Nitrogen Sources in Spineless Cactus-Based Diets for Sheep in Finishing

This study evaluated the effect of different sources of nitrogen [soybean meal (SM); cottonseed meal (CM); cottonseed + urea (CU); and urea (U)] associated with spineless cactus on intake, digestibility, performance, carcass characteristics and meat quality of conned lambs. Forty male Santa Inês sheep, uncastrated, with initial body weight of 23.00 ± 1.66 kg were distributed in random blocks design and slaughtered after 58 days of connement. Dry matter (DM) intake (1.16 ± 0.19 kg/day) did not differ (P > 0.05) between nitrogen sources, but the intake of crude protein (CP) and non-brous carbohydrates (NFC) was lower (P < 0.05) for sheep consuming CU. Sheep fed with diets containing U had lower digestibility of DM, CP and NFC (P < 0.05). The average daily gain (0.15 ± 0.04 kg/day) and the slaughter body weight (32.07 ± 2.86 kg) did not differ between nitrogen sources, but the cold carcass and loin weight was lower (P < 0.05) for sheep fed with U. The water retention capacity and meat protein content were greater (P < 0.05) for animals fed with SM. Soybean meal or cottonseed meal associated with spineless cactus-based diets for sheep meat production are recommended because it allows greater DM digestibilityand improves carcass characteristics and physicochemical composition of the meat.


Introduction
Spineless cactus has been consolidated as a food rich in high fermentation carbohydrates for ruminant diets in arid and semi-arid areas of the planet, because in addition to being an energy source, it is an important water fountain for livestock production ( Despite establishing the appropriate ber level for diets with spineless cactus (Vieira et al., 2008;Pinho et al., 2018), there is no recommendation of which N source to use in complete diets with predominance of spineless cactus. However, for the optimal growth of the ruminal microorganisms is crucial the association the energetic ingredients with an N source. Especially if we consider the need for synchronism between the degradation rates of carbohydrate sources -spineless cactus with 733 g/kg carbohydrates in fraction A + B1 -and N in the animals' rumen.
Soybean meal is the N sources with rapid ruminal degradation traditionally used in ruminant feed, but in semi-arid areas the production di culty and market competition results in high prices. Regarding this, Santos et al. (2020a) suggest that diets containing spineless cactus associated with urea can reduce freshwater intake and allow reasonable body weight gain. The cottonseed, in turn, can be both a source of ber as the N in diets with spineless cactus. Therefore, using other N sources at a lower cost could improve the viability of livestock production systems, especially in semi-arid regions, and thus enable a greater supply of sheep meat. Given the above, the objective was to evaluate the potential use of different protein sources associated with spineless cactus (Nopalea cochenillifera Salm Dyck) in diets for nishing lambs, by determining the performance and characteristics of the carcass and meat.

Material And Methods
The experiment was carried out at the Department of Animal Science of the Federal Rural University of Pernambuco (UFRPE), located in Recife-PE, Brazil. The climate is classi ed, according to Koppen, as being of the Ams' type, which is characterized by being hot and humid, with an average annual temperature of 25.2ºC.

Animals, management and diets
Forty male Santa Inês sheep, uncastrated, with an average body weight of 23 kg ± 1.66 were identi ed and treated against ecto and endoparasites and, fasted for 16 hours, being weighed and distributed in a randomized block design, with two blocks, four treatments and ten repetitions. The animals were housed in individual stalls (1.0 m x 1.8 m), provided with individual feeder and drinker. The experimental period comprised 86 days, 28 days of which were used to adapt the animals to the facilities and handling, and 58 to the period of data and sample collection.
The experimental treatments consisted of four different sources of nitrogen (soybean meal (SM); cottonseed meal (CM); cottonseed + urea (CU); and urea (U)), with roughage composed of Tifton 85 grass hay (Cynodon spp.) and spineless cactus (Nopalea cochenillifera Salm Dyck), as shown in Table 1. Rations were calculated to meet weight gains of 200 g/day (NRC, 2007). The spineless cactus, composed mostly of cladodes, was processed in a forage machine, immediately before supply; Tifton 85 grass hay ground in a forage machine with a 4 mm sieve; corn and cottonseed meal were ground; while the cottonseed, whole. Commercial urea and ammonium sulfate used was Petrobras® (Petrobras, SE, Brazil). The diet was provided as a complete mixture, twice a day (8 am and 3 pm), with 60% of the diet offered in the morning and 40% in the afternoon. The adjustment of the quantity offered was carried out every two days, in order to allow 15% of leftovers.

Nutrient intake and apparent digestibility
The amount of feed offered and the leftovers were weighed daily to calculate the voluntary intake.
Nutrients intake was calculated by the difference between the nutrient concentration in supplied feed and nutrient concentration in the leftovers. For the apparent digestibility test, feces samples were collected, directly from the rectal ampoule of the animals, for six days at 0; 2; 4; 6; 8; and 10 hours after feeding.
The feces samples were homogenized, pre-dried in a forced ventilation oven at 55ºC for 72 hours and, later, processed in a sieve mill with a 2 mm sieve for future analysis.
The estimation of fecal dry matter production (FDMP) was performed using indigestible neutral detergent ber (iNDF) as an internal indicator, obtained after an incubation period of 288-h in bovine with a permanent cannula in the rumen, according described by Valente et al. (2011). The FDMP was then estimated by the relationship between the intake of the marker and the respective percentage in the feces. The digestibility of dry matter and nutrients was calculated by the relationship between the difference between the amount of dry matter or nutrient ingested and that excreted, by the amount of dry matter or nutrient ingested, and expressed in g/kg DM.

Chemical composition
During the experimental period, samples of the leftovers, feces and the ingredients used were collected, which were pre-dried for 72 hours in a forced ventilation oven at 55ºC, identi ed and stored. At the end of the experiment, a sample was made up of leftovers from the entire experimental period, per animal, as well as the food supplied, which were processed in a mill with 1 mm diameter sieve screens, for further bromatological analysis.

Ingestive behavior
The behavioral parameters were observed using the instantaneous scanning method ("Scan sampling"), proposed by Martin and Bateson (2007), at intervals of ve minutes, for 24 hours (Johnson and Combs, 1991). In the observation intervals, the variables were determined: feeding, rumination and idling times. The total chewing time (TCT, min/day) was considered as the sum of the feeding and rumination times (FT + RUT) and the idle time, as the time when the animal was neither feeding nor ruminating. Feeding e ciency as a function of DM intake (FE DM , g DM/min) was calculated as the relationship between DM consumption and feeding time (DMI/FT); and as a function of the NDF intake (FE NDF , g NDF/min) as the fraction of the NDF intake, by the feeding time (NDFI/FT). The rumination e ciencies as a function of DM intake (RUE DM , g DM/min) and NDF intake (RUE NDF , g NDF/min), were obtained respectively through the relationships between DM intake (DMI/RU) and NDF (NDFI/RU), and the rumination time.
Performance, slaughter and meat quality During the experimental period, the animals were weighed without fasting every 14 days, to monitor development without compromising performance. The average daily gain (ADG) was calculated according to the nal and initial weights, with a 16-hour fast.
After completing the 58 days of data and sample collection, the animals were subjected to fasting solids for 16 hours. After this time, they were weighed to obtain body weight at slaughter (BWS). Slaughter operations were carried out in accordance with the current rules of RIISPOA (Brasil, 2000). Initially the animals were desensitized by the non-penetrative percussive method, suspended by the hind limbs attached to hooks, followed by bleeding from the carotid arteries and jugular veins.
Skinning and evisceration was performed head and paws were removed to record the hot carcass weight (HCW), including kidneys and pelvic-renal fat. The gastrointestinal tract was weighed lled and then emptied, washed and weighed again, to determine of the empty body weight (EBW), obtained by the difference between the BWS and the content of the gastrointestinal tract (CGIT).
Afterwards, the hot carcasses were taken to the cold chamber and kept for 24 hours at a temperature of 4ºC, with the tarsus-metatarsal joints approximately 14 cm apart, using their own hooks. Then, the cold carcass weight (CCW) was recorded, including kidneys and pelvic-renal fat; weight by cooling loss (CL = HCW -HCW) was calculated and kidney weights and pelvic-renal fat were obtained, whose values were subtracted to determine the hot (HCW) and cold (CCW) carcass weights; as well as hot carcass yields (HCY (%) = HCW/BWS x 100) and cold carcass (CCY (%) = CCW/BWS x 100).
In addition, after the cooling period, objective measurements were performed on the entire carcass, according to Cezar and Sousa (2007). From the establishment of the relationships between the measures cold carcass weight, internal carcass length, croup width and leg length, the carcass compactness indexes (CCI (kg/cm)) were calculated.
After weighing and removing the tails, the carcasses were divided sagittally and the half carcasses were divided into six anatomical regions that constituted the meat cuts: leg, loin, rib, saw, neck, and shoulder (Cezar and Sousa, 2007).
The Longissimus muscle area (LMA) was measured in the left half carcass, through a cross section between the 12th and 13th ribs, exposing the cross section of the Longissimus dorsi muscle, through its outline, using transparent plastic sheet. To read the LMA, a digital planimeter (HAFF®; model Digiplan) was used. Subcutaneous thickness fat (STF) was also mensured in Longissimus lumborum, using a digital caliper, according described by Cezar and Sousa (2007).
To determine tissue composition, the left legs were dissected according to the methodology described by Cesar and Souza (2007). With the aid of a scalpel and tweezers, subcutaneous fat, intermuscular fat, muscles, bones and other tissues (tendons, lymph nodes, nerves and blood vessels) were removed. During dissection, the top ve muscles that surround the femur (Bicepsfemuris, Semimembranosus, Semitendinosus, Quadricepsfemoris and Adductor) were removed in their entirety and, with the measurement of the length of the femur (cm), the leg muscle index (LMI) was calculated using the formula proposed by Purchas et al. (1991).
After dissecting the legs, samples of the Semimembranosus muscle were crushed in a blender to obtain a homogeneous paste, and then they were lyophilized to determine moisture (Method n. 925.04), crude protein (Method n. 981.10), ether extract (Method n. 935.38) and ashes (Method 938.08), according to AOAC (2000).
Samples of the Longissimus lumborum muscle were used for the physical-chemical analysis of the meat. The chromatic characterization was performed according to Ramos and Gomide (2009) using Minolta colorimeter, model Chroma Meter CR-400, operating in the CIE system (L *, a *, b *). The water-holding capacity (WHC) was determined according to the methodology described by Sierra (1986). For pH, 10g samples were collected, crushed and diluted with 150 ml of distilled water and agitated until the particles were evenly suspended, and the reading was performed with the aid of a potentiometer (Gomes and

Statistical analysis
The experimental design was randomized blocks, with the initial weight of the animals being the criterion for block formation, according to the following model: where Yij = the observed value of the dependent variable, µ = the overall average, the treatment effect = (i = 1 to 4), bj = the effect of block j (j = 1 to 4) and eij = the experimental error. Data were analyzed using analysis of variance (ANOVA), using the GLM procedure and SNK test (P < 0.05), using the statistical package Statistical Analysis System (SAS (Statistical Analysis Systems Institute Inc.), 2000).

Results
The nitrogen sources tested did not interfere (P > 0.05) on the DM and OM intake of the sheep, but the CP intake and NFC was lower (P < 0.05) for animals fed with cottonseed + urea (CU) followed by urea (U) ( Table 2). The NDF intake was lower (P < 0.05) for the soybean meal (SM) diet, but similar between the cottonseed meal (CM), CU and U diets. While the EE intake was higher (P < 0.05) for lambs fed SM. The average consumption of TDN was 0.71 kg/day and did not differ (P > 0.05) between diets. The sheep fed SM diet had shorter (P < 0.05) feeding, rumination and chewing times and longer (P < 0.05) idle times concerning to animals fed with other nitrogen sources (Table 3). Better e ciency of feeding and rumination of dry matter (P < 0.05) were also observed in animals fed with SM. The nitrogen sources did not in uence (P > 0.05) the nal body weight (32.07 kg), average daily gain (0.15 kg/day) and feed conversion (7.73) of the sheep (Table 4). The weight of the empty body was not in uenced (P > 0.05) by the nitrogen sources tested, but the weight of the cold carcass was higher (P < 0.05) for sheep fed with SM and CM when compared to animals fed with U (Table 5). The carcass length was similar (P > 0.05) between the diets, however the thoracic perimeter and the carcass compactness were higher (P < 0.05) in the carcasses of sheep fed with SM compared to U. The subcutaneous fat thickness and the pelvic-renal fat score did not differ (P > 0.05) between the nitrogen sources tested.
The weights of the shoulder and loin of the carcasses of the sheep fed with SM were higher (P < 0.05) than the animals fed with U. However, we did not observe an effect (P > 0.05) of the tested diets on the tissue composition and compactness index of the sheep leg (Table 6). The content of moisture, ash and ether extract of sheep meat did not differ (P > 0.05) between the nitrogen sources tested, but the protein content of meat from animals fed with SM was higher when compared to those fed with CM, COT and U (Table 7). Except for water-holding capacity, there was no in uence (P > 0.05) of the nitrogen sources tested on the pH values, color parameters and cooking losses of the meat.

Discussion
The DM intake of sheep (average of 1.16 kg/day) was high when compared to that predicted by the NRC (2007) (0.96 kg/day) for the same animal category (28 kg and gain of 0.20 kg/day). Possibly, the spineless cactus in the diets optimized the intake of the animals, reducing the powderiness of the total diet and facilitating the apprehension, by the animals, of the mixture in the trough. In a meta-analysis Knupp et al. (2019) reported that the level of 500 g/kg of spineless cactus in the dry matter of the sheep diet -a level close to that used in the present study -obtains the maximum diet intake by the animals.
The lowest intake of CP was observed for the diets CU (0.11 g/day) and U (0.14 g/day). This can be explained by the fact that the animals rejected part of the cottonseed in this diet -even with spineless cactus facilitating the mixing of the ingredients, the cottonseed was selected and despised by the animals -re ecting a lower CP intake than the concentration of this nutrient in the diet. As for the NDF intake, we observed that the animals fed with the SM diet consumed less NDF, probably due to the majority of the NDF of this diet coming from Tifton 85 hay, which was more selected by the animals due to the larger particle size in the total mixture. Costa et al. (2012) also observed a reduction in the intake of NDF and CP in sheep fed with cottonseed in diets based on spineless cactus.
The lower dietary levels of NFC observed in the CU diet possibly explain the lower consumption of NFC compared to other N sources. In addition, due the CU diet has a higher EE content, it was expected that the highest intakes of this nutrient would occur in animals submitted to this diet, a fact that did not occur. However, as previously mentioned, rejection of the cottonseed by the animals was observed during the experimental period.
The lower apparent digestibility coe cient of DM and OM for U diet, probably due to the higher NFC content derived from corn in this diet. We propose that the excess of NFC -and lower NDF contentprovided a reduction in ruminal pH and decreased the microbial population degrading brous carbohydrates, resulting in less digestibility of DM in the total diet. In line with our ndings, Santos et al.
(2020b) observed that diets containing urea as a total substitute for soybean meal alter the production of ruminal short-chain fatty acids and reduce the populations of Streptococcus bovis and Ruminococcus avefaciens.
The lower apparent digestibility of the CP for U concerning to SM and CM diets was possibly due to the greater synchronism between carbon and nitrogen. In the case of U-fed sheep, the rapid conversion of urea to ammonia and its immediate rumen absorption meant that the level of nitrogen available for microbial protein synthesis of U-fed sheep was lower than those fed with CM (Ahvenjärvi and Huhtanen, 2018).
The lower digestibility of NFC observed in the U diet is due to the nature of the NFC in this diet, which consists predominantly of corn starch. The corn starch int has a slower degrabability compared to the NFC fraction present in spineless cactus (soluble sugars, starch and fruits) (Batista et al. 2003), resulting in less digestibility of the NFC fraction of the U diet compared to the other diets that had the forage palm as the main source of NFC. Still in this context, the lower digestibility of NDF observed in the U diet, is precisely due to the depressant effect of excess NFC on pH and NDF-degrading microbiota.
The SM diet showed greater e ciency of intake mainly due to the shorter feeding time, since the consumption of DM did not differ among nitrogen sources. Batista et al., (2020) observed an increase in feeding time with the replacement of soybean meal with a mixture of cottonseed and spineless cactus for buffaloes. In addition, the lower NDF intake of animals fed with SM may explain the higher rumination e ciency observed in this experimental group.
The In addition to the fat in the carcass, the nitrogen sources tested did not in uence the amount of fat in sheep meat. However, animals fed with SM produced meat that was richer in protein, in uenced by the higher crude protein intake. Normally, the highest protein content in meat is associated with increases in myo brillar and sarcoplasmic proteins that occur due to the increase in the contents of the muscle's DM. Because, the majority content of water is maintained by capillary forces within the myo brils and these occupy about 82-87% of the volume of the muscle cell (Huff-Lonergan & Lonergan, 2005), and much as 85% of the water in a muscle cell is held in the myo brils (Hughes et al., 2014), consequently, the meat of animals fed with SM showed a higher WHC value, this was due to the direct relationship between the water content of the muscle and content and type of protein of type, this may have occurred due to a lower solubility of the proteins present in the meat of lambs fed with SM, since the moisture content was similar between nitrogen sources. The inferior WHC can negatively affect the appearance of meat, and this can in uence consumer willingness to purchase the product (Hughes et al., 2014). In addition, the possible increase in sarcoplasmic proteins (myoglobin) would result in a reduction in L* values and increments in a*, responses that were also not observed.
Regarding the physical-chemical characteristics, the similarity observed for the values of L*, a*