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Effect of yak rumen content treatments on seed germination of 11 alpine

meadow species on the Qinghai-Tibetan Plateau

Xiaojun Yu a,, Chunhua Duan a,b, Changlin Xu a, Degang Zhang a, Juan Jin a

a Key Laboratory of Grassland Ecosystem of Ministry of Education, Sino-U.S. Centers for Grazing land Ecosystem Sustainability, Pratacultural College,

Gansu Agricultural University, Lanzhou, 730070 Gansu, Chinab Fodder and Forage Station of Xuanhan County, Xuanhan, 636150 Sichuan, China

a r t i c l e i n f o

Article history:

Received 21 July 2012

Revised 9 April 2013

Accepted 25 June 2013

Keywords:

Tibetan Plateau

Yak

Rumen digestion

Seed germination

Seedling

a b s t r a c t

The Qinghai-Tibetan Plateau is located in the Third Pole of the world, characterized by a harsh environ-

ment. Despite this, the alpine meadow ecosystem have developed over a wide area but serious grassland

degradation is threatening the ecological environment on the Plateau. Recruitment of new plants to the

population, via germination and establishment, is vital to plant community survival. Previous work on

the seeds in this area has mainly focused on community-wide germination strategies, seed germination

characteristics and their correlations with seed size and seed mass. However, there have been no studies

on the effects of soaking in rumen contents on the plant seed germination characteristics of alpine mea-

dow species. The present study had two main objectives: (i) to determine the effect of fresh rumen con-

tent from yaks on seed germination characteristics and seedling growth of species common to theeastern

Tibetan Plateau alpine meadow, and (ii) to develop an effective method to enhance seed germination.

Seeds of 11 common species were collected together with fresh rumen content from three yaks that

grazed there. Seed germination tests were conducted after they had been soaked in rumen content for

one of six soaking periods (12, 24, 36, 48, 60 or 72 h). The seeds were incubated under natural light con-

ditions of 8 h light at 25 C and 16 h darkness at 15 C, for the germination period of 34 days. The results

showed that seed germination and seedling growth were affected by soaking time, seed coat complete-ness and seed type. After soaking in rumen content, the germination percentages of scarified (peeled or

with the seed coat cut through) seeds of some species (true seeds Oxytropis ochrocephala andMedicago

ruthenia var inschanica, nutlet Carex enervis, achenes Anemone rivularis and Polygonum sibiricum) and

complete seeds ofC. enervis, andA. rivularis were improved but the duration of soaking was also impor-

tant. Seed germination of caryopsisAchnatherum inebrians (a toxic grass) was significantly inhibited by

any exposure to rumen fluids. Scarified seeds generally had higher germination percentages than com-

plete ones after treatment, but with the increase in soaking time, germination percentages declined

and scarified seeds were more sensitive to the treatment than the complete seeds. After soaking in yak

rumen content, the germination indices of scarified M. ruthenia at 12 h treatment,O. ochrocephala and

achene Rumex acetosa at 1224 h treatment, nutlet Kobresia humilis at 24 h treatment, P. sibiricum at

2448 h treatment,C. enervis at 1248 h treatment and A. rivularis at 1260 h treatment were signifi-

cantly higher than the control (P< 0.05), while the germination indices of complete C. enervis seeds at

12 h and 36 h treatment, andA. rivularisat 1260 h treatment were significantly higher compared with

the control. The germination indices of other species gradually decreased with the increase in soaking

time. We concluded that yak rumen digestion could enhance, inhibit or not affect seed germination

and seedling growth of the alpine meadow species, which might influence seedling recruitment, interspe-

cific competition, and the plant community structure of the eastern Tibetan Plateau alpine meadow.

Overall, yak digestion has a positive effect on alpine meadow seed germination and seed dispersal.

2014 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

0. Introduction

The Qinghai-Tibetan Plateau is located in the Third Pole of the

world, with special conditions of challenging natural geography, a

harsh environment [1]. Despite this alpine meadow ecosystems

http://dx.doi.org/10.1016/j.chnaes.2013.06.007

1872-2032/2014 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

Corresponding author.

E-mail address: yuxj@gsau.edu.cn(X. Yu).

Acta Ecologica Sinica 34 (2014) 184190

Contents lists available at ScienceDirect

Acta Ecologica Sinica

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h n a e s

http://dx.doi.org/10.1016/j.chnaes.2013.06.007mailto:yuxj@gsau.edu.cnhttp://dx.doi.org/10.1016/j.chnaes.2013.06.007http://www.sciencedirect.com/science/journal/18722032http://www.elsevier.com/locate/chnaeshttp://www.elsevier.com/locate/chnaeshttp://www.sciencedirect.com/science/journal/18722032http://dx.doi.org/10.1016/j.chnaes.2013.06.007mailto:yuxj@gsau.edu.cnhttp://dx.doi.org/10.1016/j.chnaes.2013.06.007http://crossmark.crossref.org/dialog/?doi=10.1016/j.chnaes.2013.06.007&domain=pdf

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have developed over a wide area but is extremely fragile [2],

and serious grassland degradation is threatening the ecological

environment of the Plateau. The Alpine meadow community suc-

cession, vegetation restoration has caused widespread concern of

many scientists at present[3,4]. Plant regeneration is vital in the

processes of community succession and vegetation restoration

[5], and successful germination and establishment of the seeds

are the keys and grazing animals have role. Seed germination

directly relates to the species breeding, populations of maintaining

and expanding ecological processes, and directly affects the distri-

bution of the vegetation, community dynamics and biodiversity

[6]. The seed digestive dispersal (Endozoochory) is that the seeds

in the feces are away from the mother plants after frugivores (birds

and primates) swallowing fleshy fruit and the herbivores foraging

herbs and shrubs with seeds [7,8]. It is of great theoretical and

practical significance to clarify the endozoochorous dispersal of

seeds and other disseminules by herbivores plays an important

role in understanding plant regeneration, species diversity, com-

munity structure and succession. The actual mechanisms deserve

further study.

Partial seeds with germinability are passed out after grazed by

animals, the seed germination percentage and germination speed

are all improved[9]. In grazing ecosystems, herbivores feed, digest

and pass out plant seeds, thereby affecting both seed dispersal and

germination capacity [10]. Zhang et al. reported that after eaten

and digested by cattle and goats, Zoysia japonica seeds lost little

vitality but increased seed germination percentage [11]. Robles

and Castro [12] found that after incubation in the rumen of

herbivores(sheep and goat), seed germination ofHelianthemum

apenninum was boosted, while Li et al. [13]showed that yak and

Tibetan sheep juices significantly inhibited seeds germination of

Saussurea japonica and Saussurea iodostegia. Research has showed

that yak digestive tract effects seed germination of species of

alpine meadow in Qinghai-Tibetan Plateau, and then impacts the

population regeneration, species diversity, community structure

and succession of this region[14]. Researches have been reported

on seed germination of alpine meadow species[1519], however,there is no published work on the effect of livestocks rumen con-

tent on seed germination. In this study, we conducted the effects of

fresh yak rumen contents on seed germination and seedling

growth of 11 plant species from alpine meadow to provide a basis

for seed dispersing and management of grassland ecosystem in

Qinghai-Tibetan Plateau.

1. Materials and methods

1.1. Study area

Tianzhu Alpine Grassland Experimental Station is located to the

southwest foot of Wushao Ridge of Gansu Province with humid

climate, thin air and strong solar radiation. The annual average

temperature is 0.1 C, the lowest average temperature is

18.3 C in January while the highest is 12.7 C in July. Greater

than 0 C accumulated temperature is 1380 C, and there are still

low temperatures below 0 C in July. Annual rainfall is 416 mm

concentrated in July, August and September and annual evapora-

tion is 1592 mm. The grazing seasons are classified either as cold

or warm.

1.2. Study species

In August to September 2011, fully mature seeds of 11 common

alpine meadow species (Table 1) were manually collected. These

species are perennial and adapted to alpine meadow conditions.Most provide forage for livestock and a variety of wildlife species.

The selected species included dominant species such as Elymus

nutans,Kobresia humili,Kobresia capillifolia, as well as toxic species

Achnatherum inebrians, Oxytropis ochrocephala and Pedicularis

kansuensiswhich are poisonous to livestock either all the time or

only when green [14]. The seeds were taken to the laboratory

after collection, dried and then stored in manila envelopes at room

temperature. These seeds were then used in the experiment.

1.3. Methods

1.3.1. Yak rumen content collection and measurement

Early in December 2011, fresh rumen contents were collected

at a slaughterhouse in Tianzhu County, Gansu Province, from 3

healthy yaks raised under the same growth conditions. Rumen

contents were stored in a dark place at 25 C, and quickly trans-

ported to the lab and placed in a dark incubator at 39 C prepared

for the test see (Table 2).

Seeds of 11 species of intact and scarified disseminules (seeds of

P. kansuensis,O. ochrocephalaand M. rutheniawere cut through the

seed coat, while others had the seed coat peeled). Then all were

encased in small cotton bags (6 cm 4 cm), which were distrib-

uted evenly throughout the yak rumen content for periods of12 h, 24 h, 36 h, 48 h, 60 h and 72 h. Of these exposure times,

24 h, 48 h and 72 h are widely accepted as standards for determi-

nation of the effects on seed germination[20,21]. Rumen contents

were incubated under dark condition at 39 C to simulate the tem-

perature condition inside the animal rumen. After the soaking

tests, seeds in the cotton bags were rinsed with tap water and

put into 75% alcohol disinfection for 1 min, then washed with dis-

tilled water 4 times for the germination test.

1.3.2. Seed germination test

Three replicates of seeds from each plant species were placed

on moistened filter paper in Petri dishes for the germination tests.

Petri dishes were incubated under natural light conditions of 8 h

light at 25 C and 16 h darkness at 15 C, for the germination per-

iod of 34 days. The complete seeds untreated were as a control,

each Petri dish received 90 seeds per replicate. Every day, the per-

centage of seeds that had germinated was recorded. Seeds were

regularly watered with distilled water, a seed was considered as

germinated when the radicle was visible. Seedling length was

measured at Day 13[22,23].

1.3.3. Data analysis

Germination percentage (GP) for each treatment was calculated

after 34 days. The germination index (GI) is based on number of

seeds that germinated and the germination percentage. These

parameters were also calculated from the following formulas.

GP 100 GN=SN

where GNis the total number of germinated seed and SNis the totalnumber of seeds tested.

Table 1

Eleven common plants in alpine meadow and a guide to their disseminules.

Plant names Plant family Seed types

Achnatherum inebrians Gramineae Caryopsis

Anemone rivularis Ranunculaceae Achenes

Carex enervis Cyperaceae Nutlets

Elymus nutans Gramineae Caryopsis

Kobresia humilis Cyperaceae Nutlets

Medicago ruthenia var inschanica Leguminosae True seedOxytropis ochrocephala Leguminosae True seed

Pedicularis kansuensis Scrophulariaceae True seed

Poa crymophila Gramineae Caryopsis

Polygonum sibiricum Polygonaceae Achenes

Rumex acetosa Polygonaceae Achenes

X. Yu et al. / Acta Ecologica Sinica 34 (2014) 184190 185

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GIP

Gt=Dt

where Dtis the number of days since the day of sowing and Gtis the

number of seeds that germinated on day t[23].

All results are reported as means standard errors. The least

significant difference test was used to compare the means of seed

germination percentage, germination index and seedling length of

seeds from yak rumen content at P6 0.05. All statistical analyses

were conducted by using SPSS 16.0

2. Results

2.1. Effect of yak rumen content on seed germination percentage of 11

species

After soaking by yak rumen content, seed germination percent-

ages of 11 alpine meadow species were affected by soaking time,

seed coat completeness and seed type. Seed germination percent-

ages of scarified (peeled or cut through the seed coat) seeds as true

seedMedicago ruthenia (61.1%),O. ochrocephala (62.2%) soaked for

12 h, scarified seed as nutletsCarex enervis (80.4%, highest at 24 h)

soaked for 1236 h, intact seedC. enervis (58.5%) soaked for 24 h,both scarified and intact as achenesAnemone rivularis (62.2%, high-

est at 12 h)soaked for 1260 h, scarified seed as achenes Polygonum

sibiricum soaked for 36 h (64.4%) and 48 h (59.3%) were signifi-

cantly improved (P< 0.05). Seed germination percentages of cary-

opsis A. inebrians was significantly inhibited by any exposure to

rumen contents. With the extension of treatment time, seed germi-

nation percentages was somewhat lower than the control or even

failed. In intact treatments, seed germination percentage of nutlets

C. enervis were significantly enhanced after soaking for 24 h, seed

germination percentage of achenes A. rivularis was significantly

increased after soaking for 1260 h, and the highest germination

percentage was when exposed for 48 h. Other seed germination

percentages of 9 species were decreased with the extension of

exposure time.After soaking in rumen content, germination percentages of

caryopsisA. inebrians, E. nutans and true seed P. kansuensis declined

quickly. After soaking for 72 h, there was still seed germination of

scarified C. enervis, Poa crymophilaand P. sibiricum, intact C. enervis,

M. ruthenia, O. ochrocephala, P. crymophila and P. sibiricum, while

there was no germination of scarifiedM. ruthenia, O. ochrocephala

and P. kansuensis.. Scarified seeds generally had higher germination

percentages than complete ones after treatment, and scarified

seeds were more sensitive to the treatment than the intact ones,

such asA. rivularis, C. enervis,E. nutans andP. kansuensis.

2.2. Effect of yak rumen content on seedling growth

After soaking by yak rumen content, seedling lengths of 11 spe-cies were affected similarly to seed germination percentage. In the

scarified group, seedling shoot lengths of nutlets of K. humilis

soaked for 1248 h, nutlets of C. enervis soaked for 1272 h, and

achenes of A. rivularis soaked for 1260 h were significantly

increased (P< 0.05) (Table 3). While in intact ones, seedling shoot

lengths ofP. kansuensis soaked for 24 h,K. humilis soaked for 12

36 h, C. enervis soaked for 1248 h,A. rivularis soaked for 1260 h

were significantly greater than the control (P< 0.05).

Seedling root lengths of scarified caryopsis ofE. nutansand nut-

lets ofK. humilis soaked for 24 h, nutlets ofC. enervis soaked for 12

36 h, achenes ofA. rivularis soaked for 1260 h were significantly

enhanced (P< 0.05) (Table 4), while intactK. humilis at 24 h treat-

ment and intactA. rivularisat 1260 h treatment were significantly

higher than the control (P< 0.05). Seedling lengths of other specieswere gradually decreased with the extension of exposure time.Ta

ble

2

Seed

germinationpercentage(%)afterdifferenthours

ofyakrumencontentstreatment.

P

lantnames

Control

Scarifiedseed

Intactseed

12h

24h

36h

48h

6

0h

72h

12h

24h

36h

48h

60h

72h

A.

ine

brians

85.20.4a

61.98.5

b

26.35.8cd

19.36.9cd

14.43.2cde

1

0.70.4

de

0e

64.83.8

b

31.5

6.9c

16.74.8cde

16.75.1cde

10.0

2.8

de

0e

A.

rivu

laris

0f

62.21.7a

23.04.1c

12.20.6

de

49.31.0

b

7

.80.6e

0f

7.41.6e

6.7

0.6e

6.72.6e

15.60.6

d

10.4

3.5

de

0f

C.

enerv

is

43.31.1

d

69.64.2

b

80.45.2a

58.13.5c

44.82.6

d

4

.11.0

f

10.71.0

f

47.83.6

d

58.5

4.1c

28.50.4e

24.82.6e

8.1

2.3

f

9.61.3

f

E.

nu

tans

96.70.6a

80.70.4

b

71.55.8c

53.33.6

d

17.44.3e

0

f

0f

85.22.3

b

89.3

1.6ab

55.95.9

d

48.53.5

d

0f

0f

K

.hum

ilis

36.30.7ab

33.32.3abc

43.72.0a

36.32.1ab

19.30.7

d

0

e

0e

26.34.3

bcd

28.2

1.5

bcd

23.32.2cd

23.05.2cd

0e

0e

M

.ru

then

ia

41.10.6

b

62.27.8a

41.11.9

b

28.98.0c

2.61.0

f

0

f

0f

24.80.4cd

22.2

1.1cd

14.82.3

de

10.43.7ef

9.62.0ef

5.20.4ef

O

.ochrocep

ha

la

12.20.0

bc

61.17.8a

16.71.3

b

4.11.6

de

0e

0

e

0e

7.80.6cde

9.6

1.3

bcd

6.31.6cde

9.60.7

bcd

8.53.5cd

7.00.7cde

P.

kansuensis

55.21.0a

43.33.9

b

3.31.1e

1.50.4e

5.61.3

de

0

e

0e

53.00.4a

16.7

5.1c

14.82.

9c

10.71.0cd

0e

0e

P.

crymop

hila

45.20.7a

39.60.7ab

26.72.9

bcd

21.96.2cd

18.50.4

de

7

.00.4ef

6.71.1ef

45.23.7a

30.4

3.7

bcd

27.04.6

bcd

32.21.9

bc

21.9

3.5cd

5.60.4

f

P.

sibiricum

41.11.1cd

52.64.3abc

53.74.1abc

64.44.8a

59.34.8ab

2

2.63.7ef

17.81.1

f

33.73.6

de

34.8

2.0

de

46.71.3

bcd

39.63.5cd

22.2

1.1ef

10.41.0

f

R.

ace

tosa

84.40.6a

85.22.6a

54.41.3c

62.22.8

bc

54.42.9c

3

2.65.0

d

0e

83.70.4a

77.8

4.2ab

72.67.1abc

51.96.0cd

31.9

1.0

d

0e

Note

:0representsnilgerminationinthegerminatin

gperiod.

Lowercaselettersatthesamerow

representsignificantdifference(P