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.     

Susceptibility of Eucalyptus grandis and Acacia mearnsii seedlings to five Phytophthora species common in South African plantations

Eucalyptus grandis and its hybrids, as well as Acacia mearnsii, are important non‐native trees commonly propagated for forestry purposes in South Africa. In this study, we conducted pathogenicity trials to assess the relative importance of five commonly isolated Phytophthora spp. (Phytophthora alticola, P. cinnamomi, P. frigida, P. multivora and P. nicotianae) from the plantation environment on E. grandis and A. mearnsii seedlings. Overall E. grandis was more susceptible to the tested Phytophthora spp. than A. mearnsii. Phytophthora cinnamomi was the only pathogen that had a significant negative effect on both the host tree species, leading to a reduction in root and shoot weight as well as to death in the case of E. grandis. Phytophthora alticola and P. nicotianae exclusively affected E. grandis and A. mearnsii, respectively. This study updated the current knowledge on the pathogenicity of Phytophthora spp. on two important non‐native commercially propagated tree species from South Africa.     

Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce

Many industrial products and functional foods can be obtained from cheap and renewable raw agricultural materials. For example, starch can be converted to bioethanol as biofuel to reduce the current demand for petroleum or fossil fuel energy. On the other hand, starch can also be converted to useful functional ingredients, such as high fructose and high maltose syrups, wine, glucose, and trehalose. The conversion process involves fermentation by microorganisms and use of biocatalysts such as hydrolases of the amylase superfamily. Amylases catalyze the process of liquefaction and saccharification of starch. It is possible to perform complete hydrolysis of starch by using the fusion product of both linear and debranching thermostable enzymes. This will result in saving energy otherwise needed for cooling before the next enzyme can act on the substrate, if a sequential process is utilized. Recombinant enzyme technology, protein engineering, and enzyme immobilization are powerful tools available to enhance the activity of enzymes, lower the cost of enzyme through large scale production in a heterologous host, increase their thermostability, improve pH stability, enhance their productivity, and hence making it competitive with the chemical processes involved in starch hydrolysis and conversions. This review emphasizes the potential of using biocatalysis for the production of useful industrial products and functional foods from cheap agricultural produce and transgenic plants. Rice was selected as a typical example to illustrate many applications of biocatalysis in converting low-value agricultural produce to high-value commercial food and industrial products. The greatest advantages of using enzymes for food processing and for industrial production of biobased products are their environmental friendliness and consumer acceptance as being a natural process.     

The fate and toxicity of the flavonoids naringenin and formononetin in soil

The flavonoid class of plant secondary metabolites play a multifunctional role in below-ground plant–microbe interactions with their best known function as signals in the nitrogen fixing legume–rhizobia symbiosis. Flavonoids enter rhizosphere soil as a result of root exudation and senescence but little is known about their subsequent fate or impacts on microbial activity. Therefore, the present study examined the sorptive behaviour, biodegradation and impact on dehydrogenase activity (as determined by iodonitrotetrazolium chloride reduction) of the flavonoids naringenin and formononetin in soil. Organic carbon normalised partition coefficients, log K_oc, of 3.12 (formononetin) and 3.19 (naringenin) were estimated from sorption isotherms and, after comparison with literature log K_oc values for compounds whose soil behaviour is better characterised, the test flavonoids were deemed to be moderately sorbed. Naringenin (spiked at 50 μg g^?1) was biodegraded without a detectable lag phase with concentrations reduced to 0.13±0.01 μg g^?1 at the end of the 96 h time course. Biodegradation of formononetin proceeded after a lag phase of ～24 h with concentrations reduced to 4.5±1% of the sterile control after 72 h. Most probable number (MPN) analysis revealed that prior to the addition of flavonoids, the soil contained 5.4×10⁶ MPN g^?1 (naringenin) and 7.9×10⁵ MPN g^?1 (formononetin) catabolic microbes. Formononetin concentration had no significant (p>0.05) effect on soil dehydrogenase activity, whereas naringenin concentration had an overall but non-systematic impact (p=0.045). These results are discussed with reference to likely total and bioavailable concentrations of flavonoids experienced by microbes in the rhizosphere.     

Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the Booroola FecB gene after hypothalamic-pituitary disconnection or after treatment with a GnRH agonist

We have tested the hypothesis "that the ovulation rate in homozygous carriers (BB) and noncarriers (+2) of the Booroola FecB gene would not be different if the plasma concentrations of follicle-stimulating hormone (FSH) in the two genotypes were similar." For this purpose we used two experimental animal models: 1) the hypothalamic-pituitary disconnected (HPD) ovary-intact ewe; and 2) and GnRH agonist (i.e., Deslorelin)-treated ewe. Following HPD or Deslorelin treatment, the animals had low plasma concentrations of gonadotropins and were anovulatory. In both animal models, BB and +2 ewes were treated with exogenous pregnant mares serum gonadotropin (PMSG) and varying doses of FSH to induce preovulatory follicular growth, and human chorionic gonadotropin (hCG) to induce ovulation. HPD or Deslorelin-treated animals administered with pregnant mares serum gonadotropin without FSH followed by human chorionic gonadotropin failed to ovulate. However for both animal models, the proportion of BB and +2 ewes ovulating to various doses of FSH differed such that significantly greater proportions of +2 animals ovulated relative to the BB genotype (P < 0.05). When HPD or Deslorelin-treated BB and +2 ewes were administered identical doses of FSH, the mean ovulation rate and plasma concentrations of FSH in those animals which ovulated was the same in both genotypes. These findings confirm, at least in part, the aforementioned hypothesis. The results also demonstrated that higher ovulation rates were obtained in both genotypes as the FSH dose was increased. Collectively, these findings infer that the higher mean ovulation rate in normal intact BB ewes compared to the +2 genotype is attributable to effects of the FecB gene at the level of ovarian follicular development as well as at the level of pituitary FSH release.     

Study of the short- and long-term outcomes of 65 horses with peritonitis

The records of 65 horses with peritonitis examined at two uk referral centres over a period of 12 years were reviewed. Peritonitis was defined in terms of the horse's peritoneal fluid containing more than 5 x 10(9) nucleated cells/l. Horses that had developed peritonitis after abdominal surgery or a rupture of the gastrointestinal tract were excluded. Of the 65 horses, 56 (86 per cent) survived to be discharged. Follow-up information was obtained from practice records and telephone calls to the owners for 38 of the horses. Of these, 32 (84 per cent) had survived for at least 12 months and were considered to be long-term survivors; the others six were euthanased within 12 months. Thirteen (34 per cent) of the horses discharged had experienced complications that could have been sequelae to peritonitis and eight of the 13 were euthanased. The cause of the peritonitis was identified in 15 cases; survival rates were lowest in horses with peritonitis secondary to urinary tract involvement or intra-abdominal masses. Of the other 50 cases, 47 (94 per cent) survived to discharge, but two were euthanased owing to recurrent colic.     

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.