Developmental and epigenetic anomalies in cloned cattle.

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Loss of methylation at H19 DMD is associated with biallelic expression and reduced development in cattle derived by somatic cell nuclear transfer.

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Functional differences between the outer and inner zones of the guinea pig adrenal cortex.

The guinea pig adrenal cortex is grossly composed of two regions: an outer, yellow zone and an inner, brown zone. These zones, which represent 33% and 66% of the total adrenocortical volume, respectively, can be separated by blunt dissection. It has been previously reported that specific pregnenolone and pregnenolone sulfate binding proteins are present in the high speed supernatant fraction (cytosol) prepared from the whole adrenal cortex of the guinea pig. However, when cytosol was prepared from the separate outer and inner cortical zones, it was found that the steroid-binding proteins were concentrated in the inner zone. This correlated with the level of pregnenolone which was significantly greater in the cytosol of the inner zone where greater than 50% was found to be bound. In contrast, the concentration of cortisol was 30 times greater in the cytosol of the outer cortical zone and less than 4% was found to be bound. These data suggest that cortisol is produced primarily in the outer cortical zone, a region which comprises only one-third of the total cortical volume. On the other hand, the coexistence of pregnenolone and its binding protein in the inner cortical zone, a region which comprises two-thirds or the greatest cortical volume, indicates a different functional status for this zone. The exact hormonal control of these two vastly different regions (chromatically, morphologically, and functionally) remains to be determined. It is speculated that the inner cortical zone of the adult guinea pig adrenal is the counterpart of the fetal cortex which did not involute.

Regulation of the pituitary-adrenal axis and corticosteroid-binding globulin-cortisol interaction in the guinea pig.

Adult male guinea pigs were used to examine pituitary-adrenal function and corticosteroid-binding globulin (CBG)-cortisol interaction. A biphasic pattern in the circadian rhythm of plasma cortisol was observed, with nadirs occurring at 0800 and 2400 h and peaks and 1600 and 0400 h. An excellent correlation was noted between total and free plasma cortisol levels. In contrast, no correlation was noted between CBG binding capacity and either the total or free plasma cortisol level. The free plasma cortisol concentration ranged from 0.6-5.8 micrograms/dl, representing 6.1-14.5% of the total cortisol concentration. The CBG binding capacity ranged from 12.2-161.7 micrograms/dl, and the binding affinity was 1.3-2.2 x 10(7) M-1 at 22 C, which is 20-fold lower than that of human CBG. These results suggest that CBG in the guinea pig has a relatively minor effect on the plasma distribution of cortisol, and that free plasma cortisol is dependent on cortisol binding to nonspecific plasma proteins as well as on the total plasma cortisol concentration. It was found that the guinea pig was relatively resistant to dexamethasone suppression, requiring at least 1 mg/kg BW to obtain essentially complete suppression of the pituitary-adrenal axis. In addition, it was found that administering dexamethasone 6 h before the time that the animals were killed led to suppression, whereas giving the steroid 12 h before killing the animals was totally ineffective. Stress induced by both ether vapor and histamine injection significantly increased plasma cortisol levels. When the guinea pigs were treated with a 1 mg/kg BW dose of dexamethasone 6 h before the stimulus, however, a marked suppression of the plasma cortisol increment secondary to application of the stressful stimulus occurred.

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells.

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.

Concentrations of cyclic AMP in rabbit ovarian tissue during the preovulatory period and pseudopregnancy after induction of ovulation by administration of human chorionic gonadotrophin.

Concentrations of cyclic AMP were measured in rabbit ovaries at various times after injection of an ovulatory dose of human chorionic gonadotrophin (HCG). A biphasic increase in cyclic AMP concentration occurred during the preovulatory period, with peaks 30 min and 3-4 h after HCG injection. Concentrations of cyclic AMP had returned to those observed in ovaries of control oestrous animals before the onset of ovulation 10-12 h after administration of HCG, and remained low throughout the period of pseudopregnancy. Concentrations of cyclic AMP in the newly formed and developing corpora lutea were similar to the concentrations observed in the remainder of the tissue during this period. No significant increase in cyclic AMP concentration was observed 7-9 days after initiation of ovulation. Concentrations of ATP were also investigated during the preovulatory period. The dose-response relationship of HCG to cyclic AMP production in oestrous rabbit ovaries was investigated.

Prolonged progesterone treatment of endometrial epithelial cells modifies the effect of estradiol on their sensitivity to oxytocin.

Estradiol (E2), progesterone (P4), and oxytocin (OT) are important for the initiation of luteolysis in ruminants but the mechanisms involved are still poorly understood. The objective of this study was to determine if duration of exposure of bovine endometrial epithelial cells to P4 affected the response of the cells to E2. Endometrial epithelial cells, from cows at Days 1-3 of the estrous cycle, were cultured for 10, 17, and 21 days in the presence or absence of P4 (100 ng ml(-1)). After culture, each group of cells was incubated for a further 6, 12, 24 or 48 h with or without E2 (100 pg ml(-1)) and then incubated for 6 h with different doses of OT (2, 20, and 200 ng ml(-1)). E2 enhanced OT-stimulated PGF2 alpha secretion in cells cultured with P4 for 17 or 21 days, with a maximum effect after 24-h exposure, but not in cells cultured with P4 for 10 days. To determine the mechanism of action of E2, COX-1 and COX-2 were measured by Western blotting and OTR number was measured by saturation analysis. OT increased COX-2 (P<0.05), but there was no significant effect of E2 on the expression of either COX-1 or COX-2. E2 did, however, increase (P<0.001) the OTR number in cells cultured with P4 for 21 days, whereas it inhibited OTR in cells cultured for 10 days. These data show that E2 can stimulate PGF2 alpha secretion by increasing OTR expression in bovine endometrial cells in vitro, but only after exposure to P4.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro.

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulus-oocyte complexes (COCs) were denuded before or after maturation in vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with 35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high--possibly due to nuclear repair mechanisms. Protein profiles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specific proteins are synthesized in the nuclear region.

Arginine vasopressin stimulation of prostaglandin F2 alpha release in heifers during the estrous cycle.

The effect of arginine vasopressin on the stimulation of prostaglandin F2 alpha (PGF2 alpha) release has been examined in vivo. Fifty-eight heifers received one intravenous injection of 10 IU arginine vasopressin on either Day 0 (n = 14), Day 6 (n = 12), Day 13 (n = 14) and Day 18 or 19 or 20 (Day 18-20, n = 18) after the onset of oestrus (Day 0) to determine the effect of arginine vasopressin at different times of the oestrous cycle. Frequent blood samples were taken before and after arginine vasopressin injection for the measurement of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) by radioimmunoassay (RIA). Blood samples for progesterone determinations were taken 2 hr before and 24 hr after arginine vasopressin to monitor luteal function. The data show that arginine vasopressin causes an increase (P less than 0.005) in PGFM concentrations only at Day 18-20 of the cycle in 67% of the experimental heifers.

Control of in vitro prostaglandin F2 alpha and E2 synthesis by caruncular and allantochorionic tissues from cows that calved normally and those with retained fetal membranes.

High concentrations of PGF2 alpha and PGE2 are produced by the uterus during the early postpartum period in cows and may play an important role in both placental separation and uterine involution. In the present study, we have examined the hormonal and intracellular control mechanisms involved in PGF2 alpha and PGE2 secretion by caruncular and allantochorionic tissue in vitro. Tissue explants, obtained about 6 hr postpartum from cows that delivered normally (NFM, n = 10) or cows with retained fetal membranes (RFM, n = 4), were incubated for 6 hr and PGF2 alpha and PGE2 concentrations in the medium were determined by radioimmunoassay. Addition of oxytocin (100 microU/ml), platelet activating factor (PAF, 100 ng/ml) and epidermal growth factor (EGF, 100 ng/ml) had no effect on secretion of PGF2 alpha from the caruncle, but oxytocin and PAF did stimulate PGE2. There was no difference between groups of cows. All three substances stimulated PGF2 alpha from the allantochorion of NFM, but not RFM, cows and stimulated PGE2 secretion from the allantochorion of both groups of cows. Incubation of the tissues with cholera toxin (100 ng/ml), dibutyryl cyclic adenosine 3',5'-monophosphate (dibutyryl cAMP, 1 mM), calcium ionophore A23187 (5 microM) or phorbol ester 12-myristate-13 acetate (PMA, 100 nM) showed that PGF2 alpha secretion is essentially via the calcium-protein kinase C effector pathway. However, calcium-protein kinase C and cAMP second messenger systems appear to be involved in the secretion of PGE2. Prostaglandin secretion was sensitive to cycloheximide in both caruncular and allantochorionic tissues, suggesting that protein synthesis may be involved. In conclusion, these data show that in vitro PGF2 alpha secretion can be modulated by the agonists used only in allantochorion and is essentially via the calcium-protein kinase C effector pathway. PGE2 secretion can be modified in both caruncular and allantochorion tissues and involves both inositol triphosphate-diacylglycerol and cAMP second messenger systems.

Steroid synthesis by equine conceptuses between days 7 and 14 and endometrial steroid metabolism.

The objective of this study was to determine if changes in steroid synthesis occurred in the horse blastocyst about the time of maternal recognition of pregnancy. Embryos collected between days 7.5 and 14.5 were incubated for 8 hr in vitro in HAM's F10 containing radiolabelled pregnenolone. The steroid metabolites in the incubation medium were separated by reverse phase HPLC and the major peaks expressed as a percentage of total metabolites. It was found that there were no major changes in the profile of metabolites throughout the period of study, although there was increased conversion as the conceptuses developed. It was found that the major metabolite produced was 17 alpha-hydroxyprogesterone and not estradiol as expected. A second experiment was conducted to determine if 17 alpha-hydroxyprogesterone was metabolized by endometrial tissue. Endometrial biopsies from anestrous mares and from pregnant and nonpregnant mares at day 11 were incubated with radiolabelled 17 alpha-hydroxyprogesterone, progesterone or pregnenolone. The 17 alpha-hydroxyprogesterone, but not progesterone nor pregnenolone, was converted to a more polar metabolite in all groups. Production of this metabolite was significant greater in the anestrous mares. This metabolite has not been unidentified conclusively. Thus, results of this study show that 17 alpha-hydroxyprogesterone is the major steroid synthesized by the equine blastocyst and that this steroid is further metabolized to an unidentified steroid by the endometrium. These steroids could play a role in conceptus development or maternal recognition of pregnancy.

Follicular development and reproductive endocrinology during a synchronized estrous cycle in heifers and mature cows displaying contrasting superovulatory responses.

Ovarian follicular development and plasma concentrations of progesterone (P4), estradiol-17 beta (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared during a synchronized estrous cycle between heifers and mature cows displaying contrasting superovulatory responses. Six heifers < 2 years old with a history of good responses to superovulatory (SOV) treatment and six cows 9 to 13 years old with poor responses to SOV treatments were used. Follicular development was monitored by daily ultrasonography. Blood samples were collected two to three times daily for P4 and E2 and thrice daily for LH and FSH analysis. Intensive sampling (samples every 15 min for 6 hr) was performed at critical periods of follicular development to analyze the pulsatile secretion of gonadotropins. In both cattle groups, a transient increase (P = 0.0001) in E2 occurred 4 to 5.7 d after the preovulatory LH surge or 2.3 d before the dominant follicle reached its maximum size. FSH concentrations increased (P = 0.006) before the emergence of the second cohort of follicles and then decreased despite no change in the concentration of E2. Contrary to our expectation and despite differences between groups in terms of age, number of previous SOV treatments, and divergent responses to superovulation, follicular development was similar in both groups. However, during the luteal phase, concentrations of E2 and FSH and LH pulse amplitudes were less (P < or = 0.05) in cows than in heifers. Therefore, follicular development monitored by ultrasonography and endocrine profiles during a synchronized estrous cycle are of limited value to predict quality of embryo donors.

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture.

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.

Ovulatory response and embryo yield in superovulated holstein heifers given a priming dose of FSH-P at day 2 of the estrous cycle.

Mature Holstein heifers were given either a priming dose of follicle-stimulating hormone-pituitary (FSH-P, 10 mg) or saline on Day 2 of the estrous cycle, or no pretreatment. All animals were subsequently given a decreasing dose superovulatory treatment of FSH-P beginning between Days 8 and 14, coupled with an injection of prostaglandin F2a to induce luteolysis. Pretreatment with FSH-P had no effect on the total superovulatory response or on the number of transferable embryos collected at Day 7 of gestation. Comparison of the results of our study with previous reports in the literature may suggest that FSH-priming early in the cycle may be advantageous in promoting superovulation only when the superovulatory response of the population of animals is otherwise weak.

Effect of bacterial cell wall and lipopolysaccharide on arachidonic acid metabolism by caruncular and allantochorionic tissues from cows that calved normally and those that retained fetal membranes.

Immunoactive eicosanoids may have a role in both placental separation and uterine involution in cattle. In the present study, we examined the effects of bacterial cell wall preparation and endotoxins on in vitro prostaglandin synthesis and arachidonic acid (AA) metabolism by caruncular and allantochorionic tissues. Placentomes were obtained about 6 h post partum from cows that delivered normally (n = 10) or those with retained fetal membranes (n = 4); the tissue explants were incubated for 6 h in the presence of labeled or nonlabeled AA. Prostaglandin F(2alpha) (PGF(2alpha)) and E(2) (PGE(2)) were measured by radioimmunoassay, and labeled AA metabolites were separated by reverse phase-high pressure-liquid chromatography. There was no effect of bacterial cell wall preparations or endotoxins on in vitro caruncular PGF(2alpha) secretion. However, bacterial products increased caruncular PGE(2) secretion in both cows that delivered normally and those with retained fetal membranes. For normal delivery cows caruncular tissue, bacterial product also increased leukotriene B(4) (LTB(4)) and decreased both thromboxane B(2) (TXB(2)) and hydroxy-eicosatetranoic acids (HETE) in vitro secretion. For the allantochorion, bacterial products increased in vitro PGF(2alpha) secretion only in cows that delivered normally and increased PGE(2) secretion essentially in cows with retained fetal membranes. In general, 6 keto PGF(1alpha) was the main metabolite secreted by both allantochorionic and carucular tissues. However, in cows with retained fetal membranes, PGE(2) became the most important metabolite secreted by allantochorion, especially in the presence of endotoxin. In conclusion, these results suggest that bacteria found in the early postpartum uterus or their endotoxin affect primarily caruncular and allantochorionic PGE(2) synthesis.

Interrelationship between plasma estradiol concentration and oxytocin-induced PGF2alpha release in heifers.

The objective of this study was to determine if the increase in responsiveness to oxytocin toward the time of luteolysis was correlated with an increase in plasma estradiol in the cow. Six heifers each had a cannula placed in the jugular vein on Day 14 of the estrous cycle. Then, beginning on Day 15, growth of the largest follicles was determined by ultrasonography, and a blood sample was taken via the cannula for the measurement of progesterone and estradiol by radioimmunoassay (RIA). After the first blood sample, 3 more samples were taken at 10-min intervals, 100 IU oxytocin were injected into the vein, and a further 3 blood samples were taken at 15, 30 and 60 min after injection. The concentration of 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in these frequent samplings and was used to determine the ability of oxytocin to stimulate PGF2alpha release from the uterus. This procedure was repeated daily for at least 7 d. The results showed that the response to oxytocin increased before luteolysis and that there was a significant increase in the response to oxytocin (P<0.05) before any changes in plasma estradiol or progesterone were detected. These data show that an increase in estradiol secretion from the ovulatory follicle does not appear to initiate luteolysis.

Progesterone and luteinizing hormone profiles in heifers used as oocyte donors.

Progesterone (P(4)) and luteinizing hormone (LH) profiles were analyzed throughout the estrous cycle in 11 superovulated heifers that had follicular oocytes aspirated at different times after standing heat. It was found that high P(4) during estrus was incompatible with normal LH release, oocyte maturation and subsequent in vitro fertilizing capability. However, an LH peak was not a prerequisite for initiation of meiosis, since both metaphase I (MI) and metaphase II (MII) stages were observed in animals without an LH surge. Following follicular aspiration, the progesterone levels and the length of luteal phase were similar to those of superovulated animals that had no follicular intervention. We concluded that aspiration per se does not interfere with normal corpora lutea (CL) development in heifers when aspiration occurs after the LH surge.

Assessment of embryo potential by visual and metabolic evaluation.

Morphological evaluation of embryos is essential to the success of embryo transfer procedures and is presumed to reflect embryo metabolic activity. To investigate this assumption, correlations between morphological and metabolic parameters were determined for cultured murine morulae. After 18 h (n = 47) or 36 h (n = 48) of culture in M16, the developmental rate and quality (poor or good) of embryos were estimated, and, then, either their (14)C-glucose utilization or (35)s-methionine uptake and incorporation were measured. Retarded developing, or poor-quality embryos had lower mean glucose utilization, uptake and incorporation rates than normally developing or good-quality embryos (P < 0.05). After 18 h of culture, an association was found between developmental rate and metabolic activity, but this was not evident after 36 h of culture. Similarly, an association was found between embryo quality and metabolic activity. As expected, poor embryo quality was indicative of low metabolism throughout the culture period, but good quality did not necessarily indicate normal metabolic activity. Thus, morphological parameters do not always reflect metabolic competence, and some functional defects were not detectable by visual evaluation alone. Measuring metabolic parameters could complement visual evaluation for a better selection of embryos prior to transfer.

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses.

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.

Leukotriene B4 in cows with normal calving, and in cows with retained fetal membranes and/or uterine subinvolution.

Two experiments were performed to study the relationship between leukotriene B4 (LTB4) synthesis and placental separation and uterine involution in the cow. In experiment I, the concentration and synthesis of LTB4 by caruncular tissue was lower in cows with retained fetal membranes (RFM cows, n = 11) than in cows that expelled the fetal membranes normally (NFM cows, n = 19). The presence of bacterial cell wall, especially of alpha-hemolytic streptococci and coagulase positive staphylococci enhanced LTB4 synthesis by allantochorion only in NFM cows. In the RFM group, Escherichia coli lipopolysaccharide decreased allantochorionic LTB4 synthesis. With caruncle, only epidermal growth factor increased LTB4 production in NFM cows. In experiment II, the caruncular and endometrial secretion of LTB4 was lower in cows with subuterine involution (SUI cows, n = 5) or cows with SUI and RFM (SUI+RFM cows, n = 4) than in cows with normal uterine involution (NUI cows, n = 8). This decrease was especially noticeable in the previously gravid horn. In the three uterine involution groups, there were no differences in LTB4 synthesis by caruncular tissue taken from the previously gravid horn. However, progesterone and a bacterial suspension of E. coli reduced the synthesis of LTB4. Estradiol had no effect on LTB4 synthesis at the end of the postpartum period. These results suggest that LTB4 may play an important role in both placental separation and uterine involution in cattle and LTB4 synthesis may be modulated by endocrine and bacterial factors.