Use of propolis as a supplement in the diet of broiler chickens to increase shelf-life of breast muscle

ABSTRACT

Total antioxidant capacity (TAC) and physicochemical characteristics of meat were evaluated after supplementing different doses of propolis in broiler chickens’ diets at: 15, 30, 45 and 0 mg propolis/kg feed for Pr15, Pr30, Pr45 and Pr0, respectively. Two hundred one-day-old chickens were fed for 42 d and sacrificed. Breast meat samples were used for physicochemical determinations. Oxidative capacity was measured on days 0, 1, 3, 5, 7 and 9 of storage. There were no differences (P > 0.05) between the supplementation systems in either pH, luminosity index (L*), red (a*), yellow (b*), drip loss, cooking yield, protein, ash or moisture. Meat from chickens fed the highest propolis dose (Pr45) presented the lowest oxidative capacity (P < 0.01) throughout the study, whereas the control group (Pr0) showed the highest (P < 0.01) values until day 5. In conclusion, propolis in the chickens’ diet may decrease oxidative compounds and has no influence on the physicochemical characteristics of meat.

RESUMEN

Se evaluó la capacidad antioxidante total y las características fisicoquímicas de la carne cuando se suplementaron diferentes dosis de propóleos en la dieta de los pollos de engorde (mg propóleo/kg alimento): 15, 30, 45 y 0, para Pr15, Pr30, Pr45 y Pr0, respectivamente. Doscientos pollos de un día de edad fueron alimentados por 42 d y sacrificados. Se utilizaron muestras de carne de pechuga para determinaciones fisicoquímicas. La capacidad oxidativa se midió en 0, 1, 3, 5, 7 y 9 días de almacenamiento. No hubo diferencias entre los sistemas de suplementación (P > 0.05) con respecto al pH, el índice de luminosidad (L*), el rojo (a*), el amarillo (b*), la pérdida de goteo, rendimiento de cocción, proteínas, cenizas y humedad. La carne de pollo en la dosis más alta de propóleo (Pr45) presentó la capacidad oxidativa más baja (P > 0.01) a través del estudio; mientras que los valores más altos (P > 0.01) hasta el día 5 eran para el grupo de control (Pr0). Se concluye que el propóleo en la dieta de los pollos puede disminuir los compuestos oxidativos y no influye en las características fisicoquímicas de la carne.

KEYWORDS:

• Chicken meat
• nutritional composition
• propolis
• storage

PALABRAS CLAVE:

• carne de pollo
• composición nutricional
• propóleo
• almacenamiento

1. Introduction

Meat preservation has been carried out since prehistoric times; man has searched for strategies to increase food shelf-life for its consumption. Examples of these processes are salting, smoking, and freezing. A current challenge for the meat industry is to keep the meat fresh as long as possible, without losing its organoleptic and/or nutritional properties. Shelf-life is the period in which a food stored under pre-established optimal conditions maintains safety and acceptable sensory characteristics for the consumer (Eilert, 2005).

Food deterioration is influenced by factors such as the growth of microorganisms, amount and type of light received, and by lipid and pigment oxidation (Cortinas et al., 2005; Isaza et al., 2013). When meat is in contact with oxygen during storage, lipids begin to oxidize, causing the formation of unpleasant flavours and odours that affect the quality and acceptance of meat by the consumer (Venegas & Pérez, 2009).

The continuous increase in animal production implies long-term storage of these products before their distribution and consumption, during which the changes that cause rancidity in meat products can develop, limiting their durability (Venegas & Pérez, 2009). To solve this problem, the meat industry has implemented the use of antioxidants, which are substances of natural or artificial origin that extend food shelf-life, protecting it from deterioration caused by oxidation such as fat rancidity (Zamora, 2007). However, the use of synthetic antioxidants has been questioned because they can be harmful to health (Schwarz et al., 2001; Valenzuela & Nieto, 1996). Moreover, natural antioxidants have been used directly in meat, with the disadvantage that may affect colour and flavour (Apeleo, 2018). For this reason, alternatives have been sought using natural antioxidants directly in animal diets and exploring their influence on the meat they produce (Córdova & Iglesias, 2016; R. D. Vargas-Sánchez et al., 2019; Valenzuela & Nieto, 1996).

Due to its own characteristics, propolis is an important substance that can be fed to domestic animals to strengthen their immune system (Vargas et al., 2013) and to increase meat shelf-life (Muñoz et al., 2011), because it contains ethanolic and flavonoid extracts, which are compounds that have a high antioxidant capacity and an inhibitory effect on metal ions (Gülçin et al., 2010). However, there is limited information on propolis use in animal feed and its influence on meat characteristics during storage. Therefore, the objective of the present research was to determine total antioxidant capacity during storage and physicochemical properties of broiler chicken meat, after different doses of propolis were supplemented in the birds’ diet.

2. Materials and methods

2.1. Study area

The study was conducted at the poultry farm of the College of Veterinary Medicine and Animal Science of the Autonomous University of Tamaulipas. Located at 23°44’06” N, 99°07’51” W, at an altitude of 340 meters above sea level, the weather is warm and sub-humid (Aw1), with an average annual temperature of 24 °C, and average monthly rain precipitation of 77.22 mm (INEGI Instituto Nacional de Estadística, Geografía e Informática, 2017).

2.2. Treatments

A total of 200 sex-mixed, Ross 308, one-day-old broiler chickens was used, distributed in 50 birds per treatment as follows: treatments Pr15, Pr30, Pr45 and Pr0; treatments Pr15, Pr30 and Pr45 were supplemented with 15, 30 and 45 mg of propolis/kg of feed, respectively, whereas Pr0 served as Control, without propolis in the feed. Propolis was added to the diet by direct spray using a manual pump with a capacity of 1.9 L (Heavy Duty Sprayer).

2.3. Feeding of the birds

Chicken management was similar to that carried out in a regional conventional commercial broiler chicken farm, in which water and feed are at free access, and lighting is given 24 h per day during the entire experiment. The feeding was divided into two phases: starter, from 1 to 21 days, and finisher, from 22 to 42 days of age. The feed was formulated according to the National Research Council, NRC (1994) tables for broiler chickens. Ingredients and chemical composition of starter and finisher diets are shown in Table 1.

Table 1. Ingredients and chemical composition of starter and finisher diets.

Tabla 1. Ingredientes y composición química de las dietas de inicio y finalizado

	Starter diet	Finisher diet
Ingredient
Sorghum grain, %	58.88	65.6
Soybean meal, %	33.7	26.38
Vegetable oil, %	3.42	3.69
Premix, %*	4	4
Pigment, %	0.0	0.33
Total	100	100
Chemical composition, %
Dry matter	89.80	89.70
Crude protein	21.30	18.20
Ether extract	2.36	5.34
Ash	5.71	6.08
Crude fiber	2.90	3.04
Nitrogen free extract	67.73	67.34
ME, kcal/kg	3040	3120

Treatments for starter and finisher diets were: Pr = 15 mg of propolis; Pr = 30 mg of propolis; Pr = 45 mg of propolis; Pr0 = Control.

*Premix for starter diets: monocalcium phosphate, calcium carbonate, common salt, growth promoter (BDM and 3-nitro), sodium monensin, mineral oil, ethoxyquin, retinol (vitamin A-acetate), cholecalciferol-D3 (vitamin D3), α-tocopheryl acetate (vitamin E), vitamin K3, riboflavin (vitamin B2), cobalamin (vitamin B12), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), choline chloride (vitamin B4), butylated hydroxytoluene (BHT). Calculated to contain: 21.40% Ca; 8.10% total P; 3.40% Na; 0.80% L-lysine chlorhydrate; and 4.15% DL-methionine.

*Premix for finisher diets: contain the same supplements of starter premix; however, the finisher premix was calculated to contain: 19.80% Ca; 3.70% total P; 3.70% Na; 4.33% L-lysine chlorhydrate; and 5.15% DL-methionine.

Los tratamientos para las dietas de inicio y finalizado fueron: Pr15 = 15 mg de propóleos; Pr30 = 30 mg de propóleos; Pr45 = 45 mg de propóleos; Pr0 = Control.

*Premezcla para dietas de inicio: fosfato mono calcio, carbonato de calcio, sal común, promotor del crecimiento (BDM y 3-nitro), monensina sódica, aceite mineral, etoxinquin, retinol (vitamina A-acetato), colecalciferol-D3 (vitamina D3), acetato de tocoferilo (vitamina E), vitamina K3, riboflavina (vitamina B2), cobalamina (vitamina B12), niacina (vitamina B3), pantotenato D-calcio (vitamina B5), cloruro de colina (vitamina B4), hidroxitolueno butilado (BHT). Calculado para contener: 21.40% Ca; 8.10% total P; 3.40% Na; 0.80% L-lisina clorhidrato; y 4.15% DL-metionina.

*Premezcla para dietas de finalizado: contiene los mismos suplementos de la premezcla de inicio; sin embargo la premezcla de finalizado fue calculada para contener: 19.80% Ca; 3.70% total P; 3.70% Na; 4.33% L-lisina clorhidrato; y 5.15% DL-metionina.

2.4. Sampling

At the time of slaughter, 10 birds were selected at random per treatment. Birds were sacrificed in a manner similar to that used in commercial conditions, following the norm established in Mexico (NOM 033-SAG/ZOO-2014), desensitizing the birds with an electric stun of 0.2 amps per bird for 5 seconds, and then cutting the jugular and carotid vessels for bleeding during approximately 5 minutes; the birds were then scalded at 59 °C for 3.5 min, plucked and eviscerated. After evisceration, the carcasses were cooled in ice with water for one hour. The half of the breast was subsequently removed, packed into vacuum bags, and stored in a freezer at −20 °C to later measure the total antioxidant capacity; while the physicochemical characteristics were analyzed 24 hours post mortem.

2.5. Proximate analysis of meat

Breast meat samples from the Pectoralis major muscles were subjected to proximate analysis (moisture, ash, and crude protein) according to official methods (Association of Official Analytical Chemists, 2000). The meat samples were thawed at 4 ^°C and then maintained at room temperature for the determinations described below.

2.6. 2.5. pH

A 5-g sample of muscle was placed in a 20-mL beaker with 10 mL double distilled water. The sample was homogenized by shaking for 2 min with a polytron (Mod PT-1200, Kinematica AG, Littau, Switzerland). The homogenized sample was used for pH determination with a desk pH meter (Mod pH 1100, Oaklon, Eutech Instruments, Singapore), which was calibrated with buffer solutions at pH 4.01 and 7.06. All samples were measured in duplicate.

2.7. Colour

Colour was measured on the surface of the muscle at three different points, following Hunter’s method, using a colourimeter (Mod CR-400/410, Minolta, Tokyo, Japan) for variables L* (luminosity), a* (red-green) and b* (yellow-blue) (CIE [Commission International de l’Eclairage], 1986).

2.8. Drip loss

A 30-g sample of the muscle was weighed, hung from a thread in plastic cups and stored at 4 °C. The samples were weighed 24 h later. Drip loss at 24 h post-mortem was expressed as the percentage of weight loss of the sample relative to its initial weight (Bautista et al., 2016).

2.9. Cooking yield

Another 30-g muscle sample was weighed and placed in a cook-resistant plastic bag in a water bath at 75 °C for one hour or until an internal temperature of 70 °C was reached; the sample was weighed again. The values were expressed as a percentage of the sample weight relative to their initial weight (Guerrero et al., 2002).

2.10. Total antioxidant capacities

Total antioxidant capacity (mmol/kg in dry basis) present in meat at a given time can be measured using the DPPH (1,1 diphenyl-2-picryl-hydrazyl) inhibition coefficient (Aguirre et al., 2015). Total antioxidant capacity was measured on days 0, 1, 3, 5, 7 and 9 of storage at ° 4 C. After each corresponding storage time, 5 g of finely chopped meat were placed in disposable test tubes with 5 mL of methanol. Subsequently, the tubes were shaken in vortex for 20 seconds and immediately placed in a water bath for 10 minutes at 30 °C; this step was repeated 3 times, so that the tubes completed 30 minutes in a water bath. At the end of the last repetition, tubes were left at room temperature for 10 minutes and centrifuged for 15 minutes at 277 g-force. A 0.5-mL aliquot of supernatant from the centrifuged tubes was transferred to amber test tubes containing 0.5 mL of methanol, and 3 mL DPPH, and kept in the dark for 30 minutes, after which absorbance was measured in a spectrophotometer, and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2 carboxylic acid) concentration in meat was calculated according to Serpen et al. (2012).

2.11. Statistical analysis

The variables of physicochemical characteristics of the meat were analysed in a completely random design, using the GLM procedure of the statistical package SAS 9.4. The means were obtained and compared using the adjusted Tukey’s test.

The total antioxidant capacity of the meat was analysed using the MIXED procedure of SAS 9.4 for a completely random design with repeated means. The model included the main effects of treatments, periods and the interaction of treatment by period. The appropriate covariance structure for the analysis was determined by testing different structures, and the covariance structure with negative or near-zero values was chosen according to the Akaike and Schwartz criteria (Herrera & Garcia, 2010).

3. Results

3.1. Chemical composition and physicochemical characteristics of chicken meat

The variables of chemical composition (moisture, crude protein, ash) and physicochemical characteristics (pH, colour characteristics, drip loss, cooking yield) of meat from the Pectoralis major muscle of birds fed diets with different levels of propolis showed no differences (P > .05; Table 2).

Table 2. Physicochemical composition of meat (Pectoralis major muscle) of broiler chickens fed diets with different propolis levels.

Tabla 2. Composición fisicoquímica de la carne (músculo Pectoralis mayor) de pollos de engorde alimentados en dietas con diferentes niveles de propóleo

	Pr15	Pr30	Pr45	Control	SEM	P
Number of samples	10	10	10	10
Chemical composition, %
Moisture	73.91	73.96	73.89	73.87	0.539	0.982
Crude protein	15.76	15.90	15.86	15.75	0.394	0.739
Ash	1.07	1.05	1.07	1.01	0.094	0.369
Physicochemical characteristics
pH	6.00	6.01	6.01	6.03	0.021	0.142
L*	49.51	49.13	49.11	49.94	0.912	0.161
a*	1.74	1.73	1.72	1.74	0.078	0.891
b*	13.99	13.88	14.11	13.82	0.304	0.184
Drip loss (%)	2.69	2.58	2.58	2.65	0.284	0.748
Cooking yield (%)	83.60	83.49	83.57	83.65	0.319	0.729

Treatments were: Pr = 15 mg of propolis; Pr = 30 mg of propolis; Pr = 45 mg of propolis; Pr0 = Control.

The samples of the breast were packed into vacuum bags and stored in a freezer at −20 °C to measure the physicochemical characteristics at 24 hours post mortem.

SEM = Standard error of mean; L* = luminosity index; a* = red index; b* = yellow index; P = Probability.

Los tratamientos fueron: Pr15 = 15 mg de propóleos; Pr30 = 30 mg de propóleos; Pr45 = 45 mg de propóleos; Pr0 = Control.

Las muestras de la pechuga se empacaron en bolsas de vacío y se almacenaron en un congelador a − 20 °C para medir las características fisicoquímicas a las 24 horas post mortem.

SEM = Error estándar de la media; L* = Índice de luminosidad; a* = índice rojo; b* = índice amarillo; P = Probabilidad.

3.2. Inhibition coefficient (DPPH) in chicken meat

This coefficient varied between treatments and among the different day measurements (Table 3). Treatments Pr15 and Pr30 showed no difference (P > .05) in the value of total antioxidant capacity from day zero to day five; however, Pr15 showed a higher value than Pr30 on day seven and the opposite effect was observed on day nine (P < .01). On all sampling days, Pr45 (highest propolis dose) presented the lowest values (P < .01) of the oxidative components formed in meat. Moreover, Pr0 (control, with no propolis) had the largest (P < .01) total antioxidant capacity from day zero to day five; that is, there was a greater formation of free radicals, but on days seven and nine it had intermediate values compared to the other treatments.

Table 3. Mean DPPH inhibition coefficients (mmol/kg dry basis) in meat from chickens fed with propolis, according to treatment (T) and period (P).

Tabla 3. Medias de coeficientes de inhibición de DPPH (base seca mmol/kg) en carne de pollos alimentados con propóleo, según el tratamiento (T) y el período (P)

		Days of storage (period)						Treatment		Treatment*Period
Treatments	n	0	1	3	5	7	9	SEM	P	SEM	P
Pr15	10	3.46^bz	3.73^by	4.16^bx	5.47^bu	5.35^av	4.78^bw	0.037	<0.01	0.037	<0.01
Pr30	10	3.43^bz	3.64^by	4.26^bx	5.49^bu	5.14^bv	5.24^aw	0.037	<0.01	0.037	<0.01
Pr45	10	3.00^cz	3.35^cy	3.83^cx	4.80^cu	4.69^cv	4.03^dw	0.037	<0.01	0.037	<0.01
Pr0	10	4.44^ax	4.15^az	4.54^aw	7.45^au	5.22^bv	4.39^cy	0.037	<0.01	0.037	<0.01

Treatments were: Pr = 15 mg of propolis; Pr = 30 mg of propolis; Pr = 45 mg of propolis; Pr0 = Control, without propolis.

n = number of samples per treatment

SEM = Standard error of mean; P = Probability

The samples of the breast were packed into vacuum bags and stored in a freezer at −20 °C to later measure the total antioxidant capacity, considering the days of storage.

^a,b,c,dIn a column, means with different literals are different (P < 0.01)

^u,v,w,x,y,zIn a row, means with different literals are different (P < 0.01)

Los tratamientos fueron Pr15 = 15 mg de propóleos; Pr30 = 30 mg de propóleos; Pr45 = 45 mg de propóleos; Pr0 = control, sin propóleos.

n = número de muestras por tratamiento

T = tratamiento

T*P = interacción de tratamiento por periodo; SEM = Error estándar de la media; P = Probabilidad.

Las muestras de la pechuga se empacaron en bolsas de vacío y se almacenaron en un congelador a − 20 °C para medir posteriormente la capacidad antioxidante total, teniendo en cuenta los días de almacenamiento.

^a,b,cEn una columna, las medias con literales diferentes son diferentes (P < 0.01)

^u,v,w,x,y,zEn una fila, las medias con literales diferentes son diferentes (P < 0.01)

A treatment by time interaction was found (P < .01). There was a gradual increase in the total antioxidant capacity in all treatments, peaking on day five, later the oxidative capacity decreased, that is, the free radicals formation on days seven and nine decreased (Table 3).

4. Discussion

4.1. Chemical composition and physicochemical characteristics of broiler chicken meat

The amount of propolis in the chickens’ diet did not change the nutritional composition of meat between treatments. The moisture and protein values found in this study are similar to 68% and 18%, respectively, reported by Kumar and Aalbersberg (2006). On the other hand, Jones (1986) pointed out that the nutritional composition of meat can be modified through the diet; for example, fat level can be reduced in meat by decreasing the amount of energy or increasing the amount of protein in the diet. This effect was not shown in this study, because the diet received by all birds was similar and only the amount of propolis varied between the diets.

Based on the final pH values of meat in the four treatments of the current study, they are classified as normal meats. According to the classification of Qiao et al. (2001) and Swatland (2008), normal meats exhibit a pH value of 5.9 ± 0.12, a value close to that obtained in this study. Pale, soft and exudative meats should show less than 5.8 and dark, firm and dry meats should exhibit pH greater than 6.3.

The final pH value influences the colour variables (L*, a* b*); chicken meat with a low pH is pale, because it scatters more light back to the observer than meat with a high pH, which transmits more light into its depth. The pH therefore affects the myofibrillar refraction within the muscle fibres; colour is the main characteristic that the consumer observes at the time of purchase of the product (Swatland, 2008).

On the other hand, the level of propolis added in the diet, by not altering the final pH in meat, did not modify the colour variables. The values obtained in the present study are similar to those that classify the meat as normal, which has a desirable appearance for the consumer with an L* value between 43 and 49 (Barbut et al., 2005; Bautista et al., 2016), a b* index from 12.54 to 14.93 (Schneider et al., 2012), and an a* index of 2.62, and a firm, non-exudative, pink-red appearance (Bautista et al., 2016; Yue et al., 2010).

The dietary propolis level had no effect on drip loss and cooking yield. This can be explained because the final pH of the meat is considered a normal meat and has a direct effect in determining the number of reactive groups of proteins and their ability to bind water (Bowker & Zhuang, 2015); when pH is low (≤ 5.8) there is a higher percentage of drip loss, and lower water retention, and when it is high (≥ 6.3) the drip loss is lower, with a higher water retention capacity (Qiao et al., 2001).

4.2. DPPH inhibition coefficient in broiler chicken meat

The total antioxidant capacity in the chicken meat during storage time increased in all treatments. However, the control treatment (without propolis) showed the greatest total antioxidant capacity from day zero to day five. This behaviour can be explained by the process of lipid and protein oxidation in meat that initiates spontaneously, in which the unsaturated fatty acids of triglycerides or any other lipids react with molecular oxygen through a chain of reactions of free radicals, and are initiators and promoters of more oxidation, and therefore there is a greater formation of free radicals (Venegas & Pérez, 2009), and in turn a greater oxidative capacity. Exposure to oxygen, light sources, or other factors that influence the oxidation process (Choe & Min, 2006) allows oxidative compound formation; the amounts may vary according to the type of meat and its composition of fatty acids. This process is carried out during the shelf-life period, during its marketing at a temperature from −18 to 4 °C, as established by the Official Mexican Directive NOM-194-SSA1-2004, which was complied with during this study.

On the other hand, after death and following rigor mortis, when the muscle is already considered meat (Warner, 2016), if it is not stored at a temperature below −18 °C to stop the oxidative processes (Haščík et al., 2014), the oxidative reactions of lipids in the muscle continue increasing in the intracellular phospholipid fraction at the membrane level. These reactions are affected by the polyunsaturated fatty acid concentration, main substrates in the oxidation process, and by the presence of elements such as iron which have the ability to capture a proton of an unsaturated fatty acid; thus, these oxidative compounds increase with storage time (Laguerre et al., 2007). These biochemical reactions explain why the total antioxidant capacity increased in all treatments until day 5 of study and then decreased, maybe because less free radical formation.

Moreover, the oxidation process already occurs in the live animal, due to an imbalance between the production of reactive oxygen and the animal’s defence mechanism against oxidative stress; this process continues in muscle and fatty tissues after death, affecting the shelf-life of meat and meat products.

The use of synthetic and natural antioxidants in poultry feeding can delay the process and increase shelf-life of meat. Smet et al. (2008) reported that the use of natural antioxidant extracts such as natural tocopherols, rosemary, green tea, grape seed and tomato in the feeding of birds reduced lipid oxidation in fresh meat; in turn the increase from 100 to 200 mg/kg of natural tocopherols, rosemary, green tea, and tomato significantly reduced lipid oxidation from day 3 to day 10 of storage. Meanwhile, Sohaib et al. (2017) added quercetin dihydrate, an antioxidant flavonol compound derived from plants at levels of 25, 50 and 100 mg/kg of meat, in combination with a-tocopherol at a rate of 100 and 200 mg in chicken meat; they observed that as the dose increased, oxidation significantly decreased, by reducing the generation of malonaldehydes and total carbonyls from day 0 to day 7 of storage.

Kim et al. (2016) report a decrease in oxidative compounds when adding extracts of coffee residues in raw meat emulsions, however this effect was only shown until the third day of storage. Similar behaviour to these reports was shown in the present study by increasing the amount of propolis in the diet of birds, from 15 mg and 30 mg to 45 mg of propolis. Therefore, the use of propolis in poultry feed may be an alternative to natural antioxidant compounds to reduce the concentration of oxidative compounds in meat during storage. Vargas et al. (2013) reported that propolis prevent or delay oxidative reactions due to the anti-radical activity and to the inhibitory effect on metal ions (Gülçin et al., 2010), and due to the presence of flavonoids that have the ability to trap free radicals (R. Vargas-Sánchez et al., 2011).

In the present study, the four treatments reached the maximum antioxidant capacity on day 5; this amount was lowest in meat of birds fed 45 mg/kg of propolis, and highest in meat of chickens in the control treatment. Although the use of propolis in the diet does not delay the oxidation process, it reduces the antioxidant capacity, that is, the formation of free radicals.

5. Conclusion

Propolis inclusion in broiler chicken diets may represent an alternative to decrease the amount of oxidative compounds during storage of meat for marketing, with the advantage of being of natural origin that does not affect the physicochemical characteristics such as colour in meat, which is an important attribute that influences the acceptance of the product by the consumer, together with drip loss and water retention capacity. These are important features for higher yield in the meat processing industry.

Compliance with ethical standards

Care of the animals in this study followed the Mexican federal guidelines for animal health (DOF 25-07-2007).

Declaration

All the listed authors participated in the development of the research and in manuscript preparation.

Acknowledgments

We would like to thank the “Dirección General de Educación (SEP)-Sub Secretaría de Educación Superior Universitaria”, by the financial support. Also, we thank the department of Animal Husbandry of the Colegio de Postgraduados, Chapingo, México, for the facilities offered to carry out the laboratory analyses in their laboratories.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

We thank the “Programa para el Desarrollo Profesional Docente (PRODEP)-Dirección General de Educación Superior Universitaria (DGESU)” for funding through the “Programa Apoyo a la Incorporación de Nuevo Profesor de Tiempo Completo [UAT-EXB-304]”.