A comparison of dissected follicle numbers and follicle counts on the ovarian surface for the evaluation of ovarian follicular populations in Bos indicus cows

‘pa~~asa~ sw% ‘WWsnV

IIV ‘~‘8 a3”a!~s ~a!*aw

‘OSEZ ap2pyq

I-SI910(96)OZ~P-8LEOS IId ~661 o GWdo3 ~O’LI$/L~/OZEP-8~~0

‘pus@3 MaN 30 bi!smafun ‘sacma!q aqj 30 b1fn3q + :[a1 ‘loqme Fmpuodsa.uo3

ntl.npa.n~l~~y3yledzlll.aal :pm-3 :gzs~fj~-~~-~g + :xe~ :89[p18-~~-19

, _

Qaso~D a.n2‘lalaurwp pweur~isa uo paseq sassel3 az!s olu! uoge2!3!ssel3~!atppuv ~~03 sns?ppu! sag Jo SaueAo a4130 axyns UaqM sJaquInu30 awwgsa~apun LpJO paluasald SapJO

aq1 uo alq!s!Asa[3!~0330 slunoa xopt2npzAa a2eyns dq pasyo8ale2 ue 01 Ou!pea~ ‘LIBAO aq~30 ax3_Ins aq~ uo ease ~ua~nlsw~ 11ews B

2ZrS uunpa~

Ielolpuv a8.uq ‘wn!paw

.illaAg3adsaJ ‘6L.Opue 69'0 '()p'O ‘9L.Oa.ta~slaqurnu a[~[103

‘Ipxus u! sa13y103pai~assrppw

uo!l~IaJJo3 'S'Zf P'OP 'SA 9'1 f p’9Z pm

‘6’1 T 0’!3Z ‘SA 9’1 T p’pz

WM

axyns

30 sluno3 ~03 (A) s~ua!~!33ao3

1’0 f L’O ‘SA 1’0 T &TO‘0’1 f 9’11 ‘SA Z’O + f 9’1

‘iClaA!l~adsaJ ‘SJaquInUapg[o3 [BIO) pue S+IO%IW

‘IpXIIs U! (3s T ueaw) sap11103pavassrp pur! axJms axJms

PUe

.IO+J qqefi

PaAOUI~J

&‘Jc?[ ‘urn!paur

30 s.Iaqurnulo3 viea 'sa1x1103pal~awp

30 s~aiaunxp ~aqi!auo paseq sauoEia,e~(UIW 8 <)~%EI

[pXuS .Iaql!a u! pag_lssI?[3 Alan ~a~~![~03 ‘~.WAO qcw 52 .lapUn pa.UlSkTaUl S.laWlu?!paIXIIO3

sa1x~o3 [Iv .pa.uweatuAlan tin0 qxa30

JO

JO (rum g-p) uuupaur ‘(mm p >) .nz[nDoue %Isnado3so+UoaJals

WIOllS

SSC33X2 ‘S+IRAO

‘Jqj

UIOl3 pajaaSS!p

‘dUIO~CK+EAO .Ia]3t? ~~~~IBpaUKUI 'hIX0

I@ 30 (pa%laA'2 SaXE J.IOqSfNIt?%UOI) S.la~aUll?!p WaW

axpns

aJaM unu Z 7

dIXOI3

22

‘SaSSBIJ

aql uo aIq!s!Aulzu z 7

sa1y103

Aq pun03 sasse13 az!s asoy] u! sapg103 30 slaqwnu ~J!M pa]ela.uo~

aql 30 uop~ass!p aqdwo3

aZ!S .ICIlXIIE!p OlU! UO!lt2XJ!SS"I3 J!aI# PUIJ SM03

SYZ3~pU~SOg 30 Sa!.llZAO aql

30 a3eJms aql uo alqya sap~~~o330 siuno3 ieqlsrsaqloddqaq, au~uwxa 01 pasn alar smo~(sn3!pu~ sag ~/L--P/E) sso.13wxuq~?.t~8ugelx1 ‘sno.nzdgpw a%pax!m

9661 lSn%v

981-6~1

0~ uioy (0~1 = u) say~()

LZ pdx-v

(~661) 98 a3ua?S

uo!mpoJdax

P!=V

180

L.A. Fitzpatrick and K. W. Entwistle/Animal Reproduction Science 46 (1997) 179-186

correlated with numbers of follicles in those size classes found at dissection of the ovary for small (< 4 mm), large (> 8 mm) and total follicles but not for medium sized (4-8 mm) follicles. 0 1997 Elsevier Science B.V. Keywords: Cattle-ovary;

Ovary; Follicular

populations

1. Introduction A variety of techniques has been used to assess ovarian follicle growth and activity in cattle. Initially, reliance was on histological methods (Rajakoski, 1960; Marion and Gier, 1971; Dufour and Roy, 1985), on measuring either the size of the largest one or two follicles per ovary (or pair of ovaries) or the numbers of different-sized antral follicles within ovaries (Spicer and Echtemkamp, 1986). Another common method used has been to count and measure the diameter of follicles on the surface of ovaries (Matton et al,, 1981; Ireland and Roche, 1982; Dufour and Roy, 1985; Spicer et al., 1991) though there are few studies to support the validity or functional applicability of this technique in Bos tuurus cattle (Spicer et al., 1987), while data for Bos indicus cattle are not available. More recently, real-time ultrasonography of ovaries via a transrectal approach has been used to quantify ovarian follicles in situ, in order to define numbers and growth rates of follicles (Pierson and Ginther, 1984, 1987; Fortune et al., 1988; Lucy et al., 1992; Rhodes et al., 1995). While ultrasound imaging of ovaries has greatly facilitated the study of follicle populations and the development and regression of follicles through various size classes (Lucy et al., 1992), measures of follicle populations I 5 mm are, at best, only estimates due to difficulties experienced in visualising these size classes by ultrasonography. Such difficulties are compounded in studies using B. indicus cattle, which tend to have more small follicles than B. tuurus cattle (Segerson et al., 1984). Furthermore, many basic studies into follicular health and atresia still require direct visualisation and sampling of follicular fluid to determine ratios of steroid concentrations from various classes of follicle sizes. In this study, we examined the hypothesis that counts of follicles visible on the surface of the ovaries of B. indicus cows and their classification into size classes based on estimated diameter, are closely correlated with numbers of follicles in those size classes actually present on dissection of the ovary.

2. Materials and methods 2.1. Animals Over a 3 year period, 70 mixed-age multiparous lactating Brahman cross (3/4-7/8 Bos indicus) cows were ovariectomised via an incision in the left paralumbar fossa, as previously described (McSweeney et al., 1993) in the course of a number of studies of ovarian function.

L.A. Fitzpatrick and K. W. Entwistle/Animal

Reproduction

181

Science 46 (1997) 179-186

2.2. Follicle counting and dissection procedures Immediately after ovariectomy, mean diameters (long and short axes averaged) of all follicles 2 2 mm visible on the surface of each ovary were measured using vernier callipers. An effort was made to identify the outer perimeter of each follicle. This subjective method was used by the same individual throughout the study to assess surface diameter. The ovarian follicles were then either dissected fresh or following snap-freezing of ovaries by plunging ovaries into liquid nitrogen, and subsequent thawing. All follicles 2 2 mm were dissected from the ovaries and excess stroma removed (McSweeney et al., 1993). Diameters of dissected follicles were measured under the low power of a stereomicroscope using an ocular graticule. Measurements of long and short axes were also averaged. For each ovary, follicles were classified on the basis of diameter as either small ( < 4 mm), medium (4-8 mm) or large (> 8 mm). 2.3. Statistical

analysis

The data were analysed using the multiple regression procedure of Statistix (1990). Both the predictor (surface count) and the response (dissection count) variables were square root transformed to achieve homoscedosdicity and to maintain linear relationship.

3. Results Untransformed mean counts of surface and dissected follicles in small (< 4 mm), medium (4-8 mm), and large (> 8 mm) categories and total numbers of follicles are presented in Table 1, while the results of multiple regression analyses of transformed data are presented in Table 2. Plots of the linear regressions of transformed data for surface counts vs. dissected counts of small, medium, large and total follicles are presented in Fig. 1. Surface counts of small antral follicles visible on the surface of ovaries accounted for 92% of small follicles subsequently found at the time of dissection of the ovary. There was a high correlation ( r = 0.76) between counts of surface and dissected follicles in the small category. However, only 14% of medium follicles found at the time of dissection

Table 1 Untransformed data on surface and dissected counts (mean* SE) of small ( < 4 mm), medium large ( > 8 mm) and total follicles per ovary from postpartum Brahman cross cows (n = 70)

Number of ovaries Small follicles ( < 4 mm) Medium follicles (4-8 mm) Large follicles ( > 8 mm) Total follicles

Surface count

Dissection count

140 24.3 + 1.6 1.6kO.2 0.5+0.1 26.4 Y!Y 1.6

140 28.0f 1.9 11.6+ 1.0 0.7+0.1 40.4 + 2.5

(4-8

mm),

182

LA. Fitzpatrick and K. W. Entwistle/Animal Reproduction Science 46 (1997) 179-186

Table 2 Multiple regression analysis of counts of ovarian surface follicles vs. dissected medium (4-8 mm), and large ( > 8 mm) categories and total follicle numbers Size class

Intercept (SE)

Slope (SE)

Adjusted

Small Medium Large Total

1.257 2.187 0.225 1.507

0.801 0.823 0.801 0.931

0.583 0.162 0.474 0.622

(0.282) (0.196) (0.05 1) (0.315)

(0.057) (0.155) (0.072) (0.061)

follicles of small ( < 4 mm),

R2

df

P

139 139 139 139

< < < <

0.001 0.001 0.001 0.001

14

12 10

(a)

a

-0

1

2

3

4

5

6

9

a

7

10

a/ .-:’

:

i

.

_---z-z ---0

:

;-__.~_

1

0

1

=,

‘__;--r

:.. ,.

‘___

--

0))

___

--~~---r--_--, ‘.

I

.“’

3

2

..

3t

,, ., ...' _ / 5 2’ ,,,,. ". __-- __--_I---- ==y--1 __===--... !__---------------EM==_ .._ _411....i:m ~. : 0 2 1 0 . ..

(cl

14

W

0

1

2

3

4

5

6

7

8

9

10

Square Root (Surface Follicle Count) Fig. 1. Relationships between counts of follicles on the ovarian surface and counts of follicles subsequently dissected from the ovaries of 70 Brahman cross cows, for three diameter classes (a) < 4 mm, (b) 4-8 mm and (c) > 8 mm) and for total counts, with fitted line (-_), 95% confidence intervals (- - -_) and prediction limits (’ ‘1.

L.A. Fitzpatrick and K. W. Entwistle/Animal Reproduction Scierzce 46 (1997) 179-186

183

were detected on the surface of the ovaries and the correlation between counts of surface and dissected follicles was low (r = 0.40). Sixty-three percent of large follicles present at the time of dissection of the ovaries were visible on the surface before dissection and the correlation between these two variables was high (r = 0.69). The actual number of large follicles in the study was small, relative to the other size categories. While only 66% of total follicles > 2 mm were visible on the ovarian surface, the correlation between counts of total surface and total dissected follicles was reasonably high (Y = 0.79) (Tables I and 2: Fig. 1).

4. Discussion Counts of follicles on the ovarian surface and their classification into size categories based on diameter can be used to obtain a reasonable indication of actual populations of small, large and total follicles present in the ovaries of B. indicus cows. However. surface counts in this study did not accurately reflect actual populations of medium sized follicles. Like small follicles, medium sized follicles presented only a small translucent area on the surface of ovaries, leading to an underestimate of numbers of this follicle class. These findings are in agreement with a previous study (Spicer et al., 1987), in which ovaries from B. taurus cows were evaluated and measurements of surface diameters tended to underestimate diameters for dissected follicles < 8.0 mm. Even when a conscious effort was made to estimate follicle diameter based on size of the translucent area and the more opaque area which surrounds the follicle, many medium sized follicles were classified in the small category. Mean surface counts (k SE) of small follicles (24.4 _+ 1.6), in the present study closely approximated those obtained following ovarian dissection (28.0 & 1.91, because some small follicles which were buried in the ovarian cortex were not included in the surface count but were detected on dissection of the ovary. Those small follicles that were buried below the ovarian surface also accounted for much of the difference found between total surface follicles (26.4 f 1.6) and total dissected follicles (40.4 j-- 2.5). Spicer et al. (1987) compared diameters of follicles greater than 5.9 mm from 32 pairs of ovaries from B. tuurus cows, measured on the ovarian surface and diameters of the same follicles subsequently dissected from the ovaries. Measured diameters from both approaches yielded approximately equivalent results. In that study, measurement of surface diameters tended to underestimate dissected diameter for follicles < 8.0 mm in diameter and to overestimate dissected diameter for follicles > 12.0 mm. Furthermore, the correlation coefficient between diameters as estimated by surface and dissection evaluation in that study was 0.83, although the estimated diameters of follicles on the surface of the ovary were compared to their diameters measured after dissection, not surface count to dissected count. When diameters for surface and dissected estimations were compared, correlations were similar in that study and the present study (r = 0.79). While many studies have used counts of ovarian surface follicles, classified into a variety of size categories, as a measure of ovarian response to different treatments

184

L.A. Fitzpatrick and K. W. Entwistle/Animal

Reproduction Science 46 (1997) 179-186

Table 3 Comparison of dissected populations of small, medium, large and total ovarian follicles per ovary found in the present study (PRESENT) and in other studies of postpartum Bos tuurur (A, B and C) and Bos indicus (D) COWS

Bos taurus

Time postpartum Small follicles Medium follicles Large follicles Total follicles

(days)

Bos indicus

Aa

Bb

C

42-56 11

35-63 15

60+

60f

21

39

9 1 21

7 2 24

2 23

1 40

a (Spicer et al., 1986a). b (Prado et al., 1990). ’ (Segerson et al., 1984). d Day 17 of estrous cycle, small and medium

I5

mm, large

c.d

,,

c,d

PRESENT 30-100 = 27 = 12 =l = 40

> 5 mm.

(Matton et al., 1981; Spicer et al., 1986a,b; Wise et al., 1986; Brantmeier et al., 1987; Spicer et al., 1987; Moser et al., 1989), few appear to have validated this procedure. Unless counts of ovarian follicles on the ovarian surface of B. tuurus cows are strongly correlated to actual numbers of antral follicles by size class, and such information is apparently not available, the results of the above studies must be interpreted with some caution. Whether the poor correlation of counts of surface medium sized follicles to actual populations found in the present study with B. indicus cows holds for B. tuurus cows has not been determined. Data comparing populations of small, medium, large and total ovarian follicles per cow found in the present study and in previous studies with postpartum B. taurus and B. indicus cows are summarised in Table 3. The mean number of follicles 2 2 mm in the ovaries of these postpartum cows, was similar to previous reports for B. indicus cows (Segerson et al., 1984). While the ovaries of B. indicus cows have consistently been reported to contain larger numbers of follicles than those of B. tuurus breeds when compared within studies (Comwell et al., 1982; Segerson et al., 1984), and for comparable groups of cows between studies (Parfet et al., 1986; Spicer et al., 1986aSpicer et al., 1986b; Wehrman et al., 19911, the functional significance of these findings is not known. Given that cows of both genotypes usually only ovulate single follicles, and ovaries from B. indicus cows contain fewer follicles > 5 mm in diameter (Segerson et al., 19841, larger numbers of small follicles do not appear to result in larger numbers of follicles that become recruited or selected. These apparent genetic differences in ovarian follicular kinetics may reflect genotype differences in ovarian function. Although the patterns of follicular growth, and processes of recruitment, selection and dominance have not been studied in any detail in cows of the B. indicus genotype, the application of ultrasonographic techniques in evaluating ovarian function offers considerable promise (Rhodes et al., 1995).

L.A. Fitzpatrick and K. W. Entwistle/Animal

Reproduction Science 46 (1997) 179-186

185

5. Conclusion Data from this study lead to a query regarding the validity of using counts of follicles visible on the surface of bovine ovaries as a measure of actual ovarian follicle populations, particularly when follicles are classified into distinct size classes based on apparent follicle diameters. Total ovarian dissection, although tedious, remains the most accurate method of determining total ovarian antral follicle populations 2 2 mm in diameter.

Acknowledgements These studies were partially funded by the Australian Meat Research Corporation. We gratefully acknowledge the competent assistance of Alison Healing, Cheryl Ridd and Paula Tomkins. The assistance of Glen De’ath with statistical analyses is also acknowledged.

References Brantmeier, S.A., Bellin, M.E., Boehm, S.K., Bushmeyer, S.M., Kubajak, C.L., Dentine, M.R., Grummer, R.R. and Ax, R.L., 1987. Influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on estradiol-17B concentrations in bovine follicles. J. Dairy Sci., 70: 2138-2144. Cornwell, D.G., Tucker, J.F., Hentges, J.F., Jr. and Fields, M.J., 1982. A comparison of the reproductive performance of Brahman and Angus heifers on three levels of nutrition. J. Anim. Sci. 55 (Suppl. 1): 17. Dufour, J.J. and Roy, G.L., 1985. Distribution of ovarian follicular populations in the dairy cow within 35 days after parturition. J. Reprod. Fertil., 734: 229-235. Fortune, J.E., Sirois, J. and Quirk, S.M., 1988. The growth and differentiation of ovarian follicles during the bovine estrus cycle. Theriogenology, 29: 95-109. Ireland, J.J. and Roche, J.F., 1982. Development of antral follicles in cattle after prostaglandin-induced luteolysis: changes in serum hormones, steroids in follicular fluid, and gonadotropin receptors. Endocrinology, 111: 2077-2086. Lucy, M.C., Savio, J.D., Badinga, L., De La Sota, R.L. and Thatcher, W.W., 1992. Factors that affect ovarian follicular dynamics ill cattle. J. Anim. Sci., 70: 3615-3626. Marion, G.B. and Gier, H.T., 1971, Ovarian and uterine embryogenesis and morphology of the non-pregnant female mammal. J. Anim. Sci., 32: 24-47. Matton, P., Adelakoun, V., Couture, Y. and Dufour, J.J., 1981. Growth and replacement of the bovine ovarian follicles during the estrous cycle. J. Anim. Sci., 52: 813-820. McSweeney, C.S., Fitzpatrick, L.A., D’Occhio, M.J., Reid, D. and Entwistle, K.W.. 1993. Reducing postpartum anoestrous interval in first-calf Bos indicus crossbred heifers. I. Effects of pre- and postpartum supplementation strategies. Aust. J. Agric. Res., 44: 1063-1077. Moser, M.T., Garverick, H.A., Smith, M.F. and Youngquist, R.S., 1989. Follicular growth and endocrine patterns of prepuberal heifers administered bovine follicular fluid and (or) follicle stimulating hormone, Anim. Reprod. Sci., 18: 227-242. Parfet, J.R., Marvin, CA., Allrich, R.D., Diekman, M.A. and Moss, G.E., 1986. Anterior pituitary concentrations of gonadotropins, GnRH-receptors and ovarian characteristics following early weaning in beef cows. J. Anim. Sci., 62: 717-722. Pierson, R.A. and Ginther, O.J., 1984. Ultrasonography of the bovine ovary. Theriogenology, 21: 495-504. Pierson, R.A. and Ginther, O.J., 1987. Follicular populations during the estrous cycle in heifers. II. Influence of right and left sides and intraovarian effect of the corpus luteum. Anim. Reprod. Sci., 14: 177-186.

186

L.A. Fitzpatrick and K.W. Entwistle/Animal Reproduction Science 46 (1997) 179-186

Prado, R., Rhind, S.M., Wright, LA., Russel, A.J.F., McMillen, S.M., Smith, A.I. and McNeilly, AS., 1990. Ovarian follicle populations, steroidogenesis and micromorphology at 5 and 9 weeks postpartum in beef cows at two levels of body condition. Anim. Prod., 51: 103-108. Rajakoski, E., 1960. The ovarian follicular system in sexually mature heifers with special reference to seasonal, cyclical, and left-right variations. Acta Endocrinol. Copenhagen, 34 (Suppl. 52): l-68. Rhodes, F.M., Fitzpatrick, L.A., Entwistle, K.W. and Death, G., 1995. Sequential changes in ovarian follicular dynamics in Bos indicus heifers before and after nutritional anoestrus. J. Reprod. Fertil., 104: 41-49. Segerson, E.C., Hansen, T.R., Libby, D.W., Randel, R.D. and Getz, W.R., 1984. Ovarian and uterine morphology and function in Angus and Brahman cows. J. Anim. Sci., 59: 1026-1046. Spicer, L.J. and Echtemkamp, S.E., 1986. Ovarian follicular growth, function and turnover in cattle: A review. J. Anim. Sci., 62: 428-451. Spicer, L.J., Leung, K., Convey, E.M., Gunther, J., Short, R.E. and Tucker, H.A., 1986a. Anovulation in postpartum suckled beef cows. I. Associations among size and numbers of ovarian follicles, uterine involution, and hormones in serum and follicular fluid. J. Anim. Sci., 62: 734-741. Spicer, L.J., Convey, E.M., Leung, K., Short, R.E. and Tucker, H.A., 1986b. Anovulation in postpartum suckled beef cows. II. Association among 125-I labelled gonadotropins to granulosa and thecal cells, and concentrations of steroids in serum and various sized ovarian follicles. J. Anim. Sci., 62: 742-750. Spicer, L.J., Tucker, H.A., Convey, E.M. and Echtemkamp, S.E., 1987. Comparison of surface diameters and dissected diameters of bovine ovarian follicles. J. Anim. Sci., 64: 226-230. Spicer, L.J., Enright, W.J., Murphy, M.G. and Roche, J.F., 1991. Effect of dietary intake on concentrations of insulin-like growth factor-I in plasma and follicular fluid, and ovarian function in heifers. Domest. Anim. Endocrinol., 8: 431-437. Statistix (1990). In: Statistix 3.1. An Interactive Statistical Analysis Program for Microcomputers. Analytical Software, St. Paul, MN, USA. Wehrman, M.L., Welsh, T.H. and Williams, G.L., 1991. Diet-induced hyperlipidemia in cattle modifies the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens the onset of postpartum luteal activity. Biol. Reprod., 45: 514-522. Wise, T., Vernon, M.W. and Maurer, R.R., 1986. Oxytocin, prostaglandins E and F, estradiol, progesterone, sodium, and potassium in preovulatory bovine follicles either developed normally or stimulated by follicles stimulating hormone. Theriogenology, 26: 757-778.