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Zebu (Bos indicus) ovarian preantral follicles: morphological
characterization and development of an efficient isolation
method
C a r o l i n a M a d e i r a Lu c c i
R o d o l f o R u m p f
Jo s é R i c a r d o F i g u e i r e d o
So n i a N a i r B é o
Abstract
P r e a n t r a l f o l l i c l e s a r e a m a j o r s o u r c e o f o o c y t e s , a n d t h e i r u t i l i z a t i o n a s a n
i m p o r t a n t t o o l t o s t o r e l a r g e n u m b e r o f f e m a l e g a m e t e s f o r f u t u r e u s e in
r e p r o d u c t i v e p r o g r a m s h a s b e e n i n v e s t i g a t e d . T h e i n c r e a s i n g i m p o r t a n c e o f
s t u d i e s in t h i s s u b j e c t , t o g e t h e r w i t h t h e i m p o r t a n t r o l e o f Z e b u c a t t l e in t h e
e c o n o m y o f t r o p i c a l a n d s u b t r o p i c a l c o u n t r i e s a s w e l l a s t h e i r w e l l - k n o w n
d i f f e r e n c e s f r o m E u r o p e a n c a t t l e , l e d t o t h i s r e s e a r c h . T h e p r e s e n t s t u d y a i m s
t o d e t e r m i n e t h e b e s t s i z e i n t e r v a l f o r s e c t i o n i n g o v a r i a n t i s s u e t o i s o l a t e
p r e a n t r a l f o l l i c l e s f r o m Z e b u c o w s u s i n g a t i s s u e c h o p p e r a n d t o e v a l u a t e t h e
f o l l i c u l a r q u a l i t y a f t e r i s o l a t i o n . F u r t h e r m o r e , i t a i m s t o p r o v i d e i n f o r m a t i o n
a b o u t t h e Z e b u c o w p r e a n t r a l f o l l i c l e p o p u l a t i o n a n d u s e t h i s d a t a ( a s a
c o n t r o l ) t o e v a l u a t e t h e e f f e c t i v e n e s s o f t h e t e s t e d i s o l a t i o n m e t h o d . T e s t i n g
e i g h t d i f f e r e n t t i s s u e s e c t i o n i n g s i z e i n t e r v a l s , i t w a s p o s s i b l e t o c o n c l u d e t h a t
t h e 1 2 5 - p m - s e c t i o n i n t e r v a l i s s h o w n t o b e b e t t e r t h a n t h e i n t e r v a l s o f 2 5 , 5 0 ,
1 7 5 a n d 2 0 0 p m t o i s o l a t e p r e a n t r a l f o l l i c l e s f r o m Z e b u c o w o v a r i e s . T h e 1 2 5 -
p m i n t e r v a l a l l o w e d t h e r e c o v e r y o f 2 6 , 0 5 0 ± 1 6 1 1 ( m e a n i S . E . M . ) p r e a n t r a l
f o l l i c l e s p e r o n e - h a l f o v a r y , w h i l e t h e n u m b e r o f p r e a n t r a l f o l l i c l e s in si t u
e s t i m a t e d b y e v a l u a t i o n o f h i s t o l o g i c a l s e c t i o n s w a s 3 5 , 2 8 8 1 2 3 4 2 p e r o n e - h a l f
o v a r y . T h u s , t h e m e a n ( i S . E . M . ) r e c o v e r y r a t e ( = [ n u m b e r o f p r e a n t r a l f o l l i c l e s
i s o l a t e d / n u m b e r o f p r e a n t r a l f o l l i c l e s in si t u i n t h e s a m e o v a r y ] x 1 0 0 ) w a s
7 4 . 3 ± 4 . 3 % . T h e m o r p h o m e t r i c a l a n a l y s i s s h o w e d t h a t B o s i n d i c u s p r e a n t r a l
f o l l i c l e s a r e s i m i l a r t o B. t a u r u s p r e a n t r a l f o l l i c l e s b a s e d o n p r e v i o u s r e p o r t s . In
c o n c l u s i o n , t h i s s t u d y s h o w e d t h a t a s i m p l e , m e c h a n i c a l m e t h o d c a n b e u se d
e f f e c t i v e l y t o i s o l a t e a l a r g e n u m b e r o f i n t a c t p r e a n t r a l f o l l i c l e s f r o m Z e b u c o w
o v a r i e s , w i t h a h i g h r e c o v e r y r a t e .
1. Introduction
In th e p a s t fe w d e c a d e s , a g re a t a m o u n t o f re s e a rc h h a s a d d re s s e d th e im p o rta n c e o f s tu d y in g o v a r ia n p re a n tra l fo llic le s fo r fu tu re u tiliz a tio n in re p ro d u c tiv e p ro g ra m s . A s th e s e fo llic le s a c c o u n t fo r th e v a s t m a jo rity o f fo llic le s (9 0 % ) in th e o v a ry [1], th e y c a n p ro v id e a g re a t n u m b e r o f p o te n tia lly v ia b le o o c y te s fo r th e in v itro re p ro d u c tiv e te c h n iq u e s , s u c h as IV F a n d c lo n in g . T o r e a c h th is g o a l, h o w e v e r, it is im p o rta n t to is o la te th e s e fo llic le s fr o m th e o va ry, o th e rw is e , th e y w ill n a tu ra lly a n d in v a ria b ly d ie b y a tre s ia [1].
T h e m o s t im p o rta n t c h a ra c te ris tic o f a n is o la tio n m e th o d is to r e s c u e a la rg e n u m b e r o f fo llic le s w ith o u t c a u s in g d a m a g e to th e ir stru c tu re . In d e e d , o n e o f th e m o s t e ffic ie n t m e th o d s fo r is o la tin g a la rg e n u m b e r o f fo llic le s c o n s is ts o f u s in g a tis s u e c h o p p e r [2—4]. R e c e n tly , h o w e v e r, s tu d ie s h a v e d e m o n s tra te d th a t th e b e s t in te rv a l fo r s e c tio n in g th e o v a ria n tis s u e u s in g th e tis s u e c h o p p e r c a n v a ry a c c o rd in g to s p e c ie s [5,6 ]. In a d d itio n to is o la tin g a la rg e n u m b e r o f fo llic le s , a s u ita b le m e th o d s h o u ld p re s e rv e th e n o rm a l fo llic u la r m o rp h o lo g y a fte r is o la tio n , n o t o n ly o n th e s tru c tu ra l le v e l, b u t a lso o n th e u ltra s tru c tu r a l le v e l [7]. T h e m a in te n a n c e o f fo llic u la r q u a lity a fte r is o la tio n is e x tre m e ly im p o rta n t a n d s h o u ld b e in v e s tig a te d , b e c a u s e s tru c tu ra l o r u ltra s tru c tu r a l d a m a g e s c a n sig n ific a n tly a ffe c t fu r th e r in v itro g ro w th a n d m a tu ra tio n o f o o c y te s w ith in p re a n tra l fo llic le s [8].
In s p ite o f th e f a c t th a t th e re is a lo t o f lite ra tu re c o n c e rn in g E u ro p e a n b o v in e p re a n tra l fo llic le s , a lm o s t n o th in g is k n o w n a b o u t th is a re a in Z e b u c a ttle . A c tu a lly , Z e b u b o v in e
(Bos indicus
) d iffe r fro m E u ro p e a n b o v in e(B. taurus)
in s e v e ra l a s p e c ts re la te d to re p ro d u c tio n [ 9 - 1 4 ], a n d th e re fo re , it is im p o rta n t to s ta rt s tu d y in g th e p re a n tra l fo llic le p o p u la tio n in th is b o v in e g e n o ty p e .T h e p re s e n t s tu d y a im s to d e te rm in e th e b e s t s iz e in te rv a l fo r s e c tio n in g th e o v a r ia n tis s u e to is o la te p re a n tra l fo llic le s fro m Z e b u c o w s u s in g a tis s u e c h o p p e r a n d to e v a lu a te th e f o llic u la r q u a lity a fte r is o la tio n . F u rth e rm o re , it a im s to p ro v id e in fo rm a tio n a b o u t th e p re a n tra l fo llic le p o p u la tio n in Z e b u c o w s, a n d u s e th is d a ta (a s a c o n tro l) to e v a lu a te th e e ffe c tiv e n e s s o f th e s tu d ie d is o la tio n m e th o d .
2. Materials and methods
2.1. Ovaries
A to ta l o f 12 o v a r ie s fr o m 12 n o n p re g n a n t a d u lt Z e b u c o w s (N e lo re ) w e re u s e d in th is stud y, six p e r e x p e rim e n t. A ll th e o v a rie s w e re c o lle c te d a t a lo c a l s la u g h te rh o u s e a n d tra n s p o rte d to th e la b o ra to ry w ith in 1 -3 h . D u rin g tra n s p o rt, th e o v a rie s w e re s to re d in a th e rm o s fla s k fille d w ith P B S k e p t a t 3 2 - 3 4 °C .
2.2. Experiment 1: isolation o f preantral follicles
Sections interval adjustment button
Cutting arm with blade
Rotating Disk
Mobile Plate
t
F ig . 1. (A ) T h e tis s u e c h o p p e r in a p a n o ra m ic v iew , a n d (B ) a c lo s e u p o f th e c u ttin g a rm , th e m o b il e p la te a n d th e ro ta tin g d is k . A f te r se ttin g th e se c tio n in te rv a l p a ra m e te rs a n d tu rn in g o n th e e q u ip m e n t, th e p la te m o v e s a c c o rd in g ly fro m le ft to r ig h t w h ile t h e c u ttin g a rm m a k e s ra p id u p -a n d -d o w n m o v e m e n ts . (C ) A d ia g ra m o f th e lo n g itu d in a l (c u t 1), tr a n s v e rs e (c u t 2 ) a n d o b liq u e (c u t 3) c u ts m a d e to p ro d u c e v e ry s m a ll a n d re la tiv e ly u n ifo rm -s iz e d o v a ria n fr a g m e n ts .
S u rre y , U K ): 2 5 ( T l ) , 5 0 (T 2 ), 7 5 (T 3 ), 100 (T 4 ), 125 (T 5 ), 150 (T 6 ), 175 (T 7 ), a n d 2 0 0 |u n (T 8 ). T o s im u lta n e o u s ly te s t a ll th e tre a tm e n ts , e a c h o v a ry w a s d iv id e d in to e ig h t e q u a l p a rts th a t w e re ra n d o m ly d is trib u te d to th e e ig h t te s t tre a tm e n ts . In a ll tr e a tm e n ts , th e tis su e c h o p p e r c u t th e o v a ria n tis s u e fr o m th e c o rtic a l to th e m e d u lla r sid e a lo n g th e lo n g itu d in a l, tra n s v e rs e a n d o b liq u e a x e s, p ro d u c in g v e ry s m a ll a n d r e la tiv e ly u n ifo rm -s iz e d fr a g m e n ts . T h e o v a ria n fr a g m e n ts o b ta in e d w e re p la c e d in 5 0 m l o f P B S p lu s 5 % fe ta l c a lf s e ru m (S ig m a , St. L o u is , M O , U S A ), a n d th e n s u s p e n d e d 2 0 tim e s w ith a la rg e P a s te u r p ip e tte (d ia m e te r ^ 1 6 0 0 p m ) a n d 2 0 tim e s w ith a s m a lle r P a s te u r p ip e tte (d ia m e te r ^ 6 0 0 p m ). T h e s u s p e n s io n s w e re s e p a ra te ly filte re d th ro u g h 5 0 0 a n d 100 p m N y lo n m e s h filte rs (F ig . 2). E a c h tr e a tm e n t w a s re p e a te d six tim e s .
To e s tim a te th e n u m b e r o f is o la te d p re a n tra l fo llic le s p e r tr e a tm e n t, th e P ibe s c o n ta in in g th e s u s p e n s io n w e re g e n tly in v e rte d s e v e ra l tim e s to d is p e rs e th e fo llic le s . T h e n , tw o s a m p le s o f 1 m l e a c h w e re ta k e n fr o m e a c h tu b e a n d e x a m in e d s e p a ra te ly u n d e r a p h a s e c o n tra s t in v e rte d m ic ro s c o p e (Z e is s A x io v e r t, Je n a , G e rm a n y ).
Fig. 2. E xp erim en ta l p ro tocol to te st d iffe re n t interval o f serial se ction s in the tissue c h o p p e r to isolate preantral fo llic le s from Z eb u cow ovaries.
fo llic le s h a v e a sin g le la y e r o f c u b ic a l g ra n u lo s a c e lls , a n d se c o n d a ry fo llic le s a re o o c y te s s u rro u n d e d b y tw o o r m o re la y e rs o f c u b ic a l g ra n u lo s a c e lls.
2.3. Experiment 2: qualitative and quantitative analysis o f the specific method
T o e v a lu a te q u a n tita tiv e ly a n d q u a lita tiv e ly , th e e ffic ie n c y o f th e m e c h a n ic a l m e th o d fo r th e is o la tio n o f p re a n tra l fo llic le s , e a c h o v a ry w a s c u t lo n g itu d in a lly in to tw o h a lv e s. O n e h a lf w a s u s e d to stu d y th e p re a n tra l fo llic le p o p u la tio n in situ a n d th e o th e r w a s u s e d to is o la te p re a n tra l fo llic le s (F ig. 3). P re a n tr a l fo llic le s w e re is o la te d fr o m th e o v a ria n h a lv e s u s in g th e tre a tm e n t th a t y ie ld e d th e la rg e s t n u m b e r o f is o la te d p re a n tra l fo llic le s , fro m th e e ig h t tre a tm e n ts te s te d in E x p e rim e n t 1. T h e d ia m e te rs o f fr e s h ly is o la te d p re a n tra l fo llic le s w e re m e a s u re d u n d e r a n in v e rte d m ic ro s c o p e u sin g a n o c u la r m ic ro m e te r.
H is to lo g ic a l p ro c e s s
T is s u e c h o p p e r - b e s t in te r v a l F ix a tio n
M e c h a n ic a l d is so c ia tio n (P a s te u r p ip e tte s - 1 6 00 a n d 6 0 0 p m )
E s tim a tio n a n d c la s sific a tio n o f in situ p re a n tra l fo llic le p o p u la tio n F iltr a tio n (5 0 0 a n d 100 p m )
P rim o rd ia l P rim a r y S e c o n d a ry
C o u n tin g a n d c la s s if ic a tio n o f is o la te d fo llic le s
M o r p h o lo g ic a lly A tre tic
P rim o rd ia l P rim a r y S e c o n d a r y fo llic le s n o rm a l fo llic le s
H is to lo g ic a l e v a lu a tio n
M o rp h o lo g ic a lly A tre tic
fo llic le s n o r m a l fo llic le s
Fig. 3. E x pe rim e n ta l p ro to c o l to study Z eb u cow o va ria n p re a n tra l follic le s in situ a nd a fte r isolation p roc ed ure .
b re a k d o w n . F o r e a c h o va ry, th e re c o v e ry ra te (R R ) o f fo llic le s is o la te d b y th e m e c h a n ic a l m e th o d w a s c a lc u la te d as fo llo w s:
n u m b e r o f is o la te d fo llic le s R R p e r o v a ry = --- —— :— — — — x 100.
n u m b e r o f in situ fo llic le s
S e c tio n s w e re e x a m in e d a n d p h o to g ra p h e d u s in g a Z e is s A x io p h o t lig h t m ic ro s c o p e . U s in g a n o c u la r m ic ro m e te r, th e d ia m e te rs o f th e fo llic le s a n d th e o o c y te s in situ w e re d e te rm in e d a n d th e g ra n u lo s a c e lls w e re c o u n te d , b o th in th e s e c tio n w h e re th e n u c le o lu s o f th e o o c y te w a s o b s e rv e d (e q u a to ria l se c tio n ).
T o e v a lu a te th e ir q u a lity , fre s h ly is o la te d p re a n tra l fo llic le s w e re p re fix e d in a s o lu tio n c o n ta in in g 2 % p a r a f o r m a ld e h y d e a n d 2 .5 % g lu ta ra ld e h y d e in 0.1 M s o d iu m c a c o d y la te b u ffe r, fo r 3 0 m in a t ro o m te m p e ra tu re . T h e n , th e fo llic le s w e re e m b e d d e d in d ro p s o f 2 0 0 p i o f 4 % a g a r s o lu tio n (a g a ro s e , ty p e V II, S ig m a , S t. L o u is , M O , U S A ). T h e a g a r d ro p s w e re fix e d in th e s a m e fix a tio n s o lu tio n d e s c rib e d a b o v e f o r a n o th e r 3 h a t ro o m te m p e ra tu re . T h e d ro p s w e re d e h y d ra te d , e m b e d d e d in p a r a ffin w a x , s e ria lly s e c tio n e d a t a th ic k n e s s o f 7 p m a n d s ta in e d w ith P A S a n d h a e m a to x y lin . P re a n tra l fo llic le s w e re c la s s ifie d as m o rp h o lo g ic a lly n o rm a l o r d e g e n e ra te as d e s c rib e d a b o v e .
T o b e tte r e v a lu a te fo llic u la r m o rp h o lo g y , u ltra s tru c tu r a l a n a ly s is w a s p e r fo rm e d in s o m e is o la te d fo llic le s to v e rif y i f s lig h t d a m a g e s th a t c o u ld n o t b e o b s e rv e d a t th e lig h t m ic ro s c o p ic le v e l w e re p re s e n t. S m a ll p ie c e s o f n o n tre a te d o v a r ie s w e re p ro c e s s e d as w e ll a n d u s e d as a c o n tro l. Is o la te d fo llic le s w e re p re fix e d a n d e m b e d d e d in a g a r d ro p s as d e s c rib e d a b o v e . T h e o v a ria n p ie c e s a n d th e a g a r d ro p s w e re fix e d in a s o lu tio n c o n ta in in g 2 % p a r a f o r m a ld e h y d e a n d 2 .5 % g lu ta ra ld e h y d e in 0.1 M s o d iu m c a c o d y la te b u ffe r, fo r 3 h a t ro o m te m p e ra tu re o r o v e rn ig h t a t 4 °C . A fte r fix a tio n , th e s p e c im e n s w e re rin s e d in b u ffe r a n d p o s ffix e d in a s o lu tio n c o n ta in in g 1% o s m iu m te tro x id e , 0 .8 % p o ta s s iu m fe rric y a n id e a n d 5 m M C a C l2 in 0.1 M c a c o d y la te b u ffe r. S u b s e q u e n tly , th e s a m p le s w e re d e h y d ra te d in a c e to n e a n d e m b e d d e d in S p u rr. T h in s e c tio n s (7 0 n m ) w e re s ta in e d w ith u ra n y l a c e ta te a n d le a d c itra te , a n d e x a m in e d u s in g a J e o l J E M 100C tra n s m is s io n e le c tro n m ic ro s c o p e . A ll th e o rg a n e lle s w e re c a re fu lly o b s e rv e d , a n d s p e c ia l a tte n tio n w a s g iv e n to th e b a s e m e n t m e m b ra n e a n d th e c o n ta c t b e tw e e n o o c y te a n d g ra n u lo s a c e lls , w h e re th e m e c h a n ic a l is o la tio n m ig h t h a v e c a u s e d p h y s ic a l d a m a g e .
2.4. Statistical analysis
N u m b e r o f isola ted p re a n tra l fo llic le s (m ean ± S .E .M .) a t e ig ht d iffe re nt c ut intervals T a b le 1
T re a tm e n t (c ut in te rv al (pm )) N u m b e r o f fo llicles p e r o n e-eig h th ovary (n= 6)
M e a n ± S .E .M . R ang e
T1 (25) 3 3 16 ± 4 97 a 207 9—4806
T 2 (50) 3 6 36 ± 5 56 a 2 1 3 3 -5 7 2 4
T 3 (75) 4 1 4 0 ± 6 14 ab 6 2 1 -7 8 8 4
T 4 (100) 56 29 ± 1118 ab 2 4 0 3 -1 0 4 2 2
T 5 (125) 64 08 ± 1137 b 3 0 2 4 -1 1 0 7 0
T 6 (150) 5 1 12 ± 818 ab 2 7 0 0 -7 4 7 9
T 7 (175) 37 57 ± 699 a 1 5 9 3-5 96 7
T 8 (200) 3631 ± 375 a 2 0 2 5 -6 2 6 4
R ow s w ith d iffe re nt lette rs d iffe r sign ifican tly (P < 0.05).
3. Results
3.1. Isolation o f ovarian preantral follicles
T a b le 1 sh o w s th e o u tc o m e th a t th e e ig h t tre a tm e n ts h a d o n th e n u m b e r o f p re a n tra l fo llic le s is o la te d p e r o n e -e ig h th o va ry. T h e m e c h a n ic a l is o la tio n tre a tm e n t o f th e Z e b u c o w o v a rie s p ro v id e d a la rg e n u m b e r o f is o la te d p re a n tra l fo llic le s , irre s p e c tiv e o f th e c u t in te rv a l u s e d . H o w e v e r, a la rg e in d iv id u a l v a ria tio n w ith in th e tre a tm e n ts w a s o b s e r v e d . T h e lim its o f th is v a ria tio n w e re 62 1 (T 3 ) a n d 1 1 ,0 7 0 (T 5 ) p re a n tra l fo llic le s p e r o n e -e ig h th o v ary . T h e m e a n n u m b e r o f is o la te d p re a n tra l fo llic le s in c re a s e d fro m T1 (25 p m ) to T 5 (1 2 5 p m ) a n d d e c re a s e d a fte rw a rd s in T 6 (1 5 0 p m ), T 7 (1 7 5 p m ) a n d T 8 (2 0 0 p m ). H o w e v e r, T 5 (1 2 5 p m ) s h o w e d a sig n ific a n tly
(P
< 0 .0 5 ) h ig h e r n u m b e r o f is o la te d p re a n tra l fo llic le s o n ly w h e n c o m p a re d w ith T1 (25 p m ), T 2 (5 0 p m ), T 7 (1 75 p m ) a n d T 8 (2 0 0 p m ).3.2. Number o f preantral follicles in situ and after isolation
S in c e T 5 (1 2 5 p m ) a llo w e d th e h ig h e s t n u m b e r o f is o la te d p re a n tra l fo llic le s a n d w a s th e o n ly tre a tm e n t th a t sh o w e d a n y s ta tis tic a l d iffe re n c e w h e n c o m p a re d to th e o th e rs , th is tr e a tm e n t w a s c h o s e n to p ro c e e d w ith f o llic u la r is o la tio n in th e s e c o n d e x p e rim e n t. U s in g th e 1 2 5 -p m in te rv a l, th e n u m b e r (m e a n ± S .E .M .) o f is o la te d p re a n tra l fo llic le s p e r o n e - h a l f o v a ry w a s 2 6 ,0 5 0 ± 1611 (r a n g e 2 3 ,9 0 0 -3 0 ,8 0 0 ) . T h e n u m b e r (m e a n ± S .E .M .) o f p re a n tra l fo llic le s in situ , e s tim a te d b y e v a lu a tio n o f h is to lo g ic a l s e c tio n s w a s 3 5 ,2 8 8 ± 2 3 4 2 (r a n g e 2 9 ,8 9 9 -3 9 ,4 1 0 ) p e r o n e - h a lf o va ry. T h u s , th e m e a n ( ± S .E .M .) re c o v e ry r a te ( = [n u m b e r o f p re a n tra l fo llic le s is o la te d /n u m b e r o f p re a n tra l fo llic le s in situ in th e s a m e o v ary ] x 100) w a s 7 4 .3 ± 4 .3 % (r a n g e 6 1 .4 - 7 9 .9 % ) . O v e ra ll, th e n u m b e r o f p re a n tra l fo llic le s in s itu w a s sig n ific a n tly h ig h e r th a n th e n u m b e r o f is o la te d p re a n tra l fo llic le s in o n e - h a lf o v a ry
(P
< 0 .0 5 ).T a b le 2
M e a n n u m b e r a n d p e rc e n ta g e o f p rim o rd ia l, p rim a ry a n d se c o n d a ry fo llic le s in situ a n d iso la te d p e r o n e - h a lf o v a ry o f Z e b u c o w s (m e a n ± S .E .M .)
F o llic le c la ss N u m b e r a n d p e r c e n ta g e o f p re a n tra l fo llic le s p e r o n e - h a lf o v a ry (m e a n ± S .E .M .)
R e c o v e ry r a te
In situ Iso la te d
P rim o rd ia l P rim a ry S e c o n d a ry
3 2 2 6 6 ± 1 84 6 a A (9 1 .4 % ± 1.5) a D 2 4 0 4 ± 6 5 2 a B (6 .5 % ± 2 .4 ) a E
5 9 0 ± 196 a C (2 .1 % ± 1.6) a F
2 5 5 0 0 ± 1 6 0 2 b A (9 8 .0 % ± 0 .9 ) b D 4 0 0 ± 3 5 b B (1 .6 % ± 0 .9 ) b E 15 0 ± 2 0 b C (0 .4 % ± 0 .2 ) b F
7 9 .2 ± 3 .2 G 2 0 .5 ± 5 .2 H 3 7 .6 ± 1 6 .5 H
Total 3 5 2 8 8 ± 2 3 4 2 a (1 0 0 % ) 2 6 0 5 0 ± 1 61 2 b (1 0 0 % ) 7 4 .3 ± 4 .3
C o lu m n s w ith d iffe re n t le tte rs (a , b ) d iffe r P < 0 .0 5 . R o w s w ith d iffe re n t le tte rs (A , B , C ) d if f e r P < 0 .0 5 (c o n c e rn in g n u m b e r o f fo llic le s) . R o w s w ith d iffe re n t le tte rs (D , E , F ) d iffe r P < 0 .0 5 (c o n c e rn in g p e rc e n ta g e o f fo llic le s) . R o w s w ith d iffe re n t le tte rs (G , H ) d iffe r (c o n c e rn in g re c o v e ry ra te ) sig n ific a n tly (P < 0 .0 5 ).
th e p ro p o rtio n s o f p re a n tra l fo llic le c la s s e s o b ta in e d a fte r m e c h a n ic a l is o la tio n w e re sig n ific a n tly d iffe re n t th a n th o s e o b s e rv e d in situ
(P
< 0 .0 5 ). T h e m e a n re c o v e ry ra te s o f p rim o rd ia l, p rim a ry a n d s e c o n d a ry fo llic le s w e re 7 9 .2 , 2 0 .5 a n d 3 7 .6 % , re s p e c tiv e ly (T a b le 2 ), sh o w in g th a t th e m e c h a n ic a l m e th o d is o la te d m a in ly p rim o rd ia l fo llic le s(P
< 0 .0 5 ). "3.3. Morphometric analysis o f preantral follicles in situ and after isolation
H is to lo g ic a l a n a ly s is in situ s h o w e d th a t p rim o rd ia l fo llic le s h a v e a n o o c y te s u rro u n d e d b y o n e la y e r o f s q u a m o u s o r s q u a m o u s -c u b o id a l g ra n u lo s a c e lls ; p rim a ry fo llic le s h a v e a s in g le la y e r o f c u b o id a l g ra n u lo s a c e lls , a n d s e c o n d a ry fo llic le s h a v e a n o o c y te s u rro u n d e d b y tw o o r m o re la y e rs o f c u b o id a l g ra n u lo s a c e lls (F ig . 4). A ll th e p re a n tra l fo llic le s p re s e n te d a s tro n g ly PA S p o s itiv e b a s e m e n t m e m b ra n e s u rro u n d in g th e m . F re s h ly is o la te d
F ig . 5 . F re sh m e c h a n ic a lly is o la te d Z e b u p re a n tra l fo llic le s ( 1 7 5 x ) . Pr: p rim o rd ia l f o llic le s ; P: p rim a ry fo llic le s; a n d S : s e c o n d a ry fo llic le s.
p rim o rd ia l, p rim a ry , a n d s e c o n d a ry fo llic le s in th e s u s p e n s io n w e re s p h e ric a l w ith o n e o r m o re la y e rs o f g ra n u lo s a c e lls a ro u n d th e o o c y te , s u rro u n d e d b y a b a s e m e n t m e m b ra n e (F ig . 5 ). A fte r is o la tio n , th e o o c y te s o f p rim a ry a n d s e c o n d a ry fo llic le s w e re n o t a lw a y s c le a rly v is ib le b e c a u s e g ra n u lo s a c e lls c o v e re d th e m .
P rim o rd ia l, p rim a ry a n d s e c o n d a ry fo llic le s in situ m e a s u re d 3 6 .0 ± 0 .9 , 4 8 .5 ± 1 . 4 a n d 8 8 .4 ± 2 .9 p m in d ia m e te r , re s p e c tiv e ly , w h e re a s a fte r is o la tio n th e y m e a s u re d 2 2 .1 ± 0 .5 , 4 5 .5 ± 1 . 4 a n d 7 3 .6 ± 1 . 7 p m , re s p e c tiv e ly . T h e fo llic u la r d ia m e te rs d iffe re d s ig n ific a n tly a m o n g th e th re e fo llic u la r c la s s e s , b o th in siP i a n d a fte r is o la tio n
(P
< 0 .0 5 ). A lso , th e d ia m e te rs o f p rim o rd ia l a n d s e c o n d a ry fo llic le s w e re s ig n ific a n tly lo w e r a fte r is o la tio n th a n in situ(P
< 0 .0 5 ). A s e x p e c te d , th e o o c y te d ia m e te r in situ a n d th e n u m b e r o f g ra n u lo s a c e lls a t th e e q u a to r ia l s e c tio n d iffe re d sig n ific a n tly(P
< 0 .0 5 ) a m o n g fo llic u la r c la s s e s (T a b le 3).T able 3
M o rp h o m e tr ic a l p a ra m e te rs o f p rim o rd ia l, p rim a ry a n d se c o n d a ry f o llic le s a f te r is o la tio n a n d in situ (m e a n ± S .E .M .)
F o llic u la r c la s s e s
Iso la te d In situ
N F o llic u la r 0
(ra n g e )
N F o llic u la r 0
(ra n g e )
O o c y te 0 (ra n g e )
N u m b e r o f g r a n u lo s a c e lls
P rim o rd ia l 108 2 2 .1 ± 0 .5 A a 8 0 3 6 .0 ± 0 .9 b A 2 8.1 ± 0 .6 A 7 .3 ± 0 .4 A
(1 5 - 3 3 ) (2 5 - 5 0 ) (2 0 - 3 5 ) (1 - 1 3 )
P rim a r y 6 0 4 5 .5 ± 1.4 aB 6 6 4 8 .5 ± 1.4 a B 3 1 .7 ± 0 .7 B 1 4.6 ± 0 .7 B
(3 5 - 5 9 ) (3 5 - 7 0 ) (2 5 —45 ) (1 0 - 2 9 )
S e c o n d a ry 26 7 3 .6 ± 1.7 a C 33 8 8 .4 ± 2 .9 b C 4 3 .8 ± 1.4 C 6 2 .0 ± 4 .6 C
(6 9 - 7 9 ) (7 0 - 1 2 0 ) (3 5 - 5 6 ) (3 5 - 1 1 1 )
A m o n g th e th o u s a n d s o f p re a n tra l fo llic le s e n c o u n te re d in th e o v a ria n c o rte x , in 8 3 % (h v e /s ix ) o f th e o v a rie s so m e p o ly o v u la r p re a n tra l fo llic le s (p rim o rd ia l a n d p rim a ry ) c o u ld b e see n . T h e n u m b e r o f o o c y te s w ith in th e s a m e f o llic le v a r ie d b e tw e e n tw o a n d th re e . T h e n u m b e r o f p o ly o v u la r p re a n tra l fo llic le s w a s n o t e x p re s s e d d u e to la c k o f p a ra m e te rs o f c a lc u la tio n . In is o la te d fo llic le s , it w a s p o s s ib le to id e n tify o n ly tw o c a s e s o f p o ly o v u la r fo llic le .
3.4. Qualitative analysis o f preantral follicles in situ and after mechanical isolation
F re s h ly is o la te d p re a n tra l fo llic le s a p p e a re d h e a lth y u n d e r th e in v e rte d m ic ro s c o p e a n d n o d e n u d e d o o c y te s w e re o b s e rv e d in th is study. H is to lo g ic a l e v a lu a tio n s h o w e d th a t, a fte r m e c h a n ic a l is o la tio n , 9 3 .5 % o f p re a n tra l fo llic le s w e re m o rp h o lo g ic a lly n o rm a l, w ith a h e a lth y o o c y te s u rro u n d e d b y o n e o r m o re w e ll-o rg a n iz e d la y e rs o f g ra n u lo s a c e lls w ith o u t p y c n o tic n u c le i (F ig . 6 A ). H is to c h e m is try a lso re v e a le d th a t th e m e c h a n ic a lly is o la te d fo llic le s w e re s u rro u n d e d b y a n in ta c t b a s e m e n t m e m b ra n e . T h e p e rc e n ta g e s o f d e g e n e ra te d fo llic le s in situ a n d a fte r is o la tio n w e re 3 .9 a n d 6 .5 % , re s p e c tiv e ly . In sp ite o f th e
( B )
n u m e ric a l d iffe re n c e , th e s ta tis tic a l a n a ly s is o f th e n u m b e r o f d e g e n e ra te d fo llic le s a fte r is o la tio n a n d in situ s h o w e d th a t th e re w a s n o s ig n ific a n t d iffe re n c e
(P =
0 .2 8 4 3 ). R e tra c tio n o f th e o o c y te o r c o n d e n s a tio n o f its n u c le u s (F ig . 6 B ) c h a ra c te riz e d th e fo llic u la r d e g e n e ra tio n m o s t fre q u e n tly o b s e rv e d , b o th in situ a n d a fte r is o la tio n . G ra n u lo s a c e lls w e re a p p a re n tly u n a ffe c te d . T h e s e d e g e n e ra tiv e sig n a ls w e re d e te c te d o n ly h is to lo g ic a lly a n d n o t u n d e r th e in v e rte d m ic ro s c o p e .T h e tra n s m is s io n e le c tro n m ic ro s c o p y a n a ly s is o f is o la te d fo llic le s (F ig . 7 A ) sh o w e d th a t th e y w e re u ltra s tru c tu r a lly s im ila r to th o s e in situ (F ig . 7 B ), in d ic a tin g th a t th e y w e re n o t a ffe c te d b y th e is o la tio n p ro c e d u re . T h e o o c y te s p re s e n te d a la rg e a n d w e ll-d e m a rc a te d n u c le u s , n u m e ro u s ro u n d m ito c h o n d ria , a fe w c is te rn a e o f s m o o th e n d o p la s m ic re tic u lu m a n d G o lg i a p p a ra tu s , a n d a fe w v e s ic le s w ith in th e ir c y to p la s m . G ra n u lo s a c e lls p re s e n te d m a n y e lo n g a te d m ito c h o n d ria a n d a b u n d a n t sm o o th a n d ro u g h e n d o p la s m ic r e tic u lu m a n d G o lg i a p p a ra tu s . N o a lte ra tio n s w e re o b s e rv e d in th e c o n ta c t b e tw e e n o o c y te a n d g ra n u lo s a c e lls in is o la te d fo llic le s . G ra n u lo s a c e lls a n d o o c y te w e re c lo s e to g e th e r a n d th e b a s e m e n t m e m b ra n e w a s a lw a y s p re s e n t s u rro u n d in g th e fo llic le s , b o th in n o n tre a te d o v a ria n tis s u e a n d a fte r m e c h a n ic a l is o la tio n . T h e u ltra s tru c tu r a l a n a ly s is re v e a le d th a t th e b a s e m e n t m e m b ra n e is c o m p o s e d o f a c o m p a c t b a s a l la m in a s u rro u n d e d b y a th ic k la y e r o f c o lla g e n fibe rs.
4. Discussion
T h e r e s u lts o f th is s tu d y h a v e s h o w n th a t a la rg e n u m b e r o f Z e b u p re a n tra l fo llic le s c a n b e is o la te d u s in g a s im p le m e c h a n ic a l p ro c e d u re a n d th a t th e n u m b e r o b ta in e d is a ffe c te d b y th e c u t in te rv a l c h o s e n fo r s e c tio n in g th e o v a r ia n tis su e . T h is e ffe c t h a s a lre a d y b e e n d e m o n s tra te d in o th e r sp e c ie s, lik e c a p rin e [6] a n d o v in e [5]. In th e p re s e n t w o rk , th e g re a te s t n u m b e r o f is o la te d Z e b u c o w p re a n tra l fo llic le s w a s o b ta in e d u s in g th e 125 p m in te rv a l. In g o a ts a n d sh e e p , th e b e s t re s u lts w e re o b ta in e d w ith th e in te rv a ls o f 75 p m [6], a n d 8 7 .5 p m [5], re s p e c tiv e ly . T h e s e re s u lts su g g e st, a g a in , th a t th e m o s t s u ita b le in te rv a l fo r s e c tio n in g th e o v a ria n tis s u e u s in g a tis s u e c h o p p e r v a r ie s a m o n g s p e c ie s . In th e p re s e n t stud y, it se e m s th a t th e u s e o f c u t in te rv a ls s m a lle r th a n 125 p m m a y d e s tro y p re a n tra l fo llic le s , w h ile th e la rg e r in te rv a ls m a y re d u c e th e e ffic ie n c y o f m e c h a n ic a l d is s o c ia tio n o f th e o v a ria n fra g m e n ts w ith P a s te u r p ip e tte s a n d th u s p re v e n t p re a n tra l fo llic le r e le a s e fr o m th e o v a ria n tis su e . S p e c ie s -s p e c ific d iffe re n c e s re g a rd in g th e m o s t s u ita b le c u t in te rv a l to is o la te p re a n tra l fo llic le s c a n b e e x p la in e d b y d iffe re n c e s in o v a ria n c o m p o s itio n , w h ic h c a n in te rfe re in th e c u ttin g p ro c e s s . F o r e x a m p le , fo llic le s e m b e d d e d in a m o re fib ro u s s tro m a c a n b e m o re d iffic u lt to is o la te d a n d s m a lle r c u t in te rv a ls w o u ld b e n e c e ss a ry .
d iffe re n c e s , p re a n tra l f o llic le p o p u la tio n s m a y b e a ffe c te d b y fa c to rs su c h as b re e d [16], a g e [1 8 ,2 1 ], h o rm o n e le v e ls a n d re p ro d u c tiv e s ta g e [22], a n d n u tritio n [23].
T h e n u m b e r o f p re a n tra l fo llic le s re c o v e re d fro m Z e b u o v a r ie s u s in g th e 125 p m in te rv a l w a s 5 2 ,1 0 0 (2 6 ,0 5 0 p e r o n e - h a lf o v a ry ). O th e r re s e a rc h e rs is o la te d a la rg e n u m b e r o f o v a ria n p re a n tra l fo llic le s fro m E u ro p e a n c o w s [3 ,1 5 ,2 4 ], g o a ts [4,6 ], e w e s [2,5 ], p ig s [25], d o m e s tic c a ts [26 ,2 7 ] a n d n o n d o m e s tic fe lid s [2 8 ,2 9 ], ra b b its [30 ], ra ts [31], m ic e [32], a n d h u m a n s [33], u s in g m e c h a n ic a l [2 ^ 1 ,1 5 ,2 4 ,2 6 -2 9 ] o r e n z y m a tic [2 5 ,3 0 -3 3 ] p ro c e d u re s . C o m p a ris o n s b e tw e e n o u r re s u lts a n d th o s e o f o th e r s tu d ie s a re d iffic u lt to p e r fo rm d u e to s p e c ie s d iffe re n c e s , v a ria tio n in fo llic le c la s s ific a tio n , a n d ty p e s o f fo llic le s in c lu d e d in th e fin a l c o u n t. O n th e o th e r h a n d , th e fo llic u la r re c o v e ry r a te is a g o o d p a r a m e te r to c o m p a re th e e ffe c tiv e n e s s o f d iffe re n t is o la tio n m e th o d s . In th e p re s e n t stu dy , th e re c o v e ry r a te o f p re a n tra l fo llic le s w a s 7 4 % , a n d is h ig h e r th a n th o s e d e s c rib e d fo r g o a ts (3 6 % , 4 ) a n d s h e e p (2 7 % , 2 ). I t is im p o rta n t to n o te th a t th e r e c o v e ry r a te s h o u ld o n ly b e a p p lie d u s in g h a l f o f th e s a m e o v a ry o r a t le a s t th e c o n tra la te ra l o v a ry as a c o n tro l, s in c e th e p o p u la tio n o f th e p re a n tra l fo llic le m a y b e a ffe c te d b y o th e r fa c to rs , as m e n tio n e d a b o v e .
C o n c e rn in g th e d is trib u tio n o f th e is o la te d fo llic le s in to f o llic u la r c la s s e s , in th e p re s e n t w o rk , 9 8 .0 , 1.6 a n d 0 .4 % o f th e to ta l is o la te d fo llic le s w e re p rim o rd ia l, p rim a ry a n d s e c o n d a ry , re s p e c tiv e ly . O th e r stu d ie s s h o w e d s im ila r re s u lts in th e is o la tio n o f p re a n tra l fo llic le s u s in g a tis s u e c h o p p e r [2 ,4 ,3 4 ]. In c o n tra s t, H u ls h o f e t a l. [15 ], u s in g w a tc h m a k e r ’s fo rc e p s to is o la te p re a n tra l fo llic le s , o b s e rv e d a lo w e r p e r c e n ta g e o f is o la te d p rim o rd ia l fo llic le s (1 2 .5 % ) w h e n c o m p a re d to p rim a ry (5 7 .2 % ) a n d s e c o n d a ry (2 0 .7 % ) fo llic le s . T h e s e a u th o rs s u g g e s t th a t it m a y b e d u e to p rim o rd ia l fo llic le s b e in g tig h tly e m b e d d e d in th e tu n ic a a lb u g in e a [15 ], a n d a m ild m e c h a n ic a l tr e a tm e n t is n o t s u ffic ie n t to is o la te th e s e fo llic le s . A la rg e n u m b e r o f p rim o rd ia l fo llic le s w a s e a s ily is o la te d fr o m p ig o v a rie s u s in g a c o m b in e d m e c h a n ic a l a n d e n z y m a tic m e th o d [35], s u g g e s tin g th a t an e n z y m a tic tr e a tm e n t o f th e o v a rie s m a y b e n e c e s s a ry to is o la te th is c la s s o f p re a n tra l fo llic le s . B a s e d o n th e p re s e n t w o rk , th e u s e o f th e tis s u e c h o p p e r p ro v e s to b e a m o re p o w e rfu l m e th o d to is o la te a g re a t n u m b e r o f p rim o rd ia l fo llic le s w ith o u t a d d itio n a l e n z y m a tic tre a tm e n t. O v e ra ll, th is m e c h a n ic a l m e th o d is o la te d a b o u t 7 9 .2 ,2 0 .5 a n d 3 7 .6 % , re s p e c tiv e ly , o f th e to ta l p rim o rd ia l, p rim a ry a n d s e c o n d a ry fo llic le s p re s e n t in Z e b u c o w o v a rie s .
B e fo re th e q u a lita tiv e e v a lu a tio n o f th e is o la te d fo llic le s c o u ld b e d o n e , a c h a ra c te riz a tio n o f th e p re a n tra l f o llic le s in situ h a d to b e p e rfo rm e d . T h e re s u lts s h o w e d th a t th e m e a n d ia m e te r o f Z e b u o o c y te s a n d fo llic le s a n d th e m e a n n u m b e r o f g ra n u lo s a c e lls a t th e e q u a to ria l s e c tio n w e re s im ila r to th o s e o b s e rv e d in E u ro p e a n c o w s [3 6 -3 8 ] in a ll th re e d e v e lo p m e n ta l sta g e s (i.e . p rim o rd ia l, p rim a ry a n d s e c o n d a ry ). A lso , o u r re s u lts w e re s im ila r to th o s e d e s c rib e d fo r h u m a n s [39], b u t d iffe re n t fr o m th o s e d e s c rib e d fo r e w e s [40] a n d g o a ts [41 ]. In a d d itio n , s o m e p o ly o v u la r fo llic le s c o u ld b e o b s e rv e d in Z e b u c o w o v a rie s. T h e o c c u rre n c e o f p o ly o v u la r fo llic le s h a s b e e n re p o rte d in o th e r m a m m a ls [4 ,3 5 ,4 2 ,4 3 ]. A lth o u g h th is k in d o f fo llic le s a p p e a rs to b e n o rm a l in d iffe re n t m a m m a lia n sp e c ie s , in w h ic h th e y a re s e e n w ith a v a ria b le fre q u e n c y , th e ir ro le re m a in s u n k n o w n .
o v a ria n stro m a . A c c o rd in g to V an W e z e l a n d R o d g e rs [38 ], p rim o rd ia l fo llic le s sh o w a n o b la te s h a p e in th re e d im e n s io n s . O n c e in s u s p e n s io n th e s e fo llic le s p ro b a b ly a s s u m e a v e rtic a l p o s itio n , w ith th e p o le s u p a n d d o w n , m a k in g it p o s s ib le to m e a s u re o n ly th e s m a lle s t d ia m e te r. In c o n tra s t, in h is to lo g ic a l s e c tio n s , w e c o u ld s e e th e fo llic le s in m a n y p o s itio n s , a n d th e re fo re , ta k e th e s m a lle s t o r th e la rg e s t d ia m e te r d e p e n d in g o n th e fo llic le s o rie n ta tio n .
In re fe re n c e to th e q u a lity o f th e is o la te d p re a n tra l fo llic le s , in th is stu dy , n o d e n u d e d o o c y te s w e re o b s e rv e d , lik e ly g iv e n th e f a c t th a t th e b a s e m e n t m e m b ra n e o ffe rs m e c h a n ic a l re s is ta n c e a n d p ro te c ts th e fo llic le s fr o m p h y s ic a l d a m a g e d u rin g is o la tio n , as s u g g e s te d b y F ig u e ire d o e t al. [44 ]. H is to c h e m ic a l a n d u ltra s tru c tu ra l a n a ly s is d e m o n s tra te d th a t m e c h a n ic a lly is o la te d fo llic le s w e re s u rro u n d e d b y a n in ta c t b a s e m e n t m e m b ra n e . S im ila r re s u lts w e re o b ta in e d b y F ig u e ire d o e t al. [44 ,45 ] a n d L u c c i e t al. [4] u s in g a tis s u e c h o p p e r to is o la te b o v in e a n d c a p rin e p re a n tra l fo llic le s , re s p e c tiv e ly . In c o n tra s t, s o m e stu d ie s h a v e d e s c rib e d th a t p ro te o ly tic e n z y m e s c o m m o n ly u s e d fo r is o la tin g fo llic le s a re d e trim e n ta l to c e ll m e m b ra n e s [ 3 0 ,4 6 —4 9 1. In v itro c u ltu re o f e n z y m a tic a lly is o la te d p re a n tra l fo llic le s , w h ic h a re n o t s u rro u n d e d b y a b a s e m e n t m e m b ra n e , sh o w s a s p re a d in g o f g ra n u lo s a c e lls fro m th e o o c y te s [8 ,5 0 ]. In c o n tra s t, th is la tte r o c c u rre n c e w a s n o t o b s e rv e d w h e n m e c h a n ic a lly is o la te d p re a n tra l fo llic le s w e re c u ltu re d in v itro [4 5]. T h e c u ltu re o f p re a n tra l fo llic le s s u rro u n d e d b y a n a tu ra l b a s e m e n t m e m b ra n e h a s m a n y a d v a n ta g e s in c lu d in g p re s e rv a tio n o f f o llic u la r m o rp h o lo g y a n d m a in te n a n c e o f f o llic u la r a d h e s io n to e x tra c e llu la r c o m p o u n d s [45]. T h u s , th e p re s e n c e o f a b a s e m e n t m e m b ra n e a r o u n d th e is o la te d fo llic le s is im p o rta n t fo r fu r th e r s tu d ie s o f in v itro c u ltu re o f Z e b u p re a n tra l fo llic le s .
F u rth e rm o re , th is s tu d y s h o w e d th a t o n ly a fe w fo llic le s w e re d e g e n e ra te d in s itu (3 .9 % ), as w e ll as a fte r is o la tio n (6 .5 % ). T h e lo w p e r c e n ta g e o f d e g e n e ra te d fo llic le s o b s e rv e d in th is s tu d y c o in c id e s w ith o th e r stu d ie s [1 6 ,5 1 ]. T h e h is to lo g ic a l a n a ly s is o f p re a n tra l fo llic le s , in situ a n d is o la te d , s h o w e d th a t th e d e g e n e ra tio n is c h a ra c te riz e d b y o o c y te a lte ra tio n s w h ile p y c n o tic b o d ie s w e re n o t o b s e rv e d in g ra n u lo s a c e lls . J o rio e t a l. [51] d e s c rib e d th a t d e g e n e ra tio n o f o o c y te s is th e m o d e o f a tre s ia m o s t fre q u e n tly o b s e rv e d in p re a n tra l fo llic le s . A lth o u g h p y c n o s is o f g ra n u lo s a c e lls fre q u e n tly o c c u rs in a n tra l fo llic le s , it is a lm o s t a b s e n t in p re a n tra l fo llic le s . M o re o v e r, in a n in v itro stu d y b y B ra w -T a l a n d Y o s s e h [36] it w a s fo u n d th a t in s o m e p re a n tra l fo llic le s th e o o c y te d e g e n e ra te d o r c o m p le te ly d is a p p e a re d w h ile g ra n u lo s a c e lls lo o k e d h e a lth y a n d c o n tin u e d to p ro life ra te , sh o w in g th a t th e o o c y te is m u c h m o re s e n s itiv e th a n th e g ra n u lo s a c e lls .
In th is stu dy , th e e le c tro n m ic ro s c o p y re v e a le d th a t is o la te d fo llic le s w e re u ltr a s tr u c tu r a lly s im ila r to th o s e p re s e n t in situ . F o llic u la r d a m a g e p o s s ib ly c a u s e d b y th e is o la tio n m e th o d w a s n o t o b s e rv e d in th e p re s e n t stu dy , c o n tra ry to V an D e n H u rk e t al. [7], w h o d e s c rib e d a p o o r u ltra s tru c tu r e o f th e s m a ll p re a n tra l fo llic le s is o la te d u s in g a tis s u e c h o p p e r. S m a ll p re a n tra l fo llic le s a re v e ry s e n s itiv e to o s m o la rity c h a n g e s a n d c e n tr ifu g a tio n ( L u c c i e t a l., u n p u b lis h e d d a ta ). T h e re fo re , th is d iffe re n c e b e tw e e n stu d ie s m a y b e d u e to d iffe re n t m e th o d s u s e d to p ro c e s s th e is o la te d fo llic le s fo r u ltra s tru c tu ra l a n a ly s is , s in c e th e s a m e is o la tio n m e th o d w a s u s e d b y b o th .
th e first tim e a n e s tim a tio n o f th e p re a n tra l fo llic le p o p u la tio n in Z e b u c o w o v a rie s . T h e o b ta in e d d a ta re v e a le d th a t, d e s p ite th e d iffe re n c e s b e tw e e n
B. taurus
a n dB. indicus,
Z e b u p re a n tra l fo llic le s s e e m e d to b e v e r y s im ila r to E u ro p e a n c o w fo llic le s in n u m b e r a n d m e a s u r e m e n t c h a ra c te ris tic s .Acknowledgements
In m e m o ry o f A .R . d e B e rn . T h e a u th o rs th a n k N . D u rr e tt fo r th e E n g lis h c o rre c tio n o f th e m a n u s c rip t. T h is re s e a rc h w a s s u p p o rte d b y C N P q .
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