Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Sphingosine-1-phosphate regulation of luteinising hormone-induced steroidogenesis and proliferation of bovine theca cells in vitro.

CONTEXT: Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin. AIMS: To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells. METHODS: We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10µM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1 ; Experiment 2) and LH (0.02ngmL-1 ) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10µM; Experiment 3). KEY RESULTS: Treatment with S1P did not affect (P >0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1 ) increased (P <0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P <0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05). CONCLUSIONS: S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number. IMPLICATIONS: These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

The vascular endothelial growth factor (VEGF) system as a key regulator of ovarian follicle angiogenesis and growth.

The vascular endothelial growth factor-A (VEGFA) system is a complex set of proteins, with multiple isoforms and receptors, including both angiogenic (VEGFxxx, VEGFR2) and antiangiogenic members (VEGFxxxb, VEGFR1 and soluble forms of VEGFR). The members of the VEGF system affect the proliferation, survival, and migration of endothelial and nonendothelial cells and are involved in the regulation of follicular angiogenesis and development. The production of VEGF by secondary follicles stimulates preantral follicular development by directly affecting follicular cells and promoting the acquisition of the follicular vasculature and downstream antrum formation. Additionally, the pattern of expression of the components of the VEGF system may provide a proangiogenic milieu capable of triggering angiogenesis and stimulating follicular cells to promote antral follicle growth, whereas, during atresia, this milieu becomes antiangiogenic and blocks follicular development.

Endovascular management of a complex high-flow lower limb arteriovenous malformation: Case report and literature review.

INTRODUCTION: Lower limb high-flow arteriovenous malformations (AVM) are a rare pathology with a chronic and progressive evolution. Development in imaging and treatment has helped to improve outcome. OBJECTIVE: We report a case treated with multi-session endovascular embolization therapy with favorable results. CASE REPORT: A 44-year-old man referred with a complex high-flow lower limb AVM, previously treated with covered stents and a bypass. Six endovascular interventions were performed, combining different embolization methods. CONCLUSION: Endovascular treatment is a safe and effective option for high-flow AVMs. Embolization should be done carefully to prevent complications that could threaten the patient's limb or life.

INTRODUCCIÓN: Las malformaciones arteriovenosas (MAV) son una patología con un curso crónico y una evolución progresiva. OBJETIVO: Reportar el caso de una MAV tratada con embolización endovascular en múltiples sesiones con resultados favorables. REPORTE DE CASO: Masculino de 44 años referido con una MAV de alto flujo del miembro pélvico derecho, tratado previamente con un stent recubierto y un bypass. Se realizaron seis sesiones endovasculares combinando diferentes métodos de embolización. CONCLUSIÓN: El manejo endovascular de las MAV de alto flujo es seguro y efectivo, debe de realizarse cuidadosamente evitando poner en riesgo la extremidad del paciente e incluso la vida.

Use of pooled serum samples to assess herd disease status using commercially available ELISAs.

Pooled samples are used in veterinary and human medicine as a cost-effective approach to monitor disease prevalence. Nonetheless, there is limited information on the effect of pooling on test performance, and research is required to determine the appropriate number of samples which can be pooled. Therefore, this study aimed to evaluate the use of pooled serum samples as a herd-level surveillance tool for infectious production-limiting diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL) and Neospora caninum (NC), by investigating the maximum number of samples one can pool to identify one positive animal, using commercial antibody-detection ELISAs. Four positive field standards (PFS), one for each disease, were prepared by pooling highly positive herd-level samples diagnosed using commercially available ELISA tests. These PFS were used to simulate 18 pooled samples ranging from undiluted PFS to a dilution representing 1 positive in 1,000 animals using phosphate-buffered saline as diluent. A 1:10 dilution of the PFS resulted in positive results for IBR, BVD and EBL. Moreover, for IBR and BVD, results were still positive at 1:100 and 1:30 dilutions, respectively. However, for NC, a lower dilution (8:10) was required for a seropositive result. This study indicates that, at herd-level, the use of pooled serum is a useful strategy for monitoring infectious diseases (BVD, IBR and EBL) but not NC, using readily available diagnostic assays.

Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro.

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4-7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine-arginine protein kinase 1, SRPK1), splicing factors (serine-arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P < 0.05) when compared to cells cultured at 20% 02. VEGF164b mRNA was absent in granulosa cells from all culture conditions. The 2 and 5% 02 levels, which coincide with physiological concentrations, in the ovarian follicle, induced higher SRSF6 expression than atmospheric 02 concentrations (20%, P < 0.05). In contrast, mRNA copies for SRPK1, CLK1, SRSF1, VEGFR1 or VEGFR2 did not differ between 02 culture conditions. (P > 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P < 0.05) with 02 concentration. These results suggest that when cultured under hypoxic conditions, granulosa cells may develop an autocrine milieu that favors VEGF's biological effects on their survival and function.

Co-ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development.

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine-arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine-arginine-rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC-like kinases-1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.

Physiological responses of cultured bovine granulosa cells to elevated temperatures under low and high oxygen in the presence of different concentrations of melatonin.

Our understanding of the effects of temperature on granulosa cell (GC) physiology is primarily limited to in vitro studies conducted under atmospheric (∼20% O2) conditions. In the current series of factorial experiments we identify important effects of O2 level (i.e. 5% vs 20% O2) on GC viability and steroidogenesis, and go onto report effects of standard (37.5 °C) vs high (40.0 °C) temperatures under more physiologically representative (i.e. 5%) O2 levels in the presence of different levels of melatonin (0, 20, 200 and 2000 pg/ml); a potent free-radical scavenger and abundant molecule within the ovarian follicle. Cells aspirated from antral (4-6 mm) follicles were cultured in fibronectin-coated wells using serum-free M199 for up to 144 h. At 37.5 °C viable cell number was enhanced and luteinization reduced under 5 vs 20% O2. Oxygen level interacted (P < 0.001) with time in culture to affect aromatase activity and cell estradiol (E2) production (pg/mL/105 cells). These decreased between 48 and 96 h for both O2 levels but increased again by 144 h for cells cultured under 5% but not 20% O2. Progesterone (P4) concentration (ng/mL/105 cells) was greater (P < 0.001) under 20 vs 5% O2 at 96 and 144 h. Cell number increased (P < 0.01) with time in culture under 5% O2 irrespective of temperature. However, higher doses of melatonin increased viable cell number at 40.0 °C but reduced viable cell number at 37.5 °C (P = 0.004). Melatonin also reduced (P < 0.001) ROS generation at both O2 levels across all concentrations. E2 increased with time in culture at both temperatures under 5% O2, however P4 declined between 96 and 144 h at 40.0 but not 37.5 °C. Furthermore, melatonin interacted (P < 0.001) with temperature in a dose dependent manner to increase P4 at 37.5 °C but to reduce P4 at 40.0 °C. Transcript expression for HSD3B1 paralleled temporal changes in P4 production, and those for HBA were greater at 5% than 20% O2, suggesting that hemoglobin synthesis is responsive to changes in O2 level. In conclusion, 5% O2 enhances GC proliferation and reduces luteinization. Elevated temperatures under 5% O2 reduce GC proliferation and P4 production. Melatonin reduces ROS generation irrespective of O2 level and temperature, but interacts with temperature in a dose dependent manner to influence GC proliferation and luteinization.

Improving Underrepresented Minority Student Persistence in STEM.

Members of the Joint Working Group on Improving Underrepresented Minorities (URMs) Persistence in Science, Technology, Engineering, and Mathematics (STEM)-convened by the National Institute of General Medical Sciences and the Howard Hughes Medical Institute-review current data and propose deliberation about why the academic "pathways" leak more for URM than white or Asian STEM students. They suggest expanding to include a stronger focus on the institutional barriers that need to be removed and the types of interventions that "lift" students' interests, commitment, and ability to persist in STEM fields. Using Kurt Lewin's planned approach to change, the committee describes five recommendations to increase URM persistence in STEM at the undergraduate level. These recommendations capitalize on known successes, recognize the need for accountability, and are framed to facilitate greater progress in the future. The impact of these recommendations rests upon enacting the first recommendation: to track successes and failures at the institutional level and collect data that help explain the existing trends.

Increased incidence of DNA amplification in follicular than in uterine and blood samples indicates possible tropism of Neospora caninum to the ovarian follicle.

This study evaluated the presence of Neospora caninum in ovarian follicle aspirates and uterine flushes obtained from N. caninum seropositive dairy cows. Ninety-two cows that aborted within the previous 90 days were sampled to determine the presence of antibodies against N. caninum. Thirteen seropositive cows were chosen for collection of blood leukocytes, uterine flushes (UF; n=12) and follicular aspirates (OPU; n=13). Samples were centrifuged and the cellular sediment from the follicular fluid, uterine flushes and blood leukocytes were used for DNA extraction and PCR. Follicular aspirates had the highest frequency of DNA amplification for N. caninum (p<0.05, 92.3%; 12/13). Whereas uterine (4/12) and blood leukocyte (5/13) samples had similar (p>0.05) rate of positive results. Nonetheless, there was no agreement between blood leukocytes and follicular samples taken from the same animal (Cohen Kappa=-0.16). Similarly, blood leukocytes and uterine results had moderate agreement between them (Cohen Kappa=0.47). This study indicates that N. caninum is present in the ovarian follicle and uterus of seropositive cows, suggesting a possible risk of neosporosis transmission between females during oocyte and embryo collection and transfer. Hence, precautions should be taken to minimize the risk of N. caninum transmission. Furthermore, the high incidence of positive results in follicle samples, that exceeded that of their paired blood leukocytes, suggests a possible tropism of N. caninum for the ovarian follicle.

Oocyte quality in lactating dairy cows fed on high levels of n-3 and n-6 fatty acids.

Different fatty acid (FA) sources are known to influence reproductive hormones in cattle, yet there is little information on how dietary FAs affect oocyte quality. Effects of three dietary sources of FAs (supplying predominantly palmitic and oleic, linoleic (n-6) or linolenic (n-3) acids) on developmental potential of oocytes were studied in lactating dairy cows. A total of 12 Holstein cows received three diets containing rumen inert fat (RIF), soyabean or linseed as the main FA source for three periods of 25 days in a Latin-square design. Within each period, oocytes were collected in four ovum pick-up sessions at 3-4 day intervals. FA profiles in plasma and milk reflected profiles of dietary FA sources, but major FAs in granulosa cells were not affected. Dietary FA source did not affect plasma concentrations of leptin, insulin, IGF1, GH, or amino acids. RIF led to a higher proportion of cleaved embryos than soya or linseed, but blastocyst yield and embryo quality were not affected. It is concluded that the ovary buffers oocytes against the effects of fluctuations in plasma n-3 and n-6 FAs, resulting in only modest effects on their developmental potential.

Incidencia de patologías uterinas y fertilidad de vacas Holstein tratadas con selenio y vitamina E antes y después del parto / Incidence of uterine pathologies and fertility of Holstein cows treated with selenium and vitamin E before and after parturition

An antioxidant therapy based on vitamin E and selenium subcutaneous (sc) injections administered prior to and after parturition, was evaluated by measuring the incidence of uterine pathologies and fertility in lactating Holstein cows. Cows (n = 353) were randomly distributed into three groups: Group pre-postpartum (n = 122), cows received a sc injection of 50 mg Se and 680 IU vitamin E (10 mL of Mu-Se) on 60 and 21 days prepartum and 30 and 90 days postpartum; Group prepartum (n = 117) received a sc injection 21 days prior to parturition; control Group (n = 114), cows received sc injections of saline solution (SS, 10 mL) on 60 and 21 days prepartum and 30 and 90 days postpartum. Incidence of retained fetal membranes (RFM) in control Group (20.1%) was similar (P > 0.05) to prepartum Group (12.8%), but it differed (P < 0.05) from pre-postpartum Group (6.5%). The percentage of cows with haemorrhagic metritis (HM) was similar among groups (8.7%; P > 0.05). Proportion of cows with purulent metritis (PM) in control Group (37.7%) was similar (P > 0.05) to prepartum Group (33.3%), but reduced (P < 0.05) in pre-postpartum Group (23.7%). The global proportion of cows showing any of the evaluated pathologies was similar (P > 0.05) for control groups (44.7%) and prepartum Group (40.1%) but lower (P < 0.05) for pre-postpartum Group (27%). Pregnancy rate at 150 days postpartum (PP) was greater (P < 0.08) for pre-postpartum Group (70%) than control Group (58.9%). In conclusion, an antioxidant therapy based on vitamin E and selenium administration, 60 and 21 days prior to parturition and 30 and 90 days after parturition, reduces the incidence of uterine pathologies and improves pregnancy rate at 150 days postpartum in Holstein cows.

Se evaluó el efecto de una terapia antioxidante basada en inyecciones de selenio y vitamina E, administradas antes y después del parto, en la incidencia de patologías uterinas y fertilidad en vacas Holstein. Se utilizaron 353 vacas, las cuales se asignaron al azar a tres grupos: Grupo pre-posparto (n = 122), las vacas recibieron una inyección sc de 50 mg de selenio y 680 UI de vitamina E (10 mL de Mu-Se) los días 60 y 21 preparto y 30 y 90 posparto; Grupo preparto (n = 117), las vacas recibieron una inyección similar al grupo anterior 21 días antes del parto; Grupo testigo (n = 114), los animales recibieron una inyección subcutánea de 10 mL de solución salina fisiológica los días 60 y 21 preparto, y 30 y 90 posparto. La incidencia de retención placentaria en el Grupo testigo (20.1%) fue similar (P > 0.05) al Grupo preparto (12.8%), pero difirió (P < 0.05) del Grupo pre-posparto (6.5%). La proporción de vacas con metritis hemorrágica fue similar entre grupos (8.7%; P > 0.05). El porcentaje de vacas con metritis purulenta en el Grupo testigo (37.7%) fue igual (P > 0.05) al Grupo preparto (33.3%), pero se redujo (P < 0.05) en el Grupo pre-posparto (23.7%). La proporción global de vacas con cualquiera de las patologías evaluadas fue similar (P > 0.05) entre las vacas de los grupos testigo (44.7%) y preparto (40.1%), pero fue menor (P < 0.05) en el Grupo pre-posparto (27%). El Grupo pre-posparto tuvo mayor (P < 0.08) tasa de gestación (70%) a los 150 días posparto que el testigo (58.9%). Se concluye que la administración de selenio y vitamina E los días 60 y 21 antes del parto y 30 y 90 posparto, reduce las patologías uterinas y mejora la tasa de gestación al día 150 posparto en vacas Holstein.

Porcentaje de concepción al primer servicio en vacas Holstein tratadas con hormona bovina del crecimiento en la inseminación / Conception rate at first service in Holstein cows treated with bovine somatotrophin at the time of insemination

A single dose of bovine somatotrophin (bST) given at the time of insemination improves conception rate (CR) in repeat-breeding cows, but there is no clear evidence that this treatment is useful in first service cows. The effect of bST applied at the time of insemination on conception rate in first service Holstein cows was evaluated. This study was conducted using 435 lactating Holstein cows from 15 dairy herds located in Tizayuca, Hidalgo. Cows were divided in two groups. The bST group (n = 185) received a subcutaneous injection of 500 mg of bST; the control group (n = 250) received 2 mL of saline solution. Insulin growth factor type I (IGF-I) and insulin concentrations were measured daily on seven randomly chosen cows of each group from the day of the bST injection until day 14 post treatment. Pregnancy diagnosis was performed by rectal palpation 45 days after insemination. The effects of treatment, herd, synchronized method (spontaneous or synchronized estrus with prostaglandin F2α), parity and days in milk on conception rate were analyzed by logistic regression for binary variables. The concentrations of IGF-I and insulin were compared using ANOVA for repeated measurements. Conception rate did not varied (P > 0.05) between treatment groups [bST (36.2%) vs control (34.8%)]. There were no herd, parity, days in milk or synchronized method effects (P > 0.05) on conception rate. IGF-I and insulin were higher (P < 0.05) in the bST group than in the control group. It is concluded that a 500 mg bST injection at the time of insemination does not improve fertility in first service Holstein cows.

Una inyección de hormona bovina del crecimiento (bST) en la inseminación mejora el porcentaje de concepción (PC) en vacas repetidoras; sin embargo, no hay clara evidencia de su eficacia en vacas de primer servicio. Se evaluó si una inyección de bST en la inseminación mejora el PC al primer servicio en vacas Holstein. El experimento se hizo con 435 vacas de 15 hatos de Tizayuca, Hidalgo, México. Las vacas se dividieron en dos grupos: el grupo bST (n = 185) recibió 500 mg bST sc en el servicio; el grupo testigo (n = 250) recibió 2 mL de solución salina fisiológica (SSF) sc. Se determinaron las concentraciones del factor de crecimiento parecido a la insulina tipo I (IGF-I) e insulina diariamente en siete vacas seleccionadas al azar de cada grupo, a partir del tratamiento con bST hasta el día 14 posinseminación. El diagnóstico de gestación se hizo por palpación rectal 45 días después de la inseminación. Se realizó un análisis de regresión logística para variables binarias con el propósito de determinar los efectos del tratamiento, hato, método de sincronización (estro espontáneo o sincronizado con prostaglandina F2α), número de partos y días en leche, sobre el PC. Las concentraciones de IGF-I e insulina se evaluaron mediante un modelo lineal mixto (ANDEVA) para mediciones repetidas. El PC fue similar (P > 0.05) entre grupos [bST (36.2%) y testigo (34.8%)]. No se encontró efecto (P > 0.05) del número de partos, hato, método de sincronización, ni número de días en leche en el PC. Las concentraciones de IGF-I e insulina fueron más altas (P < 0.05) en el grupo bST que en el testigo. Se concluye que una inyección de 500 mg de bST en la inseminación no mejora la fertilidad en vacas Holstein de primer servicio.

High yield selective acylation of polyamines: proton as protecting group.

An advance in the selective acylation of polyamines having identical or similar amine functions is reported. While nucleophilicity differences between the various amine functions are slight, the corresponding conjugate acids exhibit pKa values over a significant range. We have used proton as polyamine protecting group: the monoamine resulting from single deprotonation of a polyammonium compound has allowed for high yields of selective acylation.

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one.

BACKGROUND: The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5-21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I. RESULTS: Microspreads of fetal and neonatal ovarian cells underwent immunocytochemistry for meiosis- and apoptosis-related markers. COR-1 (meiosis-specific) highlighted axial elements of the synaptonemal complex and allowed definitive identification of the stages of meiotic prophase I. Labelling for cleaved poly-(ADP-ribose) polymerase (PARP-1), an inactivated DNA repair protein, indicated apoptosis. The same oocytes were then labelled for DNA double strand breaks (DSBs) using TUNEL. 1960 oocytes produced analysable results. Oocytes at all stages of meiotic prophase I stained for cleaved PARP-1 and/or TUNEL, or neither. Oocytes with fragmented (19.8%) or compressed (21.2%) axial elements showed slight but significant differences in staining for cleaved PARP-1 and TUNEL to those with intact elements. However, fragmentation of axial elements alone was not a good indicator of cell demise. Cleaved PARP-1 and TUNEL staining were not necessarily coincident, showing that TUNEL is not a reliable marker of apoptosis in oocytes. CONCLUSION: Our data indicate that apoptosis can occur throughout meiotic prophase I in mouse fetal and early postnatal oocytes, with greatest incidence at the diplotene stage. Careful selection of appropriate markers for oocyte apoptosis is essential.

Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows.

The purpose of this study was to examine the effects of level of rumen inert fatty acids on developmental competence of oocytes in lactating dairy cows. Estrous cycles were synchronized in 22 cows on a silage-based diet supplemented with either low (200 g/day) or high (800 g/day) fat. A total of 1051 oocytes were collected by ultrasound-guided ovum pickup (OPU) in seven sessions/cow at 3-4 day intervals. Oocytes were matured, fertilized, and cultured to the blastocyst stage in vitro. Embryo quality was assessed by differential staining of Day 8 blastocysts. The high-fat diet reduced numbers of small and medium follicles. There was no effect on the quality of oocytes (grades 1-4) or cleavage rate. However, high fat significantly improved blastocyst production from matured (P < 0.005) and cleaved (P < 0.05) oocytes. Blastocysts from the high-fat group had significantly more total, inner cell mass and trophectoderm cells than the low-fat group (P < 0.05). Regression analysis showed negative effects of milk yield (P < 0.001), dry matter intake (P < 0.001), metabolizable energy intake (P < 0.005), and starch intake (P < 0.001) on blastocyst production in the low-fat group but not in the high-fat group. Within the low-fat group, blastocyst production was negatively related to growth hormone (P < 0.05) and positively related to leptin (P < 0.05). The low-fat group had higher nonesterified fatty acids than the high-fat group (P < 0.05). In conclusion, higher milk yields were associated with reduced developmental potential of oocytes in cows given a low-fat diet. Provision of a high-fat diet buffered oocytes against these effects, resulting in significantly improved developmental potential.

Selection on predicted breeding value for milk production delays ovulation independently of changes in follicular development, milk production and body weight.

Two studies were conducted to investigate the effect of selection on predicted breeding value (PBV) for milk production, and its associated metabolic and endocrine milieu, on follicular development and ovulation in the postpartum period. A total of 71 cattle from lines selected to differ in their PBV were used in two consecutive years. In Study 1 the first ovulation and commencement of normal luteal function occurred significantly earlier in low (L) line cows than high (H) line cows. In Study 2 average daily milk production during the study period did not differ (P>0.10) between cows in either the H (=31.0+/-1.5kg/day) or L (=30.2+/-1.7kg/day) PBV lines although, 305 days milk production was significantly different (P<0.01; H=6880+/-164kg versus L=5795+/-317kg). As in Study 1, first ovulation postpartum in Study 2 occurred earlier (P<0.01) in the L (day 19) versus the H line (day 28). Circulating concentrations of glucose and insulin were significantly lower, whilst concentrations of GH and BOHB were higher in cows from the high PBV line. No differences in gonadotrophin concentrations were seen between lines. Both changes in body weight and patterns of follicle development did not differ between lines. By day 15 postpartum all cows had follicles of all three-size categories (small, medium-sized and large). Small (P<0.07) and medium-sized follicle numbers increased (P<0.01) with day postpartum. However, the inclusion of predicted changes in body weight as a covariate in the analysis, demonstrated that changes in number of small and medium-sized follicles were associated with changes in body weight. In conclusion, selection on PBV for milk production is associated with a longer interval from parturition to first ovulation, independent of changes in follicular development, milk production and body weight implicating other factor(s) associated with genetic selection.

Photochemical preparation of 1,3,5,7-tetracyanoadamantane and its conversion to 1,3,5,7-tetrakis(aminomethyl)adamantane.

New adamantane derivatives 1 and 2 that bear functionalized one-carbon extensions at all four bridgehead positions have been prepared. Radical nucleophilic substitution (S(RN)1) reaction of 1,3,5,7-tetrabromoadamantane with cyanide produces 1,3,5,7-tetracyanoadamantane (1), which was reduced with borane reagents to 1,3,5,7-tetrakis(aminomethyl)adamantane (2). Improvements in the preparation of 1,3,5,7-tetrahaloadamantanes (halogen = Br, Cl, I) are also reported. [structure--see text]