Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N‐acetyl glucosamine residues and non‐sialylated galactose and N‐acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N‐linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N‐linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo‐maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.     

Induced Ovulation, Spawning, Egg Incubation, and Hatching of the Cyprinid Fish Labeo victorianus in Captivity

Two spawning inducing agents—Aquaspawn (a rapidly metabolized synthetic decapeptide gonadotropin-releasing hormone [GnRH]) and Dagin ([D-Arg⁶, Pro⁹-NEt]-sGnRH) combined with 20 mg/kg of the water-soluble dopamine receptor antagonist metoclopramide (GnRH + MET)—were tested for their efficacy in stimulating ovulation in Labeo victorianus held in its natural environment and under captive conditions. Successful ovulation, when migratory vesicle oocytes became completely transparent, was obtained with GnRH + MET, while GnRH only caused oocyte clearance up to the highly translucent phase. L. victorianus eggs were non-adhesive, semi-buoyant, and transparent at ovulation. First hatching occurred after 26 h and lasted for 8 h at 24 C. Water temperature was shown to significantly affect spawning latency and incubation time. Thus, L. victorianus could be successfully induced to spawn using a synthetic gonadotropin-releasing hormone coupled with a dopamine antagonist followed by natural fertilization in floating net cages at temperatures between 24 and 27 C.     

Climate and groundwater recharge during the last glaciation in an ice-covered region

A multitracer study of a small aquifer in northern Switzerland indicates that the atmosphere in central Europe cooled by at least 5 degreesC during the last glacial period. The relation between oxygen isotope ratios (delta18O) and recharge temperatures reconstructed for this period is similar to the present-day one if a shift in the delta18O value of the oceans during the ice age is taken into account. This similarity suggests that the present-day delta18O-temperature relation can be used to reconstruct paleoclimate conditions in northern Switzerland. A gap in calculated groundwater age between about 17,000 and 25,000 years before the present indicates that during the last glacial maximum, local groundwater recharge was prevented by overlying glaciers.     

The effect of trunk electric vibration on the growth,yield and fruit quality of peach trees (Prunus persica [L.] Batsch)

To determine the effect of electric vibration on the growth, yield and fruit quality of peach electric vibrators that provided intermittent perturbation of 6500 rpm for 15 min every 6 h were firmly attached to the trunks of peach trees. Electric vibration resulted in the reduction of shoot length by 80% but had no significant effect on fruit weight, acid content and Brix. In another experiment, electric vibrators (for 15 min every 2 h) were attached to the branches after summer pruning. The regenerated shoots from the treated branches showed more than 500% reduction in length compared to the untreated ones. Even the regenerated shoots of untreated branches nearby the vibrator showed 60% reduction in length. Ethylene production and ACC content in the shoot tips of treated branches were greater than that from control ones.     

Cloning and Characterization of Boar Epididymal Secretory Proteins by Homology to the Human

Northern blot analysis suggested that the boar epididymis produces closely related counterparts to human epididymal proteins HE1, HE3, HE4, HE5 and HE12. ‘Full‐length’ cloning by nucleic acid and amino acid sequence similarity was achieved by RT‐PCR methods in the case of the porcine counterparts of HE3 and HE4, while the homologues of HE5 and HE12, despite their cross‐hybridization during Northern blot analysis, have not yet been cloned. The two novel porcine cDNAs were derived from moderately abundant epididymal mRNAs that were 75 and 83% identical to HE3 and HE4 cDNAs, respectively. To emphasize their relationship to the corresponding HEs, they were named Se3 and Se4 cDNAs. Their open reading frames predicted small secretory proteins with 55% (Se3) and 76% (Se4) conserved amino acids. Monospecific antipeptide antibodies to HE secretory proteins identified He3‐ and HE12‐related proteins on Western blots of porcine epididymal fluid and semen. Both Northern and Western analyses indicated that the Se proteins were produced in a regionalized pattern and accumulated in the cauda fluid.     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

Maximum vs minimum harmonization: what to expect from the institutional and legal battles in the EU on gene editing technologies

New plant‐breeding technologies (NPBTs), including gene editing, are widely used and drive the development of new crops. However, these new technologies are disputed, creating uncertainty in how their application for agricultural and food uses will be regulated. While in North America regulatory systems respond with a differentiated approach to NPBTs, the Court of Justice of the European Union (EU) has in effect made most if not all NPBT subject to the same regulatory regime as genetically modified organisms (GMOs). This paper discusses from a law and economics point of view different options that are available for the EU's multi‐level legal order. Using an ex‐ante regulation versus ex‐post liability framework allows the economic implications of different options to be addressed. The results show that under current conditions, some options are more expensive than others. The least costly option encompasses regulating new crops derived from NPBTs similar to those used in ‘conventional’ breeding. The current regulatory situation in the EU, namely making the use of NPBTs subject to the same conditions as GMOs, is the most costly option. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Effects of low temperature storage on growth and transplant quality of non-grafted and grafted cantaloupe-type muskmelon seedlings

Grafting is a unique horticultural technology that allows the grower to select an alternate, compatible root system with natural disease resistance for their desired crop. Short-term storage of grafted seedlings under low temperature may extend the production window of grafted seedlings, reduce the labor input and increase production of grafted seedlings with a small propagation capacity. To evaluate the low temperature storage conditions, Cucumis melo ‘Olympic Gold’ seedlings were grafted onto Cucurbita maxima x Cucurbita moschata ‘Tetsukabuto’ rootstock and stored for a period of 2 or 4 weeks at 9, 12, or 15 °C under 12 μmol m⁻² s⁻¹ photosynthetic photon flux (PPF). The study demonstrated that grafted seedlings could be stored at 12 °C for 4 weeks without significant dry mass accumulation or effects on post-storage growth and development. Grafted seedlings stored at 15 °C for 4 weeks had a significant increase in dry mass and stem elongation; this was not observed for the non-grafted seedlings stored under the same conditions, suggesting that the rootstock enhanced the scion growth at lower temperatures than optimal for muskmelon. Storing muskmelon seedlings at 9 °C caused chilling damage but the damage was pronounced for non-grafted seedlings than grafted seedlings. ‘Tetsukabuto’ rootstock, an interspecific squash, presumably has a chilling tolerance and increased the storability of muskmelon seedlings. Further optimization is needed but there is potential for using this technique as a tool for mass production of grafted muskmelon seedlings.