Effects of Crude Rice Bran Oil and a Flaxseed Oil Blend in Young Horses Engaged in a Training Program

Simple Summary Rice bran oil and flaxseed oil contain omega-3 fatty acids with the potential to reduce post-exercise inflammation and muscle damage. This study measures interleukin-1β and creatine kinase concentrations and fatty acid profiles in lightly worked, young horses undergoing a 16-min incremental exercise test after 60 days of oil consumption. Consuming crude rice bran oil or flaxseed oil for 60 days may benefit lightly worked, young horses by reducing training-program-related increases in interleukin-1β, while a flaxseed oil blend may reduce exercise-induced increases in creatine kinase. Additionally, the flaxseed oil blend has the potential to increase plasma omega-3 and omega-6 fatty acids. Abstract Rice bran oil and flaxseed oil contain omega-3 fatty acids with the potential to reduce post-exercise inflammation and muscle damage. This study measures plasma interleukin-1β and creatine kinase and fatty acid profiles in lightly worked, young horses (Equus caballus) undergoing an exercise test after 60 days (d) of oil consumption, where the oil replaced 25% of concentrate calories. Treatments consisted of CON (no oil), FLAX (flaxseed oil blend), and RICE (crude rice bran oil). Blood was collected pre-exercise, and again at 1 min, 30 min, 24 h, 48 h, and 72 h post-IET. Data were analyzed by repeated measures ANOVA. Plasma creatine kinase activity was not different in CON during the study, greater (p < 0.05) in RICE from pre-exercise to 30 min post-exercise across all exercise tests, and lesser (p < 0.05) in FLAX at 30 min post-exercise on d 30 compared to d 0. Plasma interleukin-1β was greater (p < 0.01) in CON on d 60, but no differences were observed in FLAX and RICE throughout the study. Plasma alpha-linolenic and linoleic acids were greatest (p < 0.05) in FLAX after 30 d of inclusion, while CON horses had greater (p < 0.05) EPA across all exercise tests and DHA after 60 d. These results indicate that 60 d of inclusion of crude rice bran oil or a flaxseed oil blend may benefit lightly worked, young horses by reducing training-program-related increases in interleukin-1β, while a flaxseed oil blend may reduce exercise-induced increases in creatine kinase. Additionally, the flaxseed oil blend has the potential to increase plasma omega-3 and omega-6 fatty acids. Replacing 25% of concentrate calories with flaxseed or rice bran oil has potential benefits for young horses in training.


Introduction
Horses (Equus caballus) participate in a variety of sports, ranging from high intensity, short duration events that substantially elevate plasma lactate, such as thoroughbred with either crude rice bran oil (Riceland ® , Stuttgart, AR, USA) or a flaxseed oil blend (Soybean oil, cold-pressed organic flaxseed oil; Animed™, Winchester, KY, USA; Table 1). The oil was measured, poured over their concentrate feed, and then mixed thoroughly to ensure adherence to the pellets. On average, daily DE intake was 21 (Table 2). All horses were housed in 3.66 × 3.66 m stalls (Priefert, Mt. Pleasant, TX, USA) and were allowed 30 min of turnout time daily on a dry lot. Each horse also participated in a behavior and training class, which consisted of light groundwork 2 to 3 days a week, with no riding. Activities included lunging and round penning at walk, jog, and lope, saddled and unsaddled, as well as desensitizing to various objects. The full duration of exercise each day was approximately 30 min.
MN, USA) at 0.7% of their BW daily, which approximated 40% of DE requirements. During acclimation, Coastal bermudagrass hay was fed at 1.5 to 2% BW daily. Following acclimation to the barn and concentrate feeding, BW was obtained, and diets adjusted to feed 60% of DE requirements from hay and 40% from concentrate (NRC, 2007). An acclimation to oil inclusion started 14 d before d 0 ( Figure 1). Horses fed an oil treatment had 25% of their daily calorie requirement replaced with either crude rice bran oil (Riceland ® , Stuttgart, AR, USA) or a flaxseed oil blend (Soybean oil, cold-pressed organic flaxseed oil; Animed™, Winchester, KY, USA; Table 1). The oil was measured, poured over their concentrate feed, and then mixed thoroughly to ensure adherence to the pellets. On average, daily DE intake was 21 Table 2). All horses were housed in 3.66 × 3.66 m stalls (Priefert, Mt. Pleasant, TX, USA) and were allowed 30 min of turnout time daily on a dry lot. Each horse also participated in a behavior and training class, which consisted of light groundwork 2 to 3 days a week, with no riding. Activities included lunging and round penning at walk, jog, and lope, saddled and unsaddled, as well as desensitizing to various objects. The full duration of exercise each day was approximately 30 min.     Measurements were obtained for body weight (BW), body condition score (BCS), cresty neck score (CNS), intramuscular fat (IMF), rump fat thickness (RFT), forearm and gaskin circumference, and longissimus muscle area (LMA) at 3 wk before d 0, and again on d 30 and 60 ( Figure 1). Body weight was determined using a standard digital livestock platform scale. Body condition score was determined by using a 1-9 scale, where 1 = emaciated and 9 = obese [20]. Cresty neck scores were assigned using a 0-5 scale [21]. Several ultrasound measurements were recorded by a certified technician (Designer Genes Technologies, Inc., Harrison, AR, USA), including IMF (cm), RFT (cm), and LMA (cm 2 ). Images were also evaluated by the same certified technician, who was blinded to dietary treatments. Forearm and gaskin circumference were measured in cm using a soft tape measure around the widest point. Measurements for BCS, CNS, and forearm and gaskin circumference were obtained by a single, trained individual who was blinded to dietary treatments.

Incremental Exercise Test
An IET was conducted on an automated horse walker (Priefert, Mt Pleasant, TX, USA) 3 wk before d 0, and again on d 30 and 60 of oil inclusion ( Figure 1). All horses had free choice hay until the start of their exercise test to ensure similarities with fed states. The IET was conducted counterclockwise and consisted of 10 min at 16.1 kph, 2 min at 19.3 kph, 2 min at 22.5 kph, and 2 min at 25.7 kph or until exhaustion. Exhaustion was indicated when the horse struggled to keep pace with the automatic exerciser. Horses were then hand-walked for 30 min after completion of the exercise test. Blood samples were obtained via jugular venipuncture 4.5 h before each exercise test (fasting) and at 1 min, 30 min, 24 h, 48 h, and 72 h post-exercise. Whole blood was collected into evacuated tubes (Vacutainer, BD, Franklin Lakes, NJ, USA) coated with either lithium heparin (lipids, creatine kinase, IL-1β) or sodium fluoride/potassium oxalate (glucose and lactate) and stored on ice until centrifugation. Blood was then centrifuged for 10 min at 1500× g, and plasma was aliquoted and stored at −80 • C until further analysis. Heart rates were obtained via stethoscope, by experienced personnel, in the horse's stall directly before the IET and 1 and 30 min post-IET on d 0 and 30. On d 60, heart rates were obtained via stethoscope in the horse's stall directly before the IET and 30 min post-IET, with max heart rates obtained via heart rate monitor (KER Clockit Bluetooth Heart Rate Monitor, Polar Electro, Bethpage, NY, USA).

Sample Analysis
Plasma from collected blood was thawed at room temperature and analyzed to determine lactate, glucose, IL-1β concentrations, CK activity, and fatty acid percentages. Lactate Creatine kinase activity was obtained using the EnzyChrom™ Creatine Kinase Assay Kit from BioAssay Systems (Hayward, CA, USA) according to the manufacturer's provided protocol. The plate was covered and incubated at 37 • C inside a SpectraMax ® 190 plate reader (Molecular Devices, San Jose, CA, USA) and read at 340 nm at 20 and 40 min of incubation. The following equation was then used to calculate CK activity: The profile of fatty acids in plasma was determined by the methylation of all lipids in plasma and quantifying peaks using gas chromatography to calculate the percent that each fatty acid contributed to total plasma lipids. Fatty acid percentages were determined using the protocol created by Perfield et al. [22] and modified by Corl et al. [23] and analyzed using gas chromatography (Agilent GC system 6890N, Agilent Technologies, Santa Clara, CA, USA). The obtained peaks were identified using specific markers (Pure Methyl Ester Standards 68D and 91, Nu-Check Prep Inc., Elysian, MN, USA), and converted to a percentage of total fatty acids. While chromatographic analysis isolated and quantified multiple fatty acids, data were only analyzed for the following: palmitic acid, oleic acid, linoleic acid, alpha-linolenic acid, eicosanoic acid, eicosatrienoic acid, eicosapentaenoic acid, and docosahexaenoic acid.

Feed and Oil Analysis
Composited samples for concentrate and hay were analyzed for nutrient and fatty acid concentrations, and samples for both oils were analyzed for fatty acid concentrations (Cumberland Valley Analytical Services, Waynesboro, PA, USA).

Statistical Analysis
All data collected were analyzed using the PROC MIXED procedure of SAS (SAS Enterprise Guide 7.1), with effects for d (0, 30, 60), treatment (CON, RICE, FLAX), time point (pre-exercise, 1 min post, 30 min post, 24 h post, 48 h post, 72 h post), and all interactions using repeated measures ANOVA for the time within d, with the horse as the subject. Data for lactate, glucose, IL-1β, and CK were log-transformed to meet normal standards for these parameters, then back-transformed as geometric means with a 95% confidence interval. One FLAX horse was removed from all statistical analyses of lactate, glucose, IL-1β, and CK due to injury before the d 60 IET. Differences between simple effects were determined using Tukey tests to reduce the potential for increased type-1 error rates.
Linoleic acid (LA) had an interaction for treatment by day (p < 0.05; Table 3); however, there were no differences in simple effects. The average percentage of LA for each treatment was 48.9 ± 0.5% in CON, 49.7 ± 0.5% in FLAX, and 48.9 ± 0.5% in RICE. The average percentage of LA by day was 48.4 ± 0.4% on d 0, 49.0 ± 0.4% on d 30, and 50.0 ± 0.4% on d 60. Table 3. Mean ± SEM fatty acid profile of plasma lipids 1 in long yearlings consuming hay and a pelleted concentrate with no oil (CON) or either crude rice bran oil (RICE) or a flaxseed oil blend (FLAX) replacing 25% of concentrate calories daily for 60 d. Alpha-linolenic acid (ALA) had an interaction for treatment by day (p = 0.02), with FLAX on d 30 (2.9 ± 0.3%) having greater percentages than CON on d 30 (1.8 ± 0.3%) and RICE on d 30 (1.5 ± 0.3%; Table 3). There were no differences observed between CON and RICE on d 30 (p = 0.95). The average percentage of ALA was 2.2 ± 0.1% on d 0, 2.1 ± 0.1% on d 30, and 1.9 ± 0.1% on d 60. The average percentage of ALA for each treatment was 2.0 ± 0.2% for CON, 2.5 ± 0.2% for FLAX, and 1.7 ± 0.2% for RICE.

Plasma Lactic Acid
In CON horses, there was no effect of day (p = 0.65) or day x time interaction (p = 0.70; Figure 2A). There was a main effect of time (p < 0.001), with greater geometric mean (95% CI) concentrations at 1 min post-exercise (7. In FLAX horses, there was no effect of day (p = 0.76) or day x time interaction (p = 0.79; Figure 2B). There was a main effect of time (p < 0.001), with greater geometric mean concentrations at 1 min post-exercise (7.6 [4.6, 12.5] mmol/L) than before exercise  In FLAX horses, there was no effect of day (p = 0.76) or day x time interaction (p = 0.79; Figure 2B). There was a main effect of time (p < 0.001), with greater geometric mean concentrations at 1 min post-exercise (7.6 [4.6, 12.5]

Plasma Glucose
In CON horses, there was no effect of day (p = 0.84) or day x time interaction (p = 0.21; Figure 3A In FLAX horses, there was a tendency for a day x time interaction (p = 0.06; Figure  3B); however, there were no simple effect differences. There was no effect of day (p = 0.

Plasma Glucose
In CON horses, there was no effect of day (p = 0.84) or day x time interaction (p = 0.21; Figure 3A) on glucose concentration. There was a main effect of time (p < 0.01), with greater geometric mean (95% CI) concentrations 1 min post-exercise (5.9 [5.5, 6.4] mmol/L) than before exercise (5.1 [4.7, 5.5] mmol/L). There was also a tendency for greater geometric mean concentrations 30 min post-exercise (5.6 [5.2, 6.0] mmol/L) than before exercise (p = 0.08). There were no differences observed between 1 and 30 min post-exercise (p = 0.24). Plasma glucose geometric mean concentrations were 5. In RICE horses, there was no effect of day (p = 0.40) or day x time interaction (p = Figure 3C). There was a main effect of time (p < 0.01), with lower geometric mean co trations before exercise (

Heart Rates
On d 0, no differences were observed for heart rate (HR) before exercise (p = 0. On d 60, there were no differences observed for HR before exercise (p = 0.18), 3 post-exercise (p = 0.88), or max HR (p = 0.34; data not shown) between any of the ments. Before exercise, HR was 49.0 ± 2.9 bpm for CON, 40.0 ± 3.3 bpm for FLAX, and ± 2.9 bpm for RICE. At 30 min post-exercise, HR was 57.0 ± 5.6 bpm for CON, 55.0 bpm for FLAX, and 53.0 ± 5.6 bpm for RICE. Max HR was 215.0 ± 6.1 bpm for CON, ± 7.1 bpm for FLAX, and 218.0 ± 6.1 bpm for RICE.

Plasma Interleukin-1β
In CON horses, there was no effect of time (p = 0.86) or day x time interaction 0.31; Figure 4A) on plasma interleukin-1β (IL-1β). There was a main effect of day (p < with greater geometric mean (95% CI) activity on d 60 (  In FLAX horses, there was a tendency for a day x time interaction (p = 0.06; Figure 3B); however, there were no simple effect differences. There was no effect of day (p = 0. In RICE horses, there was no effect of day (p = 0.40) or day x time interaction (p = 0.82; Figure 3C). There was a main effect of time (p < 0.01), with lower geometric mean concentrations before exercise (5.0 [4.6, 5.5] mmol/L) than 1 min post-exercise (6.1 [5.5, 6.6]
In RICE horses, there was no effect of day (p = 0.43), time (p = 0.25), or day x time interaction (p = 0.76; Figure 4C). Plasma IL-1β geometric mean concentrations were

Discussion
The main objective of this study was to determine if 60 d of 25% calorie replacement with rice bran oil or a flaxseed oil blend would affect markers of metabolism, muscle breakdown, and inflammation post-exercise. Further objectives were to determine the effects of these oils on heart rate during exercise, plasma lipid profiles after replacement, body fat estimates, and muscling scores. It was hypothesized that crude rice bran oil, which contains omega-3 fatty acids, vitamin E, and γ-oryzanol, would lessen muscle damage and inflammation post-exercise in the oil-fed horses. The main findings of this study were that the exercise test induced anaerobic metabolism in all treatment groups, feeding a flaxseed oil blend increased linoleic acid (LA) and alpha-linolenic acid (ALA) percentages while decreasing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and replacing 25% of concentrate calories with oil did not negatively impact muscle and fat parameters.
During the course of this study, BW increased and did not differ by treatment, which supports normal growth patterns in young horses receiving adequate nutrition. On average, horses gained approximately 0.3 kg/d, which is a suggested rate of daily gain for horses aged 18 months. While our horses ranged from 18 to 30 months, the majority of horses were closer to 18 months, a fact that should partly account for the average daily gain being more representative of younger horses [24]. It has also been shown that horses tend to be at 50% of their mature BW at 1 yr of age and at 75% of their mature BW at 2 yr of age [25]. Since long yearlings and 2-year-old horses were utilized, this could indicate that the horses in the current study had the potential to add approximately 25% of their mature BW during the 60 d. Additionally, BW comes from fat and muscle, along with other components not measured in the current study. Two body fat parameters, BCS and rump fat thickness (RFT), increased during the first 30 d in all treatments, which indicates that horses in all three treatments accrued body fat. Horses increased from 9.9% body fat to 12.3% body fat, which, according to previous studies, suggests that these horses increased from the low end to the high end of the normal body fat range, regardless of treatment [26,27]. Increases in both forearm and gaskin circumference were observed from d 0 to 30, indicating that the increase in BW from d 0 to 30 was not only due to the addition of fat but also included muscle accretion. This muscle accretion could have been due in part to the role of daily exercise. It is important to note that while replacing 25% of concentrate calories with oil removes a portion of crude protein and other nutrients supplied in concentrates, the muscle parameters did not differ in RICE or FLAX compared to CON. In summary, all horses increased in BW and body fat as well as added muscle, indicating that replacing concentrate calories with oil has no negative effects on fat and muscle parameters.
Flaxseed oil has been shown to consist of roughly 50% ALA, which makes it one of the richest sources of omega-3 fatty acids [28]. For the current study, the finding that plasma ALA percentages were greater in FLAX than in CON and RICE on d 30 may indicate that 30 d of flaxseed oil blend inclusion can help to increase ALA in plasma. Our results agree with those of Hansen et al. [29], in which horses fed flaxseed oil exhibited greater plasma ALA than control horses at 8 and 12 wk of oil inclusion. As a comparison, Hansen's study added an additional 10% of flaxseed oil to the horse's energy requirements, whereas in the current study, 10% of the total energy requirement came from a flaxseed oil blend. It is important to note that the flaxseed oil fed in Hansen's study was 44% ALA, while the flaxseed oil blend in the current study was 9.2% ALA. The RICE horses consumed 58% of the amount of ALA consumed by FLAX horses, and it appears this was not a sufficient quantity to induce alterations to plasma content.
The increased ingestion of ALA did not correlate to increased plasma percentages of longer-chain omega-3 fatty acids. As shown in previous research, the conversion of ALA to EPA and DHA is limited due to competition with LA being converted to arachidonic acid [30]. Both of these fatty acids share the ∆6-desaturase enzyme, with ALA being the preferred substrate [30]. However, if LA is more prevalent in the diet, the metabolism of omega-6 fatty acids takes precedence over omega-3 fatty acid metabolism. All horses consumed more LA than ALA, which may have resulted in lower conversion rates of ALA to EPA and DHA [30]. Even after supplementation of ALA, studies have shown that there is little to no evidence of increases in circulating EPA and DHA in horses. However, in these studies, the horses consumed more ALA than LA [29,31,32]. For the current study, decreases in EPA after 30 d, as well as observations of CON horses having greater EPA and DHA than RICE and FLAX throughout the study, could also be explained by the greater amounts of LA in the FLAX and RICE treatments compared to CON, causing a decrease in the conversion rate of ALA to EPA and DHA. The increase in LA from d 0 to 30 is similar to the findings of Hansen et al. [29], in which plasma LA was greater in horses fed flaxseed oil than the control horses at 4, 8, and 12 wk of oil inclusion.
The incremental exercise test (IET) was shown to induce anaerobic exercise, as indicated by changes in plasma glucose, lactate, and heart rates. Lactate concentrations, as a measure of anaerobic threshold, are commonly used to assess the level of fitness in equine athletes [33,34]. In a study performed by Cabrera et al. [35], lactate concentrations reached the anaerobic threshold directly after high-intensity and maximum-intensity exercise and returned to aerobic levels during the recovery period, which was 10 min post-exercise. Therefore, the increase above 4 mmol/L in lactate concentrations at 1 min post-exercise across all treatments observed in the current study indicates that each horse was exercising anaerobically. Piccione et al. [33] also demonstrated a significant increase in lactic acid in show jumping horses as well as horses that participated in a 2 min treadmill running test. Both groups reached an anaerobic status, with lactate concentrations decreasing to baseline by 30 min post-exercise. For the current study, RICE lactate concentrations returned to baseline at 30 min post-exercise on d 30 and 60, with d 0 remaining higher. However, CON and FLAX remained elevated at 30 min post-exercise on d 0, 30, and 60. This could be explained by RICE containing γ-oryzanol, a mixture of ferulic acid esters that has been shown to decrease lactic acid concentrations after exercise [36].
Increases in plasma glucose concentrations for CON are consistent with a study from Ferraz et al. [37], where concentrations rose as the exercise intensity increased. Plasma glucose elevations towards the end of exercise have been related to the effects of catecholamines and glucagon on the liver, both increasing glucose release from the liver and decreasing the re-uptake of glucose by the liver [38], therefore increasing blood glucose concentrations. Some studies have indicated that fat supplementation improves glucose metabolism during exercise, which has further benefits on performance [39][40][41]. It is thought that fat supplementation drives an increase in glycogen stores or creates a glycogen-sparing effect by increasing fat utilization during exercise. These varied results may be due to a difference in fat type and amount, treatment duration in relation to metabolic adaptations, or a washout period being utilized [42]. During recovery, low free fatty acid concentrations may result in glucose being redirected for energy production. Therefore, providing a high-fat diet may spare muscle glycogen stores by increasing the availability of lipids during the recovery phase [43,44]. This could be used to explain why FLAX and RICE also had increased glucose concentrations post-exercise.
Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays an important role in mediating inflammatory responses, and production is often stimulated by strenuous exercise [45]. In the current study, increases in post-exercise IL-1β concentrations after 30 d are similar to those seen in a study conducted by Fikes et al. [46], where IL-1β showed no changes in the unstimulated state but increased after a strenuous bout of exercise. These findings and those of the current study disagree with studies where conditioning decreased the post-exercise inflammatory response [10]; however, horses in the current study were not conditioned to perform the intense exercise of the IET. The horses used for the current study were lightly exercised 2-3 days per week for 30 min per session, with some of this time being spent on non-exercise tasks such as acclimation to wearing a saddle. This could indicate that the level of conditioning utilized prior to an exercise test is related to post-exercise inflammatory responses. In addition to the effect of conditioning, studies have suggested that the most effective option to inhibit the secretion of IL-1β is to inhibit the activity of caspase-1, the protease that produces mature IL-1β from its precursor protein [47]. Yan et al. [48] showed that in mice, omega-3 fatty acid supplementation helped to inhibit caspase-1 activity as well as IL-1β secretion by inhibiting NLRP3, a cytosolic protein complex responsible for activating caspase-1. Supplemental omega-3 fatty acids have also been shown to inhibit the production of IL-1β after an induced inflammatory response in humans [49] as well as in horses [50]. In the current study, FLAX and RICE showed no difference in IL-1β concentrations during the 60 d trial compared to CON, which experienced increases. This could be explained by both rice bran oil and the flaxseed oil blend containing high amounts of omega-3 fatty acids. More research is needed to further determine the effects of omega-3 fatty acid supplementation on IL-1β concentrations in exercising horses.
Creatine kinase (CK) concentrations greater than 10,000 U/L, which is equivalent to 167 µmol/L, are commonly used as an indicator of muscle damage in horses [51]. Concentrations typically rise after strenuous exercise and return to baseline by 24 h postexercise, with a lesser rise in plasma CK indicating less muscle damage [52]. For the current study, in FLAX before calorie replacement with oil, the increase in CK activity post-exercise, followed by the decrease, corresponds to a study conducted by [53], where CK activity increased directly after exercise, then dropped down to pre-exercise values by 24 h postexercise. Observations of lower CK activity for FLAX at 30 min post-exercise after feeding 30 d of flax oil could be explained by greater amounts of ALA present in the flaxseed oil blend. In agreement, other studies have shown that CK activity is markedly lower if horses are provided a high-fat diet through the use of top-dressed flaxseed or soy-based oils that contain high amounts of omega-3 fatty acids, such as ALA [54]. This finding is potentially due to the protective effects of omega-3 fatty acids on cell membranes [55]. This could explain why CK activity remained high at 24 h post-exercise for RICE and CON, as horses on these treatments consumed lower amounts of ALA than FLAX.

Conclusions
In summary, 30 to 60 d of inclusion of crude rice bran oil or a flaxseed oil blend may benefit lightly worked, young horses by reducing training-program-related increases in interleukin-1β, while only the flaxseed oil blend may help to reduce exercise-induced increases in creatine kinase. Results also indicate that neither oil induces a loss of muscle mass or an increase in body fat. Additionally, the flaxseed oil blend has the potential to increase plasma omega-3 and omega-6 fatty acids. Future research could potentially determine the effects of a blended combination of these oils and see how it compares in young growing horses vs. mature performance horses.