VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

Sperm abnormality

ESTIMATION OF SPERM ABNORMALITY

Introduction

• Any deviation from normal morphological structure of spermatozoa is called as abnormal sperm.
• Every ejaculate will have some abnormal sperms in it.
• An increased prevalence of abnormal sperm is associated with a decrease in fertility.
• The appearance of an increased number of abnormal sperm in the ejaculate is a reflection of lesions of the testes and/or the excurrent duct system and provides a convenient clinical diagnostic aid.
• Overall length of a bull spermatozoon : 68 – 74 m
• Head - length : 8 – 10 m : width: 4 – 5 m
• Neck : 0.3 – 1.5 m
• Mid-piece : 8 – 10 m
• Tail - principle piece : 45 – 50 m : end piece : 2 – 4 m

60 per cent of the anterior head is covered by acrosome or galea capitis.

Sperm abnormality are produced by

• Inheritance
• Disease
• Adverse environmental effects
• Improper handling of semen.

The sperm abnormality increases with

• Extremes of temperature
• Reproductive infection
• Congenital testicular hypoplasia
• Acquired testicular degeneration
• Epididymal dysfunction
• Ephemeral fever
• To a subtle extent with advancing age

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Classification of Sperm Abnormality

• There are different types of classifications available. They are
  • Based on source of abnormality
  • Based on effect on fertility
  • Based on the location of abnormality

Based on source of abnormality

Primary abnormality

• They are appearing at the time of formation of spermatozoa.
• This is mainly caused due to aberration in spermatogenesis.
• These abnormalities are arising due to developmental defects in seminiferous tubules of diseased, degenerative or hypoplastic or inherited sterile conditions.
• Primary abnormalities are dangerous and they can be transmitted to their young ones also.

Secondary abnormality

• These abnormalities are acquired at the time of transport of spermatozoa through epididymis, accessory sex glands and urethra.

Tertiary abnormality

• Caused by the semen handler after collection. It is mainly due mishandling.
• Based on the effect on fertility
• Major abnormalities – Abnormalities which are having more effect on fertility is called as major abnormalities.

• Minor abnormalities - Some abnormalities will have lesser effect and is called as minor abnormalities.
• Based on the location of the abnormality

• This depends upon the region where the abnormality is located. It is divided in to head, mid piece and tail abnormalities.

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Methods of Assessing Sperm Morphology

• Sperm morphology is assessed by
  • Eosin-Nigrosin stain
  • Rose Bengal stain
  • Indian ink stain
  • Wright’s stain
  • Casaretts’s stain
  • Buffered formal saline method (Wet smear method)
• An eosin-nigrosin stain is commonly used as a morphology stain.
• The eosin-nigrosin sperm smears prepared for live and dead count is satisfactory for assessing abnormal spermatozoa.

Eosin-nigrosi staining method

Materials required

• Semen sample
• Glass slides
• Eosin stain (5%)
• Nigrosin stain (10%)
• Immersion oil
• Phase contrast microscope

Preparation of 5% eosin stain

Chemical	Quantity
Eosin powder	5 gm
2.9% sodium citrate	100 ml

• Weigh the eosin powder, put in pestle and mortar.
• Prepare 2.9% sodium citrate solution, boil it.
• Add the boiling solution to stain and grind it well. Finally filter and store it at 4 ° C.

Preparation of 10% nigrosin stain

Chemical	Quantity
Nigrosin powder	10 gm
2.9% sodium citrate	100 ml

• The preparation of the stain is as per the above method.
• After filtration the stain is stored in an air tight bottle at 4 ° C.

Staining procedure (Click here to view video demonstration)

• A drop of eosin, four drops of nigrosine and a small drop of semen are placed on a clean, grease free slide.
• Mix the semen first with eosin and then immediately with nigrosin stain.
• The mixture is taken on the edge of a slide and pulled across the top of another slide leaving a smear .

• Allow it to dry in air.
• 200 spermatozoa are counted under oil immersion at a magnification of 1000X in different areas of smear and classify them as normal, head abnormal, mid piece abnormal and tail abnormal sperms.

Calculation

Precautions

• Avoid artefacts at the time of preparation of the smear on the slide.
• Avoid cold shock and use of hypotonic solution if diluted semen is used.

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Rose bengal stain method

Preparation of Rose Bengal stain

• Rose Bengal - 3 gm
• Formalin (37-41%) - 1 ml
• Distilled water - 100 ml.
• The stain is prepared by adding and grinding the contents in pestle and mortar.

Staining method

• Prepare a thin smear of semen in a clean grease free slide and dry it
• Place slide in a jar containing Rose Bengal stain for 20 minutes
• Wash the slide in running tap water
• Dry it and examine under oil immersion microscope
• Count 200 sperms and express the results

Interpretation of result

Classification of sperm abnormalities

Head abnormalities	Neck abnormalities	Mid piece abnormalities	Tail abnormalities
Microcephalic head	Swollen neck	Coiled mid piece	Coiled tail
Macrocephalic head	Broken neck	Kinked/Bent mid piece	Terminally coiled tail
Pear shaped head		Broken mid piece	Broken tail
Tapering head		Bowed mid piece	Dag defect
Round short head		Abaxial mid piece	Stump tail
Lost acrosome		Double mid piece	Double tail
Loose head (normal & abnormal)	Immature Sperms
Knobbed acrosome	Proximal droplet
Diadem defect	Distal droplet
Double head	Teratoid

Inference

• A maximum of 20 per cent sperm abnormalities are allowed in bull semen (major 7.5% and minor 12.5%)
• Hereditary defects should not exceed 5 %
• Specific minor abnormalities should not exceed 10%

Interpretation of spermiogram

• An increased prevalence of sperm with morphologic abnormalities such as head abnormalities and retained proximal droplets may be evidence of either sexual immaturity or degenerative changes in the seminiferous epithelium of the testes.
• Abnormal sperm will usually disappear from the ejaculated as the bull ages and, in some cases, as the testes become larger.
• A bull that does not exhibit a normal spermiogram by 18 month age is a poor risk as a future breeding animal.
• In case of matured bull, it has to be differentiated whether the degenerative changes are transient or permanent which is very difficult and is complicated by the fact that 49 days are required for sperm to sperm to complete the spermatogenic cycle plus an additional two weeks are required for passage of the sperm through the epididymis.
• The presence of more that 15% major abnormalities or more than 30% total abnormalities, especially when coupled with other findings such as palpable testicular or epididymal lesions or inadequate scrotal circumference, is sufficient reason to classify a bull as questionable or, depending upon severity, as an unsatisfactory potential breeder.
• However, interpretation of a spermiogram requires information concerning the bull’s breeding history and the results obtained from a through clinical examination of the bull.

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