Blood biochemical parameters of lambs fed diets containing cactus cladodes

ABSTRACT This study aimed to evaluate the effects of partial replacement (750g/kg) of Tifton hay by two cactus cladodes (Nopalea or Opuntia) on the metabolic profile of lambs. Thirty-six uncastrated male Santa Inês lambs (22.0 ± 2.9kg initial body weight) were distributed in a completely randomized design, with three treatments and 12 repetitions. The animals were fed a control diet (Tifton hay as exclusive roughage), Miúda cactus cladodes-based diet or Orelha de Elefante Mexicana (O.E.M.) cactus cladodes-based diet. Blood samples were collected one day before (baseline) and 45 days after the introduction of the tested diets. The Miúda cactus cladodes caused an increase (P=0.055) in the serum activity of the gamma-glutamyl transferase enzyme (53.66U/L) and in the blood content of glucose and fructosamine. The O.E.M. cactus cladodes caused lower (P=0.038) serum cholesterol content (41.33mg/dL). Regardless of the variety, there was a decrease (P=0.001) in the serum content of indirect bilirubin, urea, and sodium, and increase in the serum magnesium concentration. The partial replacement of the Tifton hay by Miúda or O.E.M. cactus cladodes in lamb feeding increases the enzyme activity, indicating liver and/or kidney changes, but does not cause relevant damage to energy, protein, and mineral metabolism.


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Cactus is an important fodder option for arid and semiarid zones of the world.According to Ben Salem et al. (2002), Magalhães et al. (2019) and Diets containing cactus cladodes, compared to diets with grass hay as the only roughage, have greater ratio between non-fibrous carbohydrates:neutral detergent fiber (Silva et al., 2021a), due to the high levels of starch (Batista et al., 2003) and pectin (Pessoa et al., 2020) and low fiber content of this forage plant, representing an important energy source for animal feed (Rocha Filho et al., 2021).
Lambs fed cactus cladodes reduce voluntary water intake, as there is increase in water intake via diet (Pordeus Neto et al., 2016;Silva et al., 2021a).Additionally, cacti have oxalates in their composition (Rekik et al., 2010;Silva et al., 2021a), substances that can bind with minerals contained in food and change the metabolism of these elements in the animal organism (Rahman et al., 2013).
The study of biochemical markers in the blood is essential for understanding the possible effects that dietary components can cause in one or more organ systems and, consequently, in animal health and production.Several scientific investigations with sheep and goats fed diets containing cactus cladodes have been showing changes in blood indicators of energy, protein, mineral and enzyme metabolism (Dantas et al., 2011;Cardoso et al., 2019;Gouveia et al., 2019;Maciel et al., 2019;Silva et al., 2021b).
The Miúda (Nopalea cochenillifera Salm Dyck) and Orelha de Elefante Mexicana (O.E.M.) cactus cladodes (Opuntia stricta [Haw].Haw.) configure varieties resistant to carmine cochineal (Dactylopius opuntiae) (Vasconcelos et al., 2009;Lopes et al., 2010).On the other hand, O.E.M. has little time of use in animal feeding and there is little research to investigate the effects of this variety on the blood parameters of sheep, thus signaling the importance of evaluations in this sense.Tifton hay (Cynodon spp.) is a grass that has been successful when associated with cactus cladodes (Opuntia and Nopalea) in the sheep growth performance (Lopes et al., 2020;Cardoso et al., 2021).However, Cardoso et al. (2019), when evaluating the serum biochemistry of lambs fed diets with increasing levels (0, 150, 300 and 450g/kg, dry matter (DM) basis) of Miúda spineless cactus, associated with Tifton hay, reported decreased levels of glucose, urea, calcium, phosphorus, and magnesium.Silva (2017) observed that lamb fed diets based on different varieties of cactus cladodes showed an increase in the serum activity of the enzyme alkaline phosphatase.
Thus, it was hypothesized that diets containing cactus cladodes cause damage to the metabolic profile of sheep.Therefore, this study aimed to evaluate the effects of partial replacement of Tifton hay by two cactus cladodes varieties on indicators of energy, protein, mineral metabolism, and enzymatic activity of Santa Inês lambs in feedlot.

MATERIAL AND METHODS
The management and care of animals were performed in accordance with the guidelines and recommendations of the Committee of Ethics in the Use of Animals (CEUA) at the Federal Rural University of Pernambuco (UFRPE), under license number 142/2018.
The experiment was carried at the Department of Animal Science, UFRPE, located in Recife, Pernambuco State, Brazil.This research is a part of a larger project with a methodology based on a previous study by Lopes et al. (2020).Thus, further information regarding collection, processing and chemical analysis of feed, leftovers and feces were reported by these authors.
Thirty-six uncastrated male Santa Inês lambs, with an initial body weight of 22.0±2.9kgand approximately six months old were distributed in a completely randomized design, with twelve lambs per treatment.The animals housed in individual stalls (1.0m x 1.8m), provided with a drinking fountain and feeder.The experimental period lasted 75 days, with the first 30 days for the adaptation of the animals to the diets and the management and the remaining 45 days for data and samples collection.In the pre-experimental period, all animals were identified, vaccinated against clostridia (Covexin 9 ® , São Paulo, SP, Brazil) and treated against ectoparasites and endoparasites with doramectin 1% (Dectomax ® , Guarulhos, SP, Brazil).
The diets were provided ad libitum as a total mixed ration twice a day, at 08:00 am and 03:00 pm, and the animals had ad libitum access to water.The cactus plants were manually harvested when they were 2 years old.The processing of cactus cladodes was carried out immediately before each feeding in a forage machine (MC1n Laboremus ® , Campina Grande, Brazil), and the mixture of ingredients was conducted manually in the feeders.The Tifton hay was acquired in local commerce and ground in a forage machine with an 8-mm sieve screen.The determination of total oxalates in feed and leftovers samples was performed according to methodology described by Moir (1953).
Blood samples were collected twice, the first collection being made one day before the introduction of the experimental diets (baseline) and the second 45 days after feeding with the tested diets.They were collected by venipuncture of the jugular, four hours after the morning feeding, in vacuum siliconized tubes (Vacutainer ® ) without and with anticoagulant (sodium fluoride/EDTA), to obtain serum and plasma, respectively.The samples were immediately placed in a container containing recyclable ice.Then, they were centrifuged at 1,600 x g for 15 minutes.The serum and plasma aliquots were then stored in 2mL polyethylene tubes previously identified and frozen in a freezer at -20ºC for further analysis.
The biomarkers analyzed in the blood were alanine aminotransferase enzyme activity (ALT), aspartate aminotransferase enzyme activity (AST), alkaline phosphatase enzyme activity (AP), gamma-glutamyl transferase enzyme activity (GGT), total and direct bilirubin, plasma glucose, cholesterol, triglycerides, fructosamine, urea, creatinine, uric acid, total proteins, albumin, total calcium (Ca t ), phosphorus (P), magnesium (Mg), sodium (Na) and potassium (K).The indirect bilirubin values were estimated by subtracting the total and direct bilirubin values.Globulin was determined by the difference between the values of total serum protein and albumin.The albumin:globulin ratio and the Ca t :P ratio were calculated by dividing the values of these biochemical indicators.Ionized calcium (Ca i ) was estimated by calculation involving the values of serum Ca t (mg/dL), total protein (g/dL) and albumin (g/dL), following the recommendation suggested by the manufacturer -Labtest ® (Labtest Diagnóstica S.A., Brazil).
The experimental design was completely randomized, considering initial body weight (BW) as covariate.The data were analyzed using the GLM procedure in the software package Statistical Analysis System 9. 0 (Statistical…, 2009), according to the model below: Where Y ij = the observed dependent variable; µ = general mean; T i = effect of treatment; β(X ij − X) = covariate effect (initial BW); and e ij = experimental error.The data were submitted to ANOVA and the Tukey's test, at 5% probability, was used to compare the averages between treatments.To compare the means of blood biochemical indicators between baseline collection and after 45 days, the paired Student's t-test was applied, using the Minitab software version 17 (2014).

RESULTS
Dry matter (g/day, %BW and g/kg BW 0.75 ), crude protein, total digestible nutrients, and oxalates intakes (g/day) are shown in Table 3.The serum activity of the ALT enzyme did not differ between diets (Table 4), whose variation was from 20.58 to 24.71U/L, as well as there was no significant difference between initial and final collection (Table 5).Regarding the AST values, these did not change between collections (Table 5), as well as differences were not noticed depending on the diets, with an average value of 119.02 U/L (Table 4).AP activity was similar between animals that consumed cactus cladodes and those that were submitted to the control diet.However, when evaluating the activity of this enzyme before and after the supply of experimental diets, it was noticed that there was an increase in values in animals of all groups (Table 5).The diet containing Miúda cactus cladodes led to an increase in serum GGT activity (53.66U/L) (Table 4).Additionally, when comparing the values between collections, it was found that the varieties of cactus cladodes increased the average activity of this enzyme (Table 5).
Serum levels of direct bilirubin were not influenced by diet (Table 4).For total bilirubins, there was a 40% decrease in the concentration of this biochemical marker in the treatment with Miúda cactus cladodes (0.36mg/dL) compared to the control diet (0.60mg/dL) (Table 4).When evaluating the two collections, it is noted that the cactus cladodes varieties reduced these metabolic components (Table 5).Cactus cladodes also caused a decrease in the levels of indirect bilirubin (Table 4), as well as a decrease between the baseline collection and the test collection in the control diets and with O.E.M. cactus cladodes (Table 5).
There was no significant variation between the serum concentration of triglycerides, creatinine, uric acid, total proteins, albumin, globulin, and albumin:globulin ratio due to the diets (Table 4).Plasma glucose levels were higher in animals Arq.Bras.Med.Vet.Zootec., v.75, n.1, p.48-60, 2023 that received Miúda cactus cladodes in the diet (79.48mg/dL) (Table 4), as well as an increase in circulating glucose after the supply of all tested diets (Table 6).
Regarding the influence of the cactus cladodes on the serum cholesterol content, the O.E.M. cactus cladodes caused a lower value of this biochemical indicator (41.33mg/dL) compared to the content found in the animals of the control group (Table 4).The serum concentration of triglycerides was higher 45 days after the introduction of the tested diets, without oscillating significantly as a function of the diets (Table 6), while the Miúda cactus cladodes increased the serum fructosamine value (Table 4  and 6).At the end of the experiment, lower serum urea levels were found in animals that received cactus cladodes, regardless of variety, compared to those that received the control diet (Table 4).When compared to baseline values, the animals that subsequently consumed the Miúda cactus cladodes and the control diet had higher serum urea levels, which was not obvious with the O.E.M. cactus cladodes (Table 6).
Diets with cactus cladodes altered the metabolism of Mg and Na, causing higher serum concentrations of Mg (P=0.001) and lower Na content (P=0.001)(Table 4).The same trend was observed when comparing the averages of these minerals between collections (Table 7).

DISCUSSION
According to Kaneko et al. (2008), healthy sheep present activities of 26-34U/L and 60-280U/L for ALT and AST, respectively.In the present study, the serum activity of ALT was below the normal range and the enzymatic activity of AST fell within the physiological limits.Thus, it can be said that these enzymatic indicators do not signal hepatic damage, considering that only values above the reference range indicate possible liver cell degeneration.
The AP values determined before the introduction of the tested diets were within the normal range (Kaneko et al., 2008), which did not occur after the intake of the control diets and with cactus (Table 5).Most of the serum AP is of hepatic origin, as this enzyme is found in the cells of the biliary epithelium and in the canicular membranes of hepatocytes (González Arq. Bras. Med. Vet. Zootec., v.75, n.1, p.48-60, 2023 andSilva, 2006).In this context, Silva et al. (2021b) observed significant lesions in the hepatic parenchyma of goats fed with the same cactus cladodes varieties used in this study.However, there is a possibility that the increase in AP may also occur for bone or kidney metabolic reasons.Considering the bone origin, it is highlighted that oxalate is an anion derived from organic acid that can bind with minerals forming crystals and reduce the availability of Ca and Mg, thus stimulating bone resorption in an attempt to maintain the serum levels of these minerals (González and Scheffer, 2003;Rahman et al., 2013).
In the present study, all experimental diets showed concentrations of total oxalates in their composition (Table 2), which may have triggered hormonal actions to maintain serum calcium levels (Table 4), thus increasing the activity of AP, which represents an important indicator of bone mineral mobilization.On the other hand, oxalate levels contained in all diets can be considered safe (Table 2), considering that James and Butcher (1972) reported that diets containing up to 6% of soluble oxalates in DM did not cause acute poisoning in sheep, although they could cause mild hypocalcemia and decrease in serum Mg.
According to Tennant and Sharon (2008), even though GGT is present in the tissues of many organs (liver, kidneys, pancreas, small intestine, and bile duct epithelium), the elevation of its serum activity is observed primarily in cases of acute liver damage.This is the most specific enzyme for diagnosing cholestasis or bile duct obstruction (Gomes et al., 2008).Cholestasis consists of the decrease or interruption of bile flow and during this process the bile cells overflow, releasing some enzymes that can be detected in the bloodstream.Some clinical signs may show cholestasis, such as excess lipids in the feces (steatorrhea).In this research, differences were observed for the values of ether extract in feces (P=0.009),so that the sheep fed with Miúda cactus cladodes had 30.3g/kgDM, higher than those detected in the feces of the animals of the control group (27.4g/kgDM) and the treatment with O.E.M. cactus cladodes (23.3g/kgDM), which may have been caused by the decrease in the arrival of bile in the small intestine, thus affecting lipid digestion and increasing the concentration of this nutrient in the fecal material, reinforcing the idea that there may have been cholestasis.
After 45 days of supply of the experimental diets, the serum levels of direct bilirubin remained within the physiological limits for the sheep specie, however in the baseline collection the concentrations of this metabolite in the animals that were subsequently submitted to the control diets and with O.E.M. cactus cladodes were above the reference range (Kaneko et al., 2008).According to Barini (2007), conjugated or direct bilirubin increases in cases of hepatocellular damage or even injury or obstruction of the bile ducts.For total bilirubins, the reduction caused using cactus cladodes led to values within the ideal margin.
All indirect bilirubin values, including that of the control diet, were recorded above the normal range.According to Lassen (2007), bilirubin may increase due to pre-hepatic, hepatic, or posthepatic causes.Unconjugated or indirect bilirubin increases in cases of excessive erythrocyte destruction or by defects in the bilirubin transport mechanism within hepatocytes (Hendrix, 2005).Therefore, as there was no increase in these metabolites due to the presence of cactus cladodes in the diets, it can be inferred that the animals did not develop liver damage.Additionally, it is possible that the highest water intakes by animals that received cactus cladodes in the diet cause hemodilution of total and indirect bilirubins.
The superiority in the plasma glucose level in the animals that received Miúda cactus cladodes may be related to the higher concentration of propionic acid generated by this variety during rumen fermentation.According to Rocha Filho (2012) sheep fed diets based on Miúda cactus cladode presented fermentative profile with more propionate (greater gluconeogenic precursor) than those which consumed O.E.M. cactus cladodes (23.7 and 22.7mM/L of ruminal fluid, respectively).However, regardless of diet, the animals' blood glucose falls within the reference range for sheep (Kaneko et al., 2008).
Comparing the effect of cactus cladodes varieties, the lowest cholesterol content in animals that received O.E.M. cactus cladodes can be justified by the lower intake of total Blood biochemical… Arq.Bras.Med.Vet.Zootec., v.75, n.1, p.48-60, 2023 57 digestible nutrients.Furthermore, the presence of saponins has been detected in the cactus cladodes (Féboli et al., 2016).According to Tadele (2015), saponins decrease the intestinal absorption of glucose and cholesterol, based on intraluminal physical-chemical interactions, which may have contributed to the reduction of serum cholesterol.
Additionally, cacti cladodes, when compared to other forage plants, have shown high levels of linoleic acid (omega-6), which is a precursor to arachidonic acid that has caused hypocholesterolemic effects (El-Mostafa et al., 2014).The serum decrease in cholesterol may still be related to liver failure, since the liver is the main site of synthesis of this metabolite (Lassen, 2007).In this sense, Silva et al. (2021b) observed decrease in serum cholesterol levels and histopathological lesions in the liver of goats fed the same cactus cladodes genotypes used in the present study.According to Oh and Lim (2006), the glycoprotein (90kDa) isolated from Opuntia ficus-indica lowers plasma lipid content in mice.At the end of the experimental period, all averages were below the values considered normal (52-76mg/dL) (Kaneko et al., 2008).
The highest average of the variable fructosamine was registered in the animals that received the diet containing Miúda cactus cladodes (259.95μmol/L) in comparison with those of the control treatment and with O.E.M. cactus cladodes (244.97 and 244.45 μmol/L, respectively) (Table 4), which is related to the higher glycemia observed in animals that consumed this diet.According to Gouveia et al. (2015), the fructosamine can be defined as a stable ketoamine, formed from the nonenzymatic reaction between glucose and protein amine groups, especially albumin and IgG; and its blood concentration is controlled by the balance between the synthesis and elimination of these protein and glucose compounds.Thus, since the concentrations of total proteins, albumin, and globulin were not modified by the diets (Table 4), the highest level of fructosamine in sheep fed with Miúda cactus cladodes is associated with a higher blood glucose concentration.
According to Kaneko et al. (2008), serum urea values in sheep must be between 2.86 and 7.14mmol/L.Thus, all serum urea averages were above the reference limits.On the other hand, it should be noted that the average blood concentrations of urea (8.58 and 9.04mmol/L, in the animals submitted to diets with Miúda cactus cladodes and O.E.M. cactus cladodes, respectively), are close to the value reported by Maciel et al. (2019), which was 9.51mmol/L, when offering diets with 500g/kg of Miúda cactus cladodes and 250g/kg of Tifton hay for sheep, similar percentages to the present study.
The concentration of circulating urea has a strong connection with the amount of protein consumed, with the absorption of rumen ammonia and with the energy-protein ratio of the diet.Urea biosynthesis in the liver occurs in amounts proportional to the concentration of ammonia produced in the rumen, so that the higher the dietary protein intake, the higher the blood urea levels (Harmeyer and Martens, 1980).In this perspective, there was a higher crude protein intake by the animals that received Miúda cactus cladodes (Table 3), but with a reduction in blood urea (Table 4).Thus, diets with cactus cladodes seem to have caused a more adequate synchronism between the availability of nitrogen and energy in the rumen, which resulted in lower blood urea content.Cardoso et al. (2019), with the inclusion of increasing levels of Miúda cactus cladodes in the sheep diet, reported a linear decrease in blood urea, associating this response to the more efficient synchronism between nitrogen and energy in the rumen environment.
In addition, it is likely that the higher rate of passage of diets containing cactus cladodes (Costa et al., 2016) decreased the transformation of dietary protein into ammonia and, subsequently, into urea, reducing the circulating content of this metabolite.Bispo et al. (2007) found a linear decrease in ruminal ammoniacal nitrogen concentration with the replacement of elephant grass hay by cactus cladodes in the sheep diet.
Due to the lack of significance between serum concentrations of creatinine, uric acid, total proteins, albumin, globulin, and albuminglobulin ratio due to diets and taking into account that the means of the variables of total proteins, albumin and globulin were found according to the normal parameters for the sheep species (Kaneko et al., 2008)  said that all diets provided adequate protein supply and, possibly, did not alter the functional state of liver cells.In the case of creatinine, high levels of this metabolite in the blood reflects the rate of filtration in the kidneys and may signal kidney damage (González and Scheffer, 2002), behavior that was not observed in this study, thus indicating that there was no significant renal impairment.
In the collection after the introduction of the experimental diets, the serum concentrations of Ca t and Ca i (free) in the sheep of all groups were similar (Table 4) and were within the physiological range (Kaneko et al., 2008), however, in the baseline collection, all means indicated hypocalcemia (Table 7).The measurement of Ca i levels provides safer results when compared to Ca t , as it represents the form of calcium that is biologically active and, therefore, physiologically more relevant (Kaneko et al., 2008).According to Guedes et al. (2016), the Ca i , which corresponds to values between 50 and 60% of the Ca t , is crucial for the transmission of nerve impulses, muscle contractions, in addition to cell signalling.In the present study, Ca i averages represented 60% of Ca t (Table 4).The proportions of Ca t :P in the blood remained narrow (Table 4) and close to the ratio in which these minerals are found in the bones, which is between 1:1 and 2:1 (Pugh, 2004).
The serum level of Mg was higher in animals fed with cactus cladodes varieties (Table 4 and 7) due to the high content of Mg in this feed, whose concentration ranges from 17.0 to 18.2g/kg DM (Batista et al., 2003;Santos et al., 2009).According to Kaneko et al. (2008), normal serum Mg levels vary from 2.2 to 2.8mg/dL, lower values than those observed in this study, which were 3.21 and 3.67mg/dL in sheep that received Miúda and O.E.M. cactus cladodes, respectively (Table 4).The increase in serum Mg in sheep was also reported by Maciel et al. (2019), when observing a concentration of 2.95 mg/dL of this element, using 500g/kg of cactus cladodes, based on DM.
Na concentrations were lower in treatments containing cactus cladodes (Table 4) and decreased between collections (Table 7) due to the higher level of this mineral in Tifton hay.Santos et al. (2009) reported Na contents of 0.700 and 0.300g/kg of DM in the Tifton hay and in the Gigante cactus cladodes (Opuntia fícusindica Mill), respectively.

CONCLUSIONS
The partial replacement (750g/kg) of the Tifton hay by Miúda or Orelha de Elefante Mexicana cactus cladodes in lambs feeding increases the enzyme activity, indicating liver and/or kidney changes, but does not cause relevant damage in indicators of energy, protein, mineral metabolism, and weight gain, being these cacti a fodder resource option.However, that is possible that the metabolic changes may restrict its use, which demonstrates the need for long-term studies.
a orelha de elefante mexicana cactus cladodes, b g/kg natural matter, c neutral detergent fiber assayed with a heat stable amylase and corrected for ash and nitrogenous compounds, d non-fibrous carbohydrates, (-) not determined.

Table 3 .
Nutrients and oxalates intake, and weight gain by sheep fed cactus cladodes-based diets Silva et al. (2022)e mexicana cactus cladodes, b standard error of the mean, c body weight, d metabolic body weight; e total digestible nutrients, * values obtained byLopes et al. (2020).#valuesobtained bySilva et al. (2022).Averages in rows followed by different letters are statistically different by the Tukey's test at 5% probability.

Table 4 .
Blood biochemistry of sheep fed cactus cladodes-based diets a orelha de elefante mexicana cactus cladodes, b standard error of the mean, c alanine aminotransferase, d aspartate aminotransferase, e alkaline phosphatase, f gamma-glutamyl transferase.Averages in rows followed by different letters are statistically different by the Tukey's test at 5% probability.

Table 5 .
Enzymatic profile and serum concentration of bilirubin's of sheep before and after supply of cactus cladodes-based diets orelha de elefante mexicana cactus cladodes.Averages followed by different letters in the same column differ statistically by Student's t-test at 5% probability. a

Table 6 .
Indicators of energy and protein metabolism of sheep before and after supply of cactus

Table 7 .
Serum minerals of sheep before and after supply of cactus cladodes-based diets orelha de elefante mexicana cactus cladodes.Averages followed by different letters in the same column differ statistically by Student's t-test at 5% probability. a