Producing a quality semen sample is imperative to the success of artificial insemination in sheep. Collection of a clean ejaculate full of high quality spermatozoa and its proper extension/dilution with a standard diluent maintaining its quality during the storage period either at 4°C or ₋196°C are the most crucial steps in the production of quality artificial insemination doses. Determination of quality of semen produced is actually based on multiple parameters yet most artificial insemination centres rely on sperm motility only. Strong evidence exists in the literature that a single semen quality parameter e.g. sperm motility cannot predict the fertilizing potential of a semen sample, therefore a group of parameters have to be considered together to predict its fertility. The most important sperm quality parameters include-sperm motility, membrane viability, functional membrane viability, morphological abnormalities, acrosomal integrity superficial, acrosomal integrity molecular, level of lipid peroxidation, total viable count and sperm DNA integrity. There are three methods for semen collection- massage method, electro-ejaculation and artificial vagina (AV) method. Out of these, semen collection by AV has gained much popularity owing to the superior quality semen ejaculate obtained. There are many extenders that can be used for dilution but tris citric acid egg yolk fructose extender (TCEYF) is the most common extender used

1. Course: Andrology and Male infertility
Lecture 4: Semen collection & semen evaluation
Dr. Farooz Ahmad Lone (Ph.D Gold Medalist)
Assistant Professor
Animal Reproduction

2. Semen collection and evaluation
• Ruminants and stallion….......artificial vagina
• Boar and dog…………….digital manipulation
Evaluation
• Gross examination
• Microscopic examination

3. Q. No. 8: Sperm mitochondrial membrane potential is evaluated
by using:
• FITC-PNA
• JC-1
• CFDA
• Bodipy
Mitochondrial membrane potential is evaluated by:
• Rhodamine 123
• JC-1 (quantitative)
• Mito-tracker

4. Microscopic examination
a) Sperm motility (CASA-computer assisted sperm analyser)
b) Viability
• eosin-nigrosin staining
• SYBR14 + PI or CFDA + PI
c) Morphological abnormalities
• Eosin-nigrosin
• Williams stain (Head abnormality)
d) Acrosomal integrity
• Giemsa stain
• FITC-PNA
e) Lipid peroxidation
• Thiobarbituric acid method (TBARS method)
• BODIPY probe

5. g) Sperm DNA integrity/Chromatin status
• Tunnel assay
• SCSA
• COMET assay
• SCDT
• Toluidine blue
• Acridine orange
• Chromomycin A3
h) Cold shock resistance test
i) Methylene blue reduction test (MBRT)…measures
dehydrogenase activity in the sample
J) Fructolytic index----amount of fructose in mg utilized by 1 billion
sperm in one hour at 37°C
k) CMPT
l) Zona free hamster egg penetration test

6. Nomenclature
Volume
• Zero/none Aspermia
• Reduced Hypospermia
• Increased Hyperspermia
Sperm concentration
• Zero Azoospermia
• Reduced Oligozoospermia
• Normal Normo-zoospermia
• Increased Polyzoospermia
Sperm motility
• Decreased Astheno-zoospermia
Sperm viability
• All dead Necrozoospermia
Abnormal sperm
• High percentage Terato-zoospermia