Timing of Crude Glycerin Inclusion (Adaptation vs. Finishing) Replacing Corn in High‐Concentrate Diets of Feedlot Nellore Bulls

1. Introduction

Feedlot diets for beef cattle are increasingly based on high proportions of cereal grains and other rapidly fermentable carbohydrates. The rapid ruminal fermentation of non-fiber carbohydrates can predispose animals to digestive and metabolic disorders — subacute ruminal acidosis, rumenitis and, as a sequela, liver abscesses — which alter feeding behavior and impair performance [1,2,3]. Partial replacement of corn grain with alternative energy sources has therefore been proposed to sustain energy density while modulating the ruminal environment under high-concentrate feeding [4].

Crude glycerin (CG), the main coproduct of the biodiesel industry, is a glucogenic ingredient that can replace corn as an energy source in ruminant diets. Glycerol is the main constituent of CG, the leading coproduct of biodiesel production, which also contains variable proportions of water, mineral matter, and methanol not recovered during processing [5].

Its ruminal degradation increases the molar proportions of propionate and butyrate at the expense of acetate, an effect proportionally stronger than that of grain starch [6]. Meta-analyses in beef cattle indicate that CG up to ~200 g/kg DM does not impair — and may improve — feed efficiency, whereas higher inclusions, particularly in high-grain finishing diets, are frequently associated with depressed DMI [5,7]. In feedlot Nellore cattle, CG has nonetheless been included at up to 300 g/kg DM without loss of performance and while modifying the meat fatty-acid profile favorably [8,9], and it modulates ruminal degradability and fermentation of Bos indicus diets [10].

Despite this evidence, information remains limited on the feedlot phase — adaptation versus finishing — at which CG is best offered, and on its consequences for feeding behavior and rumen epithelial morphology in Nellore cattle. Rumen papillae undergo marked proliferative adaptation to high-concentrate diets, and the fermentable substrate and time on the diet modulate epithelial development, barrier integrity and the incidence of rumenitis [11,12,13]. Importantly, intake and efficiency need not move together: a reduction in DMI accompanied by a smaller reduction in gain implies improved feed conversion, an outcome of direct economic relevance that has rarely been examined in relation to the timing of CG inclusion.

We hypothesized that the productive and epithelial responses to CG at 300 g/kg DM would depend on the feedlot phase of inclusion — being more detrimental to intake during finishing — while attenuating the ruminal epithelial challenge. The objective was to evaluate, in feedlot Nellore bulls fed high-concentrate diets, the effects of CG inclusion during the adaptation and/or finishing periods on DMI, growth performance, feeding behavior, carcass traits, meat pH and color, rumen epithelial measurements and liver abscess incidence.

2. Materials and Methods

2.1. Experimental Site

The experiment was conducted at the beef-cattle research facilities of UNESP, Jaboticabal, São Paulo, Brazil (21°14′ S; 48°17′ W; 595 m above sea level).

2.2. Animals, Housing and Experimental Design

Sixty Nellore bulls (~18 months of age; 300.5 ± 28.5 kg initial body weight) were individually weighed, ear-tagged, vaccinated and housed in individual pens with feed bunks and drinkers. Bulls were allocated to three blocks by initial body weight (light, medium and heavy) and randomly assigned to a randomized complete block design in a 2 × 2 factorial arrangement: CG during the adaptation period (0 or 300 g/kg DM; 0CG-AP or 300CG-AP) and CG during the finishing period (0 or 300 g/kg DM; 0CG-FP or 300CG-FP), with 15 bulls per treatment sequence. The individual animal in its pen was the experimental unit.

2.3. Pre-Adaptation, Adaptation and Finishing Diets

Before the experimental period, bulls underwent a 15-day pre-adaptation on corn silage offered ad libitum to standardize the ruminal population and adapt the animals to handling and facilities. The adaptation period lasted 14 days and followed a step-up protocol with three diets of increasing concentrate proportion, fed for 4, 5 and 5 days, respectively. Concentrate accounted for 270, 440 and 650 g/kg of dietary DM in step-up diets 1, 2 and 3, and for approximately 810 g/kg in the finishing diet, calculated as the sum of all dietary ingredients other than corn silage, which was the sole roughage source. Bulls were then fed the finishing diet for 63 days, totaling 77 days on feed.

The 14-day step-up protocol follows standard practice in Brazilian feedlots and is supported by studies with Nellore cattle showing that adaptation periods shorter than 14 days compromise feedlot performance and ruminal epithelial development [14,15,16]. Crude glycerin replaced ground corn on a DM basis. Diets were formulated according to the NRC [17] and evaluated with the Large Ruminant Nutrition System 1.0.12, level 2 to be isonitrogenous (125 g crude protein/kg DM) and to meet the requirements for an average daily gain of 1.5 kg/day. Bulls were fed ad libitum twice daily at 0600 h (40% of the ration) and 1600 h (60%), with the amount offered adjusted daily on the basis of orts recorded before the morning delivery, targeting 10% orts. Concentrate and corn silage were weighed separately each day and mixed with crude glycerin at the moment of feed delivery. Crude glycerin was obtained from a commercial soybean- and sunflower-oil biodiesel plant and contained 830 g glycerol/kg, 950 g DM/kg, 11 g crude protein/kg, 60 g total salts/kg and <0.1 g methanol/kg. The ingredient and chemical composition of the diets is presented in Table 1.

2.4. Chemical Analyses

Samples of the offered diets and orts were collected throughout each experimental phase (pre-adaptation, adaptation and finishing) and composited per phase for analysis. Dry matter (#930.15), crude protein (#988.05) and ether extract (#920.39) were determined according to AOAC [18]; neutral and acid detergent fiber were determined according to Van Soest and Wine [19] using heat-stable α-amylase without sodium sulfite and expressed inclusive of residual ash. Physically effective NDF, metabolizable energy and net energy for gain were not measured; they were estimated from diet formulation using CNCPS equations and are presented in Table 1 as calculated values.

In vitro dry matter digestibility of the experimental diets was determined in a DAISY II incubator following the ANKOM procedure. Diet samples were collected weekly, dried at 55 °C in a forced-air oven for 72 h and ground through a 1-mm screen before incubation.

2.5. Feed Intake and Growth Performance

Individual DMI was recorded daily as the difference between feed offered and orts, both expressed on a DM basis. Bulls were weighed on arrival, at the end of the adaptation period and every 28 days until slaughter, after a 12-h withdrawal of solid feed. Average daily gain was calculated from initial and final body weight, and feed efficiency as the ratio between ADG and daily DMI.

2.6. Feeding Behavior

Feeding behavior was recorded by four trained observers at 5-min intervals over 24 h on day 35 of the finishing period. The variables recorded were time spent eating, ruminating (standing and lying) and resting (standing idle and lying), and the number of feed boli ingested per day. Total chewing time was obtained as the sum of eating and total rumination time. Feeding and rumination rates on a DM basis were calculated according to the equations of Bürger et al. [20]. Dry matter intake on the observation day was obtained as the difference between the amount of feed offered and the orts collected after 24 h, both expressed on a DM basis.

2.7. Slaughter, Carcass Traits and Meat Quality

At the end of finishing, bulls were transported to a commercial abattoir, where slaughter followed Brazilian animal-welfare regulations (MAPA). Animals were stunned, exsanguinated, skinned and eviscerated. Hot carcass weight (HCW) was recorded and hot carcass yield calculated; carcasses were chilled at 0-1 °C for approximately 24 h. Muscle pH was measured in the Longissimus between the 12th and 13th ribs at ~45 min and 24 h post-mortem (penetration pH meter model 205; Testo Inc., Sparta, NJ, USA). Meat color (L*, a*, b*; CIE) was determined on the exposed Longissimus surface after 30 min of blooming (Minolta CR-400; Konica Minolta, Osaka, Japan), averaging three readings.

2.8. Rumen and Liver Evaluations

Immediately after evisceration the rumen was emptied, washed and scored for rumenitis on a 0–10 scale [21], and livers were scored for abscess number and size [22]. Both organs were scored independently by three previously trained evaluators blinded to treatments, and the mean of the three scores was used as the final value.

For macroscopic morphometry, a 1-cm² fragment was collected from the dorsal cranial sac of the rumen and placed in phosphate-buffered saline (pH 7.4) as described by Daniel et al. [23]. The number of papillae per cm² of rumen wall (NOP) was determined manually, and 10 papillae were randomly collected from each fragment, scanned and measured for mean papillae area (MPA) using Image J software. The variables were defined according to Resende Júnior et al. [24], and the rumen wall absorptive surface area (ASA) was calculated as ASA = 1 + (NOP × MPA) − (NOP × 0.002), where 1 represents the 1-cm² fragment collected and 0.002 cm² the estimated basal area of a papilla; papillae area was expressed as (NOP × MPA)/ASA × 100.

For histology, a 1-cm² fragment was collected from the ventral sac of the rumen, fixed in Bouin’s solution for 12 h [25] and transferred to 70% ethanol. Fragments were routinely processed, embedded in paraffin, sectioned at 4 µm on a manual microtome (Leica) and stained with hematoxylin and eosin. The mitotic index was determined under light microscopy with a 40× objective (400× total magnification): a total of 2,000 epithelial nuclei were counted per animal, all nuclei showing mitotic figures were recorded, and the mitotic index was expressed as the percentage of mitotic figures relative to the 2,000 nuclei counted.

2.9. Statistical Analysis

Data were analyzed using the MIXED procedure of SAS (v. 9.2) as a randomized complete block design in a 2 × 2 factorial arrangement, according to the model Yijkl = μ + Bi + Aj + Fk + (AF)jk + eijkl, where Yijkl is the observation on animal l, μ the overall mean, Bi the random effect of block i (blocks formed by initial body weight), Aj the fixed effect of crude glycerin during the adaptation period, Fk the fixed effect of crude glycerin during the finishing period, (AF)jk their interaction, and eijkl the residual error. Homoscedasticity and normality of residuals were tested, and outliers and influential observations were removed when required; each variable was therefore analyzed on its own final dataset. Means were estimated by LSMEANS. Tables present the four treatment means together with the probabilities of the main effects and of the interaction; factor means are described in the text. Significance was declared at p ≤ 0.05 and tendencies at 0.05 < p ≤ 0.10.

3. Results

3.1. Dry Matter Intake and Growth Performance

Crude glycerin during adaptation increased final body weight (429 vs. 407 kg; p = 0.014), ADG (1.62 vs. 1.44 kg/day; p = 0.035) and feed efficiency (0.12 vs. 0.11; p = 0.036), without affecting DMI (p = 0.752). Crude glycerin during finishing reduced DMI by 20% (11.8 vs. 14.8 kg/day; p = 0.001) and ADG by 11% (1.44 vs. 1.62 kg/day; p = 0.024); nevertheless, these bulls converted feed into gain more efficiently (0.12 vs. 0.11; p = 0.045). Hot carcass weight and hot carcass yield were unaffected by CG in either phase (p > 0.05). No A × F interaction was detected for intake, performance or carcass traits (Table 2).

3.2. Feeding Behaviour

Crude glycerin during finishing increased time spent eating (4.7 vs. 3.5 h; p < 0.001) while markedly reducing the feeding rate (2.7 vs. 4.2 kg DM/h; p < 0.001), consistent with the lower DMI, and increased total chewing time (7.2 vs. 6.2 h; p = 0.016). It also reduced time ruminating while standing (p = 0.023) and the number of feed boli (137.8 vs. 159.0; p = 0.029), and tended to reduce total rumination time (p = 0.079). Crude glycerin during adaptation increased eating time (p = 0.024). Tendencies for an A × F interaction were observed for rumination while lying (p = 0.053) and for total rumination time (p = 0.051); no other interaction was detected (Table 3).

3.3. Rumen Epithelium and Liver Abscess

Crude glycerin during finishing reduced the epithelial mitotic index (1.65 vs. 2.17%; p < 0.001) and tended to reduce the rumenitis score (2.5 vs. 4.7; p = 0.068). Absorptive surface area tended to be lower when CG was included during adaptation (20.2 vs. 23.9 cm²/cm²; p = 0.070). Papillae number, mean papillae area and papillae area as a proportion of ASA were unaffected (p > 0.05), and liver abscess scores did not differ among treatments (Table 4).

3.4. Meat pH and Colour

Muscle pH at 45 min and 24 h post-mortem and meat colour (L*, a*, b*) were unaffected by CG during either phase (p > 0.05; Table 5); mean 24-h pH was ~6.2 across treatments.

3.5. In Vitro Dry Matter Digestibility

Crude glycerin increased the in vitro DM digestibility of the second adaptation diet (76.3 vs. 69.1%; p < 0.001) and, markedly, of the finishing diet (85.6 vs. 70.8%; p < 0.001). Digestibility of the first and third adaptation diets did not differ between diets with and without crude glycerin (p > 0.05; Table 6).

4. Discussion

In this present study, during adaptation, replacing ground corn with CG did not affect DMI and was associated with greater final body weight, ADG and feed efficiency, consistent with the provision of a rapidly available glucogenic energy source. At the same time, the concentrate proportion was still moderate and the ruminal epithelium was undergoing proliferative adaptation [11,12,13]. This contrasts with Chanjula et al. (2016), who fed with 6% CG diets and reported lower intake, which they attributed to residual methanol and its potential toxic and clinical consequences. They referred to the potential problem of methanol in GC, due to the toxicity and clinical consequences to animals. In the current study, the effect of methanol was probably minimal, since we observed an increase in ADG and FBW. The CG fed in the present trial contained less than 0.1 g methanol/kg, no clinical signs compatible with methanol toxicity, feed refusal or morbidity were recorded, and methanol entering the rumen is largely metabolised by the ruminal microbiota before systemic absorption, methanol reaching the rumen is fermented by the ruminal microbiota and quantitatively converted to methane, without disturbing ruminal fermentation. More importantly, the animals receiving CG during adaptation were heavier at slaughter and grew faster than those fed ground corn — a pattern that is inconsistent with a subclinical toxic effect. Differences in glycerin origin, inclusion level, animal species and experimental design are therefore the more plausible explanation for the divergence between studies. Rather than a toxic response, the maintained intake together with greater final body weight, ADG and feed efficiency is consistent with the provision of a rapidly available glucogenic energy source while the concentrate proportion was still moderate and the ruminal epithelium was undergoing proliferative adaptation [11,12,13].

During finishing, CG at 300 g/kg DM reduced DMI and ADG. Because the reduction in intake was proportionally larger than the reduction in gain, feed efficiency improved, and hot carcass weight and yield were preserved. This fact is economically significant in feedlots, since the highest production cost stems from nutrition.

Glycerol fermentation changes the volatile fatty acid profile towards propionate and butyrate more strongly than starch [6], and because propionate is a primary hypophagic signal in ruminants — and glycerol imposes an osmotic load — a greater propionate sup-ply combined with osmotic fill provides a coherent explanation for reduced meal size and lower DMI without acidotic epithelial damage [28,29], however, ruminal fermentation end-products were not measured here, so this interpretation rests on the literature and on the intake and behavioural responses observed, and not on direct evidence obtained in the present study. However, the behaviour observed supports this: bulls fed CG during finishing spent more time at the bunk and chewed for longer, yet ingested DM at a markedly lower rate and consumed less overall, responses compatible with early satiety rather than with impaired palatability. Ruminal fermentation end-products were not measured here, so this interpretation rests on the literature and on the intake and behavioural responses observed, and not on direct evidence obtained in the present study.

Regarding the in vitro DM digestibility of the diets. Replacing ground corn with CG raised the in vitro DM digestibility of the finishing diet by nearly 15 percentage points (85.6 vs. 70.8%), consistent with the rapid disappearance of glycerol from the rumen and with the absence of fibrous carbohydrate in this co-product; comparable improvements in in vitro DM digestibility and ruminal degradability have been reported when crude glycerin replaced corn in sheep diets [34,35]. A more digestible diet delivers more available energy per kilogram of DM consumed, which reconciles the two apparently contradictory outcomes of the finishing phase: bulls fed CG ate a fifth less yet converted feed more efficiently and produced carcasses of the same weight. The concurrent presence of urea in the CG diets may have contributed by supplying rapidly available nitrogen alongside a rapidly fermentable energy source, favouring microbial growth.

A limitation intrinsic to the diet design must be acknowledged: the CG finishing diet was also lower in neutral detergent fibre (296 vs. 386 g/kg DM), because CG replaced grain while the roughage proportion was held constant. Part of the reduction in rumination time and in the number of feed boli may therefore reflect the lower fibre supply in addition to the glucogenic fermentation per se. Diets were isonitrogenous and closely matched in estimated energy density, and the finishing 300CG diet was marginally higher in estimated ME than its 0CG counterpart, indicating that the intake response cannot be attributed to a lower dietary energy supply. Because physically effective NDF was estimated from formulation rather than measured, it is not used here to support inference on fibre effectiveness.

Despite depressing intake, CG during finishing reduced the epithelial mitotic index and tended to reduce the rumenitis score. Two non-exclusive interpretations apply. First, because all bulls were slaughtered at the end of finishing, the higher mitotic index in control animals partly reflects the larger amount of ruminally fermented starch driving epithelial proliferation. Second, replacing starch with glycerol appears to create a less aggressive epithelial environment: proteomic evidence in lambs fed high-concentrate diets shows that CG up-regulates tight-junction proteins and glycerol-related energy metabolism while down-regulating oxidative-stress and inflammation proteins [30], and high CG inclusion during adaptation and finishing did not aggravate rumen or liver lesions in lambs [31]. The rumenitis scores recorded here (2.4–4.7 on a 0–10 scale) indicate a real, moderate epithelial challenge under high-concentrate feeding, against which the reduction associated with CG is biologically meaningful.

The tendency for a lower absorptive surface area when CG was included during adaptation deserves comment, since it runs opposite to the performance response of the same factor. Absorptive surface area reflects papillae size and density at a single point in time — the end of finishing — and a smaller surface in animals that nonetheless gained more weight suggests that epithelial expansion was not the limiting step for energy capture in this system. This reinforces the broader message that epithelial morphometry and productive performance were dissociated in the present study.

The absence of effects on carcass traits and on meat pH and colour indicates that replacing ground corn with CG during either phase did not compromise carcass yield or basic meat quality, in agreement with reports in finishing bulls and steers [26,32]. The mean 24-h pH of ~6.2 is higher than the usual 5.3–5.8 range and could predispose to darker meat; however, pH was not affected by treatment, and non-stressed Nellore cattle with large muscle glycogen reserves may require longer post-mortem for complete pH decline [33]. Liver abscesses arise mainly as a sequela of acidosis and rumenitis [4]; the absence of a CG effect on liver abscess score is consistent with the moderate rumenitis scores observed.

5. Conclusions

Crude glycerin at 300 g/kg DM replacing ground corn can be included during the adaptation of feedlot Nellore bulls without depressing intake, and was associated with greater body-weight gain and improved feed efficiency. During finishing, the same inclusion depressed DMI and ADG and altered feeding behaviour, yet improved feed conversion and preserved hot carcass weight and yield, while reducing the epithelial mitotic index and tending to reduce rumenitis. Because the attenuation of ruminal epithelial lesions did not translate into greater growth, and because the tendency for a smaller absorptive surface area coexisted with better performance, epithelial responses were dissociated from productive performance. High CG inclusion during finishing should therefore be evaluated on a feed-cost basis rather than on daily gain alone, whereas its use during adaptation appears safe and advantageous. The higher in vitro DM digestibility of the crude glycerin diets provides a coherent basis for the improvement in feed conversion observed during finishing.