Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human α1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P < 0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V⁻/PI⁻), early apoptotic (Ann-V⁺/PI⁻), and late apoptotic/early necrotic sperm (Ann-V⁺/PI⁺) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V⁻/PI⁺) was significantly different (P < 0.05) between transgenic and non-transgenic boars (3.4% ± 2.7; 7.2% ± 2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r = 0.9787; P < 0.0001).     

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 μM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 ± 32.3% of control, n = 5). The ECE-1 activity (expressed as μM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 μM, 6 h) significantly increased the ECE-1 activity (0.28 ± 0.02; n = 3) compared to the control (0.07 ± 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 μM for 1 h; 0.10 ± 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 ± 0.01; n = 3) compared to control (0.08 ± 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.     

Silver(I) methanesulfonate complexes containing diphosphine ligands: Spectroscopic and structural characterization

1:1 and 2:1 adducts of diphosphine ligands R₂P(R′)_nPR₂ (dppm: R = Ph, R′ = CH₂, n = 1; dppe: R = Ph, R′ = CH₂, n = 2; dppp: R = Ph, R′ = CH₂, n = 3; dppb: R = Ph, R′ = CH₂, n = 4; dppf: R = Ph, R′ = ferrocenyl, n = 1) with silver(I) methanesulfonate have been synthesized and characterized both in solution (¹H, ³¹P NMR) and in the solid state (IR, single crystal X-ray structure analysis). The two different stoichiometries have been found to depend on the molar ratio of ligand to metal employed and the nature of the diphosphine ligand. In AgO₃SMe:dppp,dppb (1:1)₂, in the [Ag(P^P)₂Ag] arrays, the silver atoms are also bridged by anion oxygen atoms, in disparate fashion commensurate with the different Ag?Ag distances.     

Selective recognition of oncogene promoter G-quadruplexes by Mg2+

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel β-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the β-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n = 7955), swine H1N1 (n = 360) and swine H3N2 (n = 235); and highly conserved in human H1N1 (n = 2722), swine-origin pandemic H1N1 (n = 1474), human H5N1 (n = 319) and avian H5N1 (n = 2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.     

Bioactivity guided isolation of anticancer constituents from leaves of Alnus sieboldiana (Betulaceae)

The leaves of the Japanese Alnus sieboldiana have been extracted with n-hexane and then with methanol. A bioactivity-guided approach based on MTT assay for growth inhibition and quantitative real-time PCR for TNF-α inhibitory activity was taken to identify the active compounds in EtOAc soluble fraction of the methanol extract. From this active fraction, seven compounds have been isolated and four compounds (pinosylvin, galangin, quercetin and methyl gallate) have been examined for their dose-response effect on the viability of A549 cells and on TNF-α inhibitory activity. Based on MTT assay, all of the four examined compounds inhibit growth of human lung cancer cells. Among four tested compounds only galangin (3,5,7-trihydroxyflavone) significantly inhibited TNF-α gene expression in A549 cells (IC₅₀ = 94 μM). Taken together, this finding suggests that galangin may be useful in cancer prevention.     

Investigations about dissolution of cellulose in the 1-allyl-3-alkylimidazolium chloride ionic liquids

In this work, the 1-allyl-3-alkylimidazolium chloride ionic liquids were synthesized and characterized by increasing carbon atoms (n ≤ 6) of alkyl chains on a cationic 3-imidazole ring. The results indicated that 1-allyl-3-alkylimidazolium chloride with asymmetrical structure on the two sides of a cationic 3-imidazole ring (i.e., n = 1, 2, 6) exhibited alkalinity and lower thermal stabilities, and showed better solubility to the cellulose samples at 60-120 °C than those with symmetrical structures (n = 3, 4). The cellulose samples treated by 20% (w/w) ethylenediamine solution showed better solubility in 1-allyl-3-ethyl, hexyl-imidazolium chloride ionic liquids than that treated with 20% (w/w) NaOH solution at 5 °C for 72 h. XRD and TG analysis indicated that 0 0 2 plane apparent crystallite size as well as thermal stability of the regenerated cellulose samples from the ionic liquids decreased significantly compared with the untreated cellulose samples.     

Dehydroepiandrosterone-sulphate replacement improves the human plasma fatty acid profile in plasma of obese women

DHEA-S treatment is used as an anti-aging and anti-obesity hormone therapy in adults; however, it mechanisms of action are not clearly elucidated.The objective of the present work was to analyze the effect of a replacement therapy, which included a daily single oral dose of DHEA-S for three months, on the composition of human plasma fatty acids (FAs) in obese women.In the first study, a randomized, double-blind, placebo-controlled trial was conducted involving 61 postmenopausal women, who were assigned to receive 100 mg/day of DHEA-S (n = 41) or placebo (n = 20) orally for 3 months. In a second study, the effect of DHEA-S treatment on postmenopausal obese women (n = 41) was compared to that in premenopausal obese women (n = 20). Blood samples were collected at the beginning and at the end of the treatment. Plasma FAs were analyzed by gas chromatography.DHEA-S treatment produced significant changes in plasma FAs of both post- and premenopausal women with a reduction of total saturated FAs (SFA) as well as an increase in n − 6 polyunsaturated FA (PUFA). Particularly, in premenopausal women the DHEA-S treatment also increased the plasma n − 3 PUFA percentage. Regarding estimation of desaturase activity, our data showed that Δ6-desaturase was significantly decreased in postmenopausal women after DHEA-S treatment, whereas Δ5-desaturase was increased in the premenopausal group.In conclusion, DHEA-S treatment in obese women modifies plasma FA composition towards a potentially better metabolic profile, mainly by decreasing SFA and increasing n − 6 PUFA in both postmenopausal and premenopausal women.     

A novel acetyl-CoA carboxylase inhibitor reduces de novo fatty acid synthesis in HepG2 cells and rat primary hepatocytes

To identify the novel inhibitor of de novo lipogenesis in hepatocytes, we screened for inhibitory activity of triglyceride (TG) synthesis using [¹⁴C]acetate in the human hepatoma cell line, HepG2. Using this assay system we discovered the novel compound, benzofuranyl α-pyrone (TEI-B00422). TEI-B00422 also inhibited the incorporation of acetate into the triglyceride (TG) fraction in rat primary hepatocytes. In HepG2 cells, the incorporation of oleate into TG was unaffected. TEI-B00422 inhibited rat hepatic acetyl-CoA carboxylase (ACC), K_i = 3.3 μM, in a competitive manner with respect to acety-CoA but not fatty acid synthase and acyl-CoA transferase/diacylglycerol. Thus, these results suggest that the inhibition of TG synthesis by TEI-B00422 is based on the inhibitory action of ACC. The structure of TEI-B00422 is totally different from the known inhibitors of ACC and may be useful in the development of therapeutic agents to combat a number of metabolic disorders.     

Structure and luminescence of copper(I) cyanide-amine and -sulfide networks

Copper(I) cyanide reacts with various liquid amines and sulfides (L) under solvent-less conditions to form (CuCN)L_n, n = 0.5, 0.57, 0.75, 0.8, 1, 1.25, 1.5, 2. New X-ray structures are reported for L = Py (Py = pyridine, n = 0.57), 2-MePy (n = 1), 3-EtPy (n = 1.5), 2-ClPy (n = 1), 3-ClPy (n = 2), 3-MeOPy (n = 2), 4-^tBuPy (n = 1.5), piperidine (n = 1.25), N-methylmorpholine (n = 1), N,N-dimethylcyclohexylamine (n = 1), 1-methylimidazole (n = 3), Me₂S (n = 1), and tetrahydrothiophene (n = 1). The amine structures (except for the monomeric 1-methylimidazole complex) reveal 1D CuCN chains decorated with 0-2 L per metal atom. Chain structures observed include zigzag, helical and figure-8 helical. The CuCN-sulfide structures show sulfur-bridging of CuCN chains. In some cases (CuCN)L_?1.5 species are transformed to (CuCN)L under vacuum. Thermal analysis shows facile release of ligand, yielding CuCN. Most of the (CuCN)L_n products are photoluminescent, emitting in the visible region. In some cases, coordination of very similar amines results in remarkably different emission spectra.     

Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

A bioassay for the simultaneous measurement of metabolic activity, membrane integrity, and lysosomal activity in cell cultures

The aim of this study was the development of an in vitro bioassay that combines several endpoints of general cytotoxicity for the screening of compounds or mixtures of compounds with potential bioactivity. The Alamar Blue assay was employed to assess metabolic activity, the Neutral Red assay was used for the assessment of membrane function and lysosomal activity, and the lactate dehydrogenase leakage assay was employed for the assessment of membrane integrity. Each assay was performed separately and in combination using a human fibroblast cell line (MRC-5). Three fungal secondary metabolites of different chemistry that affect different cellular targets were tested as model compounds: deoxynivalenol, enniatin B1, and 2-amino-14,16-dimethyloctadecan-3-ol. The obtained inhibitive compound concentrations for the assays performed separately and in combination were not significantly different (P < 0.05, n = 9). The combination of several cytotoxicity endpoints in a single assay increases the chance that potential bioactive/cytotoxic compounds are discovered during the screening of mixtures of natural compounds (e.g., extracts from fungal cultures or plants) when one endpoint fails and, at the same time, might give some basic information on the cellular target.     

Regulation of carboxylesterase 1 (CES1) in human adipose tissue

Carboxylesterase 1 (CES1) has recently been suggested to play a role in lipolysis. Our aim was to study the regulation of CES1 expression in human adipose tissue. In the SOS Sib Pair Study, CES1 expression was higher in obese compared with lean sisters (n = 78 pairs, P = 8.7 × 10⁻¹⁸) and brothers (n = 12 pairs, P = 0.048). CES1 expression was higher in subcutaneous compared with omental adipose tissue in lean (P = 0.027) and obese subjects (P = 0.00036), and reduced during diet-induced weight loss (n = 24, weeks 8, 16, and 18 compared to baseline, P < 0.0001 for all time points). CES1 expression was higher in isolated adipocytes compared with intact adipose tissue (P = 0.0018) and higher in large compared with small adipocytes (P = 4.1 × 10⁻⁶). Basal and stimulated lipolysis was not different in individuals with high, intermediate, and low expression of CES1. Thus, CES1 expression was linked to body fat and adipocyte fat content but not to lipolytic activity.     

The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of ¹⁴C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Cerebrospinal fluid light and heavy neurofilaments in neuromyelitis optica

Background

Axonal injury is the correlate of disease progression in NMO and MS. Neurofilament (Nf) belongs to neuron specific intermediate filaments located in axons. Nf protein subunits are potential biomarkers in cerebrospinal fluid (CSF) for acute axonal injury. However, whether CSF NfH and NfL levels are elevated in NMO patients has remained unclear.

Methods

Nf light subunit (NfL) and Nf heavy subunit NfH in cerebrospinal fluid were measured by enzyme-linked immunosorbent assay (ELISA) in NMO (n = 32), MS (n = 25), and other non-inflammatory neurological disease patients (OND, n = 18).

Results

CSF pNf-H levels were increased in the NMO patients compared with OND patients (p = 0.001). CSF NfL levels in the NMO patients were also higher compared with MS patients (p = 0.001), and OND patients (p = 0.000001). When comparing NfL levels between MS and OND patients, there also significant differences (p = 0.0003). NMO and MS patients revealed a trend to an increased disability with increased CSF NfL during relapse (NMO: p = 0.006; MS: p = 0.017). There is positive relationship between CSF pNf-H and disability of MS patients (p = 0.041).

Conclusions

CSF levels of NfL are increased in NMO patients and reflect the disease severity in NMO.     

Mononuclear iron(III) complexes supported by tripodal N3O ligands: Synthesis, structure and reactivity towards DNA cleavage

A new synthetic route to the known tripodal tetradentate N₃O ligand L¹ (HL¹ = [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine) is reported. The related compounds HLⁿ (n = 2, 3) were prepared by a similar procedure. Treatment of HLⁿ (n = 1-3) with FeCl₃·6H₂O in hot methanol led to the mononuclear iron(III) complexes [Fe(Lⁿ)Cl₂] (1: n = 1, 2: n = 2, 3: n = 3). The solid-state structures of complexes 1 and 2 were determined by X-ray crystallography. [Fe(L¹)Cl₂] (1) showed effective nuclease activity in the presence of hydrogen peroxide, converting supercoiled plasmid DNA to its linear form.     

Pregnancy rates in heifers and cows with cryopreserved sexed sperm: Effects of sperm numbers per inseminate, sorting pressure and sperm storage before sorting

Field trials were conducted to increase fertility with AI of flow-sorted, sexed bovine sperm. In the first trial, a novel competitive fertilization approach was used to compare pressures (30 psi vs 50 psi) for sorting sperm. Both X- and Y-sperm were sorted to approximately 95% purity at 30 and at 50 psi; X-50 + Y-30 (and the converse) were mixed in equal numbers for AI of heifers. Fetal sex divulged which treatment produced the pregnancy; 82% of pregnancies resulted from the 30 psi treatment (P < 0.05). Based on a similar approach, a new-pulsed laser did not damage sperm any more than the previous standard continuous wave laser. In a large field trial, sorting sperm at 40 psi increased pregnancy rates in heifers relative to 50 psi (42.3% vs 34.1%, n = 367/group, P < 0.05). Storing sperm for 20 h before sorting at 40 psi decreased pregnancy rates from 42.3% (n = 367) to 36.8% (n = 368; P < 0.05). Breeding heifers with sexed sperm 55-56 h after CIDR removal and PGF_2α resulted in 34% (n = 32) pregnant, compared to 49% (n = 35) with fixed-time insemination 67-68 h after CIDR removal (P > 0.1). Lactating dairy cows pre-screened for normal reproductive tracts when OvSynch injections (GnRH, prostaglandin, GnRH) were initiated, had similar (P > 0.1) pregnancy rates to timed AI, with 10 × 10⁶ sexed sperm (43.9%, n = 57), 2 × 10⁶ sexed sperm (40.5%, n = 57) and 10 × 10⁶ unsexed control sperm (55.6%, n = 58). A final field trial with unselected, lactating dairy cows resulted in similar pregnancy rates for 2 × 10⁶ sexed sperm in 0.25 mL straws (25.0%, n = 708) and 0.5 mL straws (24.4%, n = 776), but lower (P < 0.05) than unsexed control sperm (37.7%, n = 713). Younger cows and those >84 days in milk had the highest pregnancy rates for both sexed and unsexed sperm. These studies improved sperm sexing procedures, and provided insight into appropriate commercial use of sexed sperm.