eChapter Name: Evaluation of Reproductive Toxicity Potential of Xenobiotics
9789358878363
eBook Name: EXPERIMENTAL AND EVIDENCE BASED PROTOCOLS IN PHARMACOLOGY
by Atul Prakash, Amit Shukla
Epididymal Total Sperm Count
The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003).
Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor)
Liveability and abnormal sperm
Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa.
The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain.
• A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide.
• 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop.
• Epididymal semen and stain are mixed gently using a blunt fine glass rod.
• After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide.
• The smear is examined under oil immersion objective.