Nutritional performance, hepatic and renal function in goats fed diets containing detoxiﬁ ed castor cake at different stages of pregnancy

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INTRODUCTION
In the production of goats milk, as in any animal production system, barriers are found in the nutritional management of animals, being essential the use of balanced diets, thus avoiding financial waste, especially when it comes to confi ned animals, given that the costs are higher (Araújo et al. 2020).The ideal is to use diets appropriate to the physiological stage and the level of production of the animals.However, the high cost of some foods hinders the productivity of these systems, often making them ineffi cient from a nutritional and economic point of view.
There has been a growing interest in the use of alternative food and regional (Menezes et al. 2012, Pompeu et al. 2012, Gionbelli et al. 2014) in the diets of ruminants, aiming at the reduction of costs with food supplies, among which we can highlight the castor cake, with its high protein content.However, the presence of antinutritional factors, such as the ricin, ricinina, ricinus agglutinin and complex allergenic renders it unfi t for animal feed.However, after detoxification process (Anandan et al. 2005), this problem can be circumvented requiring, therefore, studies proving its use effectively in the diet of ruminants in different physiological situations.
In this sense, the pregnant goats require special attention, because the nutrients required by the body of the female at this stage do not meet only the maintenance of the body, but mainly will be directed to the pregnant uterus and mammary gland, being that during pregnancy.There is a great variation in the metabolism of pregnant goats, in virtue of fetal growth (Konyali et al. 2007).
During the first and second third of gestation, which begins with the design, the requirements of protein, energy, minerals and vitamins may be similar to the requirements of maintenance for the goats (NRC 2007).Already in the final third of the pregnancy is observed reduction in the relative rate of growth of the fetus, possibly because the fetus is entering the linear phase of growth (Greenwood & Bell 2003, Hafez & Hafez 2004), at the same time, at this stage there is a strong mobilization of body tissues and the goats are more susceptible to the occurrence of metabolic diseases.In addition, the nutrition of the female during the gestation period will affect the production of milk, the composition and weight gain of their descendants (Liden et al. 2009), and highlighted the importance of adequate nutritional plans the goats.
On the basis of claims raised and considering the importance of diet on the pregnancy period, the aim of this research was to evaluate the possibility of use of the detoxified castor by two alkaline solutions in lactating Saanen goats and Anglo Nubian and in confinement during stages of pregnancy and observe the effects on intake and digestibility of dry matter and nutrients, nitrogen balance and hepatic and renal functions.

MATERIALS AND METHODS
All animal procedures were conducted according to the regulations of the Ethics Committee on the Use of Animals of the Brazilian Agricultural Research Corporation, National Center for Research with goats, protocol no.005/2015.The experiment was conducted at the Technological Center of production of goat milk from Embrapa Goats and Sheep (3°44'57.42"S,40°20'43.50'W)located in the town of Sobral-CE, Brazil.
Were used 24 goats, being 12 of the Saanen goats and 12 Anglo Nubian, staying in each treatment, with body weight of 42.08 ± 5.33 kg of body condition score of 3.6 ± 0.3 according to the classification of Morand-Fehr (2005), consisting of four goats with double gestation and two with single gestation in each treatment.All tests were performed on two occasions, the first when the goats were between 30 and 100 days of gestation (first and second third) and the second between 110 and 140 days of pregnancy, representing the final third of pregnancy.
The experimental design was completely randomized, in plots subdivided in time, getting in the gestational phase plots and subplots the diets.Before the trial period, the goats were evaluated in another study where it was analyzed the performance in the growth phase with the use of the detoxified castor, therefore, the goats were already adapted to the foods used, changing only the proportion of ingredients due to the demands of the category in assessment.
In pre-experimental conditions, the reproductive management adopted involved " natural, with an average duration of breeding season of 30 days.Once identified the estro, the coverage was performed by natural mounts directed.For both, were used for breeding of the same breed, without any degree of kinship with the arrays.From the coverage, the goats were placed in individual stalls, suspended and with floor ripped of 5.06 m², being 2.87 m² solarium area composed of beaten floor, provided with drinkers, feeders and salt shakers.The 35 days of gestation of the latest coverage, was held the  (2022) 94(4) e20201775 3 | 15 confirmation of pregnancy all the goats, with the use of ultrasound device KX 5000 ® .The 96 days of gestation, the access to the solarium was closed with screens of protection due to the large slope of the ramp access, staying with 2.19 m 2 per bay.
The measurement of live weight was performed fortnightly.The animals were weighed always at the same time and in fasting.The treatments consisted of three diets, a formulated with corn and soybean meal (SM) and the others were formulated with detoxified castor cake (DCC) by calcium hydroxide [Ca(OH) 2 ] and another composed by detoxified castor by sodium hydroxide (NaOH), both in total substitution of soybean meal, was used as roughage hay Tifton 85 (Cynodon spp.).The goats began to receive the diets on the day of coverage.
The diets were formulated to meet the needs of pregnant goats with two fetuses, according to the NRC (2007) for goats with body weight of 42 kg and in early pregnancy.The chemical composition of foods is presented in Table I and the proportion of the ingredients and the chemical composition of diets are shown in Table II.
The castor cake used in this study was obtained after collecting oil, by mechanically pressing castor bean seeds at temperatures between 90 and 100 °C.After mixing the cakes, reagents and water for 3 h (mixing for 10 min and resting for 30 min, alternately), the cake was placed outdoors on a plastic canvas for a period of 48 h and constantly rolled with a squeegee manual adapted for homogeneous drying.After drying, the cake was chopped using a forage machine to reduce the material size (5 mm) and to facilitate its homogenization with the other ingredients.
The concentrations of alkaline products (calcium hydroxide and sodium hydroxide) used for 100% detoxification of ricin in crude castor cakes were 90 g Ca(OH) 2 and 60 g NaOH, respectively, per kilogram, which were diluted in 2 L of water using a stationary mixer (Fischer ® MOB 400 G2) equipped with a three-phase motor, according to the methodology described by Araújo et al. (2020).No hemagglutinating activity was observed at those concentrations; i.e., ricinus agglutinin was no longer active.Therefore, these two concentrations were used to formulate the diets.Diets were supplied daily at 8h00 and 16h00, allowing a 10% leftover.Samples were collected from the bulk, concentrate, and also leftovers during the entire experimental period, then duly packaged in identified plastic bags and stored in a freezer at −18 °C.
At the end of the trial, the samples were thawed and separated between the two stages of pregnancy (first two-thirds and final third) and submitted to pre-drying in forced ventilation oven at 55 °C for 24 h.The dried samples were ground in knife mills (Wiley mill, Arthur H. Thomas, Philadelphia, PA, USA).Feed and leftovers were screened using a 1.0-mm sieve for chemical analyses, and a 2.0-mm sieve was used for evaluating apparent digestibility (Nocek, 1988).
Apparent digestibility coefficients were estimated indirectly using the internal iADF indicator.To do so, feces were collected directly from the rectal bulb for five days at different times (0, 3, 6 and 9 h after the first feeding), aiming for greater daily representativeness.Next, they were identified and stored in a freezer at − 18 °C.At the end of data collection, composite samples were made and then dried in a forced ventilation oven at 55 °C until constant weight was reached.The feces (2.0-mm) and feed samples (2.0-mm) were incubated in situ for a period of 240 h, according to the methodology described by Casali et al. (2008).
For evaluating nitrogen balance, total urine production was estimated by the concentration of creatinine in the urine.Spot urine samples were obtained approximately 4 h after feeding, Feces were collected directly from the rectal bulb for five days at different times (0, 3, 6 and 9 hours after the first feeding) for a representative sampling.Furthermore, the feces samples used for digestibility tests were collected on different days, so two samplings were performed.Due to the small amount of feces collected per day, we chose to make two separate collections.
Blood samples were collected using 9.0 mL vacutainer tubes (Grainer Bio-One, Vacuette ® Americana, SP, BRA), by puncturing the jugular vein, and 4 h after the morning feed.Two blood samples were collected from each animal; one in a tube containing an anticoagulant (EDTA) and another in a tube without the anticoagulant.The tubes with the anticoagulant were used for analyzing urea and total protein concentration, while samples without the anticoagulant were used for analyzing creatinine, total and direct bilirubin, albumin, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gammaglutamyltransferase (GGT) levels.To determine urea and total protein concentration, serum was obtained by centrifuging the tubes at 3,293 × g for 15 min, identified and stored in Eppendorf ® mini-tubes, and frozen for analysis.Blood parameters and urine creatinine were analyzed with commercial Labtest ® kits using colorimetric procedures.Blood collections were made on two occasions, the first when the goats were 60 days pregnant and the second at 120 days.
The concentration of NDF was corrected for ash as proposed by Mertens (2002), and for proteins (NDFan) according to Licitra et al. (1996).The ADL fraction was extracted with 72% sulfuric acid (Van Soest et al. 1991).Non-fibrous carbohydrate content (NFC) was calculated with an adaptation of the method proposed by Hall (2003) and NDF corrected for ashes and protein.
Total carbohydrate (TC) content was obtained using the equation proposed by Sniffen et al. (1992).
The quantity of total digestible nutrients (TDN) was calculated according to Weiss (1999).The total digestible nutrient values were converted into net energy (NE) for production (maintenance and growth) and digestible energy (DE), according to the equations suggested by the NRC (2001).The intake of TDN (TDNI) was calculated according to the methodology described by Sniffen et al. (1992), as follows: TDNI = (CPI-CPf)+2.25(EEi-EEf)+(TCi-TCf).Where, CPI, EEI, and TCI correspond to the intakes of CP, EE, and TC, respectively; and CPf, EEf, and TCf represent the respective excretion of CP, EE, and TC in the feces.In the conversion of TDNI into metabolizable energy (ME), 1 kg of TDN was assumed to be equivalent to 4.409 Mcal DE, and ME = 0.82 × DE (NRC 2001).
Data were initially subjected to normality tests (Shapiro-Wilks) and homoscedasticity tests (Levene), and to analysis of variance by the F test when the presuppositions were met, using the following model: Where Yijk is the dependent variable corresponding to the experimental observation; μ is the overall mean; αi is the fi xed effect of the diets; βj is the fi xed effect of stages of pregnancy; (αβ)ij is the interaction effect; and eijk is the random error, assuming an independent normal distribution.Interaction between stages of pregnancy and diet was only deployed when significant at 5% probability by Tukey test.Statistical analyses were performed using the GLM procedure of SAS software version 9.4 (SAS INSTITUTE 2005).

RESULTS
There was effect (P<0.05) of diets and th e stages of pregnancy on dry matter intake (DMI) and nutrients (Table III).The DMI was greater for the goats fed with SM and DCC Ca(OH) 2 , for both the values expressed in g/day and for those expressed in % of body weight (% BW), but the goats fed diets based on SM also did not differ from those who consumed a diet with DCC NaOH, when expressed in % BW.As well as the DMI, the intake of CP, EE, NDF and NDT were higher for the goats fed the diets based on SM and DCC Ca(OH) 2 .In relation to the effect of the stages of pregnancy, it was observed that, with the passing of the stages, the intake decreased signifi cantly.Similarly, the intake of CP, EE, NDF and TDN decreased in 73.55; 42.11; 235.81 and 404.43 g/day, respectively.
It has been observed that during pregnancy, the weight of the goats was amended by the diets (P<0.05).The goats fed with diet the basis of SM had greater weight in the parturition day.However, the postpartum weight was equal to the other (P>0.05).In addition, the weight of the goats generated was not infl uenced by the diets, nor was the body condition score in the pre and postpartum period and the weight of the young goats (Table IV).
There was no effect (P>0.05) of diets or stage of pregnancy on the digestibility of DM and nutrients during the gestational period of goats (Table V).
The diets influenced (P<0.05) the consumption of nitrogen and the amount of nitrogen retained (Table VI).The goats fed the diets based on SM and DCC Ca(OH) 2 consumed larger amounts of nitrogen, with average values of 17.13 and 16.78 g/day, respectively, while the goats fed with diet containing DCC NaOH consumed less quantity (15.01 g/day).Similarly, the content of nitrogen retained was lower for latter (10.28 g/day).The stages of pregnancy infl uenced (P<0.05)all the variables related to the nitrogen balance.In summary, during the fi rst two-thirds of pregnancy, the goats presented larger values, with reductions of 42.56, 49.55, 49.74 and 39.14% for the nitrogen consumed, faeces, urine and nitrogen retained, respectively.
We observed effect (P<0.05) of diets on the values of total protein, albumin, urea and gamma-glutamyl transferase (Table VII).The total protein and albumin content was in the goats that consumed the DCC NaOH diet (6.72 and 2.44 g/dL), but it did not differ from the goats fed with a diet based on DCC Ca(OH) 2 (6.49 and 2.32 g/dL), which in turn were the values of goats that consumed the soybean meal diet.Urea showed an opposite behavior values of total protein and albumin, where the goats that consumed the diet the basis of SM showed higher content (31.16 mg/dL) and those who consumed the diet DCC NaOH lower content (27.70 mg/dL), however, the goats fed with DCC Ca(OH) 2 did not differ from both diets (28.81 mg/dL).On another hand, the latter presented a higher amount of gamma-glutamyl transferase (65.78 IU/L).
In relation to stages of pregnancy, we observed effect (P<0.05) of the same on all blood parameters evaluated, with the exception of the aspartate aminotransferase (P>0.05).In summary, we observed a significant reduction in the levels of protein metabolites, which were: 18.95; 32.74; 17.06; 30.26; 17.34 for total proteins, direct bilirubin, albumin, urea, alkaline phosphatase, alanine aminotransferase, respectively, since the content of gamma-glutamyl transferase increased by approximately 30.74% in the final third of pregnancy.

DISCUSSION
The smallest DMI by goats fed with DCC NaOH may be related to the sodium content of the detoxified castor by this alkaline product (Table I), because according Yousfi et al. (2016), the greater inclusion of sodium in the diet of ruminants provides a self-adjusting effect of voluntary intake by the animals.It should be noted that the quantity of sodium in the NaOH DCC (Table I) was 32.4 times higher than that in DCC Ca(OH) 2 , which highlights even more this effect in the control of DMI.
The DMI recommended by NRC (2007) for non-lactating goats with 42 kg of body weight is of 1593,24 g DM/day for the dual pregnancies.Taking the average of consumption throughout the gestational period, without taking into account the type of pregnancy, it was found that the average DMI was 1166,34 g DM/day.This result is 26.79% lower than the recommended intake by that system.With the passing of the pregnancy, DMI decreases due to factors such as compression of the digestive tract caused by the growth of pregnant uterus and increased serum levels of estrogen (Forbes 2007).On the other hand, in absolute terms, the coefficient of digestibility of diets increases, because the passage rate tends to be lower (Lu et al. 2005).Therefore, despite the diets did not have influenced the digestibility in both periods of pregnancy, there is a slight increase of values in the final third of pregnancy (Table IV).
The effect to DMI influenced the results of consumption of EE, NDF and TDN.This result was expected, since the intake of these nutrients is directly related to the amount of DM ingested.In relation to the consumption of TDN, the highest values observed in goats fed with SM and Ca(OH) 2 TDN are justified by the greater fed with DCC NaOH, where it can be observed that the amount of nitrogen excreted in feces and urine were lower.In addition, the content of total protein and albumin was greater in the circulation of these goats (Table VII), which may be related to the greater utilization of nitrogen consumed.Under another perspective, it is important to note that in spite of the content of nitrogen retained have decreased in the final third of gestation, in percentage terms the content was higher in this phase (31.08%) that during the initial two-thirds (26.97%).
It is interesting to observe the efficiency of nitrogen use by goats fed with DCC NaOH, as they had the lowest nitrogen consumption, however the excretion of this nutrient, both in urine and faeces, was the same as goats fed with soybean meal and DCC Ca(OH) 2 (Table VI).According to Araújo et al. 2020, the DCC NaOH has a higher protein content of fraction A. Thus, it can be inferred that the DCC NaOH diet presents greater degradation of soluble proteins, favoring the availability of non-protein nitrogen, which justifies the positive nitrogen balance.
The largest proportion of nitrogen retention is derived from the intense protein metabolism of the fetus in the final third of the pregnancy, because according to Härter et al. (2017).The growth of the fetus in this period of gestation can match up to 93.6% of the total weight of the fetus, absorbing large quantities of proteins that are in circulation (Bell et al. 2005), which may be related, including, in does not change the content of total proteins in the circulation of goats fed with DCC NaOH (Table VII), despite the smaller intake of CP (Table III) during the final third of pregnancy.
The average levels of enzymes for hepatic and renal functions are within the standards for species, according to Smith & Sherman (2009).It is interesting to observe that, in spite of the diets did not affect the levels of these enzymes, there was a decrease in the final third of gestation, except the aspartate aminotransferase, which was not altered (Table VII).The reduction of these enzymes in the final third of pregnancy is common, because Huy (2005), says that all the enzymes related to liver function are usually reduced in the course of the pregnancy of mammals, due to the expansion of the extracellular fluid.This author affirms that only exception is for alkaline phosphatase, which is high during this period due to same be of placental origin, but the values observed, besides not having been influenced by the diets (Table VII) decreased in the final third of pregnancy.Despite this, the values were within the reference range for the species (Contreras et al. 2000), thus discarding a possible deficit in protein metabolism, which is common due to increased metabolic demand during pregnancy (Radin et al. 2015), which could be observed with the greatest concentration of urea in the blood, but the amount of this metabolite decreased with the passage of the pregnancy (Table VII).
In relation to the effect of the stages of pregnancy, Kalhan (2000) asserts that it is common for decrease of the concentration of urea in pregnant, because this reduction is not only a result of increased glomerular filtration, but also due to a reduction in the hepatic synthesis.With the increase of progesterone and estrogen concentrations, the activity of the enzymes decreases the urea cycle (Ismail et al. 2008), otherwise you may be related to pregnancy toxemia.Since the reduction of albumin in the final third is related to the effect of dilution, due to the increase of other plasma proteins near the delivery (Rodríguez et al. 2009).
The enzymes related to liver metabolism, only the gamma-glutamyl transferase increased in the final third of the pregnancy.According to Radin et al. (2017), the levels of this enzyme increase physiologically next to childbirth.This behavior can be explained by fact that in ruminants there is transfer of this enzyme to fetuses.Therefore, there is an increase of this compound in maternal blood concentration, so that this enzyme is transferred to their offspring, mainly through the colostrum (Kaneko 2008).In this way, you can also that diet with DCC Ca(OH) 2 presented higher content of gamma-glutamyl transferase, which can passive immunity in calves (Silva et al. 2007).

CONCLUSION
During the final third of gestation, the Saanen goats and Anglo Nubian diminish considerably the intake.The detoxified castor cake by alkaline solutions in replacement of soybean meal proved to be a viable alternative in the feeding of goats in pregnancy, because it does not affect the functionality of the liver and kidney function and the nitrogen balance.Diets formulated with detoxified castor by sodium hydroxide decrease the intake of dry matter and nutrients, but without negatively affecting the weight of the offspring.

Table I . Chemical composition of the ingredients used for the preparation of the experimental diets. Item (g/kg DM) Ingredients Tifton 85 hay Ground corn Soybean meal DCC 1 Ca(OH) 2 DCC NaOH
1DCC: Detoxified castor cake; 2 Ca(OH) 2 DCC: 0.90 g/kg Na DM and 2.25 g/kg Ca DM; NaOH DCC: 29.20 g/kg Na DM and 0.63 g/kg Ca DM.3Corrected for ash and protein.

Table II .
Proportion of ingredients and chemical composition of the experimental diets.

Table III .
Dry matter intake and nutrients of goats fed with detoxified castor cake (DCC) at different stages of pregnancy.Soybean meal; 2 MSE: Mean standard error.Averages followed by common lowercase letters in the lines and by uppercase letters in the columns do not differ from one another according to the Tukey test at 5% significance.

Table IV .
Weight and body condition score of pregnant goats fed diets containing detoxified castor (DCC) by different alkali replacing soybean meal during gestation and weight of goats generated.Mean standard error.Averages followed by common lowercase letters do not differ from one another according to the Tukey test at 5% significance.

Table VI .
Nitrogen balance of goats fed with detoxified castor cake (DCC) at different stages of pregnancy.Mean standard error.Averages followed by common lowercase letters in the lines and by uppercase letters in the columns do not differ from one another according to the Tukey test at 5% significance.

Table VII .
Blood Parameters of goats fed with detoxified castor cake (DCC) at different stages of pregnancy.Soybean meal; 2 MSE: Mean standard error.Averages followed by common lowercase letters in the lines and by uppercase letters in the columns do not differ from one another according to the Tukey test at 5% significance.