Performance , rumen development , and carcass traits of male calves fed starter concentrate with crude glycerin

The objective of this study was to assess the effects of including crude glycerin in the diet on intake, performance, rumen development, and carcass traits of dairy crossbred veal calves fed starter concentrate containing 0, 80, 160, and 240 g kg−1 crude glycerin. Twenty-eight calves with an average weight of 38.03±6.7 kg and five days of age were distributed in a completely randomized design with four treatments with seven replications. Calves were individually housed in covered stalls equipped with feeders and drinkers for 56 days. The calf response to inclusion of crude glycerin in the concentrate changed over the weeks and the inclusion level of 240 g kg−1 resulted in greater dry matter intake and average daily gain. There was no effect on the final weight and total weight gain of the animals, with mean values of 73.60 and 35.16 kg, respectively. The weight of the rumen-reticulum adjusted for body weight, empty body weight, and total stomach weight increased linearly with the inclusion of crude glycerin. Blood total protein, globulin, urea, cholesterol, gamma glutamyl transferase, aspartate aminotransferase, and alkaline phosphatase concentrations did not differ among treatments. Carcass traits and meat color were not affected. Crude glycerin can be added to dairy calf starter concentrate up to 240 g kg−1 dry matter because it benefits concentrate intake, performance, and rumen development without affecting animal health.


Introduction
Using adequate feeding and management techniques when raising male dairy veal calves allows these animals to be exploited for quality meat production and improves the efficiency of dairy farms.However, the high cost of the initial phase of rearing is one of the limiting factors for the expansion and consolidation of these animals for meat production in Brazil.Supplying solid food from the first weeks of life is the main way to stimulate the transition of the newborn animal to the condition of ruminant, accelerate rumen development, and decrease weaning age.
Using by-products in the starter concentrate to substitute noble ingredients, such as corn and soybean, can also be an alternative strategy for economic exploitation of these animals to reduce rearing costs without impairing the animal performance.Crude glycerin is a by-product derived from oil and fat transesterification that is generated in approximately 10% of biofuel production (Rahmat et al., 2010) and has aroused great interest as a food alternative for feeding ruminants due to its energetic value that is similar to that of corn (Donkin, 2008).
Information relating crude glycerin in the development of the rumen for young calves is not known, but its use as a feed additive has been reported.According to Napoles et al. (2012), crude glycerin can be used as an alternative energy source in substitution of corn in calf starter concentrate up to 100 g kg −1 of the DM without affecting ruminal or blood parameters.Adding 150 g kg −1 crude glycerin to dried milk did not affect the performance or health of Friesian calves (Drackley, 2008).The crude glycerin can replace up to 460 g kg −1 of the total lactose in milk replacers (Ebert et al., 2008;Raeth-Knight et al., 2009) or it can be used as a component of oral rehydration solution for young calves without any impacts on calf performance or health (Werner Omazic et al., 2013).Regarding carcass traits or chemical composition of the R. Bras.Zootec., 45(6): [309][310][311][312][313][314][315][316][317][318]2016 longissimus lumborum area, no significant effects were reported when crude glycerin was included at 300 g kg −1 DM in the finishing diet of steers (Mach et al., 2009;Bartoň et al., 2013;Van Cleef et al., 2014).
The objective of the present study was to assess the effect of including crude glycerin in the starter concentrate on performance, rumen development, blood parameters, and carcass traits of crossbred dairy calves.

Material and Methods
All the procedures and protocols used in the present experiment were approved by the Committee of Ethics on Animal Use of Universidade Federal do Tocantins (CEUA-UFT), under protocol no.23101.003936/2012-00.
The experiment was carried out in Rio Verde -GO, Brazil, from February to April 2011.Thirty-five crossbred Friesian-Zebu feeder calves with an average body weight 38.03±7.2kg and average age of five days were used.Seven of them were slaughtered at the start of the experiment and used as reference to calculate carcass gain, and 28 were placed in a completely randomized design with four treatments: 0, 80, 160, and 240 g kg −1 dry matter (DM) of crude glycerin in the starter concentrate, with seven replications to assess the animal performance.The concentrate was formulated according to the recommendations of NRC (2001) to meet the nutritional needs of unweaned calves (Table 1).The crude glycerin (GENPA ® -80 -nutritional energetic glycerol for feeding) used to make up the diets came from soybean oil and contained 899.8 g kg −1 dry matter, 11.9 g kg −1 DM ether extract, 78.6 g kg −1 DM mineral matter, 803.5 g kg −1 DM glycerol, 74.7 g kg −1 DM sodium chloride, less than 0.1 g kg −1 DM methanol, and 1.27 g cm −3 density.
The animals were identified with earrings, restrained by ropes and a collar in individual, movable, covered stalls.Each calf received 4 L whole milk day −1 , divided into two daily meals, at 08.00 h and 16.00 h, supplied at 38 °C in individual buckets.After the fifth day, they also received starter concentrate freely, and the quantity supplied and the orts from each animal were recorded daily to estimate the dry matter and nutrient intakes.Animals had access to fresh water ad libitum.
The animals were weighed on the fifth day of life, which was when the experiment started, and then weekly, on electronic scales, always in the morning before the diet was supplied until the eighth week of life, when the experimental period ended.At the time of the weighing, the hip and shoulder heights, hip width, body length, and thoracic girth were measured using a ruler and a tape measure.
Samples of concentrate supplied and orts were collected weekly.All these samples were pre-dried in a forced-air oven at 55 °C for 72 h, ground in a grinder with 1 mm mesh and stored for later analysis.The food samples and orts were analyzed for the contents of dry matter, organic matter, crude protein, neutral detergent insoluble nitrogen, acid detergent insoluble nitrogen, lignin, and ash according to the Association of Official Agricultural Chemists (AOAC, 1995) and neutral detergent fiber (NDF), acid detergent fiber (ADF), and ether extract according to the methodology recommended by the manufacturer of the ANKON apparatus.For the NDF and ADF analysis, 4 × 5 cm nonwoven fabric bags (TNT -100 g/cm²) were used.The non-fibrous carbohydrates (NFC) were calculated using the following equation (Sniffen et al., 1992): NFC = 100 -(%NDF + %CP + %EE + % ash).The total digestible nutrient content (TDN) was calculated following the prediction equation used by the NRC (2001).
Ten milliliters of blood were collected on the first, fourth, and eighth weeks of life by jugular puncture using vacuum tubes (Vacutainer ® ) with a lid and potassium EDTA as anticoagulant.The samples were centrifuged at 2000 x g for 20 min to obtain the serum.The serum was then separated by aspiration, divided into aliquots and placed in plastic tubes with a lid (Eppendorf ® ), labelled and frozen in a freezer for later biochemical analysis.The serum biochemical analyses (cholesterol, total protein, urea, albumin, creatinine, aspartate aminotransferase, alkaline phosphatase, and gamma glutamyl transferase) were carried out at 37 °C, using commercial reagents.The reactions were read by a Bioplus ® automatic biochemical analyzer (spectrophotometer) model Bio-2000 IL-A.The globulin concentration was calculated as the difference between total proteins and albumin.
At the eighth week, the animals were weighed and then slaughtered by brain concussion, followed by cutting the jugular and carotenoid veins according to Normative Instruction no. 3 of 01/13/2000 (Technical Regulation of Stunning Methods for Humane Slaughter of Butcher Animals).
For the morphometric assessment of the upper digestive tract, after slaughter, the abdominal cavity was opened, the four compartments (rumen-reticulum, omasum, and abomasum) were removed, and the contents of the tract were washed out with water.The volume of all the compartments was measured by tying closed the exits and filling them with water to maximum capacity, and then the volume was measured using a graded testtube.After removing the excess water from the tissues, the weight of the rumen-reticulum, omasum, abomasum, and the compartments together were measured.Samples of the cranial portion of the ventral coronary pillar in the rumen ventral sac were removed using a scalpel, preserved in 10% formaldehyde, and then the papillae height and width were assessed using a stereoscopic microscope and a millimeter ruler as proposed by Lesmeister et al. (2004).
After slaughter, the carcasses were weighed to obtain the hot carcass weight (HCW) and then placed in a cold room at 0 to 2 o C for 24 h and weighed again to obtain the cold carcass weight (CCW).The empty body weight (EBW) was obtained directly by summing the weights of the hooves, head, skin, blood, organs, viscera, internal fats, and carcass.In the right half carcass, a cut was made between the 12th and 13th ribs to expose the longissimus lumborum muscle.An outline of the area of this muscle was drawn on parchment paper and its area was measured and expressed in cm² using the ImageJ ® program.The physical composition of the carcass was estimated using the methodology of Hankins and Howe (1946).In the left carcass, half of the primal pistol cut was separated from the forequarter between the fifth and sixth ribs, and from the flank by a cut 16 cm from the spine.The pistol cut was separated into commercial or secondary cuts (rump uk trim, tail of rump, striploin, eye of round, rump cap, inside, outside, shank, cuberoll, tenderloin, and knuckle) and each piece was weighed individually.
Before analysis, the assumptions of normal distribution and homoscedasticity were checked for all the variables.The initial age was used as co-variable, and when it was not significant, the effect was removed from the model.To assess the effect of the treatments, α = 0.05 was adopted, with the mathematical model represented by: γ ij = µ + τ i + £ j + τ i *£ j + ε ij , in which γ ij = dependent variable; µ = overall mean; τ i = effect of factor i (age); £ j = effect of factor j (crude glycerin); (τ i *£ j ) = interaction between factor i and factor j; and ε ij = residual experimental error.In the regression study, the model was: γ ij = β0 + β1xi+ β2xi 2 + β3xi 3 + αj + εij, in which γij = dependent variables; β = regression coefficients; xi = substitution levels; αj = deviations from the regression; and εij = random residual error.

Results
Dry matter intake was influenced by including crude glycerin in the feeder calf concentrate (Table 2), and a linear increase was observed as the calf age increased (P<0.01).Including crude glycerin in the concentrate altered the response of the calves over the weeks, and adding 240 g kg −1 provided the highest DMI (Figure 1).Crude protein intake and total digestible nutrients showed the same response as DMI.
Average daily gain (ADG) was affected by adding crude glycerin to the feeder calf concentrate (Table 2) and a linear increase was observed as the calves became older (P<0.001).Including crude glycerin in the concentrate altered the ADG over the weeks, and the level of 240 g kg −1 inclusion resulted in the highest ADG (Figure 2).There was no effect (P>0.05) of adding crude glycerin on the final weight, total weight gain of the animals, and feed conversion (Table 3).Body measurements of the calves increased with age (P<0.01),but only the thoracic girth and hip width increased linearly (P<0.05), by 0.06 and 0.03 cm, for each gram of crude glycerin added to the concentrate, respectively (Table 4).Including crude glycerin in the concentrate did not alter (P>0.05)rumen-reticulum weight when expressed in absolute weight.However, when the weight of this component was adjusted to the BW, EBW, and total stomach weight, a linear increase was observed (P<0.05), of 0.0013, 0.0011, and 0.015 percentage points for each gram of crude glycerin added to the concentrate, respectively (Table 5).The rumen-reticulum volumes were not affected (P>0.05) by including crude glycerin (Table 5).Regression equations for the level of crude glycerin addition: DMI -dry matter intake (Ŷ = 0.426 + 0.0004x, R² = 0.96; DMI/BW -dry matter intake/body weight: Ŷ = 0.731 + 0.0006x, R² = 0.97); DMI, g kg −1 BW 0.75 : Ŷ = 19.86 + 0.018x, R² = 0.98); TDMI -total dry matter intake (milk + concentrate: Ŷ = 0.932 + 0.0003x, R² = 0.96); CPI -crude protein intake (Ŷ = 0.076 + 0.00007x, R² = 0.95); TCPI -total crude protein intake (Ŷ = 0.207 + 0.00007x, R² = 0.95); TDNI -total digestible nutrient intake (Ŷ = 0.345 + 0.0002x, R² = 0.90); ADG -average daily gain (Ŷ = 0.608 + 0.0001x, R² = 0.97).The omasum weight was not affected (P>0.05) by including crude glycerin in the concentrate when expressed in absolute weight, adjusted to the BW, or EBW; however, when it was fitted to the total stomach weight, it decreased linearly (P<0.05) by 0.002 percentage points for each gram of crude glycerin included.The abomasum weight increased linearly (P<0.01) when expressed in absolute weight, adjusted for BW, and EBW.The weight of the compartments together (RROA) was not influenced (P>0.05) by adding crude glycerin to the concentrate in any of the ways it was expressed.No effect was observed (P>0.05) on the rumen papillae height and width (Table 5).
the total stomach weight (Table 5), a 27.4% increase was observed in the rumen-reticulum of the calves fed concentrate with 240 g kg −1 crude glycerin.The proportion of the abomasum in relation to the total stomach decreased by 30.1, 27.2, 28.6, and 28.3%, respectively, for the treatments with 0, 80, 160, and 240 g kg −1 crude glycerin compared with the reference animals.Including crude glycerin in the concentrate did not change (P>0.05) the blood parameters (Table 6).However, as the animals grew older, the concentrations of creatinine, albumin, total proteins, globulins, and gamma glutamyl transferase (GGT) decreased linearly (P<0.01), while aspartate aminotransferase (AST) values increased linearly.The plasma concentrations of urea, cholesterol, and alkaline phosphatase (AP) were not affected (P>0.05) by including crude glycerin in the concentrate or by calf age.
Including crude glycerin in the concentrate did not influence (P>0.05) the feeder calf carcass traits (Table 7).The muscle percentages in the carcass of the animals that received concentrate with 0, 80, 160, and 240 g kg −1 crude glycerin added, when compared with the reference animals, presented an additional 4.5, 3.6, 4.3, and 2.5%, respectively.The percentage of bone in the carcass decreased on average by 6.14%, and the fat percentage in the carcasses  increased by 3.2, 5.6, 3.9, and 5.3%, respectively, for the treatments with 0, 80, 160, and 240 g kg −1 crude glycerin.The secondary cuts of the pistol cut -rump uk trim, tail of rump, striploin, eye of round, rump cap, inside, outside, shank, cuberoll, tenderloin, and knuckle -were not influenced (P>0.05) by including crude glycerin in the concentrate, when expressed in kg (Table 8).When the cuts were assessed for their relative participation in the special hindquarter cuts, tenderloin decreased linearly (P<0.01) with the inclusion of crude glycerin in the concentrate.

Discussion
Including crude glycerin in the concentrate led to a significant increase in DMI, perhaps because crude glycerin is viscose, hygroscopic, and has a relatively sweet flavor (Donkin and Doane, 2007), characteristics that increase the palatability of the diets.Furthermore, concentrates mixed with crude glycerin have a thick, moist texture with wellaggregated components that may have contributed to higher intake by the calves.However, previous studies did not reveal an effect on DMI (0.741 kg) when 30 and 60 g kg −1 corn were replaced with crude glycerin in pellet concentrate for dairy calves (Chester-Jones et al., 2010).
Regarding the increase in DMI as a function of age, its response is commonly observed in feeder calves in the unweaned phase, as reported in other studies (Bach et al., 2007;Khan et al., 2008;Lee et al., 2008;Bach et al., 2013), as a result of animal growth and because the intake of a fixed quantity of milk does not meet the entire maintenance and weight-gain requirements of the animals.
Research is being carried out to speed solid food intake so that animals can be weaned younger.Because the DMI was greater due to the addition of crude glycerin, the calves could be weaned earlier, which would result in lower feeding costs because concentrate is cheaper than milk or dried milk and also stimulates rumen development and contributes to supplying nutrients to animals (Baldwin et al., 2004;Suárez et al., 2006;Khan et al., 2007).Considering the intake of 700 g day −1 concentrate (Quigley III, 1996;Campos and Lizieire, 2000) to carry out weaning, the calves that received concentrate with 240, 160, 80, and 0 g kg −1 crude glycerin included could have been weaned at 40, 42, 44, and 46 days of age, respectively (Figure 1).This difference is significant when expenses with feeding and labor, milk commercialization, and animal performance are considered (Roth et al., 2009;Hulbert et al., 2011).
The greater DMI observed when crude glycerin was added to the concentrate favored the performance of the calves.However, regardless of the treatment, all animals presented considerable ideal gains (over 0.600 g day −1 ) for the unweaned phase (Hoffman, 1997), allowing the calves to reach twice their initial weight at weaning (Chester-Jones et al., 2010).Drackley ( 2008) assessed milk replacer with 150 g kg −1 crude glycerin for Friesian calves, which reduced the percentage of lactose, but did not affect the ADG, body measurements, or animal health.
The increase in thoracic girth in the calves that received concentrate with the addition of 240 g kg −1 crude glycerin was 17% greater than that of the calves fed the standard concentrate.The thoracic girth is highly correlated with the animal live weight (Heinrichs et al., 1992;Ozkaya and Bozkurt, 2009) and the increase in this measurement in the present study may be attributed to the greater performance of the animals receiving crude glycerin (Table 2).All the body measurements recorded are considered adequate and similar to data obtained in other studies with calves in the same phase (Blome et al., 2003;Lesmeister and Heinrichs, 2005;Kehoe et al., 2007;Khan et al., 2007).
The data show the positive effect of including crude glycerin in starter concentrate on rumen-reticulum development, which was probably due to the higher concentrate intake, which is considered the main stimulant to develop the rumen by the end product of its fermentation (Harrison et al., 1960;Lesmeister and Heinrichs, 2004;Coverdale et al., 2004;Khan et al., 2008;Boyd et al., 2013).The rumen-reticulum weight and volume increased ninefold in eight weeks in the animals fed concentrate with 240 g kg −1 crude glycerin, compared with the reference animals.These results show the importance of supplying solid foods for the rumen-reticulum muscular development when early weaning is desired and given the potential of crude glycerin as an alternative feedstuff to corn in calf starter concentrate.Regarding the abomasum weight, the highest values observed for this compartment in the treatments with crude glycerin were associated with greater DMI.At birth (reference), the abomasum occupied a greater proportion, but with the ingestion of solid feedstuffs the rumen-reticulum occupied a greater proportional weight, in agreement with other research with calves of equivalent ages (Khan et al., 2007;Bittar et al., 2009).Papilla development is linked to the presence of solid feedstuffs in the rumen, short-chain fatty acid production resulting from fermentation, and the stimulus caused by the feedstuffs (Sander et al., 1959;Tamate et al., 1962).
Although the blood creatinine concentration decreased as calf age increased, it was in the normal range for calves from 1 to 8 weeks (Klinkon and Ježek, 2012).A decrease in creatinine concentration with age is an indicator of normal and gradual increase in the kidney function capacity of the calves (Benesi et al., 2003;Mohri et al., 2007).Similar performance for creatinine concentrations in relation to age was reported by Khan et al. (2007), Mohri et al. (2007), and Lee et al. (2008).Creatinine levels are not deeply affected by diet (Klinkon and Ježek, 2012) and are normally used as auxiliary reference to diagnose variations in blood urea.The plasma urea levels were not altered by including crude glycerin and remained within the levels observed in other studies with calves at equivalent age (Knowles et al., 2000;Benesi et al., 2003;Klinkon and Ježek, 2012), emphasizing that the kidney functions of the calves were normal.
The total protein and globulin concentrations varied with age, but were within the range considered normal for physiological standards (Fagliari et al., 1998;Leal et al., 2003).The total protein, albumin, and globulin concentrations are important in diagnosing several diseases and disorders in the liver function (Klinkon and Ježek, 2012).The changes in the serum concentrations of total protein and globulin performed similarly as the animal age increased and were compatible with those observed by Mohri et al. (2007) for calves up to eight weeks of age.The variations in albumin concentrations with age were only observed in the animals with 240 g kg −1 crude glycerin, but remained within the reference interval for calves (2.7 to 3.9 g dL −1 ) (Knowles et al., 2000).
Serum GGT activities in the calves decreased as age increased, reaching stable values reported for eight-week old calves with normal hepatic function (Klinkon and Ježek, 2012).After colostrum is ingested, the GGT enzyme activity increases because it is absorbed through the intestinal wall and therefore its use for the assessment of the liver function is limited in the first days of life (Braun et al., 1982), but it can be used as an indirect indicator of passive immunity transference to calves (Feitosa et al., 2007), together with the total serum protein and globulin concentrations (Mohri et al., 2007).In spite of the greater serum GGT activity in the first collection (fifth day of life) recorded for the calves on the treatment with 160 g kg −1 crude glycerin, all the treatments received an adequate quantity (848.8IU L −1 ) of colostrum, considering the minimum value of 200 IU L −1 reported by Perino et al. (1993) for correct colostrum administration.
Aspartate aminotransferase activity varied with age, which is in line with Mohri et al. (2007), who observed high values 24 to 48 h after birth and an increase in AST activity from 14 to 84 days of age.In the first and second collections, the AST activity varied within the reference values established by Klinkon and Ježek (2012), indicating no liver alterations.The activity of the AP enzyme, which can also be used to indicate liver damage (González and Scheffer, 2003), ranged within the normal values, showing that the calves had no liver problem that might be related to an increase in AST activity.The cholesterol concentrations observed were in agreement with those reported by Pogliani and Birgel Junior (2007) for unweaned calves up to three months of age and were not influenced by age or treatment, which can be attributed to the constant offer of milk throughout the experimental period.
Generally, the similarity amongst treatments for the variables related to the carcass traits can be attributed to the similar weight conditions under which they were slaughtered (Kuss et al., 2005).In studies on finishing steers (Mach et al., 2009;Bartoň et al., 2013) and calves (Ramos and Kerley, 2012), similar carcass traits were described when compared with animals fed a diet without crude glycerin and diets with up to 200 g kg −1 crude glycerin.The values observed for carcass weight and yield agree with the results obtained by Brown et al. (2005) for calves slaughtered at the same weight and age as in the present study.In Brazil, there is no expressive consumption of veal such as the animals slaughtered in the present study due to economic and cultural questions.There are few studies on carcass characteristics and the meat of these animals, which hinders the establishment of a standard for this product in tropical conditions, requiring more research on the subject.
When the tissue percentages of the reference animals are considered, the decrease in the proportion of bone with an increase in age was due to the increase in the proportion of muscle and fat.This is because tissue percentages in the carcass are relative units, and an increase or decrease in the participation of a tissue in the total results in a decrease or increase in the other.Although the calves were in full development, this clearly demonstrated the changes in the tissue composition of the animals with increasing age.The higher meat yield of calves slaughtered at eight weeks makes them more attractive for commercialization.

Conclusions
Crude glycerin at up to 240 g kg −1 of the dry matter of the concentrate can be added as an alternative to the use of corn in calf feeding because it results in higher concentrate intake, average daily weight gain, and rumen development without affecting the quality of the carcass or the health of the animals.
for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico -CNPq) for their financial support.

Figure 1 -
Figure 1 -Daily dry matter intake of feeder calves receiving starter concentrates containing crude glycerin over the experimental period.

Figure 2 -
Figure 2 -Performance (average daily gain) of feeder calves receiving starter concentrate containing crude glycerin over the experimental period.

Table 2 -
Performance variables of dairy feeder calves receiving starter concentrate with addition of crude glycerin SE -standard error; CG -crude glycerin; A -age.

Table 3 -
Performance variables of dairy feeder calves receiving starter concentrate with addition of crude glycerin

Table 5 -
Morphometric measurements of the upper digestive tract of dairy feeder calves receiving starter concentrate with addition of crude glycerin SE -standard error; L -linear; Q -quadratic; RA -reference animals (n = 7).BW -body weight; EBW -empty body weight.