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The assessment of follicular parameters for the selection of oocytes recovered from superovulated heifers
Animal Reproduction Science, 23 ( 1 9 9 0 ) 181-195
181
Elsevier Science Publishers B.V., A m s t e r d a m
The assessment of follicular parameters for the selection of oocytes recovered from superovulated heifers R.B. Stubbings*, R.M. Liptrap and P.K. Basrur** Department of Biomedical Sciences, University of Guelph, Guelph, Ont. NIG 2 W1, Canada (Accepted 14 May 1990)
ABSTRACT Stubbings, R.B., Liptrap, R.M. and Basrur, P.K., 1990. The assessment of follicular parameters for the selection of oocytes recovered from superovulated heifers. Anita. Reprod. Sci., 23: 181-195. Superovulated mature Holstein heifers were ovariectomized at the expected time of ovulation in order to recover preovulatory follicles. Follicular fluid steroid concentrations from all follicles 8 mm and greater in diameter were determined and correlated with oocyte morphology and the luteinizing hormone (LH) surge in the oocyte donor. Of the 11 oocyte donors used in this study, two did not exhibit a surge ( - LH). Fluid from the aspirated follicles ranged from 0.1 to 1.4 ml in volume. Overall, 79 oocytes were recovered from 127 aspirated follicles. Heifers exhibiting an LH surge ( + LH) produced follicles with a significantly higher oocyte recovery rate ( P < 0 . 0 0 0 1 ) . Follicular fluid volume was not a significant predictor of oocyte recovery success ( P < 0.40). Using accepted morphological criteria for oocyte classification, the 79 recovered oocytes were classified as 40 mature, 22 degenerate, 3 immature, 13 bare and l not classified. Follicular fluid steroid data were available for 39 mature oocytes, of which 38 were from + LH heifers, and 21 degenerate oocytes, of which 11 were derived from + LH animals. While mature and degenerate oocytes were obtained from follicles exhibiting a range of fluid volumes and steroid concentrations, mature oocytes were more commonly derived from follicles with progesterone concentrations of greater than 100 ng/ ml, estradiol concentrations of 5-60 ng/ml, testosterone concentrations of 5-45 n g / m l and a fluid volume of at least 0.3 ml ( 10 m m diameter).
INTRODUCTION
Estradiol synthesis by the follicle is based upon the so-called two-cell theory which was first proposed by Falck (1959) and later modified and documented by studies in several species including the rat (Fortune and Armstrong, 1977) and cow (McNatty et al., 1984). Around the time of the LH *Present address: Semex C a n a d a , Guelph, Ont. N 1 G 3Z2 ( C a n a d a ) . **To w h o m c o r r e s p o n d e n c e should be addressed.
0378-4320/90/$03.50
© 1 9 9 0 - - Elsevier Science Publishers B.V.
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surge follicular estradiol concentrations reach a peak and gradually decrease as ovulation approaches. In contrast, progesterone concentration in follicular fluid is initially low, begins to increase after the LH surge and peaks just prior to ovulation. Generally, the pattern of steroid synthesis in hormonally stimulated cattle is similar to that observed in normal animals with the exception that both estrone and estradiol levels are consistently lower in follicles from superovulated animals (Fortune and Hansel, 1985). This observation was confirmed by Callesen et al. ( 1986 ) who further reported the shift from estradiol to progesterone predominance beginning from 15 to 17 h after the LH peak. Kruip and Dieleman ( 1985 ) indicated that, except in preovulatory follicles, a high progesterone concentration in follicular fluid is a sign of atresia in cattle. Steroidogenic changes in atretic bovine follicles follow a pattern similar to that observed after the preovulatory LH surge, a rising concentration of progesterone and decreasing amounts of androgen and estradiol in the follicular fluid (Tsafriri and Eckstein, 1986). It is not known if steroid concentrations in conjunction with oocyte morphology can serve to distinguish healthy from atretic follicles after the LH surge. An association has been reported between the preovulatory endocrine environment and oocyte maturation in superovulated cattle (Callesen et al., 1986). These authors concluded that a certain proportion of superovulated cows persistently show abnormal follicular and oocyte maturation. Other investigators (Moor et al., 1985) have suggested that grossly perturbed steroidogenesis and premature activation of the germinal compartment may result in asynchronous maturation of follicles and oocytes. Criteria have been established in order to categorize oocyte quality in cattle and human beings (Leibfried and First, 1979; Simonetti et al., 1985). These reports use the oocyte's investment, ooplasmic characteristics and chromatin to classify oocytes as mature, immature or degenerate. The objective of this study was to determine the relationship, in cattle, between different parameters, including the concentration of follicular fluid steroids, the presence and timing of the gonadotrophic surge and the oocyte-cumulus morphology used for the selection of oocytes. MATERIAL AND METHODS
Experimental design Eleven Holstein heifers between 14 and 24 months of age were used as oocyte donors. Animals were treated with 500 #g prostaglandin F2, analog (Estrumate, ICI Pharma, Mississauga, Ont.) to synchronize their estrous cycles so that all animals could be subjected to the experimental procedure on the same day of the week. Superovulation was induced using 38 mg of folliclestimulating hormone (FSH-P, Schering Canada, Pointe-Claire, Que.) administered every 12 h beginning Saturday p.m. (treatment day l), until
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Wednesday a.m. (treatment day 5 ). Superovulatory hormone treatment was initiated between day 9 and day 13 of the estrous cycle and prostaglandin was administered at 16.00 h on the third treatment day. Ten ml of blood were drawn from the jugular or coccygeal vein of each heifer into heparinized tubes at 08.00 h and 20.00 h of treatment days 3, 4, 6 and 7. On the expected day of estrus (treatment day 5) blood samples were collected every 4 h from 04.00 h until 20.00 h. The blood was centrifuged within 30 min of collection, and the decanted plasma was stored at - 2 0 ° C until assayed for hormones. On the morning of treatment day 6 between 09.00 h and 11.00 h (65-67 h after prostaglandin injections), the heifer was prepared for the surgical removal of the right ovary. The left ovary was left intact for other experiments. Following paravertebral anesthesia and surgical preparation of the paralumbar fossa, the ovary was exteriorized and removed after pedicle ligation. All recent ovulations and follicles 8 m m or greater in diameter were counted and aspirated and the following information recorded: follicle number, diameter in m m , volume of follicular fluid in ml, the successful recovery of an oocyte and the quality of the oocyte. Follicle diameter was determined by measuring the long axis of the follicle at the ovarian surface with a vernier caliper. Volume of follicular fluid was measured by aspirating tl{e fluid, using an 18-g needle, into a 5.0-ml graduated syringe. Fluid was flushed twice into the follicle before it was examined for an oocyte. After retrieval of the oocyte, the follicular fluid was centrifuged, decanted and stored at - 2 0 °C until hormone assay. The oocyte classification categories used were similar to those reported by Leibfried and First (1979) and Simonetti et al. ( 1985 ). Four categories were used: mature, immature, degenerate and bare. Mature oocytes exhibited the presence of the first polar body (when visible), an ooplasm which was round, even and pale in colour and a cumulus which was expanded, complete, even and pale in color. Immature oocytes exhibited an intact germinal vesicle, an ooplasm which was round, full and evenly granulated and a cumulus which was complete, greater than three cell layers thick, compact and intact. The degenerate oocytes displayed a degenerating germinal vesicle (when visible ), an ooplasm which was irregular, granular, dark in color, shrunken, vacuolated or fragmented and a cumulus which was clumping, uneven or had dark cells in scattered clumps in the matrix. Oocytes which had completely lost their cumulus were classified as bare. Hormonal assays All samples from individual cows were assayed together. Luteinizing hormone (LH) concentrations were determined using a specific double antibody radioimmunoassay for bovine LH (Niswender et al., 1969). Bovine LH with antiserum was used to establish the standard curve. All determinations were
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made using 300-Ftl aliquots of plasma. The sensitivity of the assay was 0.2 n g / ml and the variation between duplication within the assay did not exceed 7.0%. Progesterone concentrations were determined by radioimmunoassay procedures (Abraham et al., 1971; Lindner et al., 1972 ) following extraction with petroleum ether. The assay was carried out using 50/tl of follicular fluid. The sensitivity of the assay was 12.5 p g / m l and the inter-duplicate variation did not exceed 4.6%. Estradiol-I 7 8 concentrations were determined by radioimmunoassay procedures (Edqvist and Johansson, 1972; Wu et al., 1973) following extractions with di-ethyl ether. Aliquots of 50/A of follicular fluid were used for the assay. The sensitivity of the assay was 0.5 p g / m l and the variation between duplicates within the assay did not exceed 7.0%. Testosterone concentrations were measured by radioimmunoassay as described by Castro et al. (1974). Follicular fluid aliquots of 50/A were extracted using procedures as outlined for estradiol-178, the sensitivity of the assay was 8.7 p g / m l and the inter-duplicate variation did not exceed 6.8%.
Statistical analysis Regression analysing using the general linear model (GLM) procedure of Statistical Analysis System (SAS Institute Inc., Cary, NC) was used to determine the relationship between plasma steroids and plasma sampling time, and between follicular fluid steroids and follicle volume. Student's t-test and Tukey's Range test were used to determine difference in plasma sampling time (from prostaglandin injection ) for the hormone assayed, both within and between cows. CATMOD procedure was used to determine if the presence or absence of an LH surge, interacting with the follicular fluid volume, had an effect on oocyte recovery rate. RESULTS
Cows which did not exhibit an increase or decrease in LH of at least 5 ng/ ml between any two consecutive sampling times at 36-52 h after prostaglandin injection were considered not to have had an LH surge (Table 1 ). Two animals ( - LH) were in this category. The remaining nine oocyte donors ( + LH) were considered to have had a normal LH profile since they all displayed an LH surge within the mean interval from prostaglandin injection to LH peak +2 s.d. (40.4+ 11.6 h). The mean (_+s.c.) m a x i m u m LH concentration measured for these + LH cows was 17.7 + 0.6 ng/ml. The number of follicles greater than, or equal to, 8 m m in diameter and the number of corpora hemorrhagica recorded for each oocyte donor are shown in Table 2. The mean number of follicles produced by the right ovary of the 11 donors was 14.8_+2.0 with a range from 4 to 24. Most cows ( #230, # 12, # 199, # 5 ) which exhibited a high LH concentration more than 26 h before
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TABLE 1 Plasma luteinizing hormone (LH) concentrations in oocyte donor cows at intervals from 36 to 52 h after prostaglandin injection Cow #
Concentration o f L H ( n g / m l ) Sampling time after injection (h)
200 230 12 199 5 203 9 2 241 341 198
36
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1.8 2.3 2.9 4.7 4.8 9.6 17.0 0.4 1.5 1.8 0.6
1.3 1.3 1.7 1.4 2.2 4.0 3.7 19.5 4.0 1.5 0.7
0.7 1.2 1.3 1.6 1.6 1.3 2.3 16.8 17.2 1.3 0.6
aFigures in italics: maximum LH concentrations recorded indicative of an LH surge.
TABLE 2 Follicles ( > 8 mm diameter) and ovulations on the right ovary for each oocyte donor at 66 h after prostaglandin injection Cow #
Follicles (n)
Ovulations (n)
Total (n)
Interval (h) between maximum LH concentrations recorded and ovariectomy
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12 11 5 15 2 16 16 4 15 24 7 127
0 9 12 7 5 1 1 0 1 0 0 36
12 20 17 22 7 17 17 4 16 24 7 163 (14.8±2.0)
30 26-30 26-30 26-30 26-30 26 22-26 14-18 14 -" -"
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the removal of the ovary (Table 1 ) had already begun ovulation by the time of ovariectomy. Interestingly, cow # 200 which exhibited an early LH peak had not begun ovulation by 66 h after prostaglandin injection. Cows exhibiting maximum LH concentrations ( # 2 0 3 , # 9 , # 2 , #241 ) less than 26 h before ovariectomy had not, or had barely, commenced ovulation. The two cows not displaying an LH surge ( # 198, # 3 4 1 ) did not exhibit any ovulations.
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The diameters o f the 127 follicles examined were significantly correlated with follicular fluid volume ( r = 0 . 7 9 ) . The diameters ranged from 8 to 17 m m , while the fluid volumes ranged from 0.1 to 1.4 ml. Overall, 79 oocytes were recovered from the 127 aspirated follicles. The mean recovery rates for the + LH and - L H cows were 71% and 35%, respectively. The difference in recovery rate between the + LH cows and the - L H cows was highly significant ( P < 0.0001 ). Fluid volume was not a significant ( P > 0.40 ) predictor of oocyte recovery success. The 79 recovered oocytes were classified as follows: 40 mature, 22 degenerate, 3 immature and 13 bare. One was not classified. Of the 40 mature oo-
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cytes, complete follicular fluid steroid data are available for 39. O f these oocytes, only one was from a cow ( # 341 ) not displaying an LH surge while 38 were from + LH cows, with at least one oocyte originating from each animal. Of the 22 oocytes identified as degenerate, steroid information is available on 21. Of these, I l were derived from + LH cows (one each from # 9 and # 12, two from # 199 and seven from # 200). The remainder originated from - LH cows (three from # 198 and seven from # 341 ). The distribution o f mature and degenerate oocytes originating from both cow groups with respect to follicular volume and progesterone concentration
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is illustrated in Fig. 1. It can be seen that most of the oocytes from the + LH cows were mature. Many of these mature oocytes originated from follicles with fluid volumes of 0.3 ml or greater, and progesterone concentrations of 100 ng/ml or greater. In contrast, the majority of the oocytes from the - L H cows were degenerate. A good proportion of these originated from follicles yielding under 0.6 ml follicular fluid and under 150 ng of progesterone/ml. Two distinct clusters of degenerate oocytes could be seen from + LH cows. One group was found with progesterone concentrations under 100 ng/ml,
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while the other was associated with progesterone concentrations between 400 and 950 ng/ml. The distribution of mature and degenerating oocytes in relation to estradiol concentrations and follicular volume is depicted in the scatter diagram (Fig. 2 ). No degenerate ova were recovered from follicles yielding greater than 0.3 ml follicular fluid from + LH cows. Furthermore, all mature oocytes were recovered from follicles with less than 60 ng of estradiol/ml regardless of volume. The oocytes recovered from - L H cows were mainly of degenerate quality, with most arising from follicles with less than 40 ng of estradiol/ml. All oocytes recovered from follicles with estradiol concentrations greater than 250 ng/ml were degenerate. The scatter diagram (Fig. 3 ) depicts the distribution of oocytes based on fluid volume and testosterone concentrations. The observations for testosterone tend to mimic those previously recorded for estradiol. Within the + LH cows all oocytes recovered were from follicles with under 50 ng testosterone/ ml of fluid. Within the - LH cows the degenerating ova were detected in follicles of various volumes. However, the majority were found in follicles with less than 10 ng of estradiol/ml of fluid. A small group of degenerating ova was found in follicles with the highest testosterone concentrations recorded (65-96 ng/ml). The distribution of oocytes with respect to progesterone-to-estradiol ratios is shown in Fig. 4. Ratios ranged from less than 1 : 1 (i.e., estrogen-dominant follicles) to 65: 1, with only a few observations above this value. Within the + LH cows, mature and degenerate oocytes were detected at all ratios recorded. Within the - L H cows, degenerate oocytes were found at all the volumes recorded and were predominantly between 10:1 and 40: 1. Only three follicles from these cows were estrogen-dominant and all contained degenerate oocytes. DISCUSSION
Recovery rate and quality of retrieved oocytes In the present study the oocyte recovery rate was noted to be higher in animals which exhibited an LH surge compared to those which did not. Using a method similar to that used in the present study for follicle aspiration, Rodriquez et al. (1982) reported an oocyte recovery rate of 48%. Callesen et al. (1987a) have suggested that the low recovery rate from cows without an LH surge is the result of an increased oocyte fragility due to the abnormal follicular microenvironment of these oocytes. Specifically, abnormal follicular steroid concentrations were associated with degenerate oocytes. In the present study, the hormonal environment of a degenerate oocyte was often noted to be similar to those of follicles from which oocytes were not recovered. It is
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possible that the repeated flushing of each follicle may have damaged the oocyte which, once fragmented, would not be recovered. Based on the quality of oocytes recovered, it is evident that an LH surge in an oocyte donor is important for the recovery of mature oocytes. It is noteworthy however, that one apparently mature oocyte was recovered from oocyte donor # 341, which did not experience an LH surge. Additional information on this animal acquired through other experiments confirmed that this cow did not exhibit a late LH surge. This would suggest that the oocyte classified as mature was in fact degenerate. Cumulus expansion can occur during maturation and degeneration. These observations indicate that morphological criteria, while extremely useful, are not infallible. This exception apart, oocyte donors not displaying an LH surge produced degenerate oocytes exclusively. Lobo et al. (1985 ) have reported that the proportion of "dysmature" follicles containing abnormal oocytes was similar in ovulatory and anovulatory h u m a n patients. While degenerate oocytes were also recovered from cows with a normal LH surge, a larger proportion of oocytes recovered from this group were mature. An exception to this generalization was oocyte donor # 200. Even though this animal exhibited an LH surge, all oocytes recovered from her were degenerate. Perhaps the early timing of her LH surge was not properly coordinated with other events required for ovulation but, instead, induced premature maturation of all oocytes.
Follicular steroid concentrations and oocyte quality Information is scanty on follicular steroid patterns relative to a fixed reference point which in this study was prostaglandin injection. In the present study two trends were observed (Fig. 1 ). First, in the larger follicles, progesterone concentrations increased with increasing follicular fluid volume. This indicates that as follicles become larger they contain more steroidogenically active granulosa cells. Second, in the smallest follicles containing mature oocytes, progesterone concentrations reached up to and beyond those recorded in larger follicles. One possible explanation for this is that these follicles had steroidogenically active granulosa cells as a result of the LH surge. They had reached their m a x i m u m size, albeit smaller than other superovulated follicles in the study, and were destined to ovulate. This raises the question as to whether all superovulated follicles ovulate when they reach a specific size or whether they ovulate in response to the LH surge regardless of their size. Callesen et al. (1986) reported mean follicular fluid progesterone concentrations of approximately 150 n g / m l to over 500 n g / m l over a similar time after the LH peak. In animals not exhibiting an LH surge, these investigators observed a mean progesterone concentration of approximately 200 ng/ml. Using the fluid aspirated from follicles of superovulated heifers, Maurer et al. (1987 ) reported concentrations well over 1000 n g / m l during this period. Goff et al.
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(1986) noted follicular progesterone concentrations of up to 700 ng/ml of fluid recovered from the follicles of three superovulated heifers 26-33 h after the LH surge while in three other animals not exhibiting an LH peak, progesterone concentrations were as low as 100 ng/ml. Whether a rising progesterone concentration is necessary for proper oocyte maturation, or is only an indicator of "follicle health" is not clear. Epping ( 1981 ) reported that progesterone could reduce the incidence of parthenogensis. Moor and Warnes ( 1979 ) have shown that an aberrant steroid environment induced during the culture of intact follicles resulted in abnormal oocytes. In contrast to progesterone, the follicular fluid estrogen concentrations were very similar in follicles containing a range of fluid volumes (Fig. 2 ). Follicular fluid estradiol was noted to be below 60 n g / m l in follicles from which mature oocytes were recovered. Callesen et al. ( 1986 ) reported follicular fluid estradiol concentrations of approximately 70-80 n g / m l during the same period covered in the present study. These authors also reported a concentration of 55 n g / m l in follicles recovered from animals not exhibiting an LH surge. In the present study, there were a few follicles with estradiol concentrations of this order, but there were some with extremely low concentrations (less than 20 n g / m l ) . Goff et al. ( 1986 ) had reported estradiol concentrations as low as 7 n g / m l in cows exhibiting an LH surge and 60 n g / m l in animals not showing an LH peak. Results from the current study would suggest that the extremes in the concentrations recorded occurred in follicles aspirated from cows not exhibiting an LH surge and contain degenerating oocytes. Alternatively, estradiol may have a role to play in follicle/oocyte development in cows which displayed an LH surge. This is supported by the changes observed in preovulatory follicular estradiol concentrations which are highest around the time of the LH surge. Reports on the concentration of testosterone in follicular fluid recovered from supervulated animals are scanty. In the present study, testosterone concentration followed a pattern similar to that seen for estradiol (Fig. 3 ). Bousquet et al. ( 1988 ) reported increasing concentrations of testosterone with increasing time from the beginning of standing estrus, while Dieleman et al. (1983) observed decreasing androgen concentrations during the same period. The present results support the contention that the major role of androgens is to serve as a substrate for estrogen synthesis. In light of the importance of estrogen in follicle/oocyte health, the aromatization of androgens, presumably coming as androstenedione from theca interna, may actually be the determining factor for proper oocyte maturation. In the present study the progesterone-to-estradiol ratio was greater than 1 : 1 in the vast majority of follicles regardless of their origin from + LH or - LH animals. Callesen et al. (1986) reported a ratio of less than 10:1 around the time of expected ovulation in cows while Goff et al. (1986) reported ratios greater than 40: 1. In animals not exhibiting an LH surge, Callesen et al., (1986) and Goff et al. (1986) recorded mean progesterone-to-estradiol ra-
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tios of less than 1 : 1. Since follicular atresia may not affect the oocyte for some time, morphologically healthy oocytes can be recovered from atretic follicles (Tsafriri and Braw, 1984). It would be useful if a correlation between follicular fluid steroid ratios and oocyte health could be established. In the present study, this association could not be made since both mature and degenerate oocytes were found in follicles exhibiting a wide range of progesterone-toestradiol ratios. The injection of gonadotropins to initiate superovulation may induce asynchronous oocyte maturation with changes in follicular fluid steroid concentrations. Meiotically abberant oocytes and abnormal follicular steroidogenesis have been reported in superovulated rats (Yun et al., 1989). Perhaps other methods of assessing oocyte normality would be more accurate than follicular fluid steroid concentrations. Flow cytometric DNA analysis of granulosa cells may be an alternative (Callesen et al., 1987b).
Size and function of aspirated follicles Staigmiller and England (1982) reported that the ovulatory follicle of nonstimulated cattle ranges in size from as low as 10 m m to about 20 mm in diameter. Their follicular fluid volumes, based on the aspiration techniques used in this study, could be estimated to range from 0.4 to 1.4 ml. Thus, the present study indicates that a large proportion of the ovulations in superovulated heifers may originate from follicles of smaller size than those transpiring in nonstimulated cows. Little information in available on the size of stimulated preovulatory follicles. In the present study, follicular fluid volume ranged from 0.1 ml to 1.4 ml from follicles 8 m m - 1 7 mm in diameter. While some cows produced a uniform population of follicles, others produced a broad range of follicle sizes. This observation of a gradient in size of preovulatory follicles has also been recorded in human subjects by Lehman et al. (1984). However, Templeton et al. ( 1986 ) reported that, in women, despite multiple follicular development, the leading follicle in stimulated cycles ovulated at a size equal to, or even greater than, that of the ovulatory follicle in spontaneous cycles. Simonetti et al. ( 1985 ) have suggested that, in human in vitro fertilization programs, oocytes recovered from larger follicles are of better quality. It has been documented that, in normals, the largest follicle produces most of the estradiol during estrus (Staigmiller et al., 1982 ). It is proposed that if a similar hierarchy exists with stimulated follicles, then perhaps one or two of the larger follicles may produce sufficient estradiol to activate the LH surge before the remaining smaller follicles are ready for ovulation. Since their granulosa cells contain sufficient LH receptors, the smaller follicles may ovulate somewhat prematurely. However, the quality of the released oocytes may not be comparable to that of the larger follicles. The best method of determining the role of follicle size and steroid environment on oocyte quality would be to fertilize the aspirated oocytes in vitro and assess their developmental potential.
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CONCLUSION
Results of the present study indicate that a greater proportion of mature bovine oocytes will be recovered from those preovulatory follicles with a progesterone concentration greater than 100 ng/ml, an estradiol concentration of 5-60 ng/ml, a testosterone concentration of 5-45 ng/ml and a volume greater than 0.3 ml ( 10 mm diameter). Progesterone-to-estradiol ratio would appear to be of no value to predict oocyte quality. It may be possible that the current methods of visually assessing oocytes are not sufficiently accurate. Follicular steroid patterns may change at a different rate than visible morphological changes to the oocyte-cumulus complex. More objective methods of directly or indirectly assessing bovine oocytes are required. In conclusion, it is apparent that a number of factors must be considered to categorize bovine oocytes removed from preovulatory follicles. While follicular fluid steroid parameters alone may not be adequate, they may prove to be more useful to predict viability after the oocyte has been put to an end use, for example, for in vitro fertilization. REFERENCES Abraham, G.E., Swerdloff, R., Tulchinsky, D. and Odell, W.D., 1971. Radioimmunoassay of plasma progesterone. J. Clin. Endocrinol. Metab., 32:619-624. Bousquet, D., Goff, A., King, W.A. and Greve, T., 1988. Fertilization in vitro of bovine oocytes: analysis of some factors affecting the fertilization rates. Can. J. Vet. Res., 52: 277-279. Callensen, H., Greve, T. and Hyttel, P., 1986. Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology, 25:71-86. Callensen, H., Greve, T. and Hyttel, P., 1987a. Premature ovulations in superovulated cattle. Theriogenology, 28:155-166. Callensen, H., Westergaard, L., Greve, T. and Hyttel, P., 1987b. Flow cytometric DNA analysis ofgranulosa cells from preovulatory follicles in superovulated cows. Zuchthygiene, 22: 4952. Castro, A., Shih, H.H. and Chung, A., 1974. A simple radioimmunoassay of plasma testosterone without column chromatography. Steroids, 23: 625-638. Dieleman, S.J., Kruip, Th. A.M.P., Fontijne, P., De Jong, W.H.R. and Van der Weyden, G.C., 1983. Changes in oestradiol, progesterone and testosterone concentrations in follicular fluid and in the micromorphology of prevoulatory bovine follicles relative to the peak of luteinizing hormone. J. Endocrinol., 97:31-42. Edqvist, L.E. and Johansson, E.D.B., 1972. Radioimmunoassay of estrone and estradiol in human and bovine peripheral plasma. Acta Endocrinol., 71:716-730. Epping, J.J., 1981. Preimptantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro. Gamete Res., 4: 3-13. Flack, B., 1959. Site of production of oestrogen in rat ovary as studied in micro-transplants. Acta Physiol. Scand. Suppl., 163: 1-100. Fortune, J.E. and Armstrong, D.T., 1977. Androgen production by theca and granulosa isolated from proestrus rat follicles. Endocrinology, 100:1341 - 1347. Fortune, J.E. and Hansel, W., 1985. Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation. Biol. Reprod., 32: 10691079.
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Goff, A.K., Greve, T., Bousquet, D. and King, W.A., 1986. Progesterone and luteinizing hormone profiles in heifers used as oocyte donors. Theriogenology, 26: 577-586, Kruip, Th. A.M. and Dieleman, S.J.D., 1985. Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrous cycle. Theriogenology, 24: 395-408. Lehman, F., Hoppen, H.O., Schulz, C.H.R., Hamerich, U., A1-Hasani, S., Van Der Ven, H., Diedrich, K. and Krebs, K., 1984. Concentrations of steroids and prostaglandins in follicular fluids aspirated in an in vitro fertilization programme. In: R.F. Harrison, J. Bonnar and W. Thompson (Editors), In Vitro Fertilization, Embryo Transfer and Early Pregnancy. MTP Press Ltd., Boston, MA, pp. 19-24. Leibfried, L. and First, N.L., 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci., 48: 76-86. Lindner, H.R., Perel, E., Friedlander, A. and Zeitlin, A., 1972. A radioimmunoassay for plasma progesterone. Steroids, 19: 357-360. Lobo, R.A., Di Zerega, G.S. and Marrs, R.P., 1985. Follicular fluid steroid levels in dysmature and mature follicles from spontaneous and hyperstimulated cycles in normal and anovulatory women. J. Clin. Endocrinol. Metab., 60: 81-87. Maurer, R.R., Suss, U. and Wise, T.H., 1987. Oocyte quality in superovulated beef heifers with and without norgestomet implants. Theriogenology, 27: 256. McNatty, K.P., Heath, D.A., Lun, S., Fannin, J.M., McDiarmid, J.M. and Henderson, K.M., 1984. Steroidogenesis by bovine theca interna in an in vitro perfusion system. Biol. Reprod., 30: 159-170. Moor, R.M. and Warnes, G.M., 1979. Regulation of meiosis in mammalian oocytes. Br. Med. Bull., 35: 99-103. Moor, R.M., Osborn, J.C. and Crosby, I.M., 1985. Gonadotrophin induced abnormalities in sheep oocytes after superovulation. J. Reprod. Fertil., 74:167-172. Niswender, G.D., Reichert, L.E., Jr., Midgley, A.R., Jr. and Nalbandov, A.V., 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology, 84:1166-1173. Rodriquez, K.L., Hanks, D.R. and Foote, W.D., 1982. Induced oocyte meiosis in vivo in the bovine. Proc. West. Sect., Am. Soc. Anim. Sci., 33: 133-135. Simonetti, S., Veeck, L.L. and Jones, H., Jr., 1985. Correlation of follicular fluid volume with oocyte morphology from follicles stimulated by human menopausal gonadatropin. Fertil. Steril., 44:177-180. Staigmiller, R.B. and England, B.G., 1982. Folliculogenesis in the bovine. Theriogenology, 17: 43-52. Staigmiller, R.B., England, B.G., Webb, R., Short, R.E. and Bellows, R.A., 1982. Estrogen secretion and gonadotropin binding by individual bovine follicles during estrus. J. Anim. Sci., 55: 1473-1482. Templeton, A., Messinish, I.E. and Baird, D.T., 1986. Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge. Fertil. Steril., 46:113-117. Tsafriri, A. and Braw, R.H., 1984. Experimental approaches to atresia in mammals. In: J.R. Clarke (Editor), Oxford Review Reprod. Biol. Vol. 6. Oxford Univ. Press, New York, NY, pp. 226-265. Tsafriri, A. and Eckstein, B., 1986. Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally induced atresia. Biol. Reprod., 34: 783-787. Wu, C.H., Lundy, L.E. and Lee, S.G., 1973. A rapid radioimmunoassay for plasma estrogen. Am. J. Obstet. Gynecol., 115: 169-180. Yun, Y.W., Yu, F.H., Yuen, B.H. and Moon, Y.S., 1989. Effects of a superovulatory dose of pregnant mare serum gonadotropin on follicular steroid contents and oocyte maturation in rats. Gamete Res., 23: 289-298.
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The assessment of follicular parameters for the selection of oocytes recovered from superovulated heifers R.B. Stubbings*, R.M. Liptrap and P.K. Basrur** Department of Biomedical Sciences, University of Guelph, Guelph, Ont. NIG 2 W1, Canada (Accepted 14 May 1990)
ABSTRACT Stubbings, R.B., Liptrap, R.M. and Basrur, P.K., 1990. The assessment of follicular parameters for the selection of oocytes recovered from superovulated heifers. Anita. Reprod. Sci., 23: 181-195. Superovulated mature Holstein heifers were ovariectomized at the expected time of ovulation in order to recover preovulatory follicles. Follicular fluid steroid concentrations from all follicles 8 mm and greater in diameter were determined and correlated with oocyte morphology and the luteinizing hormone (LH) surge in the oocyte donor. Of the 11 oocyte donors used in this study, two did not exhibit a surge ( - LH). Fluid from the aspirated follicles ranged from 0.1 to 1.4 ml in volume. Overall, 79 oocytes were recovered from 127 aspirated follicles. Heifers exhibiting an LH surge ( + LH) produced follicles with a significantly higher oocyte recovery rate ( P < 0 . 0 0 0 1 ) . Follicular fluid volume was not a significant predictor of oocyte recovery success ( P < 0.40). Using accepted morphological criteria for oocyte classification, the 79 recovered oocytes were classified as 40 mature, 22 degenerate, 3 immature, 13 bare and l not classified. Follicular fluid steroid data were available for 39 mature oocytes, of which 38 were from + LH heifers, and 21 degenerate oocytes, of which 11 were derived from + LH animals. While mature and degenerate oocytes were obtained from follicles exhibiting a range of fluid volumes and steroid concentrations, mature oocytes were more commonly derived from follicles with progesterone concentrations of greater than 100 ng/ ml, estradiol concentrations of 5-60 ng/ml, testosterone concentrations of 5-45 n g / m l and a fluid volume of at least 0.3 ml ( 10 m m diameter).
INTRODUCTION
Estradiol synthesis by the follicle is based upon the so-called two-cell theory which was first proposed by Falck (1959) and later modified and documented by studies in several species including the rat (Fortune and Armstrong, 1977) and cow (McNatty et al., 1984). Around the time of the LH *Present address: Semex C a n a d a , Guelph, Ont. N 1 G 3Z2 ( C a n a d a ) . **To w h o m c o r r e s p o n d e n c e should be addressed.
0378-4320/90/$03.50
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surge follicular estradiol concentrations reach a peak and gradually decrease as ovulation approaches. In contrast, progesterone concentration in follicular fluid is initially low, begins to increase after the LH surge and peaks just prior to ovulation. Generally, the pattern of steroid synthesis in hormonally stimulated cattle is similar to that observed in normal animals with the exception that both estrone and estradiol levels are consistently lower in follicles from superovulated animals (Fortune and Hansel, 1985). This observation was confirmed by Callesen et al. ( 1986 ) who further reported the shift from estradiol to progesterone predominance beginning from 15 to 17 h after the LH peak. Kruip and Dieleman ( 1985 ) indicated that, except in preovulatory follicles, a high progesterone concentration in follicular fluid is a sign of atresia in cattle. Steroidogenic changes in atretic bovine follicles follow a pattern similar to that observed after the preovulatory LH surge, a rising concentration of progesterone and decreasing amounts of androgen and estradiol in the follicular fluid (Tsafriri and Eckstein, 1986). It is not known if steroid concentrations in conjunction with oocyte morphology can serve to distinguish healthy from atretic follicles after the LH surge. An association has been reported between the preovulatory endocrine environment and oocyte maturation in superovulated cattle (Callesen et al., 1986). These authors concluded that a certain proportion of superovulated cows persistently show abnormal follicular and oocyte maturation. Other investigators (Moor et al., 1985) have suggested that grossly perturbed steroidogenesis and premature activation of the germinal compartment may result in asynchronous maturation of follicles and oocytes. Criteria have been established in order to categorize oocyte quality in cattle and human beings (Leibfried and First, 1979; Simonetti et al., 1985). These reports use the oocyte's investment, ooplasmic characteristics and chromatin to classify oocytes as mature, immature or degenerate. The objective of this study was to determine the relationship, in cattle, between different parameters, including the concentration of follicular fluid steroids, the presence and timing of the gonadotrophic surge and the oocyte-cumulus morphology used for the selection of oocytes. MATERIAL AND METHODS
Experimental design Eleven Holstein heifers between 14 and 24 months of age were used as oocyte donors. Animals were treated with 500 #g prostaglandin F2, analog (Estrumate, ICI Pharma, Mississauga, Ont.) to synchronize their estrous cycles so that all animals could be subjected to the experimental procedure on the same day of the week. Superovulation was induced using 38 mg of folliclestimulating hormone (FSH-P, Schering Canada, Pointe-Claire, Que.) administered every 12 h beginning Saturday p.m. (treatment day l), until
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Wednesday a.m. (treatment day 5 ). Superovulatory hormone treatment was initiated between day 9 and day 13 of the estrous cycle and prostaglandin was administered at 16.00 h on the third treatment day. Ten ml of blood were drawn from the jugular or coccygeal vein of each heifer into heparinized tubes at 08.00 h and 20.00 h of treatment days 3, 4, 6 and 7. On the expected day of estrus (treatment day 5) blood samples were collected every 4 h from 04.00 h until 20.00 h. The blood was centrifuged within 30 min of collection, and the decanted plasma was stored at - 2 0 ° C until assayed for hormones. On the morning of treatment day 6 between 09.00 h and 11.00 h (65-67 h after prostaglandin injections), the heifer was prepared for the surgical removal of the right ovary. The left ovary was left intact for other experiments. Following paravertebral anesthesia and surgical preparation of the paralumbar fossa, the ovary was exteriorized and removed after pedicle ligation. All recent ovulations and follicles 8 m m or greater in diameter were counted and aspirated and the following information recorded: follicle number, diameter in m m , volume of follicular fluid in ml, the successful recovery of an oocyte and the quality of the oocyte. Follicle diameter was determined by measuring the long axis of the follicle at the ovarian surface with a vernier caliper. Volume of follicular fluid was measured by aspirating tl{e fluid, using an 18-g needle, into a 5.0-ml graduated syringe. Fluid was flushed twice into the follicle before it was examined for an oocyte. After retrieval of the oocyte, the follicular fluid was centrifuged, decanted and stored at - 2 0 °C until hormone assay. The oocyte classification categories used were similar to those reported by Leibfried and First (1979) and Simonetti et al. ( 1985 ). Four categories were used: mature, immature, degenerate and bare. Mature oocytes exhibited the presence of the first polar body (when visible), an ooplasm which was round, even and pale in colour and a cumulus which was expanded, complete, even and pale in color. Immature oocytes exhibited an intact germinal vesicle, an ooplasm which was round, full and evenly granulated and a cumulus which was complete, greater than three cell layers thick, compact and intact. The degenerate oocytes displayed a degenerating germinal vesicle (when visible ), an ooplasm which was irregular, granular, dark in color, shrunken, vacuolated or fragmented and a cumulus which was clumping, uneven or had dark cells in scattered clumps in the matrix. Oocytes which had completely lost their cumulus were classified as bare. Hormonal assays All samples from individual cows were assayed together. Luteinizing hormone (LH) concentrations were determined using a specific double antibody radioimmunoassay for bovine LH (Niswender et al., 1969). Bovine LH with antiserum was used to establish the standard curve. All determinations were
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made using 300-Ftl aliquots of plasma. The sensitivity of the assay was 0.2 n g / ml and the variation between duplication within the assay did not exceed 7.0%. Progesterone concentrations were determined by radioimmunoassay procedures (Abraham et al., 1971; Lindner et al., 1972 ) following extraction with petroleum ether. The assay was carried out using 50/tl of follicular fluid. The sensitivity of the assay was 12.5 p g / m l and the inter-duplicate variation did not exceed 4.6%. Estradiol-I 7 8 concentrations were determined by radioimmunoassay procedures (Edqvist and Johansson, 1972; Wu et al., 1973) following extractions with di-ethyl ether. Aliquots of 50/A of follicular fluid were used for the assay. The sensitivity of the assay was 0.5 p g / m l and the variation between duplicates within the assay did not exceed 7.0%. Testosterone concentrations were measured by radioimmunoassay as described by Castro et al. (1974). Follicular fluid aliquots of 50/A were extracted using procedures as outlined for estradiol-178, the sensitivity of the assay was 8.7 p g / m l and the inter-duplicate variation did not exceed 6.8%.
Statistical analysis Regression analysing using the general linear model (GLM) procedure of Statistical Analysis System (SAS Institute Inc., Cary, NC) was used to determine the relationship between plasma steroids and plasma sampling time, and between follicular fluid steroids and follicle volume. Student's t-test and Tukey's Range test were used to determine difference in plasma sampling time (from prostaglandin injection ) for the hormone assayed, both within and between cows. CATMOD procedure was used to determine if the presence or absence of an LH surge, interacting with the follicular fluid volume, had an effect on oocyte recovery rate. RESULTS
Cows which did not exhibit an increase or decrease in LH of at least 5 ng/ ml between any two consecutive sampling times at 36-52 h after prostaglandin injection were considered not to have had an LH surge (Table 1 ). Two animals ( - LH) were in this category. The remaining nine oocyte donors ( + LH) were considered to have had a normal LH profile since they all displayed an LH surge within the mean interval from prostaglandin injection to LH peak +2 s.d. (40.4+ 11.6 h). The mean (_+s.c.) m a x i m u m LH concentration measured for these + LH cows was 17.7 + 0.6 ng/ml. The number of follicles greater than, or equal to, 8 m m in diameter and the number of corpora hemorrhagica recorded for each oocyte donor are shown in Table 2. The mean number of follicles produced by the right ovary of the 11 donors was 14.8_+2.0 with a range from 4 to 24. Most cows ( #230, # 12, # 199, # 5 ) which exhibited a high LH concentration more than 26 h before
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TABLE 1 Plasma luteinizing hormone (LH) concentrations in oocyte donor cows at intervals from 36 to 52 h after prostaglandin injection Cow #
Concentration o f L H ( n g / m l ) Sampling time after injection (h)
200 230 12 199 5 203 9 2 241 341 198
36
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52
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1.8 2.3 2.9 4.7 4.8 9.6 17.0 0.4 1.5 1.8 0.6
1.3 1.3 1.7 1.4 2.2 4.0 3.7 19.5 4.0 1.5 0.7
0.7 1.2 1.3 1.6 1.6 1.3 2.3 16.8 17.2 1.3 0.6
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TABLE 2 Follicles ( > 8 mm diameter) and ovulations on the right ovary for each oocyte donor at 66 h after prostaglandin injection Cow #
Follicles (n)
Ovulations (n)
Total (n)
Interval (h) between maximum LH concentrations recorded and ovariectomy
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12 11 5 15 2 16 16 4 15 24 7 127
0 9 12 7 5 1 1 0 1 0 0 36
12 20 17 22 7 17 17 4 16 24 7 163 (14.8±2.0)
30 26-30 26-30 26-30 26-30 26 22-26 14-18 14 -" -"
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the removal of the ovary (Table 1 ) had already begun ovulation by the time of ovariectomy. Interestingly, cow # 200 which exhibited an early LH peak had not begun ovulation by 66 h after prostaglandin injection. Cows exhibiting maximum LH concentrations ( # 2 0 3 , # 9 , # 2 , #241 ) less than 26 h before ovariectomy had not, or had barely, commenced ovulation. The two cows not displaying an LH surge ( # 198, # 3 4 1 ) did not exhibit any ovulations.
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The diameters o f the 127 follicles examined were significantly correlated with follicular fluid volume ( r = 0 . 7 9 ) . The diameters ranged from 8 to 17 m m , while the fluid volumes ranged from 0.1 to 1.4 ml. Overall, 79 oocytes were recovered from the 127 aspirated follicles. The mean recovery rates for the + LH and - L H cows were 71% and 35%, respectively. The difference in recovery rate between the + LH cows and the - L H cows was highly significant ( P < 0.0001 ). Fluid volume was not a significant ( P > 0.40 ) predictor of oocyte recovery success. The 79 recovered oocytes were classified as follows: 40 mature, 22 degenerate, 3 immature and 13 bare. One was not classified. Of the 40 mature oo-
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cytes, complete follicular fluid steroid data are available for 39. O f these oocytes, only one was from a cow ( # 341 ) not displaying an LH surge while 38 were from + LH cows, with at least one oocyte originating from each animal. Of the 22 oocytes identified as degenerate, steroid information is available on 21. Of these, I l were derived from + LH cows (one each from # 9 and # 12, two from # 199 and seven from # 200). The remainder originated from - LH cows (three from # 198 and seven from # 341 ). The distribution o f mature and degenerate oocytes originating from both cow groups with respect to follicular volume and progesterone concentration
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is illustrated in Fig. 1. It can be seen that most of the oocytes from the + LH cows were mature. Many of these mature oocytes originated from follicles with fluid volumes of 0.3 ml or greater, and progesterone concentrations of 100 ng/ml or greater. In contrast, the majority of the oocytes from the - L H cows were degenerate. A good proportion of these originated from follicles yielding under 0.6 ml follicular fluid and under 150 ng of progesterone/ml. Two distinct clusters of degenerate oocytes could be seen from + LH cows. One group was found with progesterone concentrations under 100 ng/ml,
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R.B. STUBBINGS ET AL.
while the other was associated with progesterone concentrations between 400 and 950 ng/ml. The distribution of mature and degenerating oocytes in relation to estradiol concentrations and follicular volume is depicted in the scatter diagram (Fig. 2 ). No degenerate ova were recovered from follicles yielding greater than 0.3 ml follicular fluid from + LH cows. Furthermore, all mature oocytes were recovered from follicles with less than 60 ng of estradiol/ml regardless of volume. The oocytes recovered from - L H cows were mainly of degenerate quality, with most arising from follicles with less than 40 ng of estradiol/ml. All oocytes recovered from follicles with estradiol concentrations greater than 250 ng/ml were degenerate. The scatter diagram (Fig. 3 ) depicts the distribution of oocytes based on fluid volume and testosterone concentrations. The observations for testosterone tend to mimic those previously recorded for estradiol. Within the + LH cows all oocytes recovered were from follicles with under 50 ng testosterone/ ml of fluid. Within the - LH cows the degenerating ova were detected in follicles of various volumes. However, the majority were found in follicles with less than 10 ng of estradiol/ml of fluid. A small group of degenerating ova was found in follicles with the highest testosterone concentrations recorded (65-96 ng/ml). The distribution of oocytes with respect to progesterone-to-estradiol ratios is shown in Fig. 4. Ratios ranged from less than 1 : 1 (i.e., estrogen-dominant follicles) to 65: 1, with only a few observations above this value. Within the + LH cows, mature and degenerate oocytes were detected at all ratios recorded. Within the - L H cows, degenerate oocytes were found at all the volumes recorded and were predominantly between 10:1 and 40: 1. Only three follicles from these cows were estrogen-dominant and all contained degenerate oocytes. DISCUSSION
Recovery rate and quality of retrieved oocytes In the present study the oocyte recovery rate was noted to be higher in animals which exhibited an LH surge compared to those which did not. Using a method similar to that used in the present study for follicle aspiration, Rodriquez et al. (1982) reported an oocyte recovery rate of 48%. Callesen et al. (1987a) have suggested that the low recovery rate from cows without an LH surge is the result of an increased oocyte fragility due to the abnormal follicular microenvironment of these oocytes. Specifically, abnormal follicular steroid concentrations were associated with degenerate oocytes. In the present study, the hormonal environment of a degenerate oocyte was often noted to be similar to those of follicles from which oocytes were not recovered. It is
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possible that the repeated flushing of each follicle may have damaged the oocyte which, once fragmented, would not be recovered. Based on the quality of oocytes recovered, it is evident that an LH surge in an oocyte donor is important for the recovery of mature oocytes. It is noteworthy however, that one apparently mature oocyte was recovered from oocyte donor # 341, which did not experience an LH surge. Additional information on this animal acquired through other experiments confirmed that this cow did not exhibit a late LH surge. This would suggest that the oocyte classified as mature was in fact degenerate. Cumulus expansion can occur during maturation and degeneration. These observations indicate that morphological criteria, while extremely useful, are not infallible. This exception apart, oocyte donors not displaying an LH surge produced degenerate oocytes exclusively. Lobo et al. (1985 ) have reported that the proportion of "dysmature" follicles containing abnormal oocytes was similar in ovulatory and anovulatory h u m a n patients. While degenerate oocytes were also recovered from cows with a normal LH surge, a larger proportion of oocytes recovered from this group were mature. An exception to this generalization was oocyte donor # 200. Even though this animal exhibited an LH surge, all oocytes recovered from her were degenerate. Perhaps the early timing of her LH surge was not properly coordinated with other events required for ovulation but, instead, induced premature maturation of all oocytes.
Follicular steroid concentrations and oocyte quality Information is scanty on follicular steroid patterns relative to a fixed reference point which in this study was prostaglandin injection. In the present study two trends were observed (Fig. 1 ). First, in the larger follicles, progesterone concentrations increased with increasing follicular fluid volume. This indicates that as follicles become larger they contain more steroidogenically active granulosa cells. Second, in the smallest follicles containing mature oocytes, progesterone concentrations reached up to and beyond those recorded in larger follicles. One possible explanation for this is that these follicles had steroidogenically active granulosa cells as a result of the LH surge. They had reached their m a x i m u m size, albeit smaller than other superovulated follicles in the study, and were destined to ovulate. This raises the question as to whether all superovulated follicles ovulate when they reach a specific size or whether they ovulate in response to the LH surge regardless of their size. Callesen et al. (1986) reported mean follicular fluid progesterone concentrations of approximately 150 n g / m l to over 500 n g / m l over a similar time after the LH peak. In animals not exhibiting an LH surge, these investigators observed a mean progesterone concentration of approximately 200 ng/ml. Using the fluid aspirated from follicles of superovulated heifers, Maurer et al. (1987 ) reported concentrations well over 1000 n g / m l during this period. Goff et al.
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(1986) noted follicular progesterone concentrations of up to 700 ng/ml of fluid recovered from the follicles of three superovulated heifers 26-33 h after the LH surge while in three other animals not exhibiting an LH peak, progesterone concentrations were as low as 100 ng/ml. Whether a rising progesterone concentration is necessary for proper oocyte maturation, or is only an indicator of "follicle health" is not clear. Epping ( 1981 ) reported that progesterone could reduce the incidence of parthenogensis. Moor and Warnes ( 1979 ) have shown that an aberrant steroid environment induced during the culture of intact follicles resulted in abnormal oocytes. In contrast to progesterone, the follicular fluid estrogen concentrations were very similar in follicles containing a range of fluid volumes (Fig. 2 ). Follicular fluid estradiol was noted to be below 60 n g / m l in follicles from which mature oocytes were recovered. Callesen et al. ( 1986 ) reported follicular fluid estradiol concentrations of approximately 70-80 n g / m l during the same period covered in the present study. These authors also reported a concentration of 55 n g / m l in follicles recovered from animals not exhibiting an LH surge. In the present study, there were a few follicles with estradiol concentrations of this order, but there were some with extremely low concentrations (less than 20 n g / m l ) . Goff et al. ( 1986 ) had reported estradiol concentrations as low as 7 n g / m l in cows exhibiting an LH surge and 60 n g / m l in animals not showing an LH peak. Results from the current study would suggest that the extremes in the concentrations recorded occurred in follicles aspirated from cows not exhibiting an LH surge and contain degenerating oocytes. Alternatively, estradiol may have a role to play in follicle/oocyte development in cows which displayed an LH surge. This is supported by the changes observed in preovulatory follicular estradiol concentrations which are highest around the time of the LH surge. Reports on the concentration of testosterone in follicular fluid recovered from supervulated animals are scanty. In the present study, testosterone concentration followed a pattern similar to that seen for estradiol (Fig. 3 ). Bousquet et al. ( 1988 ) reported increasing concentrations of testosterone with increasing time from the beginning of standing estrus, while Dieleman et al. (1983) observed decreasing androgen concentrations during the same period. The present results support the contention that the major role of androgens is to serve as a substrate for estrogen synthesis. In light of the importance of estrogen in follicle/oocyte health, the aromatization of androgens, presumably coming as androstenedione from theca interna, may actually be the determining factor for proper oocyte maturation. In the present study the progesterone-to-estradiol ratio was greater than 1 : 1 in the vast majority of follicles regardless of their origin from + LH or - LH animals. Callesen et al. (1986) reported a ratio of less than 10:1 around the time of expected ovulation in cows while Goff et al. (1986) reported ratios greater than 40: 1. In animals not exhibiting an LH surge, Callesen et al., (1986) and Goff et al. (1986) recorded mean progesterone-to-estradiol ra-
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tios of less than 1 : 1. Since follicular atresia may not affect the oocyte for some time, morphologically healthy oocytes can be recovered from atretic follicles (Tsafriri and Braw, 1984). It would be useful if a correlation between follicular fluid steroid ratios and oocyte health could be established. In the present study, this association could not be made since both mature and degenerate oocytes were found in follicles exhibiting a wide range of progesterone-toestradiol ratios. The injection of gonadotropins to initiate superovulation may induce asynchronous oocyte maturation with changes in follicular fluid steroid concentrations. Meiotically abberant oocytes and abnormal follicular steroidogenesis have been reported in superovulated rats (Yun et al., 1989). Perhaps other methods of assessing oocyte normality would be more accurate than follicular fluid steroid concentrations. Flow cytometric DNA analysis of granulosa cells may be an alternative (Callesen et al., 1987b).
Size and function of aspirated follicles Staigmiller and England (1982) reported that the ovulatory follicle of nonstimulated cattle ranges in size from as low as 10 m m to about 20 mm in diameter. Their follicular fluid volumes, based on the aspiration techniques used in this study, could be estimated to range from 0.4 to 1.4 ml. Thus, the present study indicates that a large proportion of the ovulations in superovulated heifers may originate from follicles of smaller size than those transpiring in nonstimulated cows. Little information in available on the size of stimulated preovulatory follicles. In the present study, follicular fluid volume ranged from 0.1 ml to 1.4 ml from follicles 8 m m - 1 7 mm in diameter. While some cows produced a uniform population of follicles, others produced a broad range of follicle sizes. This observation of a gradient in size of preovulatory follicles has also been recorded in human subjects by Lehman et al. (1984). However, Templeton et al. ( 1986 ) reported that, in women, despite multiple follicular development, the leading follicle in stimulated cycles ovulated at a size equal to, or even greater than, that of the ovulatory follicle in spontaneous cycles. Simonetti et al. ( 1985 ) have suggested that, in human in vitro fertilization programs, oocytes recovered from larger follicles are of better quality. It has been documented that, in normals, the largest follicle produces most of the estradiol during estrus (Staigmiller et al., 1982 ). It is proposed that if a similar hierarchy exists with stimulated follicles, then perhaps one or two of the larger follicles may produce sufficient estradiol to activate the LH surge before the remaining smaller follicles are ready for ovulation. Since their granulosa cells contain sufficient LH receptors, the smaller follicles may ovulate somewhat prematurely. However, the quality of the released oocytes may not be comparable to that of the larger follicles. The best method of determining the role of follicle size and steroid environment on oocyte quality would be to fertilize the aspirated oocytes in vitro and assess their developmental potential.
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CONCLUSION
Results of the present study indicate that a greater proportion of mature bovine oocytes will be recovered from those preovulatory follicles with a progesterone concentration greater than 100 ng/ml, an estradiol concentration of 5-60 ng/ml, a testosterone concentration of 5-45 ng/ml and a volume greater than 0.3 ml ( 10 mm diameter). Progesterone-to-estradiol ratio would appear to be of no value to predict oocyte quality. It may be possible that the current methods of visually assessing oocytes are not sufficiently accurate. Follicular steroid patterns may change at a different rate than visible morphological changes to the oocyte-cumulus complex. More objective methods of directly or indirectly assessing bovine oocytes are required. In conclusion, it is apparent that a number of factors must be considered to categorize bovine oocytes removed from preovulatory follicles. While follicular fluid steroid parameters alone may not be adequate, they may prove to be more useful to predict viability after the oocyte has been put to an end use, for example, for in vitro fertilization. REFERENCES Abraham, G.E., Swerdloff, R., Tulchinsky, D. and Odell, W.D., 1971. Radioimmunoassay of plasma progesterone. J. Clin. Endocrinol. Metab., 32:619-624. Bousquet, D., Goff, A., King, W.A. and Greve, T., 1988. Fertilization in vitro of bovine oocytes: analysis of some factors affecting the fertilization rates. Can. J. Vet. Res., 52: 277-279. Callensen, H., Greve, T. and Hyttel, P., 1986. Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology, 25:71-86. Callensen, H., Greve, T. and Hyttel, P., 1987a. Premature ovulations in superovulated cattle. Theriogenology, 28:155-166. Callensen, H., Westergaard, L., Greve, T. and Hyttel, P., 1987b. Flow cytometric DNA analysis ofgranulosa cells from preovulatory follicles in superovulated cows. Zuchthygiene, 22: 4952. Castro, A., Shih, H.H. and Chung, A., 1974. A simple radioimmunoassay of plasma testosterone without column chromatography. Steroids, 23: 625-638. Dieleman, S.J., Kruip, Th. A.M.P., Fontijne, P., De Jong, W.H.R. and Van der Weyden, G.C., 1983. Changes in oestradiol, progesterone and testosterone concentrations in follicular fluid and in the micromorphology of prevoulatory bovine follicles relative to the peak of luteinizing hormone. J. Endocrinol., 97:31-42. Edqvist, L.E. and Johansson, E.D.B., 1972. Radioimmunoassay of estrone and estradiol in human and bovine peripheral plasma. Acta Endocrinol., 71:716-730. Epping, J.J., 1981. Preimptantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro. Gamete Res., 4: 3-13. Flack, B., 1959. Site of production of oestrogen in rat ovary as studied in micro-transplants. Acta Physiol. Scand. Suppl., 163: 1-100. Fortune, J.E. and Armstrong, D.T., 1977. Androgen production by theca and granulosa isolated from proestrus rat follicles. Endocrinology, 100:1341 - 1347. Fortune, J.E. and Hansel, W., 1985. Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation. Biol. Reprod., 32: 10691079.
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Goff, A.K., Greve, T., Bousquet, D. and King, W.A., 1986. Progesterone and luteinizing hormone profiles in heifers used as oocyte donors. Theriogenology, 26: 577-586, Kruip, Th. A.M. and Dieleman, S.J.D., 1985. Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrous cycle. Theriogenology, 24: 395-408. Lehman, F., Hoppen, H.O., Schulz, C.H.R., Hamerich, U., A1-Hasani, S., Van Der Ven, H., Diedrich, K. and Krebs, K., 1984. Concentrations of steroids and prostaglandins in follicular fluids aspirated in an in vitro fertilization programme. In: R.F. Harrison, J. Bonnar and W. Thompson (Editors), In Vitro Fertilization, Embryo Transfer and Early Pregnancy. MTP Press Ltd., Boston, MA, pp. 19-24. Leibfried, L. and First, N.L., 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci., 48: 76-86. Lindner, H.R., Perel, E., Friedlander, A. and Zeitlin, A., 1972. A radioimmunoassay for plasma progesterone. Steroids, 19: 357-360. Lobo, R.A., Di Zerega, G.S. and Marrs, R.P., 1985. Follicular fluid steroid levels in dysmature and mature follicles from spontaneous and hyperstimulated cycles in normal and anovulatory women. J. Clin. Endocrinol. Metab., 60: 81-87. Maurer, R.R., Suss, U. and Wise, T.H., 1987. Oocyte quality in superovulated beef heifers with and without norgestomet implants. Theriogenology, 27: 256. McNatty, K.P., Heath, D.A., Lun, S., Fannin, J.M., McDiarmid, J.M. and Henderson, K.M., 1984. Steroidogenesis by bovine theca interna in an in vitro perfusion system. Biol. Reprod., 30: 159-170. Moor, R.M. and Warnes, G.M., 1979. Regulation of meiosis in mammalian oocytes. Br. Med. Bull., 35: 99-103. Moor, R.M., Osborn, J.C. and Crosby, I.M., 1985. Gonadotrophin induced abnormalities in sheep oocytes after superovulation. J. Reprod. Fertil., 74:167-172. Niswender, G.D., Reichert, L.E., Jr., Midgley, A.R., Jr. and Nalbandov, A.V., 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology, 84:1166-1173. Rodriquez, K.L., Hanks, D.R. and Foote, W.D., 1982. Induced oocyte meiosis in vivo in the bovine. Proc. West. Sect., Am. Soc. Anim. Sci., 33: 133-135. Simonetti, S., Veeck, L.L. and Jones, H., Jr., 1985. Correlation of follicular fluid volume with oocyte morphology from follicles stimulated by human menopausal gonadatropin. Fertil. Steril., 44:177-180. Staigmiller, R.B. and England, B.G., 1982. Folliculogenesis in the bovine. Theriogenology, 17: 43-52. Staigmiller, R.B., England, B.G., Webb, R., Short, R.E. and Bellows, R.A., 1982. Estrogen secretion and gonadotropin binding by individual bovine follicles during estrus. J. Anim. Sci., 55: 1473-1482. Templeton, A., Messinish, I.E. and Baird, D.T., 1986. Characteristics of ovarian follicles in spontaneous and stimulated cycles in which there was an endogenous luteinizing hormone surge. Fertil. Steril., 46:113-117. Tsafriri, A. and Braw, R.H., 1984. Experimental approaches to atresia in mammals. In: J.R. Clarke (Editor), Oxford Review Reprod. Biol. Vol. 6. Oxford Univ. Press, New York, NY, pp. 226-265. Tsafriri, A. and Eckstein, B., 1986. Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally induced atresia. Biol. Reprod., 34: 783-787. Wu, C.H., Lundy, L.E. and Lee, S.G., 1973. A rapid radioimmunoassay for plasma estrogen. Am. J. Obstet. Gynecol., 115: 169-180. Yun, Y.W., Yu, F.H., Yuen, B.H. and Moon, Y.S., 1989. Effects of a superovulatory dose of pregnant mare serum gonadotropin on follicular steroid contents and oocyte maturation in rats. Gamete Res., 23: 289-298.