In this article we will discuss about the screening methods for anti-fertility activity of drugs in males.
1. In Vivo Methods:
Fertility Test:
Fertility test is based on the evaluation of the average litter size. Anti-fertility agents negatively affect the average litter size.
Groups of 5-10 male rats of proven fertility are treated with the drug and are paired with fertile females in the ratio of 1:3. Daily vaginal smears are examined for the presence of sperms; normally within one week all females which have passed through 1 estrus cycle would have mated. The mated animals are kept separately till the gestational period. The litters are counted and using the following formula, the average litter size is calculated.
Average litter size = Total litter/No. of females mated
If vaginal smear shows leucocytes in 10-14 days, pseudo-pregnancy is confirmed. If insemination is not detected then inhibition of libido or aspermic copulation might be the cause.
Fertility patterns can be obtained from changes in average litter size
Cohabitation Test:
This test determines the time interval for litter production after placing treated males with 2 females each. The date of mating is calculated from the date of parturition. This method is suitable for drugs known to cause sterility for several weeks.
Adult female and male albino rats of proven fertility are used for the study. They are kept for mating in the ratio of 2:1 till both females deliver litters. The date of mating is calculated from the date of parturition. The time interval for litter production after placing treated males with the two females is calculated.
Subsidiary Test:
This test determines the changes in spermatozoa count with time. The anti-fertility drugs affect the spermatozoa count negatively.
Adult male albino rats weighing between 150-250 g are used for the study. They are kept in a cage containing artificial or animal vagina. The vagina is artificially simulated by a cylindrical plastic jacket with a rubber liner filled with water at 5°C. 0.5 ml of ejaculate is diluted with saline containing traces of formalin. The resulting suspension is counted on a haemocytometer.
2. In Vitro Methods:
Spermicidal Activity:
Spermicidal drugs are diluted with normal saline and serial dilutions are made. 0.2 ml of human seminal fluid is mixed with 1 ml of spermicidal solution. Then the mixture is incubated at 37°C for 30 min. A drop of the mixture is placed immediately on a slide and at least five fields are microscopically observed under high power (× 400) for assessment of sperm morphological changes and motility. Effective agents can immobilise and kill the sperms.
Immobilisation Assay:
The cauda portion of the epididymes of a ram is isolated and minced in 0.9 % saline solution (pH – 7.5). It is filtered through a piece of cheese cloth to get a sperm suspension. For human samples, ejaculates (n = 10) from normal subjects after 72-96 h of sexual abstinence are subjected to routine semen analysis following liquefaction at 37°C. Sperm count above 100 million/ml and viability above 60% with normal morphology and rapid and progressive motility is employed for the test.
Ram epididymal sperm suspension (100-200 million/ml) or human ejaculate (100-150 million/ml) are mixed thoroughly in a 1:1 ratio with different concentration of drugs. A drop of the mixture is placed immediately on a slide and at least five fields are microscopically observed under high power (× 400) for assessment of sperm motility. The mixture is then incubated at 37°C for 30 min. and the above process is repeated.
Non-Specific Aggregation Estimation:
Different concentrations of drags are treated with ram sperm suspension in a 1:1 ratio and kept at 37°C for 1 h. One drop of the sedimented sperm is then taken from the bottom of the microcentrifuge tube, placed on a slide and the per cent aggregation examined microscopically under 400x magnification. Since the non-aggregated spermatozoa remain in the supernatant, the latter is collected and the turbidity determined spectrophotometrically at 545 nm. The aggregation is indirectly proportional to the sperm viability.
Sperm Revival Test:
This assay determines the extent of spermicidal and immobilisation capability of drags by evaluating the revival of sperm motility.
To study the revival of sperm motility, after completion of the immobilisation assay, the spermatozoa are washed twice in physiological saline. They are then incubated once again in the same medium free of drag at 37°C for 30 min. to observe the reversal of sperm motility.
Assessment of Plasma Membrane Integrity:
To assess the sperm plasma membrane, integrity ram sperm suspension (100-200 million/ml) or human ejaculated sperm (100-150 million/ml) are mixed with the drug at the minimum effective concentration, at a ratio of 1:1 and incubated for 30 min. at 37°C. Sperm samples mixed with saline in a similar manner serve as controls. For viability assessment, one drop each of 1% aqueous solution of eosin Y and of 10% aqueous solution of nigrosin was placed in a microcentrifuge tube. A drop of well-mixed sperm sample is added to it and mixed thoroughly. The mixture is dropped onto a glass slide and observed under 400x magnification.
For the hypo-osmotic swelling test (HOS), 0.1 ml of aliquot is taken from each of the treated and control sample, mixed thoroughly with 1ml of HOS medium (1.47% fructose and 2.7% sodium citrate at a 1:1 ratio) and incubated for 30 min. at 37°C. The curling tails are examined under phase contrast microscope using 100x magnification.
5-nucleotidase is released possibly due to destabilisation of plasma membrane. This can be estimated to determine the effect of the drug on the plasma membrane integrity of the sperm. The activity of 5′-nucleotidase can be determined by measuring the rate of release of inorganic phosphate from adenosine 5′-monophosphate. After incubating the sperm suspension with the drug, the sperm pellet is collected by centrifugation at 3,000 g at 37°C.
It is then washed twice in 0.9% saline and suspended in 0.1 mol/l Tris-HCl buffer (pH – 8.5) with each reaction system containing (100-200) million spermatozoa. An aliquot of 0.1 ml suspension of sperm is added to 0.9 ml of buffered substrate containing 3 mmol/l adenosine 5′-monophosphate and 50 mmol/l MgCl2 dissolved in 0.1 mol/l Tris-HCl buffer.
The tubes are incubated at 37°C for 30 min. and 0.5 ml 20% TCA (0°C-4°C) is added to the mixture to stop the reaction. The mixture is then centrifuged at 10,000 ×g at 4°C. The pellet is discarded and the supernatant kept for phosphate estimation. The activity of 5′-nucleotidase is expressed in terms of µg of phosphate released. The activity of 5′-nucleotidase is indirectly proportional to the plasma membrane integrity.
Evaluation of Acrosomal Status:
This method evaluates the acrosomal status of sperm. The acrosome is the cap-like structure on the head of the spermatozoa. It breaks down just before fertilisation, releasing a number of enzymes that assist penetration between the follicle cells that surround the ovum. The most widely studied acrosomal enzyme is the acrosin that has been shown to be associated with acrosomes of all mammalian spermatozoa. The highest substrate specificity was obtained with BAEE (N-benzoyl-L-argine ethyl ester).
Different concentrations of drugs are mixed with ram sperm suspension in a 1:1 ratio and kept at 37°C for 1 h. The suspension is centrifuged and the pellets collected. The pellets are extracted with 3 µmol/l HCl at pH 3 and the enzyme activity is measured, following the hydrolysis of 0.5 µmol/l BAEE dissolved in 0.05 mol/l Tris-HCl buffers containing 0.05 mol/l CaCl2 at pH 8.
The activity of acrosin is expressed in terms of mIU. One mIU activity means the amount of enzyme, which causes the hydrolysis of one nano mole of BAEE in one minute at 25°C. The activity of acrosin is directly proportional to the fertilising capability of sperms.
Androgenic and Anti-Androgenic Activities:
Androgenic compounds increase the weight of the testes and seminal vesicles of immature male mice. Anti-androgenic compounds suppress the increase in weight responses of testosterone. This principle is used for the screening of androgenic and anti-androgenic activity.
Immature male mice weighing around 20 g are used for the study. The drugs are administered for seven days alone and along with testosterone. Twenty-four hours after the last dose, the animals are weighed and sacrificed with an over-dose of ether. The testes and seminal vesicles are removed and weighed rapidly in a sensitive balance. The weights are compared with the control group.
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