Different Sources Of Fat In Supplements For Beef Cattle At Pasture

The aim of this study was to investigate the consequences of the fat supplementation source (free oil and rumen-protected fat) on the nutrient intake and digestion of beef cattle at pasture. Five rumen-cannulated Nelore bulls, with an average 467.8±32.8 kg of body weight (BW) and age of 26 months, were distributed in a Latin square design (5 x 5). The treatments were as follows: WF: without fat, PS: rumen-protected fat soybean oil, PA: rumen-protected fat palm oil, SO: soybean free oil, and CO: corn free oil. Nutrient intake and digestibility, ruminal pH and ammonia (NH 3 -N), serum urea, and nitrogen balance were analyzed. The supplements with different fat sources did not alter (P>0.05) the intake and digestibility of dry matter (DM), forage, organic matter (OM), crude protein (CP), neutral digestibility ber (NDF), neutral digestibility corrected ash and protein (NDFap), nonber carbohydrates (NFC) or total digestible nutrients (TDN), except ether extract (EE). An increase (P<0.05) in the intake and digestibility was observed with the inclusion of a fat supply, independent of the fat source. Differences were observed between the WF and other supplements with regard to ruminal parameters (pH and NH 3 -N) (P>0.05) and serum urea (P>0.05). The nitrogen balance was not affected by the fat source (P>0.05). Supplementation of grazing beef cattle with 2 g/kg BW low-level free oil (130 g/kg DM supplement) or rumen protection (160 g/kg DM supplement) did not interfere with the characteristic nutrient intake and digestibility. fat; PS, rumen-protected fat soybean oil; PA, rumen-protected fat palm oil; SO, soybean free oil; CO, corn free oil. 2 DM, dry matter; OM, organic matter; CP, protein; NDF, neutral detergent insoluble neutral detergent insoluble ber for contaminant and protein; carbohydrate;


Introduction
Increasing attention has been placed on enhancing the use of fat in the diet of ruminants, and according to Jenkins et al. (1993), the emphasis on ruminal lipid metabolism is associated with manipulating physicochemical events in the rumen to produce two practical outcomes: 1) control of the antimicrobial effects of fatty acids so that additional fat can be fed to ruminants without disrupting ruminal fermentation and digestion and 2) regulation of microbial biohydrogenation to change the absorption of selected fatty acids that might enhance performance or improve the nutritional qualities of animal food products.
In beef, soybean and corn oil represent sources of unsaturated fatty acids and enhance the content of CLA, the cis-9,trans-11 isomer (Duckett et al., 2009;Cooke et al., 2011;Choi et al., 2013), which has anticarcinogenic and antiatherogenic effects (Scollan et al., 2006;Shing eld et al., 2011). Early studies showed that soybean oil has the potential to provide direct energy for muscle gain and decrease in the size of adipose cells in subcutaneous tissue (Choi et al., 2013).
Using palm oil as the source of saturated fatty acid requires a longer period of supplementation time, and it may signi cantly increase carcass adiposity and has the potential to increase marbling scores without increasing the palmitic acid or reducing the oleic acid content of beef (Choi et al., 2013).
However, fat supplementation for animals at pasture may decrease the forage dry matter intake and performance of the animals. Lipids inhibit the growth of brotic bacteria and show high potential reactions with ruminal microorganism membranes, which has lethal toxicity (Patra &Yu, 2012;Huws et al., 2010).
When including fat supplements in the diet, it is important to avoid promoting negative effects on the growth of brotic bacteria; moreover, rumen-protected fat should be used because it is less harmful for ruminal bacteria and only becomes available in the intestine (Cooke et al., 2011).
The aim of this study was to investigate how the source fat (oil free or rumen protected) in supplements for beef cattle at pasture affects nutrient intake and digestion.

Experimental design and treatments
The experiment was developed during the transitional rainy-dry season at the beef cattle facility (15°47′5″ S, 56°04′ W, and 140 m above sea level) of the Sector Nutrition of Beef Cattle in Pasture, UFMT, from March 2017 to June 2017. The climate is classi ed as a tropical climate (Aw in the Köppen international system), the average maximum temperature is 32.8°C, and the minimum average temperature is 19.7°C.
Five rumen-cannulated Nelore bulls, with an median 467.8±32.8 kg of body weight (BW) and age of 26 months, were used in the experiment to evaluate the inclusion effects of fat-free or fat-protected supplements on nutrient intake and digestibility, ruminal pH and NH 3 -N, and N utilization e ciency of the The effects of different fat-free and fat-protected sources in the supplements were evaluated in the following treatments: WF: protein-energetic supplement without lipidic source; PS: rumen-protected fat soybean oil; PA: rumen-protected fat palm oil; SO: soybean free oil; and CO: corn free oil ( Table 1).
The supplements used, contain 280 g of crude protein (CP) in the dry matter, and they additional support the protein for requirements of Nellore bulls in pasture, with an average body weight of 498.5±28.2 kg and weight gain of 0.620 kg/animal/day (Valadares Filho et al., 2016). The animals were supplemented at 1.0 kg DM/animal/day at 10 a.m. In each period, the ingredients were sampled. The formulation of ingredients and chemical composition of the supplements are showed in Table 1. Every 19 days, the average sward height was randomly measured by reading 25 sampling points in each paddock with the aid of a graduated measuring stick in centimeters (Barthram, 1985). The forage mass in each paddock was estimated for each period (19 d The forage samples using in the herbage chemical composition analyses were collected manually using a plucking method (Johnson, 1978) that mimicked forage selected by grazing steers. Samples, collected from each pasture during each period, were dried to a constant weight at 55°C under forced air. Subsequently, samples were sent to the laboratory for analysis (chemical composition).

Chemical composition analysis
For the proximate analysis, supplement ingredient samples, forage samples, and feces samples were Twenty grams of each sample was ground to pass through a 1-mm screen for further analyses. where 0.98 is the true digestibility coe cient of intracellular content; NDF is the forage content of neutral detergent ber corrected for residual ash and nitrogen; and iNDF is the forage content of indigestible neutral detergent ber.

Intake estimation
Intake and nutrient digestibility were estimated throughout the period between days 15 and 18 using the marker method: titanium dioxide and indigestible NDF (iNDF). To estimate the excretion of fecal matter (as dry weight), the supplement intake and forage intake were used.
Fecal samples were collected directly from the rectum on the 16 th day from 0600 h and 1400 h, 17 th day from 0800 h and 1600 h, 18 th day from 1000 h to 1800 h, and 19 th day from 1200 h and 2000 h. The fecal samples were dried (55°C for 72 h) and composited throughout the day for each animal.
An external marker, titanium dioxide (15 g/animal/day), was used to estimate the DM fecal excretion and, estimated based on the ratio between the amount of marker supplied and its concentration in the feces where fecal iNDF = iNDF in the feces (%); supplement iNDF = iNDF in the supplement (kg/day); forage iNDF = iNDF in the forage (kg/kg); and SDMI = supplement dry matter intake (kg/day).
Samples of feces (0.5 g), forage, and supplement were placed in 5 by 5 cm polypropylene bags (nonwoven fabric, weight 100 g/m 2 ) to determine the iNDF. The samples were weighed to allow 20 mg DM/cm 2 of surface area (Nocek, 1988) and incubated in the rumen of a cannulated Nellore bull for a period of 288 h (Valente et al. 2011).

Nitrogen retained
Urine collection was ful lled on the 16 th to 18 th day of each experimental period and collected at the same time as the fecal samples by spontaneous urination. Eight urine samples were stored in the form of spot samples, kept cool in a polystyrene cooler with ice, and then formed a compost aliquot to analyze the concentration of creatinine and urea, which were analyzed using the colorimetric method according to the method of Fujihara et al. (1987) as described by Chen and Gomes (1992).
Urine volume was estimated in relation the animal body weight (kg of BC), daily creatinine excretion (mg/kg BC) and, creatinine concentration (mg/L) in the urine (Chizzotti et al., 2008). To calculate the daily creatinine excretion per kg of BW, the mean of 27.76 mg/kg LW obtained by Rennó (2003) was adopted.
Daily excretion was calculated as the product of the urea concentration and the urinary volume after 24 hours, which was then multiplied by 0.466, which corresponds to the nitrogen content in the urea (Rennó et al., 2000).
The amount of nitrogen retained was obtained based on the difference between the nitrogen ingested and the nitrogen excreted in the feces and urine.

Blood urea serum
Blood samples were collected on the 15 th day at 0600 h and 1400 h in each experimental period. Blood samples were collected of the caudal vein, by puncture, using test tubes and kept cool in a polystyrene cooler with ice. After that, serum samples were taken (centrifuge 2000 x g), send on to the laboratory and analyzed to determine the urea content.

Ruminal fermentation
The concentration of ammonia nitrogen (NH 3 -N) in the rumen uid was measured on day 19 of each period. Ruminal contents were manually obtained from several sites within the rumen at 0 (before supplementation) and 3, 6 and 9 h after supplementation. Rumen uid was obtained from strained through 2 layers of cheesecloth. Additionally, pH was measured, using a digital pH meter, immediately after collection, and samples were poured into 50-mL plastic asks, 1 mL of 9.

Statistical analysis
Tukey's test was used to analyze the forage of the GLM procedure of the SAS (Statistical Analysis System, version 9.3) software package, model: Y ij = µ + T i + e ij , where Y ij is an observation of unit j in treatment i; µ is the overall mean; T i is the random effect of treatment i, with mean 0 and variance σ 2 t ; and e ij is the random error, with mean 0 and variance σ 2 . In the analysis of variance, a value of 0.05 was considered signi cant.
The nutrient intake and digestibility and nitrogen balance were analyzed using a mixed model in the SAS software package (Statistical Analysis System, version 9.3) as follows: y lm(i) = μ + A l + P m + τ i + ε lm(i) , where y lm(i) is the observation lm(i); μ is the overall mean; A l is a random effect animal; P m is a random effect period; τ i is the xed effect of treatment i; ε lm(i) is the random error, with mean 0 and variance σ 2 ; and lmi represents ve animals, periods and treatments.
Repeated measures (ruminal pH and ammonia) analyses were performed using the mixed model of the SAS software package (Statistical Analysis System, version 9.3). Ruminal pH was analyzed using the variance structure in unstructured mode, ruminal ammonia was analyzed using the variance structure in antedependence mode, and urea serum was analyzed using the variance structure in compound symmetry mode according to the AIC (Akaike Information Criteria) and BIC (Schwarz's Bayesian Information Criteria) values. The model used was y ijk = μ + τ i + δ ij + t k + (τ*t) ik + ε ijk , where i represents ve supplements (treatments), j is ve animals (subjects), k is four times, y ijk is observation ijk, μ is the overall mean, τ i is the effect of treatment i, t k is the effect of times k, (τ*t) ik is the effect of interaction between treatment i and period k, δ ij is the random error with average 0 and variance σ 2 δ. The variance between animals (subjects) within treatment ijk was equal to the covariance between repeated measurements within animals. In addition, ε ijk is the interaction between treatment i and period k.

Results
The average forage masses for dry matter, potentially digestible dry matter, green leaves, green stems and senescence were 4,054.4, 2,668.3, 1,141.2, 1,124.5 and 1,788.7 kg/ha, respectively ( Table 2). Dry matter availability was close to the recommended value of 4,262 kg/ha, and the green leaf mass was greater than the recommended value of 1,108 kg/ha to avoid animal selectivity (Euclides et al., 1992).
The 3rd and 4th periods corresponded to April and May, and higher pdDM and green leaf mass (P<0.05) was observed in these periods, which was probably due to the higher rain levels in uencing the proportion of green leaves in the same period ( Table 2). The value and proportion of senescent leaves were higher than those of green leaves and stems in all periods due to the height of the forage of 19.6 cm.
The average content of crude protein of hand-plucked forage ( Table 2) during the experimental period was 120.5 g/kg DM, which was higher than the value of 70 g/kg DM recommended by Van Soest (1994) as the limit value of basal forage ber carbohydrates according to the rumen microorganism digestion capacity (Mathis et al., 2002). The different lipidic sources evaluated in the supplements did not alter the intake of DM, forage, OM, CP NDF, NDFap, NFC and TDN (P>0.05) ( Table 3). For ruminants at pasture, Hess et al. (2008) determined that a limit of supplemental fat of 2% of the dietary DM will prevent negative associated effects for ruminants fed high-forage diets, which was observed in the current study. The same authors a rmed that the energy density of a high forage diet will not be increased if the supplemental fat exceeds 4% of the DM.
This a rmation was con rmed in the trial of Pavan et al. (2007), who found increased fat intake of 340 to 840 g in the diet using corn oil free supplements in animals at pasture, which is higher than that observed in our experiment, and decreased forage and NDF intake. Negative effects on ber digestibility with the inclusion of fat in the diet occur because lipids inhibit the growth of brotic bacteria (Patra &Yu, 2012;Huws et al., 2010).
In this study, the lipid level of the diet based on rumen-protected fat soybean oil or oil free in the supplement was 34 g/kg DM, and greater amounts of DM had deleterious effects on nutrient intake. In Carvalho et al. (2017), supplementation of beef cattle at pasture was performed with fat sources (palm and soybean oil) that provide a fat content higher than 40 g/kg DM of diet, and they indicated that this value was su cient to decrease the DM, forage and NDF intake. Table 3. Dry matter and nutrient intake according to source of fat supplementation for grazing bulls Jenkins (1993) and Patra & Yu (2012) emphasize that the elevated content of unsaturated fatty acids (C18:2) with the use of soybean oil sources or an increase in saturated short chains (C14:0 mistiric and C:12 lauric) with the use of palm oil sources in the diet have high potential for reaction with the membrane of ruminal microorganisms, which will lead to a decrease in the microbial population and is toxic to microorganisms that ferment ber, which may reduce the intake of DM.
However, in this study, supplementation with oil-free or fat-protected levels did not have a negative effect on nutrient intake but increased (P<0.05) the fat intake in animals supplemented with fat (34 g EE/kg DM) relative to the animals that were not supplemented with fat (22 g EE/kg DM) ( Table 3). According to Doreu et al. (2009) and Ueda et al. (2003), because fat supplementation provides less than 30 g EE/kg DM of diet, the expected negative effect on the intake of forage or DM was not observed.
In relation to nutrient digestibility, DM, OM, CP, NDF, NDFap and NFC were not affected (P>0.05) by fat supplementation with free oil or rumen-protected fat (Table 4). Jenkins and Palmquist (1984) a rmed that less than 10% added fat can decrease the ruminal digestibility of structural carbohydrates by as much as 50% or more. However, this decrease was not observed here because the level of fat supplementation was consistent with the recommendation by Hess et al. (2008), who summarized previous results and indicated that an optimal inclusion rate for supplemental fat is less than 3% of DM if the goal is to maximize the intake of forage-based diets. Fat supplementation increased (P<0.05) the ether extract digestibility compared with the animals that did not receive lipidic supplementation in the diet (Table 4). A previous study showed that lipid intake greater than 500 g/day per animal could exceed the maximum intestinal capacity to digest lipids (Brandt, 1995 indicated that an increase in lipid intake over 500 g/day in the diet linearly increased the digestibility in the total gastrointestinal tract. The results of this study ( Fat supplementation may reduce the ruminal pH due to an increase in fat levels, which could enhance the glycerol content in the rumen for microbial fermentation and promote a greater release of volatile fatty acids by the potential reduction in ruminal pH (Nagaraja et al., 1997;Patra & Yu, 2012). However, in this study, the lipid levels in the diet did not change the ruminal pH ( Table 5).
The ruminal ammonia concentration (  (1977) and Leng (1990) for maximizing the fermentation rate. The curve of ruminal pH in relation to time (Figure 1a) was quadratic (P<0.05), with a minor value of reserved time of greater intake forage. The curve of ruminal NH 3 -N in relation to time ( gure 1b) was cubic (P<0.05) and presented two peaks, with the rst in relation to supplementation and second in relation to the greater intake of forage at the end of the day.
Nitrogen intake and nitrogen excretion in the feces and urine were not affected by fat supplementation (Table 6). At greater fat contents in the diet (>30 g EE/kg DM of diet), nitrogen retention may decrease because of the decreased microbial synthesis in the rumen under higher dietary lipid contents due to the disruption of ruminal degradation (Hales et al. 2017). Some lipidic sources are known to be toxic to ruminal microorganisms through the detergent action of fatty acids on the microbial cell membrane (NRC, 2016) and through inhibition of enzymatic digestion. However, in this study, the inclusion of fat in the supplement (free oil or protected fat) was not enough to promote deleterious effects. Thus, there is potential for fat use as a supplement for beef cattle at pasture at a level below 40 g EE/kg DM using fat free or rumen-protected fat sources. At this level, the lipid sources did not in uence nutrient intake, nutrient digestibility or nitrogen balance. Strategies can be explored with different sources of fat.
Studies have shown that palm oil, which is rich in saturated fatty acids (C16:0 palmitic acid), promotes an increase in the synthesis of subcutaneous fat while sources such as corn or soybean oil, which are rich in unsaturated fatty acids (C18:2 linoleic acid), promote decreased cell adiposity and alter energy for muscle growth (Choi et al., 2013).

Conclusion
Supplementation of grazing beef cattle with 2 g/kg BW low-level free oil (130 g/kg DM supplement) or rumen-protected oil (160 g/kg DM supplement) did not interfere with the characteristic nutrient intake and digestibility.