A Flow Cytometric Method for Rapid Determination of Sperm Concentration and Viability in Mammalian and Avian Semen

Abstract

A new flow cytometric method has been developed to rapidly determine sperm concentration and viability in semen from bulls and boars. Sperm viability was determined on the basis of staining with SYBR-14 and propidium iodide (PI), and this allowed detection of live (membrane-intact) sperm, dying (moribund) sperm, as well as dead cells. Fluorescent microspheres (beads) were used to determine sperm concentration. The use of SYBR-14 at 50 nM and PI at 12 micro M in combination with the FACSCount diluent in the counting tubes resulted in a uniform staining after 2.5-5 minutes at room temperature. Reagent staining was reproducible enough to allow subsequent semiautomated analysis of data using Attractors software. In experiment 1, this method was used to analyze semen from boars, rams, rats, rabbits, humans, and turkeys. In experiment 2, Attractors analysis was performed by the FACSCount AF flow cytometer, and sperm concentration determination with this system was compared with results obtained by a spectrophotometer and an electronic cell counter, which is routinely used by bull artificial insemination centers. When compared to microscopic counting in a hemocytometer, the FACSCount AF flow cytometer was two and four times more accurate than the spectrophotometer and the electronic cell counter, respectively. In addition, the FACSCount AF flow cytometer determined both sperm concentration (coefficient of variation 3.3%) and sperm viability (coefficient of variation 0.7%) with high precision.

Artificial Insemination in Pigs

“…Several studies use fluorescent dyes that stain intact or damaged spermatozoa differently, and measure the distribution of dyes in the sperm cell population by a flow cytometer (Christensen et al, 2004;Ericsson et al, 1993). In that way, viability as determined by the percentage of intact cells as well as the concentration of spermatozoa in an ejaculate can be determined by using fluorescent microspheres.…”

Effects of Kinds of Cryoprotectants on the Characteristics of Frozen Fowl Semen

“…유세포분석기(fluorescence activated cell sorting : FACS)는 주로 면역세포와 박테리아와 같은 부 유세포의 물리․생화학적 특징을 판별하고, 각 세포의 동정 및 분리를 위하여 개발되었으나, 닭 정자의 구조적 손상과 생화학적 변화도 및 물리적 특성을 관찰하는 방법으로 닭 정자 특성을 더 자세히 평가하는데 이용할 수 있는 것으로 보고되었다 (Graham et al, 1990 (Gillan et al, 2005). 죽은 정자의 막 투과성이 없어지는 현상을 이용하여 동결정 자의 융해 후 생존율을 평가하고 있으며, 정자의 생사평가 에는 SYBR-14와 PI을 주로 이용하는 생사염색법이 확립되 어 있다 (Garner and Johnson, 1995;Donoghue et al, 1996;Christensen et al, 2004 (Fig. 2), 그 결과는 Table 3과 같다.…”

Effects of N-Methylacetamide on the Viability, Fertility and Hatchability of Cryopreserved Ogye (Korean Native Black Fowl) Semen

“…또한 Lake et al (1981)은 인공수정 시 glycerol 농도를 1.501% 이하로 유지하여야 함을 보고하였으며, Hammerstedt and Graham (1992) (Sexton et al, 1980;Van Voorst and Leenstra, 1995a,b), dimethylacetamide (DMA)와 dimethylformamide (DMF)를 이용하여 닭의 정자를 동결 보존하였다 (Lake and Ravie, 1984;Tselutin et al, 1999Tereshchenko et al, 1992. Hanzawa et al (2010) (Christensen, 2004;Donoghue, 1996;Garner, 1995 …”