Quality determination of frozen semen 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.

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:

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 (5x5) secondary squares. Each secondary square is further divided into into 16 (4x4) tertiary squares. Thus each primary square is divided into 400 (25x16) 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.
i. Preparation of diluting fluid
• Eosin-Y  - 0.05 gm
• Nacl - 1.00 gm
• Formaldehyde - 1.00 ml
• Distilled water - 100 ml
ii. 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

• 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;

   Sodium citrate (g)	0.735
   Fructose (g)	1.351
   Millipore water (ml)	100
   Osmolality (mOsm Kg-1)	150

   
   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.
   

   Giemsa stain	3.8 gm
   Absolute alcohol (GR grade)	375 ml
   Glycerol (AR grade)	125 ml

5. 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:
   

   Composition:
   Solution A:
   Sodium phosphate dibasic (Na2HPO4.2H2O)		11.876 gm
   Distilled water		1000 ml
   Solution B:
   Potassium phosphate monobasic (KH2PO4.2H2O)		9.08 gm
   Distilled water		1000 ml

   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.

Suggested Reading

• Anderson, J. 1945. Semen of animals and its use for artificial insemination. Imperial Bur. Anim. Breed. & Genet., Edinburgh, Scotland.
• Angasaria, H.R., Pareek, P.K., Aminudeen, Datt, M and Purohit,G.N. 1999. Sperm quality changes during handling of bovine semen. Indian J of Dairy Sciences., 52(4): 265-267. Insemination. Bookmark Publication, Pune, India
• Bishop, D.W. 1962. Spermatozoan motility. Physiol. Rev., 42: 1
• Brucker, C. and Lipford, G.B.,1995. The human sperm acrosome reaction Physiology and regulatory mechanisms: An update. Hum. Reprod. Update., 1(1): 51-62.
• Chan, S.Y. W.: Fox, E.J., Chan, M.M.C., Tsoi, W., Wang, C., Tang, L.C.H., Tang, G.W.K. and Ho, P., 1985. The relationship between the human sperm hypo osmotic swelling test, routine semen analysis and the huma sperm zona free hamster ovum penetration assay. Fertil and Steril., 44: 668-672.
• Drevius, L.O. and Ericksson, H., 1966. Osmotic swelling of mammalian spermatozoa. Exp. Cell Res., 42: 136- 156.
• Jeyendran, R. S., van der Ven, H. H., Perez Pelaez, M., Crabo, B.G., Zaneveld, L.J.D., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod. Fertil., 70: 219-28.
• WHO laboratory manual for the examination and processing of Human semen. 2010. Fifth Edition.

Source : Saroj Rai, Rani Alex*, M.Karunakaran and R. Behera
Scientist, ICAR-National Dairy Research Institute, Karnal, *Scientist, Central Institute for Research on Cattle, Meerut