Animal Science

Effect of efficient microorganisms, native to Guantánamo, Cuba, on bioproductive and hematological indicators of pre-fattening pigs

0000-0001-5265-045XA. Valdés^{1  *}  , 0000-0002-7055-4880Yaneisy García²  , 0000-0001-7855-188XV.M. Álvarez¹  , 0000-0003-4372-5792A. Samón³  , 0000-0001-5040-5493E. Pérez⁴  , 0000-0003-1710-6322J.O Serrano⁵  , 0000-0001-9072-7577Y. Rodríguez⁴  , 0000-0002-7196-7396A. Berenguer¹ 

¹Facultad Agroforestal, Universidad de Guantánamo. Av. Che Guevara, km 1.5, Carretera Jamaica, CP. 95100, Guantánamo, Cuba.

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

³Empresa AZUMAT (Sucursal Guantánamo). Carretera Jamaica, km 5.0, CP. 95100, Guantánamo, Cuba.

⁴Facultad Agropecuaria, Universidad de Granma. Carretera Manzanillo, km 17 ½, Bayamo, CP. 85100, Granma, Cuba.

⁵Facultad de Ciencias Agropecuarias, Universidad de Ciego de Ávila. Carretera a Morón, km 9.0, CP. 65200, Ciego de Ávila, Cuba.

Abstract

To evaluate the effect of efficient native Guantanamo microorganisms (MEAG) on bioproductive and hematological indicators of pre-fattening pigs, 60 pigs (Yorkshire-Landrace/Duroc), of 33 days old and average weight of 9.9 ± 1.31 kg, were used. They were distributed, according to a completely randomized design, into four groups, with 15 repetitions each. Treatments were control (without MEAG), and addition of MEAG, in doses of 1.0; 1.5 and 2.0 mL/kg of live weight/day, orally supplied. The experiment lasted 42 d. Final live weight, weight gain, daily mean gain, feed conversion, hemoglobin, hematocrit, total leukocyte, lymphocyte, eosinophil, neutrophil, monocytes, mortality and morbidity were evaluated. Final live weight of the animals that received 2.0 mL/kg of live weight/d was 3.09 kg higher than control (P <0.05), and did not differ between doses. For weight gain, mean daily gain and dietary conversion, differences were only found between the highest evaluated dose and the remaining treatments. Animals of this group were superior to control by 3.06 kg of weight gain and 72.86 g of mean daily gain. In addition, they improved dietary conversion in 1.02 kg of consumed food/kg of weight gain. Hematological values increased with increasing doses, but within the normal range. For morbidity, differences were found between control and treated animals, without differences among the latter. It is concluded that the use of MEAG as a feed additive improves the bioproductive and hematological indicators of pre-fattening pigs.

Key words: pigs; microbial additive; productive response; health

The use of intensive animal production systems brings about imbalances in the productive performance of animals, which sometimes lead to the appearance of diseases (^{Nguyen and Nguyen 2017}). Pre-fattening category constitutes one of the most critical stages of pigs. Weaning is a crucial event in its life cycle, frequently associated with severe enteric infections and the excessive use of growth-promoting antibiotics (^{Gresse et al. 2017}). Hence, the efficient management of this event, and the subsequent fifteen days, are essential to achieve favorable productive results (^{Ayala et al. 2014}).

Currently, an alternative to increase productive yield of animals is the use of additives in their daily diet, such as biocatalysts, enzymes, probiotics, essential oils and bioactive compounds from plants and seeds (^{Sathyabama et al. 2014} and ^{Rodríguez et al. 2016}). Efficient microorganisms (EM) are considered as one of the effective and sustainable alternatives for livestock production (^{Luna and Mesa 2016}), as well as for sewage treatment and reduction of bad odors in the production of agrochemical-free food, among other multiple applications (^{Morocho and Leiva-Mora 2019}). In general, MEs are defined as a mixed culture of beneficial microorganisms, without genetic manipulation, present in natural ecosystems and physiologically compatible with each other (^{Luna and Mesa 2016}).

In Cuba, research centers, such as the Experimental Station of Pastures and Forages Indio Hatuey, in Matanzas province, and the University of Camagüey, developed mixtures of microorganisms from litter and other organic materials, obtained in areas free of the action of chemical fertilizers and herbicides, applied in pig production systems. These preparations produced beneficial effects on animal health and increases in zootechnical results (^{Rodríguez et al. 2013}, ^{Ojeda-García et al. 2016} and ^{Blanco-Betancourt et al. 2017}).

In the eastern provinces of Cuba, such as Guantánamo, scientific evidence regarding the development of efficient native microorganisms for pig farming is not sufficient. Therefore, the objective of this research was to evaluate the effect of efficient native microorganisms of Guantánamo on bioproductive and hematological indicators of pre-fattening pigs.

Materials and Methods

The research was carried out in the Unidad Porcina AZUMAT, located on the highway to Jamaica, in Manuel Tames municipality, Guantánamo province, with an epizootiology quadrant 102-147-17. Average rainfall regime is 746 mm/year, mean temperature is 25 ºC and mean relative humidity is 77%.

Experimental treatment and design. A completely randomized design was used, with 15 repetitions per treatment: control (without MEAG) and addition of MEAG, in doses of 1.0, 1.5 and 2.0 mL/kg of live weight per day, orally. Two pens were used per treatment, and each animal constituted an experimental unit.

Animals and feeding and management system. Sixty pre-fattening pig from Yorkshire-Landrace/Duroc cross were used, of both sexes (31 females and 29 castrated males), with an average age at weaning of 33 d, and an average live weight of 9.9 ± 1.31 kg. They were housed in Flat-Deck pens, with a 18 cm cement front, and a living space of 0.40 m2 per individual. The provided diet was concentrated food with corn-soy bean meal, according to nutritional requirements for the pre-fattening category (^{Rostagno et al. 2017}) and the Cuban technical procedures (^{IIP 2016}). It was offered in hanging cone feeders, twice a day, at 7:00 a.m., before the corresponding doses of MEAG were provided to animals, and at 2:00 p.m. The mean intake of concentrate was 27.94 kg. Pigs were treated with MEAG during the 42 d of the experimental stage, and they were weighed weekly to adjust the applied dose. Water was supplied ad libitum in nipple drinkers. In addition, the vaccine against cholera was applied at 35 d of life of piglets.

Preparation and characteristics of MEAG. MEAG culture was prepared according to the methodology described by ^{Tellez-Soria and Orberá-Ratón (2018)}, with the modifications described below. First, a solid product of lacto-fermentation was obtained, with the use of 30 kg of leaf litter in semi-decomposition and organic matter from the soil (1 to 6 cm from the surface), extracted from existing forests of Guantánamo province, in Cuba. This source was homogeneously mixed with 46 kg of corn meal, 10 L of sugar cane molasses and 10 L of whey. The mixture was deposited in a 200 L plastic tank, in 20 cm layers, and compacted to guarantee anaerobic conditions. The tank was hermetically closed, a valve was placed for releasing gases generated during fermentation and it was located in a warehouse with room temperature (28 ± 2 ºC) for 21 d. Later, a liquid fermentation was performed under the same temperature conditions for 7 d. For this, 10 kg of solid inoculum were taken, placed on a sieve and in a 200-L plastic tank, containing the mixture of 10 L of molasses, 10 L of whey and a sufficient quantity of drinking water without chlorine. After the fermentation time, a product with a sweet and sour odor was obtained, typical of lactic fermentations, and its characteristics are shown in table 1. For evaluating MEAG in pigs, three tanks were prepared at the same time (total capacity 600 L), which were kept in the previously mentioned warehouse during the experiment.

Table 1 Microbiological characteristics and pH of the evaluated MEAG in pre-fattening pigs1 

Indicator	Reference	Mean (n=3)	SD	CV, %
Viable yeast, CFU/mL	NC-ISO 1004:2016	1.6x1010	0.16	9.88
Filamentous fungi, CFU/mL		2.7x106	0.18	6.68
Total and fecal coliforms, CFU/mL	NC-ISO 4831:2010	Negative	---	---
Salmonella in 25 mL, CFU/mL	NC-ISO 6579:2008	Negative	---	---
Lactic acid bacteria, CFU/mL (through the method of serial dilutions and cultivation in Petri dishes with MRS agar medium, incubated 24-72 h at 37 oC)		4.2x109	0.38	9.07
pH (measured with a digital CRISON® BasiC 20,40*H 110 pHmeter, USA)		3.40	0.06	1.86

CFU: colony forming unit

¹Analyses conducted to homogeneous samples out of the three tanks at the Laboratorio Provincial de Diagnóstico Veterinario, with the use of Cuban regulations (NC) and by triplicate

SD: standard deviation

CV: Coefficient of variation

Experimental procedure for evaluating the effect of MEAG. The evaluated indicators were final live weight (FL), weight gain (WG), daily mean gain (DMG), food conversion (FC), hemoglobin, hematocrit, total leukocytes, lymphocytes, eosinophils, neutrophils, monocytes, morbidity, mortality and its causes.

FL of animals was quantified after 42 d of experimentation on a Salter scale, with a capacity of 50 kg and ± 0.01 kg precision.

WG was calculated by the difference between the FL and initial live weight (IL). This is:

[TeX:] WG = FL-IL

To determine DMG, the formula DMG = (FL-IL)/evaluation time, was applied.

FC was obtained from the formula FC = kg of consumed DM / kg of live weight increase.

During the experimental stage, the number of animals with diarrheal syndromes or dead animals was recorded, in order to determine the proportion of morbidity and mortality.

At the end of the experimentation, six animals were randomly selected per each treatment, and blood was extracted from the orbital vein with California-type needles. Samples were deposited in tubes impregnated with disodium EDTA (1.0 mg/mL of blood) and transferred to the Laboratorio Provincial de Diagnóstico Veterinario for their processing, according to the methodology described by ^{Coffin (1966)}.

Statistical analysis. Experimental data was processed with the IBM SPSS program, version 22. A simple analysis of variance (ANOVA) was carried out, with previous verification of variance homogeneity using ^{Levene (1960)} contrast test on the equality of error variances. Differences among means were found with ^{Duncan (1955)} multiple range test for P ≤ 0.05. For morbidity and mortality indicators due to digestive disorders and viability, an analysis of multiple proportions was performed using Chi-square test (χ2), with a significance level of P≤0.05. Magnitudes of differences between proportions were shown in a graphical analysis of the ANOM mean.

Results and Discussion

Table 2 shows the results of productive indicators of pre-fattening pigs with the addition of MEAG, after 42 d of experimentation. Differences (P≤0.05) were found between treatments for these indicators. FL of animals that received 2.0 mL MEAG/kg of body weight was 3.09 kg higher than control group, and did not differ among the applied doses. For the WG, DMG and FC, only differences were found between the highest evaluated dose and the remaining treatments. Animals in this group were superior to control by 3.06 kg of weight gain and 72.86 g of DMG, and improved the FC by 1.02 kg of consumed food/kg of weight gain.

Table 2 Effect of adding MEAGs on productive indicators of pre-fattening pigs of 75 d old 

Indicators	Control	MEAG doses, mL/kg of live weight			SE ±	P Value
		1.0	1.5	2.0
Final weight, kg	19.60^b	20.22^ab	20.85^ab	22.69^a	0.43	0.046
Weight gain, kg	9.70^b	10.27^b	10.87^b	12.76^a	0.33	0.005
Daily mean gain, g	230.95^b	244.60^b	258.73^b	303.81^a	8.11	0.005
Food conversion, kg/kg	3.25^a	2.97^a	2.64^a	2.23^b	0.13	0.040

^a,bMeans with different letters in the same line differ at P < 0.05 (^{Duncan 1955})

The previous results can be mainly related to the criteria of ^{Beruvides et al. (2018)} about the action of zootechnical additives on intestinal microbial ecosystem, health and productive indicators of animal. This action is attributed to the presence of viable microorganisms, organic acids, digestive enzymes and antimicrobial substances present in the additives, which favorably influence on the maintenance of microbiome eubiosis, improve intestinal health, increase absorptive, digestive and fermentative processes, and favor the synthesis of deficient nutrients in the diet. In addition, with a multifactorial approach, the aforementioned effects promote a better health state in the animal, better food conversion, increase of live weight and meat yield. Other authors also found similar effects when applying additives with mixed cultures of lactic bacteria and yeast in different categories of pigs (^{Flores-Mancheno et al. 2016}, ²⁰¹⁷ and ^{Miranda-Yuquilema et al. 2018 a}, ^b).

Variability of intergroup effect could be related to dose and application method of MEAG, as reported for microbial additives by ^{Sosa et al. (2018)}. It is possible that the application of additive per kg of body weight favored a more effective action, by using larger volumes of the preparation. This, in turn, could influence on the increase of the number of viable microbial cells in the intestinal lumen, with possible colonization or modification of intestinal microbiome composition. This effect was observed by ^{Kiros et al. (2018)} and ^{Kiros et al. (2019)}. The effectiveness, according to ^{Delgado et al. (2014)}, is only achieved when the quorum required to reach the intestine, colonize and express the own benefits of these products is added to the individual or collective stimulating potential of microorganisms involved.

^{Blanco-Betancourt et al. (2017)} observed similar results to those of this study. These authors used doses of 40, 80 and 120 mL/pig/d of EM preparation, IHplus®, obtained by a similar technology, but with inoculum from their Matanzas locality. These authors found the best results with the last doses compared to control, and reported that the dose of 40 mL/pig/ d had no incidence on productive indicators. This is possibly due to the effect of low concentration of microorganisms, and to the transit speed of digesta that, at early ages, is very fast, and does not favor the conditions for colonization of these microorganisms in the digestive system.

Hematological indicators of animals treated with MEAG are presented in table 3. Their values ​​increased as the preparation dose increased, although all were found within the reference ranges considered as normal, as reported by ^{Serem et al. (2017)}. Similar effects were found by ^{Dlamini et al. (2017)} and ^{Trevisi et al. (2017)}, who informed that lactic acid bacteria and yeasts increased digestion and absorption of the main nutrients (amino acids, vitamins, minerals and some others). This causes an increase of availability of iron in the animal and favors the central nucleus of hemoglobin, by joining with some amino acids and vitamins. Therefore, the health of the host is positively influenced.

Table 3 Effect of adding MEAGs on blood indicators of pre-fattening pigs with 75 d old 

Indicators	RR	Control	MEAG doses, mL/kg			SE±	P Value
			1.0	1.5	2.0
Hematocrit, %	24.0-45.00	30.33^b	31.67^b	35.83^a	36.00^a	0.71	0.001
Hemoglobin, g/dL	9.04-16.54	10.08^b	10.52^b	11.92^a	11.97^a	0.23	0.001
Total leucocytes, x109/L	2.55-20.0	8.75^d	10.75^c	13.83^b	16.30^a	0.66	0.000
Eosinophils, x109/L	0.10-2.70	0.38^b	0.58^b	0.93^a	1.13^a	0.07	0.000
Neutrophils, x109/L	0.00-4.80	2.88^c	3.40^b	3.65^b	4.25^a	0.12	0.000
Lymphocytes, x109/L	3.50-9.50	4.33^b	4.55^b	4.66^b	6.02^a	0.20	0.006
Monocytes, x109/L	0.00-1.60	0.42^c	0.45^c	0.62^b	0.90^a	0.05	0.000

^a,b,c,dMeans with different letters in the same line differ at P < 0.05 (^{Duncan 1955})

RR: referential ranges by ^{Serem et al. (2017)}

The improvement of nonspecific defense mechanisms of the host, and the stimulation of the production of blood cells related to adaptive or innate immune response (^{Herrera et al. 2016}), without this action being exaggerated or harmful to the host, are others of the effects associated with microbial additives. It is also possible that, by interacting with antigens, cells secrete pro and anti-inflammatory cytokines, which regulate the function of regulatory T cells. This allows achieving an effective immune system, and decreases susceptibility to various inflammations and allergies (^{Laskowska et al. 2017} and ^{Madrid et al. 2019}). It is possible that this immunomodulatory and protective effect is produced by MEAG in pigs that consumed doses of 1.5 and 2.0 mL/kg of body weight/d, and can also be related to results of morbidity and mortality indicators, shown in table 4.

Table 4 Proportion of health indicators of evaluated animal groups 

Indicator	Treatments	Number of animals	Proportion	SE ±	χ2	P Value
Morbidity	Control	10	0.67^b	0.07	10.05	0.018
	T1	6	0.40^a
	T2	4	0.27^a
	T3	2	0.13^a
Mortality	Control	1	0.06	0.02	2.07	0.558
	T1	1	0.06
	T2	0	0.00
	T3	0	0.00

^{a ,b} Proportions with different letters in the same column differ at P<0.05

T1, T2 and T3 doses of 1.0, 1.5, 2.0 mL MEAG/kg od live weight/d, respectively

χ2: Chi-square value

For morbidity, the proportion differed between animals of control group and those treated with MEAG, without differences among evaluated doses. Meanwhile, for mortality, no differences were found. Digestive disorders were death cause of animals of control group and with the applied dose of 1.0 mL of MEAG/kg of live weight/day.

According to the research, it could be affirmed that pigs treated with the mixed culture of MEAG showed fast adaptation to weaning challenges. Similar results were reported by ^{Miranda-Yuquilema et al. (2018a)}, when using biopreparations with Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus, Saccharomyces cerevisiae and Kluyveromyces fragilis, at doses of 2.5 mL/animal/d in the diet of post-weaning pigs. These authors observed that treated animals presented the least number of diarrheal disorders and deaths with respect to control (P <0.05).

^{Barreto et al. (2015)} also evaluated the action of a mixed culture of EM, with inoculum from Camagüey, ​​Cuba, for controlling and preventing pig diarrheas. Authors highlighted the role of lactic bacteria in blocking ligands for enteropathogens, which is an essential step that promotes colonization and subsequent release of enterotoxins. They also pointed out that these mixed cultures can modify the pH in the intestinal lumen (pH <4.0), which do not tolerate certain enteropathogens, due to the production of organic acids (especially lactic acid) and short-chain fatty acids (acetate, propionate and butyrate). In addition, the production of antimicrobial substances such as nisin and lactalin, as well as the production of hydrogen peroxide, may contribute to the action.

From the exposed results, it is concluded that the use of MEAG as a feed additive improves bioproductive and hematological indicators of pre-fattening pigs, with superior results, when using the dose of 2.0 mL/kg of live weight/d.

Acknowledgements

Thanks to the management of AZUMAT Pig Unit, in Guantánamo, and its workers. Likewise, gratitude is expressed to Eng. Keylis Grandales Llanes, without whose collaboration the research would not have been possible.

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Received: June 06, 2020; Accepted: July 08, 2020

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Declaración de conflicto de intereses: Los autores declaran no presentar conflicto de intereses

Contribución de los autores: Los autores declaran presentar contribución igualitaria en la concepción de la investigación, obtención y procesamiento de los datos y redacción del documento