Fertility of frozen-thawed ejaculated and epididymalsperm of Garut ram

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ingan. 1 1 1 .

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Hafez. 1.

'hrcs)

frozen (Canis rufus)

n Sci 308.

J

Acia Hungaria 15

394. Anim Reprod

Prosedur Suaa Biometrik.

Sumantri. Gramedia Pustaka Utama. esticular

dee Rumin Res 1

laturiational Asia Link Sympaslum "Rcproductiut Biorechnology for Improved Animal Breeding in Southeast Asia"

Fertility

of frozen-thawed

ejaculated

and

epididymal

sperm of Garut

ram

M. ~ i z a l ' , M. R. ~oelihere',T.

L.

~usufz, 8 , puwantara2, and P. 2. ~ i t u m o r a n d

'Department of Antma1 Science. Faculty of Agnculturc, Universrty of Pattimura, Ambon, Indonesia 2~epamncnt of Clinic, Rtptoduct~on, and Pathology. Facuhy of Veterinary Medicine.

Bogor Agricultural Univers~ty, D m a g a , Bogor Indonesia '~estarch institute for Animal Production, PO BOX 221, Bogor Indonesia Introduction

Cauda epididymal

sperm

could be used as an alternative o f gamete source for application in re-

productive technology, because the sperm is motile and have ability for fertilizing the oocyte (Axner et a]. 1999; Hafez and Hafez 2000). This sperm could be collected for life o r death ani- mal, then it might be processed for chilling or freeing. The purpose of this research was to ex-

amine and compare the quality of sperm and fertility between frozen-thawed ejaculated (EJS) and

frozen-thawed epididymal (EPS) sperm followed by artificial insemination (Al).

Materials and Methods

Semen was collectad from three Garut rams using an artificial vagina once a week. Epididymal sperm was collected from cauda epididymis of slaughtered-ram Semen was diluted with modi-

fied Tris extender containing 5% glycerol and 20% egg yolk. The modified Tris extender is con- taining 3.32 g Tris (hydroxymethyl) aminomethane, 1.86 g citric acid-monohydrate, 1 3 7 g D(-) fructose, 2.16 g lactose-monohydrate, 0.05 g glutathione, I000 IUIml penicillin, and 1000 p g m l streptomycin in 100 rnl distilled water. Semen was loaded in 0.25 ml mini straw with the final concentration of 200 million motile sperm. Semen was equilibrated at S O C for three hours. The straws were placed for 15 minutes in liquid nitrogen vapor. 10

cm

above the level of liquid nitro-

gen. The straws were then plunged into liquid nitrogen and stored at -196

C.

Straws were

thawed in a 37 "C water bath for 30 seconds prior to analysis. Quality o f processed-semen in- cluding percentages o f motile sperm, live sperm, and intacl plasma membrane (IPM: Revell and Mrode 1994) were evaluated seer diluting, equilibrating and thawing, respectively . Before in- semination, estrus af ewes were synchronized uring inaavaginal administration of CIDR-G' f o ~

1 3 days. Cervical insemination war carried out 53 hours afier withdrawal o f CIDR-G' and re- peated scven hours later with one dose, respectively. Prepancy was determined with ultraaono- graphy

(USG)

83 days afier insemination.

Results and Discussion

Results of this research showed that sperm quality of EJS is better than EPS.

Mean

percentage of motile sperm afler diluting for EJS (76.67%) was significantly (P<O.OS) higher than EPS

(70.83%). There was no significantly difference beween

U S

and EPS for mean percentages of

live sperm and IPM aRer diluting. ARer equilibrating, mean percentages of motile sperm, live spmn, and IPM for

EJS

(70.00%, 77.00%, and 74.44%) were significantly (P<O.O5) higher than

International Asla Link Symposillm "Reproductive Biotechnology for Improved Anlmd Breed~ng In S wtheu t Asia"

Table 1. Percentage of motile sperm, live sperm, and IPM 01 ejaculated and a u d a epididymal sperm of Garut ram

Parameters of Phase of semen - Treatment

semen quality Processing EJS EPS

Motile sperm (Yo) Diluting 76.67 f 2.36b 70.83 f 1.86'

Equilibrating 70.00 _f 4 . 0 8 ~ 58.33 f 2.36* Thawing 52.78

+

2 . 4 ~ ~ 45.00 f 4.08" Live sperm (%)

Diluting 82.89 f 3.35' 82.83 f 1.57"

Equilibrating 77.00 It 2 . 9 ~ ~ 70.50 f 2.87a Thawing 58.78 f 1 . 4 7 ~ 54.50 f 2.14a 1PM (%)

Diluting 83.55f1.83a 8 1 . 3 3 f 1 . 1 0 ' Equilibrating 74.44 f 2.3 1 68.33 f 1 .50a

56.2211.81b 48.83!~2.19' Thawing

4b superscrips in a row indicating significant difference

(P<0.05),

n = 6

EJS = ejaculated sperm, EPS = epididymal sperm, IPM = intact plasma membrane.

Table 2. Pregnancy and lambing rate of frozen-thawed sperm by cervical artificial insemination

Frozen- Pregnancy Lambing Number of lamb thawed rate

(Yo)

rate (%) Sinale Twin Total

EJS 7112 ( 5 ~ . 3 3 ) ~ 7/12 (58.33)' 5 2 9

EPS 4/9 (44.44)a 319 (33.33)'

3

- 3

I

_{e.b superscrips in a column indicating significant difference (P<O.D5)}

EJ S

=

ejaculated sperm,

EPS

=

epididymal

sperm.

Quality of ejaculated sperm is bcttcr than epididymal spcrnl may be causcd by its have a seminaI

plasma. The

seminal

plasma is containing several

compounds such as lipoprotein

to

protection

of

i

sperm plasma membrane (Gilbert 1988;

Johnson

and Evcritt 1995; Nolan and

Hmmerstedt

!

1997) and stimu18ting sperm

motility (Squires

et

al.

2000).

The

motility of epididymal

sperm

treated

with

seminal

plasma was

similar

to h t of ejaculated

sperm

(Squires ct al. 2000), There-

fore, fenility

of

frozcn-thawed ejaculated

sperm

is higher than frozen-thawed epididymal sperm.

Pregnancy

and

lambing rate

fw

frozen-thawed ejaculated sperm (EJS)

of

this

research

was

simi-

lar

with reported

by

McPhie

et

al. (2000).

The

same goes

for

frozen-thawed

epididymal sperm

(EPS) was similar with reported by Bravo et aI. (2000) at llamas and alpacas.

In conclusion, quality of frozen-thawed ejaculated sperm is better than frozen-thawed cauda epididymal sperm. Frozen-thawed ejaculated and caude epididymal sperm of Garut ram are suit- able for use in artificial insemination by cervical method.

lntmniional "Rcprducrive Biotechnology Improvd An~mal B

References

Farsberg S.

the 7

777.

Alarcon

RH.

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P.92. Vol. 2.

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McPhie 2000. supplementatio

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Vc

JP, Hammerdedt s~abiliry

1 1 :670-682.

Revell RA. bovine

36:77-86.

Gomez-Cuetara 2000. seminat

Procerc

Pemnuges motile sperm, sperm, and epididymrl

; Trmment - -

iity

m

f

2.3d

+

1.86"

k 4 . 0 8 ~

k

2.36'

*

2.4ab _k 4.08'

I%I -1

f f 1.57a

f 2.98b k

f 1.47~ zt 2.14'

83.55*1.83" 8 1 . 3 3 f 1 . 1 0 ~

rt rt 1-50'

Thawing

-

k 1.81b

+

2.19*

mw ( W . O S ) , n =

spenn. =

kblc 2. Pregnancy of

-

Pregnancy Lambing

(%)

""

TobI

-

7/12 (58.33)b (58.331~

4/9 (44.44r 3/9

-

-

;crips (Pc0.05)

culated

spmn,

EPS =

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dif-

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ine

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EL,

Gamez-Cuetan

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166. Abstract Vol. 2.

;perm bettor epididymal s p c n ~ ~ caused its ssminal

~lasma

containing of

me

(Gilbnt

Johnson

md

Everitt

1995;

and

Hunmastedt

motiliw

a].

epididyaul spem

m a wai that sperm et a!. Thee-

thawed ejaculated

ftonn-thawed

sperm.

rate

cjwuhsed

spurn

(WS)

research was simi-

:Phie

a].

m e

80s

fio~n-thawed

*ported el al. (2000) llamr,

of

frozen-thawed ejaculated s p m than

Asla Sympos~um "Reproduct~ve Biolcchnology Improted Breed1

timor (cewus

timore

in

~ a l l e ~

',

R,

nandarini2, 3. purwantara3, T.L.Y USU?

' ~ a c u l t ~ Nusa Kupang lndor

Darmaga, Bogor

'Department Scirncr, olAgnculturt. Universitas Sumatera Utara,

'~epartment Vcttrinary

Bogor Darmaga, Bogor Indoncsla

biology

develo~

el el al. _{Loudon et}

Woodford

manag

! five

12

fokai samplil:

Batesan ( 1

necl

aprl

lsmail(2002). sexL 'I

Tomaszweska er al. _{1 Haigh dart 1993).}

( I 00%),