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M O G U N O S T I P R I M J E N E U S T O A R S T V U H R V A T S K E
M . M a t k o v i 2 , I v a G e t z 1 , Z . M a k e k 1
1 K l i n i k a z a r e p r o d u k c i j u i p o r o d n i at v o , V e t e r i n a r s k i F a k u l t e t , Z a g r e b
2 C e n t a r z a r e p r o d u k c i j u u s t o
a r s t v u H r v a t s k e d . o . o . , Z a g r e b
S a ~e t a k
B i o t e h n o l o g i j a r a s p l o i v a n j a p o d r a z u m i j e v a z a h v a t e k o j i m a p o d k o n t r o l i r a n i m u v j e t i m a v r ai m o r a s p l o i v a n j e ~i v o t i n j a i u t j e
e m o n a g e n e t s k u s e l e k c i j u ai r e n j e m p o ~e l j n e g e n e t i k e p r e m a ~e l j e n i m p r o i z v o d n i m s v o j s t v i m a . O v e b i o t e h n o l o g i j e r a z v i j a j u s e z a d n j i h 5 0 g o d i n a p r e m a p o t r e b a m a i m o g u n o s t i m a s v a k o g r a z d o b l j a . P r v a g e n e r a c i j a b i o t e h n o l o g i j e b i l o j e u m j e t n o o s j e m e n j i v a n j e , d r u g a e m b r i o t r a n s f e r i s r o d n e t e h n o l o g i j e , t r e a p r o i z v o d n j a z a m e t a k a i n v i t r o , a
e t v r t a m a n i p u l a c i j a g e n o m o m u z p o m o t r a n s g e n e z e i k l o n i r a n j a . P r i m j e n a b i o t e h n o l o g i j e u r a s p l o i v a n j u ~i v o t i n j a o m o g u a v a 1 ) g e n e t s k i n a p r e d a k s t a d a d o b i v a n j e m v e l i k o g b r o j a p o t o m a k a o d n a j k v a l i t e t n i h ~i v o t i n j a , 2 ) m e u n a r d o n i t r a n s p o r t i t r g o v i n u z a m r z n u t i m z a m e c i m a t e t a k o s m a n j u j e r i z i k p r e n o ae n j a z a r a z n i h b o l e s t i j e r v i ae n i j e p o t r e b a n t r a n s p o r t ~i v i h ~i v o t i n j a , 3 ) p o v e a v a p o n u d u v i s o k o k v a l i t e t n e s t o k e , 4 ) o m o g u a v a a p l i k a c i j u d r u g i h t e h n o l o g i j a k o j e i m a j u d o d a t n e p r i m j e n e , k a o n p r . s e k s i r a n j e i b l i ~n j e n j e z a m e t a k a , g e n e t s k e p r e t r a g e , s e k s i r a n j e s p e r m e , p r o i z v o d n j a t r a n s g e n e s t o k e i d r u g o . O v i b i o t e h n o l o ak i z a h v a t i z a h t i j e v a j u s o f i s t i c i r a n u o p r e m u i s p e c i j a l n u o b u k u t e s u s t o g a s k u p i z a ai r o k u p r i m j e n u , a l i j e n j i h o v u
i n a k n a o d a b r a n o j s t o c i v e l i k i b r z . U g l a v n o m s v i k o j i r a d e u o v o m p o d r u
j u s l a ~u s e d a j e v r l o v a ~n o at o b r ~e p r e n i j e t i g e n o m o d m a t i
n i h s t a d a u p r o i z v o d n e j e d i n i c e .
U o k v i r u p r o j e k t a M i n i s t a r s t v a z n a n o s t i i t e h n o l o g i j e R H " P r o i z v o d n j a i n v i t r o g o v e i h z a m e t a k a " p o s t a v l j e n j e s t a n d a r d n i p r o t o k o l z a d o z r i j e v a n j e , o p l o d n j u i u z g o j z a m e t a k a i n v i t r o . T a k o e r j e o s n o v a n L a b o r a t o r i j z a a s i s t i r a n u r e p r o d u k c i j u ~i v o t i n j a p r i K l i n i c i z a p o r o d n i at v o i r e p r o d u k c i j u V e t e r i n a r s k o g f a k u l t e t a S v e u
i l i at a u Z a g r e b u , a u s u r a d n j i s a s t r u
n j a c i m a C e n t r a z a r e p r o d u k c i j u u s t o
a r s t v u H r v a t s k e d . o . o . Z a m e c i p r o i z v e d e n i u n a ae m l a b o r a t o r i j u z a d o v o l j a v a j u k r i t e r i j e z a e m b r i o t r a n s f e r . U r a d u j e i z n e s e n k r a t k i p r i k a z p r e d n o s t i p r i m j e n a b i o t e h n o l o ak i h m e t o d a u u z g o j u ~i v o t i n j a t e s u p r e d l o ~e n e m o g u e p r i m j e n e u n a ai m u v j e t i m a .
K l j u
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i : r a s p l o i v a n j e , b i o t e h n o l o g i j a , e m b r i o t r a n s f e r , u z g o j i n v i t r o , z a m e t a k .
B i o t e h n o l o g i j a r a s p l o i v a n j a
P r i m j e n a r e p r o d u k t i v n i h b i o t e h n o l o g i j a i m a d u g o r o
a n u
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a r s t v u . P r o i z v o d n a s v o j s t v a o v i s e o g e n o t i p u , u t j e c a j i m a o k o l i n e i n j i h o v o j i n t e r a k c i j i . I a k o s e u t j e c a j i o k o l i n e m o g u k o n t r o l i r a t i t a k o d a b u d u n a j p o v o l j n i , g e n o t i p j e k l j u
n i f a k t o r , a u j e d n o i r a z l o g z a at o u p r a v l j a n j e r a s p l o i v a n j e m i g r a v e l i k u u l o g u u g e n e t s k o m n a p r e t k u s t a d a . U k o n t r o l i r a s p l o i v a n j a k o r i s t e s e r a z n e m e t o d e , o d k o j i h s u n e k e n a n i s k o j t e h n o l o ak o j r a z i n i i u g l a v n o m s e o d n o s e n a r a z l i
i t e u z g o j n e o d l u k e , k a o at o s u o d r e i v a n j e d o b i z a p r v o p a r e n j e , o d a b i r p a s m i n e k r a v a z a o d r e e n u p r o i z v o d n j u , k o n t r o l a m e u t e l i d b e n o g r a z d o b l j a i d i j a g n o s t i k a g r a v i d i t e t a . K a o n a d o g r a d n j a o v i m u z g o j n i m m e t o d a m a r a z v i j e n e s u b i o t e h n o l o ak e m e t o d e k o j e i m a j u u
i n a k n a p o j e d i n a
n o j , o d a b r a n o j ~i v o t i n j i ( G o r d o n , 1 9 9 6 . ) . B i o t e h n o l o g i j a r a s p l o i v a n j a ( e n g l : A R T = a s s i s t e d r e p r o d u c t i v e t e c h n o l o g i e s ) p o d r a z u m i j e v a z a h v a t e k o j i m a p o d k o n t r o l i r a n i m u v j e t i m a v r ai m o r a s p l o i v a n j e ~i v o t i n j a i u t j e
e m o n a g e n e t s k u s e l e k c i j u ai r e n j e m p o ~e l j n e g e n e t i k e p r e m a ~e l j e n i m p r o i z v o d n i m s v o j s t v i m a . O v e b i o t e h n o l o g i j e r a z v i j a j u s e z a d n j i h 5 0 g o d i n a p r e m a p o t r e b a m a i m o g u n o s t i m a s v a k o g r a z d o b l j a . P r v a g e n e r a c i j a b i o t e h n o l o ak i h m e t o d a b i l a j e u m j e t n o o s j e m e n j i v a n j e , d r u g a g e n e r a c i j a e m b r i o t r a n s f e r , t r e a p r o i z v o d n j a z a m e t a k a i n v i t r o i s r o d n e t e h n o l o g i j e , a
e t v r t a m a n i p u l a c i j a g e n o m o m u z p o m o t r a n s g e n e z e .
K a d a s e r a z m a t r a j u m o g u e p r i m j e n e v a ~n o j e r a z l i k o v a t i g e n e t s k e p r o m j e n e p r v o g s t u p n j a k o j e s e u g l a v n o m o d n o s e n a m a t i
n a s t a d a o d o n i h k o j e s e p o s t i ~u k a s n i j e , d i s e m i n a c i j o m u p r o i z v o d n i m j e d i n i c a m a ( W i l m u t i s u r . , 1 9 9 2 . ) . G e n e t s k i n a p r e d a k o v i s i o
e t i r i v a ~n a f a k t o r a : g e n e t s k a v a r i j a c i j a o d r e u j e r a z i n u g e n e t s k o g n a p r e d k a ( p r v i f a k t o r ) , a k o j i t a k o e r o v i s i i o p r e c i z n o s t i k o j o m v r ai m o s e l e k c i j u ( d r u g i f a k t o r ) , i n t e n z i t e t u k o j i m r a d i m o s e l e k c i j u ( t r e i f a k t o r ) i o g e n e r a c i j i s k o m i n t e r v a l u (
e t v r t i f a k t o r ) . S v a k a v r s t a i m a s v o j u o p t i m a l n u r a v n o t e ~u k o j a o v i s i o n a s l j e i v a n j u o d r e e n o g s v o j s t v a k o j e g a ~e l i m o p o b o l j aa t i , o r a z i n i i b r z i n i r a s p l o i v a n j a t e o r a s p o l o ~i v o j t e h n o l o g i j i k o j o m m o ~e m o p r o m j e n i t i t u r a z i n u i n a r a v n o o e k o n o m s k o m f a k t o r u ( W i l m u t i s u r . , 1 9 9 2 . ) .
I n b r e e d i n g ( r a z m n o ~a v a n j e ~i v o t i n j a u s r o d s t v u ) s e n e m o ~e i z b j e i u p o t p u n o s t i u s t o
a r s t v u t e s u p o p u l a c i j e u s e l e k c i j i u v i j e k u o p a s n o s t i o d t o g p r o c e s a , s t o g a j e c i l j s t o
a r a p o s t i i m a k s i m a l n i d o b i t a k , a l i p r i t o m v o d i t i b r i g u o i n b r e e d i n g u .
N a m j e r a o v o g p r e g l e d n o g r a d a j e r a z m o t r i t i p r i m j e n u b i o t e h n o l o g i j e r a s p l o i v a n j a u s v i j e t u t e m o g u n o s t i i o g r a n i
e n j a u n a s .
1 . U m j e t n o o s j e m e n j i v a n j e ( U . O . ) p r i p a d a p r v o j g e n e r a c i j i b i o t e h n o l o g i j e i v e s e d u ~e o d 5 0 g o d i n a p r i m j e n j u j e u s t o
a r s t v u , s n a j v e i m u
i n k o m n a g e n e t s k i n a p r e d a k ai r e n j e m g e n o m a n a j k v a l i t e t n i j i h r a s p l o d n j a k a , s p r e
a v a n j a ai r e n j a s p o l n o p r e n o s i v i h b o l e s t i t e o m o g u a v a j u i r e l a t i v n o j e d n o s t a v n i p r i j e n o s i t r g o v i n u g e n e t s k i m m a t e r i j a l o m . U g e n e t s k o m s m i s l u o s j e m e n j i v a n j e v i ae p l o t k i n j a s j e m e n o m j e d n o g b i k a t e k o n t r o l a p o t o m s t v a p r e k o p r o g e n o g t e s t a n a j v a ~n i j i j e p o k a z a t e l j p r o i z v o d n i h s v o j s t a v a o d r e e n o g b i k a . U R H v e v i ae o d 5 0 g o d i n a p o s t o j e s t a n i c e k o j e v r ae u m j e t n o o s j e m e n j i v a n j e p o t e r e n u , p o d k o n t r o l o m H S S S , s p e r m o m z a m r z n u t o m u C R S H i d r u g i m g e n e t s k i m c e n t r i m a . U s v i j e t u t e s t a n i c e v r ae p r o m i d ~b u i d r u g i h b i o t e h n o l o g i j a , k a k o b i a n i m i r a l i s t o
a r e d a i z a u u s u s r e t n o v i m p r o i z v o d n i m i z a z o v i m a .
2 . E m b r i o t r a n s f e r ( E T ) p r i p a d a d r u g o j g e n e r a c i j i b i o t e h n o l o g i j a k o j i m a s e u t j e
e n a p o v e a n j e r a s p l o d n i h p o t e n c i j a l a n a j k v a l i t e t n i j i h ~e n s k i h ~i v o t i n j a . M O E T ( m u l t i p l a o v u l a c i j a i e m b r i o t r a n s f e r ) j e s a z r i j e l a i s t a b i l n a b i o t e h n o l o ak a m e t o d a
i j i s u p r o t o k o l i g o t o v o i s t i v e 2 0 g o d i n a , a r e z u l t a t i s u p e r o v u l a c i j e j a m
e u p r o s j e k u 5 z a m e t a k a p o k r a v i d a v a t e l j i c i p o i s p i r a n j u ( H a h n , 1 9 9 2 ; H a s l e r , 1 9 9 2 , A r m s t r o n g , 1 9 9 3 ) . U 1 9 9 4 . g o d i n i u E u r o p i s u i s p r a n e m a t e r n i c e 2 2 . 5 5 7 s u p e r o v u l i r a n i h k r a v a ( H e y m a n , 1 9 9 5 ) . G o d i n e 1 9 9 8 u k u p n i b r o j d a v a t e l j i c a s e p o v e a o n a 2 5 . 7 4 4 t e j e p r e s a e n o u k u p n o 1 2 0 . 3 6 0 z a m e t a k a ( T h i b i e r , 1 9 9 9 ) . Z a n i m l j i v j e p o d a t a k d a j e 1 9 9 8 . g o d i n e u s v i j e t u p r e s a e n o 4 4 1 . 2 9 5 z a m e t a k a , a t a j b r o j i p o d a t c i s a k u p l j e n i t i j e k o m o s a m g o d i n a p o k a z u j u d a s e o v a b i o t e h n o l o g i j a p r i m j e n j u j e u s v e ai r e m o b i m u . P o d a c i p o k a z u j u d a s e o t p r i l i k e p o l o v i c a z a m e t a k a o d o v o g b r o j a p r e s a d i u s v j e ~e m , a p o l o v i c a u z a m r z n u t o - o d m r z n u t o m s t a n j u . O v a j i z b a l a n s i r a n i o m j e r p o k a z u j e n e k e o d p r e d n o s t i o v e t e h n o l o g i j e , k a o at o j e f l e k s i b i l n o s t i p r i l a g o d l j i v o s t z a h t j e v i m a r a z l i
i t i h t r ~i at a . T e h n o l o g i j a z a m r z a v a n j a z a m e t a k a j e t a k o e r s a z r i j e l a i s t r u
n j a c i s e p o u z d a j u u n j e z i n u p r i m j e n u z a k o m e r c i j a l n e s v r h e . P o s t o t a k b r e o s t i n a k o n t r a n s f e r a s v j e ~i h z a m e t a k a i z n o s i 6 1 % , a z a m r z n u t i h 6 0 % ( T h i b i e r , 1 9 9 9 . ) . T a k o e r i m a p o d a t a k a o m e u n a r o d n o m p r o m e t u z a m e t a k a k o j i i z n o s i o k o 3 0 . 0 0 0 g o d i an j e .
O v e t e h n o l o g i j e n e p r i m j e n j u j u s e s a m o u g o v e d a : i m a p o d a t a k a i o d r u g i m v r s t a m a : u 1 9 9 8 . g o d i n i p r e s a e n o j e v i ae o d 1 5 . 0 0 0 o v
i j i h , 2 . 5 0 0 j a r e i h , 1 . 0 0 0 s r n e i h , 2 . 7 0 0 k o n j s k i h i 2 . 3 0 0 s v i n j s k i h z a m e t a k a u s v j e ~e m i l i o d m r z n u t o m s t a n j u ( T h i b i e r , 1 9 9 9 . ) . I a k o j o a n e m a p o u z d a n i h p o d a t a k a , o v e b i o t e h n o l o ak e m e t o d e p r i m j e n j u j u s e i u d r u g i h v r s t a , u k l j u
u j u i i d i v l j a
. T a j p o r a s t a k t i v n o s t i u e m b r i o t r a n s f e r u , k o j i s e b i l j e ~i u c i j e l o m e s v i j e t u , s i m p t o m j e g l o b a l i z a c i j e , n e s a m o u t e h n o l o ak i m t r e n d o v i m a , n e g o i u p o t r e b i z a r i j e ae n j e m s l i
n i h p r o b l e m a u p r o i z v o d n j i s k o j i m a s e s u s r e u s t o
a r i u c i j e l o m e s v i j e t u .
I m a n e k o l i k o r a z p r i m j e n a e m b r i o t r a n s f e r a : p r o i z v o d n j a v i ae p o t o m a k a o d n a j b o l j i h k r a v a ( M O E T p r o g r a m i ) , b l i ~n j e n j e , d o b i v a n j e p o t o m s t v a o d s e k u n d a r n o j a l o v i h k r a v a , m e u n a r o d n a t r g o v i n a z a m e c i m a t e o
u v a n j e u g r o ~e n i h p a s m i n a i v r s t a . K o m b i n a c i j o m E T s a u
i n k o v i t o m i j e f t i n o m k o n t r o l o m e s t r u s a m o ~e s e u i s t o m s t a d u p r o i z v o d i t i r a s p l o d n e b i k o v e n a m i j e n j e j e z a U . O . , b i k o v e z a t o v i b i k o v s k e m a j k e ( B e t t e r i d g e i R i e g e r , 1 9 9 3 . ) .
U m n o g i m n a p r e d n i m z e m l j a m a e m b r i o t r a n s f e r s e k o r i s t i z a p r o i z v o d n j u m l a d i h b i k o v a n a m j e r e n i h z a c e n t r e U . O . ( M o r s t i n i s u r . , 1 9 9 4 . ) , i s k o r i at a v a j u i g e n o m n a j b o l j i h b i k o v a i k r a v a . B e t t e r i d g e ( 1 9 9 5 . ) n a v o d i d a j e 2 7 , 5 % b i k o v s k i h m a j k i i 4 4 % n a j k v a l i t e t n i j i h b i k o v a u S A D - u d o b i v e n o p o s t u p k o m e m b r i o t r a n s f e r a .
M O E T p r o g r a m i u k o m b i n a c i j i s U . O . k r a v e d a v a t e l j i c e j e t e h n o l o g i j a k o j o m s e p o s t i ~e b r ~e g e n e t s k o p o b o l j aa n j e s t a d a n e g o s a m o s U . O . P r o g r a m i M O E T - a p r i m j e n j u j u s e u n u k l e u s n i m s t a d i m a g d j e s e v r ai s u p e r o v u l a c i j a d a v a t e l j i c a . K o d m l i j e
n i h k r a v a M O E T s e m o ~e p r i m i j e n i t i p r i j e n e g o u u u p r o i z v o d n j u i l i p o s l i j e p r v e l a k t a c i j e . G e n e t s k a d o b i t j e 3 0 d o 5 0 % b r ~a n a j u n i c a m a u p r o i z v o d n j i b i k o v a i p o t e n c i j a l n i m b i k o v s k i m m a j k a m a ( G o r d o n , 1 9 9 6 . ) .
M O E T s e u d r ~a v a m a u r a z v o j u s m a t r a j o a u
i n k o v i t i j i m n e g o u r a z v i j e n i m d r ~a v a m a . Z a m e t c i s u j e f t i n j i z a t r a n s p o r t n e g o o d r a s l e k r a v e , a r i z i k z a r a z a t a k o e r j e m a n j i . P r e s a e n i u d o m a e p r i m a t e l j i c e , E T z a m e t c i i m a j u p r e d n o s t i n a d o d r a s l i m ~i v o t i n j a m a , j e r p r i m a j u p a s i v n i i m u n i t e t p r e k o k o l o s t r u m a , a i m a j u f i z i o l o ak u i p r i r o d n u a d a p t a c i j u n o v o j s r e d i n i ( G o r d o n , 1 9 9 6 . ) .
O v a b i o t e h n o l o g i j a i m a i s v o j i h m a n a : v a r i j a c i j e u b r o j e v i m a n a k o n s u p e r o v u l a c i j e j o a u v j e k p o s t o j e , a t o u t j e
e n a b r ~e ai r e n j e g e n o m a n e k i h p l o t k i n j a , n e g o l i o d d r u g i h . P o r e d t o g a ai r e n j e g e n o m a j e d n o g m a t i
n o g s t a d a i m a e f e k t n a i n b r e e d i n g p o p u l a c i j e , n o g e n e t s k i s e n a p r e d a k i p a k p o s t i g n e .
3 . P r o i z v o d n j a z a m e t a k a i n v i t r o p r e d s t a v l j a t r e u g e n e r a c i j u b i o t e h n o l o g i j e . N a k o n r o e n j a p r v o g t e l e t a d o b i v e n o g o p l o d n j o m i n v i t r o j a j n i h s t a n i c a d o z r e l i h i n v i v o ( B r a c k e t t i s u r . , 1 9 8 2 . ) i n v i t r o e r a j e b r z o n a p r e d o v a l a , n o t e k j e s u s p j e an o m p r i m j e n o m
i t a v o g i n v i t r o p o s t u p k a , o d n o s n o d o z r i j e v a n j a ( I V M ) , o p l o d n j e ( I V F ) t e u z g o j a i n v i t r o ( I V C ) d o s t a d i j a m o r u l e / b l a s t o c i s t e ( L u i s u r . , 1 9 8 7 . ) , o m o g u e n k o n a
n i p r o b o j i n v i t r o t e h n o l o g i j e . R a z v o j o v e b i o t e h n o l o g i j e i m a f a n t a s t i
n e p o t e n c i j a l e k a k o z a t e m e l j n a i s t r a ~i v a n j a t a k o i z a n j i h o v u p r i m j e n u u k o m e r c i j a l n o m u z g o j u s t o k e i g e n e t s k i n a p r e d a k s t a d a . O s i m t o g a o m o g u u j e r a z v o j d r u g i h b i o t e h n o l o g i j a k a o at o s u k l o n i r a n j e i p r o i z v o d n j a t r a n s g e n i h ~i v o t i n j a .
M e u n a j z n a
a j n i j i m p r i m j e n a m a I V P u g o v e d a r s t v u s u : z a o b i l a ~e n j e n e p r e d v i d i v o g s u p e r o v u l a c i j s k o g o d g o v o r a i n e k i h p o r e m e a j a u g e n i t a l n o m t r a k t u d a v a t e l j i c a ( S i r a r d , 1 9 8 9 . ) , p r o i z v o d n j a z a m e t a k a i z j a j n i h s t a n i c a d o b i v e n i h u l t r a z v u
n o m p u n k c i j o m j a j n i k a v i s o k o v r i j e d n i h k r a v a ( O P U ) ( K r u i p i s u r , 1 9 9 1 . ) , k o n t r o l a ai r e n j a z a r a z n i h b o l e s t i ( S t r i n g f e l l o w i G i v e n s , 2 0 0 0 . ) , s k r a e n j e g e n e r a c i j s k o g i n t e r v a l a p r o i z v o d n j o m z a m e t a k a o d ~e n s k e t e l a d i p r i j e p u b e r t e t a ( D u b y i s u r . , 1 9 9 6 . ) , j e f t i n a m a s o v n a p r o i z v o d n j a z a m e t a k a i z k l a o n i
k o g m a t e r i j a l a z a t r a n f e r u m l i j e
n e k r a v e
i j a t e l a d i d e u t o v ( V a n S o o m i d e K r u i f , 1 9 9 6 . ) , p r o i z v o d n j a b l i z a n a c a u t o v n i h ~i v o t i n j a ( P u g h i s u r . , 1 9 9 4 . ) , o d r e i v a n j e p l o d n o s t i b i k o v a k o j i s e k o r i s t e z a U O ( M a r q u a n t - L e G u i e n n e i s u r . , 1 9 9 0 . ) , u b r z a v a n j e p r o g e n o g t e s t i r a n j a b i k o v a , p r o i z v o d n j a t r a n s g e n e t s k i h ~i v o t i n j a i n j e k c i j o m g e n a u p r o n u k l e a r n o m s t a d i j u i k l o n i r a n j e ( P r a t h e r i F i r s t , 1 9 9 0 . ) .
R a z v o j t e h n i k e z a p o l u
i v a n j e j a j n i h s t a n i c a i z ~i v u i h d a v a t e l j i c a u z p o m o O P U o m o g u i o j e p r i m j e n u I V P z a g e n e t s k u s e l e k c i j u ( P i e t e r s e i s u r , 1 9 9 1 . ) . P o l u
i v a n j e j a j n i h s t a n i c a o d ~i v i h v i s o k o k v a l i t e t n i h k r a v a , b e z i k a k v e p o s l j e d i c e z a d a v a t e l j i c u , i o p l o d n j a i n v i t r o s o d g o v a r a j u i m b i k o m z a p r o i z v o d n j u t e l a d i s p e d i g r e o m j e j a s n a p r e d n o s t . K l j u
n i a s p e k t i s u : m o g u n o s t d o b i v a n j a j a j n i h s t a n i c a o d v i s o k o k v a l i t e t n i h k r a v a i o p l o d n j a s a s p e r m o m r a z l i
i t i h b i k o v a , t a k o d a s e d o b i j e j e d a n f a k t o r i j a l n i d i z a j n k o j i m s e n e p o v e a v a t o l i k o i n b r e e d i n g . Z a r a z l i k u o d M O E T - a k o j i p r o i z v o d i b r a u ( i s t a m a j k a , i s t i o t a c ) , u z p o m o I V P s e o d j e d n e m a j k e m o ~e p r o i z v e s t i v i ae p o t o m a k a o d r a z l i
i t i h b i k o v a ( p o l u - b r a e ) ; t a k o s e p o s t i ~e b r ~i g e n e t s k i n a p r e d a k u z i s t i i n b r e e d i n g ( W o o l l i a m s , 1 9 8 9 . ) . U
i n k o v i t o s t o v e f a k t o r i j a l n e s h e m e j e n a j v e a k a d j e b r o j k r a v a i b i k o v a p o d j e d n a k , t a k o d a s v a k a k r a v a i m a p o t o m k e s a r a z l i
i t i m b i k o v i m a ( W o o l l i a m s i W i l m u t , 1 9 8 9 ) . Z a n a v e d e n e p r i m j e n e p o t r e b n e s u d o b r o d e f i n i r a n e , u s p j e an e m e t o d o l o g i j e , k o j e j a m
e d o b r e i s t a b i l n e r e z u l t a t e , b a r e m at o s e s a m e p r o c e d u r e t i
e , j e r z a b i o l o ak i m a t e r i j a l n i k a d a n e m a g a r a n c i j e . S t a t i s t i
k i p o d a c i I E T S - a p o k a z u j u d a u 1 9 9 8 . g o d i n i 3 1 . 3 2 7 I V P z a m e t a k a p r e s a e n o u p r i m a t e l j i c e , n a j v e i d i o u E v r o p i ( 1 4 . 1 1 3 ) , a o t p r i l i k e p o l o v i c a s u b i l i z a m r z n u t i - o d m r z n u t i z a m e t c i ( T h i b i e r , 1 9 9 9 ) . P o s t o t a k g r a v i d n o s t i n a k o n t r a n s f e r a s v j e ~i h I V P z a m e t a k a k r e e s e o d 5 0 d o 6 0 % , a k o d t r a n s f e r a z a m r z n u t i h / o d m r z n u t i h z a m e t a k a t a j j e p o s t o t a k z n a t n o n i ~i t e n e p r e l a z i 3 5 % ( G o r d o n , 1 9 9 6 . ) . M n o g a i s t r a ~i v a n j a p o k a z a l a s u d a s e I V P z a m e c i p o s v o j i m m o r f o l o ak i m i f u n k c i o n a l n i m z n a
a j k a m a r a z l i k u j u o d o n i h d o b i v e n i h i n v i v o . T o s e o d n o s i n a r a z l i k e u p r o c e s u b r a z d a n j a , m o r f o l o g i j u e m b r i o n a l n e z a m e t n e m a s e , n i ~u m e t a b o l i
k u a k t i v n o s t e m b r i o n a l n i h s t a n i c a , o s j e t l j i v o s t n a s n i ~e n u t e m p e r a t u r u t e p r e ~i v l j a v a n j e n a k o n k o n v e n c i o n a l n i h p o s t u p a k a z a m r z a v a n j a / o d m r z a v a n j a ( W a g t e n d o n k - d e L e e u w i s u r . , 2 0 0 0 . ) .
P r o i z v o d n j a v e l i k o g b r o j a E T i I V P z a m e t a k a i m o g u n o s t i m e u n a r o d n e t r g o v i n e , p o s t a v i l a j e p i t a n j e r e g u l a c i j e z a k o n s k i h n o r m a t i v a . S t o g a j e M e u n a r o d n o D r u at v o z a E m b r i o t r a n s f e r ( I E T S ) d o n i j e l o p r o p i s e v e z a n e u z s a n i t a r n u k o n t r o l u p o i z v o d n j e z a m e t a k a d a b i s e s m a n j i o r i z i k m o g u e g p r i j e n o s a z a r a z n i h b o l e s t i ( S t r i n g f e l l o w i S e i d e l , 1 9 9 8 . ) . P r o v e d e n a s u m n o g a i s t r a ~i v a n j a d a b i s e d o k a z a l o d a v i ae k r a t n o " p r a n j e " z a m e t a k a , t r e t m a n t r i p s i n o m i d o d a t a k a n t i b i o t i k a u m e d i j e o s i g u r a v a j u u
i n k o v i t o u k l a n j a n j e i l i i n a k t i v a c i j u i n f e k t i v n i h a g e n s a ( S t r i n g f e l l o w i G i v e n s , 2 0 0 0 . ) . S t o g a j e n e o p h o d n o u s t a n d a r d n i p r o t o k o l z a p r o i z v o d n j u I V P z a m e t a k a , b i l o u k o m e r c i j a l n e i l i i s t r a ~i v a
k e s v r h e , u v r s t i t i i m e t o d e k o j e p r o p i s u j e I E T S .
4 . U
i n k o v i t a p r o i z v o d n j a z a m e t a k a i n v i t r o p r e d u v j e t j e z a p r i m j e n u b i o t e h n o l o g i j e
e t v r t e g e n e r a c i j e : k l o n i r a n j a , t r a n s g e n e z e , o d r e i v a n j a s p o l a p o t o m s t v a i d r . V i s o k o t e h n o l o ak i p o t e z i s u n a j u
i n k o v i t i j i u e k o n o m s k o m s m i s l u : n j i m a s e k o n t r o l i r a b l i ~n j e n j e , s p o l t e l a d i i m o g u s e k l o n i r a t i o d r a s l e ~i v o t i n j e . N a j n a p r e d n j i m b i o t e h n o l o g i j a m a s m a t r a m o o n e g d j e s e i z r a v n o m a n i p u l i r a g e n o m o m ~i v o t i n j a d a p r o i z v e d u n e at o p o ~e l j n o i l i d a o d s t r a n i m o n e p o ~e l j n o . U g l a v n o m s v i k o j i r a d e u o v o m p o d r u
j u s l a ~u s e d a j e v r l o v a ~n o p r e n i j e t i at o b r ~e g e n o m o d n u k l e u s n i h s t a d a u p r o i z v o d n e j e d i n i c e .
U o v o m o k v i r u m o ~e s e r a z m a t r a t i k a k a v s e d o d a t n i g e n e t s k i n a p r e d a k m o ~e d o b i t i p r i m j e n o m d r u g i h b i o t e h n o l o g i j a k a o at o s u : s e k s i r a n j e , m u l t i p l i k a c i j a g e n o m a j e d n o g z a m e t k a b l i ~n j e n j e m i l i t r a n s f e r o m j e z g r e ( k l o n i r a n j e ) . U s e k s i r a n j u s u r a z v i j e n e d v i j e m e t o d e : j e d n a v r ai p r e t r a g u s p o l a p o t o m k a p u t e m d e t e r m i n a c i j e D N K s p e c i f i
n i h k r o m o s o m a Y ( T h i b i e r i N i b a r t , 1 9 9 5 . ) , d r u g a o d v a j a s p e r m a t o z o i d e k o j e n o s e Y o d n o s n o X k r o m o s o m t a k o d a s e t a s p e r m a m o ~e k o r i s t i t i u o p l o d n j i i n v i t r o i l i
a k z a U O ( J o h n s o n i W e l c h , 1 9 9 9 . ) . O v i m s e p o s t u p c i m a m o ~e i z a b r a t i s p o l p o t o m a k a i t a k o i s k o r i s t i t i u p o t p u n o s t i p o t e n c i j a l p r o i z v e d e n e m l a d u n
a d i , u g l a v n o m z a i z m j e n u u m a t i
n o m s t a d u i l i z a b i k o v s k e m a j k e ( H o h e n b o k e n , 1 9 9 9 . ) .
Z a m u l t i p l i c i r a n j e g e n o m a j e d n e ~i v o t i n j e ( i l i z a m e t k a ) k o r i s t e s e d v i j e m e t o d e : b l i ~n j e n j e ( s p l i t t i n g ) i l i t r a n s f e r j e z g r e ( n u c l e a r t r a n s f e r ) . B l i ~n j e n j e m s e m o ~e p r o i z v e s t i d o 2 t e l a d i o d j e d n o g z a m e t k a , t e s e k o r i s t i k o m e r c i j a l n o i p o b o l j aa v a r e z u l t a t e e m b r i o t r a n s f e r a ( K i p p a x i s u r . , 1 9 9 1 . ) p a j e m o g u e d o b i t i d o 1 1 0 % t e l a d i v i ae n e g o s a m o M O E T - o m . P r i m j e n o m t e h n i k e t r a n s f e r a j e z g r e t a j s e b r o j m o ~e u v e l i k e p o v e a t i , i a k o o v a t e h n o l o g i j a j o a n i j e u s a v r ae n a . O v a b i o t e h n o l o g i j a o m o g u a v a p r o i z v o d n j u i d e n t i
n i h ~i v o t i n j a ( k l o n i r a n j e ) t e i m a s p e c i f i
n u p r i m j e n u s a m o z a o d r e e n e p r o i z v o d n j e i t o u g l a v n o m u z d r u g e m a n i p u l a c i j e g e n o m o m ( t r a n s g e n e z u ) s a p l i k a c i j o m u f a r m a c e u t s k o j i n d u s t r i j i ( T u r n e r , 1 9 9 9 . ; P i e d r a h i t a , 2 0 0 0 . ) .
Z a ai r o k u k o m e r c i j a l n u p r i m j e n u , n a j v e i u t j e c a j u e m b r i o b i o t e h n o l o g i j a m a i m a e k o n o m s k i i s p l a t l i v r u t i n s k i e m b r i o t r a n s f e r , a z a m r z a v a n j e i p o h r a n j i v a n j e z a m e t a k a , t e o d m r z a v a n j e n a f a r m i i d i r e k t n i t r a n s f e r u p r i m a t e l j i c e p r i m j e n j u j e s e r u t i n s k i ( V o e l k e l i H u . 1 9 9 2 . ) .
U m l i j e
n i h k r a v a m o g u s e p r o i z v e s t i t o v n i b l i z a n c i o d j a j n i h s t a n i c a i z j a j n i k a s a k u p l j e n i h n a k l a o n i c i i o s j e m e n j e n i h i n v i t r o s a m e s n a t i m b i k o m , t a k o d a s e m o ~e g a r a n t i r a t i d a t a t e l a d i m a b a r e m 7 5 % t o v n e g e n e t i k e , a p r o i z v e d e n i s u n a n a j j e f t i n i j i n a
i n .
S v e t e h n o l o g i j e k o j e ai r e p o ~e l j n i g e n o m s u p r i m j e n j i v e u p r o i z v o d n i m j e d i n i c a m a g d j e s e ~i v o t i n j e k o r i s t e i s k l j u
i v o z a p r o i z v o d n j u , a r a s p l o d u m a t i
n i m s t a d i m a . T o s e m o ~e p r i m j e n i t i o d M O E T - a d o k l o n i r a n j a . G e n e t s k e p r e d n o s t i , o c i j e n j e n e p e r f o r m a n s e t e s t o v i m a , s l u ~e z a o d a b i r v r h u n s k i h p r o i z v o d n i h ~i v o t i n j a , p r e m a k r i t e r i j i m a i n d u s t r i j e . T e s t o v i u k l j u
u j u t a k o e r k r i t e r i j e z a u z g o j t i h ~i v o t i n j a , k a o n p r . a k l i m a t i z a c i j a n a o d r e e n u o k o l i n u , o t p o r n o s t n a o d r e e n e b o l e s t i , t e m p e r a m e n t i d r u g o . M o g u e j e t a k o e r p r o i z v e s t i k r i ~a n c e k o r i s t e i p r e d n o s t i k o j a p r u ~a h e t e r o s i s e f e k t . T a k o u z g o j e n e j e d i n i c e b i t i e u p r o i z v o d n o m s m i s l u b o l j e n e g o m a t i
n o s t a d o .
I m a n e k o l i k o s t a j a l i at a u s v e z i p r i m j e n e o v i h b i o t e h n o l o ak i h m e t o d a : e k o n o m s k i f a k t o r i s u b i t n i , a l i i s a m a g e n e t s k a m a n i p u l a c i j a . M o r a s e s h v a t i t i d a u g e n e t s k o m n a p r e t k u p o s t o j i j e d a n p u t t e d a k o r a k n a p r i j e d u n e k o m o d r e e n o m p r a v c u , t e ak o m o ~e m o v r a t i t i u n a t r a g . R e p l i k a c i j a p o ~e l j n i h g e n a u p o p u l a c i j i i m a z a p o s l j e d i c u o d s t r a n j i v a n j e n e p o ~e l j n i h g e n a , a l i i u t o m s e s k l o p u i z g u b e d r u g i g e n i k o j i s e i z g u b e i t a k o o s i r o m a au j e u k u p n i g e n e t s k i f o n d . U g l a v n o m j e m i al j e n j e d a s e p r i j e s v a k o g p o m a k a u g e n e t i c i m o r a m o d e l i r a t i r e z u l t a t . Z a m r z a v a n j e s p e r m e i z a m e t a k a , j a j n i h s t a n i c a i b i o p s i j a j a j n i k a s u s r e d s t v a z a o
u v a n j e g e n o m a i s r e d s t v o p r o t i v k a t a s t r o f a l n i h p r o m j e n a . S v e s e o v e p r o m j e n e i z v o d e u s p o r a z u m u s a g e n e t i
a r i m a k o j i k o r i s t e g e n e t s k e m a r k e r e z a s e l e k c i j u ( W i l m u t i s u r . , 1 9 9 2 ) . G e n e t s k a m a n i p u l a c i j a j e j e d i n a p o s t o j e a m o g u n o s t d a s e s t v o r i n o v a g e n e t s k a v a r i j a b i l n o s t u j e d n o j v r s t i i z v a n p r i r o d n e m u t a c i j e , p r i
e m u s e b r o j v e p o s t o j e e g p o v e a v a s e l e k c i j o m i l i s e e x n o v o p r o i z v o d i n e at o n e p o s t o j e e . G e n e t s k e m a p e i m a r k e r i z a s e l e k c i j u p o m a ~u d a s e p o ~e l j n i g e n i u v o d e u j e d n u p o p u l a c i j u p r e k o i n t r o g r e s i j e o d n e k e d r u g e p o p u l a c i j e . S e l e k c i j a p r e k o o v i h g e n e t s k i h i n d i k a t o r a p r i m j e n o m r e p r o d u k t i v n i h b i o t e h n o l o g i j a i m a v e l i k u p r e d n o s t u g e n e t s k o m n a p r e t k u j e r p o v e a v a p r e c i z n o s t s e l e k c i j e , s m a n j u j e g e n e r a c i j i s k e i n t e r v a l e i p o v e a v a i n t e n z i t e t s e l e k c i j e : s v e s e t o m o ~e p r o v e s t i u d o p u s t i v o j g r a n i c i i n b r e e d i n g a ( W i l m u t i s u r . , 1 9 9 2 . ) .
P r i m j e n a b i o t e h n o l o g i j e r a s p l o i v a n j a u R H
U R e p u b l i c i H r v a t s k o j k o m p l e t n i M O E T p o s t u p a k u v e d e n j e u n a au v e t e r i n a r s k u m e d i c i n u u o k v i r u p r o j e k t a M i n i s t a r s t v a z n a n o s t i i t e h n o l o g i j e ( M Z T ) R H " E m b r i o t r a n s f e r u g o v e d a " ( 1 9 9 3 . - 1 9 9 7 . ) , t i j e k o m k o j e g s u i s t r a ~e n i r a z l i
i t i p o s t u p c i s u p e r o v u l a c i j e d a v a t e l j i c a ( M a k e k i s u r . , 1 9 9 4 . a ) , s m r z a v a n j a g o v e i h z a m e t a k a t e t r a n s f e r a u s i n k r o n i z i r a n e p r i m a t e l j i c e ( M a k e k i s u r . , 1 9 9 4 . b ; M a k e k i s u r . , 1 9 9 4 . c ) . K a o r e z u l t a t s u r a d n j e s t r u
n j a k a K l i n i k e z a p o r o d n i at v o i r e p r o d u k c i j u V e t e r i n a r s k o g f a k u l t e t a S v e u
i l i at a u Z a g r e b u i C e n t r a z a r e p r o d u k c i j u u s t o
a r s t v u H r v a t s k e d . o . o . r o e n o j e p r v o t e l e n a k o n u s p j e an o g e m b r i o t r a n s f e r a u r u j n u 1 9 9 5 . g o d i n e ( M a k e k i s u r . , 1 9 9 6 . )
U o k v i r u p r o j e k t a M Z T " P r o i z v o d n j a i n v i t r o g o v e i h z a m e t a k a " ( 1 9 9 7 . - ) p o s t a v l j e n j e s t a n d a r d n i p r o t o k o l z a d o z r i j e v a n j e , o p l o d n j u i u z g o j z a m e t a k a i n v i t r o t e p o s t a v l j e n i t e m e l j i z a m o g u n o s t i p r i m j e n e i i s k o r i at a v a n j a p r e d n o s t i o v e b i o t e h n o l o g i j e i u n a ao j s t o
a r s k o j p r o i z v o d n j i . U s u r a d n j i s t r u
n j a k a g o r e n a v e d e n e K l i n i k e i C e n t r a z a r e p r o d u k c i j u u s t o
a r s t v u H r v a t s k e d . o . o . o s n o v a n j e L a b o r a t o r i j z a a s i s t i r a n u r e p r o d u k c i j u ~i v o t i n j a . D a n a an j e m o g u n o s t i n a ae g L a b o r a t o r i j a o b u h v a a j u : M O E T , I V P i z k l a o n i
k o g m a t e r i j a l a i O P U s u p e r o v u l a r i n i h i l i k r a v a u c i k l u s u t e e m b r i o t r a n s f e r u p r i m a t e l j i c e , k r i o p r e z e r v a c i j u z a m e t a k a t e r a n u u l t r a z v u
n u d i j a g n o s t i k u g r a v i d i t e t a u k r a v a p r i m a t e l j i c a . P o r e d t o g a i s t r a ~u j e m o i p r i m j e n u d r u g i h b i o t e h n o l o g i j a i m o g u n o s t i n j i h o v e p r i m j e n e u d r u g i h ~i v o t i n j s k i h v r s t a .
O d 1 9 9 5 . g o d i n e u v e d e n j e u n a s t a v n i p r o g r a m n a K l i n i c i z a p o r o d n i at v o i r e p r o d u k c i j u V e t e r i n a r s k o g f a k u l t e t a S v e u
i l i at a u Z a g r e b u s e m i n a r " E m b r i o t r a n s f e r i b i o t e h n o l o g i j a r a s p l o i v a n j a " , k o j i s e s e o d r ~a v a u V I I I . s e m e s t r u d o d i p l o m s k e n a s t a v e , k a k o b i s e b u d u i v e t e r i n a r i u p o z n a l i i p r i p r e m i l i z a p r i m j e n u o v i h n o v i h b i o t e h n o l o g i j a u n a ao j z e m l j i .
D a b i o v e b i o t e h n o l o ak e m e t o d e p o s t a l e p r i m j e n j i v e u n a ai m u v j e t i m a i d a b i m o g l i i s k o r i s t i t i n j i h o v e p r e d n o s t i u g e n e t s k o m n a p r e t k u s t a d a , n e o p h o d o j e o d r e d i t i ~e l j e n e p r o i z v o d n e c i l j e v e t e u s k l a d i t i p r o g a m e i r a d r a z l i
i t i h i n s t i t u c i j a u k l j u
e n i h u s t o
a r s k u p r o i z v o d n j u i s e l e k c i j u t e v e t e r i n a r s k e s l u ~b e . P r i m j e n a n a v e d e n i h b i o t e h n o l o ak i h m e t o d a n u ~n a j e z a u k l j u
i v a n j e n a ae s t o
a r s k e p r o i z v o d n j e u m e u n a r o d n e t o k o v e , p o g o t o v o n a k o n o s t v a r e n j a m o g u n o s t i u l a s k a u W T O .
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M a k e k , Z . , M . H e r a k , M . C e r g o l j , D . R u d a n , M e l i t a H e r a k , I v a B a r a c - G e t z , T . V e n d l , Z . T u
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M . M a t k o v i , I v a G e t z , Z . M a k e k
R E P R O D U C T I V E B I O T E C H N O L O G Y :
P O S S I B L E U S E I N C A T T L E - B R E EDING OF CROATIA
Abstract
Reproductive biotechnology includes manipulations of animal reproduction under controled conditions and influences on genetic selection by propagation of adventageous genetics according to desirable traits. These biotechnologies are developing for the last fifty years, according to circumstances and possibilites of each period. The first generation of biotehnology used in animal breeding was artifitial insemination, the second was embryo transfer and related technologies, the third was production of embryos in vitro and the fourth was transgenesis and cloning. The application of these new biotechnologies in cattle breeding shemes (1) facilitates genetic progress of the herd by obtaining numerous offspring from cattle of high genetic quality (2) enables international trade and transport of frozen embyos reducing the need for long-distance cattle movement and avoiding the risk of disease agents transmission (3) permitts high quality breeding stock to be available for sale in much larger numbers then were previously possible (4) enables application of other developing technologies, such as embryo sexing and embryo spliting, genetical tests, sperm separation techniques and gender preselection, production of transgenic cattle and others. The adoption of these procedures require sofisticated equipment and special trainings and therefore their use is expensive, but their effect on selected herd is considarable and fast. Researchers in this area agree that it is extremely important to transfer genome rapidly from nucleus herds to production units.
Under the project financed by Ministry of science and technology "Production of bovine embryos in vitro" the standard protocol for maturation, fertilization and culture in vitro was developed. Laboratory for assisted animal reproduction was founded in the Clinic for obstetrics and reproduction, Faculty of Veterinary Medicine University of Zagreb, in collaboration with experts from the Center for reproduction and animal breeding of Croatia d.o.o. Bovine embryos produced in our laboratory are of good quality and suitable for embryo transfer or cryopreservation. This paper reviews the application of these new biotechnologies in cattle breeding shemes and possible use in our stockbreeding conditions.
Key words: reproduction, biotechnology, embryo transfer, culture in vitro, embryo.
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