ORIGINAL ARTICLE

Characterization of in situ ruminal degradabilty of dry matter in new varieties of drought tolerant Cenchrus purpureus

Caracterización de la degradabilidad ruminal in situ de la materia seca de nuevas variedades de Cenchrus purpureus, tolerantes a la sequía

J. L. Ledea,^I O. La O,^II J. V. Ray,^I

ABSTRACT

Two Creole cows, 450 ± 10 kg live weight, cannulated in rumen were used, with the purpose of studying the kinetics of in situ ruminal degradability of dry matter of leaves and stems of new varieties of drought tolerant Cenchrus purpureus (CT-601, CT-603 and CT-605) obtained by tissue culture at different regrowth ages (60, 80, 100 and 120 d).For that, they were introduced in duplicate bags in the rumen for 0, 4, 6, 8, 12, 24, 48 and 72 h. The results of the ruminal fermentation were fitted to the exponential equation (a + b) *(1-e^ˆ(-c*t)).  For ages 60, 80 and 100 d, the CT-603 had the highest degradability of the potentially degradable fraction (a + b) of the leaves dry matter, with 81.2, 97.8 and 77.5 %, respectively. For 120 d, was the CT-605 which showed better performance of this fraction, with 84.7%. For the stems, at 60 d, the CT-605 showed 94.6% degradation of the fraction a + b, while the CT-601 and CT-603, at 80 d, showed values of this fraction of 75.2 and 75.1 %, respectively. For 100 and 120 d, CT-601 and CT-603, with 59.7 and 88.8 %, respectively, were highlighted.  The protein tenors began to decrease below 7% from 80 d of regrowth. For DM degradability in leaves and stems, each variety showed values that exceeded 50% degradation. It is recommended using the ages of 80 and 100 d, in both botanical fractions, to supply ruminants.

RESUMEN

Se utilizaron dos vacas criollas, de 450 ± 10 kg de peso vivo, canuladas en rumen, con el propósito de estudiar la cinética de la degradabilidad ruminal in situ de la materia seca de hojas y tallos de nuevas variedades de Cenchrus purpureus (CT-601, CT-603 y CT-605), obtenidas por cultivo de tejido, tolerantes a la sequía en diferentes edades de rebrote (60, 80, 100 y 120 d).  Para ello, se introdujeron por duplicado las bolsas en el rumen durante 0, 4, 6, 8, 12, 24, 48 y 72 h. Los resultados de la fermentación ruminal se ajustaron a la ecuación exponencial (a + b)* (1-e^ˆ(-c*t)). Para las edades de 60, 80 y 100 d, el CT-603 presentó la mayor degradabilidad de la fracción potencialmente degradable (a+b) de la materia seca en hojas, con 81.2, 97.8 y 77.5 %, respectivamente. Para los 120 d, fue el CT-605 el que mostró mejor comportamiento de esta fracción, con 84.7 %. Para los tallos, a los 60 d, el CT-605 mostró 94.6 % de degradación de la fracción a+b, mientras que el CT-601 y CT-603, a los 80 d, presentaron valores de esta fracción de 75.2 y 75.1%, respectivamente. Se destacaron, para los 100 y 120 d, las variedades CT-601 y CT-603, con 59.7 y 88.8 %, respectivamente. Los tenores de proteína comenzaron a disminuir a partir de los 80 d, por debajo de 7 %. Para la degradabilidad de la MS en hojas y tallos, cada variedad presentó valores que superaron 50 % de degradación. Se recomienda utilizar las edades de 80 y 100 d, en ambas fracciones botánicas, para suministrar a los rumiantes.

INTRODUCTION

As a family, Poaceas are more tolerant to warm environments. Among them, Cenchrus genus, known as Pennisetum purpureum until Chemisquy et al. (2010) showed by the use of morphological, atomic techniques and their combination that the Cenchrus genus absorbs the Pennisetum and Odontelytrum genus. They are known to be very efficient plants, because they have the C3 and C4 photosynthetic pathways. They also highlight as being which provide better response in the synthesis of organic compounds at high temperatures, relative humidity, transpiration, evaporation, between other climatic variables (Vazquez and Torres 2007).

Specifically in Cuba, as in other tropical and subtropical countries, climate variation and rainfall distribution, mainly, influence on the quality and productivity of grasses and forages that are used for animal feeding, which is a limitation in most of the livestock systems (Pérez 2013). To stabilize the forage production in Cuba, specially in the dry season, in the 2000s begins the program of obtaining Cenchrus purpureus cultivars, from CT-115. As a result of this program, drought tolerant clones were obtained. Herrera (2000), when carry out the molecular characterization of these plants, by the electrophoretic determination of more than five isoenzymatic systems, showed that were varieties. This was reaffirmed because those varieties mantained their initial characteristics for more than five generations.

In agronomic studies performed in the eastern region of Cuba, the new drought tolerant varieties (CT-601, CT-603 and CT-605) surpassed its ancestor in the agronomic response (Díaz 2007, Almaguer 2012, Arias 2012). The evaluation of these varieties was conducted in ecosystems degraded by the intense seasonal drought, characteristic of the Cauto Valley, where 90 % of livestock production in eastern Cuba (Fajardo & Carbonell 2008) is developed.

Due to the structural characteristics of the cell wall, grasses do not exceed 50 % of ruminal degradation (Cáceres 1985, Ramírez 2010), stimulated by the accumulation of structural carbohydrates that, depending on the effect of climatic variables and their combination, they will be used as power source or deposited in the cell wall with structural function (Fortes 2014), in the form of cellulose, hemicellulose and lignin. Then, they were converted into lignocellulosic compounds (Valenciaga 2007), which retard the ruminal degradation process.

Palma and Landi (2012) reported that the degradation process of dry matter is one of the indicators assumed as favorable response in the effectiveness of the enzymatic hydrolysis or enzyme-substrate combination. This process suggests the degradation level, microbial efficiency and the sufficient of junction points for the enzymatic action, which is a basic principle for microbial efficiency, from the histological characteristics of the substrate (Martínez et al. (2002). According to Valenciaga (2007), the high fiber contents of Poaceas reduce the junction points. Furthermore, they are involved in the enzymatic combination (Martínez et al. 2002), which make difficult the process of microbial adherence to the active centers and produce saturation effect because they are occupied.

The objective of this study was to characterize the in situ ruminal degradability and the degradation dynamics of dry matter, contained in leaves and stems of new varieties of C. purpureus, drought tolerant at different regrowth ages, obtained by tissues culture.

Experimental procedure. The study was conducted in the Estación Experimental de Pastos y Forrajes, belonging to the Instituto de Investigaciones Agropecuarias "Jorge Dimitrov" from Granma province. This facility is located in Cauto plains, 10 ½ km from Bayamo city. The plant material is originate from the seed bank, established five years ago in 200 m² plots, composed of  10 furrows, spaced at 1 and 0.75 m between plants. For the banks establishment, the department of Pastos y Forrajes from the Instituto de Ciencia Animal provided the seeds.

At the moment of sampling in each furrow, the ages to be evaluated (60, 80, 100 and 120 d) were distributed in an intercropped way. The furrows were fractionated into subplots, 4.50 m linear, with four repetitions and one meter of border effect at the beginning and end of each furrow, conditions that were due to an agronomic study which was performed in unison. Taking advantage of this design, they were taken from each repetition five plants in each regrowth age, for a total of four samples for 60 d (three for 80 and 100 d, and two for 120 d). In each sample, 300 g were taken in green basis of each botanical fraction (leaves and stems) that was removed with stainless scissors. Subsequently, these fractions were homogenized in dry basis to determine the chemical composition and in situ ruminal degradability. Then, they were dried in a forced air oven (MEMMERT brand) at 100 °C for one hour and then, at 60 °C until reaching constant weight, as recommended by Lezcano and Gonzalez (2000). Finally, in a knife mill, they were reduced to 2 mm to determine the in situ ruminal degradability (100 g), and at 1 mm to determine the chemical composition (200 g). The cultivar used was Cenchrus purpureus, specifically the varieties CT-601 CT-603 and CT-605.

 It was fertilized with organic matter of bovine origin, at a rate of 25 t ha^-1 during establishment cut. For this, a wheelbarrow was weighed and it was filled with the corresponding amount, depending on the area and fertilizer dose to be use. During the filling process of the wheelbarrow, the spades with the desired weight were counted. Then, this fertilizer was applied along the furrow. The weighing of the wheelbarrow with organic matter was performed only once. Then, with the number of spades that reached the desired weight, it was proceeded to apply the fertilizer to the rest of furrows.

Chemical composition. The residual dry matter content of (rDM), organic matter (OM), crude protein (CP) and neutral detergent fiber (NDF) in leaves and stems was estimated using Goering and van Soest (1970) techniques, AOAC (2012).

Statistical analysis. The statistical procedures were performed, according to completely randomized design with four repetitions. The normality of data was checked by the Kolmogorov-Smirnov test (Massey 1951) and means were compared by multiple Keuls test (1952), for each regrowth age in each variety.

 In situ ruminal degradability. For the evaluation of in situ ruminal degradability, two Creole cows cannulated in rumen (450 ± 10 kg live weight) were used which receives forage grasses ad libitum, with free access to water and mineral salts .Five grams of sample were weighed per  polyethylene bag. The samples had particle size of 2 mm. After having found the uniformity of the internal dimensions using a millimeter ruler and an electron microscope (Microscope Model N-800M with camera HDCE-50 B) which allowed to determine the size and quantity of pores.cm², uniform dimensions were found for all bags (14.0 cm long x 8.5 cm wide internal dimensions, 48 μm porosity and 1044 pores.cm²). Then, they were washed and dried in an oven (BINDER brand) at 60 °C for one hour and weighed on an analytical balance (SARTORIUS brand) of 0.001 g precision to determine the weight of each of the bags included in the experiment. Finally, they were grouped per organ to introduce them in the rumen and doubled for each sampling time and animal. Leaves samples were fermented first, and stems samples at seven days. This interval corresponded to a resting time for the animal. The bags all together were introduced into the rumen for ruminal fermentation and extracted at 0, 4, 6, 8, 12, 24, 48 and 72 h. Each bag was hand washed with running water till water was clear once extracted from the rumen. Later, they were placed in aluminum trays and dried in an oven (BINDER brand) at 60 °C for 72 h. Then, they were transferred to a desiccator for 30 minutes and proceed to weighing. The difference between the initial weight of the sample placed in the nylon bags and the residue weight, after ruminal incubation, was used to determine the degraded DM in the rumen. The solubility at zero hour was obtained when incubating two bags with each food in the rumen for 15 minutes and then they were treated like the rest.

The estimate of ruminal degradation was performed using the interactive process MARQUARDT algorithm, using the procedure for non-linear models PROC NLIN from the Statgraphics Centurion software (Stat Point Technologies 2010). The exponential model proposed by Ørskov and McDonald (1979) was used to estimate the degradative characteristics. It was assumed that the degradation curve of the DM in the time followed a first order kinetic process, described as follows:

P=A         for t₀ = 0         

(a+b)*(1-e^{^(-c*t)}) t > t₀  

P:   Is the ruminal degradation of the evaluated indicator in the staying time t in the rumen.

a: Intercept (soluble fraction, if time is not  limiting).

b: degradable fraction, if time is not limiting(t).

c: degradation rate of fraction (b).

t: incubation time .

a+b: Potential degradation

A: easily soluble fraction.

The rapidly soluble fraction is obtained in several ways. In this study it was determined by introducing the bag in the rumen, allowing the ruminal fluid to penetrate inside. Then it was suddenly extracted and continued the procedure described for the in sacco degradability.

Table 1 shows the chemical composition of leaves and stems of new varieties of C. purpureus in study. Only significant differences (p ≤ 0.05) were obtained in protein tenors at the age of 60 d, effect which is given by the variety. These differences are due to the crossbreeding from the use of biotechnological tools, with the objective of achieving genetic variability, as reported by Ramírez (2010) because Cenchrus spp planting in Cuba came from the same genetic pattern. The  crude protein values obtained in the CT-603 and CT-605 were higher to those reported by Dórea et al. (2013) in the elephant silage, and as reported by Leo et al. (2012) in 11 tropical plants. They also exceeded the Loyola et al. (2015) results in natural grasses for both seasons, and those who reported Montiel et al. (2011) in grain sorghum. However, they were lower than Silva et al. (2011).Requires special attention the low crude protein content of the CT-601 for 60 d, as well as the decrease of concentration of this metabolite, from the 80 d of age for all varieties.

According to Minson (1990), tenors lower than 7 % not allow the efficient and complete use of the forage carbohydrates, so degradability could be affected by this concept. This is an aspect that should be considered for the cultivation of new varieties in the vertisol of Cauto Valley, which need to supply some protein food that provides to the microorganisms enough nitrogen for the development of the cellulolytic microflora, and the subsequent degradation of highly fibrous content.

The high tenors of NDF are due to the low nitrogen content, as Dominguez et al. (2012) referred. These authors stated that when there are low levels of nitrogen soluble carbohydrates synthesized are used, to be deposited in the cell wall as structural compounds. This thickness of these walls is increased, which makes little rough succulent this structure. This effect can affect the palatability and choice of food.

In the parameters of the in situ ruminal degradability of dry matter in leaves, the fraction a values ranged between -4.07 and 28.69 %, represented by the variety CT-603 and CT-605 respectively, there were high variability during the ages and varieties studied (table 2, figure 1). These values were lower than those reported by Pedraza (2000) in botanical fractions composed of leaves + petioles of Gliricidia sepium varieties and those of Valenciaga et al. (2001) in Cenchrus purpureus leaves. They were also below from those reported by Valenciaga (2007) in CT-115 (21.06-30.0%) and by Ferreira et al. (2014) (26.9-30.7%), who inoculated with three different strains (Enterococcus and Streptococcus JB1 and HC5) for processing as silage. They were similar to those of Silva et al. (2011) (21.9-28.6%), who added vinasse for their fermentation. Valenciaga (2007) and Trujillo et al. (2009) agreed that changes in the cell wall stimulated the variability of the results obtained in their researchers, which were in correspondence with the differences between the evaluated ages.

The fraction b had a similar performance to the soluble fraction, but with ranges of 28.8 to 85.3 %, which were higher than those obtained by Pedraza (2000) (37.4-48.06%), Valenciaga et al. (2001) (4.7-47.4%) and Valenciaga (2007) (34.05-39.8%). Also they exceeded those reported by Cardenas (2013). However, they were lower than Debela et al. (2011), in leaves of the variety Sesbania sesban and Desmodium intortum in southern Nigeria. However, the result of a + b was between 47.07 and 97.89 %, which manifested a higher performance than reported by Valenciaga et al. (2001), who obtained only 75.8 % degradation of this fraction. Also exceeded Valenciaga et al. (2006) results, when evaluating the resting time of the CT-115 and its effect on ruminal degradability of the lignocellulosic compound. These authors obtained for the characteristics of DM degradation at 95 d of age values much lower than those of this research.

The results presented in table 2 were above to those reported by Lara et al. (2010) in Dichanthium aristatum at different regrowth ages, and those informed by Valles et al. (2016), who grouped the Cenchrus genus and regrowth ages to evaluate their performance in the ruminal degradability of dry matter. Likewise, they were also better than Montiel et al. (2011), when evaluating the DM degradability of seeds of different sorghum hybrids, with minimum and maximum percentage values of 28.6- 34.7, respectively. However, they were lower than those reported by Debela et al. (2011).

In Cuba, the nutritional assessments that have been made to different varieties and clones of the Cenchrus genus (King grass, Mott, Taiwan, Merkerón, CT, CRA and CIAT) by Herrera et al. (1990) and Fernandez et al. (2001) have showed DMD values between 50 and 60 %, range which coincides with that obtained in the new C. purpureus varieties drought-tolerant, and with those reported by Minson (1990). According to the opinion of this author, this result is due to the complex structure of these varieties and the relations established between them as the age is advancing, stimulated by the effect of climate and growth. According to Minson (1981), Barahona and Sanchez (2005) tropical grasses hardly exceed 50 % of ruminal degradability.

In the kinetics of DM degradation of leaves of the varieties under study, can be observed different exponential performances. After12h, there was a lag of kinetics. CT-603 was the best for 60 and 80 d of age and CT-605 and CT-601 were the best for 100 and 120 d of age with 95 % of reliability. In this regard, Palma and Landi (2012) stated that the ruminal degradability of DM is one of the indicators assumed as favorable response in the effectiveness of the enzymatic hydrolysis, that is, the substrate-enzyme combination, in correspondence with which produce methane, as an end product of fermentation of structural carbohydrates (Beauchemin et al. 2008).

These results exceed those of Valenciaga et al. (2001), who stated that from 48 h of incubation there was not exponential performance of degradability. Similar results obtained Pedraza (2000) in different botanical fractions of Jupiter (Gliricidia sepium). Benavides et al. (2012) also reported a response in the dynamic of DM degradation in forage maize, from 24h, which was lower than in this study. This shows that the tissues of new varieties favor the microbial action early, mainly hydrolytic microorganisms (aminolytic and cellulolytic) which, according to Martínez et al. (2002), they are the first to actuate on the substrates present in the rumen. And then they are adding to rest of microorganisms from chemotaxis and established symbiosis.

The fraction b had high degradability percentages, ranging between 48.96 and 68.28%, higher values than those reported by Debela et al. (2011), who referred to stems of Sesbania sesbian and Desmodium intortum values of 44.5 and 41.5 % respectively, caused, according to these authors, by lignin tenors.  However, Pérez (2013) reported for tropical grasses lower amount of lignin than for legumes, but their distribution in the plant causes lower use than in Fabaceas, which stimulated degradation rates from 1.1 %.h (CT-605, 60 d) to 5.1%.h (CT-603, 60 d). These degradation percentages are lower than those obtained by Debela et al. (2011), due to the characteristics of the cells of this organ in this variety. Pérez (2013) stated that the stems have two to three thicker and lignified cell walls than the leaves.

The fraction a + b was favored by the high degradative action of fraction b, which allowed degradation ranges between 50.92 and 94.66 %. These results were higher than the 52.7 % reported by Martínez et al. (2002) in forage sorghum, and those obtained by La O et al. (2006), when evaluating the stem of six varieties of creeping legumes with different ruminal replacement constants.

In the degradation dynamics, the ages of 60 and 80 d were the ones that showed the best performance in the dry matter degradation, without subjectively suggesting that the ages of 100 and 120 d showed wrong performance, although they show the effects of the age on microbial activity. These results coincide with those obtained by Naranjo (2002), Soto et al. (2005) and Ferreira et al. (2014), and they were higher to those of Galindo et al. (2014), who evaluated the foliage of shrub grasses and obtained higher activity at 8h after incubating the samples. These authors reported that the presence of secondary metabolites may have influenced on the time of colonization, a criterion that coincides with that of Pedraza (2014), who understands the presence of these compounds as a barrier that affects the colonization process.

Although in the tropical grasses the presence of secondary metabolites is not reported as a chemical barrier that can affect rumen degradability, it must be taken into account. Ledea (2015), in Cuba, in an area of the Cauto Valley from Granma province, degraded by intense drought, identified by phytochemical screening in leaves of Cenchrus vc. CT-115 free amino acids, alkaloids, coumarins and anthocyanidins in the ethanolic extract, while in the aqueous only applied for moderate presence of flavonoids and bitter principles. In the state of Rio, in Nigeria, Okaraonye and Ikewuchi (2009) identified cyanogenic glycosides, oxalates, phosphates, saponins, tannins, alkaloids, tannins and flavonoids in the same genus. Therefore, it is possible that not only the effect of chemical composition and transformations in the structure and morphology of the evaluated organs influenced on the lateness of the ruminal microorganisms colonization, but that could be secondary metabolites synthesized by these plants. This is due, above all, to the environment influence, so it is recommended to deepen    into this line of research.

For the genus, variety, and organ, degradability is affected by the morphological and histological characteristics of the cell wall, which are accentuated as age advances, as an inherent process of growth that is increased by the incidence of climate. However, Cáceres (1985) stated that not always the nutritive value of a grass decreases as the age advances. In his studies, this author obtained differences between the tenors of dry matter, protein and fiber, not so for organic matter and crude energy. However, the results of this study demonstrate a performance that coincides with those reported by Ramírez (2010), when evaluating four tropical grasses in the Cauto Valley, where there were observed deterioration of agronomic, chemical and nutritional performance, as the age of the plant increased.

It is concluded that the new varieties of C. purpureus evaluated (CT-601, CT-603 and CT-605) show the ruminal fermentation pattern of tropical grasses, but with low crude protein content, in leaves and stems in evaluated ages. In both organs, 50% of degradation was exceeded, with evolution in the degradative dynamics that surpassed 72 h, decreasing at 120 d in the studied fractions.

It is recommended the introduction of new varieties into livestock production flows, so that they can be used for grazing, from 80 to 100 d, and at 80 to 120 d, as forage. It is necessary their association with shrub or herbaceous legumes to improve the chemical quality of biomass.

ACKNOWLEDGMENTS

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J. L. Ledea, Instituto de Investigaciones Agropecuarias “Jorge Dimitrov”. Estación Experimental de Pastos y Forrajes, km 10½, Carretera Bayamo-Tunas. Bayamo, Granma, Cuba. Email: ledea@dimitrov.cu