Mechanisms linking under-nutrition and ovarian function in beef heifers.

Prolonged reduction in energy intake in beef heifers has been reported to suppress ovulation but the mechanisms involved are poorly understood. The objective of this study was to examine whether changes in the pattern of LH secretion following each of three different tests predicted the functional state of the hypothalamo-pituitary-ovarian (H-P-O) axis. Test 1 examined the ratio of LH secretion during the 1h before and 2h after naloxone (NAL) administration. The other two tests assessed the LH surge following an exogenous oestradiol positive feedback signal (Test 2) or exogenous progesterone priming (Test 3). In phases 1 and 3, each of 8 weeks duration, the heifers were fed 100% of their maintenance energy requirements. In phase 2, of 9 weeks duration, they were fed 50% of their maintenance energy requirements. Oestrus was induced in all heifers by PG administration at the start of the experiment. Heifers were administered a naloxone challenge of 50, 100, 200 or 400mg naloxone hydrochloride i.v. (one dose per heifer) during the mid-luteal period of phase 1 and all four naloxone treated heifers received 400mg naloxone hydrochloride at the end of phases 2 and 3. Doses of 10, 20 or 40 mg oestradiol benzoate (EB) i.m. were each administered to two of the remaining heifers during the mid-luteal period of phase 1. One heifer on each dose of oestradiol benzoate in phase 1 had the same dose administered at the end of phases 2 and 3. The progesterone challenge was administered to three heifers by insertion of a PRID for 12 days starting in the middle of phase 2. In Test 1, the ratio of LH secretion before and after naloxone administration in phase 1 was 1:1 (50mg), 1:4 (100mg), 1:4 (200mg) and 1:9 (400mg) (50mg versus 100mg and 100mg versus 200mg doses, P<0.05); 50mg versus 400mg doses, P<0.001). In phase 2, this ratio was 1:1 and there was no response to 400mg dose of naloxone in any of the four heifers. In phase 3, the ratio depended on the ovarian activity in the heifer and ranged from 1:1 to 1:4 (P<0.05). In Test 3, a positive oestradiol feedback signal was detected in cyclic heifers in phases 1-3 but not in the acyclic heifer in phase 2. Heifers challenge with exogenous progesterone did not have oestradiol or LH values above threshold levels. We conclude that all three tests successfully predicted the functional state of the hypothalamo-pituitary-ovarian axis. In nutritionally undernourished beef heifers onset of ovarian acyclicity is either preceded or accompanied by the loss of a positive feedback signal (Test 2) and progesterone priming ability (Test 3), and that a plasma LH ratio of > or =1:2 following naloxone challenge (Test 1) is a sign of recovery of the functional state of the hypothalamo-pituitary-ovarian axis.

Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus).

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH.

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers. Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH. In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.

Cattle, pig and sheep oocytes isolated from healthy cumulus-oocyte complexes were pooled, within species, to provide samples of immature denuded oocytes with intact zona pellucida (n = 1000 per sample) for determination of fatty acid mass and composition in total lipid, constituent phospholipid and triglyceride. Acyl-containing lipid extracts, transmethylated in the presence of a reference penta-decaenoic acid (15:0), yielded fatty acid methyl esters which were analysed by gas chromatograph. Mean (+/- SEM) fatty acid content in samples of pig oocytes (161 +/- 18 micrograms per 1000 oocytes) was greater than that in cattle (63 +/- 6 micrograms; P < 0.01) and sheep oocytes (89 +/- 7 micrograms; P < 0.05). Of 24 fatty acids detected, palmitic (16:0; 25-35%, w/w), stearic (18:0; 14-16%) and oleic (18:1n-9; 22-26%) acids were most prominent in all three species. Saturated fatty acids (mean = 45-55%, w/w) were more abundant than mono- (27-34%) or polyunsaturates (11-21%). Fatty acids of the n-6 series, notably linoleic (18:2n-6; 5-8%, w/w) and arachidonic acid (20:4n-6; 1-3%), were the most abundant polyunsaturates. Phospholipid consistently accounted for a quarter of all fatty acids in the three species, but ruminant oocytes had a lower complement of polyunsaturates (14-19%, w/w) in this fraction than pig oocytes (34%, w/w) which, for example, had a three- to fourfold greater linoleic acid content. An estimated 74 ng of fatty acid was sequestered in the triglyceride fraction of individual pig oocytes compared with 23-25 ng in ruminant oocytes (P < 0.01). It is concluded that the greater fatty acid content of pig oocytes is primarily due to more abundant triglyceride reserves. Furthermore, this species-specific difference, and that in respect of polyunsaturated fatty acid reserves, may underlie the contrasting chilling, culture and cryopreservation sensitivities of embryos derived from pig and ruminant (cattle, sheep) oocytes.

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment.

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.

The response of bovine granulosa cells to different gonadotrophins in culture.

Previous studies with bovine granulosa cells cultured in vitro indicated that follicle-stimulating hormone (FSH) stimulated differentiation and progesterone production of granulosa cells in a dose-dependent manner, this was due mainly to an increase in the number of differentiated cells. The objectives of the present study were to investigate (1) whether the response of bovine granulosa cells in culture to luteinising hormone (LH) and equine chorionic gonadotrophin (eCG) was similar to the response to FSH, and (2) whether granulosa cells derived from different cattle breeds responded similarly to gonadotrophin stimulation. Pairs of ovaries were recovered postmortem from Charolais (38) and Hereford (41) crossbred post-pubertal heifers, and granulosa cells were aspirated from 5-8 mm follicles. In two simultaneous experiments, granulosa cells (2-3 x 10(5) viable cells) were cultured with different gonadotrophins (oFSH or oLH in Experiment 1; oFSH or eCG in Experiment 2). Cell culture was for 4 days at 37 degrees C in a humidified atmosphere of 5% CO2 in air in 1 ml of serum-free culture medium. Progesterone production, total DNA and the protein content of granulosa cells on Day 4 of culture were determined. Log10 data were analyzed by analysis of variance and multiple linear regression. In Experiment 1, both FSH and LH stimulated progesterone production (ng microgram-1 DNA) and protein content (microgram microgram-1 DNA) of granulosa cells in a dose-dependent manner (P < 0.01). The relative potencies of FSH to LH (milli micron/milli micron) were found not to be different from unity. In Experiment 2, progesterone production and the protein content of granulosa cells were stimulated by both FSH and eCG in a dose-dependent manner (P < 0.001). The progesterone response curves (log/log) were linear up to 1-10 milli microns FSH and 1-10 iu eCG, and were Y = 1.67 + 0.093 FSH and Y = 1.60 + 0.091 eCG for progesterone production. Calculated on a milli micron/iu basis, FSH was found to be 5.8 times more potent than eCG (P < 0.05) in terms of stimulating progesterone production. Granulosa cells derived from Hereford crosses were more sensitive (P < 0.001) than those from Charolais crosses to gonadotrophin stimulation (31 and 42 times for FSH and eCG, respectively, in terms of progesterone production, and 4.8 and 3.1 times for FSH and eCG, respectively, in terms of protein content). The response curves for both FSH and eCG were similar within each breed. The slopes of the progesterone response curves, and the protein responses were similar for all the gonadotrophins. In conclusion, these results imply that FSH; LH and eCG have similar effects on the differentiation and progesterone production of bovine granulosa cells from 5-8 mm follicles cultured in vitro. Furthermore, granulosa cells from different breeds cultured in vitro had different sensitivities to gonadotrophin stimulation.

Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin.

Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2-3 x 10(5) viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/micrograms DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations.

A sequence-tagged site map of human chromosome 11.

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.

Effects of clomiphene citrate on early pregnancy in guinea-pigs.

Clomiphene citrate (2 mg/kg body wt) given on the day of mating can block or interrupt pregnancy in guinea-pigs. Corpus luteum function, uterine histology, implantation and embryo development were studied in clomiphene-treated and control animals on Days 5, 9 and 20 of pregnancy. Following treatment, only 25% of the females were regularly pregnant, presenting large and healthy foetuses. The other females examined showed either pregnancy with embryos undergoing resorption or no sign of pregnancy. In these females, corpus luteum size was reduced, progesterone concentrations were very low and the endometrial glands and the epithelium were often altered. It is concluded that clomiphene causes a reduction in fertility by altering the uterus and, by directly or indirectly inducing luteolysis, causes later pregnancy loss.

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function in the guinea pig.

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function was investigated in the guinea pig. Certain comparisons were made with testosterone, the naturally occurring androgen, administered as the phenylpropionate ester. Two milligrams trenbolone acetate per kg given subcutaneously on alternate days for 20 days blocked oestrous cyclicity and ovulation in 9 of 10 animals. A similar effect was shown by 2.2 mg of testosterone phenylpropionate. Treatment of trenbolone acetate-treated animals with exogenous gonadotrophins suggested that the production of follicle-stimulating hormone had been suppressed. Signs of abnormality were seen in the livers of animals receiving 2 mg trenbolone acetate and 2.2 mg testosterone phenylpropionate.

The measurement of insulin-like growth factor 1 in sheep plasma.

A method is described for the radioimmunoassay (RIA) of insulin-like growth factor 1 (IGF-1) in neutralised formic acid-ethanol extracts of sheep plasma. The ability of the acid-ethanol pretreatment to remove the IGF-1 binding proteins (BPs), which interfere in the assay has been examined. Comparative plasma IGF-1 concentrations determined by the method correlated closely (P less than 0.001) with corresponding values where BPs were removed by acid gel filtration. The method has been applied to studies in which sheep were given exogenous growth hormone and indicated that plasma IGF-1 levels respond rapidly to the onset and termination of treatment.

Plasma and pituitary thyroid-stimulating hormone levels in bromocriptine-treated New Zealand genetically hypertensive and normotensive rats.

1. The effects of a 13-day intraperitoneal (i.p.) infusion of bromocriptine, delivered by osmotic pump, on plasma and pituitary thyroid-stimulating hormone (TSH) levels were investigated in New Zealand genetically hypertensive (GH) rats and their normotensive (NT) controls. 2. In both the GH and NT rats, bromocriptine significantly reduced plasma TSH level but did not have any significant effect on pituitary TSH content. 3. No significant difference was found in the plasma TSH level and pituitary TSH content between the vehicle-treated GH and NT rats. 4. These results suggest that there are no differences between the GH and NT rats with regard to the activity of the central dopaminergic system influencing TSH release and also that TSH does not play a role in the hypertension of the GH rats.

Changes in saline and water intakes in bromocriptine-treated genetically hypertensive and normotensive rats.

1. We have previously reported on the effects of a 13-day intraperitoneal infusion of bromocriptine delivered by osmotic pump on blood pressure, plasma and pituitary PRL levels in genetically hypertensive (GH) rats and their normotensive (NT) controls. This paper reports further on that study in describing the changes in saline and water intakes in rats as a result of bromocriptine (BRC) treatment. 2. In the GH rats, bromocriptine did not have any significant effect on saline or water intake. 3. In the NT rats, bromocriptine significantly decreased saline intake and increased water intake. 4. The saline intake in the vehicle-treated GH rats was significantly lower than that in the vehicle-treated NT rats while the water intake was not significantly different. 5. These results indicate that differences exist between the GH and NT rats with regard to their saline and water intakes and their responses to chronic bromocriptine treatment. The changes in saline and water intakes in the GH rats seem to be different from those seen in the spontaneously hypertensive rat in another study.

Plasma and pituitary thyroid-stimulating hormone and their responses to bromocriptine in the Japanese strains of hypertensive and normotensive rats and the F2 generation of the hybrid.

Plasma thyroid-stimulating hormone (TSH) concentration and pituitary TSH content were examined in bromocriptine (BRC)- and vehicle-treated male Spontaneously Hypertensive Rat (SHR), its normotensive control, the Wistar-Kyoto (WKY) rat and the F2 generation of the SHR/WKY hybrid. The hybrid rats were divided into three groups according to their systolic blood pressures (BP) as follows--group I: BP less than 140 mmHg; group II: BP = 140-160 mmHg and group III: BP greater than 160 mmHg. Plasma TSH concentrations were not significantly different between the vehicle-treated SHR and WKY nor between the three groups of vehicle-treated hybrid rats. The pituitary TSH content was significantly higher in the vehicle-treated SHR than in the vehicle-treated WKY while it was not significantly different between the three groups of vehicle-treated hybrid rats. Plasma TSH concentrations in the BRC-treated SHR, WKY and three groups of hybrid rats were significantly lower than the concentrations in the corresponding vehicle-treated rats. Pituitary TSH content were significantly lower in the BRC-treated SHR, group I and group III rats but not in the BRC-treated WKY and group II rats when compared to their respective vehicle-treated controls. The results of this study suggest that TSH is not involved in the maintenance of systolic BP in the SHR, WKY and hybrid rats.

Effects of dopamine on blood pressure and heart rate after microinjection into the hypothalamus of spontaneously hypertensive and normotensive Wistar-Kyoto rats.

In anaesthetized spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats, 0.0125-0.4 mg/kg dopamine (DA) given intracerebroventricularly (icv) produced dose-related decreases in mean arterial blood pressure (MAP) and heart rate (HR), whereas intravenous DA produced dose-related increases in MAP and HR. SHR was significantly more responsive to icv DA compared with the normotensive controls. DA (0.02 mg/kg) significantly reduced MAP in a biphasic manner, when microinjected into the arcuate nucleus or third ventricle of SHR, with no significant changes in HR. No such effect occurred in WKY rats. Metoclopramide given concurrently with DA into the third ventricle attenuated the hypotensive response to DA. Saline injection had no significant effect on MAP and HR. The hypotensive responses to DA were not confounded by the spread of injected DA into the adjacent hypothalamic areas. These results support the hypothesis that there exists an abnormal sensitivity in SHR to centrally administered DA and that the arcuate appears to be the brain site involved in this abnormality.

Blood pressure, plasma and pituitary prolactin responses to bromocriptine in New Zealand genetically hypertensive and normotensive rats.

1. The effects on blood pressure (BP), plasma and pituitary prolactin (PRL) of a 13 day intraperitoneal infusion of bromocriptine (BRC) delivered by osmotic minipump were investigated in genetically hypertensive rats (GHR) and their normotensive (NT) controls. 2. In the GHR, the mean BP in the BRC-treated group over the 13 day period of study was significantly lower than in the vehicle-treated group. In the NT rats, the mean BP in the BRC-treated group over the 13 day period was also significantly lower than in the vehicle-treated group. 3. Mean plasma PRL concentration in the GHR and NT rats were comparable. In the GHR, the mean plasma PRL concentration taken on day 13 was significantly lower in the BRC-treated group than in the vehicle-treated group. In the NT rats, the mean plasma PRL concentration taken on day 13 in the BRC-treated group was, however, not significantly different from that in the vehicle-treated group. 4. The mean pituitary PRL content was not significantly different in the GH and NT rats. There was a greater suppression of pituitary PRL content in the BRC-treated GHR than in the BRC-treated NT rats compared with their respective vehicle-treated groups. 5. The results raise the possibility that PRL may have an indirect role in the pathogenesis of the hypertension of the GHR.