The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa

The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10⁶ sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Low-Dose Insemination in Pigs: Problems and Possibilities

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.     

Management of a stingray barb laceration and suspect envenomation in a dog

This case report describes the successful management of a stingray laceration and suspected envenomation using a combination of opioid analgesia, heat compression, antimicrobial therapy, surgical debridement and closure. Stingray envenomation in the dog is a rare clinical presentation and is yet to be documented in the Australian veterinary literature. Envenomation can be markedly painful and may cause swelling and local tissue necrosis. No consensus on treatment guidelines has been published. Diagnostics and treatments performed are outlined with recommendations on a management plan for future cases.     

Direct effects of induced parturition on subsequent reproductive performance of dairy cows from commercial herds in south-western Victoria

SUMMARY Reproductive performance was compared between cows whose previous parturition was induced and non-induced cows with similar calving dates, in 49 winter-calving, pasture-fed, commercial dairy herds in south-western Victoria. Parturition was induced in winter when most cows were between 27 and 35 weeks of pregnancy. Reproductive performance was assessed during the next mating period after induction which was mainly in spring of the same year. Percentages of cows in induced and untreated groups that were not pregnant after the mating period (9.0% and 7.2%, respectively) did not differ significantly. Induction tended to increase the percentage of cows of unknown pregnancy status. Mean percentages for induced and untreated groups were 11.5% and 7.9%, respectively. Induced and untreated groups calved at similar intervals after the planned start of calving in the following year, and the percentages of groups that required induction in that year did not differ significantly. The direct effects of induced parturition on reproduction were therefore concluded to be minimal. In seasonal calving herds, improvements in reproductive performance could be expected among cows whose calving dates were altered substantially by induction, due to increased intervals from calving to mating start date .     

High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants

Background  

Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells.     

Visualization of three-dimensional chaos

Most chaotic mixing experiments have been restricted to two-dimensional, time-periodic flows, and this has shaped advances in theory as well. A prototypical, bounded, three-dimensional flow with a moderate Reynolds number is presented; this system lends itself to detailed experimental observation and allows for high-precision computational inspection. The flow structure, captured by means of cuts with a laser sheet (experimental Poincare section), was visualized with the use of continuously injected fluorescent dye streams and revealed detailed chaotic structures with high-period islands.