Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics

Background  

The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4.     

Comparative studies of the influence of sward conditions on the ingestive behaviour of cows and sheep

Separate groups of non-lactating cows and wether sheep grazed at similar herbage allowances for two successive 5-d periods on swards that had previously been grazed frequently or infrequently with the intention of creating differences in canopy structure. Measurements were made of sward structure and composition, ingestive behaviour and diet composition. The preliminary treatments had little effect upon either sward conditions or animal behaviour.
Herbage mass was reduced from 4020 kg dry matter (DM) ha⁻¹ to 3290 kg DM ha⁻¹ on average over a 5-d grazing period as a consequence of the relatively low grazing pressure imposed. This resulted in a mean decline in intake per bite of 28%, and the changes for cattle and sheep did not differ significantly. However, there was a marked difference in the other behavioural responses of the two species; in the sheep biting rate fell and grazing time increased with declining herbage mass, particularly in the sward previously grazed infrequently, whereas the changes in the cattle were small.
Differences in the botanical composition of the herbage eaten by cattle and sheep were minor, but there was a small but consistent advantage to the sheep in the digestibility of the herbage eaten.
In the first of the two periods the variation in surface height after grazing was substantially greater for sheep-grazed than for cattle-grazed swards, indicating more patchy grazing by the sheep.
Estimates of daily herbage organic matter (OM) intake calculated from ingestive behaviour variables were high (means 38 and 32 g (kg LW) ⁻¹ for cattle and sheep respectively) and usually declined substantially over a grazing period.     

Influence of the FecXR Allele in Heterozygous Ewes on Follicular Population and Outcomes of IVP and ET using LOPU‐Derived Oocytes

Ewes heterozygous for the FecX^R allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX^R allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild‐type (++) ewes were subjected to 2 laparoscopic ovum pick‐up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH‐treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (?0.2 mm in untreated and ?0.8 mm in FSH‐treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus–oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture.