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

Abstract

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 × 10(6) 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.

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted, frozen–thawed spermatozoa in field conditions

“…; Rodenas et al . ), the technique for AI and the high‐dose insemination of the number of sex‐sorted sperm preclude the practical application of this procedure at the farm level. The present study was to perform a successful procedure for AI with sex‐sorted dog spermatozoa in farm conditions.…”

“…All of the ejaculates used in this study met the following quality criteria: at least 75% total motility, 60% progressive motility and 80% normal morphology (Rodenas et al . ).…”

“…The sperm-rich fractions (SRFs) of the ejaculates were collected into pre-warmed sterile specimen cups. All of the ejaculates used in this study met the following quality criteria: at least 75% total motility, 60% progressive motility and 80% normal morphology (Rodenas et al 2014).…”

Storage of sexed boar spermatozoa: Limits and perspectives

“…Analyzing five ejaculates from three of the bad sperm sorter boars, the percentage of the ejaculates not exhibiting a well-defined split ranged between 20% and 70% (intraboar variability) [10]. Such variability in pigs, unlike other species (dogs, horses) [12,13], is not influenced by the percentage of non-viable spermatozoa in the semen samples but is closely related to ejaculate sperm concentration. Ejaculates are diluted to achieve the optimal sperm concentration for Hoechst 33342 (Ho) staining; during the staining step, samples from ejaculates with low sperm concentration would have a high proportion of seminal plasma that may alter Ho entrance into the sperm cell thereby affecting the effectiveness of DNA staining [10,11].…”

Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa

“…Sperm viability was evaluated by simultaneous cytometric assessment of the plasma and acrosomal membrane’s integrity using a triple-fluorescence procedure, as described previously by Ródenas et al [20] and modified slightly. Briefly, 2 × 10 6 spermatozoa were transferred to tubes containing 50 µl of TFC-Ext, 4 µl of H-42 (0.05 mg/ml in PBS), 1 µl of propidium iodide (PI, 0.5 mg/ml in PBS, Invitrogen; Molecular Probes, Eugene, OR, USA) and 2 µl of fluorescein-conjugated peanut agglutinin (PNA-FITC, 20 µg/ml in PBS).…”