Carcass characteristics of lambs fed diets with increasing levels of crude glycerin

Objective An experiment was conducted to evaluate the effects of increasing levels of crude glycerin (0%, 6%, 12%, and 18%) used as a substitute for corn in lamb feed on the quantitative characteristics of the carcass. Methods A total of 40 crossbred Santa Inês lambs that were four months old with a mean initial weight of 21.0±0.8 kg were randomly distributed in four treatments with ten replicates. The animals were slaughtered after 66 days of confinement. The effects of crude glycerin as a replacement for corn in the diet of the lambs on the carcass characteristics, commercial cut weight and yield and carcass measurements were studied. Results There was an increasing linear effect for body weight at slaughter with the replacement of corn by crude glycerin. The dry matter and metabolizable energy intakes, weight of the empty body, hot carcass weight and cold carcass weight showed a quadratic effect, with maximum crude glycerin levels estimated at 10.9%, 9.8%, 10.83%, 11.78%, and 11.35%, respectively. The initial pH was not influenced by the replacement of corn for crude glycerin, while the final pH presented a quadratic effect. The other parameters of the carcass and the weights and yields of commercial cuts were not influenced. There was also no effect of the diets on carcass morphometric measurements, except for the thoracic perimeter and the carcass compactness index, which presented quadratic and linear effects, respectively. Conclusion Crude glycerin can replace up to 18% of corn because it favours muscle tissue deposition without promoting changes in the main carcass characteristics of lambs.


INTRODUCTION
Among the various agroindustrial byproducts currently used in ruminant feed, the most important are those derived from biodiesel production. Glycerin (C 3 H 8 O 3 ) is the main co product generated in the production of biodiesel and approximately 10% of the total volume of biodiesel produced corresponds to glycerin [1]. This coproduct results from the forma tion of methyl esters of fatty acids from triglycerides [2].
Given that feed is the largest cost in livestock production, the use of alternative foods as coproducts of biodiesel can be a viable alternative in economic and nutritional terms. Gly cerin can be used as an energy ingredient in feed, replacing grains that are more expensive, to increase the profitability of the activity [3].
Glycerol is absorbed by the ruminal epithelium, metabolized in the liver and directed to gluconeogenesis by the action of the enzyme glycerol kinase, which converts it to glucose. Part of glycerol can be fermented to propionate in the rumen, which in turn is metabolized to oxaloacetate via the Krebs cycle in the liver and can be used to form glucose by the gluconeogenic route. Thus, crude gly cerine (CG) has a potential application as a gluconeogenic substrate for ruminants [4]. In addition to serving as a source of energy, glycerol may also have positive effects on amino acid retention by inhibiting the activity of the enzymes phospho enolpyruvate carboxykinase and glutamate dehydrogenase, which results in saving glucogenic amino acids, favouring the deposition of body protein [5].
Diet directly influences the consumption and digestibility of nutrients and consequently, the performance of animals, as well as body and carcass composition. The valuation of the carcass depends on the relationship between body weight and age at slaughter, among other factors. The goal is to obtain higher weights at younger ages to meet consumer market demands [6]. Some studies have evaluated the effects of CG on the diets of sheep and cattle by substituting concentrated feeds and/or associated with urea [79], and improvements were achieved or these changes did not impair weight gain and carcass characteristics.
Thus, the objective of this study was to evaluate the replace ment of corn with CG in lamb feed on the quantitative and qualitative characteristics of the carcass.

Animal care
The experiment was carried out at the Animal Evaluation Center with Small Ruminants III of the Animal Science Department of the Federal Rural University of Pernambuco (UFRPE), Recife PE, Brazil. The experimental protocols were approved by the Institutional Animal Care and Use Com mittee of the Federal Rural University of Pernambuco (CEUA UFRPE) under license number 059/2016.

Animals, experimental design and experimental diets
We used 48 Santa Ines noncastrated male lambs that were four months old and had an average initial body weight of 21.0±0.8 kg. Initially, lambs were identified, dewormed with Ivermectin, immunized against clostridia and supplemented with an ADE vitamin compound. The animals were confined in individual pens with feeders and water suppliers. Initially, lambs were submitted to a period of 24 days to adapt to the experimental diets. After the adaptation period, the animals were randomly assigned one of four experimental treatments, which consisted of four concentrations of CG (0%, 6%, 12%, and 18% of dry matter [DM]) as a substitute for ground corn, with ten replicates per treatment.
The ingredients used were tifton hay, ground corn, CG, soybean meal, urea, ammonium sulfate, mineral supply, limestone calcitic and dicalcium phosphate (Table 1). Crude glycerine was obtained from the production of biodiesel from cottonseed oil at the Bioenergy Unit of the Center for Strategic Technologies of the Northeast, Caetés (PE), Brazil. The diets were formulated with the aim of an average daily gain of 250 g, according to the nutritional recommendations of the National Research Council [10] (Table 2).

Experimental procedures and sampling
The diets were provided as a complete mixture in individual feeders twice daily at 08:00 (50%) and 16:00 (50%), allowing 15% leftovers. Weekly samples were collected from the sup plied diets (offer and refusal), and then the ingredients and leftovers were predried in a forced ventilation oven at 55°C for 72 hours and ground in a Willey type mill with a 1 mm sieve. They were then submitted to further chemical analysis for their content of DM, organic matter, crude protein (CP), and ethereal extract (EE) which was determined according to the AOAC [11] with methods numbers 967.03, 942.05, 981.10, and 920.29, respectively. Neutral detergent fibre (NDF) was determined according to Mertens [12], with corrections for protein and ash according to the methodologies described by Licitra et al [13] and Mertens [12], respectively. The con centrations of glycerol, methanol and sodium of the CG were determined by gas chromatography. The total digestible nutrients (TDN) was estimated as sug gested by Weiss [14], using TDN = (CPd+NFCd+NDFapd+ [EEd×2.25]), where CPd, digestible crude protein; NDFapd, digestible neutral detergent fibre corrected to protein and ash; NFCd, digestible nonfibrous carbohydrate; EEd, digestible ether extract). The digestible energy (DE) was estimated as DE (Mcal/kg) = 0.04409×TDN (%). The conversion of DE to metabolizable energy (ME) was estimated as DE×0.82. The DM intake, ME intake, feed conversion (FC) and feed effi ciency used in the results and discussion of the present study were obtained by Andrade et al [15].
After 66 days of feeding, the lambs were fasted for 16 hours, weighed to obtain body weight at slaughter (BWS) and then slaughtered by cerebral concussion followed by jugular and carotid venesection. Preharvest handling was in accordance with good animal welfare practices and our slaughtering procedures followed the Sanitary and Industrial Inspection Regulation for Animal Origin Products [16].
After obtaining BWS, bleeding, evisceration and head and limb removal were performed, and the carcass was weighed to obtain the hot carcass weight (HCW). The gastrointestinal tract was also weighed when it was full and then when it was empty, and it was weighed again to obtain the weight of the contents of the gastrointestinal tract and to calculate the empty body weight (EBW). The yields of the hot (HCY) and bio logical (BY) carcass were determined using the formulas HCY (%) = (HCW/BWS)×100 and BY (%) = (HCW/EBW)×100.
Subsequently, the carcasses were taken to the cold room with an average temperature of 4°C where they remained for 24 hours, and after this cooling period they were weighed to obtain the cold carcass weight (CCW). Cold carcass yield (CCY) and cooling losses (CL) were determined according to the equations CCY (%) = (CCW/BWS)×100 and CL (%) = (HCW-CCW/HCW)×100.
After the cooling period, morphometric measurements were performed on the carcasses. The carcass compactness index (CCI) and the leg compactness index (LCI) were also calculated using the following equations: CCI (kg/cm) = CCW/ internal length carcass; and LCI (cm/cm) = hind width/leg length [17]. After which each carcass was divided sagittally. The left halves of the carcasses were sectioned in six ana tomical regions that constitute meat cuts according to the methodology adapted from Cezar and Souza [17]: neck, shoul der, rib, saw, loin, and leg.
To obtain the longissimus muscle area in the left halves of the carcasses, a cut was performed between the 12th and 13th ribs to expose the longissimus dorsi muscle. This area was hatched on a transparent plastic sheet and was measured later using a digital planimeter (HAFF, Digiplan model; Pfron ten, Baviera, Germany). In the same muscle, we measured subcutaneous fat thickness using callipers. The carcass pH was measured at 45 minutes and 24 hours post mortem in the semimembranosus muscle using a portable pH meter (Testo, model 205; Testo Instrument Co. LTD., Berlin, Ger many).

Statistical analysis
The experimental design was a randomized block with four treatments and ten replicates. The initial weight of the animals was the criterion for the formation of blocks according to the model: where Y ij = observed value of the dependent variable; μ = gen eral mean; T i = treatment effect (i = 1 to 4); b j = effect of block j (j = 1 to 4) and e ij = experimental error. The variables studied were interpreted by analysis of variance and regression anal ysis, considering the level of 5% probability for the type I error, using general linear model and REG procedures for L linear and Q quadratic effect of the SAS software package [18].
There was no influence (p>0.05) of the corn replacement levels by CG on hot and cold carcass yields, BY, or CL % ( 3). There was no effect (p>0.05) of the diets on the longissimus muscle area or subcutaneous fat thickness ( Table 3). The ini tial pH (45 min) after slaughter was not influenced (p>0.05), but the final pH (24 h) presented quadratic behaviour (p< 0.05), with a minimum value of 5.28 that was estimated at the CG level of 12% (Table 3).

Weights and yields of commercial cuts
The weight and yields of the meat cuts (neck, shoulder, ribs, saw, loin, and leg) were not influenced (p>0.05) by replacement (Table 4).

Morphometric measurements
There was a quadratic effect (p<0.05) on the thoracic perim  eter, with a maximum length of 71.63 cm, estimated at the CG level of 12.20% (Table 5). The other morphometric mea surements were not influenced (p>0.05) ( Table 5). The LCI was not influenced (p>0.05) by the replacement of corn with CG, but an increasing linear effect (p<0.05) was found for the CCI (Table 5).

DISCUSSION
The observed behaviour for the EBW, HCW, and CCW vari ables may be related to DM intake and ME intake due the fact that glycerol from CG may have increased dietary en ergy efficiency by the microorganisms in the rumen and consequently favoured the synthesis of tissues in the body of the animal. The BWS values are between the intervals of studies on sheep of the same age that were fed CG in con finement and were slaughtered when they were between 25 and 38 kg [7,8,19,20]. In the present study the animals were slaughtered at an average of 35.21 kg and presented a linear behaviour that increased with the inclusion of CG in the diets, with a better performance of the animals as the amount of corn replaced by CG increased. The values found for the CL % of the carcasses were within the range cited by Martins et al [21] which is between 1% and 7%. Lage et al [19] reported lower HCY (44.32%) and CCY (42.92%) values when they evaluated the effects of CG (36.2% glycerol). However, their CG inclusions were lower, mainly the glycerol content present in glicerin, reflecting the metab olism and utilization of glycerol by the animals. In addition, the carcass yield is directly affected by carcass weight. The performance presented (Table 3) by the animals in confine ment was reflected in the HCW, which presented satisfactory values and influenced the significant performance in carcass yield.
The longissimus muscle area that we obtained can be con sidered satisfactory. Lage et al [19] evaluated the effects of inclusion of CG with 36.2% of glycerol in the diet of confined sheep slaughtered at a mean of 32.72 kg and found an average of 12.1 cm 2 , while Rego et al [8] and Carvalho et al [20] ob tained much higher values of 13.66 and 14.44 cm 2 . According to Cezar and Souza [17] the determination of the longissimus muscle area measured as the longissimus dorsi muscle has tra ditionally been used as a good estimate of carcass musculature and is directly correlated with the muscle/bone relationship in the most important cuts of the carcass, which exerts an important influence on the classification of the carcass and the evaluation of the final price of the meat. The absence of an influence of the diets on subcutaneous fat thickness could be related to the time of confinement, which was of 90 days for animals that were four months old. Consequently, slaughter occurred before the adipose tissue began its major deposition because according to Gerrard and Grant [22] adipose tissue develops last, after peak muscle growth. According to Osório et al [23] subcutaneous fat thickness is associated with several factors, including race, gender, diet, the duration of the feeding period and confine ment. Therefore, it was not related to the energetic level of the diets, but to the chronology of body development, meaning the animals did not reach their maximum body development.
The behaviour observed for the final pH (24 h) may have occurred as a result of the increased ME intake with increas ing CG levels. A hypothesis for this behaviour would be that the increase in muscle glycogen reserves, which were con verted to lactic acid later, thereby reduced the final shell pH. Silva Sobrinho et al [24] found that the final pH value of sheep meat can range from 5.5 to 5.8. Therefore, the pH that we found at 24 hours was close to this range, indicating no pre slaughter stress.
Regarding the commercial cuts, the leg was the cut of greater weight and, consequently, higher yield (Table 4). Ac cording to Silva Sobrinho [25] this becomes important because it is a region with greater muscularity and a greater yield of the edible part. In addition to the leg, the shoulder and the loin are the most valued commercial cuts of the carcass. Thus, when greater yields of these cuts are obtained, the carcass has a higher value. In this research, the three cuts in question comprised 60.08% of the yield. These results correspond to those reported by FurushoGarcia et al [26] where the shoul der and leg represented more than 50% of the carcass, and these cuts are the best predictors of the total contents of the carcass tissues. Body development occurs in the following sequence: bones, muscle and fat. The bone structure is almost fully developed, in dimension, in the first months of life of the animal, followed by muscle hypertrophy and finally the deposition of adipose tissue [22]. The morphometric results observed in this study demonstrate that the linear and circular measurements of the carcass did not vary statistically between the inclusion levels of CG in the diet.
The increasing linear effect observed on the CCI is related to the CCW, which also increased linearly with replacement. The values found are within the range of the indexes for sheep that are documented in the literature, indicating good depo sition of muscle tissue per unit length. The higher the CCI, the greater the deposition of muscle tissue per unit area and the better the carcass will be evaluated. Thus, the replacement of corn by CG favours the deposition of muscle tissue.

CONCLUSION
Crude glycerin can replace up to 18% of corn and favour the muscle tissue deposition. Therefore, it presents itself as an excellent alternative food, contributes to obtaining more valued carcasses and consequently, corresponds to the final value of commercialized meat.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.