Plasma concentrations for several androgens and estrogens were determined in male domestic pigs from birth to eight months of age. Samples (N = 6) of blood were collected from Yorkshire males at weekly intervals from birth to four weeks, and thereafter at monthly intervals to eight months. Radioimmunoassays were done without extraction from plasma for dehydroepiandrosterone sulphate, androstenedione and estrone sulphate. Other steroids were measured after solid-phase extraction, separate elution of unconjugated and conjugated fractions, and solvolysis of sulpho-conjugated steroids (testosterone, 5α-androstane-3β, 1 7β-diol, epiandrosterone, 19-nortestosterone and estradiol-177β). All steroids showed a peak in plasma levels at 2–4 weeks after birth. Concentrations remained low from 2–5 months and rose markedly thereafter. Most steroids were present in much greater quantities as sulpho-conjugated compounds. Concentrations of testosterone sulphate and testosterone were similar (9.4 μmol/l) at three weeks but the sulphated form predominated after six months of age. This study shows that during postnatal development the testes of the domestic pig are remarkably active in steroidogenesis with a peak at 2–4 weeks after birth. Also, the range of steroid products seen at this stage is comparable to that shown by the mature boar.
To allow improved prediction of daily herbage intake of dairy cows in rotational grazing systems, intake behaviour was assessed throughout the day in 24-h paddocks. Herbage intake in 16 lactating Holstein±Friesian cows was assessed using the short-term (1-h) weight gain method at four predetermined natural meal times throughout the day (early morning, T1; late morning, T2; mid-afternoon, T3; and early evening, T4). The study comprised two 4-day experiments, each with a cross-over design of four blocks. In both experiments, cows grazed a 24-h paddock daily, and the effect of the immediately previous grazing experience on intake behaviour was investigated throughout the day, taking account of daily¯uctuations in the short-term physiological condition of the cows. Experiment 1 was carried out to investigate overall grazing behaviour during meals as a sward is progressively depleted during the day, with intake being assessed within the paddock and, hence, on a depleted sward. Experiment 2 similarly investigated the effect of sward depletion and physiological condition throughout the day on intake, but cows were removed to fresh, undefoliated swards during intake measurement periods; thus, intake rate was not in¯uenced by differences in sward condition. Intake behaviour from both experiments was compared to establish the effect on herbage intake of changes in sward state and non-sward factors. In Experiment 1, sward surface height, available herbage mass, proportion of leaf and green leaf mass declined as the day progressed. Bite mass declined with sward depletion, and mean intake rate was 1á64 kg dry matter (DM) h ±1 , which was signi®cantly lower at T3 (P < 0á01) than during other meals. In Experiment 2, plot sward conditions did not change throughout the day, and intake behaviour also remained constant, with a mean intake rate of 2á11 kg DM h ±1 . Mean bite depth as a proportion of pregrazing extended tiller height was constant throughout the day (mean 0á32). The results show that, although cows grazed throughout the day on progressively depleted swards, indicative of rotationally grazed paddocks (Experiment 1), bite mass declined linearly and intake behaviour was variable. However, where intake was assessed on high-quality, undefoliated swards (Experiment 2), intake behaviour was similar regardless of the time of day and the immediately previous experience. There was some indication of an interaction between the effects of the sward and the physiological condition of the animal on herbage intake.
Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [(3)H]-labeled E(2) or E(1), and with [(14)C]-E(1) in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E(2) right harpoon over left harpoon E(1)) by trophoblast varied with development; it favored E(2) in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [(3)H](2)O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E(2) in both free and sulfate fractions. It was concluded that local metabolism of E(2) is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.
Breeding soundness evaluation (BSE) is the primary assessment for determining the reproductive potential of male animals. This method, however, cannot be used to evaluate semen frequently or to predict future semen quality. Computerized analysis of ultrasonographic images provides information on histophysiological changes in male reproductive organs. We hypothesized that: (i) semen parameters would correlate with ultrasonographic characteristics of the distal region (cauda) of the epididymis and (ii) testicular ultrasound images and/or circulating testosterone concentration would predict future semen quality in the ram. Six adult rams underwent BSE and scrotal ultrasonography approximately 60 d apart (average duration of the spermatogenic cycle) both during the breeding (December and February) and non-breeding (June and August) seasons. An inverse correlation was found between pixel intensity (numerical pixel values) of the epididymes and percentage of sperm in semen with normal morphology (r = -0.46, P < 0.05). Pixel heterogeneity (standard deviation of pixel values) correlated negatively with percentage of sperm with normal morphology (r = -0.42, P < 0.05) and directly with percentage of spermatozoa with abnormal tails (r = 0.43, P < 0.05). Pixel heterogeneity of testicular parenchyma obtained approximately 60 d prior to semen evaluation inversely correlated with percentage of sperm with normal morphology (r = -0.73, P < 0.01) and sperm progressive motility (r = -0.76, P < 0.01), and directly with percentage of sperm with abnormal tails (r = 0.72, P < 0.01) and loose heads (r = 0.79, P < 0.01). We concluded that scrotal ultrasonography combined with computer-assisted analyses of epididymal and testicular echotexture in the ram was a valuable method for determining certain current and future semen parameters, respectively.
Dehydroepiandrosterone (DHEA) and 3 beta-hydroxyandrosta-5,7-dien-17-one (7-dehydro-DHEA) are secreted in large quantities by the remarkably hypertrophied fetal gonads of both sexes in the pregnant mare. Their secretion serves as the fetal component of a feto-placental unit for oestrogen production in equine pregnancies. They are secreted in large amounts but show a decline in late pregnancy when the fetal gonads regress and levels of oestrogens in the mare fall as a consequence. We have examined the levels of these precursor steroids in the newborn foal in the first days after birth. DHEA and 7-dehydro-DHEA were measured in peripheral plasma in a direct RIA with a DHEA antibody which cross-reacts with 7-dehydro DHEA (> 150%). Subsequent studies were performed with solid-phase extraction, separation of unconjugated from conjugated steroids, and HPLC fractionation followed by RIA. Detection on HPLC at 254 and 280 nm was compared with results from RIA. It was concluded that DHEA is the major steroid produced by the gonads at birth. The concentrations are highly variable in the first day of postnatal life (70.45 +/- 63.06 ng/ml, n = 52) and decline rapidly to < 2 ng/ml (n = 6) at 96 h after birth. At this time the sulphate form is also seen, with an increasing ratio of DHEAS/DHEA as the value for total DHEA falls. The mechanism and significance of the apparent abrupt decline in gonadal steroidogenesis in the newborn foal remain unknown.
REPRODUCTION RESEARCHThe in vitro metabolism of cortisol by ovarian follicles of rainbow trout (Oncorhynchus mykiss): comparison with ovulated oocytes and pre-hatch embryos Mao Li, Heather L Christie and John F Leatherland Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1Correspondence should be addressed to M Li who is now at Department of Integrated Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; Email: mao@uoguelph.ca AbstractMid-vitellogenic stage rainbow trout (Oncorhynchus mykiss) ovarian follicles (both intact and yolk free (YF)), ovulated oocytes and embryos were co-incubated with [2,4,6,7-3 H]cortisol for 18 h to determine the degree and nature of the metabolism and biotransformation of the glucocorticoid. There was evidence of the conversion of cortisol to the less biologically potent glucocorticoid, cortisone, and the formation of glucocorticoid sulphates (both cortisol and cortisone) for all cell and tissue samples, suggesting the presence of 11b-hydroxysteroid dehydrogenase (11b-HSD) and glucocorticoid sulphotransferase (GST) activity at all stages; however, GST activity was particularly marked in both intact and YF ovarian follicles, suggesting an important role of follicles in limiting the exposure of oocyte to maternal cortisol. As there was no evidence of 11b-HSD or GST activity in ovarian fluid, the findings affirm that ovarian follicles (probably the thecal and granulosa cells) provide a barrier against the transfer of cortisol to the oocytes by forming sulphated steroids, whereas ovulated oocytes and early embryos have a more limited capacity to either metabolize or conjugate cortisol and are therefore more vulnerable at the post-ovulatory and early embryonic stages to increases in exposure to the glucocorticoid. (2012)). The relationship between the maternal stress response, maternal hypercortisolism and reproductive and embryonic outcomes is complex and still the object of ongoing research; however, some of the outcomes are most likely caused by metabolic or immune system-related actions of cortisol that indirectly impact the maternal involvement in the reproductive process; however, other responses can be attributed to the direct actions of cortisol on the developing embryo or fetus. In mammals, the antral fluid of the ovarian follicle regulates the exposure of the pre-ovulatory oocyte to cortisol, and the placental tissues act as a partial barrier to the transfer of maternal cortisol to the embryo and fetus. Because these protective barriers are not present in oviparous vertebrate species, there is the potential for the transfer of maternal cortisol to the oocytes. Consequently, if the oocytes and early embryos are unable to metabolize cortisol, embryogenesis may be compromised (Leatherland et al. 2010. The antral fluid of the ovarian follicles in mammals contains relatively high concentrations of unconjugated cortisol, the levels of which are regulated within narrow limits by the actions of two forms of 1...
Steroid metabolism in target tissues has relevance in assessing biological response. We have investigated the metabolism of testosterone and estrogens in the reproductive tract and accessory sex glands in the boar. Seminal vesicles were taken from four 6-mo-old animals; and seminal vesicles, prostate, vas deferens, and regions of the epididymis were taken from two mature boars (10 and 24 mo old). Tissues were incubated in 5 ml medium (TC-199) at 34 degrees C under 5% CO(2) and 95% air for 2 h with (3)H-labeled testosterone, estrone, and estradiol-17beta. Aliquots of spent media were taken to measure radioactivity before separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. Sulfoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis, respectively. Profiles of metabolites for free and hydrolyzed fractions were obtained from gradient HPLC with acetonitrile:water on a reversed-phase C(18) column. No clear evidence of conjugation was seen for testosterone metabolites. 5alpha-Dihydrotestosterone was the principal metabolite, but the amounts formed depended on the source, with little from the epididymal tissues and seminal vesicles, but greater quantities from the vas deferens (>25%) and prostate (>30%). The most noteworthy feature of estrogen metabolism was the extent of conjugation by all tissues. Almost all radioactivity in the conjugate fractions for the epididymis and vas was present as sulfates. Glucuronidates were seen for the prostate and were the dominant form of conjugation (about 60%) for the seminal vesicles. A striking parallel existed for the profiles of estrogen metabolites from all tissues for unconjugated and hydrolyzed fractions. Only in quantitative terms were some distinctions noted. These overall findings underscore a need to consider local metabolism of steroid hormones in target tissues of the male reproductive system.
Sixteen cases of spontaneous pregnancy loss (11 of singletons and five of pairs of twins) are described. The losses occurred between gestation Days 13 and 25 in 12 mares being monitored almost daily by transrectal ultrasonography (for measurement of conceptus growth) and blood sampling (for determination of maternal plasma progesterone concentrations as evidence of luteolysis) in experimental studies of early pregnancy. In 10 of the 16 cases the uterus was flushed and eight conceptuses were recovered for morphological assessment. Five of the 11 losses of singletons occurred before Day 16 and, with one exception, were preceded or accompanied by luteolysis. The remaining six singleton pregnancies failed after Day 16, with two cases evidencing luteolysis beforehand. Thus, overall, 6/11 singleton losses were associated with luteolysis while 5/11 were not. The five cases of simultaneous loss or degeneration of twin conceptuses all occurred on Day 19 or 20, preceded by luteolysis in only one case. These observations suggest that while the causes of spontaneous early pregnancy failure are multifactorial, luteolysis might contribute to the problem more often than has been previously contended.
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