Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 µM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Ovicidal, larvicidal and pupicidal efficacy of silver nanoparticles synthesized by Bacillus marisflavi against the chosen mosquito species.

Microbial synthesis of silver nanoparticles is more advantageous and is eco-friendly to combat the various vectors that cause diseases in humans. Hence, in the present study a Bacillus strain is isolated from marine habitat and is evaluated for its ability to synthesize silver nanoparticles (AgNPs) and its efficacy evaluated against the immature stages of selected mosquito species. The effective candidate was confirmed to be Bacillus marisflavi after 16S rRNA sequencing. The synthesis of AgNPs was confirmed by UV-Vis spectrophotometer. Atomic Force Microscopic (AFM) analysis showed spherical nanoparticles. Size analysis using Scanning Electron Microscope (SEM) showed particles of nano size averaging 78.77 nm. The diameter of the particles analyzed by Dynamic Light Scattering (DLS) showed 101.6 nm with a poly-dispersive index of 0.3. Finally the elemental nature of the nanoparticles was identified by Fourier-transform infrared spectroscopy (FTIR). LC50 and LC90 values for the ovicidal, larvicidal and pupicidal efficacy of the AgNPs against the egg, larvae and pupae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi respectively were evaluated. The present study revealed that the nanoparticles have an excellent toxic effect against the disease transmitting vector mosquitoes. Hence, the rapid synthesis of AgNPs would be an appropriate eco-friendly tool for biocontrol of vector mosquitoes.

Zidovudine inhibits telomere elongation, increases the transposable element LINE-1 copy number and compromises mouse embryo development.

PURPOSE: Millions of pregnant, HIV-infected women take reverse transcriptase inhibitors, such as zidovudine (azidothymidine or AZT), during pregnancy. Reverse transcription plays important roles in early development, including regulation of telomere length (TL) and activity of transposable elements (TE). So we evaluated the effects of AZT on embryo development, TL, and copy number of an active TE, Long Interspersed Nuclear Element 1 (LINE-1), during early development in a murine model. DESIGN: Experimental study. METHODS: In vivo fertilized mouse zygotes from B6C3F1/B6D2F1 mice were cultured for 48 h in KSOM with no AZT (n = 45), AZT 1 µM (n = 46) or AZT 10 µM (n = 48). TL was measured by single-cell quantitative PCR (SC-pqPCR) and LINE-1 copy number by qPCR. The percentage of morulas at 48 h, TL and LINE-1 copy number were compared among groups. RESULTS: Exposure to AZT 1 µM or 10 µM significantly impairs early embryo development. TL elongates from oocyte to control embryos. TL in AZT 1 µM embryos is shorter than in control embryos. LINE-1 copy number is significantly lower in oocytes than control embryos. AZT 1 µM increases LINE-1 copy number compared to oocytes controls, and AZT 10 µM embryos. CONCLUSION: AZT at concentrations approaching those used to prevent perinatal HIV transmission compromises mouse embryo development, prevents telomere elongation and increases LINE-1 copy number after 48 h treatment. The impact of these effects on the trajectory of aging of children exposed to AZT early during development deserves further investigation.

BPA and BPS affect the expression of anti-Mullerian hormone (AMH) and its receptor during bovine oocyte maturation and early embryo development.

BACKGROUND: Exposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized. METHODS: This study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence. RESULTS: Our findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2-4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2-4 cells, 8-16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05). CONCLUSION: This study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.

Small-molecule compounds boost genome-editing efficiency of cytosine base editor.

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

Research Note: Effects of the in ovo injection of organic zinc, manganese, and copper and posthatch holding time before placement on broiler body temperature during grow out.

Effects of the in ovo injection of organic microminerals (OM) (zinc, manganese, and copper) and posthatch holding time (HT) on the daily body temperature (bt) of broilers during grow out were determined. The hatching eggs from a Ross 708 breeder flock at 32 wk of age were incubated under standard commercial conditions. At 17 d of incubation, eggs were randomly allocated to 3 in ovo OM injection treatment (TRT) groups, and at 21 d of incubation, male hatchlings were randomly allocated to 2 posthatch HT treatment groups. Eggs were either not injected or were in ovo injected with diluent only or diluent containing the OM mixture. A 0-hour HT group had immediate access to water and feed, and a 24-hour HT (24HT) group contained birds that were kept in transport baskets in their pens without access to feed and water for 24 h before being released. Fifteen male birds were placed in each of 36 litter floor pens in a temperature-controlled facility. Approximately 2 birds in each of 6 replicate pens belonging to each TRT-HT combination had temperature transponders inserted subcutaneous in the mid-dorsal region of the neck. All birds were brooded under standard commercial conditions and had ad libitum access to feed and water after their respective HT. The bt of the same birds were determined daily at the same time each day beginning at hatch and ending on 39 d of posthatch age (AGE). There were no significant main or interactive effects involving TRT or HT for bt. However, there was a significant (P ≤ 0.0001) main effect because of AGE. A general increase in bt occurred during the 39 d grow out period. At hatch, bt was 40.54 ± 0.056°C and at AGE 39 was 41.46 ± 0.055°C. Under standard brooding conditions, a general increase in bt occurred in the Ross 708 broilers. However, these birds did not exhibit a significant bt response to TRT or a 24HT before placement.

Knockdown of CYP301B1 and CYP6AX1v2 increases the susceptibility of the brown planthopper to beta-asarone, a potential plant-derived insecticide.

The cytochrome P450 monooxygenases of insects play crucial roles in the metabolic detoxification of insecticides. Our previous finding showed that two cytochrome P450 genes, both CYP301B1 and CYP6AX1v2, in the BPH underwent overexpression due to ß-asarone. In this study, we investigated the molecular characteristics, expression patterns and functions of these two cytochrome P450 genes. The results showed that CYP301B1 had the highest expression level in the eggs, while CYP6AX1v2 was expressed in macropterous female adults. Moreover, the expression level of CYP301B1 in the head was higher than that in the integument, fat body and gut. The expression level of CYP6AX1v2 in the fat body and gut was higher than that in head and integument. Importantly, silencing CYP301B1 and CYP6AX1v2 separately could increase the sensitivity, resulting in significant higher mortality of BPH following treatment with ß-asarone. Our findings indicated that CYP301B1 and CYP6AX1v2 could contribute to the resistance of BPH to ß-asarone, and these two genes may be involved in the detoxification metabolism of ß-asarone in BPH.

Brief communication: rapid elimination of egg stickiness using sodium hypochlorite in wels catfish Silurus glanis.

The wels catfish Silurus glanis is valuable fish for aquaculture. Its production relies mainly on artificial reproduction. One of the crucial steps determining success of the reproduction is elimination of egg stickiness after fertilization. To date, the catfish egg de-adhesion is usually carried out using proteolytic enzymes. Here, we prove a novel method based on oxidation of the egg surface by means of sodium hypochlorite. An effect of different exposure times and concentrations on the egg adhesiveness and damage was tested in the first trial. The selected concentration of sodium hypochlorite 0.3 mg · l-1 with exposure time 40 s was used for comparison with the conventical de-adhesion method using alcalase treatment. The fertilization and hatching rates reached very satisfactory outcome in both treatments (98.3 ± 0.7% vs 97.5 ± 2.2% and 86.6 ± 8.3% vs 91.3 ± 8.5% in alcalase- and sodium hypochlorite-treated embryos, respectively) without any statistical differences. Thus, the de-adhesion method using sodium hypochlorite can be recommended as a suitable method for wels catfish eggs. The method is simple, cheap, very fast, and the treated eggs are disinfected.

Vitrification of Porcine Oocytes and Zygotes in Microdrops on a Solid Metal Surface or Liquid Nitrogen.

Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2 µL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop® sheets or in microdrops were equally effective.

Improvement in the performance and inflammatory reaction of Ross 708 broilers in response to the in ovo injection of 25-hydroxyvitamin D3.

The effects of the in ovo administration of vitamin D3 (D3) and its metabolite, 25-hydroxyvitamin D3 (25OHD3), on the performance, breast meat yield, and inflammatory responses of broilers fed commercial diets were investigated. Live embryonated Ross 708 broiler hatching eggs were randomly assigned to one of the following 5 in ovo injection treatments at 18 d of incubation: 1) noninjected; 2) diluent; diluent containing 3) 2.4-µg D3, 4) 2.4-µg 25OHD3, or 5) 2.4-µg D3 + 2.4-µg 25OHD3. A 50-µL solution volume of each prespecified treatment was injected into each egg using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly assigned to each of 30 floor pens. The BW, BW gain, feed intake, and feed conversion ratio of the birds were determined in each dietary phase. At 14, 28, and 39 d of posthatch age (doa), plasma α-1-acid glycoprotein (AGP) levels in 1 bird in each of 6 replicate pens per treatment were determined at 14 and 39 doa. The pectoralis major and minor weights of those same birds were also determined. The remaining birds were processed at 43 doa, and the weights of their processing parts were determined. At 39 doa, the in ovo injection of 25OHD3 alone decreased plasma AGP concentrations in comparison with the noninjected, diluent, and D3-alone treatment groups. In addition, birds that received 25OHD3 alone had a greater BW at 42 doa than birds in the noninjected, diluent, and D3-alone treatment groups. At 39 and 43 doa, breast meat yield was increased in response to the in ovo injection of 25OHD3 alone in comparison to all other treatments. These results indicate that the in ovo injection of 2.4 µg of 25OHD3 resulted in an improvement in the performance and inflammatory responses of broilers. A reduction in the inflammatory response subsequent to the in ovo injection of 2.4 µg of 25OHD3 may have led to an increase in broiler performance.

Treatment with Laevo (L)-carnitine reverses the mitochondrial function of human embryos.

PURPOSE: Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development. METHODS: To examine the effect of L-carnitine on mitochondrial function in morulae, 38 vitrified-thawed embryos at the 6-11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM L-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without L-carnitine was prospectively analyzed. RESULTS: Addition of L-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in L-carnitine-supplemented medium after single embryo transfers. CONCLUSION(S): L-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

Effects of a dry hydrogen peroxide disinfection system used in an egg cooler on hatchability and chick quality.

A sanitation method that could continually clean and disinfect the air and surfaces in a hatchery could provide a second layer of microbial reduction on top of routine cleaning and disinfection. A gaseous dry hydrogen peroxide (DHP) system has been used in other facilities for this purpose and could have potential for use in chicken hatcheries. Because the DHP is a true gas and can permeate through the entire hatchery space, contact with eggs during storage and incubation could potentially interfere with normal hatching processes. Therefore, the aim of this study was to evaluate the effects of the DHP system on hatching parameters and chick quality. A total of 3,960 hatching eggs were collected from an ∼40-week-old Ross 308 broiler breeder flock and distributed in 2 treatments: treated and nontreated. For the treated group, the egg cooler was cleaned, and 1 DHP generator was placed inside. Two other DHP generators were placed in the common area outside as well. Both areas were treated for 7 D before placement of eggs, and then eggs were collected and placed inside the cooler over a 4-day period. Eggs were then stored for an additional 3 D after the last collection. Dry hydrogen peroxide levels were recorded each day during storage. For the nontreated group, all DHP machines were removed from the cooler and external room, and the egg cooler was cleaned. Eggs were collected in the same way for the control group as the treated group. After storage, eggs were placed into a single stage Natureform incubator. The eggs exposed to DHP showed higher (P < 0.05) hatchability of fertile eggs and lower (P < 0.05) early embryonic dead than eggs from the nontreated group. No other parameters evaluated were different between groups. Based on this work, the DHP treatment of fertile eggs had no detrimental effect on any performance parameter, with potential positive effects seen on hatch of fertile eggs and early embryonic dead embryos.

Clove essential oil in the sanitation of fertile eggs.

The aim of this study was to evaluate the efficacy of sanitizing fertile eggs with clove essential oil as an alternative to paraformaldehyde; effects on the reduction in eggshell microbial count, incubation yield, and neonatal chick quality were measured. A total of 1,460 brown fertile eggs with a mean weight of 58.64 ± 0.49 g (from 37-wk-old CPK [Pesadão Vermelho] breeder hens) were collected under aseptic conditions and randomly distributed into 4 treatments (nonsanitized and sanitized with grain alcohol, clove essential oil, and paraformaldehyde) before incubation. The count of total aerobic mesophilic bacteria was significantly lower after spraying with clove essential oil (2.30 ± 0.24 log10 CFU/mL) than on nonsanitized eggs (3.49 ± 0.34 log10 CFU/mL) or on eggs sprayed with grain alcohol (3.09 ± 0.14 log10 CFU/mL) but did not differ significantly from the count in the paraformaldehyde group (2.23 ± 0.29 log10 CFU/mL). The hatchability of fertile eggs differed significantly between the studied treatments. The mean values for the eggs treated with clove essential oil (84.69 ± 1.65%) and paraformaldehyde (81.87 ± 3.92%) were statistically similar but were higher than the negative control (74.03 ± 3.58%) and grain alcohol (73.59 ± 2.87%) values. In the Pasgar© score assessment, it was determined that the clove essential oil (9.21 ± 0.89%) had a superior effect on the physical quality of the chicks compared with the effects of the other treatments. Clove essential oil is effective and safe for eggs intended for incubation. Its use as an alternative to paraformaldehyde in the sanitation of fertile eggs is strongly recommended.

Effect of a commercial product containing canthaxanthin for in ovo feeding to broiler embryos on hatchability, chick quality, oxidation status, and performance.

In ovo feeding has been indicated to improve hatchability, newly hatched chick quality, and broiler performance. The aim of this study was to investigate the effect of in ovo feeding of a commercial canthaxanthin product (CCX) containing lignosulphonate, corn starch, canthaxanthin, dextrin (yellow), and ethoxyquin through assessing incubation results, newly hatched quality and oxidation status and broiler performance at 1 to 14 d of age. A total of 780 egg were distributed in a randomized complete block design with 5 treatments (levels of CCX: 0.0, 0.35, 0.45, 0.55, and 0.65 mg/0.5 mL of sterilized and distilled water) and 156 eggs per treatment. The blocking factor was setters. At 17.5 d of embryo development, in ovo injected treatments were applied, using a manual needle. The in ovo feeding of CCX resulted in lower hatching rates (P < 0.05) and a longer hatching window (P < 0.05) as compared with noninjected CCX treatment. The CCX injection did not affect the bursa and spleen percentage of newly hatched chick (P > 0.05). In addition, a higher percentage of chicks with poor physical quality score (<71.0 points) was obtained among the chicks from eggs injected with 0.55 and 0.65 mg of CCX (P < 0.05). There were higher total proteins and catalase activity in the livers of the chicks injected with CCX. Broiler chicks in the control group (0.0 mg of CCX) presented higher BW and BW gain during 1 to 7 and 7 to 14 d of after hatch (P < 0.05). The viability (%) of chicks at 1 to 14 d of after hatch decreased with inoculation greater than 0.45 mg of CCX in ovo (P < 0.05). Although the CCX shown an improvement in oxidation status of chicks, the hatchability and performance of broilers decreased. We concluded that a commercial CCX is not recommended for injection in ovo, and furthers studies should carried out to elucidate the use of pure canthaxanthin.

Manganese requirements of broiler breeder hens.

The present research was conducted to assess Mn requirements of broiler breeder hens. One hundred and twenty Cobb 500 hens, 22 wk of age, were individually allocated in cages. After fed a Mn-deficient diet (22.2 ppm), hens were randomly placed in treatments having 6 increments of 30-ppm Mn. All trace minerals were from laboratory grade sources being Mn from Mn sulfate (MnSO4H2O). Treatments were fed for 4 periods of 28 d. There were no interactions between dietary Mn and period for any evaluated response (P > 0.05). Requirements of Mn for hen day egg production and settable egg production were 115.8 and 56.6 ppm and 122.1 and 63.6 ppm (P < 0.05), respectively, using quadratic polynomial (QP) and broken line quadratic (BLQ) models, whereas total eggs and total settable eggs per hen had Mn requirements estimated at 115.7 and 56.6 and 121.8 and 61.7 ppm (P < 0.05), respectively. Number of cracked, defective, and contaminated eggs decreased, whereas hatchability, hatchability of fertile eggs, eggshell percentage, and eggshell palisade layer increased when hens were fed diets having 48.5 to 168.2-ppm Mn (P < 0.05). Maximum responses for egg weight and eggshell percentage were 117.7 and 63.6 ppm as well as 131.6 and 71.0 ppm (P < 0.05), respectively, using QP and BLQ models. Breaking strength and egg specific gravity had Mn requirements estimated at 140.2 and 112.7 ppm as well as 131.3 68.5 ppm (P < 0.05), whereas eggshell palisade layer and eggshell thickness were maximized with 128.8 and 68.8 ppm and 140.2 134.2 ppm, respectively, for QP and BLQ models (P < 0.05). Maximum yolk Mn content values were obtained using 118.0- and 118.4-ppm Mn by QP and BLQ models, respectively. The average Mn requirements estimated for QP and BLQ models is 128.4 and 92.3 ppm Mn (18.7 and 13.5 mg/hen/d), respectively, which is much lower than what has been currently recommended in commercial production.

Effects of manganese and Bacillus subtilis on the reproductive performance, egg quality, antioxidant capacity, and gut microbiota of breeding geese during laying period.

This experiment was conducted to investigate the effects of manganese (Mn) and Bacillus subtilis (BS) on the production performance, egg quality, antioxidant capacity, and gut microbiota of breeding geese during laying period. A total of 120 forty-six-week-old breeding geese (Wulong) were randomly assigned to 1 of 6 treatment diets formulated to supply 10, 20, and 30 mg/kg Mn with 5 × 109 CFU/kg or 2.5 × 109 CFU/kg BS for a 10-wk trial. Results showed that dietary supplementation with 20 and 30 mg/kg Mn could decrease the daily feed intake (DFI) of geese. Moreover, 30 mg/kg Mn significantly increased the laying rate. Besides, although Mn addition had no obvious effect on egg quality, 5 × 109 CFU/kg BS was found to elevate the hatching egg hatching rate and eggshell thickness. For the serum hormones, 30 mg/kg Mn promoted estradiol secretion, while 5 × 109 CFU/kg BS increased the level of follicle-stimulating hormone. Furthermore, 20 and 30 mg/kg Mn and 5 × 109 CFU/kg BS significantly enhanced the total antioxidant capacity by increasing the activity of total superoxide dismutases or decreasing the content of malondialdehyde. Dietary supplementation with 5 × 109 CFU/kg BS also increased the intestinal villus height and upregulated the abundance of Fusobacteria, Fusobacteriaceae, Fusobacterium, and Faecalibacterium in cecal content. In addition, 20 and 30 mg/kg Mn elevated the levels of Bacteroidetes, Bacteroidaceae, Bacteroides, and Ruminococcaceae but decreased Streptococcaceae. Importantly, an interaction effect was observed between Mn and BS on the DFI, egg mass, average egg size, and the abundance of Bacteroides as well as Faecalibacterium. In conclusion, dietary inclusion of Mn and BS could improve the production performance, egg quality, antioxidant capacity, intestinal structure, as well as gut microbiota. Supplementation of 30 mg/kg Mn and 5.0 × 109 CFU/kg BS provided the optimal effect.

The Effect of GH Administration on Oocyte and Zygote Quality in Young Women With Repeated Implantation Failure After IVF.

Growth hormone (GH) has been shown to improve implantation and live birth rates in women of >40 years of age treated by in vitro fertilization (IVF). This effect was initially attributed to a GH effect on oocyte quality, but later studies showed that GH can also improve uterine receptivity for embryo implantation. As to younger women with previous failures of embryo implantation after IVF, data reported in the literature are ambiguous. This retrospective study focused on this latter category of women, comparing the numbers and morphological appearance of oocytes recovered from women with two previous IVF failures, aged between 30 and 39 years and treated with GH, with a comparable group of women without GH treatment. These results were complemented with the analysis of morphological markers of zygote and embryo quality and IVF clinical outcomes in both groups. The oocytes, zygotes and embryos from women treated with GH showed better morphological scores, and their uterine transfer resulted in more implantations, pregnancies and live births, as compared with the untreated group. It is concluded that the improvement of IVF outcomes in women with previous repeated IVF failures by exogenous GH administration is, at least partly, related to an increase in oocyte developmental potential. The statistically evident improvement of oocyte and embryo quality is the main finding of this study. Its weakness is its retrospective nature.

The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.

Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes.

Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

The effect of methionine and folic acid administered in ovo on the hematological parameters of chickens (Gallus gallus domesticus).

Methionine (Met), an essential amino acid in poultry diets, when overdosed may cause hyperhomocysteinemia, which is mainly a trigger for cardiovascular diseases in humans. Homocysteine is neutralized (remethylated) in the presence of folic acid (FA), which also plays an important role in hematopoiesis and participates in the synthesis of DNA, and its deficiencies may result in the development of neural tube defects. One of the basic tools in studying the impact of both xenobiotics and nutrients on the animal organism is hematological analysis. Thus, the aim of this study was to determine the effect of in ovo supplementation with Met and FA on the hematological parameters of broiler chickens. On the 17th day of incubation, embryonated eggs (Ross 308) were injected with 5 or 25 mg of Met per egg (M5 and M25), 3 and 15 mg of FA per egg (F3 and F15), or a mixture of these 2 compounds (M5/F3 and M25/F15). The broilers were reared in accordance with welfare regulations and fed with commercial diets ad libitum. Blood samples were collected on the first, seventh, and 35th day of rearing (D1, D7, and D35), and complete hematological analysis was performed. The observed changes in red blood cell parameters probably result from physiological changes occurring during bird growth. Mean erythrocyte volume decreased with the age of chickens in the control, M5, and M25 groups, but not in those supplied with FA. Among supplemented groups, the number of white blood cells on D1 was lower only in group M5 than in the sham (C) group. The analysis of leukograms showed no significant differences between the groups. Comparing D1 with D7 in the group injected with a higher dose of Met and FA (MF25/15), a statistically significant increase in the percentage of lymphocytes and a significant decrease in the percentage of heterophils were observed. In addition, in the group injected with a higher FA dose (F15), there was statistically significant reduction in the percentage of eosinophils and a significant increase in the percentage of monocytes at day 7 compared with day 1. It seems that Met supplementation led to temporary immunosuppression in the animals.