Plasma lipopolysaccharide elevations in cattle associated with early-stage infection by Fasciola hepatica

Fasciolosis is an endemic zoonotic parasitic disease with significant impacts on human health and both animal health and production. Early post-infection impacts on the host remain unclear. The objective of this study was to determine the changes, if any, to levels of endotoxin in cattle plasma in response to early-stage infection with Fasciola hepatica. Thirty-six (36) commercial bred cattle were experimentally infected with approximately 400 viable metacercariae. Plasma lipopolysaccharide (endotoxin) levels were examined on 24 occasions from 0 h before infection to 336 h after infection using the Limulus Amoebocyte Lysate chromogenic end point assay and compared with that of six (6) uninfected control animals. Peak lipopolysaccharide levels in infected animals were reached at 52 h after infection and returned to pre-infection levels at time 144 h after infection. Infected animals had significantly elevated lipopolysaccharide levels between 24 and 120 h after infection when compared to uninfected animals. The mean change in endotoxin units (EU)/mL over time after infection was statistically significant in infected animals. Elevations of lipopolysaccharide occurred in all infected animals suggesting a possible repeatable and titratable endotoxemia conducive to therapeutic agent model development.     

Genetic mapping and utilization of quantitative trichome-mediated insect resistance in potato

Introgression of trichome-mediated insect resistance from the wild speciesSolanum berthaultii has become a major focus of the potato improvement program at Cornell University during the past twelve years. Several quantitative characters are involved in this resistance which is effective against a wide range of pest types. Correlative biochemical assays have been developed to assay specific components of the resistance, and the effects of the resistance on the target pests have been studied. Quantitative laboratory assays and specific measurements of insect behavior and biology have increased the precision of selection and enable the investigation of the genetic control of the resistance.We are currently using restriction fragment length polymorphisms (RFLPs) for genetic mapping of factors controlling the trichome traits fromS. berthaultii. Backcrosses to both the wild and the cultivated species parents have been evaluated for phenotypes contributing to the resistance mechanism, including trichome density, sucrose ester and polyphenol oxidase production by the trichomes, and the enzymatic browning reaction responsible for insect entrapment. Genetic maps are being developed for these progenies, using RFLP markers previously mapped in potato. Field and greenhouse trials under insect infestations are also being conducted with the mapping progeny. Our goal is to locate genes responsible for quantitative insect resistance by correlating RFLP variation at mapped loci with the trichome phenotypes and insect resistance. Genetic markers for these traits will be useful in transfer of the effective wild chromosomal segments into and among tetraploid potatoes, and for a better understanding of the resistance mechanism.     

Production of interspecific F1 hybrids, BC1, BC2 and BC3 populations between Lycopersicon esculentum and two accessions of Lycopersicon peruvianum carrying new root-knot nematode resistance genes

Two accessions of Lycopersicon peruvianum Mill. (PI270435, PI126443) carrying Mi-2 and Mi-3, respectively, new root-knot nematode resistance genes, were selected as the male parents for crosses with L. esculentum Mill. in order to produce interspecific hybrids. Crossability barriers between these two distantly related species were circumvented by ovule culture. A total of ten interspecific F₁ hybrid plants were produced. The hybrid nature of the putative F₁ plants was confirmed by a comparison of several morphological characteristics and a PCR-based assay. Eight of ten hybrid plants were backcrossed with L. esculentum to generate a total of 98 BC₁ progeny. Two lines were advanced to the BC₂ and BC₃ levels. Based on these results, ovule culture was found to be an effective method for the production of novel interspecific F₁ and BC₁ plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative fine mapping of fruit quality QTLs on chromosome 4 introgressions derived from two wild tomato species

Despite their unsuitability for agricultural production, the wild relatives of crop species represent a largely untapped resource of novel QTLs potentially useful for crop plant improvement. In this regard, previous introgression studies, involving several different wild tomato species, have shown that the long arm of chromosome 4 contains QTLs for many horticulturally important traits including soluble solids content, fruit shape, lycopene content and biochemical composition. However, these earlier studies were unable to determine how many genes control these traits and whether genes affecting the same character from different wild species are allelic or not. In an effort to shed light on these issues,we have constructed a series of lines containing small, overlapping introgressions for portions of the long arm of chromosome 4 from L. peruvianum and L. hirsutum and tested these lines in replicated field trials. The results provide evidence for multiple, non-allelic loci controlling soluble solids and fruit weight. They also show that the loci controlling some traits (e.g. fruit shape, fruit weight, epidermal reticulation) co-localize to the same portions of chromosome 4, a result that maybe attributed to pleiotropy and/or gene dense areas with lower than average recombination. The implications of these finding for molecular breeding and utilization of exotic germplasm are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of the SP22 Sperm Protein in Santa Inês and Dorper Rams

The sperm membrane protein referred to as SP22 has been identified in different species and, at least in rats, is highly correlated with fertility. The goals of this study were to identify and to quantify the SP22 protein on spermatozoa from adult rams (Dorper and Santa Inês breeds), and to correlate its levels to morphological and kinematics parameters. SP22 on ram sperm was effectively quantified by both enzyme‐linked immunosorbent assay (ELISA) and fluorescein isothiocyanate immunostaining analysis and the two methods were significantly correlated (R² = 0.70). Clustering analysis of motility parameters obtained by computer‐assisted semen analysis system was used to establish that three distinct kinematic subpopulations with different vigour and progressiveness coexistent within ejaculate. While there were significant differences in the distribution of the three subpopulations in the rams, there was no significant correlation between the proportion of each subpopulation in the rams and the SP22 levels. Quantification of SP22 immunostaining intensity was not correlated with any of the sperm parameters. However, SP22 levels obtained by ELISA were negatively correlated with morphological abnormalities and positively correlated with membrane integrity (three variable R² = 0.47). Future breeding studies are now needed to validate that this protein is a biomarker of fertility in this species.     

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus–oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.     

Preliminary Trial: Motility Comparisons of a Unique Freezing Technology (UFT) to Liquid Nitrogen Mist Methodology for Cryopreservation of Porcine Spermatozoa

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.     

Molecular expression and identification of caprine estrogen receptor gene 1 for fertility status in bucks

Estrogen and its receptors are essential for sexual development and reproduction. Oestrogen receptor alpha (ERα) is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene ESR1 (oestrogen receptor1) responsible for better fertility. ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. The present study was undertaken to investigate the expression and presence of ESR1 gene in fertile and low-fertile male goat breeds. We identified ESR1 gene through various molecular tools. Genotyping was carried out by high resonance melting analysis using Roche Light Cycler 480(LC-480) system and found three different genotypes. Genotypic frequency-AA (blue-0.67), BB(Red-0.2), AB(Green-0.08) with allele frequency A(0.71 and B (0.29). The predominance of this gene in head of epididymis in fertile bucks was confirmed by SDS-PAGE, Western blotting and immunohistochemistry. From the results, we corroborated that the present study provides a useful and effective way to predict male fertility in goat breeds, which in turn increases the percentage of fertility in flock leading to more number of offspring in a kidding season.