A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.     

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350 Gy of X‐rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non‐irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non‐irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X‐rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.     

Effects of the butyric acid‐producing strain Clostridium butyricum MIYAIRI 588 on broiler and piglet zootechnical performance and prevention of necrotic enteritis

The objective of this study was to assess the effects of a probiotic strain Clostridium butyricumMIYAIRI 588 (CBM588) on broiler and weaned piglet health and zootechnical performance. Five field studies were carried out in broilers and five in weaned piglets under European feed additive guidelines. Each study followed a randomized blocked design with two treatments: Control (basal diet) and CBM588 supplemented groups. The zootechnical performance parameters selected were body weight, daily gain, feed intake and feed efficiency (feed:gain). Broilers fed diets with CBM588 gained significantly more weight (+2%, p < .001) and exhibited significantly better feed efficiency (?1.6%, p < .001) in comparison with Controls. Similarly, analysis of pooled data of weaned piglet trials showed that CBM588‐fed piglets were significantly heavier than Controls (+2.6%, p = .014), exhibited significantly higher mean daily gain (+4.7%; p = .004), and significantly improved feed efficiency (?4.2%, p = .001). In addition to the zootechnical efficacy studies, the preventive effect of CBM588 on necrotic enteritis (NE) was assessed in a natural challenge model in broilers where CBM588 reduced the incidence and severity of NE lesions. These data indicate the potential of CBM588 to improve broiler and weaned piglet zootechnical performance, and to make a positive contribution to animal health.     

Mycoplasma hominis ssp. associated endocarditis with myocardial necrosis in an alpaca (Vicugna pacos) in Manitoba in 2011

Severe endocarditis with myonecrosis, moderate to severe pleural and pericardial effusions, and mild ascites were found on necropsy in 3 alpacas. Mycoplasma hominis ssp. was detected on polymerase chain reaction (PCR) of fresh affected endocardial tissue in 1 alpaca.     

Effect of different commercial spawning agents and thermal regime on the effectiveness of pikeperch, Sander lucioperca (L.), reproduction under controlled conditions

Pikeperch, Sander lucioperca (L.), has been identified as one of the most perspective candidates for diversification of freshwater aquaculture. However, some aspects of production are still being developed, and controlled reproduction is one of the bottlenecks. The aim of the present study was to compare the effectiveness of different commercial spawning agents in the induction of final oocyte maturation (FOM) and ovulation in wild spawners. Within the study, four spawning agents [human chorionic gonadotropin (hCG), mixed human and horse gonadotropin (PG-600), carp pituitary (CPH) and mammalian GnRH analogue combined with metoclopramide (Ovopel)] in different thermal regimes (13 and 15 °C) were tested. In both thermal regimes, the highest (P < 0.05) ovulation rate among the treatment groups was obtained after stimulation with hCG (100 % in both cases). Latency time was the shortest in groups where CPH was used (2–3 and 3–4 days for 15 and 13 °C) and was similar in the remaining groups (3–4 and 4–5 days for 15 and 13 °C, respectively). Embryo survival was the highest in groups treated with hCG (78.9 and 81.3 % at hatching stage for 15 and 13 °C, respectively). Hormonal stimulation did not significantly affect spermiation rate or spermatozoa motility (P > 0.05). Based on the obtained results, hCG can be recommended for induction of FOM and ovulation in pikeperch. In addition, the thermal regime within the tested range seemed to have no effect on the reproduction outcome, and the application of lower temperature only prolonged the time of ovulation.     

Effect of gonadotropin hormonal stimulation on out-of-season propagation success of different year classes of indoor-reared pikeperch (Sander lucioperca (L.))

Three trials of hormonally induced out-of-season spawning of cultured pikeperch were performed. Two preparations containing human chorionic gonadotropin (hCG) (groups Bg-4 and Pl-4) were applied in experiment I. Injections of preparations containing hCG either alone (group P1-3) or in combination with pregnant mare serum gonadotropin (PMSG) of different producers (groups Sr-3 and Pg-3) were applied in experiment II. Spawning effectiveness was determined for four age groups (2+ to 5+) of pikeperch stimulated with hCG (groups Pl-2–Pl-5) in experiment III. The females from all the experimental groups were stimulated once with a single dose of hormone at 300 IU kg^?1 body weight (BW). Females from the control group were injected with a 0.9 % solution of NaCl (group C). The working fecundity (CF; % of BW), latency time (LaT; h), and eyed-egg survival (EES, %) were determined. No progress in oocyte maturation was noted among the females from group C. Significant differences were noted in LaT, which was lower in group Pl-3 than in groups Sr-3 and Pg-3. The youngest fish (2+) matured significantly more slowly and exhibited significantly lower values of the CF and EES indexes in comparison with older fish (LaT 102 vs 75–80 h; CF 5.4 vs 10.4–11.2 % BW; EES 47 vs 70–77 %). The hormonal preparations used had a positive impact on the ovulation and spermiation of the cultured pikeperch. No improvement in the effectiveness of out-of-season spawning was noted when combined preparations of hCG and PMSG were used.     

Growth performance, feed intake and morphology of juvenile European catfish, Silurus glanis (L.) fed diets containing different protein and lipid levels

The study was carried out with juvenile European catfish to evaluate the effects of commercial diets on growth, feed utilization and changes of selected morphometric traits of fish. Three diets containing different levels of crude protein and lipid: 37 and 12 %, 45 and 15 %, 45 and 20 %, respectively, were fed to triplicate groups of European catfish for 50 days. The fish were fed continuously (24 h day^?1) by automatic belt feeders for fish with a clock drive. Biometric characteristics of fish included the determination of plasticity traits using modified Pravdin method. Fish growth rate and final body weight were statistically higher in variant 45/20 diet (specific growth rate of 2.6 % day^?1). The intergroup significant differences were recorded for food conversion ratio (FCR) and protein efficiency ratio (PER), during the particular decades of the experiment. For the whole period of the growth test, no significant differences in FCR were observed, but the most favorable values of PER were obtained in variant 37/12. Protein retention in fish body ranged from 18.7 to 21.0 %, and fat retention was significantly more variable, ranged from 87.5 to 121.3 %. Fish survival rate recorded during the growth test was higher in all variants (above 95 %). Statistically significant increases in protein and lipid content were found in fish fed diets 45/15 and 45/20. The tested diets have no effect on changes in fish body proportions. The most effective in juvenile European catfish rearing was diet with 45 % of crude protein and 20 % of lipid.     

Tagging juvenile European whitefish (Coregonus lavaretus (L.)) with passive integrated transponders—Impact of fish size on growth performance and tag retention

The study examined the effect of PIT tagging and size on the growth, survival, food conversion, tag retention and wound healing in juvenile European whitefish. Three size classes of juvenile whitefish (class S—body weight (b.w.) approx. 4.0 g; class M—b.w. approx. 8.0 g; class L—b.w. approx. 13.6 g) were tagged with PIT implanted intraperitoneally (TROVAN^®, United Kingdom). These groups formed S‐P, M‐P and L‐P respectively. Fish from the control groups (groups S‐C, M‐C and L‐C) were not tagged. Whitefish from the tagged and control groups were reared for 28 days in recirculating aquaculture systems. Only in the fish from the smallest group (group S‐P) was tagging confirmed to have a negative impact on growth rate and survival, which, after 28 days, was 70% in comparison with 94.4% in group S‐C. The rate of wound healing in all whitefish groups was similar. After 28 days following PIT implantation, all wounds were healed. Short‐term PIT retention (28 days) for all the groups was > 90%, and no differences were noted among groups. In summary, it is recommended that whitefish be PIT‐tagged using the intraperitoneal method after they have attained a body weight > 8 g. Tagging smaller specimens of this species leads to higher mortality.     

Effect of visible implant elastomers on the growth,survival and tag retention in juvenile Atlantic sturgeon (Acipenser oxyrinchus) in laboratory conditions

This study identifies the possibility of using visible implant elastomers (VIE) to tag juvenile Atlantic sturgeon (Acipenser oxyrinchus). The mean body weight was 2.4 ± 0.4 g, and mean body length 5.5 ± 0.5 cm. Markers in three colours were used: blue, orange and green. Implant elastomers were injected in the rostrum and in the tail shaft. The experiment was conducted over a period of 10 weeks and included also a control group, that is, no tagging. No significant effect of tagging on the specific growth rate, feed conversion ratio and condition factor of juvenile Atlantic sturgeon was observed. The fish in all groups increased their body weight more than 10 times, reaching body weight of 25–28 g. The final survival in all of the groups was approximately 40%. The final tag retention varied depending on the implant colour. The highest retention of 100% was obtained with orange VIE and was significantly different from blue and green. Tag retention was dependent on the place of marking (P > 0.05). A correlation was observed between the applied dye and the place of marking (P = 0.0166). The best results were obtained when marking with orange VIE in the rostrum as all markers were visible after 70 days even without the use of UV light. According to our research, marking with VIE does not negatively affect growth and survival of juvenile Atlantic sturgeon and can be recommended for fish with an initial body weight of approximately 2.5 g in the case of short‐term research.