Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.
Abstract: The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after collection. Sperm incubated in both skim milk and 1% BSA-supplemented diluents had equivalent total (47 and 49.5%, respectively) and progressive (4.73 and 5.67%, respectively) sperm motilities after 45 min, and comparable acrosome integrity (65.9 and 67.9%, respectively). For Experiment 2, the protein source was optimised by comparing the characteristics of sperm stored and incubated in five diluents supplemented with skim milk, BSA, fatty acid and endotoxin free BSA (I-BSA), KnockOut™ Serum Replacement, and β-lactoglobulin, respectively. The I-BSA diluent was superior to skim milk for motility maintenance during incubation (74.0 vs 63.7%). The effect of diluent on sorting was investigated in Experiment 3 using a range of H33342 concentrations and incubation durations. The clear (1% BSA) diluent improved the split ratio compared with the opaque (skim milk) diluent (0.17 vs 0.08), with an optimum staining time of 45 min using 0.09 mM H33342. In conclusion, a diluent containing 1% fatty acid free, low endotoxin BSA in lieu of skim milk improved the sorting efficiency and motility characteristics of stallion sperm after storage for 18 h.
Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.
Publication Date: 2010-12-07 PubMed ID: 21144575DOI: 10.1016/j.theriogenology.2010.10.001Google Scholar: Lookup
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- Journal Article
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- Non-U.S. Gov't
Summary
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The study proposed a novel, clear, diluent formula that improves the sex-sorting efficiency of stallion sperm compared to traditional milk-based diluents.
Overview of Research
This research aimed at formulating a clear diluent that improves the sex-sorting efficiency of stallion sperm. Their efforts were concentrated on increasing the flow cytometric sorting efficiency, which had previously been reduced due to the use of an opaque, skim milk-based diluent. The study was divided into three different experiments.
Experiment 1: Consideration of Different Diluents
- The researchers compared sperm incubated in five different diluents — no protein, skim milk, 0.25% Cohn’s Fraction V BSA, 0.5% BSA, or 1% BSA.
- Sperm were stored for 18 hours at 15 degrees Celsius, or examined shortly after being collected, before exposure to the diluents.
- The study found that sperm incubated in skim milk and 1% BSA had similar total and progressive sperm motilities and comparable acrosome integrity after 45 minutes of incubation.
Experiment 2: Optimization of Protein Source
- The second experiment involved the use of five different diluents — skim milk, BSA, fatty acid and endotoxin-free BSA (I-BSA), KnockOut™ Serum Replacement, and β-lactoglobulin.
- It was realized that I-BSA diluent was superior for maintaining sperm motility during incubation compared to skim milk.
Experiment 3: Effect of Diluents on Sorting
- In Experiment 3, the aim was to investigate the effect of the diluent on sorting, using different concentrations and incubation periods of H33342.
- The results revealed that the clear solution (1% BSA) improved the split ratio — the division of sperm into differently gendered divisions — when compared with the opaque solution (skim milk).
- Optimum staining time was noted as 45 minutes using 0.09 mM H33342.
Conclusion
The study concluded that the newly formulated diluent containing 1% fatty acid free, low endotoxin BSA was superior to traditional skim milk diluents as it improved sorting efficiency and motility characteristics of stallion sperm even after storage for 18 hours. This development opens up avenues for more effective sex selection in horse breeding.
Cite This Article
APA
Gibb Z, Morris LH, Maxwell WM, Grupen CG.
(2010).
Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.
Theriogenology, 75(4), 610-619.
https://doi.org/10.1016/j.theriogenology.2010.10.001 Publication
Researcher Affiliations
- Faculty of Veterinary Science, The University of Sydney, Camperdown, NSW, Australia. zamira.gibb@sydney.edu.au
MeSH Terms
- Acrosome / drug effects
- Animals
- Flow Cytometry / methods
- Flow Cytometry / veterinary
- Horses
- Male
- Serum Albumin, Bovine / pharmacology
- Sex Chromosomes
- Sex Preselection / methods
- Sex Preselection / veterinary
- Sperm Motility / drug effects
- Spermatozoa
Citations
This article has been cited 3 times.- Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II. Animals (Basel) 2021 Nov 20;11(11).
- Anel-Lopez L, Ortega-Ferrusola C, Álvarez M, Borragán S, Chamorro C, Peña FJ, Morrell J, Anel L, de Paz P. Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm. BMC Vet Res 2017 Jun 26;13(1):200.
- Yadav SK, Gangwar DK, Singh J, Tikadar CK, Khanna VV, Saini S, Dholpuria S, Palta P, Manik RS, Singh MK, Singla SK. An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm. Vet World 2017 May;10(5):498-504.
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