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A n i m a l R e p r o d u c t io n S c i e n c e , 35 (1994) 145-1500378 -432 0/94/ 07.0 0 1994 - Elsevier Science B.V. All rights reserved

Short Com m unication145

Te chniqu e for e ff ic ient recovery o f sheep oocy tesby lapar oscop ic fo l l i cu locentes i sH . Baldassarre , D .G . de M atos, C.C. Furnus, T.E. Castro, E .I. Cabrera Fisch er

Funda cion M argari ta Pe rez Companc, Centro de Invest igaciones Reproductivas, Perez Companc,Florida 234 4 Piso, 1334) Buen os Aires, Arg entina(Accepted 24 June 1993)

A b s ~ a ~A simple and inexpensive pipette for in vivo recovery of sheep oocytes by folliculocentesis wasdeveloped. Two experiments were conducted to assess the recovery rate. In Experiment 1, 20 Mer-ino Corriedale ewes were heat synchronized using intravaginal sponges containing 60 mg of me-droxyprogesterone acetate (MPA) for a period of 12 days. In Experiment 2, 26 Merino Corriedaleewes were synchronized with CIDR devices for 12 days, with the original device being replaced bya new one on Day 10 (2 days before withdrawal). In both experiments, ewes were superovulated with

a total dose of 16 nag of follicle stimulating hormone (FSH) given in six decreasing dosage injections,starting 48 h before spo nge/CIDR removal. Folliculocentesis was performed 36-40 h after spongeremoval, and 16-20 h after CIDR removal. Overall, an average of 12.5 follicles per ewe were punc-tured and 10.2 oocytes per ewe were recovered (recovery rate 81.9%). The differences between thetwo experiments in terms of follicles punctured per ewe (10.8 vs. 13.7) and oocytes recovered perewe (8.5 vs. 11.5) were not statistically significant (P >0 .0 5) . The follicle size did not significantly(P > 0.05 ) affect the recovery rate, although there was a tendency for higher rates of recovery from 1-5 mm follicles (88.5%) compared with follicles over 5 mm (79.5%).

Introduct ionSince the b i r th of the f i r s t ca l f f rom in v i t ro fe r t i li za t ion (Bracke t t e t a t.,1982 ) , li ve of fspr ing f rom oocytes subjec ted to in v i t ro ma tura t ion ( IV M ),fe r t il iz a t i on ( IV F) , and cu l t u re ( IV C) , have been p roduced i n th ree rum i -na nt spec ies (ca t tl e , sheep , and goa t s ) . Never the less , mo st work repor t ed insheep has been accompl i shed wi th aba t to i r -der ived oocytes (Nai t ana e t a t . ,1992) , w h i le ve ry li tt le w ork w i t h i n v i vo de r i ved i m m at u re oocy t es has beenrepor t ed (Terv i t e t a l. , 1992) .The poten t i a l appl i ca t ion of in v i t ro t echnology for matura t ion , fe r t i l i za -t ion an d cu l ture of dom est i c an im al oocytes, par t i a l ly depend s on the deve l-opm en t o f r e li ab le , r epea tab l e and e f f ic i en t t e chn i ques fo r r ecove ry o foocy t e sf rom g enet i ca l ly va luable an im al s . Thi s pa per d escr ibes a t echniqu e tha t ap-

*Corresponding author.S S D I 0378-4320 (93) 01255-7

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146 1t . Baldassarre / An im al Reproduction Science 35 1994) 145-1 50p e a r s t o s a t is f y t h e s e r e q u i r e m e n t s f o r t h e r e c o v e r y o f o v i n e o o c y t e s b y l a p a -r o s c o p i c f o l l i c u l o c e n t e s i s .M a t e r ia l s a n d m e t h o d sA n i m a l s

F o r ty - si x M e r i n o C o r r i e d a l e a d u l t e w e s w e r e u s e d i n t h is s tu d y . T h e y w e r em a i n t a i n e d o n p a s t u re u n t i l 48 h b e f o r e s y n c h r o n i z a t i o n d e v i c e w i t h d r a w a l ,w h e r e a f t e r t h e y w e r e h o u s e d i n a s h e d a n d m a i n t a i n e d o n g o o d q u a l i ty h a y( a d l i b i t u m ) u n t i l t h e d a y o f fo U i c u l o ce n t es i s. T h e y w e r e d e p r i v e d o f fo o d f o r1 2 h b e f o r e s u r g e r y i n p r e p a r a t i o n f o r a n e s t h e s i a .Trea tmen t

I n E x p e r i m e n t 1 , 2 0 e w e s w e r e s y n c h r o n i z e d u s in g i n t r a v a g i n a l s p o n ge sp r e p a r e d i n o u r l a b o r a t o ry , c o n t a i n i n g 6 0 m g o f m e d r o x y p r o g e s t e r o n e a ce t a te( M P A ) . I n E x p e r i m e n t 2 , 2 6 ew e s w e r e sy n c h r o n iz e d w i t h C I D R ( C a r te rH o l t H a r v e y , H a m i l t o n , N e w Z e a l a n d ) d e v ic e s . I n b o t h c a se s, sy n c h r o n iz a -t i o n d e v i c e s w e r e r e m o v e d a f t e r a p e r i o d o f 1 2 d a y s , b u t i n C I D R - t r e a t e de w e s , t h e o r i g i n a l d e v i c e w a s r e p l a c e d w i t h a n e w o n e 4 8 h b e f o r e w i t h d r a w a l( T h o m p s o n e t a l ., 1 9 9 2 a ) . A l l e w e s w e r e t r e a t e d w i t h a to t a l d o s e o f 1 6 m g o fF S H - P ( S c h e r i n g L a b o r a t o r y , U S A ) , d i v i d e d i n t o s ix d e c r e a si n g d o s a ge i n-

j e c t i o n s t h a t w e r e a d m i n i s t e r e d a t 1 2 h i n t e rv a l s , s ta r t in g 4 8 h b e f o r e s p o n g e /C I D R r e m o v a l . F r o m t h e t i m e d e v i c e s w e r e w i t h d r a w n , t h e e w e s w e r e a ll o-c a t e d w i t h t e a s e r r a m s f o r h e a t d e t e c t io n .Equipmen t

T h e l a p a ro s c op y e q u i p m e n t u se d ( R i c h a r d W o lf , G e r m a n y ) w a s c o m -p o s e d o f a 7 m m t e l es c o p e , l ig h t c a b le , li g h t so u r c e , 7 m m t r o c a r f o r la p a r o -s c o p e , a t r a u m a t i c g r a s p i n g fo r c e p s, a n d t w o 5 m m ' s e c o n d p u n c t u r e ' tr o c a rs .T h e f o l l ic l e p u n c t u r e s e t w a s c o m p o s e d o f a p u n c t u r e p i p e t te , t u b i n g , a c o l-l e c t io n t u b e , a n d a v a c u u m l in e . T h e p u n c t u r e p i p e t te w a s h a n d m a d e a t o u rl a b o r a t o r y u s i n g a c r y s ta l P V C p i p e t t e ( 5 m m e x t e r n a l d i a m e t e r , 2 m m i n t e r-n a l d i a m e t e r ) , a n d a n h y p o d e r m i c n e ed l e t h a t w a s c u t to a l e n gt h o f 5 m ma n d f ix e d i n t o t h e t i p o f t h e p i p e t t e w i t h i n s t a n t c e m e n t . T h e n e e d l e s iz e u se di n th i s s t u d y w a s 16 G , w h i c h h a d b e e n f o u n d t o b e t h e b e s t d u r in g t e c h n i q u ed e v e l o p m e n t . T h e c o n n e c t i o n t u b i n g w a s a 15 c m l e n g th o f s il ic o n t u b i n gw i t h a n i n t e r n a l d i a m e t e r o f 5 ra m . I t c o n n e c t e d t h e p u n c t u r e p i p e t t e t o t h ec o l l e c t i o n t u b e . T h e c o l l e c t i o n t u b e ( O v u c a p , I M V , L 'A i g l e , F r a n c e ) h a s a ni n le t a n d o u t l e t a v a il a b le i n t h e c a p . O n e w a s c o n n e c t e d t o t h e p i p e t t e a n d t h eo t h e r t o a v a c u u m l in e . In t h i s s tu d y , v a c u u m w a s p r o v i d e d w i t h i n o u r l ab o -r a to r y , b u t a n y v a c u u m p u m p s h o u l d w o r k i f i t p r o v i d e s t h e a p p r o p r i a te p re s-

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H. Baldassarre ~A nim al Reproduction Science 35 1994) 145 -15 0 1 4 7

s ur e. T h e v a c u u m p r e ss u re , m e a s u r e d a s d r o p s o f c o l le c t io n m e d i a p e r m i n u t ee n t e r i n g t h e c o ll e c ti o n t u b e , w a s a d j u s t e d to 5 0 - 7 0 d r o p s m i n - i . T h e c o m -p l e t e p u n c t u r e s e t w a s w a s h e d a n d r i n s e d t e n t i m e s i n t i ss u e c u l t u r e q u a l i t yd i s t il l e d w a t e r , a n d o n c e i n c o l l e c t i o n m e d i u m b e f o r e u s e . T h e c o l l e c t i o n m e -d i u m w a s m o d i f i e d D u l b e c c o ' s P B S ( G i b c o , G r a n d I s la n d , N Y , U S A ) s up -p l e m e n t e d w i t h 0 .0 5 m g m l - 1 h e p a r i n ( S ig m a , S t. L o u i s, M O , U S A ) , 1 ( v /v ) o f a n t i b i o t ic - a n t i m y c o t i c ( G i b c o ) , a n d 1 ( v / v ) f et al c a l f s e r u m ( F C S )( S i g m a ) . T h e c o l le c t io n t u b e c o n t a i n e d 10 m l o f t h is m e d i u m t o r e c e iv e t h eo o c y t e s .Technique

T h e e w e s w e r e r e s t r a i n e d i n a c r a d l e i n t h e c l as si c al p o s i ti o n f o r l a pa r o s-c o p i c a r t if i c ia l i n s e m i n a t i o n ( E v a n s a n d M a x w e l l , 1 9 8 7 ) , u n d e r g e n e r a la n e s t h e s i a . T h e t h r e e p r e v i o u s l y d e s c r i b e d t r o c a r s w e r e i n s e r t e d a n d t h e a b -d o m i n a l c a v i t y w a s f i l le d w i t h f i l te r e d a ir . T h e o v a r y w a s e x p o s e d f o r v is u a l-i z a t i o n a n d p u n c t u r e o f t h e f o l li cl es , b y p u l l i n g t h e f i m b r i a i n d i f f e r e n t d ir e c -t i o n s w i t h t h e g r a s p i n g f o r ce p s . O u r a i m w a s t o e n t e r t h e f o l li c le f r o m t h e s i d e ,w i t h t h e n e e d l e i n a d i r e c t i o n p a r a l l e l t o t h e b a s e o f th e f o ll ic l e. I f t h i s w a s n o tp o s s ib l e , p u n c t u r e w a s p e r p e n d i c u l a r to t h e f o ll ic l e w a l l. O n c e t h e n e e d l e w a si n s i d e t h e f o l l ic l e , i t w a s g e n t l y r o t a t e d t o e n s u r e t h a t a s m u c h o f t h e f o l l i cl ec o n t e n t s a s p o s s i b le w a s a s p i r a t e d .T h e c o n t e n t s o f fo l li c le s l a r g e r a n d s m a l l e r t h a n 5 m m i n d i a m e t e r w e r ea s p i r a t e d t o d i f f e r e n t c o l l e c t i o n t u b e s , i n o r d e r t o e s t a b li s h d i f f e r e n c e s i n t e r m so f r e c o v e r y r a t e a n d o o c y t e q u a l i t y r e l a t iv e t o f o ll ic l e s iz e . T h e c o m p l e t e p r o -c e d u r e t o o k a p p r o x i m a t e l y 2 0 m i n p e r e w e .Oocyte evaluation

T h e f o l l i c u la r a s p i r a n t w a s p o u r e d i n t o a p e tr i d i s h a n d o b s e r v e d u n d e rs t e r e o m i c r o s c o p y . O n c e o o c y t e s w e r e l o c a t e d , t h e y w e r e t r a n s f e r r e d t o a n -o t h e r pe t ri d is h c o n t a i n i n g H e p e s b u f f e re d T C M 1 99 ( S i g m a ) s u p p l e m e n t e dw i t h 1 0 F C S . O o c y t e s w e r e g r a d e d in t o t h e fo l l o w i n g c la ss es : I , c o m p l e t ec u m u l u s / c o r o n a c o m p l e x ; II , i n c o m p l e t e c u m u l u s / c o r o n a c o m p l e x ; II I, su r-r o u n d e d b y c o r o n a o n l y ; I V , n o s u r r o u n d i n g c e ll s.Statistical analysis

R e s u l t s w e r e a n a l y z e d b y A N O V A u s i n g c o m p u t e r s o f t w a r e( C S S : S t a t i s t i c a T M M o d u l e C , S ta t so f t, T u l s a , O K ) . D a t a p e r t a i n i n g to t h e re -c o v e r y r a te w e r e a n a l y z e d a f t e r a r c s i n e t r a n s f o r m a t i o n , w h i l e s u p e r o v u l a t i o nd a t a w e r e s q u a re r o o t t r a n s f o r m e d .

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1 4 8 I -I . Ba ldassa rre / A n i m a l R e p r o d u c ti o n S c i e n c e 3 5 1 9 94 ) 1 4 5 - 1 5 0

R e s u l t s

A t o t a l o f 5 7 5 f o l li c le s w a s a s p i r a t e d , o f w h i c h 4 71 o o c y t e s w e r e r e c o v e r e d ,g iv ing a re co ve ry ra te o f 81 .9 +_ 18 .3 (m e a n ___S D ) . T h e i n d i v i d u a l r es u lt sf o r t h e 4 6 e w e s a s p i r a t e d i n d i c a t e t h a t t h e r e c o v e r y r a t e w a s la r g e r t h a n 8 0i n 3 0 / 4 6 ( 6 5 . 2 ) , b e t w e e n 5 0 a n d 8 0 i n 1 4 / 4 6 ( 3 0 . 4 ) , a n d l es s t h a n 5 0i n 2 / 4 6 ( 4 . 3 ) .T h e o v e r a l l re s p o n s e t o s u p e r o v u l a t i o n w a s 1 2.5 + 5.5 f o l li c le s p u n c t u r e dp e r e w e , o f w h i c h 1 0.2 + 5 .1 o o c y t e s p e r e w e w e r e r e c o v e r e d .D i f f e r e n c e s i n t e r m s o f f o ll ic l es a s p i r a t e d p e r e w e , o o c y t e s r e c o v e r e d p e re w e , a n d r e c o v e r y ra t e b e t w e e n b o t h e x p e r i m e n t s a r e s h o w n i n T a b l e 1. T h e s ed i f f e r e n c e s w e r e n o t s t a t i s t i c a ll y s ig n i f i c a n t ( P > 0 . 05 ) .T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e ( P > 0 .0 5 ) i n t h e r e c o v e r y r a t e b e t w e e nt h e f o ll ic u l a r s iz e s s t u d i e d ( l a rg e r a n d s m a l l e r t h a n 5 m m d i a m e t e r ) , a s s h o w ni n T a b le 2 . H o w e v e r , w e f o u n d t h a t l a rg e ( m o r e t h a n 5 m m d i a m e t e r ) f oll i-c l es w e r e m o r e d i f fi c u l t t o a s p i r a t e , a s th e i r c o n t e n t s a p p e a r e d t o b e d e n s e r .A l s o , s o m e l a r g e f o ll ic l es t e n d e d t o ' e x p l o d e ' w h e n p u n c t u r e d , t h u s s o m e f o l-l i c u l a r c o n t e n t s w e r e lo s t .W i t h r e s p e c t t o o o c y t e q u a l i t y ( T a b l e 3 ) , m o s t o o c y t e s h a d p a r t i a ll y l o stT a b l e 1C o m p a r a t i v e r e s u lt s ( m e a n + S D ) o f t o t a l f o li el e s a s p i r a t e d ( T F A ) , t o t a l o oc y t es re c o v e r e d ( T O R ) ,a n d r e c o v e r y r a t e ( R ) , b e t w e e n e w e s t r e a t e d w i t h s p o n g e s ( E x p e r i m e n t 1 ) o r C I D R s ( E x p e r i m e n t2 )E x p . N o . o f T F A T O R R

ewesr t X r / X

1 2 0 2 1 7 1 0 . 8 + 5 . 0 a 1 71 8 . 5 4 . 8 b2 26 358 13 .7 + 5 .6 a 300 11 .5 + 4 .9 bTo ta l 46 575 12 .5 + 5 .5 471 10 .2 + 5 .1

78 .89 + 23 .7 c8 3 . 8 + 1 2 .5 c8 1 . 9 + 1 8 . 3

V a l u e s i n t h e s a m e c o l u m n w i t h t h e s a m e s u p e r s c r i p t d o n o t d i f f e r s i g n i fi c a n t ly ( P > 0 . 05 ) .T a b l e 2C o m p a r a t i v e r e s u lt s o f t o t a l fo ll ic le s a s p i r a t e d ( T F A ) , t o t a l oo c y te s r e c o v e r e d ( T O R ) , a n d r e c o v e r yr a t e ( R ; m e a n + S D ) , b e t w e e n f o ll ic le s s m a l l e r a n d l a r g er t h a n 5 m m i n d i a m e t e rF o l l ic l e s i z e T F A T O R R( m m )< 5 156 138> 5 4 1 9 3 3 3T o t a l 5 7 5 4 7 1

88.5 + 15.9 a7 9 . 5 + 2 1 . 2 a8 1 . 9 + 1 8 . 3

V a l u e s in t h e s a m e c o l u m n w i t h t h e s a m e s u p e r s c r i p t d o n o t d i f f e r s i g n i fi c a n t ly ( P > 0 . 0 5 ) .

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H. Baldassarre / Animal Reproduction Science 35 1994) 145-150

Table 3Quality o f oocytes recovered from di fferent follicle sizes studied surrounding

149

Follicle size Grade I Grade I1 Grade III Grade IV(mm) n % n % n % n %< 5 33 26 a 20 16 b 66 52 ~ 8 6 d> 5 86 26 ~' 42 13 b 186 56 ~ 17 5 d1In term s of the surrounding cells. The grading scheme was as follows: I, comple te cumulu s/c oronacomplex; II, in complet e cumu lus /co rona complex; III, sur rounded by corona only; IV, no surround-ing cells.Values in the same col umn with the same superscri pt do not differ significantly (P > 0.05).their cumulus cells in transit from the follicle to the collection tube. Folliclesize did not influence oocyte grading (P > 0.05 ).Discussion

The results obtained here indicate an interesting future for the applicationof IVM, IVF and IVC technology to genetic improvement programs in sheep.The recovery rate is apparently better than that reported in cattle by trans-vaginal ultrasound guided follicular aspiration (Pieterse et al., 1991 ), a tech-nique that is not suitable for sheep because of ovary/follicle size.Of concern was the loss of cellular investments surrounding oocytes follow-ing aspiration. We believe most of the cumulus cells are lost in the transit ofoocytes from the follicle to the collection tube. If this is the case, cumulus cellscould be preserved by means of refining the vacuum pressure and/or workingwith earlier stage follicles in order to have more compact cumulus/oocytecomplexes. The retention of cumulus cells is of importance, as it has beenshown by others (Kim et al., 1990; Lonergan et al ., 1992; Madison et al.,1992 ) that the level of cellular investment correlates with embryonic devel-opment in vitro following IVM/IVF.Further research must be undertaken to establish the most appropriate timefor follicular aspiration, and to study the maturability and fertilizability ofthese in vivo collected oocytes.Acknowledgements

We are grateful to Dr. Daniel Moses for his statistical analysis of the data,and to Dr. Jeremy Thompson (Ruakura Agricultural Center) for reviewingthe manuscript.

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150 H. Baldassarre / Animal Reproduction Science 35 1994) 145-150

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