Stallion AI Services performs tests on all semen collected at the centre to make sure only the best quality semen is used for AI. More indepth evaluations are also carried out on frozen semen and this service is also available for stallion owners wishing to know the quality of their stallions’ semen.
There have been many recent advances in the evaluation of equine sperm over the last decade. This is particularly relevant as the demand for Artificial Insemination to replace natural service increases. Accurate semen evaluation is critical for a successful semen processing laboratory where quality control and quality assurance is paramount to the overall acceptance of equine A.I.
The following summarises some of the standard tests now being applied in professional semen processing laboratories such as Stallion A.I. Services.
Raw Semen Characteristics
When semen is first collected from a stallion, it is important to evaluate the sperm thoroughly so that it can be processed accurately and discarded if it is likely to impede the chance of a successful pregnancy.
Progressive Linear Motility (PLM)
A small amount of semen is placed onto a microscope slide and observed under low power on a phase contrast microscope. A subjective estimate of the number of live sperm swimming in a straight line is recorded. Semen that scores less than 50% PLM is not generally considered suitable for freezing.
Although this test is considered as the industry standard for assessment of semen quality, it is very subjective and often depends on the generosity of the person scoring the sample. More objective tests that score progressive linear motility are available in the form of computer assisted analysis otherwise known as ‘sperm trackers’. The several systems available (such as the ‘Hamilton-Thorne sperm tracker’ and the ‘Cell Motion Analyser’) use a similar concept. They identify sperm that are swimming in a straight line, sperm that are circling and sperm that are motionless and calculate a ratio accordingly.
Sperm morphology can tested using two methods. In the first and simpler the sperm is mixed with a solution of Nigrosin and Eosin, which stains the sperm, a dark purple colour. The dyed sperm are then smeared across a microscope slide and dried so that the sperm are fixed in position.
In the second and more indept the semen is examined using Fluorescent microscopy. Individual sperm are examined under a high powered microscope to identify the presence of abnormal defects.
There are a range of defects that can be exhibited by sperm. The defects affect one of the four domains which make up a sperm cell; namely the head (part of the sperm containing DNA), the mid-piece (part of the sperm containing energy producing mitochondria), the tail (for forward motility) and the acrosome (a separate membrane on the head of the sperm responsible for binding to and fertilising the egg).
Below are examples of some defects that can occur:
· Detached head defect (head is separated from tail)
· Tapered head defect
· Pyriform head defect (spoon shaped heads)
· Cytoplasmic droplet defect (residual cytoplasm on mid-piece, a sign of an underdeveloped sperm cell)
· Bent tail defect (typically caused by a swelling in the tail due to testicular dysfunction).
· Knobbed head defect (a folding of the acrosome membrane)
Sperm characterised by any of the above defects will not be capable of fertilisation.
The number of sperm present in an ejaculate depends on the volume of semen and the concentration of sperm. The volume of semen is either measured with a graduated measuring cylinder or the volume can be calculated by weighing the mass of semen in grams and dividing by 1.04 (average density of equine semen in g/ml).
The sperm concentration can be measured by several techniques:
· Haemacytometer count. There are a variety of different protocols for this technique. The following outlines one of them. A sample of semen diluted 1:20 with sodium fluoride is pipetted into a counting chamber on a thickened glass slide called a heamacytometer. The counting chamber is etched with a grid so that the number of sperm in an exact volume can be counted accurately across the grid. An equation is used to convert the number of sperm counted into a sperm concentration measured in million sperm/ml.
· Spectrophotometer. Semen is diluted 1:200 in a plastic cuvette with a saline solution and placed in a device called a spectrophotometer. The spectrophotometer emits a beam of filtered light at 535nm wavelength, which passes through the diluted sample. The device can be calibrated against haemacytometer counts so that the amount of light absorbed by the sample correlates to the sperm concentration. This is recorded as an arbitrary unit that can be converted into a sperm concentration using a calibration curve.
· Coulter Counter. Sperm are passed through a light source in single file and light sensors accurately count the number of sperm in a sample. This is the most accurate (and probably most expensive) method of calculating sperm concentration.
The sperm count is calculated by multiplying volume with sperm concentration.
Post Thaw Quality Control
Historically the industry standard for sperm quality post thaw is simple, subjective motility evaluation. This is a good indicator of samples that are dead and therefore unlikely to fertilise but that is where the correlation with fertilising ability of a frozen thawed inseminate ends. Since the advent of epi-fluorescent microscopy and flow cytometry, more objective analysis of sperm with better correlation to fertilising ability has become available.
The best evaluation of fertility is still actual pregnancy rate and this can only be based on breeding history of a stallion over a period of time.
The fluorochromes will emit light of various colours (fluorescence) when excited with light at various wavelengths.
This fluorescence can be observed under a fluorescent microscope to identify certain characteristics of the sperm cell.
Below is a range of examples of fluorescent stains that can be used to illuminate specific sperm characteristics:
This dual fluorescent stain can be used as an objective evaluation of live:dead ratio. The 6-CFDA will move across the entire sperm membrane and once inside will undergo esterification. The ester-fluorochrome complex remains locked inside the sperm unless the cell membrane is degenerate (as is the case with a dead sperm) when the stain will be able to leak out of the sperm. Propidium Iodide is a DNA stain that will only move across a dead sperm membrane. When excited with visible blue light the 6-CFDA will fluoresce green and the Propidium Iodide will fluoresce red. The live:dead ratio can be calculated by simply counting the green sperm v the red sperm.
This stain is also useful for observing the morphology of live sperm post thaw, as only the live sperm are green.
Other dual fluorescent stains that will produce a similar assay include:
· SYBR-14/Propidium Iodide (Green live : Red dead)
· Acridine Orange/Propidium Iodide (Green live : Red dead)
· Hoechst 33342/Propidium Iodide (Blue live : Red dead)
PNA is a peanut derivative that will actively bind to the sperm acrosome (the part of the sperm responsible for binding to and fertilising the egg). FITC is a fluorochrome that will in turn bind to PNA and fluoresce green when excited with visible blue light. Therefore, a conjugate of FITC and PNA can be used to stain acrosomes so that they become visible under a fluorescent microscope. The status of the acrosome can then be examined to determine whether or not the sperm are capable of binding to and ultimately fertilising an egg. If the sperm acrosomes are missing or ‘reacted’ then the sperm are known as prematurely capacitated and are not capable of fertilisation.
JC-1 is a fluorochrome that will actively bind to respiring mitochondria in the mid-piece of the sperm and will fluoresce orange when exited with UV light. This is a measurement of mitochondrial function of sperm and relates to motility of the sperm. Impaired mitochondrial function is a hereditary condition and can lead to infertility.
Sperm Chromatin Assay
By staining the DNA with a fluorochrome dye called Acridine Orange, the amount of intact, double stranded DNA can be compared to the ‘unzipped’ single stranded RNA.
RNA fluoresces Orange and double stranded DNA fluoresces Green.
Stallions that exhibit more than 20% single stranded RNA are considered to be sub-fertile.
All of the above tests can be more accurately carried out on a device known as a flow cytometer. Using laser technology and hi-tech light sensors, this device is capable of counting a very large number of sperm (several million) in a short space of time. This leads to a very objective and accurate analysis.
The same technology is used to analyse X-bearing and Y-bearing sperm for sperm sexing.