Different Methods of Assessing Semen Quality before use for AI in Domestic Animals

Different Methods of Assessing Semen Quality before use for AI in Domestic Animals

 Male fertility and semen evaluation is of paramount importance as its semen is used to breed a number of females. Many of the semen collection centers have made it mandatory to evaluate the semen in accordance to Central Monitoring Units (CMU) for Semen Station. Assessing the quality before semen is further used reduces time and resources involved. Semen should be evaluated as soon as possible after collection to avoid the changes due to exposure of light, chemicals, lubricants etc. Henceforth, vitality, motility and morphology of the spermatozoa are the initial quality parameters judging semen quality.

INFERTILITY IN MALE ANIMALS

Male infertility is the key-determining factor that has to be primarily taken into consideration with relation to fertilization. Infertility is noticed both in male and female animals, but it is very crucial in case of male animals. Because, the semen collected from an individual male animal is used for Artificial Insemination to a number of female animals. Hence, assessing the fertility status of male animal is of prior importance. There are various ways to examine the semen quality. This includes physical examination, chemical examination, microscopic examination and biological tests.

Sperm Cell

A typical sperm cell has three distinguishable regions viz. head, middle-piece and tail. In addition to the normal spermatozoa, one finds a variety of abnormal, immature, degenerated cells etc. which deviates from the normal cell structure, from tapering, double head, double tail, mega cells etc. Although a high degree of abnormal cells above 25% in bulls is undoubtedly associated with sub fertility. The shape of the sperm head is ovoid in bull, ram, boar and rabbit. The anterior part of the nucleus is covered by a cap like structure called acrosome. After being deposited in the female genital tract, the spermatozoa traverse a long distance (prior to fertilization) during which some important functional and structural changes of spermatozoa takes place of which capacitation is of utmost importance. Capacitation helps to bring about some changes in the intact plasma membrane, which is followed by the release of acrosomal enzymes viz., hyaluronidase, acrosine etc leading to sperm binding and penetration through the zona pellucida and fusion with oocyte. Most workers examined the acrosome abnormalities by using Giemsa stain technique.

Besides acrosome intactness, membrane integrity is important not only for sperm metabolism but also plays a tremendous role in fertilization because a correct change in the properties of membrane is required for sperm capacitation , acrosome reaction and binding of the spermatozoa to the egg surface for which biochemically active membrane is required. The test is based on the principle that when the sperms are subjected to a hypo osmotic solution, the cells with intact membranes take up water apparently without a significant enlargement of their area, thus forcing the flexible apparatus of the tail to bend and coil. Viable sperm in a hypotonic solution has been shown to develop bent and coiled tails whereas dead sperm had straight tails probably associated with cell lysis. Therefore it was hypothesized that the ability of the sperms to swell in hypotonic solution indicates its membrane integrity and normal function activity.

As per the guidelines given by the Central Monitoring Units (CMU) for Semen Station the cut off level for the frozen semen samples are:

PHYSICAL EXAMINATION

Physical parameters of semen vary greatly with individual species. So, species difference has to be taken into consideration. Physical examination indicates the gross characteristics of semen.

1. Volume of semen: Volume of semen is measured by suing a graduated tube or a small cylinder. Individual animal species has got individual difference in semen volume.

2. Appearance of semen: Normal freshly collected semen has a cloudy white color. Intensity of cloudiness is directly correlated with sperm density. If the semen is yellow in color, it’s an indication of any infection and the condition can be called as pyospermia. Brown and red color of semen indicates that the semen is mixed with blood, which may be because of any traumatic injuries in male reproductive tract and the condition is termed as hemospermia.

3. Consistency of semen, i.e., viscosity of semen: Viscosity of semen is measured only after liquefaction. A glass rod is dipped into the semen sample and it is raised to measure the length of the semen thread. If thelength is more, it indicates that the viscosity is less and vise-versa. High viscosity of semen indicates infection in genital tract especially prostrate and seminal vesicles.

4. pH of the seminal fluid: pH of the semen can be measured by using pH paper, pH meter and indicator dyes. pH is mainly dependent on sperm concentration, semen collection and quality of semen. A drop of semen is spread evenly on pH paper and after 30 seconds; the color of impregnated zone is compared to calibration strip to read the pH.

CHEMICAL EXAMINATION

Chemical examination includes various tests that utilize various chemicals to analyze the fertilization strategy

1. Resistance to cold shock: This test is done to assess the freezability or preservability and fertilizing ability of the semen of a particular animal. Spermatozoa are subjected to cold stress by exposing the semen to ice. The percentage of live sperm after exposure to cold shock is counted. Examination or counting of live sperm has to be done immediately after collection. One ml of semen is taken in a clean test tube and it is placed on a beaker with crushed ice for 10 minutes. After 10 minutes, determine the live sperm % and sperm motility. Compare the observed values with the previous values and estimate the freezability of the desired semen sample.

2. Millovanov’s resistance test: It is the test that is used to assess the resistivity of spermatozoa towards 1% Sodium Chloride solution. The measurement of resistance is denoted as the millitre of 1% Sodium Chloride solution required stopping the progressive motility of spermatozoa in 0.02 ml of semen. Check the progressive motility of sperm before starting the experiment. Then, pipette out around 0.02 ml of semen into a conical flask. Add around 10 ml of 1% Sodium Chloride solution and assess the progressive motility. Likewise, keep on adding 10 ml of 1% NaCl solution and check for progressive motility upon each addition. Addition of NaCl has to be done upon complete ceasing of progressive motility.

3. Methylene Blue Reduction Test: The principle behind this test is that libertation of hydrogen ions during metabolism of sperm will reduce the intense blue coloured methylene blue dye to a colorless leucomethylene blue. The dehydrogenase enzyme is the key determinant behind the release of hydrogen ions. The time taken for color change is directly proportional to the concentration and motility of spermatozoa. If the semen sample is having high degree of motility and concentration, it leads to release of more hydrogen ions. This makes the time required for color change from blue color to colorless at a faster rate. Take a 5ml test tube which is clean, add fresh semen sample of around 0.2 ml and add 0.8 ml of egg yolk citrate diluent. To this, add methylene blue dye of around 0.1 ml and mix the contents. Place the test tubes in a water bath which is maintained at 46.5 C. Note down the time taken for a change in color from blue color to colorless.

4. Resazurine reduction test: This test is considered as an indicator to assess the metabolic activity of semen. As like methylene blue reduction test, dehydrogenase enzyme activity is the key factor behind this test. The dehydrogenase helps in the liberation of hydrogen ions that makes the blue color of Resazurine to pink color at first and finally to a colorless solution. High quality good semen sample tends to reduce the blue color of Resazurine to pink within a minute and it requires only 4 minute making it to colorless.

5. Fructolysis index: It is defined as the amount of fructose which is utilized by 109 number of spermatozoa in one hour at a temperature of 37 C. The amount of fructose metabolized in the semen sample is directly proportional to the metabolic activity of spermatozoa. The rate of fructose utilization has got direct relationship with the semen quality. Measuring the disappearance of sugars and the lactic acid accumulation by a number of spermatozoa under specific condition and in a specified time is termed as fructolysis. Always, a significant correlation is found between fructolysis and sperm concentration.

6. Oxygen Utilization Test: Active sperms can take-up the maximum volume of oxygen per unit time. Volume of CO2 produced by the spermatozoa per unit of time divided by the volume of O2 consumed in the same unit of time is termed as respiratory quotient (ZO2). The ZO2 value of Bull semen is found to be 21. This test indicates live percentage of spermatozoa and spermatozoa activity.

7. Pyruvate Utilization Test: Semen quality could be graded in relation to the O2 consumption after the addition of pyruvate and pyruvate plus 2:4 dinitrophenol. To reduce the exogenous metabolism to a low level, fluoride is added to the sample. Upon addition of pyruvate, there will be an increase in oxygen consumption in high fertile bull semen and low fertile bull semen. Upon addition of 2:4 dinitrophenol, uptake will be increased up to two folds and there will be no increase in low fertile bulls. Monometric equipment can be used to measure oxygen uptake.

MICROSCOPIC EXAMINATION

Microscopic examination indicates analyzing the sperm characteristics using microscope

1. Presence of other cells: The following are the materials may be present inside the semen. From the animals, the foreign materials may be dung, pus, urine, hair and dust. Semen can also be contaminated by sand, insects, bedding materials and dried dung, if the animals are not properly cleaned. While using Artificial Vagina, semen can be contaminated with lubricant jelly, dusting powder and water.

2. Sperm motility (individual motility and mass activity): It is the percentage of spermatozoa that are progressively motile. Sperm which swims forward briskly in a straight line is considered as progressive motile sperm. Mass activity is considered as a collective movement of sperms in a wave like motion. Mass activity is measured by placing a semen drop on a prewarmed glass slide and place a cover slip over it and it is examined under low power microscope.

3. Sperm concentration by using a hemocytometer chamber: Semen has to be diluted with a diluent mixed with eosin stain. Aspirate the semen up to the notified mark in a diluting pipette and aspirate the diluting fluid. Clean the tip of diluting pipette and discard the few drops. Charge the haemocytometer by releasing the fluid below the coverslip which is placed over the hemocytometer. Avoid formation of air bubble and wait for 1-2 minutes to settle. Examine the charged hemocytometer under low power of microscope followed by high power microscope. Count the number of sperms in the desired chamber of hemocytometer. Total number of sperms has to be calculated by taking dilution factor into consideration.

4. Sperm vitality (live and dead sperm %) by using various stains: Place a drop of eosin dye and four drops of Nigrosin stain and add a small drop of semen on a grease free, clean slide. Mix the eosin and semen first and then mix it immediately with Nigrosin. The mixture should be taken on a slide edge and make a smear by pulling it across the top of another slide. Allow it to dry by moving in air. 200 spermatozoa are counted under the oil immersion of microscope in different areas of smear.

5. Sperm morphology (sperm abnormalities): Place a drop of eosin dye and four drops of Nigrosin stain and add a small drop of semen on grease free, clean slide. Mix the eosin and semen first and then mix it immediately with Nigrosin. The mixture should be taken on a slide edge and make a smear by pulling it across the top of another slide. Allow it to dry by moving in air. 200 spermatozoa are counted under the oil immersion of microscope in different areas of smear. Abnormal structures include bent tails, decapitated head, tail piece broken, mid piece broken and diadem effect. The sperm percentage with normal shape and size is determined. A major abnormality which has to be taken into consideration includes head, middle piece and tail abnormalities.

6. Sperm membrane integrity by hypo-osmotic swelling test: Integrity of plasma membrane is a key determining factor for maintenance of fertility. Capacitation and acrosome reaction are the essential physiological process which sperm has to achieve to attain fertilization. There is a need for a test which analyse the membrane integrity of spermatozoa. Under normoosmotic conditions, there won’t be any shrinkage and swelling of spermatozoa. Under hypo osmotic conditions, transport of fluid occurs in an intact cell membrane between outside and inside the cell. Bulging or tail bending will occur because of fluid influx. Higher rate of Hypo-osmotic swelling percentage indicates that the semen is having higher fertility rate. Hypo-osmotic swelling is directly proportional to the Freezability of semen.

7. Acrosome Integrity Test: Take grease free, clean and pre-warmed slide and add a drop of diluted semen (1:5 to 1:10 in 2.9 per cent sodium citrate) and air dry the smear. Immerse the slide in 5% formaldehyde for 30 minutes for fixing the smear. Wash the slide in running tap water and air dry the smear. Then immerse the slide in Giemsa solution for about 3 hrs at 37C. Finally wash the slide with running tap water and dry the smear. Examine the slide under oil immersion of phase contrast microscope and count the 200 sperms. Check for the integrity of acrosome and the fresh semen should have 80% of acrosome integrity and frozen semen should have 65%. Hereditary defects like diadem defect and knobbed acrosome should not exceed 5%.

Common Laboratory Evaluation Techniques

Macroscopic assessment of the semen quality

1. Colour – milky white, creamy and lemon colour
2. Volume – read from semen collection vials which is 15ml graduated sterile tubes
3. Mass activity – A drop of post thaw semen samples semen is spread uniformly over a clean grease free glass slide maintained at 37ºC by a thermostatic plate. Motility is graded under low power (10x) according to the mass movement of the spermatozoa. The motility is judged according to its progressiveness.
4. Sperm concentration – Today, the Spectrophotometer is the most widely used method for sperm cell concentration analysis. It is easy, quick and reliable. To run an evaluation, a cuvette or microcuvette is filled with the ejaculate sample (properly diluted) and placed into the spectrophotometer where the optical density of the sample is read. The sperm cell count is then calculated based upon the optical density. On the contrary, the haemocytometer is considered a gold standard for assessing the sperm numbers. After diluting semen in clear diluents, a haemocytometer can be used to determine the concentration of sperm in an ejaculate. Each RBC chamber (primary square) is divided into 25 (5×5) secondary squares. Each secondary square is further divided into into 16 (4×4) tertiary squares. Thus each primary square is divided into 400 (25×16) tertiary squares. These total 400 tertiary squares have a total area of 1mm². When a drop is charged under the cover slip in a Neubauer cell counting chambers, the thickness of the film on chamber is 0.1mm. Thus, the total volume of the semen covering 400 tertiary squares in RBC chamber is 0.1 mm³.

1. Preparation of diluting fluid

1. • Eosin-Y  – 0.05 gm
   • Nacl – 1.00 gm
   • Formaldehyde – 1.00 ml
   • Distilled water – 100 ml

1. Procedure: Mix the semen thoroughly gently so that a representative sample is obtained. Take a drop of semen on a glass slide and suck the semen up to 0.5 mark of RBC counting pipette of haemocytometer. Clean the tip of RBC pipette with finger tip. Draw diluting fluid in the same pipette up to 101 mark (above the bulb). Roll the pipette between the palms of hand for 2-3 minutes. Release few drops of diluted semen and repeat the mixing. Focus the Neubaur´s counting chamber under microscope first under low power and then under high power objective. Place the coverslip on the counting chamber of haemocytometer. Transfer a very small drop of semen on neubaur´s counting chamber to charge the slide just below the cover slip. Overflowing of semen on the slide is to be avoided. Wait for 2-3 minutes to allow the sperm to settle down. Examine the charged haemocytometer slide under high power objective and count the number of sperm cells in left top, right top, right bottom, left bottom and central secondary squares that are meant for counting RBC.
   iii. Calculation

1. • No. of sperms counted in 5 secondary square = n
   • No. of sperm in 25 secondary square = n x 5
   • No of sperms in 0.1mm³ of undiluted semen = n x 5 x 200
   • No of sperms in 1cc of undiluted semen  = n x 5 x 200 x 1000=  n x 10 x 1000000

Microscopic assessment of the semen quality

1. Incubation Test – The frozen semen straw is carefully taken out from the liquid nitrogen can, shaken and put in the thawing unit at 37° C. After a minute, the straw is assessed for the post thaw motility under a microscope. The remaining straw is left in the thawing unit for an hour after which the motility is assessed in the similar way. Ensure that the temperature is maintained at 37° C.
2. Sperm viability and Sperm morphology – The staining solution for the one-step technique contains 0.67 per cent eosin-Y and 10 per cent nigrosine is used. Thus, 0.67 g of eosin-Y yellow and 0.9 g sodium chloride is dissolved in 100 ml distilled water under gentle heating. Then 10 g of nigrosine is added. Boil the solution and allow to cool it to room temperature (20°C) after which it is filtered through filter paper (Whatman filter paper no. 40) and store in a dark and sealed glass bottle. Before use, bring the temperature of staining solution to room temperature.
   Procedure: Semen samples are kept at 37°C for 30 min before analysis. Forty microlitre (ml) of neat semen is mixed in a micro-centrifuge tube with 400 ml eosin-nigrosine staining solution. Keep the suspension for one minute at room temperature (27°C). Then, a 12 µl droplet is transferred with the pipette to a labeled microscope slide (pre-warmed to 37°C) for preparing smear. Duplicate smears are made from each sample. After smearing it is allowed to air dry at room temperature. About 200 sperms are assessed under bright field 100X oil immersion objectives. Sperms that are white (unstained) are classified as non-eosinophilic and those that show any pink or red coloration is classified as dead (eosinophilic), with the sole exception for sperm with a slight pink or red appearance restricted to the neck region (‘leaky necks’), which are assessed as non-eosinophilic.
3. Morphological assessment – The same slide of eosin-nigrosine stain is used for screening morphological abnormalities. A drop of oil is applied to the cover-slip and the semen is examined on 100X objectives under bright field. If the preparation is too thick, examination will be difficult because many of the sperm head will be lying on their edges rather than flat. Each cell, even in thin preparations, is usually not totally in one focal plane and one must therefore focus up and down slightly on each cell.  About 200 spermatozoa should be counted in different fields and percentage of abnormal spermatozoa is calculated.
4. Hypo-osmotic swelling test (HOST) – Hypo-osmotic solutions of 150 mOsmol l-1 is prepared as follows;

Procedure: One ml of hypo-osmotic solution, having an osmotic strength of 150 mOsm Kg^-1 is mixed with 0.1 ml of semen and incubated at 37°C for one hour. Following incubation, a drop of well mixed solution take on a clean dry glass slide and cover with a cover-slip. Sperm tail curling is recorded as an effect of swelling due to influx of water. A total of about 200 spermatozoa are counted in different fields on 40X objectives under DIC phase contrast. The total proportion of swollen spermatozoa is calculated by dividing the number of reacted cells by the total spermatozoa counted in the same area and multiplying the figure by 100.

Acrosome integrity: Staining is carried out as described by Hancock (1952).
Preparation of Giemsa stain : Ground Giemsa stain with absolute methanol in a pastle and mortar. To this add glycerol. Stain mixture is stored at 37⁰ C for one week. During this storage period, shake it for 2 minutes each day.
Preparation of Soreson’s phosphate buffer:

The desired pH (7.0) can be obtained by adding enough solution B to solution A (61.2 ml) given below to make a volume of 100 ml. Preparation of final stain solution: Dilute 3.0 ml of Giemsa stain with 2.0 ml Sorenson’s M/15 phosphate buffer (pH=7.0) and 35 ml distill water. The staining solution is stored in refrigerator. Fresh stain is prepared every week.
Staining procedure : A thin smear of extended semen is prepared on a grease free, clean and dry slide. The smear is air-dried at room temperature for at least 10 minutes in a current of warm air. Smear is fixed by immersion in buffered formal saline (10 percent) for 15 minutes. Then it is washed in running tap water for 15-20 minutes and dry. Again immerse the slide in buffered Giemsa solution for 90 minutes and rinse briefly in distilled water and dry. Study the dry smears at 100X oil immersion objectives. About 200 spermatozoa are counted for acrosomal status after staining.

Conclusion

Although, manual semen analysis using a light microscope and motility estimation based on the use of best guess has been the standard method for analysis in most quality control laboratories for years which are much prone to technical errors. However, semen evaluation is important, as it allows us to identify semen samples that are likely to have poor fertilizing potential. However, the only way that we will be able to develop assays that can be consistently correlated with fertility is to develop unbiased assays that measure multiple sperm attributes simultaneously. We can use those laboratory assays to develop a test to predict fertility. Therefore, the essential practice should be accurate, quick, reproducable and affordable.

Although so much time and effort is being put into developing new laboratory assays of spermatozoa, the results of a particular laboratory assay and fertility varies. Particularly when evaluating a single sperm attribute, we can determine which samples are likely to have poor fertilizing ability (those with few motile sperm in them, for instance), but we cannot determine if the sample will be fertile. For example, all the motile sperm in a sample may also possess damaged acrosome and will be infertile for an attribute not evaluated, and this will cause the correlation between motility and fertility to be low.

However, semen evaluation is important, as it allows us to identify semen samples that are likely to have poor fertilizing potential. However, the only way that we will be able to develop assays that can be consistently correlated with fertility is to develop unbiased assays that measure multiple sperm attributes simultaneously. We can use those laboratory assays to develop a test to predict fertility.

 Compiled  & Shared by- This paper is a compilation of groupwork provided by the

Team, LITD (Livestock Institute of Training & Development)

 Image-Courtesy-Google

 Reference-On Request

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