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by Lentiviral Vector
S UN Ke -ning
, ZH U H ua -bin , LIN Feng , WANG Dong , H AO H ai -sheng ,
DU Wei -hua 1, ZHAO Xue -ming 1
(1. Institute of A nimal Sciences , Chinese A cademy o f Ag ricultural Sciences , Beijing 100193, C 2. Co llege o f
Animal Science and V eterina ry M edicine , Henan A g ricultur al U nive rsity , Zheng zho u 450002, China )
　　A bstract :In or der to increase the quality o f transg ene vec to rs and the transg enic efficiency , the co nstr uctio n and the dev el -o pment cour se of the lentiviral v ecto r w ere rev iewed . In o rder to increase the biosafety , leng then the loaded frag ment o f targ et DN A , and ex tend the ho st cell o f the le ntiviral vecto r , all accesso ry genes o f lentivirus w ere deleted , cer tain ex og eno us reg ula -to ry sequences w ere intro duced , and the envelo pe glyco pr otein w as replaced also , w hich gives the lentivira l v ector a br oad r ang e of tar get cells and a higher biosafety , transfe r efficiency and expre ssio n efficiency . What ' s mo re , the develo ped transfection me tho d laid the founda tion for integ ra tion and optimization o f transgenic technolog y . T he integ ra tion and optimize between lentiviral vecto r and o ther transgene technologies will help to create a simple , ef ficient and eco nomical transg ene technology . 　　Key words : l strategy of v ector co nstr uctio n
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寻找更多 ""1Department of Fitotecnia, State University of North Fluminense Darcy
Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
2Department of Zootecnia, State University of North Fluminense Darcy
Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil
Email: .br
Copyright & 2014 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License
http://creativecommons.org/licenses/by/4.0/
This study was conducted to evaluate the physiological quality of forage seeds after
passage through the gastrointestinal tract of cattle, as well as the emergence of
seedlings derived from these seeds. Three Fabaceae species were used: tropical kudzu,
leucaena and calopo. One hundred grams of seeds of each species were offered, separately,
to five bovines. After the start of the dispersion, the faeces of the animals were
collected every 6 hours, in a total of 72 hours, being taken to the greenhouse immediately
after each collection. The study evaluated the total number of seedlings in the
stool, the lengths of roots and aerial parts, as well as the natural and dry seedling
weight thirty days after the disposal of the faeces in the greenhouse. The experimental
design consisted of randomized blocks, with twelve treatments (sampling periods)
and five replications (cattle). The best performance for the total number of seedlings
in the faeces of cattle was observed in the kudzu species. The length of the root,
length of the aerial part and dry matter seedling weight was observed in the leucaena
species, followed by kudzu. The calopo showed low results for all variables due
to the high degradation of the seeds.
Keywords:Pueraria phaseoloides, Leucaena leucocephala, Calopogonium
mucunoides, Dispersal, Physiological Quality
1. Introduction
Domestic animals, such as bovines, contribute to the maintenance of ecosystems,
providing services such as seed dispersal and nutrient cycling or simply assuring
their economical sustainability
Seed dispersal has its importance to the diversity of our pastures. The inclusion
of Fabaceae in Poaceae—exclusive pastures represents a food source from a nutritional—for
they present high protein content and good digestibility—and strategic—for
they store green food in the dry season—point of view. Besides these, other advantages
can be mentioned, such as nitrogen fixation, nutrient recycling and the recovery
of pastures or degraded areas.
However, a major obstacle in the adoption of mixed pastures of Poaceae and Fabaceae
is the cost of their deployment to the operating system
Studies that emphasize seed dispersal by animals in pastures have real importance,
for not much is known about this process. Furthermore, there is still little knowledge
about the proportion of seeds that remain viable after passing through the digestive
tract of animals
In the process of seed ingestion by animals, damages are caused through chewing
and digestion, and, in these conditions, a higher or lower percentage of germination
depends on the size and shape of the ingested seeds. Reference
states that preliminary studies have shown that small, rounded seeds
have a higher likelihood of surviving ingestion and passing through the gastrointestinal
tract (GIT) than bigger, elongated seeds.
In addition to the factors regarding the passage through GIT, seed germination in
faecal plates can be influenced by the fermentation of the stool sample, by high
fungal or bacterial contamination, by the positioning of the seeds in the faecal
matter, and by the dehydration of the faecal matter.
The seeds that survive the passage through GIT are benefitted by the great amount
of nutrients available in the faeces
. Subsequently,
these nutrients are still useful to seedlings. On the other hand, seed germination
and seedling establishment can be inhibited due to the toxicity and hydrophobic
nature of the faecal matter
The importance of natural sowing is acknowledged in the renovation and persistence
of the species in pastures. Therefore, it is necessary to understand more deeply
the behavior of seeds excreted by bovines in order to evaluate the dynamics of forage-seed
dissemination
According to these considerations, this research was conducted with the objective
of evaluating the physiological quality of seeds of the forages: tropical kudzu
(Pueraria phaseoloides Benth), leucaena (Leucaena leucocephala), and calopo (Calopogonium
mucunoides), after the passage through GIT, as well as the emergence of seedlings
from these seeds.
2. Material and Methods
This study was carried out with seeds from three forage species of the Fabaceae
family: tropical kudzu (Pueraria phaseoloides Benth), leucaena (Leucaena leucocephala),
and calopo (Calopogonium mucunoides), whose plots were previously analyzed regarding
germination and vigor, following the recommendations of the Rules for Seed Testing
Five bovines, with average weight of 238 kg, were accommodated in individual stalls
to facilitate the collection o faeces. The period for the animals to adapt to the
housing and diet alterations was of seven days. The feeding was carried out individually,
following a 90:10 voluminous-concentrate ratio, with 1 kg/day of corn meal and soy
meal concentrate, 10 kg/day of tifton grass hay—which was divided into two daily
feedings—, and water at will. The diet was balanced following the nutrient requirements
of small ruminants—NRC— to suppress the maintaining
requirements.
One hundred grams of seeds of each Fabaceae were mixed with 1 kg concentrate and
given to the animals, once a day, in the morning (6:00 a.m.) on the first day only.
This amount corresponds to, approximately, 8695 kudzu tropical seeds, 1530 leucaena
seeds, and 6896 calopo seeds
, all intact.
The faeces were collected every 6 hours after the total ingestion of the seeds (12,
18, 24, 30, 36, 42, 48, 54, 60, 66, and 72 hours). The rejects from the first 6
hours were disregarded, for no seeds were observed in this period in preliminary
essays. For the control test (0 hour), four repetitions were carried out, with one
hundred seeds of each species in direct contact with the commercial substrate Basaplant,
in plastic trays, without going through GIT.
After collection, the faeces were packaged in tagged plastic bags and taken to the
greenhouse, where they were disposed in rectangular plastic trays (61 cm length,
40 cm width, 10 cm height), identified and punctured to enable the drainage of water
excess. The trays were covered with a layer of the commercial substrate Basaplant
to simulate soil. The faeces were deposited in the trays over the substrate according
to the collecting periods and stayed for thirty days. During this time, the samples
were watered at least twice a day. The averages for maximum and minimum temperatures
in the greenhouse for the experimental period were of 32.2˚C and 20.3˚C,
respectively.
The emerged seedlings were counted daily, and, at the end of the thirty days, they
were removed from the faeces and weighted in their integrity (aerial part and roots)
for the natural weight. For the dry weight, the seedling were packaged in tagged
paper bags and disposed in a forced ventilation greenhouse, at 70˚C, for 72
3. Statistical Analyses
The species were analyzed separately, and the chosen experimental delineation consisted
in a randomized block design, with twelve treatments (collecting periods) and five
repetitions (bovines). The results were submitted to the analysis of variance and
to Duncan’s test, with a significance of 5% for the grouping of the averages, through
the statistical software Assistant.
4. Results and Discussion
In , it is observed that the kudzu species
presented a growing number of seedlings emerged in faeces in the periods from 18
to 30 hours after the ingestion of seeds by the bovines, gradually decreasing from
(a)(b)(c)(d)(e)
Figure 1. Number of emerged plants (a), length of
the root (cm) (b), length of the aerial part (cm) (c), natural matter (g) (d), and
dry matter (g) (e) of kudzu seedlings from seeds that went through bovine GIT.
period onwards. These results suggest that 30 hours of permanence of kudzu seeds
in bovine rumen are effective in augmenting their germination, which evinces the
overcoming of the intergumentary dormancy they present. The higher emergence of
kudzu seedling in the faeces may be connected to shape and texture of the tegument.
According to
, seeds of smaller length suffer
less damage when passing through GIT, and spherical seeds and seeds with smooth
skin hinder the damages during the chewing process.
In a similar study about the dispersion of Fabaceae seeds in the faeces of bovines
in open fields,
also found a higher number
of kudzu plants when comparing to macrotiloma, to calopo, and to estilosantes. The
author relates these results to the efficiency in overcoming dormancy and degradation
in the passage of the seeds through bovine GIT. Additionally,
stated that the passage of the seeds through the digestive system of
birds that spread seeds increases the percentage of germination and the vigor of
Cecropia pachystachya in the cerrado region in central western Brazil.
In an emergency essay on Fabaceae seedlings in bovine faeces,
observed an emergence of zero for dwarf pigeon peas. For
broad fava beans and calopo, the root protrusion was observed, but not the development
of the seedlings. For leuceana, there was an emergence of 10%, but the seedlings
were considered abnormal. Reference
that the bovine faeces may have not been in a favorable medium for the initial growth
of the seedling of those species and that the medium might have been toxic for the
seedlings, mainly due to the possible pH variations and to nitrogenous compounds
created by microbial activity.
For the three studied legumes, we could not observe any seedling in the period of
12 hours, which indicates that the dispersion had not begun in full intensity (Figure 1(a),
Regarding the length of the root and the aerial part of the seedlings, there was
a significant difference between the periods after the ingestion of the seeds for
all three species. For kudzu, the greater root length were observed between 18 and
60 hours. The greater aerial part length were observed between 18 and 66 hours.
For both variables, the results obtained with the dispersion, did not differ from
control ( and ).
show that, for kudzu seedlings, the greater natural matters and dry
matter were observed between 18 and 60, and 42 and 54, respectively, after the ingestion
of seeds, decreasing afterwards. In the dispersion times of 42, 48 and 54 hours,
the kudzu seedlings obtained dry matter significantly greater than the control group.
The values for this variable only decreased after 54 hours and onwards, coinciding
with the decrease in the emergence of seedlings, which indicates the dispersion
was equally starting to decrease or that some seeds in the faeces might have already
been dead. According to
, the digestion is
the main cause of the disappearance of the seeds given to the animals and of the
lost of its germinative capacity.
From the results of the number of emerged seedlings and natural matter of kudzu
seedlings, it was possible to verify that the bovine faeces did not prevent the
emergence and the development of this Fabaceae species. The results agree with those
, which affirmed that growth
of the seedlings was not hindered from being surrou on the
contrary, faeces may provide the plants with greater vigor due to the liberation
of nutrients
In , it is observed that, for the leucaena
seeds, the number of emerged seedlings was gradually growing 18 until 30 hours after
the ingestion of the seeds until its maximum was reached, decreasing from this point
onwards. The leucaena seeds expelled between 18 and 54 hours after the ingestion
were the ones that originated bigger seedlings, both for the aerial part and for
the root ( and ). These results are similar to those found by
, which observed that the leucaena seeds that went
through bovine GIT originated higher seedlings than the intact seeds. For the Cajanus
cajan seeds, the passage through bovine GIT reduced the growth rate for the height
of the seedlings. The effect was less accentuated in calopo seeds.
Greater natural matter occurred at 18, 24, 30, 36, 42 and 54, and dry matter occurred
at 18 until 54 hours after the dispersion of the seeds, with gradual decrease from
this point on for both variables. The dry matter decreased after 36 hours of dispersion,
coinciding with the fall in seedling emergence ( and ).
For the calopo, the greater number of emerged seedlings was observed for the intact
seeds, i.e., that did not went through animal GIT and, therefore, there was no significant
effect between the dispersion periods ().
These results agree with those found by
with estilosantes in bovine faecal plates, which attributed the low emergence percentage
of the estilosantes seedlings to the high degradability in the passage through GIT
of the animals.
The calopo seedlings originated from the seeds that went through the bovine gastrointestinal
tract were smaller and less developed, i.e., less heavy when compared the seedling
originated from intact seeds, evidencing once again the negative effect caused by
the passage through GIT, but for seedlings emerged 24 hours after the
(a)(b)(c)(d)(e)
Figure 2. Number of emerged plants (a), length of
the root (cm) (b), length of the aerial part (cm) (c), natural matter (g) (d), and
dry matter (g) (e) of leucaena seedlings from seeds that went through bovine GIT.
ingestion of seeds, whose length of the roots did not differ from control. For the
length of the aerial part, seedlings emerged 24, 30, 36 and 42 hours after the ingestion
of seeds did not differ from control (
The passage of the calopo seeds through bovine GIT should not constitute a favorable
medium to the initial growth of the seedlings, as can be seen by the values of natural
and dry matters, whose lengths were smaller from control ( and ), bearing in mind
the low or inexistent germination in such medium, which may be toxic to the seedlings
due to pH variations and to nitrogenous compounds created by microbial activity.
These authors believe that the residence time of the seeds in the faeces has a direct
relation with their mortality, in addition to the total dehydration of the faecal
plate in two days
. Nevertheless, in
open field conditions, the stomping, the rain, and, mainly the activity of coprophage
beetles may modify the substrate and create a microenvironment that is favorable
to the germination of seeds and to the growth of the seedlings deposited in the
faeces, in such a way that they do not act as a limiting factor for dispersion [16] .
In , there is the individual response of
the studied species. Even though kudzu had the largest number of seeds provided
to the animals (8695) and a larger number of emerging seedlings (675), leucaena
was the one
(a)(b)(c)(d)(e)
Figure 3. Number of emerged plants (a), length of
the root (cm) (b), length of the aerial part (cm) (c), natural matter (g) (d), and
dry matter (g) (e) for calopo seedlings from seeds that went through bovine GIT.
. Number of seeds
given to the animals and of emerged plants, percentage of emerged seedlings, length
of the root, length of the aerial part, natural matter and dry matter for kudzu,
leucaena and calopo seedlings from seeds that went through bovine GIT.
with the greatest percentage of seedling emergence (12.5%) regarding the amount
of seeds available to the animals (1530). These results suggest that the leucaena
seeds present larger resistance to the conditions encountered in bovine GIT due
to the cutaneous hardness inherent to the species. After passing through bovine
GIT, the leucaena seeds produced seedlings with larger root length and greater dry
matter weight, while the length of the aerial part was similar to that of the kudzu
seedlings.
In a similar study,
reports that the highest
percentage of recovery was obtained for kudzu seeds (23%) and that the lowest percentage
of recovery was observed with the Butterfly Pea seeds (16%); the study attributes
this lower recovery to the larger size of the seeds presented by the species. Reference
obtained a recovery rate of 50% for the
seeds of five species of the Cistaceae family after the passage through bovine GIT
and attributed this high percentage to the small size and hardness of the seed coat,
which protected them from damage during the chewing and ruminating.
tendered two hundred
intact seeds of each one of the following legumes: Leucaena leucocephala, Clitorea
ternatea, Calopogonium mucunoides, Neonotonia wightii, and Macrotiloma axilare.
The percentage of emerging seedlings in the faeces was low, ranging between 2.5%
and 8.63% for the total amount of ingested seeds. In contrast,
tendered 9000 seeds of each one of the legumes to cattle:
Leucaena leucocephala (Lam.) of Wit, Cajanus cajan (L.) Huth, and Cal
it also recovered 12% of the Cajanus cajan seeds and 17.5% of the Calopogonium mucunoides
seeds. These authors considered the values of this low recovery and attributed to
it the damage that the seeds suffered during the ingestion (chewing) and passage
through animal GIT.
The passage through bovine GIT severely affected the capacity of the calopo seeds
to germinate. As they went through the animal GIT, the seeds increased size due
to the imbibition process and presented a rupture on the tegument. Thus, the low
percentage of seedling emergence could be related to the softening and deterioration
of seeds, caused by hydration within the GIT, which would lead to the loss of viability
due to the action of the gastric juice
5. Conclusions
Leucaena seeds are less susceptible to damages caused by chewing and degradation
in bovine GIT.
The greatest total number of seedlings emerged in faeces was observed in the kudzu
The greatest results for the length of the aerial part and the root and for dry
matter, observed in the leucaena species, followed by kudzu.
The passage of the calopo seeds through bovine GIT should not constitute a favorable
medium to the dispersal.
Acknowledgements
We thank the “Research Support Foundation of the State of Rio de Janeiro” (FAPERJ).
Clarification
The proceedings were approved and carried out according to Protocol 207, registered
in the Ethics and Animal Use Committee of UENF.
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