Semen analysis and bull fertility

Semen evaluation:

• Semen is also termed as seminal fluid. It is the fluid that is released from the male reproductive tract and comprises of sperm cells that can fertilize the female’s eggs.
• Among the cells, sperm are unique in form and function.
• Mature sperms are the terminal cells that are incapable to go further division or differentiation.
• One of the standard methods for evaluation of the fertility of breeding males is by examination of semen.

Evaluation of semen of bull and fertility of bull:

• For the evaluation of semen of bull, physical examination and a vaccination/ health certification is required.
• Generally, the minimal standards required for a classification of a specimen of bull semen are:
  • a. over 500 million sperm per ml.
  • b. >50% of motile sperm leads to forward progression.
  • c. Normal morphology is confirmed by >80%of spermatozoa.
• We can conclude that the bull is sterile only if the motile sperm are completely absent and the reproductive system has been properly examined.

Appearance and volume of bull semen:

• Bull semen should have a comparatively uniform and opaque appearance.
• Opaque appearance is the indicator of high sperm cell concentration.
• However, translucent samples consist of few sperm.
• The sample should be devoid of contaminants such as hair, dirt etc.
• Semen which is curd like in appearance comprising of chunks of material are avoided i.e. it suggests infection.
• Sometimes, presence of riboflavin results in yellow appearance of semen which is usually considered harmless.
• Repeated ejaculation leads in lower average volume.
• If the two ejaculates are extracted consecutively, the second generally has the lower volume.

Measurement of Sperm concentration:

• The measurement of sperm concentration can be done by using colorimeter, hemocytometer, or spectrophotometer.
• A hemocytometer is a microscopic slide with accurate scored chambers.
• The number of sperm per chamber are then counted.
• It is more tedious job but has greater preciseness.
• In contrast to that, spectrophotometer and colorimeter have a merit of being fast and accurate.
• The calibration of machine is done at 550nm.
• For the dilution of the ejaculate, 2.9% sodium citrate and 5ml of 10% formalin per litre.
• A standard curve that measures concentration versus 0.5% increment of light transmittance provides a range that is required to measure concentration.
• However, in presence of contamination, photometers are not accurate.
• The results can confound if the cloudy extenders are added prior to the estimation of concentration.
• The concentration of sperm ranges from 2X 10^8 sperm/ml in young bulls to 1.8 X 10^9 sperm/ml in mature bulls.

Measurement of Sperm motility:

• The measurement of the sperm motility requires subjective estimation of the viability of the spermatozoa and the effectiveness of motility.
• Light microscopic analysis is widely used.
• Sperm motility assessment is performed with raw and prolonged semen.
• Raw semen assessment is an indicator of sperm performance in its own accessory gland fluid.
• Greater sperm concentration will hinder the calculation of motility in the raw form, making it hard to distinguish individual patterns of motility.
• An aliquot of semen should be extended in a good quality extender (i.e. concentration of 25X 10⁶ sperm/ml) to overcome this restriction.
• The motility of sperm is highly prone to environmental factors (as such excessive heat or cold).
•  Hence, the semen needs to be shielded from harmful agents or circumstances, prior to study.

Process of sperm motility assessment:

• On a glass slide, a drop of extended semen is placed and smeared with other slide.
• Then, it is observed under microscope with a built-in-stage warmer and phase-contrast optics.
• To estimate sperm motility, magnification of 200X or 400X is usually used.
• The parameters of motility consists of:
  • a. Percentage of motile sperm (normal is 70%-90% motile).
  • b. Percentage of increasingly motile sperm.
  • c. velocity of sperm (based on an arbitraty scale on between 0-4).
  • d. Longevity of sperm motility in raw semen (at room temperature 20^oC to 25^oC) and in extended semen (at room temperature, or refrigerated temperature 4^oC to 6^oC).
• Several patterns of motility are observed.
• In diluted semen, general patterns of sperm motility appear in a long semiarc pattern.

Several factors that affect sperm motility:

• The extender might alter motility slightly, generally by enhancing velocity measures.
• A high percentage of sperm may show circular motility pattern, which generally resolves after 5-10 minutes in the extender.
• If the sperm are swimming in a tight circular motion, it indicates that they could have been in presence of cold shock.
• There is correlation between motility patterns and infertility or subfertility of males.
• For unbiased evaluation of sperm motility, various techniques have been developed.
• Some of the procedures are time-lapse photomicrographs, frame-by-frame playback videomicrography, spectrophotometry, and computerized analysis.

Abnormalities in morphology of sperm:

• Each semen sample comprises of few abnormal sperm cells.
• The abnormalities in morphology of sperm is directly related to the fertility of livestock.
• One of the causes for the abnormality is heat stress.
• Heat stress results in large number of damaged sperm.
• Long exposure of high ambient temperature in combination of high humidity may cause sterility in male for upto 6 weeks.
• During the recovery duration, high number of abnormal sperm are observed in ejaculates.
• The effects of heat stress can be reduced by supplying sufficient shade and clean cool water.
• In the condition, when abnormal sperm cells exceed 20%, it results in infertility.
• The microscopic magnification is determined by the type of abnormality being evaluated.
• The classification of morphologic abnormalities are as:
  • 1. Primary
  • 2. Secondary
  • 3. Tertiary
• Primary abnormalities are related with sperm heads and acrosome.
• Secondary abnormalities is defined by the presence of the droplet on the midpiece of the tail.
• Other tail defects are shown by tertiary abnormalities.
• The use of eosin-nigrosin stain analyzes the sperm morphology.
• Wright’s and William’s stains can also be used.
• By the use of high microscopic magnification, the examination of stained slides are done.
• Among 150 spermatozoa examined with abnormal sperm were classified into 5 categories:
  • a. tailless
  • b. abnormal heads
  • c. abnormal tail formations.
  • d. formation of abnormal tail with a proximal cytoplasmic droplet.
  • e. Formation of abnormal tail with a distal droplet.

Evaluation of quality of semen:

• The technique for sperm quality evaluation from frozen semen are linked with motility and intact acrosome.

Procedure:

• In a 95^oF water bath, two straws (0.5ml-0.25ml) are thawed.
• After 45 seconds, one straw is removed and a paper towel is used to dry it. Note: Drying is essential as water can be harmful to sperm.
• Towards the end of the cotton plug, the contents are shaken.
• The other end of straw is cut off and the semen is released into a small clean disposable test tube by cutting a small opening just below the cotton plug.
• On a warm side with a cover slip, a small drop of semen is placed.
• Microscopic analysis is used to perform initial motility readings.
• After incubation of the second straw for 3h at 95^oF, a microscope with interference optics (1000X) and oil immersion is used to analyze the % of intact acrosomes and abnormalities of the sperm.
• The anterior two-thirds portion of the sperm head is covered by the acrosome.
• The acrosome comprises of the enzymes that facilitate the capacitated sperm to penetrate the egg.
• After the 3hrs of incubation, there is direct correlation with the percent intact acrosomes, and fertility.

Ancillary tests to evaluate quality of sperm

a. Electron microscopy:

• In light microscopy, limited magnification is found, and hence the evalution of sperm morphology is limited.
• By the use of scanning and/or transmission electron microscopy, abnormalities in sperm structure can be detected precisely.
• These two microscopic techniques provide details of high-resolution and allow closer examination of sperm morphology.
• Scanning Electron microscopy  observes the 3D visualization of the entire sperm.
• Transmission electron microscopy allows to view cross-section of sperm revealing ultra-structural detail.

b. Sperm chromatin structure assay (SCSA):

• The procedure termed as flow cytometry is employed for the evaluation of structural integrity of the sperm chromatin.
• It does so by measuring the relative amounts of double stranded and single stranded DNA in sperm populations.
• SCSA has been considered fruitful for the identification of some forms of subfertility in bulls and stallions.
• In a few minutes, SCSA can screen 5,000 to 10,000 sperm.

c. Other fluorescent probes:

• The investigation of new fluorescent probes is being carried on for the evaluation of their reliability for sperm function tests by the use of microscope or flow cytometer.
• These assays can provide detailed information on specific sperm features such as plasma membrane integrity, acrosomal integrity, and mitochondrial integrity, and mitochondrial potential.
• Hence, these assays can prove to be very useful accessory tests.

d. Antisperm antibody assay:

• The maturation of sperm completes in the adluminal compartment of the seminiferous tubules in order to circumvent immunologic attack.
• If the sperm escapes from the adluminal compartments of the seminiferous tubules, it triggers an immune response following the formation of antisperm antibodies.
• For this immunologic attack of sperm, the precipitating factors include lacerations, neoplasms, biopsies, trauma, or degenerative changes of the testes.
• Antisperm antibodies hinder the fertilization process in case of humans, however, the mechanism of action stays unresolved.
• Antisperm antibodies can play as a factor to affect the fertility of stallions.
• Primarily, the autoantibody response to sperm is of the IgG type in serum and of IgA type in the seminal plasma.
• For the presence of auto antibodies, evaluation of serum, seminal plasma, and sperms of stallions is lately being investigated.
• The best predictive fertility index is provided by the tests that evaluate the actual binding of antibodies to sperm.

e. Biochemical analysis of seminal plasma/ Secretions of female reproductive tract:

• On post-thaw motility of cryo-preserved sperm, the protein concentration, specific protein composition of seminal plasma, electrolyte concentration do not supply abundant predictive information.
• Osteopontin and lipocalin-type prostaglandin D synthase are the 2 proteins of epididymal origin that serve as markers of bull fertility potential.
• The accessory glands secrete deoxyribose-1 like enzyme and 2 tissue inhibitor of metalloproteinases at ejaculation and bind to sperm as they traverse the male reproductive tract, and are also linked with greater bull fertility.
• The components of the female reproductive tract secretions such as glycosaminoglycans bind to bull sperm and cause acrosome reactions invitro.
• There is a high correlation between % acrosome reacted sperm triggered by glycosaminoglycans and non-return days.