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A. C. O. Evans, G. P. Adams and N. C. Rawling

The aim of this study was to characterize changes in ovarian follicle dynamics in relation to changes in hormone secretion in heifer calves from birth to 8 months of age. The position and diameter of ovarian follicles ≥4 mm in diameter were recorded, the number of ovarian follicles ≥2 mm in diameter counted, and blood samples collected daily for periods of 18 days, starting at 2, 8, 14, 24 and 34 weeks of age in ten heifers. The mean age at first ovulation was 52.8 ± 1.6 weeks. At all ages ovarian follicular development occurred in a wave-like manner, as in mature cattle. The maximum diameter of the dominant and the largest subordinate follicles increased between 2 and 34 weeks of age (P < 0.05); however, the greatest increase occurred between 2 and 8 weeks of age. There was a similar increase in the numbers of small and large ovarian follicles (P < 0.05). The duration of detection of dominant follicles (number of days visible at a diameter of ≥4 mm) also increased between 2 and 34 weeks of age (P < 0.05). The emergence of waves of follicular development was preceded by peaks in plasma FSH concentrations (P < 0.05) at 2 weeks of age but this was less clear at other ages. There was a rise in circulating concentrations of gonadotrophins between 4 and 14 weeks of age. We concluded that in heifer calves as young as 2 weeks of age ovarian follicles grew in a wave-like fashion, similar to those of adult cattle. We speculate that the early rise in gonadotrophin secretion stimulated the increase in numbers of follicles and follicle diameters observed, indicating an early critical step in reproductive development.

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A. C. O. Evans, W. D. Currie and N. C. Rawlings

Summary. The pattern and opioidergic control of the secretion of gonadotrophins in prepubertal heifer calves were examined. Ten age-matched Hereford heifer calves were weighed and a blood sample was taken every 2 weeks from 2 to 25 weeks of age and then weekly until 60 weeks of age. At 60 weeks, a fertile bull was introduced and at 75 weeks of age pregnancy diagnosis was performed by transrectal ultrasonography. At 4, 12, 18, 24 and 32 weeks of age, the opioid antagonist naloxone was injected (i.v., n = 5; 1 mg kg−1 body weight) each hour for 12 h. Control heifers received sterile saline at the same ages. Blood samples were collected every 12 min for the 12 h treatment and serum samples were analysed for luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Samples taken once every 2 weeks from 2 to 60 weeks were analysed for LH, FSH and oestradiol, and weekly samples were taken for progesterone determination. There was no effect of naloxone on the age at puberty, which was 56·2 ± 0·7 weeks at a body weight of 388·5 ± 8·0 kg. The mean age at conception was 63·4 ± 0·5 weeks. On the basis of samples taken every other week, serum concentrations of LH were high at 10 weeks and between 40 and 60 weeks of age. From the periods of intensive blood collection, the early rise in mean serum concentrations of LH appeared later at 12 and 18 weeks of age and was caused by a rise in LH pulse amplitude. Serum FSH concentrations were increased between 20 and 22 weeks and oestradiol concentrations at 22, 56 and 58 weeks of age. At 4 weeks, naloxone increased mean LH concentrations, pulse amplitude and pulse frequency (P < 0·01) and thereafter only decreased LH pulse amplitude at 18 weeks (P < 0·05) and increased LH pulse frequency at 24 weeks (P < 0·05). The FSH secretion was pulsatile at all ages and naloxone only increased FSH pulse amplitude at 4 weeks.

From these data we conclude that (i) there is an early transient increase in gonadotrophin secretion in prepubertal heifers, (ii) significant opioidergic inhibition of gonadotrophin secretion occurs only in very young heifers and (iii) a decrease in endogenous opioid inhibition of LH secretion, particularly LH pulse amplitude, allows for the early rise in LH secretion.

Keywords: puberty; gonadotrophins; naloxone; heifer

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A. C. O. Evans, G. P. Adams and N. C. Rawlings

Changes in the pattern of follicular growth and development, and the associated endocrine changes, were examined in prepubertal heifers approaching their first ovulation. Ten, age-matched (± 3 days), Spring-born Hereford heifers were examined daily by transrectal ultrasonography for 17 days beginning 12 weeks before the first ovulation, and daily from just before the first ovulation until the completion of one normal duration ovulatory cycle. On each day of ultrasound examination, the position and diameter of corpora lutea and follicles ≥ 3 mm in diameter were recorded, and one blood sample was collected. Blood samples were also collected every 15 min, for 12 h, at 20, 12 and 4 weeks before the first ovulation, to assess the pulsatile nature of LH and FSH secretion. The first ovulation occurred at 56.0 ± 1.2 weeks of age, at a body weight of 391.9 ± 12.0 kg. Waves of follicular development, similar to those of adult cows, were seen at all ages, and in all heifers, the first ovulation was followed by an ovulatory cycle of short duration (7.7 ± 0.2 days) and then by a normal duration ovulatory cycle (20.3 ± 0.5 days). The maximum diameter of the dominant, or largest subordinate, follicles did not increase as the first ovulation approached, or during the subsequent ovulatory cycles. Similarly, there were no differences between follicle growth rates (1.4 ± 0.1 mm day−1) or regression rates (1.2 ± 0.1 mm day−1) as the animals matured, and the interwave interval increased up to the first ovulation. Waves of follicular development were associated with peaks in FSH concentrations at 12 weeks before the first ovulation. The short duration, ovulatory cycle was associated with low progesterone concentrations and small corpora lutea (mean maximum values 2.75 ± 0.66 ng ml−1 and 19.9 ± 2.0 mm, respectively), compared with normal cycle durations (10.15 ± 0.58 ng ml−1 and 25.8 ± 0.8 mm). Mean serum oestradiol and LH concentrations and LH pulse frequency increased as the first ovulation approached, but FSH concentrations did not. We conclude that, in heifers before the first ovulation, growth and regression of large follicles occur in a wave-like pattern, with characteristics and associated patterns of gonadotrophin secretion similar to those seen in adult, cyclic animals. Hence, in late prepubertal heifers, factors controlling follicle growth are in place, and there are no changes in these parameters in the three months preceding the first ovulation.

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G. P. Adams, A. C. O. Evans and N. C. Rawlings

Eleven age-matched (±4 days) Hereford heifers were examined by transrectal ultrasonography daily for 18 days beginning 20 weeks (5 months) before puberty (first ovulation) to determine the suitability of the transrectal ultrasound technique for imaging the ovaries of prepubertal heifers and to test the hypothesis that ovarian follicular development occurs in waves in prepubertal heifers. Satisfactory ovarian images were obtained during preliminary ultrasound examinations conducted 4 weeks before the observational period (that is 32 weeks of age), during which a semirigid probe extension was used to allow external manipulation of the intrarectally placed ultrasound transducer. Daily examinations commencing at 36 weeks of age were accomplished by intrarectal placement of the operator's hand and transducer, without complication, in all 11 heifers throughout the observational period. Periodic increases in the number of follicles detected (day effect, P < 0.02) were inversely related to the diameter of the largest follicle (r = −0.3, P < 0.03). Portions of three anovulatory follicular waves were detected in all heifers during the observational period (first and third waves in part and second wave in whole). Individual follicles destined to assume a dominant or subordinate position in a wave were retrospectively identified and monitored beginning at a diameter of 4–5 mm. The interval between the emergence of dominant follicles of successive waves (interwave interval) was 8.0 ± 0.4 days and the interval between successive maxima in the number of follicles per heifer per day was 8.1 ± 0.5 days. The growing phase of the dominant follicles best fit a quadratic curve. The growing phase of the largest subordinate follicles, and the static and regressing phases of dominant and subordinate follicles best fit simple linear expressions. Periodic surges in serum concentrations of FSH (day effect, P < 0.0001), but not of LH (day effect, not significant), were associated with follicular wave dynamics. FSH surges (increase and decrease, respectively, best fit quadratic curves) spanned a mean of 3 days and reached maximum values 0.9 ± 0.3 days before emergence of the wave. Results supported the hypothesis that follicular development occurs in waves in prepubertal heifers. Mechanisms controlling the well-ordered phenomena of wave emergence, follicle selection and follicle regression, similar to those of sexually mature heifers, were present in 36-week-old prepubertal heifers.

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R. K. Chandolia, A. C. O. Evans and N. C. Rawlings

In bull calves serum concentrations of LH, FSH, and to a lesser extent testosterone, are increased transiently, between 6 and 20 weeks of age. The function of gonadotrophin and testosterone secretion in this period of growth and development was tested by injecting five Hereford bull calves with a GnRH agonist (15 mg Leuprolide acetate) i.m. at 6, 10 and 14 weeks of age; five vehicle treated calves acted as controls. On the basis of blood samples taken every 15 min for 10 h, at 12 weeks of age, mean serum concentrations of LH, FSH and testosterone and LH and FSH pulse frequency and amplitude were decreased (P < 0.05) by Leuprolide acetate. At 24 weeks of age, mean serum concentrations of LH, and LH and FSH pulse frequency in Leuprolide acetate treated calves exceeded (P < 0.05) that seen in control calves. On the basis of blood samples taken every other week, treatment with Leuprolide acetate decreased mean serum concentrations of FSH and testosterone at 14, 16 and 18 weeks of age compared with control calves and delayed the peak of the early increase in LH secretion from 20 to 24 weeks of age (P < 0.05). Scrotal circumference between 22 and 50 weeks of age, pixel units from ultrasound images of the testes, testis mass at castration at 50 weeks of age, and numbers of spermatids and pachytene spermatocytes were all lower in Leuprolide treated calves than in controls. A transient increase in secretion of LH, FSH and testosterone in young bull calves before 20 weeks of age may, therefore, be a critical step in the initiation and timing of testicular development in bull calves.

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A. C. O. Evans, W. D. Currie and N. C. Rawlings

The control of gonadotrophin secretion during the early developmental period in the bull is poorly understood; opioidergic regulation may be involved. The opioid antagonist naloxone was administered to bull calves (i.v. n = 5; 1 mg kg−1 body weight h−1) for 12 h at 4, 12, 18, 24 and 32 weeks of age. Control animals (n = 5) received sterile saline solution. During the treatment period, 4 ml blood samples were collected every 12 min. The calves were also weighed and blood samples collected every two weeks from 2 to 48 weeks of age. Scrotal circumference was measured from 20 to 48 weeks of age to determine age at puberty. Serum samples were assayed for concentrations of LH, FSH, testosterone and oestradiol. On the basis of blood samples taken every two weeks, serum concentrations of LH increased to a peak at six weeks of age and then decreased; serum concentrations of FSH reached a peak at 20 weeks of age. Serum concentrations of testosterone and oestradiol increased with scrotal circumference from 20 to 48 weeks of age. On the basis of blood samples taken every 12 min for 12 h, we concluded that the early rise in serum LH concentrations was due to an increase in LH pulse frequency. LH pulse amplitude decreased from 4 to 18 weeks of age and FSH pulse amplitude from 4 to 32 weeks of age. Naloxone treatment increased LH pulse frequency at 4 and 24 weeks of age but not at 12, 18 or 32 weeks of age. LH pulse amplitude was depressed and mean serum concentrations of LH were increased by naloxone at 4 weeks of age. At 12 weeks of age, LH pulse amplitude and mean and basal serum LH concentrations were increased by naloxone. At 18 weeks, only mean concentrations of LH were increased by naloxone. The only effect of naloxone on FSH secretion was to increase FSH pulse amplitude at 24 weeks of age. We conclude that there is opioidergic inhibition of LH secretion in the young bull calf, but that between 12 and 18 weeks of age a decrease in opioidergic inhibition on LH pulse frequency contributes to the overall increase in mean circulating concentrations of LH at this time.

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J. P. Ravindra, N. C. Rawlings, A. C. O. Evans and G. P. Adams

Transrectal ovarian ultrasonography was performed daily in eight ewes during one interovulatory interval, using a 7.5 MHz, rigid, human prostate transducer, and a realtime B-mode scanner to record the numbers, diameters and position of all follicles ≥ 2 mm in diameter and the corpora lutea in both ovaries. Blood samples were taken once a day and were analysed for concentrations of FSH, progesterone and oestradiol. During the interovulatory interval of 17.2 ± 0.4 days, antral follicles (follicles > 2 mm in diameter) emerged on all days except for days 1, 5, 15, 16 and 17. A significant increase in the numbers of follicles emerging was seen on days 2 and 11. The ovulatory follicle (6.9 ± 0.1 mm diameter) was retrospectively traced to emergence on day 11.1 ± 0.3 and grew over a period of 4.1 ± 0.1 days at a growth rate of 1.2 ± 0.04 mm day−1. The largest nonovulatory follicles of the same period grew at the same rate as ovulatory follicles and regressed over a period of 2.6 ± 0.2 days at a rate of 1.2 ± 0.07 mm day−1. The mean diameter of the largest follicles seen on each day of the oestrous cycle was lowest on the day of ovulation (2.9 ± 0.2 mm), increased from day 3 to day 5 (4.1 ± 0.4 mm) and again from day 11 to the day before ovulation (6.9 ± 0.1 mm; P < 0.05). The mean number of antral follicles ≥2 mm in diameter increased over the oestrous cycle from 4.5 ± 0.4 on day 3 to 7.2 ± 0.7 on day 11 and showed a sharp decline starting on day 15, to a low of 3.5 ± 0.3 on the day of ovulation (day 17.2 ± 0.4). The corpus luteum could be identified by day 3 of the oestrous cycle in all the ewes, at a mean diameter of 11.5 ± 0.3 mm. The diameter increased to 13.3 ± 0.6 mm on day 5, and declined from day 11 to a diameter of 7.5 ± 0.3 mm on the day of ovulation. Apart from increases before ovulation, there were no clear associations between serum concentrations of FSH or oestradiol and the pattern of follicular growth and regression. We concluded that follicle emergence appeared on many days of the oestrous cycle of ewes, with two phases of increased emergence. There was no discernible connection between follicle emergence and FSH secretion; the overall pattern of growth and regression of follicles was not as distinctly wave-like as in cattle. Follicular dominance was noted only just before ovulation, again, in contrast to cattle.

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K Bender, S Walsh, A C O Evans, T Fair and L Brennan

There has been a marked decline in the fertility of dairy cows over the past decades, and metabolomic analysis offers a potential to investigate the underlying causes. Metabolite composition of the follicular fluid, which presents the intrafollicular environment, may be an important factor affecting oocyte maturation and subsequent early embryo development. The aim of the present study was to investigate the metabolic differences between follicular fluid from the dominant follicle of lactating cows and heifers using gas chromatography mass spectrometry (GC–MS)-based metabolomics. Follicular fluid and serum were collected from cows and heifers over three phases of follicle development: newly selected dominant follicles, preovulatory follicles prior to oestrus and post-LH surge follicles. Analysis of the fatty acids revealed that there were 24 fatty acids and 9 aqueous metabolites significantly different between cows and heifers. Of particular interest were the higher concentrations of saturated fatty acids (palmitic acid, P=0.001; stearic acid, P=0.005) in follicular fluid from cows and higher docosahexaenoic acid levels (P=0.022) in follicular fluid from heifers. Analysis of the metabolite composition of serum revealed that follicular fluid had a unique lipid composition. The higher concentrations of detrimental saturated fatty in cows will have a negative impact on oocyte maturation and early embryo development. Overall, the results suggest that the follicle microenvironment in cows potentially places their oocytes at a developmental disadvantage compared with heifers, and that this may contribute to well-characterised differences in fertility.

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K. M. Battye, A. J. Ammit, C. O'Neill and G. Evans

Summary. Embryos were collected from superovulated ewes on Day 2 (2–8 cell), Day 4 (8–16 cell) and Day 6 (morula/early blastocyst). Two embryos were cultured in 1 ml of one of four media: (i) Ham's F10 + 4mg bovine serum albumin (BSA)/ml, (ii) synthetic oviduct fluid medium + 20% human serum, (iii) Quinn's human tubal fluid medium (HTF) + 3 mg BSA/ml or (iv) HTF + 10% acid-treated fetal calf serum for 24 h. They were transferred to fresh media of the same type and their further development was monitored. A quantitative bioassay and radioimmunoassay was used to measure the concentration of platelet-activating factor (PAF, 1-o-alkyl-2-acetyl-snglyceryl-3-phosphocholine) produced. Following extraction and partial purification, 21/95 (22·1%) of the embryo-conditioned media samples had PAF concentrations greater than that measured in corresponding control media. This was designated as embryo-derived PAF and the corresponding cultures were termed 'PAF-positive'. PAF was produced by embryos at all three developmental stages examined and in each of the four media used, and the average amount of PAF produced was 60·9 ± 9·8 pmol/embryo/24 h. However, neither the developmental stage of the embryo, nor the type of media affected the proportion of PAF-positive cultures nor the amount of PAF produced during culture. Thus, it is demonstrated for the first time that early ovine embryos can secrete PAF in vitro, and that there is considerable variability in their capacity for PAF secretion.

Keywords: platelet-activating factor; embryo; in vitro; sheep

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K E Ryan, S M Casey, M J Canty, M A Crowe, F Martin and A C O Evans

Dominant follicles are those that continue to develop and have the potential to ovulate while subordinate follicles regress. Characteristics of dominant follicles include a larger diameter, higher intrafollicular estradiol, and lower IGF-binding protein (IGFBP)-4 concentrations compared with other cohort follicles. Follicle development is regulated by endocrine hormones that act via intracellular signaling pathways. Here, we show the differences in Akt, Erk, c-Jun N-terminal protein kinase, and p-38 signaling pathways between dominant and subordinate follicles at the dominance stage of the follicle wave. However, earlier in the follicle wave (dominant follicle selection), there were only differences in the levels of Akt and Erk signal transduction proteins among dominant and subordinate follicles. Using this profile of Akt and Erk protein expression in granulosa and theca cells of selected dominant follicles compared with subordinate follicles, we suggest a predictive model to identify future dominant and subordinate follicles from the pool of otherwise similar cohort follicles at the time of follicle wave emergence. We conclude that the Erk and Akt signal transduction pathways are important for dominant follicle selection and development and, furthermore, that the observed differences in these pathways mark the future dominant follicle from subordinate follicles before differences in follicular diameter, follicular fluid estradiol, and IGFBP-4 concentrations are apparent.