ANIMAL SCIENCE

Effect of diet supplementation with meal of Agave tequilana stems on hematological indicators and blood biochemistry of fattening rabbits

Maidelys Iser¹  , M. Valdivié²  , D. Sanchez³  , M. Rosales³  , D. Más⁴  , Y. Martínez^{5  *} 

¹Universidad de Granma, Bayamo, Granma, Cuba

²Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba

³Universidad de Guadalajara, Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA), Departamento de Producción Animal, Guadalajara, Jalisco, México

⁴Laboratorio de Nutrición Animal, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro 76230, México

⁵Escuela Agrícola Panamericana, Valle de Yeguare, San Antonio de Oriente 96, Honduras

ABSTRACT

To evaluate the effect of diet supplementation with Agave tequilana stem meal on indicators of blood health and blood biochemistry of fattening rabbits, 64 animals of New Zealand x California breed, with 35 days old and for 60 days, were located according to a completely randomized design, with eight repetitions and two animals per repetition. At the end of the experiment, six rabbits were taken per treatment for laboratory analysis. Treatments consisted of a basal diet and three diets with supplements of 0.5, 1.0 and 1.5% of stem meal of A. tequilana. Supplements up to 1.5% did not change the clinical blood indicators (P˃0.05) and all values were within the normal ranges for the category and animal species under study. Also, the daily use of A. tequilana stem meal reduced proportionally (P <0.05) the prepandrial values of urea nitrogen, glucose, total lipids, triacylglycerides, cholesterol, very low density lipoprotein, low density lipoprotein, high lipoprotein density and atherogenic index. In addition, serum creatinine concentration decreased with the 0.5 and 1.5% supplementation (P <0.05) compared to the basal diet and the treatment with 1.0%, although the latter treatments were different among them. This natural product based on A. tequilana stem meal, used as a diet supplement during the productive life of fattening rabbits (95 days), indicated hypoglycemic and lipid-lowering properties, without affecting blood health indicators.

Key words: Agave tequilana; blood biochemistry; health indicators; rabbit fattening

INTRODUCTION

Modern cuniculture production is characterized by high productive intensity, in which animals are subjected to different stress situations. These, in turn, cause imbalances in the intestinal microbiota, with the development of pathogenic microorganisms, immunosuppression, inefficient food conversion, high mortality and decreased zootechnical response (^{Jiya et al. 2018}). For the above reasons, for decades, growth-promoting antibiotics have been used (^{Liu et al. 2016}). Nowadays, functional foods and nutraceutical products have been used for replacing or decreasing the indiscriminate use of these sub-therapeutic antibiotics (^{Ebrahimi et al. 2016}). Specifically, prebiotics are considered as a viable alternative from a technical, economic and biological point of view due to the security of their inclusion and their zero residuality (^{Liu et al. 2017}).

In this sense, plants belonging to the Agave genus have been considered as medicinal sources due to their high concentration of fructans and other chemical substances with anti-inflammatory action, so their use in animal diets promotes production and health of the host (^{Iser et al. 2016a} and ^{Padilla et al. 2018}). ^{Sánchez et al. (2015)} and ^{Chavez et al. (2019)} showed that the addition of Agave tequilana stem meal (HTAT) in pig and poultry diets increased the population of cecal beneficial bacteria and modified the harmful serum lipids, respectively. Also, previous results showed that the use of up to 1.5% of HTAT increased the productive performance and quality of fattening rabbit carcass (^{Iser et al. 2016b}).

However, there is little scientific evidence that refers to the effect of this natural product (HTAT) on blood hematological and biochemical analyzes of rabbits. According to ^{Iser et al. (2016a)} these blood indicators are particularly affected by diet and nutraceutical additives, and it also allows to quickly assess the possible beneficial or harmful effects of the use of new natural products in animals. The objective of this study was to evaluate the effect of diet supplementation with Agave tequilana stem meal on the hematological indicators and blood biochemistry of fattening rabbits.

MATERIALS AND METHODS

Experimental location. This study was carried out at the Estación de Pruebas de Comportamiento of the facilities of the Instituto de Biotecnología Animal, “Rancho Cofradía” of the Universidad de Guadalajara, located at km 7.5 of the road to San Isidro Mazatepec, Tlajomulco de Zúñiga municipality, Jalisco, Mexico.

Animals, diets and experimental treatments. A total of 64 male rabbits of the New Zealand x California crossing with 35 days of age, with initial live weight of 769 ± 2 g were used. They were identified by marking with indelible ink and two rabbits/cage were located for 60 days, according to completely randomized design with four treatments, eight repetitions and two animals per repetition. For sample size, it was taken into ccount the reports of ^{de Blas and Mateos (2010)}.

Four treatments were used: T1: basal diet (DB) as a control; DB + 0.5% of Agave tequilana stem meal (HTAT); DB + 1.0% of HTAT and DB + 1.5% of HTAT. A. tequilana stem meal was supplied by the Centro Universitario de Ciencia Biológicas y Agropecuarias (CUCBA), Universidad de Guadalajara, Jalisco, Mexico. According to the manufacturer (CUCBA), this natural product contains 94.10% of dry matter, 2.17% of crude protein, 0.34% of ether extract, 4.01% of ash, 79.65% of total carbohydrates and 43.24% of fructans. Results of ^{Sánchez et al. (2015)} were used for selecting supplementation levels of this natural product in diets for rabbits.

The basal diet for rabbits was prepared according to the nutritional requirements indicated by ^{de Blas and Mateos (2010)}. The commercial concentrate was produced in an industrial feed mill, with 2.5 mm grain size, as established for this animal species (^{de Blas and Mateos 2010}). Ingredients and nutritional contributions of the diet are shown in table 1.

Table 1 Ingredients and nutritional contributions of the diet for fattening rabbits (35 to 95 days old) 

Ingredients	Content (%)
Wheat straw	17.4
Alfalfa hay	12.0
Barley grain	19.0
Wheat bran	24.0
Sunflower meal (crude protein 30 %)	12.0
Soy bean meal (crude protein 44 %)	11.0
Soy bean oil	2.88
Sodium chloride	0.50
Monocalcium phosphate	0.50
L-lysine	0.09
L-threonine	0.08
DL-methionine	0.05
Premix^1	0.50
Calculated contributions (%)
Crude protein	16.7
Digestible energy (MJ/kg)	9.92
Neutral detergent fiber	29.1
Lysine	0.77
Methionine + cystine	0.59
Threonine	0.65
Ashes	5.37

¹Each kg contains: vitamin A 12 000 IU, vitamin D3 2000 IU, vitamin B2 4160 IU, Niacin 16 700 IU, pantothenic acid 8200 IU, vitamin B6 3420 IU, folic acid 0.980 g, vitamin B12 16 mg, vitamin K 1560 IU, Vitamin E 16 g, BHT 8.5 g, cobalt 0.750 g, copper 3.5 g, iron 9.86 g, manganese 6.52 g, sodium 0.870 g, zinc 4.24 g, selenium 6.67 g

Experimental conditions. Rabbits were located in metal cages 76 x 76 x 45 cm long, wide and high, respectively. Food was supplied ad libitum twice a day (8:00 am and 4:00 pm) in galvanized tubular feeders and availability adjustments were made, based on the difference between supply and rejection. Water was also offered ad libitum in automatic nipple water troughs located in the cages. No medications were offered, nor veterinary therapeutic care was provided during the entire experimental stage. However, the health status of animals was daily checked (presence of diarrhea, vomiting, depression, sneezing, tearing and coughing) and possible deaths were recorded.

Hematological indicators and blood biochemistry. At the end of their productive life (95 days old), six rabbits were randomly selected per treatment and sacrificed by the jugular vein bleeding method, at the slaughterhouse of Instituto de Biotecnología Animal Rancho “Cofradía” de la Universidad de Guadalajara, Jalisco, Mexico. Before the sacrifice, the animals were fasted for 12 hours, only with water ad libitum (^{Iser et al. 2016b}).

Out of the sacrificed rabbits per each treatment, 10 mL of blood were taken. To obtain blood serum, samples were let to rest for one hour in 20 mL vials, then centrifuged (Eppendorf centrifuge) at 10,000 rpm and 20 °C for 25 min. To obtain blood plasma, blood was deposited in 2 mL tubes and sodium heparin was added at a ratio of 2: 1. Both samples were stored at -20 °C, until further analysis in the laboratory.

In blood serum, colorimetric methods were used for determining glucose using the LabAssay ™ Glucose kit (Wako Pure Chemical Industries Ltd., Chuo-Ku, Osaka, Japan), creatinine using the Creatinine-PAP test kit (Boehringer Mannheim GmbH, Germany), urea nitrogen with the Enzymatic Kit Urea-ammonium (Boehringer Manheim GmbH, Germany), triglycerides by the MAK266-1KT Triglyceride Quantification Colorimetric / Fluorometric Kit (Sigma-Aldrich St. Louis, MO, USA), total cholesterol with the MAK043-1KT Cholesterol Quantitation Kit (Sigma-Aldrich St. Louis, MO, USA), for very low density lipoproteins (VLDL) and high density lipoproteins (HDL) with the Quantitation Kit (Sigma-Aldrich St. Louis, MO, USA) and the MAK045-HDL kit, respectively, and total lipids were determined with the MAK055-total lipids-CAL kit, and a Humalyzer 2000 ultraviolet spectrophotometer (Germany) was used. To determine the concentration of low density lipoprotein (LDL) and atherogenic index (AI), the formulas of ^{Friedewald et al. (1972)} and ^{Dobiášová (2004)} were used, respectively:

${\mathrm{C}}_{\mathrm{L}\mathrm{D}\mathrm{L}}={\mathrm{C}}_{\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{m}\mathrm{a}}-C_{\mathrm{H}\mathrm{D}\mathrm{L}}-\frac{TG}{5} IA=\frac{\mathrm{L}\mathrm{D}\mathrm{L}}{\mathrm{H}\mathrm{D}\mathrm{L}}$

In blood plasma, leukocytes were analyzed by blood smear and Giemsa dye; hemoglobin, by the Hemotest method; hematocrit according to ^{Wintrobe (1962)} and total proteins by Biuret (^{Gornall et al. 1949}), analyzed by means of a Shimadzu UV-Visible 160 A spectrophotometer (Japan). Erythrocytes and platelets were determined by Neubauer chamber method and by the automatic blood count method.

Mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MHC) and mean corpuscular volume (MCV) were determined by the following formulas:

$\mathrm{M}\mathrm{C}\mathrm{H}\mathrm{C}\mathrm{ }=\mathrm{ }\frac{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{H}\mathrm{b}\mathrm{ }(\mathrm{g}/100\mathrm{m}\mathrm{L})\mathrm{ }\mathrm{*}\mathrm{ }100}{\text{Ht (\%}\text{)}} \mathrm{M}\mathrm{C}\mathrm{V}\mathrm{ }=\mathrm{ }\frac{ \mathrm{H}\mathrm{t}\mathrm{ }\left(\mathrm{\%}\right) * 10}{\text{No. er}\text{y}\text{t}\text{h}\text{roc}\text{y}\text{t}\text{e}\text{s (mill}\text{i}\text{ons/}{mm}^3\text{blood)}} \mathrm{M}\mathrm{C}\mathrm{H}\mathrm{ }=\mathrm{ }\frac{\text{(}\mathrm{H}\mathrm{g}\mathrm{b}\mathrm{ }\text{*}\text{10)}}{\text{leucocyt}\text{e}\text{s}}$

Hematological studies were carried out in the laboratory of the Centro de Investigación de Patología Animal, Departamento de Medicina Veterinaria, División de Ciencias Veterinarias de la Universidad de Guadalajara, Jalisco, Mexico.

Statistical analysis. Data was processed using one-way analysis of variance (Anova) in a completely randomized design. In the necessary cases, ^{Duncan (1955)} test was applied. The statistical software SPSS version 20.0.1. 2012 was used.

RESULTS AND DISCUSSION

Table 2 shows that hematological indicators of fattening rabbits were not altered (P> 0.05) when up to 1.5% of HTAT was added in the diets. They were also within the normal physiological ranges for the species and breed under study (^{Giusti et al. 2012}). Currently, hematological indicators are taken as health indicators in humans and animals. A variation of them may indicate bacterial, viral, parasitic and fungal infections, as well as poisoning, dehydration and blood clotting problems (^{El-Ratel et al. 2017}). These results confirm that animals were maintained throughout the experimental period without visible clinical symptoms.

Tabla 2 Effect of diet supplementation with Agave tequilana stem meal on hematological indicators of fattening rabbits (95 days old) 

Items	Agave tequilana stem meal (%)
	0	0.5	1.0	1.5	SE±	P Value	R Value^1
Erythrocytes (millions/mm^3)	6.53	6.42	6.44	6.48	0.27	0.093	4.5-7.0
Leucocytes (thousands/mm^3)	6.69	6.45	6.46	6.40	0.46	0.209	6.0-9.3
Hb (g/dL)	13.44	13.90	13.52	13.32	0.89	0.312	8-15
Ht (%)	40.50	41.60	40.70	40.34	0.98	0.266	30-50
MCH (Pg)	20.08	21.55	20.92	20.81	0.88	0.868	19-30
MCV (fL)	62.02	64.79	63.19	62.25	1.15	0.744	40-80
MCHC (g/dL)	33.18	33.41	33.22	33.01	0.27	0.206	32-38
Platelets (thousands/mm^3)	549.20	547.40	550.40	555.40	3.87	0.208	400-700
TP (gm/dL)	7.28	7.24	7.60	7.66	0.22	0.448	5.2-7.8

MCH: mean corpuscular hemoglobin; MCV: mean corpuscular volume, MCHC: mean corpuscular hemoglobin concentration; TP: total protein; Ht: hematocrits; Hb: hemoglobin.

^{1Giusti et al. (2012)}

In general, the introduction of a new food and/or additive in animal diets, especially those that do not have enzymatic affinity cause changes in polymorphonuclear leucocytes (neutrophils and eosinophils), by activating the immune system to eliminate the foreign body and/or the possible toxic and allergenic compound (^{Ghasemi et al. 2010} and ^{Giusti et al. 2012}). HTAT with a high fructan concentration and the presence of secondary metabolites (^{Chávez et al. 2019}) did not cause adverse symptoms with its nutraceutical addition in the diet of rabbits and did not decrease the defenses (white blood cells). The hemoglobin value indicates that the addition of HTAT may not have affected iron absorption, as, according to ^{Martínez et al. (2013)}, the tannins found in HTAT (^{Velázquez et al. 2019}) prevent the absorption of this mineral, which induces iron deficiency anemia.

In addition, the hematocrit value reflects that animals were subjected to suitable hydration conditions, this indicator increases due to a hemoconcentration due to water deficit (^{Martínez et al. 2013}). In this sense, ^{Iser et al. (2016a)} found similar results when supplementing up to 1.5% of Agave fourcroydes stem meal in rabbit diets. This demonstrates that the daily use of HTAT in the diet (up to 1.5%) during the productive life of rabbits does not cause adverse reactions.

Table 3 shows that the diet supplementation of three levels of Agave tequilana stem meal statistically modified (P <0.05) all the biochemical (blood) indicators measured in fattening rabbits. The use of this natural product (HTAT), up to 1.5% in the diet, proportionally reduced (P <0.05) urea nitrogen, glucose, total lipids, triacylglycerides, cholesterol, VLDL, LDL, HDL and AI. In addition, serum creatine concentration decreased with HTAT, mainly with T2 and T4.

Table 3 Effect of diet supplementation with Agave tequilana stem meal on blood biochemistry and atherogenic index of fattening rabbits (95 days old) 

Items (mg/dL)	Agave tequilana stem meal (%)
	0	0.5	1.0	1.5	SE±	P Value
Urea nitrogen	39.20ª	34.00^b	30.52^c	25.22^d	0.74	˂0.001
Glucose	129.80ª	105.50^b	95.20^c	81.60^d	1.42	˂0.001
Creatinine	0.98ª	0.76^c	0.83^b	0.78^c	0.02	˂0.001
TL	585.20a	553.80^b	539.00^c	512.00^d	2.48	˂0.001
TAG	180.60ª	142.60^b	144.40^b	133.68^c	1.89	˂0.001
Cholesterol	213.60ª	185.60^b	180.60^b	172.40^c	2.40	˂0.001
VLDL	41.80ª	36.40^b	31.00^c	31.20^c	0.87	˂0.001
LDL	184.60ª	85.60^b	86.00^b	67.00^c	1.88	˂0.001
HDL	65.44^ab	67.20ª	63.80^b	52.20^c	0.91	˂0.001
AI	2.82^a	1.27^b	1.34^b	1.28^b	0.03	˂0.001

^a,b,c,d Means with different letters differ at P<0.05 (^{Duncan 1955})

TL: total lipids; TAG: triacylglycerides, HDL: high density lipoproteins, LDL: low density lipoproteins, VLDL: very low density lipoproteins and AI: atherogenic index

According to ^{Bossart et al. (2001)}, blood urea nitrogen is the final product of protein catabolism and it is often used as an indicator of renal and hepatic function, as well as a measure of relative hydration status of animals. A decrease in this biochemical indicator (table 3) with the addition of HTAT indicated that this product increases protein efficiency of the diet, by controlling urea synthesis and liver hydration (^{Li et al. 2011}).

On the other hand, serum creatinine is one of the main blood biochemical indicators. This indicator is associated with kidney functioning, and a high concentration may indicate acute, chronic kidney diseases and kidney failure (^{Kin et al. 2016}). Although rabbits remained without apparent diseases, a decrease in this biochemical indicator with HTAT by 0.22 mg/dL could be the starting point for future research in animals and humans (table 3).

Many reports indicate that foods rich in fructans, such as agaves, reduce serum glucose, by increasing the secretion of glucagon-like peptide 1 (GLP 1) in the endocrine L cells of the intestine. In addition, they stimulate insulin secretion from pancreatic β cells and inhibition of secretion of glucagon from α cells (^{Tappenden et al. 2003} and ^{Urías et al. 2008}). ^{Hernández et al. (2016)} found a hypoglycemic effect when using Agave tequilana extracts in the diets of obese laboratory mice.

Although there are contradictions about the importance of reducing serum glucose in animals, especially since glucose is an important energy source for several metabolic processes, according to ^{Yin et al. (2010)}, a decrease in serum glucose suggests a high efficiency in the use of glucose and protein. In addition, ^{Delzenne and Williams (2002)} reported that a reduction of this carbohydrate favors the decrease of blood circulation of harmful lipids. It was demonstrated that HTAT has an important hypoglycemic effect, since it decreased serum glucose by 48.2 mg/dL with respect to the control.

Since the 20th century, rabbits have been used as a model to study the influence of food, additives or drugs on the reduction of harmful lipids (^{Niimi et al. 2016}). A relevant fact in this study is that the addition of HTAT in the diet of rabbits has an important hypolipidemic effect, due to the reduction of prepandial serum harmful lipids. According to ^{Liu et al. (2016)}, a combination of several physiological, biochemical and microbiological mechanisms is necessary for the reduction of these lipids. Research has shown that HTAT with high concentration of fructans and some beneficial secondary metabolites (^{Chavez et al. 2019}) improves the growth of cecal lactic acid bacteria (LAB) and, in turn, the intestinal health of monogastric animals (^{Sánchez et al. 2015} and ^{Chávez et al. 2019}), which could influence on the decrease in serum cholesterol by 41.2 mg / dL with respect to control.

It is known that LABs increase VFA production, which decreases the enzymatic activity of HMG-CoA that synthesizes endogenous cholesterol. This causes a decrease in the circulation of this lipid due to lower intestinal absorption (^{Barclay 2010}). In this sense, ^{Shehata et al. (2016)} demonstrated that probiotic strains are capable of assimilating the cholesterol present in the medium and lowering these blood levels. In this sense, ^{Hernández et al. (2016)} found that HTAT has a hypocholesterolemic effect in mice with dyslipidemia. Also, the use of compounds such as inulin, fructans and other prebiotics in diets for non-ruminant species, reduce this lipid in blood (^{Pérez et al. 2017}).

Generally, a decrease in circulating cholesterol affects a lower concentration of LDL, with high content of esterified cholesterol and low in apolipoprotein (^{Martínez et al. 2015}). In addition, ^{Navab et al. (2001)} report that a decrease of serum cholesterol increases the hepatic expression of LDL receptor and the uptake of these lipoproteins by the liver. Perhaps this caused a decrease of LDL by 117.6 mg/dL, compared to the control.

In addition, triacylglycerides decreased due to the effect of HTAT by 46.92 mg/dL, compared to the control. According to ^{Clarke et al. (2002)}, foods rich in fructans and essential fatty acids induce triglyceridemia, since they decrease liver lipogenesis and stimulate the oxidation of fatty acids in liver and muscle. Likewise, a reduction in the circulation of VLDLs was found, consisting mainly of this simple lipid (^{Bennett et al. 1995}). Little is known about the action of nutraceutical additives in VLDL. However, some studies state that VLDL have a direct relationship with C-reactive protein, which is activated by innate immunity (^{Shrivastava et al. 2015}). It seems that the circulation of this lipoprotein will depend on the immunological status of rabbits, however, other studies are necessary to corroborate this hypothesis.

In general, a lower concentration of LDL increases HDL, something that did not occur in this experiment. This natural product (HTAT) reduces both serum lipoproteins, although with greater emphasis on LDL, which has the highest percentage of esterified cholesterol and harmful effect (^{Martínez et al. 2015}). However, HTAT reduced atherogenic index by 1.01 compared to basal diet. This indicator showed the effectiveness of HTAT for the decrease in harmful lipids. Currently, there are no indicators of atherogenic indexes for rabbits. However, a decrease in this index should favor the health of these growing animals.

This natural product based on Agave tequilana stem meal, used as a diet supplement during the productive life of fattening rabbit (95 days), showed hypoglycemic and hypolipidemic properties, without affecting hematological health indicators. This article confirms that Agave tequilana stem meal is an effective natural product to be used as a diet supplement in fattening rabbit diets.

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Received: April 24, 2019; Accepted: July 11, 2019

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