11. Preparation of Semen for Morphological Examination

Why is the preparation of semen for morphological examination important?

The preparation of semen for morphological examination is important because it allows for the evaluation of spermatozoa structure and helps determine the severity of testicular degeneration, hypoplasia, and congenital defects. It is used to assess whether the sample falls within the normal range and provides insights into the potential fertility of the individual.

What are the staining techniques used for morphological examination of spermatozoa?

There are two main staining techniques used for the morphological examination of spermatozoa:


1. Fixed smears
2. Unstained wet mount

Describe fixed smears

Fixed smears stained with eosin-nigrosin and basic fuchsin: This method involves fixing the smears, staining them with a combination of eosin and nigrosin, and using basic fuchsin as a counterstain.

Describe unstained wet mount

Unstained wet mount fixed in 0.2% glutaraldehyde: In this technique, a wet mount of the semen sample is prepared without staining, and the cells are fixed using a 0.2% glutaraldehyde solution.

How should the semen sample be prepared for morphological examination?

As most bull samples contain a high number of sperm cells, the sample should be diluted to allow for the observation of individual sperm cells on a microscope slide. A dilution of 0.1ml semen added to 1.0ml clear isotonic diluent (such as saline, ringer, or phosphate buffers) is typically used.

What is the procedure for preparing smears for morphological examination using the Karas method with modification by Kordel?

1. Prepare a thin smear of the semen sample and let it air-dry.
2. Fix the smear in a formol-saline solution for 3-5 minutes.
3. Stain the smear in an eosin solution for 3-5 minutes.
4. Gently wash the smear in distilled water until the red color disappears.
5. Stain the smear in an oak's bark concentration solution for 5-10 minutes.
6. Wash the smear in flowing water.
7. Air dry the smear.

What are some important considerations when performing morphological examination of spermatozoa?

* Smears should be made, fixed, and dried before the semen becomes cold to prevent artifacts.


* Extended semen containing substances like egg yolk or milk is not satisfactory for morphological examination.
* Fertile bulls typically show around 90% morphologically normal spermatozoa.
* The process of interpreting sperm morphology is retrospective, as abnormalities may have occurred days or weeks prior to ejaculation. Repeated collections can help with accurate interpretation.