Pastos y Forrajes Vol 42n1 del 2019

Vol. 42, No. 1, January-March 2019 / NRS 0099
ISSN 0864-0394 (printed version) / ISSN 2078-8452 (online version)
Quarterly journal. Official organ of the Ministry of Higher Education for pastures and forages | 1978
MISSION: to disseminate research results,
development of technologies and innovation,
related to the farming sector.
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for national and foreign researchers, professors
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promote rural development, decision-makers
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and producers.
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papers, review papers, short communications,
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flections) which contribute to the knowledge of
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TOPICS
• Introduction, evaluation and dissemination of
plant genetic resources related to the farming
sector.
• Agroecological management of production
systems.
• Sustainable livestock production.
• Conservation of forages and agroindustrial
byproducts for animal feeding.
• Agroforestry for animal and agricultural
production.
• Integrated food and energy production
systems in rural areas.
• Utilization of alternative medicine in tropical
farming systems.
• Adaptation to and mitigation of the climate
change in farming ecosystems.
• Economic, managerial and social aspects of
farming production.
• Extension, agricultural innovation and
technology transference.
• Rural and local development.
ESTACIÓN EXPERIMENTAL DE PASTOS Y FORRAJES INDIO HATUEY
EDITORIAL COUNCIL
Editor-in-Chief | Dra. Tania Sánchez Santana
Assistant Editor | M.Sc. Nayda Armengol López
Editor-Agricultural Sciences | Dra. Marta Hernández Chávez
Editor-Veterinary Sciences | Dr. Javier Arece García
EDITORIAL COMMITTEE
Dr.C. Jesús Suárez Hernández | Dra.C. Maybe Campos Gómez
Dra.C. Marlen Navarro Boulandier | Dra.C. Maykelis Díaz Solares
Dr.C. Jesús M. Iglesias Gómez | Dr.C. Hilda B. Wencomo Cárdenas
Dr.C. Anesio R. Mesa Sardiñas | Dr.C. Luis A. Hernández Olivera
Dra.C. Hilda C. Machado Martínez | Dr.C. Osmel Alonso Amaro
Dr.C. Giraldo J. Martín Martín | Dra.C. Odalys C. Toral Pérez
Dr.C. Luis Lamela López | M.Sc. Onel López Vigoa
Dra.C. Mildrey Soca Pérez | M.Sc. Milagros de la C. Milera Rodríguez
Dr.C. Félix Ojeda García | M.Sc. Yolai Noda Leyva
Dr.C. Gertrudis Pentón Fernández | M.Sc. Juan C. Lezcano Fleires
SCIENTIFIC COMMITTEE
Dra. Sonia Jardines González | Universidad de Matanzas, Cuba
Dra. Angela Borroto Pérez | UNIVERSIDAD DE CIEGO DE ÁVILA, Cuba
Dr. Aníbal E. Fernández Mayer | Instituto Nacional de Tecnología
Agropecuaria, Argentina
Dr. Argemiro Sanavria | Universidad Federal Rural de Rio de Janeiro, Brasil
Dr. Tyrone J. Clavero Cepeda | Universidad de Zulia, Venezuela
Dr. José M. Palma García | Universidad de Colima, México
Dr. Oscar Romero Cruz | Universidad de Granma, Cuba
Dr. Carlos J. Bécquer Granados | Estación Experimental de Pastos y Forrajes
de Sancti SpÍritus, Cuba
Dr. Rodobaldo Ortíz Pérez | Instituto NACIONAL de Ciencias agrícolas, CUBA
Dr. Pedro C. Martín Méndez | Instituto de Ciencia Animal, Cuba
Dr. Pedro P. del Pozo Rodríguez | Universidad Agraria de La Habana, Cuba
Dr. Redimio Pedraza Olivera | Universidad de Camagüey, Cuba
Dr. Rafael S. Herrera García | Instituto de Ciencia Animal, Cuba
Dr. Pedro José González Cañizares | Instituto Nacional de ciencias agrícolas, CUBA
Dr. Ángel Arturo Santana Pérez | Universidad de Granma, Cuba
SUPPORT COMMITTEE
Editing and correction
M.Sc. Alicia Ojeda González
Design and editing
Dailys Rubido González
Miresleidys Rodríguez Rizo
Translation
B.A. Nidia Amador Domínguez
Cover design
B.A. Israel de Jesús Zaldívar Pedroso

Vol. 42, No. 1, January-March 2019
Revista Trimestral. Órgano oficial del Ministerio de Educación Superior para el área de los pastos y forrajes
Quarterly journal. Official organ of the Ministry of Higher Education for pastures and forages
PASTURE AND FORAGE
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© 2019. Estación Experimental
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CONTENT
| REVIEW paper |
Intensive rational grazing as alternative for low-emission animal husbandry
Milagros de la Caridad Milera-Rodríguez, Rey Leovigildo Machado-Martínez,
Osmel Alonso Amaro, Marta Beatriz Hernández-Chávez and Saray
Sánchez-Cárdenas................................................................................................3
| scientific paper |
Adaptation of grasses associated with Lotus uliginosus Schkuhr in the
high Colombian tropic
Edwin Castro-Rincon; Juan Evangelista Carulla-Fornaguera and Edgar Alberto
Cárdenas-Rocha..................................................................................................13
Influence of in vitro plant size and substrate type on the acclimatization of
Morus alba L.
Ángel Espinosa-Reyes, Juan José Silva-Pupo, Marisel Bahi-Arevich and
DariannisRomero-Cabrera...............................................................................23
Effect of IHPLUS®
on the germination process of Sorghum bicolor L. (Moench)
Maykelis Díaz Solares, Yunel Pérez Hernández, Jessika González Fuentes, Inelvis
Castro Cabrera, Leticia Fuentes Alfonso, Madyu Matos Trujillo and
Maryla Sosa del Castillo....................................................................................30
Effect of the inoculation of beneficial microorganisms and Quitomax®
on
Cenchrus ciliaris L., under conditions of agricultural drought
Carlos José Bécquer Granados, Pedro José González Cañizares, Urbano Ávila
Cordoví, José Ángel Nápoles Gómez, Yaldreisy Galdo Rodríguez, Ivón
Muir Rodríguez, María Hernández Obregón, Maribel Quintana Sanz and
Fernando Medinilla Nápoles.............................................................................38
Weeds-beneficial entomofauna ecological relation in silvopastoral
systems of western Cuba
Osmel Alonso-Amaro, Juan Carlos Lezcano-Fleires and Moraima Suris-
Campos.................................................................................................................46
Evaluation of the nutritional value of forages in a silvopastoral system
Onel López-Vigoa, Luis Lamela-López, Tania Sánchez-Santana, Yuseika
Olivera-Castro, Roberto García-López, Magaly Herrera-Villafrancaand
Manuel González-Ronquillo.............................................................................54
Utilization of shrimp (Litopenaeus vannamei) waste meal in heifers
RobertoMartínez-León, Roberto García-López, Juan Eulogio Guerra-Liera
and Delfin Gutiérrez-González........................................................................64
Learning results in the process of local development management in a
Matanzas municipality
Taymer Miranda-Tortoló, Hilda Machado-Martínez, Juan Carlos Lezcano-Fleires,
Antonio Suset-Pérez, Katerine Oropesa-Casanova, Frank David Tirado-
García, Luis Lamela-López and Iván Lenin Montejo-Sierra..........................69
Bibliometric analysis about the published studies on Morus alba L.
Maida D. Peña-Borrego, Diannelis Fermoselle-Cumbá, Yuri Freddy Peña-Rueda,
and Carlos Bécquer-Granados..........................................................................76

Pastos y Forrajes, Vol. 42, No. 1, January-March, 3-12, 2019 / Intensive rational grazing 3
Review paper
Intensive rational grazing as alternative for low-emission animal husbandry
Milagros de la Caridad Milera-Rodríguez, Rey Leovigildo Machado-Martínez, Osmel Alonso
Amaro, Marta Beatriz Hernández-Chávez and Saray Sánchez-Cárdenas
Estación Experimental de Pastos y Forrajes Indio Hatuey, Universidad de Matanzas. Ministerio de Educación Superior, Central
España Republicana, CP 44280, Matanzas, Cuba
Correo electrónico: mmilera@ihatuey.cu
https://orcid.org/0000-0001-8531-3425
Abstract
In order to review essential aspects of the contribution of intensive rational management to low-emission animal
husbandry, a set of effects related to climate change and its repercussion on the soil-plant-animal relation were considered,
in which emphasis is made on the results obtained in Cuba. The world faces extreme climate events, such as: drought,
flooding, temperature increase, among others. In turn, the decrease in land productivity, soil degradation and pastures,
decrease of animal production, make countries more vulnerable to climate change. The world agriculture represents
14 % of greenhouse gas emissions; while Cuba is responsible for 18 % of the total emissions. The studies conducted
on intensive rational grazing have had positive repercussion in different countries of the area. There are results on the
rational management of different cultivated grasses and its effect on the stability of floristic composition, dry matter
availability, pests and diseases, nutrient recycling, soil biota and underground phytomass. From the contribution of the
ecological management of intensive grazing systems to develop low-emission animal husbandry, results are provided
that represent a resilient choice in the face of climate change and a contribution to the food self-sufficiency of countries.
In this sense, the challenge is the transition or reconversion of conventional systems to agroecological, resilient systems,
which allow to decrease CO2
-eq emissions and increase sinks.
Keywords: adaptation, climatic change, species
Introduction
FAO estimates that the world food production
in 2050 should increase by 49 % compared with
2012 to feed an increasing population and with
changing habits (FAO, 2017).
The increases in human population and poverty,
changes in diet preferences for animal-derived
products in the developing world and the increase
of the use of arable lands for biofuels will have to
be managed within the context of climate change.
It is estimated that between 2005 and 2015, 26 %
of the total damages and losses caused by climate
disasters in developing countries occurred in agri-
culture. During this period, drought caused 30 % of
the agricultural losses caused by natural disasters,
which is equivalent to 29 000 millions USD (FAO,
2017).
Animal husbandry in the world is responsi-
ble for 14,5 % of the anthropogenic emissions of
greenhouse gases (GHG). They amount to 7,1 thou-
sand million tons of CO2
-eq per year. The GHG
emissions in the animal husbandry sector can be
reduced by 14-41 %, through the adoption of im-
provements in: the diet, concentrate feed quality,
animal health, management of herd manure and
efficient energy use (FAO, 2017). In Cuba, agri-
culture represents 18 % of the total emissions, and
from it enteric fermentation produces 45 % of the
GHG emissions (CITMA, 2015).
The use of intensive animal production
systems on agroecological bases is stated as a
strategic possibility to mitigate the anthropogenic
GHG emissions; which include intensive rational
grazing systems, from the postulates proposed by
Voisin (2012), based on several factors, such as: no
utilization of agrotoxicals; stimulation of natural
cycles; utilization of pasture at the optimum resting
time and with sufficient reserves in the root to allow
vigorous regrowth. In addition, the plant is used
when it has the nutrients to feed livestock and thus
maximizes the harvest of organic matter per area
unit and is managed with the carrying capacity in
that space. This flexible management contributes
to eliminate overgrazing and the disappearance of
the cover of adapted species, protects the soil and
strengthens the root system, for which it contributes
to the adaptation and mitigation.
Due to the above-explained facts, worldwide
sustainable development is promoted as a viable
choice for mankind’s survival in harmony with na-
ture. In that sense, the objective of this review paper
was to review the essential aspects of the contribu-

4 Pastos y Forrajes, Vol. 42, No. 1, January-March, 3-12, 2019 / Milagros de la Caridad Milera-Rodríguez
tion of intensive rational grazing system manage-
ment to develop low-emission animal husbandry,
where the results obtained in Cuba are emphasized.
Some essential elements of Voisin’s Rational
Grazing (VRG)
The studies conducted by the French scientist
André Voisin in temperate climate and the argu-
ments taken from the scientific contributions of
previous years in Germany and other countries,
allowed him to enunciate the main principles for
pasture management under those conditions.
The way in which these management princi-
ples, their basis and logics were formulated, provid-
ed them with a certain universality character, but
they did not escape the reality of being formulated
for a specific type of pastureland and ecological,
technological and cultural environment; however,
their application is possible if the principles of for-
mulation are taken into consideration.
In the four laws formulated by Voisin (2012),
the author assigns equal importance to the pasture
and to the animal.
In those related to pasture, their application
depends on the edaphoclimatic characteristics;
nevertheless, the manager is the one in charge of
determining the optimum moment for grazing and
no similar recommendations could be offered for
the different conditions.
Voisin (2012) defined that grazing is the meet-
ing of the cow with the grass, in balance so that
none affects the survival of the other, because the
cow selects from the pasture the nutrients for the
maintenance, growth, production and reproduction,
but in turn the grass needs to be consumed, tram-
pled, fertilized, in order to start a new growth cycle
and the cow stimulates that growth with the saliva,
feces and urine.
Pasture areas with adequate management have
large carbon reserves. In this sense, carbon balance
on Earth is related to four reservoirs: oceans, at-
mosphere, geological and land system with 50 Pg
as biomass-vegetation and 1 550 Pg as soil organic
carbon (SOC). In the land ecosystem, the largest
component is SOC, followed by inorganic carbon
(750-950 Pg C). The SOC makes up about two
thirds of the fixed C in land ecosystems; thus, the
soil represents a large carbon store in nature (Bur-
bano-Orjuela, 2018).
On the other hand, the management methods
included in the laws involve new concepts of nature
balance, with neither exploitation nor deterioration
and their application should be contextualized.
The first two laws were related to the pasture
and the other two to the animal. In the ones enun-
ciated for the pasture management, the most im-
portant principle is the resting between a grazing
moment and the other to reach the highest produc-
tivity, and the occupation time of one paddock by
the animals.
• First Law: For a grass, sheared with the animal’s
teeth, to achieve its maximum productivity, su-
fficient interval must have elapsed between two
successive grazings, in order to allow the grass
to accumulate in its roots the necessary reserves
for a vigorous spurt of re-growth and to produce
its blaze of growth.
• Second Law: The occupation period of one pa-
ddock should be sufficiently short for a grass
sheared on the first day of occupation not to be
cut again by the teeth of these animals before
they leave the paddock.
In the laws related to the animal Voisin brings
to the fore the requirements and ration quality to
cover them, key aspects to guarantee that the ani-
mal expresses its potential.
• Third Law: The animals with the greatest nutri-
tional requirements must be helped to harvest
the greatest quantity of grass of the best possible
quality.
• Fourth Law: For a cow to give regular milk
yields she must not stay any longer than three
days in the same paddock. Yields will be at their
maximum if the cows do not stay in one paddock
for more than one day.
Another aspect to which great importance was
ascribed was the stocking rate. In this regard, in
his book “Grass productivity” (Voisin, 2012), he
stated: the farmer who embarks on rational graz-
ing generally states first: How many animals can I
load? I answer: I do not know, I cannot know. It is
necessary to establish a time plan, from this plan
the surface plans and the possible stocking rates
could be deduced… If rational grazing is convenien-
tly performed you will be led in successive years
to increase considerably the global stocking rate in
your pastures.
This idea, along with other principles such
as resting, permanence time or paddock jumping
(flexibility),speakontheonehandoftruetechnological
laws of rational grazing and, on the other hand, of the
deep ecological vocation that promotes balance and
moderation in the relations with nature, for which their
application can contribute to low-carbon systems. The

interpretation and innovation under the conditions of
each site is the key to success.
In Cuba the corollaries and laws enunciated by
the French scientist were known since his books
were edited in the early 1960’s, and the technology
was assumed, to a large extent, as something
finished, whose success depended only on knowing
and applying it well. The expectations around it were
very high and although actions were undertaken to
follow up the management of the technology that
was being implemented, the studies in this regard
were neither systematized nor was his work further
analyzed in the few research centers that existed at
that time.
In 1990 the country had lost the trade with the
socialist block and was undergoing double embar-
go, because of which fertilizers, fuel, feed sup-
plements and medicines, among other products,
drastically decreased. At that time, the topic of
Voisin’s rational grazing was taken up again, which
was successful in some dairy farms, but the lack
of minimum resources and training of the farmer
as protagonist did not allow it to triumph at large
scale; yet, a research program was conducted with
the participation of different scientific institutions
in which the system approach prevailed and com-
mendable results were reached in the studies of the
soil-plant-animal relation.
Considerations of the impact of animal
husbandry on the climate change and possible
actions to compensate it
World animal husbandry is responsible for two
thirds of the GHG emissions (FAO, 2017). Cattle
management is the main warrant, and the emissions
depend on the actions that are developed. Among
the most important ones are: the diet that is offered,
maintaining unproductive cows and replacement
animals that are not utilized later, not processing
manure adequately and using imported cereals with
a large carbon footprint.
In this sense, cattle cannot be accused as
the main responsible for emissions, because the
management practices in feeding aimed at deviating
fermentation towards propionate production without
affecting ruminant production, as well as providing
the increase of intake or digestion rate or reducing
feed permanence in the rumen, decrease methane
production per unit of digested forage (Sosa et al.,
2007). It is also stated that when intensive feeding
systems are used, lower methane amounts are
produced when compared with extensive systems
(Clemens and Ahlgrimm, cited by Sosa et al., 2007).
On the other hand, although industrial agri-
culture has been the prevailing model in different
geographic zones, small farmers produce most
foodstuffs and promote economic development
in Africa, Latin America and Asia. Five hundred
million of these small farmers feed more than two
thousand million people worldwide (Altieri et al.,
2014).
Although the industrial systems have reported
gains in productivity in the last 50 years, they have
also had indisputable negative impacts on the envi-
ronment. For such reason, the future challenges for
production systems depend on the redesign of in-
dustrial animal husbandry systems and the increase
of production in small farms through a set of al-
ternative practices to industrial agriculture. Agro-
ecology offers a holistic framework to face these
issues and their interconnections at different scales
(Dumont et al., 2014).
Latin America had a reduction of the emissions
in 2000 with regards to 1990; however, at present
it has 9,9 % of the emissions of t CO2
eq per capita
with regards to the world, in the land change and
use (Loaiza-Ceró et al., 2015).
The reconversion and/or agroecological tran-
sition is a transformation process of conventional
production systems, towards agroecological basis
systems, which comprises not only technical, pro-
ductive and ecological elements, but also socio-
cultural and economic aspects of the farmer, his/
her family and community (Vázquez and Martínez,
2015). In Cuba, during 30 years, conventional
agriculture was practiced; however, during the last
20 years, a process of agroecological transition has
occurred (Vázquez, 2018), in which more than one
hundred thousand farmer families participate with
results in the agroecological movement (Machín,
2016).
The sustainable intensification of animal hus-
bandry in the face of climate change is a challenge.
It represents a different concept from the intensifi-
cation that has been practiced with the conventional
systems that attempt to “increase” food production
and “face” the extreme events of climate change
at the expense of ecosystem and natural resource
sustainability, in order to solve the problems which,
precisely, have been generated by the same techno-
logical approach with external dependence on basic
products and contamination, among others. Agri-
culture in transition towards sustainability pursues
food production, from self-sufficiency, diversity,
frequency, innocuousness and quantity (Vázquez,
2018).

The decrease of vulnerability and the increase of
resilience (adaptation), requires a social construction,
because it is in the farm, according to the soil and
climate of the site, where the plant and animal spe-
cies are selected. In this construction, research-
er-farmer innovation processes intervene, which
with adequate management can contribute to re-
duce the GHG emissions and increase the removal
of CO2
.
Contribution of the intensive rational grazing
systems to low-carbon animal husbandry
In the studies conducted in Cuba with
an intensive rational management, where the
agroecological management of the soil and the
flexibility of animal rotation prevailed, encouraging
results were found in the studies carried out on the
soil and the species that populated the system.
Milera et al. (2016), when using the grasses
Andropogon gayanus Kunth cv. 621; Megathyrsus
maximus (Jacq.) B.K. Simon & S.W.L. Jacobs
cv. Likoni, Cenchrus ciliaris L. cv. Biloela,
Urochloa mutica (Forssk.) T.Q. Nguyen cv. Aguada
[=Brachiaria purpurascens (Raddi) Henrard] and
Cynodon nlemfuensis Vanderyst cv. Tocumen,
in a system with intensive rational management,
without application of fertilizers and the presence
of herbaceous legumes, observed favorable results
in the chemical and biological composition of the
soil, recycling related to the discharge of excreta in
the paddock, as well as the underground phytomass,
floristic composition, persistence, availability
and bromatological composition of the species,
phenology, pests and diseases.
In this study the most important aspects in the
pastureland management received priority: grazing
system, necessary resting time (expressed as the
optimum moment for the entrance of the animals
to the paddock or plot), the intensification or high
instantaneous stocking rate that would allow high
discharge of excreta, and global stocking rate. For
such purpose, there was a large number of pad-
docks, in an area well covered by pastures and with
sufficient available biomass.
The new grazing design took into considera-
tion the optimum moment to be grazed and from it
the resting time and quantity of paddocks were de-
termined; this logic had not been practiced before
in management trials.
To introduce the cattle in the system with
grasses, a set of factors were considered which were
evaluated through a ranking-visual observation
method to determine the optimum grazing moment.
Due to their importance, the most important steps
are summarized.
The measurements were carried out from five
points or moments of grass growth, depending on
visual observation. The analysis of measurements
and trip notes allowed to select the best moment:
• Point 1. Departure of the animals: At that point
the grass is not very high, but refuse is very im-
portant (neither a lot of grass nor very little), that
is, the soil must not be uncovered.
• Point 2. Slow growth and color change: the effect
of selection by the animal on the consumed leaf
disappears and plant growth and height start.
• Point 3. Linear growth: increase of the leaf bio-
mass, more intense color, widened leaves, more
height.
• Point 4. Increase of leaf biomass: more dense
area with higher yield; in erect pastures the leaf
apex is bent down by the weight, there is little
inflorescence.
• Point 5. Maturity: Basis with wilted leaves,
maturity, change of color and more than 10 %
flowering.
In this sense, the number of floral stems in
A. gayanus started to appear since September,
but in low percentages (September-November) in
points 3 and 4, and it increased at point five; never-
theless, in December flower stems were observed at
points 3, 4 and 5.
The number of live shoots (Sl) and total
shoots (St) and the respective variables calculated
depending on them (density of live shots, live
shoots-diameter ratio, live shoots-dead shoots ratio,
total shoots-diameter ratio and density of total
shoots), were significantly higher when the pasture
was rotated a higher number of times. Thus, it was
confirmed that with shorter frequencies and high
instantaneous rates used a beneficial stimulus was
originated in shoot production. This indicates that
the defoliation produced by the animals did not
damage at any moment the potentially apt buds
and buds-sprout to support the sequence of sprout
formation, which were produced, even, in higher
quantity (Machado et al., 2012).
Fariñas et al. (2017), from visits and measure-
ments with a systemic approach and taking into
consideration all the factors that have incidence on
the management of the grazing system, obtained re-
sults which coincide with the ones analyzed in this
paper, not only in the approach but in the flexibility
and rationality to organize the pasture management.

Regarding the best moment or optimum point
for the grass harvest, it was concluded that point 4
turned out to be the best for introducing the animals
in the paddock. The stay was for one day with high
instantaneous stocking rates, but there was a residue
left that allowed vigorous regrowth. With regards
to the utilization, it was taken into consideration to
offer availability per animal which, according to
the season, could stay in the paddock the whole day
or only an amount of hours; neither the pastures nor
the cows were affected, that was the premise used
from the beginning.
According to Marín et al. (2017), the moment
for the beginning of rotation was determined by
the height. These authors stated that the rotational
grazing is characterized by managing with high
pressure when a high availability of pasture is
reached upon the entrance of the animals to the
paddock and this mass decreases remarkably
upon the departure of the animals due to the high
utilization. Then an optimum pre-grazing height
will be reached which maximizes the short-term
ingestion rate (STIR). That is why they recommend
a pre- and post-grazing height and concluded that
the maximum STIR leads to higher daily intake
and animal performance, and in turn improves the
intensity of the GHG emissions per unit of animal
product or per area.
From the optimum moment method its
principles can be adopted, but not its quantitative
results related to age, height, stocking rate, because
they all start from the manager’s experience and
the observation of the availability and quality
of certain species, which are conditioned by the
edaphoclimatic characteristics of the site and type
of variety, growth habit, stocking rate used, among
other factors.
The careful management to achieve a quality
offer, besides maintaining the persistence of the
species, is what contributes to the adaptation and
sustainability of the system; nevertheless, adapta-
tion cannot be extended, or imported, it is reached
when the adequate management in the long term is
performed in each site.
As could be observed, the determination of the
optimum moment or point depends on managing
flexibly each paddock with the necessary resting,
taking into consideration the different species un-
der evaluation within the system.
Pastures have their own physiological and mor-
phological characteristics, which allow the specific
adaptation for their growth and quality; however,
when changes occur under the climate conditions
(under the temperature, radiation, rainfall and its
distribution), pastures experience morphological
modifications in their yield and quality.
Milera et al. (2016) observed that the species
that showed a higher proportion with regards to the
number of established paddocks were A. gayanus,
M. maximus and C. ciliaris. In the first year of
evaluation, M. maximus showed the highest number
of paddocks under grazing, with 3, 4, and 5 rotations,
followed by A. gayanus with the highest percentage
in 3 rotations and similar result was obtained by
C. ciliaris; however, M. maximus was the one that
needed less time for recovery (38 days). In the
second year M. maximus and C. ciliaris had the
highest percentage of paddocks with 4 rotations, but
C. ciliaris needed more time of recovery (56 days)
and in the third year A. gayanus and M. maximus
reached 6 and more than 6 rotations in certain
paddocks, but C. ciliaris, besides needing more
recovery time, was below in yield and covered area.
The highest resting time coincided with the
end of the rainy season and during the dry season,
which had beneficial incidence on the accompany-
ing herbaceous legumes. On these plants the long
resting times had a beneficial effect on persistence,
seed production, germination, development and
dissemination.
Overgrazing is prevented when the necessary
resting period for recovery is used, which has direct
implication in adaptation because it increases the
species persistence, their covered area, structure,
quality; soil erosion and vulnerability of the eco-
system to the climate change were prevented, with
positive incidence on biodiversity and resilience.
The soil cannot be analyzed as independent
unit; in this case, the soil with established pasture
species and animals that consume them form an
ecosystem with biotic and abiotic characteristics.
When analyzing the general performance of
nutrients in the above-mentioned area, stability
could be observed in the system fertility and the
discharge of excreta was higher when the rotations
planned in each species were fulfilled. This is
highly important if the scarcity and high costs of
mineral fertilizers are taken into consideration, as
well as the negative consequences their use can
cause on the ecological environment.
The return of mineral elements through
the animal excretions is highly important in
sustainable systems. Soil fertility was not affected
by the grazing system or by the absence of mineral

fertilizers, and at the end of the experimental period
a trend to increase was observed in the phosphorus
and potassium contents in the areas established with
cv. Likoni and A. gayanus, respectively (Milera et
al., 2016).
In the evaluations conducted in a commercial
dairy farm, Guevara (2005) did not observe
significant variations in the pH or the K contents;
nevertheless, P showed a decrease in the paddocks
with C. nlemfuensis and an increase in the ones with
M. maximus; while the organic matter increased.
However, another important aspect to be considered
in the systems with high grazing intensity, besides
the nutrient return, is the N that returns through the
litter, because the last one is the guarantee of humus
formation.
The species showed a vertical structure
characteristic of tropical pastures [leaf (l)-stem (s)-
dead material (dm)]. In the annual average, all of
them showed a higher percentage in favor of leaves
in the strata of more than 30 cm of height, accessible
by the animals (l-72, s-2, dm-26 %). In the stratum
of 20-30 cm of height, although the leaf percentage
was also high, the dead material was favored (l-38,
s-10, dm-52 %) and showed a high component of
stems with regards to the higher stratum; and in the
0-20 cm stratum the stem and dead material were
benefitted (l-17, s-19, dm-64 %), but it was lower
than the ones reported in conventional systems by
Hernández et al. (1992).
The leaf-stem-dead material ratio in all the
studied species showed the best structure in favor
of leaves in the stratum of more than 30 cm and
in the rainy season; nevertheless, A. gayanus
and M. maximus were higher than C. ciliaris, C.
nlemfuensis and U. mutica.
In the annual average in all the strata there was
presence of dead material and on the soil, senescent
material cover. Intensive-flexible management, to
graze each paddock at the optimum moment and
with high stocking rate in one day of permanence,
had a positive effect on structure in the five species,
with no detriment for the litter.
The soil cover by different plant species and
litter, the non-application of chemical fertilizers
and the discharge of excreta due to the high instan-
taneous stocking rate, benefitted the main groups
that make up the macrofauna, which besides con-
tributing to burying the OM improve structure and
porosity.
Soil management can affect its carbon content
and lead, although paradoxical in principle, to the
soil becoming an important emitter of GHG instead
of being a carbon sink. For such reason, appropriate
practices and adequate management should be
used which recover and maintain soil potentiality
and influence the amount of carbon they can store
(Burbano-Orjuela, 2018).
The soil organic carbon (SOC) for a depth of
0,30 m is 677 Pg; 993 for 0,50 m, and 1 505 for 1 m.
Approximately 55 % of SOC lies below the depth
of 0,30 m. The agroecosystem soils have their SOC
reserves depleted and low efficiency in the use of
inputs to reach the agronomic yield (Rattan, 2018).
The agroecological management used contribu-
ted to improve the biological indicators, in the case
of soil biota, the results were very encouraging and
served as basis for other studies. The soil fauna
is an appropriate bioindicator given its relatively
sedentary habits and presence throughout the year,
its ease of measurement and its high sensitivity and
fast response to environmental stress (FAO, 2015).
A total of 540 individuals, belonging to 2 Phy-
la, 5 classes and 8 orders, were collected. At the be-
ginning of the studies in the grazing of A. gayanus,
the insects represented 75,7 % of the fauna and the
rest were oligochaetes, and at the end the quantity
of insects decreased to 35,5 %, due to the remarka-
ble increase of oligochaetes (32,1 %) and to the ap-
pearance of other groups, for example: millipedes,
isopods and arachnids (Sánchez et al., 1997).
Earthworms participate directly or indirectly in
the physical, chemical and biological soil processes;
they and macroinvertebrates also act through their
functional controls in the regulation of important
soil functions.
They select in ingestion a large quantity of
organic and mineral material, and their activity
leads to the production of structures that participate
directly in the soil physical properties such as:
increase of porosity and aeration, improvement of
water conductivity and there is better structural
stability that includes the formation of macro-
aggregates and micro-aggregates (Jiménez-Jaén et
al., 2003).
Earthworms also participate in the soil physical
structure because they produce large quantities of
organic-mineral aggregates. In a short time inter-
val, of a few hours, the earthworm digestion breaks
the organic residues and releases some nutrients,
such as nitrogen (N) and phosphorus (P), which can
then be assimilated by plants (Jiménez-Jaén et al.,
2003).

In general, grasslands have more earthworms
than the crops that leave less quantity of residue
on the field. Soil management, by not affecting the
accumulation of SOC, acts on the earthworm num-
ber and weight. The soil organic C participates in
the biological properties, basically acting as energy
source for the soil heterotroph organisms.
In general terms, the techniques that use ade-
quate soil management, such as: minimum tillage,
adequate crop rotation on the same land, mulch or
cover between crop strips, optimum use of water
in irrigation systems, application of organic and
organic-mineral fertilizers, adequate management
of resting and use of silvopastoral systems, have
caused improvements in the nutritional status of
plants and in soil care, with favorable effects on the
saving of chemical fertilizers and on the increase of
crop yield (Crespo, 2018).
Hernández et al. (2011), when evaluating in five
pasturelands with rotational management, found
that the fine roots or rootlets (< 0,2 mm of diameter)
showed significant differences in all the soil layers;
while in the detritus the differences were in the
surface layers, 0-20 cm, and the rhizomes were
found only in the 0-5 cm layer. It was observed that
in the 10-20 cm layer all the components differed
statistically.
In turn, in this same study the average under-
ground phytomass of the five studied pasturelands
was 788 g m-2
; 30,8 % belonged to the roots, 15,3 % to
the rhizomes and 53,9 % to the detritus. The com-
ponents of the underground phytomass were con-
centrated in the soil surface layers. From the total
underground phytomass 70 % was in the 0-10 cm
layer and 81 % of the total was found in the total
phytomass, in the first 5 cm; while the roots repre-
sented 81 %.
The total underground phytomass in all the
studied soil profiles fluctuated between 355 and
1 162 g m-2
. The highest values were in the erect
pasture species, M. maximus and U. mutica,
which did not differ significantly between them,
and the lowest value was shown by A. gayanus,
differing from the others. The creeping legumes C.
nlemfuensis and B. purpurascens differed between
them and from the other erect species, which shows
the particular behavior of each one of the species
that in turn belong to different plant genera. M.
maximus showed a high proportion of fine roots
that allow it higher capacity to take up water and
nutrients from the soil (Hernández et al., 2011).
Although it is not an endemic species of Cuba, it is
one of the oldest and best adapted to the different
edaphoclimatic conditions of the country.
On the other hand, Machado et al. (2011) classi-
fied 75 species from several plant families in a Voisin
rotational grazing which showed stability in general,
because some of them were not perennial and others
did not withstand the grazing intensity in the experi-
mental period. The remarkable diversity of species
that characterized the plant biomass contributed by
the grasses, the companion herbaceous legumes that
exceeded 20 % of the total existing flora and other
species which were in association with the grasses,
can be selected by the animals and remain with high-
er stability than when they constitute pure crops, or
when they are not adequately managed.
In this study, a total of 21 species were classified
in the legume family, which increased at the end of
the experimental period. In this sense, the species
Calopogonium mucunoides and Alysicarpus vagi-
nalis decreased their population, but increased their
distribution frequency in the paddocks. However,
Centrosema, Neonotonia, Teramnus and Indigofera
mucronata not only increased their population in
the paddocks where they were already established,
but also the distribution frequency (presence in
other paddocks in which they had not been present)
throughout the area.
In general, herbaceous legumes had an in-
creased evolution in the paddock and the frequency
of appearance and distribution in other paddocks
where they were not present at the beginning of the
studies. Among the most outstanding ones were:
Neonotonia wightii, Teramnus labialis and Cen-
trosema pubescens, because their seed production
occurs during the dry season, and the management
carried out facilitated the fructification and seed
maturation, as well as dissemination(Milera et
al., 2016). The species biodiversity achieved in the
system, not only increased, but also contributed to
the biomass availability and quality of the ingested
feedstuff.
According to López-Vigoa et al. (2018), when
Leucaena leucocephala and N. wigthii were includ-
ed in addition to the improved grass, a significant
increase of the ration quality and digestibility was
observed in the pastureland.
In turn, in a study conducted in forage legumes
by Gurbuz (2009), a negative correlation condensed
tannins and methane gas production (r2
= -0,882),
carbon dioxide gas production and total gas pro-
duction (r2
= -0,883 and r2
-0,867) were found,
respectively, which could be due to an effect of

methanogenesis, structural variation and biological
activity of tannins. Although the herbaceous legumes
used by this author were not the same as the ones
used by Milera et al. (2016), this is an important
aspect that should be taken into consideration in
grazing systems.
The legumes favored the selective abilities of
the grazing animals; yet, the intensification without
fertilization requires an association of grasses with
legumes in a significant percentage that has inci-
dence not only on the productive performance, but
also on the carbon capture and decrease of emis-
sions, and the best way to achieve it is with herba-
ceous and tree legumes.
When analyzing the availability according to
the resting days of the paddock, since 20 and until
60 days, it reached average values of 3,4 t DM/ha/
rotation, and with regards to the season, in the rainy
one it was higher (4,3 t DM/ha/rotation) than the
dry season (3,1 t DM/ha/rotation), with significant
differences for p<0,01. The protein contents were
between 9,11 and 9,7 % of DM, in M. maximus and
A. gayanus; C. ciliaris in the dry season reached 8,7
%. In cultivated grasses managed in conventional
grazing, if no fertilizers are applied, these results
are not achieved, which proves that it is possible
to maintain the grass-legume association and
recycling when intensive grazing is used, with
adequate management.
From the phytosanitary point of view, the con-
tribution to adaptation and mitigation of the effects
of climate change was given by the presence and
negligible affectation by insects and fungal disease
causing microorganisms, mainly. This was the re-
sult of the management which was performed on
the monocrop grasses (A. gayanus cv. CIAT-621,
M. maximus cv. Likoni, C. nlemfuensis cv. Tocu-
men, C. ciliaris cv. Formidable and U. mutica cv.
Aguada) in the intensive rational grazing system to
which they were subject, standing out the resting
time of the pastures; the stocking rate (global and
instantaneous), which implied higher trampling at a
certain moment; the grazing-browsing hours by the
animals; and the fact of assigning the same value to
the pasture species as to the cows, considering the
essential importance of this type of feedstuff in the
cattle diet.
Hence with these measures changes occurred
in the system, which in addition to exerting certain
influence on the pest organisms, for example:
existence of lower availability of leaf mass to be
consumed by the pest at a certain moment; the loss
of a host pasture par excellence for not withstanding
intensive grazing; the short resting time of some
pasture species, which causes the animal to pass
through the same paddock with higher frequency
than when it is grazed with the traditional system;
and finally, the fact of not applying fertilizers or
inorganic pesticides (Alonso et al., 2011). They could
have also favored the decrease of the emissions
by maintaining the biological balance in the
pastureland; and as there were no mass explosions
of pests that deteriorated it, given by the prevailing
plant diversity such as for example the most vigorous
pastures and weeds, within all the agrobiodiversity,
according to the criterion expressed by Nicholls
and Altieri (2017).
As the areas with species biodiversity (grasses-
legumes) and the grazing rational management
are maintained, the emission of CH4
/kg of animal
product, the soil erosion and compaction will be
lower, the species persistence will be higher and
pest affectations will be lower, which in turn has
economic implications because they improve
productions and incomes, contributing to the food
security of the family and the locality.
Conclusions
Species biodiversity in rotational grazing with-
out the application of agrotoxicals not only contributes
to healthier landscapes, but has a positive effect on
the quality and innocuousness of the ration, better
utilization of the biomass by the animals and con-
tributes to a lower quantity of phytopathogens.
The intensive rational grazing of the pasture-
land can contribute significantly in the evolution
of the edaphic biota, better utilization of organic
matter by the plants, humidity holding, prevents
compaction, has incidence on carbon capture and
holding, for which this type of management favors
the soil-plant-animal relation and resilience to the
climate change.
There are results in diversified production
systems which preserve natural resources, use
adapted plants and animals, and show productive
efficiency. The challenge is to continue working in
the transition or reconversion from conventional
systems to resilient, agroecological systems, which
allow to increase food self-sufficiency from the
diversity, quantity, frequency and innocuousness in
the productions.

Acknowledgements
The authors thank the Pastures and Forages Re-
search Station Indio Hatuey which contributed to
the conduction of these studies.
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Received: October 28, 2018
Accepted: December 29, 2018

Pastos y Forrajes, Vol. 42, No. 1, January-March, 13-22, 2019 / Adaptation of grasses associated 13
Scientific Paper
Adaptation of grasses associated with Lotus uliginosus Schkuhr in the
high Colombian tropic
Edwin Castro-Rincon1
; Juan Evangelista Carulla-Fornaguera2
and Edgar Alberto Cárdenas-Rocha2
1
Corporación Colombiana de Investigación Agropecuaria, AGROSAVIA. (Nariño, Colombia)
2
Universidad Nacional de Colombia. Facultad de Medicina Veterinaria y de Zootecnia. (Bogotá, Colombia)
Correo electrónico: ecastro@agrosavia.co
https://orcid.org/0000-0001-9841-8242
Abstract
The objective of this study was to evaluate the agronomic performance of 11 grass accessions associated with the
legume Lotus uliginosus Schkuhr, at two cutting ages, in the high Andean tropic of Colombia. The trial was conducted
at the Marengo Animal Husbandry Center, of the National University of Colombia, in the Mosquera municipality,
Cundinamarca, Colombia. Eleven grass accessions associated with the legume and a pure control (Cenchrus clandestinus
Hochst. ex Chiov), were evaluated, using a randomized block design with split-strip arrangement and three repetitions.
The biomass production was measured when cutting at 45 and 70 days in the rainy and dry seasons. In general, L.
uliginosus as well as the associated grasses showed good growth in the establishment stage. The total aerial biomass
production was higher in the rainy season and with the 70-day frequency, especially in the associations with Festuca
rubra, Festuca arundinacea, Bromus catharticus, Anthoxantum odoratum and Holcus lanatus. The grasses F. rubra,
F. arundinacea, B. catharticus and C. clandestinus (naturalized) stood out for their high aerial biomass production,
with a higher production than that of the pure control. It is concluded that the associations with the best performance
were F. arundinacea and C. clandestinus, the naturalized as well as the introduced one, which stood out for their higher
resistance to pests and diseases during the establishment stage, showing the potential of this type of system in the high
Colombian Andean tropic.
Keywords: biomass, plant establishment, forage legumes
Introduction
The agroecological zone of the high Colombian
Andean tropic, whose particular microclimate
characteristics favor specialized milk production,
dedicates 300 000 hectares to pasture production,
formed in 80 % by kikuyu (Cenchrus clandestinus
Hochst. ex Chiov), and in lower proportion ryegrass
(Lolium sp.), oat (Avena sativa L.), cock’s foot
(Dactylis glomerata L.), Yorkshire fog (Holcus
lanatus L.), clovers (Trifolium spp. L.) and alfalfa
(Medicago sativa L.) (Cardenas, 2003; Sánchez and
Villaneda, 2009).
With the above-mentioned materials studies
have been conducted in which new forages (native
and introduced) have been evaluated, in order to
maintain high good-quality biomass productions
throughout the year at minimum costs, searching
for species with low fertilization requirements,
high resistance to pests and diseases, adaptation,
persistence and palatability (Ochoa et al., 2016).
In Colombia different pastures have been
evaluated such as ryegrass (Lolium sp.), clovers, cock’s
foot and kikuyus, and a large variety of materials
adapted to the conditions of the high Colombian tropic
has been reported (Cadena et al., 2019).
Among the evaluated foreign materials Lotus uligi-
nosus Schkuhr stands out, promising legume for its
establishment in dairy systems in the high Colombian
Andean tropic; thus, it has been proposed in several
evaluation studies, in associations with grasses (Castro
et al., 2009; Santacoloma-Varón et al., 2017).
In this sense, Morales et al. (2013) found good
performance in the milk quality and production in
cows that grazed in an association of F. arundina-
cea and L. uliginosus under conditions of the high
Colombian tropic, when comparing it with pure C.
clandestinus pastures.There are no abundant re-
cords of evaluated and released materials for the
country in the case of associations of grasses and
legumes.
The objective of this research was to evaluate
the agronomic performance of 11 grass accessions
associated with the legume Lotus uliginosus, at two
cutting ages, in the high Colombian Andean tropic.
Materials and Methods
The trial was conducted at the Marengo Animal
Husbandry Center, located on the San José lane,
Mosquera municipality, Cundinamarca, Colombia.

14 Pastos y Forrajes, Vol. 42, No. 1, January-March, 13-22, 2019 / Edwin Castro-Rincon
The municipality is located at 4º 42’ North latitude
and 74º 12’ West longitude, at an altitude of 2 650
m.a.s.l. The average temperature is 13 ºC, with
fluctuations between 0 and 20 ºC, and presence of
frost in January, February and early August. The
mean annual rainfall is 528,9 mm. The months with
average rainfall equal to or higher than 50 mm were
considered as the rainy season, that is, April-May
and October-November; while in the dry season the
months in which at least 50 mm as average did not
occur were included, and it was in correspondence
with January, February and June.
The soils belong to the Tibaitatá series, which
have been formed from heterogeneous materials
with variable influence from volcanic ashes. They
show little evolution, are generally deep, vary
from well-drained to poor-drainage ones and have
moderate fertility. Their texture is clay loam, pH is
4,9 and organic matter, 6,8 % (Orduz, 2014).
Treatments and experimental design. Eleven
grass accessions associated with L. uliginosus
and a pure control (Cenchrus clandestinus) were
evaluated. The accessions were selected from the
Plant Genetic Resource Unit (PGRU) of the National
University. The evaluated accessions were: Bromus
catharticus (Vahl) var. Banco, Festuca arundinacea
(Schreb.) var. Festorina, Dactylis glomerata (L.) var.
Knaulgrass, Festuca pratense (Huds) var. Preval,
Holcus lanatus (L.) (naturalized), Anthoxantum
odoratum (naturalized), Cenchrus clandestinus
(pure control, naturalized), Phleum pratense,
Cenchrus clandestinus 1 (naturalized), Cenchrus
clandestinus 2, (introduced), Dactylis glomerata,
var. Varaula, all of them associated with Lotus
uliginosus (legume to be associated). A randomized
block design was used with split-strip arrangement,
where the experimental unit was each plot that
contained each accession. The main plot was the
accession and the strip, the cutting frequency (45
and 70 days). The trial had a total of 12 plots, each
one with three repetitions.
Land preparation. It was prepared with one
month of anticipation, using rotovator once, chisel
plow twice and light harrow twice; at the same
time the soil acidity was corrected with 2 000 kg
of lime/ha. The total area of the experiment was
950 m2
, with plots of 2,5 by 5 meters with 1 meter
distance between plots, where 5 rows of grass and
two alternate rows of legume were planted through
seedlings from greenhouse.
Post-establishment stage. One hundred eighty
days after sowing the plots, a homogenization
cut was made with scythe at 10 cm from the soil,
simulating grazing. From this moment the post-
establishment stage was considered as started, in
which cuttings were performed every 45 and 70
days, according to the recommendations made
for the species by Correa et al. (2016) and Vargas-
Martínez et al. (2018).
Fertilization. The fertilization recommended for
the establishment of pastures, according to the report
by Bernal-Eusse (1994) and Silva (1986), was used.
N was applied (50 kg/ha; applied only to the pure
control) upon establishment, K (25 kg/ha), P (30 kg/
ha) and Mg (25 kg/ha). The control was fertilized af-
ter each production cutting with 50 kg N/ha.
Measured variables
During the establishment. The period com-
prised between transplant and the following 180
days was considered as establishment, in which
the following variables were measured every eight
days:
• Vigor: In scale from 1 to 5, which was conside-
red as the degree of adaptation to the environ-
ment (1: very low, 2: low, 3: regular, 4: good, 5:
excellent).
• Legume cover (%): Area covered by the plant in
a 1-m2
frame.
• Grass height (from the soil to the petiole of the
tallest leaf) and radius of the legume (cm; from
the center to the distal end).
• Pests and diseases: In scale from 0 to 5; 0: wi-
thout affectations and 5: severely affected.
• Phenology (grasses): Beginning of flowering:
when 30 % of the plot showed inflorescence, it
was determined that the grass started flowering.
Beginning of the maturation stage: when 30 % of
the plot showed mature seed.
Forage production (kg DM/ha). The forage
production was measured in the post-establish-
ment stage. In this sense, for the cuttings of aerial
biomass production each experimental unit or plot
was divided into two halves, where the first was cut
at 45 days and the second at 70 days after the ho-
mogenization cutting. The forage contained within
a frame of 1 square meter was cut and it was then
estimated for one hectare.
Statistical analysis. The qualitative variables
(all the adaptation and phenology ones) were ana-
lyzed through descriptive statistics; while the bio-
mass production was analyzed by the PROG GLM/
ANOVA (SAS V 9.2). The means were compared
through Tukey’s test.

Results
Establishment stage
Vigor. The grasses F. rubra, D. glomerata, and
C. clandestinus (control) stood out for their vigor;
while B. catharticus and P. pratense showed the
lowest values. L. uliginosus showed similar values
independently from the grass, except when it was
associated with H. lanatus and D. glomerata var.
Knaulgrass, where the vigor was higher (table 1).
Grass height. The average height was 27,3
cm, with significant differences among accessions
(p<0,001), being higher in B. catharticus and H. la-
natus with 39,7 and 42,3 cm, respectively; while A.
odoratum and D. glomerata var. Knaulgrass showed
the lowest height (20,0 and 21,3 cm, respectively).
Pests and diseases. The highest incidence of
pests and diseases was shown in F. pratense and
D. glomerata, associated to the rust (Puccina sp.)
attack; while in B. catharticus and H. lanatus
damage on the inflorescence was observed, related
to smut. In L. uliginosus a damage caused by slugs
was visible at the beginning of the establishment
stage,butitdidnothavehighincidenceandtheplants
recovered quickly. For the case of C. clandestinus
no pest and disease incidence appeared (table 1).
Legume cover. In all the accessions good
legume cover was shown, with an average value
of 90 %, and the association with P. pratense, A.
odoratum and F. pretense stood out with 95 % of
cover; however, the association with D. glomerata
reached 75 % of legume cover (table 1).
Phenology. Only eight of the 11 grass
accessions associated with L. uliginosus showed
flowering; B. catharticus and H. lanatus were the
ones that had a shorter period between transplant
and the emergence of inflorescence, with 28 days.
The grass with the largest period was D. glomerata,
with 49 days before flowering; while in the
control C. clandestinus (naturalized, control), the
associations with C. clandestinus (introduced) and
C. clandestinus (naturalized), the early flowering
emergence was not visible, and thus it was not
evaluated. For the case of the presence of mature
Table 1. Measured variables during the establishment in the grass accessions associated with L. uliginosus
and the Cenchrus clandestinus control.
Treatment
Grass Legume
Height2
, cm P & D3
Vigor4
Vigor Cover5
, % P & D
1
C. clandestinus (naturalized, control) 24,0def
0,0 4,6 - - -
B. catharticus 39,7a
1,6 4,3 4,7 90 0,0
F. rubra 25,7bcd
0,0 4,8 4,6 90 0,3
D. glomerata 29,0b
2,1 4,7 4,7 75 0,1
F. arundinacea 28,7bc
0,3 4,6 4,7 90 0,0
P. pratense 26,0bcd
0,0 4,2 4,7 95 0,0
C. clandestinus 1 (introduced) 23,4def
0 4,5 4,7 90 0,1
A. odoratum 20,0f
0,0 4,5 4,6 95 0,0
H. lanatus 42,3ª 1,5 4,5 4,7 90 0,0
D. glomerata (var. Knaulgrass) 21,3ef
0,0 4,5 4,7 90 0,0
F. pratense 23,0def
2,2 4,4 4,6 95 0,0
C. clandestinus 2 (naturalized) 24,7cde
0,0 4,5 4,7 90 0,0
Mean 27,3 0,6 4,5 4,7 90 0,04
SE ± 0,88*** - - - - -
1
There was no bug incidence, but there were frosts in the establishment stage.
2
Height at the end of the establishment stage (180 days since sowing).
3
P & D =Incidence of pests and diseases from 0 to 5 (0: without affectations and 5: severely affected).
4
Vigor in scale from 1 to 5 (1: very low, 5: high).
5
Cover at the end of the establishment stage
Different means in the same column significantly differ at p<0,05, according to Tukey’s test. ***p<0,001.

seed H. lanatus stood out with the shortest time
period in seed maturation, which occurred 14 days
after flowering.
Post-establishment stage
Grass height in the rainy season. For this
variable in the regrowth frequency of 45 days an
average of 31,3 cm was obtained, where signifi-
cant differences were observed among the grasses
(p<0,001). B. catharticus stood out with 57,5 cm;
while A. odoratum and F. pratense showed the low-
est values (16,5 and 17,0 cm, respectively). In the re-
growth frequency of 70 days, a height was reached
in the grasses of 37,0 cm with significant differences
(p<0,001); the highest value was 63,5 cm in B. ca-
tharticus and had the lowest value (19,0 cm) in F.
pratense (table 2).
Grass height in the dry season. During the dry
season, the average height at 45 days of regrowth
was 27,5 cm, with significant differences (p<0,001)
among grasses. The highest height was reached by
B. catharticus (51,5 cm). In turn, P. pratense and
C. clandestinus (introduced) showed the lowest
values (15,0 and 15,5 cm, respectively). In the re-
growth frequency of 70 days significant differences
(p<0,001) also appeared and average height of 32,3
cm was achieved. In turn, B. catharticus stood out
with a height of 58,5 cm and C. clandestinus (intro-
duced), with the lowest height (table 2).
Total aerial biomass production in the rainy
season (RS). In the cutting frequency of 45 days a
total aerial biomass production in the RS of 1 347 kg
DM/ha was observed, with significant differences
for p<0,001 (table 3), and the associations with F.
rubra, F. arundinacea and A. odoratum stood out
with 1 764, 1 745 and 1 680 kg DM/ha, respectively;
meanwhile, C. clandestinus (naturalized, control)
showed the lowest production with 624 kg DM/ha.
For the 70-day frequency, the total aerial
biomass production in the RS had a value of 1 476 kg
DM/ha; significant differences (p<0,001) were
observed for production among grass species (ta-
ble 4). F. rubra and F. arundinacea reached the
highest productions with 2 576 and 2 365 kg DM/ha,
respectively; while F. pratense showed the lowest
one with 168 kg DM/ha. On the other hand, C.
clandestinus(naturalized,control)showedavalueof
1 834 kg DM/ha, which was above the average.
Significant differences (p < 0,001) were found in
the legume cover in the associations, standing out
Table 2. Average height (cm) in the grass accessions associated with L. uliginosus
and the Cenchrus clandestinus control, during the rainy and dry seasons.
Treatment
Rainy season Dry season
45 days 70 days 45 days 70 days
C. clandestinus (naturalized, control) 25,5c
31,0c
22,5c1
25,5d
B. catharticus 57,5a
63,5a
51,5a
58,5a
F. rubra 38,0b
46,0b
34,0b
39,5c
D. glomerata 40,5b
46,5b
39,5b
46,5b
F. arundinacea 41,0b
48,5b
33,5b
38,5c
Ph. pratense 19,0cd
20,5ef
15,0d
19,0e
C. clandestinus 1 (introduced) 22,5cd
28,5cd
15,5d
19,5e
A. odoratum 16,5d
22,5ef
17,0cd
23,5ed
H, lanatus 52,0a
62,5a
46,0a
51,0b
D. glomerata (var. Knaulgrass) 20,0cd
24,5de
18,5cd
21,0ed
F, pratense 17,0d
19,0f
17,0cd
20,0e
C. clandestinus 2 (naturalized) 25,5c
31,5c
20,5cd
25,5d
Mean 31,3 37,0 27,5 32,3
SE ± 0,39*** 0,28*** 0,31*** 0,26***
Different letters in the same column significantly differ for p<0,05, according to Tukey’s test.
***(p<0,001).

Table 3. Total aerial biomass production (kg DM/ha)1
in the grass accessions associated
with L. uliginosus and the C. clandestinus control during the rainy season2
.
Treatment
Cutting frequency
45 days 70 days Sig3
C. clandestinus (naturalized, control) 624b4
1 834bc4
S
B. catharticus + Legume 1 200ab
2 959abc
S
F. rubra + Legume 1 764a
3 974ab
S
D. glomerata + Legume 982ab
2 692abc
S
F. arundinacea + Legume 1 745a
3 793ab
S
P. pratense + Legume 1 417ab
2 224abc
S
C. clandestinus 1 (introduced) + Legume 1 424ab
1 685bc
NS
A. odoratum + Legume 1 680a
4 583a
S
H. lanatus + Legume 1 371ab
3 465abc
S
D. glomerata (var. Knaulgrass) + Legume 1 311ab
1 220c
NS
F. pratense + Legume 1 279ab
1 817bc
S
C. clandestinus 2 (naturalized) + Legume 1 365ab
1 329c
NS
Mean 1 347 2 631 ***
SE ± 66,26*** 214,57***
1
kg DM/ha= kg DM of grass + kg DM of legume, 2
average production of one cutting per each
frequency, 3
Significance = Indicates whether there is (S) or there is not (NS) significant difference
between cutting frequencies for each accession.
Different letters in the same column differ for p<0,05 according to Tukey’s test, ***p < 0,001.
Table 4. Biomass production of the grass (kg DM/ha)1
and legume percentage during the rainy
season2
.
Treatment
Cutting frequency
45 70
kg DM/ha Legumes, % kg DM/ha Legumes, %
C. clandestinus (naturalized, control) 624abcd2
- 1 834abcd2
-
B. catharticus 625abcd
57,3abc2
1 861abcd
50,9cd2
F. rubra 1 003a
60,6abc
2 576a
50,1cd
D. glomerata 812abc
22,1d
2 156abc
27,8d
F. arundinacea 805abc
64,1abc
2 365ab
48,9cd
Ph. Pratense 261d
35,0cd
837ab
-
C. clandestinus 1 (introduced) 605abcd
62,8abc
868abcd
57,3bcd
A. odoratum 832ab
62,2abc
2 318ab
58,4d
H. lanatus 822abcd
48,bcd
1 992abcd
45,8abcd
D. glomerata (var. Knaulgrass) 431bcd
79,9ab
475bcd
67,8c
F. pratense 328cd
81,2ab
168d
94,4abc
C. clandestinus 2 (naturalized) 952a
35,4cd
753abcd
53,0d
Mean 667 60,8 1 476 58
SE ± 44,57*** 3,77*** 168,07*** 3,44***
***(p<0,001), 1
kg DM/ha= kilograms of grass DM, 2
average production of one cutting per each frequency.

in F. pratense with the highest percentage (94,4 %)
and in D. glomerata with the lowest (27,8 %).
Total aerial biomass production in the dry sea-
son (DS). Regarding the total aerial biomass pro-
duction in the DS in the cutting frequency of 45
days, an average of 663 kg DM/ha was obtained,
and significant differences were found (p<0,001)
among associations (table 5). F. pratense and B.
catharticus stood out for their higher productions,
with 988 and 832 kg DM/ha, respectively; while C.
clandestinus (naturalized, control) had the lowest
production with 348 kg DM/ha.
At the 70-day cutting frequency a grass aerial
biomass production of 1 184 kg DM/ha was ob-
tained; and significant differences (p<0,001) were
found among the associations (table 5). The highest
biomass productions were recorded in B. catharti-
cus and H. lanatus with 2 009 and 1 721 kg DM/
ha, respectively; while the lowest production was
obtained in the association with C. clandestinus
(introduced).
When comparing the total aerial biomass pro-
duction between the two cutting frequencies, sig-
nificant differences (p<0,01) were found (table 5).
The biomass production in the 70-day cutting was
higher, except in P. pratense, C. clandestinus 1 (in-
troduced), D. glomerata (var. Knaulgrass) and F.
pratense.
Aerial biomass production in the grass in the
DS. In the grass biomass production during the
DS in the cutting at 45 days, an average of 510 kg
DM/ha was obtained, with significant differences
(p<0,001) among accessions (table 6). The highest
production was obtained in B. catharticus being the
one with the (741 kg DM/ha); while P. pratense had
the lowest production, with 337 kg DM/ha. For the
legume cover significant differences (p<0,01) were
found among the associations (table 6). The aver-
age was 33,2 %, in which the legume stood out in
the association with D. glomerata var. Knaulgrass
(54,1 %); while the associations with B. catharticus
and D. glomerata had the lowest values (15,0 and
15,9 %, respectively).
In the 70-day frequency a general average of
917 kg DM/ha was reached, with significant diffe-
rences (p<0,001) for the grass biomass produc-
Table 5. Total aerial biomass production (kg DM/ha)1
in the grass
accessions associated with L. uliginosus and the Cenchrus
clandestinus control during the dry season2
.
Treatment
Cutting frequency
45 days 70 days Sig3
C. clandestinus (naturalized, control) 348c4
1 182abcd4
S
B. catharticus + Legume 832ab
2 009a
S
F. rubra + Legume 804ab
1 682abc
S
D. glomerata + Legume 772abc
1 114abcd
S
F. arundinacea + Legume 776abc
1 715ab
S
Ph. pratense + Legume 639abc
865bcd
NS
C. clandestinus 1 (introduced) + Legume 501bc
578d
NS
A. odoratum + Legume 545bc
1 152abcd
S
H. lanatus + Legume 647abc
1 721ab
S
D. glomerata (var. Knaulgrass) + Legume 596abc
675d
NS
F. pratense + Legume 988a
768cd
NS
C. clandestinus 2 (naturalized) + Legume 507bc
749d
S
Mean 663 1 184*** **
SE ± 40,63*** 76,48***
** p < 0,01, ***p < 0,001. 1
(kg DM/ha) = kg DM of grass + kg DM legume.
2
Average of two cuttings. 3
Sig = Indicates whether there is (S) or there is not (NS)
significant difference between cutting frequencies for each accession (P<0,05). 4
The
means followed by equal letters in the same column are not significantly different (P<0,05),
according to Tukey’s test.

tion (table 4). B. catharticus (1 537 kg DM/ha),
F. arundinacea (1 512 kg DM/ha) and H. lanatus
(1 421 kg DM/ha) stood out with the highest produc-
tions; while C. clandestinus (int.) and D. glomerata
(var. Knaulgrass) had the lowest production with
350 and 370 kg DM/ha. C. clandestinus (naturalized,
control) showed a production above the average
with 1 182 kg DM/ha.
Regarding the legume proportion, significant
differences (p < 0,001) were found among associations
(table 6), with an average of 33,2 and 32,9 % for the
cutting frequency of 45 and 70 days, respectively.
The legume stood out when it was associated with
D. glomerata (var. Knaulgrass), with the highest
proportion, followed by P. pratense and F. pratense;
while the one associated with D. glomerata showed
the lowest proportion.
Discussion
Establishment stage. During the establishment
stage all the accessions were observed to have an
adequate adaptation to the environment, but in the
treatments with F. arundinacea and D. glomera-
ta, the grass showed problems due to Puccina sp.
(rust), which coincides with reports made in other
studies, where it is proven that these materials and
other grasses are susceptible to rust incidence (No-
votná et al., 2017).
On the other hand, L. uliginosus showed an
excellent performance in this stage; although it
showed slow growth during the beginning of the
experiment, which coincides with the report by
Marley et al. (2006), under similar conditions to
the ones in this study (Castillo et al., 2017). The
difference in the values found for grass height at the
end of the establishment stage is directly associated
with the growth physiology of each species (because
grasses of erect, semi-erect and prostrate habit were
evaluated), which is reflected on the results that
were reached in such variable (table 1).
Regarding the legume cover, it was observed
that all the accessions, with the exception of D.
glomerata, had good growth. In some cases the
grass was partially displaced, as in the associations
with P. pratense, F. pratense and A. odoratum. This
is related to the prostrate growth habit of this legume
(Castillo et al., 2017).
Table 6. Aerial biomass production of the grasses (kg DM/ha)1
and proportion of legume (%)
during the dry season2
.
Treatment
Cutting frequency
45 70
kg DM/ha Legume ,% kg DM/ha Legume, %
C. clandestinus (naturalized, control) 348de3
- 1 182abc3
-
B. catharticus 741a
15,0c3
1 537a
23,9ab3
F. rubra 645abc
25,3bc
1 307ab
28,8ab
D. glomerata 690ab
15,9c
1 031abcd
9,3b
F. arundinacea 599abcd
22,5bc
1 512a
18,3ab
P. pratense 337e
41,7abc
482cd
48,2a
C. clandestinus 1 (introduced) 360d
49,6ab
350d
44,3a
A. odoratum 394d
38,5abc
791abcd
39,0ab
H. lanatus 499bcd
28,3abc
1 421a
24,1ab
D. glomerata (var. Knaulgrass) 346e
54,1a
373d
49,2a
F. pratense 726a
47,8ab
443cd
47,8a
C. clandestinus 2 (naturalized) 437cd
23,2bc
574bcd
27,6ab
Mean 510 33,2 917 32,9
SE ± 33,52*** 2,92** 68,78*** 2,53**
NS
Not significant (p>0,05), **(p<0,01), ***(p<0,001).
1
(kg DM/ha) = kg of grass DM, 2
Average of two cuttings, 3
The means followed by equal letters in the same
column are not significantly different (P<0,05), according to Tukey’s test.

With regards to the grass phenology, the spe-
cies such as B. catharticus, H. lanatus and F.
arundinacea showed early flowering and abundant
seed production, characteristic that can be useful to
multiply these materials in field and identify the op-
timum moment of pasture grazing and management
during the establishment.
Post-establishment stage
Total aerial biomass production. The asso-
ciations showed higher biomass production in the
two cutting frequencies during the rainy season. In
turn, in biomass production at 45 days in the RS
higher values were observed than those reported by
other authors, such as Corredor (1986), who found
productions of 930 kg DM/ha in F. arundinacea
associated with T. repens in this period.
In some studies the reports are expressed in
kg DM/ha/year; the possible number of cuttings
to be performed during the year according to the
frequencies should be taken into consideration. In
this sense, cuttings every 45 days represent 8,1 cut-
tings/year that are equivalent to 10 910,7 kg DM/
ha/year; while cutting every 70 days is equivalent
to 5,2 cuttings/year, which would mean 13 681,2 kg
DM/ha/year.
Taking the above-explained facts into con-
sideration, reports have been found like those by
Mendoza (1988), with productions of up to 14 440
kg DM/ha/year in C. clandestinus associated with
T. repens, higher value than the ones observed in
this study, in the DS as well as the RS (table 3).
Nevertheless, under grazing conditions these spe-
cies are used with lower resting periods than 35
days in some cases (Posada-Ochoa et al., 2013; Di-
maté-Gil, 2016).
On the other hand, Navas (1972) observed in
B. catharticus, associated with T. repens values
of 2 400 kg DM/ha/year, lower value than the one
found in the DS for the two cutting frequencies
of this study. Also in the Mosquera zone, Cundi-
namarca, in Colombia, productions are reported in
F. arundinacea pastures with L. uliginosus of up
to 2 282 kg DM/ha with cuttings at 45 days, higher
than the value observed in this study (Castro et al.,
2009), but lower than others reported in the western
Bogotá Savanna in P. clandestinus + L. uliginosus
pastures (4 012 kg DM/ha) and in F. arundinacea +
L. uliginosus (4 168 kg DM/ha), which was due to
the fact that they were well prepared and adequately
fertilized lands (Morales et al., 2013).
In other zones, Leep et al. (2002), found in F.
arundinacea associated with L. corniculatus 1 000
kg DM/ha/year; on the other hand, in L. uliginosus
used as protein bank for grazing in sheep, higher
productions than 1 300 kg de MS/ha have been
found (Piaggio et al., 2015).
Biomass production in the grass. ICA (1987)
suggested that under good management conditions
F. arundinacea and B. catharticus can produce
from 20 000 to 30 000 kg DM/ha/year, which coin-
cides with more recent studies (Castro et al., 2009;
Morales et al., 2013).
In other latitudes similar, or sometimes high-
er, productions have been reported than the ones in
this study for the pure grasses; this is the case of F.
arundinacea, with productions between 2 500 and
3 500 kg DM/ha (Malcolm et al., 2015; Carlsson et
al., 2017). Also in Dactylis glomerata, alone and as-
sociated with Lolium perenne and Trifolium repens,
productions have been observed of 20 100 kg DM/
ha/year in the mixture and 12 100 kg DM/ha/year
in monocrop, which proves the benefit of associat-
ing grasses and legumes (Maldonado et al., 2017).
Most of the reported productions of the grass
are higher than the ones in this study; however, it
must be taken into consideration that the sampling
was carried out jointly for the grass and the legume
per square meter, and not for the grass alone as in
the control. This explains the inferiority of the pro-
ductions with regards to studies conducted in pure
grasses. However, when comparing the total aerial
biomass production of this study, in most cases it
was higher than the ones reported in other works.
Regarding the legume proportion, it was higher
in the rainy season than in the dry season, which
coincides with the report by Corredor (1986), with
29,7 % in the DS and 44,8 % in the RS in association
of T. repens with F. arundinacea. In addition, the
low proportion of legume in the association with
D. glomerata can be directly related to allelopathic
factors of the grass on this species, as reported by
Chung and Miller (1995) and Aldana et al. (2016) in
different grasses, including F. arundinacea and D.
glomerata, where they observed how the aqueous
extracts of these plants affected the germination and
growth of M. sativa crops. In other studies similar
or higher productions are reported with legume
proportions from 30 to 50 %, using D. glomerata
alone and associated with L. perenne and T. repens,
with production of up to 21 000 kg DM/ha/year in
the proportion of 40 % legume (Rojas-García et al.,
2016).

Conclusions
The associations that had the best performance
were F. arundinacea and C. clandestinus, the
naturalized as well as the introduced one, which
stood out for their higher resistance to pests and
diseases during the establishment stage, showing
the potential of this type of system as a viable
option for the high Colombian Andean tropic.
The associations that stood out for their
higher aerial biomass production were F. rubra,
F. arundinacea, and A. odoratum, for the two
evaluated cutting ages. The legume proportion
was higher than 40 % in all the associations for the
two cutting frequencies, except with D. glomerata,
where it was barely found in 25 %, value below the
average in each season.
Acknowledgements
The authors thank the National University, Bo-
gotá campus, for funding this study, as well as the
Colombian Corporation of Animal Husbandry Re-
search (AGROSAVIA) for co-authoring it.
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Received: October 26, 2018
Accepted: December 4, 2018

Pastos y Forrajes, Vol. 42, No. 1, January-March, 23-29, 2019 / Plant size and substrate type on Morus alba 23
Scientific Paper
Influence of in vitro plant size and substrate type on the acclimatization
of Morus alba L.
Ángel Espinosa-Reyes, Juan José Silva-Pupo, Marisel Bahi-Arevich and Dariannis Romero-Cabrera
Centro de Estudios de Biotecnología Vegetal, Facultad de Ciencias Agropecuarias, Universidad de Granma,
Carretera a Manzanillo km 17½, CP 85100, Bayamo, Granma, Cuba
E-mail: aespinosar@udg.co.cu
https://orcid.org/0000-0002-9918-641X
Abstract
The objective of this study was to evaluate the influence of size and substrate type on the acclimatization of in
vitro Morus alba L. plants. For such purpose, three trials were conducted: in the first one in vitro plants were evaluated
grouped according to their sizes, which constituted the treatments (T1: 1,5-2,5 cm; T2: 2,6-3,5 cm and T3: higher
than 3,5 cm); in the second one, plants with a length between 2,5 and 3,0 cm were selected and three formulations of
substrate mixtures were evaluated: T1: soil (70 %)-cattle manure (20 %)-zeolite (10 %), T2: soil (45 %)-cattle manure
(45 %)-zeolite (10 %), T3: soil (90 %)-zeolite (10 %); and in the third trial the substrate type was evaluated on the in
vitro plant growth under nursery conditions, with the same treatments. A simple variance analysis was carried out, and
Tukey’s multiple range comparison test was applied for p ≤ 0,05. The statistical package Infostat (2017) on Windows®
was used. The increases in sprout length and survival (93,6 %) were significantly higher when in vitro plants from
1,5 to 2,5 cm long were used. Using the substrate mixtures T1 and T2 a survival higher than 80 % was obtained; just
like higher length, number of leaves and leaf size in the in vitro plants in the initial stage of acclimatization and under
nursery conditions. It is concluded that with the use of in vitro plants of size between 1,5 and 2,5 cm and the substrates
of treatments T1 and T2 during acclimatization, high survival and higher growth and development of the M. alba
plants were achieved.
Keywords: growth, tissue culture, survival, in vitro plants
Introduction
Mulberry (Morus alba L.) was introduced in
Cuba with forage purposes for animal feeding,
and it has been proven to have excellent nutritional
qualities for feeding different animal species (No-
da-Leyva y Martín-Martín, 2017). This plant has
high adaptive capacity to different edaphoclimatic
conditions, can produce between 10 and 12 t of dry
matter per hectare per year, contains from 20 to
25 % crude protein, and dry matter digestibility is
higher than 80 % (Martín et al., 2014). In addition, it
is renowned for its commercial value in the cosmetic
and medicinal industry, and its physical-chemical
antioxidant and hypoglycemic properties have been
widely used in drug production (Huh et al., 2017).
Mulberry propagation is generally done by
stakes; however, depending on the cultivar there
are certain aspects –such as the low survival and
multiplication rate, as well as the rooting difficul-
ty– which limit the propagation of this plant species
with productive purposes (Castro-Ramírez, 2010).
The in vitro propagation of plant species has
emerged as a valuable alternative for the propaga-
tion of species of economic and ornamental interest,
because it allows the production of large quantities
of plants in a relatively short time period; it is an
excellent tool for the preservation and recovery of
species that have significantly decreased their popu-
lations and for breeding.
Mulberry breeding programs are aimed at the
increase of foliage yields. Nevertheless, due to the
high heterogeneity and long periods for plant re-
generation, conventional breeding techniques are
limited; for such reason it has been necessary to
complement them with modern biotechnological
techniques, such as tissue culture, molecular DNA
recombination techniques and molecular markers
(Vijavan et al., 2014).
The success of tissue culture techniques de-
pends essentially on having a well-established
protocol that includes the different stages of the
process, such as plant propagation, rooting and ac-
climatization (Resende et al., 2015).
In Cuba diverse research works have been con-
ducted aimed at establishing an efficient protocol
of in vitro mulberry propagation, among which the
ones conducted by Salas et al. (2005) and Salas

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