Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

Mass selection of wheat for grain filling without transient photosynthesis

Summary Post-anthesis chemical desiccation of wheat (Triticum aestivum L.) plants in the field eliminates transtent photosynthesis by killing all green tissues, thus revealing the plant's capacity for grain filling from stored stem reserves, as the case is for post-anthesis stress such as drought or leaf diseases. This study was conducted to investigate whether mass selection for large kernels under chemical desiccation would lead to the improve ment of grain filling in the absence of transient photosynthesis.Six crosses of common spring wheat were subjected to three cycles of mass selection from F₂ through F₁ when selection was performed for large kernels by sieving grains from plants that were erther chemically desiccated after anthesis, or not (controls). The resulting 36 bulks (six crosses by three selection cycles by two selection environments) were compared with their respective F₂ base populations, when tested with and without chemical desiccation.Selection for large kernels under potential conditions (without chemical desiccation) did not improve kernel weight under potnetial conditions, evidently because these materials were lacking in genetic variation for kernel weight under potential conditions. In four of the crosses, 3rd cycle selection for large kernels under potential conditions decreased kernel weight under chemical desiccation. On the other hand, selection for large kernels under chemical desiccation was effective in improving kernel weight and test weight under chemical desiccation, depending on the cross and the selection cycle, with no genetic shift in mean days to heading or mean plant height. Selection for large kernels under chemical desiccation was also effective in some cases in increasing kernel weight under potential conditions. The results are interpreted to show that selection under potential conditions and under chemical desiccation operate on two different sources for grain filling, namely transient photosynthesis and stem reserve utilization, respectively. In order to expose genetic variability for stem reserve utilization to selection pressure, transient photosynthesis must be eliminated, as done by chemical desiccation in this study.     

The drought response of landraces of wheat from the northern Negev Desert in Israel

Summary Diverse landraces of wheat, collected from the semi-arid (150 to 250 mm of total annual rainfall) Northern Negev desert in Israel were considered as a potential genetic resource of drought resistance for wheat breeding. These materials were therefore evaluated for their reponses to drought stress in agronomical and physiological terms. Up to 68 landraces, comprising of Triticum durum, T. aestivum, and T. compactum were tested in two field drought environments, in one favourable field environment, under post-anthesis chemical plant desiccation which revealed the capacity for grain filling from mobilized stem reserves, under a controlled drought stress in a rainout shelter and in the growth chamber under polyethylene glycol (PEG)-induced water stress. Biomass, grain yield and its components, harvest index, plant phenology, canopy temperatures, kernel weight loss by chemical plant desiccation, growth reduction by PEG-induced drought stress and osmotic adjustment were evaluated in the various experiments.Landraces varied significantly for all parameters of drought response as measured in the different experiments, which was in accordance to their documented large morphological diversity. Variation in grain yield among landraces under an increasing drought stress after tillering was largely affected by spike number per unit area. Kernel weight contributed very little to yield variation among landraces under stress, probably because these tall (average of 131 cm) landraces generally excelled in their capacity to support kernel growth by stem reserve mobilization under stress. Yield under stress was reduced with a longer growth duration of landraces only under early planting but not under late planting. Landraces were generally late flowering but they were still considered well adapted phenologically to their native region where they were always planted late.Landraces differed significantly in canopy temperature under drought stress. Canopy temperature under stress in the rainout shelter was negatively correlated across landraces with grain yield (r=0.67^**) and biomass (r=0.64^**) under stress. Canopy temperature under stress in the rainout shelter was also positively correlated across landraces (r=0.50^**) with canopy temperature in one stress field environment. Osmotic adjustment in PEG-stressed plants was negatively correlated (r=–0.60^**) with percent growth reduction by PEG-induced water stress. It was not correlated with yield under stress in any of the experiments. In terms of yield under stress, canopy temperatures and stem reserve utilization for grain filling, the most drought resistant landrace was the Juljuli population of T.durum.     

The combined effects of temperature and GnRHa treatment on the final stages of sexual maturation in Atlantic salmon (Salmo salar L.) females

Atlantic salmon (Salmo salar L.) females (2 SW), maturing for the first time, were reared under one of three temperature regimes (high: 14.3 ± 0.5°C; natural: 10.6 ± 1.0°C; and cold: 6.9 ± 1.0°C) in combination with one of two experimental treatments; an injection of GnRH analogue (GnRHa) contained in biodegradable microspheres, or a sham injection (microspheres only). The six experimental groups were then reared under simulated natural photoperiod for 4 weeks. Blood samples were drawn for analysis of plasma steroid levels and the fish were inspected for ovulation weekly. Batches of stripped eggs were incubated in triplicate incubators in raceways until the eyed stage. Treatment with GnRHa resulted in a substantial advancement and synchronization of ovulation at all temperatures, while exposure to cold water also appeared to advance ovulation slightly. While 75% (warm and cold) to 90% (natural) of GnRHa fish ovulated during the 4-week trial, only 30% of sham-treated females exposed to cold water, and none of the sham-treated fish held at higher temperatures, ovulated during this period. Survival rates of embryos to the eyed-stage were significantly higher for broodstock exposed to cold water. Plasma levels of testosterone (T), 17β-oestradiol (E₂), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) were all significantly affected by treatment with GnRHa and, to a lesser extent, temperature. The efficiency of GnRHa in counteracting the negative effects of high temperature on ovulation and the associated changes in circulating sex steroids suggest that temperature inhibition operates at least in part at the brain or pituitary.