Dietary inclusion of ruminally protected linseed oil as a means to mitigate heat and slaughter-induced stress in feedlot cattle

Abstract

There is evidence of a relationship between increased energy intake and the development of metabolic inflammation and insulin resistance (IR), and between the aforementioned metabolic state and impaired tolerance to heat stress. Based on the anti-inflammatory properties and mitigating effects on IR and stress of n-3 polyunsaturated fatty acids (n-3 PUFA), an experiment was performed to evaluate the effect of n-3 PUFA supplementation to feedlot-finished steers during summer on animal performance, physiological and biochemical variables associated with glucose metabolism, heat and preslaughter-induced stress, and meat quality. A total of 48 Angus steers (388 ± 2 kg) were fed one of three corn-based finishing diets containing (dry matter basis) 0% added oil (CON; negative control), or 1.90% of sunflower oil-calcium salt (SUN; positive control), or 1.92% of linseed oil-calcium salt (LIN). There was a trend (P = 0.08) for greater dry matter intake (DMI) and greater (P = 0.02) average daily gain (ADG) in LIN-fed animals compared with the average between those that received the CON or SUN diets, whereas no differences (P ≥ 0.34) were observed between the latter. No other performance, physiological, or carcass variables were affected (P ≥ 0.12) by treatment. Blood glucose and insulin were similar (P ≥ 0.14), though the homeostatic model assessment (HOMA) which gauges IR tended (P = 0.06) to be reduced for LIN-fed animals compared with the average between those that received the CON or SUN diets. Blood insulin and HOMA increased linearly (P ≤ 0.01) with days on feed. An interaction between the study phase (feeding period or slaughter) and treatment was observed (P ≤ 0.05) for glucose and cortisol. While the magnitude of glucose increase (P < 0.01) from the end of the feeding period to slaughter was greater for CON- and SUN-fed animals compared with LIN-fed ones, cortisol increased (P < 0.05) only in animals that received CON or SUN diets. Meat quality attributes were not affected (P ≥ 0.16) by treatment. The concentration of n-3 PUFA was greater (P < 0.01) and n-6:n-3 ratio was lesser (P < 0.01) in meat from LIN-fed animals compared with that resulting from the average between the animals that received the negative (CON) or positive (SUN) control diets. Results suggest that n-3 PUFA supplementation mitigated metabolic alterations associated with IR and preslaughter-related stress. It may have also improved tolerance to heat, resulting in greater DMI and ADG of steers fed a high-energy diet during summer. Results also indicate that glucose metabolism and heat stress tolerance worsen with time when feeding concentrate-based diets.

Lay Summary

Polyunsaturated fatty acids (n-3 PUFA) of diets have anti-inflammatory properties and mitigating effects on insulin resistance (IR) and stress in steers. This study evaluated the effect of n-3 PUFA supplementation to feedlot-finished steers during summer on animal performance, physiological and biochemical response, and meat quality. We found that n-3 PUFA supplementation mitigated metabolic alterations associated with IR and slaughter-related stress. It may have also improved tolerance to heat, resulting in greater dry matter intake, and average daily gain of steers fed a high-energy diet during summer.

The dietary inclusion of n-3 polyunsaturated fatty acids to feedlot-finished steers mitigated metabolic alterations related to insulin resistance, improving both production and nutritional aspects of beef.

Introduction

During recent past years, different aspects related to animal nutrition have been evaluated beyond their effects on animal performance and product quality. According to Carrillo et al. (2016), dietary manipulation has been considered as an important nutritional welfare tool, able to influence the animal as a whole entity. In this regard, greater feed and energy intake may result in nutrient overload, increased body fat accretion, and obesity. This metabolic condition is positively associated with cellular stress and chronic inflammation, a state known as metabolic inflammation (MI; Xu et al., 2003) and with the development of insulin resistance (IR) in several species (McCann and Reimers, 1986). Furthermore, there is evidence of a relationship between the development of MI and IR and reduced vasodilation and perspiration during heat-induced stress in humans (Petrofsky et al., 2005), as well as between stress and animal performance and meat quality (Digiacomo et al., 2014; Warner et al., 2014). Additionally, slaughter has been proven to increase plasma cortisol as a response to increased stress (Pighin et al., 2013).

Nutritional strategies are being discussed in order to relieve animal stress and to minimize its negative impact on meat production and quality. Reduced feed intake has been identified as a behavioral response to reduce metabolic heat load in heat-stressed animals (Sejian et al., 2018). In that scenario, increasing dietary fat, fed either ruminally protected or unprotected, has been suggested as a strategy to maintain energy intake and further decrease metabolic heat in livestock animals (Renaudeau, 2012; Conte et al., 2018). Additionally, several studies have indicated that, as opposed to n-6 polyunsaturated fatty acids (n-6 PUFA), n-3 polyunsaturated fatty acids (n-3 PUFA) have important anti-inflammatory effects and insulin sensitivity modulation properties in different animal species and humans (Pighin et al., 2003; Nagao and Yanagita, 2008; Schmitz and Ecker, 2008). In light of the beneficial effects of n-3 PUFA on the response to stress in monogastric animals (McAfee et al., 2019), dietary inclusion of n-3 PUFA has been considered in prophylaxis for heat stress in humans and ruminants (Caroprese et al., 2011; Shah et al., 2016). However, to the best of our knowledge, supplementation with n-3 PUFA to beef cattle-fed high-energy diets as a means to mitigate the negative effects of heat exposure on animal performance and meat quality has not yet been tested. Consequently, and based on the anti-inflammatory properties of n-3 PUFA and their mitigating effects on IR and stress, we hypothesized that the biological and productive response of cattle exposed to heat load and slaughter-induced stress would be improved by n-3 PUFA supplementation compared with n-6 PUFA and no fat supplementation and that it would remain similar between the last two. Therefore, the objective of the study was to evaluate the effect of n-3 PUFA supplementation to feedlot-finished steers during summer on animal performance, physiological and biochemical variables associated with glucose metabolism as well as with heat and slaughter-induced stress, and meat quality.

Materials and Methods

Animal care and handling procedures were approved by the Institutional Animal Care and Use Committee (CICUAE#03-18) of the National Institute of Agricultural Technology (INTA). The experiment was conducted from January 1 to March 30, 2015 at the General Villegas Experimental Station of INTA, Buenos Aires, Argentina (−34.866242, −62.781375).

Dietary treatments and feeding protocol

A total of 48 Angus steers (388 ± 2 kg) were evenly assigned by weight to one of three dietary treatments. Within each treatment, animals were randomly pair-grouped and housed in 24 open soil-surfaced pens (360 m²) without shade, where water was always available. Treatments consisted in corn-based diets containing (dry matter [DM] basis) 0% added oil (negative control; CON), or 1.90% of ruminally protected sunflower oil-based product (78% lipid content; positive control; SUN), or 1.92% of ruminally protected linseed oil-based product (77% lipid content; LIN; Table 1). Both oil-based products were commercial calcium salts (Tecnuar LLC, Rosario, Santa Fe, Argentina); degree of effective ruminal protection was labeled by the manufacturer at 50%. Doses were chosen based on results from Kronberg et al. (2011) and Suksombat et al. (2016). Diets were formulated to generate a rumen degradable protein (RDP) balance equal to zero and to meet or exceed metabolizable protein requirements at expected dry matter intake (DMI) and average daily gain (ADG), according to Level 1 of the NRC (2000) model. The CON diet was prepared daily in a mixer and delivered in the bunks of each pen; oil products were then top-dressed and hand-mixed with the ration in the bunks of the corresponding pens.

Table 1.

Composition¹ (dry matter basis) of diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added and fed to finishing beef cattle during summer

Item	Dietary treatment2
		SUN	LIN
Dry-rolled corn, %	78.77	77.27	77.26
Sorghum-sudangrass silage, %	18.05	17.71	17.70
Urea, %	1.22	1.20	1.20
Dry supplement, %3	1.96	1.92	1.92
Sunflower oil-based calcium salt, %4	0	1.90	0
Linseed oil-based calcium salt, %4	0	0	1.92
Chemical composition 5
Organic matter, %	95.5	95.2	95.1
Crude protein, %	11.5	11.3	11.3
Neutral detergent fiber, %	17.0	16.7	16.7
Ether extract, %	5.6	7.0	7.0
NEg, Mcal/kg6	1.49	1.53	1.53

CON diet was delivered in the bunks of each pen; oil products were then top-dressed and hand-mixed with the ration in the bunks of the corresponding pens. Therefore, ingredient and chemical composition for SUN and LIN diets were proportionally calculated based on CON ingredient composition and analyzed chemical composition, considering the addition of 1.90% or 1.92% oil product, respectively.

CON, negative control; SUN, positive control; LIN, treatment.

Contained 0.08%, 34.2%, 0.08%, 0.15%, and 0.01% of K, Ca, P, Mg, and S, respectively; 3, 457, 846, 2,459, 6, 1,160, 45, and 1,500 ppm of Co, Cu, Fe, Mn, Se, Zn, I, and monensin, respectively, and 165,167, 22,523, and 1,502 IU/kg of vitamin A, D, and E, respectively.

Analyzed lipid content was 78% and 77% (dry matter basis) and n-3 polyunsaturated fatty acid concentration was 8% and 38% for sunflower and linseed oil-based products, respectively.

Performed in duplicate on a composite derived from 27 samples (3 samples collected per week).

Net energy for gain calculated based on analyzed diet total digestible nutrient content and NRC (2000) equations.

Steers were adapted to the CON diet from day −14 to day −1 by increasing feed delivery at a rate of 0.5 kg/d (as fed) until feed crumbs were found. From day 1 to day 63, each group of animals received its corresponding dietary treatment. Animals were fed once daily at 0900 hours. Bunk scores (0 = licked; 1 = feed crumbs; 2 = more than crumbs) were recorded before feeding. Feed offer was increased 0.5 kg per animal (as is) when the bunk scored 0 or was kept as the previous day when the bunk scored 1. When the bunk scored 2, and the weight of the refusal (visually estimated) was below 5% of feed offered, feed offer was determined as that of the day before minus half the weight of the refusal. In this case, refusal was kept in the bunk, and the amount of feed delivered was calculated as targeted feed offer minus refusal. Refusals greater than 5% were removed, weighed, and sampled immediately, and steers were offered the same amount of feed as the previous day. Otherwise, refusals were kept in the bunk and removed, weighed, and sampled once a week to estimate DMI, which was calculated as the difference between total feed delivered and refused.

Cattle handling, sample and data collection, and analytical methods

Temperature, humidity, solar radiation, and wind speed were recorded hourly using a s032-01 SuperVis meteorological station (Siap+Micros LLC, Treviso, Italy). These records were used to calculate the hourly temperature–humidity index (THI; Mader et al., 2004).

Ruminally protected oil products were sampled and analyzed prior to the beginning of the experiment to determine the lipid content according to Sukhija and Palmquist (1990) with modifications. Fatty acid (FA) profile was determined in the lipid extract after saponification with 4% KOH in ethanol absolute. Methyl esters of FA were prepared and determined using GC equipment (Varian CP 3800, Varian Inc., Walnut Creek, CA) fitted with a flame ionization detector as described in Garcia et al. (2008). Lipid, n-6 PUFA, and n-3 PUFA contents were measured at (DM basis) 78% and 77%, 51% and 23%, and 8% and 38% for sunflower and linseed oil-based products, respectively.

To determine total digestible nutrient (TDN), crude protein (CP), and neutral detergent fiber (NDF) contents that were used as inputs by the NRC (2000) model when formulating the diets, diet ingredients were sampled prior to the beginning of the experimental period. During the latter, diet samples were collected once a week in the beginning, in the middle, and at the end of the bunk line. Feed ingredients were sampled once a week to determine DM content and adjust daily feed offer. After completion of the experimental period, diet samples were composited and analyzed to determine the chemical composition (Table 1). The TDN content was assumed to be equal to in vitro DM digestibility, measured after 30 h in the incubator (DaisyII; ANKOM, Macedon, NY) as suggested by Goering and Van Soest (1970). The CP content was determined by the improved Kjeldahl method, through which the sample is digested in sulfuric acid, ammonia is distilled, and excess acid is titrated (method 46-129 of the AACC, 1995). The NDF content was determined using thermostable alpha-amylase, sodium sulfite, and a fiber analyzer (ANKOM200/220; ANKOM, Macedon, NY) as suggested by Goering and Van Soest (1970). The RDP balance was estimated using book-referenced RDP and effective NDF (eNDF) values (NRC, 2000), except for sorghum-sudangrass silage. In this case, eNDF (particle size larger than 1.18 mm; Smith and Waldo, 1969; Mertens, 1985) concentration was measured at 95.8% on a 200-g sample, using a particle separator fitted with a 1.18-mm sieve (Nasco, Fort Atkinson, WI), following Heinrichs (2013) procedure, and determining the weight and NDF concentration on each fraction.

To estimate initial and final body weight (BW), animals were individually weighed before feeding on days 1 and 61. On the same days, the longissimus dorsi muscle (LM) area and 12th rib fat thickness were recorded by ultrasonography.

Based on the hypothesis that IR and consequently, impaired heat tolerance, may develop with days on feed (DOF), blood samples were collected in EDTA-containing tubes from the jugular vein on days 1, 29, and 61 before feeding to determine glucose, insulin, and cortisol concentrations. Plasma and serum were separated from blood samples and stored at −20 ± 1 °C. Glucose concentration in plasma was determined by GOD/POD Trinder Color Test (Wiener, Rosario, Argentina) without deproteinization. Serum concentrations of insulin and COR were measured using commercial kits provided by DiaSource (DiaSource S.A., Belgium). Intra and inter-assay coefficients of variation were 3% and 6% for insulin, and 3% and 5% for cortisol, respectively. Also on days 1, 29, and 61, rectal temperature was recorded by carefully introducing a digital thermometer approximately 4 cm into the rectum until the temperature reading was stable. In order to test the effect of dietary treatment under more challenging environmental conditions (Table 2), additional blood samples were collected on day 22, in a time frame during which THI was classified as “Alert” (74 < THI < 79). Similarly, respiratory rate was recorded on days 10, 11, and 36 in time frames during which THI was classified as “Danger” (79 < THI < 84, Mader et al., 2004; Table 2); the amount of respiratory movements during three 15-s periods was recorded, for which each animal was observed without disturbing or removing it from the pen. Concentrations of glucose and insulin were used to calculate the homeostatic model assessment (HOMA), which is considered as a model to determine IR (Warner et al., 2014). After being transported 350 km and a 12-h rest at a commercial abattoir lairage (with free access to water), animals were humanely slaughtered on day 64 and carcass weight was recorded. Concentrations of glucose, insulin, and cortisol were also determined in blood samples collected during slaughter in EDTA-containing tubes and compared with those determined at the end of the feeding period (day 61), in order to evaluate differential effects of slaughter-related stress across treatments. Additionally, hematocrit was immediately determined by the micro-hematocrit method in blood samples collected during slaughter. Profile of FA was determined in blood samples collected on day 1 and in blood and meat samples collected on day 64.

Table 2.

Temperature–humidity index (THI) in time frames of different experimental days during which measurements were recorded and/or samples were collected

Day	Time frame, h	THI	Classification1	Measurement/sample2
1	1000–1300	57	Normal	BW, LMa, 12RFT, blood, Temp
10	1500–1600	81	Danger	Respiratory rate
11	1350–1550	83	Danger	Respiratory rate
22	1300–1600	78	Alert	Blood
29	1000–1300	74	Normal	Blood, Temp
36	1430–1530	80	Danger	Respiratory rate
61	1000–1300	67	Normal	BW, LMa, 12RFT, blood, Temp
64	0800–1000	56	Normal	Blood, HCW

Based on The Livestock Weather Safety Index for heat stress (Mader et al., 2004).

BW, body weight; LMa, longissimus dorsi muscle area; 12RFT, 12th rib fat thickness; Temp, rectal temperature.

At 48 h after slaughter, a rib section encompassing the 10th to 12th ribs was removed from the left side of each carcass, identified, and transported to laboratory facilities. At 72 h, each section was separated in 2.5-cm wide steaks, which were identified, vacuum packaged, and stored at −20 °C for subsequent analysis. Steaks were allowed to thaw for 24 h at 4 °C prior to analysis.

Instrumental color, muscle pH, and water holding capacity (WHC) were determined on steaks from the 12th rib as described in Coria et al. (2019). Steaks from the 11th rib were deboned, trimmed of subcutaneous fat and epimysium, weighed, and placed in a preheated electric grill until they reached a final internal temperature of 71 °C. Cooked steaks were weighed after cooling for 20 min at 20 °C. Cooking loss (CL) was expressed as the percentage of weight loss relative to the initial weight of the sample. For the textural profile analysis (TPA) test, each cooked steak (2.5-cm thickness) was sampled obtaining eight 1.25-cm wide cores parallel to muscle fibers. A TA.XT Plus texture analyzer (Stable Micro Systems LLC, Surrey, UK) was used to assess the TPA. Each core of the sample underwent two cycles of 70% compression (1.0 mms-1 test crosshead speed) using a P/35 35mm-diameter probe. Force-by-time data were used to calculate mean values for the TPA parameters (hardness, chewiness, cohesiveness, and springiness) for each steak (Ruiz de Huidobro et al., 2005).

Total intramuscular fat content was determined by the Soxhlet method on two 5-g samples of steak from the 10th rib as described in Garcia et al. (2008). An additional 5-g sample from the same steak was processed following the method suggested by Folch et al. (1957). The resulting chloroform extract was used to determine the FA profile as described earlier.

Experimental design and statistical analyses

All statistical analyses were performed with SAS software using the SAS Studio interface through SAS On Demand for Academics (SAS Institute Inc., 2021). Data were analyzed using the MIXED procedure considering a completely randomized design with eight replications (pens) with two animals per replication (pen = experimental unit). For pen-based variables (DMI and gain-to-feed ratio), the statistical model included the effect of pen within dietary treatment as the random error. For variables individually determined (BW, ADG, carcass characteristics, biochemical variables, respiratory rate, rectal temperature, quality attributes of meat, and blood and meat FA profile), the statistical model additionally included the effect of the animal within pen and dietary treatment which represented the random error (St-Pierre, 2007).

For variables determined repeatedly on the same subject, a repeated measure structure was considered. Degrees of freedom were calculated requesting the Satterthwaite option. To select the best covariance structure for each variable, unstructured matrices of variances, covariances, and correlations were requested. Based on this information, inadequate structures were ruled out. When more than one structure was potentially suitable, the final selection was made based on information criteria. Unstructured covariance matrices were selected for glucose and insulin concentrations, rectal temperature, and respiratory rate while heterogeneous compound symmetry and first-order autoregressive matrices were chosen for HOMA and cortisol, respectively.

Backfat thickness and LM area recorded on day 1 were used as covariates in the analyses of backfat thickness and LM area recorded on day 61, respectively. Similarly, the initial blood FA profile measured on day 1 was used as a covariate in the analyses of the final blood and meat FA profile determined on day 64. The treatment effect was evaluated through preplanned orthogonal contrasts (CON vs. SUN and [CON + SUN]/2 vs. LIN). In order to test if the biochemical profile was similar across treatments at the beginning of the experimental period, glucose, insulin, and cortisol blood concentrations, as well as HOMA measured on day 1 were compared across treatments. The effect of DOF was evaluated using linear trend contrasts, for which coefficients for unequally spaced sampling times were obtained through the IML procedure. Body temperature and respiratory rate were not expected to change with DOF but THI. Therefore, the effect of the latter, treatment, and their interaction was evaluated. Because the order in which animals entered the stun box could not be evenly allotted across treatments, that order was included as a covariate in the analysis of data collected during and after slaughter to minimize any possible confounding effects due to changes in peri-slaughter conditions. Additionally, the association between the position in the entry line into the stun box and glucose, cortisol, or hematocrit determined in blood samples collected during the slaughter (day 64) was evaluated using the CORR procedure. The possible differential effect of slaughter-related stress across treatments was evaluated by contrasting the biochemical profile measured at the end of the feeding period (day 61) and the slaughter day (day 64) within the dietary treatment. Effects were considered significant when P-values were less than or equal to 0.05 and were considered trends when P-values were between 0.05 and 0.10.

Results and Discussion

Performance and physiological response variables

In 36 out of the 64 d of the experimental period, there were hourly THI values that classified, at least, as “Alert” (>74); 95% of these records were observed between 0010 and 1900 hours. Within this time frame, 34% of the THI records were greater than 74.

There was a trend (P = 0.08; Table 3) for greater DMI in animals fed the LIN diet compared with the average between those that received the CON or SUN diets, while no difference (P = 0.34) was observed between the latter. This result probably explains greater (P = 0.02) ADG in LIN-fed animals compared with the average between CON- or SUN-fed animals, and similar ADG between the latter (P = 0.86). Calculated supplemental oil intake resulted in 0, 147, and 155 g/d for animals fed CON, SUN, and LIN, respectively. No other performance or carcass variables were affected (P ≥ 0.12) by dietary treatment.

Table 3.

Performance and carcass characteristics of finishing cattle fed corn-based diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added

Item	Dietary treatment1			SED	Contrast P-value
	CON	SUN	LIN		CON vs. SUN	CON+SUN vs. LIN
n	8	8	8	-	-	-
Initial body weight, kg	390	387	388	6	0.66	0.91
Final body weight, kg	459	455	462	9	0.71	0.53
Dry matter intake, kg/d	10.2	9.9	10.5	0.3	0.34	0.08
Average daily gain, kg/d	1.14	1.13	1.30	0.08	0.86	0.02
Gain-to-feed ratio	0.114	0.116	0.125	0.007	0.79	0.12
Final 12th rib fat thickness, mm	5.65	5.25	5.80	0.41	0.37	0.31
Final Longissimus dorsi muscle area, cm2	63.1	64.0	64.2	1.8	0.64	0.71
Hot carcass weight, kg	247	248	252	4	0.79	0.21

Diets contained (dry matter basis): CON, 0% added oil (negative control); SUN, 1.90% of ruminally protected sunflower oil-based product (positive control); LIN, 1.92% of ruminally protected linseed oil-based product.

Regardless of treatment (treatment, P = 0.77; treatment × THI, P = 0.59), rectal temperature was affected (P < 0.01) by THI, being greater when THI was measured at 74 than when THI resulted in 57 or 67 (Figure 1A). The respiratory rate was recorded in time frames during which THI was classified as “Danger” and all records fell within the 90-to-120 bpm range, which is classified as “Moderate” (Mader et al., 2004). The respiratory rate was not affected (P ≥ 0.44) by the dietary treatment (113, 109, and 116 ± 8 bpm for CON, SUN, and LIN, respectively) or the interaction between THI and treatment (P = 0.61). However, respiratory rate was greater (P < 0.01) when THI was measured at 83 compared with 80 and 81 (Figure 1B).

Figure 1.

Rectal temperature (A) and respiratory rate (B) of feedlot finishing cattle as affected by the temperature–humidity index (Mader et al., 2004). Means with uncommon letters differ (P ≤ 0.05).

Biochemical profile

Concentrations of glucose, insulin, and cortisol as well as HOMA determined on day 1 of the experimental period were similar (P ≥ 0.38) among treatments. No significant (P ≥ 0.44) interactions between dietary treatment and DOF resulted from the analysis of the aforementioned variables. Blood glucose and cortisol were not affected by dietary treatment or DOF (P ≥ 0.26; Table 4). In general, cortisol concentrations were elevated compared with those observed in a previous study performed by this work team during winter, using the same facilities as well as similar animals and diets as in the present study (Pighin et al., 2015). Blood insulin and, consequently, HOMA, increased linearly (P < 0.01) with DOF. Blood insulin was similar (P ≥ 0.14) among treatments. However, HOMA tended (P = 0.06) to be reduced in animals fed the LIN diet compared with the average between those that received the CON or SUN diets and was similar (P = 0.46) between the latter. In agreement with increased HOMA over time, Joy et al. (2017) reported increased IR with advancing DOF when feeding high-energy diets with or without a high-lipid, canola-containing byproduct pellet to finishing cattle. The HOMA value observed for animals fed the LIN diet might suggest greater insulin sensitivity and it was close to HOMA calculated based on glucose and insulin concentrations reported for grazing animals (Aoki et al., 2006). On the other hand, HOMA values measured for animals fed CON or SUN diets were closer to those calculated based on glucose and insulin concentrations reported for animals fed diets containing 70% concentrate (Kneeskern et al., 2016). Taken together, these results may indicate that the addition of ruminally protected linseed oil tended to increase DMI and increased ADG; despite a trend for greater DMI, numerical reductions in blood glucose (P = 0.26) and insulin (P = 0.14) were observed, which resulted in a trend for reduced HOMA.

Table 4.

Biochemical profile of finishing cattle fed corn-based diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added as affected by days on feed

Item	Dietary treatment1			SED	Days on feed			SED	Contrast P-value2
	CON	SUN	LIN		1	29	61		CON vs. SUN	CON+SUN vs. LIN	Day, linear
n	8	8	8	-	8	8	8	-	-	-	-
Glucose, mM	4.38	4.58	4.30	0.18	4.43	4.41	4.41	0.13	0.29	0.26	0.91
Insulin, μIU/mL	30.6	30.6	25.7	3.8	23.1	29.7	34.1	2.4	0.99	0.14	<0.01
HOMA3	5.9	6.4	4.9	0.8	4.5	5.9	6.8	0.5	0.46	0.06	<0.01
Cortisol, μg/dL	57.2	62.1	60.5	4.7	63.4	56.5	60.0	4.9	0.31	0.84	0.51

Diets contained (dry matter basis): CON, 0% added oil (negative control); SUN, 1.90% of ruminally protected sunflower oil-based product (positive control); LIN, 1.92% of ruminally protected linseed oil-based product.

Nonsignificant dietary treatment × days on feed interaction for all variables (P ≥ 0.44).

Homeostatic model assessment, a model to determine insulin resistance (Warner et al., 2014); HOMA was calculated as (Glucose [mM] × Insulin [μIU/mL])/22.5.

In agreement with the results described above, blood concentrations of glucose, insulin, and cortisol determined on day 22, when THI was classified as “Alert” (Table 2), were similar (P ≥ 0.11) among treatments (4.27, 4.44, and 3.99 ± 0.34 mM of glucose, 32.0, 33.4, and 25.8 ± 5.5 μIU/mL of insulin, and 61.1, 56.8, and 58.1 ± 8.0 μg/dL of cortisol for CON, SUN, and LIN, respectively). Reduced (P = 0.02) HOMA was measured in animals fed LIN diet (4.5 ± 1.0) compared with the average between those fed CON and SUN diets (6.1 and 6.8 ± 1.0 for CON and SUN, respectively), while similar (P = 0.49) HOMA was measured in the last two groups.

As stated previously, sustained great energy intake may result in the development of MI and IR. Under this metabolic state, cytokine and series-2 prostaglandin secretion, and blood glucose as well as glucose concentration in insulin-independent tissues increase. These events lead to a decreased synthesis of nitric oxide, a well-known vasodilator in blood vessels (Vinik et al., 2003; Bansal et al., 2005; Förstermann and Sessa, 2012). This evidence may partially explain the relationship between the development of MI and IR and reduced vasodilation and perspiration during heat exposure (Petrofsky et al., 2005), as well as reduced heat stress tolerance when feeding high-energy diets to feedlot cattle. Moreover, improved glucose metabolism, stress response, DMI, and ADG in animals fed linseed oil may relate to the positive association between n-3 PUFA and synthesis of the anti-inflammatory series-3 prostaglandin (Schmitz and Ecker, 2008), along with a relationship between n-3 PUFA and enhanced production of adiponectin, a proven insulin sensitizer (Nagao and Yanagita, 2008).

A differential effect of preslaughter-related stress among treatments was observed for blood glucose and cortisol. In that regard, the magnitude of the increase (P < 0.01) in glucose concentration measured at slaughter (day 64) compared with that measured at the end of the feeding period (day 61) was greatest (study phase × dietary treatment, P = 0.01) for CON (1.5-fold increase), intermediate for SUN (1.33-fold increase), and lowest for LIN-fed animals (1.28-fold increase; Table 5). Concentration of cortisol increased (P < 0.05) at slaughter in animals fed CON or SUN diets but remained unchanged (P = 0.62) in those that received LIN diet (study phase × dietary treatment, P = 0.09). In sum, these findings suggest an attenuated response to preslaughter-related stress of animals fed LIN diet. Concentrations of insulin measured during the feeding period and slaughter day were similar (P = 0.41), irrespective of dietary treatment (study phase × dietary treatment, P = 0.57). Preslaughter-related stress may explain cortisol and glucose increases in CON- and SUN-fed animals, since cortisol increase might be the result of increased glucose synthesis via hepatic gluconeogenesis. Mechanisms by which linseed oil may mitigate the effects of emotional, short-term stress such as that resulting from slaughter in ruminants have not been reported. However, they may involve the regulation of stress mediators, such as catecholamines and proinflammatory cytokines (McAfee et al., 2019). Hematocrit was similar (P = 0.59) between animals fed the LIN diet (50.6 ± 1.5%) and the average resulting from those that received the negative (CON, 50.7 ± 1.5%) or the positive control diet (SUN, 52.1 ± 1.5%), as well as between the latter (P = 0.36). The position in the entry line into the stun box was positively associated (P ≤ 0.02; Table 6) with cortisol and hematocrit concentrations and tended to correlate (P = 0.07) with insulin concentration measured at slaughter, while no significant association (P = 0.32) was observed with glucose concentration. Direction and significance of these correlations are suggestive of greater stress experienced by cattle entering the stun box later. Although only 40 min elapsed between the first and last animal in the line, it is possible that sights, smells, and noises generated by animals in early line positions may have contributed to this stress response.

Table 5.

Biochemical profile measured at the end of a feeding period (day 61; FDG) and the slaughter (SLG) day (day 64; SLG) of finishing cattle fed corn-based diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added

Item	Dietary treatment1						P-value
	CON		SUN		LIN
	Study phase		Study phase		Study phase
	FDG	SLG	FDG	SLG	FDG	SLG	SED2	Study phase	Treatment	Study phase × treatment
n	8	8	8	8	8	8	-			-
Glucose, mM	4.22a	6.32b	4.69a	6.25b	4.10a	5.23b	0.24	<0.01	0.04	0.01
Insulin, μUI/mL	34.9	34.7	36.9	31.4	30.6	25.7	5.01	0.23	0.34	0.73
Cortisol, μg/dL	55.0a	69.1b	61.1a	77.3b	63.9	60.4	7.04	0.03	0.26	0.09

Diets contained (dry matter basis): CON, 0% added oil (negative control); SUN, 1.90% of ruminally protected sunflower oil-based product (positive control); LIN, 1.92% of ruminally protected linseed oil-based product.

Standard error of the difference between study phases within dietary treatment.

Means with uncommon superscripts differ within dietary treatment (P ≤0.05).

Table 6.

Pearson correlation coefficients between position in the entry line into the stun box and biochemical profile variables at slaughter (day 64), and between cortisol concentration and meat hardness of feedlot finishing steers

Item	Position in entry line		Meat hardness
	r	P-value	r	P-value
Glucose	0.15	0.32	-	-
Insulin	0.27	0.07	-	-
Cortisol	0.33	0.02	0.34	0.02
Hematocrit	0.84	<0.01	-	-

Meat quality attributes

Color, WHC, CL, and texture quality traits were not affected (P ≥ 0.16; Table 7) by dietary treatment. Results from Daly et al. (2007) and Kronberg et al. (2011) are similar with those from the present study. Daly et al. (2007) studied meat quality from grazing animals supplemented with 290 or 415 g/d of sunflower oil plus 0 or 85 g/d of fish oil during refrigerated display. The authors reported no differences in meat redness (a* parameter) among dietary treatments regardless of storage time. Interestingly, the authors reported that metmyoglobin proportion was slightly higher in meat from the oil-supplemented group compared with that from grazing animals. Kronberg et al. (2011) reported similar 12th-rib lean color characteristics at 24 h postmortem in meat from grazing animals supplemented with ground flaxseed or with ground corn and soybean meal mixture. Also, the authors reported no effect of supplementation in Warner-Bratzler shear force measurements. No significant (P ≥ 0.52; Table 7) differences in muscle pH were observed among dietary treatments. Abnormal pH (>6.0) is usually associated with preslaughter events, sexual condition, and breed (Oliveira et al., 2012). Even though an increase in cortisol and glucose levels were observed at slaughter for CON- and SUN-fed animals, suggesting an acute response to challenging preslaughter events, pH values fell within the normal range for beef cattle (5.4 to 5.8).

Table 7.

Quality attributes of meat from finishing cattle fed corn-based diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added

Item	Dietary treatment1			SED	Contrast P-value
	CON	SUN	LIN		CON vs. SUN	CON + SUN vs. LIN
n	8	8	8	-	-	-
24-h pH	5.55	5.53	5.50	0.06	0.72	0.52
Water holding capacity, %	33.2	34.7	33.4	1.5	0.29	0.69
Cooking losses, %	30.7	31.1	30.1	1.0	0.68	0.40
Total intramuscular fat	2.6	3.2	3.3	0.4	0.17	0.26
Meat color2
L*	36.3	37.6	37.3	1.4	0.34	0.81
a*	19.5	19.8	19.9	0.7	0.70	0.71
b*	10.4	10.8	10.2	0.6	0.43	0.45
Chroma	22.2	22.6	22.3	0.8	0.60	0.80
Fat color2
L*	68.2	68.4	66.9	1.1	0.80	0.16
b*	14.4	14.1	14.7	0.7	0.71	0.56
Chroma	15.2	14.8	15.5	0.8	0.63	0.44
TPA3 parameters
Hardness, N	59.7	62.1	61.2	4.9	0.62	0.95
Chewiness, N	12.9	13.4	12.9	0.8	0.54	0.74
Cohesiveness	0.477	0.470	0.472	0.010	0.46	0.83
Springiness	0.457	0.461	0.454	0.009	0.65	0.51

Diets contained (dry matter basis): CON, 0% added oil (negative control); SUN, 1.90% of ruminally protected sunflower oil-based product (positive control); LIN, 1.92% of ruminally protected linseed oil-based product.

CieLab system L* = luminosity from 0 (black) to 100 (white); a* = index from green (−) to red (+); b* = index from blue (−) to yellow (+); Chroma = (a*2 + b*2)1/2.

Textural profile analysis.

Plasma concentration of cortisol is influenced by previous and current exposures to stressors, and several studies have investigated the association between cortisol concentration and meat quality. A positive correlation (r = 0.34; P = 0.02) between cortisol, averaged across sampling dates, and meat hardness was observed, which may underline the impact of stress during a finishing period and slaughter event (Table 6). Mechanisms independent of pH may possibly be involved in this association. It has been postulated that increased concentrations of catecholamines and/or cortisol perimortem may activate nitric oxide synthesis, which in turn may modify the activity of the calpain/calpastatin system, restraining the development of tenderness of beef without altering final pH (Pighin et al., 2015). Total intramuscular fat did not differ among treatments (P ≥ 0.17; Table 7).

As expected, no differences (P ≥ 0.23) in blood FA profile determined on day 1 of the experimental period were observed among treatments. Even though blood FA profile at slaughter was similar among treatments (P ≥ 0.33; Table 8), dietary treatments led to differences in meat FA profile. In that regard, negative and positive control treatments differed (P = 0.01) and tended (P = 0.06) to differ in monounsaturated FA (MUFA) concentration and MUFA:saturated FA (SFA) ratio, respectively, being greater in SUN compared with CON-fed animals. This result may relate to a greater concentration of dietary lipids in SUN (7.0%; Table 1) compared with CON (5.6%), as a consequence of sunflower oil inclusion. Conversely, the concentration of PUFA and n-3 PUFA tended (P ≤ 0.09) to be reduced in animals that received the SUN diet. Concentrations of SFA, PUFA, and n-3 PUFA were greater (P < 0.01) in meat from LIN-fed animals compared with the concentration averaged between animals that received the negative (CON) or positive (SUN) control treatments (Table 8). On the other hand, MUFA, n-6:n-3 ratio, and MUFA:SFA ratio were reduced (P < 0.01) in meat from LIN-fed animals. These results are consistent with increased n-3 PUFA concentration in ruminally protected linseed oil-based product, singular (38%) compared with the sunflower oil-based one (8%). Dietary treatments did not differ (P ≥ 0.11) in any other FA-profile parameters.

Table 8.

Fatty acid profile determined at slaughter (day 64) in blood and meat samples from finishing cattle fed corn-based diets without oil added (CON) or with sunflower (SUN) or linseed oil (LIN) added

Item, %	Sample
	Blood						Meat
	Dietary treatment1			SED	Contrast P-value		Dietary treatment			SED	Contrast P-value
	CON	SUN	LIN		CON vs. SUN	CON+SUN vs. LIN	CON	SUN	LIN		CON vs. SUN	CON+SUN vs. LIN
N	8	8	8	-	-	-	8	8	8	-	-	-
SFA2	36.2	34.5	35.4	2.4	0.49	0.99	44.4	43.7	45.9	0.6	0.30	< 0.01
MUFA3	14.6	15.8	15.8	1.5	0.48	0.68	49.5	50.9	47.8	0.5	0.01	< 0.01
PUFA4	47.7	49.0	48.6	3.3	0.70	0.94	5.82	5.29	6.26	0.28	0.09	< 0.01
CLA5	0.11	0.12	0.12	0.04	0.79	0.89	0.28	0.31	0.27	0.02	0.17	0.33
n-3 PUFA6	4.98	4.46	4.73	0.57	0.39	0.99	1.80	1.53	2.21	0.13	0.07	< 0.01
n-6 PUFA6	43.1	44.2	43.5	3.2	0.73	0.96	3.77	3.43	3.78	0.19	0.11	0.27
n-6:n-3 ratio	9.21	10.44	9.86	1.39	0.38	0.98	2.11	2.26	1.75	0.10	0.18	< 0.01
MUFA:SFA ratio	0.42	0.46	0.44	0.04	0.33	0.95	1.11	1.17	1.04	0.02	0.06	< 0.01
PUFA:SFA ratio	1.37	1.46	1.43	0.17	0.60	0.90	0.13	0.12	0.14	0.01	0.19	0.13

Diets contained (dry matter basis): CON, 0% added oil (negative control); SUN, 1.90% of ruminally protected sunflower oil-based product (positive control); LIN, 1.92% of ruminally protected linseed oil-based product.

Saturated fatty acids: include short-chain fatty acids.

Monounsaturated fatty acids.

Polyunsaturated fatty acids.

Conjugated linoleic acid.

n-3 PUFA include the n-3 fatty acids 18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3; n-6 PUFA: includes the n-6 fatty acids 18:2n-6, 20:4n-6, and 22:4n-6 in meat samples, plus the 18:3n-6 in blood samples.

Results from this study suggest that intake of 155 g/d of ruminally protected linseed oil mitigated metabolic alterations associated with IR and improved the performance of steers fed a high-energy diet during summer. These effects might have led to greater DMI and ADG in animals fed linseed oil, without affecting meat quality traits. Meat FA profile changed in agreement with differences in FA profile between oil-based products. In addition, results suggest that glucose metabolism and heat stress tolerance worsen with time when feeding concentrate-based diets.

Glossary

Abbreviations

ADG

average daily gain

BW

body weight

CL

cooking loss

CP

crude protein

DM

dry matter

DMI

dry matter intake

DOF

days on feed

FA

fatty acids

HOMA

homeostatic model assessment

IR

insulin resistance

LM

longissimus dorsi muscle

MI

metabolic inflammation

MUFA

monounsaturated fatty acids

NDF

neutral detergent fiber

n-3 PUFA

n-3 polyunsaturated fatty acids

n-6 PUFA

n-6 polyunsaturated fatty acids

RDP

rumen degradable protein

SFA

saturated fatty acids

TDN

total digestible nutrients

THI

temperature–humidity index

TPA

textural profile analysis

WHC

water holding capacity

Conflict of interest statement

There is no conflict of interest.

Ethics statement

Animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of INTA (CICUAE#03-18).

Software and data repository resources

This research is under INTA Digital Repository regulations.