Changes In Peripheral Concentrations Research Articles

Molecular mechanisms regulating follicular recruitment and selection

Ovarian follicular growth and development is an integrated process encompassing both extraovarian signals, such as gonadotrophins and metabolic hormones, and intraovarian factors. Follicular development has been classified into gonadotrophin-independent and -dependent phases. In the latter, FSH provides the primary drive for follicular recruitment and LH is required for continued development of follicles to the preovulatory stage. A transient increase in circulating FSH precedes the recruitment of a group of follicles, and these recruited follicles are characterized by expression of mRNAs encoding P450scc and P450arom in granulosal cells. As follicles mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism(s) involved in selection of follicles for continued growth. Indeed, changes in the pattern of expression of mRNA for gonadotrophin receptors and steroid enzymes within follicular cells appear to be closely linked to changes in peripheral concentrations of gonadotrophins. The mechanism of selection of dominant follicles still requires clarification, but seems to be linked to the timing of mRNA expression encoding LHr and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in granulosal cells. Additional intraovarian systems, including the ovarian IGF and activin/inhibin systems, also exert a role. For example, it appears that the development of follicular dominance in cows is associated with the FSH-dependent inhibition of the expression of mRNA encoding insulin-like growth factor binding protein 2 (IGFBP-2) in granulosal cells. In conclusion, the integration of these endocrine signals and intraovarian factors within follicles determines whether follicles continue to develop and become dominant or are diverted into apoptotic pathways leading to atresia.

36 CHANGES IN PLASMA CONCENTRATIONS OF PROGESTERONE AND ESTROGENS DURING GESTATION IN COWS WITH STILLBORN SOMATIC CELL CLONED CALVES

Cloning of mammals by nuclear transfer frequently results in gestational or neonatal failure with a variety of abnormalities that are likely caused by inappropriate epigenetic reprogramming. Early diagnosis of fetal abnormality is important for efficient production of cloned animals. Sex steroids are produced in the bovine placenta and their levels in the blood might be useful as a measure of fetal well being, as they are in humans. The objective of this study was to investigate whether changes in peripheral concentrations of progesterone and estrogens reflect fetal conditions. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were transferred into the uterine horn ipsilateral to the ovary bearing the CL of 29 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (Day 0). Blood was collected at regular intervals from Day 40 until parturition. Plasma progesterone, estrone, oestradiol-17β, and estrone sulfate were measured by RIA in 4 recipient cows. 2 vaginally delivered healthy calves weighed 35 and 36 kg on days 278 and 280, respectively. The other 2 delivered stillborn calves weighed 42 and 31 kg by Caesarean section on days 267 and 287, respectively. Steroid profiles were compared with each other and with those in a cow made pregnant by embryo transfer. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. Stillborn calves were subjected to necropsy and histopathological analysis. Significant differences in steroid levels were observed individually and temporarily. In the cow bearing the former stillborn, progesterone and oestradiol-17β concentrations tended to be lower during mid and late gestation, respectively. Large offspring syndrome, ventricular septal defect, pulmonary oedema, hepatic fibrosis, and placental dysplasia were found in the calf by necropsy and histopathological analysis. In the cow bearing the second stillborn, plasma progesterone concentrations were temporarily increased from days 220 to 230 and reached a peak of 16 ng mL–1, which was approximately 3 times as high as before (5.0 ± 0.9; mean ± s.d.), then followed by a similar transient increase in estrogens during days 240 and 250 (170 pg mL–1, 16 pg mL–1, and 21 ng mL–1 at peak of estrone, oestradiol-17β, and estrone sulfate, respectively). Thereafter, oestrogen concentrations stayed low until parturition. As internal organs of the stillborn calf were severely autolysed with polyhydramnios at birth, the fetus might have been dead around Day 250. These results imply that changes in peripheral steroid concentrations in some cases reflect fetal condition.

Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow

We investigated changes in peripheral concentrations of inhibin A, total inhibin, steroids, and gonadotropins throughout the intact estrous cycle of the sow in relation to ovarian changes determined by daily transrectal ultrasonography. All visible follicles of 3 mm or more in diameter were classified as small (> or =3 and <6 mm) or large (> or =6 mm). Follicular recruitment was identified in two periods of the cycle: one from the late luteal to the follicular phase, characterized by an increase in the number of small follicles followed by the appearance of large follicles; and another during the early luteal phase, consisting only of increased numbers of small follicles. Plasma concentrations of inhibin A increased (P<0.05), coinciding with the two periods of follicle emergence. Estradiol (E(2)) levels increased (P<0.05) during the follicular phase, but not during the early luteal phase. An inverse relationship (P<0.01) between the patterns of inhibin and FSH concentrations was noted around the two periods of follicle emergence, but there was no relationship (P> or =0.1) between the patterns of plasma E(2) and FSH during the early luteal phase. In conclusion, measurement of plasma inhibin A levels combined with ultrasonographic examination of the ovaries revealed two periods of synchronous follicular growth during the sow's estrous cycle. The results strongly suggest that inhibin A functions as a negative feedback regulator of FSH secretion throughout the estrous cycle, whereas E(2) appears to influence FSH secretion only during the follicular phase.

Embryonic and Early Foetal Losses in Cattle and Other Ruminants

Embryo survival is a major factor affecting production and economic efficiency in all systems of ruminant milk and meat production. For heifers, beef and moderate yielding dairy cows, does and camelids it appears that fertilization generally lies between 90% and 100%. In high-producing dairy cows there is a less substantive body of literature, but it would appear that it is somewhat lower and perhaps more variable. In cattle, the major component of embryo loss occurs before day 16 following breeding with some evidence of greater losses before day 8 in high-producing dairy cows. In cattle late embryo loss, while numerically much smaller than early embryo mortality loss, nevertheless, causes serious economic losses to producers because it is often too late to rebreed females when they repeat. In multiple ovulating small ruminants, the loss rate is positively related to ovulation rate. Systemic concentrations of progesterone, during both the cycle preceding and following insemination, affect embryo survival rate with evidence that too high or indeed too low a concentration being negatively associated with survival rate. Uterine expression of mRNA for progesterone receptor, oestradiol receptor and retinol-binding protein appears to be sensitive to changes in peripheral concentrations of progesterone during the first week after artificial insemination. Energy balance and dry matter intake during 4 weeks after calving are critically important in determining conception rate when cows are inseminated at 70-100 days post-calving. Concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates though sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy balance must be developed. There is sufficient genetic variability within the Holstein breed for fertility traits. Alternative dairy breeds such as the Jersey or Norwegian Red could also be utilized. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilization and the establishment of pregnancy, but also, in the future, identify genes responsible for improved embryo survival. Its incorporation into breeding objectives would increase the rate of genetic progress for embryo survival.

Embryo survival in dairy cows managed under pastoral conditions

Efficient pasture-based milk production systems require a compact calving pattern aligned to the onset of the grazing season, a 365-day calving interval and low culling rates for infertility. Achievement of these targets requires high herd reproductive performance. While high genetic merit Holstein cows produce more milk in grass-based systems their fertility is compromised. Management of the modern high genetic merit Holstein dairy cow presents a major challenge in pasture-based systems of production. It appears that the extent of early embryo loss is greater (up to 20% points greater) in the modern high-producing dairy cow and that a much higher proportion of the embryos die before day 7 following insemination in contrast to heifers and lower yielding cows. About 7–8% of pregnancies are lost between days 30 and 90 of gestation with no evidence that loss rate is related to cow genetic merit, parity or level of production. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival rate with evidence that too low or indeed too high a concentration of progesterone been negatively associated with embryo survival rate. Peripheral concentrations of both progesterone and oestradiol are lowered by increased plane of feed intake due to increased metabolic clearance rate of the steroids, which is related to liver blood flow. It appears that high producing dairy cows have an increased risk of embryo death as a result of lowered peripheral concentrations of progesterone as a consequence of increased hepatic metabolism of progesterone. Uterine expression of mRNA for progesterone receptor, oestradiol receptor and retinol binding protein mRNA appears to be sensitive to changes in peripheral concentrations of progesterone during the first week after AI. It would appear that energy balance and dry matter intake during the 4 weeks, immediately after calving are critically important in determining conception rate when cows are inseminated at 70–100 days post-calving. Concentrate supplementation of cows at pasture during the breeding period has minimal affects on conception rates though sudden reduction in dietary intake should be avoided. For pasture-based systems of milk production more balanced breeding strategies, with greater emphasis on fertility and feed intake must be developed.

Leptin in horses: tissue localization and relationship between peripheral concentrations of leptin and body condition.

Obesity has been a major concern in the horse industry for many years, and the recent discovery of leptin and leptin receptors in numerous nonequine species has provided a basis for new approaches to study this problem in equine. The objectives were to: 1) clone a partial sequence ofthe equine leptin and leptin receptor genes so as to enable the design of primers for RT-PCR determination of leptin and leptin receptor gene presence and distribution in tissues, 2) develop a radioimmunoassay to quantify peripheral concentrations of leptin in equine, 3) determine if peripheral concentrations of leptin correlate with body condition scores in equine, and 4) determine if changing body condition scores would influence peripheral concentrations of leptin in equine. In Experiment 1, equine leptin (GenBank accession number AF179275) and the long-form of the equine leptin receptor (GenBank accession number AF139663) genes were partially sequenced. Equine leptin receptor mRNA was detected in liver, lung, testis, ovary, choroid plexus, hypothalamus, and subcutaneous adipose tissues using RT-PCR. In Experiment 2, 71 horses were categorized by gender, age, and body condition score and blood samples were collected. Sera were assayed for leptin using a heterologous leptin radioimmunoassay developed for equine sera. Serum concentrations of leptin increased in horses with body condition score (1 = thin to 9 = fat; r = 0.64; P = 0.0001). Furthermore, serum concentrations of leptin were greater in geldings and stallions than in mares (P = 0.0002), and tended to increase with age of the animal (P = 0.08). In Experiment 3, blood samples, body weights, and body condition scores were collected every 14 d from 18 pony mares assigned to gain or lose weight over a 14-wk interval based on initial body condition score. Although statistical changes (P = 0.001) in body condition scores were achieved, congruent statistical changes in peripheral concentrations of leptin were not observed, likely due to the small range of change that occurred. Nonetheless, serum concentrations of leptin tended to be greater in fat-restricted mares than in thin-supplemented mares (P = 0.09). We conclude that leptin and leptin receptors are present in equine tissues and that peripheral concentrations of leptin reflect a significant influence of fat mass in equine.

Changes in peripheral concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha induced by progesterone in swine.

This experiment was designed to examine the effects of progesterone on endogenous and oxytocin-induced secretion of prostaglandin F2 alpha (PGF2 alpha) in sows. Peripheral concentrations of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were used as an indirect measure of uterine PGF2 alpha secretion. Eight sows received twice daily injections of progesterone (160 mg/injection) on d 1 to 5 after estrus. Another eight sows received comparable injections of a corn oil injection vehicle. Each sow then received i.v. injections of oxytocin (30 IU) on d 10, 12, and 15 after estrus. Concentrations of PGFM were determined in jugular venous blood samples collected at -60, -45, -30, -15, 0, 2, 5, 10, 15, 30, 45, 60, 90, and 120 min after each oxytocin injection. The mean concentrations of PGFM in samples collected before injection of oxytocin (baseline), the magnitude of the PGFM response to oxytocin, and the area under the PGFM response curve (AUC) were calculated for the three oxytocin challenges administered to each sow. Baseline, magnitude, and AUC were low on d 10 after estrus and similar for the two treatment groups. On d 12 baseline, magnitude, and AUC remained low in the control sows; however, all three response variables increased in sows that received progesterone. By d 15, all three variables were high and similar in both treatment groups. In conclusion, progesterone, administered early in the estrous cycle, seems to promote premature secretion of PGF2 alpha as indicated by the high basal concentrations of PGFM observed before injection of oxytocin on d 12.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle of mares

Summary A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin α-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin α-subunit standard curve and porcine follicular fluid dose-response curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached ≤25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect of day for concentrations of ir-inhibin (P

Roles of pattern of secretion of luteinizing hormone and the ovary in attainment of puberty in ewes lambs

Four experiments were conducted to examine (1) how patterns of luteinizing hormone (LH) change as lambs approach first estrus, (2) whether mimicry of these changes by exogenous LH will initiate events of the pubertal process, (3) aspects of possible roles of the ovary and estradiol in the initiation of an ovulatory surge of LH in prepubertal lambs and (4) the time requirements for the presence of the ovary in induction of that surge of LH. In 19 lambs (Exp. 1), concentrations of LH in plasma were higher at puberty than 7 weeks earlier. Increased concentrations of LH in samples taken every 20 min for 6 hr during the luteal and follicular phases preceding first estrus were attributed to increased amplitude, but not increased frequency, of episodic pulses of LH. In Exp. 2, endocrine events which are included in the natural onset of ovarian cyclic activity (i.e. surge of LH and subsequent rise in progesterone) were initiated in 5 6 , 4 6 , 1 5 , 1 6 , and 3 6 lambs, receiving either injections of 7.5 μg/hr, 15 μg/hr, 30 μg/2hr, 45 μg/3hr or constant infusions of 15 μg/hr of purified ovine LH, respectively. However, these lambs did not exhibit estrus or cyclic ovarian activity. In Exp. 3, the mechanism by which hourly injections of LH prompted surges of LH was determined to be mediated through ovarian stimulation and not through a direct effect of exogenous LH on the hypothalamo-pituitary axis. All the intact (n=5) but no acutely (two weeks) ovariectomized (n=5) lambs had a preovulatory-like surge in response to exogenous LH. Further, (Exp. 4) it was shown that the ovarian signal must be maintained or evoked within 6 hr preceding the surge of LH, because lambs (n=20) ovariectomized at or 9, 18 or 27 hr after initiation of the exogenous LH with the one exception failed to show a surge of LH. It could not be demonstrated that the ovarian signal involved changes in peripheral concentrations of androstenedione, testosterone or estradiol-17β, although patterns of LH in ovariectomized lambs were responsive to both negative and positive feedback effects of estradiol (n=6, Exp. 3) and in general estradiol levels were increasing prior to the induced surge of LH.

Endocrine Responses of Cows Subjected to Controlled Voltages During Milking

For the relative influence of central and of peripheral inhibitive mechanisms on the milk-ejection reflex, dairy cows were subjected to control voltages during milking. Integrated 1-min jugular vein samples were taken at −20, −12, −9, −2, −1, 2, 4, 6, 10, and 20min relative to the start of milking for measurement of norepinephrine, epinephrine, dopamine, oxytocin, and prolactin. Cows were subjected to voltages that resulted in body currents of 5mA. For the response of cows to continuously applied voltage, the voltage remained on for 20min starting 10min prior to milking. For the response to intermittently applied voltage, the voltage was on for 5 of every 30s.Milk yield and milking time were decreased in cows subjected to stimulation by intermittent voltage. This decrease could not be explained by changes in peripheral concentrations of either oxytocin or catecholamines. Plasma prolactin did decrease; however, it seems unlikely the prolactin would affect the milk-ejection reflex. We hypothesize that this decrease in milk yield resulted from neural mediated changes within the mammary gland.