIALS AND METHODS Therefore, developing nutritional strategies to miti- gate the progression of joint inflammation and as- Horses and Dietary Treatments sociated degenerative joint disease may reduce the need for oral or intra-articular pharmaceuticals, All procedures and handling of horses were ap- which act primarily to alleviate symptoms of pain proved by the Institutional Animal Care and Use associated with the disease. Committee (AUP# 2015-0380). Twenty-seven ma- A potential alternative is the use of dietary ture horses (n = 25 geldings, n = 2 mares) from an es- bioactive proteins (BP) derived from bovine spray- tablished herd at Parsons Mounted Cavalry (Texas dried plasma proteins (SDP) to reduce the inflam- A&M University, College Station, TX) were util- matory effects associated with excess joint load ized in a randomized design for a 34-d trial. Horses and trauma. These proteins are a diverse mixture were housed in a dry-lot track (9 m × 1600 m) for of functional components with biological activity voluntary exercise. All horses were allowed ad lib- independent of their nutritional value, including itum access to coastal Bermudagrass hay (Cynodon immunoglobulins, growth factors, biologically ac- dactylon) obtained from a single cutting and water. tive peptides, and other factors with biological ac- Horses were fed a commercially available con- tivity within the intestine (Campbell et  al., 2010). centrate (14% CP pellet; Cargill, Elk River, MN) Spray-dried plasma was found to be more suc- twice daily in 3 m × 3 m individual box stalls every cessful than antibiotics in reducing the expression 12  h. Horses underwent moderate exercise for the of pro-inflammatory cytokines in pigs challenged duration of the study in accordance with Parsons with Escherichia coli K88 (Bosi et al., 2004). Beski Mounted Cavalry protocol, consisting of basic et al. (2016) observed that broiler chickens receiving horsemanship maneuvers at the walk, trot, and SDP supplemented in starter diets had lower con- canter 5 d/wk for approximately 60 min each day. centrations of immunoglobulins in serum, indi- before the start of the experimental period, cating that stimulation of the immune system was horses were stratified by body weight (BW; 560  ± reduced, and more resources could be allocated to 52  kg), body condition score (BCS; 5.21  ± 0.42), maintenance and development. Furthermore, in and age (13 ± 3 yr) and randomly assigned to one weaned rats challenged with Staphylococcus aureus of three dietary treatment groups that included a superantigen B, dietary supplementation with SDP supplement containing either soybean meal or two reduced immune activation of Peyer’s patches and levels of spray-dried bioactive proteins (LIFELINE mesenteric lymph nodes (Perez-Bosque et al., 2004). Equine Elite, BioThrive, APC, LLC, Ankeny, IA). More recent studies performed in horses suggest Dietary pelleted supplements were top-dressed that BP improves gait kinematics in mature horses, (230  g/d) on the commercially available concen- specifically via improved mean stride length (SL) in trate feed (Table 1) and consisted of a control the forelimb and hindlimb as levels of BP increase soy protein-based supplement without BioThrive (Coverdale and Campbell, 2014). Systemic levels (CON; n  =  9), 40  g BioThrive (40BP; n  =  9), or of key inflammatory biomarkers in serum provide 80  g BioThrive (80BP; n  =  9) with BioThrive re- an indication of total metabolic activity originat- placing soy protein in the respective supplement. ing from all joints in the body (Sumer et al., 2006). Investigators remained blinded to treatments until To date, limited work has been performed to de- completion of data analysis, and treatments were termine the effects of dietary BP supplementation coded alphabetically and pre-weighed to 230  g. Translate basic science to industry innovation Bioactive proteins on equine performance Table 1.  Nutrient analysis of dietary treatments 4°C for 20 min. The supernatant was transferred to including commercial concentrate and top-dressed pour off tubes and placed into a freezer at −20°C supplement for subsequent analysis. a b b Concentrate CON 40BP 80BP Gait Kinematics Dry Matter, % 90.0 91.9 93.0 91.8 DM Basis, % Gait analysis was performed every two weeks 17.9 32.6 34.4 38.8 CP on day 0, 14, and 28, and 24  h post-parade on NDF 28.7 32.9 32.4 32.7 day 34. Horses were assigned a single handler for ADF 16.6 23.0 22.4 21.4 each gait analysis throughout the study. Horses Ca 1.17 1.58 1.68 1.74 performed three passes at both the walk and trot P 0.99 1.09 0.99 0.86 over a 10 m path on solid dirt footing. Reflective 14% CP pelleted feed (Cargill, Elk River, MN). adhesive markers were placed on major joints on LIFELINE Equine Elite (APC, LLC, Ankeny, IA). the right front and right hind limb to allow for visibility and calibration for later analysis. Front Dietary treatments began on day 1, and no signifi- limb markers included the greater tubercle of the cant refusals were recorded after day 3. Horses were humerus, lateral humeral epicondle, ulnar carpal allowed 1  h to consume dietary treatments before bones, lateral metacarpal epicondyle, middle pha- orts were collected. lanx-proximal phalanx junction, and proximal pha- On day 33, horses that remained consistent in lanx-distal phalanx junction. Hind limb markers exercise (CON: n  =  6; 40BP: n  =  8; 80BP: n  =  7) included distal phalanx-middle phalanx junction, underwent an exercise stressor via participation middle phalanx-proximal phalanx junction, prox- in the Battle of Flowers Parade in San Antonio, imal phalanx- third metacarpal junction, tarsal TX. Horses have hauled 290 km in a stock-type bones, and lateral femoral epicondle. Video footage trailer with approximately 1 h rest before walking was collected and recorded using EquineTec soft- a 6.5 km parade on concrete followed by a 290 km ware (EquineTec, Monroe, GA) installed on an HP return in the same stock-type trailer. Previous re- Pavilion m6 laptop with a 2.3 GHz microprocessor search has indicated limited stress to joints on a and AMD Rodeon HD 7660G video graphics sandy surface compared to asphalt and has also (Hewlett-Packard, Palo Alto, CA). indicated changes to gait kinematics on an asphalt surface when compared to sand (Chateau et  al., Sample Analysis 2010; Chateau et al., 2013). Further, transport has been shown to induce inflammation in both cattle Serum collected was analyzed for tumor necrosis and horses (Wessely-Szponder et  al., 2015; Van factor α (TNF-α) and interleukin-1β (IL-1β) using Engen and Coetzee, 2018). commercially available ELISA kits (R&D Systems, Inc., Minneapolis, MN) developed for use in the horse. Assays were run according to the kit insert. The inter- Sample Collection and intra-assay CV was ≤ 10% for both TNF-α and Weekly, BW and BCS were determined using IL-1β. Plasma samples were used for blood chemistry a calibrated platform scale and three independent profiles where abnormal hematology may indicate in- evaluators, respectively (Henneke et  al., 1983). ternal disease independent of joint disease. Samples Plasma and serum samples were collected every 7 were analyzed by the Texas Veterinary Medical d from day 0 to 28, and 24  h post-parade on day Diagnostic Lab at Texas A&M University (College 34. Approximately 50 mL of whole blood was col- Station, TX) for total serum protein (TSP), albumin, lected via jugular venipuncture. Samples intended phosphorus (P), glucose, blood urea nitrogen (BUN), for plasma analysis were collected into 10  mL creatinine, creatine kinase (CK), total bilirubin (TB), evacuated tubes containing 0.1  mL 15% buffering bilirubin direct (BD), gamma-glutamyl transferase solution and 15  mg K EDTA (BD Vacutainer, (GGT), albumin:globulin (A/G) ratio, and globulins. Franklin Lakes, NJ) and immediately placed in Horses with values outside of normal ranges were ex- ice. Samples intended for serum analysis were col- cluded from cytokine analysis to ensure that expres- lected into 10  mL evacuated non-additive tubes sion of systemic inflammatory markers was reflective (BD Vacutainer, Franklin Lakes, NJ) and allowed of joint disease and no other inflammatory processes. to clot for 30 min before processing to allow blood One horse from each treatment group, CON, 40BP, to clot. All samples were centrifuged at 2,000 × g at and 80BP were removed from cytokine analysis due Translate basic science to industry innovation Fikes et al. to abnormal blood chemistry parameters not ex- with 80BP having greater BCS on day 14 compared plained by joint disease. to both 40BP and CON. Horses receiving 80BP maintained BCS through day 28, while CON and 40BP increased BCS such that there was no signifi- Statistical Analysis cant difference between dietary treatments on d 28. All data were analyzed as a randomized design All horses, regardless of dietary treatment, gained using the PROC MIXED procedure of statistical BW (P < 0.01) until day 28 and lost BW to day 34 analysis software (SAS) (SAS Inst., Inc., Cary, NC) while BCS remained constant. with effects for treatment, day, and treatment × day interaction with linear and quadratic contrasts. The Gait Kinematic Parameters model uses RANDOM and REPEATED state- ments to account for variability between animals. Stride length:   A trend towards a treatment × Day 0 was used as a covariate for gait kinematics. time interaction (P  =  0.10, Fig. 3A) was observed Effects were considered significant if P ≤ 0.05, with in stride length (SL) of the forelimb at the walk, a trend towards significance if P ≤ 0.10. largely driven by an increase in SL in horses re- ceiving 80BP while horses receiving CON and 40BP RESULTS decreased from d 14 to d 28. A  trend towards a linear effect (P = 0.09) was also demonstrated with CON SL at 155 ± 3.83 cm on d 28, 40BP at 168 ± Physical Measurements 5.0 cm, and 80BP at 168.9 ± 3.56 cm. No significant There was no effect of BP supplementation on effects were observed for SL of the forelimb at the BW (P  =  0.76; Fig. 1); however, there was a treat- trot (P = 0.21, Fig. 3B). ment × time interaction for BCS (P < 0.01; Fig. 2) At the walk, a trend towards a treatment × time interaction (P  =  0.07; Fig. 4A) was observed for hindlimb SL, resulting from decreases in SL from day 14 to 28 in CON and 40BP while 80BP increased. From day 28 to 34, 40BP and 80BP de- creased while CON increased. There was a linear Figure 1. Bodyweight (kg) over time (d) in horses receiving a pel- leted concentrate with control, 40BP, or 80BP g/d of spray-dried bio- active proteins (LIFELINE, APC, LLC, Ankeny IA). Main effects included treatment, time, and treatment × time. Figure 3. Stride length (cm) of the forelimb at the walk (A) and Figure 2. Body condition score (BCS) over time (d) in horses re- trot (B) over time (d) in horses receiving a pelleted concentrate with ceiving a pelleted concentrate with either control, 40BP, or 80BP g/d either control, 40BP, or 80BP g/d of spray-dried bioactive proteins of spray-dried bioactive proteins (LIFELINE, APC, LLC, Ankeny (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- a,b,c a,b,c IA). Main effects included treatment, time, and treatment × time. ment, time, and treatment × time. Within d, superscripts denote a Within d. difference between dietary treatments (P < 0.05). Translate basic science to industry innovation Bioactive proteins on equine performance Figure 4. Stride length (cm) of the hind limb at the walk (A) and trot (B) over time (d) in horses receiving a pelleted concentrate with Figure 5. Range of motion (degrees) of the knee at the walk (A) either control, 40BP or 80BP g/d of spray-dried bioactive proteins and trot (B) over time (d) in horses receiving a pelleted concentrate (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- with either control, 40BP, or 80BP g/d of spray-dried bioactive proteins a,b,c ment, time, and treatment × time. Within d, superscripts denote a (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- a,b,c difference in dietary treatment (P < 0.05). ment, time, and treatment × time. Within d, superscripts denote a difference in dietary treatment (P < 0.05). increase in SL of the hindlimb at the walk-in treated affected hock range of motion at the walk quad- horses compared to CON (P = 0.05). Treatment did ratically (P  <  0.01) demonstrated by 40BP having not affect SL of the hindlimb at the trot (P = 0.17; the highest ROM at 33.2 ± 1.24°, followed by CON Fig. 4B). at 28.6 ± 1.45°, and 80BP having the lowest ROM Range of motion.  Range of motion (ROM) in at 28.0  ± 1.36°. Hock ROM at the trot was influ- the knee at the walk exhibited a trend towards a enced by time (P  =  0.05 Fig. 6B), but not treat- treatment × time interaction (P = 0.10, Fig. 5A), due ment (P  =  0.39) with all horses decreasing ROM to the increased range of motion for treated horses over time. compared to CON on day 34. Horses in the CON Blood chemistry parameters.  Dietary supple- dietary treatment decreased ROM from day 14 to mentation of BP did not influence blood chemistry 34, while treated horses increased. Horses receiving parameters (P > 0.10, Table 2); however, treatment × 80BP increased ROM throughout the study, with time interactions were observed for TSP, albumin, ROM reaching 32.5 ± 2.27° on day 28 and 34.6 ± globulins, and glucose (P  <  0.09). The interaction 2.27° on day 34. The 40BP dietary treatment group for TSP (P = 0.04; Fig. 7A) resulted from a decrease increased ROM to 34.4 ± 2.16° on day 28 and de- in TSP in CON with a corresponding increase in creased to 33.6 ± 2.16° on day 34. Supplementation BP supplemented horses to day 28. The interaction of BP did not influence knee ROM at the walk or of treatment × time for albumin (P < 0.01; Fig. 7B) trot (P  =  0.56, P  =  0.38, respectively). No signifi- is described by a decrease in CON to d 28 while al- cant effects were observed for knee ROM at the trot bumin increased in treated horses. Globulins tended (P = 0.64; Fig. 5B). to express a treatment × time interaction (P = 0.09; A significant treatment × time interaction Fig. 7C) where 40BP and 80BP horses increased to (P < 0.01, Fig. 6A) was observed for hock ROM at day 28 and CON horses decreased. Finally, a treat- the walk described by an increase in ROM in 80BP ment × time interaction for glucose (P = 0.02; Fig. horses from d 28 to d 34, a decrease from d 28 to 7D) is described by lower plasma blood glucose on 34 in 40BP horses, and relatively constant values day 34 in 80BP horses compared to CON or 40BP. in CON horses. Dietary supplementation of BP Translate basic science to industry innovation Fikes et al. Serum inflammatory markers.  A treatment × expression of IL-1β. Dietary treatment of BP time interaction was observed for IL-1β (P < 0.01; did not affect serum levels of TNF-α (P  =  0.51; Fig. 8A) is described by CON and 40BP horses Fig. 8B); however, TNFα increased over time re- increasing to d 34 while 80BP horses decreased gardless of dietary treatment (P = 0.05), with all horses increasing from d 28 to d 34. Time also influenced IL-1β (P < 0.01) as all treatments de- creased to day 28. DISCUSSION This study examined the effect of dietary BP supplementation on gait kinematics and systemic inflammatory markers in horses over time and fol- lowing an extensive transport and aerobic exer- cise challenge. Previous research in horse suggests that dietary BP improves stride length which indi- cates increased levels of comfort (Coverdale and Campbell, 2014). This study is the first in horses to measure systemic markers of inflammation with gait kinematics in response to dietary BP supple- mentation and exercise. Increased BW over time to day 28 may be ex- plained by the change from group feeding to indi- vidual feeding at the initiation of the study to ensure each horse was fed to meet NRC requirements for mature horses undergoing light exercise (NRC, 2007). Minor loss in BW between day 28 and 34 is likely due to trailering and participation in the parade challenge on day 34 of the study. Decreased Figure 6. Range of motion (degrees) of the hock at the walk (A) and trot (B) over time (d) in horses receiving a pelleted concentrate BW may be related to body water loss via sweat and with either control, 40BP, or 80BP g/d of spray-dried bioactive pro- subsequent dehydration as horses were weighed teins (LIFELINE, APC, LLC, Ankeny IA). Main effects included 24  h following transport (Friend, 2000). A  study a,b,c treatment, time, and treatment × time. Within d, superscripts denote differences in dietary treatment (P < 0.05). performed by Friend (2000) observed a similar Table 2. Least square treatment means of blood chemistry including total serum protein (TSP), albumin, phosphorus (P), glucose, blood urea nitrogen (BUN), creatinine, total bilirubin (TB), bilirubin direct (BD), creatine kinase (CK), serum glutamine oxaloacetate transaminase (SGOT), globulins, albumin:globulin ratio (A:G), and gamma-glutamyl transferase (GGT) in plasma of horses receiving a pelleted concentrate with either 0 g/d, 40 g/d, or 80 g/d of bioactive proteins P-values Parameter 0 g/d 40 g/d 80 g/d SEM Treatment Time Treatment x Time TSP, g/dL 6.45 6.48 6.51 0.13 0.96 0.01 0.04 Albumin, g/dL 2.9 2.99 2.94 0.04 0.33 <0.01 <0.01 P, mg/dL 2.9 2.88 2.93 0.08 0.89 <0.01 0.99 Glucose, mg/dL 91.33 90.74 91.1 1.29 0.95 <0.01 0.02 BUN, mg/dL 15.52 16.44 15.86 0.62 0.57 <0.01 0.17 Creatinine, mg/dL 1.37 1.43 1.41 0.05 0.72 <0.01 0.17 TB, mg/dL 0.7 0.76 0.61 0.05 0.15 <0.01 0.09 BD, mg/dL 0.27 0.28 0.27 0.01 0.72 <0.01 0.56 CK, U/L 302.73 307.67 306.29 24.35 0.99 0.27 0.48 SGOT, U/L 274.83 255.61 278.9 12.76 0.4 0.63 0.38 Globulins, g/dL 3.55 3.49 3.57 0.15 0.92 0.09 0.09 A:G 0.81 0.89 0.84 0.04 0.42 0.89 0.11 GGT, U/L 14.05 11.51 11.89 1.35 0.37 0.02 0.15 Translate basic science to industry innovation Bioactive proteins on equine performance Figure 8. Serum concentrations of interleukin-1β (IL-1β; A; pg/ mL) and tumor necrosis factor α (TNFα; B; pg/mL) over time (d) in horses receiving a pelleted concentrate with either control, 40BP, or 80BP g/d of spray-dried bioactive proteins (LIFELINE, APC, LLC, Ankeny IA). Main effects included treatment, time, and treatment × a,b,c time. Within d, superscripts denote differences between dietary treatments for serum IL-1β (P < 0.05). initial loss in BW during transport, which was sus- tained but not increased for 24 h in horses with ac- cess to water. Improvements in SL in the forelimb to day 28 at the walk were similar to those observed by Coverdale and Campbell (2014, 2015). This study included a challenge component that incorpor- ated trailering and riding on day 33, leading to the overall trends being altered from previous studies that did not include a trailering and exercise chal- lenge. Forelimb and hindlimb SL at the walk had a trend toward significance in this study compared to forelimb and hindlimb SL at the trot. This may be explained by the varying biomechanics of move- ment for the different gaits. Because the walk lacks a suspension phase (Clayton, 1989), training and coordination of the individual horse play a smaller role. This would allow any potential benefits of BP Figure 7. The effect of dietary supplementation of control, 40BP or 80BP g/d of spray-dried bioactive proteins (LIFELINE, APC, supplementation to become more evident and less LLC, Ankeny IA) on total serum protein (A; g/dL), albumin (B; g/dL), reliant on individual athletic ability. globulins (C; g/dL), and blood glucose (D; mg/dL). Main effects in- Bioactive proteins generally modulate the a,b,c cluded treatment, time, and treatment × time. Within d, superscripts denote a difference in dietary treatment (P < 0.05). immune response to physiological stress in a Translate basic science to industry innovation Fikes et al. challenging environment (Bosi et  al., 2004; Perez- involved in blood clotting and inflammation com- Bosque et  al., 2016). However, SL for both limbs prising many key components of the immune at the walk decreased on day 34. The inconsistent response (Miglio et  al., 2019). Because SDP con- response may be explained by other factors influ- tains functional factors such as immunoglobulins, encing SL after the challenge, such as muscle and growth factors, and active peptides (Campbell periarticular tissue soreness and behavior factors et al. 2010b), it is possible that levels of total serum during the challenge (Rawson et al., 2001; Lee et al., protein, albumin, and globulins would increase in 2002; Braun and Dutto, 2003). Additionally, dur- treated horses either by differences in dietary crude ing gait kinematic analysis, horses were recorded protein (CP; Leveille and Sauberlich, 1961) or bio- at the walk before moving into the trot, potentially active protein stimulation of the common mucosal acting as a warm-up to exercise, allowing the horse system to result in increased globulin concentra- to become more fluid in the movements at the trot. tions (Walther and Sieber, 2011). While little work has been done investigating the Decreasing or sustained levels of total serum effects of a walking warmup on equine gait kine- protein, albumin, and globulins to day 34 in treated matics, in humans, walking improves gait biomech- horses compared to CON, whose levels increased anics thus it is likely that similar effects are present from day 28 to 34 following the day 33 challenge, in this study (Schieber et  al., 2019). Furthermore, may indicate modulation of the immune response in the instance of muscle soreness following trans- following physiological stress of transport and ex- port and exercise stress, the active walking warmup ercise. Furthermore, previous studies have observed may have reduced any incidence of delayed onset modulatory effects of SDP on plasma glucose re- muscle soreness (Andersen et al., 2013). sulting from alterations in SGLT1 expression in a The interaction observed for knee ROM at the challenging environment and an increase in glucose walk but not at the trot may also be attributed to uptake by insulin-sensitive tissues (Moreto and differing gait biomechanics as well as individual Perez-Bosque, 2009; Oseguera-Toledo et al., 2014). athletic ability. Previous studies did not observe Our study shows comparable results following the trends or significance in knee ROM at either gait day 33 challenge where horses receiving 80BP had (Coverdale and Campbell, 2014, 2015). The re- lower plasma blood glucose than CON or 40BP. sponse observed in the present study for this par- In other species, SDP has demonstrated the ameter of gait kinematic analysis to the challenge ability to regulate cytokine expression (Bosi et al., on day 33 indicates that treated horses improved in 2004; Perez-Bosque et al., 2016). Following an acute performance compared to CON. Articular ROM lipopolysaccharide challenge in swine, SDP de- relates more closely to periarticular joint comfort creased mucosal expression of TNF-α (Bosi et al., than SL, suggesting that bioactive protein supple- 2004). However, in the current study, serum TNF- mentation was more effective in improving articular α levels were not influenced by dietary treatment. ROM than SL following a challenge (Bischoff et al., This is likely due to the use of a physical stressor 2003; Ahvazi et al., 2014). rather than a pathogen-induced challenge and the Currently, there is no literature on gait kine- use of a sustained physical challenge rather than matic analysis of hock ROM as it relates to SDP an acute response. Pathogen recognition functions supplementation. It is unknown why horses re- through acquired immunity, while acute physical ceiving 40 g/d BP showed improved hock ROM at stress functions primarily through the innate im- the walk to day 28 compared to horses receiving mune system. This change could explain the differ- 80  g/d BP. However, hock ROM at the walk re- ence in response between the current study and the sponded to the day 33 challenge similarly to knee study performed by Bosi et al. (2004). Furthermore, ROM at the walk with horses receiving 80  g/d BP rather than testing an acute response, sampling in having improved articular ROM in response to the the present study was designed to test the response challenge, further suggesting that BP are more ef- to a sustained physical challenge and resulted in im- fective at improving articular ROM than SL after proved hock ROM suggesting anti-inflammatory an exercise challenge. effects. Total serum protein (TSP) is a combined Similar to the Perez-Bosque et al. (2016) mouse measure of all proteins in the blood, including al- model, the current study demonstrates decreased bumin and globulins. Albumin is a blood protein levels of IL-1β in horses receiving 80BP following produced by the liver and is integral in the main- the day 33 challenge. Horses receiving 80BP had tenance of osmotic pressure (Miglio et  al., 2019). increasing values of TNF-α to day 28 and 34 Globulins include antibodies and other proteins with decreasing values of IL-1β. This suggests an Translate basic science to industry innovation Bioactive proteins on equine performance improved ability to adapt to the stress-related im- Conflict of interest statement. The authors af- mune response before IL-1β is expressed. Because firmatively acknowledge that they were free from IL-1β is the major cytokine associated with joint influence by any funding sources or their employees disease, this also suggests that dietary SDP’s ob- that would result in any conflict of interest. served improvement of articular ROM may be explained by the reduced expression of pro-inflam- REFERENCES matory IL-1β. Ahvazi,  S.  P., H.  Sadeghi, and M.  Gholami. 2014. 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Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses

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Effect of bioactive proteins on gait kinematics and systemic inflammatory markers in mature horses † † †,1, ‡ † † K. K. Fikes, J. A. Coverdale, J. L. Leatherwood, J. M. Campbell, T. H. Welsh, Jr., C. J. Hartz, † † ‖, †, M. Goehring, A. A. Millican, A. N. Bradbery, and T. A. Wickersham † ‡ Department of Animal Science, Texas A&M University, College Station, TX 77843; APC, LLC, Ankeny, IA 50021, USA; and Department of Animal and Range Sciences, Montana State University, Bozeman, MT 59717, USA ABSTRACT:  Twenty-seven mature Quarter IL-1β. Data were analyzed using PROC MIXED horses were used in a randomized design to de- of SAS. A trend towards treatment × time inter- termine the effects of bioactive protein sup- action was observed in ROM of the knee at the plementation on gait kinematics and systemic walk (P  =  0.10), due to the increasing ROM for inflammatory markers in a 34-d trial. Treatments 40BP and 80BP as time increased and decreasing consisted of oral doses of 230 g/d of pelleted sup- ROM for CON. A  treatment × time interaction plements containing 0  g (CON; n  =  9), 40  g of was observed (P < 0.01) for hock ROM at a walk bioactive protein (40BP; n = 9; LIFELINE, APC, resulting from CON and 80BP decreasing from LLC, Ankeny, IA), and 80 g of bioactive protein day 14 to 28 with 40BP increasing, while from day (80BP; n  =  9) daily. Horses were fed a commer- 28 to 34 ROM at a walk decreased for 40BP and in- cial concentrate at 0.5% BW (as-fed) and received creased for 80BP. The main effect of treatment on ad libitum coastal bermudagrass (Cynodon dac- hock ROM at the walk was quadratic (P  <  0.01) tylon) hay daily. On day 33, horses consistent in and characterized by higher ROM values for exercise (CON, n  =  6; 40BP, n  =  8; 80BP, n  =  7) 40BP compared to CON or 80BP. Dietary treat- participated in a trailering and riding challenge. ment lengthened (P  =  0.04) SL of the hind limb Kinematic gait analysis was performed on day 0 at the walk for 40BP and 80BP compared to CON for use as a covariate, and on day 14, 28, and 34 to on both days 14 and 28. A significant treatment × allow for the determination of potential time and time interaction was observed in the expression dosage effects. Video footage was collected and of IL-1β (P  <  0.01) and can be explained by analyzed using gait analysis software (EquineTec, lower concentrations of IL-1β for 80BP on day Monroe, GA) for the determination of stride 34 compared to the other treatments, with 40BP length (SL) and range of motion (ROM). Blood being intermediate and CON being the highest. was collected via jugular venipuncture on days 0, Increased articular ROM with decreased expres- 14, 28, and 34 for determination of systemic ex- sion of IL-1β may indicate potential anti-inflam- pression of tumor necrosis factor (TNF)-α and matory effects of 80 g/d of bioactive proteins. Key words: bioactive proteins, equine, gait kinematics, inflammation © The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu- tion, and reproduction in any medium, provided the original work is properly cited. Transl. Anim. Sci. 2021.5:1-10 doi: 10.1093/tas/txab017 Corresponding author: leatherwood@tamu.edu Received June 16, 2020. Accepted February 4, 2021. 1 Fikes et al. INTRODUCTION in horses on the relationship between blood chem- istry parameters, serum inflammatory biomarkers, The performance potential of equine athletes and performance gait kinematics. Therefore, the is dependent upon the ability of articular joints objective of this study was to determine the effect to maximize the range of motion to create long, of dietary BP supplementation on gait kinematics fluid strides. Data gathered through gait kinematic and systemic inflammatory markers in horses over software are closely associated with local and sys- time and following an extensive transport and ex- temic inflammatory responses which when elevated ercise challenge. We hypothesized that dietary BP may the limit range of motion, stride length, and will reduce basal inflammation and the inflamma- overall comfort of movement for the performance tory response to a transport and exercise challenge horse. Furthermore, excessive inflammation results resulting in improved gait kinematics and overall in long-term damage to the joint and subsequent performance potential. osteoarthritis, which is the greatest single economic loss to the equine industry (Frisbie et  al., 2006). MATERIALS AND METHODS Therefore, developing nutritional strategies to miti- gate the progression of joint inflammation and as- Horses and Dietary Treatments sociated degenerative joint disease may reduce the need for oral or intra-articular pharmaceuticals, All procedures and handling of horses were ap- which act primarily to alleviate symptoms of pain proved by the Institutional Animal Care and Use associated with the disease. Committee (AUP# 2015-0380). Twenty-seven ma- A potential alternative is the use of dietary ture horses (n = 25 geldings, n = 2 mares) from an es- bioactive proteins (BP) derived from bovine spray- tablished herd at Parsons Mounted Cavalry (Texas dried plasma proteins (SDP) to reduce the inflam- A&M University, College Station, TX) were util- matory effects associated with excess joint load ized in a randomized design for a 34-d trial. Horses and trauma. These proteins are a diverse mixture were housed in a dry-lot track (9 m × 1600 m) for of functional components with biological activity voluntary exercise. All horses were allowed ad lib- independent of their nutritional value, including itum access to coastal Bermudagrass hay (Cynodon immunoglobulins, growth factors, biologically ac- dactylon) obtained from a single cutting and water. tive peptides, and other factors with biological ac- Horses were fed a commercially available con- tivity within the intestine (Campbell et  al., 2010). centrate (14% CP pellet; Cargill, Elk River, MN) Spray-dried plasma was found to be more suc- twice daily in 3 m × 3 m individual box stalls every cessful than antibiotics in reducing the expression 12  h. Horses underwent moderate exercise for the of pro-inflammatory cytokines in pigs challenged duration of the study in accordance with Parsons with Escherichia coli K88 (Bosi et al., 2004). Beski Mounted Cavalry protocol, consisting of basic et al. (2016) observed that broiler chickens receiving horsemanship maneuvers at the walk, trot, and SDP supplemented in starter diets had lower con- canter 5 d/wk for approximately 60 min each day. centrations of immunoglobulins in serum, indi- before the start of the experimental period, cating that stimulation of the immune system was horses were stratified by body weight (BW; 560  ± reduced, and more resources could be allocated to 52  kg), body condition score (BCS; 5.21  ± 0.42), maintenance and development. Furthermore, in and age (13 ± 3 yr) and randomly assigned to one weaned rats challenged with Staphylococcus aureus of three dietary treatment groups that included a superantigen B, dietary supplementation with SDP supplement containing either soybean meal or two reduced immune activation of Peyer’s patches and levels of spray-dried bioactive proteins (LIFELINE mesenteric lymph nodes (Perez-Bosque et al., 2004). Equine Elite, BioThrive, APC, LLC, Ankeny, IA). More recent studies performed in horses suggest Dietary pelleted supplements were top-dressed that BP improves gait kinematics in mature horses, (230  g/d) on the commercially available concen- specifically via improved mean stride length (SL) in trate feed (Table 1) and consisted of a control the forelimb and hindlimb as levels of BP increase soy protein-based supplement without BioThrive (Coverdale and Campbell, 2014). Systemic levels (CON; n  =  9), 40  g BioThrive (40BP; n  =  9), or of key inflammatory biomarkers in serum provide 80  g BioThrive (80BP; n  =  9) with BioThrive re- an indication of total metabolic activity originat- placing soy protein in the respective supplement. ing from all joints in the body (Sumer et al., 2006). Investigators remained blinded to treatments until To date, limited work has been performed to de- completion of data analysis, and treatments were termine the effects of dietary BP supplementation coded alphabetically and pre-weighed to 230  g. Translate basic science to industry innovation Bioactive proteins on equine performance Table 1.  Nutrient analysis of dietary treatments 4°C for 20 min. The supernatant was transferred to including commercial concentrate and top-dressed pour off tubes and placed into a freezer at −20°C supplement for subsequent analysis. a b b Concentrate CON 40BP 80BP Gait Kinematics Dry Matter, % 90.0 91.9 93.0 91.8 DM Basis, % Gait analysis was performed every two weeks 17.9 32.6 34.4 38.8 CP on day 0, 14, and 28, and 24  h post-parade on NDF 28.7 32.9 32.4 32.7 day 34. Horses were assigned a single handler for ADF 16.6 23.0 22.4 21.4 each gait analysis throughout the study. Horses Ca 1.17 1.58 1.68 1.74 performed three passes at both the walk and trot P 0.99 1.09 0.99 0.86 over a 10 m path on solid dirt footing. Reflective 14% CP pelleted feed (Cargill, Elk River, MN). adhesive markers were placed on major joints on LIFELINE Equine Elite (APC, LLC, Ankeny, IA). the right front and right hind limb to allow for visibility and calibration for later analysis. Front Dietary treatments began on day 1, and no signifi- limb markers included the greater tubercle of the cant refusals were recorded after day 3. Horses were humerus, lateral humeral epicondle, ulnar carpal allowed 1  h to consume dietary treatments before bones, lateral metacarpal epicondyle, middle pha- orts were collected. lanx-proximal phalanx junction, and proximal pha- On day 33, horses that remained consistent in lanx-distal phalanx junction. Hind limb markers exercise (CON: n  =  6; 40BP: n  =  8; 80BP: n  =  7) included distal phalanx-middle phalanx junction, underwent an exercise stressor via participation middle phalanx-proximal phalanx junction, prox- in the Battle of Flowers Parade in San Antonio, imal phalanx- third metacarpal junction, tarsal TX. Horses have hauled 290 km in a stock-type bones, and lateral femoral epicondle. Video footage trailer with approximately 1 h rest before walking was collected and recorded using EquineTec soft- a 6.5 km parade on concrete followed by a 290 km ware (EquineTec, Monroe, GA) installed on an HP return in the same stock-type trailer. Previous re- Pavilion m6 laptop with a 2.3 GHz microprocessor search has indicated limited stress to joints on a and AMD Rodeon HD 7660G video graphics sandy surface compared to asphalt and has also (Hewlett-Packard, Palo Alto, CA). indicated changes to gait kinematics on an asphalt surface when compared to sand (Chateau et  al., Sample Analysis 2010; Chateau et al., 2013). Further, transport has been shown to induce inflammation in both cattle Serum collected was analyzed for tumor necrosis and horses (Wessely-Szponder et  al., 2015; Van factor α (TNF-α) and interleukin-1β (IL-1β) using Engen and Coetzee, 2018). commercially available ELISA kits (R&D Systems, Inc., Minneapolis, MN) developed for use in the horse. Assays were run according to the kit insert. The inter- Sample Collection and intra-assay CV was ≤ 10% for both TNF-α and Weekly, BW and BCS were determined using IL-1β. Plasma samples were used for blood chemistry a calibrated platform scale and three independent profiles where abnormal hematology may indicate in- evaluators, respectively (Henneke et  al., 1983). ternal disease independent of joint disease. Samples Plasma and serum samples were collected every 7 were analyzed by the Texas Veterinary Medical d from day 0 to 28, and 24  h post-parade on day Diagnostic Lab at Texas A&M University (College 34. Approximately 50 mL of whole blood was col- Station, TX) for total serum protein (TSP), albumin, lected via jugular venipuncture. Samples intended phosphorus (P), glucose, blood urea nitrogen (BUN), for plasma analysis were collected into 10  mL creatinine, creatine kinase (CK), total bilirubin (TB), evacuated tubes containing 0.1  mL 15% buffering bilirubin direct (BD), gamma-glutamyl transferase solution and 15  mg K EDTA (BD Vacutainer, (GGT), albumin:globulin (A/G) ratio, and globulins. Franklin Lakes, NJ) and immediately placed in Horses with values outside of normal ranges were ex- ice. Samples intended for serum analysis were col- cluded from cytokine analysis to ensure that expres- lected into 10  mL evacuated non-additive tubes sion of systemic inflammatory markers was reflective (BD Vacutainer, Franklin Lakes, NJ) and allowed of joint disease and no other inflammatory processes. to clot for 30 min before processing to allow blood One horse from each treatment group, CON, 40BP, to clot. All samples were centrifuged at 2,000 × g at and 80BP were removed from cytokine analysis due Translate basic science to industry innovation Fikes et al. to abnormal blood chemistry parameters not ex- with 80BP having greater BCS on day 14 compared plained by joint disease. to both 40BP and CON. Horses receiving 80BP maintained BCS through day 28, while CON and 40BP increased BCS such that there was no signifi- Statistical Analysis cant difference between dietary treatments on d 28. All data were analyzed as a randomized design All horses, regardless of dietary treatment, gained using the PROC MIXED procedure of statistical BW (P < 0.01) until day 28 and lost BW to day 34 analysis software (SAS) (SAS Inst., Inc., Cary, NC) while BCS remained constant. with effects for treatment, day, and treatment × day interaction with linear and quadratic contrasts. The Gait Kinematic Parameters model uses RANDOM and REPEATED state- ments to account for variability between animals. Stride length:   A trend towards a treatment × Day 0 was used as a covariate for gait kinematics. time interaction (P  =  0.10, Fig. 3A) was observed Effects were considered significant if P ≤ 0.05, with in stride length (SL) of the forelimb at the walk, a trend towards significance if P ≤ 0.10. largely driven by an increase in SL in horses re- ceiving 80BP while horses receiving CON and 40BP RESULTS decreased from d 14 to d 28. A  trend towards a linear effect (P = 0.09) was also demonstrated with CON SL at 155 ± 3.83 cm on d 28, 40BP at 168 ± Physical Measurements 5.0 cm, and 80BP at 168.9 ± 3.56 cm. No significant There was no effect of BP supplementation on effects were observed for SL of the forelimb at the BW (P  =  0.76; Fig. 1); however, there was a treat- trot (P = 0.21, Fig. 3B). ment × time interaction for BCS (P < 0.01; Fig. 2) At the walk, a trend towards a treatment × time interaction (P  =  0.07; Fig. 4A) was observed for hindlimb SL, resulting from decreases in SL from day 14 to 28 in CON and 40BP while 80BP increased. From day 28 to 34, 40BP and 80BP de- creased while CON increased. There was a linear Figure 1. Bodyweight (kg) over time (d) in horses receiving a pel- leted concentrate with control, 40BP, or 80BP g/d of spray-dried bio- active proteins (LIFELINE, APC, LLC, Ankeny IA). Main effects included treatment, time, and treatment × time. Figure 3. Stride length (cm) of the forelimb at the walk (A) and Figure 2. Body condition score (BCS) over time (d) in horses re- trot (B) over time (d) in horses receiving a pelleted concentrate with ceiving a pelleted concentrate with either control, 40BP, or 80BP g/d either control, 40BP, or 80BP g/d of spray-dried bioactive proteins of spray-dried bioactive proteins (LIFELINE, APC, LLC, Ankeny (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- a,b,c a,b,c IA). Main effects included treatment, time, and treatment × time. ment, time, and treatment × time. Within d, superscripts denote a Within d. difference between dietary treatments (P < 0.05). Translate basic science to industry innovation Bioactive proteins on equine performance Figure 4. Stride length (cm) of the hind limb at the walk (A) and trot (B) over time (d) in horses receiving a pelleted concentrate with Figure 5. Range of motion (degrees) of the knee at the walk (A) either control, 40BP or 80BP g/d of spray-dried bioactive proteins and trot (B) over time (d) in horses receiving a pelleted concentrate (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- with either control, 40BP, or 80BP g/d of spray-dried bioactive proteins a,b,c ment, time, and treatment × time. Within d, superscripts denote a (LIFELINE, APC, LLC, Ankeny IA). Main effects included treat- a,b,c difference in dietary treatment (P < 0.05). ment, time, and treatment × time. Within d, superscripts denote a difference in dietary treatment (P < 0.05). increase in SL of the hindlimb at the walk-in treated affected hock range of motion at the walk quad- horses compared to CON (P = 0.05). Treatment did ratically (P  <  0.01) demonstrated by 40BP having not affect SL of the hindlimb at the trot (P = 0.17; the highest ROM at 33.2 ± 1.24°, followed by CON Fig. 4B). at 28.6 ± 1.45°, and 80BP having the lowest ROM Range of motion.  Range of motion (ROM) in at 28.0  ± 1.36°. Hock ROM at the trot was influ- the knee at the walk exhibited a trend towards a enced by time (P  =  0.05 Fig. 6B), but not treat- treatment × time interaction (P = 0.10, Fig. 5A), due ment (P  =  0.39) with all horses decreasing ROM to the increased range of motion for treated horses over time. compared to CON on day 34. Horses in the CON Blood chemistry parameters.  Dietary supple- dietary treatment decreased ROM from day 14 to mentation of BP did not influence blood chemistry 34, while treated horses increased. Horses receiving parameters (P > 0.10, Table 2); however, treatment × 80BP increased ROM throughout the study, with time interactions were observed for TSP, albumin, ROM reaching 32.5 ± 2.27° on day 28 and 34.6 ± globulins, and glucose (P  <  0.09). The interaction 2.27° on day 34. The 40BP dietary treatment group for TSP (P = 0.04; Fig. 7A) resulted from a decrease increased ROM to 34.4 ± 2.16° on day 28 and de- in TSP in CON with a corresponding increase in creased to 33.6 ± 2.16° on day 34. Supplementation BP supplemented horses to day 28. The interaction of BP did not influence knee ROM at the walk or of treatment × time for albumin (P < 0.01; Fig. 7B) trot (P  =  0.56, P  =  0.38, respectively). No signifi- is described by a decrease in CON to d 28 while al- cant effects were observed for knee ROM at the trot bumin increased in treated horses. Globulins tended (P = 0.64; Fig. 5B). to express a treatment × time interaction (P = 0.09; A significant treatment × time interaction Fig. 7C) where 40BP and 80BP horses increased to (P < 0.01, Fig. 6A) was observed for hock ROM at day 28 and CON horses decreased. Finally, a treat- the walk described by an increase in ROM in 80BP ment × time interaction for glucose (P = 0.02; Fig. horses from d 28 to d 34, a decrease from d 28 to 7D) is described by lower plasma blood glucose on 34 in 40BP horses, and relatively constant values day 34 in 80BP horses compared to CON or 40BP. in CON horses. Dietary supplementation of BP Translate basic science to industry innovation Fikes et al. Serum inflammatory markers.  A treatment × expression of IL-1β. Dietary treatment of BP time interaction was observed for IL-1β (P < 0.01; did not affect serum levels of TNF-α (P  =  0.51; Fig. 8A) is described by CON and 40BP horses Fig. 8B); however, TNFα increased over time re- increasing to d 34 while 80BP horses decreased gardless of dietary treatment (P = 0.05), with all horses increasing from d 28 to d 34. Time also influenced IL-1β (P < 0.01) as all treatments de- creased to day 28. DISCUSSION This study examined the effect of dietary BP supplementation on gait kinematics and systemic inflammatory markers in horses over time and fol- lowing an extensive transport and aerobic exer- cise challenge. Previous research in horse suggests that dietary BP improves stride length which indi- cates increased levels of comfort (Coverdale and Campbell, 2014). This study is the first in horses to measure systemic markers of inflammation with gait kinematics in response to dietary BP supple- mentation and exercise. Increased BW over time to day 28 may be ex- plained by the change from group feeding to indi- vidual feeding at the initiation of the study to ensure each horse was fed to meet NRC requirements for mature horses undergoing light exercise (NRC, 2007). Minor loss in BW between day 28 and 34 is likely due to trailering and participation in the parade challenge on day 34 of the study. Decreased Figure 6. Range of motion (degrees) of the hock at the walk (A) and trot (B) over time (d) in horses receiving a pelleted concentrate BW may be related to body water loss via sweat and with either control, 40BP, or 80BP g/d of spray-dried bioactive pro- subsequent dehydration as horses were weighed teins (LIFELINE, APC, LLC, Ankeny IA). Main effects included 24  h following transport (Friend, 2000). A  study a,b,c treatment, time, and treatment × time. Within d, superscripts denote differences in dietary treatment (P < 0.05). performed by Friend (2000) observed a similar Table 2. Least square treatment means of blood chemistry including total serum protein (TSP), albumin, phosphorus (P), glucose, blood urea nitrogen (BUN), creatinine, total bilirubin (TB), bilirubin direct (BD), creatine kinase (CK), serum glutamine oxaloacetate transaminase (SGOT), globulins, albumin:globulin ratio (A:G), and gamma-glutamyl transferase (GGT) in plasma of horses receiving a pelleted concentrate with either 0 g/d, 40 g/d, or 80 g/d of bioactive proteins P-values Parameter 0 g/d 40 g/d 80 g/d SEM Treatment Time Treatment x Time TSP, g/dL 6.45 6.48 6.51 0.13 0.96 0.01 0.04 Albumin, g/dL 2.9 2.99 2.94 0.04 0.33 <0.01 <0.01 P, mg/dL 2.9 2.88 2.93 0.08 0.89 <0.01 0.99 Glucose, mg/dL 91.33 90.74 91.1 1.29 0.95 <0.01 0.02 BUN, mg/dL 15.52 16.44 15.86 0.62 0.57 <0.01 0.17 Creatinine, mg/dL 1.37 1.43 1.41 0.05 0.72 <0.01 0.17 TB, mg/dL 0.7 0.76 0.61 0.05 0.15 <0.01 0.09 BD, mg/dL 0.27 0.28 0.27 0.01 0.72 <0.01 0.56 CK, U/L 302.73 307.67 306.29 24.35 0.99 0.27 0.48 SGOT, U/L 274.83 255.61 278.9 12.76 0.4 0.63 0.38 Globulins, g/dL 3.55 3.49 3.57 0.15 0.92 0.09 0.09 A:G 0.81 0.89 0.84 0.04 0.42 0.89 0.11 GGT, U/L 14.05 11.51 11.89 1.35 0.37 0.02 0.15 Translate basic science to industry innovation Bioactive proteins on equine performance Figure 8. Serum concentrations of interleukin-1β (IL-1β; A; pg/ mL) and tumor necrosis factor α (TNFα; B; pg/mL) over time (d) in horses receiving a pelleted concentrate with either control, 40BP, or 80BP g/d of spray-dried bioactive proteins (LIFELINE, APC, LLC, Ankeny IA). Main effects included treatment, time, and treatment × a,b,c time. Within d, superscripts denote differences between dietary treatments for serum IL-1β (P < 0.05). initial loss in BW during transport, which was sus- tained but not increased for 24 h in horses with ac- cess to water. Improvements in SL in the forelimb to day 28 at the walk were similar to those observed by Coverdale and Campbell (2014, 2015). This study included a challenge component that incorpor- ated trailering and riding on day 33, leading to the overall trends being altered from previous studies that did not include a trailering and exercise chal- lenge. Forelimb and hindlimb SL at the walk had a trend toward significance in this study compared to forelimb and hindlimb SL at the trot. This may be explained by the varying biomechanics of move- ment for the different gaits. Because the walk lacks a suspension phase (Clayton, 1989), training and coordination of the individual horse play a smaller role. This would allow any potential benefits of BP Figure 7. The effect of dietary supplementation of control, 40BP or 80BP g/d of spray-dried bioactive proteins (LIFELINE, APC, supplementation to become more evident and less LLC, Ankeny IA) on total serum protein (A; g/dL), albumin (B; g/dL), reliant on individual athletic ability. globulins (C; g/dL), and blood glucose (D; mg/dL). Main effects in- Bioactive proteins generally modulate the a,b,c cluded treatment, time, and treatment × time. Within d, superscripts denote a difference in dietary treatment (P < 0.05). immune response to physiological stress in a Translate basic science to industry innovation Fikes et al. challenging environment (Bosi et  al., 2004; Perez- involved in blood clotting and inflammation com- Bosque et  al., 2016). However, SL for both limbs prising many key components of the immune at the walk decreased on day 34. The inconsistent response (Miglio et  al., 2019). Because SDP con- response may be explained by other factors influ- tains functional factors such as immunoglobulins, encing SL after the challenge, such as muscle and growth factors, and active peptides (Campbell periarticular tissue soreness and behavior factors et al. 2010b), it is possible that levels of total serum during the challenge (Rawson et al., 2001; Lee et al., protein, albumin, and globulins would increase in 2002; Braun and Dutto, 2003). Additionally, dur- treated horses either by differences in dietary crude ing gait kinematic analysis, horses were recorded protein (CP; Leveille and Sauberlich, 1961) or bio- at the walk before moving into the trot, potentially active protein stimulation of the common mucosal acting as a warm-up to exercise, allowing the horse system to result in increased globulin concentra- to become more fluid in the movements at the trot. tions (Walther and Sieber, 2011). While little work has been done investigating the Decreasing or sustained levels of total serum effects of a walking warmup on equine gait kine- protein, albumin, and globulins to day 34 in treated matics, in humans, walking improves gait biomech- horses compared to CON, whose levels increased anics thus it is likely that similar effects are present from day 28 to 34 following the day 33 challenge, in this study (Schieber et  al., 2019). Furthermore, may indicate modulation of the immune response in the instance of muscle soreness following trans- following physiological stress of transport and ex- port and exercise stress, the active walking warmup ercise. Furthermore, previous studies have observed may have reduced any incidence of delayed onset modulatory effects of SDP on plasma glucose re- muscle soreness (Andersen et al., 2013). sulting from alterations in SGLT1 expression in a The interaction observed for knee ROM at the challenging environment and an increase in glucose walk but not at the trot may also be attributed to uptake by insulin-sensitive tissues (Moreto and differing gait biomechanics as well as individual Perez-Bosque, 2009; Oseguera-Toledo et al., 2014). athletic ability. Previous studies did not observe Our study shows comparable results following the trends or significance in knee ROM at either gait day 33 challenge where horses receiving 80BP had (Coverdale and Campbell, 2014, 2015). The re- lower plasma blood glucose than CON or 40BP. sponse observed in the present study for this par- In other species, SDP has demonstrated the ameter of gait kinematic analysis to the challenge ability to regulate cytokine expression (Bosi et al., on day 33 indicates that treated horses improved in 2004; Perez-Bosque et al., 2016). Following an acute performance compared to CON. Articular ROM lipopolysaccharide challenge in swine, SDP de- relates more closely to periarticular joint comfort creased mucosal expression of TNF-α (Bosi et al., than SL, suggesting that bioactive protein supple- 2004). However, in the current study, serum TNF- mentation was more effective in improving articular α levels were not influenced by dietary treatment. ROM than SL following a challenge (Bischoff et al., This is likely due to the use of a physical stressor 2003; Ahvazi et al., 2014). rather than a pathogen-induced challenge and the Currently, there is no literature on gait kine- use of a sustained physical challenge rather than matic analysis of hock ROM as it relates to SDP an acute response. Pathogen recognition functions supplementation. It is unknown why horses re- through acquired immunity, while acute physical ceiving 40 g/d BP showed improved hock ROM at stress functions primarily through the innate im- the walk to day 28 compared to horses receiving mune system. This change could explain the differ- 80  g/d BP. However, hock ROM at the walk re- ence in response between the current study and the sponded to the day 33 challenge similarly to knee study performed by Bosi et al. (2004). Furthermore, ROM at the walk with horses receiving 80  g/d BP rather than testing an acute response, sampling in having improved articular ROM in response to the the present study was designed to test the response challenge, further suggesting that BP are more ef- to a sustained physical challenge and resulted in im- fective at improving articular ROM than SL after proved hock ROM suggesting anti-inflammatory an exercise challenge. effects. Total serum protein (TSP) is a combined Similar to the Perez-Bosque et al. (2016) mouse measure of all proteins in the blood, including al- model, the current study demonstrates decreased bumin and globulins. Albumin is a blood protein levels of IL-1β in horses receiving 80BP following produced by the liver and is integral in the main- the day 33 challenge. Horses receiving 80BP had tenance of osmotic pressure (Miglio et  al., 2019). increasing values of TNF-α to day 28 and 34 Globulins include antibodies and other proteins with decreasing values of IL-1β. This suggests an Translate basic science to industry innovation Bioactive proteins on equine performance improved ability to adapt to the stress-related im- Conflict of interest statement. The authors af- mune response before IL-1β is expressed. Because firmatively acknowledge that they were free from IL-1β is the major cytokine associated with joint influence by any funding sources or their employees disease, this also suggests that dietary SDP’s ob- that would result in any conflict of interest. served improvement of articular ROM may be explained by the reduced expression of pro-inflam- REFERENCES matory IL-1β. Ahvazi,  S.  P., H.  Sadeghi, and M.  Gholami. 2014. 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Journal

Translational Animal ScienceOxford University Press

Published: Feb 8, 2021

Keywords: bioactive proteins; equine; gait kinematics; inflammation

References