Hypo-Osmotic Swelling Test for Membrane Integrity Evaluation
of Frozen-Thawed Water Buffalo (Bubalus bubalis, Linn.) Spermatozoa


Danilda Hufana-Duran1,2,3*, Rachelle P. Mallari2, Devon P. Suba2,
Peregrino G. Duran1, Evaristo A. Abella2, and Felomino V. Mamuad1

1Reproductive Biotechnology Laboratory, Philippine Carabao Center,
Science City of Muñoz, Nueva Ecija, Philippines.
2Department of Biological Sciences, Central Luzon State University,
Science City of Muñoz, Nueva Ecija, Philippines
3Prometeo, Universidad Tecnica de Babahoyo, Los Rios, Ecuador

corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it. / This email address is being protected from spambots. You need JavaScript enabled to view it.


Studies were conducted to establish the standard procedure of hypo-osmotic swelling test for frozen-thawed water buffalo spermatozoa. Hypo-osmotic swelling test is used in assessing plasma membrane integrity of spermatozoa of human and other livestock species. Plasma membrane integrity is a requisite of a fertile sperm. Standardization of hypo-osmotic swelling solutions and methodologies has not yet been done for frozen-thawed water buffalo spermatozoa; thus, the objective of this study was to identify assay conditions specifically for water buffalo. The following studies were conducted: Effective exposure time (0, 15, 30, 45, 60, 75, 90, 105, and 120 min) and temperature (25°C or 37°C), examination of the best osmolality of solution (0, 50, 100, 150, 200, 250, and 300 mOsm), evaluation of sugar (fructose vs. sucrose) better for HOS solution, and assessment of the repeatability and consistency of the test. Results showed optimum sperm reaction at 150 mOsm at 60 min but no significant difference was observed to 15 to 60 min exposure at 37°C in 50 to 200 mOsm using either fructose or sucrose as hypo-osmotic swelling solutions. Using the optimum HOST treatment procedure using 150 mOsm fructose-sodium citrate solution at 37ºC for 45 min, a coefficient of variation ranging from 0.05 to 0.09 among bulls was observed. Results demonstrated a standard hypo-osmotic swelling test procedure that is simple, accurate and consistent with good repeatability to predict the functional membrane integrity of buffalo spermatozoa.



Water buffalo is known for its inferior reproductive potential compared to cattle. In carrying out artificial insemination (AI) and in vitro fertilization (IVF), the efficiency rate is lower in water buffalo compared to cattle and the quality of spermatozoa significantly affects the success rate (Hufana-Duran et al. 2005). Though frozen semen use for AI and IVF has undergone rigorous semen quality evaluation prior to processing, the post-thaw quality needs to be reassessed to ensure that the sperm processing, storage and transport processes have not affected the . . . . . read more



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