Detailed motility evaluation of boar semen and its predictive value for reproductive performance in sows

Reliable estimates of boar fertility potential from semen evaluation could be a valuable tool for boar selection. The aim of this study was to investigate the morphology and the detailed motility parameters of diluted boar semen and to relate these to their predictive value concerning conception and farrowing rate, litter size and the number of live born piglets. In addition, the optimal time for evaluation of the motility of preserved semen with respect to its predictive effect on fertility was determined. One hundred ejaculates from 38 boars were evaluated morphologically by eosin-nigrosin staining and different motility characteristics were assessed using Computer Assisted Semen Analysis (CASA). The motility was determined at 15, 45 and 120 minutes after incubation at 37 degrees C. The conception rate, farrowing rate, litter size and number of live born piglets were registered from 276 sows inseminated with these ejaculates. Different regression models were used to evaluate the predictive value of the semen characteristics on these fertility parameters, taking into account the effect of herd, parity and weaning to estrus interval. The motility characteristics of the spermatozoa varied significantly during the 15 to 120 minutes of incubation. The longer the incubation time, the more the velocity parameters along the actual cell path decreased, while the parameters of straightforward movement increased. The predictive value of individual semen parameters on conception and farrowing rate was very small. The predictive value of certain associations of different semen parameters, on the other hand, was significant. The percentage of motile spermatozoa had a significant (P<0.05) and positive effect on the total number of piglets born (litter size) and on the number of live born piglets, independent of the time of measurement (X-2 0.38-1.00 and 0.41-1.00, respectively). Accurate evaluation of the motility of a semen dose is therefore imperative for estimating its predictive value relating to fertility. In conclusion, since the time of evaluation after warming the samples significantly influences the motility parameters, CASA measurement should be done when the cells are completely acclimatized to 37 degrees C. On the basis of the available data, a 45 min incubation period appeared to be sufficient. The percentage of motile spermatozoa, as assessed by CASA on diluted semen, offers detailed predictive information regarding litter size, irrespective of the time of measurement

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal

Background: The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objectives: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Study design: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. Results: Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. Main limitations: The relatively low number of equine oocytes and embryo transfer procedures performed. Conclusions: For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence

In vitro production of bovine embryos derived from individual donors in the Corral® dish

Background: Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral (R) dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral (R) dish, each donor group in a separate quadrant. Results: In two experiments, the Corral (R) dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral (R) dish used during IVM and IVC than when only used during IVM (12.9% +/- 2.10 versus 22.8% +/- 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination. Conclusions: In the present study, the Corral (R) dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral (R) dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral (R) dish is used during IVM and IVC

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters.</p> <p>Methods</p> <p>Multilevel images of embryos on day 1, day 2 and day 3, were analysed using a standard scoring system. The kappa coefficient was calculated to measure intra- and inter-observer variability before and after training sessions.</p> <p>Results</p> <p>Good to excellent intra-observer agreement was present for most parameters exceptions being scoring the position of pronuclei and the presence of a cytoplasmic halo on day 1, multinucleation on day 2 and the size of fragments on day 3. Inter-observer agreement was only good to excellent for the number of blastomeres on day 2 and day 3 and the orientation of the cleavage axes on day 2. Training sessions had a positive impact on inter-observer agreement.</p> <p>Conclusion</p> <p>In conclusion, assessment of morphological characteristics of early stage embryos using multilevel images was marked by a high intra-observer and a moderate inter-observer agreement. Training sessions were useful to increase inter-observer agreement.</p

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis

BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis

Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications

<p>Abstract</p> <p>Background</p> <p><it>Acidithiobacillus ferrooxidans </it>is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, γ-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth. It thrives at extremely low pH (pH 1–2) and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. The lack of a well-developed system for genetic manipulation has prevented thorough exploration of its physiology. Also, confusion has been caused by prior metabolic models constructed based upon the examination of multiple, and sometimes distantly related, strains of the microorganism.</p> <p>Results</p> <p>The genome of the type strain <it>A. ferrooxidans </it>ATCC 23270 was sequenced and annotated to identify general features and provide a framework for <it>in silico </it>metabolic reconstruction. Earlier models of iron and sulfur oxidation, biofilm formation, quorum sensing, inorganic ion uptake, and amino acid metabolism are confirmed and extended. Initial models are presented for central carbon metabolism, anaerobic metabolism (including sulfur reduction, hydrogen metabolism and nitrogen fixation), stress responses, DNA repair, and metal and toxic compound fluxes.</p> <p>Conclusion</p> <p>Bioinformatics analysis provides a valuable platform for gene discovery and functional prediction that helps explain the activity of <it>A. ferrooxidans </it>in industrial bioleaching and its role as a primary producer in acidic environments. An analysis of the genome of the type strain provides a coherent view of its gene content and metabolic potential.</p