Gonadal Maturation, RNA/DNA Ratio of Mature Eggs, and the Effect of Refrigeration on Egg Viability and Sperm Motility in Korean Walleye pollock Theragra chalcogramma

296

Copyright © 2017 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815

서 론

어류의정상적인 수정란을얻기 위해서는정상적으로 성숙 된복강내성숙란을확보하여야한다

.

난낭에서배란된배란 란은산란되지않으면과숙으로진행되어수정되지않는과숙 란이된다

(Lahnsteiner, 2000).

^{자연이나 수조내에서암수의} 교미로수정란을확보하는경우에는난낭에서배란된배란란 은교미행동에의해체외로산란되어수정이되지만

,

인공번식 을통한수정란확보를위해서는인위적인방법에의해채란 시간을정해서성숙란을얻어야만한다

.

성숙란이과숙으로진 행하게되는기간은어종에따라다른데

, sturgeon Acipencer sp.

는

2

일

, Rainbow trout Salmo gairdneri

^{는성숙란이산란되}

지 않아도

7

^{일까지과숙으로의 이행이안 된다}

(Rottmann et al., 1991).

하지만

bighead carp Hypophthalmichthys nobilis, common carp Cyprinus carpio, grass carp Ctenopharyngodon idello

은

30

분에서

80

분사이에과숙으로의이행이매우빠르 게진행된다

.

심지어

Striped bass Morone saxatilis

^는

15-30

분 이내에과숙에의해수정이되지않는상태가된다

.

^인공성숙 에는최근일본과우리나라를중심으로종묘생산되고있는극 동산뱀장어의경우수정시키기가장좋은시간을산정하기위 해

17α, 20β-dihydroxy-4-pregnen-3-one (DHP)

주사후

15

시 간으로정하고있다

(Ohta et al., 1996).

하지만확률을보다높

이기위해성숙란을가진어미에

DHP

^{주사를한후수컷과합}

사하여자연채란이유도된직후인공채란을하기도한다

(No-

동해안 북부 자망에서 어획된 명태( Theragra chalcogramma)의 생식소 성숙과 포획 후 경과시간에 따른 성숙란의 RNA/DNA ratio 및 냉장보관 정자의 활력

서주영·권오남 ¹ *

강원도한해성수수산자원센터, ¹강원양식생물연구소(주)*

Gonadal Maturation, RNA/DNA Ratio of Mature Eggs, and the Effect of Refrigeration on Egg Viability and Sperm Motility in Korean Walleye pollock Theragra chalcogramma

Joo-Young Seo and O-Nam Kwon

¹

*

Fisheries Resource Center for Cold Water, Gosung 24747, Korea

1Gangwon Aqua-cultural Biology Institute Co. Ltd., Gangneung 25435, Korea

We conducted a study to 1) determine the indicators of gonadal maturity in male and female Korean walleye pollock Theragra chalcogramma for the purposes of artificial insemination; 2) establish the RNA/DNA ratio of mature eggs in this species; and 3) monitor the effect of refrigerated storage on egg viability and the motility of sperm collected from dead adult males. During the spawning season, the color of female gonads changed from orange to transparent, and that of male gonads changed from pale orange to milky white. The DNA content and RNA/DNA ratio of mature eggs were maintained without significant changes for approximately 6 h when eggs were preserved at 4°C. Sperm could be obtained from both milt and undiluted semen. Sperm obtained from milt ceased moving on the second day after isolation, while over 60% of sperm obtained from semen showed movement until the 13th day. Seven attempts were made to artificially inseminate mature eggs, of which two resulted in successful fertilization. The successful inseminations produced 94,000 and 5,000 fertilized eggs, respectively. This study shows that artificial insemination of walleye pollock is a viable strategy when natural propagation is not possible.

Key words: Walleye pollock, Theragra chalcogramma, Artificial fertilization, Viability, Sperm activity

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

https://doi.org/10.5657/KFAS.2017.0296 Korean J Fish Aquat Sci 50(3) 296-301, June 2017

Received 20 September 2016; Revised 21 March 2017; Accepted 14 June 2017

*Corresponding author: Tel: +82. 33. 610. 2038 Fax: +82. 33. 610. 2038

E-mail address: [email protected]

자망에서 어획된 명태로부터 수정란 확보

297

mura et al., 2013).

^{그리고인공채란을위해암수를별도관리} 한다면성숙란의확보도중요하지만활성을갖는정충을성숙 란이확보된그시점에가질수있어야만한다

.

명태

(Theragra chalcogramma)

는

1981

년

16

만

6

천톤의어획고를보였고이후

급격히감소하여

2000

년이후완전히자취를감추었다

(Kang

et al., 2013).

^{그리고이자원의변화는}

1970

^{년대후반부터감}

소경향이확인되어

(Lee, 1991)

현재에는방류와같은적극적인

자원회복을위한노력이필요하게되었다

.

현재우리나라명태 는자연채란을위해서확보된어미의양과암수비율이맞지않 고

,

암컷의수조내폐사로지속적인수정란확보가불확실한 것이현실이다

.

^결국매년

400-600

^마리정도

(1

^톤가량

)

^의성어 가자망에잡히는데산란기에잡히는

100

여마리의명태선어 에서도수정란을확보하려는노력도해야할필요가있다

.

하지

만명태는다회산란어종으로

10-20

회에걸쳐산란기동안나

눠서산란하기때문에

(Bacheler et al., 2010)

성숙란을가지고 있는명태의비율은그만큼줄어들게된다

.

^{또한자망에포획되} 어폐사한시간의경과에따라성숙란이라고해도수정율이낮

아지는과숙으로진행되어버린다

. 2014

년이후국내외에서명

태선어로부터수정란을확보하려는많은노력이있었지만수 정란이원활히확보되지못한이유가여기에있다고판단된다

.

따라서본연구에서는암수생식소의상태를

Williams (2007)

^의 형태와조직학적발달양상을바탕으로채란

/

채정가능여부를 확인하고

,

폐사한암컷으로부터채취한성숙란을

4℃

저장시 간의경과에따른활력평가

(Buckley, 1984)

를

RNA, DNA

함 량과

RNA/DNA ratio

를통해확인하였다

.

그리고채취한정충 의활력을현미경상에서확인하였고

,

^{최종적으로명태선어로} 부터확보한성숙란과냉장보관된정충을이용한수정을통해 부화자어를얻었다

.

재료 및 방법

본연구에사용한명태어미는

2014

년

2

월에서

5

월까지포 획된총

203

마리중성숙한암컷

7

마리와수컷

45

마리였다

.

암 컷으로부터복부압박법에의해성숙란을채취하였다

.

그리고 수컷으로부터암컷과동일한방법으로정액을얻은후복개하 여생식공근처의정소일부를채취하여정자의활력을보았 다

.

채취한성숙란은압박법에의한채란으로생식소내에서투 명하게변한알만을사용하였으며

,

냉장보관

(4℃)

경과시간별

10

개내외의알로핵산함량과단백질량을측정하였다

. DNA

와

RNA

^가산

(acid)

^과염기

(base)

^{에각각추출되는원리를이용한}

Kwon and Adachi (2008)

의방법과수용성단백질측정을위한

Bradford (1976)

의방법을이용하여

pg/mg protein

의단위로 계산하여표시하였다

. 3

마리암컷의성숙란을이용하였으며

3

회이상분석하여결과로나타내었다

.

그리고전체

45

마리수컷 의정액에서정충활성이있었던

10

^{마리와정소의표면과내부} 에서활성이있었던

7

마리를사용하였다

.

이들로부터확보된정

충의정액내밀도는개체별로차이는있었지만

600-800×10

⁴

sperms/mL

^{로조사되었다}

.

^{정충은해수에노출후}

1

^분이내에 대부분활력을잃어버리기때문에해수에노출후

5

초이내의 순간활력을현미경

(Olympus CH40, Olympus, Japan)

으로확 인하여

100%

에서

5%

까지

5%

단위로

, 5%

이하에서는

1%

단 위로기록하여결과로정리하였다

.

그리고인공수정은활력을 갖는정액을가지고있을때성숙한암컷으로부터성숙란을확 보한당일인공수정에의해

7

회에걸쳐실시하였다

.

단매수 정시정자활성을현미경으로확인하고

10%

이상의활력을 보이는정충을사용하였다

.

통계처리는채취후경과시간에따 른성숙란의

RNA, DNA

함량과이들의

ratio

의평균값에대해 서

one-way ANOVA test

^{를실시하고}

, Duncan (1955)

^의다중 검정으로처리

,

평균간의유의성을검정하였다

.

모든통계처리 는유의확률

95%

범위에서

SPSS

프로그램

(Ver. 20.1)

을이용 하여분석하였다

.

결 과

고성

,

속초지역에서

2014

년

1-3

월동안포획된명태암컷생 식소는

Fig. 1

과같다

.

산란기직전성숙란

(A)

은연한고동색

(

간혹선홍색

)

^{으로생식소표면에혈관이선명하게보이고}

,

^내 부성숙란은분리되지않았다

.

성숙하여산란기직전의생식소

(B)

는선홍색으로성숙과정에서투명한구멍이난것과같았 고

,

내부알은뭉쳐있다

.

그리고산란중인생식소

(C)

는투명 한반점들이많아졌으며생식소를절개하였을때내부에서투 명한알이분리되어있었으며

,

^{생식소표면은선홍색을띄었다}

.

산란후의생식소

(D)

는크기가줄어있으며외부는반투명하 고내부는투명하고선홍색의알이혼재되어있었다

.

같은지 역과시기에포획된명태수컷생식소는

Fig. 2

와같다

.

산란기 전수컷의생식소

(A)

는연한선홍색으로꼬여있다

.

성숙하면서 정소

(B)

^{는희어지고주위혈관들로인해붉은빛이있다}

.

^그리 고정세포

(

정충

)

가만들어지는유백색의정액이흘러내리는완 숙정소

(C)

에서정액을확인할수있다

(C

의화살표

).

같은지역 에서

2014

년

2

월에포획된암컷의완숙란은채취후

4℃

에서 저장시간에따른핵산

(RNA

와

DNA)

함량과비율

(ratio)

의변 화를

Fig. 3

^{에나타내었다}

.

^완숙란내

RNA

^함량은

1.0±0.03 pg/mg protein

으로채취후

16

시간경과시까지

1.0±0.01 pg/

mg protein

으로유의적인차이가없었지만

(P>0.05),

^이후유의 적으로감소하였다

. DNA

함량은채취직후

1.4±0.01 pg/mg

protein

으로채취후

2

시간까지유의적인차이를보이지않았지

만

(P>0.05),

^{이후감소하여채취후}

8

^{시간째완숙란마다차이} 를보여

0.9±0.26 pg/mg protein

으로확인되었다

.

이후

24

시 간경과시유의적으로감소하였다

(P<0.05). RNA/DNA ratio

는함량이낮아지는

DNA

함량에반비례하여

8

시간까지

0.76- 0.80

의비율을보였고

(P>0.05),

^{이후유의적으로증가하여}

24

시간경과시

2.2±0.29

^{로나타났다}

(P<0.05).

수컷

45

마리중

26

마리에서채취한정액과정소의일부분에 서얻은정충의

4℃

에서보관시경과시간에따른활력은

Fig. 4

에나타내었다

. 4

마리에서의정액이채취당일

20%

이상의활 력을보였고

,

정소에서는

2

마리가

20%

이상활력을보였지만

, 2

일경과후활력이급격히줄었다

.

반면보관된정액은최대

13

일까지

60%

^{이상의활력을보였다}

.

^{그리고채취된알과보관된}

정충을이용해서수정란의확보현황은

Fig. 5

에나타내었다

. 1

회에약

15

만개의투명한성숙란에서

9

만

4

천개의수정란을얻 어서

7

만마리의 부화자어를얻었으며

, 2

회차에는

5

천마리의

부화자어를얻었다

.

하지만

3

회차부터는성숙란은확보했었지 만

,

정충의양과활력이

1%

이하로낮아짐으로인해수정란을 전혀얻지못했다

.

고 찰

어류성숙란의수정후정상적인 발생율은배란직후수정 시키지않는다면노출수온과경과시간에수정율이영향을받 Fig. 1. Development from pre-spawning to spent ovary in Korean walleye pollock Theragra chalcogramma adult male caught Between January and March 2014 by gill net in nature. A, ovary opaque; B, just before spawning; C, spawning; D, spent. The arrow of "E" indicated the matured eggs inside ovary. Bar indicated 5 cm.

Fig. 2. Development from pre-maturation to ejaculation testicle in Korean walleye pollock Theragra chalcogramma adult male caught Be- tween January and March 2014 by gill net in nature. A, before maturation stage; B, just before maturation; C, ejaculation stage. The arrow of E indicated the spermatogenesis inside testicle. Bar indicated 5 cm.

자망에서 어획된 명태로부터 수정란 확보

299

는다

(Bahre Kazemi et al., 2009).

본연구에서는고성과속초 지역의자망에서포획된명태어미로부터성숙란과정충을확 보하여다회에걸쳐수정을시도하였다

.

어미로부터채취한성 숙란은일정시간이경과하면수정율이떨어져서더이상발생 하지못하는데

,

어종에따라

30

분이내에과숙으로진행되는

striped bass, Morone saxatilis

^{도 있고}

, 7

일이 경과해서야과 숙으로진행되는

rainbow trout Salmo gairdneri

도있다

(Rott- mann et al., 1991).

우리나라동해안에서는산란기인

12-4

월사 이에비정기적으로어획되는관계로수컷으로부터정액을채취 하여보존하고암컷의성숙란이나오기를기다려야만하는문 제들이있다

.

또한자망명태암컷이폐사한지몇시간이되었는 지확인이안되는상황에서모든성숙란을수정시켜부화자어 를얻을수는없었다

.

정상성숙란을확보하였다고하여도수정 시킬수있을정도의활력을가지고있는정충의확보가안되었 다면불가능한일이다

.

다행히

Rottmann et al. (1991)

과

Bahre

a a a a a ab

b

0.00 0.40 0.80 1.20

0 5 10 15 20 25

A a a a b

c

d e

0.00 0.40 0.80 1.20 1.60

0 5 10 15 20 25

B

a a a a ab

bc

c

0.00 0.50 1.00 1.50 2.00 2.50

0 5 10 15 20 25

C

RNA, DNA (pg/mg protein) RNA/DNA ratio

Elapsed time (h)

0 20 40 60 80 100

0 2 4 6 8 10 12 14 16 18 20

M ov em en t ( % )

Elapsed time (day)

1 6

9 13

15 17

18 21

22 23

A No. of Pollock

0 10 20 30 40 50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Movement (%)

Elapsed time (day)

6 9

13 15

17 18

26

B No. of Pollock

0 5 10 15 20 25

1st 2nd 3rd 4th 5th 6th 7th

N o. o f eg gs (X 10

4

)

No. of egg collection

Collected eggs Fertilized eggs Hatched eggs

Fig. 3. Nucleic acids content (pg/mg protein) due to elapsed reservation time (hour) of maturated Korean walleye pollock Theragra chal- cogramma eggs reserved on 4℃. A, RNA content (pg/mg protein); B, DNA content (pg/mg protein); C, RNA/DNA ratio. Different super- scripts on spots indicate a significant difference; double superscripts (like ab, bc) indicate no significant difference with either component:

ab is not significantly different from either a or b, etc.

a a a a a ab

b

0.00 0.40 0.80 1.20

0 5 10 15 20 25

A a a a b

c

d e

0.00 0.40 0.80 1.20 1.60

0 5 10 15 20 25

B

a a a a ab

bc

c

0.00 0.50 1.00 1.50 2.00 2.50

0 5 10 15 20 25

C

RNA, DNA (pg/mg protein) RNA/DNA ratio

Elapsed time (h)

0 20 40 60 80 100

0 2 4 6 8 10 12 14 16 18 20

M ov em en t ( % )

Elapsed time (day)

1 6

9 13

15 17

18 21

22 23

A No. of Pollock

0 10 20 30 40 50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Movement (%)

Elapsed time (day)

6 9

13 15

17 18

26

B No. of Pollock

0 5 10 15 20 25

1st 2nd 3rd 4th 5th 6th 7th

N o. o f eg gs (X 10

4

)

No. of egg collection

Collected eggs Fertilized eggs Hatched eggs

Fig. 4. Movability (%) due to elapsed reservation time (day) of spermatozoa obtained from semen (A) and milt (B) of maturated Korean walleye pollock Theragra chalcogramma male.

Kazemi et al. (2009)

의낮은수온에서서식하는어종에서길 지는않지만과숙으로의이행이느리다는것에착안하여명태 채란과수정작업을할수있었다

. Seoka et al. (2003)

은뱀장어

(Anguilla japonica)

^{에서수정후부상란과침강란의생화학적} 특성을밝혀수정되지않는성숙란이발달과정에서부터문제가 있다는것을밝혔다

.

과숙에의한변화는결국수정율에서부화 후기형율까지영향을미치는데

(Mohagheghi Samarin et al.,

2010; Samarin et al., 2011),

과숙이진행되면서난내

ATPase

의활력이높아지고

(Sago, 2008), triglyceride, glucose

^의함량 이낮아지면서

aspartate aminotransferase

활성이급격히높아 지는경향을보이기도하고

(Mohagheghi Samarin et al., 2010),

vitellogenin, lectin

및

apolipoprptein A 1-1

과같은난성숙인 자의축적도있다

(Rime et al., 2004).

하지만수정율저하로오 는알의과숙은위와같은물질들의 함량변화가체강액에서 먼저일어나게된다

.

한편

Nomura et al. (2013)

은뱀장어

(A.

서주영

ㆍ

권오남

300

japonica)

^{성숙란의채란후저장시간경과에따라낮아지는}

수정율과부화율을보고하면서

4

시간이후정상비율이낮아 지는것이체내에서생식소로지속적인간유래

vitellogenin

의 공급

(Kwon and Adachi, 2008)

에의해서만의원인은아니라 고밝혔다

.

^또한

snapper Pagrus auratus

^{의성숙란의과숙을막} 기위해동물세포배양용

L-15

배지

(L-15 Medium, Leibovitz)

에 번식관련 호르몬

(testosterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one and human chorionic

gonadotrophin)

을 함께 처리하여도 수정율의 보존에는 효과

가없었다

(Hobby and Pankhurst, 1997).

^{결국과숙되면서난} 내산화반응에따라

polysaccharide acids

와

lipid

를함유하는 수많은소낭

(vesicle)

의생성

(Lahnsteiner, 2000)

이되고

yolk mass

와

perivitelline space (

난황주위간극

)

의변화가조직학적 으로관찰

(Mohagheghi Samarin et al., 2010)

되기때문에모 든과숙에의한결과들이나오는것으로판단된다

.

^이와같은 연구결과들을바탕으로본연구에서의

Fig. 3

과

Fig. 4

의결과

를보면명태성숙란은

6

시간이후급격히

DNA

손상

(4

시간

째

1.29±0.032 pg/mg protein, 8

시간째

0.92±0.259 pg/mg

protein)

에의한수정율저하가예상되고

,

정충은수컷의상태

에따라

13

^일까지

60%

^{이상의활력을유지시킬수있었다}

.

^생 산량도많지않은수컷배우체

(

정충

)

는

Routray et al. (2006)

와

Kang et al. (2009)

에서와같이동결보존된정충을수정에이용 되기도한다

.

하지만우리나라에서는명태인공정장실험을위 한충분한정장액을확보하지못하고삼투압

,

이온농도를최적 화시키지못해서소량의정액을원액냉장보존할수밖에없다

.

하지만성숙한수정란이확보되더라도저장된정충의활력이 낮다면

Fig. 5

의

3-7

회차채란에서부화율

“0%”

는당연한결과 라고판단된다

.

하지만본연구에서와같이폐사한

major carp, Labeo rohita

^{에서정충을채취하여}

0℃

에서

8

시간까지는수정 가능한정충을얻을수있음이

Routray et al. (2006)

^에의해보

고되어명태와같이다회산란으로인해한꺼번에얻을수있는 정충의양이적은종류에서는기술개발의필요성은있다고할 수있다

.

본연구에서는죽은지몇시간이되었는지모르는자 망에서잡힌명태선어를이용한것이고

,

개체별로실험이되어 서통계처리조차도하지못했지만

,

전체

45

마리의수컷중에

10

^{마리에서정액을얻어서실험할수있었다}

.

^{그리고정원세포}

들사이에있는정충들에서도낮지만채취당일사용할수있을

정도의

20%

이상의활력을갖는정충들을얻을수있었다

.

하지

만

Fig. 5

에서와같이수정란확보에따라부화까지시킬수있

는수정란의확보율이저조한이유는수컷으로부터확보한정 충의양과활력저하가가장큰원인이었다고판단되지만

,

^성숙 란의과숙정도도영향을미쳤을것으로판단된다

.

결과적으로 확보되는정충의양과성숙란의과숙정도에따라정상수정란 확보량이결정날수밖에없다는것이다

.

이와같은과정을거 쳐

2014

년도명태산란기에

9

만

5

천개의수정란을얻었고

,

부 화율은계산되지않았지만부화자어를확보하였다

.

^{근본적으로} 본연구에서는폐사체를이용해서인공채란을하였지만명태는 다회산란어종이기때문에

,

가능하다면살아있는명태어미들 을이용해서자연산란에의한수정란확보가가장바람직한명 태의종묘생산방법이라고판단된다

.

사 사

이논문은

2015

년해양수산부재원으로한국해양과학기술진

흥원수산실용화기술개발사업의지원을받아수행된연구임

(

동해안명태의종묘생산및방류기술개발

).

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0 5 10 15 20 25

d e

0.00 0.40 0.80

0 5 10 15 20 25

a a a a ab

0.00 0.50 1.00 1.50 2.00

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RNA, DNA (pg/mg protein) RNA/DNA ratio

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M ov em en t ( % )

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