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A. T. GREGOIRE, H. RAMSEY and A. ADAMS

Summary.

Administration of oestradiol benzoate, 5 to 200 μg, intramuscularly daily for 3 days, results in glycogen deposition in the uterus and cervix of castrated Wistar rats. The relation between dose of oestrogen and amount of glycogen deposited is not, however, linear nor did all portions of the tract respond in a similar manner. The glycogen level of the vagina was not affected by the hormone administration, while progesterone lowered the glycogen content of all portions of the tract.

The glycogen metabolism of the mammalian female genital tract is briefly reviewed and discussed.

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A. T. GREGOIRE, D. H. DRISCOLL and A. ADAMS

Various attempts have been made to utilize the absorptive properties of the vagina for administration of therapeutic drugs. The vaginal absorption of organic elements, their subsequent isolation in urine, serum or manifestation of their endocrine effects, has been demonstrated with phenylmercuric acetate in the human (Eastman & Scott, 1944) and rat (Laug & Kunze, 1949), and with insulin in the dog (Fischer, 1923) but not the human (Woodyatt, 1922). Penicillin is absorbed within 60 min by the human vagina (Lovelady, Randall & Hosfeld, 1946; Goldberger, Walter & Lapid, 1947), while vaginal absorption of sulphanilamides exhibits daily variation (Carrington, Rohrer, Jones & Moore, 1944).

While investigating oestrogen metabolism, the uptake of tritiated oestrogens by spayed mouse genital tissues after intravenous (Stone, Baggett & Donnelly, 1963), subcutaneous (Stone, 1963) or

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N. R. Adams, C. M. Oldham and R. A. Heydon

Summary. Ewes which had been exposed to oestrogenic clover for 3 years showed an elevated ovulation rate but no difference in numbers of primordial follicles compared with normal ewes.

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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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O A Bogle, M H Ratto and G P Adams

An ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas (induced ovulators) and cattle (spontaneous ovulators) suggests that OIF is a conserved constituent of seminal plasma among mammals. In this study, three experiments were designed to determine the biological effects of OIF in different species. In experiment 1, superstimulated prepubertal female CD-1 mice (n=36 per group) were given a single 0.1 ml i.p. dose of 1) phosphate-buffered saline (PBS), 2) 5 μg gonadotropin-releasing hormone (GNRH), 3) 5 IU hCG, or 4) llama seminal plasma. The proportion of mice that ovulated was similar among groups treated with GNRH, hCG, or seminal plasma, and all were higher than the saline-treated group (P<0.001). In experiment 2, female llamas (n=8 or 9 per group) were intramuscularly treated with 1) 2 ml PBS, 2) 1 ml diluted llama seminal plasma, 3) 3 ml equine seminal plasma, or 4) 3 ml porcine seminal plasma. Experiment 3 was the same as experiment 2 except that the dose of equine and porcine seminal plasma was increased to 8 and 10 ml respectively. All llamas that were treated with llama seminal plasma ovulated and none that were treated with saline ovulated (P<0.0001). The proportion of llamas that ovulated in response to equine and porcine seminal plasma was intermediate. We conclude that the mechanism for the biological response to OIF is present in prepubertal CD-1 mice and that OIF is present in equine and porcine seminal plasma.

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J. Singh, R. A. Pierson and G. P. Adams

Nulliparous heifers (n = 58) were studied to determine whether computer-assisted quantitative echotexture analysis of ultrasound images reflects the functional and histomorphological characteristics of the corpus luteum. The ovaries of heifers were examined daily by transrectal ultrasonography from day −2 (day 0 = ovulation) until the day of ovariectomy during metoestrus (day 3; n = 8), early dioestrus (day 6; n = 9), mid-dioestrus (mean, day 10; n = 7), or pro-oestrus (mean, day 18; n = 8; Expt 1). High resolution ultrasound images of corpora lutea were obtained in vitro, and were digitized and analysed using custom-developed computer algorithms optimized for ultrasonography. Cryostat sections of corpora lutea were examined for lipid distribution, and corpora lutea were homogenized to determine the content of progesterone, total protein, cholesterol and triglyceride. In Expt 2, heifers (n = 26) were ovariectomized as in Expt 1, and ovaries were prepared for histomorphometric evaluation. Pixel values (brightness of picture elements) of ultrasound images of corpora lutea were characterized as high during metoestrus, low during early and mid-dioestrus, and increasing again during pro-oestrus (P < 0.05). Changes (P < 0.001) in volume density of luteal cells were characterized as increasing from metoestrus (40.7 ± 0.4%) to mid-dioestrus (55.8 ± 2.8%) and decreasing again at pro-oestrus (41.5 ± 0.9%). The proportion of blood vascular components decreased (P < 0.001) progressively from 31.0 ± 1.0% in metoestrus to 15.6 ± 1.1% in pro-oestrus. Pixel values of ultrasound images of corpora lutea were correlated with luteal (r = −0.72, P < 0.05) and plasma (r = −0.71, P < 0.03) progesterone concentration, and to the volume densities of luteal cells (r = −0.75, P < 0.02) and connective tissue (r = 0.69, P < 0.03). Estimates of triglyceride, protein and cholesterol content of corpora lutea were not correlated with pixel values of ultrasound images. Protein and cholesterol content did not change while triglyceride concentration increased during pro-oestrus (P < 0.05). Results support the hypothesis that ultrasound images reflect luteal and plasma progesterone content, and histomorphological characteristics of the corpus luteum.

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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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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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J. Singh, R. A. Pierson and G. P. Adams

Heifers were studied to determine whether computer-assisted quantitative echotexture analysis of ultrasound images reflect functional and endocrine characteristics of dominant and subordinate follicles at specific stages of development. Heifers were examined using transrectal ultrasonography each day until ovariectomy on day 3 (n = 8) and day 6 (n = 9) of wave 1, day 1 of wave 2 (n = 7), or after onset of pro-oestrus ≥ 17 days after ovulation (n = 8) to obtain growing, early-static, late-static and regressing dominant follicles of wave 1, subordinate follicles, preselection follicles and preovulatory dominant follicles. Ultrasound images of the follicles were obtained in vitro and analysed using custom-developed computer algorithms. Mean pixel (picture element) values (grey-scale: black = 0, white = 255) for the follicle wall and stroma increased (P < 0.05) progressively from the growing to the regressing phases of the dominant follicle of wave 1. The antrum and wall of subordinate follicles had higher (P < 0.05) mean pixel values than that of the corresponding dominant follicles. Pixel heterogeneity (a measure of variation of grey-scale values of pixels) of images of the follicle antrum and wall increased (P < 0.05) progressively during the early-static to regressing phases. A progressive increase (P < 0.05) in the slope of the regression line of pixel values for the follicle wall was detected from the growing to the regressing phases of the dominant follicle of wave 1. The regression line of the wall of the preovulatory dominant follicle had the lowest (P < 0.05) slope. Oestradiol concentration in the follicular fluid decreased (P < 0.05) from the growing to the late-static phase, while a marked decrease (P < 0.05) in the androstenedione concentration was recorded between the growing and the early-static phases of the dominant follicle. Progesterone content did not increase until follicles were in the final stages of regression. Pixel heterogeneity of the antrum and wall, and the slope of the follicle wall regression line were negatively correlated (P < 0.001) with oestradiol and the oestradiol:progesterone ratio in follicular fluid. The results of this study support the hypothesis that echotexture characteristics of ultrasound images of the follicle antrum and wall are correlated with the functional and endocrine status of a follicle.

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N. W. MOORE, C. E. ADAMS and L. E. A. ROWSON

Summary.

Single blastomeres of two-, four- or eight-cell rabbit eggs devoid of the zona pellucida, enclosed in their own zona, or injected into an evacuated host zona were transferred to the Fallopian tubes of recipient does.

No single blastomeres devoid of the zona survived, while 30%, 19% and 11% of those of two-, four- and eight-cell eggs enclosed in their own zona developed into normal viable young.

Thirty-three out of ninety-seven single blastomeres of four-cell eggs injected into host zonae showed limited development, undergoing one or more cleavages, during the first 2 days after transfer. However, none implanted or developed into 6-day blastocysts. Failure to survive may have been due to leucocytic invasion of the zona and subsequent destruction of the blastomeres.

The developmental potential of individual blastomeres is briefly discussed in relation to the production of identical offspring.