Peripheral Plasma Progesterone Concentrations Research Articles

Intra-ovarian dynamic blood flow in pseudopregnant rabbits during prostaglandin F2α-induced luteolysis.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4days) and mid-luteal (9days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40min that was followed 50min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6hr later to reach basal values after 24hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

An earlier rise in systemic progesterone and increased progesterone in the uterine vein during early pregnancy are associated with enhanced embryonic survival in the ewe

Improved livestock production efficiency through greater embryonic survival (ES) is of economic and animal welfare benefit. Physiological characterization of animals that are extreme outliers for ES provides a valuable opportunity to identify a naturally occurring mechanism by which this trait may be enhanced. The objective was to determine the likely cause for the lifetime history of enhanced or reduced ES in a line of ewes selected for high fecundity. To address this question, progesterone concentrations in peripheral plasma as well as ovarian and uterine venous plasma samples were compared between groups of ewes with a lifetime history of either enhanced or reduced ES. The ability of the uterus to synthesize progesterone de novo at Day 5 of gestation was also tested. Ewes with enhanced ES had an earlier rise in progesterone concentration after estrus, irrespective of pregnancy status. In addition, there were increased concentrations of progesterone in the uterine vein in enhanced ES compared with reduced ES ewes on Day 5 of gestation (8.3 ± 0.8 ng/mL and 3.9 ± 1.4 ng/mL, respectively, P < 0.05). However, there were no differences in ovarian venous plasma (enhanced ES, 1725 ± 166 ng/mL; reduced ES, 1665 ± 268 ng/mL) at Day 5 of gestation. Although the endometrial tissue of some ewes (3/8) at Day 5 of gestation expressed three of the key genes necessary for regulation of de novo synthesis of progesterone, expression was not present exclusively in either of the two ES groups and therefore was unlikely to explain differences in the uterine vein progesterone concentrations between the enhanced and reduced ES groups. Collectively, the earlier rise in progesterone concentrations in peripheral plasma during the first week of gestation in the enhanced ES animals was independent of the presence of an embryo. Moreover, increased progesterone concentrations were also observed in the uterine vein at Day 5 of gestation of the enhanced ES ewes. It is proposed that the difference in uterine vein progesterone concentration was likely due to the differences in ovarian venous blood supply rather than de novo synthesis by the uterus.

Mechanisms of Infertility Associated with Clinical and Subclinical Endometritis in High Producing Dairy Cattle

Clinical and subclinical endometritis are common causes of infertility and subfertility in high producing dairy cattle, delaying the onset of ovarian cyclic activity after parturition, extending luteal phases and reducing conception rates. Escherichia coli and Arcanobacterium pyogenes cause endometrial damage and inflammation. Components of microbes, such as lipopolysaccharide (LPS), are detected by Toll-like receptors on endometrial cells, leading to secretion of cytokines, chemokines and antimicrobial peptides. Long luteal phases associated with endometritis are probably caused by a switch in endometrial prostaglandin production from prostaglandin F2a (PGF) to prostaglandin E2. In addition, LPS impairs the function of the hypothalamus and pituitary, and directly perturbs ovarian granulosa cells steroidogenesis, providing mechanisms to explain the association between uterine disease and anovulatory anoestrus. Cows with uterine disease that ovulate have lower peripheral plasma progesterone concentrations that may further reduce the chance of conception associated with endometritis.

Defining Postpartum Uterine Disease and the Mechanisms of Infection and Immunity in the Female Reproductive Tract in Cattle1

Uterine microbial disease affects half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Infection with Escherichia coli, Arcanobacterium pyogenes, and bovine herpesvirus 4 causes endometrial tissue damage. Toll-like receptors on endometrial cells detect pathogen-associated molecules such as bacterial DNA, lipids, and lipopolysaccharide (LPS), leading to secretion of cytokines, chemokines, and antimicrobial peptides. Chemokines attract neutrophils and macrophages to eliminate the bacteria, although persistence of neutrophils is associated with subclinical endometritis and infertility. Cows with uterine infections are less likely to ovulate because they have slower growth of the postpartum dominant follicle in the ovary, lower peripheral plasma estradiol concentrations, and perturbation of hypothalamic and pituitary function. The follicular fluid of animals with endometritis contains LPS, which is detected by the TLR4/CD14/LY96 (MD2) receptor complex on granulosa cells, leading to lower aromatase expression and reduced estradiol secretion. If cows with uterine disease ovulate, the peripheral plasma concentrations of progesterone are lower than those in normal animals. However, luteal phases are often extended in animals with uterine disease, probably because infection switches the endometrial epithelial secretion of prostaglandins from the F series to the E series by a phospholipase A2-mediated mechanism, which would disrupt luteolysis. The regulation of endometrial immunity depends on steroid hormones, somatotrophins, and local regulatory proteins. Advances in knowledge about infection and immunity in the female genital tract should be exploited to develop new therapeutics for uterine disease.

314 EARLY PREGNANCY DIAGNOSIS USING A NOVEL TRANSRECTAL ULTRASONOGRAPHY PROTOCOL IN JAPANESE BLACK COWS

The present study aims to establish a novel practical protocol for early pregnancy diagnosis in cows by using transrectal ultrasonography. The protocol is based on measurements of corpus luteum (CL) cross-sectional area (CL c-s area) change performed at 2 separate days before the coming estrus after AI. Fourteen cows were inseminated artificially, and transrectal ultrasonographical observation of the ovaries and blood collection for measurement of peripheral plasma progesterone (P4) concentration were carried out daily from Days 12 to 23 (Day 0 = the day of onset of estrus). Thereafter, cows were routinely diagnosed for pregnancy at Day 30 by transrectal ultrasonography. The largest CL c-s area was obtained at Day 14 in both pregnant and non-pregnant cows. Seven out of 8 non-pregnant cows showed significant CL c-s area regression between Days 14 and 20 (422 ± 112 v. 249 ± 63 mm2), whereas no regression was observed between Days 14 and 20 in pregnant cows (416 ± 65 v. 402 ± 78 mm2). The regression in the CL c-s area between pregnant and non-pregnant cows was significantly different during Day 18 (424 ± 65 v. 288 ± 88 mm2) to Day 23 (402 ± 71 v. 139 ± 64 mm2). P4 concentration was significantly low (less than 1 ng mL–1) at Day 20 in 3 out of 8 non-pregnant cows, whereas the pregnant cows showed significant increase of P4 between Days 14 and 20 (2.6 ± 0.2 v. 3.4 ± 0.5 ng mL–1). The pregnant cows showed significantly higher P4 concentration starting from Day 18 than non-pregnant cows. However, in non-pregnant cows, 4 cows returned to estrus on Day 20 or after, 3 cows showed no signs of estrus, and 1 cow came in estrus as early as Day 18 after AI. In conclusion, the results of the present study suggest that measuring the change in the CL c-s area at Days 14 and 20 makes it possible to detect the non-pregnant cows at Day 20 after AI. However, it was also indicated that measuring the change of P4 concentrations on the same days did not always successfully detect non-pregnant cows. The new protocol based on CL c-s area regression rate can detect almost certainly non-pregnant cows at Day 20 after AI. It is suggested that this method is advantageous in research and industrial breeding.

Comparison of the Immulite and RIA assay methods for measuring peripheral blood progesterone levels in Greyhound bitches

Determination of optimal breeding time in bitches earmarked for single insemination only is based on measurement of peripheral blood serum or plasma progesterone concentration. In this paper a comparison is made between radioimmune assay (RIA) and chemoluminescent assay (Immulite) for determination of P4 concentrations in the bitch. The Immulite assay is shown to be an accurate and reliable method for serum or plasma P4 measurement. It compares favourably with other methods in terms of turn-around time, cost and accessibility for veterinarians in practice.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F 2α (PGF) or prostaglandin E 2 (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers ( n = 7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

Antioxidant defense system during endometrial receptivity in the guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator

The role of the antioxidant defense system during endometrial receptivity, a phenomenon crucial for implantation and decidualization, and the effect of ormeloxifene, a selective estrogen receptor modulator, were investigated in the guinea pig, a laboratory mammalian species with interstitial implantation and a long functional luteal phase during each estrous cycle. A sharp rise in the activity of superoxide dismutase (SOD) in both antimesometrial (AM) and mesometrial segments and peroxidase in the AM segment of the uterus was observed on the day of maximal endometrial receptivity. Pretreatment with ormeloxifene resulted in loss of endometrial responsiveness, as evidenced by inhibition of trauma-induced decidualization and the activity of ornithine decarboxylase, a marker of tissue growth and repair. This was associated with a decrease in SOD and estradiol dehydrogenase activities, with corresponding increases in estrone dehydrogenase activity and stimulation of uterine luminal epithelial cell height and a distension of the uterine and glandular lumen. A decrease in peroxidase activity was observed only in the AM segment of the uterus on the imminent day of maximal endometrial receptivity. No effect on peripheral plasma progesterone concentration or surface ultrastructure was evident. These findings demonstrate that SOD plays an important role, with peroxidase having a supplementary role, in the first line of defense against superoxide anion radicals during the period of maximal endometrial receptivity in the guinea pig. Inhibition of endometrial receptivity and decidualization by ormeloxifene administered during the pre-receptive phase appears to be due to a depressed antioxidant defense system via dysregulation of redox-sensitive signaling, resulting in altered cellular toxicity due to increased superoxide radicals, and might contribute to the contraceptive action of ormeloxifene. This might be related to its estrogen antagonistic activity and/or decreased bioavailability of estradiol at a cellular level due to its increased metabolism to biologically less-active estrone via activation of estradiol-17 beta-hydroxysteroid dehydrogenase and suppression of estrone-17 beta-hydroxysteroid dehydrogenase.

Oestrous cycle of captive southern hairy-nosed wombats ( Lasiorhinus latifrons) in South Australia, Australia

There is limited information available on the oestrous cycle of female southern hairy-nosed wombats ( Lasiorhinus latifrons). This is mainly due to an extremely poor breeding success in captivity and the difficulty in routine recapturing of these cryptic, semi-fossorial animals in the wild. The aim of this study was to characterise the oestrous cycle of this species by monitoring peripheral plasma concentrations of progesterone and oestradiol, assessing changes in vaginal cytology, pouch condition and the urogenital sinus. Eight adult female wombats were monitored during the breeding season (July–December) over 2 years (2002–2003). Samples were collected up to three times a week. Vaginal smears contained several cell types, categorised by morphology, as either superficial epithelial cells or parabasal–intermediate cells. Leucocytes were also counted. Plasma progesterone profiles showed a mean oestrous cycle length of 36.33 ± 0.67 days with a peak progesterone concentration of 139.53 ± 10.62 nmol/L. Levels of oestradiol peaked at a mean level of 467.33 ± 44.32 pmol/L on average 5 days before a rise in plasma progesterone values. The proportion of epithelial cells in vaginal smears varied throughout the cycle, with a high percentage of superficial epithelial cells observed during the follicular phase. During periods when progesterone concentrations were high, a greater percentage of parabasal–intermediate cells was observed. In conclusion, this study has characterised the oestrous cycle of the southern hairy-nosed wombat and confirmed that changes in vaginal smears together with pouch and urogenital sinus details could be used to determine signs of oestrus in this species.

Peripheral Plasma Progesterone: A Marker for Determining Cyclicity in Yaks (Poephagus grunniens L.)

An attempt was made to determine cyclicity in yaks using plasma progesterone during the breeding and non-breeding seasons. Fifteen non-lactating yaks were used in this experiment. During the breeding season (July to November), blood samples were collected from 8 yaks at least twice weekly until estrus was observed and then at 2 days interval for 30 days. During the non-breeding season (February to March), blood samples were collected from the same number of yaks at 2-day interval for 30 days. Progesterone was determined in plasma samples by radioimmunoassay. During the breeding season, plasma progesterone at estrus was basal (< or = 0.2 ng/ml). Concentrations increased thereafter with a sharp increase during the late luteal phase, typically reaching peak levels around day 15. Concentrations then declined rapidly over the following 4 days, reaching basal levels at estrus. A high proportion (66.7%) of potential estrous periods (based on progesterone concentrations) went undetected, indicating that silent or weak estrus was a prominent problem in yak cows. During the non-breeding season, three animals were found to be cycling as determined by the patterns of plasma progesterone. Yet, behavioral symptoms of estrus were not observed in any of these yak cows. We conclude that peripheral plasma progesterone concentrations can be used to monitor cyclicity in yak cows effectively.

Oestrous cycle of the common wombat, Vombatus ursinus, in Victoria, Australia.

Wild-caught female common wombats from Victoria, Australia, were studied in captivity to investigate the oestrous cycle by assessing vaginal cytology and peripheral plasma progesterone concentrations. Eight wombats, five adults (21-29 kg) and three subadults (19-23 kg), which were held for between 2 weeks and 11 months did not cycle in captivity. Their progesterone concentrations were consistently low (< or = 6.9 nmol L(-1)) and vaginal smears contained predominantly superficial epithelial cells. Three wombats (21-27 kg), held in captivity for >1 year, regularly cycled (when bodyweights exceeded 23.5 kg). Information gathered from four consecutive cycles in each of these three wombats revealed a follicular phase with low progesterone concentrations (< or = 6.9 nmol L(-1)) and vaginal smears with a high percentage of superficial epithelial cells alternating with periods of high progesterone concentrations (range 41.6-123.8 nmol L(-1)) and smears in which parabasal-intermediate epithelial cells predominated. The average length of the monitored oestrous cycles was 47.2 days (35-60 days). The follicular phase lasted ~19 days and the luteal phase lasted ~28 days. In conclusion, wombats can cycle regularly in captivity even under conditions of intensive monitoring.

Oestrous cycle of the common wombat, Vombatus ursinus, in Victoria, Australia

Wild-caught female common wombats from Victoria, Australia, were studied in captivity to investigate the oestrous cycle by assessing vaginal cytology and peripheral plasma progesterone concentrations. Eight wombats, five adults (21–29 kg) and three subadults (19–23 kg), which were held for between 2 weeks and 11 months did not cycle in captivity. Their progesterone concentrations were consistently low (≤6.9 nmol L–1) and vaginal smears contained predominantly superficial epithelial cells. Three wombats (21–27 kg), held in captivity for &gt;1 year, regularly cycled (when bodyweights exceeded 23.5 kg). Information gathered from four consecutive cycles in each of these three wombats revealed a follicular phase with low progesterone concentrations (≤6.9 nmol L–1) and vaginal smears with a high percentage of superficial epithelial cells alternating with periods of high progesterone concentrations (range 41.6–123.8 nmol L–1) and smears in which parabasal–intermediate epithelial cells predominated. The average length of the monitored oestrous cycles was 47.2 days (35–60 days). The follicular phase lasted ~19 days and the luteal phase lasted ~28 days. In conclusion, wombats can cycle regularly in captivity even under conditions of intensive monitoring.

Regulation of nitric oxide synthase isoforms and role of nitric oxide during prostaglandin F2alpha-induced luteolysis in rabbits

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF(2alpha) at day 9 of pseudopregnancy. At 12 h after PGF(2alpha) administration, luteal mRNA encoding eNOS decreased (P0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P0.05) two- to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P0.01) within 6 h after PGF(2alpha) treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P0.01) from 302.4+/-42.3 pg x mg(-1) at day 9 to 58.6+/-8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of Nomega-nitro-L-arginine methyl ester (0.6 g l(-1) per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF(2alpha) administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P0.01) than in controls at 24-48 h after PGF(2alpha) administration (4.5 to 3.2 ng x ml(-1), respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF(2alpha)-induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

Regulation of nitric oxide synthase isoforms and role of nitric oxide during prostaglandin F2alpha-induced luteolysis in rabbits.

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF(2alpha) at day 9 of pseudopregnancy. At 12 h after PGF(2alpha) administration, luteal mRNA encoding eNOS decreased (P<or=0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P<or=0.05) two- to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P<or=0.01) within 6 h after PGF(2alpha) treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P<or=0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P<or=0.01) from 302.4+/-42.3 pg x mg(-1) at day 9 to 58.6+/-8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of Nomega-nitro-L-arginine methyl ester (0.6 g l(-1) per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF(2alpha) administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P<or=0.01) than in controls at 24-48 h after PGF(2alpha) administration (4.5 to 3.2 ng x ml(-1), respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF(2alpha)-induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

Relationship between Peripheral Plasma Inhibin and Progesterone Concentrations in Sahiwal Cattle (Bos Indicus) and Murrah Buffaloes (Bubalus bubalis)

The present study investigated peripheral plasma immunoreactive inhibin (ir-inhibin) concentrations in relation to the stage of oestrous cycle and progesterone concentrations in cycling Sahiwal cattle (Bos indicus) and Murrah buffaloes (Bubalus bubalis). Blood samples were collected once daily for thirty-two consecutive days from cattle and buffaloes (5 each) during winter months of January and February. Mean (±S.E.M.) plasma ir-inhibin concentrations ranged from 0.40±0.01 to 0.59±0.03 ng/ml in cattle and from 0.29±0.03 to 0.52±0.05 ng/ml in buffaloes. In cattle, ir-inhibin concentrations increased from 0.47±0.07 ng/ml on day -4 (day 0=day of oestrus) to reach a maximum value of 0.59±0.03 ng/ml on day -2. Thereafter, ir-inhibin concentrations showed a decline to reach a low of 0.40±0.01 ng/ml on day 11 of the oestrous cycle. In buffaloes, ir-inhibin concentrations increased from 0.38±0.04 ng/ml on day -4 to reach a maximum concentration of 0.52±0.05 ng/ml on day -2. Ir-inhibin concentrations then declined to reach a low of 0.29±0.03 ng/ml on day 9 of the cycle. In both cattle and buffaloes, ir-inhibin concentrations which were lowest (0.43±0.02 and 0.34±0.02 ng/ml, respectively) during the mid-luteal phase of the oestrous cycle increased (p<0.05) to 0.52±0.03 and 0.44±0.04 ng/ml, respectively, during the late luteal phase, and then further to the highest value of 0.53±0.02 and 0.49±0.04 ng/ml, respectively, during the perioestrus phase, following which these declined to 0.50±0.02 and 0.39±0.03 ng/ml, respectively, during the early luteal phase. The variations in peripheral plasma ir-inhibin profile in both the species appear to be related to the changes in characteristics of follicular populations during the oestrous cycle. Peripheral plasma ir-inhibin concentrations were negatively correlated with progesterone concentrations in cattle (r=-0.51, p<0.01) and buffaloes (r=-0.30, p<0.01) indicating that the corpus luteum is not a source of peripheral ir-inhibin in these species. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 1 : 6-10)

Reproductive characteristics of cloned heifers derived from adult somatic cells.

This study examined the onset of puberty, follicular dynamics, reproductive hormone profiles, and ability to maintain pregnancy in cloned heifers produced by somatic cell nuclear transfer. Four adult somatic cell-cloned heifers, derived from a 13-yr-old Holstein cow, were compared to 4 individual age- and weight-matched heifers produced by artificial insemination (AI). From 7 to 9 mo of age, jugular venous blood samples were collected twice weekly, and from 10 to 11 or 12 mo of age, blood sampling was carried out every other day. After the heifers reached puberty (defined as the first of 3 consecutive blood samples with peripheral plasma progesterone concentrations of >1 ng/ml), ultrasound examination of ovaries and jugular plasma sample collection were carried out daily for 1 estrous cycle. Cloned heifers reached puberty later than controls (mean +/- SEM, 314.7 +/- 9.6 vs. 272 +/- 4.4 days and 336.7 +/- 13 vs. 302.8 +/- 4.5 kg for clones and controls, respectively; P < 0.05). However, cloned and control heifers were not different in estrous cycle length, ovulatory follicle diameter, number of follicular waves, or profiles of hormonal changes (LH, FSH, estradiol, and progesterone). Three of the 4 clones and all 4 control heifers became pregnant after AI. These results demonstrate that clones from an aged adult have normal reproductive development.

Feed restriction and insulin treatment affect subsequent luteal function in the immediate postovulatory period in pigs: progesterone production in vitro and messenger ribonucleic acid expression for key steroidogenic enzymes.

Progesterone production and release in vitro, and mRNA expression for key steroidogenic enzymes, were studied in luteal tissue recovered in the immediate postovulatory period from cyclic gilts allocated to one of three treatments: moderate feed restriction during the first (RH) or second week of the estrous cycle, with (HR+I) or without (HR) concomitant injections of long-acting insulin. Time of feed restriction affected neither progesterone production or release, nor mRNA expression for several key steroidogenic enzymes. However, luteal tissue from RH but not from HR gilts responded to LH stimulation by increasing progesterone production and release (P: < 0.05). Insulin treatment increased progesterone production and release, restored luteal tissue responsiveness to LH, up-regulated steroidogenic enzyme mRNA expression, and down-regulated the tissue inhibitor of metalloproteinase-I mRNA expression in HR+I compared with HR gilts (P: < 0.05). In vitro progesterone production and gene expression were affected by time of tissue collection after ovulation in RH and HR gilts but not in HR+I gilts, and were correlated with temporal changes in oviductal and peripheral plasma progesterone concentrations. Inherent differences in luteal function therefore appear to mediate latent effects of nutrition and insulin treatment on circulating progesterone concentrations in the critical postovulatory period in gilts.

Effect of an oxytocin antagonist on prostaglandin F2 alpha secretion and the course of luteolysis in sows.

The role of oxytocin (OT) in the regulation of prostaglandin F2 alpha (PGF2 alpha) secretion during luteolysis in gilts was studied using a highly specific OT antagonist (CAP-581). In Experiment 1 gilts on Days 14 to 19 of the oestrous cycle in Latin square design were used, to determine the dose and time of application of OT and CAP. In Group I (n = 6) gilts were treated intravenously with saline or with 10, 20 and 30 IU of OT. Concentrations of the main PGF2 alpha metabolite i.e. 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were measured in blood samples as uterine response to the treatment. Twenty IU of OT was the most effective to stimulate PGFM release and this dose was used after CAP treatment in gilts of Groups II, III and IV. Gilts of Group II (n = 3) were injected into the uterine horns (UH) with saline (5 ml/horn) or CAP (2 mg, 3 mg and 4 mg; half dose/horn) and OT was injected (i.v.) 30 min thereafter. Any of the CAP doses given into the UH affected PGFM plasma concentrations stimulated by OT. In Group III (n = 4) gilts were infused (i.v.) for 30 min with CAP (9 mg, 14 mg and 18 mg/gilt) followed by 20 IU of OT. All doses of CAP effectively inhibited OT-stimulated PGF2 alpha release, therefore 9 mg was selected for the further studies. Gilts of Group IV (n = 4) received OT 4, 6 and 8 h after CAP to define how long CAP blocks the OT receptors. Concentrations of PGFM increased after any of this period of time. Thus, we concluded that 9 mg of CAP infused every 4 h will effectively block OT receptors. In Experiment 2, gilts (n = 4) received CAP as a 30-min infusion every 4 h on Days 12-20 of the oestrous cycle. Control gilts (n = 3) were infused with saline. CAP infusions diminished the height of PGFM peaks (P < 0.05). Frequency of the PGFM (P < 0.057) and OT (P < 0.082) peaks only tended to be lower in the CAP-treated gilts. Peripheral plasma concentrations of progesterone (P4) and oestradiol-17 beta (E2) and the time of luteolysis initiation as measured by the decrease of P4 concentration were the same in CAP- and saline-treated gilts. The macroscopic studies of the ovaries in gilts revealed lack of differences between groups. We conclude that OT is involved in the secretion of luteolytic PGF2 alpha peaks but its role is limited to controlling their height and frequency. Blocking of OT receptors did not prevent luteolysis in sows.

Measurement of gestagen concentration in feces using a bovine milk progesterone quantitative test EIA kit and its application to early pregnancy diagnosis in the sow.

We attempted to measure the gestagen concentration in the feces of pigs by using a commercial bovine milk progesterone quantitative test EIA kit, and investigated the possibility of applying of this method of gestagen concentration measurement to early pregnancy diagnosis in the sow. Feces were collected from the rectum of the pig, and 0.5 g of the feces was placed in 20 ml of distilled water, stirred, and centrifuged. The supernatant was used as the fecal solution for measurement of gestagen. The procedure used for measuring gestagen in feces was the same as that for the measurement of progesterone in milk, except that a standard fecal gestagen solution (0.5-30.0 ng/ml) was prepared by the authors in the laboratory. The sensitivity of measurement using this method was 0.80 ng/ml, or 32.0 ng/g of fecal weight. The recovery was 105.2-105.6%. Intra-assay coeffecients of variation (CVs) were 2.8-8.5%. The interassay CVs were 7.4-10.2%. Gestagen concentrations in feces measured by the present method and progesterone concentrations in peripheral plasma, collected at the same time as the feces were highly correlated (r = 0.98, p < 0.001). The criteria for diagnosis of pregnancy based on the fecal gestagen level was positive for a gestagen level of > or = 200 ng/g and negative for a gestagen level of < 200 ng/g. When fecal gestagen measurements were applied to early pregnancy diagnosis in 149 sows, the accuracy of diagnosis from day 21 to day 25 after the last mating was 96.2% for positive cases (102/106) and 95.3% for negative cases (41/43). Thus, the results of this study show the quantitative measurement of the fecal gestagen concentration in the sow using a bovine milk quantitative test EIA kit is a practical method for early pregnancy diagnosis.

The incidence of luteal activity, as determined by peripheral plasma progesterone concentration, before the onset of the breeding season in the Rasa Aragonesa breed of sheep

It has been reported in several sheep breeds that the first oestrus of the breeding season is preceded by at least one ovulation unaccompanied by oestrous behaviour. This is associated with seasonal variations in sensitivit)' to oestrogen (Karsch el al., 1978), and progesterone could be involved in the initiation of the fldl-length oestrous cycle (I'Anson & Legan, 1988). Since the occurrence of silent ovulations has been previously demonstrated, the aim of this experiment was to study the pattern of progesterone secretion at the end of seasonal anoestrus, and its capacin' to establish the first oestrous bebaviour of the breeding season in the Rasa Aragonesa breed of sheep. A threshold of 0.5 ng ml -~ of progesterone was selected as an indication of luteal activity (Schirar el al., 1989). From approximately 60 days before the expected onset of the breeding season at 41 ° 40'N (5-21 August; Forcada el al., 1992), blood samples were collected evm T 6 days fi'om 144 anoestrous ewes. Oestrus was detected daily using aproned rams, and the blood salnpling continued in an individual ewe until first detected in oestrus. Only those samples collected fi'om 34 days (day-34) to 1 day before the first detected oestrus (day-1) (nvo complete oestrous cycles) were nsed. Animals were fed as a group to support their maintenance requirements. Plasma progesterone concentrations were determined by radioimmunoassay (Forcada et aL, 1992). For the purpose of statistical analysis, samples (n=744) were grouped every two consecutive days (-34+ -33; ...; -2+ -1 days beiore first oestrus). Mean (_+sE.x0 plasma progesterone concentl-ations were compared by analysis of variance, with the time of sampling as treatment effect and individual treated as a random effect. Data were recoded on the basis of progesterone concentration ( 0.5 ng ml <, 1). The hypothesis that the probability of plasma samples with progesterone concentrations lower than 0.5 ng ml -~ was equal to 1 was checked using a t)inomial test, with a test proportion of 0.999 (as the maximum value accepted by