VersaStep trocar hernia rate in unclosed fascial defects in bariatric patients.

OBJECTIVE: Use of the VersaStep trocar system (US Surgical, Norwalk, CT) has the perceived advantage of minimal trocar-related hernias in patients undergoing Roux-en-Y gastric bypass surgery (RYGB). We performed a retrospective review of our last 747 consecutive operative procedures using these trocars. METHODS AND PROCEDURES: The patient population was 747 consecutive patients who underwent laparoscopic RYGB at Duke University Health System Weight Loss Surgery Center from January 2002 through April 2005. A total of 3735 radially expanded trocar sites were used. VersaStep trocars were used in all cases. The port configuration included one supraumbilical Hasson port, two 12-mm ports, and three 5-mm ports. The Hasson port was closed with a figure-of-eight number 1 Polysorb suture. All other trocar sites had no fascial closure. Intestinal anastomoses were created with a linear stapler in all of the laparoscopic cases, with hand suturing of the residual enterotomy. The fascial incisions were therefore not extended to accommodate an EEA stapler. The charts were reviewed for occurrence of subsequent trocar site hernias. RESULTS: There were no hernias at any of the VersaStep trocar sites-an incidence of 0%. There were nine incisional hernias at the Hasson port site which later required surgical repair-an incidence of 1.20%. CONCLUSIONS: There were no hernias detected at any of the 1494 12-mm or 2241 5-mm VersaStep trocar sites, despite lack of suture closure. At the Hasson port site, there was a hernia incidence of 1.20%. In the bariatric RYGB population, routine suture closure of the fascia or muscle is not necessary when using radially expanding VersaStep trocars.

Nitrogenase IX. Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro.

Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays. Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase. The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A. and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity. In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase. The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities. Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed.

Nitrogenase XI: Mössbauer studies on the cofactor centers of the MoFe protein from Azotobacter vinelandii OP.

We have studied the MoFe protein from Azotobacter vinelandii OP with Mössbauer spectroscopy in applied magnetic fields up to 50 kG. The results are as follows. (1) The Mössbauer spectra of the S = 3/2 centers, which reside on the cofactor of nitrogenase, have been decomposed into six subcomponents. This suggests that each center contains 5-7, most probably 6, Fe atoms, thus confirming our earlier conclusions which were based on the quantitation of EPR data and on the assumption that the MoFe protein contains (30 +/- 2) Fe atoms. (2) Analysis of the high-field data shows that three subsites are characterized by a positive magnetic hyperfine coupling constant, A0, while A0 is negative for the other three sites. This observation demonstrates that the S = 3/2 centers are spin-coupled structures. (3) The zero-field splitting parameter D = +(6 +/- 1.5) cm-1 obtained from the Mössbauer data is in good agreement with our earlier EPR results, D approximately +5.5 cm-1. (4) The resolution of the Mössbauer spectra of the MoFe protein can be dramatically increased by employing Fourier transform deconvolution techniques. This allows a clear demonstration of spectral component S.

Nitrogenase XII. Mössbauer studies of the MoFe protein from Clostridium pasteurianum W5.

We have studied the molybdenum-protein (MoFe protein) from Clostridium pasteurianum with Mössbauer spectroscopy in the temperature range from 1.5 to 200 K in magnetic fields up to 55 kG. Except for some small differences in the hyperfine parameters the results for the C. pasteurianum protein are essentially the same as those published previously for the protein from Azotobacter vinelandii, i.e. (30 +/- 2) Fe atoms partition into two identical cofactor centers M (each center most likely containing six Fe atoms and one Mo atom), four P-clusters (each center containing four Fe atoms), and one iron environment labeled S (about two Fe atoms per holoenzyme). We have analyzed the spectra of the cofactor centers in three distinct oxidation states, Formula: (see test). The diamagnetic (electronic spin S = 0) state MOX is attained by oxidation of the native, EPR-active (S = 3/2) state MN. The reduced state MR is observed in steady state under nitrogen fixing conditions; high-field Mössbauer studies show that the cofactor centers are paramagnetic (integer electronic spin S greater than or equal to 1) in the state MR. We have evaluated the complex high-field spectra resulting from the P-clusters in the oxidized state POX. The analysis shows that one iron site is characterized by a positive hyperfine coupling constant A0 while the other three sites have A0 less than 0. A slightly modified set of parameters also fits the high-field data of the MoFe protein from A. vinelandii. Finally, we will present a discussion summarizing our principle results obtained to date for the proteins from A. vinelandii and C. pasteurianum.

Mössbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and Mössbauer spectroscopy. Low temperature Mössbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has Mössbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The Mössbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.

Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction.

EPR absorption-derivative lineshapes have been computed and least-squares fitted to the spectrum of the intermediate derived from 5'-deoxy-5'-adenosylcobalamin in the ribonucleotide reductase reaction. A Gaussian-type intrinsic lineshape was assumed and the effects of inhomogenous broadening, rotation of coordinate axes of the A-tensor relative to the g-tensor, angular dependence of transition probability and ligand hyperfine splitting have also been investigated. When the overall spectrum was computed as the sum of the lineshapes corresponding to two distinct Co(II) species, A and B, each having rhombic symmetry, the least squares procedure converged to a much better fit than with a single species, and matched almost all of the features of the experimental spectrum. The magnetic properties of A and B were compared with those of a series of other Co(II) complexes by a plot of g - g versus A - A. The results eliminate cobalt with 5-coordination to nitrogen for A and B, and suggest low-spin cobalt complexes having strongly distorted 6-fold coordination. The possibility that the sixth, symmetry-decreasing ligand is the oxygen molecule is excluded by the chemistry of the system and by the EPR properties of previously reported cob(II)alamins. It is suggested that the sixth ligand is a carbonyl, amide or sulfhydryl group of an enzyme sidechain which is inserted off-axis into the coordination position so as to exert the observed symmetry-lowering effect.

Nitrogenase. VIII. Mössbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using Mössbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of Mössbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and Mössbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic Mössbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The Mössbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has Mössbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.

Mössbauer studies of cytochrome c' from Rhodospirillum rubrum.

Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with Mössbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field Mössbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins.

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.

Nitrogenase X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with Mössbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the Mössbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the Mössbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.

Effect of presynchronization using prostaglandin F2alpha and a milk-ejection test on pregnancy rate after the timed artificial insemination protocol, Ovsynch.

The objective of this research was to determine if PGF2alpha-induced milk letdown (ML) is an accurate indicator of luteolysis, allowing cows to be synchronized to begin the Ovsynch protocol (GnRH-7d-PGF2alpha-2d-GnRH-24h-AI) at the most beneficial time of the estrous cycle (days 5-9), and determine if this would improve pregnancy rate (PR). Lactating Holstein cows between 55 and 70 days in milk were used to evaluate the ML test and PR after the Ovsynch protocol, when initiated on the basis of the test result (PROSYNCH). PROSYNCH cows (n = 60) had one teat cannulated to test for ML and were treated with 500 microg cloprostenol, PGF2alpha analogue (PG). Cows with ML were started on Ovsynch 10 days later, and those without started 3 days later. Cows in the control group (OVSYNCH, n = 64) were injected with physiological saline and observed for ML. This group was started on Ovsynch 10 days after saline treatment. Milk samples were collected thrice weekly to determine progesterone concentrations. ML indicated luteolysis with a sensitivity of 98% and a specificity of 60%. The positive and negative predictive values were 83 and 92%, respectively. Pregnancy rates were 48% for PROSYNCH and 52% for OVSYNCH (P = 0.72). When data from both groups were combined, PR was greater in cows that started the Ovsynch protocol in stage 2 of the estrous cycle (days 5-9, 67%) than all other stages (stage 1: days 1-4, 35%; stage 3: days 10-16, 45%; stage 4: days 17-21, 42%; P < 0.01). The proportion of animals with ovulation after GnRH#1, luteolysis after PGF2alpha, and ovulation after GnRH#2 were all greater in the PROSYNCH group (77% versus 55%, P < 0.02; 83% versus 66%, P < 0.03; 97% versus 84%, P < 0.03, respectively). Therefore, the ML test indicated luteolysis with sufficient precision to time the initiation of the Ovsynch protocol between days 5 and 9 of the cycle, however, this did not alter PR compared to starting the protocol randomly throughout the cycle. Initiating the Ovsynch protocol between days 5 and 9 of the cycle increased PR, and improved the efficacy of each injection.

The effect of a single administration of cephapirin or cloprostenol on the reproductive performance of dairy cows with subclinical endometritis.

This study examined the effect of a single administration of cephapirin iu or cloprostenol im on the reproductive performance of dairy cows with subclinical endometritis. Cows (n = 228) at 20-33 days in milk (DIM) from two commercial dairy farms, determined to be normal for clinical endometritis (based on absence of abnormal uterine discharge on vaginoscopic examination) were enrolled. At enrollment, a thorough reproductive examination was performed, including rectal palpation, ultrasonography (US) and endometrial cytology (EC). The case definition for subclinical endometritis was the presence of >18% neutrophils on EC examination or fluid in uterus (FIU) on US examination. All cows were randomly assigned to receive one of three treatments: 500 mg benzathine cephapirin iu, 500 microg cloprostenol im, or control (no treatment). Reproductive performance was monitored for a minimum of 8 months after treatment. Cows with subclinical endometritis treated with cephapirin or cloprostenol had a significantly increased relative pregnancy rate compared to control [hazard ratios 1.89 (P = 0.01) and 1.70 (P = 0.05), respectively]. In conclusion, a single treatment with cephapirin or cloprostenol at 20-33 DIM significantly improved the reproductive performance of cows with subclinical endometritis.

A new class of inhibitors of human leucocyte elastase.

Studies of the inhibition of elastases at a molecular level have resulted in the identification of protected dipeptides which are reversible and highly specific inhibitors of human leucocyte elastase (HLE). These have been further developed by increasing their hydrophilicity and potency to give a new family of elastase inhibitors, typically N alpha-(1-adamantanesulphonyl)-N epsilon-(4-carboxybenzoyl)-L-lysyl-L-alanyl-L-valinal. These compounds are active in pharmacological models designed to detect compounds of potential therapeutic value in the treatment of emphysema.

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene D4 receptor antagonists. 4. Addition of chromone moiety enhances leukotriene D4 receptor binding affinity.

The combination of the benzopyran-4-one ring, a moiety found in the prototype leukotriene antagonist, FPL 55,712, with the (2-quinolinylmethoxy)phenyl group led to a significant increase in leukotriene receptor binding affinity. This modification resulted in a 10,000-fold improvement in binding affinity compared to FPL 55,712. Compound 7 (RG 12553), with a Ki value of 0.1 nM, has higher affinity than the natural agonist LTD4 and is one of the most potent LTD4 antagonists reported. The structure-activity relationships of this series of potent leukotriene antagonists are discussed.

The development of a novel series of (quinolin-2-ylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 3. Structural variation of the acidic side chain to give antagonists of enhanced potency.

This paper is the third in a series outlining the development of orally active sulfido peptide leukotriene antagonists containing a (quinolin-2-ylmethoxy)phenyl moiety. In this work the systematic variation of the acid side chain substituents led to dramatic and reproducible changes in the oral activity of these compounds, presumably due to alterations in their pharmacokinetic properties. The most potent compound identified, 5-[4-[4-(quinolin-2-yl-methoxy)phenyl]-3-methylbutyl]tetrazole (32), represents a convergence of good in vitro antagonist activity and a 3-10-fold improvement in oral potency over the current clinical candidate 2. The new findings from these optimization studies are as follows: oxygen substitution in the acid side chain was not necessary for antagonist activity, in vitro and in vivo activity was enhanced by alkyl or phenyl substitution on the gamma-carbon of the acid side chain of para-substituted (quinolin-2-ylmethoxy)phenyl derivatives, and free rotation about the side chain carbon atom adjacent to the (quinolin-2-ylmethoxy)phenyl ring was required for activity. The lead compound of this report (32) is a competitive inhibitor of [3H]LTD4 binding to receptor membrane purified from guinea pig lung (Ki = 12 +/- 3 nM) and of the spasmogenic activity of LTC4, LTD4, and LTE4 in guinea pig lung strip. Dosed orally in guinea pigs, this compound blocks LTD4-induced bronchoconstriction (ED50 0.8 mg/kg) and antigen-induced systemic anaphylaxis (ED50 = 1.2 mg/kg).

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 1. Initial structure-activity relationships.

This series of reports describes the development of orally active, highly potent, specific antagonists of the peptidoleukotrienes containing a (2-quinolinylmethoxy)phenyl moiety. Described in this first report are the structure-activity relationships that led to a more than a 20-fold improvement of the potency and selectivity of the initial chemical lead (RG 5901). From this series of compounds, RG 7152 (16) was identified and selected for further evaluation in the clinic as an antiasthmatic agent. Compound 16 competitively inhibits [3H]LTD4 binding to membranes from guinea pig lung (Ki = 38 +/- 6 nM) and the spasmogenic activity of LTC4, LTD4, and LTE4 in parenchymal lung strips from guinea pigs. Unlike the original lead (RG 5901), compound 16 does not inhibit 5-lipoxygenase from guinea pig PMNs. Following oral administration to guinea pigs, 16 blocks LTD4-induced dermal permeability (ED50 = 6.9 mg/kg), LTD4-induced bronchoconstriction (ED50 = 1.1 mg/kg), antigen-induced bronchoconstriction (ED50 = 2.5 mg/kg), and anaphylactic-induced mortality (ED50 = 16 mg/kg). These studies on structure-activity relationships indicate that there is a requirement for an acidic function and the presence of the (2-quinolinylmethoxy)phenyl moiety in a specific geometric arrangement.

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene D4 receptor antagonists. 2. Effects of an additional phenyl ring on receptor affinity.

This series of reports describe the development of orally active, highly potent, specific antagonists of the peptidoleukotrienes containing a (2-quinolinylmethoxy)phenyl moiety. The compounds reported in this paper contain an additional phenyl ring, which has significantly improved the receptor affinity. The effect of changes in the linkage between the two phenyl rings as well as the orientation of the acidic functional group on biological activity are discussed. Many of these compounds have high affinity to the sulfidopeptide leukotriene D4 receptors with Ki values ranging between 2 and 20 nM and are orally active. Compound 27 [RG 12525, 5-[[2-[[4-(2-quinolinylmethoxy)phenoxy]- methyl]phenyl]methyl]-1H-tetrazole] represents the best combination of in vitro and in vivo biological activity in this series and has been selected for further evaluation in clinical studies of asthma.

Ro 32-3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo.

1. Ro 32-3555 (3(R)-(cyclopentylmethyl)-2(R)-[(3,4,4-trimethyl-2,5-dioxo-1- imidazolidinyl)methyl]-4-oxo-4-piperidinobutyrohydroxamic acid) is a potent, competitive inhibitor of human collagenases 1, 2 and 3 (Ki values of 3.0, 4.4 and 3.4 nM, respectively). The compound is a selective inhibitor of collagenases over the related human matrix metalloproteinases stromelysin 1, and gelatinases A and B (Ki values of 527, 154 and 59 nM, respectively). 2. Ro 32-3555 inhibited interleukin-1 alpha (IL-1 alpha)-induced cartilage collagen degradation in vitro in bovine nasal cartilage explants (IC50 = 60 nM). 3. Ro 32-3555 was well absorbed in rats when administered orally. Systemic exposure was dose related, with an oral bioavailability of 26% at a dose of 25 mg kg-1. 4. Ro 32-3555 prevented granuloma-induced degradation of bovine nasal cartilage cylinders implanted subcutaneously into rats (ED50 = 10 mg kg-1, twice daily, p.o.). 5. Ro 32-3555 dosed once daily for 14 days at 50 mg kg-1, p.o., inhibited degradation of articular cartilage in a rat monoarthritis model induced by an intra-articular injection of Propionibacterium acnes. 6. Ro 32-3555 is a potential therapy for the treatment of the chronic destruction of articulating cartilage in both rheumatoid and osteoarthritis.

Assessment of immunoregulation by cultured, pre-attachment bovine embryos.

The possibility that the viability of bovine embryos might be predicted by measuring their release of immunoregulatory substances during culture has been investigated. Bovine embryos between days 2 and 19 of gestation were cultured for 24-48 h, the embryo-conditioned medium was harvested and studied for suppression of PHA-stimulated bovine leukocyte cultures. Medium incubated in the absence of any conditioning tissues served as control. Artefactual immunosuppression was detected in incubated control material that could be attributed, in part, to the mixing of different tissue culture media, the type of plastic-ware employed for incubation and supplementation of media with additional L-glutamine. It was observed that day-2 to day-9 bovine embryos, cultured in medium able to support the lymphocyte proliferation assay, did not release immunosuppressive substances. Medium conditioned by day-10 to day-12 embryos produced variable immunosuppression while that conditioned by trophoblastic vesicles derived from day-14 to day-19 embryos was consistently highly suppressive. Since bovine embryo transfer is normally conducted at 6-8 days of gestation, it is unlikely that measuring the immunosuppressive products released from bovine embryos will be of value for predicting their viability.