Multi-trait association mapping for phosphorous efficiency reveals flexible root architectures in sorghum.

BACKGROUND: On tropical regions, phosphorus (P) fixation onto aluminum and iron oxides in soil clays restricts P diffusion from the soil to the root surface, limiting crop yields. While increased root surface area favors P uptake under low-P availability, the relationship between the three-dimensional arrangement of the root system and P efficiency remains elusive. Here, we simultaneously assessed allelic effects of loci associated with a variety of root and P efficiency traits, in addition to grain yield under low-P availability, using multi-trait genome-wide association. We also set out to establish the relationship between root architectural traits assessed in hydroponics and in a low-P soil. Our goal was to better understand the influence of root morphology and architecture in sorghum performance under low-P availability. RESULT: In general, the same alleles of associated SNPs increased root and P efficiency traits including grain yield in a low-P soil. We found that sorghum P efficiency relies on pleiotropic loci affecting root traits, which enhance grain yield under low-P availability. Root systems with enhanced surface area stemming from lateral root proliferation mostly up to 40 cm soil depth are important for sorghum adaptation to low-P soils, indicating that differences in root morphology leading to enhanced P uptake occur exactly in the soil layer where P is found at the highest concentration. CONCLUSION: Integrated QTLs detected in different mapping populations now provide a comprehensive molecular genetic framework for P efficiency studies in sorghum. This indicated extensive conservation of P efficiency QTL across populations and emphasized the terminal portion of chromosome 3 as an important region for P efficiency in sorghum. Increases in root surface area via enhancement of lateral root development is a relevant trait for sorghum low-P soil adaptation, impacting the overall architecture of the sorghum root system. In turn, particularly concerning the critical trait for water and nutrient uptake, root surface area, root system development in deeper soil layers does not occur at the expense of shallow rooting, which may be a key reason leading to the distinctive sorghum adaptation to tropical soils with multiple abiotic stresses including low P availability and drought.

Evaluation of the agricultural potential of the serpentinite rock as a soil remineralizer.

Magnesium is one of the essential elements for the plant growth. However, when the supply of magnesium is required exclusively, few economically feasible options are available. Serpentinite represents an alternative source of magnesium, although little is known about its potential and efficiency under tropical soil conditions. This work aimed to evaluate the use of serpentinite as a soil remineralizer, as well as magnesium fertilizer. The study was conducted in a greenhouse, using a completely randomized design, with seven treatments and four replications, as follows: three levels of serpentinite, mix of serpentinite and phonolite, and the controls with dolomitic limestone and without fertilization. Two plant species (Zea mays L. corn hybrid BRS - 1055 and Phaseolus vulgaris L. common bean variety BRS - Estilo) and two contrasting soils (clayey and sandy texture), were used in pots. Results showed that serpentinite's free silica and toxic element contents fitted the legal requirements. No statistically significant difference was observed for the plant dry matter weight production in the serpentinite and dolomitic limestone control, as well as in the pure serpentinite and the mix with phonolite treatments. The serpentinite was able to supply and to fullfil magnesium requirements for growth and development of corn and bean plants.

Effect of a single or two doses of an anti-GnRH vaccine on testicle morpho-functional characteristics in Nelore bulls.

The aim of this study was to compare testicle morpho-functional characteristics in bulls undergoing a single or two immunizations against GnRH. Nelore (Bos taurus indicus) bulls were randomly allocated into three experimental groups: G1 (n=12), a single 400 µg dose of anti-GnRH vaccine on day 0; G2 (n=11), a first 400 µg dose of anti-GnRH vaccine on day 0 followed by a second (boost) dose 30 days later; and control group (CG, n=12), 1 mL saline 0.9% at day 0. Every 30 days, from day 0 until slaughter at day 90, the bulls were weighed and underwent testicular biometry, semen collection and analysis, and blood sample collection for testosterone measurement. Immediately after slaughter, the testicles were removed and transport at 15°C to the laboratory for histopathological analysis. There was a decrease in testicular height (P=0.0476), width (P=0.0021), and in scrotal circumference (P=0.0001), after either a single (G1) or two (G2) immunizations against GnRH. Both G1 and G2 had lower testosterone concentrations than CG from day 60 on (P<0.01), but in G2, it was also lower than in G1 at day 90 (P=0.0006). All sperm parameters were affected by active immunization against GnRH (P<0.05), and in G2, averages were lesser (P<0.05) than in G1 from day 60 on. No signs of seminiferous tubule degeneration were found in any sample from the CG, contrasting with 75.0% and 100.0% of the samples from G1 and G2, respectively. In summary, immunocastration affected testicle morpho-functional characteristics in bulls in a time- and dose-dependent way.

Vascular and morphological features of the corpus luteum 12 to 20 days after timed artificial insemination in dairy cattle.

Our objective was to retrospectively compare pregnant versus nonpregnant cattle in terms of vascular and morphometric changes in corpora lutea between d 12 and 20 following timed artificial insemination (TAI). Crossbred (Gir × Holstein) lactating dairy cows (n = 136) and heifers (n = 111) were bred after synchronizing ovulations using an estradiol plus progesterone (P4)-based protocol. Corpus luteum (CL) characteristics (area, echotexture, blood flow) were recorded at 48-h intervals from d 12 to 20 following TAI using an ultrasound equipped with color Doppler. Blood samples were collected to determine CL function (plasma P4). Pregnancy diagnosis was performed at d 30. Quantitative assessment of colored pixels within the CL was performed using ImageJ software (National Institutes of Health, Bethesda, MD) and echotexture was quantified using custom software. Continuous variables such as luteal tissue area (LTA), CL blood flow (CLBF), adjusted CLBF (ratio LTA:CLBF), mean pixel value (MPV), pixel heterogeneity (HETER), and plasma P4 were analyzed retrospectively as repeated measures (d 12 to 20) in pregnant versus nonpregnant females using PROC MIXED (SAS Institute Inc., Cary, NC). Main effects were pregnancy status, day of cycle, and their interaction. Further analyses used only data from d 16, because this was the earliest time point of deviation between CLBF of pregnant and nonpregnant animals. We created quartiles for each variable and calculated the risk of pregnancy within quartile. Differences were determined using the chi-squared test. Plasma P4 was significantly higher in prospective pregnant versus nonpregnant cattle on d 18 and 20, whereas LTA differed only on d 20. On d 16, CLBF and adjusted CLBF diverged between pregnant and nonpregnant, followed by a progressive reduction in the latter until d 20. Mean pixel value was not affected by pregnancy status, but HETER was lower on d 20 in pregnant than in nonpregnant cattle. Likelihood of pregnancy increased from quartile (Q)1 (lowest values) to Q4 (highest) of CLBF (Q4 vs. Q1, odds ratio = 32.8, 95% confidence interval: 9.6 to 112.1) and adjusted CLBF [Q4 vs. Q1, odds ratio = 25.4, 95% confidence interval: 8.1 to 80.4), whereas a lower risk of pregnancy was observed only for animals within Q1 of plasma P4 [Q4 vs. Q1, odds ratio = 3.1, 95% confidence interval: 1.3 to 7.2). Day 16 quartiles of LTA, MPV, and HETER did not affect odds of pregnancy. In conclusion, we identified distinct CLBF patterns as early as 16 d after TAI and confirmed that CL function is lost by a reduction in blood flow, which precedes physical regression.

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.

Consequences of assisted reproductive technologies for offspring function in cattle.

Abnormal fetuses, neonates and adult offspring derived by assisted reproductive technologies (ART) have been reported in humans, rodents and domestic animals. The use of ART has also been associated with an increased likelihood of certain adult diseases. These abnormalities may arise as a result of an excess of or missing maternally derived molecules during invitro culture, because the invitro environment is artificial and suboptimal for embryo development. Nonetheless, the success of ART in overcoming infertility or improving livestock genetics is undeniable. Limitations of invitro embryo production (IVEP) in cattle include lower rates of the establishment and maintenance of pregnancy and an increased incidence of neonatal morbidity and mortality. Moreover, recent studies demonstrated long-term effects of IVEP in cattle, including increased postnatal mortality, altered growth and a slight reduction in the performance of adult dairy cows. This review addresses the effects of an altered preimplantation environment on embryo and fetal programming and offspring development. We discuss cellular and molecular responses of the embryo to the maternal environment, how ART may disturb programming, the possible role of epigenetic effects as a mechanism for altered phenotypes and long-term effects of ART that manifest in postnatal life.

Duplicate and conquer: multiple homologs of PHOSPHORUS-STARVATION TOLERANCE1 enhance phosphorus acquisition and sorghum performance on low-phosphorus soils.

Low soil phosphorus (P) availability is a major constraint for crop production in tropical regions. The rice (Oryza sativa) protein kinase, PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was previously shown to enhance P acquisition and grain yield in rice under P deficiency. We investigated the role of homologs of OsPSTOL1 in sorghum (Sorghum bicolor) performance under low P. Association mapping was undertaken in two sorghum association panels phenotyped for P uptake, root system morphology and architecture in hydroponics and grain yield and biomass accumulation under low-P conditions, in Brazil and/or in Mali. Root length and root surface area were positively correlated with grain yield under low P in the soil, emphasizing the importance of P acquisition efficiency in sorghum adaptation to low-P availability. SbPSTOL1 alleles reducing root diameter were associated with enhanced P uptake under low P in hydroponics, whereas Sb03g006765 and Sb03g0031680 alleles increasing root surface area also increased grain yield in a low-P soil. SbPSTOL1 genes colocalized with quantitative trait loci for traits underlying root morphology and dry weight accumulation under low P via linkage mapping. Consistent allelic effects for enhanced sorghum performance under low P between association panels, including enhanced grain yield under low P in the soil in Brazil, point toward a relatively stable role for Sb03g006765 across genetic backgrounds and environmental conditions. This study indicates that multiple SbPSTOL1 genes have a more general role in the root system, not only enhancing root morphology traits but also changing root system architecture, which leads to grain yield gain under low-P availability in the soil.

In vivo collection of follicular fluid and granulosa cells from individual follicles of different diameters in cattle by an adapted ovum pick-up system.

BACKGROUND: Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters. METHODS: In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle. RESULTS: In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05). CONCLUSIONS: The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.

Use of ultrasound biomicroscopy to evaluate induced ovarian follicular growth and ovulation in mice.

Recent advances in image technology, including significant gains in spatial resolution, have made realtime sequential ovarian evaluations possible in small rodents, allowing longitudinal (continued) studies of the ovarian cycle and reducing the required number of experimental animals. The aim of this study was to evaluate exogenous stimulated follicular growth in mice using high-resolution ultrasound technology. Female mice (n = 15) received a 5 IU intraperitoneal injection of equine chorionic gonadotropin (eCG) and 48 h later a 5 IU injection of human chorionic gonadotropin (hCG), and were allowed to mate thereafter. In experiment 1, animals (n = 7) were evaluated every 6 h, from 3 to 51 h after eCG injection, with an ultrasound biomicroscopy (UBM) equipped with a realtime 45 MHz microvisualization probe (RMV 707b). The ovaries were identified and follicular population quantified, and follicles were classified according to the diameter as small (≤449 µm) or large (≥450 µm). A significant change in the distribution of follicle population according to category was observed only 45 h after eCG injection (P < 0.05). In experiment 2, animals (n = 8) were evaluated every 2 h, from 2 h to 10 h after hCG treatment. The largest follicles reached a maximum size (596.7 ± 106.0 µm) 5.8 ± 2.3 h after hCG injection. As expected, the population of large follicles decreased thereafter, indicating the progress of ovulations, but large follicles were still detected late after treatment (10.1 ± 1.1 h). In conclusion, UBM can be used to evaluate follicle dynamics in superstimulated mice (C57BL/6 and BALB/c); significant changes in follicle distribution only occur at later stages after eCG stimulation; and hCG-induced ovulations may not occur synchronously in mice.

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes.

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.