Semen Evaluation

Question 1

indications of semen evaluation (8)

Answer 1

1. Breeding Soundness Certification.
2. Before breeding/purchase for breeding
3. Diagnosis and/or prognosis of reproductive disorders.
4. Characterize semen samples for trade.
5. Processing insemination doses (fresh/ chilled/ frozen semen).
6. Guide clinical and management decisions.
7. Assessment of treatment / sperm production (e.g., toxicology and nutrition studies).
8. Frozen straw quality control

Question 2

semen collection with an artificial vagina; stimulates, species used in, pros, cons

Answer 2

• Simulates natural mating (shaping, texture, warmness, slipperiness, attitude).
• Commonly in bulls, stallions, rams, and goats.

Pros
* Comfortable for male, yields physiological semen

Cons
* Requires training to mount dummy/teaser,
* Not suitable for all species or individuals (uncooperative or physical limitations)

Question 3

semen collection with an electroejaculation; stimulates, species used in, pros, cons

Answer 3

• Probe into the male rectum,
• Delivers mild electrical stimuli to induce ejaculation.
• Commonly in bulls, rams, and sometimes in wildlife species.

Pros
* Does not require prior training,
* Useful for collecting males that are unable or unwilling.

Cons
* May cause discomfort or stress to the animal.
* Semen quality sometimes lower compared to other methods

Question 4

semen collection with an manual massage; stimulates, species used in, pros, cons

Answer 4

• Massages the animal’s penis to induce ejaculation.
• Commonly in Dog, Boar, Poultry.

Pros
* Does not require specialized equipment.
* Can be performed without the need for a teaser animal.

Cons
* Requires skill and experience
* May be time-consuming

Question 5

what are sperm cells highly sensitive to? (7)

Answer 5

• Temperature: sudden changes, abnormal hot/cold
• pH: Acidic or alkaline environments damage sperm cells.
• Osmotic Pressure:Hypotonic or hypertonic cause swelling or shrinkage
• Light Exposure: Prolonged exposure to light, especially UV light.
• Toxic Substances: Detergents, disinfectants, and heavy metals
• Time: sperm loses viability over time.
• Mechanical Stress: vigorous agitation

Question 6

things to do with semen handling (9)

Answer 6

1. Use clean, non-reactive materials (e.g., AVs, tubes, syringes, slides, pipettes).
2. Pre-warm all tools and equipment (~37–38°C).
3. Avoid sudden temperature changes.
4. Handle semen gently.
5. Keep samples away from light and air.
6. Avoid contact with water, detergents, or chemicals.
7. Train staff on correct techniques.
8. Update lab protocols regularly.
9. Record details for every animal and sample

Question 7

macroscopic evaluation of semen (4)

Answer 7

o Volume
o Colour
o Consistency
o pH

Question 8

routine evaluation of semen (5)

Answer 8

o Concentration
o Motility
o Morphology
o Viability
o Culture

Question 9

advanced evaluation of semen (4)

Answer 9

o DNA Fragmentation
o Mitochondrial Activity
o Oocyte penetration
o Proteomic and Genomic

Question 10

features of volume evaluation and influence

Answer 10

• Stallions (gel-free) 30–100 ml, Bulls: 2–10 ml, Dog (sperm-rich) 0.5–3 ml.
• Influence of species, breed, individual, method & frequency of collection, teasing, season

Question 11

normal vs abnormal colours of semen

Answer 11

• Normal: Milky white to creamy
• Abnormal Colors:
  o Pink/Red: Hemospermia (trauma/ inflammation reproductive tract)
  o Yellow: Urospermia, indicating urine contamination.
  o Greenish: Suggests pyospermia (infection).
  o Translucent: Low sperm concentration

Question 12

normal vs abnormal consistency of semen

Answer 12

Normal
* Semen appears slightly viscous and turbid, with a uniform texture.

Abnormal
* Watery: low sperm conc., excessive collection frequency , testicular hypofunction.
* Thick or Gel-Like: stallions excess seminal plasma proteins or prolonged abstinence.

Question 13

normal vs abnormal pH of semen

Answer 13

Normal
* Stallions: 7.2–7.8…..Bulls: 6.8–7.2
* Dog: 6.5–7.0 for the sperm-rich fraction.

Abnormal pH
* Acidic: Urospermia or inflammation
* Alkaline: Infections or contamination

Question 14

what is a hemocytometer used for

Answer 14

are the most common, the use of chambers in which cells can be
observed and counted within a known area, then allowing the calculation of the number of
sperm per unit of volume

Question 15

pros and cons of hemocytometers

Answer 15

Pros: Low cost and widely accessible. Direct visualization allows confirmation of sperm morphology and exclusion of debris.

Cons: Labor-intensive and time-consuming. Requires skilled technicians to ensure accuracy.
Errors can arise from improper dilution, uneven sample mixing, or misinterpretation of grid boundaries

Question 15

what is a Spectrophotometer used for

Answer 15

estimate sperm conc. based on the optical density or turbidity of a semen sample. Sperm conc. is directly proportional to its optical density when measured at a specific wavelength of light. As sperm cells scatter light, the higher the sperm concentration, the greater the light scattering

Question 16

pros and cons of Spectrophotometers

Answer 16

Pros
* Quick and efficient for high sample volumes.
* Relatively inexpensive equipment.
* Minimal sample handling compared to manual methods.

Cons
* Indirect measurement can be influenced by non-sperm (debris or other cells).
* Requires calibration for each species
* Suitable for raw but not extended semen (extender interfere light)

Question 17

what is a nucleocounter used for

Answer 17

a fluorescent dye (propidium iodide) to stain sperm DNA. Measures
fluorescence intensity to count sperm within a defined volume. First, dilute semen sample with detergent that damage the sperm membrane and allow PI to bind DNS

Question 18

pros and cons of a nucleocounter

Answer 18

Pros
* Highly accurate and specific to sperm, excluding non-sperm components.
* No calibration required, as sample preparation follows a standardized protocol.
* Quick analysis (approximately 30 seconds per sample).

Cons
* Requires proprietary consumables (diluents and disposable cassettes).
* Influence of somatic cells (contains DNA, ex: WBC, Epithelial cells)
* Higher cost compared to hemocytometers.
* Limited to species pre-validated by the manufacturer

Question 19

how to evaluate gross motility of sperm

Answer 19

• Single drop of undiluted semen + no coverslip is evaluated at 10×.
• Affected by the concentration of the sample.
• Swirling, Waves, Eddies – only in species with high sperm conc. (bull, ram, buck)

Question 20

motility; normal, motile vs non motile sperm for fertilization

Answer 20

• Historically, the most common test of sperm quality (easy to-do)
• Normal: bull 30-60% stallion 60%, Dog 70%
• Non-motile sperm definitely unable to Fertilize the ova.
• Motile sperm may/may not be able to Fertilize the ova (Motility poorly correlated
  with fertility, but still important parameter).
• Many morphologic abnormalities are motile!

Question 21

motility is a function of _____ and ____

Answer 21

midpiece and tail

Question 22

gross motility scores (4)

Answer 22

Very Good (VG): rapid dark swirls and eddies
Good (Good): slower swirls and eddies
Fair (F): no swirls, but prominent individual motion
Poor (P): little or no individual cell motion

Question 23

what is individual motility influenced by

Answer 23

Morphology, temperature, time between collection & evaluation, detergents, lubricants, latex, water, contamination with dirt, epithelial cells

Question 24

two main aspects to assess for individual motility

Answer 24

total motility, progressive motility

Question 25

visual inspection of sperm and classification

Answer 25

* Subjective assessment of sperm motility * Phase-contrast microscopy * Estimate if 50, 75, 25% of the sperm that are in motion

Question 26

what is computer assisted semen analysis used for

Answer 26

* use automated software combined with microscopy and video imaging to analyze parameters such as sperm motility (progressive and total), velocity, concentration, morphology, and other kinematic traits (straightness, and linearity) * Useful in research arena . objectivity and repeatability measurements.

Question 27

what is the most useful and important aspect of sperm quality? methods

Answer 27

* Sperm morphology is the most useful and important aspect of the semen quality Not time dependent, can fixed evaluated at a later * The eosin–nigrosin (EN) staining For (Morphology+ Viability) * Eosin is a vital stain penetrates dead sperm (pink) , * Nigrosin provides a dark background for contrast

Question 28

__% normal sperm is the threshold for a satisfactory sample

Answer 28

70%

Question 29

categories of sperm morphology

Answer 29

Each sperm is placed into 1 category, only: Normal or Abnormal head, midpiece, and tail defects

Question 30

types of abnormal sperm morphology (9)

Answer 30

A) Normal sperm B) macro, micro, tapered, pyriformhead , constricted, degenerate head C) Acrosomal defect D) Proximal and distal cytoplasmic droplets E) segmental aplasia, rough, enlarged mitochondrial sheath, bent midpiece, double midpiece/double head F) Bent tail (or hairpin tail), G) Coiled tail, with/ without encircling the head I) Premature germ cells with a single /multiple nuclei

Question 31

what is the dag defect

Answer 31

characterized by a figure -8- appearance of the midpiece. caused by gossypol toxicosis

Question 32

features to become a satisfactory potential breeder (4)

Answer 32

-Passed a physical exam. -Met the minimum requirements for scrotal circumference based on his age/breed -Has at least 30% (SFT) or (60%) WCABP progressive sperm motility. -Produces at least 70 % normal sperm