Malays. Appl. Biol. (2006) 35(1): 43–48
DETECTION OF ACROSOME IN MOUSE SPERM INCUBATED IN VITRO USING FLUORESCENCE STAINING TECHNIQUE
NOORAAIN, H.1, ABDULLAH, R.B.2* and DURRIYYAH, S.H.A.2
1Faculty of Applied Sciences, University Technology MARA, 40450, Shah Alam, Selangor, D.E. , Malaysia
2 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia
*E-mail:
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ABSTRACT
The protocol for detecting acrosome in mouse sperm was developed by using two types of fluorescent dyes namely fluorescein conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine conjugated Lens culinaris agglutinin (TRITC-LCA). The results showed that mean percentages of acrosome-intact sperm incubated in vitro were considerably low. After one-hour incubation there were 43.20±2.83, 31.81±8.44 and 30.90±4.15% for ICR, C57/6J and F1 sperm, respectively. Intact acrosome in ICR sperm was significantly reduced (P<0.05) to 16.95±4.23% after two-hour incubation, however, insignificant reduction (P>0.05) was found for C57/6J and F1 sperm, which were 30.03±2.06 and 20.93±3.81%, respectively. The results showed that both dyes FITC-PSA and TRITC-LCA suitably stained the mouse sperm and produced insignificant difference (P>0.05) in the percentages of acrosome-intact sperm, which were 40.03±4.20 and 49.79±4.63%, respectively. One-hour incubation was adequate to capacitate sperm in vitro. Low acrosome intact due to extended period of incubation and morphologically abnormal sperm could compromise in vitro fertilization rates.
ABSTRAK
Protokol untuk mengesan akrosom dalam sperma mencit telah dihasilkan dengan menggunakan dua jenis pewarna berpendafluor, iaitu fluorescein berkonjugasi Pisum sativum agglutinin (fluorescein conjugated Pisum sativum agglutinin, FITC-PSA) dan rhodamine berkonjugasi Lens culinaris agglutinin (rhodamine conjugated Lens culinaris agglutinin, TRITC-LCA). Kajian menunjukkan bahawa purata peratusan sperma berakrosom intak yang dieram secara in vitro adalah agak rendah. Setelah satu-jam eraman terdapat 43.20±2.83, 31.81±8.44 dan 30.90±4.15% bagi sperma ICR, sperma C57/6J dan sperma F1, masing-masing. Setelah dua-jam eraman, purata peratusan bagi sperma ICR telah menurun secara signifikan (P<0.05) ke 16.95±4.23%. Walau bagaimanapun, penurunan yang tidak signifikan (P>0.05) didapati bagi sperma C57/6J dan sperma F1, iaitu 30.03±2.06 dan 20.93±3.81%, masing-masing. Keputusan kajian juga menunjukkan bahawa kedua-dua pewarna FITC-PSA dan TRITC-LCA adalah sesuai untuk mewarnakan sperma mencit dan tidak menunjukkan perbezaan yang signifikan (P>0.05) dalam peratusan sperma berakrosom intak, iaitu 40.03±4.20 dan 49.79±4.63%, masing-masing. Satu-jam eraman adalah memadai untuk mengkapasitasi sperma secara in vitro. Bilangan sperma berakrosom intak yang rendah disebabkan oleh eraman yang berlanjutan dan sperma bermorfologi tidak normal mungkin menjejaskan kadar persenyawaan in vitro.
Key words: Sperm, acrosome, lectin, Pisum sativum agglutinin, insemination.
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