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FULL PAPER TheriogenologyDynamic Changes in Plasma Concentrations of Gonadotropins, Inhibin, Estradiol-
17β and Progesterone in Cows with Ultrasound-guided Follicular Aspiration
Atsushi TOHEI1), FangXiong SHI1–3), Manao OZAWA1), Kei IMAI4), Hiroto TAKAHASHI4), Itsuo SHIMOHIRA4), Toshiyuki KOJIMA4), Gen WATANABE1,2) and Kazuyoshi TAYA1,2) 1)Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183–8509, 2)Department of Basic Veterinary Sciences, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501–1193, Japan, 3)College of Animal Sciences, Zhejiang University, Hangzhou 310029, China and 4)National Livestock Breeding Center, Ministry of Agriculture, Forestry and Fisheries, Nishigo, Fukushima 961–8601, Japan (Received 9 March 2000/Accepted 14 September 2000) ABSTRACT. To elucidate the effects of ultrasound-guided transvaginal follicular aspiration, plasma concentrations of FSH, LH, inhibin,estradiol-17β and progesterone, and folliculogenesis were examined in Holstein cows. Four clinically healthy cows with regular estrouscycles were scanned by ultrasound per rectum once a week for 9 weeks before the commencement of follicular aspiration. All visiblefollicles were divided into 3 categories based on their sizes (2 ≤ small < 5 mm; 5 ≤ medium < 10 mm, large ≥ 10 mm). The follicularaspiration was started at random during the estrous cycle and conducted under epidural anesthesia induced with 5 ml of 2% lidocaineonce a week for 6 weeks. The average number of total visible follicles ≥ 2 mm in diameter at 7 days after aspiration (21.7 ± 7.4, n=24)was similar to that before starting aspiration (26.7 ± 10.5, n=36). Plasma inhibin and estradiol-17β declined and fell to a trough on 1.5days and returned to pre-aspiration values by 5 days after aspiration. Plasma concentrations of FSH increased and reached peak levelsbetween 1 and 1.5 days after aspirations. Plasma concentrations of LH also increased and reached peak levels between 0.5 and 1.5 daysafter aspirations. Both plasma FSH and LH had returned to pre-aspiration levels by 5 days after aspirations. Plasma concentrations ofprogesterone did not change with the follicular aspiration. These results demonstrate that follicular aspiration decreases plasma concen-trations of inhibin and estradiol-17β, which in turn leads to a rise in plasma concentrations of FSH and LH. It is suggested that markedincreases in plasma concentrations of FSH and LH after the aspiration stimulate the development and maturation of a new cohort of fol-licles within one week in cows.
KEY WORDS: bovine, estradiol, follicular aspiration, gonadotropin, inhibin.
J. Vet. Med. Sci. 63(1): 45–50, 2001 The inability to obtain a large number of viable embryos vantages with the use of this method. For example, the ova from selected female cattle populations restricts genetic or embryo collection can only be performed with long inter- improvement. Females are born with a large store of primor- vals between hormonal treatment [29], and results from dif- dial follicles in ovaries, of which a predetermined number of ferent studies were not consistent due to individual animal follicles are stimulated at a time to undergo further develop- variation in response to exogenous gonadotropins [28, 37].
ment [38]. If the oocytes in these follicles are normal then Ultrasound-guided follicular aspiration has been per- there is scope for increasing the reproductive potential of formed in human gynecology since the 1970's [24, 25, 36, selected females since less than 1% of follicles reach the ovu- 39], but great progress have been achieved only in veterinary latory stage [38]. A more homogeneous and reliable source medical practice in the past decade [9, 22, 23, 33, 40]. The of oocytes could be obtained by growing primordial and pre- collection of bovine oocytes by follicular aspiration during antral follicles in vitro [32, 44]. Recent improvements in our the estrous cycle, followed by in vitro maturation, fertiliza- understanding of the factors involved, together with tion, and culturing up to the transferable stage of embryonic improved culture systems, indicate that this is feasible [45].
development, is a useful alternative way to superovulation In the past decade, remarkable progress has been made in for producing embryos. Some previous studies have reported improving the procedures for embryo production in vitro that oocytes after aspiration developed well without exoge- using oocytes obtained from ovaries of cattle at slaughter- nous gonadotropin [9, 23], and have suggested that the nega- houses [7, 11, 26, 46]. Oocytes from slaughtered animals tive feedback effects of inhibin and estradiol-17β may be have provided an important source of embryos for research removed by follicular aspiration, which in turn causes purposes, but they have limited value in the production of increased secretion of gonadotropins in cattle [9]. Ultra- embryos for use in livestock improvement programs. Con- sound scanning and ultrasound-guided follicular aspiration siderable effort has been made by cattle breeders to get off- are quite useful to study the process of follicle selection and spring from genetically superior cows. Furthermore, dominance [10, 11], which is poorly understood at present hormonal induction of multiple ovulation and embryo trans- [27], however, the endocrine response following ultrasound- fer are also widely used. Multiple ovulation induced by guided follicular aspiration has not been well documented in exogenous gonadotropins can be repeated in the same animal several times per year, however, there are also some disad- In the present study, ultrasound-guided transvaginal folli- A. TOHEI ET AL. cular aspiration was performed on Holstein cows once homologous double-antibody RIA as described previously weekly, and plasma concentrations of FSH, LH, inhibin, [14] using bovine 32-kDa inhibin for radioiodination and estradiol-17β and progesterone before and after follicular anti-bovine inhibin serum (TNDH-1). The sensitivity was 39 aspiration were examined.
pg/tube. The intra- and inter-assay coefficients of variationwere 6.8% and 13.5% .
MATERIALS AND METHODS Statistics: All data were expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Duncan's Experimental design: Four clinically healthy Holstein Multiple Range test. A value of P<0.05 was considered to be cows between 8–10 yr of age and weighing 700–800 kg, with statistically significant. regular estrous cycles and non-milking and non pregnancy,were used. All cows were scanned by ultrasound per rectum once a week for 9 weeks before the commencement of folli-cular aspiration. Then, follicles were aspirated under epidu- Follicular development with follicular aspiration: The ral anesthesia induced with 5 ml of 2% lidocaine, once a number of follicles was measured weekly using the ultra- week for 6 weeks. Blood samples (10 ml) were collected sound-guided technique 9 times before starting follicular from the jugular vein at -12, 0, 12, 24, 36 and 120 hr after aspiration. At seven days after follicular aspiration the pro- each follicular aspiration. The samples were centrifuged cedure was repeated for another 5 weeks. The average num- (2000 × g for 15 min at 4°C) and the sera were removed and ber of total visible follicles ≥ 2 mm in diameter at 7 days after stored at –20°C until assay for FSH, LH, inhibin, estradiol- aspiration (21.7 ± 7.4, n=28) was similar to that before start- 17β and progesterone.
ing aspiration (26.7 ± 10.5, n=36). The aspiration did not sig- Ovarian observations and follicular aspiration: All visi- nificantly affect the average number of follicles of each size ble follicles were divided into 3 categories based on their (small, 17.5 ± 8.9 vs. 14.6 ± 7.1; medium, 7.3 ± 4.2 vs. 5.2 ± sizes (2 ≤ small < 5 mm; 5 ≤ medium < 10 mm, large ≥ 10 2.6; large, 1.9 ± 1.1 vs. 1.9 ± 1.2) (Table 1).
mm). Follicular aspiration was conducted as described in Plasma concentrations of FSH, LH, inhibin, estradiol-17β previous studies [21,22] with an ultrasound monitor (Aloka and progesterone with follicular aspiration: Similar changes SSD-1200, Tokyo, Japan) and a 7.5 MHz convex array trans- in plasma concentrations of FSH, LH, inhibin and estradiol- ducer attached to a specially designed puncturing device with 17β following follicular aspirations were observed in all indi- a 17 g stainless steel needle guide. All follicles ≥ 2 mm in vidual cows throughout the experimental period (Fig. 1).
diameter were aspirated with a vacuum pressure (100 mm The changes in plasma concentrations of progesterone in Hg) through the stainless steel needle 59 cm in length into a experimental cows still showed cyclic fluctuation following 50 ml centrifuge tube via 100 cm of teflon tubing; PBS con- follicular aspirations. Plasma concentrations of inhibin were taining 80 µg/ml heparin was used for rinsing the needle and 700–900 pg/ml before aspiration, and were lowest (420–680 pg/ml) at 36 hr after aspirations (P<0.01) (Fig. 2e). The con- Radioimmunoassays (RIA) for FSH, LH, inhibin, estra- centrations of estradiol-17β in plasma were 6.5–10.0 pg/ml diol-17β and progesterone: Plasma concentrations of FSH before aspiration. After aspiration they decreased and were measured by RIA as described previously [2] using reached their troughs (2.5–4.0 pg/ml) at 24 hr after aspiration anti-bovine FSH β-subunit serum (USDA-5-pool), USDA- (P<0.01) (Fig. 2b). Plasma estradiol-17β decreased more FSH-BP3 for radioiodination, and USDA-bFSH-B1 as a ref- quickly than plasma inhibin, but both inhibin and estradiol- erence standard. Plasma concentrations of LH were mea- 17β returned to pre-aspiration values by 120 hr after aspira- sured by RIA [6] using anti-bovine LH serum (USDA-309- tion. Plasma concentrations of FSH increased and reached 684P), USDA-bLH-I-1 for radioiodination, and USDA- peak levels (from 10.5–18.3 ng/ml to 19.0–47.5 ng/ml) bLH-B1 as a reference standard. The sensitivity of the assays between 24 hr and 36 hr after aspiration (P<0.01) (Fig. 2d).
were 6 pg/tube for FSH and 0.24 ng/tube for LH. The intra- A negative correlation between plasma concentration of and inter-assay coefficients of variation were 5.3% and 9.8% inhibin and FSH was observed after aspirations (r=–0.232, for FSH and 6.5% and 11.5% for LH, respectively.
n=144, P<0.01). Plasma concentrations of LH also increased Plasma concentrations of estradiol-17β and progesterone after aspiration, and reached peak levels (from 290–320 pg/ were determined by a double antibody RIA system using 125I- ml to 556–806 pg/ml) between 12 hr and 48 hr after aspira- labeled radioligands as described previously [16, 30, 43].
Antisera against estradiol-17β (GDN 244) and progesterone Table 1. Size distributions of follicles in cows before and after follic- (GDN 377) were kindly provided by Dr. G. D. Niswender(Animal Reproduction and Biotechnology, Colorado State University, Fort Collins, CO, U.S.A.). The sensitivity of the assays were 0.62 pg/tube for estradiol-17β and 2.5 pg/tube for progesterone. The intra- and inter-assay coefficients ofvariation were 6.0% and 9.8% for estradiol-17β, 3.5% and 13.4% for progesterone, respectively.
Results represented as mean ± SEM in 4 cows once a week for 9 weeks Plasma concentrations of inhibin were measured by a before starting aspiration and at 7 days after aspirations for 6 weeks.
ENDOCRINE RESPONSE TO FOLLICULAR ASPIRATIONS FSH and LH. Marked increases in plasma concentrations ofFSH and LH may stimulate development and maturation of anew cohort of follicles within one week in the cow after theaspiration.
In other studies, the most popular aspiration schedule used has been weekly [34] starting on Day 3 or 4 (Day 0 = estrus)of the estrous cycle, which corresponds with the emergenceof the first follicular wave [1, 15]. In the present study, theaverage number of total follicles were not different after aspi-ration, though apparently all visible follicles (≥ 2.0 mm) hadbeen aspirated from the ovaries. Both plasma concentrationsof inhibin and estradiol-17β decreased after aspiration, andreturned to their pre-aspiration levels by 120 hr. However,plasma estradiol-17β decreased more quickly than plasmainhibin after aspiration, this may be because of their differentmetabolism rates. Plasma concentrations of FSH and LH, onthe other hand, increased after aspiration, and returned totheir pre-aspiration levels by 120 hr. These results indicatethat a new cohort of follicles had developed within one week,following the aspiration of follicles.
It has been known that inhibin is mainly secreted by gran- ulosa cells of antral follicles in the ovary, and has the abilityto suppress FSH secretion in mammals [5]. Immunoneutral-ization of inhibin results in increases in plasma concentra-tions of FSH and the rate of ovulation in cattle [12, 16–18,42], indicating that inhibin has an important role in the regu-lation of FSH secretion in this species [5, 12, 15–18]. It hasalso been reported that a negative correlation exists betweenplasma concentrations of estradiol-17β and FSH during thefollicular phase [3, 19, 35, 41], and that estradiol-17β hassome inhibitory effects on FSH secretion in cows. However,it was reported that injections of estradiol-17β antiserum didnot result in any changes in FSH secretion in cows [16].
Therefore, a marked increase in FSH secretions observedafter follicular aspiration in the present study was probablydue to the reduction in plasma inhibin concentrations. Ova- Fig. 1. Circulatory profiles of FSH (a), LH (b), inhibin (c), estradiol- riectomy in cattle results in an increase in frequency of 17β (d; Estradiol) and progesterone (e) in a representative cow.
release of LH pulses [4, 20]. This post-ovariectomy increase Follicular aspirations were conducted once a week for 6 weeksthroughout the experimental period. Arrows indicate the times of in frequency of LH pulses can be inhibited by administration of estradiol-17β [4]. Thus, the increase in plasma levels ofLH following the aspiration in the present experiment may be tion (P<0.05) (Fig. 2a). Both plasma FSH and LH returned mainly because of the removal of negative feedback of estra- to the pre-aspiration levels by 120 hr after aspiration. No sta- diol-17β on the pituitary gland. Corpora lutea usually have a tistical changes in plasma concentrations of progesterone definite life span in cows [8, 31], but follicular aspiration did were observed following follicular aspiration (Fig. 2e). not affect the plasma concentrations of progesterone in thepresent experiment, probably indicating that follicular aspi- ration dose not affect the function of corpora lutea.
In conclusion, the present study clearly demonstrates that This is the first report to describe the dynamic changes in follicular aspiration decreases plasma concentrations of plasma concentrations of gonadotropins, inhibin, estradiol- inhibin and estradiol-17β, thereby resulting in an increase in 17β and progesterone in cows with ultrasound-guided follic- plasma concentrations of FSH and LH. Marked increases in ular aspiration. The results clearly demonstrate that follicu- plasma concentrations of FSH and LH after aspiration, may lar aspiration decreases plasma concentrations of inhibin and stimulate the development and maturation of a new cohort of estradiol-17β, resulting in attenuation of the feedback actions follicles within one week in cows. of inhibin and estradiol-17β on FSH and LH secreted by thepituitary gland. Thus, the removal of inhibition by ovarian ACKNOWLEDGMENTS. We are grateful to Mr. Z. Q. Shi hormones results in an increase in plasma concentrations of (Zhejiang University, China) for analyzing the data, Dr. D.J.
A. TOHEI ET AL. Fig. 2. The effects of follicular aspirations on plasma concentrations of LH (a), estradiol-17β (b; Estradiol), progesterone (c), FSH (d) and inhibin (e) in cows. Values are represented as mean ± SEM of 24 observa-tions in 4 cows. In each panel, results are expressed as percentage value of time -12 hr (before follicularaspiration). *, P<0.05; **, P<0.01 compared with the value of before aspirations (Duncan's multiple-rangetest).
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