Hormonal and physical changes associated with bovine conceptus development.

Conceptus (placental membranes, fetal fluids and fetus) development was characterized between Days 27 and 111 of gestation. Progestagens, oestrone, oestradiol, oestrone sulphate and prostaglandins (PG) F were measured in maternal plasma and allantoic and amniotic fluids. Protein concentrations are described for fetal fluids. The early increase in placental membrane weight from 1.12 g (27 days) to 58.45 g (50 days) was associated with oestrogen production presumably of conceptus origin. Oestrogens increased significantly in allantoic and amniotic fluids throughout the period studied with oestrone being the primary free oestrogen, rising from 2 pg/ml (Day 33) to 144 ng/ml by 111 days in allantoic fluid. Changes in plasma oestrogens of the maternal circulation were not detected until after Day 70 at which time oestrone concentration was greater than that of oestradiol. Fetal fluid concentrations of progestagens, oestrone sulphate and PGF were not related to maternal plasma levels and a sequestration of these hormones by the allantois is postulated.     

Effect of intrauterine application of oestradiol-17 beta and prostaglandin E-2 on the porcine oestrous cycle and uterine endocrinology.

Silastic beads were inserted into the uterine lumen on Day 10 after oestrus. Gilts received beads containing oestradiol-17 beta only, oestradiol benzoate, or oestradiol-17 beta+prostaglandin (PG) E-2. Oestrous cycles were slightly longer in treated than in untreated pigs (20.2 +/- 0.4 days), and durations were 22.6 +/- 1.3, 26.2 +/- 1.7 and 23.2 +/- 1.8 days for oestradiol-17 beta, oestradiol benzoate and oestradiol-17 beta+PGE-2 treatments, respectively (P greater than 0.05). Thus, PGE-2 and an oestrogen such as oestradiol benzoate that persist for a longer period cannot prolong the cycle more than oestradiol-17 beta alone. Additional cyclic gilts underwent similar treatments with beads containing oestradiol-17 beta, oestradiol-17 beta+PGE-2 or cholesterol, and cannulation of one utero-ovarian vein on Day 10. Blood samples were collected from the catheter every 15 min from 08:00 until 11:00 h and from 20:00 until 23:00 h for 5 consecutive days starting the day after surgery and peripheral plasma samples were also collected daily. On Day 16, beads containing oestradiol-17 beta were surrounded by endometrial folds whereas cholesterol beads were free. Concentrations of plasma progesterone did not vary significantly from Days 11 to 16 in gilts treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2, but decreased in cholesterol-treated gilts. Concentrations of plasma oestrone and oestradiol-17 beta were more than ten times higher in gilts treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2 than in cholesterol-treated gilts on the day after bead insertion, but decreased rapidly to values comparable to those in cholesterol-treated gilts by Day 14. In contrast, concentrations of oestrone sulphate remained high until Day 16. Concentrations of PGE-2 in the utero-ovarian vein plasma did not differ (P greater than 0.05) between treatments but those of PGF-2 alpha were higher (P less than 0.004) in gilts treated with cholesterol than in those treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2. It is postulated that insufficient oestradiol-17 beta is released by the beads toward the end of a 'recognition period' to prolong the cycle for more than 3-6 days.     

Changes in the plasma concentrations of free and conjugated oestrogens in heifers after treatment to induce superovulation and the relationship with number of ovulations

Oestradiol-17 beta and conjugated oestrone, oestradiol-17 beta and oestradiol-17 alpha were measured in peripheral plasma of heifers treated with PMSG/PGF-2 alpha to induce superovulation. Changes in the concentrations of each hormone were synchronous, the highest level being near oestrus. For a given number of ovulations the hormone with the highest concentration was total oestradiol-17 alpha, then came total oestrone, total oestradiol-17 beta and oestradiol-17 beta. For each oestrogen, the maximum preovulatory concentration measured was significantly correlated with the number of ovulations; the regression line for total oestradiol-17 alpha was twice as steep as that for oestradiol-17 beta. It is concluded that in animals treated to induce superovulation assay of total oestradiol-17 alpha gives a better induction of the number of follicles induced to ovulate than does the more conventional assay of oestradiol-17 beta.     

Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.     

Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-17 beta on the epithelial cells

Epithelial and stromal cells of guinea-pig endometrium were separated by enzymic digestion, isolated by successive centrifugation, and maintained in culture as pure cell types for 5 days on growth medium. On Day 5, ultrastructural studies were performed on the two cell types, demonstrating that epithelial cells can grow as a monolayer composed of cohesive groups of polygonal cells (1.3 X 10(5) cells/cm2), while stromal cells were mostly fibroblastic. The effect of hormones was studied on the epithelial cells in culture. The monolayer was cultured into harvest medium for 3 days to ensure the complete removal of endogenous steroids, then these cells were incubated with 2 X 10(-9) M-oestradiol-17 beta for 3 days. There was a rise in the progesterone receptor level, varying from 1.3 to 10.8 times. The three enzymes known to interfere with oestradiol-17 beta metabolism were present in the epithelial cells grown in our culture conditions. By incubation with oestrone sulphate for 3 days it was demonstrated that, in cultured epithelial cells, oestrone sulphate is converted into oestradiol-17 beta sulphate, and oestrogen sulphates are hydrolysed to active oestrogens.     

On the significance of in situ production of oestrogens in human breast cancer tissue

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Levels of circulating and urinary oestrogens during pregnancy in the marmoset monkey (Callithrix jacchus)

The levels of immunoreactive oestrone, oestradiol-17 beta and oestriol in plasma and urine were measured during early, mid- and late pregnancy in the marmoset monkey. In plasma, unconjugated oestrone remained less than 2% of total (conjugated plus unconjugated) oestrone throughout gestation, whereas unconjugated oestradiol-17 beta increased from 3% of the total value in early and mid-pregnancy to 35% in late pregnancy. The reversal in the unconjugated oestrone: oestradiol-17 beta concentration ratio from early (12:1) to late (0 . 15:1) pregnancy occurred despite the continuing predominance of oestrone in terms of total hormone. Total oestriol was measurable but in relatively low concentrations. Oestradiol conjugate was the predominant urinary oestrogen metabolite measured at each stage of pregnancy. The pattern of urinary oestrone and oestradiol-17 beta reflected plasma levels of total hormone, rather than unconjugated hormone, showing no further increase after mid-pregnancy. In contrast, oestriol increased throughout pregnancy and to a proportionately greater extent than oestrone or oestradiol-17 beta, but at lower absolute levels. High-pressure liquid chromatography of urine extract indicated the presence of considerable amounts of oestrogen immunoreactivity not accounted for by oestrone, oestradiol-17 beta and oestriol and with a retention time similar to that of 16 alpha-hydroxyoestrone. Gas chromatography and mass spectroscopy provided further evidence to suggest that 16 alpha-hydroxyoestrone is an abundant urinary oestrogen metabolite during pregnancy in the marmoset monkey.     

Developmental changes of amino acids in ovine fetal fluids

We recently reported an unusual abundance of arginine (4-6 mM) in porcine allantoic fluid during early gestation. However, it is not known whether such high concentrations of arginine are unique for porcine allantoic fluid or whether they represent an important physiological phenomenon for mammals. The present study was conducted to test the hypothesis that arginine is also the most abundant amino acid in ovine allantoic fluid. Allantoic and amniotic fluids, as well as fetal and maternal plasma samples, were obtained from ewes between Days 30 and 140 of gestation. Glycine was the most abundant amino acid in maternal uterine arterial plasma, representing approximately 25% of total alpha-amino acids. Alanine, glutamine, glycine, plus serine contributed approximately 50% of total alpha-amino acids in fetal plasma. Fetal:maternal plasma ratios for amino acids varied greatly, being less than 1 for glutamate during late gestation, 1.5-3 for most amino acids throughout gestation, and greater than 10 for serine during late gestation. Marked changes were observed in amino acid concentrations in amniotic and allantoic fluids associated with conceptus development. Concentrations of alanine, citrulline, and glutamine in allantoic fluid increased by 20-, 34-, and 18-fold, respectively, between Days 30 and 60 of gestation and were 24.7, 9.7, and 23.5 mM, respectively, on Day 60 of gestation (compared with 0.8 mM arginine). Remarkably, alanine, citrulline, plus glutamine accounted for approximately 80% of total alpha-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed approximately 60% of total alpha-amino acids in allantoic fluid on Day 140 of gestation. These novel findings of the unusual abundance of traditionally classified nonessential amino acids in allantoic fluid raise important questions regarding their roles in ovine conceptus development.     

Circulating oestrogen concentrations during pregnancy in the African elephant (Loxodonta africana)

Oestrone, oestradiol-17 beta and oestriol were measured in plasma samples from non-pregnant and pregnant African elephants shot in the wild. Enzymic hydrolysis of plasma showed that approximately 90 and 96% of the total (i.e. conjugated plus unconjugated) concentrations of oestrone and oestradiol-17 beta, respectively were represented by conjugated hormones. Unconjugated oestrogens remained low (less than 50 pg ml) in all samples, with no distinction between non-pregnant and pregnant animals. Levels of total oestrone during pregnancy varied between 160 and 594 pg/ml but were not significantly different from non-pregnant values. Total oestradiol-17 beta concentrations were significantly elevated during pregnancy (P less than 0 X 01) and, despite considerable individual variation (193-1428 pg/ml), were consistently higher than non-pregnant values after 6 months of gestation. The elevated levels of oestradiol-17 beta resulted in a reversal of the total oestradiol-17 beta: oestrone concentration ratio at about 6 months of pregnancy. Concentrations of total oestriol did not exceed 103 pg/ml. An indirect method of measurement indicated that oestradiol-17 beta sulphate was probably the most abundant circulating oestrogen during pregnancy in the African elephant.     

Changes in oestrogen-synthesizing ability of preovulatory bovine follicles relative to the peak of LH

Preovulatory bovine follicles (n = 28) were collected at different times after the onset of standing oestrus until shortly before ovulation. In-vitro conversion of tritiated androstenedione in the presence of NADPH by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) conversion into oestradiol-17 beta was high and production of oestrone was about 8-fold lower. During phases 1 (0-6 h after the LH peak) and 2A (6-14 h after the LH peak) the production of oestradiol and oestrone remained constant; the percentage of remaining androstenedione increased. In phase 2B (14-20 h after the LH peak) conversion into oestradiol and oestrone had decreased to about one third correlating with a higher percentage of remaining androstenedione. In phase 3 (20 h after the LH peak until ovulation) conversion into oestradiol and oestrone remained constant. The ratio between the production of oestrone and oestradiol remained constant throughout the phases of preovulatory development (0.13), indicating a concurrent inhibition of aromatase and 17 beta-hydroxysteroid dehydrogenase activities. Conversion into 19-hydroxyandrostenedione showed a pattern similar to that of oestradiol, and testosterone was produced in minute quantities. The results indicate that in preovulatory bovine follicles eventual inhibition of aromatization takes place at about 14 h after the preovulatory LH peak.     

Steroid hormones in uterine washings and in plasma of gilts between days 9 and 15 after oestrus and between days 9 and 15 after coitus

Between Days 9 and 15 after oestrus, concentrations of pregnenolone, pregnenolone sulphate, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, oestrone and oestrone sulphate in free uterine fluid collected from non-pregnant gilts were greater than respective values in plasma (P less than 0.05). The total contents of pregnenolone, progesterone, DHEA, testosterone, oestrone and oestradiol in washings from pregnant uteri exceeded (P less than 0.05) respective non-pregnancy levels during this same period. Concentrations of pregnenolone, pregnenolone sulphate, DHEA, DHEA sulphate, androstenedione, oestrone, oestrone sulphate and oestradiol in free uterine fluid recovered from gravid uteri were also higher (P less than 0.05) than respective plasma values. By contrast, the progesterone concentration in uterine fluid from pregnant animals was lower (P less than 0.001) than the plasma value. Concentrations of DHEA, DHEA sulphate, androstenedione and oestrone sulphate in plasma of pregnant gilts between Days 9 and 15 after mating exceeded (P less than 0.05) the respective concentrations in unmated gilts between Days 9 and 15 after oestrus. Plasma levels of pregnenolone sulphate were lower (P less than 0.05) in the pregnant animals. We therefore suggest that the endometrium of the pig can concentrate steroid hormones in uterine fluid and that increases in steroid levels in this milieu between Days 9 and 15 after coitus reflect steroidogenesis by embryonic tissues and modification of enzyme activities within uterine tissues under the influence of progestagens. The pool of steroid sulphoconjugates present in uterine fluid between Days 9 and 15 post coitum could serve as an important precursor source for progestagen, androgen and oestrogen synthesis by tissues of pig embryos before implantation.     

Plasma concentrations of 15-keto-13,14-dihydro-PGF-2 alpha, oestrone sulphate, oestradiol-17 beta and progesterone in pregnant guinea-pigs treated with polychlorinated biphenyls

Guinea-pigs treated by gavage with a total dose of 100 mg polychlorinated biphenyls (PCB: Clophen A50) during Days 17-61 of gestation had higher plasma concentrations of 15-keto-13,14-dihydroprostaglandin F-2 alpha, oestrone sulphate and oestradiol-17 beta during the later stages of gestation than did vehicle-treated guinea-pigs. No changes were observed in plasma progesterone concentrations. Our results provide no support for the hypothesis that an enzyme-induced decrease in progesterone concentrations is the main cause of the fetal death observed in PCB-treated guinea-pigs.     

Direct effects of oestradiol-17 beta and prostaglandin E-2 in protecting pig corpora lutea from a luteolytic dose of prostaglandin F-2 alpha

Implants containing vehicle or oestradiol-17 beta (10 mg) were placed into pairs of corpora lutea (CL) with and without prostaglandin F-2 alpha (PGF-2 alpha) (100 micrograms) on Day 11 and CL were collected on Day 19, in cyclic gilts (Exp. 1). The results demonstrated that CL implanted with PGF-2 alpha with or without oestradiol-17 beta had a markedly lower (P less than 0.01) weight (mg) and progesterone concentration (ng/mg) than CL with vehicle-or oestradiol-17 beta-implanted or unimplanted CL, which were similar (149 and 7.2 vs. 304 and 49.6, respectively). In Exp. 2, CL implanted with vehicle, oestradiol-17 beta or PGE-2 remained fully functional until Day 19, whereas CL implanted with oestradiol-17 beta +/- PGF-2 alpha and PGE-2 + PGF-2 alpha exhibited lower (P less than 0.05) weight and progesterone concentrations; CL implanted with PGE-2 + PGF-2 alpha were heavier (P less than 0.05) and tended (P less than 0.10) to have greater progesterone concentrations than CL implanted with oestradiol-17 beta + PGF-2 alpha. In Exp. 3, a dose-dependent (P less than 0.05) effect of PGE-2 on preventing regression induced by PGF-2 alpha was observed on Day 19. These data demonstrate a direct effect of PGE-2, but not of oestradiol-17 beta in protecting the CL against luteolysis induced by PGF-2 alpha.     

Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer

Aromatase distribution in membranes of preimplantation horse and donkey conceptuses was compared by measuring the incorporation of [3H]androstenedione into oestrone and oestradiol-17 beta. In the donkey conceptus, aromatase activity was similar in all the tissues examined (yolk sac, chorionic girdle and allantochorion), whereas in the horse it was generally lower and showed the relationship chorionic girdle greater than yolk sac greater than allantochorion. A higher proportion of labelled precursor was incorporated into oestradiol-17 beta by extra-embryonic tissues of the donkey compared with those of the horse. In contrast to previous results, aromatase in the chorionic girdle did not decline progressively before its migration into the endometrium on Day 36 to form the endometrial cups. The chorionic girdle of a donkey conceptus carried in the uterus of a mare failed to invade the surrogate horse endometrium and aromatase activity was still high in this tissue at Day 42. Aromatase distribution in 2 transferred donkey-in-horse conceptuses resembled that of the fetal, rather than the maternal, genotype indicating a lack of effect of the maternal environment.     

Influence of the fetal pituitary-adrenal axis on fetal and maternal progesterone and unconjugated oestrogen concentrations in the pig

Fetal and maternal plasma progesterone and unconjugated oestrone and oestradiol-17 beta concentrations were measured in intact pig fetuses and those in which the pituitary had been destroyed. Progesterone concentrations were significantly higher (P less than 0.05) and oestrogen concentrations significantly lower (P less than 0.01) in hypophysectomized fetuses than in intact fetuses. When fetuses in one uterine horn only were hypophysectomized, oestrogen concentrations in the uterine vein draining this horn were lower than those from the contralateral vein. The results indicate that both fetal and maternal oestrogen concentrations are influenced by the fetal pituitary. When dexamethasone was infused (at 27 micrograms/h for 96 h) into 5 chronically-catheterized hypophysectomized fetuses no changes in peripheral fetal progesterone or oestrone were observed.     

Demonstration of oestrogens in developing pig trophectoderm and yolk sac endoderm between days 10 and 16

Segments of individual blastocysts collected on Days 10, 12, 14 and 16 were examined microscopically to observe yolk-sac development and treated immunocytochemically to localize oestrogens in specific membranes. Mesoderm was present beneath the embryonic disc of ovoid blastocysts on Day 12. The mesoderm spread beyond 1 cm from the disc on Day 14, producing a splanchnic yolk-sac membrane extending across the blastocoelomic cavity, but no mesodermal cells had yet reached 5 cm. By Day 16, proliferation of mesoderm and development of the yolk sac had progressed beyond 20 cm from the disc in most of the specimens examined. Incubation of ultrathin sections with sheep antiserum to oestrone or oestradiol-17 beta followed by rabbit anti-ovine IgG-gold complex and subsequent counting of gold particles retained over the tissues gave a weakly positive reaction for oestrone in trophectodermal cells on Day 10. The most intense reaction for oestradiol-17 beta was also present in the trophectoderm and yolk-sac endoderm on Days 12, 14 and 16.     

Differences in amniotic fluid and maternal serum cytokine levels in early midtrimester women without evidence of infection

The amniotic fluid cytokine profile has been shown to be indicative of various disease states, and changes may be associated with preterm labor or infection. Anti-inflammatory cytokine profiles may be essential for successful normal pregnancy. However, there are currently few normative data on the concentration of cytokines in amniotic fluids during pregnancy. The aim of this study was to provide new amniotic fluid cytokine data for future comparative studies in disease states, notably in utero viral infections, and to compare these with maternal serum levels. Amniotic fluid was obtained from 100 pregnant women undergoing elective amniocentesis at the Royal Hospital for Women, Randwick. Concentrations of 27 cytokines were simultaneously measured in amniotic fluid and a subset of matching maternal sera (n=33) using a multiplex bead-based immunoassay system (Bio-Plex, Bio-Rad). To exclude infection, nested multiplex PCR targeting 17 known congenital infectious agents were performed on all amniotic fluid and maternal serum samples, and serological testing was also performed against some of these agents. Maternal serum concentration was positively correlated with amniotic fluid levels for MIP-1beta (r=0.39, P=0.027). IL-1ra was positively correlated to maternal age (r=0.210, P=0.036), and mean IL-5 levels were significantly higher in amniotic fluids from pregnancies with male fetuses than those with female fetuses (P=0.036). Normal amniotic fluid concentrations for five cytokines (IL-6, IL-8, IP-10, MCP-1, IL-1ra) were found to be significantly elevated over maternal serum concentrations in matched pairs (P<0.05). Concentrations of 12 cytokines (eotaxin, IFN-gamma, IL-9, IL-12, IL-15, IL-17, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, VEGF, PDGF bb) were significantly elevated in maternal serum compared to paired amniotic fluid at midtrimester (P<0.05). Amniotic fluid may be more representative of the fetal cytokine profile than cytokine analysis on antenatal sera as it represents predominantly fetal urinary and respiratory secretions. This study provides new normative data for multiple cytokine levels in amniotic fluid and maternal sera at 14-16 weeks gestation, and is a valuable tool for future diagnostic and comparative studies.     

Proteomic analysis of bovine conceptus fluids during early pregnancy

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.