Evaluation of Methods to Determine Sperm Density for the European eel,Anguilla anguilla

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer‐assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G‐forces, were tested and the best G‐force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 ×  g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA‐1 and CASA‐2), CASA‐2 showed a very high accuracy to haemocytometer counts (r  =  0.93), but low precision (CV: CASA‐2 = 28.4%). FCM was tested with and without microfluorospheres (FCM‐1 and FCM‐2), and relationships to haemocytometer counts were highly accurate (FCM‐1: r  =  0.94; FCM‐2: r  =  0.88) and precise (CV: FCM‐1 = 2.5; FCM‐2 = 2.7%). Overall, CASA‐2 and FCM‐1 feature reliable methods for quantification of European eel sperm, but FCM‐1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.     

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.     

Variation in seed protein digestion of different pea (Pisum sativum L.) genotypes by cecectomized broiler chickens: 1. Endogenous amino acid losses, true digestibility and in vitro hydrolysis of proteins

The aim of this study was to investigate the cause of variation in the digestibility of pea protein in poultry and to find a tool to select genotypes with high digestibility potential by using an in vitro hydrolysis assay. Eight pea genotypes were selected for their difference in seed protein content and composition. To reduce the variation due to tannins and particle size, seeds from these 8 genotypes were dehulled and micro-ground. They were incorporated as the only protein source in 8 different experimental isoproteinaceous diets with similar metabolisable energy content. The amino acid digestibility was studied in cecectomized chickens. A balance method was used to obtain apparent digestibility, and the isotope dilution technique was used to determine endogenous losses and true digestibility, after feeding a double labelled test meal containing chromic oxide and ¹⁵N-labelled peas. The 8 diets showed differences in apparent amino acid digestibility. The average apparent digestibility for all amino acids varied between 79.5 and 86.3%, with the highest values for arginine (85.2 to 90.8%) and glutamic acid (85.2 to 90.5%), and the lowest values for cystine (63.3 to 69.7%) and tryptophan (69.1 to 80.3%). This variability of apparent amino acid digestibility was due to variations in endogenous losses and true digestibility among the 8 pea genotypes. The average endogenous losses as determined for 9 amino acids ranged from 3.6 to 5.4% of ingested amino acids, with the highest value for threonine (8.0 to 11.0%). The average true digestibility varied between 84.4 and 90.2%, with the highest values for lysine (89.0 to 95.0%), and the lowest for isoleucine (81.0 to 88.7%) and valine (82.4 to 88.7%). In vitro hydrolysis of protein from micro-ground seeds was performed for the 8 pea genotypes using three proteases (pepsin, trypsin and chymotrypsin). The quantity of small peptides (< 3 kDa) that appeared after the combined hydrolysis with pepsin (3 h) followed by trypsin and chymotrypsin (15 min) was significantly correlated with the average true digestibility of the 8 genotypes (R = 0.74; P < 0.05).     

Exogenous paraoxonase‐1 during oocyte maturation improves bovine embryo development in vitro

Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml^?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml^?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.     

Reproductive biology of pond reared Nile tilapia, Oreochromis niloticus L.

The reproductive performance of female Nile tilapia, Oreochromis niloticus L., reared in ponds under tropical conditions was studied. The experiments used stock matured to the age of 2–4 months and a size of 30–50 g. Oocyte development proved a better indicator for the determination of the size at first maturation than the development of the gonado-somatic index. The Nile tilapia became ‘stunted' in the sense that early breeding took place, but the somatic growth of the stock still continued. This result indicates that the earlier breeding of Nile tilapia is not a real bottleneck for the expansion of this production sector. No relationship was found between the condition factor and the size at first maturation or the relative fecundity. This led to the conclusion that aggravation of the living conditions is not a major cause of earlier breeding of pond-reared O. niloticus.     

Factors Affecting Plasma Pregnancy-associated Glycoprotein 1 Concentrations Throughout Gestation in High-producing Dairy Cows

This study was designed to establish the factors, if any, which could affect plasma pregnancy-associated glycoprotein-1 (PAG-1) expression in a study population of 87 pregnant, high-producing dairy cows. The factors examined were: semen providing breed (Holstein-Friesian vs Limousin), outcome of gestation (male vs female newborn, and singleton vs twin pregnancies), lactation number, milk production at pregnancy diagnosis, plasma progesterone concentration, season of gestation (warm period, March–November vs cool period, December–February), and day of gestation (40, 90, 120, 150, 180 and 210). Pregnancy was diagnosed by transrectal ultrasound on day 40 post-insemination and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The relative contributions of the different factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. No significant effects of the herd, foetal sex, milk production, lactation number and plasma progesterone concentrations were observed. In contrast, twin pregnancy, the use of Limousin semen and conception during the cool period were correlated with significantly increased plasma PAG-1 concentrations throughout gestation. Our data indicate that both cow well-being during early placental development, determined in our conditions by reduced heat stress when conception occurred in the cool season, and crossbreed pregnancies lead to improved PAG-1 production throughout the gestation period.     

Demographics of cattle positive for Mycobacterium avium subspecies paratuberculosis by faecal culture,from submissions to the Cork Regional Veterinary Laboratory

The demography of bovine infections caused by Mycobacterium avium subspecies paratuberculosis (MAP) in Ireland is poorly defined. The objective of this study was to describe the demographics of cattle positive to MAP on faecal culture, based on submissions to the Cork Regional Veterinary Laboratory (Cork RVL) from 1994 to 2006. The study focused on all available faecal samples from adult cattle with non-responsive chronic diarrhoea that were submitted by private veterinary practitioners to Cork RVL for MAP culture. For each MAP-positive by faecal culture animal, data were collated from Cork RVL and Cattle Movement Monitoring Scheme (CMMS) records. Johne's disease (JD) was confirmed in 110 animals from 86 herds by the Cork RVL between 1994 and 2006, with a rate of positive cases between 15% and 18% over last four years of the study. Two breeds (Holstein/Friesian or Limousin) made up 78% of submissions. Movements were assessed for the 57 study animals with available movement information, 90% died within one year of the test and 26% tested positive in the herd they were born into. The study provides preliminary information about movement trends and demographics of animals with MAP positive submissions. Although the study area is restricted, it includes the most intensive (and economically-important) dairy region in Ireland. The demographics of JD infection from the study area are in agreement with international reports. Further work is required to determine demographic trends, incidence and prevalence of JD throughout Ireland. It is hoped this work may contribute to the development of a surveillance strategy for MAP by regional veterinary laboratories.